Drug Screening Methods

Drug Screening Methods

THIRD EDITION

Editor
SK Gupta PhD DSc
Professor Emeritus
Department of Pharmacology
Delhi Institute of Pharmaceutical Sciences and
Research, University of Delhi
Pushp Vihar, Sector-3, New Delhi, India
&
National Advisor
Pharmacovigilance Programme of India (PvPI)

Formerly
Professor and Head
Department of Pharmacology
All India Institute of Medical Sciences
Ansari Nagar, New Delhi (India)
and
Dean and Director General
Institute of Clinical Research, India

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© 2016, SK Gupta

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Drug Screening Methods

First Edition: 2004

Second Edition: 2009
Third Edition: 2016

ISBN: 978-93-5152-982-8

Printed at
 Dedicated to
My Family,
Research Associates and Students...
contributors

Anna Krasilnikova Klaas Poelstra

Faculty of Medicine Department of Pharmacokinetics and Drug
Universiti Teknologi Mara Delivery, University Centre for Pharmacy
Sungai Buloh, Malaysia University of Groningen
Groningen, The Netherlands
Binit Kumar PhD
Kresge Eye Institute, School of Medicine Leonie Beljaars
Wayne State University Department of Pharmacokinetics and
MI, USA Drug Delivery
University Centre for Pharmacy
Deepa Trivedi PhD University of Groningen
HCL Technology Groningen, The Netherlands
Sector 60, Noida (UP), India
Monisha Sharma PhD
E Anand MD MSD Pharmaceuticals Private Limited
Clinigene International Ltd (Merck & Co. Inc)
Bengaluru (Karnataka), India Gurgaon, India
Hanuman Prasad Sharma Nabanita Halder PhD
Dr Rajendra Prasad Centre for Dr Rajendra Prasad Centre for Ophthalmic
Ophthalmic Sciences Sciences
All India Institute of Medical Sciences All India Institute of Medical Sciences
Ansari Nagar, New Delhi, India Ansari Nagar, New Delhi, India

Ipseeta Ray Mohanty PhD Namratta Manhas

Department of Pharmacology Division of Pharmacology
MGM Medical College Central Drug Research Institute
Sector-16, Kamothe Lucknow (UP), India
Navi Mumbai (Maharashtra), India
Niranjan Galpalli PhD
Jagdish Jaiswal PhD Lupin Bioresearch Center
Auckland Cancer Society Research Centre Pashan, Pune (Maharashtra), India
Faculty of Medical and Health Sciences Pradeep Tyagi PhD
The University of Auckland Department of Urology
Auckland, New Zealand University of Pittsburgh
Pittsburgh, PA 15217, USA
Jai Prakash PhD
Indian Pharmacopoeia Commission (IPC) Priya Mathur PhD
Ministry of Health and Family Welfare, GOI School of Optometry
Raj Nagar, Ghaziabad (UP), India University of California
Berkeley, USA
Jai Prakash Bansal PhD
Department of Pharmacokinetics and Drug Rachna Gupta MD
Delivery, University Centre for Pharmacy Department of Pharmacology
University of Groningen University College of Medical Sciences
Groningen, The Netherlands GTB Hospital, Shahdara, Delhi, India
viii Drug Screening Methods

Rajan Mittal MD SK Gupta PhD DSc

Dr Reddy’s Laboratory Ltd Professor Emeritus
Hyderabad (Andhra Pradesh), India Department of Pharmacology
Delhi Institute of Pharmaceutical Sciences and
Rajani Mathur PhD Research, University of Delhi
Department of Pharmacology Pushp Vihar, Sector-3, New Delhi, India
Delhi Institute of Pharmaceutical Sciences and and National Advisor
Research, University of Delhi Pharmacovigilance Programme of India (PvPI)
Pushp Vihar, Sector-3, New Delhi, India
Srinivasan Senthilkumari PhD
Ram Raghubir PhD Department of Ocular Pharmacology
Division of Pharmacology Aravind Medical Research Foundation (AMRF)
Central Drug Research Institute #1, Anna Nagar
Lucknow (UP), India Madurai (Tamil Nadu), India
Ravi Saklani SS Agrawal PhD
Department of Pharmacology Amity University
Delhi Institute of Pharmaceutical Sciences and Sector 125
Research, University of Delhi Noida (UP), India
Pushp Vihar, Sector-3, New Delhi, India
Subrata Pore PhD
Renu Agarwal PhD Department of Urology
Faculty of Medicine University of Pittsburgh
Universiti Teknologi Mara Pittsburgh, PA 15217, USA
Selangor, Darul Ehsan, Malaysia
Sujata Joshi PhD
Rishi Sharma PhD Department of Pharmacology
Division of Pharmacology All India Institute of Medical Sciences
Central Drug Research Institute Ansari Nagar
Lucknow (UP), India New Delhi, India
RK Bhardwaj PhD Suresh L Mehta
Rutgers University Division of Pharmacology
New Jersey, USA Central Drug Research Institute
Lucknow (UP), India
Rohit Saxena MD
Dr Rajendra Prasad Centre for Ophthalmic Sushma Srivastava PhD
Sciences Department of Pharmacology
All India Institute of Medical Sciences Delhi Institute of Pharmaceutical Sciences and
Ansari Nagar, New Delhi, India Research, University of Delhi
Pushp Vihar, Sector-3, New Delhi, India
Shiladitya Sengupta PhD
Harvard Medical School T Velpandian PhD
Brigham and Women’s Hospital Dr Rajendra Prasad Centre for
MIT, Rm 317, 65 Landsdowne Street Ophthalmic Sciences
Cambridge, MA, USA All India Institute of Medical Sciences
Ansari Nagar, New Delhi, India
Shirish Dongare MPharm
Department of Pharmacology V Kalaiselvan PhD
Delhi Institute of Pharmaceutical Sciences and Indian Pharmacopoeia Commission
Research, University of Delhi Ministry of Health and Family Welfare, GOI
Pushp Vihar, Sector-3, New Delhi, India Raj Nagar, Ghaziabad (UP), India
PREFACE to the third edition

I am extremely pleased to introduce the much-awaited third edition of the book Drug
Screening Methods. Both the previous editions of this book were very well received by the
students and professionals from the academia and industry, from across the world. The
book was a pioneering endeavor in 2004 and was written keeping in mind the widespread
need and limited availability of the consolidated screening methods for biological systems
in drug discovery and development. Hence, the techniques of practical importance were
discussed in both the 1st and 2nd editions.
I received numerous valuable suggestions and comments for enhancing the quality of the
book after publication of first edition and accordingly an improved version was published
as the 2nd edition. Subsequently, personal critical evaluation and feedback from readers
encouraged me to take up the areas, which were left untouched in both the previous editions,
and present this third edition of Drug Screening Methods.
Several modifications, additions and deletions have been made in this edition keeping in
view the need of the hour. All chapters have been revised to include the latest key studies and
updated techniques. One new chapter has been written on antiviral drugs other than HIV.
We have again strived to maintain a balance of including the most important information for
researchers while keeping the essential backbone. As before, we strongly encourage readers
to refer to the primary literature for further details and references not included here. I hope
that the clarity of the text makes up for any limitations in its comprehensiveness. I hope
that like previous editions, the readers will benefit and appreciate this edition of the book
and Drug Screening Methods will continue to be an invaluable resource for postgraduate
students, industry professionals and others engaged in the research and drug development
at various laboratories.
I would be failing in my duty if I do not express my sincere thanks to Dr Sushma Srivastava,
Senior Scientist, for having taken up the responsibility with a smile on her face at all the
times and for her editorial assistance from the stage of 1st edition to present 3rd edition of
this book. She coordinated effectively and efficiently in bringing out this book.
I am grateful to all the contributing authors, including the new ones, who made an
enterprise of this magnitude possible due to their hard work and dedication. As an editor,
I express my heartfelt gratitude to the contributors of the chapters. Their names and positions
are mentioned.
The publication of the book has been possible due to the hard work put in by the staff
of Jaypee Brothers Medical Publishers, New Delhi. The good work done deserves our
appreciation to Shri Jitendar Vij, Group Chairman of Jaypee Brothers Medical Publishers,
one of the largest Medical Publishers in the world.

SK Gupta
PREFACE to the first edition

Drug discovery and development is a challenging field of research requiring the

synchronized efforts of medicinal chemists, natural chemists, pharmacologists, toxicologists
and clinicians, to name a few. The ultimate target of this team is to generate a safe and
biologically active drug that is capable of stalling, if not reversing, the pathological events
leading to disease condition. In the drug discovery program, the battery of tests and assay
systems that evaluate the efficacy and safety of the novel molecule in biological system
occupy the center stage. Thus, these assays or ‘screening methods’ are critical as it is on
their basis that the molecule sees the light of the day or is consigned to obscurity.
Advances in the field of drug discovery program has seen the graduation of simple
screening methods of yore to automated methods utilizing molecular techniques spanning
across in vitro, in vivo and clinical systems. There has been an explosion of available
information making it essential for the professionals working in this area of research to keep
pace with the advancing times. It has become an uphill task for researchers focusing on a
particular field to scan realms of literature regarding developments in other fields. This often
curtails synchronized research and delays results. At this juncture, it has become of utmost
importance to consolidate our present knowledge and review its validity and ascertain the
future direction.
In view of this widespread need and limited availability of consolidated screening
methods for biological systems, the Herculean task of penning a book on Drug Screening
Methods was undertaken. It is hoped that the First Edition of this book will enjoy a broad
readership ranging from professionals to students of pharmacology.
Keeping in tune with the practically used techniques used in both academia and
pharmaceutical industry, this book outlines numerous methods utilizing molecular and
fluorescence techniques. A complete section has been devoted to the concept and basic
techniques involved in novel drug discovery program. It is hoped that this will at least
provide a theoretical exposure to students of pharmacology who may tomorrow be in a
position to use them practically. Special attention has been given to novel methods based
on genetically modified animals that simulate the human condition as these help in not
only understanding the pathogenesis, but also develop targets for therapeutic modalities.
Additionally, an overview has been provided for major fields of investigations like drug
absorption and metabolism, cancer biology, angiogenesis, apoptosis, AIDS and autoimmune
disorders.
As a pharmacology teacher for over 40 years now, I have always felt the need for
simple screening procedures that explicitly demonstrate the nuances of sympathetic,
parasympathetic nervous system and gut motility to the students, and I hope by way of this
xii Drug Screening Methods

book, this lacuna has been addressed. As a researcher, the fields of ocular and cardiovascular
pharmacology have been very close to my heart and special care has been taken to develop
chapters regarding these areas. Considering the renewed interest in outsourcing novel
drugs from natural source, a separate chapter dealing with assay procedures for screening
the pharmacological activity of herbal drugs has been written.
Every drug has a therapeutic effect, that is desired, along with an unwanted side effect.
This book would be incomplete and lend a myopic view to drug discovery program without
the methods for assessing safety of novel molecules. Toxicity studies are critical as it is on
their basis that the toxicity and efficacy profile of a novel molecule can be weighed and its
future determined.
It is obvious that an enterprise of this magnitude has been possible due to the cooperation
and assistance of a large number of individuals across the globe. As the editor, I express
my heartfelt gratitude to the authors of the chapters, dedicated team of editorial board and
the production staff at M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, who all
joined to convert this dream project into reality.
SK Gupta
CONTENTS

1. Newer Tools for Drug Screening 1-19

SK Gupta, Rajani Mathur
Cassette Dosing 1
Virtual Screening 5
Microassays 5
Application of Modern Analytical Techniques in Biological Systems 9
Cell Free Assays 11
Microbe-based Screening Assays 11
Receptor Screens 12
Nano Screens 12

2. High Throughput Screening for Drug Discovery 20-38

T Velpandian, Hanuman Prasad Sharma
What is High Throughput Screening (HTS)? 20
HTS—Positive and Negative Screening 21
In Vitro Interaction Studies (Array Based) 21
Fluorescence Techniques in HTS 22
Flow Cytometry for Cell-based Assay 24
Role of Mass Spectrometry in the High Throughput Screening 25
In Vivo Imaging of Drug Action (Near Infrared Imaging) 26
Virtual Screening (In Silico Drug Development or Dry Lab) 27
HTS-Scintillation Methods in Drug Screening 28
Unconventional Rapid Pharmacological and Toxicity
Testing Models 29
Hts Pharmacokinetic Studies 31
Parallel Artificial Membrane Permeability Assay (PAMPA) 31
High Speed Drug Synthesis Process ‘Combinatorial
Chemistry’ (Combichem) 33
Natural Products Combichem—A Newer Dimension 34
Other Roles of HTS 35

3. Models for Studying Stem Cell Therapy 39-70

Rishi Sharma, Ram Raghubir
Stem Cells: An Introduction 39
Types of Stem Cells 39
xiv Drug Screening Methods

Stem Cell Therapy for Neurological Disorders 43

Stem Cell Therapy for Cardio- and Cerebrovascular Disorders 53
Conclusion and Future Perspectives 55

4. Antistroke Agents 71-87

Suresh L Mehta, Namratta Manhas, Ram Raghubir
Types of Stroke 72
Selection of Model 73
Target Selection for Drug Screening 77
Methods of Assessment 79

5. Anti-HIV Agents 88-103

Anna Krasilnikova, Rajan Mittal, Rachna Gupta
In Vivo Models 88
Nonhuman Primate Models for AIDS 88
Severe Combined Immunodeficiency (SCID) Mice Models 91
Mo-MSV Induced Tumors in Nmri Mice 94
Transgenic Animal Models 94
In Vitro Models 95
In Vitro Model Systems for HIV in the CNS 98
DIV-BBB Model 99

6. Angiogenesis 104-120
Ipseeta Ray Mohanty, Shiladitya Sengupta
Tumor Angiogenesis 104

7. Sexual Dysfunction 121-131

T Velpandian, Rajani Mathur
Models for Male Erectile Dysfunction 122
Animal Models for Female Sexual Dysfunction 128

8. Antifertility Agents 132-151

SS Agrawal
Methods for Females 132
Methods for Males 145

9. Antiobesity Agents 152-169

Rachna Gupta
Animal Models for the Study of Antiobesity Drugs 152
In Vitro Assays on Isolated Adipose Tissue Cells 161
 Contents xv

10. Anticancer Agents 170-190

Jai Prakash, SK Gupta
In Vitro Methods 171
In Vivo Methods 177
Methods Involving Cell Line/Tumor Pieces Implantation 184

11. Screening Methods for Renal and Liver Fibrosis 191-208

Jai Prakash Bansal, Leonie Beljaars, Klaas Poelstra
The Pathogenesis of Renal Fibrosis 191
The Pathogenesis of Liver Cirrhosis and the Crucial Role of
Hepatic Stellate Cells (Hsc) 192
Models of Renal Fibrosis 193
Models of Liver Fibrosis 198

12. Techniques for the Detection of Apoptosis 209-225

Ipseeta Ray Mohanty, Deepa Trivedi
Proapoptotic and Antiapoptotic Agents 209
Categories of Cellular Changes that Form the Basis of
Apoptosis Assays 210
Detection of Apoptosis Based on Morphology 210
Detection of Dna Fragmentation 213
Terminal Deoxyribonucleotidyl Transferase–Mediated
dutp Nick End Labeling (Tunel) 216
Flow Cytometry 216
Annexin V Staining 218
Alterations in Plasma Membrane Permeability 219
Enzyme Assays 220
Recent Advances 221

13. Anticataract Agents 226-244

Sushma Srivastava, Sujata Joshi
Experimental Models of Cataract 226
In Vitro Models 227
In Vivo Models 233

14. Evaluation of Pharmacological Activity of Herbal Medicines 245-253

Rajani Mathur

15. Agents for Immune-based Disorders 254-265

Rajani Mathur
xvi Drug Screening Methods

16. Antihypertensive Agents 266-277

Ipseeta Ray Mohanty
In Vitro Models 266

17. Antiglaucoma Agents 278-291

Sushma Srivastava
Experimental Models of Glaucoma 280
Other Models 287

18. Antiarrhythmic Agents 292-304

Ipseeta Ray Mohanty
Cell Culture Technique 292
In Vitro Models 293
In Vivo Models 295

19. Cardiotonic Agents 305-320

Ipseeta Ray Mohanty
In Vitro Method 306
In Vivo Models 307
Developing New Therapeutic Targets in CHF 316

20. Antianginal Agents 321-333

Ipseeta Ray Mohanty
In Vitro Models 321
In Vivo Models 325

21. Antiplatelet Agents 334-345

Pradeep Tyagi, V Kalaiselvan
Antiplatelet Agents 334
Importance of Antiplatelet Drugs 335
Targets for Newer Drugs 335
In Vitro Screening Methods 336
In Vivo Screening Methods 340

22. Drugs Acting on Sympathetic Nervous System 346-359

Renu Agarwal, Rachna Gupta, RK Bhardwaj
In Vivo Methods 347
In Vitro Methods 351

23. Drugs Acting on Parasympathetic Nervous System 360-368

Renu Agarwal, Rachna Gupta, RK Bhardwaj
In Vivo Methods 361
In Vitro Models 364
 Contents xvii

24. Neuromuscular Blocking Agents 369-379

Renu Agarwal, Priya Mathur
Evaluation in Laboratory Animals 370
Evaluation in Man 375
Characterizations of Depolarizing and Nondepolarizing
Blockers 377

25. Antiviral Drugs 380-392

Anna Krasilnikova
In Vitro Models 381
In Vivo Models 386

26. Antipsychotic Agents 393-403

Rajani Mathur
In Vitro and Ex Vivo Models 394
In Vivo Models 395
Computational Models of Dopamine Function
and Psychosis 401

27. Antidepressant Agents 404-411

Rajani Mathur
In Vivo Methods 405

28. Antiepileptics 412-435

Ipseeta Ray Mohanty, Rajan Mittal
In Vitro Methods 413
In Vivo Methods 418

29. Anti-Alzheimer Agents 436-452

Rajani Mathur, Monisha Sharma
In Vitro and Ex Vivo Models 441
In Vivo Models 444
Behavioral Paradigms to Assess Learning and Memory 446

30. Antiparkinsonian Agents 453-467

Rajani Mathur
In Vitro and Ex Vivo Models 455
Behavioral Models of Parkinsonism 457

31. Antimigraine Agents 468-475

Rajan Mittal, Sushma Srivastava
In Vitro Models 469
In Vivo Models 471
xviii Drug Screening Methods

32. Analgesic Agents 476-494

Rachna Gupta
Mechanism and Modulation of Pain/Nociception 476
Animal Models of Acute Pain 477
Models Using Thermal Stimulus 477
Models Using Electrical Stimulus 479
Models Using Chemical Stimulus 480
Models Using Mechanical Stimulus 482
Animal Models of Chronic Pain 483
Models of Cancer Pain 486
In Vitro Methods 487
Role of Vasoactive Intestinal Polypeptide (Vip)
and Pituitary Adenylate Cyclase- Activating
Peptide (Pacap) and Nociceptin in Analgesia 490

33. Anti-inflammatory Agents 495-513

Pradeep Tyagi, Subrata Pore, Jai Prakash
In Vitro Methods 498
In Vivo Methods 501

34. Ocular Inflammation 514-526

Sushma Srivastava, Rohit Saxena
Experimental Models for Ocular Inflammation 515

35. Antiulcer Agents 527-539

Jai Prakash, Rajan Mittal, RK Bhardwaj
In Vitro Methods 527
In Vivo Methods 529

36. Agents Affecting Gut Motility 540-545

Rajan Mittal, RK Bhardwaj
In Vitro Models 540
In Vivo Models 543

37. Antiemetic Agents 546-558

Renu Agarwal
In Vivo Models 547
In Vitro Models 555
 Contents xix

38. Absorption and Metabolism 559-568

Jai Prakash, RK Bhardwaj, Rajan Mittal
Absorption Studies 559
In Vitro Models 559
In Situ Model 562
Metabolism Studies 563
In Vitro Models 563

39. Hormones of Pituitary, Thyroid, Parathyroid,

Adrenal Cortex, Ovary and Testes 569-612
Nabanita Halder, Renu Agarwal
Growth Hormone 569
In Vitro Studies 570
Immunoassays 573
Immunofunctional Assay (IFA) 574
In Vivo Methods 574
Prolactin 576
Corticotropin 577
In Vitro Studies 578
In Vivo Models 579
Gonadotropins 581
Oxytocins 582
Vasopressin 584
Thyroid Hormones 586
In Vitro Methods 586
In Vivo Methods 587
Parathyroid Hormone 588
In Vitro Methods 589
In Vivo Methods 589
Adrenal Steroids 591
Glucocorticoids 592
In Vitro Methods 592
In Vivo Methods 594
Mineralocorticoids 596
In Vitro Methods 596
In Vivo Methods 596
Androgens 597
In Vitro Methods 597
In Vivo Methods 598
xx Drug Screening Methods

Progesterone 600
In Vitro Methods 601
In Vivo Methods 601
Estrogens 604
In Vitro Assay 604
In Vivo Methods 605

40. Antidiabetic Agents 613-644

Binit Kumar, Shirish Dongare, E Anand
Models for IDDM 614
Models for NIDDM 619
Transgenic and Knock-out Animals 623
Normoglycemic Animal Models 625
In Vitro Methods on Isolated Organs, Cells and Membranes 627
Models to Study Insulin Secretion from β Cells 629
Experimental Models for Diabetic Complications 634

41. Diabetic Retinopathy 645-658

Sushma Srivastava, Rajani Mathur, Binit Kumar, Ravi Saklani
Magnitude of Disease 647
Present Therapy for Diabetic Retinopathy 647
In Vitro Models 648
Ex Vivo Models 650
In Vivo Models 650

42. Ocular Toxicity 659-670

Deepa Trivedi, Sushma Srivastava
In Vivo Irritation Testing 659
In Vitro Methods 662

43. Phototoxicity 671-682

Jagdish Jaiswal, Niranjan Galpalli
Alternative Designs in the In Vivo Methods 674
Photosensitization Models 675
In Vitro Assays 680

44. Anti-asthmatic Agents 683-696

Ipseeta Ray Mohanty
In Vitro Models 684
In Vivo Models 687
 Contents xxi

45. Cell-Based Assays in High-Throughput Screening of Drugs 697-708

Srinivasan Senthilkumari
Types of Cell-based Assays 697
Components of Cell-based Hts Assays 698
Types of Cell Culture Systems 699
Detection Methods 701
Recent Developments in 3D Cell-based Hts Assays
for Drug Discovery 705
Cell-based Hts in Commercial Drug Development 705

Index 709
 CHAPTER

1
Newer Tools for Drug Screening
INTRODUCTION
The last quarter of the century has seen transformation of the pharmaceutical industry. The
advances in the field of information technologies have driven basic research to evolve into
fast-track drug discovery and development program that is target oriented while still beating
the clock. There is competition within the industry to continuously generate the next billion-
dollar selling compound. Market estimates project that the journey of a new molecule from
laboratory to market takes well over seven years at an average cost of over $600 m. Conse­
quently there is thrust on developing advances in technologies aiming to reduce the time
and the costs involved in bringing a new drug to market. This has lead to the introduction of
revo­lutionary technological advances such as combinatorial chemistry, biochemical assays,
genomics, proteomics, miniaturization, automation, robotic systems and computerization,
which have cumulatively increased the speed of lead generation manifold.
Just to cite a case, by using way of traditional drug development techniques, it took nearly
half-a-century to tap the cholesterol biosynthesis pathway and develop statin drugs, as
cholesterol lowering agents. On the other hand, molecular-revelations regarding the role of
the HER-2 receptor in breast cancer led to the development of the chemotherapeutic agent,
Herceptin® within three years. The advanced techniques of in silico molecular modeling, high
throughput screening and genomic and proteomic databases, were instrumental in the quick
discovery and development of Herceptin.1
Some of the screen paradigms that are being rigorously adopted by pharmaceutical
companies to speed up the development of next blockbuster drug are discussed in this chapter.

CASSETTE DOSING
As compared to generation of 100 leads that were produced a decade ago, combinatorial
chemistry now enables the chemist to produce thousands of leads each year.2 The problem
is no longer of too few drug candidates emerging from the discovery process – in fact the
number of quality lead compounds that emerge is much higher. It is being understood
that rather than drug discovery, it is drug development that is proving to be the bottleneck
(Fig. 1.1). The battery of preclinical tests such as toxicity, bioavailability and pharmacokinetics
are extremely critical, time-consuming and costly. Prioritization of leads with regard to
2 Drug Screening Methods

Figure 1.1: Bottleneck at drug development stage reducing the speed of generation of novel drugs

these studies becomes a stumbling block in the selection of viable targets from millions of
compounds. Due to several constraints, classical methods are not high throughput and have
often impeded the quick development of new drug candidates. This has generated need for
high throughput drug development program to be set-up and started in full throttle.3
Although pharmacokinetic evaluation is an essential component of drug discovery program,
it has proved to be a serious bottleneck during the ‘hit-to-lead’ and lead optimization phases
of drug discovery and development program. The prioritization of leads with regard to drug
metabolism and pharmacokinetics (DMPK) assumes importance as it determines the selection
of viable targets from millions of compounds. A compound with favorable pharmacokinetics
is more likely to be efficacious and safe. Early elimination of pharmacokinetically ineligible
candidates helps to sift grain from chaff. For this critical juncture in drug development program,
absolute reliance on in vitro tests cannot be advocated. As in vitro tests can never replicate the
complex biological system, they serve as confirmatory tests rather than preliminary tests.4
Rodent species (mice, rats) have been classically used for in vivo pharmacokinetic studies.
As there is a limitation to the amount of blood samples that may be withdrawn per animal,
different animals have to be used for each time point, leading to high animal usage. In addition,
inter-individual variation of various pharmacokinetic parameters is commonly encountered
(up to two-fold) due to the differences in the expression of drug metabolism enzymes and
genetic polymorphism. These further complicate data interpretation and slow the progress.3
Advances in drug development technology have provided the solution to the problems by
providing alternate methods that help the discovery scientists to predict pharmacokinetics
within the constraints of real physiological environment, but at the pace of in vitro or in
silico methods. Cassette dosing is an elegant, inexpensive, nonlabor/time intensive novel
technique that has been developed with the aim to rapidly assess pharmacokinetics of a large
number of compounds (Table 1.1). Scientists at Glaxo Wellcome have been the pioneers in its
development.3
 Newer Tools for Drug Screening 3

In theory, cassette dosing or CD or ‘N-in-one dosing’ involves the simultaneous

administration of several compounds (5-10) to a single animal followed by rapid sample
analysis for the compounds and their metabolites by liquid chromatography/tandem mass
spectrometry. It is a highly recommended technique as it enhances the efficiency in terms of
time, money, manpower while reducing animal usage.3

Table 1.1: Estimated reduction in preclinical screening time after adopting cassette dosing
Stage of drug discovery program Currently time taken (months) Estimated time taken with
cassette dosing (months)
Preclinical 15 12
Phase I 18 14
Phase II 22 12
Phase III 31.5 17.5
Total estimated time 7.2 years 4.5 years

Cassette dosing is based on the concept of serial bleeding that involves withdrawal of
blood samples from the same animal for all the time points, which is estimated to not only
dramatically reduce the number of animals used but also increased the quality of the kinetic
data for compounds compared in that animal. Furthermore, the volume of blood samples that
are withdrawn/animal/time point is miniscule. Literature search brings to fore descriptions
of analytical methods that have used only 10-20 μl of whole blood using capillary LC/MS/
MS. The onus for the success of this approach has to be granted to the superlative technique
of LC/MS/MS, as it would not have been possible with other classical methods of analysis
(spectrophotometry and chromatography).
Cassette dosing poses an analytical challenge as it involves simultaneously assaying many
compounds in a single sample. In the absence of theoretical guidance, a set of intuitive
assumptions has developed regarding the nature of the errors and how to avoid them.3 These
assumptions are:
1. Drug-drug interactions only occur when one of the dosed compounds is a potent inhibitor
of drug-metabolizing enzymes.
2. One may guard against competitive inhibition of a shared metabolic enzyme by keeping
doses small.
3. The size of the cassette (n) is limited only by the sensitivity of the assay and the solubility of
the compounds.
4. Errors can be detected by including a benchmark compound with known pharmacokinetic
characteristics.
5. Drug-drug interactions can lead only to false positives, which will be discovered later, and
6. Even if the absolute values are wrong, the correct rank order will be observed.
Although cassette dosing is an advantageous technique in terms of resources and
throughput, there are possible complications associated with this approach. The technique
of cassette dosing has been under critical review and is subject to high level validation. While
using the technique of cassette dosing, inherent limitations of the technique have to be
accounted for. Firstly, the potential for compound interactions is increased manifold. 5
4 Drug Screening Methods

Although cassette dosing has been reported to yield useful results when used as a screen,
especially to rank-order drug candidates, it has been shown to be fraught with both theoretical
and experimental large errors. Consequently, under no circumstances can the pharmacokinetic
parameters derived from cassette dosing be accepted as accurate. Potentially affected
parameters include F, CL, AUC, t½, mean residence time, Vd. High-clearance compounds have
the greatest potential for screening errors (i.e., false-positive, false-negative). To detect errors,
a second dosing episode could be opted, that may in itself defeat the productivity gained from
cassette dosing.5
A better way to detect errors is to include a benchmark compound with known in vivo
pharmacokinetics. To minimize the potential for errors, one should use the smallest doses
detectable and keep the total number of co-administered compounds small.4
Even the looming limitation of drug-drug interactions entailed with cassette dosing is being
overcome. With advances in IT and availability of predictive databases, approaches such as
structure-metabolism relationship (SMR) are gaining importance. Knowledge about ligand
structure, ligand-active site interactions and stereoelectronic factors involved in metabolic
transformations, the metabolic pathways that may be involved and corresponding potential
metabolites formed can be predicted. Recently, METAPRINT, a metabolic fingerprint has
been developed to facilitate in the design of cassette dosing experiments. These approaches
will supplement cassette dosing and go a long way in reducing any confounding information
especially with regard to drug-drug interactions in cassette dosing.6
In another development, a novel method of serial bleeding has been developed to withdraw
blood samples from the same animal for all the time points in a pharmacokinetic study. This
helps to not only dramatically reduce the number of animals used, but also increased the
quality of the kinetic data for compounds compared in that animal. Following protocol has
been developed with the approval of Animal Use and Care Committee for conducting in vivo
experiments.
Male Swiss Webster mice, 7 weeks old (body weight 28-36 g), are used for pharmacokinetic
studies. Using a stratified randomization procedure the animals are either administered
vehicle, standard or test drug. The route of drug administration may be oral, intraperitoneal
or subcutaneous, as per the protocol. After administration, serial tail bled blood samples
(5 µl) are collected using heparinized tip at various time points (5 min to 24 h). The samples are
transferred to a microcentrifuge tube, weighed with an analytical balance and vortexed with
purified water and internal standard. 7
The samples are extracted with organic solvent (ethyl acetate, methanol or acetonitrile). The
organic layer is transferred to a microcentrifuge tube, and dried under nitrogen. The residues
can be reconstituted in minimum volume (up to 25 µl) of appropriate solvent (methanol).
Aliquots are injected onto LC/MS/MS system for analysis.7
The technique has the following distinct advantages:
•• Minimizes the number of animals used
•• Significantly reduces trauma to the animal that is associated with sample withdrawal
•• Marked reduction in inter-individual variation in pharmacokinetic parameters
•• Reduces the amount of drug used
•• Sample processing time is markedly minimized.
 Newer Tools for Drug Screening 5

However, care has to be taken, that the small animals are handled with care as serial bleeding
may alter the physiological state of the animal. For example, micro sampling increases the
amount of inflammatory eicosanoids in blood and may decrease the proportion of cellular
components in a sample.7

VIRTUAL SCREENING
The rate of synthesis of compounds has increased exponentially owing to the advances in
combinatorial chemistry. These compounds have been housed in huge virtual libraries.
Numerous such databases have been created, each housing over 109 compounds, in each. The
obvious question, which arises, is how can this enormous database be filtered to bring forth
compounds of utility? To achieve this goal, miniaturized and automated assays have been
developed that limit cost, material, time and manpower requirement. As a natural extension
high speed computer systems running specialized softwares have been developed that are
capable of screening the molecules from the libraries against identified targets.8
As the first step the appropriate technique of X-ray crystallography, Nuclear Magnetic
Resonance (NMR) are used to determine the 3-D structure of the macromolecular target.
This is followed by application of 2D QSAR (2-Dimensional Quantitative Structure Activity
Relationship), wherein, the chemical structure is quantitatively correlated against a biological
activity so as to predict its biological activity. The compounds are superpositioned on the target
site as a function of energy and potential. On the basis of this screen, compounds exhibiting
favorable kinetics are selected for “fine tuning” and the rest are eliminated.
While searching a virtual library, maximum output can be generated, if information under
following heads is available:
•• Information about other known ligands (substrates, agonists, antagonista, etc.) that are
bioactive at the target.
•• Detailed structural and functional information about the target site on the site, binding
thermodynamics, etc.
•• Lastly, a thorough knowledge in rules of conformational analysis and a medicinal chemistry
‘instinct’ proves to be beneficial.8

MICROASSAYS
Modern chemical and biological techniques have revolutionized the synthesis of new
chemical entities, and have set a pace that was unimaginable with traditional methods of
synthesis.
9-12
With the advancements in modern science and DNA recombinant biotechnology,
various novel and sensitive procedures have been developed to determine the side effects,
pharmacological action, toxicity, and efficacy of biologically active and clinically important
compounds, derived from herbal origin or prepared synthetically. This has provided a much
needed impetus to drug development program. Conventional bioassays have been replaced
by sensitive ELISA, reverse transcriptional polymerase chain reaction (RT-PCR), ribonuclease
protection assays, cDNA microarrays, etc. some of which are briefly described below.
6 Drug Screening Methods

Radioimmunoprecipitation
This elegant procedure is employed to quantitatively estimate the gene expression at the
translation level using radiolabeled 35S-methionine.13-15 The labeled sample is generated
as radioimmunoprecipitate which is counted above background using liquid scintillation
counter.8

Immunoblotting
SDS-polyacrylamide gel electrophoresis is performed to study protein, enzyme, neuro­
transmitter or hormone expression of the immunoprecipitated lysates. Slab gel electrophoresis
is performed and the autoradiograms are densitometrically analyzed.13

RNA Extraction
Cellular monolayer is trypsinized to detach from the bottom of the flask and the cell pellet
is obtained by centrifugation. The pellet is suspended in guanidine isothiocyanate (GITC)
solution (composition: 0.1 M dithiothretol, 4 M guanidine isothiocyanate, 0.5% (v/v) N-lauryl
sarcosine, 20 mM sodium acetate, pH 4.0) and treated according to standard protocol. RNA is
pelleted by centrifugation. One µl of the purified RNA sample is diluted to 1 ml. Readings are
taken at 260 nm and 280 nm to determine the A260/A280 ratios (pure RNA provides a ratio
between 1.6 and 1.8). RNA is resolved in 1% agarose gel containing ethidium bromide at a
current strength of 30 mA for 2-3 h (80 volts for 1 h). The gels are visualized on UV eluminator.

Reverse Transcription (First Strand cDNA Preparations)

Reverse transcription of 1 µg of RNA is conducted using either 50-100 ng of poly(A) mRNA
or 5-10 µg of total RNA. The volume is adjusted to 38 µl with DEPC-treated water. Three µl of
oligo-dT primers (100 ng/µl) or 3 µl of random primers (100 ng/µl) are added and the contents
are mixed gently. Both control and experimental tubes are incubated at 65°C. The tubes are
cooled slowly at room temperature (10 min) to allow primers to anneal to RNA. First strand
cDNA is synthesized by adding the following reagents in the control and experimental tubes
in sequence. Five µl (10X) first strand buffer, 1 ml of RNase block (Ribonuclease inhibitor,
40 U/µl), 2 µl of 100 mM dNTPs. One µl of MMLV-RT (50 U/µl). The tubes are mixed gently and
incubated at 37°C for 1 h followed by incubation at 90°C for 5 min. The first strand cDNA is kept
on ice for use in PCR amplification protocol.

Amplification of First Strand cDNA

One to five µl of first strand cDNA is transferred in autoclaved 500 µl PCR tubes. In control
PCR amplification reaction tubes, 10 µl of Taq-DNA polymerase buffer is added along with
0.8 µl of 100 mM dNTPs, 3 µl of control primer set (100 ng/µl) and double distilled water to
adjust the final volume to 99.5 µl. In the experimental PCR reaction mixture following reagents
are administered in a sequence, 10 µl of 10X Taq DNA polymerase buffer, 0.8 µl of 100 mM
dNTPs, 2 µl of 10 µM oligonucleotide (primer 1: Forward: and 2 µl of 10 µl of oligonucleotide)
(primer 2. Reverse) primers are added. The final volume of the reaction mixture is adjusted
to 99.5 µl. Both control and experimental amplification reaction mixture tubes are placed
in a DNA Thermal cycler. Each PCR amplification reaction is heated to 91°C for 5 min and
 Newer Tools for Drug Screening 7

then immediately cooled at 54°C for 5 min. This step is essential to maximize thermal cycling
performance. The control and experimental PCR amplification reaction tubes are removed
from the Thermal cycler, briefly microcentrifuged and 0.5 µl of Taq-2000 (DNA polymerase)
(5 U/µl) to each reaction tube is added. The reaction tubes are briefly centrifuged again. The PCR
amplification reaction mixture is overlaid with a drop of mineral oil to prevent evaporation of
reaction components during thermal cycling. The PCR amplification reaction tubes are placed
in the thermal cycler and processed for amplification. For PCR amplification, programmed
thermal cycler is used and experimental parameters are established that are optimal for the
oligonucleotide primer set employed. Amplification is usually done depending on the primer
length, GC content, and its sequence (usually 25-40 cycles of denaturation for 1 min at 94°C,
annealing for 1 min at 54°C, and extension of 2 min at 72°C). The final reaction is done using
72°C for 10 min to complete the amplification. The reaction products are kept at 6°C before the
analysis. Ten µl of each PCR amplification reaction is taken from below the mineral oil layer
into separate lanes of 1.2% agarose. One Kb DNA ladder is used as molecular weight marker.
The amplified products are analyzed densitometer.

Cell Transfection
Cell transfection studies are conducted on healthy cells at subconfluent stage. Usually we have
used the cells between 4-5th passage. One µg of antisense oligonucleotide to µ-synuclein:
5’-CCT-TTT-CAT-GAA-CAC-ATC-CAT-GGC-3’, Reverse Sense: 5’-GCC-ATG-GAT-GTG-TTC-
ATG-AAA-GG-3’; Scrambled: 5’-TAG-CTC-GCT-ACG-TAA-TCA-CCA-CT-3’. Metallothionein-1
antisense: CAC-AGC-ACG-TGC-ACT-TGT-CCG-CCG-CCG-CTT-TGC-AGA-CAC-AGC-C, MT-1
Forward: GTT-CGT-CTC-ACT-GGT-GTG-AGC, MT-1 Reverse: AAA-AGA-AAT-CGA-GGA-
AAT-GGC (GIBCO/BRL Life Technologies, USA), mixed with 8 µl of enhancer, and 25 µl of
Effectine transfection reagent as per manufacturer’s recommendations. The transfected cells are
authenticated using Radioimmunoprecipitation, immunoblotting, and RT-PCR using specific
primer sets of genes. Spontaneous and drug-induced apoptosis is studied using heat shock,
staurosporine (1 µM), serum deprivation, ceramide or other apoptogens including toxic drugs.

Multiple Fluorochrome Comet Assay

This sensitive assay is performed to determine mitochondrial and nuclear DNA damage
simultaneously in a single cell in response to various environmental neurotoxins or physio­
logical stress. It can provide basic information regarding condensed, partially condensed,
partially fragmented and fully fragmented DNA based on the charge associated with each
molecular species of DNA. Thus, this procedure provides information regarding single cell
apoptosis. Multiple fluorochrome Comet assay provides more quantitative information
regarding the extent of genotoxicity of a compound and that can be determined by
quantitatively estimating the Comet tail length, tail intensity, and tail diameter. This procedure
is particularly useful to determine the levels of DNA damage in an alkaline medium and is
employed particularly in the field of genotoxicology. It is a convenient and more sensitive
method for multiple processing and drug screening. It is an assay at the inter-phase between
molecular biology and cellular biology and is little more sensitive and specific as compared to
conventional DNA fragmentation assay performed on agarose gels. This method is very useful
in comparative pharmacological analysis of excitoneurotoxins as well as drugs.14
8 Drug Screening Methods

Triple Fluorochrome Analysis

Triple fluorochrome analysis is conducted to assess various stages of apoptosis at the plasma
membrane and cytoplasmic level, using 100 nM acridine orange, which stains specifically
RNA and proteins, and is very useful to detect apoptotic bodies; to estimate mitochondrial
membrane potential and mitochondrial apoptosis, JC-1 and decifer are employed, whereas
nuclear apoptosis is detected by fluorochrome.
The neurons are grown in eight-chambered microscopic slides, exposed to either toxins or
drugs overnight for seven days, and incubated at 37°C for 45 min in a mixture of fluorochromes.
The monolayer is washed with Dulbecco’s phosphate buffered saline and the cellular monolayer
is mounted, air-dried in the dark chamber, and observed under Fluorescence microscope,
equipped with immunofluorescence imaging system. The fluorescence images are digitized
using Digital camera and analyzed. For obtaining a detailed analysis of apoptosis and its
intermediary events, images captured with different filters are combined and plotted.15,16

Multiprobe Ribonuclease Protection Assay (MRPA)

MRPA is a highly sensitive procedure to simultaneously quantify several mRNA species in
a single sample of total RNA and can be used for comparative analysis of different mRNA
species, which can be compared between samples. MRPA can be performed on total RNA
preparations by standard methods from either frozen tissue or cultured cells without further
purification of polyA+ RNA. It is highly sensitive procedure for the detection and quantification
of gene transcripts, induced in response to neurotoxic insult. It was discovered based on the
knowledge about DNA-dependent RNA polymerase from bacteriophage SP6, T7 and T3 and
information of their promoter sequences. To synthesize high specific activity RNA probes from
DNA templates as they have high degree of fidelity for the promoters, polymerize RNA at high
rates, efficiently transcribe long segments, and do not require high concentrations of rNTPs.
So a cDNA fragment of interest can be subcloned into a plasmid that contains bacteriophage
promoters and the construct can be used as a template for the synthesis of radiolabeled and
antisense RNA probes. We use T7 polymerase-directed synthesis of high specific activity
32P labeled anti-sense RNA probe set. The probe set is hybridized in excess to target RNA
in solution after which free probe and other single stranded RNA are digested with RNase.
The remaining RNase protected probes are purified, and resolved on denaturing PAGE and
quantified by autoradiography or phosphor imaging. Quantity of each mRNA species in the
original RNA sample can be determined based on the intensity of the properly sized-protected
probe segment. The procedure takes usually three days.
Day 1: For probe synthesis and overnight hybridization, Day 2: RNase treatment, purification
of protected probe, and Gel Electrophoresis, and Day 3: Autoradiography or phosphor imaging.

cDNA Microarrays for Differential Gene Expression

This is a relatively new research procedure employed to study differential multiple gene
expression under the influence of drugs, neurotransmitters, enzymes, or hormones, etc. under
investigation. As many as 30,000 genes can be analyzed with this procedure. cDNA Microarray
scanning requires cDNA microarray scanner with computer software to investigate the role of
various genes involved in drug-induced apoptosis and antioxidants-induced antiapoptosis.
 Newer Tools for Drug Screening 9

The procedure works hand in hand with DNA sequencing for high throughput screening for
exploring point mutations of nuclear and/or mitochondrial origin. Plastic microarrays are
relatively economical, and they can be utilized to investigate as low as 1200 genes of interest
from the biological samples. cDNA microarray scanning is relatively sensitive procedure and
can pinpoint minor yet subtle changes in the gene expression in response to environmental
neurotoxins as well as pharmacological drugs of clinical importance (such as anticarcinogenic
or antiapoptotic agents). The main objective of these technical procedures is to develop cDNA
chips for clinical diagnosis, better prognosis, and effective treatment of various diseases with
low undesirable effects.

APPLICATION OF MODERN ANALYTICAL TECHNIQUES IN BIOLOGICAL

SYSTEMS

Atomic Absorption Spectroscopy

This method is employed to estimate the concentration of various metal ions of physiological
significance, such as Na, K, Ca, Cl, Fe, Cu, and Zn from the biological fluids, tissue extracts,
and from the microdialysates. Various atomic absorption spectrometers equipped with
graphite furnace and computer software are now available which can analyze more than
four metal ions from as small as 20 µl biological sample. Tissue or cell extracts are prepared
in 0.3 N perchloric acid by sonication at low wattage and microcentrifuged at 14,000 rpm
at 4°C. The supernatants are filtered through the syringe tip filters and the filtered extracts
are directly utilized in the atomic absorption spectrometer or high performance liquid
chromatography with electrochemical, UV, or fluorescence detector capabilities. Perkin-
Elmer Atomic Absorption Spectrometer is equipped with Graphite furnace (which is
operated in an argon environment), an autosampler, and computer software for the online
data analysis and preparing concentration reports. Usually, pyrolysis at 1,300-1,700°C, and
atomization of samples at 2,400-2,800°C, is employed, depending on the metal ion under
investigation.13-15

Coulter Counting
This procedure is very simple and requires a photocell, which estimates the number of particles
present in the photocell. Beckman-Coulter Company (USA) has developed this procedure to
determine total number of cells following treatment with a drug. Although it is a single step
method and requires only 1:20 dilution of a sample, it does not decipher between live and
dead cells. Therefore, Coulter counting is supplemented with hemocytometer reading made
using a microscope and Trypan blue exclusion method (the live cells exclude trypan blue,
while dead cells are stained with trypan blue).

Fluorescence Activated Cell Sorting (FACS)

This biophysical equipment is utilized to determine differential expression of as many as 6
genes with a capability of estimating 6 more physical parameters of physiological interest,
such as number of cells undergoing apoptosis and necrosis, and the number of live cells
10 Drug Screening Methods

simultaneously based on the side scatter, forward scatter, and granularity. The FACS machine
(flow cytometer) employs lasers (such as He-Ne, Argon lasers, Ruby lasers and Cadmium
lasers) microbeams for the determination of fluorescence properties of cells. For measuring
intracellular free ionized calcium, UV detectors are employed. The equipment can also sort
out genetically engineered cells by a turbo sorter facility. Fluorescence activated cell sorting
(FACS) machines are now being utilized to prepare stable transfectants using vectors encoding
for green, red, or yellow fluorescence proteins. This approach eliminates the need to perform
in vitro reporter gene analysis using either X-Gal or luciferase reporter gene assays. pEGFP-N-1
vectors are thus very convenient to study the behavior of various pharmacological agents on
genetically engineered cell lines. This approach is being utilized particularly in gene therapy
labs. FACS machine is also utilized to detect the efficacy of anticancer drugs based on the
extent of DNA damage (apoptosis and/or necrosis) they produce in vitro. This machine is
also utilized to determine at which phase of the DNA cycle (G1-S, G2-M), the drug might
have induced its maximum effect. The cells can be synchronized using chemicals influencing
the DNA cell cycle at a particular phase. In addition, this machine is being utilized to study
mitochondrial membrane potential, and to determine the production of free radicals in
response to a particular drug/agent. Although very useful, this equipment is very costly.
Moreover, its maintenance cost is also quite expensive. In addition, the cost of fluorochromes
adds to its limited use in many labs all over the world. This equipment requires qualified and
trained persons to handle and interpret the experimental data.

Positron Emission Scanning

With the advancements in linear accelerators, cyclotrons, and targets, the preparation of
short-lived positron emitters has been facilitated. This procedure is noninvasive and provides
information regarding brain regional physiology and biochemistry such as synthesis of DNA,
RNA, and proteins in vivo. Furthermore, cyclotron-generated positron emitters (15O, t½ 120
sec, 13N t½ 10 min, 11C t½ 20 min, and 18F t½ 110 min) are now being utilized in basic research
and clinical practice to study brain regional metabolism and molecular neuroimaging of
genes of interest. 18F-DOPA is being employed to examine nigrostriatal dopamine transporter
activity, and to discover pleasure centers involved in drug addiction for substances of abuse.
18F-deoxyglucose is being employed for detecting the stages of malignancies. High-resolution
microPET scanners (Concorde Microsystems Inc, Knoxville, TN, USA) are now being used
to discover new and clinically effective neuroprotective drugs and for an early diagnosis
of diseases. CTI Corporation (USA), Physics for Medicine (USA), Seimens-Gamma-Med
(German), and Digi-Rad have recently developed high-resolution CT-SPECT fusion scanners
for research purpose as well as for clinical applications.

Magnetic Resonance Imaging and Spectroscopy

High-resolution magnetic resonance imaging is utilized in basic and applied research. In
particular high-resolution magic angle spinning nano-NMR probes require as small as
40 µl of biological samples in 750 MHz magnets. This equipment can analyze and quantitate
biological samples with microgram to nanogram concentrations. Running cost of this
equipment could be as high as $800 per hr. This equipment is used to determine the concen­
tration, purity, and structural formula of as many as 80 metabolites from the aliphatic and
 Newer Tools for Drug Screening 11

aromatic regions. Biological compounds including DNA, RNA and proteins contain carbon,
hydrogen, and nitrogen atoms. Their nuclei spin at a particular frequency, which can be picked
up and detected to evaluate their clinical significance. Usually, brain regional metabolism
of amino acid neurotransmitters and metabolites can be explored by these advanced and
sophisticated techniques. Various resonances are picked up and computer-analyzed using
Fast Fourier transformation (FFT) analysis. From the position of the resonance peak, we
identify the compound/metabolite, and from the peak height, we determine the concentration
of the compound/metabolite. These advanced research tools are also utilized to determine
the structure and purity of various unknown compounds and molecular designing of drugs.
Various pharmaceutical industries are interested to determine the structural formula of their
product before evaluating its therapeutic potential. High-resolution magnetic resonance
imaging is performed on small animals (rats, mice) to determine any space-occupying lesion
(such as cyst, infarct, tumor, edema). This equipment measures regional proton density per
unit area, which is altered during edema or during water accumulation in the brain in response
to neuronal injury or following neurotoxic insult. Thus, MRI is used to correlate and confirm
information derived from PET and computerized axial tomography conducted employing soft
X-rays.

CELL FREE ASSAYS

Cell-free assays include simple to very complex systems. These biochemical assays include
enzyme assays, protein-protein interactions and membrane receptor ligand and soluble
receptor-ligand binding assays. The advantages of this kind of assay system includes ready
accessibility of the compounds to the target, easy identification of the target of the compound
without any ambiguity, a well-defined mechanism of action, the possibility of developing
inexpensive screens, the easy adaptability to newer technologies, amenability to minia­
turization, and ready automation.17 These assays can be classified as heterogeneous and
homogeneous assays. Heterogeneous assays are multistep assays that include steps of
incubations, washings, filtrations, reading of signals etc. as in ELISA. On the other hand, one
pot assays that do not involve any transfer or wash steps are known as homogeneous assays,
such as, chromogenic, absorbance or fluorescence based assays.18

MICROBE-BASED SCREENING ASSAYS

Microbe-based screening has been used to screen antibacterial agents and cytotoxic anti-
cancer agents. Advance biotechnology techniques are used to clone and express target proteins.
The mammalian proteins are expressed in microbial cells and stored therein inclusion bodies.
These proteins are not useful for the microbial cells. The insoluble aggregates are isolated,
dissolved and refolded and if needed, subjected to post-translational modifications. The
advantages of this type of assay systems include low cost, simple technique and high yield.
Commonly used microbial systems include E. coli, Saccharomyces and Yeast. Microbial systems
have been adapted to identify agonists and antagonists of GPCRs, detail the mechanism of
action of immunosuppressants such as cyclosporin and FK506, screen for K+ channel openers
and blockers and many more.19-21
12 Drug Screening Methods

RECEPTOR SCREENS
Langley and Ehrlich described the concept of receptor-ligand interaction.22 The concept of
receptor has been overhauled since then, to include cell membrane, nuclear, ion, voltage
gated, tyrosine kinase, tyrosine phosphatase, hematopoietic cytokine, peptide, extracellular
calcium sensing, cAMP receptors. The receptors as ligand target represent more than 60% of
all drug discovery targets.23
Receptor-ligand binding assays using high affinity radiolabeled ligand provide a direct
screening approach for detecting specific/non-specific agonists/antagonists. This technique
also finds application for quantitation of the potency of competing agents, investigating
functional signal transduction pathways, monitoring molecular changes within a single cell,
determine functions of orphan receptors.24

NANO SCREENS
The face of drug discovery process has been imparted a dramatic lift by combinatorial chemistry,
robotics, miniaturization. At the screening rates enabled by ultra-high throughput screening
(uHTS) applications, reagent consumption poses as the limiting factor. This calls for means to
reduce the cost by reducing the volume of reagent required. This form of miniaturization has
given birth to nano screens, wherein assay protocols have been validated using nano volumes
(including pipetting, dispensing, and compound retrieval).
To achieve this modular platforms have been specially designed to handle high precision
liquid handling and sensitive detection. In these operation systems, piezo technology is used
to focus the liquid droplets into accurate volume and well. Moreover, the inherent advantage
of amplification by the fluorescence detection systems is utilized at the read-out. NanoStore,
EVOscreen, EVOTEC are some of the examples of systems adept at microseparation, detection
and analysis.25

Patch Clamp Technique: Single Channel Recording

In vitro techniques are crucial to understand the normal physiological processes of synthesis
and release, which are ongoing in individual cells. The pivotal patch clamp technique being
widely applied today was developed from a series of experiments in frog muscle. Current was
passed through individual ion channels activated by ACh and measured with high resolution.
This was shown to be a discrete pulse-like event with duration of a few milliseconds.
Fluctuation and relaxation measurements of end-plate currents led to the conclusion that the
rate of channel opening increases with agonist concentrations, and that the channel, once
open, closes spontaneously.26
The patch clamp technique has revolutionized cellular physiology since its introduction in
the early 1980s. It allows investigators to assess: (i) ionic currents of a whole cell, including the
molecular level of single ion channels, (ii) cell membrane potential and (iii) including fusion
of a single secretory vesicle.27 When combined with microfluorimetry and digital imaging
techniques, the method can be used to measure the spatio-temporal aspects of intracellular
levels and distribution of ions such as calcium, sodium, chloride, changes in pH, production
of signaling molecules and movement of these molecules. These techniques help to investigate
 Newer Tools for Drug Screening 13

signaling pathways at the cellular or molecular level in (cell lines, primary and transformed
tissue cultures, brain slice preparations transient and/or stable transfections of cells and in
primary cells derived from transgenic animals.
Conventional fine tip microelectrodes impale the cell in order to measure potential across
the cell membrane, while patch clamp electrodes are too large to be inserted into a cell. The
patch pipette is stuck onto the surface of a cell membrane instead of piercing it (Fig. 1.2). If a
patch pipette is placed onto the cell surface and gentle suction is applied, a bubble shape of
membrane is drawn into the patch pipette. The edges of this patch of membrane adhere tightly
to the glass of the patch pipette. The electrical resistance of this seal between pipette glass
and membrane is so high (a giga-ohm seal, or gigaseal) that the small patch of membrane
underneath the patch pipette is by comparison a low resistance pathway and thus the favored
route for current flow. This small patch of membrane may be voltage clamped to a series of
potentials and the conductance of the patch calculated from the amount of current required
to move from one potential to another. The patch of membrane under the pipette is very small.
If the radius of a patch clamp pipette is 1�m, then the area of the patch under the pipette will
be about 3 square picometres. Therefore, opening or closing of a single ion channel will cause
a significant alteration in the overall conductance of the patch. Mostly, ion channels are either
open or closed and they switch very rapidly from one state to the other. Therefore, the opening
of a single ion channel causes an abrupt increase in the conductance of the patch of membrane
beneath the pipette. The patch clamp technique involves a step-like increase in current. At a
given voltage and ionic environment, the size of the current deflection is directly proportional
to the conductance of this channel; the larger the deflection, the greater the conductance. If
two channels open simultaneously, then the current is exactly twice as large. Ion channels may
be distinguished from one another on the basis of this characteristic unit conductance, the
duration of each opening (open time) and on the probability of the channel being open (open
probability) under specific experimental conditions.
A patch of membrane from the cell can be removed without breaking the gigaseal and thus
measure ion channel openings in an isolated patch of membrane. Besides, the single channel
recording modes, the patch clamp technique may be applied to measure the currents that
result from ion movements across the membrane of the whole cell. This mode of operation is
known as the whole cell configuration. The first step in achieving this configuration is to obtain
a high resistance contact between the pipette and the cell membrane (gigaseal). However, the
patch of membrane under the pipette, which was the focus of attention in the single channel

Figure 1.2: Patch electrode on cell membrane

14 Drug Screening Methods

experiments is, in whole cell experiments, ruptured by application of a short pulse of negative
pressure. The tight seal between pipette glass and cell membrane persists and the low resistance
route for current flow is now into the cell and across entire cell surface membrane. A second
feature of the whole cell configuration is that, following disruption of the patch of membrane
under the pipette, the interior of the patch pipette is continuous with the cell interior. Thus, the
solution filling the patch pipette will enter into and equilibrate with the cell interior. Small ions
equilibrate within seconds of breaking through into the whole cell configuration.28
The application of the patch clamp technique has provided so many insights into cellular
physiology that its originators, Bert Sakmann and Erwin Neher were awarded the Nobel Prize
for Physiology and Medicine in 1991.

Advances in Patch Clamp Technique

More than ten years have passed since the slice-patch-clamp technique was established as a
powerful method for the analysis of central synaptic transmission. Although this technique
was earlier restricted only to young animal preparations, we can now even apply it to slices
obtained from adult animals.
In addition, the advances have made paired whole-cell recording from two or more synap­
tically connected neurons, recording from dendrites or some presynaptic terminals, possible.
Whole-cell patch-clamp technique can help to achieve complete biophysical characterization
of an individual neuron, intrinsic, synaptic and spiking properties of cells in either superficial
or deep structures, determining the synaptic receptive fields of single cells in anesthetized or
awake head-fixed and even freely moving preparations so that synaptic activity may be linked
directly to sensory processing and behavior.29
Future trends include developments facilitating microscopic analysis such as investigating
glutamatergic sensitivities of single dendritic spines in combination with two-photon
photolysis of a caged-glutamate compound and physiological function-oriented manner such
as investigation of pain perception mechanisms using in vivo patch clamp technique.30

Microdialysis Technique
The neurobiologists wish to follow moment-by-moment, the sequence of biochemical events
in various parts of the brain during a behavior. In this regard, various in vitro methods such as
incubation of tissue slices and subcellular components have been very successful. Nevertheless,
there is a definite need for a chemical technique comparable to the techniques of physiology
where functional events can be followed closely over time. So far, the most successful in
vivo “chemophysiological” techniques have been ventricular perfusions, cup perfusions on
the surfaces of the brain, and push-pull perfusions carried out under stereotactic control in
various parts of the nervous system.
The technique of microdialysis is a very important tool for in vivo studies in neuro­
psychopharmacology, toxicology, drug delivery, pharmacokinetics and endocrinology. Micro­
dialysis is an extension of the push-pull technique because the perfusion fluid is circulating
inside a semipermeable membrane instead of freely in the tissue. Substances in the extracellular
fluid will diffuse into the perfusate, while substances included in the perfusate will diffuse into
the tissue. This idea was first applied by Delgado and then by Ungerstedt who introduced the
 Newer Tools for Drug Screening 15

use of hollow fibers continuously perfused by a physiological liquid. Physiological processes

may be closely followed in anesthetized as well as awake animals.31

Principle
Principle of dialysis has been applied for sampling the extracellular fluid of brain, thereby
circumventing the problems associated with perfusion solution coming into direct contact
with brain tissue. This technique is based on the principle of an artificial blood vessel surgically
inserted into the tissue. The diffusion of chemical substances will occur in the direction of
the lowest concentration. In this way, substances may be recovered from the organ or added
to the organ depending upon their relative concentration in the perfusion fluid. There will
be bidirectional molecular and ionic traffic between the interior of the microdialysis probe
and the surrounding tissue. This provides the unique possibility of carrying out an entire
pharmacological experiment within less than a cubic millimeter of tissue.31

Factors Affecting the Microdialysis

A unique feature of microdialysis is the possibility to compare in vitro experiments with in vivo
experiments. The in vitro experiments may be performed on a solute contained in a simple
beaker. The recovery of individual substances may be studied by determining the relative
concentration in the perfusate in comparison with the concentration in the outside medium.
The concentration will depend upon the properties of the membrane (most notably its
molecular cut-off and its thickness), the speed of the perfusion, and the initial concentration
of the compound in the perfusate. By using an appropriate size membrane and a sufficiently
low perfusion speed, it is possible to reach 100% recovery.6
The dialysis membrane also acts as a filter to prevent the diffusion of large molecules from
extracellular fluid into the perfusion medium. This provides certain advantages for the analysis
of transmitter content in the dialysate. First, the membrane can prevent large molecules such
as enzymes from entering the perfusion solution and thereby halt the continuous enzymatic
degradation of neurotransmitters once they have entered the perfusion solution. Also, by
virtue of its ability to exclude molecules from the perfusion solution, the membrane partially
purifies samples prior to their analysis.32

Dialysis Probe
The development of the loop probe provided a means of reducing the extent of surgically
induced injury. This probe consists of a loop of dialysis membrane, which is implanted
vertically into the brain via a single hole in the skull (Fig. 1.3). Still less damage is produced by
a vertical concentric style dialysis probe. This probe consists of a single piece of dialysis tubing
blocked off at one end with glue; the inlet and/or outlet portions of the probe pass down into
the dialysis tubing.
Adaptations in microdialysis probe designs have made it possible to obtain samples from
the extracellular fluid of a variety of tissues with high temporal resolution. The resulting small
volume samples, often with low concentration of the analyte(s) of interest, present a particular
challenge to the analytical system. Rapid separations can be coupled online with microdialysis
to provide near real-time data.33
16 Drug Screening Methods

Figure 1.3: Dialysis probe

Analysis of Sample
In vivo microdialysis in itself is only a sampling technique. The ability to measure compounds
within dialysate is entirely dependent upon the sensitivity of an appropriate analytical method.
In addition to the postoperative time at which samples are collected, other variables include
the ionic composition of the dialysate, and the rate of perfusion affect sample content in the
dialysate. Pharmacological tools have been used to compensate for inadequate sensitivity by
increasing the level of substance to be analyzed. For example, acetylcholinesterase inhibitors
have been used to enable detection of ACh in dialysate.34 Another approach has been to prelabel
neurons by infusing isotopes of the transmitter or precursors and assaying the radiolabeled
compounds.35

Duration of Experiment
Implantation of the dialysis probe results in several reactions within the CNS tissue. Knowledge
of the time course of these events is critical in determining the interval during which
microdialysis experiments can be performed with minimal interference from tissue reactions.
In general it is thought that dialysis experiments should not be performed either very soon
(< 10 h) or very long (several days to weeks) after probe implantation. The optimal interval
for performing microdialysis experiments is approximately 16-48 h after implantation of the
dialysis probe. Efforts have been made to develop methods whereby sampling can be carried
out over many days in a single subject using either chronic implantation of a dialysis probe or
implantation of a guide cannula followed by multiple insertions of a probe over days. However,
these have generally been unsuccessful.
 Newer Tools for Drug Screening 17

Microdialysis versus Other Techniques

This technique has advantages over the blood sampling since it provides protein free samples
ready to analyze without any loss of blood, permits more frequent sampling and offers an
option of simultaneous drug delivery at the same site. Microdialysis can be considered to be
superior to biosensors as more than one chemical system can be analyzed while circumventing
the electrode contamination.

Present Status of Microdialysis Technique

Microdialysis is now used extensively for the study of several neurotransmitters in the CNS.
The two main areas of application of microdialysis are the recovery of endogenous substances
(neurotransmitters, catecholamines, neurotrophic factors, cAMP) and the infusion of drugs
through the microdialysis cannula (retrodialysis). Clinical applications of microdialysis
includes monitoring of ischemic injury, subarachnoid hemorrhage, trauma and epilepsy.36
Permutation and combination of novel membrane sampling techniques, ultrafiltration
procedures and functional imaging (PET, MRI) have allowed numerous applications of the
basic technique in pharmacokinetics, metabolism and/or pharmacodynamics.37,38 Semi-
invasive techniques like microdialysis can be used to measure concentrations of the free,
active drug or endogenous compounds in tissues and organs, determine transdermal drug
distribution, tissue pharmacokinetics. Thus it gains importance as a widely used sampling
technique in clinical drug monitoring, drug development, therapy and disease follow-up.
It can play pivotal role in rationalizing drug dosing regimens and influencing the clinical
decision-making process.39

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16. Sharma SK, Carlson E, Ebadi M. Neuroprotective actions selegiline in inhibiting 1-methyl,
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 CHAPTER

2
High Throughput Screening
for Drug Discovery
INTRODUCTION
The increasing burden of new diseases like cardiovascular disorders, diabetes, immune system
related disorders, diverse microbial infections are demanding new drugs to control them in an
improved manner. The rise in the occurrence of treatment failure has also increased the strain
of the drug discovery scientists for the discovery of novel compounds with better treatment
outcomes. As in the case of antibiotics, an innovation gap of almost 50 years came from the
rapid discovery era of beta lactam class of antibiotics, still patients are being treated with the
congeners of the antibiotics discovered almost 80 years ago. Rapid discoveries of novel drug
targets have increased the use of high throughput screening in a potential manner to meet the
pace with biochemical or pathological findings.
In the domain of drug research target identification, purification and assay development
constitute the initial step. In the next step, the potential compounds are screened against the
identified target. Conventionally this has been a slow and tedious manual process requiring
huge investment of manpower, time and money. Traditional techniques like test tube analysis
and chemical analysis of the intermediate biomolecules may lead to the loss of the precious
samples and may not be sensitive enough to capture the minute information that may help in
the due course. Consequently, the conventional drug discovery program has been called as a
slow process.

WHAT IS HIGH THROUGHPUT SCREENING (HTS)?

The last two decades have seen astonishing innovations in technology that have helped
the manual low speed screening to evolve into an automated, microprocessor controlled
robotic process called ‘High Throughput Screening (HTS)’. This recent process is a synergy of
chemistry, biology, engineering and informatics. HTS has helped to speed up conventional
languid process and now over 50,000-1,00,000 compounds can be screened per week, against
the validated biological target. Further advancements are making it possible to screen 10,000-
100,000 compounds within 24 h time and the process is called as ultra-high throughput
screening (uHTS). High throughput synthesis of large number of test compounds in a lesser
time is also a reality now. Simultaneous development in other areas such as drug synthesis,
toxicity screening, drug metabolism and pharmacokinetics studies (DMPK) are helping the
process to achieve its ultimate speed in new drug development (NDD). After the fundamental
 High Throughput Screening for Drug Discovery 21

biological research, the effective target is identified and validated for its function. Then the
method development takes a few months to initiate screening process. In HTS, the trend is
to replace radio labeling by simple luminescence and fluorescent techniques. Since the yield
of target protein or purified biological target is generally low (often obtained only in a few
milligrams), technologies that permit screening with reduced volumes (3-20 µl) and reduced
protein/ligand have played a pivotal role in facilitating HTS. Development of detection
techniques having ultra high precisions are used in these assays to give more valuable
information about the ligand-protein interaction. The conventional 96-well plates have been
replaced with 384-well plates and subsequently by 1536-well plates with low internal volume
to make the screening possible at a high speed. Nowadays, rapid development of negative
screening method is also in place for earlier detection of the ligand affinity with known targets
leading to adverse reactions, drug interaction, altered intestinal permeation, etc. Revolution
in every individual process towards drug development is bridging the gap between older
concepts with newly optimized robust and technology enabled methods for its quick and
economic implementation in drug discovery platform.

HTS—POSITIVE AND NEGATIVE SCREENING

In the drug development screening Hit and Lead are two frequently used terminologies. Hit is
explained as: “a molecule with confirmed activity from primary HTS assay with a good profile
in secondary assays and with confirmed structure,” and Lead is explained as: “a hit series
exhibiting the Structure Activity Relationship (SAR) and demonstrating activities both in vitro
and in vivo.”

IN VITRO INTERACTION STUDIES (ARRAY BASED)

In vitro assays have now been miniaturized from the test tubes to well plates having capacity
of assaying samples starting from traditional 96 well format to 1536 wells. Microfluidics has
impacted the interaction study area in a surprising way and has drastically decreased the
need of both reagents and samples. Chemical analysis on the microarray plate for the quick
and economic identification of the biological target is evolving as a good tool.1 The accuracy
and speed of the system has made these techniques a great success. Still the system is not
completely free from errors
1. Different chemicals have different functional groups
2. Difference in the diffusivity of the samples
Chemical microarray technology has come up with great successes in the evaluation of
chemical–protein interactions, enzyme activity inhibition, target identification, signal pathway
elucidation and cell-based functional analysis. Their success can easily be seen from the
Genomic and proteomic studies where almost 10,000 targets for the drug activities have been
found out; however, less than 500 of these targets have turned up in approved drug candidates.
Chip based microarray: Chip based microarrays have started becoming popular these days.
They are made in the same way as semiconductors. The microfluidics helps in the mixing of
samples with the targets bound on the chip, finally signals are read by the analyzers that can be
fluorescent or simple UV or visible signals.
22 Drug Screening Methods

Other microarrays: So many other microarrays are also available in the market. These arrays
are specific for some of the important biomolecules in the physiological or pathological
pathways, e.g. Cytokine based kits, angiogenesis responsive bimolecular kits, transcription
factor analysis kits, etc. They work on the principle of the binding of the biological samples with
the antibodies bound to the array membrane, incubation, binding to the secondary antibodies
and finally tagging of the secondary antibody with the signal emitting molecules such as dyes.

FLUORESCENCE TECHNIQUES IN HTS

Fluorescence (FU) based detection of the compounds, analytes or genetic materials has
now become a well-established technique (Table 2.1). In HTS the interaction of ligand with
the biological compartment is elucidated by luminescence-based binding assays. Various
fluorescence techniques like Fluorescence Anisotropy (FA), Fluorescence Correlation
Spectroscopy (FCS), Fluorescence Intensity (FI), Fluorescence Lifetime Imaging Microscopy
(FLIM), Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection
Fluorescence (TIRF) and Time Resolved Resonance Anisotropy (TRRA) are used. Along with
these techniques, certain specific nano bead-based techniques like Scintillation Proximity
Assay (SPA), Amplified Luminescence Proximity Homogeneous Assay (ALPHA) are also used
(Fig. 2.1).
Detection system for the fluorescent based assays has also developed hand in hand with
the techniques. Currently the instruments are available which are capable of screening a single
reaction well for different modes starting from UV, Visible to FU and chemiluminescence.
These instruments are known as multimode readers (Fig. 2.2). These instruments are cartridge
based and having the advantage of replacing the cartridge as per the need. FU microscopes

Figure 2.1: cAMP detection using ALPHA screen principle. The ALPHA screen kit includes streptavidin coated donor
beads and acceptor beads conjugated with an antibody to cAMP. Biotinylated cAMP is also included as a positive control
and for competition with unlabeled cAMP
 High Throughput Screening for Drug Discovery 23

Figure 2.2: Photograph showing multimode reader (Courtesy – Ocular Pharmacology & Pharmacy; Dr RP Centre, AIIMS)

are also playing a big role in the drug development. FU microscopes help in the identification
of the entry of the fluorescent tagged compound at the desired site in the cell. FU microscope
also helps in the identification of the drug effect on to a particular cellular component or
biomolecule. Immunofluorescent technique depends on the FU microscope for the detection
of the fluorescent bound antibody on the tissue sectioned glass slide.

Table 2.1: Fluorescent techniques used in drug discovery process

Technique Principle of working Area of application

Fluorescence Fluoroprobe is exited using plane polarized light and emission Receptor ligand binding
anisotropy (FA) is recorded parallel to plane polarized light. Binding of the assay2,3
or fluorescence fluoroprobe with receptor would restrict the rotational mobility
polarization (FP) of it and would exhibit a higher polarization.
Time resolved Records the change in the rotational diffusion of phosphorescent- Screening of chemical
fluorescence labeled membrane receptor when they bind to a ligand. Also compounds4
anisotropy (TRFA) delivers information on protein structural fluctuations
Fluorescence Used for mass-Dependent and independent fluorescent assays. Screening of chemical
correlation Can be used to study the protein-ligand interactions on the compounds
spectroscopy (FCS) membrane bound receptors in live cells
Fluorescence Works on the interaction of two different chromophores via Interaction of the
resonance energy dipole-dipole mechanism, excited donor chromophore transfers chemicals or drugs with
transfer (FRET) its excitation energy to a closely located acceptor chromophore biomolecules5
Time resolved Combines the advantage of fluorescence resonance energy Interaction of the
Fluorescence transfer along with the time chemicals or drugs with
resonance energy biomolecules6,7
transfer (TR - FRET)
Fluorescence Based on the excitation of the fluorescent molecule bound to the Cellular or tissue structural
microscopy cellular surface and visualizing under a magnifying object lens analysis
Real time PCR Works on the ability of the dyes to emit fluorescence signals via Gene expression analysis
binding to a double stranded DNA
Fluorescence Lifetime Sample is illuminated with a pulsed laser and the lifetime of the Analysis of the cellular
Imaging Microscopy fluorescent probe is determined from the phase shift between components8
(FLIM) the modulation of the excitation light and the emission of
fluorescence
24 Drug Screening Methods

FLOW CYTOMETRY FOR CELL-BASED ASSAY

A flow cytometer (FCM) is an instrument which illuminates the cells or particles as they flow
in form of droplets in front of a light source and then reads the illumination. FCM was first
described as an analytical tool in 1960. After that FCM changed a lot for it’s utilization in the
drug discovery and diagnosis.
Principle of working: FCM has several components which work in tandem to produce
desirable results.
1. Light source: Nowadays most of the FCMs contain laser light as a source for the illumination.
Lasers are preferred because of their intense and narrow beams. They may be gas lasers
(e.g. argon ion laser or helium neon laser) or solid state lasers (e.g. red or green diode laser,
new blue and violet lasers). These lasers would be generating defined and fixed type of
wavelengths (e.g. 488 nm for argon ion laser). Usually a flow cytometer would be having
one laser light source but advance machines may have more as well.
2. Fluidics: In FCMs particles need to be suspended in the fluid so that a single particle can be
analyzed at a time. Concentrated cell suspension may form aggregates of the cells, even so
called single cell suspension cells may form clumps. Hence fluidics is designed in a way to
decrease the chances of aggregation and to facilitate similar illumination of each cell. The
strategy used by Crosland-Taylor for the confinement of the cells in a focused, narrow flow
stream (known as hydrodynamic focusing) is applied to achieve this. The sample stream is
injected into the rapidly flowing stream (sheath stream) in a “flow cell” which has a narrow
exit orifice. This difference in the diameter increases the velocity of the entire stream and
confined the path of the cells tightly to the center of the laser beam.
3. Detectors: Photodiode is fitted in the line of the illuminating laser beam with an obscuration
bar in front of it. The laser light which has been bent by the cell can only reach the detector
after avoiding the obscuration bar, it is known as forward scatter. The forward scatter light is
not well defined in the terms of biology or chemistry of the cells because of the confusion in
the refractive indices of the live or dead cells and the medium. One lens is fitted to the right
angle to the direction of the laser beam and light collected by it is known as side scatter light
or 90o light scatter..
•• Applications: Since the development of the first FCM in 1960s, a tremendous work has
been carried out in the hematological discoveries. One or two color FCMs helped in the
identification of cellular (T-cells) and humoral (B-cells) cells lineages, followed by CD3,
CD4 and CD8 identification by multicolor flow cytometer.9,10 These days flow cytometry
has also been utilized in the vaccine development by intracellular cytokine staining assays
and further cell sorting for their genomic as well as transcriptional analysis.11 In previous
10 years possibilities of the in vivo flow cytometry has also been tried by several research
groups from around the world. By using very high speed, high resolution, continuous CCD
or CMOS cameras, images have been taken from the rat mesenteric blood and lymphatic
vessels.10 Several compounds have been tested in the in vivo flow cytometric analysis of the
blood and lymphatic vessels. Natural property of the lymph vessel and valve function has
also been studied by the in vivo flow cytometry.12

THERMAL SHIFT ANALYSIS FOR HTS

Thermal shift analysis of the proteins has now become a good tool for the identification of
the ligand interaction with the proteins in a rapid and high throughput manner. This method
 High Throughput Screening for Drug Discovery 25

needs a fluorescent dye which associates and dissociates from the protein with the change
in the temperature of the reaction. It is based on the principle of binding of the ligand to a
protein and stabilizing or destabilizing its structure which ultimately changes the melting
temperature (Tm) of the protein. Sypro orange is such a dye which binds to the protein in a
temperature dependent manner. Fluorescence of sypro orange is quenched in the aqueous
environment but when protein unfolds and expresses its hydrophobic core then dye binds
to it and starts emitting fluorescence. Finally fluorescence is monitored and plotted versus
temperature. Thermal shift analysis can be used to assess the following:
1. Role of inhibitor in the binding affinity of the ligand with protein.
2. Stability of the protein at different pH and salt conditions.
3. Ligand screening for the binding with the protein for the lead identification.
Thermal shift assay has helped in the identification of the inhibitors of the carbonic
anhydrase enzyme.13,14 Carbonic anhydrase is a zinc metal containing enzyme which is involved
in the dehydration of the bicarbonate. This enzyme is also involved in the several pathologies
like glaucoma, epilepsy, Alzheimer’s and Parkinson’s disease hence serves as a potential
drug target. Thermal shift assay has also helped in the identification of the estrogen receptor
antagonists (Toremifene and tamoxifen) as anti-cryptococcal agents. The assay showed that
these drugs directly bound to the calmodulin (cam1) which was purified from Cryptococcus
neoformans and prevented it from binding to its substrate calcineurin (Cna1) and blocked
its activation.15 Further thermal shift assay helped in the evaluation of the mitogen activated
protein kinase inhibitor 4 (MAP2K4) which activates pro-invasion signaling pathways in the
prostate cancer.16

ROLE OF MASS SPECTROMETRY IN THE HIGH THROUGHPUT SCREENING

Mass spectrometry is an important tool in the clinical and medical development area. Almost
hundred years have passed when Nobel laureate Sir J. J. Thomson first described about mass
spectrometer in Cambridge Philosophical Society. From the time of its initial development,
mass spectrometer changed a lot but became suitable for the biological field only after the
development of the electrospray ionization (ESI) technique by John Fenn which fetched him
a Nobel Prize in 2002. After this development mass spectrometer evolved as an important
tool in the field of biology. Coupling the separation capabilities of the liquid chromatography
with the structural analysis abilities of mass spectrometer gave a third dimension for the quick
and accurate identification and quantification of the compounds. Information dependent
acquisition (IDA) available with some of the mass spectrometers gives the freedom of the
identification of the unknown from the pool of several molecules by available mass spectras
of the known one. Thus the mass spectrometer has now become an essential tool for the drug
development in a rapid and accurate manner.
Phytochemical extract analysis: Analysis of the natural product extracts has always been a
tedious task. This process needs the identification of the gross chemical group, purification,
enrichment and structure elucidation of the final compound. Once the process is done, the
compound is again screened pharmacologically. This process is having one drawback that
compound recovered at the end may fail to show any pharmacological activity. The reason
behind this is that the separated and enriched compound might not be responsible for the
pharmacological activity found with crude extract at the beginning. Mass spectrometry
has given a solution for the above problem—information dependent scans of the extract
26 Drug Screening Methods

and identification of the hit. It works on the basis of the available structural and functional
(pharmacological) information for the similar natural product group compounds. Once
the pharmacological activity on the crude extract is done, the library is constructed for the
compounds similar in the pharmacological activity and from the similar natural product
group. The extract is screened for the diverse mass ranges (from few Daltons to almost one
thousand Dalton) and the fragmentation pattern of the similar molecular mass compounds
(hit) is matched with the available mass spectra of the known compounds. The same technique
was used by Kyadari et al for the identification of lyngbyatoxin-A and malyngamide-J in the
marine algae extracts.17
Purity analysis of the drugs or synthesized compounds: Mass spectrometry can very well
be used for the purity analysis of the compounds. Purity analysis of the drug formulation
or the synthesized molecules gives important information regarding the improvement in
the formulation or synthesis process. In case of drug formulation impurities may lead to
therapeutic failure or even adverse events due to toxic metabolites or contaminants. The
stability and impurity profile of latanoprost drug formulation was evaluated by Velpandian et
al using LC-MS/MS.18

IN VIVO IMAGING OF DRUG ACTION (NEAR INFRARED IMAGING)

It is a newly added method to evaluate the drug action and quantification without killing the
animal. Rapid development in this technology is expected to revolutionize the in vivo studies
that warrant the killing of the experimental animals. Fundamental of this technique is using
molecules capable of emitting light in higher wavelength near IR region thereby, it gains the
capability of crossing biological membrane and to be detected by sensitive camera turned for
near IR region. For this application an exogenous contrast agent like indocyanine green (ICG) is
used. In vivo near IR fluorescence imaging has been successfully utilized in vascular mapping,
and tissue perfusion studies, to image vasculature of brain, for conducting angiograms of
eye, imaging tumors, biodistribution studies, gastrointestinal tract (using endoscopic optics),
inflammation, atherosclerosis, cell death, and osteoblastic activity, etc.19 For all the studies
excitation was achieved by using pulsed diode laser having different fluence rate above
700 nm and emission was recorded using powerful cooled CCD capable of monitoring near IR
emission radiation.
Recently, GFP (Green fluorescent protein) or Ds Red2 genes are making a rapid entry in
the biological research. For tumor imaging Ds Red2 expressing CR8 Lewis-lung carcinoma
cells were injected into nu/ne mice. Seven to ten days after inoculation of cells Cy-annexin V
conjugate was injected intravenously to the mice bearing tumors and submitted for imaging
near infrared signal. Using cyclophosphamide, Petrovsky et al20 evaluated the usefulness of
active Cy-annexin that can be used as a near infrared probe to image apoptosis from outside
an intact living animal and facilitate the antiproliferative drug screening.

Other Whole Body Imaging Techniques

Single photon emission computed tomography (SPECT) is a highly sensitive nuclear
medicine tomographic imaging technique using gamma rays. It is very similar to conventional
nuclear medicine planar imaging using a gamma camera. However, it is able to provide true
 High Throughput Screening for Drug Discovery 27

3D information. This information is typically presented as cross-sectional slices through the

organism, but can be freely reformatted or manipulated as required.
SPECT uses gamma-emitting nuclides, which undergo a radioactive decay with the
emission of gamma radiation. In order to attain a nuclear stability, the nucleus of an unstable
atom undergoes a process of transition in which neutrons are converted to protons and vice
versa. This process leads to the formation of daughter nucleus in an excited state. This state is
unstable; therefore, it reverts back to the ground state and loses some form of energy in terms
of electromagnetic radiation or gamma radiation, e.g. Technetium-99m (99mTc), Iodine-123
(123 I), cobalt-60 (60 Co).
SPECT imaging involves the administration of radiotracers into the biological system and
the radiations emitted from the radiotracer in the body are captured by the gamma cameras
with sodium iodide crystal detector and photomultiplier tube (PMT) in which the radiations
are converted into scintillating photons and amplified and the images at different angles
are reconstructed by the computer in such a way that the imaging of the organ of interest is
achieved.
Recently, SPECT has been utilized in the study of involvement of drug efflux transporters
like multidrug resistance associated protein (MRP), P-glycoprotein (P-gp) and lung resistance
protein (LRP) as a contribtuting factor for multidrug resistance in cancer therapy in patients.21
In research, due to the ease of small animal imaging with SPECT, it is been utilized to evaluate
and to visualize the modulation of P-gp or MRP using (99m) Tc - sestamib as a model substrate.
For the first time from our laboratory, SPECT imaging has been utilized to explore the
intraocular uptake of fluoroquinolones following systemic administration by such drug efflux
transporter (Fig. 2.3) in the eye using ciprofloxacin labelled99mTc as a radiotracer in rabbits.22
This study was carried out in presence of GF 120918, which is a P-gp modulator and verapamil,
a known P-gp as well as OCT inhibitor.

VIRTUAL SCREENING (IN SILICO DRUG DEVELOPMENT OR DRY LAB)

It is totally an indirect approach using advanced computer technology to screen newer
compounds based on virtual coordinates of receptor and ligands. Computer-aided molecular
design (CAMD) approach involves computational analysis of large data set in order to
highlight those compounds most likely to be active in the actual assay, so that a focused

Figure 2.3: Pictures of SPECT-CT showing the intraocular uptake of radiolabeled ciprofloxacin after blocker (verapamil)
treatment. Dual head SPECT–CT with collimator-low energy (Courtesy: Dept. of Ocular Pharmacology & Pharmacy, AIIMS
and INMAS, New Delhi)
28 Drug Screening Methods

Figure 2.4: Computer-aided molecular design approach

subset of compounds can be selected. CAMD covers a wide range of technologies leading
to very fast property predictions through more computationally elaborate modeling of drug-
receptor binding. Using receptor-based properties, such as binding affinity and receptor
selectivity, CAMD calculates to propose a broad range of properties that are likely to be useful
in drug design—from physical properties like molecular size and solubility to indicators of
developmental issues like metabolic fate and toxicity, etc. Therefore, virtual screening can
filter out undesirable compounds on the basis of a wide variety of criteria, depending on the
problem in hand. Virtual screening needs the 3D molecular structure of the receptor along with
the 3D structure of ligands to perform docking. However, this approach is highly applicable for
the lead optimization process since for many newly isolated receptor’s conformational data
may not be available. Moreover, the techniques like X-ray crystallography and NMR studies are
necessary for the information about spatial arrangement and virtual coordinates of targets and
ligands.23 Figure 2.4 showing the docking of ciprofloxacin on DNA gyrase using CaChe (Ver.6.1,
Fujitsu, Japan).
The applicability of computer models has also used completely empirical and statistical
model like the Rule of Five or Lipinski’s rule. According to this rule, a drug like compound looks
like a molecule with a molecular weight less than 500, OH and NH groups less than 5, the sum
of N and O atoms less than 10 and log P value less than 5 for a better absorption in the intestine.

HTS-SCINTILLATION METHODS IN DRUG SCREENING

Apart from fluorescent techniques, the advancements in using the radioactive compounds to
understand molecular mechanisms are also very interesting. One of such attempts is named
as scintillation proximity assay (SPA). This system uses the principle that an antibody or a
receptor molecule, which is bound to a bead, emits light when beta emission from an isotope
occurs in close proximity, i.e. when a radiolabeled ligand binds to the bead with receptor or
antibody (Fig. 2.5). Amersham has introduced several advancements in these techniques
leading to make it as a successful high throughput screen. The basic technology of the SPA is
based on the fluorescent signal produced by a scintillant-dyed polystyrene or polyvinyl toluene
microsphere that could be excited by the proximity of a radiolabeled molecule. The scintillant-
dyed microspheres are in the size range of < 2 µm in diameter. The recent improvements in this
technique are yttrium silicate and yttrium oxide beads which enable the use of higher number
 High Throughput Screening for Drug Discovery 29

Figure 2.5: Principles of scintillation proximity assay (SPA)

of wells in which the signal can be picked up by highly sensitive readers containing CCD
camera instead of classical photomultiplier tubes. So far, this technology is applied in several
attempts in drug discovery processes like evaluation of kinetics of protein kinase inhibitors,
to evaluate neurotransmitter transporter inhibitors, to evaluate novel farnesyltransferase
inhibitors, to identify poly (ADP-ribose) polymerase-1 inhibitors (enzyme involved in DNA
repair), evaluation of HIV-I reverse transcriptase inhibitors, etc. SPA is a homogeneous, rapid,
versatile and amenable to automation, which can also simplify the screening protocol in the
drug discovery process.

UNCONVENTIONAL RAPID PHARMACOLOGICAL AND TOXICITY

TESTING MODELS
Zebrafish (Danio rerio) and Gold Fish (Carassius auratus auratus)
Drugs working on the central nervous system (CNS) are having a choice of being tested on
the marine organisms like fishes. Antipsychotic class of drugs have been discovered without
having any target approach. Still mechanism is unknown for many of them. For these classes
rational approach may not work but behavioral approaches may work for the screening of
newer compounds. Gold fishes have mainly been used for the CNS depressant and stimulant
drug analysis. It was an attempt of the Vijayakumar et al24 to develop a model for the screening
of the drugs for their effect on CNS. Gold fishes were exposed to the low concentration of known
CNS depressant and stimulant drugs and their swimming pattern (optomotor response) was
assessed along the gyro-dots of different colors. Similarly, toxicity of the ethambutol on the eye
was also evaluated using the gold fishes.
Zebrafishes are used mainly for the angiogenic and anti-angiogenic effects of the compounds
on the vasculature of this marine organism. The reason of the extensive use of the zebrafish is
the development of optically clear embryo provides the opportunity of the visual inspections
and very high egg laying capacity of the female zebrafish.25 Zebrafish has been utilized for the
better understanding of the Alzheimer’s disease.26 Zebrafish has been used in the hematology
30 Drug Screening Methods

research because of the conserved genetic factors regulating blood development and
visualization of the circulating erythrocytes with only a dissecting microscope. Their organ
regeneration capacity made them popular model in the regeneration experiments like heart
regeneration.27 As zebrafish express cytokines, macrophages, neutrophils, dendritic cells, mast
cells, eosinophil, T-cells, and B-cells; it has also been used in the host pathogen interaction
studies as well.28

Chick (Gallus gallus domesticus)

Use of chick for the drug screening is now a well applied model in the drug development.
Extraembryonic membrane serving gas exchange known as Chorioallantoic membrane has a
network of blood vessels (Fig. 2.6A). Chorioallantoic membrane can be used for the evaluation
of the angiogenic or antiangiogenic potential of the compounds. One important factor for the
use of chick for these studies is the non-requirement of the ethical approval. Fertilized egg can
be obtained from the hatchery and can further be developed as per need in the incubator at
the laboratory. Several marine isolates have been studied for their anti-angiogenic effect on
the chorioallantoic membrane of the chick.29
The size of the lens and the time taken for the development of the model has made
hydrocortisone-induced cataract in the chick a good model (Fig. 2.6B). This model has been
used for the screening of the larger groups of the compounds for their anti-cataract activity on
the lens. Several polyherbals have also successfully been studied using the same technique.30,31

Caenorhabditis elegans
Currently in toxicity and pharmacological studies C. elegans are being utilized extensively.
Availability of whole Genome sequence, transparency of the tissues at all the developmental
stages, availability of large mutant species and shorter life span has made C. elegans a very good
model for biological as well as disease and drug effect research. C. elegans was used for the
evaluation of the Genistein for its life span enhancement ability by Lee et al (2015). Genistein
is a dietary phytoestrogen present in the seeds of Vigna angularis cultivated in East Asia. The
phytoestrogen was tested on the nematode C. elegans in both normal and stress conditions

A B
Figure 2.6: Photographs showing use of chick for pharmacological screening of compounds: (A) Black arrows showing
developing blood vessels in the chorio allantoic membrane of the chick under a coverslip (black circle). (Courtesy: Gupta
et al (2014); (B) Figures a,b and c are showing normal lenses; d—stage 2 cataract; e—stage 3 cataract; f—stage 5 cataract;
Courtesy Velpandian et al (2003)
 High Throughput Screening for Drug Discovery 31

produced by heat and oxidation. Genistein could able to enhance the life span of the nematode
in both the conditions.32 In another study the growth retardation effect of caffeine on the fetus
was assessed on the larvae of the C. elegans by Min et al. They found that the caffeine was able
to hinder development at most of the stages in a dose dependent manner.33

HTS PHARMACOKINETIC STUDIES

HTS-absorption Studies
Oral route is a preferred way of drug administration. In the drug development process it is
very important to screen the drugs for gastrointestinal absorption. Conventionally, it is a very
lengthy and time-consuming process. Moreover, this process also requires a large number of
animals. Intestinal absorption is mainly due to the concentration dependent diffusion. If the
drug passes through paracellular route, then there is not much of a hurdle. Some compounds,
like cyclosporine and digoxin, are substrates of well recognized P-gp transporters, which
belong to the ATP Binding Cassette (ABC) transporters having Walker’s motives. P-gp
substrates were found to have variable bioavailability. Therefore, it is necessary to screen the
drug candidates during the developmental stage to ensure that they are not substrates for P-gp.
Colon cancer cell lines (CaCo) grow confluently and form a monolayer upon polycarbonate
support or collagen coated polycarbonate support.34 They are quite suitable for performing
intestinal permeation studies and to elucidate the drug candidates susceptible for P-gp efflux
mechanism in the intestine. Moreover, they also express CYP3A4 enzyme allowing prediction
of intestinal metabolism. This process can be automated to examine the intestinal absorption
characteristics of drugs in a shortest possible period to decrease the incidence of failure in the
Phase I clinical trial in human volunteers.

PARALLEL ARTIFICIAL MEMBRANE PERMEABILITY ASSAY (PAMPA)

In order to assess the intestinal permeation, apart from cell-based assays like CaCo monolayer
cells, PAMPA is an alternative. It is a passive-permeability screen with focus on the simulation
of transcellular processes. It is reported to be an excellent compliment to cellular models in
absorption, distribution, metabolism, excretion (ADME) screening of research compounds. It
is a fast, versatile and low-cost method that is compelling and biologically relevant model of
transport. Its principle is based on the application of physicochemical properties of molecules
to predict intestinal permeability.35 In PAMPA, a 96 well microtiter plate completely filled with
aqueous buffer solutions is covered with a hydrophobic filter coated with lipids in an organic
solvent solution in a sandwich construction. Typically, Hwang et al36 prepared the membrane
by wetting with 5 µl of the artificial membrane solution (0.8% egg lecithin in n-dodecane). The
donor and recipient solutions consisted of phosphate-buffered saline (pH 5.5 or 7.4) with 2%
DMSO (donor contained 200 µM test compound in each well). These plates were incubated
for 2 hours at room temperature with gentle shaking, following which drug concentrations in
the receiving solutions were assayed by HPLC. Percentage transport across the lipid bilayer
(%T) was calculated by %T= 100 x Ar/Ad (Ar and Ad correspond to the HPLC peak areas of
the receiving solution and initial donor solution). Using this method they have predicted their
model drug supposed to be absorbed via passive diffusion across the human gastrointestinal
32 Drug Screening Methods

tract following oral administration. Different membranes used for the prediction of absorption
from intestinal,36 blood brain barrier37 and skin38 are listed in the Table 2.2.

Table 2.2: Membranes used for prediction of absorption in PAMPA

Membranes used Predicted absorption in PAMPA

Egg-lecithin + N-dodecane Intestinal permeability
Egg-lecithin + 1,9, decadiene Intestinal permeability
Composition of lipids + 1,7-octadiene Intestinal permeability
Porcine polar brain lipid + dodecane Blood-brain barrier permeability
70% silicone + 30% isopropyl myristate Skin permeability

HTS Metabolism Studies

In order to increase the speed of metabolism studies or to decrease the animal utilization in
the metabolism studies, in vitro techniques were developed. Isolated human or animal liver
microsomes are incubated along with the drug of interest and at periodical interval the aliquots
are subjected for LC-MS or LC-NMR to elucidate the metabolites. Sometimes the major
metabolites are isolated and subjected to primary in vitro screening to elucidate whether they
are active metabolites or not. Liver microsomes are prepared by the homogenization of a liver,
followed by centrifugation of the homogenate at 10000 g to obtain a supernatant fraction known
as S10 fraction. The S10 fraction at 100,000 g pellets out smooth endoplasmic reticulum where
the enzymes responsible for Phase I oxidation, including the CYP450 monooxygenases reside.
However, most of the Phase II enzymes are cytosolic and are absent from the liver microsomes.
Apart from microsomes, isolated hepatocytes (or hepatocytes-derived microsomes), cDNA-
expressed enzymes and liver slices are also used to study the metabolic stability of drugs. The
screening of metabolic stability with liver microsomes or human hepatocytes can be performed
in 96-well plates to enhance throughput.

HTS-cytochrome (CYP) Inhibition and Induction Studies

During the drug development process, pharmacokinetic drug-drug interaction can also
be an undesirable factor. For example, ketoconazole, a potent inhibitor of CYP3A4, causes
drug-drug interaction with drugs that are substrates for the same enzyme. Cryopreserved
hepatocytes retain both Phase I and II drug metabolizing enzyme activities and, therefore,
are used extensively for drug interaction studies. To get more information about the newly
developed compound’s metabolic interaction in specific cytochrome, liver microsome
incubation studies are carried out using specific inhibitors. For this study phenacetin
(for CYP1A2), coumarin (for CYP2A6), tolbutamide (for CYP2C9), s-mephenytoin (for
CYP2C19), dextromethorphan (for CYP2D6), chlorzoxazone (for CYP2E1) and testosterone
(for CYP3A4) are used. For HTS enzyme induction studies, the mechanism of CYP3A4
induction has been defined. Compounds, which are capable of inducing CYP3A4, also induce
pregnane-X-receptor (PXR), that bind to the response element in the CYP3A4 gene called
the pregnane-X-receptor response element (PXRE). An HTS method has been developed
using a genetically engineered cell line that expresses a PXRE-luciferase reporter gene. In this
method, the induction of CYP3A4 by the xenobiotic-mediated binding of PXR to PXRE leads
 High Throughput Screening for Drug Discovery 33

to the activation of luciferase synthesis, which can be quantified using a chemiluminescent

substrate (luciferin).

HTS Hepatotoxicity Studies

The isolated hepatocytes are also used to study the possible xenobiotic induced hepatotoxicity
in vitro. After the incubation of hepatocytes along with the drug candidate, the hepatocytes
are subjected for measurement of ATP content in microtiter plates using chemiluminescence
with the help of a luciferin-luciferase assay. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide] assay is performed to quantify mitochondrial toxicity. LDH
measurement, neutral red uptake test, thymidine uptake test and estimation of glutathione
give more information on the possible hepatotoxicity of the xenobiotics.

HIGH SPEED DRUG SYNTHESIS PROCESS ‘COMBINATORIAL

CHEMISTRY’ (COMBICHEM)
High-speed primary screening for ‘hits’ require the support from chemistry to feed the prolific
requirement. This is achieved by combinatorial chemistry using parallel synthesis technology.
Traditional synthesis of organic compounds is a very slow process as compared to the present
day technologies adopted for high speed combichem synthesis to meet the requirement of
compound libraries for HTS and lead optimization. The first combinatorial chemistry methods
were presented by Geysen and coworkers39 for peptide synthesis. Later, this has been utilized
for classical heterocyclic and other small molecule organic compounds.40 The intervention of
microwave flash-heating chemistry in dramatically reducing reaction times (reduced from
days and hours to minutes and seconds) has been well recognized.41 Conventional round
bottom flask is replaced with polymer (like TentagelTM) attached reactants in columns to make
easy elution after the reaction. This process can also reduce the time involved in purification of
the final product from the unreacted reactants. Automated organic synthesizers are on rapid
development to further support this effort.

High Speed Natural Product Isolation Techniques

Natural products (NP) are the most successful sources of leads to the evolutionary development
of biologically active metabolic byproducts. Out of 520 new drugs developed and approved
between 1983-1994, 39% of them were either NP or their derivatives.42 However, the long-pending
problems with the NP are the isolation and identification of the active ingredients as well as
ensuring their pharmacological properties. Seasonal, regional, geographical, taxonomical
variations and processing methods make traditional activity guided NP development very
long. Many pharmaceutical giants stopped using whole natural product extract for biological
activities. Due to the development of successful two-dimensional high performance isolation
units like SEPBOX (SEPIAtec, GmbH, Germany) for multiparallel HPLC, it is now possible to
load up to 5 gm of NP extract and to isolate all compounds in 70-80% purity within 24 hr and in
amounts sufficient and in microtiter formats amenable for direct HTS.
This SEPBOX system works by using gradient elution and polarity based trapping in solid
phase extraction (SPE) trap columns. After the successful entrapment of compounds, they are
34 Drug Screening Methods

eluted to obtain pure compounds. Coupling the SEPBOX to photodiode array (PDA) detector
(enhanced UV detector), and evaporative light-scattering detector (ESD) in series enables
the identification and quantification of the significant compounds. NMR or mass spectro­
scopy is further used for the complete identification and structure elucidation. NP database
containing about 10000 different structurally characterized compounds is being commercia­
lized [Chapman and Hall Dictionary of Natural Products (DNP), Ver. 7:2, 8:2 on CD-ROM and
Antibase, Ver 3.0] using a combination of mass spectroscopy and 1 and 2 D NMR.
A study was conducted jointly by Aventis Pharma AG (Vitrysur Seine, France) and
AnalytiCon Discovery (Berlin, Germany) to test the feasibility of high-throughput profiling,
isolation and structure-elucidation technology for NP. A project named Megabolite was
initiated to get minimum 5 mg samples of 4000 pure compounds from microbes and plants.
They have used the above (SEPBOX) technology for the isolation of the active compounds.
The plants were selected from the families of Leguminosae, Euphorbiaceae, Umbelliferae,
Solanaceae, Borganiaceae, Compositeae, Labiatae, Apocynaceae, Rubiaceae, Meliaceae and
Araliaceae. A total of 2242 compounds from microorganisms and 1758 compounds from
plants were isolated in the course of the project. About 2400 pure compounds isolated from
NP were tested against biological targets using HTS assays (scintillation proximity assay and
homogeneous time-resolved fluorescence). The compounds isolated from NP gave a higher
confirmation rate as compared to the Rhone-Poulenc Rorer synthetic compound collection
(>50000 single compounds) and combinatorial library (> 50000 single compounds). Therefore,
the development of advanced HPLC techniques can go beyond the synthetic process in making
drugs with higher success rate in screens.43

NATURAL PRODUCTS COMBICHEM—A NEWER DIMENSION

Crude natural product isolation and testing their activity using different pharmacological
assays is a way to identify thehit from the mixture of the biodiverse compounds. An outstanding
example of the richness of the resources available is the Natural Products Repository of the
National Cancer Institute, comprising >180,000 extracts from > 50,000 organisms. Other
organizations maintain similar, often focused libraries of natural product extracts, including
the Spanish Fundación Medina, with a large collection of microbial samples, the Korean
Research Institute of Bioscience and Biotechnology, with a Korean plant extract bank (http://
www.kribb.re.kr/eng/sub02/sub02_07_02.jsp), and the Eskitis Institute in Australia, with
Australian plants and marine invertebrates.44,45
It is a combined strength of natural products optimization with combinatorial chemistry
for getting the successful bioactive ligands to quicken the success rate. The most powerful
bioactive compounds obtained from natural sources could be due to the revolutionary
transformation of prey-predator concept to acquire unique feature to escape from being
destroyed. For example, Datura is not eaten by cattles only due to the presence of the bioactive
alkaloids. Similarly, thousands of examples can be explained from plants for having bioactive
compounds. One of the key factors for this secret is the enrichment of stereochemistry within
the scaffolds of such molecules. This classically differs from man made compounds having
heterocyclic rings as basic blocks. Simple alkaloid morphine is obtained from opium poppy
takes 40 organic synthetic steps to synthesis. The major structural differences between
 High Throughput Screening for Drug Discovery 35

natural and combinatorial compounds originate mainly from properties introduced to make
combinatorial synthesis more efficient. These include the number of chiral centers, the
prevalence of aromatic rings, the introduction of complex ring systems, and the degree of the
saturation of the molecule as well as the number and ratios of different heteroatoms. As drug
molecules derive from both natural and synthetic sources, they cover a joint area in property
space of natural and combinatorial compounds. A principal component analysis compares
random selection of combinatorial compounds from commercial suppliers against a natural
product and drug database.46,47

OTHER ROLES OF HTS

Role of high throughput techniques in the analysis of the toxins: Currently the increasing
utilization of the natural sources especially for sea foods has posed a potential risk of the
exposure of the consumers to marine toxins. Pollution of drinking water from the marine
toxins is also a big problem. Palytoxins, Ciguatoxins, Cyclic imines and Tetrodotoxins are few
of the marine toxins which are having their impact on the sea food as well as on the water also.
Scientists have started using the techniques of the high throughput screening for the rapid
identification of the contaminants. European Commission and the European Food Safety
Authority (EFSA) have emphasized the potential of these toxins. Earlier Mouse bioassays
(MBAs) have been used for years as important tools to manage seafood safety. Currently the
debate over utilization of the animals for the toxicological analysis has also impacted marine
toxin testing. For the lipophilic marine toxin identification the LC-MS/MS technique is the
reference used by the European Union in an approach to completely remove the use of MBA.
Immunoassay techniques have also been employed for the identification of the marine toxins.
Cell based approaches using the Ouabain and Veratridine as agonist or antagonists have been
utilized for the identification owing the action of toxins on the physiology, morphology or
viability of the cells. Biosensors have also shown promising approaches for the detection of the
marine toxins.48

CONCLUSION
High throughput screening is one of the most prominent domains in terms of drug discovery
which has a pivotal contribution for a new molecule to turn into a therapeutic entity. Emergence
of new diseases and failing treatments for existing diseases has increased the importance of high
throughput screening in a greater extent. Availability of multiplex systems and cellular imaging
systems has made the live screening of the compound a reality. The approach has again turned
towards natural sources for the discoveries of the new molecules in a high throughput manner.
Rapid development in the instrumentation has also helped to fasten up the process of drug
discovery. The combinatorial approach of the drug development scientists and engineering
experts has made the never approaching dream a reality. Learning experiences and out of box
thoughts are still waiting so many changes in this area. This amalgamation of technology in
every field is expected to increase to multiple folds in the next decade and would be creating
a method through which more and more safe drugs would be discovered for almost all the
diseases.
36 Drug Screening Methods

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30. Velpandian T, Gupta P, Ravi AK, et al. Evaluation of pharmacological activities and assessment of
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38 Drug Screening Methods

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 CHAPTER

3
Models for Studying
Stem Cell Therapy
STEM CELLS: AN INTRODUCTION
The science of stem cell therapy, evolved through an imperative phase of research and
development, could possibly bring about unprecedented cures and palliative treatments.
Successful culturing of human stem cells may further illustrate the feasibility and possibility
offered by stem cell derived therapies. Although, the new drug development and advancements
in the biomedical sciences has provided enormous benefits to the individuals as well as
society, yet devastating illnesses such as heart diseases, diabetes, cancer, and neurological
disorders such as Alzheimer’s disease renders enduring challenges to the health and well-
being of people world over. Such disorders have imposed a high economic and psychological
burden virtually on every citizen directly or indirectly. The total costs of treating diabetes,
for example is approaching $100 billion in the United States alone. The existing evidence
from animal studies suggests that stem cells can be made to differentiate into cells of choice,
and that these cells will act properly in their transplanted environment. The transplants of
hematopoietic stem cells for the treatments of cancer, in human beings have been used for
years now. Ex-vivo expansion and transplantation of limbal epithelial stem cells to the corneas
to treat blinding ocular surface disease was one of the first stem cell therapies to be exploited
clinically. It is only through controlled scientific research that the role of stem cell biology in
benefiting various degenerative, metabolic, cardiovascular and cerebrovascular disorders can
be evaluated in animal models as a proof-of-concept and that the true promise of stem cell
therapy will become a boon for incurable diseases.

TYPES OF STEM CELLS

There are several types of stem cells viz:
•• Embryonic stem cells
•• Fetal neural stem/progenitor cells
•• Adult neural stem/progenitor cells
•• Mesenchymal stem cells
•• Umbilical cord blood cells
•• Adipose tissue.
40 Drug Screening Methods

Embryonic Stem Cells

Human embryonic stem cells (ESCs) are pluripotent or endlessly dividing cells that represent
an inexhaustible source of precursor cells as they can differentiate into any cell type. The
capacity of multipotency and self-renewal makes these cells a valuable experimental tool.
The development of ESCs from the human blastocysts and their subsequent differentiation
involves a complex interplay of several technical manipulations.1-7 The human ES cell lines
were derived from embryos produced by IVF for infertility treatment. Cryopreserved embryos
were thawed, cultured to the blastocyst stage (5-6 days) and the pluripotent stem cells from
the ICM were isolated. The stem cells were most commonly cultured on mouse embryonic
fibroblast feeder cell layer to prevent differentiation and to promote the proliferation of
the stem cells.8 Several lineages have been developed using this technique, for example,
endothelial and hematopoietic progenitor cells,9 and neural stem cells.10 Different strategies
have also been adopted to eliminate the use of mouse feeders and maintain the human ESCs
in an undifferentiated state.11-18
Since, the key factor responsible for the differentiation of ESCs is still unknown, several trial
and error approach have been adopted to direct them to differentiate into a desired lineage
as shown in Figure 3.1. There are evidences that most of the undifferentiated cells in the EBs
express receptors for the growth factors.19 Hence, supplementation of the human ES cell culture

Figure 3.1: Tissues of endodermal, mesodermal and ectodermal origin derived from embryonic stem cells
after in vitro differentiation
 Models for Studying Stem Cell Therapy 41

with different growth factors altered the expression profile of an array of tissue-restricted gene.
With this technique a variety of cell and tissue types have been derived from ESCs (Table 3.1).
Although, many studies using embryonic or fetal stem cells have shown cell maturation and
successful engraftment, yet there are several difficulties in using human ESCs. The first one is
the ethical issue surrounding the use of human ES cells.50 Secondly, ES cells are allogenic, and
immunosuppressive therapy might be needed. This problem could be alleviated by establishing
ES cell banks containing the full range of major histocompatibility antigens.51 However, this
may be a futuristic approach since the establishment of human cell lines52 has been more
difficult than murine ones.53 Thirdly, increased cell death due to ischemia has been observed,
when ES cell-derived cardiomyocytes were grafted into a normal myocardium. Finally, human
ESCs have also been shown to have the potential to form teratomas, when injected into an
immunocompromised mouse.52 These drawbacks have lead researchers to seek alternative
undifferentiated cells for cell replacement therapy.

Fetal Neural Stem/Progenitor Cells

Neuronal progenitor cells (NPC) derived from ventral mesencephalon (VM) are particularly
suited as the target population for genetic and cellular therapy of neurological disorders.
Recently, continuously dividing immortalized cell lines of neural stem cells (NSCs) have been
generated by introduction of oncogenes and these immortalized NSC lines have advantageous
characteristics for basic studies on neural development and cell replacement therapy or gene
Table 3.1: Differentiated cell types from mouse and human ESCs in vitro

S. No. Mouse Human

1. Adipocytes 20
Hematopoietic colony-forming cells43
2. Astrocytes 21
Dopaminergic neurons44
3. Cardiomyocytes22 Myocytes45
4. Chondrocyte 23
Pancreatic cells46
5. Primitive and Definitive hematopoietic cells24 Different cells of spinal cord47
6. Dendritic cells 25
Cardiomyocytes48
7. Endothelial cells26 Endothelial cells49
8. Keratinocytes 27

9. Lymphoid precursor cells28

10. Mast cells29
11. Neurons30, 31
12. Oligodendrocytes32, 33
13. Osteoblasts34
14. Insulin secreting cells35, 36
15. Smooth muscle cells37
16. Skeletal muscle cells38
17. Melanocytes39
18. Hepatocytes40, 41
19. Pneumocytes42
42 Drug Screening Methods

therapy studies because: (a) NSC cell line can be expanded to large numbers in culture in
short time (24–36 h doubling time); (b) NSC cells are homogeneous, since they were generated
from a single clone; (c) stable expression of therapeutic genes can be achieved readily.54-57
Immortalized NSCs were shown to have genetically manipulated in vitro, survive, integrate
into host tissues and differentiate into both neurons and glial cells after transplantation to
the intact or damaged brain.58,59 More recently, new lines of immortalized human NSCs, were
developed with the ability to self-renew, differentiate into cells of neuronal and glial lineages
both in vivo60 and in vitro.61 Following transplantation into the brain of animal models of focal
ischemia,62, 63 intracerebral hemorrhage,64 Huntington disease65,66 and Parkinson’s disease.67
HB1.F3 human NSCs successfully integrated into host brain parenchyma and provided
functional recovery in these experimental animals.

Adult Neural Stem/Progenitor Cells

Recent, studies have confirmed that the adult mammalian brain, including that of primates
and humans, harbors stem cell populations. Adult NPC in the nigrostriatal dopaminergic
system have the ability to respond to the lesion or when exposed to appropriate environmental
signals to repopulate missing cells in neurodegenerative disorders such as Parkinson’s
disease.68-71 Manipulations of NPCs in culture can be made for therapeutic transplantation or
for mobilizing endogenous precursors to repair diseased or injured brain.72,73

Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs), also referred to as marrow stromal cells, can be isolated
from several tissues but easily accessible bone marrow (BM) seems to be the most common
source. These cells isolated from BM can be induced in vitro and in vivo to differentiate
into neuronal cell population.74 The multilineage potential of MSCs,75,76 their ability to elude
detection by the host’s immune system as expression of costimulatory molecules like B7-1
(CD80), B7-2 (CD86) and CD40 are lacking in them, which are necessary for instigation of
T-cell proliferation.77-79 Recently, it was shown that human MSCs isolated by their adherence to
plastic were transplanted80,81 or infused82-86 into the corpus striatum and successful engraftment
was achieved.

Umbilical Cord Blood Stem Cells (UCBSC)

The multipotent-stem-cell-rich blood found in the umbilical cord has proven useful, as they
are less prone to rejection than either BM or peripheral blood stem cells. This is probably
because the cells have not yet developed the features that can be recognized and attacked
by the recipient’s immune system. Also, because umbilical cord blood lacks well-developed
immune cells, there is less chance that the transplanted cells will show immune reaction with
the recipient’s body. Both the versatility and availability of umbilical cord blood stem cells
makes them a promising source for transplant therapies.

Adipose Tissue
Primary cultures of adipose tissue are a heterogeneous collection of hematopoietic cells,
pericytes, endothelial cells, and smooth muscle cells. Several passages in cultures yields
stromal cells that exhibit cell-surface markers consistent with mesenchymal stem cells.87,88
 Models for Studying Stem Cell Therapy 43

STEM CELL THERAPY FOR NEUROLOGICAL DISORDERS

Chronic degenerative diseases and traumatic injuries are responsible for a decline in neuronal
function and transplantation of stem cells or their derivatives, and mobilization of endogenous
stem cells within the adult brain has been proposed as future therapies for such neural
disorders (Table 3.2), and many kinds of cells including ESCs and NSC have been considered
as candidates for transplantation therapy (Fig. 3.2).

Parkinson’s Disease
Parkinson’s disease (PD) is caused by the degeneration of dopaminergic bundle originating
from SN pars compacta (SNpc) leading to diminished tone exerted by dopamine which elicits
the characteristic parkinsonian movement disorder. Currently, the stem cells from various
sources are induced to acquire a mesodiencephalic dopaminergic (mdDA) phenotype.
The protocols summarized in Figure 3.3 briefly describe the methods for in vitro expansion,
manipulation of genetic as well as culture conditions of stem cells. Out of the various PD
models, 6-OHDA and MPTP induced PD has been widely used to assess the therapeutic
potential of stem cells.

In Vivo Models
a. 6-OHDA Model: The 6-OHDA is the first chemical agent discovered that has specific
neurotoxic effects on catecholaminergic pathways.139 To specifically target the nigrostriatal
DA pathway, 6-OHDA must be injected steriotactically into the SN, the nigrostriatal tract or
the striatum.140 Following injections, DA neurons start degenerating within 24 h after, and
striatal dopamine is depleted 2 to 3 days later. 141
There is ample evidence for the involvement of oxidative stress in 6-OHDA-induced
neurotoxic effects.142-147 The 6-OHDA lesion model has been used to ascertain the efficacy
of antiparkinsonian compounds.148 Additionally, this experimental model has been useful
for evaluating the efficacy of cell transplantation, and for testing neurotrophic factors,
compounds that promote survival of the degenerated dopaminergic nigral neurons in
PD.149
b. The MPTP Model: Inadvertent injection of MPTP resulted in clinical symptoms remarkably
similar to sporadic PD in humans150 by selectively affecting DA neurons.151 The mechanism
of action of MPTP suggests a role of mitochondrial dysfunction in typical PD.152,153 MPTP
administration is one of the most common animal models used to study PD and various
doses and regimens of MPTP administration are used by different laboratories.154-156

Gene-knockout and Transgenic Animals

Recent transgenic strategies have generated several mouse lines with mutations in the DA
system.157,160 D2-receptor-deficient mice,161 in contrast to D1162 and D3163 receptors exhibit
PD-like symptoms without neuronal death and Lewy bodies’ formation. Mutations in the gene
encoding a-synuclein, found in Lewy bodies, are related to PD in some species.164,165 However,
transgenic mice overexpressing either wild-type or mutant asynuclein have generated
inconsistent results.166,167
Table 3.2: Use of different stem cells in various animal models of neurological disorders 44
Types of stem cells Cerebral stroke Alzheimer’s disease Parkinson’s Huntington’s Amyotrophic lateral
Models Species Models Species Models Species Models Species Models Species
Embryonic stem MCAO R89,90 Nucleus M103 6-OHDA R106 Quinolinic R116 Transgenic M129
cells basalis of Model acid induced animals
Meynert lesion in
(NBM) lesion striatum
Embryonic 6-OHDA R107 Transgenic R130
carcinoma cells Model animals
Adult neural stem/ MCAO SprhR91, Transgenic M104 6-OHDA R108-110 Quinolinic M117-119 Transgenic R131
Drug Screening Methods

Progenitor cells R92, 93 animals Model acid induced animals

R120-123
-lesion in
striatum,
Transgenic
animals
Fetal neural stem/ MCAO M94, 95, CM96, Nucleus M105 6-OHDA R111-113 Quinolinic R124-126, 66 Transgenic R132-134
Progenitor cells R97, MG98 basalis of Model acid induced animals
Meynert -lesion in
(NBM) lesion striatum
Mesenchymal MCAO R99 6-OHDA R80, 86, 114, Quinolinic R127, M128 Familial M135, R136
stem cells or Model, MPTP- M81, 115 acid induced ALS model,
Marrow stromal Model -lesion in Transgenic
cells striatum animals
Hematopoietic Motoneuron M137
stem cells degeneration
model
Umbilical cord MCAO R100, 101 6-OHDA Transgenic R138
Model animals
Adipose tissue MCAO M102 6-OHDA
derived stem cells Model

MCAO-middle cerebral artery occlusion; R-rat; M-mouse; 6-OHDA-6-hydroxy dopamine; Sprh R-Spontaneously hypertensive rats; CM-cynomolgus monkeys;
MG-Mongolian gerbils; MPTP-1-methyl-4-1, 2, 3, 6- tetrahydropyridine; ALS-amyotrophic lateral sclerosis
 Models for Studying Stem Cell Therapy 45

Figure 3.2: Different sources of stem cell employed for cell replacement therapy for the treatment of various
neurological disorders

In Vitro Models
Cultures of dissociated mesencephalic neurons from fetal rats168 and human neuroblastoma
SH-SY5Y cells169 are suitable models of PD.
The restorative potential of successful transplantation of stem cells may be assessed
by subjecting the animals to the battery of neurobehavioral tests such as amphetamine/
apomorphine-induced rotation,170 rotarod test,171 cylinder test172 and elevated body swing
test.173

Alzheimer’s Disease
Alzheimer’s disease is a neurological disorder characterized by the presence of neurofibrillary
tangles, neuritic plaques and dystrophic neurites in susceptible areas of the brain. The
adult mammalian brain contains populations of stem cells that can proliferate and then
differentiate. The highest concentration of such neural progenitor cells (NPC) is located in
the SVZ and provide a cellular reservoir in the aged and AD brain, both the pool of NPC and
46 Drug Screening Methods

Figures 3.3A to C: Schematic representation of protocols for the generation of dopaminergic neurons for the treatment
of parkinson’s disease from different sources of stem cells viz Embryonic stem cells (A), Fetal neural progenitor cells (B),
Adult neural progenitor cells (C)
 Models for Studying Stem Cell Therapy 47

Figures 3.3D and E: Mesenchymal stem cells (D) and umbilical cord blood cells (E).
Abbreviations: SCDIA-stromal cell-derived inducing activity; FGF-2-fibroblast growth factor-2; SFM-serumfree medium;
DA-dopaminergic; TH-tyrosine hydroxylase; GTPCH-1-guanidine triphosphate cyclohydrolase- 1; NPC-neural progenitor
cells; VM-ventromedial; DMEM-Dulbecco’s Modified Eagles’ Medium; FCS-fetal calf serum; EGF-epidermal growth factor;
bFGF-basic fibroblast growth factor; TGF-tissue growth factor

their proliferative potential is markedly diminished. It was reported that Abeta itself can impair
neurogenesis in the SVZ/cerebral cortex of adult mice and in human cortical NPC in culture.
The proliferation and migration of NPC in the SVZ of APP mutant mice, and in mice receiving an
intraventricular infusion of Abeta, were greatly decreased compared to control mice. Studies of
NPC neurosphere cultures derived from human embryonic cerebral cortex showed that Abeta
can suppress NPC proliferation and differentiation, and can induce apoptosis. The adverse
effects of Abeta on neurogenesis were associated with a disruption of calcium regulation174 and
high oxidative stress.175 It has been recently discovered that Seladin-1 (selective Alzheimer’s
disease indicator-1) abundantly expressed by stem cells is an antiapoptotic gene, which
is down-regulated in brain regions affected by Alzheimer’s disease (AD).176,177 However,
recently it has been shown that alpha-secretase-cleaved fragment of the amyloid precursor
protein (sAPPalpha), a potent neurotrophic factor, potentiates the NGF/retinoic acid (RA)
induced transdifferentiation of bone marrow-derived adult progenitor cells (MAPCs) into
neural progenitor cells and, more specifically, enhances their terminal differentiation into a
cholinergic-like neuronal phenotype.178 Further, a neurosteroid alloprognanolone (APalpha)
was discovered, which showed potential to promote neurogenesis in the aged brain and restore
neuronal populations in brains recovering from neurodegenerative disease or injury.179,180
Mammalian presenilins (PS) consist of two highly homologous proteins, PS1 and PS2.
Because of their indispensable activity in the gamma-secretase cleavage of APP to generate
Abeta peptides, inhibition of PS gamma-secretase activity is considered a potential therapy for
Abeta blockage and AD intervention. In addition to its well-established role in gamma-secretase
cleavage, presenilin (PS) also plays a role in regulating the stability of cytosolic beta-catenin, a
protein involved in Wnt signaling in familial Alzheimer’s disease, mutation in the presenilin-1
(PS1) or APP gene and abnormally elevated levels of FGF-2181 is responsible for the development
of early-onset of vascular pathology182 and altered neurogenesis in the adult hippocampus.183,184
As mentioned above, the pathological changes seen in AD display an extremely
problematic situation for cell replacement. The widespread damage found in the AD brain
48 Drug Screening Methods

usually incorporates distinct regions of brain and hence it is unlikely that the mechanisms for
instructing transplanted NSCs to differentiate into new neurons will be intact. Moreover, the
endogenous neurogenesis itself has been severely hampered by the Abeta plaques which may
be an important reason for running out of the nitch required for the growth and development
of newly transplanted neurons. However, genetically modified stem cells could be used to
deliver growth factors that can modify the course of the disease.

Models for Alzheimer’s Disease

Nucleus Basalis of Meynert (NBM) Lesion

Clinically, degeneration of the nucleus basalis of Meynert (nbM), from which cholinergic
fibers project to the cerebral cortex, leads to the development of dementia of Alzheimer type
(DAT).185-191 To obtain more selective lesion in the substantia innominata (SI), ibotenic acid
injection rather than kainic acid injection or electrocoagulation techniques192,193 are employed.

Transgenic Animals
Overexpression of the gene encoding the beta-amyloid precursor protein (APP) may have a
key role in the pathogenesis of both Alzheimer’s disease (AD). Because it has been difficult to
overexpress APP by using conventional transgenic technologies, Lamb et al194 have recently
used yeast artificial chromosome(s)/ESC (YAC-ES) technologies to create a dosage imbalance
and overexpression of APP in mice.
A 650 kb YAC that contained the entire unrearranged 400 kb APP gene was transferred into
ES cells by lipid mediated transfection; ES cells that expressed human APP were introduced
into mouse blastocytes to generate a number of chimeric mice. Subsequent breeding efforts
resulted in mice that harbor human sequences in the germ line.

Huntington’s Disease
Huntington’s disease (HD) is an autosomal dominant genetic disease, which results in
progressive neuronal degeneration in the neostriatum and neocortex, and the mechanism
by which this leads to neuronal cell death is still a mystry. Huntingtin (Htt) gene plays a
fundamental role in embryogenesis,195 growth and development and, is essential for the normal
nuclear (nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria,
endoplasmic reticulum, Golgi and recycling endosomes) organelles and for proper regulation
of the iron pathway.196 One proposed mechanism by which mutant htt causes dysfunction
of striatal enkephalinergic neurons is the downregulation of BDNF.197 NSC may become the
tissue/cell source necessary for developing the therapeutic potential of neural transplantation
(Fig. 3.4).

Models for Huntington’s Disease

1. Quinolinic Acid Induced Lesion in Striatum
The leading animal models of HD have involved injection of neurotoxins into the striatum
of rats, which causes local destruction of this area of the brain. Neurotoxins such as kainic
 Models for Studying Stem Cell Therapy 49

Figures 3.4A and B: Schematic representation showing use of ESC (A) and adult neural stem cells (B) for the generation
of neural progenitors cells for the treatment of Huntington’s disease
Abbreviations: ESC-embryonic stem cells; NGF-nerve growth factor; BDNF-brain derived neurotrophic factors

Figure 3.5: Schematic representation showing in vivo transgenic mice model carrying a mutated human gene for
huntingtin (htt) and a protocol for producing neuronal and glial population with HD phenotype for in vitro screening of
drugs and stem cell therapy for Huntington’s disease

acid198 and ibotenic acid,199 lead to excitotoxic death. In 1983, Schwarcz group found that
injection of quinolinic acid (QA) into the striatum of rats produced a similar excitotoxic
effect.200-206
2. Transgenic Models
The transgenic mouse model R6/2 expresses exon 1 of the human huntingtin gene with >150
CAG repeats, which produces mutant HD protein with an expanded poly-glutamine tract.
For in vitro models, neuronal stem cell system deriving from transgenic HD R6/2 neonatal
brains can be used as a renewable source for neurons and glia to facilitate studies of HD
neuropathology and therapies. These R6/2 stem cell cultures can be cryopreserved and
revived. Thawed neural progenitors can be expanded, established as continuous cell lines,
and induced to differentiate into glia and neurons207, 208 (Fig. 3.5). The restorative potential
of successful transplantation of stem cells may be assessed by subjecting the animals to the
battery of neurobehavioral tests such as amphetamine/apomorphine-induced rotation,170
water-maze test,209 beam-walk test,210 staircase (skilled-limb use) test.211
50 Drug Screening Methods

Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease)

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease affecting the
spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular
weakness and atrophy, leading to paralysis and death within 3–5 years of diagnosis and
hence demands for an effective treatment. Stem-cell transplantation is an attractive strategy
for neurological diseases and early successes in animal models of neurodegnerative disease
generated optimism about restoring function or delaying degeneration in human beings.
Therefore, autologous or allogeneic stem cells, undifferentiated or transdifferentiated and
manipulated epigenetically or genetically, could be a candidate source for local or systemic
cell therapies in ALS (Fig. 3.6). The attraction of cell implantation or transplantation is that
it might help to overcome the inability of the CNS to replace lost neurons. It is also clear that
neural implantation will yield little benefit if the donor cells fail to integrate functionally into
the recipient CNS circuitry. The recent breakthroughs in stem cell research provide possibilities
for neural implantation and cell replacement therapy for patients with ALS.

Models for Amyotrophic Lateral Sclerosis

1. Transgenic Model
Mutations of human Cu, Zn superoxide dismutase (SOD) are found in about 20 percent of
patients with familial ALS. Expression of high levels of human SOD containing a substitution of
glycine to alanine at position 93 in chromosome 4, causes motor neuron disease in transgenic
mice. The mice became paralyzed in one or more limbs as a result of motor neuron loss from
the spinal cord and died at 5 to 6 months of age.
The familial mouse model of ALS was prepared as described in Figure 3.7, and the completed
construct was then excised with Sal I and electroeluted from agarose for microinjection212-214
into zygotes.
There are evidences of widespread regenerative response in the spinal cord of amyotrophic
lateral sclerosis transgenic (Tg) mice.215-217 However, this regenerative response appears to
be largely unproductive and it has been shown that proliferation and migration of neural
precursor cells to the ventral horns is greatly activated in symptomatic Tg mice and is further
enhanced by EGF and FGF2 treatment.31 (Fig. 3.8)
2. In Vitro Model
In vitro model has been developed for studying the molecular and cellular mechanisms that
underlie the neurodegenerative disease ALS213 (Fig. 3.9). ESCs derived from mice carrying
normal or mutant transgenic alleles of the human SOD1 gene were used to generate motor
neurons by in vitro differentiation. These motor neurons could be maintained in long-term
coculture either with additional cells that arose during differentiation or with primary glial
cells. Motor neurons carrying either the nonpathological human SOD1 transgene or the
mutant SOD1(G93A) allele showed neurodegenerative properties when cocultured with
SOD1(G93A) glial cells. The glial cells carrying a human SOD1(G93A) mutation have a direct,
non-cell autonomous effect on motor neuron survival. More generally, ESC-based models of
disease provide a powerful tool for studying the mechanisms of neural degeneration. These
phenotypes displayed in culture could provide cell-based assays for the identification of new
ALS drugs or alternative stem cell based therapy.
 Models for Studying Stem Cell Therapy 51

Figure 3.6: Generation of cholinergic motor neurons for ALS

Abbreviation: SOD1-superoxide dismutase-1
52 Drug Screening Methods

Figure 3.7: Mutagenesis of the mouse SOD-1 gene. The transgene was constructed by introducing a point mutation at
the indicated position (*) in exon 4. Nucleotides shown in boldface type form a recognition sequence for Fsp I generated
by the mutagenesis procedure

Figure 3.8: Administration of growth factors promotes endogenous regenerative process in ALS mouse model

Figure 3.9: In vitro model system for studying the mechanisms of neural degeneration and identification of
new ALS drugs or alternative stem cell based therapy
 Models for Studying Stem Cell Therapy 53

STEM CELL THERAPY FOR CARDIO- AND CEREBROVASCULAR

DISORDERS
Congestive heart failure (CHF) due to myocardial infarction (MI, or heart attack) often has scar
tissue in the heart, which limits the heart’s ability to pump blood which may be characterized by
dyspnea, edema and disability. In MI, the infarcted tissue does not contribute to the generation
of mechanical activity and the compensatory forced contractile movements uncouples the
excitation contractile function of surrounding viable myocardium through a series of events
known as left ventricular remodeling. Treating physicians may now have the opportunity to
successfully replace scarred heart tissue with healthy muscle via intracardiac injections of
stem cells taken from the patient’s own body. By using a catheter and transplanting the stem
cells into scarred tissue, myocardium can be regenerated with limited risk to the patient. Since,
the transplanted stem cells are harvested from the patient’s own body, the cells are compatible
with the body, avoiding possible immune system and tissue compatibility complications.

Myocardial Infarction
There is growing evidence, which suggests that the heart has the ability to regenerate through
the activation of resident cardiac stem cells or through the recruitment of stem cell population
from other tissues such as bone marrow. Hence, stem cell therapy appears as a promising new
approach for myocardial repair.
Experimental models were extensively used to study the stem cell potential in regenerating
cardiomyocytes.218 Therapeutic intervention like supply of endothelial stem cell219 or angiogenic
factors like the cytokine vascular endothelial growth factor (VEGF) and the bFGF have also
been successfully tested.220 Lyseggen et al221 used an open chest dog heart model to document
echocardiographic indices of potential use during evaluation of reperfused myocardium. This
model provides means to evaluate post-ischemic viability and therefore whether reperfusion
was successful on the basis of echocardiography, sonomicrometry, intraventricular pressure
measurements, tissue staining for necrosis and coronary perfusion measurements using
radionucleotid microspheres for microvascular perfusion.

In Vivo Model
Coronary Artery Ligation in Mice
Ischemia is achieved by occluding LAD by using 8-0 silk suture, with a 1 mm section of
polyethylene (PE)-10 tubing placed on top of the LAD, 1 to 3 mm from the tip of the normally
positioned left atrium. After occlusion for a specified duration, reperfusion is allowed to
occur by releasing the ligature and removing the PE-10 tubing. Blood flow is confirmed
by visualization of the return of a bright red color in the previously pale region. Hearts are
harvested after desired period of reperfusion.222, 223

In Vitro Models
a. Isolated perfused heart: Hearts are excised from heparinized mice and prepared according
to the standard Lagendorrf preparation for retrograde perfusion.224 The hearts are subjected
to global ischemia by clamping the perfusion line. After ischemia of desired duration, the
perfusion line is released, and hearts are reperfused. Cardiac contractile performance and
coronary flow are recorded during stabilization, ischemia, and reperfusion.
54 Drug Screening Methods

b. Cardiomyocytes: Cardiomyocytes can be isolated acutely from hearts of all species and
manipulated to simulate ischemia. By applying confocal laser scanning microscopy-based
cell imaging technique to study isolated adult rat cardiomyocytes, mitochondrial function
and integrity intact cells.225
c. Cell culture techniques: Neonatal mice heart cell cultures can be harvested from genetically
engineered mice hearts; transfection techniques as well as silencing RNA technique can
be used in cell cultures. HL-1 cells are immortalized cells derived from an atrial tumor
(myoma) in female mice.226, 227 Compared to the use of primary isolation of ventricular
myocytes, the advantages of having a good immortalized cell culture model for use in
ischemia studies involve the following: (i) no animal use and care, (ii) no costly and lengthy
preparation time before any experiments can take place, (iii) no variable and limited yield
for high-throughput approaches and (iv) no problems with heterogeneous cell population.
The HL-1 cells and the following generations of these cells have been used successfully
for ischemia related experiments; overall results correlate well with results obtained, for
example, isolated hearts.228-231

In Silico Models
There is limited tradition for in silico models in ischemic heart research. One exception is the
electrophysiological modeling of the consequences of changes in ion balance with ischemia.
Important input in these models includes potassium loss to the extracellular space, intracellular
proton accumulation and opening of ATP dependent potassium channels, cellular electrical
uncoupling and delayed conduction, diastolic calcium overload and increase in cellular
sodium.232,233

Cerebral Stroke
The major causes of stroke include ischemia and intracerebral hemorrhage (ICH) and it affects
multiple different neuronal phenotypes like oligodendrocytes, astrocytes, and endothelial
cells. Medical therapy against stroke remains limited, hence prompting an alternative
approach.234 No approach has been successful in replacing the lost neurons, improving the
deteriorated functions, or reducing the long-term sequelae.235 There have been previously
several reports of cell transplantation in the brain of ischemic animal models. Stem cells from
various sources such as ESCs, MSCs,236 immortalized NPCs237,238 and adult stem cells were
grafted into the ischemic brain as shown in Figure 3.10, and reduced the neurological deficits
induced by experimental brain ischemia. Specific types of cells with restricted fates may limit
their use as potential sources for implantation in stroke. For maximal functional recovery,
however, regenerative therapy may need multifaceted approaches, which may include cell
replacement, trophic support, protection from oxidative stress, and the neutralization of the
growth-inhibitory components for endogenous neuronal stem cells.

Models for Cerebral stroke

To instigate better understanding of the underlying pathology, and neurological and behavioral
consequences of cerebral ischemia, it is mandatory that physiologically controlled and highly
reproducible in vivo models be developed.239-257
 Models for Studying Stem Cell Therapy 55

Several methods have been adopted to achieve clinical manifestations as close as possible,
as shown in Table 3.3.

Table 3.3: Some common models of cerebral ischemia

Types of model Representative models Notes

Focal ischemia Middle cerebral artery occlusion Several species used:
• Transient • Uses clips, intraluminal thread and snare
• Permanent • Uses intraluminal thread, clips and coagulation
• Thrombotic • Injection of either microspheres or clots into
cerebral vessels including MCA
Global ischemia Bilateral carotid occlusion • Primarily in gerbils
Two vessel plus hypotension • Normally in rats
Four vessel occlusion • Normally in rats
Hemorrhagic Infusion of collagenase into brain

CONCLUSION AND FUTURE PERSPECTIVES

The hope of stem cell-based therapy against irreversible cellular damage in organs with
limited regenerative capacity has opened up new vistas for the treatment of several debilitating
disorders. However, American Association of Advancement of Science (AAAS) and the Institute
of Civil Society (ICS) have recognized that varied ethical, religious, legal and social view points
are to be considered before carrying out research on tissues derived from embryonic and fetal
stem cells. Although, the concept of transdifferentiation and reprogramming had resolved
several long-standing issues such as teratoma formation, immune rejection, etc. yet successful
functional and anatomical integration of transplanted cells with the host environment needs
several technical manipulation. These include isolation of desired stem cells from a mixed
population and alteration in growth medium during in vitro differentiation in order to obtain a
repertoire of specific types of cells for replacement. Moreover, transfer of new genetic material
to stem cells and expression of the gene product in daughter cells is an exiting approach to
make cells compatible for the host environment. Recently, researchers have explored self-
repair mechanism in organs earlier thought of as post-mitotic one, by identifying resident stem
cells which could also be induced to migrate to the damaged site for repair. However, there is
still a long way to go before exploiting them clinically since many basic issues remain to be
resolved, and we need to move forward with caution while undertaking clinical trials. Hence,
overall stem cell-based therapy is a promising, yet to improve, strategy for the treatment of
several degenerative disorders.
To aid in further progress toward the clinics, animal models that closely mimic the human
disease have been used. Such models will allow us to assess and balance potential risks and
benefits of stem cell therapies before their application in humans. Moreover, transgenic animals
expressing wild-type or mutated genes which may be responsible for any disease phenotype
are currently being used which provide an important opportunity to study the involvement
of specific gene/s in particular disorder and derive a relation between genetic mutations and
environmental exposures. Moreover, current in vivo test methods are laborious, costly and
56 Drug Screening Methods

Figure 3.10: Protocols for generation neural and glial population for the treatment of cerebral stroke from different
sources of stem cells viz ESCs (A), Fetal neural progenitor cells (B), Adult neural progenitor cells (C), Mesenchymal stem
cells (D)
Abbreviations: BMSCs-bone marrow derived stem cells; BDNF-brain derived neurotrophic factors; G-CSF Granulocyte
colony stimulating factor; bFGF-basic fibroblast growth factor; SCF-stem cell factor
 Models for Studying Stem Cell Therapy 57

require use of large numbers of laboratory animals which necessitates the use of in vitro model
system. Likewise, we need to improve noninvasive in vivo imaging technologies so that we can
track down the migration of stem cells to the damaged site and monitor regenerative processes
subsequent to stem cell-based approaches in animals and humans.

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 CHAPTER

4
Antistroke Agents
INTRODUCTION
Stroke, or “brain attack,” is a clinical syndrome caused by impairment of cerebral blood flow
(CBF) leading to acute cerebral deficit. It is the second commonest cause of death with an
estimated 5·7 million deaths in 2005 and 87% of these deaths were in poor and developing
countries.1-4
Brain receives effective perfusion about 55 ml/100 g/min through finely distributed blood
vessels and their collaterals in order to cope with the high metabolic demands of the brain.
The inability of brain unlike other organs to store oxygen and energy substrates makes it highly
sensitive to CBF changes and any disruption of blood supply to the brain tissue if persists for
more than three to four minutes, neuronal cells are at the thresholds of damage.5
Neuronal injury in stroke is thought to results following reduction in the blood supply
predominating at the center of ischemic zone (core) and at the penumbra of ischemic damage,
and is dependent on the severity and duration of ischemic insult. The oxidative damage does not
occur in isolation but in a complex interplay between excitotoxicity, apoptosis, inflammation
and extent of reperfusion.6 The cellular and molecular basis of these events is highlighted by us
in a recent review with a focus on numerous molecular targets.7
The energy failure following CBF reduction results in neuronal depolarization and
subsequent activation of glutamate receptors. This in turn alters ionic gradient across
membranes and activate complex survival/damage signaling mechanisms. However, a
particular threshold exists for various cellular/molecular events leading to failure of synaptic
function, when CBF falls below 16-18 ml/100 g/min and at a lower threshold (10-12 ml/100
g/min), the membrane failure ensues due to breakdown of cellular ionic homeostasis.5,8
Further, the most brain lesions that develop after cerebral ischemia evolve from an initial
stage of reversible to an infarct at the core, where most neurons become necrotic, while
cellular damage in penumbral zone is mediated via apoptotic pathway although secondary
necrosis may occur if the CBF is not restored early. Necrosis occurs in an indiscriminate
fashion due to loss of osmotic homeostasis and rupture of plasma membrane. On the other
hand apoptosis becomes activated following cerebral ischemia by variety of death signals
such as production of free radicals and tumor necrosis factor, deficiency of growth factor,
DNA damage, and mitochondrial dysfunction.9,10 It is characterized by a series of well-defined
distinct morphological and biochemical changes.11-13
72 Drug Screening Methods

Apoptosis is fundamentally mediated by caspases, a family of cysteine proteases that are

synthesized as proenzymes and cleaved into active form. Caspase activation ultimately leads
to break down of cellular framework, DNA cleavage and chromatin condensation, which is
the hallmark of apoptosis. The caspase cascade is initiated either by death receptor belonging
to tumor necrosis factor receptor (TNFR) superfamily proteins (extrinsic), mitochondrial
dysfunction or endoplasmic stress (intrinsic).12,14,15 The crosstalk between extrinsic and
intrinsic apoptosis occurs at various stages.
Neuroprotection remains a key goal for stroke therapy and rt-PA is the only current
pharmacological option clinically available.16 However, the use of rt-PA has been limited only
to less than 5% of ischemic stroke patients owing to its narrow therapeutic window and chances
of hemorrhagic transformation. Further, the announcement by AstraZeneca of withdrawal of
nitrone radical trapping agent disodium 2,4-disulfophenyl-N-tert-butylnitrone (NXY-059)
from clinical trials due to the poor results at most of the endpoints is the latest setback to the
development of effective neuroprotective therapy and has joined the list of unsuccessful 114
compounds that have gone under clinical trials. To avoid this ever increasing widespread
failure of neuroprotective drugs, a meeting of experts from academia and industry was held to
formulate the guidelines for the preclinical evaluation of candidate neuroprotective drugs.17,18
Despite these efforts in translational research, the failure of NXY-059 and other have further
raised the doubts on future trial.18-20 Recommendations of the Stroke Therapy Academic
Industry Roundtable (STAIR) for development of preclinical neuroprotectant for clinical
advancements.17,18
•• The candidate drug should be evaluated in permanent and transient focal occlusion models
and subsequently in primate models.
•• Time window studies confirming efficacy at both pre- and post-treatment dose regimen,
dose response effect over a reasonable time window.
•• Appropriate physiological monitoring of animals undertaken.
•• Histological and functional short and long-term outcome should be assessed with prolonged
survival and protection of subcortical as well as cortical structures.
•• Results should be replicated in different animal models and in various laboratories.
•• Data should be published both positive and negative in peer-reviewed journal.
Moreover, a single neuroprotective agent working on only one aspect of the ischemic
cascade is not likely to exert a substantial beneficial effect on the size of infarct or functional
outcome. Thus, it is speculated that combination therapies21,22 or single agent that acts on
multiple pathways of the ischemic cascade23 might have a greater chance of success in
providing neuroprotection.

TYPES OF STROKE
Stroke may be classified as ischemic accounting for ~85% of all stroke cases and hemorrhagic
only by 15% (Fig. 4.1). Ischemic stroke is induced by total hypofusion as during cardiac arrest,
near drowning, carbon dioxide poisoning, massive bleeding or focal loss of regional CBF due
to atherosclerotic or embolic blockade of an artery. Whereas, hemorrhagic stroke occurs, when
there is focal loss of blood flow may be due to vasospasm, which is caused by rupture of blood
vessels within the brain (primary subarachnoid) or on the surface (primary intracerebral).
 Antistroke Agents 73

Figure 4.1: Schematic presentation of different types of stroke

SELECTION OF MODEL
The contribution of the various signaling mechanisms to the extent of the final infarct in
human is still not very clear, therefore, in vitro and in vivo models have been developed to
mimic human stroke pathophysiology to develop the effective neuroprotective strategies.
Their inclusion in the stroke and related study depends upon the anatomic homogeneity,
complexity, reproducibility or simulation of physiological and pathophysiological events.
These models can be classified as focal or global, complete or incomplete and transient or
permanent (Table 4.1).
However, appropriate selection of model system is imperative prerequisite for drug
development for any pathology including stroke. Therefore, designing any neuroprotective
strategy, STAIR recommendations should be taken into consideration in order to achieve a
drug with fruitful outcome (Fig. 4.2).

In Vitro Models
The effect of cerebral ischemia in vitro can be visualized using primary culture of cerebral
capillary endothelial cells, astrocytes, microglia, neurons (cortical, hippocampal, striatal,
cerebellar granule cells, etc.), cell lines (HT22, PC12, SY5Y, etc.). The cells, for example,
postmitotic neurons, from different brain regions such as cortex, striatum, hippocampus, etc. of
16 to 18 day old mouse or rat embryos can be isolated and grown for several days as 10 to 14 days.
These cells are then deprived from oxygen and glucose referred as oxygen-glucose deprivation
(OGD) model and induced chemical hypoxia through 3NPA, CN- and deoxyglucose. Moreover,
stroke related changes can be also studied in vitro using organotypic cortical or hippocampal
brain slices (acute and culture),24-29 since, it is believed that complexity in a system decreases
the screening capacity and high throughput screening is easiest to perform in cell free
systems. Therefore, cultured brain slices have advantages over cell culture as it retain many
74 Drug Screening Methods

Table 4.1: In vitro and in vivo models

Model type Method of induction Model system Purpose of study

Anoxia, hypoxia, O2 and/ glucose deprivation, endothelin-1 Cell and slice (cortical Excitoxicity,
OGD and cyanide (CN–) treatment and hippocampal) neuroprotection
culture and death, neuronal
maturation and
plasticity
Cell damage Treatment with staurosporine, C2-ceramide, Neuronal and other cell Apoptosis, calcium
etoposide, tunicamycin, serum deprivation, culture (HT22, PC12, signaling, inflammation,
cyanide (CN–), bleomycin sulfate, sodium SY5Y, etc.) oxidative stress and
nitroprusside (SNP), thapsigargin, lipopoly- candidate agent
saccharide, chromogranin A, veratridine, screening
sodium cyanide, sodium azide cytochrome
oxidase inhibitor H2O2, iron, photochemical
stress, glutathione depletion, superoxide,
naphthazarin, antimycin A, rotenone,
3-morpholinosydnonimine
Focal permanent MCAo, internal carotid artery ligation, Mice, rat, gerbil, Basic stroke
ischemia intracarotid perfusion with wax, polyvinyl cat, rabbit, dog and pathophysiology study
acetate, single/multiple clip, electrocautery, monkey and neuroprotective
thrombic or embolic. compound screening
Focal transient MCAo, internal carotid artery ligation, single/
ischemia multiple clip, electrocautery, thrombic
or embolic (microsphere, macrosphere,
autologous clot, fibrin clot, polyvinyl
acetate) Chemical by endothelin-1, Rose
Bengal photochemical dye, arachidonic acid,
adenosine 5-phosphate and epinephrine,
FeCl3, subarachnoid hemorrhage
Global ischemia Respiratory block by cyanide (CN-), asphyxia, Mice, rat, gerbil, Stroke pathophysiology
complete carbon dioxide, nitrogen, electrocauterization cat, rabbit, dog and study and
by clip, tourniquet, balloon compression, monkey neuroprotective
decapitation, ligation, intracranial pressure compound screening
elevation, cardiac arrest, aortic occlusion,
drowning, neck puff.
Global ischemia 2, 3 or 4-VO, intracranial hypertension,
incomplete hemorrhage, cervical compression.

essential organizational features of the host tissue, such as neuronal connectivity, relatively
well preserved cellular stoichiometry and complex glial-neuronal interactions.26 These slices
are made ischemic by incubating with 95% N2/5% CO2 without glucose and can be replaced
with 95% O2/5% CO2 to simulate a reperfusion period. Therefore, cellular damage that occurs
depends on the duration and severity of the insult and can be detected by morphological,
biochemical or molecular markers.
These models can be used to screen putative neuroprotective agents at high throughput
scale for various parameters such as ionomycin cell lesion, glutamate mediated excitotoxicity,
rotenone induced oxidative stress, lipopolysaccharide induced inflammation, serum
withdrawal and apoptosis with apoptosis inducing agents (staurosporine, ceramide, etoposide,
tunicamycin, etc.). Further, advances in automation, genomics and proteomics technology
have also considerably amplified the number of potentially interesting targets that can be used
for drug development.
 Antistroke Agents 75

Figure 4.2: Schematic view of primary and secondary screening for antistroke test compounds

In Vivo Models
Besides in vitro model of stroke, several in vivo focal and global animal models have been
developed in various animal species (mice, rats, gerbils, rabbits, cats, dogs, monkeys and
baboons). However, the rat and mice strain are usually preferred for stroke study. The use of
rat in stroke and related studies owes its resemblance to human cerebrovascular anatomy and
physiology, easiness in handling due to moderate size, homogeneity in various strains and
importantly reproducibility of stroke damage. Mice bear the special advantage of being its
easiness for genetic manipulation.30 Therefore, its application in studying various molecular
mechanisms has been largely attributed. Despite rodents models, it is recommended
that neuroprotective studies must be replicated in higher animals particularly nonhuman
primates17 due to their closer resemblance to brain of human in term of behavior and
sensorimotor function. These model systems, therefore, can be used to screen molecules with
thrombolytic, neuroprotective and neurorestorative potential.

Focal Ischemic Model

Experimentally focal cerebral ischemia can be induced by occluding middle cerebral artery
(MCA) with intraluminal suture or with a vascular clip either transiently or permanently,
mimicking stroke in humans.31 Depending upon the type of occlusive method used, the damage
occurs in cortex or striatum. Further, occlusive model can also simulate the reperfusion injury
situation in humans, which may be due to lysis of a thrombo-embolic clot.
76 Drug Screening Methods

Middle Cerebral Artery Occlusion (MCAo)

The middle cerebral artery occlusion (MCAo) in rat is considered to be the most reliable
and acceptable model of stroke. It is less invasive and can be used to perform transient or
permanent ischemia in controlled manner. Focal cerebral ischemia is induced by inserting
suture to the origin of middle cerebral artery (MCA) through the proximal external cerebral
artery and advanced via internal carotid artery (ICA) until it blocks blood flow to MCA. The
intraluminal suture MCAo model is most widely used in rats and mice. It was introduced by
Kozuimi et al.,32 and thereafter modified to avoid the subarachnoid hemorrhage and premature
reperfusion by coating the suture either with silicone or poly-L-lysine.33-35 These factors besides
length and diameter of the suture affect the lesion size. This model has also been extended and
used in genetically altered transgenic and knockout mouse strains. The ischemia can be given
for a variable duration of time 60, 90, and 120 min or permanent and then suture removed to
produce reperfusion. Advantageously, this technique does not require craniotomy and can be
done in a high throughput manner.36 It can simulate the clinical situation of stroke in humans
and is therefore, often used in the preclinical evaluation of putative neuroprotective agents.
The cerebral damage following MCAo has been demonstrated in striatum and overlying
frontal, parietal cortices (Fig. 4.3).
Similar type of damage would be seen in human thrombotic-embolic occlusion of the
MCA.37 Damage to these functionally varied brain loci is likely to produce complex motor,
sensory, autonomic and perhaps cognitive deficits depending upon a number of factors that
includes mainly the intensity and duration of ischemic insult. Therefore, it is important to
study neurobehavioral alterations and histologic endpoints over a limited time frame. Hence,
determining the true potential of an intervention requires a set of behavioral analysis that can
depict the true functional outcome in terms of sensorimotor and cognitive deficits.

Embolic Model
Embolic models of focal cerebral ischemia can be produced by injecting homologous small
clot or fibrin-rich autologous clots, collagen, viscous silicone, polyvinylsiloxane, retractable
silver ball and heterologous atheroemboli into extracranial arteries such as the external
carotid artery to reach the more distal intracranial arteries most commonly in rats but also
in larger animals although many studies often used non-clot embolus models produced
by microsphereinduced embolization.38-44 Similarly, ischemia may also be produced by
photoactive dye most often Rose Bengal that when exposed to radiations generate singlet

Figure 4.3: TTC stained brain slices after different time point of ischemia/reperfusion
 Antistroke Agents 77

oxygen and leads to focal endothelial damage and platelet activation. Thus, thromboembolic
ischemia developed may produce damage around the territory affected by occlusion. The
advantages of these types of models are their potential to test thrombolytic, neuroprotective
and indeed combination therapies.
In addition to above focal models, endothelin-1, a potent vasoconstrictor can be used to
induce stroke by applying endothelin-1 directly onto the exposed MCA or adjacent to the MCA
by stereotaxic intracerebral injection. The lesion developed, thus, mimic ischemic damage
produced by MCAo.

Global Ischemic Model

The global model on the other hand is more relevant to human cardiac arrest and involves
brief bilateral occlusion of carotid and vertebral arteries. It is believed that global ischemia
follows selective neuronal necrosis, whereas focal cerebral ischemia is more likely to develop
infarction with pannecrosis. The various global ischemic model viz., two-vessel occlusion
(2-VO) by transient bilateral common carotid artery (CCA) occlusions plus hypotension,
fourvessel occlusion (4-VO) by transient bilateral CCA occlusions plus permanent vertebral
artery occlusions, decapitation without recirculation, neck tourniquet, neck cuff inflation,
ventricular fibrillation, cardiopulmonary resuscitation (CPR), has been developed to study
the various aspects of brain damage. The global cerebral ischemia is generally induced for a
brief period (5-15 min) to allow near complete cessation of CBF, followed by reperfusion. The
2-VO in rat and gerbil is the simplest and most often used global model for screening novel
neuroprotactants although 4-VO is can also be valuable to drug discovery and development.31,45
Gerbil lacks the posterior communicating arteries necessary to complete the circle of Willis,
which allows collateral blood flow in humans and rats, therefore, gerbil is the preferred animal
to study the effect of ischemia on hippocampal area.
The foremost property of a potentially therapeutic compound should be to restore or
maintain the normal brain function. Thus, a systematic study of the effect of a putative
neuroprotective agent at various dose regimens, administered before ischemia (pretreatment)
or after ischemia and or reperfusion (post-treatment) such as, it can be applied at 0.0, 3.0, 6.0,
12.0, etc. hours post-ischemia, is required. It is also important to determine the therapeutic
window of the agent administered, since any drug with broader therapeutic window will
obviously have greater chances to combat the fatal disorder and will be available to larger
population affected by stroke unlike rt-PA. Various putative agents with antiexcitotoxicity,
anti-inflammatory, free radical scavenger/antioxidant, antiapoptotic, restorative/regenerative,
calcium antagonists, adrenergic modulators, antihypertensive, nootropic/stimulator, throm­
bolytic properties can be screened using these models (Fig. 4.4).

TARGET SELECTION FOR DRUG SCREENING

The difficulty in distinguishing and evaluating early the reversible cellular changes from
irreversible brain damage needs the identification of appropriate potential reliable soluble
markers of brain damage in blood and cerebrospinal fluid (CSF) that can be used as a fast
screening tool during early ischemic period46,47 such as neuronal specific enolase (NSE),
myelin basic protein (MBP), S100 protein and a brain endothelial membrane protein viz.
78 Drug Screening Methods

Figure 4.4: Shows various approaches of targeting cerebral stroke

thrombomodulin or a similar molecule, based upon ischemic injury to different brain cell types.
NSE is a dimeric isoenzyme of the glycolytic enzyme enolase, localized mainly within neurons
and cells of neuroendocrine origin. MBP, on the other hand, is an abundant myelin membrane
proteolipid produced by oligodendroglia cells. S100 is an abundant cytosolic calcium-binding
protein isoforms, primarily found in glial and Schwann cells. The characteristic features: low
molecular size, high organ specificity and a high degree of solubility of isoforms S100ß of S100
bear the desirable advantage to be considered as a potential biochemical marker. Further,
thrombomodulin is an endothelial cell membrane-bound glycoprotein that binds thrombin,
producing an anticoagulant effect. The deregulation of these marker help to presume the
stroke induced injury to brain cells and their presence in blood plasma/serum and CSF make
them available for early damage prediction and drug evaluation.
These markers can be easily detected with immunoassays involving western blotting,
immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). ELISA bears the
special attention since it is rapid, highly sensitive and specific, and can estimate nano to
picogram concentration in serum. The basic principle of an ELISA is to use an enzyme bound
to antibody (Ab) to detect the antigen (Ag) present in the sample. The reaction can be read
with the ELISA plate reader upon conversion of colorless substrate (chromogen) to a colored
product by enzyme, indicating the presence of Ag:Ab complex.
 Antistroke Agents 79

METHODS OF ASSESSMENT
In Vitro Methods
Receptor Binding Assay
Glutamate mediated excitotoxicity through glutamate receptors, such as NMDA receptor,
is one of the early event in cerebral ischemic cascade. Characterizations and anatomical
distribution of these receptor can be analyzed with radioligand receptor binding assays. The
receptor-based screening system normally uses a cell-free system or cells from a mammal.
Radioligand binding assay require preparation of receptor of interest that is incubated with
a suitable radioligand for an appropriate period of time and then radioactivity bound to the
receptor is determined. The three major types of experiment that can be performed with this
assay are saturation, kinetic and inhibition. A saturation curve is generated by keeping the
amount of receptor constant and varying the concentration of radioligand. This experiment is
widely used to measure receptor density (Bmax) and affinity of the receptor for the radioligand
[dissociation constant (Kd)] through quantitative autoradiography or phosphor-imaging and
β/ω emission detector. The kinetic experiment on the other hand involves variable time,
constant receptor and radioligand, therefore, used to determine the reverse rate constant. The
inclusion of variable nonradioactive competitor in the experiment can be used to determine
the affinity (Ki) of that drug for the receptor by radioligand.48 So, what is required for the assay
is crude or isolated receptors and appropriate radiolabeled or unlabeled agonist/ antagonist.
Most receptor preparations can be stored at–20°C or at –80°C for extended periods of time. The
important factors that should be considered to perform assay are time, buffer, concentration
of radioligand, receptor and nonradioactive drug. This is also important to determine the
specific binding to the receptor from nonspecific binding in the presence of an appropriate
excess of unlabeled drug. However, the selectivity of a receptor may vary depending on the
post-translational modifications, its state of presence, i.e. homodimers or heterodimers and
association with other proteins. However, when experiments are properly conducted and
appropriately interpreted, these assays can be an extremely powerful tool for the study of
receptors although in vivo imaging are beginning to allow receptor binding to be studied at
the level of intact organs or whole animals including humans and can be thus used to screen
potential unlabeled neuroprotective compounds.

Methods of Oxidative Stress Measurement

Flow Cytometric Determination of ROS
The reactive oxygen species (ROS) is one of the hallmark mechanisms of brain damage
following cerebral ischemia. Its estimation is therefore critical for both evaluating the cerebral
damage and effectiveness of antioxidants. ROS generation can be detected by flow cytometry
in cells using 2’-7’-dichlorofluorescein (DCF) and ethidium (ETH), which are the oxidation
products of 2’, 7’-dichlorodihydrofluorescein (DCDHF) and dihydroethidium (DHE) with a
sensitivity for H2O2/NO-based radicals and O2– respectively.49 DHE reacts with ROS and forms
red fluorescent ETH. ETH binds to DNA, causing amplification of the red fluorescence signal.
For the assay, single cell suspension or primary culture form various brain loci viz., cortex,
striatum and hippocampus can be obtained by a combination of mechanical and enzymatic
80 Drug Screening Methods

(collagenase or trypsin) dissociation methods. The enzymatic dissociation is terminated by

washing the cells with phosphate buffered saline (PBS). Isolated 1 × 106 cells may be incubated
with DCF or DHE in the dark and subjected to flow cytometeric estimation for signal recording
at 530 nm (FL-1 channel) or 610 nm (FL-2 channel).
TBARS Reactive Substances
Brain tissue contains large amounts of polyunsaturated fatty acids (PUFA), which are
particularly vulnerable to free radical attacks. An important indicator of the lipid peroxidation
is conjugated diene (CD), formed from the molecular rearrangement of the carbon-centered
radicals,50 malondialdehyde (MDA) or its enol form, 4-hydoxynonenal. These products are
toxic to neurons and white matter as it can induce apoptosis.51,52 Therefore, specific quantitation
of these markers is useful to reveal the extent of lipid damage as a result of oxidative stress in
stroke.
Lipid peroxidation can be measured by the thiobarbituric acid test for malondialdehyde.53-55
The pink colored MDA-TBA complex formed at low pH and high temperature may be measured
at 532 nm by spectrophotometer.
Brain Glutathione Level
Glutathione is a central component in the antioxidant defences of cells present in
both cytoplasmic and mitochondrial compartments. It is a tripeptide (g-L-glutamyl-L-
cysteinylglycine) that is the reductant for glutathione peroxidase. Oxidation of the cysteine
sulfhydryl groups joins two glutathione (GSH) molecules with a disulfide bridge to form
glutathione disulfide (GSSG). Glutathione, directly detoxify reactive oxygen species and
also act as a substrate for several peroxidases.56,57 Normally, the brain maintains a high ratio
of GSH/GSSG for antioxidant defence. Depletion of total glutathione and a decreased GSH/
GSSG ratio are markers for oxidative stress in ischemic brain. Brain glutathione levels can be
measured by the 5, 5’dithiobis-(2-nitrobenzoic acid) (DTNB)-glutathione reductase coupled
assay essentially.58 The yellow colored chromogen formed by reaction of GSH with DTNB may
be read at 412 nm with spectrophotometer.
Superoxide Dismutase (SOD)
SOD activity can be measured according to the method described by Fridovich59,60 based
on the principle that xanthine and xanthine oxidase generate superoxide radicals that react
with p-iodonitrotetrazolium violet (INT) to form a red formazan dye that can be measured at
505 nm or the inhibition of autoxidation of epinephrine.61 SOD activity is expressed as U/mg
protein in tissue samples.
Catalase
Similarly, catalase is one of the antioxidant enzymes responsible for scavenging free radical
and can be used to as a marker of oxidative stress. Catalase activities can be determined by
measuring the decrease in hydrogen peroxide concentration at 230 nm62-64 and is expressed as
U/mg protein in tissue samples.

Cerebral Infarcts Analysis Using 2,3,5-Triphenyltetrazolium Chloride (TTC)

Cerebral infarct depicts the region of brain tissue under irreversible cellular damage and
can be analyzed using TTC staining (see Fig. 4.2). For the staining, animals are sacrificed
 Antistroke Agents 81

after completion of ischemia or ischemia/reperfusion. The brain should be removed

immediately and cut into 2 mm thick coronal slices. The brain slices should be immersed
in 0.5-2% solution of 2,3,5-triphenyltetrazolium chloride in normal saline/PBS at 37 °C for
15-30 min and then fixed in 10% phosphate-buffered formalin at 4°C.65,66 Images of brain
can be captured with digital camera-based imaging and infarct volume can be analyzed by
image-analysis software system, by multiplying the area of the lesion with the slice thickness.
Enlargement of the infracted tissue by edema results in overestimation of infarct volume,
therefore, calculation of corrected infarct volume may be used to compensate for the effect
of brain edema.67,68
Assessment of Cellular Damage by Hematoxylin and Eosin (H and E) Staining
The cellular changes following cerebral ischemia/reperfusion injury can be analyzed
with hematoxylin and eosin (H and E) staining. This is a differential stain used for detailed
histological morphology analysis of intact, injured, necrotic and apoptotic cells in thin brain
tissue sections with light microscopy. Hematoxylin is a base that preferentially stains acidic
(nucleus containing DNA and RNA) components of the cell blue, whereas, eosin stain the
basic components of cell (cytoplasm) pink. The paraffin embedded or fresh frozen cryostat
brain sections are stained with H and E. The sections are dehydrated, mounted with DPX
(Deoxyplasticizer Xylene) and examined under light microscope. Histological features that can
be used to identify the ischemic lesion includes: diffuse pallor of the eosinophilic background,
alterations in the shape, size and stainability of cellular perikarya, and vacuolation (sponginess)
of the neuropil.69
Annexin V/PI Apoptosis/Necrosis Detection Assay
Apoptosis and necrosis are the two routes of cell loss resulting due to metabolic derangement,
genomic fragmentation and ultimately a collapse of cellular infrastructure. Therefore, various
changes, viz. morphological, biochemical and genomic are important factors to discriminate
between apoptosis and necrosis during progression of ischemic lesion, since, it is believed that
penumbral region, which is the site of apoptotic damage may be rescued with the application
of appropriate therapy. One of the methods, Annexin V/PI apoptosis/necrosis detection assay,
is used to detect the early changes due to apoptotic cell damage. It is based on the observation
that during early stage of apoptosis induction, phosphatidylserine (PS) is translocated from
the inner leaflet to the outer leaflet of the plasma membrane. PS exposure acts as a signal for
elimination of apoptotic cells by phagocytic cells such as microglial in case of brain. Annexin
V is a 35 kDa Ca2±–binding protein with high affinity to PS.70 Its conjugation to fluorochromes
allows for the use of this assay with fluorescent microscopy, flow cytometry and real-time
imaging although dyes for light and electron microscopy are also available for the use of
annexin V. However, annexin V can also label necrotic cells rather makes this method less
specific. But this assay, when used with vital dye propidium iodide (PI), is quite sensitive in
detection of apoptosis at early stage.

TUNEL Staining
The most commonly used technique to quantify apoptosis in cells, fresh or fixed tissues is
the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.71,72 It takes
advantage of the multiple free DNA ends generated by activated endonucleases to insert
82 Drug Screening Methods

labeled dUTP that can be later detected by light or fluorescence microscopy. The enzyme,
terminal deoxynuclotidyl transferase (TdT) specifically binds to 3’-OH ends and incorporate
X-dUTP (X = biotin, DIG, or fluorescein) at sites of DNA breaks following proteolytic treatment
of histological sections. Termini modified nucleotides, Avidin-peroxidase amplifies the
signal and allows for examination of labeled cells under light or fluorescent microscopy, flow
cytometry, or immunohistochemistry. However, a serious drawback of TUNEL remains in its
ability to stain, in certain conditions, necrotic cells that can also have free DNA ends, especially
after oxidant and toxic injury. But use of vital dye propidium iodide (PI) particularly in single
cells study makes TUNEL assay a sensitive technique to detect apoptosis, although it should be
confirmed with other reliable test methods.

Electron Microscopy
The differences in the specific morphological changes such as apoptosis or necrosis and
changes in early and late apoptosis can be detected using electron microscopy (EM). It is a highly
specific and sensitive test for the detection of apoptosis and can be enhanced by staining DNA
fragmentation by using EM-TUNEL. Cells or brain tissue need to be fixed, stained, dehydrated,
embedded, and cut into thin sections with an ultramicrotome. Sections are picked up on
copper grids, post-stained, then viewed under electron microscope. This technique although
is cumbersome, expensive time consuming and requires specialized training but is the gold
standard in apoptosis detection.

In Vivo Methods
Neurobehavioral Assessment
The neurobehavioral break-down correlation to extent of histologically-determined stroke
damage in animal models is the key event in drug development program. However, it is not
always easy to interpret the behavioral recovery due to neuroprotective agent administration
and from self-improvement with time. Nevertheless, inclusion of behavioral end-points
in experimental stroke studies represents an important step for preclinical intervention.
Therefore, to investigate interventions that at least show clinical promise, an extensive battery
of behavioral tests are required. At a minimum, the tests should be able to distinguish among
the levels of injury and also should also be capable of detecting beneficial effects during
recovery phase. Additionally, whenever possible, procedures should be used that are not
influenced by recurrent testing.
Gross neurological dysfunction of animals following cerebral ischemia (MCAo) can
be evaluated based on the assessment of spontaneous motor functions on a 5-point scale
(0: no deficit; 1: failure to extend left forepaw fully; 2: circling to the left; 3: falling to the left
(hemiparesis); 4: non-spontaneous walking) as demonstrated by Longa et al.33 or score that
includes testing of lateral instability as 5: no deficit; 4: consistent flexion of the left forelimb; 3:
reduced resistance to lateral push toward the paretic side; 2: circling to the left when held by
the tail; 1: spontaneous circling to the left; 0: no spontaneous motion.73
In addition other behavioral tests that assess MCAo-induced deficits such as versions of
the water maze (radial arm maze (RAM) or morris water maze (MWM)), hemi-neglect and
sensorimotor integration, forelimb use for vertical-lateral exploration, vibrissae-evoked
 Antistroke Agents 83

forelimb placing and passive and active avoidance can be performed to assure the proper
evaluation of putative neuroprotective agents.74-76 However, it is especially important to
distinguish between a test compound’s ability to speed up normal recovery from its ability to
produce recovery of functions otherwise lost.

Imaging of Cerebral Ischemic Damage

Magnetic resonance imaging (MRI)
The infarct volume produced as a result of final outcome following ischemic stroke can be easily
demarcated by various histological techniques, however, in vivo magnetic resonance imaging
(MRI) not only measure the final infarct size, but it is also applicable to investigate the temporal
evolution of ischemic lesion, differentiation of ischemic over hemorrhagic transformation and
prediction of stroke severity. Neuroimaging, particularly with diffusion and perfusion MRI has
been advanced to the extent that even the minute disturbances in brain following stroke in
humans as well as experimental animals can be detected. Diffusion weighted imaging (DWI) is
the most sensitive and accurate method for stroke detection and when linked with perfusion-
weighted imaging (PWI), it can be used to review the functional status of the ischemic brain.
Further, MRI when used together with the X-ray computed tomography (CT) may be helpful
to demarcate the core and penumbra. Although, the frequent use of imaging techniques is not
possible for routine drug screening but may be used as a standard for drug effect conformation.
Therefore, it is presently considered as a gold standard in therapeutic decision making and is
used as one of the paradigm in clinical trial studies for evaluation of neuroprotective agents.77,78

Molecular Techniques
Other methods that can be used to detect apoptosis are: DNA laddering, caspase assay,
immmuno-detection of apoptotic proteins such as caspases, apoptosis inducing factor (AIF),
poly (ADP-ribose) polymerase (PARP), Bcl-2 family proteins etc.79 Besides these methods,
other fast screen methods may also be used and developed in vitro and in vivo so that stroke
research, particularly development of antistroke drugs becomes a reality. Studying in such
lines, recently, Wang and colleagues80 have developed an ultra-high-content chemical genetic
screening assay of rat brain slices transfected with the gene for yellow fluorescent protein and
subjected to OGD. The yellow fluorescent protein labeled a subset of cortical neurons that
acted as sentinels in providing a measure of neuronal survival. They screened a collection of
1,200 small-molecules with known pharmacologies for potential neuroprotective properties.

CONCLUSION
Cerebral stroke is a heterogeneous neurological disorder with a complex pathophysiology and
serious long-term disability. Therefore, proper understanding of the molecular mechanisms of
stroke damage is imperative for bioevaluation of antistroke agents that interferes with specific
or multiple mechanisms of injury, which may serve as critical determinant in drug discovery
and development. However, the failures of various neuroprotective agents in clinical settings
warrant the rigorous assessment of test substance having thrombolytic, neuroprotective
and neurorestorative properties. The various novel putative antistroke agents can be tested
84 Drug Screening Methods

preclinically using suitable in vitro and in vivo models mimicking closely the clinical situation.
The testing of short- and long-term functional outcome should also be assessed in primate
models as well besides rodents for optimizing efficacy and clinical safety of candidate drugs as
per STAIR recommendations.

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 cHAPTER

5
Anti-HIV Agents

Introduction
Acquired immunodeficiency syndrome (AIDS) is a viral disease caused by human
immunodeficiency virus type-1 (HIV-1) and type-2 (HIV-2). Both of these are retroviruses.
Since the detection of first case of AIDS in USA in 1981,1 AIDS has spread world over acquiring
pandemic proportions. It is estimated that 6 new infections occur every minute2 and globally
there are over 33 million cases of HIV infection, with AIDS now being the fourth leading cause
of death worldwide.3
HIV infection is characterized by an acute HIV syndrome, which occurs 3-6 weeks after the
primary infection, followed by a period of clinical latency and finally clinical disease after a
long latency of 8-10 years. The characteristic feature of AIDS is decline in CD4+ T-cell count,
which leads to severe immunodeficiency and death due to a variety of opportunistic infections.
Although a large number of highly effective antiretroviral drugs are available but their
high cost, need for lifelong therapy, toxicity and emergence of resistant strains of virus have
limited the use of these drugs. Also, in spite of development of various vaccine strategies,
none have proved to be efficacious either in the preclinical or in the clinical phases of vaccine
development.4,5
Therefore, there is an urgent need for safer anti-HIV drugs and a cheap and effective
vaccine. The answer to all these issues can be acquired by extensive research in suitable in vivo
and in vitro screening procedures. Some of the important animal models and in vitro methods
employed in the screening of antiretroviral drugs and vaccine are described here.

In viVo Models
Nonhuman Primate Models for AIDS
Nonhuman primates have provided us with invaluable information about AIDS pathogenesis.
Depending on the virus and species of the nonhuman primate, a disease resembling AIDS can
be produced in these animals. Decline in CD4+ cell count and viral load are used as surrogate
markers for AIDS in these models.6 These animals can be utilized for understanding the
hitherto unknown aspects about AIDS or for preclinical evaluation of potential anti-retroviral
compounds and vaccine strategies.
 Anti-HIV Agents 89

HIV-1 virus, as such, causes infection only in chimpanzees and pigtailed macaques, besides
human beings. However, with the discovery and isolation of simian immunodeficiency virus
(SIV), a lentivirus, from a rhesus macaque by MD Daniel, et al7 and subsequent demonstration
that certain SIV strains could cause a disease very similar to AIDS, most of the studies regarding
AIDS have been conducted in macaques using SIV as surrogate virus for HIV-1. SIV has
been obtained from a number of primate species like African green monkeys, chimpanzees
and sooty mangabeys.8-10 Molecular cloning of SIV and demonstration of pathogenecity of a
molecular clone, SIVmac239, has been a big advancement in the study of AIDS pathogenesis.11
In spite of all the similarities between SIV and HIV-1, the two differ in their envelope
glycoproteins, which can lead to differing immune responses.12 Therefore, in order to study
the behavior of HIV-1 genes in SIV animal models, SIV and HIV-1 hybrid virus, i.e. SHIV
(simian human immunodeficiency virus) was designed. It consists of HIV-1 genes like env,
tat or rev inserted into SIV genetic background.13,14 SHIV has been shown to be pathogenic
for pigtailed macaques and rhesus monkeys.15,16 Biggest advantage with SHIV infection is that
since the envelope glycoproteins are of HIV-1 origin, SHIV infection of macaques can be used
to evaluate HIV-1 envelope based vaccines.13-17
HIV-2 virus has also been used to study AIDS pathogenesis by infection of a variety of
primates like baboons (Papio cynocephalus), pigtailed macaques (Macaca nemestrina),
rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis) and the
mangabey monkeys.2,18-20
At present we have a large number of nonhuman primate models of AIDS utilizing different
animal species or strains of the virus. Some important models include the following:

Chimpanzees (Pan Troglodytes)

Chimpanzees have been proposed to be the most likely origin of AIDS in humans.21 They offer
the best animal model for studying AIDS as they have been shown to be infectable with HIV-
1.22 Although infectable with HIV-1, the primary infection in chimpanzees is associated with
only mild symptoms and no clinical disease or immune impairment is apparent.23 However,
Novembre et al have reported the development of AIDS in a chimpanzee over 10 years post­
infection. The disease was characterized by plasma viremia, CD4+ T-cell decline, opportunistic
infections and the infection could be transmitted to other chimpanzees by blood transfusion.24
Shibata et al have reported the cultivation of a HIV-1 strain, HIV-1DH12, from a patient of AIDS
that can establish infection in chimpanzees. They isolated viruses from 23 patients by cell
free infection of fresh activated human peripheral blood mononuclear cells (PBMCs). Out of
these, three strains viz. HIV-1DH12, HIV-1 DH20 and HIV-1DH29 could infect the chimpanzee PBMCs.
Further screening with PBMCs from other chimpanzees showed that HIV-1 DH12 produced rapid
and highly cytopathic infection and exhibited a dual tropism for T-cells and macrophages.
Injection of HIV-1DH12 isolate into a chimpanzee led to establishment of infection within 1 week,
with virus being recoverable by cocultivation of chimpanzee PBMCs with human PBMCs.
The animal also developed lymphadenopathy and disseminated rash during the acute phase
and levels of viremia during the acute phase and 6 months post-infection were similar to that
observed in humans.25
Chimpanzees have greater than 98% genetic identity to the humans26 and are infectable
with HIV-1 virus. Therefore, they have played a crucial role in the development of our current
90 Drug Screening Methods

concepts about HIV-1, like modes of transmission, the immune response to infection and
various other aspects about AIDS pathogenesis. Chimpanzees are also suited for preclinical
evaluation of antiretroviral drugs and vaccines.
However, being endangered species, the number of chimpanzees available for study is
limited and their maintenance is expensive. Thus ethical and practical problems can limit their
use.

Pigtailed Macaques (Macaca Nemestrina)

Pigtailed macaques are the only nonhuman primates, other than chimpanzees, which can be
infected with HIV-1. Agy et al showed that HIV-1 infection could lead to acute and persistent
infection in pigtailed macaques. The animals developed lymphadenopathy and rash over the
abdominal and inguinal regions, HIV DNA could be detected in PBMC and seroconversion
was observed in these animals.27 However, Frumkin et al observed that although pigtailed
macaques could be infected with HIV-1, the virus could not be recovered from the infected
animals after 2 months and there were no disturbances in immune system.28 It has, therefore,
been proposed that use of HIV-1/2 chimeric viruses or SHIV chimeras for infecting these
animals may be able to overcome the block in replication normally seen in these animals.18-29
Pigtailed macaques are also infectable with HIV-2.18
Pigtailed macaques have been used to study different routes of transmission of HIV‑1 like
vertical, oral, anal and intravenous.30,31 These animals have also been employed in preclinical
studies of antiretroviral drugs like AZT.28 These animals can give important insights into the
acquired immune response to HIV-1 and have been used for anti-HIV vaccine testing.32
The advantages offered by pigtailed macaques are that they are not endangered species,
available in large number and are not expensive to maintain.

Baboons (Papio Cynocephalus)

Baboons have been shown to develop a disease, very similar to human AIDS, by HIV-2. Virus
isolate and baboon subspecies dictate the development of AIDS like disease in baboons
with disease being produced by HIV-2UC2 isolate in hamadryas subspecies of baboons.17 The
disease progression in baboons is similar to that observed in the chronic HIV-1 infection
in humans and is characterized by an acute phase, lasting up to 20 weeks, followed by a
clinically healthy phase lasting 4-7 years and subsequently development of AIDS like disease.
Lymphadenopathy, petechial rash over abdomen and back of the animal, high plasma viremia,
a decline in CD4+ cell count and subsequent development of cytotoxic T-lymphocyte (CTL)
response and neutralizing antibodies characterize the acute phase. In the clinically healthy
phase, the plasma viremia is low but the onset of AIDS like disease is associated with increase
in plasma viremia, fall in level of CD4+ T-cell counts and it manifests as gingivitis, hair loss,
interstitial pneumonia, cachexia and Kaposi’s sarcoma like lesions on limbs.2
Due to similarity of disease progression in the humans and baboons, it is possible to
study pathogenesis of AIDS in baboons including the immunobiology of viral latency,
mechanisms of disease progression, the lymphatic tissue pathogenesis, the dynamics of viral-
host interactions, the acquired immune response and also the modes of transmission of HIV.
Baboons are especially suitable for preclinical assessment of efficacy of antiviral drugs and
vaccines.2
 Anti-HIV Agents 91

A variety of advantages are offered by baboons as animal model for AIDS:

•• Baboons are not endangered species and available in sufficient numbers to carry out studies.
•• They are physiologically and genetically related to humans33 and have similar immune
response, so are more suitable for preclinical vaccine efficacy studies.
•• Since the HIV infection in baboon subspecies are virus isolate specific, mechanisms of
innate and acquired resistance to HIV infection can be studied in these animals. This will be
helpful in developing new targets for vaccine and therapeutics.
However, lack of infectivity of baboons with HIV-1 is a significant disadvantage of these animals.

Rhesus Macaques (Macaca Mulatta)

Asian rhesus macaques are readily infectable with SIV and have been widely used for AIDS
studies. Rhesus monkeys are also infectable with HIV-220 and SHIV.34 With marked similarity
in the disease caused by SIV and HIV-1, rhesus macaques infected with SIV have been used to
assess almost all aspects about the pathogenesis of AIDS like modes of transmission, genetics
of lentiviruses, the host immune response, potential antiretroviral drugs and vaccination
strategies.17
However, as referred to above, SIV differs from HIV-1 in its envelope glycoproteins, so
efficacy of envelope-based vaccines in these animals may not be reflective of their efficacy in
humans. Also since protease inhibitors are not effective against SIV proteases, therefore, it is
not possible to study combination therapy in these SIV infected animals.6

Severe Combined Immunodeficiency (SCID) Mice Models

SCID mice were first described in 1983 by Bosma et al.35 These mice have an autosomal
recessive mutation that impairs the rearrangement of B and T-cell receptor genes and thus
immunocompetancy of these animals.35 As a result, the SCID mice are not able to produce
antibodies or reject foreign tissue grafts. It is, therefore, possible to transplant human tissues
into these mice without the elicitation of graft-versus-host disease. Taking this lead a variety of
human immune tissues capable of being infected with HIV-1 have been xenotransplanted into
the SCID mice to study important aspects of the HIV-1 infection like its pathogenesis, modes
of transmission, safety and efficacy of antiretroviral drugs and vaccines, etc.

SCID-hu Mice (Thy/Liv Model)

SCID-hu mice are constructed by co-transplantation of human fetal thymus and liver fragments
under the renal capsule of 2-3 months old SCID mice. These liver and thymic fragments fuse to
form a conjoined organ in which the fetal liver acts as a source of multipotent hemopoietic stem
cells while thymus provides the appropriate environment for maturation of T and B-cells.36
Human thymopoiesis can take place for over a year in these mice and phenotypically normal
and functional T and B-cells of human origin can be detected in the peripheral circulation
of these mice.36-38 These T-cells, in the process of maturation, become tolerant to the major
histocompatibility complex (MHC) background of the mice and thus do not mount any host
vs graft attack.39
92 Drug Screening Methods

The SCID-hu mice are infected by direct injection of HIV-1 isolate into the graft tissue,
3-4 months after transplantation. The pathological changes induced by HIV-1 infection can
then be studied by analyzing the grafts and the circulating peripheral T-cells at varying times
after infection using various methods like in situ hybridization, immunohistochemistry, PCR,
ELISA, etc. The animals can be treated with anti-HIV drugs at any time before or after the
infection and their effects are studied using suitable analytical parameters.37
The advantages offered by SCID-hu mice for studies on AIDS include:
•• SCID-hu mice provide excellent in vivo environment for studying the efficacy and toxicity of
antiretroviral drugs. A number of anti-HIV drugs like zidovudine, nevirapine and bicyclam
have been shown to be efficacious in this model.40-42
•• SCID-hu mice can be used to identify new targets for therapeutic intervention.
•• Active human thymopoiesis can occur for extended periods of time in these mice.
However, some potential disadvantages of these mice are:
•• Due to the immunocompromized nature of these animals, the upkeep of these animals
requires very stringent, pathogen-free conditions thus posing difficulties in maintenance.
•• Good surgical skills are required for construction of these mice.
•• The study may be confounded by physiological variables of the mice. Cytokines and other
growth factors required for growth and optimal function of the engrafted cells are lacking
in these mice.
•• HIV infection can only be produced by intragraft injection and the infection is mainly
limited to the graft tissue. Thus peripheral immune system cannot be studied.
•• The data obtained from these mice may not be generalizable to adults as only human fetal
tissue is transplanted.
• Some scid/scid mice have been shown to produce antibodies and mature lymphocytes
due to somatic reversion events. This may interfere with engraftment.43,44

Modifications
In order to enhance the efficacy of Thy/Liv graft in populating the peripheral lymphoid organs
with T-cells, Kollmann et al transplanted the fetal human thymus and liver fragments under
both the renal capsules of the mice. This led to increased number of circulating T-cells and
offered the advantage of infection of the animal by intraperitoneal route, in addition to the
intragraft route. Disseminated infection was also observed in these mice.45

Hu-PBL-SCID Mice
Hu-PBL-SCID mice are constructed by injecting human peripheral blood leucocytes (PBL)
into the SCID mice. The PBL are taken from a HIV-1 negative, Ebstein Barr Virus (EBV)-
negative donor and injected intraperitoneally into the mice, 2 weeks prior to exposure with
the virus isolates. The cells, being of adult human origin, get activated in the mice and thus are
easily infectable with HIV-1 virus.46 Intraperitoneal injection of HIV-1 isolate leads to high titers
of plasma viremia and depending on the tropism of the virus injected, a decline in the CD4+
T-cells. Thus this model provides good in vivo environment for studying the pathogenesis of
HIV.
 Anti-HIV Agents 93

Further, Mosier et al have shown that donors who have been vaccinated with anti-gp160
vaccines can pass on the resistance to the hu-PBL-SCID mice.47 These mice can therefore serve
as a model for preclinical evaluation of anti-HIV vaccines. Hu-PBL-SCID mice can also be
used to study the efficacy of antiviral drugs and immunomodulators.48

Human-CBL-nSCID Mice
Human-Cord Blood Leukocyte-neonatal SCID mice were developed to mimic pediatric
HIV infection. These mice are constructed by intraperitoneally injecting human cord blood
leukocytes (hu-CBL) into 1-4 days old neonatal C.B-17 scid/scid mice. These cells contain
immature T and B-cells, more CD4+ than CD8+ T-cells and few memory cells. Following
injection, these cells engraft in the spleen, lymph nodes and are also present in high number
in the peripheral circulation and can be detected for upto 8 weeks postimplantation. Two
weeks after the CBL injection, the mice are injected with HIV-1 isolates, intraperitoneally.
At various time points after infection, blood samples can be collected to study the viral
antigenemia and effect of HIV-1 on the blood cell counts or the animals can be sacrificed and
their organs subjected to various analytical procedures like coculturing and PCR analyses to
establish pathological changes induced by HIV-1 infection. These mice can be used to study
the peculiarities of pediatric AIDS and test strategies for prevention of HIV-1 infection of the
neonates.49

NOD/SCID BLT Mice

Non-obese diabetic (NOD)/SCID bone marrow-liver-thymus (BLT) mice are constructed by
transplanting of human bone marrow, liver and the thymus under the kidney capsule. NOD/
SCID mice are characterized by reduced NK cell levels and transplantation of the liver, and
thymus enhances the reconstitution of the human immune system. This model is used for the
studies of HIV latency and could be very helpful in antiretroviral drug screening.50

U-937-SCID Mice
U-937-SCID mice are constructed by injecting human U-937 tumor cells, subcutaneously, into
4-5 week old C.B-17 scid/scid female mice. In order to increase the take of grafted tissue the
mice are treated with either polyclonal sheep antimurine IFNα/β or antimouse granulocyte
mAb given 1 day before and on days 3 and 7 after tumor cell injection. Various protocols are
followed for HIV-1 infection of these mice. The infection can be accomplished by injecting
either in vitro HIV-1 infected human PBMCs or cell free HIV-1 isolates simultaneously with the
tumor cells or the tumor cells can be injected with cell free HIV-1, 20 days postimplantation.
A long lasting HIV-1 infection (for up to 3 months), with high levels of infectious particles at
the tumor site and p24 antigenemia, is seen. It was shown that treatment of these animals with
AZT could dramatically reduce the p24 antigenemia and serum level of HIV-1 RNA copies as
well as suppress the HIV-1 infection at the tumor level.51
This model offers the advantage of high reproducibility, ease of establishment of the tumor
cells, high levels of viremia, well defined viral kinetics and long-lasting HIV-1 infection which
can be very valuable for the screening of potential antiretroviral compounds. This model also
allows the serial implantation of the tumor cells from HIV-1 infected and drug treated mice to
other naïve mice and thus long-term selection of resistant infections can be studied.51
94 Drug Screening Methods

MO-MSV INDUCED TUMORS IN NMRI MICE

Antiretroviral compounds can be screened by studying the inhibitory effect of these
compounds on Moloney-MSV (Mo-MSV) induced tumor formation in NMRI mice. Briefly,
inject 2-3 day old NMRI mice with 100 focus-forming units of Mo-MSV subcutaneously in
the left leg and treat the animals with test compound for 5 days. Treatment is started 2-4 hr
prior to the inoculation with MSV. Antiretroviral potential is estimated by noting the effect
on time to appearance of visible tumor formation and time to death of animal. In normal
course, the virus causes tumor formation within 4-5 days and leads to death within 10-12 days
postinfection.52-54

Transgenic Animal Models

Transgenic Mice
Transgenic mice expressing individual HIV-1 viral genes like gag, pol, nef, env or even full
length HIV-1 DNA have provided substantial evidence about the role played by these genes
in the pathogenesis of AIDS. Some of the important insights gained using transgenic mice are
described below.
Hanna et al constructed transgenic mice expressing full-length proviral DNA of HIV-1.
These animals developed severe immune disease resembling AIDS in humans. The disease
was characterized with thymic atrophy, depletion of CD4+ cells, architectural changes in
lymphoid organs, wasting and failure to thrive, diarrhea, interstitial lymphocytic pneumonitis,
tubular interstitial nephritis and early death.55 It has further been shown that nef gene is a major
determinant of pathogenecity of HIV virus and severity of disease is positively correlated with
the level of expression of nef gene.56
The role of tat gene in the development of Kaposi’s sarcoma has been confirmed by studies
on tat transgenic mice.57 It has also been shown that tat causes upregulation of TNFα in
activated T-cells, which may play a role in AIDS pathogenesis.58 env gene has been shown to be
important in the development of neurological lesions associated with AIDS.59,60
The potential role of transgenic mice in the development of anti-HIV drugs that target
specific genes like nef or tat is rapidly emerging and these animals will play a significant role in
the future in the better understanding of HIV pathogenesis as well as in drug development.

Humanized Transgenic Mice

There are several transgenic murine lines humanized to enhance their ability to maintain HIV
replication. Such models can be infected up to a year and are used as chronic models of HIV
infection.
The Rag2-/-γc-/- mice are double knockout mice that lack T, B and NK cells. This allows easy
transplantation of human stem cells which are often rejected by NK cells. Humanization with
hematopoietic stem cells, leads to development of human T, B, and dendritic cells in the blood,
lymphoid and mucosal tissues and allows mice to be infected intrarectally or intravaginally
that mimics natural way of HIV transmission.61
 Anti-HIV Agents 95

Transgenic Rats
Human CD4 and CCR5 (hCD4/hCCR5)-transgenic rats are genetically constructed on an
outbred Sprague–Dawley rats by the introduction of hCD4 and hCCR5 vectors. hCD4/hCCR5
rats express the HIV-1 receptor complex on CD4 T cells, macrophages, and microglia and are
susceptible to HIV-1 infection expressing viral gene products and even displaying a low-level
viremia.62 This model has shown to be well suited for the screening of new antiviral compounds
targeting viral entry or reverse transcriptase.63

Transgenic Rabbits
Transgenic rabbits expressing human CD4 receptors have been developed. These can be used
to study the pathogenesis of disease as well as for testing of potential antiretroviral drug.44

In vitro Models
Mono Mac 1-Cell Line
Mononuclear cells (monocyte/macrophage) are major targets for HIV. Mono Mac 1-cell
line exhibits all the properties of mature monocytes including phagocytosis, surface marker
expression, etc.64 Therefore, Mono Mac 1 is being considered as an appropriate model system
to study the HIV-1 infection of the mononuclear cells.65
Procedure: Mono Mac 1-cell line is cultured in RPM1 1640 medium containing 10% heat
inactivated fetal bovine serum, penicillin-streptomycin, L-glutamine, nonessential amino
acids and sodium pyruvic acid. Bacterial Lipopolysaccharides (LPS) or phorbol 12-myristate
13-acetate (PMA) are added in the culture for differentiation of Mono Mac 1-cells into a more
mature phenotype i.e. macrophage. After this flow cytometric analysis is performed and
untreated and LPS treated cells are incubated for 30 min on ice with saturation of monoclonal
antibodies against CD4 (SIM.d), CCR5 (clone 2D7), CXCR4 (clone 12G5), CCR3 (clone 7B11)
and CD14 (clone CRIS-6). Cells are again incubated for 30 min on ice with concentration of a
secondary antibody of R-phycoerythrin-conjugated goat antimouse immunoglobulin G. Cells
are resuspended in phosphate buffered saline (PBS). Controls consist of commercial isotype-
matched murine monoclonal antibodies.66
After this virus stocks are prepared using NL4-3 luciferase backbone (pNL4-3-luc-ER)
and pc DNA/Amp based vectors coding for HIV-1 ADA (R5), JR-FL(R-5) or amphotropic
murine leukemia virus (AMLV) envelope proteins.66 Infectivity experiments are done using
recombinant luciferase–encoding and infectious viruses to confirm increased virus production
in differentiated Mono Mac 1 cells.
To perform viral infection assay, treated and LPS-treated cells are seeded in 96–well dishes
in culture medium and infected with luciferase-encoding virions (10 ng of p24). After 72 hr
supernatant is removed and incubation is performed with cell culture lysis buffer [triphosphate,
dithiothereitol (DTT), triton, glycerol] for 30 min at room temperature. An aliquot (20 ml)
from lysate is mixed with luciferase assay buffer. Luciferase activity is measured by microplate
96 Drug Screening Methods

luminometer. After that undifferentiated Mono Mac 1-cells are infected with HIV-1 (X4) or
HIV-1 (R5) (100 ng of p24) in culture medium for at least two hours at 37°C. Cells are washed
with ice-cold PBS and resuspended in cold RPMI 10 consisting of pronase for 5 min at 40°C.
Later pronase is eliminated from cells by washing these cells with ice-cold RPMI 10 (with 10%)
Fetal Bovine Serum and with ice-cold PBS. Semiquantitative PCR analysis is done to detect
viral (HIV-1) DNA levels.67

Visna Virus Model

Visna virus is a nononcogenic retrovirus that causes chronic diseases of sheep including
pneumonia, neurological diseases, etc. and affects mononuclear cells.68,69 As this virus is
closely related to HIV and nonpathogenic to humans, it is used to study HIV replication in
vitro and to inhibit reverse transcriptase as well as to identify novel potential agents for the
treatment of HIV. 70, 71
Method: A line of sheep choroids plexus (SCP) cells is established from a newborn lamb
as described by Sundquist and Larner72 and monolayer cultures are maintained in EMEM
containing 10% fetal bovine serum. Visna virus (strain 1514) is used to infect the cells. SCP
cell monolayers are washed with PBS and viruses adsorbed at 37°C for 1 hr in serum free
EMEM. After this EMEM is added with 1% heat inactivated lamb serum and gentamicin is
added. Medium is changed after 2 days and incubated till marked virus induced pathology
is observed. After that cells and supernatant are stored at –70° C. Titration of virus is done in
six-well SCP cell cultures by plaque assay. Using phosphate-buffered saline cells are rinsed
and infected with virus (200 µl vol). After one hour these cells are cultured in EMEM, which
contains 1% heat-inactivated lamb serum, 25 µg of gentamicin/ml and 0.5% agarose. After 6
days, cell monolayers are stained with 1% crystal violet in 20% ethanol.
Plaque reduction assays are performed in six-well SCP plates.73 Viruses that can produce
100 to 200 plaques are exposed to test compound for 10 days and then plaques are counted.
Cytopathic effect (CPE) inhibition assays are performed in 96-well SCP microtiter plates.74 Cells
are infected prior to addition of test compound. After 5 days of incubation, CPE in untreated
cultures and treated cultures are compared. Reverse transcriptase assay is performed for
reverse transcriptase activity.75

Human Lymphocytes
CEM Cells
Lymphoblastoid leukemia cell line (CEM) was obtained from a Caucasian patient with acute
T-lymphoblastic leukemia and is widely used.
HIV induces giant cell formation in these cell cultures. Antiretroviral activity can be assessed
by observing the inhibition of giant cell formation. Briefly, 2-3 × 105 cultured cells per ml are
seeded in wells of a 96 well microtiter plate. The test compounds are added after dissolving
in cell culture medium. Four days after infecting the cells with 100 CCID50 (50% cell culture
infective dose) of HIV-1 or HIV-2, giant cell formation is observed microscopically.53
 Anti-HIV Agents 97

MT-4 Cells
MT-4 cells are human T cells isolated from a patient with adult T-cell leukemia. Protection
against HIV induced cytopathogenicity in MT-4 cells is an important in vitro screening test
for antiretroviral drugs. Briefly, MT-4 cells are infected with 100 CCID50 of HIV viruses in a
microtiter plate and incubated at 37°C for 90 min. Now, 5 × 104 infected MT-4 cells are transferred
to wells of a 96-well microplate that contain 100 microliter of various concentrations of test
compounds. These are then incubated for 5 days at 37°C. Whole of the procedure is carried
out in parallel with mock-infected MT-4 cells. After the 5 days incubation, cells from both the
groups are stained with trypan blue and number of viable cells determined in a blood cell
counting chamber. Concentration of compound that protects 50% of HIV infected cells (ED50)
and concentration of compound that reduces viability of mock-infected cells by 50% (CD50)
are determined.52,76
C8166 Cells
C8166 cells are primary umbilical cord blood T lymphocytes immortalized by human T cell
leukemia virus (HTLV-1) from adult T cell leukemia. These cells are commonly used as an in
vitro model for antiretroviral drug screening, because of their ability to form syncytia in the
presence of HIV. Briefly, C8166 cells (4 × 104 cells per well), infected with HIV are seeded on
96-well plate in the absence or presence of various concentrations of tested compounds in
triplicate. Cells are incubated at 37°C for different time periods and the cytopathic effect (CPE)
is measured by counting the number of syncytia under an inverted microscope. Then EC50 is
calculated.77,78

Murine C3H-3T3 Embryo Fibroblast Cells

Antiretroviral compounds can be screened for activity against MSV by studying their inhibitory
effect on Moloney-MSV (Mo-MSV) induced transformation of murine C3H-3T3 embryo
fibroblast cells. Procedure, as described by Balzarini et al (1989) is briefly described. 5 × 105
C3H-3T3 embryo fibroblast cells per ml are seeded into wells of a microplate and grown to
confluency. Twenty four hours later, these are infected with 80 focus forming units of Mo-
MSV for 60-90 min at 37°C. Subsequently, the medium in the microplate is replaced with
fresh culture medium containing various concentrations of the test compound. Six days later,
transformation of cell culture is examined under microscope and ED50 (concentration of
compound that inhibits Mo-MSV induced cell transformation by 50%) is calculated.52,53,76

Human PBMC Cultures

Peripheral blood mononuclear cells are collected from healthy donors and cultured for 3 days
at 37°C after stimulating with phytohemagglutinin. Subsequently 0.5 × 106 cells are seeded
into wells of a 48 well microplate. Test compounds are added in various concentrations after
dissolving in cell culture medium and the wells are infected with HIV-1 virus. On day 12, the
cell supernatant is collected and analyzed for HIV-1 core antigen using a p24 antigen ELISA
kit. EC50 (concentration of compound required to inhibit viral replication, i.e. p24 production
in PBMC culture by 50%) and EC90 (concentration of compound required to inhibit viral
replication, i.e. p24 production in PBMC culture by 90%) are calculated.53
98 Drug Screening Methods

Human Embryonic Stem Cells

H9 (WA09) is one of the most widely used human embryonic stem (HES) cell lines. HES cells
are derived from the inner cell mass of blastocyst-stage human embryos and are widely used
for antiviral drug screening.79

In vitro Model Systems for HIV in the CNS

Blood-Brain Barrier (BBB) Models

The failure of blood-brain barrier (BBB) structural integrity and its functions play a pivotal role
in the development of HIV induced CNS destruction. Various in vitro models of the BBB have
been developed to reproduce the key physical and biochemical properties of the mammalian
BBB. These models are used to investigate various aspects of HIV infection including the
infectivity of the endothelial cell of the BBB by HIV and mechanisms of transendothelial
migration of monocytes.80

Macaque BBB Model

Microvascular brain endothelial cells (MBEC) are isolated from rhesus macaque at necropsy 80
and cultured in medium containing M 199, 10% FCS, 5% human serum, 15 mg/ml endothelial
cell growth supplement, insulin-transferring-selenium premix and antibiotics. After one week,
distinct colonies are transferred to 2% gelatin coated tissue culture flasks.
Autologous astrocytes are cultured.81 Small pieces of brain are incubated for 30 min in the
presence of trypsin and DNAse. Fetal calf serum is added to inhibit trypsin. Cells are then
passed through a 120 µm nylon mesh, pelleted (1000 rpm) and plated at 105/ml in M199, 5%
FCS. Medium is replaced on the next day. EDTA washing and quick trypsinization is performed
for removal of astrocytes. Astrocytes are subcultured at a ratio of 1:4.
MBEC are grown onto 3 µm PET filter inserts precoated with fibronectin in 2% gelatin
solution. After 5 days, monolayers are stained with hematoxylin and eosin to observe
confluence and astrocytes are then transferred onto the lower layer of the filter. Astrocytes are
allowed to adhere for 2 hr in a humidified atmosphere and MBEC are added to the upper well
and astrocytes to the lower well. After 5 days in culture, immunocytochemical analysis is done
to confirm the presence of the markers, i.e. GLUT-1, CD 147.82

Cultured Brain Microvascular Endothelial Cells

In this model human endothelial cells are taken from umbilical cord and astrocytes from
human cerebrum. These cells are cocultured on opposite sides of a porous (3 mm diameter
pores) tissue culture support. Astrocytes penetrate the tissue culture barrier through these
pores and come in contact with endothelial cells. In this model system BBB protein, brain type
glucose transporter and glutamyl transpeptidase are expressed.83

Modification
In this model human umbilical cord endothelial cells and rat fetal astrocytes are used and
cocultured.84
 Anti-HIV Agents 99

DIV-BBB Model
Brain or peripheral microvascular endothelial cells (BMEC) are cultured in hollow fiber
capillaries inside a sealed chamber and endothelial cells are grown intraluminally in the
presence of astrocytes, which are cultured albuminally. The hollow fiber cartridge system
is made up of artificial capillaries that are in contact with luminal pulsatile flow. A high
transendothelial electrical resistance of 1500-2000 ohms/cm2 is used that mimics in vivo. In
this model HIV infection is maintained for a longer period in BMEC.85,86
This model is used to study the mechanisms of viral persistence, viral entry into the CNS
through the BBB and viral interference with tight junction formation.

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 CHAPTER

6
Angiogenesis
INTRODUCTION
Blood vessels perfuse the entire body and are structurally and functionally highly
heterogeneous in order to meet the metabolic demands of tissues that are exposed to different
microenvironments. The basic components of blood vessels include an endothelial cell
monolayer known as the endothelium, which is surrounded by organ-specific mural cells and
an extracellular matrix.1,2 These vessels serve many crucial physiological roles, including the
separation of blood and tissues, regulation of leukocyte adhesion, regulation of organ blood
flow, coagulation, vasodilation and angiogenesis.2
In all vertebrates, both vasculogenesis and angiogenesis are required for normal
development of the vascular system in the developing embryo. While vasculogenesis entails
de novo formation of blood vessels from the mesoderm during early embryogenesis, all other
vessels arise from this vasculature by angiogenesis, a process that persists throughout adult
life. In the adult, angiogenesis is essential to maintain homeostasis, such as during wound
healing and menses, whereas uncontrolled angiogenesis underlies a plethora of diseases.3
As such, excessive angiogenesis can lead to tumor growth and metastasis, psoriasis, retinal
vasculopathy and arthritis,4-7 whereas insufficient angiogenesis is observed in ischemia,
strokes and myocardial infarctions.8-11 At the molecular level, angiogenesis is mediated to a
large extent by endothelial cell-specific tyrosine kinase receptors, such as Flk-1/KDR and Tie-
2, receptors for vascular endothelial growth factor (VEGF) and the novel angiopoietin family
(Ang-1, -2, -3 and -4), respectively.12

TUMOR ANGIOGENESIS
In 1991, Judah Folkman et al. were the first to propose that tumor growth and metastasis
correlate with the extent of angiogenesis.13 This finding revolutionized anticancer therapeutics,
as it suggested that blood vessels, rather than the cancer cells themselves, should preferentially
be targeted in tumor settings. Effectively, it has since been established that without angio­
genesis, tumors can not grow more than 2 mm in diameter.14-16 Tumor angiogenesis entails the
same sequence of events as physiological angiogenesis, namely endothelial cell invasion into
the surrounding matrix, proliferation, migration and tube formation; however, these events
proceed in an uncontrolled and excessive manner.15 Due to the complexity of this disease, as
 Angiogenesis 105

well as its associated morbidity and mortality, most in vitro and in vivo angiogenesis models
have focused on investigating tumor angiogenesis.
Although in vitro models of angiogenesis are crucial to quantitate discrete steps of
angiogenesis, e.g. migration, proliferation, etc., they can not mimic the effects of the
microenvironment and spatiotemporal cues on vessel phenotype that occur in vivo. For
instance, it is well-established that microenvironment cues help define the type and number of
vessels being formed, explaining why for instance, liver vessels are functionally and structurally
distinct from cardiovascular vessels.17 In addition, due to the complex 3-dimensional vessels
structures observed in vivo, effective modeling of vessels is only possible in whole animals versus
cell cultures. A main limitation of animal models of angiogenesis, as compared to cell culture
models, involves lack of sensitivity, accuracy and reproducibility in quantifying angiogenesis.
These limitations are mainly due to the fact that the heterogeneous microenvironment can
not be controlled and kept static in animal models as it can under tissue culture conditions.
In addition, in vivo approaches are generally much more time consu­ming and expensive than
cell culture assays. Hence, there is an urgent need to improve existing in vivo models, or even
create new ones, so that animal assays will one day be as cost and time-effective, quantitative
and reproducible as in vitro assays. Being able to modulate angiogenesis in laboratory animals
is crucial for understanding the underlying mechanisms of angiogenesis in order to find
suitable targets which will translate to humans.
This review will describe the most established models, as well as emerging ones, focusing
on their respective advantages and limitations. For clarification purposes, these models are
classified into four categories: (i) tissue excision models, (ii) implant models, (iii) translucent
models, and (iv) non-mammalian models.

Tissue Excision Models

The models presented here require that a piece of tissue be removed from the animal either
after (xenografts, cancer tissue arrays) or prior (ischemic hind limb model) induction of
angiogenesis.

Xenografts
One of the most popular methods for studying tumor angiogenesis and anticancerous drugs
are xenografts, whereby cells or tissues from one animal (donor) are transplanted into an
immunodeficient animal from another species (recipient).18 When transplantation involves
the same tissue type between donor and recipient, then this is termed an “orthologous
xenograft”. Xenograft models allow morphometric and immunohistological measurements
to be performed which reflect the role of the heterogeneous microenvironment on the
vasculature.19 For instance, injecting breast tumor cells in the mammary gland of mice results
in less angiogenesis than when these cells are injected in the cranial window.20 These results
are clinically relevant, as they imply that various types and stages of human cancers can be
mimicked by alternating the grafts sites, which in turn will help identify markers of disease
progression.
106 Drug Screening Methods

Xenografts can also be performed between mammalian and non-mammalian systems, such
as between mammals and fish, by grafting human or mice tumor cells close to the subintestinal
vessels of 2 days-old zebrafish embryos.17 This model is useful for studying the mechanisms of
tumor angiogenesis and antitumor drugs at a large scale, as zebrafish are easily manipulated
and can be studied hundreds at a time (see below). Although extensively used, this method is
time-consuming, but does not require extensive surgical skills.

Syngeneic Grafts
Syngeneic grafts, also called allografts,21 are performed between a donor and recipient
belonging to identical species, thus negating the need for an immunodeficient recipient.22
For example, Lewis Lung Carcinoma cells or B16/F10 melanoma cells can be implanted into
C57/Bl6 mouse, and leads to robust tumor growth with vigorous angiogenesis. This property
makes the syngeneic graft the ideal in vivo model for studying the complex interactions that
occur between tumor cells and host cells.22 More recently, this model has found widespread
application for investigating murine stem cell transplantation for therapeutic strategies during
cardiovascular diseases and diabetes.21,23
The main advantages of tumor allografts, as compared with xenografts, are that they are less
costly24 and metastasis can be induced more readily.22,25 The latter is explained by the lack of
natural killer cells in nude mice, which often impairs the metastatic potential of the injected
tumor cells. This limitation, however, has been partly addressed by injecting immunodeficient
mice with human peripheral blood or bone marrow cells from the donors (e.g. humanized
mice) and then challenging them with the donor tumor cells or tissue.26 The main disadvantage
of allografts when compared with the latter is that human tumors can not be studied, hence
limiting its clinical usefulness, especially in terms of drug responsiveness.22,26 In order to study
as many parameters of cancer as possible, it would, hence, be advantageous to combine both
xenografts and allografts, allowing the investigator to obtain more data than using either model
alone.

Cancer Tissue Arrays

A new twist on immunohistochemistry, inspired by DNA microarrays in that they allow for
high throughput analyses, was recently introduced as “cancer tissue arrays”. These arrays have
gained considerable popularity in the past 7 years and have proved a valuable tool in the
discovery of potential tumor markers. Each array can accommodate up to 600 “spots” of cancer
tissues or corresponding non-neoplastic controls (of around 0.6 diameter per spot) which are
embedded in paraffin on a glass slide.27,28 This model has the advantages of analyzing clinically
relevant human samples, and to simultaneously allow for the analysis of many different types
of tumors, or different grades of the same tumor type, thus bypassing the needs for animal
models in some cases and saving considerable time and money.27,28
However, as histochemistry is semiquantitative, tissue arrays still need to be validated
using more sensitive techniques, such as ELISA.27 Although not well-documented, it should
be noted that these arrays can introduce gender and ethnic biases, as it does not reflect
population heterogeneity and it is a well-known fact that tumor markers differ among sexes
and ethnic groups.29 Although tissue arrays are currently used for cancer research, it will be
 Angiogenesis 107

interesting to construct protein arrays to study other diseases in the future, such as diabetes
and cardiovascular diseases.

Ischemic Hind Limb Model

The models described thus far are mainly useful to study diseases of excessive angiogenesis,
hence their usefulness in cancer studies. However, models to study insufficient angiogenesis
remain scarce. The best known and used of such models is the ischemic hind limb muscle
model, which has successfully been used since 1994 to study ischemia-induced vessel
regression.30 In this model, a segment of the proximal femoral artery and vein are excised
from one leg of the animal, generally a rabbit or rodent, and the remaining collateral vessels
(emanating mainly from the internal and external iliac artery) are ligated in order to induce
unilateral hind limb ischemia.30-32 This procedure yields ischemia-induced vessel regression
in about 10-14 days, thus mimicking cardiovascular disease-induced ischemia in patients.
To investigate the roles of pro-angiogenic factors in treating ischemic symptoms, various
angiogenic agents or drugs can be administered at low doses to the animal using various routes
(e.g. local injection in the collateral vessels or oral administration) and vascular recovery
monitored in real-time. Although this is a technically-demanding and time-consuming assay,
it has the advantages of being highly quantitative and reproducible. In addition, since vessel
growth is easily quantified in the live animal using angiography and Doppler flowmetry, for
instance, this minimizes the number of animals, and hence, the costs associated with this
assay.

Implant Models
Various models have been developed in order to deliver continual and slow release of
angiogenic factors or tumors cells implanted at localized sites. These models rely on the
encapsulation of these factors or cells in avascular matrices prior to implantation in the
animal, allowing vessel infiltration in the matrices to be easily recovered from the implant sites
and quantified by microscopy. These assays require an inert matrix devoid of endogenous
angiogenic/angiostatic activities and are useful to quantitate not only the effects of the
angiogenic modulators or tumors, but also to assess the pharmacokinetic profiles of drugs.33,34
As described in the next section, these types of models offer much diversity simply by playing
with the nature and design of the sponge.

Sponge Implant Model

In this model, a sterile polyester sponge containing the angiogenic modulator under study
is attached to a cannula and subcutaneously implanted in the animal (usually a mouse or
rat), with only the cannula protruding from the skin (Fig. 6.1).18,35,36 The protruding cannula
can then be easily injected with a tracer (the radioisotope133Xe) and vessel infiltration into
the sponge, indicative of blood flow and hence angiogenesis, is quantified by measuring
radioisotope (133Xe) clearance from the sponge or from the tail blood.18,36 In addition,
the sponge can be excised and vessel density correlated to blood flow histologically. This
108 Drug Screening Methods

Figure 6.1: Angiogenesis observed in a sponge implanted

subcutaneously in the subscapular region in the mouse.
Angiogenesis is quantified by measuring the clearance of a dye or
radioactive tracer from the sponge or by staining the sections with
an antibody against von Willebrand factor that acts as a marker for
endothelial cells. Other markers can also be used

assay offers several advantages. For instance, the small size of the sponges allows them to
be implanted in various sites in the animal, thus allowing vascularization to be assessed
in heterogeneous microenvironments. This model is technically simple, inexpensive and
the exposed canula allows for repeated real-time measurements to be performed. A major
limitation of this model, however, is that since sponges are encapsulated by granulation
tissue at the site of implant, inflammation-induced angiogenesis is a main source of false-
positive results. Also, sponge-to-sponge compositional variability, by influencing the degree
of inflammation, can also create false positive results.18 Radioactivity is another limitation,
due to the increased risk of toxicity as well as the increased monetary cost associated with it.
This latter issue has been remedied by substituting 133 Xe with the fluorometric dye sodium
fluorescein.34

Matrigel Plug
Matrigel is an extracellular-rich tumor basement membrane extract which becomes rapidly
vascularized in vivo without the requirement for surgery, thus allowing vessel infiltration
into the Matrigel plug to be quickly visualized (Fig. 6.2).35,37 Matrigel is a solution at 4oC, but
polymerizes at body temperature into a gel-like structure. Its major disadvantages are that the
composition of Matrigel is poorly defined, histological analysis of the plug is time-consuming
due to its inhomogeneous structure and low reproducibility, since different Matrigel plugs tend
to have variable 3-dimensional structures.38 These disadvantages can be partly minimized by
 Angiogenesis 109

Figure 6.2: Matrigel implant model. Effect of PI3K and MAPK inhibitors on HGF/SF-induced angiogenesis in matrigel
implants in vivo. Photomicrographs depict neovasculariza­tion induced by HGF/SF and inhibitory effects of PD98059 and
LY294002. Upper panel shows gross morphology; lower panel shows cross section with blood vessels delineated with
immunolabeling (FITC) for von Willebrand factor. Nuclei were coun­terstained with propidium iodide and appear blue.
Images were captured with resolution of 512 × 512 pixels

using growth factor reduced Matrigel and quantifying blood flow in the plugs by ultrasound
measurements, rather than by histology.39

Disc Assay (DAS)

DAS consists of polyvinyl alcohol sponges sandwiched between two “Millipore” filters.33,40 It
is a generally useful model for investigating tumor-mediated angiogenesis, but is very lengthy
(7-12 days to detect significant angiogenesis).
110 Drug Screening Methods

The Directed in Vivo Angiogenesis Assay (DIVAA)

DIVAA involves semi-closed silicone cylinders plugged at one end with a stainless steel rod
or silicone (angioreactor). The angioreactor is filled with the angiogenic agents under study,
which are themselves encapsulated in Matrigel. Following subcutaneous implantation of the
angioreactor in the animal, a fluorogenic dye such as FITC-dextran is systemically injected in
the tail vein. Vessel density is then quantified in the angioreactor by measuring fluorescence.
For all its complexity, this is a poorly accurate model due to the subjectivity associated with
vessel quantification, the use of Matrigel, the length of the assay (it typically takes 9 days to
detect significant angiogenesis), the low amount of angiogenic factor that can be added to
the bioreactor (18 µl) and the fact that this model only quantifies the effect of pro-angiogenic
agents, but not inhibitors of angiogenesis nor xenografts.41,42

Hollow Fiber Solid Tumor Model

The hollow fiber solid tumor model was initially created at the National Cancer Institute to
screen for anticancer drugs prior to performing xenograft experiments, in order to minimize
the cost associated with the latters.43-45 In this model, well-characterized tumor cell lines are
first propagated in biocompatible polyvinylidene fluoride (PVDF) fibers, in order to reproduce
the environmental heterogeneity found in tumors in vivo. These PVDF fibers are implanted in
mice in order to induce an angiogenic response after which, an intraperitoneal (ip) adminis­
tration of the anticancer drug under study is administered to the animal. At the end point of
the experiment, the fiber is removed and analyzed for drug concentration, chemosensitivity
and vessel infiltration. Only drugs successfully inhibiting tumor-associated angiogenesis are
subsequently tested in xenografts. Although this is a promising model covering a wide range of
variables for cancer drug screening, it is costly, extremely lengthy (14 days for in vitro studies,
28-32 days for in vivo studies), requires extensive expertise in both cell culture and animal
techniques and often culminates in false negatives results.43,45 Due to these limitations, it has
scarcely been used since being introduced in 1994 and has instead been substituted with more
straight-forward assays including the matrix implants (see below).43
Altogether, the usefulness of sponge implant models depends on the experimental needs
of the investigator. For instance, the Matrigel plug allows for the most rapid quantification
of tumor angiogenesis, but provides less reproducibility than the other models. Hence,
combining implant models could offer more advantages than using each method alone.
Effectively, it was recently demonstrated that combining the sponge implant model with the
Matrigel plug increases sensitivity of vessel quantification.46 The main disadvantage that these
implant models all share is that they do not allow for real-time observations to be made. This
disadvantage is partly remedied in the subsequent section.

Translucent Models
These models rely on physiological or artificially-induced translucent structures in order to
facilitate vessel visualization and quantification, usually in real-time. In general, quantification
of vessel growth in these models has been greatly aided by imaging using intravital microscopy.
Intravital, meaning “in the living animal”, is highly useful for real-time measurements in
chamber and windows models, but is also used to quantify vessels in tissues taken from
 Angiogenesis 111

sacrificed animals.47 Intravital microscopy, often combined with video and computer
technology techniques (e.g. videomicroscopy), relies on oblique trans-illumination and
fluorescence epi-illumination in order to quantify vessel architecture according to vascular
volume per unit area.47-49

Hamster Cheek Pouch Model

The first established model of naturally-occurring transparent structure is the hamster cheek
pouch assay, which has been used since 1950.50-52 This is a localized model of angiogenesis
which is mainly used to investigate angiogenic mechanisms related to buccal cancers. This is
an ideal model to study tobacco-related carcinogenesis, where the carcinogen under study is
directly placed on or inside the cheek pouch. Although vessel growth can be quantified in real-
time, the animal generally has to be sacrificed for measurements to be taken, as the pouch is
not readily accessible.

Corneal Micropocket Model

The corneal micropocket model, introduced in 1974 in rabbits, provides a more readily
accessible organ to study real-time angiogenesis than the cheek pouch. This assay relies
on the fact that the cornea is an avascular tissue under homeostatic conditions, and hence,
inducing angiogenesis can be easily quantified in real-time using light microscopy.53,54 This
is accomplished by making several incisions (pockets) in the corneal stroma and implanting
pellets containing the angiogenic modulators or drugs under study in these pockets. Since
the cornea is initially isolated from potential angiogenic factors present in blood, this model
has the immense advantage of only allowing the factors present in the pellets to be studied.
However, this model is costly, although this can be partly remedied using smaller animals such
as mice or rats, is highly time-consuming and requires intricate surgical expertise, especially in
smaller corneas (e.g. rats versus rabbits).53 For these reasons, only a small number of animals
can be studied for a given experiment. In addition, great care must be taken by the surgeon to
minimize an inflammatory response, as this would lead to artifactual angiogenesis.

Mesentery Model
The mesentery is a thin connective membrane, which extends from the dorsal body wall to the
small gut. Under physiological conditions, it is sparsely vascular and transparent, similarly to
the cornea, thus allowing vessel density to be easily quantified by light microscopy.55 In the
mesenteric window model of angiogenesis, the animal is usually injected ip with a mast cell-
activating agent in order to induce inflammation-mediated angiogenesis. Other methods used
to stimulate angiogenesis include ip injections of inflammatory mediators (e.g. interleukins)
or potent growth factors (e.g. VEGF), as well as perforating the mesenteric window.56-58 The
mesentery, along with the small intestine, is then collected and spread out on objective slides
at the desired time points. The small intestine is excised and the membra­nous part of the
mesentery examined under the microscope in order to quantitate microvessel density. The
main advantage of this model is that the large numbers of mesenteric windows per animal,
for example up to 45 windows in the rat, allow multiple observations to be made in only one
animal. The main disadvantages of this method are that it relies on inflammation-induced
112 Drug Screening Methods

angiogenesis, thus restricting the types of angiogenic inducers that can be studied, and that
the animal must be injected with the angiogenic inducer for several days in order to detect
angiogenesis, thus increasing the time and monetary cost of this assay.

Chamber and Rat Dorsal Air Sac Models

In the chamber assay, a piece of tissue is excised from the animal in order to expose translucent
structures where angiogenesis can be easily visualized. This assay is generally performed on
rabbit ears or on the dorsal skinfold or cranium of rodents.18,38,59 Following the removal of
skin or skull from the animal, a matrix containing the tumor cells or angiogenic factors under
study is layered on the exposed surface and covered by a glass coverslip, hence the name
of this assay.18,38 This assay has a major advantage over most other in vivo models, in that it
quanti­fies angiogenesis in 3-dimensions, thus more accurately representing the intricate
complexity of blood vessels than 2-dimensional measurements.49 The main disadvantages of
this model are that it is invasive, technically demanding, costly and can result in false positives
due to significant inflammation-induced angiogenesis following implantation of the glass
window.18,38,60
The rat dorsal air sac assay (also called pouch or blister assay) creates an artificial trans­parent
chamber by subcutaneously injecting air in the back of the animal, resulting in a size­able air
sac within 8 to 10 days.61 Neovascularization in the air sac is then induced by im­plan­tation
of angiogenic factors or tumor cells, either in solvent or matrices, under or into the sac.61,62
Although this model is invasive and time-consuming, it is technically simple to perform.

Angiomouse/Metamouse
Thus far, the major limitations with the above models are that animals are subjected to intense
discomfort or even sacrificed in order for angiogenesis to be quantified, and that false positive
results often occur (e.g. artificial chambers). These limitations are due to the scarcity of
physiological translucent structures, to inflammation induced by artificially created chambers
and to the requirement of surgically altering the animal in order to expose the tissues of
interest (e.g. mesentery). In order to remedy these limitations, a novel experimental model,
the Angiomouse (or Metamouse) was recently introduced.
In this model, mice are either injected systemically or grafted with human or rodent tumor
cells stably expressing green fluorescent protein (GFP) and the metastasized tumor cells
visualized in real-time, without the need to sacrifice or further alter the animal.63,64 This is
a very promising model where many variations are possible. For instance, conditional GFP
promoters can be used in order to investigate the roles of specific growth factors during cancer
progression, as was recently done using the VEGF promoter.49 Growth and infiltration of tumor
cells into the mouse vasculature can be measured in real-time using a trans-illuminated epi-
fluorescence microscope, a multiphoton laser-scanning microscope or a fluorescence light
box.49,63 The tumor cells can be visualized in depths and appear intensely green, whereas the
mouse’s endogenous vasculature appears as well-defined dark networks. This model offers
the immense advantage of allowing for rapid, technically simple, non-invasive and real-time
whole-body measurements of tumor metastasis for prolonged time periods and, unlike most
models presented thus far, does not induce an inflammatory response.63 The main limitation
 Angiogenesis 113

of this method is that the short wavelength of GFP (520 nm) is highly scattered by surrounding
tissues, thus reducing sensitivity of the assay.63 Although the experimental set-up is initially
more expensive than most other methods, in the end, the reduced numbers of animals (since
no sacrifice is necessary) and analysis (no need to excise tissues and count vessels) make this
an ideal and cost-effective method for studying tumor angiogenesis and for testing anti-tumor
drugs.64

Non-mammalian Models
The models reviewed thus far were performed on various mammals (e.g. mice, rats or
rabbits) in order to closely mimic human vascular phenotypes and genotypes. However, non-
mammalian models can also provide valuable angiogenesis tools, especially with regards to
studying developmental angiogenesis. These animal models include the extensively used
chick chorioallantoic membrane (CAM) and Danio rerio (zebrafish) models, as well as
the Xenopus Laevis tadpole, and more surprisingly, the common medicinal leech, Hirudo
medicinalis.
As opposed to the larger mammalian models previously described, these small animal
models of angiogenesis are technically simple to maintain and manipulate, as these animals
are easily bread and produce large quantities of offsprings in a rapid amount of time. These
models hence allow high throughput experiments to be performed, explaining their use for
high scale genomics. Their main disadvantages are that they are embryonic by nature, and
hence, care must be taken when extrapolating results to adult vasculatures. In addition, the
zebrafish model in particular is costly as it requires expensive housing. The use of these animals
as angiogenesis models is hereby presented in terms of chronological order.

Chick Chorioallantoic Membrane (CAM)

CAM is one of the most widely used in vivo angiogenesis model, owing mainly to its
requirements for minimal technical handling, as well as time (eggs hatch in 18 days) and
money.65 It involves removing a small part of the fertilized egg’s shell (windowed method) in
order to expose the extraembryonic CAM membrane. The membrane is then overlaid with the
angiogenic modulators, cancer cells or tissues grafts under study and vessel density is assessed
by light microscopy.65,66 Limitations with this model are that some angiogenic factors produce
opposite phenotypes on CAM vessels as compared with mammalian vessels, resulting in non-
negligible artefacts.18,38 These limitations are due to the fact the CAM model is an embryonic
avian model, whose vasculature hence has different physiological requirements than adult
mammalian vessels. Furthermore, overestimation of vascular growth often occurs in CAM
models, resulting from non-specific angiogenesis induced by inflammation or changes in
oxygen tension. Another limitation with this method is the highly subjective quantification
of vessel growth, due to the presence of an existing vasculature prior to the addition of
carriers or grafts, which make it harder to distinguish newly formed vessels from pre-existing
ones.65 Numerous methods have been developed to overcome the latter limitation, including
morphometric analyses of vessels, metabolic labeling of endothelial cells with thymidine, or
more recently, mathematical modeling based on fractal 2-dimensional analyses or imaging
using 3-dimensional probing techniques.65‑69
114 Drug Screening Methods

Xenopus laevis
Although the Xenopus laevis tadpole is not as widely used as the zebrafish model, one model
by no means replaces the other, and using both models could actually complement angiogenic
studies. Early embryos are partly transparent (e.g. tail fin), thus allowing angiogenesis to be
monitored in real-time.70,71 Compared to zebrafish, tadpoles have several disadvantages,
explain­ing why they have been largely replaced by the zebrafish model. As such, as opposed
to zebrafish, albino or pigment-deficient (e.g. not completely albinic) tadpoles are required for
better overall visualization of the vasculature, handling of the tadpoles is more time-consuming
and tadpoles transgenics are not as easily generated, requiring alternative approaches such
as retrovirus infection, which can lead to deleterious side effects.70 However, tadpoles have
the major advantage over zebrafish, as well as many other species, of being exceptionally
disease-resistant, thus minimizing long-term costs. Xenopus are also better suited to study
lymphogenesis than zebrafish. Hence, combining zebrafish and Xenopus models would give
an overall better estimate of vascular and lymphatic mechanisms than using either model
alone.

Danio rerio
Among all animal models of angiogenesis, the small (3-4 cm long in adults) tropical zebrafish
has become the most widely used model since 1999.70 This is primarily due to the unique
characteristics of zebrafish embryos of being completely translucent, thus allowing highly
accurate quantification of the vasculature to be made in real-time. This quantification is further
improved by fluorescent techniques, such as injecting fluorescent markers in the cardinal vein
of zebrafish or generating GFP stable transfectants (Fig. 6.3).70,72 Although more genetically
divergent from humans than tadpoles, increasing evidence suggests that most zebrafish genes
have human orthologues.70 In addition, zebrafish is one of the easiest system to modify for both
forward and reverse genetics, translating in a plethora of knock-down and transgenics models
of vascular pathologies by simple injection (e.g. morpholino antisense oligonucleotides) into
single to four–cell stage embryos.72 It is also a model of choice for drug screening, as the drug
can simply be added to the fish embryo culture media and the vascular phenotype monitored
by intravital microscopy.73 The main disadvantage of this system is that zebrafish are highly
susceptibility to parasites, which can alter embryo development and easily spread between
laboratories.74

Angiogenesis in the Regenerating Zebrafish Fin

Adult zebrafish have a remarkable regenerative capability. Many tissues which may not be
regenerated in mammals are quickly regenerated in zebrafish. Among these are the heart,

Figure 6.3: Fluorescence image of 48-hour post-fertilization zebrafish larvae expressing GFP under a
Fli-1 promoter. Fli is preferentially expressed on the endothelial cells
 Angiogenesis 115

retina, maxillary barbell and fins.75 Importantly, as they regenerate, new blood and lymph
vessels grow into the regenerating tissue – which enables studies on regenerative angiogenesis.
One commonly used assay in the adult zebrafish, based on this principle is the regenerating
tail fin. After amputation, the tail fin will re-grow and after approximately 1 month, the fin
is back to its original size.76 This process of fin regeneration encompasses many of the same
mechanisms as in human wound healing and regeneration, and is therefore a good model
of regenerative angiogenesis. As the fin is largely transparent, and as morpholinos can be
introduced by microinjection and electroporation, this assay is almost as versatile as the
zebrafish developmental angiogenesis assays, the major difference being that it is performed
in an adult animal. Also in the fins, the vasculature is remarkably simple, and as the fin grows
back various levels of vascular remodeling can be observed and therefore studied in detail.75
This regeneration model is probably the most commonly used, adult zebrafish angiogenesis
model, and is considered to be complementary to the developmental angiogenesis models.

Hirudo Medicinalis
The leech Hirudo medicinalis, well-known for its blood-sucking feeding habits, was recently
proposed to be a promising model of angiogenesis for the following reasons: its vasculature
responds to treatment of human pro- (e.g. VEGF) and anti-angiogenic factors, as well as to
the anti-cancer drug mitomycin, following injection in the body wall.77 These results indicate
a surprisingly high degree of vascular homology between leeches and mammalian systems.
Although this model has not gained popularity in the scientific community, we propose it
could serve as a complementary method to quantify angiogenesis, as it is a simple system
which is initially largely avascular, like the cornea, and which can be studied in both embryos
and adults.77,78 Blood vessel growth in the leech is also faster than in other animal models, as its
whole body wall (2 mm thick) can be vascularized in only 24 hours. In addition, leeches are very
easily manipulated. For instance, to monitor endothelial cell proliferation in real-time, leeches
are simply dipped in BrdU-containing solution and then examined by electron microscopy.77
Since leeches feed on blood, it is also tempting to speculate that adding angiogenic modulators
or drugs in blood, rather than injecting them, would provide easier and quicker methods of
assaying their angiogenic potentials.

Genetic Models

Ob/ob Mice
In 1950, obese mice carrying the mutation obese (ob) were described for the first time.79 The
ob mutation was later shown to be located in the gene coding for a hormone known as leptin.
Leptin is important in the regulation of appetite and food intake. Leptin signaling is mediated
via binding to the leptin receptor (Ob-R) and subsequent signaling to the hypothalamus. Via this
pathway, food uptake, energy expenditure as well as fat and glucose metabolism are regulated.
Heterozygotes on the other hand do not display any phenotype as the mutation is recessive.
The leptin deficient mouse can be used as an excellent model to study the role of angiogenesis
in adipose tissue expansion.80 Obesity in these mice can be prevented by treatment with anti-
angiogenic drugs. Since these mice are comparable to morbidly obese humans regarding the
116 Drug Screening Methods

obesity phenotype, using this model might be helpful to identify potential novel targets to treat
obesity and obesity-related metabolic disorders in the future.

Db/db mice
The autosomal recessive mutation diabetes (db) was first described in 1966 in the mouse strain
C57BL/KsJ.81 These mice are deficient for the leptin receptor. Animals which are homozygous
for this mutation, exhibit a phenotype that resembles human diabetes mellitus. This mutant
strain is also characterized by an obese phenotype. Furthermore, homozygous mutants are
infertile and hyperglycemic while heterozygotes are phenotypically indistinguishable from
wild type littermates. These mice are excellent models for studying mechanisms of obesity-
related diabetes and insulin insensitivity, and the role of angiogenesis in this regard.

CONCLUSION
In conclusion, with so many different in vivo angiogenesis models available, it can become
overwhelming to know which one to use. The choice of model would depend on laboratory
resources, technical expertise, type of disease being investigated and budget. The ultimate goal
would be to combine the high throughput potential and ease of use of zebrafish to mammalian
models, in order to more sensitively and reproducibly quantify angiogenesis, ultimately saving
time and money. Furthermore, studying angiogenesis in vivo should not rely on a single, global
experimental approach, but rather, should combine assays depending on the parameters (e.g.
antitumor drugs and metastatic potential of tumor xenografts) and/or disease (e.g. tumor
versus infarctions) under study. Just as each disease in humans has distinct, yet overlapping
phenotypes, so should animal models of angiogenesis.

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 CHAPTER

7
Sexual Dysfunction
INTRODUCTION
Although sexual dysfunction has been well recognized both in male and female, male
erectile dysfunction has been studied in greater detail as compared to female. After the
introduction of phosphodiesterase V inhibitors, drug therapy for sexual dysfunction created
a lot of commercial interest in developing newer and safe drugs. Penile erection is a complex
neurohumoral-hemodynamic phenomenon.1,2 Stimulation of sacral parasympathetic or
cavernous nerves causes erection. Various neurotransmitters (acetylcholine, norepinephrine)
and neuropeptides (vasoactive intestinal peptide, substance P) are known to be key mediators
of erection.3,4 Nonadrenergic, noncholinergic (NANC) neurotransmission is also a critical
player in penile erection. During sexual arousal nitric oxide is produced in corpus cavernosum
leading to relaxation of smooth muscle and engorgement of sinusoids with blood and penile
erection (Fig. 7.1).5-7 Male erectile dysfunction (MED) is multifactorial in origin wherein;
stress, hypertension, hypercholesterolemia, diabetes mellitus, cigarette smoking and aging
have been implicated (Fig. 7.2).8-10 Different treatment options available are: psychosexual
counseling, use of external vacuum devices, vascular surgery, penile prosthesis, intracavernous

Figure 7.1: Interaction between endothelial cells and smooth muscle of the corpora
cavernosa leading to erection
122 Drug Screening Methods

Figure 7.2: Causes of male erectile dysfunction

injection therapy and medication. Majority of the drugs currently used are the result of
serendipitous discovery rather than systematic research. Vascular smooth muscle relaxants
like papaverine, prostaglandin E1 or an adrenoceptor antagonists like phentolamine, when
injected intracavernously, cause increase in arterial inflow of blood, distention of sinusoids
and consequently penile erection. However, these drugs do not act through any of normal
physiological pathways or address the underlying pathology of erectile dysfunction and are
ridden with numerous morbid side-effects like pain during injection, priapism, prolonged
erection and cavernous fibrosis.
Metabolic syndrome (MetS) is a cluster of risk factors (hyperglycemia/diabetes, abdominal
obesity, hypertriglyceridemia, low HDL cholesterol and hypertension), which identifies
subjects at high risk for type 2 diabetes mellitus (T2DM) and CVD. Recently, MetS has been
associated with ED and unresponsiveness to PDE5 inhibitors. The prevalence of ED in
subjects with MetS ranges from 27% to 80% and is strictly associated with the number of MetS
components and endothelial function impairment.11
The models for mimicking female sexual dysfunction (FSD) are more complex than male
sexual dysfunction. FSD is currently categorized according to disorders of desire, arousal,
orgasm and sexual pain.12 The female sexual response cycle is initiated by nonadrenergic/
noncholinergic neurotransmitters (vasoactive intestinal polypeptide, nitric oxide) that
maintain vascular and non-vascular smooth muscle relaxation resulting in increased pelvic
blood flow, vaginal lubrication, and labial engorgement. Furthermore, hormonal status may
influence female sexual function.13 The advancement of research defining the physiological,
pathophysiological and psychological mechanisms of these disorders, and to develop
treatments for female sexual dysfunction, has been hampered by the paucity of experimental
paradigms and animal models.
Bioassay and animal models have been developed in mice, rats, rabbits and dogs to
understand the underlying physiology and pathology of erectile dysfunction, respectively.

MODELS FOR MALE ERECTILE DYSFUNCTION

Isolated Tension Studies in Organ Bath
Sexually mature male (22 to 26 weeks old) New Zealand rabbits weighing between 3.75–4.5 kg
are used for the bioassay studies. Under intramuscular anesthesia of ketamine/xylazine and
nembutal (25 mg/kg), the penis is isolated at the level of attachment of the corporal bodies to
 Sexual Dysfunction 123

the ischium. The corpus cavernosum (total length—20 mm) is sharply dissected free from the
tunica albuginea. Two longitudinal strips with a resting length of about 10 mm are made from
its proximal portion, within Tyrode’s solution gassed continuously with 95% oxygen and 5%
CO2 and maintained at 37°C. The tissue is mounted into the organ bath of 10 ml capacity and
allowed to equilibrate for 30 minute under 2 g mechanical tension. The other end of the tissue
is connected to a force displacement transducer and the change in muscle tension is recorded
using polygraph.
Each strip is prestimulated with 100 µl of 200 µM phenylephrine to produce maximal
contraction. Field stimulation method using a field stimulator is used to deliver biphasic
square wave pulses of 80 V, 1 msec duration and 1–64 Hz frequencies. The relaxant effect of test
agents is studied on the phenylephrine-stimulated contraction.14
Similarly, human corpus cavernosum strips obtained from patients undergoing penile
prosthesis implant or penectomy can also be used to test in vitro effect of drugs on human
tissue.15 As a modification, corpus cavernosum of diabetic rabbit can also be used to understand
the pathology of diabetic impotence and explore pharmacological interventions for it.16
An obvious limitation of in vitro models is the complete physiological mechanisms of
cellular transport, tissue distribution and metabolism. Hormonal effects do not come into
play and the results need to be interpreted with caution. In isolated tissue preparations, the
turnover rate of cyclic nucleotide is low and a high concentration of phosphodiesterase (PDE)
inhibitors is required to achieve significant response. PDE inhibitors show better efficacy in in
vivo systems than in this model.17

In Vivo Model for Vasculogenic Impotence

Penile erection is a consequence of three simultaneous hemodynamic events, viz: increased
arterial flow, sinusoidal relaxation and venous outflow restriction. Vasculogenic impotence is
an important clinical issue, which demands thorough investigation. In such patients, vascular
smooth muscle relaxants, like papaverine, prostaglandin E1 or a-adrenoceptor antagonists like
phentolamine, have proved to be efficacious. Studies have been designed to evaluate the effect
of impairment of each hemodynamic event alone, or in combination, on erectile function.
Adult male Sprague-Dawley rats weighing 200–300 g are used for the experiment. On the
day of experiment, the animals are anesthetized with intraperitoneal pentobarbital sodium (50
mg/kg). Trachea, femoral artery and vein are cannulated for maintaining airway, measuring
arterial pressure and drug administration, respectively.
With a midline perineal incision, followed by blunt dissection of the overlying striated
muscles, entrance to the tunica albuginea of the crus corpus cavernosum is achieved. Needle
prefilled with heparinized saline, attached to polyethylene catheter and connected to a pressure
transducer, is inserted into corpus cavernosum to monitor intracavernous pressure. The
needle has to be accurately inserted to avoid it from protruding from the corpus cavernosum.
Standard drug like papaverine or prostaglandin E1 can be used to evaluate the robustness
of the set-up. Test drugs and vehicle are injected intracavernously either subsequent to or in
combination with papaverine for pharmacological evaluation. The dose response at various
concentrations can be plotted to achieve dose response curve and ED.50 Peak effect, peak time,
initiation of response, duration of response and the ratio of peak intraca­vernous pressure to
systemic blood pressure are the other paradigms which are evaluated.18
124 Drug Screening Methods

For electrical stimulation, cavernous nerve should be selected. The appropriate submaximal
electrical stimulation correlates the increase or decrease in intracavernous pressure for the
evaluation. On electrical stimulation, the difference between basal and peak intracavernous
pressure, ratio of peak intracavernous to systemic blood pressure and the slopes of tumescence
and detumescence are evaluated.
Rats provide suitable model for the evaluation of penile erection in small laboratory animals.
Although penile elongation and intracavernous pressure can be recorded, erectile angle is not
feasible due to congenital angulation of penis in the rat. Also, the engorgement during erection
is not prominent.18
Monitoring intracavernous pressure is an objective, accurate and quantitative assessment
for penile erection. Moreover, in this model neuraxis is intact and neurophysiological and
pharmacological studies can be conveniently conducted.18

Model for Arteriogenic Impotence

Penile vascular smooth muscle relaxation increases the inflow of arterial blood and distension
of sinusoids leading to erection. Low cavernosal artery perfusion pressure can lead to
erectile dysfunction. Arteriogenic impotence is clinically manifested as slowly developing,
inadequately rigid erections for satisfactory sexual intercourse.19 To elucidate the effect of
obstruction of internal iliacs on erectile function, models with acute and chronic occlusion
have been designed in dogs. In the acute model, the penile artery can be clamped with non-
crushing clamps unilaterally or bilaterally.20
Arteriogenic impotence is induced in adult male dogs weighing between 15–20 kg. The penile
arteries are ligated just proximal to the crus of the penis. The cavernous vein and penile artery
are anastomosed proximal to the site of ligation and the pudendal vein is ligated proximally
to prevent the diversion of arterial blood away from the penis. The intracavernosal pressure
is recorded through a 21–gauge butterfly needle inserted into the cavernosum and connected
to a pressure transducer. Electrodes are planted around cavernous nerves and erection is
induced by electrostimulation. Alternatively, erection may also be induced by intracavernous
injection of papaverine. The effect of the drugs is studied by comparing the erectile response
before and after cavernous vein arterializations. The following parameters are studied: blood
flow rate through penile artery and intracavernosal pressure. After completion of the drug
treatment, selective pudendal artery angiography can also be conducted. The animals are
sacrificed under excess ether and the tissue is assessed histopathologically.21
Acute mechanical ligation of both right and left internal iliacs, or pudendal arteries in
dogs, reduces the cavernosal blood supply by 50% and 60% fall in intracavernosal pressure.20,22
However, chronic obstruction of penile blood vessels has minimal effect on erectile function
due to the development of collaterals around the penis.20

Model for Atherosclerotic Impotence

Studies have associated obstruction of the arterial supply to the corporal bodies due to
atherosclerotic vascular disease to impotence in humans.23 Rabbit model, simulating erectile
dysfunction due to atherosclerotic lesions, has been validated by Azadzoi and Goldstein.24
Six to seven months old male White New Zealand rabbits, weighing between 3 and 3.5 kg
are used for the experiment. The animals are divided into two groups. The control group is
 Sexual Dysfunction 125

fed with normal diet. The test group is administered pentobarbital anesthesia and a catheter
is passed through femoral arteriotomies into the abdominal aorta. A balloon is inflated with
0.2 ml normal saline to fit the abdominal aorta, and subsequently withdrawn to femoral
artery. The exercise is repeated thrice and endothelial injury inflicted on the iliacs. After
balloon de-endothelialization, the animals receive 1.6% cholesterol mixed with 4% peanut oil
for 8 weeks. After 8 weeks, the arterial blood is analyzed for cholesterol, triglycerides, high
density lipoprotein, low density lipoprotein along with angiographic analysis. The percentage
of arterial narrowing of iliacs is evaluated and intracavernosal effects are also studied.
Under anesthesia, the carotid artery is cannulated and connected to pressure transducer to
monitor systemic blood pressure. A 21-gauge needle connected to catheter is inserted into
the corporal body for recording intracavernosal pressure. A second 21-gauge needle is placed
into contralateral corporal body for intracavernosal drug administration. Penile erections
are induced by 5 mg intracavernosal papaverine. The test and vehicle are injected either
concomitantly or subsequent to papaverine administration. The parameters studied are: ratio
of intracavernosal to systemic blood pressure, systemic systolic and diastolic arterial blood
pressure. After the conclusion of the experiment, the animals are sacrificed and the tissue is
studied histopathologically.24
Attempts to induce atherosclerotic arteriogenic impotence in dogs using ligatures or
occluding rings, have failed to achieve any hemodynamic impairment or local vascular
and erectile abnormalities.24 The rabbit model is more appropriate to study the effect of
hypercholesterolemia and atherosclerosis on erectile function. Moreover, the effects manifest
within 6 weeks in rabbits, while dogs and monkeys may take one to five years to exhibit the
same.25 However, there are some pathologic differences evident in the atherosclerotic lesions
between rabbits and humans.24

Model for Post-traumatic Arteriogenic Erectile Dysfunction

Pelvic or perineal trauma from bicycle accidents are known to cause focal lesion of the common
penile or cavernous artery, leading to post-traumatic arteriogenic erectile dysfunction. Rat
model has been developed to simulate traumatic arteriogenic erectile dysfunction by ligating
each internal iliac artery of the animal.26
The experiment protocol requires 3-month-old male Sprague-Dawley rats weighing
between 350–400 g. Under intraperitoneal pentobarbitone (35 mg/kg) anesthesia, a midline
laparotomy is done and iliac vessels identified. In the test group the internal iliac artery is
ligated at its origin, whereas, in the sham group only exploration is conducted. Intracavernous
pressure is recorded using a needle inserted into crus and connected to a pressure monitor.
The wound is closed and animal observed for 6 weeks.
The cavernous nerve is stimulated using bipolar platinum wire electrodes. The exposed end
of the electrodes is hooked around the nerve and stimulated with positive electrode positioned
proximally and the negative electrode 2-3 mm distally. The stimulus parameters used are 1.5 V,
frequency 20 pulses/second pulse width 0.2 msec and duration 50 second.
After the study, the animals are sacrificed and the tissue can be used for immunohisto­
chemical staining and electron microscopy.
Arteriogenic erectile dysfunction develops immediately in these animals with no gross
ischemic changes in pelvic organs or genitalia. This model has many advantages. The nerves of
126 Drug Screening Methods

the rat can be easily identified and stimulated. The rats are resilient and less prone to infection
and anesthesia related complications. However, as the internal iliac artery of the rat is small,
the surgical procedure has to be conducted under operating microscope with constant
monitoring of intracavernous pressure.26

Cavernous Nerve Injury Model of Erectile Dysfunction

The rat model of cavernous nerve (CN) injury has been developed in an effort to define the
functional and structural consequences of neural trauma in the corpus cavernosum. However,
many methods were reported in the literature to induce cavernous nerve injury in rats viz.
neurotomy,27 nerve crush,28 and hemostat nerve crush bilateral nerve freezing,27 etc. Mullered
et al.29 induced cavernous nerve injury using various techniques in an effort to compare the
hemodynamic sequelae of these injuries. They compared the effect of different procedures
on the intracavernosal pressure in adult male Sprague-Dawley rats to standardize this model.
1. Control: laparotomy only.
2. Exposure: laparotomy and exposure of cavernous nerves bilaterally without nerve
manipulation.
3. Neurotomy: bilateral neurotomy.
4. Bulldog crush: bilateral nerve crush with bulldog vascular clamp.
5. Hemostat nerve crush: bilateral nerve crush with a hemostat.
Ten days later, a second surgery was performed during which systemic mean arterial
pressure (MAP) and intracavernosal pressure (ICP) was measured in response to cavernosal
nerve stimulation proximal to the site of injury. This study reported that ICP/MAP ratios were
significantly reduced in all cavernosal nerve injury groups as compared with control group.
No significant difference existed in ICP/MAP ratios between the injury groups. Moreover, the
rates of tumescence and detumescence were significantly reduced in all groups as compared
with the control group, without any significant difference in the magnitude and consistency
of hemodynamic alterations. Using the cavernous nerve injury model, Allaf and co-workers
studied the effect of erythropoietin in promoting the recovery of the cavernous nerve injury. 30

Model for Venous Incompetence Induced Impotence

Venous incompetence leads to organic impotence and was first reported by Lowsley and Bray
in 1936.31
Adult healthy male mongrel dogs weighing between 20 and 30 kg are used in this study. The
animals are anesthetized using intravenous pentobarbitone (30 mg/kg). An intravenous line
for fluid infusion (2 ml/kg/hr) is also given. Surgery involves a midline incision and prostate
exposure. The cavernous nerve is identified by electrostimulation and bipolar cuff electrodes
are placed around it for inducing erection. Saline prefilled needle is carefully inserted into
the corpus cavernosum and connected to a pressure transducer to monitor intracavernous
pressure. Ipsilateral pudendal artery is cannulated to monitor blood flow. Systemic blood
pressure is monitored via catheter placed in femoral artery.
After all cannulations are made, a stabilization time of 30 minute is allocated and baseline
reading of following paradigms recorded: peak and maintenance of penile blood flow, latency
period to achieve maximal intracavernous pressure, rise in intracavernous pressure and
 Sexual Dysfunction 127

duration of detumescence. The cavernous nerve is stimulated to elicit erection and above-
mentioned paradigms are recorded again.
To simulate the pathological condition of erectile dysfunction due to venous leakage,
different sized heparinized needles are inserted in corpus cavernosum and venous blood is
allowed to leak. Cavernous nerves are re-stimulated and the aforementioned paradigms are
re-estimated. In addition, the volume of blood lost is also recorded. The effect of test, vehicle
and standard is recorded and the data analyzed statistically.
This model helps to gain insight into the physiology and interplay of various mechanisms of
penile erection. In a healthy animal with robust arterial flow, minor venous leakage does not
have prominent impact on erectile response. This model sheds light on the assessment criteria
set for the diagnosis of vasculogenic impotence in humans.

Psychical Model of Erectile Dysfunction

It is now well established hypothesis that stress deters sexual function and this hypothesis
has been demonstrated in animal model of psychical erectile dysfunction wherein, emotional
stress has been correlated to the sexual performance of the animals.32 Male rats with normal
sexual function were divided into normal group and model group randomly according to their
weights. Stress was applied to the rats in the model group by suspending them upside down in
midair over the water and irritated repeatedly. Two weeks later, the sexual abilities of all rats, i.e.
the times of mounting and intromitting the estrus female rats, the latent period of mounting,
intromission and ejaculation, were recorded, and the number of rats that had sexual activities
were also counted. They have also measured hemorheology indices of the rats. This study has
also compared the sexual behavior of demasculinized rats subjected to the same stress. The
authors observed that as compared to normal rats, the latency of mounting and intromission
of the model rats were longer (p <0.01), with the shortening of latency of ejaculation (p<0.05).
They have also reported that intromission times of model rats were lower than that of the
normal rats (p <0.01). Compared with the normal rats, the sexual activity incidence of the
model rats (mounting: 58.3%, intromission: 33.3%, ejaculation: 16.7%) was significantly lower
than that of the normal rats (100%) (p <0.01). But, there was no significant difference in the
sexual ability between the model and the demasculinized rats (p >0.05). This could be one of
the available models to study the drug, which can reduce stress on the sexual behavior.

Model for Diabetes Mellitus Induced Impotence

Due to unhealthy lifestyle, the prevalence of diabetes and its complications is increasing.
Diabetes mellitus is a significant risk factor in the development of erectile function and animal
models have been developed to study this complications.
Neonate C57BL6 (bl6) mice are randomly divided into different groups of treated and
control. The treatment-group rats are fed a high-fat (45% of total calories) diet for either 4,
8, 12, 16, or 22 weeks. The animals in control group are fed normal diet. At the end of the
study, under excess ether anesthesia, the corporal tissues from the animals is harvested and
studied for various parameters including vasoreactivity, endothelial and smooth muscle cell
content. Corporal tissue from mice with diet-induced diabetes mellitus demonstrate many
of the major functional, structural, and biochemical changes found in humans with erectile
128 Drug Screening Methods

dysfunction. This model serves as a valuable tool for studying the role of diabetes mellitus in
the pathogenesis of ED.33

High-fat Diet (HFD)-induced Metabolic Syndrome and Complication of

Erectile Dysfunction
Male New Zealand white rabbits are provided with either high-fat-diet (0.5% cholesterol and
4% peanut oil) or regular diet for 12 weeks. The animals develop alterations in hormonal and
endothelial function that chronically manifests as erectile dysfunction. The penile tissue from
rabbits from the different groups can be compared on the basis of morphology and function of
endothelial and smooth muscle cells of the vascular bed and cavernous spaces.11,34

Genetically Modified Mice

The development of sterility in the inbred male mice homozygous for the stubby gene mutation
has been reported. Chubb and Henry reported the utilization of stubby gene mutated mice as
an animal model for the study of impotence.35 The autosomal gene mutation is reported to be
a responsible factor for the primary effect on male sexual behavior in this model. The utility of
this model was not found in subsequent literature for the screening of MED.

ANIMAL MODELS FOR FEMALE SEXUAL DYSFUNCTION

Female sexual dysfunction (FSD) is a significant and highly prevalent problem that affects a
substantial number of women that causes personal distress and has negative effects on quality
of life and interpersonal relationships. The experimental models of female sexual dysfunction
(FSD) have involved a range of in vitro to in vivo methodologies. Specifically, the in vitro and
in situ models include vaginal or clitoral smooth muscle preparations, histological evaluation
and vaginal blood flow assessments.

Vaginal or Clitoral Smooth Muscle Preparations (Bio-Assay)

Female rabbits were anesthetized with sodium thiopental (30 mg/kg intravenously) and
exsanguinated. The entire clitoris including vagina was excised rapidly. The vaginal wall
was dissected free from the clitoral body. The clitoral cavernosum tissue was then carefully
dissected free from the surrounding tunica albuginea under dissecting microscope and two
strips along the longitudinal axis (about 0.5 × 0.5 mm) were obtained. During the preparation,
care was taken not to damage the functional endothelium or overstretch the tissue. A strip
of the rabbit clitoris was vertically placed in 2 ml organ chamber, with one end connected
with a cotton thread to the prong of force transducer and the other end secured with a cotton
thread to a holder for isometric tension measurements. The bath chambers contained the
appropriate HEPES buffer solution (37°C) and constantly aerated with 100% O2. Isometric
force was measured and recorded using a polygraph. After mounting, strips were equilibrated
for 60 minute with several adjustments of length until a baseline force stabilized at 1 g and
oxygenated medium was replaced every 20 minute. This preparation was contracted with
phenylephrine and relaxed with acetylcholine. Using the above procedure and preparation,
Park and coworkers36 studied the effect of angiotensin peptides in the regulation of clitoral
cavernosum smooth muscle tone.
 Sexual Dysfunction 129

Pelvic Nerve Electrical Stimulation in Female Dogs

Angulo et al37 evaluated the effects of vardenafil on the increase of blood flow into the vagina
and clitoris induced by Pelvic Nerve Electrical Stimulation (PNES) in female dogs. Application
of PNES produced consistent and frequency-related increased blood flow into the vagina and
clitoris of anesthetized female dogs. The magnitude and duration of the blood flow responses
to PNES were variable among the different animals but remained stable over time within the
same animal. In this model, they have evaluated the effect of vardenafil, a phosphodiesterase
–V inhibitor, by monitoring blood flow produced by PNES into the vagina and clitoris after
drug administration. Similarly, effect of sildenafil facilitated female genital sexual arousal was
studied in female rabbits38 after pelvic nerve stimulation. After pelvic nerve stimulation at 4,
16, and 32 Hz, hemoglobin concentration and oxygen saturation in female genital (vaginal,
labial, clitoral) tissues is evaluated by laser oximetry. Clitoral blood flow is recorded by laser
Doppler flowmetry, vaginal luminal pressure by a balloon catheter pressure transducer and
vaginal lubrication by tampon.

CONCLUSION
Continued upsurge in the insight towards human sexual functions, behavior and innovative
approaches in understanding molecular mechanisms in this field, triggered researchers to
develop and validate suitable in vitro and in vivo models. The models enable effective scree­
ning of molecules for the management of male and female sexual dysfunction.

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3. De Groat WC, Steers WD. Neuroanatomy and neurophysiology of penile erection. In Tanagho EA,
Lue TF (Eds): Contemporary Management of Impotence and Infertility. Baltimore : Williams and
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4. Polak JM, Gu J, Mina S, et al. Vipergic nerves in the penis. Lancet 1981;2:217-9.
5. Kim N, Azadzoi KM, Goldstein I, et al. A nitric oxide-like factor mediates nonadrenergic,
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8. Goldstein I, Feldman MI, Deckers PJ, et al. Radiation-associated impotence: a clinical study of its
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10. Levine FJ, Greenfield AJ, Goldstein I. Arteriographically determined occlusive disease within the
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11. Maneschi E, Vignozzi L, Morelli A, Mello T, Filippi S, Cellai I et al. FXR activation normalizes insulin
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12. Hale TM, Heaton JP, Adams MA. A framework for the present and future development of
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13. Verit FF, Yeni E, Kafali H. Progress in female sexual dysfunction. Urol Int 2006;76(1):1-10.
14. Liu SP, Hass MA, Horan P, et al. Physiological effects of macrocycle 1 on the rabbit corpus
cavernosum. Pharmacology 1998;56:144-9.
15. Stief CG, Uckert S, Becker AJ, et al. The effect of the specific phosphodiesterase (PDE) inhibitors on
human and rabbit cavernous tissue in vitro and in vivo. J Urol 1998;159:1390-3.
16. Utkan T, Yildirim MK, Yildirim S, et al. Effects of the specific phosphodiesterase inhibitors on
alloxan-induced diabetic rabbit cavernous tissue in vitro. Int J Impot Res 2001;13:24-30.
17. Nicholson CD, Challiss RA, Shahid M. Differential modulation of tissue function and therapeutic
potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends
Pharmacol Sci 1991;12:19-27.
18. Chen KK, Chan JY, Chang LS, et al. Intracavernous pressure as an experimental index in a rat model
for the evaluation of penile erection. J Urol 1992;147:1124-8.
19. Vardi Y, Siroky MB. Hemodynamics of pelvic nerve induced erection in a canine model.I. Pressure
and flow. J Urol 1990;44:794-7.
20. Aboseif SR, Breza J, Orvis BR, et al. Erectile response to acute and chronic occlusion of the internal
pudendal and penile arteries. J Urol 1989;141:398-402.
21. Breza J, Aboseif SR, Lue TF, et al. Cavernous vein arterialization of vasculogenic impotence: an
animal model. Urology 1990;35:513-8.
22. Takagane H, Matsuzaka J, Aoki H, et al. Hemodynamic studies of penile erection in dogs blood flow
changes in the corpus cavernosum caused by arterial ligation. Proceedings of the sixth Biennial
International Symposium on Corpus Cavernosum Revascularization and Third Biennial World
Meeting on Impotence, Boston, Massachusetts, 13, October, 1988.
23. Virag R. Impotence: a new field in angiology. Int Angio 1984;3:217-9.
24. Azadzoi KM, Goldstein I. Erectile dysfunction due to atherosclerotic vascular disease: The
development of an animal model. J Urol 1992;147:1675-81.
25. Wissler RW, Vesselinovitch D. Evaluation of animal models for the study of the pathogenesis of
atherosclerosis. In International Symposium on the State of Prevention and Therapy in Human
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26. Lee MC, El-Sakka AL, Graziottin TM, et al. The effect of vascular endothelial growth factor on the rat
model of traumatic arteriogenic erectile dysfunction. J Urol 2002;167:761-7.
27. Kato R, Wolfe D, Coyle CH, Wechuck JB, Tyagi P, Tsukamoto T et al. Herpes simplex virus vector-
mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury. Gene
Ther 2009 Jan;16(1):26-33.
28. Hayashi N, Minor TX, Carrion R, Price R, Nunes L, Lue TF. The effect of FK1706 on erectile function
following bilateral cavernous nerve crush injury in a rat model. J Urol 2006 Aug;176(2):824-9.
29. Mullerad M, Donohue JF, Li PS, Scardino PT, Mulhall JP. Functional sequelae of cavernous nerve
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30. Allaf ME, Hoke A, Burnett AL. Erythropoietin promotes the recovery of erectile function following
cavernous nerve injury. J Urol 2005 Nov;174(5):2060-4.
31. Lowsley OS, Bray JC. The surgical relief of impotence: Further experiences with a new operative
procedure. JAMA 1936;107:2029-35.
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Nan Ke Xue 2006;12:43-5;49.
33. Xie D, Odronic SI, Wu F, Pippen A, Donatucci CF, Annex BH. Mouse model of erectile dysfunction
due to diet-induced diabetes mellitus. Urology 2007;70:196-201.
34. Morelli A, Vignozzi L, Maggi M, Adorini L. Farnesoid X receptor activation improves erectile
dysfunction in models of metabolic syndrome and diabetes. Biochim Biophys Acta 2011
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36. Park JK, Kim SZ, Kim JU, Kim YG, Kim SM, Cho KW. Comparison of effects of angiotensin peptides
in the regulation of clitoral cavernosum smooth muscle tone. Int J Impot Res 2002;14:72-80.
37. Angulo J, Cuevas P, Cuevas B, Bischoff E, Sáenz de Tejada I. Vardenafil enhances clitoral and vaginal
blood flow responses to pelvic nerve stimulation in female dogs. Int J Impot Res 2003;15:137-41.
38. Min K, Kim NN, McAuley I, Stankowicz M, Goldstein I, Traish AM. Sildenafil augments pelvic nerve-
mediated female genital sexual arousal in the anesthetized rabbit. Int J Impot Res 2000;12:S32-9.
 CHAPTER

8
Antifertility Agents
INTRODUCTION
Antifertility agents are the agents, which prevent the fertility by interfering with various normal
reproductive mechanisms, in both males and females. Development of newer methods/agents
for fertility control and research in this direction are imperative, particularly, in the developing
nations. If an ideal contraceptive were available, that contraceptive would be 100% effective,
safe, and easy to use; its effect would be reversible. It would be aesthetically and personally
acceptable in a variety of social, political and religious settings. It would be suitable culturally
in terms of local attitudes concerning sexuality, reproduction, menstruation and the roles and
responsibilities of men and women, and it would be applicable in terms of the health status of
widely differing populations. It would be affordable, readily available and legal. Finally, it would
be appropriate for use at all stages of reproduction. As yet, no single method of contraception
meet all of these criteria, but each of the presently available methods meet at least a few, and
some methods meet many of them.1

METHODS FOR FEMALES

Female antifertility agents might be acting through following mechanisms:
1. Inhibition of ovulation.
2. Prevention of fertilization.
3. Interference with transport of ova from oviduct to endometrium of the uterus.
4. Interference with the implantation of fertilized ovum.
5. Distraction of early implanted embryo.

Antiovulatory Activity

HCG-induced Ovulation in Rats

Immature female albino rats do not ovulate spontaneously and do not show cyclic changes of
the vaginal epithelium. Priming with human chorionic gonadotropin (HCG) induces follicular
maturation, followed by spontaneous ovulation 2 days later. Injection of antiovulatory drugs,
prior to the induction procedure will prevent ovulation. This principle is used for the screening
of anti-ovulatory agents.2
 Antifertility Agents 133

Procedure: Immature female albino rats 24-26 days of age are used for the experiment. The
animals are treated with various test drugs in a different dose levels. After the administration
of the test drug, HCG is given exogenously for ovulation. After 2 days, animals are sacrificed,
ovaries are dissected out, preserved in 10% buffered formalin and subjected to histopathological
evaluation. The results are compared with the control group.

Cupric Acetate-induced Ovulation in Rabbits

The rabbits are reflex ovulators. They ovulate within a few hours after mating or after mechanical
stimulation of vagina or sometimes even mere presence of males, or administration of certain
chemicals like cupric acetate. In this method, cupric acetate is used for the induction of
ovulation. The rabbit ovulates within a few hours after injection of cupric acetate (0.3 mg/kg
i.v. of 1% cupric acetate in 0.9% saline). Injection of antiovulatory drugs, 24 hours before the
induction procedure prevents ovulation.3
Procedure: Sexually mature female albino rabbits, weighing 3-4 kg, are used for the study.
Animals are kept in isolation for at least 21 days to ensure, they are not pregnant and to prevent
the induction of ovulation by mating. They are treated with test drug and 24 hours later cupric
acetate is given. The rabbits are sacrificed and ovaries are examined 18-24 hours later. The
total number of ovulation points on both ovaries are recorded for each animal. Then the
ovaries and uterus are excised out and preserved in 10% buffered formalin and subjected to
histopathological evaluation (Fig. 8.1).4

Estrogenic Activity
A primary therapeutic use of estrogen is in contraception. The rationale for these preparations
is that excess exogenous estrogen inhibits FSH and LH, thus prevents ovulation.

In Vivo Methods
Vaginal Opening
This assay is based on the principle that vaginal opening occurs in immature female albino
mice and rats by treating with estrogenic compounds. The sign of complete vaginal opening is
observed as a sign of estrogenic activity.

Figure 8.1: Ovulation point in rabbit ovary (HE × 40 × )

134 Drug Screening Methods

Procedure: Immature female animals (18-day-old mice, 21-day-old rats) are used for the
study. The test and standard drugs are administered to the animals intramuscularly in cotton
seed oil. The time of complete vaginal opening can be observed as a sign of estrogenic activity.3
Assay for Water Uptake
The principle of the assay is based on the observation that the uterus responds to estrogens by
increased uptake and retention of water. A peak for water uptake is observed at 6 hours after
administration.3
Procedure: Ovariectomized animals may be used, because this assay employs the uterine
weight increase as the response, the uterus must remain intact during ovariectomy. It is simpler
to use immature 18-day-old mice or 22-day-old rats obtained 2 days prior to the beginning of
the experiment. The animals are randomly grouped. The control group is given 0.1 ml of cotton
seed oil subcutaneously. The estrogen control group is given a range doses (0.01–0.1 μg) to
establish a dose response curve. The test compound is given to groups in the initial test at a
high and low dose. In subsequent tests it is given over a range of doses to provide the dose
response curve. All doses are given in 0.1 ml of cotton seed oil. Five hours after treatment,
the animals are killed by cervical dislocation and the uteri are quickly excised. The operation
is begun by a longitudinal slit through the skin of abdomen and through the body wall. The
uterus is picked up with the forceps and severed from vagina. The uterine horns are separated
from the connective tissues and are then cut at constriction near the ovary. The uteri are kept
damp by placing them on (not wet) filter paper and by covering them with moist filter paper.
They are then rapidly weighed in a sensitive balance. The uteri are dried in an oven at 100°C, for
24 hour and are reweighed. The percentage increase in water over control can be calculated,
and can be compared with the values of other groups.
Ovariectomy: The animals are slightly anesthetized with ether. A single transverse incision is
made in the skin of the back. That incision can be shifted readily from one side to the other,
so as to lie over each ovary in turn. A small puncture is then made over the site of the ovary,
which can be seen through the abdominal wall, embedded in a pad of fat. The top of a pair of
fine forceps is introduced and the fat around the ovary was grasped, care being taken not to
rupture the capsule around the ovary itself. The tip of the uterine horn is then crushed with a
pair of artery forceps, and the ovary together with the fallopian tube is removed with a single
cut by a pair of fine scissors. Usually, no bleeding is observed.
The muscular wound is closed by absorbable sutures and outer skin wound is closed by
nylon suture.
Four-day Uterine Weight Assay
This assay is based on the observation that estrogens cause an increase in protein synthesis,
and thus, bring about an increase in uterine weight. A peak in uterine weight is observed in
about 40 hours.3
Procedure: Immature or adult ovariectomized albino mice or rats can be given test drug
intramuscularly in cotton seed oil for three consecutive days. On the fourth day, animals are
killed by cervical fracture, the uteri are rapidly excised, and the uterine contents are gently
squeezed out (results are unreliable if the uterine contents are not removed). The uteri are
weighed immediately in the wet state. The uteri may be dehydrated in an oven at 100o C for 24
 Antifertility Agents 135

hour and reweighed to obtain the dry weight increase. The log dose, plotted against the wet
weight, produces a sigmoid curve, and the ED50 can be determined for comparison of the test
compound with estradiol.
Vaginal Cornification
On the basis of the observation of cyclic vaginal cornification in guinea pigs by Stockard and
Papanicolaou (1917),5 the Allen-Doisy (1923)6 found the vaginal cornification in rodents.
This assay is based on the fact that rats and mice exhibit a cyclical ovulation with associated
changes in the secretion of hormones, this lead to the changes in the vaginal epithelial cells. The
estrus cycle is classified into proestrus, estrus, metestrus and diestrus. Drugs with estrogenic
activity change the animals into estrus stage.
Procedure: Adult female albino rats having regular estrus cycle are used for the study. Animals
are treated with various test and standard drugs. Changes in the vagina can be observed by
taking vaginal smears, and examining these for cornified cells, leukocytes and epithelial cells
in the normal animals, and treated animals twice daily over a period of 4 days. The drug, which
changes the animals into estrus stage skipping other stages, is considered to have estrogenic
activity.3,7
Stages of the estrus cycle in rats: The estrus cycle is a cascade of hormonal and behavioral
events, which are highly synchronized and repetitive.
The short and precise estrus cycle of the laboratory rats has been a useful model for
reproductive studies. The laboratory rat is a spontaneous ovulating, nonseasonal, polyestrus
animal. It ovulates every 4–5 days throughout the year unless interrupted by pregnancy or
pseudopregnancy. A century ago, the English scientist Walter Heape described the progressive
stages of the estrus cycle. The cycle itself is divided into four stages, centered around the period
proceeding estrus “proestrus”, which signifies the period of follicular growth in the ovary, and
he termed the period succeeding estrus “metestrus” and recovery period following ovulation
and “diestrus”, a period when the ovarian secretions from the corpus luteum prepare the uterus
for implantation. The estrus cycle of a rat is usually completed in 4–5 days.
Proestrus: It is the beginning of new cycle. The follicles of the ovary start to mature under
the influence of gonadotropic hormones and estrogen secretion start increasing; the smear is
characterized by nucleated epithelial cells, the stage last for about 12 hours.
Estrus: In this stage, the uterus is enlarged and extended due to fluid accumulation, estrogen
secretion is at its peak. In estrus stage, the smear shows presence of squamous cornified cells
(hexagonal or pentagonal cells). Estrus means period of heat and is characterized as a period
of sexual receptivity, when the female allows copulation. During this stage, there is increased
running activity. This stage lasts for 12 hours.
Metestrus: The ovary contains corpora lutea, secreting progesterone. This stage is indicated by
the presence of a mixture of cornified epithelial cells and leukocytes indicating the postovulatory
stage and desquamation of the epithelial cells. Metestrus stage lasts for about 21 hours.
Diestrus: The corpus lutea regress and the declining secretion of estrogen and progesterone
causes regression of the uterus. The smear shows only leukocytes. This stage is the longest
phase of the estrus cycle and has duration of about 57 hours (Figs 8.2 to 8.7).
136 Drug Screening Methods

Figure 8.2: Rat vaginal smear at proestrus stage showing nucleated epithelial
cells as seen in 40X

Figure 8.3: Rat vaginal smear at estrus stage showing cornified epithelial
cells as seen in 10X

Figure 8.4: Rat vaginal smear at metestrus stage showing both cornified epithelial
cells and leukocytes as seen in 10X
 Antifertility Agents 137

Figure 8.5: Rat vaginal smear at diestrus stage showing only leukocytes
as seen in 10X

Figure 8.6: Rat vaginal smear of mated animal showing cornified epithelial
cells and sperms as seen in 20X

Figure 8.7: Rat vaginal smear of mated animal showing

sperms as seen in 40X
138 Drug Screening Methods

Preparation of vaginal smears: Hold the animal with the ventral side up, a drop of normal
saline is inserted into the vagina with a Pasteur pipette. Care must be taken to avoid damage
or injury to vagina so as to prevent pseudopregnancy. The drop of normal saline should be
aspirated and replaced several times and transferred to a microscopic slide and allowed to dry.
The smears are fixed by placing the slide in absolute alcohol for 5 second, allowing it to dry,
and staining it with a 5% aqueous methylene blue solution for 10 minutes. The excess stain is
washed off with tap water and the slide is dried and observed using low power of microscope.8
Chick Oviduct Method
The weight of the oviduct of young chicken is increased dose-dependently by natural and
synthetic estrogen. This principle is used for the screening of estrogenic compound.7
Procedure: Seven days old pullet chicks are injected subcutaneously, twice daily with solutions
of the test compound in various doses for 6 days. Doses between 0.02 and 0.5 μg 17 β-estradiol
per animal serve as standard. Six to ten chicks are used for each dosage group. On the day after
the last injection, the animals are sacrificed and weight of the body and oviduct is determined.

In Vitro Methods
Estrogenic Receptor-binding Assay
Estrogenic receptor binding assay uses the principle of competitive binding of labeled and
unlabeled estrogen on the estrogenic receptors. Estrogenic compounds displace the labeled
estrogen in a concentration dependent manner from the estrogen receptor.7
Procedure: Cytosol preparation: Uteri from 18-day-old female albino mice are removed and
homogenized at 0°C in 1:50 (w/v) of Tris-sucrose buffer in a conical homogenizer. Human
endometrium from menopausal women is frozen within 2 hours of hysterectomy and stored in
liquid nitrogen until use. The frozen endometrium is pulverized and homogenized in 1:5 (w/v)
of Tris-Sucrose buffer. Homogenates are centrifuged for 1 hour at 1,05,000 g. Determination
of specific binding in mouse uterus cytosol as a function of steroid concentration, incubation
time and temperature. Triplicate aliquots of 125 ml of cytosol are incubated with 5 or 25 nM
labeled steroid either for 2 or 24 hour at 0°C or for 2 or 5 hours at 25°C in the absence (total
binding) or presence (non-specific binding) of a 100 fold excess of radio inert steroid. Bound
steroid is measured by dextran coated charcoal (DCC) adsorption.
Dextran-coated Charcoal (DCC) Adsorption Technique
A 100 μl aliquot of incubated cytosol is stirred for 10 minutes at 0°C in a micro titer plate with
100 μl of DCC suspension (0.625% dextran 80,000, 1.25% charcoal Norit A) and then centrifuged
for 10 minutes at 800 g. The concentration of bound steroid is determined by measuring the
radioactivity in a 100 μl Aliquot of supernatant.
For calculation of relative binding affinity, the percentage of radioligand bound in
the presence of competitor compared to that bound in its absence is plotted against the
concentration of unlabeled competing steroid.
Potency Assay
This assay determines the affinity of the test compound for estrogen receptor sites in the uterus
(rats, rabbits, mice). The uptake of titrated estradiol by immature uteri must be established,
and then the inhibition of this uptake by pretreatment with a test compound will indicate the
 Antifertility Agents 139

estrogenic potency of the compound. The procedure for this assay is based on the work of
Terenius (1965, 1966)9,10 and Johnsson and Terenius (1965).11
Procedure: Four immature female mice (20-day old) are killed. The uteri are quickly excised
and are placed in Krebs’-Ringer phosphate buffer. Pieces of diaphragm are taken from each
animal to serve as control tissue for nonspecific uptake of estradiol. The uteri are divided at
the cervix into two horns; in this way one horn is used as the control and the other for testing
the compound. The tissues are placed in vials containing 5.0 ml of Krebs’-Ringer phosphate
buffer, incubated, and shaken at 37°C with 95% oxygen, and 5% carbon dioxide is bubbled
through. The radiochemical purity of the 3H-estradial can be checked chromatographically.
Buffer solution of radioactive estradiol is made up so that each 5 ml of buffer contains 0.0016
μg of radioactive estradiol (0.25 μCi). A stock solution can be made and kept refrigerated for up
to 6 weeks. The excised tissues are treated as follows:
•• Control: Four pieces of diaphragm are incubated and shaken with 5 ml of buffer solution
for 15 minutes at 37°C and are then shaken for 1 hour with 5 ml of buffer containing the
radioactive estradiol and 2% w/v bovine albumin.
•• Experimental: Four uterine horns are incubated and are shaken in 5 ml of buffer at 37oC
for 15 minutes. Then they are incubated and shaken with 5 ml of buffer containing 2% of
albumin and radioactive estradiol at 37°C for 1 hour. Both control and experimental tissue
are removed and washed with buffer at 37°C for 5 minutes, kept in damped filter paper, and
weighed. The tissues are then prepared for counting. Samples of 100 μl of the incubation
solution are also taken for counting.
Treatment of tissues for counting: The tissues are dried for constant weight and the dry
weight is recorded. Each piece of tissue is placed in a glass counting vial and incubated at
60°C in a shaking water bath with 0.5 ml of hyamine hydrochloride 10x until the tissue has
completely dissolved. If the solution is discolored 50 μl of 20% hydrogen peroxide may be
added. 50 μl of concentrated HCl and 15 ml of phosphor solution are added to each vial. The
vials are allowed to equilibrate in the packed liquid scintillation counter and counts are taken.
Counting efficiency is determined by the addition of an internal standard. The results are
expressed as disintegrations per minute per unit of wet weight (dpm/mg). Test compounds
can be incubated with the labeled estrogen in assaying their effectiveness in competing for the
receptors in the uterus.3

Anti-estrogenic Activity
In Vivo Methods
Antagonism of Physiological Effects of Estrogen
Anti-estrogenic compounds will inhibit some or all of the physiological effect of estrogen such
as water uptake of uterus, uterotrophy and vaginal cornification. This principle is used for the
screening of anti-estrogenic activity.7
Procedure: The assay techniques used for anti-estrogens are modifications of the estrogenic
assays. The dose of estrogen used is that which is required to produce 50% of the maximum
possible response. The test compound can be injected simultaneously or at varying times
before or after the estrogen. The procedure for assays of water uptake, uterotrophy and vaginal
140 Drug Screening Methods

cornification are followed as described earlier, except that the test compounds are given along
with the estrogen.

In Vitro Methods
Aromatase Inhibition
This assay is based on the principle that compounds which inhibit aromatase (estrogen
synthase) possess anti-estrogenic activity. Anti-estrogenic activity of compounds can be
evaluated indirectly by evaluating aromatase-inhibiting ability.7
Procedure: Ovarian tissue from adult golden hamsters is used. Estrus cycle is monitored for
at least three consecutive 4 days estrus cycle prior to the experiment. The experiments for
evaluating inhibitor effects are performed with ovaries obtained from animals sacrificed on
day 4 (pro-estrus). The ovaries are excised freed from adhering fat tissue and quartered. The
quarters are transferred into plastic incubation flasks with 2 ml of Kreb’s Ringer bicarbonate salt
(KBR) solution pH 7.6, containing 8.4 mM glucose. The flasks are gassed with O2/CO2 (95%/5%)
tightly closed and placed in a shaker/water bath (37°C) for incubation of the fragments. The
incubation media are replaced with fresh KBR after pre incubation for 1 hour. The ovaries are
further incubated for 4 hour in the presence or absence of inhibitors. 4-OH androstendione is
used as standard in concentrations between 0.33 and 330 μM/L. At the end of the experiment
the incubation media are removed and centrifuged. In the supernatant estrogen, progesterone
and testosterone are determined by radioimmunoassays. The data of control and test groups
are compared with suitable statistical analysis.

Progestational Activity
In Vivo Methods
Pregnancy Maintenance Test
Progesterone is responsible for the maintenance of pregnancy. This principle is used for the
screening of progestational compound.7
Procedure: Ovariectomy is done on day 5/10/15 of pregnancy in different groups of pregnant
rats. The animals are treated with different test and standard drugs. Pregnant rats are killed
5/10/15 days later. An average of living fetuses at the end of the experiment is compared with
the standard and the control group (without ovariectomy). The ED50 of progesterone is 5 mg/
day in rat and less than 0.5 mg/day in mouse.
Proliferation of Uterine Endometrium in Estrogen-primed Rabbits (Clauberg Mcphail Test)
Female rabbits weighing between 800–1,000 g are primed with estradiol and followed by the
administration of progestational compound, leading to the proliferation of endometrium
and converted into secretary phase. This principle is used for the screening of progestational
compounds.3,7,13
Procedure: Female rabbits weighing 800–1,000 g are primed with injection of estradiol 0.5
mcg/ml in aqueous solution daily. On day 7 drug treatment is begun. The total dose is given
in five equally divided fraction daily over 5 days. Twenty-four hours after the last injection,
animals are killed and uteri are dissected out and frozen sections of segment of middle portion
 Antifertility Agents 141

of one horn is prepared and examined for histological interpretation. For interpretation of
progestational proliferation of endometrium, beginning of glandular development may be
graded 1 and endometrium consisting only of glandular tissue may be graded 4.
Carbonic Anhydrase Activity in Rabbit’s Endometrium
There is a linear dose response relationship between dose of progestogens and carbonic
anhydrase activity in rabbit endometrium. This principle is used for the screening of
progestational compounds.3,7
Procedure: Immature female albino rabbits are used in this study. The animals are primed
with estradiol and followed by the administration of test and standard drugs. After the drug
treatment, animals are sacrificed and uteri are removed. The endometrial extract of the uterus
is evaluated for the carbonic anhydrase activity calorimetrically.
Deciduoma Reaction in Rats
This study is based on the principle of maternal/placental tumor formation by progestational
drugs in traumatized uterus of ovariectomized rats. This phenomenon is used for the screening
of progestational compounds.3
Procedure: The ovariectomized adult female albino rats weighing between 150–200 g are
used for the study. The rats are primed with four injection of 1 μg of estrone/estradiol. This
is followed by 9 days of drug therapy. On day 5, one uterine horn is exposed and 1 mg of
histamine dihydrochloride is injected into the lumen. Twenty-four hours after the last dose of
drug, animals are killed, uterine horns are cut off and weighed and histologically examined.
Prevention of Abortion in Oxytocin Treated Pregnant Rabbits
Intravenous administration of oxytocin to the pregnant rabbits on 30th day of pregnancy
causes abortion. Prior administration of progestational compound prevent the abortion. This
principle is used for the detection and screening of progestational compounds.
Procedure: Ten units of oxytocin are administered intravenously to pregnant rabbits on day 30
of pregnancy. Twenty-four hours before oxytocin, test and standard drugs in oil are injected.
Control animal not receiving drug abort within 2–30 minutes after oxytocin. The drugs, which
are having progestational activity, prevent abortion.12

In Vitro Methods
Progesterone Receptor-binding Assay
Progesterone receptor binding assay uses the principle of competitive binding of labeled and
unlabeled progesterone on the progesteronic receptors. Progesteronic compounds displace
the labeled progesterone in a concentration dependent manner from the progesterone
receptor.7,14,15
Procedure: Human uteri obtained after hysterectomy is frozen in liquid nitrogen and stored
at –80°C until use. For cytosol preparation uterine tissues are minced and homogenized with a
homogenizer at 0–4°C in ice-cold buffer composed of 10 mM KH2PO4, 10 mM K2HPO4, 1.5 mM
EDTA, 3 mM NaN3, 10% glycerol, pH 7.5 (PENG buffer). The homogenates are then centrifuged
at 10,500 g at 4°C for 30 minutes. The supernatant is taken as cytosol.
142 Drug Screening Methods

The cytosol preparations are incubated with 3H-R5020 as radioligand at a concentration of

8 nmol/L and increased concentrations (1 × 10-10 to 1 × 10-5 mol/L) of the competitor steroid
overnight at 4°C. Then unbound steroids are adsorbed by incubating with 0.5 ml of DCC (0.5%
Norit A, 0.05% dextran T400 in PENG buffer) for 10 minute at 4°C. After centrifugation (10
minute at 1,500 g at 4°C) 0.5 ml of the supernatant is withdrawn and counted for radioactivity.
To calculate the relative binding affinity, the percentage of radioligand bound in the presence
of competitor compared to that bound in its absence is plotted against the concentration of
unlabeled competing steroid.

Anti-progestational Activity
The anti-progestational compound inhibits some or all the physiological effect of progesterones.
This principle is used to screen the anti-progestational activity.7 The procedure for assay of
Clauberg/McPhail and deciduoma formation is followed, except that the test compounds are
given along with the progesterone.

Anti-progestational Activity in Immature Rabbits (Mc Ginty Test)

Mc Ginty method is used to determine the local progestational activity of the test drugs. It
determines the degree of endometrial proliferation and transformation in immature rabbits,
initially primed with estradiol and subsequently treated with the test substances. Antagonistic
properties may be evaluated by the co-administration with progesterone.
Procedure: Local progestational test involves a direct injection of progesterone into a
uterine segment. The test is performed in immature rabbits (700–950 g) primed for 6 days
with estrogen. Estradiol at a dose of 1 μg/kg/rabbit is injected by subcutaneous route to
the rabbits of positive control and test groups.16 The rabbits of negative control group
received only the vehicle (0.5% CMC) by subcutaneous route for 6 days. On the 7th day,
the rabbit is anesthetized by intramuscular injection of ketamine hydrochloride (dose
35 mg/kg). The abdomen is opened and the uterus is exposed by laparotomy. The upper
middle segment of each horn is ligated without disturbance of blood circulation. In the
positive control group, 0.1 ml of progesterone was injected alone in the right uterine horn
(concentration 0.1 mg/ml).17 In the opposite horn, only 0.1 ml of the vehicle is injected
(0.5% C.M.C). In the test groups, a solution of the progesterone and the test drugs in
0.5% C.M.C is injected into the left lumen of segment through the lower ligature, which
is drawn right after the injection. Progesterone is injected alone in the right uterine horn.
In the negative control group, progesterone alone is injected in the right uterine horn. In
the opposite horn, only vehicle is injected. After the injection, the lower ligature can be
tightened. Three days later, the animals were sacrificed. The uteri were excised, weighed,
fixed in buffered formalin. The sections of the horn are evaluated histologically according
to Mc Phail scores.17,18
Evaluation: Compared to the vehicle treated animals, the rabbits treated with estradiol
benzoate have an increased uterine weight. In estimating the degree of proliferation the six
sections of each uterus are examined and the average proliferation judged as accurately as
possible, taken as the result. Variation in different parts of the same uterus often exceeded one
 Antifertility Agents 143

Figure 8.8: Uteri of immature rabbits showing the standard scale of progestational proliferation (x17) (1) no treatment;
(2) estrin only, reaction 0; (3-6) estrin followed by progestin, reaction 1,2,3 and 4, respectively. (Adopted from original
article “The assay of progestin” by Mc Phail, 1934)13

stage. The average of all reactions in a group of animals is reached as the proliferation index.
Inhibition by test compounds for the proliferation ability is the index of antiprogestational
activity. The following Mc Phail scores are used for evaluating the degree of proliferation
(Fig. 8.8).
Scores:
Score 0—Ramification of the uterine mucosa but no proliferation (estrogen treatment only).
Score 1—Slight proliferation of the uterine mucosa.
Score 2 —Medium proliferation of the uterus mucosa, slight additional ramification.
Score 3—Pronounced proliferation of the uterine mucosa.
Score 4—Very pronounced, proliferation of the uterus mucosa, pronounced proliferation of
the uterus mucosa, pronounced ramification.
The scores from each dosage group are averaged.
144 Drug Screening Methods

Anti-implantation Activity
Procedure: Female albino rats of established fertility in proestrous or estrous stage are mated
with matured male rats of established fertility (in the ratio female 3:1 male). Each female is
examined for the presence of spermatozoa in the early morning vaginal smear. The day on
which this sign of mating is seen is taken as a day 1 of pregnancy. The female is then separated
and caged singly and drug is administered orally to the animals once daily on specific days of
pregnancy at different concentrations. On day 10th of pregnancy, the animals are laparotomized
and the number of implants present in both the uterine horns as well as the number of corpora
lutea (CL) on each ovary is counted. The animals are allowed to complete the gestation period
(21–23 days) and the number of litters delivered, if any are counted (Fig. 8.9).19,20
Pre-post- and anti-implantation activity are calculated using the following formula.21
Pre-implantation loss = No. of CL on 10th day–No. of implants on 10th day.
Post-implantation loss = No. of implants on 10th day–No. of litters delivered.

No. of CL – No. of implants

% Pre-implantation loss = × 100
No. of CL

No. of implants – No. of litters

%
Post-implantation loss = × 100
No. of implants

No. of CL – No. of litters

%
Antifertility activity = × 100
No. of CL

Procedure for laparotomy: The animal is lightly anesthetized with ether and limbs are tied to a
rat board (waxed) with the ventral side up. The hair on the area around the midline abdominal
region are clipped with curved scissor and the region is cleaned with 70% alcohol. An incision
of 2 cm length is made along the midline to expose the viscera. The superficially lying coils of
ileum are lifted to expose the two uterine horns. The horns are examined for implantation sites.
Implants are visible as clear swellings on the uterine horns giving the uterine tube a beaded
appearance. Embryos with bright-red dish aspect and a clear margin are considered to be

Figure 8.9: Rat uterus showing normal implants on 10th day of pregnancy
 Antifertility Agents 145

healthy. Those with dull blue color, with no clear margin and orientation with some exudates
are considered resorbing. The number of implants and resorption sites per horn are counted.
The ovaries, which lie on the upper end of the uterine horns, show corpora lutea as yellow
spots over the surface. The number of corpora lutea present on each ovary is also noted.
After counting, the organs are replaced back. A small quantity of neosporin powder is
sprinkled over the organs to prevent any infection. The incision through muscular layer is
closed with continuous suture using absorbable catguts and skin layer with continuous sutures
using silk thread. An antiseptic, povidone iodine solution is applied on the sutured area after
wiping with 70% alcohol. After laparotomy, the rats are transferred to a warm place till they
recover from the anesthesia.

Abortifacient Activity
Procedure: Adult female albino rabbits are used for the study. The pregnancy date is counted
from the date of observed mating. The existence of pregnancy may be confirmed by palpation
after 12th day of pregnancy. Intra-amniotic and intraplacental injections are performed on
rabbits under ether anesthesia on day 20 of pregnancy. The uterus is exposed through the
midline incision, a particular site is chosen for injection and its various parts are identified by
transillumination from a strong source of light. Then material is injected in 0.1 ml of solvent
into the amniotic fluid or in 0.05 ml of solvent into the placenta. Alternatively, the drugs
can be given through any route and duration from day 20 of pregnancy. The effect of drug is
determined by looking for vaginal bleeding, changes in weight, abdominal palpation and by
postmortem examination.12,22

METHODS FOR MALES

Developing male antifertility agent involves interference with spermatogenesis without loss of
libido and secondary sexual characteristics.

Emergent Spermatozoa Made Nonfunctional/Oligospermia/Aspermia

In Vivo Methods
Cohabitation Test
This test determines the time interval for litter production after placing treated males with 2
females. The date of mating is calculated from the date of parturition. This method is suitable
for drugs known to cause several weeks sterility.
Procedure: Adult female and male albino rats of proven fertility are used for the study. They
are kept for mating in the ratio of 2:1 till both females deliver the litters. The date of mating is
calculated from the date of parturition. The time interval for litter production after placing
treated males with two females is calculated.
Fertility Test
Fertility test is based on the evaluation of average litter size. Antifertility agents negatively
affect the average litter size.
146 Drug Screening Methods

Procedure: Groups of 5–10 male rats of proven fertility are treated with drug and are paired
with fertile females in the ratio of 1:3. Daily vaginal smears are examined for the presence of
sperms. All females passed through 1 estrus cycle must have mated. The mated animals are
kept separately till the completion of the gestational period. The litters are counted, and using
the following formula average litter size is calculated:
Total no. of litters
Average litter size =
No. of females mated

If vaginal smear shows leukocytes for 10–14 days, pseudopregnancy is confirmed. If

insemination is not detected, then inhibition of libido or aspermic copulation might be the
cause. Fertility patterns can be obtained from changes in average litter size.12
Subsidiary Test
This test determines the changes in spermatozoa count with time. The antifertility drugs affect
the spermatozoa count negatively.
Procedure: Adult male albino rats weighing between 150–250 g are used for the study. They are
kept in a cage containing artificial or animal vagina. Artificial vagina is the cylindrical plastic
jacket with the rubber liner, filled with water at 5°C. 0.5 ml of ejaculate is diluted with saline
containing traces of formalin. Resulting suspension counted on hemocytometer.12

In Vitro Methods
Spermicidal Activity
Procedure: Spermicidal drugs are diluted with normal saline and serial dilutions are made
in 0.2 ml of human seminal fluid with 1 ml of spermicidal solution. Then the mixture is
incubated at 37°C for 30 minutes. A drop of the mixture is placed immediately on a slide and
at least five fields were microscopically observed under high power (400X) for assessment
of sperm morphological changes and motility. Effective agents can immobilize and kill the
sperms.23
Immobilization Assay
Procedure: The cauda portion of epididymes of ram is isolated and minced in 0.9% saline
solution (pH 7.5) and filtered through a piece of cheese cloth to get sperm suspension. For
human sample, ejaculates (n=10) from normal subjects, after 72–96 h of sexual abstinence,
are subjected to routine semen analysis following liquefaction at 37°C. Sperm count above 100
million/ml and viability above 60% with normal morphology, rapid and progressive motility is
employed for the test. Ram epididymal sperm suspension (100 million/ml to 200 million/ ml)
or human ejaculate (100 million/ml to 150 million/ml) is mixed thoroughly in 1:1 ratio with
different concentration of drugs. A drop of the mixture is placed immediately on a slide and
at least five fields were microscopically observed under high power (400×) for assessment of
sperm motility. The mixture is then incubated at 37°C for 30 minutes and the above process is
repeated.23
 Antifertility Agents 147

Nonspecific Aggregation Estimation

Procedure: Different concentrations of drugs are treated with ram sperm suspension in
1:1 ratio and kept at 37°C for 1 hour. Then from the bottom of the micro centrifuge tube,
one drop of the sedimented sperm is placed on a slide and the percent aggregation was
examined microscopically under 400X magnification. Considering that the non-aggregated
spermatozoa will remain in the supernatant, the latter is collected and the turbidity determined
spectrophotometrically at 545 nm. The aggregation is indirectly proportional to the sperm
viability.23
Sperm Revival Test
This assay determines the extent of spermicidal and immobilization capability of drugs by
evaluating the revival of sperm motility.
Procedure: To study the revival of sperm motility, after completion of the immobilization
assay, the spermatozoa are washed twice in physiological saline and incubated once again in
the same medium, free of drug at 37°C for 30 minute to observe the reversal of sperm motility.23

Assessment of Plasma Membrane Integrity

Procedure: To assess the sperm plasma membrane integrity, ram sperm suspension (100
million/ml to 200 million/ml) or human ejaculated sperm (100 million/ml to 150 million/ml)
are mixed with drug at the minimum effective concentration, respectively, at a ratio of 1:1 and
incubated for 30 minutes at 37°C. Similarly, sperm samples in saline served as the controls.
For viability assessment, one drop each of 1% aqueous solution of eosin Y and of 10% aqueous
solution of nigrosin was placed in a micro centrifuge tube. A drop of well-mixed sperm sample
is added to it and mixed thoroughly. The mixture is dropped onto a glass slide and observed
under 400X magnification.
For hypo-osmotic swelling test (HOS) 0.1 ml of aliquot is taken from each of the treated and
control sample, mixed thoroughly with 1 ml of HOS medium (1.47% fructose and 2.7% sodium
citrate at 1:1 ratio), incubated for 30 minutes at 37°C and the curling tails were examined under
phase contrast microscope using 100X magnification.
5-nucleotidase is released possibly due to destabilization of plasma membrane. This can
be estimated to find the effect of drug on plasma membrane integrity of sperm. The activity of
5’-nucleotidase can be determined by measuring the rate of release of inorganic phosphate
from adenosine 5’-monophosphate. After incubating the sperm suspension with drug, the
sperm pellet is collected by centrifugation at 3,000 g at 37°C. Then it is washed twice in 0.9%
saline and then suspended in 0.1 mol/L Tris-HCl buffer (pH 8.5) with each reaction system
containing 100–200 million spermatozoa. An aliquot of 0.1 ml suspension of sperm is added
to 0.9 ml of buffered substrate containing 3-mmol/L adenosine 5’-monophosphate and
50 mmol/L MgCl2 dissolved in 0.1 mol/L Tris-HCl buffer. The tubes are incubated at 37°C for
30 minutes and 0.5 ml 20% TCA (0°C–4°C) is added to the mixture to stop the reaction. The
mixture was then centrifuged at 10,000 g at 4°C. The pellet is discarded and the supernatant
was kept for phosphate estimation. The activity of 5’-nucleotidase was expressed in terms of μg
of phosphate released. The activity of 5’-nucleotidase is indirectly proportional to the plasma
membrane integrity.23
148 Drug Screening Methods

Evaluation of Acrosomal Status

Acrosome is the cap like structure on the head of spermatozoa. It breaks down just before
fertilization, releasing a number of enzymes that assist penetration between the follicle cells
that still surround the ovum. This method evaluated the acrosomal status of sperm. The most
widely studied acrosomal enzyme is the acrosin that has been shown to be associated with
acrosomes of all mammalian spermatozoa, and the highest substrate specificity was obtained
with BAEE (N-benzoyl-L-arginine ethyl ester).
Procedure: Different concentrations of drugs are mixed with ram sperm suspension in 1:1
ratio and kept at 37°C for 1 hour. The suspension is centrifuged and the pellets are collected.
The pellets are extracted with 3 mmol/L HCl at pH 3 and the enzyme activity is measured,
following the hydrolysis of 0.5 mmol/L BAEE dissolved in 0.05 mol/L Tris HCl buffer containing
0.05 mol/L CaCl2 at pH 8. The activity of acrosin is expressed in terms of m IU. One m IU activity
means the amount of enzyme, which causes the hydrolysis of one nanomole of BAEE in one
minute at 25°C. The activity of acrosin is directly proportional to the fertilizing capability of
sperms.23

Androgenic Activity
In Vivo Methods
Chicken Comb Method
This assay is based on the principle of growth of cap on comb by androgenic compounds. This
method has been useful for the isolation and structural elucidation of natural androgens.24
Procedure: In the beginning of the assay, the sum of the length plus height of each individual
comb is determined by measurement with a millimeter rule placed directly on the comb. The
capons are injected daily intramuscularly for 5 consecutive days with a solution or suspension
of the test compound or the standard in 1 ml olive oil. Twenty four hours after the last injection,
the comb is re-measured and the growth of the comb is expressed as the sum of the length
and height in millimeter. Groups of eight animals are used for at least two doses of the test
compound and the compound. The weight of control and test group is compared with suitable
statistical analysis.7
Weight of Ventral Prostate, Seminal Vesicles and Musculus Levator Ani
This assay is based on the principle that the androgens affect the secondary sex organs in male
individual. In the rats, the growth of the ventral prostate, the seminal vesicle and the musculus
levator ani depend on the presence of male sexual hormones.
Procedure: Immature male rats weighing about 55 g are orchidectomized. The animals
are treated with the test compounds in various doses orally in 0.5 ml 0.5% carboxymethyl
cellulose or 0.2 ml sesame oil suspension daily over a period of 10 days. Testosterone given
subcutaneously in doses of 0.02, 0.1 and 0.5 mg per animal, or methyl testosterone in doses
of 0.25, 1.5, and 5 mg per animal, serve as standard. Controls receive the vehicle only. On the
eleventh day, the animals are sacrificed and the seminal vesicles, the ventral prostate, and the
musculus levator ani carefully dissected and weighed. The weight of control and test group is
compared with suitable statistical analysis.7
 Antifertility Agents 149

Nitrogen Retention
The assay is based on the principle that the anabolic agents induce positive nitrogen balance
in the rats. Anabolic agents decrease the nitrogen excretion in the castrated rats fed a liquid
diet and in nitrogen balance.25
Procedure: Twenty-five-day-old rats are castrated and kept untreated for 67 days, reaching
about 300 mg body weight on normal laboratory diet. After 67 days they are changed to liquid
diet force-feeding regime. Besides carbohydrates and fat, the diet contains casein and brewer’s
yeast as nitrogen source. At the start, the rats receive 10 ml per day, and this is increased to 26
ml per day. This feeding is continued for 30 days with simultaneous administration of the test
drug once a day. Twenty-four hour urine specimens are collected 3 times weekly and analyzed
for total nitrogen.7

Anti-androgenic Activity
In Vivo Methods
Chicken Comb Method
This assay is based on the principle of inhibition of growth of capon comb by anti-androgenic
compounds.
Procedure: One to 3 days old male or female white leghorn chicks are housed at constant
temperature in a heated incubator. Testosterone is incorporated into the finely ground chick
starting mash at a concentration of 80 mg per kilogram food. The chicks are placed on this diet
for day one. The test compound is dissolved in sesame oil. Each day for 4 days 0.1 ml of the oil
solution is injected subcutaneously. Control chicks receive only the vehicle. Twenty-four hour
after the last injection, the animals are sacrificed, the combs removed and after blotting of
the cut edge, weighed rapidly to the nearest 0.5 mg. The weight of control and test groups are
compared using suitable statistical method.7
Antagonisim of Effect of Testosterone on Weight of Ventral Prostate, Seminal Vesicles and
Musculus Levator Ani
In the rats, the growth of the ventral prostate, the seminal vesicle and the musculus levator ani
is stimulated by testosterone, anti-androgenic compound inhibits this effect.
Procedure: Male rats weighing 50–70 g are castrated and one day after surgery, the rats are
injected once daily for 7 days with 0.15 mg testosterone propionate in 0.1 ml sesame oil.
The test compound also dissolved or suspended in sesame oil at various doses and injected
subcutaneously daily at a separate site for 7 days. Controls receive testosterone injections only.
On 8th day, the animals are sacrificed and weights of ventral prostate, seminal vesicles and
musculus levator ani weighed. The weight of control and test group is compared with suitable
statistical analysis.7
Anti-androgenic Activity in Female Rats
This assay is based on the principle of antagonism of the anti-androgens against the tropical
effect of testosterone on uterine and preputial growth.
150 Drug Screening Methods

Procedure: Female rats weighing 40–45 g are ovariectomized, one week later the treatment
is started over a period of 12 days with daily subcutaneous injection of 0.3 mg testosterone
propionate and various doses of the antagonist. Controls receive testosterone propionate only.
On the 13th day, the animals are sacrificed and the uteri and preputial glands weighed. Weight
increase of female accessory sexual organs due to testosterone treatment is dose despondent
reduced by an anti-androgens.7

REFERENCES
1. Sciarra JJ. The continuing need for contraceptive research. Fertil Steril 1981;36:697–8.
2. Kostyk SK, Dropcho EJ, Moltz H, Swartwout JR. Ovulation in immature rats in relation to the time
and dose of injected human chorionic gonadotropin or pregnant mare serum gonadotrophin. Biol
Reprod 1978;19:1102-7.
3. Turner RA. Screening methods in pharmacology. New York: Academic Press 1971:85-118.
4. Kapoor M, Garg SK, Mathur VS. Antiovulatory activity of five indigenous plants in rabbits. Ind J Med
Res 1974;62:1225-7.
5. Stockard CR, Papanicolaou GN. The existence of a typical estrus cycle in the guinea pig with the
study of its histological and physiological changes. Am J Anat 1917;22:225-83.
6. Allen E, Doisy EA. An ovarian hormone. Preliminary report on its localization, extraction and partial
purification and action in test animals. J Am Med Ass 1923;81:819-21.
7. Vogel HG, Vogel WH, Editors. Drugs Discovery and Evaluation. Berlin: Springer 1997.
8. Agrawal SS, Paridhavi M. Herbal Drug Technology. Hyderabad: Universities Press 2007.
9. Terenius L. Uptake of radioactive estradiol in some organs of immature mice. Acta Endocrinol
(Copenh) 1965;50:584-96.
10. Terenius L. The uptake of radioactive isomers of synthetic estrogens in various organs in immature
mice. Acta Endocrinol (Copenh) 1966;53:84-92.
11. Jonsson CE, Terenius L. Uptake of radioactive estrogen in the chicken oviduct and some other
organs. Acta Endocrinol (Copenh) 1965;50:289-300.
12. Ghosh R. Modern concept on pharmacology and therapeutics. (24th ed). Calcutta, Hilton and Co,
1991.
13. Mc Phail MK. The assay of progestin. J Physiol 1934;83:1545-56.
14. Verma U, Laumas KR. Screening of antiprogestins using in vitro human uterine progesterone
receptor assay system. Steroid Biochem 1981;14:733-40.
15. Bayard F, Damilano S, Robel P, Baulieu EE. Cytoplasmic and nuclear estradiol and progesterone
receptors in human endometrium. J Clin Endocrinol Metab 1978;46:635-48.
16. Brooks JR, Babiarz EA, Berman C, Primka RL, Rasmusson GH. Some endocrinological and anti-
fertility properties of an androstane cyanohydrin derivative in the rat. Biol Reprod 1978;18:186-92.
17. Mc Ginty DA, Anderson LP, Mc Collough NB. Effect of local application of progesterone on the
rabbit uterus. Endocrinology 1939;24:829-32.
18. Tayama T, Mtoyama T, Ohono Y, Ide N, Turusaki T, Okada H. Local progestational and
antiprogestational effects of steroids and their metabolites on the rabbit uterus. Jpn J Feril Steril
1979;24:48-51.
19. Agrawal SS, Aravinda S. Anti-implantation activity of H2 receptor blockers. Indian J Pharmacol
1995;27:40-42.
20. Shafiq N, Malhotra S, Pandhi P. Comparison of nonselective cyclo-oxygenase (COX) inhibitor and
selective COX-2 inhibitors on preimplantation loss and duration of gestation: an experimental
study. Contraception 2004;69:71-75.
 Antifertility Agents 151

21. Agrawal SS, Gatak N, Arora RB. Antifertility activity of roots of Abrus precatorius Linn. Pharmacol
Res Commun 1970;2:159-62.
22. Turner RA. Screening methods in pharmacology. New York: Academic Press, 1965:270-73.
23. Chakrabati K, Pal S, Battacharyyya AK. Sperm immobilization activity of Allium sativum L. and
other plant extracts. Asian J Androl 2003;5:131-5.
24. Dorfman RI. Studies on the bioassay of hormones. The assay of testosterone propionate and
androsterone by a chick inunction method. Endocrinology 1948;48:1-6.
25. Stafford RO, Bowman BJ, Olson KJ. Influence of 19-nortestosterone cyclopentyl-propionate on
urinary nitrogen of castrate male rats. Proc Soc Exp Biol Med 1954;86:322-6.
 CHAPTER

9
Antiobesity Agents
INTRODUCTION
When energy intake exceeds energy expenditure, obesity develops. Obesity is characterized by
an excessive development of fat mass, which is a consequence of increased size of adipocytes
and/or increased number of adipocytes. It increases the risk of developing diabetes,
hypertension, dyslipidemia, certain forms of cancer and osteoarthritis, etc.
Obesity is a multifactorial disease. It arises as a result of interaction among numerous
behavioral, environmental and genetic factors and associated with the dysregulation of energy
homeostasis, normally maintained by the hypothalamic neuroendocrine/neurotransmitter
network. Signaling factors, like leptin and various neuropeptides, are important components
of this complex network. Leptin is synthesized and secreted primarily from adipocytes
and acts centrally in the hypothalamus by binding to the leptin receptor. Circulating levels
of leptin are highly correlated with the level of body fat. Insulin and glucocorticoids can
stimulate production of leptin by adipocytes. Low plasma concentrations of leptin and insulin,
e.g. during fasting and weight loss, increase food intake and decrease energy expenditure by
stimulating neuropeptide Y (NPY) synthesis, and perhaps by inhibiting sympathetic activity
and other catabolic pathways. High leptin and insulin concentrations, e.g. during feeding
and weight gain, decrease food intake and increase energy expenditure through release of
melanocortin and corticotropin-releasing hormone (CRH). Stimulation of the leptin receptor
can lead to changes in the expression of a variety of neuropeptides.
Neuropeptides that are involved in energy homeostasis can be orexigenic (appetite stimula­
ting) and anorectic as shown in Table 9.1.1 The involvement of most of these neuropeptides
in maintaining energy homeostasis was deduced from transgenic animal studies and
spontaneous mutations.
β3-adrenoceptor also has an important role in the regulation of lipid metabolism and
obesity. However, the physiological function of β3-adrenoceptor in humans has not yet been
established.

ANIMAL MODELS FOR THE STUDY OF ANTIOBESITY DRUGS

The animal models of obesity have been either of spontaneous origin or the result of
experimental manipulation of the environment or hypothalamic centers that regulate food
 Antiobesity Agents 153

Table 9.1: Orexigenic and anorectic neuropeptides involved in energy homeostasis

Orexigenic peptides Anorectic peptides
NeuropeptideY (NPY) Corticotropin-Releasing Hormone
Agouti-related peptide (AgRP) Melanocyte Stimulating Hormone
Orexins A and B Cholecystokinin
Galanin Glucagon-like peptide1
β endorphin Calcitonin gene-related peptide
Norepinephrine Bombesin
Growth Hormone-Releasing Hormone
Melanin concentrating hormone

intake and energy balance. The identification of gene mutations that cause obesity and the
use of transgenic techniques have provided new insights into the physiologic and molecular
mechanisms underlying obesity. However, much remains to be studied in this complex field
of research.
In animal models of obesity some parameters are studied:
•• Food intake
•• Body weight
•• Adipose tissue cell size and number
•• Body composition
•• Locomotor/physical activity
•• Plasma lipids, insulin and glucose levels.
Food intake and weight gain: Food intake and weight gains are recorded in control and
experimental group daily at a fixed time, preferably in the morning. For measurement of food
intake, spilled food is collected by suspending a paper under the cage and the amounts of
spillage are also determined after drying. Food intake measurements are also important to
study the anorectic activity of various compounds.2
Adipose tissue cell size and number: Number and size of adipose tissue cell is determined by
osmium fixation method.3
Body composition: Body composition is estimated by determining the water content of the
carcasses by oven drying at 95°C for 6–9 days until constant weight is reached. Lipid content
is measured in gonadal and retroperitoneal fat pads. For this, adipose tissue is homogenized
with a 2:1 chloroform-methanol mixture and extract is washed by addition of water. The
resulting mixture separates into two phases. Lower phase consists of pure lipid extract, which
is measured.4,5
Locomotor/physical activity: Generally, locomotor/physical activity is reduced in obese
animal.
154 Drug Screening Methods

Diet-induced Obesity
Procedure
Adult female rats, weighing approximately 230 g, are housed individually in wire mesh cages
under controlled temperature and artificial light/dark cycle. Animals are divided into two
groups. First group receives ordinary purina chow and the other group is given in addition
to chow, a high fat diet, sweetened condensed milk and a number of supermarket foods like
cookies, cheese, milk chocolate, peanut, butter, etc. (cafeteria diet). Body weight, food intake,
locomoter activity and serum insulin levels are measured and compared in both the groups.
After three months, rats are sacrificed by decapitation and adipose tissue cell size and number,
body composition and lipid content in fat pads are determined, and comparison is made
between the groups.6,7

Hypothalamic Obesity
Hypothalamus regulates food intake by interaction of a lateral feeding center and a medial
satiety center. Ventromedial hypothalamic lesions are found to increase food intake followed
by increased body weight and obesity after 3–4 months. Injury to hypothalamus can be
produced by surgical methods and by chemical agents (such as gold thioglucose, mono
sodium glutamate, etc.) in the animals especially rodents, which can result into obesity.

Surgically-induced Hypothalamic Obesity

Procedure
Female Sprague Dawley rats, weighing approximately 190 g are fed a high fat diet for 5–9 days.
After this, rats are anesthetized with 35 mg/kg pentobarbital sodium along with 1 mg atropine
methyl nitrate administered intraperitoneally.
To produce hypothalamic lesions, bilateral knife cuts are stereotaxically made in the
hypothalamus of rats. Incision bar is positioned at –3.0 mm and parasagittal cuts are made
in between the lateral and medial hypothalamus using a retractable wire knife.8 The cuts are
 Antiobesity Agents 155

made 1.0 mm lateral to the midline and extended from 8.5 to 5.5 mm anterior to the ear bars
and from 3.0 mm dorsally from the base of the brain.
Sham-operated rats serve as control.9,10 Food intake, body weight and other parameters are
recorded for comparison in between the groups.

Chemically-induced Hypothalamic Obesity

Monosodium Glutamate-induced Hypothalamic Obesity in Mice
Mice are injected daily with mono sodium-L-glutamate subcutaneously in the doses of
2 g/kg for 5 consecutive days in early stages of life. In control group, physiological saline is
administered.
All mice are housed under maintained temperature and artificial light/dark cycle and
provided with chow and water ad libitum. Food consumption, weight gain and other
parameters (as mentioned above) are recorded and comparison is made between the groups.11
Modification:Young albino rats between the age of 2–40 days are used and treated with either
single or repeated doses of 0.1–6.0 mg/g body weight of monosodium-L-glutamate.12
Gold Thioglucose Induced Hypothalamic Obesity in Mice
Six weeks old Swiss albino mice of either sex are given a single intraperitoneal injection of
gold-thioglucose in the doses of 30–40 mg/kg. After that body weight is recorded for 3 months
and compared with untreated controls.13
Modification: Gold thioglucose implants in the hypothalamus of rats, are used to induce
obesity.14
Other Chemical Compounds
Besides these, several other chemical compounds have been reported to produce obesity in
mice, such as single intraperitoneal injection of bipiperidyl mustard in doses between 5–50
mg/kg and single intracerebral injection with 4-nitroquinoline-1-oxide.15,16

Virus-induced Obesity
Mice infected with canine distemper virus, a morbillivirus antigenically related to measles
virus, develop obesity. Obesity is seen after 8–10 weeks of viral infection. Canine distemper
virus targets certain brain structures, including the hypothalamus followed by virus-induced
disruption of critical brain catecholamine pathways as a result of which obesity is developed.17
Other viruses that can cause obesity in animals18-22 are shown in Table 9.2.
Table 9.2: Viruses causing obesity in animals

Viruses Animals
Canine distemper virus Mice
Borna disease virus Rats
Rous-associated virus-7 Chickens
Avian adenovirus SMAM-I Chickens
Ad-36 (human adenovirus) Chickens, mice, nonhuman primates
The exact mechanism of obesity caused by these viruses is unclear.23
156 Drug Screening Methods

Genetic Models of Obesity

Obesity is a phenotype that is readily observable, and mice with naturally arising mutations
causing this phenotype have been extensively characterized. Genetic models can be
monogenic, polygenic and transgenic.24

Monogenic Models (Table 9.3)

Mice with naturally arising mutations causing obesity have been extensively characterized.The
oldest of these mutations is the agouti gene, whereas the most well-known are mutations in
the genes of the hormone leptin and its receptor, ob and db, respectively. Naturally occurring
single gene mutations producing obesity (Table 9.4) form the basis for a candidate gene
approach to identify the genes responsible for human obesity. Several human counterparts of
these rodent obesity syndromes have been identified.

Table 9.3: Monogenic and polygenic models of obesity

Monogenic Polygenic
Yellow obese (Aya) mouse Japanese K K mouse
Obese (ob/ob) mouse NZO mouse
Diabetes (db/db) mouse Otsuka-Long_Evans-Tokushima-Fatty rats
Fat mouse BSB model
Tubby mouse AKR/J × SWR/J model
Fatty (fa/fa) rat M16 mouse
Obese SHR rat
JCR: LA- Corpulent rat
WDF/TA-FA Rat

Table 9.4: Obesity mutations in rodents and mutant proteins

Mutation Gene Inheritance (Autosomal) Mutant protein

Mouse Agouti Ay Dominant ASP
Diabetes db Recessive Lepr
Fat fat Recessive Carboxypeptidase
Obese ob Recessive Phosphodiesterase
Rat Fatty fa Recessive Lepr

Yellow Obese (Aya) Mouse

The yellow obese mouse was discovered as early as 1883 by Lataste and in 1905 by Cuenot.25,26
An agouti mutation results into obese yellow mice. Yellow obese mouse is the only example
of obesity inherited through a dominant gene, located on chromosome 2 at linkage group 5,
the agouti locus. Since genes controlling obesity and the agouti coat colors are closely linked,
the obesity is associated with a change of pigmentation of hair from black to yellow. This
association allows the early identification of pre-obese mice as soon as the coat hair begins to
 Antiobesity Agents 157

Figure 9.1: Yellow obese (Aya) mice

grow. The homozygous (Ay/Ay) alleles are lethal in utero and several different alleles (Avy/Avy,
Aiy/Aiy) have appeared at the agouti locus, in which the degree of obesity is linked directly to the
level of yellow pigmentation in the coat.
Yellow (Aya) mice exhibit moderate form of obesity and diabetes. Body weight starts
increasing at the time of puberty (8–12 week) and reaches maximum up to 40 g.27,28
In 1992, agouti gene was cloned and it was the first obesity gene characterized at the
molecular level.29 The molecular categorization of agouti was responsible for elucidation of
the melanocortin system’s involvement in weight regulation, due to its resemblance to agouti-
related protein activity in the hypothalamus. The melanocortin receptor family comprises
five G-protein-coupled proteins, melanocortin 1 receptor (Mc1r) to Mc5r, which demonstrate
tissue-specific patterns of expression. Mc4r is expressed in the hypothalamus and plays a key
role in the regulation of feeding and metabolism and is normally antagonized by agouti-related
protein.30

Obese (ob/ob) Mouse

The obese mouse, first described by Ingalls et al. in 1950, inherits its obesity as autosomal
recessive mutation on chromosome 6. This obese (OB) mutation has been maintained as
inbred stock on the C57BL/6J strain. Obese mouse is characterized by obesity, hyperglycemia
and insulin resistance. Animal is visually detectable as obese by 25–28 days.31,32
The mutant gene responsible for the phenotype in Lepob mice encodes a protein termed
leptin, which is deficient in these animals. The gene encoding human leptin has been studied
extensively.

Figure 9.2: Ob/ob mice

158 Drug Screening Methods

Diabetes (db/db) Mouse

The diabetes (db) mutation arose in the C57BL/KsJ strain on chromosome 4. It is an autosomal
recessive gene with full penetrance. Diabetes mouse exhibits marked obesity and hyperglycemia
associated with insulin resistance. Overt obesity is observed shortly after weaning, after that body
weight increases rapidly.33 db/db mice have a mutation in leptin receptor as mentioned above.
Fat Mouse
The fat mouse (fat/fat or Cpefat/ Cpefat) is a model of a late-onset form of obesity. Obesity is
inherited as autosomal recessive mutation known as fat mutation, located on chromosome
8 as coding for carboxypeptidase E (CPE). CPE is involved in the final stages of processing
of insulin, POMC and other hormones. Mouse is characterized by pronounced early onset
hyperinsulinemia, obesity and infertility. Fat mouse develops obesity between 6–8 weeks of
age and attains body weight of 60–70 g by 24 weeks.34-35
Tubby Mouse
The tub mutation was discovered for the first time in a C57BL/6J male mouse and tubby colony
was bred from this mouse. Mutation is autosomal recessive. Tubby mouse exhibits slow
onset obesity; means phenotype cannot be recognized until 9–12 weeks of age and average
weight is 46 g at 24 weeks. This animal also develops sensorineural hearing loss and retinal
degeneration.36

Figure 9.3: Tubby mouse

Fatty (fa/fa) Rat

The Zucker fatty (fa/fa) rat is the best known and most widely used rat model of genetic obesity.
The fa mutation was discovered by Zucker and Zucker (1961) in crosses between Sherman and
Merck stock M rats. The affected rats are characterized by obesity, hyperinsulinemia and insulin
resistance. The obesity is inherited as an autosomal recessive mutation. Rats homozygous for
the fa allele become noticeably obese by 3–5 weeks of age.37, 38

Figure 9.4: Fatty (fa/fa) rat

 Antiobesity Agents 159

Obese SHR Rat

Obese SHR rat was developed by mating a spontaneous hypertensive female rat (Kyoto-Wistar
strain) with a normotensive Sprague-Dawley male rat. After several generations of selective
inbreeding, rats exhibited obesity, hypertension and hyperlipidemia.39
JCR: LA- Corpulent Rat
JCR: LA-corpulent rat was developed as a substrain from obese SHR rats. This rat is characterized
by obesity, hyperinsulinemia with impaired glucose tolerance and hyperlipidemia.40
WDF/TA-FA Rat
WDF/TA-FA rat is also known as Wistar Fatty rat. This strain was established by transfer of the
fatty (fa) gene from the Zuker rat to the Wistar Kyoto rat. WDF/TA-FA rat is characterized by
hyperinsulinemia, glucose intolerance, hyperphagia and obesity.41

Polygenic Models (Table 9.3)

The polygenic models of obesity have allowed identification of multiple genetic loci within
individual strains that modify obesity, plasma cholesterol levels, specific deposition of body
fat depots, and propensity toward development of obesity on a high-fat diet. These polygenic
models more closely resemble the human obesity phenotypes than single gene models. These
models are described below:
Japanese KK mouse
Japanese KK is one of the most suitable polygenic mouse models of obesity. Konello et al. first
described it in Japan in a strain of mice bred for large body size mice. The dominant gene for
yellow obesity (Ay) has also been transferred into the Japanese KK strain and the resultant
animals are named as yellow KK mice (KK-Ay). In KK mouse onset of obesity is late, body
weight begins to rise after 2–3 months of age and slowly attains a maximum up to 40–50 g by
6–9 months of age. The mouse also develops hyperinsulinemia, hyperphagia and moderate
hyperglycemia.42
NZO Mouse
The New Zealand obese (NZO) mouse was first described in 1953 by Bielschowsky and
Bielschowsky.36, 43 NZO mouse develops obesity, mild hyperglycemia and insulin resistance.
The adult NZO attains a body weight of 50–70 g by 6–8 months. After this age, weight gain
occurs slowly. By 6 months of age this mouse develops renal disease and other autoimmune
disorders.44

Figure 9.5: NZO mouse

160 Drug Screening Methods

Otsuka-Long-Evans-Tokushima-Fatty (OLETF) Rats

OLETF rat strain is developed from outbred colony of Long-Evans rats by selective breeding.
It has since been maintained at the Tokushima Research Institute, Japan. OLETF rat is
characterized by mild obesity, hyperglycemia, polyuria and polydipsia. Weight begins to rise
two weeks after weaning.45
The M16 Mouse
The M16 mouse is an outbred animal model of polygenic obesity. Selection for increased 3–6
weeks postweaning gain in the M16 line of mice was first reported by Hanrahan et al.46 M16 is
the result of long-term selection for 3–6 weeks weight gain from an ICR base population. M16
mice are heavier, fatter, hyperphagic, hyperinsulinemic, and hyperleptinemic relative to ICR.
M16 mice represent an excellent polygenic model to study the genetics of growth, obesity,
and type 2 diabetes. Relaxed selection over to facilitate gene discovery and pathway regulation
controlling early onset polygenic obesity and type 2 diabetic phenotypes. These phenotypes,
observed at young age (early onset), closely mimic current trends in humans.47

Transgenic or Knockout Animal Models

In these animal models, genes regulating energy homeostasis are manipulated. Gene
manipulations are targeted either to a specific tissue or to more ubiquitous expression such as:
Knockout of β3 adrenergic receptor gene in white and brown adipose tissue.48
Knockout of uncoupling protein in brown adipose tissue: Transgenic toxigene approach is
used to create transgenic mice. In this approach, targeted expression of a gene for diphtheria
toxin A chain is used to knockout selectively functional activity of brown adipose tissue. By
linking the gene to uncoupling protein gene expression, brown adipose tissue thermogenic
function is abolished and mice become obese and develop hyperphagia.49
Knockout mice lacking steroidogenic factor I (SF-I): Knockout (KO) mice lacking
steroidogenic factor I (SF-I) exhibit a phenotype that is characterized by abnormalities of
ventromedial hypothalamic nucleus, gonadal agenesis and impaired gonadotropin expression.
SF-1 KO mouse become obese by 8 weeks of age and represents late-onset obesity.50
Barden and colleagues incorporated a type II glucocorticoid receptor antisense RNA
construct into mice and focused its expression primarily to neural tissues by linking the
construct to a human neurofilament gene promoter sequence 4. All transgenic offspring
developed obesity (two fold increase in body weight) by 6 months of age.51,52
Knockout of the Mc4r gene in mice is observed to result in early-onset obesity, non–insulin-
dependent diabetes, and other obesity-associated syndromes. These symptoms are in parallel
to the yellow agouti mouse syndrome, indicating that agouti expression in the hypothalamus
inhibits Mc4r function, leading to obesity.53
Targeted deletion of the Mc3r gene also results in a late-onset obesity phenotype, but
regulation of appetite and metabolism remains intact. Mc3r transgenic mouse are also
susceptible to diet-induced obesity.53
NPY Y5 receptor knockout mice have demonstrated that mutants lacking this receptor
develop mild late-onset obesity characterized by increased food intake, weight gain, and
increased body fat mass.54
 Antiobesity Agents 161

Serotonin receptor type-2C null mutant mouse is developed to understand serotonin

receptor functions, since this single-gene-mutation produced a mouse which is predisposed to
obesity. The serotonin type-2C null mutant exhibits excessive feeding behavior (hyperphagia)
without concurrent hyperinsulinemia. The hyperphagia observed in the null mutants preceded
development of obesity and reduced sensitivity to insulin and leptin.55
Melanin-concentrating hormone overexpression in transgenic mice: Melanin-
concentrating hormone (MCH) is known to play an important role in feeding behavior. In
the brain, MCH is expressed in the lateral hypothalamus and responds to nutritional signals,
including fasting and leptin deficiency. Thus, expression is increased with fasting.
Transgenic mice that overexpress MCH (MCH-OE) in the lateral hypothalamus, around
two-fold higher levels than normal mice, are produced on the FVB genetic background.
Homozygous transgenic animals are found to be heavier at 13 weeks of age when fed a high-fat
diet than wild-type animals along with higher systemic leptin levels. MCH-OE animals were
hyperphagic, hyperleptinemic, and had higher blood glucose levels. MCH-OE animals also
develop hyperinsulinemia and insulin-resistance.56 When transgene was bred onto obesity-
prone genetic background, the C57BL/6J, heterozygote C57BL/6J mice expressing the transgene
showed increased body weight on a standard diet, confirming that MCH overexpression can
lead to obesity.57
Other transgenic mice models of over expression of genes include—over expression of
corticotropin releasing factor gene, GLUT-4 gene, human agouti-related protein complementry
DNA.58-60
Transgenic mice overexpressing 11beta HSD-1: Model of visceral obesity: Glucocorticoids
can be produced locally from inactive 11-keto forms by an enzyme 11beta hydroxysteroid
dehydrogenase type 1 (11beta HSD-1). Increased glucocorticoid levels are known to
produce visceral obesity. Transgenic mice overexpressing 11beta HSD-1 selectively
in adipose tissue (similar to that found in adipose tissue of obese humans) are created.
These mice had elevated levels of corticosterone in adipose tissue and developed visceral
obesity. The mice also exhibited insulin-resistant diabetes, hyperlipidemia, hyperphagia
and hyperleptinemia.61

IN VITRO ASSAYS ON ISOLATED ADIPOSE TISSUE CELLS

To Study Metabolic Activity in Brown Adipose Tissue
Brown adipose tissue (BAT) is an important site for energy expenditure or thermogenesis.
BAT activity is associated with uncoupling proteins (UCP), which are mitochondrial carrier
proteins and dissipate the hydrogen ion gradient in the oxidative respiration chain, releasing
energy as heat. UCP1 is expressed in brown adipose tissue, UCP2 is widely expressed and
UCP3 is found in skeletal muscle and brown adipose tissue. Brown adipose tissue activity is
increased by leptin activity through the sympathetic nervous system. Uncoupling proteins
and GLUT 4 are indicators of thermogenic activity of brown adipose tissue.
162 Drug Screening Methods

Assay for Uncoupling Protein and GLUT 4 in Brown Adipose Tissue

Procedure
Male fatty (OLETF fatty) rats, 10 weeks of age are given subcutaneous injection of test
compound or solvent once daily. Rats are sacrificed after 14 weeks of treatment and brown
and white adipose tissue are removed. Uncoupling proteins and glucose transporter 4 (GLUT-
4) are determined by protein analysis or Western blot analysis.62
To carry out this analysis, adipose tissue is homogenized in 5–10 volumes of a solution,
which contains 10 mmol/1 Tris-HCl and 1 mmol/1 EDTA (pH 7.4) for 30 sec with a Polytron.
After that centrifugation is performed at 1,500 g for 5 min and the infranatant (fat-free extract)
is used to measure the proteins. The fat free extracts (10 µg protein of brown adipose tissue,
20 µg of white adipose tissue) are solubilized. The solubilized tissue is subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter.
After blocking with 5% non-fat dry milk, the filter is incubated with rabbit antiserum against
rat uncoupling protein or GLUT. The rabbit antisera against rat uncoupling protein and
GLUT4 are prepared by immunizing purified rat uncoupling protein63 and a 12-amino acid
peptide corresponding to GLUT4 (residues 498–509, TELEYLGPDEND), respectively, that is
coupled with keyhole limpet hemocyanin.64 Then the filter is incubated with [125I] protein A.
Autoradiography is done by exposing dry blot to an X-ray film and to an imaging plate of BASI
000 (Fuji film) for quantitative analysis.

Assay to Study Activity of β3 Agonists

β3 adrenoceptors have been cloned and well characterized in animals and humans. They are
expressed mainly in brown adipose tissue and in white adipose tissue. In obese rodents, β3
agonists induce weight loss, without reducing food intake, which may be due to increased
thermogenesis in brown adipose tissue, increased lipolysis in white adipose tissue and
suppression of leptin gene expression and serum leptin levels.

c-AMP Response Element-luciferase Receptor Gene Assay for β3 Adrenoceptor

Using the human β3 adrenoceptor gene, a Chinese hamster ovary (CHO), cell transfection
system is developed. Human β1, β2 and β3 adrenoceptors are expressed in CHO cells. These cells
are transfected with cAMP response element-luciferase plasmids using electroporation with
a single 70 ms, 150 V pulse.65 The transfected cells are seeded at a density of 40,000/well in
96-well microtiter plates and allowed to grow for 20 h. After 20 h the cells receive varying drug
concentrations (10–11 to 10–4 M) for 4 h, which result in lysis of the cells. Luciferase activity
is measured using the LucLite assay kit. Changes in light production are measured using a
Topcount luminometer.66

Assay for Neuropeptide Y (NPY)

Neuropeptide Y is the most widely distributed neuropeptide in the brain as well as in the
peripheral nervous system, playing an important role in the weight regulation. NPY is a potent
stimulant of appetite. Six receptors of neuropeptide Y have been identified: Y1, Y2, Y3, Y4, Y5,
 Antiobesity Agents 163

and Y6. Neuropeptide Y antagonists (Y5, Y1) are being evaluated as new therapeutic targets for
the treatment of obesity. Bioassays have also been performed for neuropeptide Y receptors
characterization.67

To Study Role of Leptin in Obesity

Ob gene product has been identified and named as leptin. It is expressed in adipose tissue and
enters the brain where it functions to reduce food intake, serum glucose and insulin levels and
increases metabolic rate, ultimately leading to a reduction in body weight. Leptin mediate its
effects through a specific receptor OB-R (lepr). Five different isoforms of leptin receptors have
been identified. In OB/OB mice, obesity is due to mutation in Ob (lep) gene associated with
deficiency of leptin, whereas in db/db mice and fa/fa rats, mutations in the leptin receptor
(lepr) lead to obesity. Human homologue of mouse obese gene has been cloned and it is 84%
homologous to the mouse protein (leptin). Leptin and leptin mRNA levels are correlated with
the percentage of body fat. Generally, obese rodents and humans show increased levels of
these in blood.

In Vitro Assay for Leptin mRNA Level in Adipose Tissue

Leptin mRNA levels in adipose tissue are determined by Northern blot analysis.68

Procedure
Male Wistar rats around 230 g body weight are administered with test drug for 14 days and then
sacrificed by decapitation. Liver, epididymal white adipose tissue and brown adipose tissue
(intracapsular) are rapidly removed and freezed in liquid nitrogen. Total RNA is extracted from
frozen tissues by a guanidiniumthiocyanate-phenol/chloroform method.69 The RNA (10 µg per
lane) is fractionated by horizontal gel electrophoresis and transferred to a positively charged
nylon membrane and fixed with UV-light.
Prehybridization is performed at 42°C in prehybridization solution containing 7% sodium
dodecyl sulfate, 50% formamide, 5X saline-sodium citrate buffer, 2% blocking reagent, 50 mM
sodium phosphate (pH 7.0) and 0.1% N-laurylsarcosine for 45 min. After that hybridization
is performed at 42°C in prehybridization solution containing oligonucleotide probe
(25 ng/ml) specific to leptin, malic enzyme or 185 RNA. This is followed by post-hybridization
washes, i.e. twice for 15 min in 0.1% saline-sodium citrate buffer/0.1% sodium dodecyl
sulfate (at room temperature) and twice for 5 min in 2% saline-sodium citrate buffer, 0.1%
sodium dodecyl sulfate (at 48oC). The membranes are washed with washing buffer. At room
temperature, they are blocked with blocking buffer by incubation for 30 min and again
incubated (at the same conditions as above) with blocking buffer containing a polyclonal
antibody against digoxigenin conjugated to alkaline phosphate. Then washing is done twice
with washing buffer for 15 min. The membranes are rinsed with detection buffer (1 M Tris-
Cl. pH 9.5, 0.1 M NaCl) for 5 min and immersed in CDP star solution for 5 min. Membranes
are exposed to Kodak XAR film for 15 min to 1 h.70 The mRNA level is determined by using
Peak Fit software.
164 Drug Screening Methods

Radioimmunoassay for Measurement of Plasma Leptin

Procedure
The peptide VPIQKVQDDTKTLIKTIVT represents the first twenty amino acids of the predicted
sequence of mature leptin protein (leptin 1-20), which is synthesized with 9-fluorenyl methyl
oxycarbonyl-(Fmoc)-protected L-amino acids on an applied biosystems peptide synthesizer
and purified by HPLC on a Dynamax column developed with a 0.1% trifluoroacetic acid (TFA)/
H2O/acetonitrile (8-40%) gradient. By using carbodiimide as coupling reagent the peptide is
conjugated to thyroglobulin. Rabbits are immunized by injections given intradermally and
boosted at 4 weeks intervals.
A synthetic peptide identical to the one used for antibody generation, except for a tyrosine
residue added to the C-terminal end, is labelled with 125I using the oxidizing agent iodogen and
purified on HPLC. For radioimmunoassay, the buffer consists of Tris-HCl 1 M, pH 7.4; 0.1%
Triton X-100, and 0.01% NaN3. Unlabelled ob peptide is used as standard. 100 µl of standard
or unknown, 200 µl buffer, 100 µl 125I–leptin peptide (approximately 5000 cpm) is added to
polystyrene tubes and incubation is performed for 48 h at 4oC. Separation of antibody-bound
and unbound peptides is done by addition of goat-rabbit immunoglobulin antibody followed
by centrifugation.
The precipitates are counted on a computer-linked gamma-counter and the leptin
concentrations of the samples are recorded using the “RIA-Calc” program.71,72

Isolated Adipocytes Cell-lines for Leptin and Leptin m-RNA

Rat Preadipocytes
Epididymal fat pad is removed after killing the rats by decapitation. The fat is minced and
digested with collegenase [1 mg/ml in Hanks balanced salt solution]. Stromalvascular cells
from adipose tissue are isolated by centrifugation at 800 g for 10 min and plated in 100 mm
culture in Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 100 IU/ml penicillin
and 100 µg/ml streptomycin (Basal medium). After 12 h, the adherent cells are washed 3 times
with Hanks balanced salt solution, treated with trypsin and counted. Cells are plated for 12 h
before replating to permit separation of the preadipocytes from other cell types. 95% cells
isolated by this procedure are preadipocytes.73

Rat Primary Cultured Mature Adipocytes

Male Sprague-Dawley rats weighing 180–200 g are killed by decapitation. The epididymal fat
pads are removed, minced and digested with collagenase1. Cell preparation is performed
at 37oC in Krebs-Ringer bicarbonate HEPES buffer (pH 7.4) containing 10 mmol/L NaHCO3,
30 mmol/L HEPES and 1% bovine serum albumin. Incubations are performed at 37oC in
DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 5% bovine serum albumin,
glutamine, 200 mmol/L adenosine, 50 µg/ml gentamicin and 100 mg/dl glucose. Isolated
adipose cells are distributed equally into plastic dishes to a final incubation volume of 10 ml.
Using these primary cultured mature adipocytes, leptin protein level and leptin m-RNA level
can be measured.74
 Antiobesity Agents 165

3T3-L1 Adipocytes
3T3-L1 adipocytes are derived from mouse fibroblast 3T3 line. 3T3-L1 cells are cultured in basal
medium (Dulbecco’s Modified Eagle’s Medium) (DMEM), fetal bovine serum (10%), penicillin
(100 units/ml and streptomycin 100 µg/ml) for 2 days post-confluence. To induce differentiation
cells are exposed to basal medium supplemented with MDI [methylisobutylxanthine (120 µg/
ml), dexamethasone (0.39 µg/ml) and insulin (10 µg/ml)]. Cells are washed 2 days later and
exposed to insulin (2.5 µg/ml) with basal medium.75

Human Mesenchymal Stem Cells (hMSCs): New Model for

the Study of Human Adipogenesis
Janderov et al. evaluated human mesenchymal stem cells (hMSCs) as an in vitro model for
human adipogenesis. hMSCs can be differentiated into adipocytes by exposure to insulin,
dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine three times for three days
each. hMSCs differentiated into adipocytes to a different extent depending on the experimental
conditions. Differentiation medium based on medium 199 and containing 170 nM insulin, 0.5
mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin, 1 mM dexamethasone, and 5% fetal
bovine serum was optimal. The gene expression during adipogenic conversion is assessed by
reverse-transcription polymerase chain reaction, real-time reverse-transcription polymerase
chain reaction, and Western blotting.76

CONCLUSION
The development of animal models for obesity is important in the development of treatments
for obesity. The mouse is an ideal model because it has similar genetics and development as
humans and genetic manipulation techniques are now routine.
Obesity is a particularly challenging medical condition to treat because of its complex
etiology involving behavior, energy expenditure, and genetics. No single animal model could
be able to represent all these factors. However, research using rodent models of obesity has led
to a significant expansion in our knowledge of the physiological mechanisms of this disease.
Various animal models have been developed for better understanding of the anatomical,
neurochemical and endocrine systems regulating food intake and energy expenditure.
Behavioral and environmental factors are represented in animal models of dietary obesity.
Hence, dietary obesity model could be more appropriate model for human obesity. As far
as genetic models are concerned, they are useful in providing tools to study the genetics of
obesity because identification of the genes involved in rodent genetic obesity has implications
for understanding the genetics of human obesity. Human geneticists have used the candidate
gene approach, using known genes identified from their obesity effects on animal models, in
the search for potential human obesity genes. Other genetic mutations in candidate genes
have been found in obese humans, including mutations in the genes encoding leptin, leptin
receptor, and carboxypeptidase E. However, the frequency in obese humans exhibiting these
single genetic mutations is exceedingly low, and therefore, are not suspected to be the key
genetic factors responsible for the most common forms of obesity.
166 Drug Screening Methods

The genetic alterations leading to obesity appear to be much more complex than single-
gene mutations, since few cases of single-gene mutations in obese human subjects have
been identified. Many individuals may have a combination of genetic alterations, which may
predispose them to obesity.
Since, human obesity is polygenic having significant genetic heterogeneity, therefore
polygenic models are known to be good models to characterize genetic mechanisms of
complex human obesity. Genetic models have revealed several genes involved in human
obesity that help us providing new therapeutic targets for obesity treatment. For example:
leptin, leptin receptor, α-melanocyte stimulating hormone, melanocortin 4-receptor, Agouti-
related protein, TUB, neuropeptide Y receptor, β3adrenoreceptor, uncoupling proteins that are
being extensively studied and being evaluated for their role in the treatment of obesity.
Recombinant leptin is also being developed as both an appetite suppressant and a mobilizer
of fat mass. This is a powerful approach, since this therapy has the potential to decrease
appetite, increase metabolic rate, and reduce body fat levels. In the future, small molecule
therapeutics may be developed as leptin receptor stimulators (agonists). These therapeutics
may be more effective in combating obesity, since obese human subjects have decreased
receptor responsiveness to leptin, despite having hyperleptinemia.77 It will be important to
identify the molecular determinants causing leptin insensitivity in order to identify future drug
targets.

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 CHAPTER

10
Anticancer Agents
INTRODUCTION
Cancer is a disease characterized by uncontrolled proliferation of cells that have transformed
from the normal cells of the body. On a worldwide basis cancer represents the single largest
cause of death both in men and women. Oral cavity cancer is amongst the most prevalent
cancers worldwide and incidence rates are higher in men than women.1 Apart from this, other
cancers burden is also very high. As per latest press release of WHO (International Agency for
Research on Cancer),2 with 528,000 new cases every year, cervical cancer is the fourth most
common cancer affecting women worldwide, after breast, colorectal, and lung cancers with
almost 70% of the global burden falling in areas with lower levels of development, and more
than one-fifth of all new cases are diagnosed in India.
There are more than 100 different types of cancer.3 It is a multifactorial disease, the biology
of which is not yet fully understood. However, the induction of proto-oncogenes and inhibition
of tumor suppressor genes has been implicated in the pathogenesis of cancer. Apart from this,
angiogenesis plays an important role in the pathogenesis of cancer and is a common target
for most chemopreventive agents. Angiogenesis is a highly coordinated process regulated by
a variety of molecules. Vascular endothelial growth factor (VEGF) is the major regulator of
tumor associated angiogenesis in lung adenocarcinoma, responsible for promoting tumor
growth and metastasis.4 VEGF-A is considered to be the most prominent angiogenic factor in
human lung cancer. The over expression of pituitary tumor-transforming gene-1 (PTTG1) has
also been reported in a variety of tumors.5 Down regulation of Forkhead transcription factor
(FOXO1) has been associated with promotion of cell proliferation in cervical cancer.6 As a
result of continuous research newer molecular targets are being identified and it has facilitated
the anticancer drug development process.
The cancer cells can invade the adjacent and distant tissues via the circulation. In advanced
stages cancer patient may die as a result of either improper diagnosis and treatment or
treatment failure. One of the causes of treatment failure is the development of resistance to
anticancer agents. The mechanisms of cellular resistance to anticancer agents have been dealt
in detail by Kruh et al. 20037 and therefore not explained here. Cancer is one of the thrust
area for which effective drugs at affordable prices are not available as yet probably due to lack
in understanding the cancer pathophysiology. For such a dreadful disease, anticancer drugs
have been developed from a variety of sources ranging from natural products (plants and
 Anticancer Agents 171

microbes) to synthetic molecules. Over the past decade, drugs for cancer have become a large
therapeutic market, third only after central nervous system and cardiovascular drugs, and it is
continuously growing. The number of blockbuster anticancer drugs with sales of $1 billion or
more increased from 19 in 2007 to 24 in 2008.8
However, the widely used drugs that are cancer chemotherapeutic agents suffer from
drawback of high toxicity such as bone marrow suppression, alopecia, nausea and vomiting
and are not within the reach of a common man. Therefore, the challenging task at this moment
is to identify the quick and novel methods that can identify and develop, which can be of
therapeutic value in human cancers. This urgently necessitates screening of a large number of
compounds. For this purpose both, the in vitro and in vivo models are employed for systematic
screening of an anticancer drug. In this chapter, screening methods for anticancer drug
discovery are described with a focus on their strength and limitations.

IN VITRO METHODS
Though animal models provide more predictable results, in vitro testing is still preferred prior
to in vivo testing of a potential chemotherapeutic agent. There are following advantages of in
vitro models over in vivo models.
1. These are less time consuming.
2. More cost effective.
3. Small quantities of, and large number of compounds can be tested.
4. These are easier to manage.
In addition, in vitro cultures can be cultivated under a controlled environment (pH,
temperature, humidity, oxygen/carbon dioxide balance, etc.) resulting in homogenous
batches of cells and thus minimizing experimental errors.
The in vitro methods are not free from disadvantages also and they often furnish false
positive results (compounds show no activity in vivo) and false negative results (compounds
show no activity in vitro but show activity in vivo as they need to be biotransformed in vivo to a
pharmacologically active compound).4 A second pitfall is that the role of pharmacokinetics in
determining drug effects cannot be evaluated in vitro. In addition, geometry of solid tumors in
vivo is very different from that of cells growing in vitro in suspension or monolayer cultures.

Ideal Characteristics of an in Vitro Screening Method

An ideal in vitro screening method should be simple, economical, reproducible, rapid and
sensitive. The assay should be applicable to large number of tumor types and test compounds.
The choice of the cell lines should be representative of clinical situation as close as possible.
The range of drug concentrations used in vitro should be comparable to that expected for in
vivo treatment. The assay should be able to process a large number of samples quickly and in
an automated fashion. Data acquisition should be simple, easily interpreted and applied. At
present no such system is available. Even the most extensively studied assays will more often
identify agents that will not work in an individual patient. However, the chemosensitivity assays
contribute to an active area of research and are routinely used for the screening of anticancer
drugs.9
172 Drug Screening Methods

The goal of a screening assay is to test the ability of a compound to kill cells, at the same
time, the assay should be able to discriminate between replicating cells and non-replicating
cells (quiescent cells that are dead or dying (apoptosis). Different assays take advantage of
various properties of cells as mentioned below.

S. No. Cell properties Assay

1. Enzymatic properties Tetrazolium salt assay (MTT)
2. Protein content/synthesis Sulphorhodamine B assay
3. DNA content/synthesis 3
H-Thymidine uptake Newer fluorescent analogues
with flow cytometry
4. Membrane integrity Dye exclusion tests
5. Clonogenic properties Clonogenic assay
6. Cell division Cell counting assay

Tetrazolium Salt Assay (Microculture Tetrazolium Test or MTT)

MTT assay is an internationally accepted in vitro method for anticancer drug screening. Though
viable cells can be measured using several other staining procedures also but these procedures
suffer from drawbacks that they require washing steps thereby increasing processing time and
sample variation. The multiwell plate scanning spectrophotometers can quickly measure large
number of samples with a high degree of precision and accuracy. Ideally, a colorimetric assay
for living cells should utilize a colorless substrate that is modified to a colored product by any
living cell, but not by non-viable or dead cells or culture medium. However, the MTT assay
utilizes a color reaction as a measure of viable cells.10 The assay is dependent on the cellular
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazolium salt
to a blue formazan product by the mitochondrial dehydrogenase of viable cells/metabolically
active cells. The intensity of blue colored formazan produced is directly proportional to the cell
viability.
The cells from a particular cell line when in log phase of growth are trypsinized, counted
in a hemocytometer and adjusted to appropriate density in a suitable medium and then
inoculated in different multiwell plates (usually 96-well plates). The cells are treated with
various concentrations (in replicates) of drugs for specified duration (usually 1 to 4 days),
after which MTT dye is added in each well and plates are incubated at 37°C for 4 h in a CO2
incubator. The plates are then taken out of incubator and dark blue colored formazan crystals
are thoroughly dissolved in isopropanol/DMSO at room temperature. The plates are then read
on an ELISA Reader at 570 nm. The percent cell viability with respect to control is calculated
using the formula:

% Cell viability = OD of treated cells × 100

OD of control cells
This assay has been successfully used by us.11 The DMSO as a solvent rapidly solubilizes
the serum as well as formazan and use of spectrophotometric grade DMSO gives stable
“background” absorbance levels. Other solvents like isopropanol, propanol, hexane and
dimethylformamide though used, do not solubilize serum at concentrations exceeding
0.0625 %.12
 Anticancer Agents 173

Figure 10.1: Dose response curve

The advantage of this assay is that it can be run on microtiter dishes on hundreds of cell
samples at one time so that the various drug concentrations can be used to get an idea of the
dose response relationship for each drug tested. As a result, this assay can be adopted for the
determination of IC50 of drugs (concentration of drug required to inhibit 50% cell growth)
(Fig. 10.1).
Further this assay is relatively simple and therefore easy to perform. It can be used for
both adherent and suspension cell lines. This method is cheap, requires low number of cells,
manageable and a large number of drugs can be quickly screened for antiproliferative activity.
However, the assay suffers from the drawback of giving false results due to the inclusion of cells
that might be metabolically active but not capable of dividing (nonreplicating). In addition,
drugs whose mechanism of action might spare mitochondria may not yield positive results in
this assay, especially for short incubation times. Also, use of DMSO warrants safe handling by
laboratory personnel.

Sulphorhodamine B Assay
The sulphorhodamine B (SRB) assay measures whole-culture protein content, which should
be proportional to the cell number.13 Cell cultures are stained with a protein staining dye, SRB.
SRB is a bright pink anionic dye that binds to basic amino acids of cells. Unbound dye is then
removed by washing with acetic acid, and protein-bound dye extracted using unbuffered. Tris
base for determination of optical density in a computer-interfaced, 96-well microtiter plate
reader. Since, dead cells either lyse or are lost during the procedure, the amount of SRB binding
is proportional to the number of live cells left in a culture after drug. This assay can be used
to measure the cellular protein content of both adherent and suspension cultures. Screening
capacity, reproducibility and quality control all appear to be enhanced in this assay relative
to the tetrazolium salt assays. The assay is more cumbersome and time consuming compared
174 Drug Screening Methods

to the MTT assay. Non-replicating and dead cells might contribute to the total protein and
interfere with the results.11

H-thymidine Uptake Assay

3

In this assay, tumor cell suspensions are exposed to the drug continuously for 5 days, after
which a radio-labeled precursor (3H-thymidine) is added during the final 48 hours of the
assay to label proliferating cells. The replicating cells will incorporate [3H]-thymidine into
their DNA, which can then be determined either by autoradiography or by liquid scintillation
counting. Autoradiographic determination of the [3H]-thymidine, though, is time-consuming
but it provides information on tumor growth kinetics.14 This can generate DNA histograms,
which can provide information on the ploidy status of the cells. This assay looks at cells, which
have actively replicating DNA and hence are viable. Non-replicating or dead cells will not be
counted in this case. The assay can be used for both adherent and suspension cell- lines. The
assay suffers from the drawback of using radioactivity and being labor intensive. This assay
is rapid, relatively inexpensive, and feasible in the majority of tumor types. However, it will
not differentiate between malignant and nonmalignant cells and might lead to false-negative
predictions if lethally damaged cells undergo a final division.

Fluorescence
Fluorescent dyes may be used in conjunction with microscopic evaluation methods as an in
vitro chemosensitivity assay.15 Cells are exposed to fluorescent-labeled precursors after drug-
exposure. The replicating cells will incorporate labeled precursor into their DNA and the
resulting fluorescence is then measured by flow cytometry. This assay also looks at actively
replicating cells and hence dead or nonreplicating cells are not counted. In addition, the assay
does not involve the use of radioactivity and is useful for adherent and suspension cell-lines.
Also using flow cytometry it is possible to determine that in what phase of the cell cycle the
cells are. The quantitation of apoptotic cells is also possible. However this, assay requires
the data to be analyzed by an expensive and sophisticated fluorescence activated cell-sorter
(FACS) instrument. Because of technical difficulties in applying flow cytometry to primary
tumor specimens, data on the predictive value for clinical response for this assay are too scarce
to permit definitive conclusions.

Dye Exclusion Tests

Early attempts to use exclusion of vital dyes like trypan blue, eosin, or nigrosin to predict
chemosensitivity were unsuccessful. These assays relied on the structural integrity of the
cells. Dead cells would have lost membrane integrity and hence would take up vital dyes
like trypan blue. This method was mainly used because of its technical simplicity and ease
of handling a number of specimens.16 More recently, Weisenthal and colleagues have used a
novel combination of fast green dye and eosin-hematoxylin [called the differential staining
cytotoxicity (DiSC) assay] with more promising results, particularly in patients with hematologic
malignancies such as chronic lymphocytic leukemia (CLL).17-19 No prospective trials of these
assays have yet been performed, however, to demonstrate their ability to predict for response
or lack of response. The DiSC assay is drug sensitive assay, which relies on structural integrity
 Anticancer Agents 175

of the cells. In this assay, cells are incubated with drugs for 4 days. Dead cells are stained in
suspension with fast green dye with or without nigrosin. The specimen is centrifuged and disks
of cells are collected in the microscopic slides. Live cells are then stained with hematoxylin-
eosin. As control duck erythrocytes are used. The end point of the study is the morphologic
identification of tumor-cell cytotoxicity compared with the internal control standard of duck
erythrocytes. The DiSC assay measures cell kill in both dividing and nondividing tumor cell
population.

Clonogenic Assays
A concern in the use of antiproliferative assays is that they measure growth inhibition rather
than cell killing. This is particularly important for drugs that act by arresting cells at checkpoints
in the cells cycle. Checkpoint arrest is a survival response of cells that allows repair of DNA
damage and is therefore not directly related to the induction of cell death. Thus, cells that act by
arresting cells at checkpoints may show lower IC50 but increased survival. Clonogenic survival
assays on the other hand, measure loss of tumor cell reproductive viability (i.e. the ability of
a single cell to form colonies). It is the most direct method of measuring cytotoxic activity of
a drug. In clonogenic assays single-cell suspension are prepared from tumor biopsies and
exposed to anticancer agents to be tested. Cells are then rinsed and plated in a semisolid
medium (agar or methyl cellulose), a medium that precludes proliferation of nonmalignant
cells in the specimen.20 After 14 to 28 days, some cells will have undergone several divisions
and will have formed tumor colonies, which can be quantified in a visual or semi-automated
fashion. Non-replicating and dead cells are not counted in this case. The number of colonies
from the treated cells is compared with the number of colonies from the untreated control cells
and the fraction of control growth provides an index of drug activity. Traditional clonogenic
systems suffer from a number of significant technical problems like long incubation time (at
least 14 days) before results can be made available to the clinician. The assay is labor-intensive,
costly, and cannot be used for suspension cell-lines.

Cell Counting Assay

Cells are cultured in the presence of drug for 2-5 culture-doubling times, after which the cell
number is estimated using a hemocytometer or a cell counter. The assay is easy to perform,
rapid and can be used for both adherent and suspension cell lines. However, dead and
non-replicating cells can be counted in this assay by the cell counter. The IC50 values can be
calculated in all the above assays.

3D Tumor Models
3D in vitro models have revolutionized cancer research in recent years due to its biomimetic
property and the ability to accurately depict the in vivo situation for drug screening. 3D models
are advantageous over the complexity of animal models and the spatial limitations of the cell
culture models.
In 3D cancer models appropriate matrix components found in vivo can be obtained.
Cancer cells can be cocultured in a spatially relevant manner with endothelial cells and other
cells associated with the in vivo scenario. It makes it possible to monitor and control the
176 Drug Screening Methods

oxygen levels to mimic the levels of angiogenic factors released by cancer cells in response
to hypoxia in native tumors. 3D models provide promising in vitro platform for aggregation
and clustering of cancer cells, migration and proliferation, release of angiogenic factors and
formation of hypoxia within tumor masses which assist in preclinical evaluation of the efficacy
and molecular mechanism of the anticancer drugs.21
An automated assay for 3D models, such as SpheroChip system has been developed by
Kwapiszewska et al. (2014).22 It is a relatively new microfluidic-based platform for long-term
3D cell culture and analysis compatible with commercially available microplate readers.
This chip provides a continuous in situ monitoring of cultured tumor spheroids cultured
on a chip where the dynamic changes in the metabolic activity of the cells can be observed
after subsequent drug doses. They compared the penetration of doxorubicin, quantum dots,
and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-
dimensional culturing system.
Ma et al. (2012)23 developed a flexible and highly reproducible method using three-
dimensional (3D) multicellular tumor spheroids derived from HeLa cell to quantify
chemotherapeutic and nanoparticle penetration properties in vitro. They compared the
penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid
versus HeLa cells grown in a traditional two-dimensional culturing system. Their data revealed
that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular
characteristics (such as resistance to chemotherapeutics) often found in human solid tumors
which could not be captured using conventional two-dimensional cell culture techniques.
The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid
screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle
delivery into solid tumors.

4D Tumor Models
In a more recent research, Mishra et al. (2014)24 developed an ex vivo lung cancer model (four
dimensional, 4D) that forms perfusable tumor nodules on a lung matrix mimicking human
lung cancer histopathology and protease secretion pattern. In their study they compared the
gene expression profile (Human One Array v5 chip) of A549 cells, a human lung cancer cell
line, grown in a petri dish (two-dimensional, 2D), and of the same cells grown in the matrix
of ex vivo model (4D). They also compared the 3D expression profile with that of 4D. Gene
ontology (GO) analysis showed up-regulation of several genes associated with extracellular
matrix, polarity and cell fate and development. The ex vivo 4D model may be a good mimic of
natural progression of tumor growth in lung cancer patients with larger tumors having worse
rate of survival.

National Cancer Institute’s in Vitro Screening Program

There are nine broad categories of cancer cells covered in NCI-60, a panel of 60 diverse human
cancer cell lines. The drugs are screened against this diverse panel of cell lines, including lung,
colon, CNS, leukemia, pancreatic, melanoma, prostate, ovarian, cancer of breast and kidney
cancer cell lines, at five different doses and allowed to incubate for 48 hours.25 Drug-resistant
tumors are specifically included in the screen. These include the human breast carcinoma
 Anticancer Agents 177

selected for multiple drug resistance (MDR) and P-388 murine leukemia resistant to natural
products, both of which potentially provide additional identification of new agents with
particular activity against potentially resistant tumors.26,27 As many as 200 compounds per
week, or 10,000 per year can be tested in the screening facilities. National Cancer Institute
(NCI) has planned to implement full-scale screen with a capacity for testing new substances at
a rate > 10,000 per year against a broadly representative panel of 100 or more human tumor cell
lines.28 If the drug is unique in some way-kills preferentially one or more of the tumor cell lines,
has unique structure or mechanism of action, or can kill tumors at a very small concentration,
testing will proceed to the next stage. About 2% of those screened will be recommended for the
next stage of testing in mice. Blower et al. 2007 have studied the MicroRNA expression profiles
for NCI-60 cancer cell panel and have incorporated the resulting data into the CellMiner
program package for integrative analysis.29 They showed that cell line groupings based on
microRNA expression were generally consistent with tissue type and with cell line clustering
based on microRNA expression. However, mRNA expression seemed to be somewhat more
informative among tissue types than was microRNA expression.
In addition, there did not seem to be a significant correlation between microRNA expression
patterns and those of known target transcripts. Comparison of microRNA expression patterns
and compound-potency patterns showed significant correlations suggesting that microRNAs
might play a role in chemoresistance. These investigators have suggested that combined with
gene expression and other biological data using multivariate analysis, microRNA expression
profiles may provide a critical link for understanding mechanisms involved in chemosensitivity
and chemoresistance.

IN VIVO METHODS
In vivo models are advantageous over in vitro models in the sense that they detect host-
mediated activity, are relatively predictable and estimate therapeutic ratio. However, as
compared with in vitro systems, their sensitivity is low, are costly, time consuming and large
number of samples cannot be handled and are difficult to manage. After all, animal models are
used both for toxicological studies and for detecting preclinical anticancer efficacy. They are
able to detect agents irrespective of their mechanism of action. The drugs with high degree of
efficacy and broad spectrum of activity in animal models are usually expected to be effective
in clinical cancer, however, there are exceptions also30 which could be due to metabolic
differences and heterogeneity of cancer cells between human and rodents. Despite these
differences animal models are widely used to support the results obtained from in vitro studies.
The most promising candidate compound is tested in more than one animal model. Dose
response relationship, combined effect of drugs, modes of their anticancer action and organ
specificity are established. Varied drug dosage forms, doses and animal strains and animals
of a particular age group may be used. The selected animal models should be representative
of high incidence of human cancers.31 The in vivo anticancer drug screening methods are
described under the following headings:
A. Chemically induced tumor models
B. Models involving cell line/tumor pieces implantation.
178 Drug Screening Methods

Chemically Induced Tumor Models

Chemical carcinogens are well-known to account for about 80% of all cancers and are
used to induce cancer in animal models. Carcinogens require metabolic activation before
inducing carcinogenesis. The epidemiological studies indicate that human carcinogenesis
occurs through multiple steps in the same way as in mouse skin. The concept of multistep
carcinogenesis was first of all developed in rodent skin models in 1940s and applies to cancers
to many species and cell types. Experimental carcinogenesis involves following three steps:
i. Initiation is due to exposure to carcinogens transforming the normal cell to a cancer cell.
ii. Promotion is due to the triggering of uncontrolled growth of the transformed cell.
iii. Malignant conversion is caused due to unlodging of cancer cells from the original site, its
transportation by circulation and the establishment of secondary tumors in the body.
The exact sequence of cellular, biochemical and molecular genetic events may differ
between tissues and species, the overall concept seems to be directly applicable to clinical
cancer and thus in future multistage mouse skin carcinogenesis model will be of immense
utility for further understanding the mechanisms of epithelial carcinomas in human beings.32
The with over one-half of the chemotherapeutic drugs currently in use. This institute is
systematically experiment is well designed; dose of the carcinogen as well as drug treatment
schedule is standardized by conducting pilot studies. This helps in accurate evaluation of the
test compound.

National Cancer Institute’s in Vivo Screening Program

National Cancer Institute (NCI) is playing an active role in the development of anticancer drugs
for last over 50 years with over one-half of the chemotherapeutic drugs currently in use. This
institute is systematically screening a large number of compounds for anticancer activity using
in vitro (cancer cells grown in culture dishes in the laboratory) and in vitro systems (animal
models).
About 80,000 compounds have been screened by NCI since 1990. The chemoprevention
branch of NCI has been using the following chemically induced tumor models, which represent
high incidence of human cancers.31

DMBA-induced Mouse Skin Papillomas

This is a classical two-stage experimental carcinogenesis model. Mouse skin is generally most
sensitive to epidermal carcinogenesis. Rats, hamsters and rabbits are less sensitive and guinea
pig is very resistant.32 SENCAR mice are highly sensitive to DMBA-induced skin tumors. Swiss
albino mice are relatively less susceptible to tumor induction. DMBA acts as an initiator and
12-O-tetradecanoyl-phorbol-13-acetate (TPA) is used as a promoter to induce skin papillomas
and squamous cell carcinomas. Mice are topically applied a single dose of 2.5 µg DMBA in
acetone on the shaved back, followed by 5-10 µg of TPA in 0.2 ml acetone twice weekly on the
same site starting one week after DMBA application. Papillomas begin to appear after 6 to 7
weeks of application of TPA. Weekly observations are made to monitor tumor development
till the experiment terminates after 18 weeks. Percent tumor incidence and multiplicity of
treatment group is compared with DMBA control group. Drug under test can be administered
either topically or by oral route. The tumor incidence in this model is usually about 100% in
 Anticancer Agents 179

Figure 10.2: DMBA treated Swiss albino mouse (24th week) with papillomas. 100 nmol DMBA/100 µl acetone was
applied topically on the depilated back of mouse twice weekly for 8 weeks

DMBA controls. In various laboratories, however, repeated topical application of DMBA alone
has also been shown to induce carcinogenesis.33,34 The development of papillomas in DMBA
treated Swiss albino mouse (24th week) is represented in Figure 10.2.

N-methyl, N-nitrosourea (MNU)-induced Rat Mammary Gland Carcinogenesis

MNU induces hormone dependent tumors. In MNU-induced rat mammary gland cancer
model, single intravenous injection of 50 mg/kg body weight of MNU (pH 5.0) is given to
Sprague-Dawley rats, usually at 50 days of age. In some tests, carcinogen has been administered
to 120 days old animals (a better model). The incidence of tumors produced in this model is
75-95% within 180 days post-carcinogen. MNU-induced tumors are invasive and predominantly
adenocarcinomas. Because MNU does not require metabolic activation, this model cannot
detect inhibition of carcinogen activation. This model is a better model of human breast
cancer. Drug efficacy is measured as percent reduction in adenoma incidence, multiplicity or
percent increase in adenocarcinoma latency compared with that of carcinogen control. The
usual tumor multiplicity ranges from 2-4 in this model. Tumor latency is about 65 to 80 days.

DMBA-induced Rat Mammary Gland Carcinogenesis

In DMBA-induced model, female Sprague-Dawley rats are given single intragastric injection
of 12 mg/kg DMBA at 50 days of age. This dose results in 80-100% incidence of total mammary
tumors (adenocarcinoma, adenoma and fibroadenoma) within 120 days post-carcinogen.
This model can detect the agents/drugs inhibiting carcinogen activation (for example, those
inhibiting cytochrome P-450).
DMBA produces encapsulated tumors with high incidence (adenomas and fibroadenomas).
In addition, tumors are associated with activated ras gene. Drug efficacy is measured as percent
reduction in adenoma incidence, multiplicity or percent increase in adenocarcinoma latency
compared with that of carcinogen control. The usual tumor multiplicity ranges from 3-4.4 in
DMBA controls. Tumor latency is about 65 to 80 days.
180 Drug Screening Methods

Mechanism of mammary tumorigenesis: The molecular mechanism of mammary tumori­

genesis is not yet fully understood. Currier et al. 2005 have examined oncogenic signaling
pathways that are activated in mammary tumors in mice treated with the DMBA.35 In female
FVB mice given 6 doses of 1 mg of DMBA by weekly gavage beginning at 5 weeks of age, all of
the mice developed tumors by 34 weeks of age (median 20 weeks after beginning DMBA); 75%
mice had mammary tumor, DMBA induced mammary tumors exhibited elevated expression
of the aryl hydrocarbon receptor (AhR), c-myc, Cyclin D1 and hyperphosphorylated
retinoblastoma (Rb) protein. The elements of Wnt signaling pathway, the NF-kB pathway
and the prolyl isomerase Pin-1 were found to be frequently upregulated in the tumors when
compared to normal mammary gland controls suggesting that environmental carcinogens can
produce long lasting alterations in growth and anti-apoptotic pathways, leading to mammary
tumorigenesis.
In another study, Wang et al. (2014)36 were able to induce stable breast premalignant lesions
in SD (Sprague-Dawley) rats by administration of DMBA (15 mg/kg, administered three times)
followed by administration of female hormones (estrogen and progesterone) 5-day cycle. The
premalignant breast lesions were confirmed by ultrasound and palpation.

MNU-induced Tracheal Squamous Cell Carcinoma in Hamster

In this model, 5% solution of MNU in normal saline is administered once a week for 15 weeks
using specially designed catheter, which exposes a defined area of the trachea of male Syrian
golden hamsters to the carcinogen. Fifteen weeks MNU administration produces tumors in
40-50% animals within 6 months. Test drug efficacy is measured as percentage reduction of
tumor incidence compared with carcinogen control.

MNU-induced Prostate Cancer in Gerbils

Goncalves et al (2013)37 developed a new rodent model of chemically induced prostate
carcinogenesis in which prostate cancer progression occurs differentially in the dorsolateral
(DL) and ventral lobes (VL). In their study adult gerbils were treated with MNU alone or
associated with testosterone for 3 or 6 months of treatment. DL developed tumors exclusively
in the periurethral area and showed intense AR, PCNA (proliferating cell nuclear antigen), and
MGMT immunostaining. Moreover, VL lesions emerged throughout the entire lobe. MNU-
induced lesions presented markers indicative of an aggressive phenotype: lack of basal cells,
rupture of the smooth muscle cell layer, loss of E-cadherin, and high MGMT staining. The
investigators propose this as a good model for prostate cancer as it allowed the investigation of
advanced steps of carcinogenesis with shorter latency periods in both lobes.

N, N-Diethylnitrosamine (DEN)-induced Lung Adenocarcinoma in Hamster

In this model, 17.8 mg DEN/kg body weight twice weekly by subcutaneous injection for 20
weeks starting at age 7 to 8 weeks usually produces tracheal tumors in 90-100% and lung tumors
in 40-50% of male Syrian hamsters. The studies have shown that lung tumors originate from
pulmonary Clara and endocrine cells while the tracheal tumors are derived from the basal
cells of the respiratory epithelium. The percentage reduction in tumor incidence in treatment
group is compared with that of control group animals.
 Anticancer Agents 181

1,2-Dimethylhydralazine (DMH)-induced Colorectal Adenocarcinoma in Rat

and Mouse
Intraperitoneal injection of DMH, a procarcinogen, produces colorectal adenocarcinoma
both in rats and mice. DMH is first activated to azoxymethane (AOM) and then to ultimate
carcinogen methylazoxymethanol (MAM). In rat model, a single subcutaneous dose of 30
mg/kg body weight given to 7-week-old F-344 male rats produces colon adenomas and
adenocarcinomas within 40 weeks. The total tumor incidence is approximately 70%. In the
mouse model, 9-11-week-old female CF1 mice are injected intraperitoneally MAM four times
in 11 days (low dose) and 8 times in 22 days (high dose). Colon tumors have been reported to
appear within 38 weeks after dosing.

Azoxymethane (AOM)-induced Aberrant Crypt Foci in Rat

Azoxymethane is a commonly used colon carcinogen in rodents inducing aberrant crypt foci.
Aberrant crypt foci are single and multiple colonic crypts containing cells exhibiting dysplasia.
These are potential precancerous lesions and are being evaluated as intermediate biomarkers
for colon cancers in rodents. These crypts foci can be produced by single injection of 30 mg
AOM/kg body weight in rats. Drug treatment schedules can vary depending on the selection.
At the end of the treatment, animals are sacrificed and frequency of aberrant crypt foci are
determined by histopathologic examination (larger size and increased stain uptake, increased
distance from luminal to basal surface of crypt cells and enlarged pericryptical zone compared
with normal crypts after staining with methylene blue.

N-Butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)-induced Bladder Carcinoma

in Mouse
OH-BBN induces urinary bladder invasive transitional cell carcinomas that are morphologically
similar to that of human variant of advanced urinary bladder transitional cell carcinomas. In
this model male BDF (C57 BL/6 × DBS/2-F1) mice are administered 8 weekly doses of 7.5 mg
OH-BBN by intragastric instillation beginning at 50 days of age. Drug effectiveness is measured
as percent reduction in incidence of transitional cell carcinoma compared with carcinogen
control group. This model induces about 40% tumor incidence during 180 days of experimental
period in control animals.

Other Models
Apart from animal models used at NCI, a few other important models of chemical carcinogenesis
are as follows.

DMBA-induced Oral Cancer in Hamster

Oral cancer can be induced in male Syrian hamsters by painting right buccal mucosa, 3
times/week for 16 weeks with 0.5% solution of DMBA in liquid paraffin (approximately 10 µl
containing 100 µg). Tumor size, number and tumor burden of drug treated animals can be
38
compared with that of control animals at the termination of the experiment.
182 Drug Screening Methods

DMBA Sustained release suture technique

Wami et al. (2001)39 induced squamous cell carcinoma in cheek pouch of Syrian hamsters using
DMBA sustained-release suture technique followed by application of a promoter (arecaidine).
In 80.6% of hamsters, squamous cell carcinoma reached a size of 100 mm2. The mortality was
observed in 15.8% and severe inflammation in 3.6% animals. This model is useful to study
upper aerodigestive tract tumors.

3-Methylcholanthrene-induced Fibrosarcoma Tumors in Mouse

3-Methylcholanthrene is also known as 20-Methylcholanthrene (MCA). In this model, single
dose of 200 µg of MCA/100 µl of DMSO is injected subcutaneously into the thigh region. Tumor
incidence occurs approximately 6 to 7 weeks after MCA injection and reaches approximately
90% within 15 weeks. Tumor growth delay and tumor volume of control animals are compared
with that of drug treated animals. Weekly fibrosarcoma incidence is also observed in both
treated and control animals during the 15-week experimental period.
MCA has been shown to produce 100% fibrosarcoma incidence within 15 weeks.40 Almost
similar incidence has been observed in our studies also.41 The MCA injected mice were
diagnosed as having fibrosarcoma in the subcutaneous tissue with focal areas of necrosis (Fig.
10.3).

Figure 10.3: 20-Methylcholanthrene induced fibrosarcoma in the subcutaneous tissue of mouse with focal areas of
necrosis (N) (H and E × 120). Mouse was injected 200 µg of MCA/100 µl DMSO into the thigh region subcutaneously and
sacrificed at the end of 15th week for histopathological analysis

3-Methylcholanthrene-induced Skin Tumors in Mouse

Skin tumors can be induced with 0.05 ml of 0.3% twice weekly topical application of MCA on the
shaved back of ddY mice till appearance of visible tumors. Tumor incidence and multiplicity
in drug treated and control group is observed during the 15 weeks period of the experiment.
Tumor incidence in this model has been found to be approximately 85%.42
 Anticancer Agents 183

Benzopyrene-induced Forestomach Tumors in Mouse

Benzopyrene, a tremendously potent carcinogen, occurs widely as an environmental pollutant.
The presence of methyl group constitutes the principal pathway of its metabolism, which is
similar to a number of other polycyclic hydrocarbons found in the environment.
Benzopyrene induces tumors when given by gavage to mice (1 mg of benzopyrene in
0.1 ml peanut oil) twice weekly for 4 weeks. Tumor incidence and tumor burden in drug
treated animals can be observed and compared with carcinogen control animals at the end
of the experiment. It has been shown to induce 100% incidence of fore stomach tumors in
benzopyrene control mice.33

Hepatocellular Carcinoma
Hepatocellular carcinoma (HCC) can be readily induced using various chemicals.43 There are
several animal models of HCC which are well established, including those which are naturally
occurring such as wood chucks infected with the wood chuck hepatitis virus and Long-
Evans cinnamon rats with a copper- storage condition analogous to Wilson’s disease 44,45 and
chemically-induced carcinogenesis.46 Wood chuck infected with wood chucks hepatitis virus
is the only reliable animal model of HCC44 however; these animals are difficult to maintain.
Di Bisceglie et al. (2005) have used male B6C3F1 mice and induced hepatocellular carcinoma
and associated lesions just by single intraperitonial dose of 120 µg/kg of ENU (Ethylnitrourea)
and killed the animals 60 weeks after injection of ENU.47
The advantages of using these animals are:
1. Maintenance is easy.
2. For the sake of consistency of results.
3. HCC is more common in males than females.
4. Model is suitable for screening chemopreventive agents (substantial incidence of HCC).
5. Long duration of study (60 weeks) comparable to human situation as HCC most often
develops after prolonged necroinflammation and fibrosis resulting from viral hepatitis or
non-viral liver disease.
Estrogen receptors have been demonstrated by Alagaratnam et al. 1987 in chemically
induced HCCs in rodents.48
Hepatocarcinogenesis in MDR2 knockout mice: It is a model of inflammation associated hepato-
cellular carcinoma. MDR-KO mice lack P-glycoprotein responsible for phosphatidylcholine
transport across the bile canalicular membrane. Absence of phospholipids from bile results in
bile regurgitation and portal inflammation followed by development of hepatocyte dysplasia
and hepatocellular carcinoma.49-51
Katzenellenbogen et al. (2006) have shown induction of antioxidant protection systems and
stimulation of hepatocyte DNA replication in the liver of MDR2-KO mice at the age of 3 months.
PCNA and cyclin D1 expression was highly increased. However, the hepatocyte mitotic activity
was blocked at this stage. In the later stage of the disease, although inflammation was less
prominent, and the total antioxidant capacity of liver tissue returned to normal level, mitotic
activity of hepatocyte was increased.52
184 Drug Screening Methods

High fat diet induced NAFLD/NASH model in mouse

Nakamura et al. (2013)53 have shown that long-term (60 weeks) high fat (HF) diet induced
obesity and insulin resistance in C57BL/6J male mice leading to non-alcoholic steatohepatitis
(NASH) caused hepatocellular carcinoma. The results showed the natural course of non-
alcoholic fatty liver disease (NAFLD)/NASH i.e. steatosis development in healthy livers, release
of cytokines triggering inflammatory process and oxidative stress which leads to hepatocellular
degeneration, fibrosis and tumorigenesis. This model also showed some atypical features as
compared to human NASH.

Pancreatic Cancer Models

Dramatic progress has been made over the last few years in the development of animal models
of pancreatic cancer, in particular the genetically engineered mutant mouse models (GEMs)
of exocrine pancreatic cancer. Thirteen GEMs of exocrine pancreatic dysplasia and neoplasia,
including a new model of chronic pancreatitis were presented by 11 investigators during a
3-day conference cosponsored by the National Cancer Institute Mouse Models of Human
Cancer Consortium and the Abramson Cancer Centre of the University of Pennsylvania, at
Philadelphia, Pennsylvania, in December 2004. The models were characterized histologically
and immunohistochemically. However, it is yet unproven whether GEMs will more closely
predict therapeutic efficacy in pancreatic cancer patients than the current FDA standard
of tumor xenografts. The infrastructural facilities similar to those necessary for the proper
conduct of investigational human clinical trials have also been suggested.54

Angiogenesis Assays
Various in vitro and in vivo angiogenesis assays used in anticancer drug research have been
reviewed recently by Phung and Dass (2006)55 involving testing on other transplantable tumors.
Some other cell lines which can be inoculated.

METHODS INVOLVING CELL LINE/TUMOR PIECES IMPLANTATION

1. If the specified number of cells of a particular cell line are inoculated into sensitive mouse
strain, tumors can be developed rapidly as compared to chemical carcinogen-induced
tumors and time can be saved using this model system. Usually, L-1210 and P-388 cell lines
are used. These cell lines are derived from mouse lymphocytic leukemia and have 100%
growth fraction and tumor implanted animal dies on reaching the tumor burden to 109 cells.
So, the death time of the animal implanted with specified number of L-1210 or P-388 cells
can be predicted. The effective drug would retard the tumor growth and increase the life
span of the animal. A drug, which prolongs the lifespan of the animal by 20%, is taken for
subsequent studies involving testing on other transplantable tumors. Some other cell lines
which can be inoculated to induce tumors are B-16 (melanoma), Lewis lung carcinoma
and sarcoma-180, etc. The host mouse strain for above type of cell lines is BDF1 except
Swiss for sarcoma-180. P-388 and L-1210 cell lines are inoculated intraperitoneally, B-16
as intraperitoneally and subcutaneously, Lewis lung carcinomas as intramuscularly and
sarcoma-180 as subcutaneously. The experiment takes about 10 days for completion.56
 Anticancer Agents 185

Mean survival time (MST) is calculated as:

MST of treated animals (T)
T/C % = × 100
MST of control animals (C)

For sarcoma 180 tumors, reduction of tumor size (tumor weight) is used to find out the
tumor inhibiting activity of solid tumors as follows:
Average tumor weight of the treated animals (T)
Tumor inhibiting activity (TIA) = × 100
Average tumor weight of the control animals (C)

2. Hollow-fiber technique: Small hollow fibers (tubes 1 millimeter in diameter and 2

centimeters long made of a plastic, polyvinylidene fluoride), containing cells from human
tumors are inserted underneath the skin and in the body cavity of the mouse. Each
candidate drug is administered at two dosages and is tested against 12 target tumor cells
in different hollow fibers. A total of about 20 compounds per week are screened by this
method. Compounds that retard the growth of the cells are recommended for the next level
of testing. The average length of this test is four days.
The assays using hollow fiber techniques have been optimized for human cancers
originating from the lung, breast, colon, ovary, brain gastric and hepatocellular cancer
mouse cell lines. The gastric and hepatocelluar cell lines have been found to be useful in
screening small molecules.57
3. Use of xenografts: Human tumors are injected directly below the skin of mice. Candidate
drugs, which have evidence of activity in the hollow fibers, are administered to the mice at
various dosages, and those compounds that kill or slowdown the growth of specific tumors
with minimal toxicity to the animal will proceed to the next stage of testing. The average
length of this test is about 30 days.

Spheroid culture of LuCaP 147-induced prostate cancer

Saar et al. (2014)58 for the first time developed a prostate cancer model by serially passaging
spheroid cultures of several LuCaP 003147 xenografts, demonstrating capabilities for high-
throughput drug screening and anticancer agents-induced cell cycle arrest and apoptosis
in spheroid cultures. Their study showed that cells formed tumors when re-introduced
into mice, providing an authentic in vitro-in vivo preclinical model of a subtype of
prostate cancer with a hypermutator phenotype and an SPOP mutation.

Integration free-induced pluripotent stem cells (iPSCs) model

Liu et al. (2014)59 derived an integration free-induced pluripotent stem cells (iPSCs) from an
FA (Fanconi anaemia) patient without genetic complementation and reported in situ gene
correction in FA-iPSCs and generated isogenic FANCA (Fanconi anaemia, complementation
group A)-deficient human embryonic stem cell (ESC) lines. This model can be used as a
drug-screening platform by identifying several compounds that improve hematopoietic
differentiation of FA-iPSCs.
4. Nude mouse model: Nude mice have been widely used to test the tumorigenicity of cells
or for testing of anticancer drugs. These mice are immunologically incompetent because of
absence of thymus. They neither show mitotic response in mixed lymphocyte reaction, nor
186 Drug Screening Methods

generate cytotoxic effector cell. Lack of helper T and suppressor T cells alters the antibody
response of the animals to antigen. They do not show contact sensitivity and do not reject the
transplanted material. They are required to be maintained under strictly sterile conditions
and in a warm environment (26-28°C). Some other points regarding their use are:
a. Certain tumors like melanomas and colon carcinomas grow very well in nude mice,
whereas prostate carcinomas and most types of leukemia grow very poorly.
b. Large numbers of cells, usually > 106 are required to be inoculated beneath the skin to get
a successful tumor take.
c. Metastases are rarely observed.
d. Overall maintenance is very expensive.
5. Newborn rat model: Newborn rat pups can be used for transplantation of tumors as an
alternate for nude mice because they are cost effective and their maintenance is easy.
Transplantable tumor cell line can be maintained with ease using rat pups. Rat pups are
especially useful to study neural tumors. As an example, 1 × 106 viable C6 glioma cells in
10 µl of phosphate buffered saline can be injected into the left side of the pup under sterile
conditions. The animals are checked for palpable tumors twice weekly. C6 glioma tumors
transplanted in Sprague-Dawley rats come to palpable stage within 15-20 days.60
6. Transgenic mouse model: Cancer is known to be a disease of genome and a large number
of human cancers arise from mutations in one or more oncogene or tumor suppressor
gene. Therefore, inactivation of a particular gene within specific tissues of adult mouse may
confer an excellent model of somatic mutational events characteristic of human cancer. The
genetically engineered mouse may serve both as a model of disease as well as a model for
possible gene therapy. Such mice can be generated either by pronuclear injection of DNA
or by gene targeting.61
Metamouse, a genetically engineered animal has got US patent in the recent past. In
this mouse, tumor pieces of patients are transplanted into the organ of primary growth,
in contrast to conventional models in which single cell suspension from tumor cell line is
injected underneath the skin of nude mice. In this particular mouse, metastasis and weight
loss occurs in the same way as that in humans. The conventional mouse models rarely
show metastasis (because metastasis either occurs very slowly or not at all). This limitation
contributes to the failure of so many drugs, which are active in such systems to become the
approved anticancer drugs. In words of Tetsuro Kubota, tumors require cell-to-cell contact
to grow. In suspension form tumor breaking enzymes are expected to destroy the cell
surface receptors mediating such cell-cell contact to grow. Certain human cancers, which
can develop well in metamouse, include liver, pancreas, head and neck, bladder, stomach,
ovarian, colon cancer and lymphomas. It is the only relevant model of mesothelioma.
The metastasis of breast and prostate cancer is very slow in this mouse. The important
indication of metamouse is to test new routes, doses and indications of old drugs. It can
serve as surrogate marker in cancer patient and prognosis of the patient is also possible.
The model is very useful to clinicians to make better therapeutic choice by first testing the
drugs on patients tumor grown in metamouse. The disadvantages of such studies are that
they require prolonged time.62
 Anticancer Agents 187

CONCLUSION AND FUTURE PERSPECTIVES

Basic research in cancer biology has provided new targets for cancer drug development and
has brought older targets into sharper focus, leading to new and novel approaches to cancer
prevention and treatment. The conventional methods of drug screening are continuously
being refined or replaced with newer methods and thereby accelerating the drug development.
Newer smart approaches are now being sought to screen potential anticancer drugs in the
postgenomic era. The trend is directed towards the application of robotics and miniaturization
to achieve high throughputs.63 The pharmaceutical companies are focusing their efforts
towards more rational screening of agents that target a specific predefined locus of action. In
cancer field for example, the efforts are being directed to identify molecular abnormalities,
which are responsible for cancer causation and progression. Of particular interest are signal
transduction pathways involved in cell proliferation, which are deregulated in most human
cancers.64 In addition, relevant genes that are mutated or aberrantly expressed in cancer
cells and their corresponding proteins targets are increasingly being realized as interesting
candidates. Some success in this area has been achieved with discovery of inhibitors of EGF-
receptor tyrosine kinase.65 3D and 4D models of personalized therapy as well as the genetically
engineered mouse models have revolutionized the cancer research and help understanding
the tumorgenesis process in a better way than earlier and thus facilitating the anticancer drug
discovery for clinical benefit. It is inevitable that in the years to come we will see high technology,
high-speed, high-volume and information-intensive approaches to the identification of novel
targets genes, proteins and drugs. However, the importance of basic research in the molecular
biology and pharmacotherapy of cancer remains critical.

ACKNOWLEDGMENT
The encouragement and guidance from Dr GN Singh, Indian Pharmacopoeia Commission,
Ghaziabad, during the course of preparation of this manuscript is gratefully acknowledged.
The efforts of Amandeep Bhatia in preparing the manuscript is acknowledged.

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 CHAPTER

11
Screening Methods for Renal
and Liver Fibrosis
INTRODUCTION
Fibrotic diseases are characterized by an increasing amount of connective tissue deposited in
the afflicted organ. Fibrosis can occur in a variety of organs including the liver, kidney, lung,
intestine, heart and skin. The underlying mechanism of fibrosis in all of these particular organs
involves the proliferation of mesenchymal cells that posses a myofibroblast—like phenotype
and the subsequent deposition of interstitial collagens and other extracellular matrix proteins
by these cells leading to progressive scarring and loss of organ function.1,2 In general, the
fibrotic process is a physiological response to chronic and persistent tissue injury and the
pattern shows similarities with the wound healing process, which occurs after acute damage
to an organ. In the current chapter, we will focus on fibrosis in the kidney and in the liver and
we will discuss the most widely used models for screening of experimental drugs.

THE PATHOGENESIS OF RENAL FIBROSIS

Renal fibrosis is a final common process of many chronic renal diseases. It is characterized
by over-deposition of the extracellular matrix, which eventually leads to the end-stage renal
disease.3 Several renal disorders such as diabetic nephropathy, chronic glomerulonephritis,
tubulointerstitial fibrosis and hypertensive nephrosclerosis can result into end-stage renal
disease.4 Nephrons, the major unit of kidney, comprise of two major parts, i.e. the glomerulus
and the tubulus. Renal fibrosis can be initiated through the injury either at glomerular
cells such as mesangial cells, basement membrane, podocytes or at tubular cells. In many
pathological conditions, systemically-produced immune complexes bind to glomerular cells
and cause inflammation called glomerulonephritis. Persistent occurrence of such events can
lead to glomerulosclerosis during which process scar tissues is formed.5
Proteinuria, i.e. excessive excretion of proteins in urine, is a hallmark of renal injury. After
a renal insult, either by immunologic or non-immunologic events, several molecules such as
albumin, immunoglobulins, complement factors, growth factors, angiotensin-II, cytokines
and high glucose concentrations filter through the glomerulus and subsequently activate renal
tubular cells.6 In addition, hypoxia, oxidative stress, and many other factors which are induced
during pathological conditions, stimulate pro-inflammatory and profibrotic pathways in
tubular cells. In response of these activating agents, tubular cells produce growth factors,
192 Drug Screening Methods

chemokines and adhesion molecules. Chemokines such as monocyte chemoattractant

protein-1 (MCP-1) attract monocytes/macrophages from the systemic circulation. These
infiltrated macrophages subsequently produce several growth factors such as transforming
growth factor- beta (TGF-β) which stimulate native fibroblast cells. Recent evidence has
shown that tubular cells can be activated with TGF-β and undergo epithelial mesenchymal
transdifferentiation (EMT) and transform into fibroblasts.7 Activated fibroblasts called
myofibroblasts generate extracellular matrix (ECM) proteins including fibronectin and
collagens in the interstitial space which eventually cause tubulointerstitial fibrosis.8 In this
chapter, we will explain the most commonly used animal models for renal fibrosis to test
antifibrotic drugs. Although these models are representative for renal fibrosis, many issues
such as type and etiology of the disease may differentiate for their applicability.

THE PATHOGENESIS OF LIVER CIRRHOSIS AND THE CRUCIAL ROLE OF

HEPATIC STELLATE CELLS (HSC)
Liver cirrhosis is a slowly progressing disease that develops gradually over a period of about
20 years without major symptoms in these patients. In contrast to renal function, which
can be monitored by urinary protein excretion, there is a lack of early markers to screen for
liver fibrosis. The disease is initiated after repeated and chronic damage to hepatic cells.
This damage can be caused by viruses (hepatitis B and C virus), chronic alcohol abuse, toxic
injury, biliary problems, but it is also induced by genetic and metabolic liver disorders. Liver
cirrhosis, the end-stage of the fibrotic process, is characterized by insufficient liver functioning.
In general, three different processes contribute to the gradual loss of liver function; [1] chronic
inflammation that causes damage to hepatocytes and activates other resident hepatic cells, [2]
capillarization that leads to portal hypertension and shunting of blood which impairs proper
blood flow through the liver sinusoids, and [3] fibrosis that leads to the excessive deposition of
scar tissue within the liver which affects the liver architecture and nutrient exchange.9-11
Various hepatic cell types are involved in the processes of initiation and perpetuation of
fibrosis. Often, inciting stimuli damage the hepatocytes that subsequently release factors that
activate other resident hepatic cells such as Kupffer cells and sinusoidal endothelial cells.
Simultaneously, inflammatory cells are recruited from the general circulation. All these cells
in turn produce various cytokines and growth factors that activate the HSC and these cells will
subsequently proliferate and differentiate into myofibroblasts. It is the chronic activation of
this inflammatory process that leads to an irreversible accumulation of extracellular matrix
constituents. Several autocriene and paracriene positive feedback loops further stimulate
activation of sinusoidal endothelial cells, Kupffer cells, HSC and myofibroblasts. Eventually, this
leads to a perpetuation of the disease independently of the presence of the inciting stimulus.
Nowadays, the HSC are considered as the crucial cells in the development of liver fibrosis and
are therefore viewed upon as the central target cell for drugs aiming at an attenuation of the
fibrogenic process.12,13 HSC and myofibroblasts are the major producers of collagens and other
extracellular matrix constituents in the fibrotic liver. These myofibroblasts are of various origin
from inside and outside the liver: transformed HSC, portal fibroblasts, stem cells,14-16 and
recent evidence indicate origination from epithelial cells such as hepatocytes and bile duct
epithelial cells.17,18 This latter process is called epithelialmesenchymal transition (EMT).
 Screening Methods for Renal and Liver Fibrosis 193

Since, the current pharmacotherapeutic treatment of fibrosis in the clinic is not effective, the
need for novel therapies to treat liver fibrosis is high. Amongst others, research groups nowadays
focus on the development of potent and safe antifibrotic agents that interfere with the various
activities of HSC. These include inhibition of HSC proliferation and activation, inhibition of
cytokine and growth factor production, inhibition of matrix production, promotion of matrix
degradation, inhibition of vasoconstriction and promotion of HSC apoptosis. A number of
comprehensive overviews of these novel experimental therapies have been published.10,13,19,20
To evaluate the effects of potential antifibrotic compounds, animal models are indispensable.
The disease involves namely many hepatic cell types and several different processes act in
concert with each other, which will occur only in vivo. Furthermore, animal models allow
easy testing of compounds in various stages of the disease provided that the proper model is
chosen. In addition, in vitro models can be useful to study cell-specific aspects of the drug-of-
interest. However, final efficacy studies should always be performed in vivo since all hepatic
cell types contribute to the disease progression and their contribution to the disease changes
in time. Therefore, in section 2.2 various animal models will be discussed that are used to asses
drug efficacy in liver fibrosis, but also two in vitro systems with its options and limitations will
be outlined.

MODELS OF RENAL FIBROSIS

Since, progression of fibrosis is a long-term process, in most of the animal models renal fibrosis
develops during weeks to months. There have been many attempts to design quick models to test
antifibrotic drugs in short time. In addition, several animal models have been described on the
basis of the etiology of the renal disease. Renal injury can be induced by physical/surgical damage
(e.g. subtotal nephrectomy, ureteral obstruction and renal ischemia/reperfusion injury) and
biochemical stimuli (e.g. adriamycin and puromycin) as well as it can be instigated by metabolic
diseases (e.g. diabetes mellitus and hyperlipidemia) or immune-mediated diseases (anti GBM-
nephritis, anti-Thy1 nephritis, and Heymann nephritis). In addition, these models can lead to
fibrosis after injuring primarily either the glomerulus (subtotal nephrectomy model), the tubule
(ureteral obstruction model) or both (protein overload model). A specific animal model can be
selected to screen an antifibrotic compound for its activity in specific type (s) of renal fibrosis,
e.g. glomeruloscelerosis, tubulointerstitial fibrosis or both. In addition, antifibrotic drugs can
display their pharmacological activity with different modes of action; therefore different animal
models can be selected on the basis of etiology to the disease. For instance, the anti-hypertensive
drug carvedilol has been found to inhibit fibrosis by reducing blood pressure in spontaneously
hypertensive rats with adriamycin nephropathy.21 Moreover, selection of animal model may
depend on the duration of the disease, since some models develop renal fibrosis rapidly e.g.
ureteral obstruction model (1-2 weeks) while other takes several weeks e.g. adriamycin, Anti-
Thy1 or protein overload induced nephropathy. Furthermore, reproducibility in the level of
disease is an important criterion to choose the animal model.

Unilateral Ureteral Obstruction Model

Unilateral ureteral obstruction (UUO) model is characterized by tissue loss, atrophy of tubular
epithelial cells, and the development of tubulointerstitial inflammation and fibrosis.22 This
194 Drug Screening Methods

Figure 11.1: Schematic presentation of the ureteral obstruction model showing the placement of two ligations at the
ureter (A) and the renal ischemia-reperfusion model showing the blockade of renal artery and vein with clips (B)

model resembles conditions seen in human obstructive nephropathy. In the UUO model, one
ureter is ligated to block the flow of urine from kidneys to urinary bladder. The accumulated
urine in kidneys activates renal tubular cells, which eventually leads to fibrosis in the obstructed
kidney whereas the contralateral kidney can regulate renal function at some extent. The UUO
model is widely performed in mice and rats.
Animals are preferably anesthetized with general anesthesia (2 % isoflurane in 2:1 O2/N2O,
1 L.min-1) for their rapid recovery after operation. Alternatively, ketamine chloride (100 mg/
kg) and xylazine sulfate (10 mg/kg) intraperitoneally can be used. Left kidneys and ureter
are exposed via a flank-incision. Then the ureter is ligated near the hilum at two sites with
silk suture (i.e. 4-0) for reliable ligation (Fig. 11.1). Abdominal muscle and skin are stitched
separately layer-by-layer. After recovery from anesthesia, animals are put back in cages and
animals are treated with postsurgical analgesic buprenorphine (0.05 mg/kg, subcutaneously)
after every 12 h for 48 h. In this model, renal fibrosis is visible on day 3 at the earliest and
extensive fibrosis occurs after 2-3 weeks.23 This animal model has been used from 3 days to 21
days but 14 days is an optimum time to test antifibrotic drugs. Samples from the obstructed
kidney can be used to determine the extent of fibrosis whereas the contralateral kidney can
serve as a control. However, one should be careful in using contralateral kidneys as control
as they are not completely normal. Immunohistochemical analyses are performed on kidney
sections to determine tubulointerstitial collagen deposition (collagen I and III), activation
of fibroblasts (alpha-smooth muscle actin, α-SMA) and tubular cell apoptosis (TUNEL or
caspase-3 assay). Yamate et al.24 have explained some crucial immunostainings in long-term
UUO model. Kidney cortex pieces can be used to study gene expression for inflammatory
(MCP-1, RANTES, etc.) and fibrotic (TGF-β1, α-SMA, TIMP-1, procollagen-Iα1) factors.
This model is quite consistent and takes relatively little time to develop fibrosis. However,
this model is only suitable to study antifibrotic effects on tubulointerstitial fibrosis and not on
glomerulosclerosis.

Subtotal Nephrectomy
The 5/6 subtotal nephrectomy (SNx) or subtotal renal ablation is a model for chronic renal
disease which develops proteinuria, hypertension, and ECM deposition with a fall in
glomerular filtration rate. SNx is induced by right nephrectomy and partial left nephrectomy
(upper and lower poles excision).25,26
 Screening Methods for Renal and Liver Fibrosis 195

In general, male Wistar rats are used for this model. To perform 5/6 subtotal nephrectomy,
rats are operated by incising at the flank under anesthesia (halothane or isoflurane). The right
kidney is exposed and the adrenal gland is separated from the upper pole, and the kidney is
decapsulated. The renal pedicle is ligated and the right kidney is removed. Thereafter, the left
kidney is also exposed through a flank incision. The adrenal gland is separated from the upper
pole and the kidney is decapsulated. Ligatures are placed around the upper and lower poles
and the poles are excised. Abdominal muscle and skin are stitched separately layer-by-layer
with silk sutures. After recovery from anesthesia, animals are put back in cages. Animals are
treated with postsurgical analgesic buprenorphine (0.05 mg/kg, subcutaneously) after every
12 h for 48 h. In SNx model, animals develops mild tubular atrophy at day 7 which gradually
increases until day 150 with 75% tubules damage. Glomerular damage and interstitial ECM
accumulation are visible after 15 days which increase at the highest level after 150 days.27
Moreover, 12 weeks time point has been widely used to test antifibrotic drugs. The progression
of the disease can be followed by monitoring proteinuria and renal functions.
This model has advantages to analyze both glomerular and tubular damage in the same
model and the development of the disease can be followed during the study by analyzing
proteinuria and renal function. But it has drawbacks of tedious surgical procedure, high
variability and long time for establishment of the disease.

Adriamycin-induced Nephropathy
Adriamycin or doxorubicin-induced nephropathy model is characterized to develop
proteinuria, focal segmental glomerulosclerosis and tubulointerstitial fibrosis.28 This model
mimics the proteinuric condition in patients and is considered as an experimental analogue
of human focal glomerulosclerosis. Adriamycin is an anti-neoplastic agent and causes
nephrotoxicity through many mechanisms such as by inducing oxidative stress and by
affecting water and urea transporters in renal medulla.29 This model is usually performed in
mice and rats.
Animals are injected intravenously with a single dose of adriamycin (2 to 7 mg/kg in rats
and 10 to 13 mg/kg in mice)28,30,31 The rate of the renal damage and the development of fibrosis
increases with the increase of the adriamycin dose. To examine the extent of disease, urine
is collected at different time points and analyzed for urinary protein levels. Animals can be
stratified on the basis of the proteinuria levels. The renal damage starts after 1 to 2 weeks and
progresses continuously for several weeks. During this period of time, renal functions drop
and ECM proteins gradually accumulate in glomeruli and tubulointerstitium (Fig. 11.2). At the
end of the studied time point, animals are sacrificed and kidneys are studied for tubular injury,
inflammation and fibrosis using the immunohistochemical and gene expression analyses as
discussed earlier. This model has been used to evaluate several drugs such as angiotensin
converting enzyme inhibitor captopril, angiotensin receptor antagonist losartan and different
kinase inhibitors.32-34 In addition, we have used this model to examine the affectivity of renal-
specific delivered captopril-lysozyme conjugate on proteinuria.35
Adriamycin-induced nephropathy model has many advantages, e.g. it mimics the clinical
situation of proteinuria; it is easy to induce; it develops both glomerular and tubulointerstitial
fibrosis; variation can be reduced by stratifying animals on the basis of their proteinuria levels;
and development of the disease can be followed during the study by measuring proteinuria
196 Drug Screening Methods

Figure 11.2: Pictures of immunohistochemical staining (red color) for collagen-III in normal kidney (A) and in kidneys with
adriamycin-induced nephropathy (B). A single dose of adriamycin (5 mg/kg) was administered in Wistar rats and after 4
weeks staining was performed. Collagen-III deposition in tubulointerstitium was substantially enhanced in adriamycin
treatment rats. Sections were counter-stained with hematoxyllin. g = glomerulus. Magnification 20 × 10

and renal function. However, a major drawback of this model is that it takes a long time to
develop fibrosis and consequently needs long-term treatments.

Protein-overload Rat Model

After many renal insults, plasma proteins filtrate through glomeruli and activate tubular
cells, which eventually induce inflammation and fibrosis. To mimic this phenomenon, in the
protein-overload model, a large amount of a protein is administered to animals repeatedly
which causes excessive protein filtration through glomeruli and eventually leads to tubular
damage.36-38 This model has been studied in mice and rats but rats are used more often.
Rats are incised at the flank under anesthesia (isoflurane) and the right kidney is removed.
Muscle and skin are stitched layer-by-layer with silk sutures. Animals are treated with
postsurgical analgesic buprenorphine (0.05 mg/kg, subcutaneously) after every 12 h for 48 h.
After uninephrectomy, rats are administered with daily injections of bovine serum albumin (1
to 2 g) intraperitoneally for several weeks. In this model fibrosis develops slowly and the model
has been used to evaluate antifibrotic drugs from 2 to 7 weeks. Similar to the adriamycin-
induced nephropathy model, induction of disease can be studied by measuring proteinuria,
renal function tests. After sacrificing animals, kidneys are studied for tubular damage,
inflammation and fibrosis using immunohistochemical and gene expression analyses.39,40
Since, protein overload model develops fibrosis by causing both glomerular and tubular
damage, this model is suitable to study antifibrotic effects on both type of tissues. However,
daily intraperitoneal injections of protein for several weeks makes this model labor-intensive.

Renal Ischemia/Reperfusion-induced Fibrosis

Ischemia/reperfusion (I/R)-induced renal fibrosis is one of the main factors which leads to the
chronic allograft nephropathy, a main cause for allograft failure.41 I/R injury is well-known to
induce tubular damage and inflammation particularly in renal medulla. In I/R injury, renal
blood flow is hampered temporarily for 45 to 60 min and then perfused. This process induces
 Screening Methods for Renal and Liver Fibrosis 197

hypoxia within kidneys which severely affects medulla, the poorly perfused region in kidney.
In addition, I/R injury enhances endothelial cells-leukocyte interaction that leads to leukocyte
entrapment in interstitium.42 Mice and rats have been widely used for this model.
Animals are preferably anesthetized with general anesthesia (2% isoflurane in 2:1 O2/N2O,
1 L.min-1) and left kidney is exposed via incision in the flank. The renal artery and vein are
exposed carefully and clamped individually under microscope. Then a third clamp is placed
together at both the renal artery and the vein to completely stop the renal blood flow (see Fig.
11.1). Body temperature is maintained using a heating pad. Saline kept on 37°C is instilled in
the peritoneal cavity occasionally. After 45 or 60 min, clamps are removed and reperfusion
of the kidney is assessed by the restoration of normal color. Then, the abdominal cavity is
closed by stitching muscle and skin layer-by-layer with silk sutures. Animals are treated with
postsurgical analgesic buprenorphine (0.05 mg/kg, subcutaneously) after every 12 h for 48
h. In this model, inflammation is prominent in the initial phase of the disease whereas the
activation of fibroblasts and the interstitial accumulation of ECM proteins can be detected
already after 3-4 days.43,44 After 3 weeks; the expression of fibrotic markers such as collagens
and fibronectin increases significantly. In addition, many studies have used I/R injury with
uninephrectomy to mimic the partial condition of an chronic allograft nephropathy.41
This model is quite reproducible and is employed often to study the effect of drugs on
tubular damage and inflammation after a short time (3-4 days).

Anti-Thy1 Antibody-induced Glomerulosclerosis

Anti-Thy1 antibody-induced glomerulonephritis is an experimental animal model representing
immunoglobulin A (IgA) nephropathy in patients. In this model, a monoclonal antibody

Figure 11.3: Pictures of immunohistochemical stainings (red color) for desmin, alpha-smooth muscle actin collagen
type I and collagen type III in normal kidney (upper panel) and in kidneys with anti-Thy1 IgG-induced renal fibrosis
(lower panel). A single dose of anti-Thy 1 IgG was administered in Wistar rats and after 21 days staining was performed.
The number of fibroblasts (desmin and actin staining) as well as the interstitial matrix deposition (collagens staining)
was substantially increased after administration of anti-Thy 1 IgG. Sections were counterstained with hematoxyllin. g =
glomerulus. Magnification 20 × 10
198 Drug Screening Methods

is administered to animals, which binds to a Thy1-like antigen on the surface of mesangial

cells of the kidney.45 This causes complement-dependent and nitric oxide-dependent lysis
of mesangial cells.46 Since, anti-Thy1-induced glomerulonephritis resolves over 4 weeks in
animals if two kidneys are present, uninephrectomy is performed to incite chronic progressive
glomerulosclerosis.47,48
Male or female Wistar rats (120-180 g) are used commonly in this model. Uninephrectomy
is performed as described in the subtotal nephrectomy and unilateral ischemia-reperfusion
injury models in this chapter. Three days after surgery, a dose of anti-Thy1 monoclonal antibody
(5 mg/kg) is injected intravenously. After 16 weeks, proteinuria is well established and the
expression of fibrotic proteins is highly upregulated in tubulointerstitium and glomerulus (Fig.
11.3). In addition, the inflammatory events can be studied in this model, as the infiltration of
macrophages and T-lymphocytes is prominent in this model.48
The anti-Thy1 model is a reproducible model and can also be used to study the effects on
glomerulonephritis in short period of time (< 1 week) as a reversible model, in which anti-
Thy1 antibody is injected without performing uninephrectomy.49

MODELS OF LIVER FIBROSIS

Several approaches to induce fibrosis in animals are described and these models can be
divided according to their stimulus from inciting injury.18,50 Liver fibrosis models are
associated with (1) toxic damage (hepatocytes: CCl4, dimethylnitrosamine (DMN),
galactosamine; bile duct epithelial cells: thioacetamide (TAA), (2) immunological-induced
damage (heterologous serum and experimental schistosomiasis), (3) biliary damage
[common bile duct ligation (BDL) or occlusion] or (4) alcohol-induced damage (baboon
ethanol diet or Tsukamoto/French model in rats). Nowadays, fibrosis-related models are
developed that have their origin in fatty liver disease (5). Fatty liver disease, in particular
the ‘malignant’ inflammatory form non-alcoholic steatohepatitis (NASH), can progress to
liver fibrosis and cirrhosis. It is strongly associated with obesity and diabetes, two modern
health problems in Western countries. Of the existing animal models for fatty liver disease,
as reviewed by Anstee et al.,51 the genetic leptin-deficient (ob/ob) or leptin-resistant (db/
db) mice and the dietary methionine/choline-deficient models are used in the majority
of published research. Progressive fibrosis was reported only in the methionine/choline-
deficient models in 100% of the mice.
BDL and CCl4 are the most widely used rodent models in liver fibrosis research to assess the
effectivity of experimental drugs on the pathogenesis, because these models represent features
of human pathogenesis. Therefore, these models are the best characterized with respect to
histological, biochemical, cell, and molecular changes associated with the development of
fibrosis (Fig. 11.4). In the past years, there is a tendency in fibrosis-related research to shift
from rat to mice models, and most of the models originally described for rats are now applied
in mice. Moreover, new testing models arise due to the development of transgenic or knock-out
mice models, which were developed to elucidate the pathogenesis and common pathways in
liver fibrosis.52 Examples of knockouts with spontaneous formation of liver fibrosis are mdr2-
/- mice53,54 lhx2-/- mice,55 and the mice models for NASH mentioned above.51
 Screening Methods for Renal and Liver Fibrosis 199

Figure 11.4: Pictures illustrating the enhanced matrix deposition (collagen type III immunostaining) in two important rat
models of liver fibrosis as compared to normal livers. (A) normal rat liver (B) rat liver 3 weeks after bile duct ligation, and
(C) rat liver 8 weeks after CCl4 intoxication. Magnification 4 × 10

Figure 11.5: Schematic illustration of two different models of liver fibrosis: (A) CCl4 intoxication and (B) Bile duct ligation
(BDL). CCl4 causes hepatocyte damage predominantly in zone 1 (as illustrated by the PAS stained liver at the left). Ligation
of the bile duct causes damage to bile duct epithelial cells and damage is seen in portal area (as illustrated with the
collagen I+III stained liver at the right).
Abbreviation: cv=central vein; bd=bile duct; ha=hepatic artery; pv=portal vein. Magnification 20 × 10

Acute and Chronic Models with Carbon Tetrachloride (CCl4)

CCl4 intoxication results in hepatocyte necrosis and apoptosis with damage predominantly
in zone III (around central vein) of the liver. The mechanism behind this hepatocyte damage
is the activation of CCl4 by cytochrome P450, which results in the formation of a trichloromethyl
radical in these cells and this free radical initiates lipid peroxidation (Fig. 11.5A).
The damage to hepatocytes by CCl4 is reflected by high plasma alanine transaminase (ALT)
and aspartate transaminase (AST) levels after CCl4 administration. CCl4 causes also fatty
200 Drug Screening Methods

changes in the hepatocytes. This initial damage is followed by hepatic stellate cell activation
and tissue fibrosis.
The CCl4 model is associated with tremendous inflammation, a feature that is also often
seen in livers of patients with liver fibrosis. Disadvantages of this model are the variations
obtained in disease induction in the animals and the relatively high rate of mortality after CCl4
administration (≥ 20%).
In animal models, CCl4 treatment is used to obtain different stages of the fibrotic process,
ranging from early damage and HSC activation until advanced cirrhosis.50 The fibrotic stage
obtained in the rodents depends on the number of injections of CCl4 that are administered. The
models for CCl4 that are used in liver fibrosis research, as summarized in Table 11.1, represent
(1) acute damage (72 hours after a single injection of CCl4) with HSC activation, (2) early and
established fibrosis (4-6 week of twice-weekly CCl4 dosing), (3) early cirrhosis (8 week of twice-
weekly CCl4 dosing), and (4) advanced micronodular cirrhosis (12 week of twice-weekly CCl4
dosing). In addition, for each of these models (5) spontaneous recovery from fibrosis can be
studied after cessation of dosing of CCl4.56,57 This latter model is a valuable model to determine
drug induced acceleration of recovery from established fibrosis after removal of the inciting
stimulus. This is similar to treatment situations in patients with liver fibrosis in case their
inciting stimulus can be eliminated; for instance after alcohol abstinence or after antiviral
therapy against hepatitis virus infections.
CCl4 is administered to the animals via intraperitoneal, subcutaneous, or oral
administration, or by inhalation. For intraperitoneal injections, CCl4 is diluted in olive
oil and given in dosages of 0.5-1.0 ml/kg to rats and mice. Often, supplementation of
phenobarbital in drinking water (resulting in induction of hepatocyte cytochrome P450) is
used to get more reproducible fibrosis development and to accelerate the speed of fibrosis
development. Usually, phenobarbital concentrations of 0.3-0.4 g/I in drinking water are
used and started 1 week before the initial exposure to CCl4. In case of inhalation of CCl4, the
animals are placed in an inhalation chamber twice a week with a progressively increasing
exposure time (1-5 min). Also with this procedure, supplementary phenobarbital in
drinking water is added. To reduce early toxicity and mortality, some research groups vary
with the dose of CCl4 in time. In these cases, gradually increasing dosages in the first weeks
are administered to the rats.58

Table 11.1: Various rodent models used in liver fibrosis research based on CCl4 intoxication, that
represent different stages of liver fibrogenesis
Model CCl4 injections Time period of injections Stage of disease
1 1 72 hours Acute damage, hepatocyte regeneration and HSC
activation
2 2x/week 4-6 weeks Early and established fibrosis
3 2x/week 8 weeks Early cirrhosis
4 2x/week 12 weeks Micronodular cirrhosis
5 2x/week 6-12 weeks, cessation for Regression models
> 4 weeks
 Screening Methods for Renal and Liver Fibrosis 201

Bile Duct Ligation (BDL)

The second well-studied experimental animal model of liver fibrosis is the bile duct ligation
model.59,60 This model corresponds with the human pathology of biliary cirrhosis, such as
extrahepatic biliary atresia and primary sclerosing cholangitis. Ligation of the bile duct causes
acute epithelial damage, and the detergent action of the subsequently released bile salts in the
liver is likely associated with the solubilization of plasma membranes and hepatocyte cell death.
This latter is visualized by elevated ALT and AST levels in plasma, in particular immediately
after ligation (first week). Characteristics of obstruction of the bile is the appearance of bile
products, such as bilirubin, into the blood circulation, which causes jaundice in these animals.
The initial damage is followed by a massive expansion of the bile duct epithelial cells and
periductal myofibroblasts, which can be referred to as portal expansion (stage I). In total,
this results in marked liver enlargement, which can be up to twice the weight as compared to
normal. Then, bile duct epithelial cells and myofibroblasts in the portal tract are progressively
expanding which results in a gradual remodeling of the liver architecture by linking adjacent
portal tracts (biliary cirrhosis stage IV).
To ligate the bile duct, the abdomen of the rat is opened under general anesthesia (preferably
N2O/O2/halothane inhalation to allow quick recovery from narcosis) to identify the common
bile duct. The bile duct runs from the hilum of the liver, where the hepatic ducts meet, through
the pancreas, into the lower end of the duodenum. Of note, the rat has no gall bladder in contrast
to other rodents. Three ligatures are placed and tied around the bile duct: two close to the
liver and one close to the duodenum (Fig. 11.5B). The first ligatures will prevent formation of a
reservoir of bile outside the liver. After tight closure, the bile duct is cut between the second and
third ligation in order to prevent restoration of the bile flow by bile duct formation around the
ligature. Subsequently, the abdomen is closed again and analgesics can be given to the rats. We
use a local anesthetic compound (Marcaine® which contains bupivacaine), but also systemic-
acting analgesics are sometimes administered (e.g. Temgesic® (containing buprenorphine).
For mice, the procedure is a little bit more complicated because a mouse possesses a gall
bladder, and attention should be paid to tightly ligate the whole duct, in general, more than
three ligatures are needed, to prevent rupture of the bladder and subsequent problems.
Already in the first days after ligation, proliferation of bile duct epithelial cells, activation
and proliferation of HSC and myofibroblasts, and deposition of extracellular matrix can be
observed microscopically starting in the portal areas of the liver (zone 3). After one week, a
fibrous expansion of the portal areas is visible and after about 10-14 days, portal-portal bridging
is visible. Three to 4 weeks after ligation, these rats develop advanced cirrhosis characterized
by extensive proliferation of the bile ducts, around which the activated and transformed HSC
are detectable (markers: a-smooth muscle actin and PDGFbeta receptor) and around which
the interstitial collagens (types I and III) are deposited (Fig. 11.6). At this stage, only small
islands of hepatocytes are still present (see Fig. 11.4).
A major advantage of the BDL model is the relatively fast development of fibrosis (within
3 weeks) in rats. Furthermore, the model is quite reproducible, and the mortality due to the
ligation procedure in rats is low (<10%). Disadvantages of the BDL model are the limited
inflammation associated with this type of fibrosis development and the excessive expansion
of bile duct epithelial cells. Another drawback with regard to drug screening is that the BDL-
induced disease is difficult to reverse with experimental drugs, and a reason for this may be
202 Drug Screening Methods

Figure 11.6: Liver fibrosis induced 3 weeks after ligation of the bile duct (BDL3). Pictures of immunohistochemical
stainings (red color) for cytokeratin-7 (marker of bile duct epithelial cells), collagen types I and III (matrix formation),
desmin (marker for all HSC), and alpha-smooth muscle actin, and PDGFbeta receptor (markers for transformed HSC and
myofibroblasts) in normal livers (upper panel) and in BDL3 livers (lower panel). The number of bile duct epithelial cells,
myofibroblasts (desmin, actin, and PDGFR staining) as well as the interstitial matrix deposition (collagens staining) was
substantially increased after ligation of the bile duct. Sections were counterstained with hematoxyllin. Magnifications
20 × 10

because the initiating stimulus (ligation of the bile duct) remains present during treatment
periods and causes continuous damage as subsequent fibrosis that troubles the potential
treatment effects.

Dimethylnitrosamine (DMN)
DMN induces liver damage leading to fibrosis and cirrhosis. Characteristic for this model
is that ongoing administration of this toxic compound finally leads to the development
of hepatocellular carcinoma in rodents. DMN induces liver injury by initiating damage to
the hepatocyte. It is metabolized primarily in hepatocytes by Cytochroom P450 (isotype
2E1) to more toxic compounds with formation of reactive oxygen species in hepatocytes
and subsequently this will lead to lipid peroxidation. In contrast to the hepatotoxin
CCl4, DMN administration does not cause fatty changes, steatosis, in the hepatocytes.
To induce the fibrosis, DMN (10 microliter/kg body wt., i.p.) is given 3 days a week for 3
weeks to rats.61,62
After administration of DMN, hemorrhagic necrosis is evident in centrolobular part (zone
III) of the liver. Incomplete septa appear after 7 days and micronodular cirrhosis is developed
after 3 weeks of treatment with DMN. Increased numbers of HSC and myofibroblasts are found
in the formed septa. Influx of inflammatory cells, mainly lymphocytes, is noted early in DMN-
induced liver injury.
Advantages of this model are that the disease induction is quite reproducible in the animals,
and this model is associated with a prominent inflammatory reaction. Furthermore, this
model can be used to study the transition from cirrhosis to hepatocellular carcinoma, and the
influence of drugs on this process.
 Screening Methods for Renal and Liver Fibrosis 203

HSC in Culture (In Vitro System)

HSC are key players in fibrosis and these cells predominantly orchestrate the development of
the disease. To assess the antifibrotic efficacy of experimental drugs, these primary cultured
cells are useful in assessing specific effects on HSC activities. In particular, the primary
isolated HSC are valuable in drug research, because in vitro they spontaneously transform
into myofibroblasts, and this transformation process is associated with cellular activation,
proliferation and matrix production resembling cellular activities that also happen in
vivo. This transformation does not occur in the various HSC cell lines that are also used in
literature.63 Immediately after isolation they represent a quiescent state, e.g. as present in the
normal healthy liver, with vitamin A droplets as their main characteristic. During culture on
plastic for about 10-14 days a cell with myofibroblast-like features is obtained (Fig. 11.7).64 This
transformed cell displays different cellular activities as compared to the original isolated one.
The procedure to isolate HSC is well described by various fibrosis research groups.65-67
Briefly, HSC are isolated from livers of normal rats weighing at least 500 g in order to achieve
a good separation from the other hepatic cells. The liver is digested with pronase, collagenase
and DNase by in situ perfusion. Pronase is essential in the isolation, yet it affects the viability of
other hepatic cells (i.e. hepatocytes) and therefore this procedure can only be used to isolate
HSC from the liver. After several centrifuge steps, the cell suspension is subjected to a Nycodenz
gradient to collect the HSC on top of the Nycodenz layer. The separation is based on the low
density of the HSC as compared to other liver cells, as a consequence of their high cellular lipid
content. Instead of Nycodenz, also other compounds are used, e.g. Stractan, Metrizamide, or
Percoll, to separate the HSC from the other cells by density gradients. The yield of HSC after
collagenase/pronase digestion and Nycodenz separation is about 20-40 × 10E6 cells per rat
liver. The yield of HSC obtained from a mouse liver is much smaller, and to isolate and purify
proper amounts of HSC, about 5 mice have to be used at the same time in one total isolation
(Geerts, personal communication).

Figure 11.7: Representative photographs of rat hepatic stellate cells at 3 days (quiescent HSC) and 10 days (activated
HSC) after isolation from a rat liver. Note the vitamin A droplets in the quiescent HSC and the change in morphology
during culture (activated HSC)
Photos are obtained in the lab of Prof. A. Geerts (Brussels, Belgium)
204 Drug Screening Methods

The purity after isolation can be confirmed by phase contrast microscopy or by staining of
the cells with markers for hepatic cell types. The isolated cells are cultured in DMEM containing
10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 10-14 days in culture, the
cells display an activated phenotype as assessed by light microscopy (change in morphology,
Fig. 11.7) and acquire the presence of alpha-smooth muscle actin.64,68
Additionally, it is also possible to isolate HSC from human livers. Often, (parts of ) human
livers are used that are unsuitable for transplantation or are derived from tumor-free parts of
the human liver and dissected after partial hepatectomy. Roughly, two methods are used to
isolate human stellate cells: (i) out-growth of the cells by culturing small pieces of the liver
in medium, and (ii) a combined digestion with collagenase/pronase, after which HSC were
separated from other liver nonparenchymal cells by centrifugation over density gradients
similar to the rat procedure. Of note, the first method will yield a combination of various (myo)
fibroblastic cells including HSC and myofibroblasts. These cells are subsequently cultured in
DMEM supplemented with 5% Fetal Calf Serum and 5% Human Serum. The myofibroblastic
nature of the cells can be microscopically evaluated, and tested for the expression of a-smooth
muscle actin.

Liver Slice System

A second in vitro test system which was recently developed to assess effects of antifibrotic drugs
is the liver slice preparation.69 Drug studies with tissue slices (8 mm diameter, 250 μm thickness
that is about 10-12 cell-layers thick) containing stellate cells in their natural environment that
maintain their in vivo cellular functional and anatomic relationships, may provide additional
information about the hepatocellular specificity of the experimental drug and their effects on
all hepatic cells.
Two models have been developed in the past years: one in which CCl4 induced the activation
of quiescent HSC in normal rat liver slices,70 while in the second model fibrotic rat liver slices
were used that were harvested from rats with BDL-induced liver fibrosis and cultured for 1-2
days.69 In both models, markers of HSC activities were shown to be induced (AB-crystallin,
HSP47, alphaSMA and collagen expression). In particular, the slice preparations from
fibrotic livers are promising tools for the testing of antifibrotic drugs in vitro in their natural
multicellular, fibrotic milieu, which cannot be achieved in vitro using single cell cultures or
co-culture systems.71
Liver slices are prepared as follows: Livers are excised from rats and stored in UWsolution
(University of Wisconsin transplantation fluid) until the slice procedure starts. Slices were
prepared in ice-cold Krebs–Henseleit buffer saturated with carbogen (95% O2/5% CO2) and
containing 25 mM glucose, 25 mM NaHCO3 and 10 mM Hepes using the Krumdieck tissue
slicer. To equilibrate the tissue, slices are pre-incubated for 1 h in Williams Medium E with
glutamax-I supplemented with 25 mM d-glucose and 50 μg/ml gentamicin (WEGG) under
carbogen-atmosphere at 37°C in six-well culture plates while gently shaken. After that, slices
are transferred to six-well culture plates containing fresh medium and incubated under
carbogen atmosphere at 37°C for maximally 48 h while gently shaken.
A disadvantage of this system is the limited time period of study. Slices, in particular fibrotic
slices, are viable for maximally 48 hours after preparation, which limits the time to study
effects of experimental drugs. Another limitation of this method is the absence of blood flow
 Screening Methods for Renal and Liver Fibrosis 205

through the slice. Advantages of the slice system is that they can be prepared from normal,
fibrotic or cirrhotic tissue, and this in vitro system easily allows drug testing in human liver
material. Furthermore, the HSC is present in its natural environment including extracellular
matrix components and all other resident hepatic cells, which allows testing of multicellular
interactions.

CONCLUSION
Animal models are indispensable in the testing of antifibrotic compounds, because of the
complex multicellular processes that determine the development of the fibrosis in kidneys
as well as in livers. Even the importance of various processes varies during the course of the
disease. For both renal and liver fibrosis, various animal models exist, as described in this
chapter. Therefore, to evaluate the effects of potential antifibrotic compounds it is essential to
choose the proper model and the proper stage of the disease because this will largely determine
the outcome of the study. In addition, specific disease markers to describe the fibrotic stage and
determine the effects of potential antifibrotic compounds are crucial and therefore additional
research searching for new and good discriminating disease markers is needed. The tendency
in research seen in the past years to perform efficacy studies in mice instead of rats, is related
to the occurrence of specific knock-out mice. However, mice models will also be helpful to
assess the effects of for instance biological mediators, such as cytokines and growth factors, on
the development of fibrosis, and in particular after chronic administration of these mediators.

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cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver
fibrogenesis. Lab Invest 2003;83:163-73.
16. Russo FP, Alison MR, Bigger BW, et al. The bone marrow functionally contributes to liver fibrosis.
Gastroenterology 2006;130:1807-21.
17. Sicklick JK, Choi SS, Bustamante M, et al. Evidence for epithelial-mesenchymal transitions in adult
liver cells. Am J Physiol Gastrointest Liver Physiol 2006;291:G575-83.
18. Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a
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(Suppl 3): S300-05.
20. Wu J, Zern MA. Hepatic stellate cells: a target for the treatment of liver fibrosis. J Gastroenterol 2000;
35:665-72.
21. Jovanovic D, Jovovic D, Mihailovic-Stanojevic N, et al. Influence of carvedilol on chronic renal
failure progression in spontaneously hypertensive rats with adriamycin nephropathy. Clin Nephrol
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22. Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J Physiol Renal Physiol
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23. Moon JA, Kim HT, Cho IS, et al. IN-1130, a novel transforming growth factor-beta type I receptor
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2006;70:1234-43.
24. Yamate J, Okado A, Kuwamura M, et al. Immunohistochemical analysis of macrophages,
myofibroblasts, and transforming growth factor-beta localization during rat renal interstitial fibrosis
following long-term unilateral ureteral obstruction. Toxicol Pathol 1998;26:793-801.
25. Muchaneta-Kubara EC, Sayed-Ahmed N, el Nahas AM. Subtotal nephrectomy: a mosaic of growth
factors. Nephrol Dial Transplant 1995;10:320-27.
26. Oldroyd SD, Miyamoto Y, Moir A, et al. An IGF-I antagonist does not inhibit renal fibrosis in the rat
following subtotal nephrectomy. Am J Physiol Renal Physiol 2006;290:F695-F702.
27. Muchaneta-Kubara EC, el Nahas AM. Myofibroblast phenotypes expression in experimental renal
scarring. Nephrol Dial Transplant 1997;12:904-15.
28. Tamaki K, Okuda S, Ando T, et al. TGF-beta 1 in glomerulosclerosis and interstitial fibrosis of
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29. Manabe N, Kinoshita A, Yamaguchi M, et al. Changes in quantitative profile of extracellular matrix
components in the kidneys of rats with adriamycin-induced nephropathy. J Vet Med Sci 2001;63:
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30. Vielhauer V, Berning E, Eis V, et al. CCR1 blockade reduces interstitial inflammation and fibrosis in
mice with glomerulosclerosis and nephrotic syndrome. Kidney Int 2004;66:2264-78.
31. Ryuzo M, Soares V. Effect of mycophenolate mofetil on the progression of adriamycin nephropathy.
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32. Li J, Campanale NV, Liang RJ, et al. Inhibition of p38 mitogen-activated protein kinase and
transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis
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 Screening Methods for Renal and Liver Fibrosis 207

33. Kim HJ, Ryu JH, Han SW, et al. Combined therapy of cilazapril and losartan has no additive effects
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34. Mansour MA, El-Kashef HA, Al-Shabanah OA. Effect of captopril on doxorubicin-induced
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35. Windt WA, Prakash J, Kok RJ, et al. Renal targeting of captopril using captopril-lysozyme conjugate
enhances its antiproteinuric effect in adriamycin-induced nephrosis. J Renin Angiotensin
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36. Eddy AA. Interstitial nephritis induced by protein-overload proteinuria. Am J Pathol 1989;135:719-
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37. Eddy AA, Giachelli CM. Renal expression of genes that promote interstitial inflammation and
fibrosis in rats with protein-overload proteinuria. Kidney Int 1995;47:1546-57.
38. Nagasawa Y, Takenaka M, Kaimori J et al. Rapid and diverse changes of gene expression in the
kidneys of protein-overload proteinuria mice detected by microarray analysis. Nephrol Dial
Transplant 2001;16:923-31.
39. van Timmeren MM, Bakker SJ, Vaidya VS, et al. Tubular kidney injury molecule-1 in protein-
overload nephropathy. Am J Physiol Renal Physiol 2006;291:F456-64.
40. Shimizu H, Maruyama S, Yuzawa Y, et al. Anti-monocyte chemoattractant protein-1 gene therapy
attenuates renal injury induced by protein-overload proteinuria. J Am Soc Nephrol 2003;14:1496-
1505.
41. Yang B, Jain S, Pawluczyk IZ, et al. Inflammation and caspase activation in long-term renal ischemia/
reperfusion injury and immunosuppression in rats. Kidney Int 2005;68:2050-67.
42. Bonventre JV, Zuk A. Ischemic acute renal failure: an inflammatory disease? Kidney Int 2004;66:480-5.
43. Prakash J, Sandovici M, Saluja V, et al. Intracellular delivery of the p38 mitogen-activated protein
kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] in
renal tubular cells: a novel strategy to treat renal fibrosis. J Pharmacol Exp Ther 2006;319:8-19.
44. Furuichi K, Wada T, Iwata Y, et al. Interleukin-1-dependent sequential chemokine expression and
inflammatory cell infiltration in ischemia-reperfusion injury. Crit Care Med 2006;34:2447-55.
45. Bagchus WM, Hoedemaeker PJ, Rozing J, et al. Glomerulonephritis induced by monoclonal anti-Thy
1.1 antibodies. A sequential histological and ultrastructural study in the rat. Lab Invest 1986;55:680-7.
46. van GH, Albrecht EW, Heeringa P, et al. Nitric oxide inhibition enhances platelet aggregation in
experimental anti-Thy-1 nephritis. Nitric Oxide 2001;5:525-33.
47. Sakai N, Iseki K, Suzuki S, et al. Uninephrectomy induces progressive glomerulosclerosis and
apoptosis in anti-Thy1 glomerulonephritis. Pathol Int 2005;55:19-26.
48. Kramer S, Loof T, Martini S, et al. Mycophenolate mofetil slows progression in anti-thy1-induced
chronic renal fibrosis but is not additive to a high dose of enalapril. Am J Physiol Renal Physiol
2005;289:F359-68.
49. Sadlier DM, Ouyang X, McMahon B, et al. Microarray and bioinformatic detection of novel and
established genes expressed in experimental anti-Thy1 nephritis. Kidney Int 2005;68:2542-61.
50. Constandinou C, Henderson N Iredale JP. Modeling liver fibrosis in rodents. Methods Mol Med
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51. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver disease and steatohepatitis
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52. Weiler-Normann C, Herkel J Lohse AW. Mouse models of liver fibrosis. Z Gastroenterol 2007;45:43-50.
208 Drug Screening Methods

53. Van Nieuwkerk CM, Elferink RP, Groen AK, et al. Effects of Ursodeoxycholate and cholate feeding
on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. Gastroenterology
1996;111:165-71.
54. Popov Y, Patsenker E, Fickert P, et al. Mdr2 (Abcb4)-/- mice spontaneously develop severe biliary
fibrosis via massive dysregulation of pro- and antifibrogenic genes. J Hepatol 2005;43:1045-54.
55. Wandzioch E, Kolterud A, Jacobsson M et al. Lhx2-/- mice develop liver fibrosis. Proc Natl Acad Sci
U S A 2004;101:16549-54.
56. Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis.
Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J
Clin Invest 1998;102:538-49.
57. Issa R, Zhou X, Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis:
evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology
2004;126:1795-808.
58. Zhu J, Wu J, Frizell E, et al. Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits
fibrogenesis in an in vivo model of liver fibrosis. Gastroenterology 1999;117:1198-204.
59. Kountouras J, Billing BH, Scheuer PJ. Prolonged bile duct obstruction: a new experimental model
for cirrhosis in the rat. Br J Exp Pathol 1984;65:305-11.
60. Hinz S, Franke H, Machnik G, et al. Histological and biochemical changes induced by total bile duct
ligation in the rat. Exp Toxicol Pathol 1997;49:281-88.
61. Jezequel AM, Mancini R, Rinaldesi ML, et al. Dimethylnitrosamine-induced cirrhosis. Evidence for
an immunological mechanism. J Hepatol 1989;8:42-52.
62. Jezequel AM, Mancini R, Rinaldesi ML, et al. A morphological study of the early stages of hepatic
fibrosis induced by low doses of dimethylnitrosamine in the rat. J Hepatol 1987;5:174-81.
63. Gutierrez-Ruiz MC, Gomez-Quiroz LE. Liver fibrosis: searching for cell model answers. Liver Int
2007;27:434-9.
64. Geerts A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate
cells. Semin Liver Dis 2001;21:311-35.
65. Weiskirchen R, Gressner AM. Isolation and culture of hepatic stellate cells. Methods Mol Med 2005;
117:99-113.
66. Geerts A, Niki T, Hellemans K, et al. Purification of rat hepatic stellate cells by side scatter-activated
cell sorting. Hepatology 1998;27:590-8.
67. Friedman SL, Roll FJ, Boyles J, et al. Hepatic lipocytes: the principal collagen-producing cells of
normal rat liver. Proc Natl Acad Sci U S A 1985;82:8681-5.
68. Rockey DC, Boyles JK, Gabbiani G, et al. Rat hepatic lipocytes express smooth muscle actin upon
activation in vivo and in culture. J Submicrosc Cytol Pathol 1992;24:193-203.
69. van de BM, Groothuis GM, Meijer DK, et al. Precision-cut fibrotic rat liver slices as a new model to
test the effects of antifibrotic drugs in vitro. J Hepatol 2006;45:696-703.
70. van de Bovenkamp M, Groothuis GM, Draaisma AL, et al. Precision-cut liver slices as a new
model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. Toxicol Sci
2005;85:632-8.
71. van de Bovenkamp M, Groothuis GM, Meijer DK, et al. Liver fibrosis in vitro: Cell culture models
and precision-cut liver slices. Toxicol In Vitro 2007;21:545-57.
 CHAPTER

12
Techniques for the
Detection of Apoptosis
INTRODUCTION
Apoptosis is naturally occurring cell death. It is physiological in nature, and also known as
‘programmed cell death’ (PCD). It is an active, genetically controlled process that removes
unwanted or damaged cells.1-3 It is essential for the appropriate development and function of
multicellular organisms.
Dysregulated apoptosis results in excessive or insufficient cell death. It is fundamental to the
initiation and progression of many human diseases. Diseases associated with increased cell
survival/inhibition of apoptosis have been thoroughly reviewed by Thatte and Dahanukar.4.
It is implicated in neurodegenerative disorders,5 AIDS,6 autoimmune disorders,7 other viral
diseases,8 etc.
The other form of cell death is pathological in nature and is termed as necrosis. Cell death
can be distinguished as necrosis and apoptosis, on the basis of morphology and biochemistry.9
During necrosis there is rapid breakdown of the membrane systems within the cell. In contrast
apoptosis is the result of several well-orchestrated events that require RNA and protein
synthesis induced by certain stimuli.10,11 It is characterized by a series of morphological features
and biochemical events. The cell breaks up into membrane-bound fragments of various sizes
—known as apoptotic bodies in which internal organelles are preserved.12,13
As apoptotic bodies are always membrane bound, there is no exposure of intracellular
contents. The membrane undergoes changes such as exposure of normally hidden sugar
moeities like N-acetyl glucosamine14 to signal neighboring macrophages to phagocytose these
apoptotic bodies. The lack of inflammation is, therefore the hallmark of apoptosis in contrast
to necrosis.

PROAPOPTOTIC AND ANTIAPOPTOTIC AGENTS

The manipulation of the apoptotic pathways either by a pharmacological agent or by genetic
engineering may be useful in the management of various diseases. The proapoptotic agents are
promoters of apoptosis while antiapoptotic agents are inhibitors of apoptosis. The promoters
and inhibitors identified are genes and they encode proteins, which either activate or inhibit
the apoptotic process.4,15-18
The widespread importance of apoptosis in the field of biology and medicine has led to
active research in the area. Many different techniques have been developed and are used for
210 Drug Screening Methods

the detection of apoptosis. However, none of these are full proof to detect specifically apoptosis
and can detect necrosis also. Thus, it is important to use more than one technique to study
apoptosis.

CATEGORIES OF CELLULAR CHANGES THAT FORM THE BASIS OF

APOPTOSIS ASSAYS
Surface Morphology and Composition
•• time-lapse surface morphology
•• membrane permeability: impermeable dyes (PI)
•• permeable DNA stains (DAPI, Hoechst)
•• phospholipid externalization (Annexin V binding).

Nuclear Events and DNA Cleavage

•• nuclear morphology: segmentation of chromatin and nuclei DNA cleavage by gels
•• large fragments and internucleosomal cleavage DNA cleavage in situ
•• detection of strand breaks in situ nick-translation (ISNT)
•• TUNEL (terminal transferase)
•• anti-single-stranded DNA antibody
•• hairpin oligos (double-stranded breaks)
•• cell dissolution (pre-GI peak) (apoptotic bodies containing DNA).

Cytoplasmic Biochemical Activation Events

•• caspase cleavage products
•• caspase activity (caged fluorophores, FRET)
•• PARP activity transglutaminase activity
•• death antigens.

Mitochondrial Function and Integrity Permeability

•• transition (vital dyes)
•• mitochondrial antigens (accessibility)
•• metabolic activity
•• cytochrome c release and alterations.

DETECTION OF APOPTOSIS BASED ON MORPHOLOGY

As described previously, apoptosis is characterized by distinct morphology. During the
process of apoptosis the cell shrinks, detaches from the neighboring cells. This is followed by
chromatin condensation and nuclear fragmentation into multiple chromatin bodies, leading
to the formation of apoptotic bodies, which are phagocytosed, by neighboring cells. These
morphological changes can be assessed by light microscopy, fluorescence microscopy or by
transmission electron microscopy (TEM).
 Techniques for the Detection of Apoptosis 211

Light Microscopy
This is one of the most simple, easy and economic method for the quantitation of the apoptotic
cell. The tissue sections are stained with routine hematoxylin and eosin stain 19 and observed
under an ordinary light microscope. The chromatin condensation characteristic of apoptosis
is evident in these cells as “pyknosis”, i.e. very dense staining of chromatin by hematoxylin.
Giemsa stain can also be used as it differentiates nuclear structures. Preparation of the stains
and staining procedure is given in Table 12.1. Periodic acid Schiff-staining is less desirable
because its dark, dense coloration fails to distinguish pyknosis and nuclear chromatin
condensation. This technique has the advantage of being economic, as it requires a basic
microscope and simple reagents. Also, large number of cells from culture and tissue can be
examined at the same time. However, the limitation is that the nuclei of quiescent cells might
be identified mistakenly as pyknotic nuclei. Apoptosis occurring after administration of certain
chemotherapeutic regimens in vivo can be readily detected by this technique.20

Table 12.1: Preparation of stains and staining procedure

Stains Solutions Procedures

Hematoxylin/ Hematoxylin (Mayer) 2.0 g/l 0.3% Eosin Y Dip the slide with tissue section in Hematoxylin for
Eosin stain in 70% ethanol 15-30 sec, wash under running tap water for 5 min,
then dip in eosin for 5 sec followed by dehydration
in 70, 95 and 100% ethanol (The time of dehydration
will vary depending upon the tissue and its thickness).

Geimsa Geimsa: 0.8 g Geimsa, 0.1 g Geimsa (after filtration) for 30 sec – 5 min, then washing
Eosin Y in 100 ml 1:1 v/v in Geimsa buffer for 5 min, followed by dehydration in
glycerol and methanol buffer: 9 ml 0.1 M 70, 95 and 100% ethanol (The time of dehydration will
citric acid in 25% methanol + 11 ml 0.2 M vary depending upon the tissue and its thickness).
Na2HPO4 in 25% methanol + 380 ml DW,
pH 6.4 with HCl

Fluorescence Microscopy
Apoptosis can also be detected in tissue sections and cultured cells, by examination under
fluorescence microscope. The fixed, permeabilized cells are stained with specific DNA stains
such as acridine orange, propidium iodide, Hoechst 33258, 4’, 6-diamidino-2-phenylindone
(DAPI), etc. Nuclear changes such as chromatin condensation and nuclear fragmentation are
readily visible by this technique. The advantages and disadvantages are similar to that of light
microscopy.
Cells fixed on the glass coverslips are gently rinsed two times with phosphate buffered
saline (PBS) and then fixed and permeabilized with methanol: water (4:1) for 15 min. After
rinsing with PBS the coverslips are stained with propidium iodide (5 µg/ml) for 5 min in the
dark and mounted on glass slides in glycerol: PBS (1:1). Instead of propidium iodide, Hoechst
dye 33258 (2.5 µg/ml) can be used to assess apoptosis by nuclear staining (blue) after fixation
of cells in 4% paraformaldehyde. The glass slides are preferentially examined under confocal
laser scanning microscopy. Apoptotic nuclei are hyper fluorescent, condensed or fragmented,
and smaller compared to normal nuclei (Fig. 12.1). The nuclear hyper fluorescence and the
212 Drug Screening Methods

Figure 12.1: Fluorescence microscopy image of 15 weeks gestational age human fetus brain primary cell culture. Cells
were stained with Hoechst staining after induction of apoptosis with anti-Fas agonist antibodies. The labeled cells exhibit
condensed nuclear fluorescence while viable cells exhibit diffuse nuclear fluorescence. (Reproduced with permission from
J Cell Mol Med 2001; 5: 179-87)

nuclear size change can be utilized with appropriate video-image analysis systems to provide
a semi-automatic scoring system for apoptotic cells.

Electron Microscopy
Electron microscopy is a reliable qualitative method of characterizing apoptosis both in
tissue sections and cell culture. Condensation of nuclei within nuclear margin, blebbing, and
shrinkage of the cytoplasm, intact mitochondria and other membranes can be observed in the
apoptotic cell under electron microscope.
A mixture of apoptotic and necrotic features are often observed in the sample and therefore
the data should be interpreted carefully. Sometimes under tissue culture conditions, secondary
necrosis may occur when the ability to remove apoptotic cells by phagocytes is insufficient
or compromized. Therefore cells with apoptotic, fragmented nuclei and secondary plasma
membrane lysis are frequently observed. Additionally, one should also keep in mind that the
same insult can lead to temporally distinct phases of apoptosis and necrosis. For instance
Bonfoco and colleagues21 found that mild insults with glutamate or nitric oxide/superoxide to
cerebrocortical neurons in vitro led to apoptosis of delayed onset, whereas intense exposure
led to more rapid necrosis within a few hours. For cerebellar granule cell neurons in culture,
it was found that exposure to glutamate induced a wave of necrosis in a subpopulation of the
cells, followed by delayed apoptosis in another subpopulation.22
 Techniques for the Detection of Apoptosis 213

The brief procedure for the preparation of tissue for electron microscopy is: tissue samples
or cultures are fixed in 1% glutaraldehyde diluted in 0.1 M phosphate buffer at pH 7.2. The
tissue is cut in 1 µm slices and fixed for 5 h at room temperature and then postfixed with 1%
osmium tetroxide in PBS for 1 h. After dehydrating samples in acetone and embedding them in
epon, thin sections are cut on an ultratome, counterstained with uranyl acetate and examined
with an electron microscope.
Electron microscopy is not a suitable technique for quantitation because (i) at a given point
of time only few cells are observed, and (ii) the active phase of apoptosis is usually brief.19

DETECTION OF DNA FRAGMENTATION

DNA from cells undergoing apoptosis displays a characteristic series of bands known as
nucleosomal ladder after agarose gel electrophoresis. This fragmentation pattern results from
the preferential cleavage of DNA in the linker regions between nucleosomes double-stranded
nuclease that cuts DNA randomly. The nucleases are activated during apoptosis resulting in
DNA fragmentation. A variety of techniques described below have been developed based on
the detection of fragmented DNA by nucleases.

Conventional Agarose Gel Electrophoresis

DNA can be prepared for agarose gel electrophoresis from whole cells or tissues by a number
of treatment protocols. One commonly used protocol involves SDS (Sodium dodecyl sulfate)
extraction and protease digestion followed by extraction with phenol to remove peptide
fragments. Application of the resulting total cellular DNA to the agarose gel with suitable
separation properties, e.g. 1-2% (w/v) agarose, readily demonstrates a ladder of ~180 bp
fragments and integer multiples thereof in many models of apoptosis. A brief protocol that
can be followed is: Cells (1 × 106) are suspended in 500 µl of TE buffer (10 mM Tris-HCl pH
7.6, 1 mM ethylenediamine tetra acetic acid, pH 8.0) and lysed in 500 ml lysis buffer (3% SDS,
50 mM Tris, pH 12.6) at room temperature for 10 min.23 Alternatively, cells (1 × 106) can be
suspended in 500 µl of extraction buffer (10 mM Tris, pH 8.0; 10 µM EDTA, pH 8.0; 75 mM
NaCl; 0.5% SDS and 150 µg/ml proteinase k) and incubated at 50°C for 3 h.24 After incubation,
microfuge the sample for 8 min at room temp. DNA is precipitated from the supernatant by
2 vol. of 100% ethanol with 0.1 M NaCl. The precipitated DNA is washed with 70% ethanol
and then treated with DNAse-free RNAse for 60 min. The DNA sample is separated on 2.0%
agarose gel electrophoresis and stained by ethidium bromide and photographed under UV
illumination. The advantage of this technique is the relatively low cost of the equipment and
reagents as well as its simplicity.
In many articles in the literature, agarose gel electrophoresis has often been the single
criterion used to detect apoptosis. This is not advisable because it has been shown that DNA
laddering is a rather late event in the apoptotic process.25-27 Therefore, though DNA laddering
is typical for apoptosis, its absence cannot be used to exclude apoptosis.22
This technique is relatively insensitive and inherently qualitative rather than quantitative.
It can distinguish between the nucleosomal ladder of DNA fragments that is characteristic of
apoptosis and the random DNA fragmentation that accompanies necrosis, but it is difficult to
214 Drug Screening Methods

determine the percentage of the total DNA present in the nucleosomal ladder in a particular
lane.28

Quantitation of DNA Fragments by Cell Fractionation

Two techniques have been developed to quantify the amount of DNA fragmentation. One of
these is based on the observation that double-stranded DNA fragments of less than 10-20 kb
can be extracted from nuclei when cells are lysed under nondenaturing conditions in buffer
containing EDTA.29,30 After treatments to initiate apoptosis, cells are suspended in 20 mMTris
(pH 7.0 or 8.0) containing EDTA and a neutral detergent. After incubation at 4°C for 10-30 min,
the intact chromatin is pelleted at low to moderate speed, leaving the nucleosomal fragments
in the supernatant. DNA in the two fractions is then assayed using colorimetric or fluorimetric
techniques. Alternatively, if DNA in the cells is uniformly radiolabeled (i.e. greater than one
cell cycle) prior to stimulation of apoptosis, DNA in the various fractions is quantitated by
scintillation counting.
These approaches allow relatively precise quantitation of the amount of DNA that has been
fragmented to oligomers of nucleosome-sized fragments. This information is useful in the time
course studies of nuclease activation during apoptosis.30 Using this simple technique for DNA
extraction and assay, large number of samples can be examined. However, these techniques
cannot be used to differentiate apoptosis from necrosis as DNA fragments resulting due to
necrosis are also extracted. The other drawbacks include: (i) high molecular weight DNA
fragments are not extracted under the denaturing conditions and are not detected and (ii)
often these results in underestimation of the amount of DNA damage to the cells. For example,
if 25% of DNA is extracted, it is not clear whether 25% of the total cells or 25% of DNA in each
cell is undergoing apoptosis. The limitations diminish the utility of this approach as a high
throughput screening process.

Detection of DNA Fragmentation by Filtration Assays

The rate at which deproteinated DNA flows through the pores of filters is related to the size
of the DNA fragments. A variation of this technique has been applied for the quantitation of
oligonucleosomal DNA fragmentation.31 In brief, the DNA in cells is radiolabeled for atleast
one generation time. After a suitable postlabeling incubation to chase radiolabeled out of
newly synthesized DNA, which has aberrant retention properties on filters, cells are treated
with an apoptosis-inducing stimuli. At the desired point(s) in time, cells are then applied to the
filters and lysed by addition of an aliquot of deproteinising buffer (e.g. sodium sarkosyl). Low-
molecular-weight DNA fragments will flow through the filter with the lysing solution, whereas
high-molecular-weight DNA will be retained on the filter unless it is eluted with a large volume
of neutral or alkaline buffer pumped through the filter. It is possible to estimate the amount of
DNA that has been degraded to low-molecular-weight fragments by quantitating the amount
of radiolabel flowing through the filter during cell lysis.
The technique is relatively simple, rapid and the equipment required (a suitable filter
manifold and a scintillation counter) is widely available. The approach can also be used
for high-throughput screening. However, few precautions should be taken while using
this technique. The DNA should be radiolabeled with 14C – labeled nucleotide rather than
 Techniques for the Detection of Apoptosis 215

3
H‑labeled nucleotide because the radiation form 3H – labeled nucleotide induces apoptosis
in some cell types.32 The labeling step should be followed by sufficient incubation period with
radiochemical- free medium to “chase” all the radiolabel into the mature DNA. The limitation
is that the technique allows precise quantitation of the amount of DNA that is converted to
fragments small enough to pass through the filters during cell lysis; it does not distinguish
internucleosomal DNA degradation that accompanies necrosis.

Field-inversion Gel Electrophoresis (FIGE)

Apoptosis is often accompanied by formation of high-molecular-weight DNA fragments
(50–200 kb) in addition to nucleosomal ladder33 by the action of endonuclease on the
DNA. The high-molecular-weight DNA fragments are most commonly detected by FIGE.
And oligonucleosomal fragmentation is traditionally detected by conventional agarose gel
electrophoresis as explained earlier. An elegant combination of these two techniques involving
the recovery of small fragments from the plugs used for the FIGE, allows for the simultaneous
detection of both high and low-molecular-weight DNA fragments.
To examine DNA by FIGE, cells are treated with an inducing stimulus, encapsulated
in agarose (to protect the DNA from shearing during subsequent manipulation), lysed in a
deproteinising detergent such as SDS, treated with proteinase k and embedded in the wells of
an agarose gel. The DNA is then subjected to an alternating electric field rather than the fixed
field utilized in conventional electrophoresis. In this alternating field, larger DNA fragments
change migration directions more slowly than smaller fragments. As a consequence, the
differences in fragment mobility in pulsed fields, termed “reptation” (reptile like movement
through the gel matrix) contribute to separation of high-molecular-weight fragments by
size.34 Molecular weight standards utilized with this technique include oligomers of large viral
genomes (e.g. T phage) as well as yeast chromosomes.
The advantage of this technique is its ability to detect the infrequent DNA strand breaks
that appear to occur in several models of apoptosis. Because of the widespread and early
occurrence of these breaks, it has been suggested that they might be a universal feature of the
apoptotic process. However, it is important to note that similar DNA fragmentation has been
reported in cells undergoing necrotic cell death as well.35
A detailed protocol for the detection of apoptosis in neurons in culture by FIGE as described
by Ankarcrona and colleagues22 is: neurons are gently removed from culture dishes and
suspended in a solution containing 0.15 M NaCl, 2 mM KH2PO4, pH 6.8, 1 mM EGTA and 5 mM
MgCl2. An equal volume of liquefied 1% low melting point agarose gel solution is then added
to the suspension, while gently mixing. Next, the mixture is aliquoted into gel plug casting
forms and allowed to cool and solidify on ice for 10 min. The resulting agarose blocks are
transferred into a solution containing 10 mM NaCl, 10 mM Tris-HCl, pH 9.5, 25 mM EDTA, 1%
lauroyl sarcosine and 200 µg/ml proteinase k, and incubated for 24 h at 50°C with continuous
agitation. The plugs are then rinsed three times over 24 h at 4°C in 10 mM Tris-HCl, pH 8.0, 1
mM EDTA. Subsequently, the plugs are stored until used for electrophoresis at 4°C in 50 mM
EDTA, pH 8.0. FIGE is performed with horizontal or vertical gel chambers equipped with a
constant temperature cooling system. The temperature control is very important. Normally,
the electrophoresis is run at 180 V in 1% agarose gel in 0.5 TBE (45 mMTris, 1.25 mM EDTA, 45
mM boric acid, pH 8.0) at 12°C. The ramp rate changes from 20-30 sec for the first 6 h, 10-20 sec
for the second 6 h and 8–10 sec for the next 12 h applying a forward to reverse ratio of 3:1.
216 Drug Screening Methods

TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE–MEDIATED

dUTP NICK END LABELING (TUNEL)
A major disadvantage of all the electrophoretic methods for detecting DNA damage is the
inability to determine the number of cells that are affected by the apoptotic process. The
limitation has been overcome by the development of in situ end-labeling techniques such as
TUNEL. It is based on a simple principle. Terminal deoxyribonucleotidyl transferase (TdT)
catalyses the addition of nucleosides at a free 3’ OH end of DNA, including the 3’ ends produced
by endonuclease action during apoptosis. When the reaction is performed using Co+2 as the
divalent cation, the enzyme will transfer a nucleoside to a blunt 3’ end, a 3’ protruding end or
a 3’ recessed end, albeit with differing efficiencies. A secondary reaction with antibodies or
other detection systems is used to detect the nicks. Thus, the TUNEL technique is a method to
visualize strand breaks in DNA and can be applied to both tissue sections and cell cultures.36
Kits for TUNEL and related assays are commercially available. A detailed procedure to be
followed for the assay is supplied with the kit. The precautions to be taken for TUNEL are: the
slides should be held in glass slide racks and immersed in glass staining dishes. The solutions
should be freshly made. Either superfrost slides or the slides scrubbed with TESPA or poly-L-
lysine at least 2 days prior to application should be used to place the tissue sections. 5-10 mm
thick paraffin sections of the tissue should be prepared.
A general protocol that can be followed for TUNEL is: Slide-mounted tissue sections are
placed on a slide warmer at 45°C for 60 min followed by 15 min each in methanol and 3%
hydrogen peroxide respectively. The slide is then incubated for 15 min in 0.3% Triton-X 100 in
phosphate buffered saline and rinsed with running de-ionized water. The slide is incubated at
37°C for 1.5-2 h in 100 µl buffer constituted by 20 µl 5 × terminal transferase reaction buffer +
2 µl (40 U) terminal deoxytransferase + 2 µl (2 nmol) biotinylated deoxy UTP and 76 ml sterile
distilled water. The slide is then rinsed three times in PBS for 10 min each. Later, the slide is
incubated in avidin biotinylated peroxide complex (1:100 dilution) for 2 h at room temperature
or alternately for 30 min at 37°C. The slide is rinsed in PBS and incubated for 10 min with 25 mg
3, 3’—diaminobenzidine (Sigma) per 50 ml PBS, with 25 µl 3.0% hydrogen peroxide. The slide
is again rinsed with demonized water, dehydrated, defatted and covered with coverslip. The
slide is then observed for apoptotic cells under light microscope.
The TUNEL technique has several advantages. The cell morphology and staining intensity
can be examined simultaneously; it is therefore possible to compare the time course of DNA
fragmentation with morphological changes in individual cells. It can also determine whether
DNA degradation is occurring in a small subpopulation of cells as opposed to the entire cell
population. However, several groups have reported that cells undergoing necrotic cell death
will also be stained by the TUNEL technique.37,38 Therefore, TUNEL must be considered a
sensitive and convenient method for detecting apoptotic cells as long as alternative techniques
are also used to confirm that the cells are actually undergoing apoptosis rather than necrosis.

FLOW CYTOMETRY
The flow cytometer measures the amount of fluorescence that is associated with single cells.
An aliquot of cell suspension is aspirated into the machine, and the fluid is atomized into
droplets so small that the average droplet has 0.05-0.2 probability of containing single cell.
 Techniques for the Detection of Apoptosis 217

These droplets then flow single file in front of a laser and a series of detectors. As each droplet
encounters the laser, photomultiplier tubes detect the light that is scattered as well as the
fluorescence emission that results from excitation by the laser.
Several approaches for the study of apoptosis using flow cytometer have been developed.
The flow cytometer offers several advantages over microscopic methods. Because of the rapid
response of the photomultiplier tubes, it is possible to analyze the fluorescence intensity of
hundreds of cells per second. Also, because of the wide dynamic range of photomultipliers, it
is possible to precisely quantitate the fluorescence intensity over a 10,000-fold range.
Flow cytometry also has some limitations: it requires relatively expensive and sophisticated
equipment that is not easily available; and it is not suitable for the analysis of tissues or even
tissue culture cell lines that fail to yield a single-cell suspension.
The flow cytometry techniques that have been developed are described below.

Dye Uptake
The techniques that rely on altered membrane permeability for detection of apoptosis can
also be adopted to flow cytometry. The cells can be incubated in dye like Hoechst 33258 or
7-aminoactinomycin D at 4°C and then subjected to flow cytometry. Quantitation of the
number of cells that are weakly fluorescent provides an indication of the number of cells
undergoing apoptosis.39
The principle of the method is that these dyes are excluded from cells at 4°C. Cells that
have ruptured readily take up these agents and therefore fluoresce strongly. Whereas,
cells undergoing apoptosis take up small amounts of these compounds at 4°C despite the
presence of an intact plasma membrane. As a result, cells undergoing apoptosis are weakly
fluorescent. It is extremely important to keep cells at 4°C during the entire incubation with
the dye because above its phase transition temperature these dyes freely penetrate the
plasma membrane.
The advantages of this method are that it is possible to rapidly quantitate the number of cells
undergoing apoptosis in a large population of cells. And if these fluorochromes are combined
with fluorescently tagged antibodies they permit the precise quantitation of apoptosis in a
subset of cells (e.g. CD4—expressing lymphocytes in a larger cell population). The disadvantage
of this technique is that the dye uptake is not absolutely specific for apoptosis. Other changes
within cells (e.g. ATP depletion) might also allow small amounts of these dyes into the cells. The
dye uptake assays appear to offer a rapid and powerful technique for investigating apoptosis in
a mixed population of cells as long as this potential limitation is kept in mind.

Decrease in DNA Staining

DNA fragmentation that occurs during apoptosis can also be quantitated by flow cytometry
besides cell fractionation technique described previously. For this, the cells treated with an
inducing stimulus are fixed with ethanol, extracted with an aqueous buffer lacking divalent
cations reacted with propidium iodide or Hoechst 33258, and subjected to flow cytometry.
This technique allows precise determination of the number of cells showing DNA
fragmentation. But it does not distinguish between apoptotic and necrotic cells and therefore
it can potentially overestimate the number of cells undergoing apoptosis.
218 Drug Screening Methods

Altered Size
Early shrinkage of the cell occurs in apoptosis due to loss of water. Because larger cells scatter
light more effectively than smaller cells, cell size can be monitored by changes in light scatter
properties. The cells treated with an inducing stimulus are fixed at different time and subjected
to flow cytometry. Apoptotic cells are distinguished as those particles that have less forward
scatter than control cells.
Although, it is true that apoptotic cells are smaller than nonapoptotic cells, this parameter
alone is probably not sufficient as a single discriminator for distinguishing the two populations.40

Loss of Mitochondrial Transmembrane Potential (ψm)

The predominant localization of Bcl-2 and some of its family members to the mitochondrion
indicates a potential role of it in apoptosis. Mitochondria loose transmembrane potential early
in the course of apoptosis.
The electrochemical gradient that establishes across the inner mitochondrial membrane
during normal oxidative phosphorylation provides energy for concentration of membrane-
permeant cationic dyes against concentration gradients. The accumulation ratio of these
dyes in mitochondria relative to the extramitochondrial space is directly related to the
transmembrane electrochemical gradient. The active concentration of these dyes in
mitochondria reflects normal oxidative phosphorylation, and the failure to accumulate these
dyes reflects a loss of mitochondrial transmembrane potential.41 These observations, which
were initially made using isolated mitochondria, form the basis for flow cytometry‑based
methods of assessing mitochondrial transmembrane potential. In these experiments, cells
treated with inducers of apoptosis are incubated briefly with membrane-permeant cationic
dyes e.g. 5–5’, 6–6’-tetrachloro-1, 1’, 3–3’—tetraethylbenzimidazolcarbocyanine iodide or 3–3’—
dihexyloxacarbocynine iodide, and immediately subjected to flow cytometry. Cells containing
normal ψm contain larger amounts of these dyes due to their concentration in mitochondria. The
appearance of dim staining with this technique reflects a loss of mitochondrial transmembrane
potential.
It has been suggested that this change in ψm might be the first identifiable biochemical
change in cells undergoing apoptosis. However, this change is not universally identifiable
and is not specific for apoptosis. Changes in ψm have been extensively documented in cells
undergoing necrosis and can result from any treatment that alters mitochondrial electron
transport or proton transport e.g. metabolic poisons such as cyanide or azide. Accordingly,
changes in ψm must be interpreted cautiously.

ANNEXIN V STAINING
In healthy cells, sphingomyelin is predominantly present in the outer layer and
phosphatidylserine in the inner layer. During the course of apoptosis, this asymmetry is lost.
Phosphatidylserine becomes accessible on the surface of cells undergoing apoptosis, and can
interact with annexin V, a polypeptide that binds strongly and specifically to phosphatidylserine.
These observations form the basis for histochemical and flow cytometry-based methods of
detecting cells undergoing apoptosis.42,43
 Techniques for the Detection of Apoptosis 219

Figure 12.2: Fluorescence microscopy image of 15 weeks gestational age human fetus brain primary cell culture. Cells
were stained with annexin V- FITC after induction of apoptosis with anti-Fas agonist antibodies. Labeled cells exhibit
condensed nuclear fluorescence while viable cells exhibit diffuse nuclear fluorescence. (Reproduced with permission
from J Cell Mol Med 2001; 5: 179-87)

The cells treated with an inducing stimulus can be fixed using a nonpermeabilizing
fixative (e.g. formaldehyde), treated with fluorochrome-coupled annexin V, and examined
by fluorescence microscopy or subjected to flow microfluorimetry. Alternatively, cells can be
stained without fixation. In either case, cells undergoing apoptosis will be fluorescently labeled
(Fig. 12.2), whereas other cells will not.

ALTERATIONS IN PLASMA MEMBRANE PERMEABILITY

Cells that undergo apoptosis are either phagocytosed by neighboring cells or the cells/cell
fragments lose membrane integrity. The loss in membrane integrity can be detected by agents
that are unable to penetrate viable cells or by the ability to exclude charged dyes such as trypan
blue or propidium iodide. Alternatively, it can also be detected as loss of cytoplasmic contents,
e.g. 15Cr or lactate dehydrogenase.
The advantages of these methods are that they are quantitative in nature and can be used
to examine a large number of cells. However, the disadvantages are: they do not distinguish
between apoptosis and necrosis and therefore must be applied only when other techniques
have established the apoptotic nature of cell death; also these techniques detect only the
change in the later stage of cell death and therefore cannot be used to study earlier changes
in the process of cell death; these are not suitable to study changes in individual cells; these
techniques are not useful in those apoptotic models that involve phagocytosis of the cells e.g.
most in vivo models; and lastly these techniques require viable cells.
220 Drug Screening Methods

Vital Dyes
Vital dyes have been one of the most rapid, simple, inexpensive and useful tools for
distinguishing between viable and nonviable cells both in tissue culture and in living tissues
in situ. Trypan blue, erythrosin or nigrosin are unable to enter normal cells but are readily and
irreversibly taken up by cells that have lost plasma membrane integrity.44
Before applying these dyes, it is important to remember that loss of plasma membrane
integrity occur both in apoptosis and necrosis. Besides, because loss of membrane integrity
occur relatively late in apoptosis, staining with vital dyes will miss “doomed” cells in the early
stages of apoptosis, and will therefore underestimate the number of apoptotic cells. Also dyes
such as trypan blue are themselves cytotoxic depending on concentration and/or time.

Release of Sequestered Compounds

The integrity of the plasma membrane can also be assessed by monitoring the release of
compounds that are actively sequestered by living cells. Incubation of cells in medium
containing diacetyl fluorescein or 15Cr, leads to active uptake and concentration of these
compound within cells. Measuring the release of such compounds into culture medium has
been used as a means of quantitatively assessing cell death.45 In a related approach, quantitative
information has also been derived by measuring activity of cytoplasmic enzymes (e.g. lactate
dehydrogenase or adenylate kinase) released into culture medium by dying cells.46
However, these methods are again of little value in understanding the early events of
apoptosis. The assays of cytoplasmic enzyme release must be carefully controlled to take into
account differences in enzyme activity between cell types or loss of tissue culture sera. Again
the method is nonspecific for detection of apoptotic cell death and therefore should be used
only in conjunction with the methods that can clearly discriminate between the two forms of
cell death.

ENZYME ASSAYS
The identification of morphological and biochemical changes that occur during apoptosis
has led to continued efforts to study the enzymatic activities responsible for those changes.
Several enzyme activities have been reported to be increased in cells undergoing apoptosis.
These include tissue transglutaminases,47 deoxyribonuclease48 and ICE family proteases.49, 50
Studies of these enzymes have generally involved three different approaches: direct assays for
activities that are thought to be altered; application of enzyme inhibitors to assess the effect of
perturbing enzyme activity; and genetic approaches involving overexpression (transfection)
or underexpression (antisense oligoneucleotides or genetic knockouts) to assess the effect of
altering enzyme content.
In setting up assays for enzyme activities, several considerations must be kept in mind. First,
the assay should be as specific as possible. Second, if assays are being performed to compare
relative amounts of enzyme activity present under different conditions, the assays should be
performed under conditions in which the product formed is directly related to the amount of
enzyme present. Finally, the activity should be investigated under conditions that approximate
the intracellular milieu during apoptosis. Adherence to these principles can help eliminate
misinterpretations of data.
 Techniques for the Detection of Apoptosis 221

RECENT ADVANCES
Ribble and colleagues have recently described a simple and rapid technique for quantifying
apoptosis in 96-well plates.51 The authors have modified ethidium bromide and acridine
orange staining assay that can be performed entirely in a 96-well plate format. The technique
can be used to quantify apoptosis of suspension cells as well as adherent cells. The technique
eliminates the detaching and washing steps, which drastically reduces the time needed to
perform the test, minimizes damage to adherent cells, and decreases the possibility of losing
floating cells.

Lipid Proton MR Spectroscopy

The first MRI technique applied to the detection of apoptosis was lipid proton magnetic
resonance spectroscopy.52 These studies described apoptosis-specific changes, including
a selective increase in CH2 (methylene) relative to CH3 (methyl) mobile lipid proton signal
intensities at 1.3 and 0.9 ppm, respectively. The rise in CH2 resonance occurred with a wide
range of apoptotic drugs as well as apoptosis associated with serum (growth factor) deprivation.
The CH2/CH3 ratio also had a strong linear correlation with other markers of programmed cell
death, including fluorescent annexin V cytometry and DNA ladder formation. Although, there
was an increase in the methylene resonance, there was no detectable change in total lipid
composition or new lipid synthesis, suggesting an increase in membrane mobility as opposed
to increased amounts of lipids within cells.53

Diffusion Weighed MRI

Diffusion-weighted MRI (DWI) is an alternative MRI modality that can image apoptosis
in response to radiation and chemotherapy without the need for a contrast agent.54 DWI
generates image contrast by using the diffusion properties of water within tissues. Diffusion
can be predominantly unidirectional (anisotropic) or not (isotropic) and can be restricted
or free depending on the amount of water in the extracellular (relatively unrestricted) or
intracellular (restricted) compartments. Diffusion-sensitized (weighted) images can be
acquired with magnetic gradients of different magnitudes, generating an apparent diffusion
coefficient (ADC) map. As increases in cellularity are reflected as restricted motion, DWI has
been used in cancer imaging to distinguish between tumor (restricted microenvironment)
and peritumoral edema (unrestricted). DWI may also be valuable in monitoring treatment,
where changes due to cell swelling and apoptosis are measurable as changes in ADC. The
magnitude of changes, however, is small (i.e. < 50% of control), and it may be difficult to
separate tumor shrinkage, necrosis, and other processes that can occur with therapy.55
Therefore, more studies are needed to confirm the validity of DWI as a marker of therapeutic
efficacy in the clinic.

Detection of Apoptosis with Ultrasound

High-frequency ultrasound (40 MHz or greater) has been used to detect the unique specular
reflections of apoptotic cells in vitro and in vivo. Backscatter from apoptotic nuclei is up
to 6-fold greater than that from nonapoptotic cellular nuclei. The specific nuclear features
222 Drug Screening Methods

resolved at 40 MHz include fragmentation of DNA and chromatin condensation, which occur
relatively late in the apoptotic cascade. Unfortunately, the significant energy loss with the soft
tissues at these higher frequencies currently limits high-frequency ultrasound to the study
of the skin and other superficial structures. High-frequency ultrasound, however, could be
quite useful for the study of apoptosis in the brain (and possibly other organs) of neonates
and young infants. The open fontanels of neonates provide excellent sonographic windows for
the high-frequency ultrasonographic study of apoptosis known to be associated with hypoxic
ischemic injury.56 In fact, in the most recent study (2005), Tunis et al. developed statistical
methods at a frequency of 20 MHz that enable the monitoring of structural changes within a
very low percentage of apoptotic cells in a tissue, raising the possibility of using this technique
in vivo, particularly in the premature neonatal brain, which is at high risk for periventricular
leukomalacia (PVL).57

CONCLUSION
It is very important to remember that none of the above techniques are highly specific for
the detection of apoptosis. Therefore, it is mandatory to use more than one technique(s)
to establish the biochemical nature of cell death as apoptotic. The techniques that can be
employed to study apoptosis will depend upon the type of the experiment, expected end result
and feasibility to use a particular technique with reference to the tissue under investigation as
well as facilities available.

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 Techniques for the Detection of Apoptosis 225

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 CHAPTER

13
Anticataract Agents
INTRODUCTION
Cataract is the clouding of lens, now considered to be the inevitable consequence of aging and
can only be repaired by surgery. Cataract, responsible for 50% or more of blindness globally,
remains the leading cause of visual impairment in all regions of world, despite improvements
in surgical outcomes.1-3 More than 28,000 new cases are reported daily worldwide.4 Several
factors like genes, socioeconomic status, illiteracy, malnutrition, diarrhea, diabetes, myopia,
renal failure, hypertension, sunlight exposure, smoking, steroids, etc. are responsible for the
development of cataract. Out of 38 million blind persons, 8.9 million are in India and 5.12
million are blind due to cataract.4 The annual incidence of cataract blindness is about 3.8
million. Annually 2.7 million cataract operations are carried out, yet they are not adequate to
clear the backlog.5 During the last two decades, extensive research inputs have been made to
delineate the etiology of cataract. Efforts have been directed to delay the onset and slow down
the progression of cataract by a variety of agents.These agents have been classified into different
categories; their anticataract properties have been evaluated in different experimental models
of cataract.
Cataract, a multifactorial disease, occurs mainly due to the formation of large protein
aggregates in the lens. Research has shown that post-translational modifications of lens
crystallins such as oxidation, glycation, Schiff’s base formation, carbamylation, transamidation,
phosphorylation and proteolysis lead to clouding of the lens (Fig. 13.1).6-8
Drugs effective in modulating the altered metabolism and lens pathology may delay the
progression of protein aggregation and opacification. The agents that are claimed to be effective
in vitro and in vivo models of cataract have been classified as aldose reductase inhibitors, non-
steroidal anti-inflammatory drugs, calpain inhibitors, antiglycatics, antioxidants of natural/
synthetic origin and a group of miscellaneous agents having different chemical structure. In
this chapter, a few commonly used in vitro and in vivo experimental models are described.

EXPERIMENTAL MODELS OF CATARACT

Experimental models are indispensable tools for the better understanding of physiology and
pathogenesis of cataract. Cataractogenesis can be interrupted in experimental models at
intervals to study the intermediate structural changes to establish or determine the causative
 Anticataract Agents 227

Figure 13.1: Various pathways involved in cataractogenesis

mechanism. Several in vitro and in vivo models that mimic human senile cataract have been
developed indicating crucial targets to intervene and prevent the lens damage. Many of these
models however, share few common final steps during the development of cataract.

IN VITRO MODELS
In vitro models include:
•• Cell culture
•• Organ culture.

Human Lens Epithelial Cell (HLEC) Culture

Most of the studies related to etiology and mechanism of cataract are conducted on animals. In
spite of being very closely related to human senile cataract these studies fail to show the exact
mechanism. Studies with culture of human lenses are also not possible as enough number
of lenses of same age and sex are not easily available. Besides the whole lens is processed for
biochemical estimations which results in several fold dilution of the lens epithelial cells. This
is the outermost layer and metabolic unit of lens, which is constantly abused by the changing
climatic conditions. This layer has various antioxidant enzymes and helps in maintaining the
lens homeostasis. Culturing the HLEC makes the studies on etiology and various pathways
easier as it offers superior control of physiological environment, accessibility for biochemical
monitoring. It is used as a screening model for anticataract agents.9
Procedure: Human eyes obtained from the cadaver within 8 h of death are dissected for
obtaining the lens epithelium. The anterior capsule with the adhering subcapsular layer of
epithelial cells is dissected out and spread on the surface of the culture flask. 4 ml of Dulbecco’s
228 Drug Screening Methods

Figure 13.2: Human lens epithelial cells: (A) Normal HLEC cultured in DMEM alone; (B) HLEC cultured in DMEM
under oxidative stress

modified eagle’s medium (DMEM), supplemented with 20% fetal calf serum, is added to the
flask. The flask is kept in a CO2 incubator maintaining a temperature of 37°C. The cells are
subcultured when they are semiconfluent. To evaluate the potential of anticataract agent the
equal number of cells are distributed in different flasks. Cells in one of the flasks are incubated
in normal conditions and others are incubated under stress conditions generated by addition
of oxidants in the medium. The anticataract agent is added to one of the flasks containing the
stress inducer. As per the requirement of the study incubation period may vary. Following
incubation morphological and biochemical variations can be evaluated and compared to test
the anticataract potential of the agent (Fig. 13.2). 10,11
Recently the human lens epithelial cell line SRA01/04 is being used for studying various
pathways and drug targets involved in opacification of lens.12 Studies using human lens
epithelial cell line have shown that the epithelial mesenchymal transition (EMT) plays a key
role in anterior subcapsular cataract (ASC) and posterior capsular opacification (PCO).13,14
The Jagged/Notch pathway has been reported to be essential in EMT during embryonic
development, fibrotic diseases and cancer metastasis. However, the function of Jagged/Notch
signaling in LEC EMT is unknown. With the hypothesis that crosstalk between Notch and
TGFβ2 signaling could induce EMT in LECs, the studies were conducted and it was shown that
inhibition of the Jagged/Notch signaling may have therapeutic value in the prevention and
treatment of ASC and PCO.

Human Capsular Bag Model

Posterior capsular opacification is a common complication of cataract surgery. PCO develops a
secondary loss of vision in significant number of patients.15 Modern cataract surgery generates
a capsular bag, which consists of a portion of the anterior capsule and the entire posterior
capsule. The capsular bag remains in situ separating the aqueous and vitreous humors, and,
in most cases, houses an IOL. Some of the epithelial cells in spite of the trauma of the surgery
remain in the anterior capsule and grow and reach the IOL surface occupy regions of the outer
anterior capsule, and colonize the previously cell-free posterior capsule. These cells can finally
reach to visual axis hampering the vision if the changes to the matrix and cell organization are
severe. This may lead to the need of corrective laser surgery.16-18 Studies have reported that
 Anticataract Agents 229

the rate of PCO can be reduced by improving the design of IOL. These IOLs are manufactured
using various range of materials and can affect the progression of PCO because of their contact
with the capsule, creating a barrier effect.19 The IOL designs were tested in rabbit models
because of the similarities in lens size, however, the response of injury was more severe in
rabbits. Development of human capsular bag model to test the new IOL designs is a valuable
tool and reduces the use of animals.
Method: The method for preparing the human capsular bag is an adaptation of the method
earlier described by Liu et al.20 Whole donor eyes are used after ethics committee approval for
performing sham cataract surgery with anterior capsulorhexis, nucleus hydroexpression and
aspiration of lens fibers.
Sham cataract surgery is done in a laminar flow hood on whole donor eyes obtained within
48 hours of death. The lenses are washed briefly with EMEM. A small rhexis in the anterior
surface of the lens capsule is created and central fibrous mass from donor globes is removed.
The capsular bag thus obtained can house an IOL when needed. The capsular bag containing
an IOL is subsequently removed from the eye separating the zonules with utmost care. It is
then transferred to a tissue culture dish with anterior side facing down for better physical
interaction between the IOL and capsule. The bag is secured to the dish with entomological
pins and maintained in EMEM supplemented with 2% human serum, 10 ng/ml TGF-β2, and
50 μg/ml gentamicin (Sigma) for a period of four weeks. In the partner capsular bag, no IOL
was implanted. Capsular bags are compared using phase-microscopy, immunocytochemistry
with fluorescence microscopy.15, 21, 22

Organ Culture
Induction of cataract in isolated animal lenses maintained in organ culture has become
a convenient, quick and appropriate method for testing the anticataract efficacy of an
agent. Opacification of lens is induced by generating oxidative stress/hyperglycemic/
hypergalactosemic conditions around the lens by supplementing the culture medium with a
variety of exogenous substances.23, 24
In general enucleated animal lenses are individually maintained in 2 ml of physiologically
competent tissue culture medium (TC-199/MEM) supplemented with 10% fetal calf serum
at 37°C and 5% CO2 atmosphere in a 24 well Falcon plate. The lenses are incubated for 2 to
16 hours prior to the initiation of the cataractogenic insult. Opaque lenses, if any, damaged
during the extraction procedure are discarded. Supplementing the medium with different
agents causing oxidation of lens proteins/lipids directly/indirectly induces cataractogenesis.
Lenses incubated in high sugar containing medium mimic sugar cataract. To carry out the
anti-cataract screening process, transparent lenses are divided into different groups as per the
requirement of the study. Few lenses are incubated in the plain culture medium, representing
normal group and few in culture medium supplemented with stress inducing agent to serve
as control. Efficacy of test drug is evaluated by maintaining treated groups, the medium of
which as the control is additionally supplemented with different concentration of test drug.
Some lenses are also incubated in culture medium containing only the test drug. Lenses
belonging to these four groups are further incubated for the period varying from 24 to 72 h
depending upon the experimental conditions. Morphological changes and the changes in
230 Drug Screening Methods

various biochemical parameters occurring in the different groups are compared to evaluate
the efficacy of the test drug. Morphological changes in terms of opacity are categorized as faint
peripheral, cortical, dense cortical or nuclear opacities. The biochemical parameters include
levels of glutathione (GSH), malondialdehyde (MDA), polyol and enzyme activities such as
aldose reductase, sodium potassium ATPase, superoxide dismutase (SOD), catalase (CAT),
glutathione peroxidase (GSHPx) and glutathione S-transferase (GST), etc.

Oxidative Stress-induced Experimental Cataract

Oxidative mechanisms play an important role in many biological phenomena including
cataract formation. Formation of the superoxide radical in the aqueous humor, lens and
its derivatization to other potent oxidants may be responsible for initiating various toxic
biochemical reactions leading to the formation of cataract (Fig. 13.3). In vitro, such cataracts
are induced by agents like selenium, H2O2, photosensitizers and enzyme xanthase oxidase.
Lenses exposed to 100% oxygen showed optical and structural changes. 25

Selenite-induced Cataract
Selenium, an essential nutrient but a hazardous element, plays a critical role in maintaining
the normal physiological conditions of lens. Selenite-induced cataract models have been
used to quantify and characterize events that occur in the lens prior to the formation of nuc­
lear cataract. The critical sulfhydryl (SH) group on Ca2+ ATPase is oxidized by selenium and
opens ion channels in the lens epithelial membrane allowing the influx of calcium from
the aqueous humor. Elevated calcium levels activate calpain, a cytosolic calcium activated
protease, by autolysis, thereby exposing a buried sulphydryl group in the active site of calpain.
Elevated calcium ions translocate calpain to the plasma membrane where phosp­holipids
lower the calcium activation requirement for calpain. The membrane provide locali­zation
site for hydrolysis of substrates. High levels of activated cytosolic calpain are also avai­lable for
proteolysis. The N-terminal extensions on soluble β-crystallin dimer (βL) are clea­ved by calpain.

Figure 13.3: Free radical generation in the eye and endogenous defense
 Anticataract Agents 231

This probably exposes charge groups on the βL that interacts to form in­so­luble aggregates.
Hydrophobic interactions may also occur. Hydrolysis of cytoskeleton and membrane proteins
by calpain causes further leaking and cell disruption that finally leads to light scattering and
opacity.26,27 Selenium has been used to induce opacity both in iso­lated lenses and in young rats
employing various dosage form and routes of application.27-29
Procedure: In vitro cataract is produced by supplementing the tissue culture medium with 25
to 100 μM sodium selenite in which freshly enucleated transparent rat lenses are incubated at
37°C. This causes membrane damage and faint cortical opacities within 24 h.

Photochemically-induced Cataract
Photochemically induced oxidative insult is reported by Spector et al.30 Riboflavin, a
photosensitizer is supplemented in the culture medium to induce cataract in cultured
lenses. Micro quantities (4-200 µM) of riboflavin lead to severe physiological damage and
opacification within 24 h after exposure to light. The initial membrane damage is evidenced
by a disturbed cation ratio between lens water and the medium of incubation. Riboflavin on
getting photosensitized generates free radicals in a sequence of reactions.
Procedure: Lenses are maintained in organ culture for 24 to 72 h as described earlier. The
lenses are divided into four groups and incubated in the dark and light both in presence and
absence of riboflavin. The lenses are exposed to light with two 15-W day light fluorescent
lamp placed at 8 inches above the cluster plate. The culture medium is replaced every 24 h.
Riboflavin shows no effect on the lens in the absence of light, and light without riboflavin has
no significant effect. Opacification starts in the equatorial zone and gradually extends towards
the center of the lens.31

Enzymatically-induced Cataract
Supplementation of culture medium with 1 mM xanthine and 0.1 unit of xanthine oxidase,
which acts as substrate and enzyme respectively, leads to generation of superoxide radical. The
lenses suffer severe oxidative damage and turn opaque within 24 h when incubated in culture
medium at 37°C.23

Hydrogen Peroxide-induced Cataract

Incubation of lenses in medium, containing 50-500 µM H2O2, produces cataract. Opacification
starts in the equatorial region within 24 h. The entire superficial cortex becomes opaque by
96 h. Due to the high instability of H2O2 the medium is changed every 2 h during the first eight
hours.31,32

Sugar-induced Cataract
Diabetes is one of the most important risk factors of cataract. Enzyme aldose reductase (AR)
has been implicated to play a pivotal role in sugar cataract formation. AR acts on the sugars
like glucose, galactose and xylose and converts them into their respective alcohols. These
alcohols (polyols) are not directly involved in the cataractogenic mechanism. If they are found
within the lens they accumulate to high levels and produce osmotic effects. Since polyols are
232 Drug Screening Methods

not capable of diffusing out easily from the lens nor metabolize rapidly, they accumulate in
lens causing hypertonicity. Increase in intralenticular tonicity draws water into the lens fibers
causing them to swell. Sugar cataracts have been produced in enucleated lenses in vitro.33,34
Procedure: Transparent and undamaged lenses are incubated in a basic culture medium with
fetal calf serum for 24 to 48 h. The experimental groups are formed as described earlier. In
the control group the medium is supplemented with glucose (30 mM), galactose (30 mM) or
xylose (20 mM). Lenses develop opacity in the subcapsular region on day 1 and in the central
region on day 2. Biochemical analyses reveal raised polyol, malondialdehyde levels and water
content, and decreased glutathione levels in these lenses.

Steroid-induced Cataract
Steroid induced experimental cataract is produced in vitro by incubating the transparent
lenses in the medium containing methyl prednisolone (1.5 mg/ml). To screen anticataract
effect of any agent different experimental groups are formed as described earlier. The test agent
and methyl prednisolone added alone and together to the medium form drug control, control
and treated groups respectively. Early cataract around the equator is produced within 24 h
of incubation. Incubation period may be extended to 48 h for dense opacity.35 Morphological
changes and modulation in biochemical parameters between the groups may show the
potential of the anticataract agent.

Naphthalene-induced Cataract
Naphthalene is insoluble in the culture medium hence naphthalene metabolites are used for in
vitro studies of which naphthalene dihydrodiol shows similar morphological and biochemical
effect on the cultured lenses as observed in naphthalene fed rats.36
Procedure: TC-199 medium modified by Zigler and Hess is used for the pre incubation of
lens.37 Stock solution of naphthalene dihydrodiol is prepared in 20% ethanol at 2.5 × 10–3 M
con­centration. The stock solution is diluted 1:100 to obtain the final concentration of 2.5
× 10–5 M. The final osmolarity of the solution is 295-300 mOsmol. Rat lenses are incubated in
TC-199 medium containing naphthalene metabolite solution. Medium is renewed daily till 72
h. Lenses remain clear during the initial 24 h but form shell like opacity around the nuc­leus by
48 h. Opacification becomes more peripheral and widespread after 72 h. At 48 h, under such
conditions of incubation, development of opacity mimics the in vivo naphthalene cataract.36

Ca++ - induced Cataract

Human and animal cataract lenses contain higher levels of calcium than normal lenses. The
homeostasis of ions such as Na+ and K+ is affected by the abnormal calcium metabolism.
Influx of calcium into the lens activates cysteine proteinase, calpain I and II, which in turn
degrades cytoskeleton components and the lens crystallins and eventually causes crystalline
aggregation resulting in cataract formation.38, 39
Procedure: In this model the control group contains the lenses incubated in the medium
enriched with 20 mM Ca2+ or 1 × 10–2mM A23187 calcium ionophore. The treatment group
lenses are cultured in the calcium and the test drug-containing medium. Incubation period
 Anticataract Agents 233

can range from 24-72 h. Light scattering intensities can be compared as described by Siew and
Bettelheim.40

Ultraviolet-induced Cataract
Epidemiological studies have shown a link between exposure to UV-B radiation in sunlight
and development of cataract. Experimental studies confirm that ultraviolet radiation (UVR)
induces cataract. Ultraviolet (UV) light damages the lens by increasing free radicals. Choh et al
tested the antioxidant property of a Chinese herb goji berry using in vitro model.41
Procedure: Bovine lenses are dissected and cultured for 24 hours in the culture medium.
Lenses are placed in the medium with or without test drug, then placed into an incubation
chamber equipped with UVB light (2.0 J/cm2) for two hours. Control lenses are placed in
lightproof cardboard boxes before being added to the UV irradiation chamber. Optical
quality is assessed using a scanning laser monitor. Lenses are scanned prior to UV-irradiation
(baseline) and at different intervals post radiation for a period of two weeks. The absorbance of
the culture medium with and without test drug is determined at 280- 320 nm UV range.

IN VIVO MODELS
Several animal models of cataract are established for the screening of anticataract agents.
These models include sugar, oxidative stress, radiation-induced cataract, etc.

Sugar Cataract Models

Sugar cataract is produced in rats by feeding them high sugar diet such as galactose28,42,43
or impairing their insulin production using agents like streptozotocin or alloxan. Albino
rats (Wistar/Sprague Dawley) are used to evaluate the mechanism of diabetes related
cataractogenesis in animals. The eyes of the rats are first examined through a slit lamp to see
any abnormality in lens or cornea and if found the rats are discarded. The rats are grouped with
a comparable weight distribution. The commonly used sugar cataract models are described
below.

Galactose-induced Cataract
Rats of either sex, weighing 50 to 60 g are fed 30% galactose in diet. Diet and water are given ad
libitum. The rats are divided into two groups, control and treated. The test agent is administered
orally or topically in the treatment group. The eyes of the rats are examined weekly by using
a slit lamp to see the cataractogenic changes. The vacuoles start appearing at the periphery
of the lens within a week’s period, which may be attributed to globular degeneration of the
fiber cells. The vacuoles gradually increase in number and size as they extend towards the
centre. This takes about 14 days. All these changes are not visible through unaided eye. During
the third week opalescence of the lens is visible and it becomes totally opaque in 30 days time
(Fig. 13.4). Different stages of cataract are graded as given below.44
•• Stage 0 — Lenses similar to normal lenses
•• Stage I — Lenses showing faint peripheral opacity
•• Stage II — Irregular peripheral opacity with slight involvement of the lens in the center
234 Drug Screening Methods

Figure 13.4: Slit lamp photographs of various stages of galactose-induced cataract in rats. Normal: Clear transparent lens;
Stage I: Peripheral vacuoles in the lens; Stage II: Vacuoles involving the center of the lens; Stage III: Faint opalescence visible
with the naked eye; Stage IV: Mature nuclear cataract. [Reprinted from Nutrition, Vol. 19(9), Suresh Kumar Gupta, Deepa
Trivedi, Sushma Srivastava, Sujata Joshi, Nabanita Halder and Shambhu Dayal Verma. ‘Lycopene attenuates oxidative
stress induced experimental cataract development: An in vitro and in vivo study’, pp 794-9, 2003, with permission from
Elsevier]

•• Stage III — Faint opalescence visible with the naked eye

•• Stage IV — Mature nuclear cataract
•• Stage V — Opacity involving entire lens.
Morphological changes in the lens are compared in all the groups to evaluate the efficacy
of the drug. Biochemical changes in lens related to galactose cataract may also be measured at
different stages of opacification to support the findings.
A dog model of cataract has also been reported by Sato et al.45 Nine-month-old beagles are
fed a daily diet of 450 g of standard chow containing 30% galactose for 9 months. The dogs
developed cataract in 39 months.

Alloxan-induced Cataract
Alloxan is a cyclic urea analog, which produces permanent diabetes in laboratory animals.
According to one of the mechanisms proposed it is a highly reactive molecule that is readily
reduced to dialuric acid, which is then auto oxidized back to alloxan resulting in the production
of H2O2, O2, O2-, and hydroxy radical. In vivo administration of alloxan induces DNA strand
 Anticataract Agents 235

breaks in isolated islets and in islets. However, the other mechanism reveals the ability of
alloxan to react with protein sulfhydryl groups on hexokinase, a signal recognition enzyme
in the pancreatic β-cell that couples changes in the blood glucose concentration to the rate
of insulin secretion. By this mechanism, inhibition of glucokinase and other SH-containing
membrane proteins on the β-cell would eventually result in cell necrosis within minutes.
Procedure: Rats of Wistar/Sprague Dawley strain weighing 150 to 200 g are given subcutaneous
injection of alloxan @ 100 to 175 mg/kg body weight. After approximately 12 weeks cataractous
changes are observed. Alloxan-induced cataract can also be produced in rabbits (2.0 to 3.5 kg)
by infusing 150 mg/kg alloxan monohydrate in the ear vein. Morphological examinations of
lens of all the animals in age matched normal, control and treated groups for the presence of
opacity helps in evaluating the efficacy of the drug.46-48

Streptozotocin Cataract
Diabetes related cataractogenic changes are seen in the animals injected with STZ.49-51 The
chemical structure of streptozotocin (STZ) has a glucose molecule with a highly reactive
nitrosourea side chain, which supposedly initiates its cytotoxic action. The glucose moiety
directs this agent to the pancreatic β-cells. There it binds to the membrane receptor to generate
structural damage. At the intracellular level three major phenomena are responsible for
β-cell death (i) process of methylation, (ii) frees radical generation and (iii) nitric oxide (NO)
production. The damage caused to β-cells alters the sugar metabolism leading to diabetes.
Induction of cataract: Albino rats (Wistar/Sprague Dawley) of 150 to 200 g body weight are
used. Diabetes is induced by intraperitoneal injection of streptozotocin 50 to 70 mg/kg body
weight. Care should be taken not to puncture the intestine. Streptozotocin is dissolved in
0.02 M sodium citrate buffer. The solution is filtered through a 0.22 µM Millipore filter into a
sterilized container placed on ice. The sterile solution thus prepared is used within 10 minutes
of preparation. The nondiabetic rats are injected with sterilized buffer alone. Blood glucose
levels of each rat are estimated after 3 days. STZ injected rats having blood glucose level <150
mg/dl are reinjected with fresh solution of STZ and tested again for blood glucose.50 Progression
of cataract stages is observed through a slit lamp. The initiation of cataract occurs 15 days after
STZ injection. The sutures become prominent and the fully mature cataract appears nearly in
110 days depending upon the age of the rat at the time of injection. The cataractogenic changes
can be compared with the age matched buffer injected control rats.
Hegde and Varma (2005) preferred mice to rats for streptozotocin cataract induction
because of the reported similarity between the lenses of the mice and humans in respect of
AR deficiency and the similarity in morphological changes. They induced cataract in mice by
injecting streptozotocin intraperitoneally. Morphological changes similar to those in humans
such as shrinkage, elongation, lobulization of the nuclei of the lens epithelial cells were
observed.51
Care of diabetic rats: Diarrhea often occurs in diabetic rats and they drink large amount of
water and produce high urine volume. Bedding is changed frequently to keep the rats dry.
The rats should be kept dry or else there is a risk of loosing body heat. Diabetic rats should be
provided with plenty of water.
236 Drug Screening Methods

Figure 13.5: Selenite-induced cataract in rat pups: (A) 16-day-old rat pup showing normal eye with clear lens; (B) Rat pup
of the same litter injected subcutaneously with sodium selenite showing nuclear cataract

Selenite-induced Cataract
Selenite cataract is the most reliable and reproducible experimental model for initial screening
of potential anti-cataract agents. Selenite cataract resembles human cataract in many ways
such as vesicle formation, increased calcium, insoluble protein, decreased water-soluble
proteins and reduced glutathione (GSH), etc. However, selenite cataract shows no high
molecular weight protein aggregation or increased disulfide formation and is dominated by
rapid calpain-induced proteolytic precipitation while senile cataracts may be produced by
prolonged oxidative stress.23,24
Induction of cataract: Selenite nuclear cataract is usually produced in neonatal albino
rats (10 to 14 days) by a single subcutaneous injection of 19 to 30 µmoles/kg body weight of
sodium selenite (Na2SeO3).26,27,52-54 Young rats are housed together with their mother. Mother
is fed normal diet and water ad libitumand and she suckles the pups. The young rats of same
age are grouped into three. One group is left as normal group. Rest of the two groups, control
and treated, are given subcutaneous injections of sodium selenite in the scruff of the neck/
abdomen of young rat. Treatment group is given the anticataract agent intraperitoneally four
hours prior to the sodium selenite administration. On the 16th day when the eyes of the young
rats first open severe bilateral nuclear cataracts are seen (Fig. 13.5). The eyes of the treated
group can be compared with the control group to evaluate the efficacy of the drug.28,52
Repeated injections of smaller doses of selenite29 or oral administration are also
cataractogenic.55

Naphthalene Cataract
Naphthalene cataract model is thought to be a good model for human age related cataract.
Van Heyningen and Pirie56 studied naphthalene cataract in rabbits and proposed the following
metabolic pathway. The injected naphthalene is oxidized in the liver first to an epoxide and
then is converted into naphthalene dihydrodiol. This stable compound on reaching the eye
gets converted enzymatically to dihydroxynaphthalene. Being unstable at physiological pH,
1,2-dihydroxynaphthalene spontaneously autoxidises to 1,2 naphthoquinone and H2O2. Rees
and Pirie57 provided further evidence that 1,2 naphthoquinone is a highly reactive compound.
It alkylates proteins, glutathione and amino acids, and generates free radicals.
 Anticataract Agents 237

Induction procedure: Albino male rats (125 to 150 g) are used for this model. Naphthalene
solution (10%) is prepared in mineral oil by heating at 60°C for 30 min. The rats are dosed
with this solution with an 18-gauge needle at 0.5 g per kg per day for three days and 1.0 g
per kg per day thereafter. A group of rats is administered with same amount of mineral oil to
serve as controls. The drug treatment is given orally by gavage one hour prior to naphthalene
administration. Morphological changes in the eyes of the rats are observed through slit lamp
examination after dilating the pupil. The lenses are examined and graded twice a week during
the first two weeks and thereafter at weekly interval. One week after the administration spoke
like opacities in the cortex is seen. By the third week in the deep cortex region an opaque shell
is visible which becomes denser and slightly deeper with time.36

Hyperbaric Oxygen-induced Cataract

Hyperbaric oxygen (HBO) has been shown to produce cataract in rabbits58 and guinea pigs.59-61
Old guinea pigs of 17-18 months age are subjected to 2.5% atmospheres of 100% oxy­gen for
2.5 h three times per week on alternate days till seven months. This induces high mole­cular
weight aggregate formation in the nucleus resulting in increased nuclear light scattering.

Glucocorticoid-induced Cataract in Developing Chick Embryo

Steroid cataract is reviewed by Urban and Cotlier.62 Formation of steroid–adduct protein,
induction of transglutaminase and reduction of ATPase activity may lead to cataract. Nishigori
et al. (1987)63 suggested that steroid cataract are produced by the activities of glucocorticoids
and progressed by way of production of oxidative stress similar to other types of cataract.
Induction of cataract: Fertile white Leghorn eggs are incubated in an incubator at 35.5°C and
68% relative humidity. The onset day of incubation is called day 1. To 15-day-old embryos,
hydrocortisone succinate sodium (HC; 0.25 μmol in 0.2 ml sterilized water) is administered
through a small hole in the eggshell over the air sac. The puncture is sealed with a cellophane
tape and the eggs are incubated for 48 h. Anticataract agent is applied in the same way as HC.
For the control embryos same quantity of sterilized water is administered. Lenses are removed
from the chick embryo after 48 h of HC administration and the severity of opacity is visually
classified according to Nishigori et al. (1983).64

L-Buthionine –S, R-Sulfoximine (BSO)-induced Cataract

Glutathione is present in mammalian lens in high concentrations and is involved in the
protection of lens against oxidation. In most of the cataracts the decrease in its level is observed.
BSO, a specific inhibitor of GSH, generates cataract in suckling mice when repeatedly
injected. Four subcutaneous injections of BSO are administered per day to the mouse pups on
postnatal days 7 and 8 at intervals of 2.5 h. The dosage of each injection is 4 µmol per gram body
weight (20 ml g-1 of a 0.2 M solution of BSO prepared in 0.10 M NaCl). Initiation of opacification
occurs on day nine, i.e. within 24 h. The progression of the cataract in 24 h is divided into
four stages (i) developing floriform, (ii) mature floriform, (iii) degenerate floriform and (iv)
amorphous translucent cataract. Dense cortico-nuclear opacities develop within several
days.65
238 Drug Screening Methods

N-Methyl-N- Nitrosourea-induced Cataract

Cataract is induced by a single intraperitoneal injection of 100 mg/kg N-methyl-N-nitroso urea
(MNU) in 0, 5, 10, 15, or 20-day-old male and female Sprague Dawley rats. In 0, 5, 10 and 15
day old MNU–treated rats, mature cataract develops at 7, 14, 19 and 30 days respectively after
dosing. In 20-day-old MNU-treated rats, only subcapsular cataract has been seen 30 days after
dosing. Therefore the rats exposed to MNU at an earlier age develop cataract more rapidly
and severely. The pathogenesis of MNU-induced cataract is associated with DNA adducts
formation in the lens epithelial cell nuclei leading to apoptosis by up regulation of Bax protein,
down regulation of BCl-2 protein and activation of caspases-3.66,67
Women have a high incidence of cataract and epidemiological data suggests that the
increased risk may be caused by a lack of estrogen in postmenopausal year. Effect of estrogen
on MNU-induced cataractogenesis is very well documented and serves as a good model for
age-related cataractogenesis.

Transforming Growth Factor β (TGF-β)-induced Cataract

Hales, et al produced experimental cataract in nine months old Wistar rats by injecting
approximately 60 ng TGF-β into the vitreous.69 TGF-β stimulates lens epithelial cells to undergo
aberrant morphologic and molecular changes that mimic the changes observed in human
posterior subcapsular and cortical cataract.

Smoke-induced Cataract
Avunduk et al. induced cataract in Wistar rats by cigarette smoke.70 Cigarette smoke contains
trace and heavy metals.71,72 The increased metal contents in lens causes lens damage by the
mechanism of oxidative stress forming oxygen radicals via metal catalyzed Fenton reaction.73
In other words cigarette smoke-induced cataractogenesis is associated with the accumulation
of iron and calcium in the rat lens.
Procedure: Male Wistar rats of 200 to 350 g weight range are equally divided into control
and test groups. The rats are fed with standard rat chow and water ad libitum. The test group
rats are exposed to cigarette smoke for 2 h/day continuously for 60 days using an exposure
system as described by Chen et al.74 Control rats are exposed to room air in identical chambers.
Morphological and histological changes between the groups are compared; elemental
concentration of the lenses of both experimental and control rats can be measured and
compared.

UV-induced Cataract
In vivo studies have been conducted to show the effect of UV radiations on eye. There is,
however, a lack of data on the age dependence in experimental UVR cataract.
Procedure: Anesthetized albino rats are exposed in vivo to UV-B radiation. The UVR source
is a mercury lamp with water filter and a double monochromator set to 300 nm and 9 nm
full bandwidth at half maximum. The dose ranges between 0.1 and 20 kJ/m2. The exposure
time is 15 minutes. One eye in each rat is irradiated. Before the irradiation the rats receive
pupil-dilating eye drops. The animals are kept between 6 h and 32 weeks after exposure. The
 Anticataract Agents 239

extracted lenses are photographed. The UVR-induced cataract is produced after one week of
the exposure. One or eight weeks after exposure the forward light scattering in the lenses is
determined. The lens is placed in a cuvette filled with salt solution. The probing light from the
dark-field illumination will in the case of a perfectly transparent lens pass through the lens and
not reach the photo detector. If there are scattering centers in the lens, the probing light will
scatter and reach the photo detector.75

Gamma Rays-induced Cataract

Karslioglu et al.76 irradiated rats with gamma rays using Cobalt-60 teletherapy unit with a single
dose of 5 Gy. Cataract was graded according to Chylack’s classification.77

Microwave-induced Cataract
Microwave radiation has been reported to produce posterior subcapsular and cortical cataracts
in rabbits and dogs within a short span of time at intensities no more than ten folds above safety
limits.78-81 It is believed that microwave radiation have primarily heating effect therefore, there
is no need of long-term dosing. Foster, et al conducted single dose experiments in rabbits.78

Thermal Cataract
Kramer et al. have produced cataract in rabbits. They circulated hot water intraocularly
and maintained the retrolental temperature in the range of 43 to 45°C. Biomicroscopic and
light microscopic examination revealed changes similar to microwave-induced cataract.
This supports the assumption that microwave-induced cataractogenesis is due to the local
production of elevated temperature.82

Mechanical Stimulation and Cataract

Mechanical stimulation in the eyes of rabbits (in vivo) and in isolated rat lenses (in vitro)
resulted in high frequency opacification of the lens. The rabbit eyes and the isolated lenses were
given vibration from an electric massage machine and tapping. The opacity thus produced was
in anterior or posterior subcapsular region.83

Hereditary Cataract Model

Hereditary cataract models have been described in many species such as rat, guinea pig,
dog, sheep, cattle and birds. Spontaneous hereditary cataract models of dominant trait are
found in mouse, such as cataract Fraser mice.84-86 These models are useful in understanding
the physiology of eye lens and the pathogenesis of cataracts. Hereditary dominant cataracts
are induced by irradiation or by a chemical mutagen. Representative spontaneous hereditary
recessive mutations are Nakano cataract mouse and Deer mouse. 87,88
Kolosova et al. 2003 have suggested that senescence accelerated OXYS rat strain develop
cataract spontaneously with progressive macular degeneration, lenticular changes correspond
to human senile cataract.89. Deletion of GPR 48 can cause age-related cataracts by decreasing
the resistance of lens epithelial cells to oxidative stress, which may be related to altered
expression of several antioxidant defense enzymes.90
240 Drug Screening Methods

CONCLUSION
The efficacy of an anticataract agent can be tested in the above-mentioned models. The
galactose, streptozotocin and alloxan induced models mimic sugar cataracts in humans and
are used for evaluating aldose reductase inhibitors. Galactose fed rat model is a popular model
as it is easily produced than streptozotocin and alloxan induced models. The rate of mortality
is high in streptozotocin and alloxan induced models. The utmost care of the animals has to
be taken. Among the models described above selenite model is the widely accepted model
for the screening of anticataract agents. It is reproduced easily within a short span of time. It
resembles the age-related cataract in humans. Though naphthalene cataract model resembles
human cataract still it is not preferred as the rate of mortality is high and reproducibility is less.
Hereditary mouse models are more suitable to understand the mechanism of cataractogenesis,
however, the cost and maintenance of these mice is high.

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 cHAPTER

14
Evaluation of Pharmacological
Activity of Herbal Medicines
Introduction
Traditional or alternative or complementary systems of medicine are popular not only in
developing but also in developed countries. Their popularity can be attributed to various
historical and cultural reasons. According to WHO estimates, around 80% of population in
developing countries relies on plant derived traditional medicines. Current statistics indicate
that the global market for medicinal plants is to the tune of US $62 billion and the demand is
growing rapidly. Global resurgence of the interest in herbal drugs has led to the need of their
mass production. Consequently, large-scale production has necessitated that standards for
their quality, efficacy and safety be clearly defined.
Despite their widespread usage, phytomedicines have not been evaluated scientifically
with regard to their safety and efficacy. Moreover, the government policy regarding their
registration and regulation remains obscure. With the increasing incidence of metabolic
diseases and age-related degenerative disorders that are associated with oxidative processes
in the body, the use of herbs has gained much attention; but without careful documentation in
well-defined clinical trials, they remain equivocal.1 It is because of this loophole that spurious,
illicit and substandard herbal drugs find their way into the market.2 International agencies
like World Health Organization (WHO), United Nations Industrial Development Organization
(UNIDO), International Centre for Science and High Technology (ICS) and Asia Pacific Centre
for Transfer of Technology (APCTT) have also emphasized on the need of ensuring quality
control of medicinal plant drugs by applying suitable standards employing modern techniques.
Owing to the aforementioned reasons the WHO was requested, to compile a list of
medicinal plants and establish their international specifications, during the IVth International
Conference of Drug Regulatory Authorities (ICDRA) held in Tokyo in 1986. The guidelines for
the assessment of herbal medicines were prepared by WHO and adopted by the VIth ICDRA
held in Ottawa, Canada in 1991. The objective of these guidelines was to define basic criteria
for the evaluation of quality, safety, and efficacy of herbal medicines and thereby to assist
national regulatory authorities, scientific organizations and manufacturers to undertake an
assessment of the documentation/submissions/dossiers in respect of such products.
As a general rule in this assessment, traditional experience means that long-term use as
well as the medical, historical and ethnological background of these products shall be taken
into account. The definition of long-term use may vary according to the country but should
246 Drug Screening Methods

be at least several decades. Therefore, the assessment should take into account a description
of a herbal medicine in the medical/pharmaceutical literature or similar sources, or a
documentation of knowledge on the application of such medicines without a clearly defined
time limitation. It may be noted that the traditional description of the herbal preparations
are often in vernacular prevalent in those times. Consequently, the description of the plants
and their therapeutic uses need to be carefully understood and interpreted, so as to avoid
misinterpretation.
WHO defines herbal medicines as finished, labeled medicinal products that contain
as active ingredients the aerial or underground parts of plants, or other plant material, or
combinations thereof, whether in the crude state or as plant preparations. Plant material
includes juices, gums, fatty oils, essential oils and any other substance of this nature. Herbal
medicines may contain excipients in addition to the active ingredients. Medicines containing
plant material combined with chemically defined, isolated constituents of plants, are not
considered to be herbal medicines.
WHO has defined certain related terms to provide consistency and international acceptance
for the evaluation and research on herbal medicines.

Definitions
Herbs
Herbs include crude plant material such as leaves, flowers, fruits, seeds, stems, wood, barks,
roots, rhizomes or other plant parts, which may be entire, fragmented or powdered.

Herbal Materials
Herbal materials include, in addition to herbs, fresh juices, gums, fixed oils, essential oils,
resins, and dry powders of herbs. In some countries, these materials may be processed by
various local procedures, such as steaming, roasting, or stir baking with honey, alcoholic
beverages or other materials.

Herbal Preparations
Herbal preparations are the basis for finished herbal products and may include comminuted
or powdered herbal materials, or extracts, tinctures and fatty oils of herbal materials. They
are produced by extraction, fractionation, purification, concentration or other physical or
biological processes. They also include preparations made by steeping or heating herbal
materials in alcoholic beverages and/or honey, or in other materials.

Traditional Use of Herbal Medicines

Herbal medicines include herbs, herbal materials, herbal preparations, and finished herbal
products. Traditional use of herbal medicines refers to their description in ancient literature and
long historical use of these medicines. Their use is well established and widely acknowledged
to be safe and effective, and may be accepted by national authorities.
 Evaluation of Pharmacological Activity of Herbal Medicines 247

Therapeutic Activity
Therapeutic activity refers to the successful prevention, diagnosis and treatment of physical
and mental illnesses, improvement of symptoms of illnesses, as well as beneficial alteration or
regulation of the physical and mental status of the body.

Active Ingredients
Active ingredients refer to ingredients of herbal medicines with therapeutic activity. In
herbal medicines where the active ingredients have been identified, the preparation of these
medicines should be standardized to contain a defined amount of the active ingredients, if
adequate analytical methods are available. In cases where it is not possible to identify the
active ingredients, the whole herbal medicine may be considered as one active ingredient.
Standardization of the presumed active constituents of the drug is perhaps not the
best approach, as only in few cases a single component can be held responsible for the
pharmacological effect. In majority of the cases, the activity is a synergistic effect of several
compounds. In addition, number of factors such as age, origin, time of collection, method of
drying, etc. also play a critical role in determining the proportion of the various compounds
and ultimately, the effect.

Assessment of Quality
Pharmaceutical Assessment
This should cover all important aspects of the quality assessment of herbal medicines sufficient
to make reference to a monograph in pharmacopoeia. The procedures described should be in
accordance with good manufacturing practices. The identity and quality of the plant material
or preparation must be according to the following headings.

Information for Fresh, Dried, and Processed Plant Materials

i. Name and characteristics of crude plant materials
• Name of the plant material in Latin, native languages, and English.
• Scientific name and the family to which it belongs.
• Part and condition (dried, fresh, sliced or decorticated, etc.) of the plant used.
• Time and method of collection, preliminary preparation, drying, and processing.
• Description and distribution of plant habitat; growing wild or cultivated (including
possible pesticide used). Drawings or photographs of the plants should be provided.
• Characterizing compounds of the plant materials, which may also be the biologically or
therapeutically active principles, should be quantified and described with their structural
formulae.
• Foreign matter (such as stem, rachis fragments in the leaves or leaflets, leaf fragments
in the flowers etc.), foreign mineral matter (such as sand and soil adhering to the plant
material), impurities and microbial content should be defined or limited.
• Voucher specimens, representing each lot of plant material processed, should be
authenticated by a qualified botanist and should be stored for at least a 10-year-period. A
lot number should be assigned and this should appear on the product label.
248 Drug Screening Methods

ii. Authentication of plant preparations

Plant preparations include comminuted or powdered plant materials, extracts, tinctures, fatty
or essential oils, expressed juices and preparations whose production involves fractionation,
purification or concentration. A method for identification and assay of the plant preparation
along with a description of the physical and chemical tests for the identification of the
plant substances should be provided. If identification of an active principle is not possible,
“chromatographic fingerprint” of the mixture is needed to ensure consistent quality of the
preparation.
iii. Packaging, labeling, and storage of finished products
The manufacturing procedure and formula needs to be described in detail. A method for
quantification of the plant material in the finished product should be defined. For imported
finished products, confirmation of the regulatory status in the country of origin is needed. The
WHO Certification Scheme on the quality of pharmaceutical products moving in international
commerce should be applied.
iv. Product information for the consumer
Product labels and package inserts should be understandable to the consumer or patient and
should include necessary information on the correct use of the product.
v. Promotion
Advertisements and other promotional material directed to health personnel and the general
public has to be consistent with the approved package information.
vi. Stability
The physical and chemical stability of the product should be tested under defined storage
conditions and the shelf life has to be established.

Assessment of Safety
Long-term use of a medicine without any evidence of risk may indicate that it is harmless,
it is not always definite how far one can rely on it as an assurance of innocuity. Preclinical
toxicological studies can be undertaken to study safety of herbal medicines. In addition, side-
effects may be documented according to normal pharmacovigilance practices.

Toxicological Studies
Standard methods of nonclinical toxicological studies are indicated below. All tests are not
necessarily required for each herbal medicine intended for human use.

Acute Toxicity Test

•• Animal species: Some regulatory agencies recommend that at least two species be used,
one of them to be selected from rodents and the other from non-rodents.
•• Sex: In at least one of the species, males and females should be used.
•• Number of animals: In the case of rodents, each group should consist of at least five animals
per sex. In the case of non-rodents, each group should consist of at least two animals per
sex.
 Evaluation of Pharmacological Activity of Herbal Medicines 249

•• Route of administration: Ordinarily, the oral route is sufficient as this is the normal route of
clinical administration. In cases where it is proposed to administer the herbal preparation
to a subject by the parenteral route, it may be sufficient to use this route for animal testing.
•• Dose levels: A sufficient number of dose levels should be used in rodents to determine
the approximate lethal dose. In non-rodents, sufficient dose levels should be used for the
observation of overt toxic signs.
•• Frequency of administration: The test substance should be administered in one or more
doses during a 24-hour period.
•• Observation: Toxic signs and severity, onset, progression and reversibility of the signs
should be observed and recorded in relation to dose and time. As a general rule, the animals
should be observed for at least 7 to 14 days. Animals dying during the observation period,
as well as surviving to the end of the observation period should be autopsied. If necessary,
a histopathological examination should be conducted on organs or tissues showing
macroscopic changes at autopsy.

Long-term Toxicity Test

•• Animal species: At least two species be used, one a rodent and the other a non-rodent.
•• Sex: Equal number of males and females should be used.
•• Number of animals: In case of rodents, each group should consist of at least ten males and
ten females. In case of non-rodents, each group should consist of at least three males and
three females.
•• Route of administration: Expected clinical route of administration should be used.
•• Administration period: The period of administration of the test substance to animals will
depend on the expected period of clinical use (Table 14.1). It may vary from country to
country, according to its individual regulations.

Table 14.1: Commonly used ranges of administration periods

Expected period of clinical use Administration period for the toxicity study
Single administration or repeated administration for less than one week 2 weeks to 1 month
Repeated administration, between one week to four weeks 4 weeks to 3 months
Repeated administration, between one to six months 3 to 6 months
Long-term repeated administration for more than six months 9 to 12 months

The test substance should be administered seven days a week. Administration periods for
the toxicity study must be recorded in each result.
•• Dose levels: At least three different dose levels should be used. One dose should be a no
effect dose, the other should exert-overt toxic effects. Within this range one more dose may
be included to observe a dose-response curve for toxic effects.
•• Observations and examinations: Following points should be considered
a. General signs, daily body weight, food, and water intake.
b. Hematological and blood chemistry examination should be done before the start of the
drug treatment and compared with that during the administration period and before
autopsy values.
250 Drug Screening Methods

c. Renal and hepatic function tests to be performed before, during and after drug
administration period.
d. Other function tests include—ECG, visual, and auditory tests.
e. Animals found dead during the examination should be autopsied as soon as possible.
•• Recovery from toxicity: This is observed in animals that are allowed to live for varying lengths
of time after cessation of the period of administration of the test substance.

Local Toxicity Test

This is a sensitization test for dermatological preparations. Usually, guinea pigs are considered
the most suitable experimental animals and test methods like patch test, Buehler test, Draize
test, Freund’s complete adjuvant test, Maximization test, Open epicutaneous test, Optimization
test and Split adjuvant test, etc. are usually undertaken.

Special Toxicity Tests

i. Mutagenicity test: Reverse mutation test in bacteria, chromosomal aberration test with
mammalian cells in culture, micronucleus test with rodents are some of the standard tests
which can provide any inkling regarding the toxicity profile of the drug.
ii. Carcinogenicity test: Animals are screened in two phases—preliminary and full scale
carcinogenicity studies with the same test substance. In preliminary studies, effect
of single and repeated doses are observed in a small number of animals and the data
from this study is used to determine the dose of the test drug to be used in the full scale
carcinogenicity studies. For full-scale carcinogenicity test at least two species of animals
are employed. The parameters to be observed are—development of tumor type, frequency
of development, onset of development, variety of organs involved, etc.
iii. Reproductive and developmental toxicity test: The effect of the test drug is observed on
fertility, incidence of spontaneous malformation and susceptibility to substances known
to affect reproduction and development.

Assessment of Efficacy
Herbal medicines are currently being used either as first line of medical care or in conjunction
with conventional treatment. Citation of existing literature is sufficient to substantiate claims
of benefits of herbal medicine being traditionally used. However, in case, any replacement,
addition or deletion of traditionally used ingredients from herbal drug product is made, or old
product is marketed for new indication, or traditional method of preparation is altered, the
“new” herbal product needs to be extensively investigated preclinically and clinically along
with post-marketing surveillance.3

Activity
The pharmacological and clinical effects of the active ingredients and, if known, their
constituents with therapeutic activity should be specified or described.
 Evaluation of Pharmacological Activity of Herbal Medicines 251

Evidence Required to Support Indications

According to WHO guidelines the requirements for proof of efficacy of traditional medicines
depends on the kind of indication. For treatment of minor and nonspecific indications, some
relaxation in requirements for proof of efficacy may be justified, taking into account the extent
of traditional use. The relaxation applies to prophylactic use also. Individual experiences of
physicians, traditional health practitioners or treated patients should be taken into account.3

Combination Products
Many herbal medicines are combination of several active ingredients, and as experience of
the use of traditional remedies is often based on combination products, assessment should
differentiate between old and new combination products. In the case of traditionally used
combination products, the documentation of traditional use (classical texts of Ayurveda,
traditional Chinese medicine, Unani, Siddha, etc.) and experience may serve as evidence of
efficacy.3
An explanation of a new combination of well-known substances, including effective dose
ranges and compatibility, should be required in addition to the documentation of traditional
knowledge of each single ingredient. Each active ingredient must contribute to the efficacy
of the medicine. For study of those herbal medicines, which are used under the principles
of traditional medicine, animal models may need to be established according to those
principles.3

Clinical Trials
The principles of the clinical trials of herbal medicines are similar to those applied to synthetic
drugs. Clinical trials of herbal medicines have two objectives—to validate the safety and efficacy
claim and to develop new herbal medicines or examine a new indication for an existing herbal
medicine. Clinical trial is conducted in a step-wise approach in four phases and the entry point
into the phase may be determined by the nature and history of the herbal medicines being
studied.3

Phase I
New compound or a new formulation is administered for the first time to a small number of
healthy volunteers and their tolerance to the herbal medicine is noted. An indication of the
intended dose that may be used safely in subsequent phases is decided.

Phase II
Studies are conducted on a limited number of patients to determine clinical efficacy and
safety. The dosage schedules established in such studies are then used for a more extensive
clinical study.

Phase III
Larger patient groups are usually studied at several centers to validate preliminary evidence of
efficacy obtained in earlier studies.
252 Drug Screening Methods

Phase IV
Studies performed after the dosage form is available in the market for general use. This is also
known as post-marketing surveillance. Hence, the main purpose of such studies is to detect
toxic events that may occur so rarely that they are not detected earlier.

Pharmacovigilance of Herbal Drugs

There is an urgent need to develop pharmacovigilance practices for herbal medicines. The
current model of pharmacovigilance and its associated tools are inadequate for monitoring
safety of herbal medicines.4 The matter is further complicated due to the practise of co-
administration of herbal and allopathic medicines leading to herb-drug interactions. In such
cases, it becomes essential to delineate the mechanism responsible, like activity of metabolic
enzymes, active transporters pharmacokinetic profiles etc.5
Despite concerted efforts to stimulate reporting of suspected ADRs associated with herbal
medicines, such as extending the scheme to unlicensed herbal products, and including
community pharmacists as recognized reporters, numbers of herbal ADR reports received
remain relatively low. Although, Spontaneous Reporting Schemes form the backbone of
pharmacovigilance, it is fraught with an inherent limitation of under-reporting. This limitation
is more pronounced for herbal medicines, since users typically do not seek professional advice
about their use of such products, or report if they experience adverse effects.
Worldover, herbal medicines are being sold under the garb of neutraceuticals and thereby,
circumvent any such targeted monitoring. The herbal sector in the UK has taken the initiative
for adverse effect monitoring of herbal drugs by means of spontaneous reporting by the herbal-
medicine practitioners. Presently, other tools that are routinely used in pharma­covigilance,
such as prescription-event monitoring, computerized health-record databases, offer limited
utility in monitoring the safety of herbal medicines, as these drugs are usually sold OTC.
Proposed European Union legislation for traditional herbal medicinal products will require
manufacturers of products registered under new national schemes to comply with regulatory
provisions on pharmacovigilance. In the longer term, other improvements in safety monitoring
of herbal medicines may include modifications to existing methodology, patient reporting and
greater consideration of pharmacogenetics and pharmacogenomics in optimizing the safety of
herbal medicines.6 Further, it needs to be re-inforced to earnestly take up reporting of adverse
events associated with herb use in special population such as pediatric and lactating.7,8

References
1. Tapsell LC, Hemphill I, Cobiac L, Patch CS, Sullivan DR, Fenech M. Health benefits of herbs and
spices: the past, the present, the future. Med J Aust 2006;185(4 Suppl):S4-24.
2. Kinsel JF, Straus SE. Complementary and alternative therapeutics: rigorous research is needed to
support claims. Annu Rev Pharmacol Toxicol 2003;43:463-84.
3. Research guidelines for evaluating the safety and efficacy of herbal medicines. World Health
Organization, 1993.
4. Shaw D1, Graeme L, Pierre D, Elizabeth W, Kelvin C. Pharmacovigilance of herbal medicine. J
Ethnopharmacol 2012; 140(3):513-8.
 Evaluation of Pharmacological Activity of Herbal Medicines 253

5. Gouws C, Steyn D, Du Plessis L, Steenekamp J, Hamman JH. Combination therapy of Western drugs
and herbal medicines: recent advances in understanding interactions involving metabolism and
efflux. Expert Opin Drug Metab Toxicol 2012;8(8):973-84.
6. Barnes J. Pharmacovigilance of herbal medicines: a UK perspective. Drug Saf 2003;26:829-51.
7. Gardiner P, Adams D, Filippelli AC, Nasser H, Saper R, White L. A systematic review of the reporting
of adverse events associated with medical herb use among children. Glob Adv Health Med 2013;
2(2):46-55.
8. Budzynska K, Gardner ZE, Low Dog T, Gardiner P. Complementary, holistic, and integrative
medicine: advice for clinicians on herbs and breastfeeding. Pediatr Rev 2013;34(8):343-52.
 CHAPTER

15
Agents for Immune-based
Disorders
INTRODUCTION
The main objective of the human immune system is to discriminate self from non-self
(infectious invaders/microbes; dysregulated self/tumors). This requires active and efficient
detector and effector mechanisms capable of identifying and destroying the non-self in order
to preserve the self. Loss or suppression of immune system functionality can lead to disease
conditions like cancer, bacterial, viral or protozoan infections. In contrast, overactive immune
system can also cause significant health care problems like autoimmune diseases [rheumatoid
arthritis (RA), diabetes mellitus (DM), systemic lupus erythematosus (SLE), multiple sclerosis
(MS)], allergic and hypersensitivity conditions. New evidence correlates obese condition with
chronic state of low-grade inflammation leading to the pathogenesis of several inflammatory
conditions including rheumatic autoimmune and inflammatory diseases.1
Immunosuppressive or immuno-stimulant therapies, aiming at immune system mediated
diseases have evolved. Antibiotics and antimetabolites have also been successfully used for
management of infections and cancer, respectively but not without accompanying side effects.
On the other hand, drugs for immunosuppression including glucocorticoids, cyclosporine,
tacrolimus and sirolimus have proved to be highly efficacious for management of organ
transplant cases, RA and other autoimmune disorders. Unfortunately, these drugs have also
proved to be extremely disabling with life threatening side effects like growth retardation,
osteopenia, hyperglycemia, nephropathy, hypertension, poor wound healing and increased
risk of infection. Consequently, there has been an exponential rise in the need for biological
molecules that act specifically to overcome the considerable side effects of non-specifically
acting anti-inflammatory and immunosuppressive drugs. Recently, macrophages have been
identified for their positive impact upon tissue remodeling following injury. The pivotal role
of macrophages can initiate transition from a pro-inflammatory state to a regulated/anti-
inflammatory state and reduce scar tissue formation.2
Helpful tools in the analysis of drug effects include high-throughput screening techniques
such as microarrays, which are used in transcriptomics and pharmacogenomics. Although we
are far from using these extensive and costly tests in our daily clinical routine, their application
in basic research nevertheless takes us closer to individualized therapeutic strategies, in which
the optimal therapeutic regimen is identified for each individual patient.
 Agents for Immune-based Disorders 255

In Vivo Models
It is imperative to screen novel molecules for management of immune-based disorders and
the challenge lies in developing suitable animal models that can help to accurately predict the
activity of these agents. Depending on the goal, susceptibility of the animal to the pathogen
and innate immune system, various animals like mice, rabbits, goat, sheep and even horse
have been used as experimental animals of choice.
Of all the animals, mice have been found to be most useful, as not only they are easy
to handle and have a rapid breeding cycle but also because they are genetically well
characterized. Genetically identical strains have been developed by inbreeding brother and
sister littermates for 20 generations to yield 98% homozygous progeny that are called syngeneic
mice. Thus, mice colonies serving as models for cancer types (4T07cg for metastatic breast
tumor), diabetes (NOD mice) and other immune-based disorders have been developed to
understand underlying pathology of the disease conditions and screen novel drug therapies
(Fig. 15.1).3,4
Another approach has been to microinject cloned foreign genes (transgenes) into
mouse embryos to produce transgenic mice. This helps to assess the variation in biological
effects induced by the gain of a single gene. Transgenic mice have been of value in studying
the immunopathology of human major histocompatibility class II (MHC-II) associated
autoimmune diseases like RA, MS and DM by helping to identify the target antigens that are
involved in the initiation of these diseases. Many of the mice develop aspects relevant to the
human diseases, either spontaneously or following immunization with the relevant antigen,
thus providing an in vivo disease model, that may be used as a tool for further understanding
the disease mechanisms and testing novel immunotherapies.5,6
Change in phenotype due to loss of single gene function can also be studied by developing
knockout mice. In order to develop these mice, the normal gene is replaced with the mutant
allele in the cultured embryonic stem (ES) cells. The recombinant ES are then transferred to
recipient blastocyst and implanted into foster mother. Finally, the chimeric offsprings that
are heterozygous for the disrupted gene are mated to produce homozygous knockout mice

Figure 15.1: Syngeneic mouse-Non-obese diabetic (NOD) mouse as model for screening
immune-based disorders
256 Drug Screening Methods

Figure 15.2: Developing knockout mouse

(Fig. 15.2). These animals have been used to develop novel treatment modalities against
cancer, viral infections and autoimmune diseases.7,8
An autosomal recessive mutation resulting in severe combined immunodeficiency
disease (SCID) developed spontaneously in a strain of mice called CB-17. These CB-17
SCID mice fail to develop mature T and B cells and consequently are severely compromised
immunologically. Hence, these animals must be housed in a sterile (germ-free) environment
if they are to survive, as they cannot fight off microorganisms of even low pathogenecity. They
have proven to be ready recipients of foreign cells and grafts from other strains of mice or even
other species. DNA-based vaccine strategies and vectors meant for the treatment of human
immunodeficiency virus are being actively developed using this model.9
Animal models have been used in the drug development process for the identification of
targets for therapeutic intervention and to provide proof of a therapeutic principle. Preclinical
animal models for immune-based disorders are briefly described here.

Asthma
One of the major immune disorders is asthma wherein triggering of the immune responses
leads to the manifestation of the clinical symptoms. The events leading to clinical manifestation
of the disease involve recognition of microbial components (Chlamydia, Mycoplasma, bacteria
as well as virus and air-borne particles) and their activation through receptors and initiation of
the cascade leading to induction of adaptive immune responses. The immune responses and
efficacy of therapeutic modalities have been extensively studied using inbred mouse strains
like C57BL/6, 129/SvEv.10
Six- to eight-weeks old 129/SvEv mice are maintained under sterile laboratory conditions
and chow. The mice are immunized with subcutaneous 50 µg of ovalbumin (OVA) alone or in
combination with 50 µg of ISS-ODN (TCCATGACGTTCCTGATGCT) or synthetic lipopeptide
Pam3Cys (50 µg) on days 0 and 7. The mice are intranasally challenged with 5 µg of OVA 7
 Agents for Immune-based Disorders 257

days and 1 day before sacrifice. On the 21st day the mice are tested for airway responsiveness
to methacholine (3-24 mg/ml) and a bronchoalveolar lavage for the differential lung cell
count is performed. Mediastinal lymph nodes are digested with DNAseI/collagenaseVII and
restimulated with OVA for T cell ctytokine analysis. ELISA can be used to estimate OVA specific
immunoglobulins and interferons in the sera. The modulation of these parameters after drug
treatment can evidence the efficacy of the intervention at the cellular and molecular level.10
Based on their anatomical similarity and responsiveness of the airway with that of
humans, rabbits serve as useful species for screening inflammatory mediators, lung disease
pathophysiology and anti-asthmatic agents. Intraperitoneal administration of antigen
in combination with adjuvant to neonatal rabbits like soluble bovine serum albumin in
conjunction with Corynebacterium parvum adjuvant within the first 24 h of life chronically,
initiates immune response via production of antigen-specific IgE antibodies. This protocol
can be modified, to include other sensitizing agents like ovalbumin, lipopolysaccharide,
house dust mite, and ragweed pollen for administration to rabbit neonates and study for
various parameters such as comparison of anatomical structures of lung (bronchioles,
tracheobronchial capillary bed), lung volume, airway epithelial layer, presence/absence of
mucus producing cells, etc.11

Immunoinflammatory Disorders of the Central Nervous System (CNS)

Multiple sclerosis is a spectrum of chronic immunoinflammatory diseases of the CNS with
multifactorial etiology and a complex pathogenesis. The pathological hallmark of MS is
focally demyelinated lesion within the white matter and cortex along with accompanying
inflammation, gliosis, axonal pathology and remyelination.12 While many experimental animal
models are available, the use of relevant model that aptly mimics the different forms of the
disease is essential. Understanding the cascade can help to identify gene and protein targets
for therapy. Experimental autoimmune encephalomyelitis (EAE) in mice and non-human
primates shows similar pathogenesis as the clinical disorder.
Experimentally autoreactive T cells alone or T cells plus autoantibodies are used to
induce the CNS lesions. For inducing the pathology, emulsion containing Freund’s adjuvant
in combination with CNS preparations such as brain, spinal cord or myelin antigen are
inoculated. EAE is initiated by activation of a pre-existing repertoire of myelin reactive CD4+ T
cells in peripheral lymphoid organs. The activated cells pass through the blood-brain barrier.
Advances in genetic engineering and the subsequent ease of producing animals with
dysfunctional (knock-out/knock-in) genes have helped in probing the disease. Intraperitoneal
infection of transgenic mice (C57BL/6, Biozzi ABH) with avirulent A7(74) strain of Semliki-
Forest virus or Theiler’s virus induces EAE. These protocols have allowed studies on MS-
associated immunological responses. Neurovirulent strains of virus induce fatal encephalitis
to cause persistent infection and demyelination of the CNS, spinal cord and brainstem.13
Non-human primates are attractive models for studying EAE as they share phylogenetic
proximity with humans. Based on this, intervention strategies for MS are being actively explored
using non-human primate animal models. The common marmoset, a small neotropical
primate with significant genetic and immunological similarity to humans provides an excellent
model that approximates chronic MS by the clinical and neuropathological presentation.14
Attempts to evoke MS like syndrome in chimps by inoculation of MS brain material have
258 Drug Screening Methods

yielded intriguing results on the viral origin of MS.15 Macaque also develops neurological
deficits associated with CNS inflammation after infection with healthy CNS tissue.16 Based
on these findings, reproducible EAE models have been established for the study of genetic,
immunological, pathological features of MS.17

Viral Diseases
Non-human primates are complex species including Lemur, Lorisers, Tarsiers, Marmosets,
Tamarins and Monkeys. They serve as important models to study human diseases especially
of immune origin, as they share a high degree of genetic similarity, susceptibility to pathogens
(HIV, HBV, malarial parasite), cascade of pathological events and drug responsiveness. They
have proven to be extremely useful for studying mechanisms of immune pathology, exploring
interventional therapies and vaccine strategies. However, their use in research requires special
ethical permission and dedicated care, attention and housing conditions.18
Hepatitis B Virus
Hepatitis B virus (HBV) is infectious to chimps, however, the impact of infection is minimal
as compared to humans as they initiate a strong polyclonal cytotoxic T-lymphocyte response
immediately.19 In humans, the response remains restricted and is robust only in chronically
infected patients.17 In the experiment conducted by Bertoni and co-workers, two chimps were
inoculated with a terminally redundant copy of the HBV. Both developed acute and self-limited
HBV infection. The host interferon-α levels were monitored and associated with decreasing
HBV titer. This showed that the chimps could mount an effective response to the infection on
the basis of their innate immune response.20 Further studies exploring the mechanism could
provide insight into the cascade of events and management strategies for humans.
Chronic hepatitis B (HBV) infection is one of the most common causes of chronic active
hepatitis, and dramatically increases the risk of developing hepatocellular carcinoma (HCC).
Because of the narrow host range of this virus, there are very few useful animal models of HBV
infection. However, transgenic technology affords the opportunity to produce mouse models
of the condition. Moreover, because transgenic technology inevitably involves integration of
the donor DNA, this particular characteristic of HBV DNA in HCC can readily be achieved in
transgenic mice.21 Healthy carrier state in hepatitis B virus (HBV) infections, transgenic mice
expressing HBV genes were produced according the following protocol. Briefly, fertilized one-
cell eggs were microinjected with subgenomic fragments of HBV DNA containing the coding
regions for the HBV surface antigen (HBsAg) and pre-S and X antigens. Either the normal
(HBV) or metallothionein promoters were used to obtain expression of the HBV genes. There
was no evidence of viral replication or tissue pathology. The integrated HBV DNA sequences
were inherited in a normal Mendelian fashion. Three of 16 transgenic mice expressed HBV-
encoded gene products to which they were immunologically tolerant. Expression was not
tissue specific and may be influenced by the genomic integration site and cellular factors.
Both HBsAg and pre-S antigen were detectable within the cytoplasm of hepatocytes and renal
tubular epithelial cells. High serum concentrations of HBsAg were detectable and the secreted
product appeared authentic as judged by mean density, morphology, mean particle diameter,
polypeptide composition, and antigenicity.21
HBx gene of HBV has also been implicated in HCC and has been introduced under the control
of its own promoter into mice, so as to develop a model for HCC.22 High expression of HBx
 Agents for Immune-based Disorders 259

has been documented in liver, kidney and testis. By 4 months of age focal areas of hepatocyte
abnormalities were observed, and definite tumors were diagnosed by 8 to 10 months.
In summary, the species barrier to HBV infection can be overcome in mice by direct
microinjection of HBV genes. The genes integrate, and when HBsAg is expressed at high levels,
liver cell injury and HCC develop. A serous limitation when working with transgenic mice is
that they pose a threat of zoonotic transmission of diseases. It is possible that an animal with
the entire genome in every cell could produce mature virions and release them into its serum.
In fact, some transgenic mice that carry the entire HBV genome carry core antigens in blood.23
To date, transmission of disease specific to expression of a transgene from a transgenic animal
to man has not been reported. However, it is important to study new proposals involving
insertion of human pathogens to determine if risk exists.
Hepatitis C Virus
For the development of effective therapies against hepatitis C virus (HCV), it is essential to
develop suitable small animal models. Although the chimpanzee has been a valuable model to
study HCV-host interactions but its use is severely hampered by financial and ethical constraints
pertaining to large animals. Alternatively, human-liver chimeric mice have been developed as
well-characterized tools for the efficacy assessment of antiviral interventions and are widely
accepted and used. Transgenic mouse has also been developed immunocompetent mouse
model, and is appropriate for the evaluation of both antivirals and murine vaccine responses.24
Human Immunodeficiency Virus
Studies indicate that human-immunodeficiency virus (HIV-1 and 2) are products of multiple
infections from simian immunodeficiency virus (SIV) infected primates.25 The difference in
response to HIV infection by primates provides clues regarding viral interaction with receptors
and co-receptors on the cell surface. Infected chimp in large maintain normal CD4+ lymphocytes
and do not develop clinical immunodeficiency. The viral-host immune interaction evidences
the events leading to AIDS after infection with HIV. It is possible that chimps express favorable
homologues that have protective effect.26
Chimps deal more effectively with viral infections. The viral load is lower with minimal sequel
of events after MHC recognition. Subtle differences in peptide recognition and responses may
account for the efficiency of viral clearing. These models are effective for studying cell-cell
interactions and mediation of immune response leading to differences in handling of viral
challenges.

Rheumatoid Arthritis
Rheumatoid arthritis (RA) is characterized by chronic inflammatory infiltration of the
synovium, leading to eventual cartilage and bone destruction. It is a common autoimmune
disease, the treatment for which is rarely curative and often tied to major side effects. RA
differs from other autoimmune diseases in several aspects, as RA patients often develop
extra-articular disease manifestations such as rheumatoid nodules, rheumatic lung disease,
and vasculitis, suggesting a more generalized autoimmune process. Genetic susceptibility to
RA is associated with human leukocyte antigen (HLA) class II alleles, that share a collection
of positively charged amino acids at positions 70–72 of the DRB1 chain, called the ‘shared
epitope’.27
260 Drug Screening Methods

Humanized mouse models of RA have been developed that allow screening of new, less
toxic, vaccine-like treatments for RA.28 Novel screening techniques such as high-throughput
screening tools such as microarrays, are also being extensively used. Their application in basic
research will takes us closer to individualized therapeutic strategies, in which the optimal
therapeutic regimen is identified for each individual patient.
The primary disease phenotype, detected in several different tumor necrosis factor (TNF)‑α
transgenic mice with constitutive expression of human TNF, is an inflammatory arthritis
similar to RA.29 This phenotype is essentially preserved when these mice are backcrossed to a
severe combined immune deficiency background, which lacks the develop­ment of B cells and
T cells, indicating that arthritis can develop without the participation of lympho­cytes.30-32
Spontaneous RA-like arthritis was also observed in a murine TCR transgenic model,
(KRN×NOD).33 This TCR is specific for bovine ribonuclease in the context of I-Ak. The transgenic
TCRab-positive cells are completely deleted in I-Ak mice. The TCR transgene-positive animals
develop arthritis within the first few weeks of life after the first cross to the nonobese diabetic
mouse strain (NOD).33
Human T cell leukemia virus type-I (HTLV-I) is the etiologic agent of adult T cell leukemia
and has also been suggested to be involved in other diseases such as chronic arthritis or
myelopathy. To elucidate pathological roles of the virus in disease, transgenic mice were
produced that carry the HTLV-I genome. At 2 to 3 months of age, many of the mice developed
chronic arthritis resembling rheumatoid arthritis. Synovial and periarticular inflammation with
articular erosion caused by invasion of granulation tissues was marked. These observations
suggest a possibility that HTLV-I is one of the etiologic agents of chronic arthritis in humans.34
T cells have well established diverse role in pathogenesis of RA and mouse models
simulating the same have been developed. In SKG mice, point mutation in the gene encoding
ZAP70, a tyrosine kinase involved in T-cell receptor signal transduction, results in aberrant TCR
signalling. Such self-reactive T cells are understood to lead to the development of spontaneous
autoimmune arthritis.35

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrigs disease, is an age-related
neurodegenerative disorder that primarily involves motor neurons. Although the majority of
ALS cases are sporadic, a subset of affected individuals inherits the disease. Rosen and others
(1993) observed that a subset of individuals with familial, autosomal dominantly inherited
ALS (FALS) harbor mutations of the Cu/Zn superoxide dismutase (SOD-l) gene. These
findings indicate a causative relationship between altered SOD-1 activity and motoneuron
degeneration. Because humans with ALS are generally not identified until muscle weakness
sets in, early medical intervention and prevention is difficult. For these reasons it is highly
desirable to develop an animal model of FALS.36
Transgenic mouse model of ALS is one of the most important screening model. When a
mutation is introduced into the 4th exon of a 15 kb mouse genomic clone, it leads to an alteration
in the glycine residue (GGC) at position 86 of the protein, that simulates the mutation found
in some families with FALS. The sequence change not only creates a mouse counterpart of a
pathogenic human gene sequence, it introduces a recognition sequence for FspI restriction
endonuclease. This construct can be microinjected to produce transgenic mice. In mouse lines
 Agents for Immune-based Disorders 261

with high levels of transgene mRNA in the central nervous system (CNS), phenotype that is
manifested is development of motor paralysis, degenerative changes of motoneurons within
the spinal cord, brainstem, and neocortex. These animals constitute a potentially valuable
animal model of ALS.37
The transgenic mice produced are of the highly inbred FVB/N strain and are housed under
uniform conditions. The appearance and progression of symptoms is very consistent and all
mice die between the ages of 94 and 117 days. This is a sensitive model that can be used for
testing of potential therapeutic agents or environmental factors involved in the disease.38

Recombinant DNA Technology

Recombinant DNA technology is an amalgam of research techniques aimed at gene cloning
and DNA sequencing for producing recombinant proteins and providing immunologists with
defined components to study structure-function correlation of the immune system at the
molecular level.
DNA cloning is a means of amplifying a given DNA fragment and producing unlimited
amounts of identical DNA fragments called “cloned DNA”. In order to clone DNA, the desired
fragment is inserted into an autonomously replicating DNA molecule, called “cloning vector”
like bacterial virus (bacteriophage), insect virus and mammalian retrovirus. For producing
DNA clones, firstly the vector genes are removed. The desired DNA insert is then incorporated
with the remnant vector genome. The expression of the gene is now under the control of the
vector promoter region.
Similarly, clones of mRNA can also be produced. First mRNA is isolated from cells and
transcribed into complementary DNA or cDNA with the help of reverse transcriptase enzyme.
Copies of cDNA are produced by inserting it into an appropriate vector. DNA sequences within
vectors representing all the mRNA sequences derived from a cell or tissue is called a cDNA
library. The genes from the library are screened to identify, purify and sequence the fragments.
Thus, with the advances in technology, it is possible to decipher nucleotide sequences of the
corresponding gene if the amino acid sequence is known.
The techniques of protein biochemistry have been very crucial for immunology and have
made it possible to elucidate the structure of various immunoglobulins and their functional
interactions.39 Basic techniques like electrophoresis, radio immunoassay, polymerase chain
reaction (PCR) and Western blotting have proved to be of central importance and are briefly
described here.

Immunoelectrophoresis
Electrophoresis is a separation technique that uses electricity to separate the sample mixture
on the basis of size. In gel electrophoresis, the gel acts as a sieve across which the sample
moves from the negative to the positive end of the machine. The smaller sized molecules move
faster and farther across the sieve than the larger ones. Staining the gel helps to analyze the
sample contents quantitatively and qualitatively. Using this technique, DNA, RNA or protein
samples can be analyzed. Similarly, antigen mixtures can be separated by electrophoresis
and then identified by double immunodiffusion and this qualitative technique is known
as immunoelectrophoresis. Here, after the sample components have been separated by
electrophoresis, antiserum is added. Consequently, antibody and antigen diffuse towards
262 Drug Screening Methods

each other and produce lines of precipitation. The technique is being widely applied to detect
the presence or absence of proteins in serum.40

Radioimmunoassay
Radioimmunoassay (RIA) is a sensitive technique for detecting antigens and antibodies that
was developed by two endocrinologists, S. A. Berson and Rosalyn Yalow in 1960 as an assay for
the quantification of insulin in plasma sample. The protocol involves labeling of antigen with
125
I or 131I. A mixture of the radioactive antigen and antibodies against that antigen is prepared.
Known amounts of unlabeled (“cold”) antigen are added to samples of the mixture. These
compete for binding sites of the antibodies. At increasing concentrations of unlabeled antigen,
an increasing amount of radioactive antigen is displaced from the antibody molecules. The
antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the
radioactivity of each is measured. From the data, a standard binding curve can be prepared
to quantify the test sample. The technique is being applied to detect the presence of marker
proteins from the sera and plasma of the patients.41

Enzyme-linked Immunosorbent Assay

Enzyme-linked immunosorbent assay or ELISA is similar in principle to RIA but depends on
an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts
with a colorless substrate to generate a colored reaction product. A number of enzymes
have been employed for ELISA including alkaline phosphatase, horseradish peroxidase and
β-galactosidase. These assays are sensitive, safe and less costly.42 There are many different
types of ELISAs, which can detect the presence of protein in serum or supernatent. One of the
most common types of ELISA is the so-called “sandwich ELISA.” It is so termed because the
antibody that is being detected, gets sandwiched between an antigen and a chromogenically-
conjugated antibody. The technique is being experimentally and clinically used to analyse
levels of matrix metalloproteinases (MMP), inflammatory and collagen degradation markers,
tissue inhibitor of MMPs (TIMP) levels and circulating proteasomes in the systemic circulation
and synovial fluid of patients with a variety of autoimmune diseases.43,44

Polymerase Chain Reaction (PCR)

PCR is an in vitro method for DNA amplification that was invented by Kary Mullis in 1985 for
which he was awarded the Nobel Prize in 1993. It is a simplified version of the process that
normally occurs during cell division. DNA sample obtained from hospital tissue specimen,
single hair strand, or a drop of blood can be amplified and analysed by PCR. The three-step
protocol of PCR involves thermal degradation of DNA, followed by primer annealing and
lastly, primer extension. The entire procedure can be carried out in specialized equipment
called “thermocycler”. At the temperature of 94-98°C, within 8 min, the DNA of the sample
can be degraded such that the double helical structure opens up into single strands. Now,
oligonucleotides of 18-30 bases or “primer” is introduced into the reaction mixture and heated
from 5 to 60°C for 1-2 min. The primer is complementary to the DNA template flanks and
anneals to the template. The final step involves addition of DNA polymerase or Taq protein,
which helps to produce multiple copies of the identified flank. The purity of the product can
 Agents for Immune-based Disorders 263

be determined by electrophoresis, HPLC or direct sequencing. The technique is being used for
diagnosing disease condition, polymorphism, developing transgenic animals, gene therapy
or quantification of biomarker in disease tissues to provide insights into the mechanisms of
action of novel therapeutic agents.45

Western Blotting
Western blotting is a technique that helps to identify specific protein from a complex mixture.
In contrast, Southern blotting is for identifying DNA fragments and Northern blotting for
messenger RNAs (mRNA). Samples are prepared from tissues or cells that are homogenized in
a buffer that protects the protein of interest from degradation. The sample is electrophoretically
separated using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred
to a nitrocellulose membrane for detection. The membrane is incubated with a generic protein
(such as milk protein) to bind to any remaining sticky places on the membrane. A primary
antibody is then added to the solution, which is able to bind to its specific protein. A secondary
antibody-enzyme conjugate, which recognizes the primary antibody, is added to find locations
where the primary antibody is bound. A number of autoimmune diseases have shown genetic
linkage, and Western blotting helps to detect them.46 For example, SLE is characterized by
autoantibody production against different nuclear molecules, including those involved in pre-
mRNA processing. The presence of autoantibodies in the sera of patients can be determined
using Western blotting and proves to be a crucial diagnostic tool.47

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 cHAPTER

16
Antihypertensive Agents
Introduction
Hypertension is a complex multifactorial disease and one of the leading causes of mortality
and morbidity due to stroke, heart attack and kidney failure. Because the etiology of essential
hypertension is not known and may be multifactorial, the use of experimental animal models
may provide valuable information regarding many aspects of the disease, which include
etiology, pathophysiology, complications and treatment. As new insights in to the pathogenesis
of hypertension are revealed, new models are being developed to produce hypertension in
animals. In this chapter, a brief overview of the most widely used animal models, their features
and their importance is provided. Nevertheless a cautious approach is mandatory when the
experimental findings in these models is extrapoliated to human hypertension.

In vitro models

1. Endothelin Receptor Antagonism in Porcine Isolated Hearts

Potent long lasting contractions of isolated blood vessel strips and increase blood pressure in
vivo is elicited by endothelin peptides. Endothelins have been implicated in the pathophysiology
of cardiovascular disorders. In this model, isolated porcine coronary artery is used since the
smooth musculature of artery is considered to contain the ETA receptors.
From porcine hearts left anterior descending coronary arteries are isolated. The
endothelium-denuded arteries are cut into spiral strips about 10 mm long and 1 mm wide. The
intimal surface of the spiral rings is then rubbed gently with filter paper to remove the vascular
endothelium. Each strip is suspended in an organ bath containing Krebs-Henseleit solution
bubbled with 95% O2/5% CO2 at 37oC. Once the isolated preparation is stabilized reference
contraction is isometrically obtained with 50 mM KCl. Concentration-response curves for
ET-1 are obtained by cumulative addition of ET-1. Twenty minutes before the addition of ET-1
the endothelin receptor antagonist/test drug is added to the organ bath and concentration
response curve is recorded. The pA2 values and slopes are obtained by analysis of Schild plots.1
 Antihypertensive Agents 267

2. Monocrotaline Induced Pulmonary Hypertension

Monocrotaline is a hepatotoxic and pneumotoxic agent used in rats to induce pulmonary
hypertension. It is a pyrrolizidine alkaloid derived from Crotaloria spectabilis and its single
injection leads to progressive pulmonary hypertension followed by right ventricular hypertrophy
and cardiac failure. Ultrastructural changes such as degeneration and fragmentation of
endothelial cells, perivascular edema, extravasation of red blood cells and muscularization
of pulmonary arteries and arterioles are also observed. Monocrotaline administration in rats
may result in severe right ventricular hypertrophy accompanied by ascitis and pleural effusion.
Sprague Dawley rats (200 to 225 g) are fed with the test drug for one week prior to single
subcutaneous injection of 100 mg/kg monocrotaline. The animals are sacrificed 4, 7 or 14 days
latter and their hearts and lungs are excised from thoracic cavity. The left ventricle and left lung
are weighed. Their pulmonary artery segments, main pulmonary artery, right extra pulmonary
and an intra pulmonary artery are also isolated. Each vessel is suspended between stainless
steel hooks in tissue baths containing Krebs-Hensleit buffer aerated with 95% O2 and 5% CO2 at
37oC. At the end of the experiment vessel segments are blotted, weighed and their dimensions
are measured. Cross-sectional area of artery is determined from tissue weight and diameter.
After 1 h arteries are made to contract to KCl (6 ×10-2 M). Maximum active force generated by
an artery is plotted as a function of applied force and changes in isometric force are monitored
using force displacement transducers and recorded on a polygraph. Contractile and relaxant
agonist responses are assessed in pulmonary arteries. Cumulative concentration–response
curves to KCl, angiotensin II, norepinephrine are plotted. Contractions are expressed as active
tension development, force generated per cross-sectional area. Both contractile and relaxation
responses are plotted as a function of negative logarithm of agonist concentration.2 T-test for
grouped data is used to compare differences in mean responses.

Rat Models of Hypertension

1. Reno-vascular Hypertension
Experimentally, renal hypertension can be produced by constriction of the renal artery which
activates peripheral Renin angiotensin aldosterone system (RAAS) and sympathetic nervous
system. In response to decreased blood flow, renin is secreted by kidneys. Renin converts
angiotensinogen to angiotensin-I, which is further converted to angiotensin-II by angiotensin
converting enzyme (ACE). Angiotensin-II is a potent vasoconstrictor and also causes release
of aldosterone leading to salt and water retention resulting in increased blood volume and
hypertension . The various methods to produce renal hypertension include:
A. Two-kidney one clip (Goldblatt hypertension, 2K1C)
In the two-kidney one clip method, constriction of only one renal artery is carried out, while
the contralateral kidney is left intact. In rats clamping the renal artery for 4 h can induce acute
renal hypertension by activation of renin-angiotensin system. After re-opening of the vessel,
accumulated renin is released into circulation leading to acute hypertension. This test is used
to evaluate antihypertensive activities of drugs.3,4
268 Drug Screening Methods

Sprague Dawley rats (300 g) are anesthetized with hexobarbital sodium (100 mg/kg,
intraperitoneally). The trachea is cannulated to facilitate spontaneous respiration. Through
a pressure transducer connected to carotid artery, blood pressure is measured. Jugular vein
is cannulated for administration of test compound. A poly vinyl chloride (PVC) coated clip
is placed into the left hilum of the kidney and fixed to the back muscles. The renal artery is
occluded for 3.5-4 h. Ganglionic blockade is performed with pentolinium and after obtaining
stable reduced blood pressure values, the ‘renal arterial clip’ is removed. As a consequence
of elevated plasma renin level, blood pressure rises. Test compound is administered by
intravenous route. Blood pressure is monitored continuously.
Increase in blood pressure after re-opening of renal artery and reduction of blood pressure
after administration of test compound is determined. Percent reduction of blood pressure
values under drug treatment is calculated as compared to pretreatment values.5-7
B. Chronic renal hypertension in rats (1-kidney-1-clip method)
Constriction of the renal artery is done on one side and on the other side the contralateral
kidney is removed. As discussed earlier, ischemia of the kidneys induces hypertension.
Various modifications of the technique have been described for several animal species. The
1-kidney-1-clip method is one of the most effective modifications in rats in which one kidney
is removed.
Sprague Dawley rats (200 to 250 g) are anesthetized with pentobarbitone sodium (50 mg/
kg, intraperitoneally). In the left lumbar area, a flank incision is made parallel to the long
axis of the rat. The renal artery is dissected, cleaned and a U shaped silver clip is slipped
around it near the aorta. The size of the clip is adjusted so that the internal gap ranges from
0.25 to 0.38 nm. The right kidney is removed after tying off the renal pedicel. Four to five
weeks after clipping, blood pressure is measured and rats are divided into different groups of
different doses. For individual dose each animal is used as its own control. Test compounds
are administered for 3 days. Pre-drug and 2 h post-drug blood pressure readings are taken.8,9
Antihypertensive activity of test drug is determined by comparing treatment blood pressure
value with day 1, pre-drug BP Comparisons are made using the paired t-test for evaluation of
statistical significance.
C. Chronic renal hypertension in rats [Two kidney two clip (2K2C) method]
In the two kidneys two clip method (2K2C) hypertension; constriction of aorta or both renal
arteries is undertaken. When the aorta or both renal arteries are constricted, there is severe
renal ischemia caused by renal clipping, occasioning the activation of renin-angiotensin and
the sympathetic nervous system and the elevation of serum vasopressin, leading to increased
BP. The 2K2C, with a high incidence of spontaneous stroke, can be used as independent of
a genetic deficiency. The lesioned small artery or arteriole with thrombotic occlusion is the
main cause of cerebral infarction in 2K2C, and this may be similar to lacunar infarction in the
human brain. Indeed, one of the most common causes of renal hypertension in human beings
is such a patchy ischemic kidney disease.
Sprague Dawley rats (300 g) are anesthetized; trachea, carotid artery and jugular vein
are cannulated. The renal arteries are located, U shaped silver clip is slipped around them
 Antihypertensive Agents 269

or near the aorta. Either the renal arteries or the aorta is occluded using renal arterial clips.
Test compound are administered by intravenous route via the jugular vein. Blood pressure is
monitored continuously. Percent reduction of blood pressure values under drug treatment is
calculated as compared to pre-treatment values.10
2. Neurogenic Hypertension
Evidence suggests that the central nervous system participates in the genesis of hypertension.
Neurogenic hypertension can be defined as a permanent increase in BP resulting from a
primarily neural change. One of the most important negative feedback in the control of BP
originates from baroreceptors located in the carotid sinus and aortic arch. Denervation of
sino-aortic baroreceptors (SAD) is the neurogenic model of hypertension most often used.
A. Blood pressure in pithed rats
The pithed rat model is devoid of neurogenic reflex control that may modulate the primary
drug effect; it is frequently used to evaluate drug action on the cardiovascular system.
Male Wistar rats (250 to 350 g) are anesthetized with halothane. The carotid artery is
cannulated for monitoring blood pressure and blood sampling. The trachea is cannulated and
the animal is maintained on artificial respiration using a ventilation pump (60 cycles/min). The
jugular vein is also cannulated for the administration of test drug. Pithing is done by inserting
a steel rod, 2.2 mm in diameter and 11 cm in length, through the orbit and foramen magnum
down the whole length of the spinal canal. Inspired air is oxygen-enriched by providing a flow
of oxygen across a T-piece attached to the air inlet of the ventilation pump. Thirty minutes
after pithing, a 0.3 ml blood sample is withdrawn from the carotid cannula and analyzed for
pO2, pCO2, pH and bicarbonate concentration using blood gas analyzer. Through the carotid
artery blood pressure and cardiac frequency is recorded. To measure α1 and α2 antagonism,
first dose response curves are registered with phenylephrine, a selective α1 agonist (0.1-30
μg/kg, intravenously) and BHT 920, a selective α2 agonist (1-1000 μg/kg, intravenously). The
test drug is administered intravenously and the agonist dose response curves are repeated 15
min later. The curve of blood pressure response to agonist is obtained. Dose response curves
are plotted on a logarithmic probit scale. Potency ratios are calculated from the dose response
curves.11
3. Dietary Hypertension
It is known that long-term exposure to a special diet (high salt, fat or sugar) results in dietary
hypertension in some rats. The presence of oxidative stress and inactivation of Nitric oxide
(NO) in rats maintained on the high-fat or high-sugar diet, may contribute to the development
of hypertension by enhanced generation of reactive oxygen species (ROS). The reduction in
NO availability in the high-fat and high-sugar diet-fed animals was associated with marked salt
sensitivity, as evidenced by a significant rise in BP on the high-salt diet. Dietary intake of fats
and carbohydrate, particularly the intake of simple sugars and the resultant effects of plasma
insulin, adipokine and lipid concentrations, may affect cardiomyocyte size and function,
especially with chronic hypertension.10
A. Fructose-induced hypertension in rats
Blood pressure increases by intake of either sucrose or glucose and result in the development
of spontaneous hypertension or salt hypertension in rats. Fructose feeding also causes insulin
resistance, hyperinsulinemia and hypertriglyceridemia in normal rats.
270 Drug Screening Methods

Wistar rats (200 to 250 g) are housed per cage on a 12 h light and dark cycle and fed water and
chow diet ad libitum. Drinking water consists of 10% fructose solution. Fluid intake, food intake
and body weight of each rat are measured every week during the course of drug treatment.
Systolic blood pressure and pulse rate is measured using the tail-cuff method. Blood samples
are collected before and every second week during treatment and plasma glucose, insulin,
triglycerides are measured.12 One way or two way analysis of variance followed by Newman-
Keuls test is used for the statistical analysis.
B. Increased salt induced hypertension in rats
Physiologically, normal kidney has the ability to excrete easily the daily salt load without
allowing a marked rise in extracellular volume. However, general epidemiological data
have shown that higher the average sodium intake in a given population, the greater will
be the prevalence of hypertension. Chronic ingestion of excess salt produces hypertension
in rats, which mimics human hypertension morphologically. High salt intake hypertension
has been produced in rats by replacing drinking water with 1-2% sodium chloride for 9-12
months.
Wistar rats (200 to 250 g) are fed chow diet ad libitum. Drinking water is replaced with 1-2%
sodium chloride solution. Fluid intake, food intake and body weight of each rat are measured
every week during the course of drug treatment. Systolic blood pressure and pulse rate is
measured using the tail-cuff method. Blood samples are collected before and every second
week during treatment.13
4. Endocrine Hypertension
Mineralocorticoids cause retention of sodium and water in the body until escape diuresis
occurs due to increased pressure on the kidneys. No further retention of sodium and water
occurs, but general level of body sodium and water is slightly raised. Selye et al. was the first
to demonstrate that deoxycorticosterone acetate (DOCA) produces hypertension in rats.
There is increased DOCA-induced reabsorption of salt and water leading to increased blood
volume and hence increased BP. There is also increased secretion of vasopressin leading to
water retention and vasoconstriction. In addition, altered activity of RAAS leads to increased
sympathetic activity.
A. DOCA-salt rats
Mineralocorticoid induces hypertension by causing increase in plasma and extracellular
volume. Salt loading and unilateral nephrectomy in rats further increases the hypertensive
effect. The administration of DOCA, in combination with a high salt diet and unilateral
nephrectomy induces a low renin form of hypertension, which can be opposed to the other
artificial model, where renin level is high.14
Male Sprague Dawley rats (250 to 300 g) are anesthetized with ether. The left kidney is
removed through a flank incision. DOCA (20 mg/kg) is dissolved in olive oil and injected
subcutaneously to rats, twice weekly for four weeks. Drinking water is replaced with 1% NaCl
solution. Blood pressure starts to rise after 1 week and systolic value reaches around 160
to 180 mm Hg after 4 weeks. DOCA pellets or implants in silastic devices can also be used
instead of repeated injections. Test drug is administered orally for one month. Blood pressure
 Antihypertensive Agents 271

is measured before and after the administration of the test drug and their values are compared
to evaluate the antihypertensive effect.
End organ damage:. The animals are sacrificed and their hearts weighed. Renal changes are
seen with proteinuria and glomerulosclerosis. This rat model also demonstrates endothelium
dependent relaxations.
5. Psychogenic Hypertension
It has been reported that elevation of BP resulting from repeated exposure to stressful situation
may lead to a state of persistent hypertension. Other types of stress that may be applied include
emotional stimuli, psychosocial stress, immobilization stress and electric stimuli. However,
the degree and stability of hypertension may not be comparable to other types of hypertension.
The stress-induced hypertension is associated with either normal or suppressed PRA values,
suggesting that the hypertension in these animals is not renin-dependent. As stress plays an
important part in development of human hypertension, this model is very frequently used to
study the pathophysiology of hypertension.
Air-jet stimulation-induced hypertension
Borderline hypertensive rats (BHR) are useful for psychogenic hypertension. BHRs
are exposed to daily sessions of either short (20 min) or long (120 min) duration air-jet
stimulation. BP is monitored at regular intervals using tail cuff method. It is generally
observed that the BHR develop hypertension within 2 weeks in comparison to home cage
controls. Animals exposed to 120 min stress sessions have significantly higher systolic
BP relative to the 20 minute group. Its deleterious effects depend on the critical period
of exposure, duration and type, as all these factors may alter functions of the basic auto-
regulatory stress response components in the hypothalamic–pituitary–adrenal axis,
sympathoadrenal medullar system, rennin–angiotensin–aldosterone system (RAAS) and
sympathetic nervous system.15
6. Genetic Hypertension
A. Salt-sensitive Dahl rats
The salt-sensitive Dahl rats develop severe and fatal hypertension when fed high salt diets,
whereas salt resistance Dahl rats do not develop such severe hypertension upon salt loading.13
Also when fed normal salt diets, the salt sensitive rats become hypertensive, demonstrating
that this is a model of genetic hypertension, with the extra feature of salt sensitivity.14
Sprague Dawley rats (250 to 300 g) are used for this study. The drinking water is replaced
with 8% NaCl saline solution. High Dahl salt diet is prepared in the laboratory by mixing salt
with the regular diet. The animals are fed the prepared diet and 8% NaCl solution ad libitum.
The test group rats are administered the drug orally for 1 month. Blood pressure changes are
recorded. After the completion of the experimental duration, animals of both groups (test and
sham control) are sacrificed. Their hearts are removed and total cardiac mass, weight of left
and right ventricle is measured and compared. Upon salt feeding (8% NaCl), blood pressure
rises steeply, to levels slightly higher than found in spontaneously hypertensive rats (upto
32%). The ability of the test drug to reverse these changes is studied.
272 Drug Screening Methods

End organ damage: Cardiac failure occurs at 4 to 5 months of age in the salt sensitive Dahl
rats. Also, renal changes are more severe than spontaneously hypertensive rats, with severe
early proteinuria. In this model endothelium dependent relaxations are found to be impaired.
B. Spontaneously hypertensive rats (SHR)
By breeding a strain of spontaneously hypertensive Wistar rats with a female having slightly
raised blood pressure, Okamoto and Aoki obtained a strain of rats with spontaneous
hypertension, the SHR.17 Blood pressure rises around 5 to 6 weeks of age and steadily increases
to reach systolic blood pressure of 180 to 200 mm Hg. The SHR develop many features of
hypertensive end organ damage including cardiac hypertrophy, cardiac failure and renal
dysfunction. However, they do not exhibit gross vascular problems. Apart from depressed
endothelium dependent relaxations, they have no tendency to develop strokes, and do not
develop macroscopic atherosclerosis or vascular thrombosis. The SHR stroke prone (SHR-
SP) is a further developed substrain, with even higher levels of blood pressure, and a strong
tendency to die from stroke.18 The SHR have been widely used to evaluate genetic factors in
hypertension, yielding a wide variety of genes that seem to co-segregate in various crosses
which is not always confirmed.19
End organ damage : The untreated SHR exhibit cardiac hypertrophy and develop heart failure
between the age of 18 to 24 months. However, not all rats exhibit signs of heart failure after 24
months so that despite the uniformity of the model, individual differences are seen. Impaired
endothelium dependent relaxations have been consistently found, although rats until 13 to 15
weeks of age may sometimes have normal endothelium dependent relaxation. Renal damage
has also been found in older SHRs. Comparisons between the sham rats (untreated) and
treated rats are made on the basis of above-mentioned parameters as well as blood pressure
recordings. The ability of the test drug to reverse/delay the changes seen in sham control group
is studied and antihypertensive potential of test drug is evaluated.
Methods to measure BP in rats
A. Tail cuff method in rats
The indirect tail cuff method allows the measurement of blood pressure without any surgical
procedure. The method is analogous to sphygmomanometry in humans. The indirect tail cuff
method is widely used to evaluate the influence of antihypertensive drugs in spontaneously
and experimentally induced hypertensive rats.
Charles River Male Spontaneously hypertensive rats (300 to 350 g) are anesthetized with 0.8
ml of 4% chloralhydrate solution. Both kidneys are exposed. A silver clip (0.2 mm diameter)
is placed into both renal arteries, kidneys are reposed and wound is closed by suture. After 5
to 6 weeks, operated animals attain renal hypertension with systolic blood pressure of 170 to
200 mm Hg. To measure blood pressure, a tubular inflatable cuff is placed around the base of
the tail and a pizoelectric pulse detector is positioned distal to the cuff. The cuff is inflated well
above suspected systolic blood pressure until the pulse is obliterated. Thereafter, pressure in
the cuff is slowly released and as the pressure reaches the systolic blood pressure, the pulse
reappears which is detected and subsequently recorded on a polygraph. The test substance is
administered intraperitoneally once a day over a period of 5 days. Blood pressure and heart
rate are measured (predose and 2 h post-drug) at days 1, 3 and 5. Percent decrease in systolic
 Antihypertensive Agents 273

blood pressure after administration of the test drug and the duration of effect is determined.20
Statistical significance is assessed by the Student’s t-test. Scores for percentage decrease in
systolic blood pressure and for the duration of the effect are allotted.
B. Indwelling catheter for measurement of blood pressure in conscious rats
The method allows direct measurement of blood pressure in conscious rats eliminating the
influence of anesthesia on cardiovascular regulation. 7 cm long cannula are prepared by
cutting PE 10 and PE 20 tubing respectively. A stylet wire is inserted into the PE 10 tubing and
PE 20 tubing is also slipped over the stylet wire. The tube ends are heated in a current of hot
air and fused together. Using ridges the cannula is anchored to the animals tissue. In order to
make a ridge, the stylet wire is left inside the cannula and the cannula is heated in a jet of hot
air. When the polyethylene at the point of heating becomes soft, the cannula is pressed slightly
and the ridge is formed.
Male Sprague Dawley rats weighing 300 g are anesthetized with pentobarbitone sodium
(45 mg/kg, intraperitoneally). Through a midline incision, the abdominal aorta is exposed,
a trocar is passed through the psoas muscles adjacent to the segment of the aorta. Then the
cannula is inserted into the trocar and the trocar is withdrawn from the body. The end of the
cannula thus comes out from the neck, being anchored by silk sutures to the neck skin and to
the psoas muscle. The cannula is filled with heparin solution and the end which is projecting
out from the neck skin, is blocked with a tight fitting stainless steel needle. The other end
of the cannula is implanted into the aorta. The aorta is wiped with cotton tipped applicator
stick over the bifurcation, occluded above this segment and punctured with a bent 27 gauge
hypodermic needle. The tip of the PE 10 catheter is inserted through the needle and advanced
up the aorta. The intestines are replaced and wounds sutured. The rats are allowed to recover
for one week.
After 1 week the occluding stainless steel needle is removed and the cannula is flushed with
heparin solution. To restrict the movement of the rat, it is placed in a small cage. The cannula
is connected to a Statham P 23 Db pressure transducer and blood pressure is recorded on a
polygraph.21 Test drugs are administered either subcutaneously or orally. Recordings are taken
before and after administration of the drug over a period of 1 h. Changes of blood pressure
are measured and the maximal changes of each group are averaged and compared with the
standard.

Dog Models of Hypertension

1. Chronic renal hypertension
Dogs (8 to 12 kg) are anesthetized intravenously with 15 mg/kg thiopental. Midline abdominal
incision is made, one kidney is exposed and wrapped in cellophane and then replaced. The
contralateral kidney is exposed and artery, vein and ureter are ligated and kidney is removed.
The abdomen is then closed and sutured back. After six weeks of surgery, blood pressure
is measured. Blood pressure is recorded either by indirect tail cuff method or by direct
measurement through the carotid artery. Test drugs are administered for 5 days. On day 1
readings are taken every 2 h, just before, and 2 and 4 h after oral treatment. On day 3 and 5
blood pressure is recorded before, 2 and 4 h after drug treatment.
274 Drug Screening Methods

The starting value is the average of the 2 readings before application of the drug. Subsequent
readings are subtracted from this value and recorded as fall of blood pressure at the various
recording times.22-24
2. Neurogenic hypertension
Baroreceptors situated in the carotid sinus and aortic arch play an important part in the
regulation of blood pressure. Stimulation of the afferent buffer fibers exerts an inhibitory
influence on the vasomotor center. A persistent rise in blood pressure is observed on sectioning
the baroreceptors. Thus, by this procedure acute neurogenic hypertension is induced in dogs.
Adult dogs (10 to 15 kg) are anesthetized using 15 mg/kg thiopental, 200 mg/kg sodium
barbital and 60 mg/kg sodium pentobarbital.25 Femoral vein is cannulated for the administration
of test compounds. Left ventricular pressure and dP/dt are recorded through common carotid
artery using Millar microtip pressure transducer. Pmax and cardiac output are also calculated. The
carotid sinus nerves are isolated, ligated and sectioned and a bilateral vagotomy is performed to
induce neurogenic hypertension. After 30 min equilibration period, a bolus of test compound
is administered by intravenous route. Heart rate, arterial pressure, left ventricular pressure, Pmax
and dP/dt are monitored for 90 min. Changes in cardiovascular parameters are expressed as
percentage of the values before and after administration of the drug.26

Monkey Model of Hypertension

1. Renin inhibition in monkeys
Blood pressure is mainly regulated by the renin angiotensin system and can be influenced
by several ways including inhibition of renin. Renin is an aspartyl protease that hydrolyses
angiotensinogen to release angiotensin I. Angiotensin I is subsequently converted to
angiotensin II by angiotensin converting enzyme. Renin inhibitors developed so far have a
high specificity for primate renin and cause only weak inhibition of renin in subprimate
species. This suggests that most common laboratory animals such as rats and dogs are not
suitable for in vivo evaluation of renin inhibitors.
Marmosets (Callithrix jacchus) of 300 to 400 g are fed pellet diet supplemented with fruits.
The animals are anesthetized two days prior to the experiment and catheters are implanted
in the femoral artery for measurement of blood pressure. Lateral tail vein is cannulated for
the administration of test compounds. Furosemide (5 mg/kg) is injected intravenously 30
min before the experiment in order to stimulate renin release. During the experiment, the
marmosets are sedated with diazepam (0.3 mg/kg, intraperitoneally) and kept in restraining
boxes. Mean blood pressure is recorded continuously, and heart rate is measured at fixed
intervals. The test compound and standard drug are injected by intravenous infusion or
administered orally. Blood pressure is recorded after 30 min of intravenous infusion. Changes
from pretreatment values after various doses of drug are compared. Dose-response curves can
be established.27

Transgenic Models
1. Transgenic rats overexpressing the mouse Ren-2 gene {TGR (mRen 2) 27}
The introduction and overexpression of the mouse Ren-2 gene in the rat leads to severe
hypertension, lethal in the homozygous rats. Two important features characterize this rat
 Antihypertensive Agents 275

model: firstly, it is genetic, inherited form of hypertension where the single genetic event is
known, and secondly, despite the known genetic alteration, the exact mechanism underlying
hypertension remains elusive. In this rat hypertension, is related to an increased renin activity.
The severity of hypertension depends partly upon the genetic background of the rats used for
breeding the TGR (mRen2) 27. An accelerated and malignant type of hypertension occurs
when these rats are bred with Sprague Dawley rats.28
End organ damage: 70% of the heterozygous rats survive at least until the age of 5 months.
Before that age they develop marked cardiac hypertrophy and impairment of endothelium
dependent relaxations.
The ability to specifically introduce genetic constructs and thereby breed transgenic
animals, has opened new possibilities for hypertension research.29 The transgenic rat that was
obtained after introduction of the entire mouse Ren2d gene.30 In this hypertensive model,
the hypertension and ensuing end organ damage depends upon increased local angiotensin
II formation and is exquisitely sensitive to renin angiotensin system (RAS) inhibition. Other
transgenic models have been obtained where the introduction of both renin and human
angiotensinogen increases blood pressure in mice and rats.31
In the knockout models, genes for ANF and NO-synthase have been knocked out. The
ANF knockout rats resulted in salt sensitive hypertension whereas the knockout of the type A
receptor for ANF resulted in salt independent hypertension in rats. These models can be used
to screen various anti-hypertensive agents.

Discussion and Conclusion

A brief overview of the most widely used animal model strains, and their characteristics has
been provided. The most important lesson from a direct comparison of these animal models
is that despite the well-known heterogeneity of hypertension, the outcome of hypertension
can be similar in some respects: rats from all models exhibit cardiac hypertrophy and all
demonstrate impaired endothelin dependent relaxations of isolated arteries. However, the
most severe form of end organ damage such as heart failure, stroke and kidney failure occurs
in only a subset of hypertensive rats.
Not all classes of antihypertensives are equally effective in all rat models of hypertension:
endothelin receptor antagonists are not effective in SHR, but have beneficial effects in DOCA-
salt model.
Thus, it seems that rat models of hypertension mainly share high blood pressure, but
otherwise display a wide variety of biochemical disturbances, with equally varying course and
prognosis. The course and prognosis seem to depend on three main factors:
1. The mechanical stress which is the absolute level of blood pressure, and when above a
certain threshold will always cause severe organ damage.
2. The biochemical stress which is an important modifier of the course of hypertension.
3. The ability to recruit coping or adaptive mechanisms.
These models of hypertension provide ample opportunity not only to investigate the
mechanisms involved in the pathogenesis of hypertension, but also to learn about the critical
balance between stress and coping which eventually determines prognosis.
276 Drug Screening Methods

Summary

In vitro models In vivo models

Rat Models
Reno-vascular Neurogenic Diet induced Endocrine Psychogenic Genetically
induced induced induced induced
Endothelin Two-kidney Blood Fructose DOCA-salt Air-jet Salt-sensitive
receptor one clip pressure in induced rats stimulation Dahl rats
antagonism in (Goldblatt pithed rats induced
porcine isolated hypertension, hypertension
hearts 2K1C)
Monocrotaline Chronic renal Increased Spontaneously
induced hypertension salt induced hypertensive
pulmonary in rats rats (SHR)
hypertension (1-kidney-1-
clip method)
Chronic renal
hypertension
in rats (Two
kidney two
clip (2K2C)
method
Dog model of hypertension
• Chronic renal hypertension
• Neurogenic hypertension
Monkey model of hypertension
• Renin inhibition in monkeys
Transgenic model of hypertension
• Transgenic rats overexpressing the mouse Ren-2 gene [TGR (mRen 2) 27]

References
1. Calo G, Gratton JP, Orleans-Juste P, et al. Pharmacology of endothelins: vascular preparations for
studying ETA and ETB receptors. Moll Cell Biochem 1996;154:31-7.
2. Altiere RJ, McIntyre MJ, Petrenka J, et al. Altered pulmonary vascular smooth muscle responsiveness
in monocrotaline-induced pulmonary hypertension. J Pharmacol Exp Ther 1986;236:390-5.
3. Goldbatt H, Lynch J, Hanzal RF, et al. Studies on experimental hypertension & the production of
persistent elevation of systolic blood pressure by means of renal ischemia. J Exp Med 1934;59:347-79.
4. Okakomoto AK. Development of a strain of spontaneously hypertensive rat. Jap Circ J 1963;27:282-93.
5. Baura ALA, Green AF. Antihypertensive agents. In: Laurence DR, Bacharach AL, ed. Evaluation of
drug activities: Pharmacometrics. London & New York: Academic Press, 1964:431-6.
6. Cerqua S, Samaan A. Cure of experimental renal hypertension. Clin Sci 1939;40:113-8.
7. Swales JD, Tange JD. The influence of acute sodium depletion on experimental hypertension in the
rat. J Lab Clin Med 1971;78:369-79.
8. Brunner HR, Kirshman JD, Sealey JE. Hypertension of renal origin: evidence for two different
mechanisms. Science 1971;174:1344-6.
9. Leite R, Salgado MCO. Increased vascular formation of angiotensin II in one kidney one clip
hypertension. Hypertension 1992;19:575-81.
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10. Badyal DK, Lata H, Dadhich AP. Animal models of hypertension and effect of drugs. Indian J
Pharmaco 2003;35:349-62.
11. Curtis MJ, Mc Leod BA, Walker MJA. An improved pithed rat preparation: the actions of the optical
enatiomers of verapamil. Asia Pacific J Pharmacol 1986;1:73-8.
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18. Yamori Y. Development of spontaneously hypertensive rat (SHR) the stroke prone hypertensive SHR
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Ganten D, de Jong W, volume eds. Birkenhager WH, Reid JT, series eds. Handbook of Hypertension,
Experimental and Genetic models of Hypertension. Amsterdam: Elsevier Press, 1994:26-9.
19. Kreutz R, Struk B, Rubatta S. Role of alpha, beta and gamma subunits of epithelial sodium channels
in a model of polygenic hypertension. Hypertension 1997;29:131-6.
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tension 1997;29:428-34.
 CHAPTER

17
Antiglaucoma Agents
INTRODUCTION
Glaucoma is a progressive optic neuropathy characterized by visual field changes and cupping
of optic disc. Elevated intraocular pressure (IOP) is one of the important risk factor. The rise
in IOP is due to the increase in aqueous formation, low rate of outflow or a raised pressure
in the episcleral veins. An obstruction to the circulation of the aqueous at the pupil or to its
drainage through the angle of the anterior chamber causes glaucoma. The normal IOP of an
individual ranges from 10-20 mm Hg and can rise up to 60 mm of Hg in glaucoma patients.
Raised IOP of this magnitude can result in loss of vision. Optic nerve axons of the eyeball
become compressed at the optic disc due to elevated IOP. This compression probably blocks
the axonal flow of cytoplasm from the neuronal cell bodies in the retina to the extended optic
nerve fibers entering the brain. It results in lack of nutrition of fibers and ultimately causes
death of the neurons. Compression of retinal artery may increase the neuronal damage due to
reduction in retinal nutrition.1
The global estimate of blindness was over 37 million with glaucoma accounting for slightly
more than 12% of the blind patients worldwide as per WHO report in 2005.2 In 2013, the number
of people, in the age range of 40–80 years, with glaucoma worldwide was estimated to be 64.3
million, increasing to 76.0 million in 2020 and 111.8 million in 2040.3 India has a high burden
of blind (23.5%) in the world and 13% of the global blindness due to glaucoma is in India.
Many population based surveys carried out in the west and in Asia have shown that glaucoma
remains undetected in nearly 50% of the cases and hence glaucoma-related blindness and
disability is often underestimated.4,5
Glaucoma is generally classified as: (i) primary (ii) developmental and (iii) secondary. The
commonest form of glaucoma is primary glaucoma; it can be open angle glaucoma or angle
closure glaucoma. In primary open angle glaucoma (POAG) the angle of the anterior chamber
is always open, at all stages of disease, and aqueous has access to the outflow channels at all
times, whether the tension is normal or elevated. There is increased resistance to outflow in the
corneoscleral meshwork, whereas in primary angle closure glaucoma no abnormal resistance
to outflow in the corneoscleral meshwork is observed. The sole cause of elevated tension is
closure of the angle. The iris obstructs the access of aqueous humor to the outflow channels
(Fig. 17.1).
 Antiglaucoma Agents 279

Figure 17.1: Pathway of aqueous flow

Prompt and effective management of glaucoma is necessary to reduce the incidence of

cases of bilateral blindness due to progressive glaucoma. Biological revolution in medicine has
provided new avenues for therapeutic intervention. Newer and innovative treatment strategies
are being considered for the control of elevated IOP by the use of synthetic and herbal drugs in
glaucoma.
The primary goal in the management of glaucoma is to lower IOP below 20 mm Hg in the
patients with mild changes in the optic disc and below 15 mm Hg in the patients with more
severe changes. Surgical intervention aiming at increasing the aqueous humor outflow is
undertaken when IOP remains uncontrolled even with multiple drug therapy.
Tables 17.1 and 17.2 show the groups/drugs widely used in the treatment of glaucoma.

Table 17.1: Topical drugs

Group of drugs Formulations
Cholinergic agonists Pilocarpine, carbechol, physostigmine
Adrenergic agonists Forskolin, isoproterenol, salbutamol, epinephrine,
brimonidine
Adrenergic antagonists Timolol, Levobunolol, betaxolol, atenolol, metipranolol
Prostaglandin analogues Latanoprost, Unoprostone, travoprost, bimatoprost
Carbonic anhydrase inhibitors Trifluoromethazolamide, aminozolamide, dorzolamide,
brinzolamide
280 Drug Screening Methods

Table 17.2: Systemic drugs

Group of drugs Formulations

Carbonic anhydrase inhibitor Acetazolamide, methazolamide, dichlorphenamide
Hyperosmotic agents Glycerol, mannitol
Miscellaneous Cannabinoids, prostaglandins, ACE inhibitors, melatonin, calcium channel
blockers, haloperidol, etc.

A potential antiglaucoma agent needs thorough screening, which can be done in various
in vitro and in vivo experimental models. To study the mechanism of glaucoma and the efficacy
of the antiglaucoma agent, animal models mimicking human forms of glaucoma are used.

EXPERIMENTAL MODELS OF GLAUCOMA

Studies of glaucoma are carried out in animals having close resemblance of eye structures
with human eye. The methods of increasing the IOP should be easy to carry out, produce a
reliable increase in IOP, allow tonometric monitoring in experimental animals, and minimize
secondary ocular changes like inflammation. Several animal models are used for the screening
of IOP lowering drugs. The most common and feasible model under present circumstances,
when there is a great concern for animals used in research, is a rabbit model. The surface area
and other structures of the eye of a rabbit resemble human eye and they can be housed and
handled easily.
Intraocular pressure is measured by tonometery using methods such as:
1. Schiotz method: The eyes are anesthetized and then schiotz tonometer, a small handy
instrument is placed on the cornea and pressed lightly to measure the pressure (Fig. 17.2).
2. Applanation method: A flouresein-stained strip is touched to the side of the eye that helps
in the examination. The dye is washed out along with the lacrimation. The instrument is
fitted on a slit lamp. The probe of the tonometer is made to touch the cornea by moving the
instrument forward towards the eye of the patient (Fig. 17.3).
3. Non-contact method: The IOP is measured without any contact of the tonometer to the eye.
A puff of air is released when the eye is focused which indents the eye. The force required for
indenting the eye is measured (Fig. 17.4).
The IOP is elevated beyond the limits by the following methods:
i. Ocular injections
ii. Reducing the serum osmolarity
iii. Application of lasers and
iv. Steroids.

Ocular Injections
There are reports on the production of glaucoma in the experimental animals by injections of
a variety of agents like alpha chymotrypsin into the posterior chamber and, methylcellulose,
autologous ghost red blood cells, kaolin, prostaglandin, alkali into the anterior chamber of eye
which results in elevated IOP.
 Antiglaucoma Agents 281

Figure 17.2: Shiotz tonometer Figure 17.3: Goldman applanation tonometer

Figure 17.4: Noncontact tonometer

Alpha Chymotrypsin-induced Experimental Glaucoma

Alpha chymotrypsin is a proteolytic enzyme secreted by pancreas. It is specific for peptide
bonds containing uncharged amino acid residues. Intravitreal injection of alpha chymotrypsin
into owl monkeys and rabbits elevates the ocular pressure by 5 to more than 25 mm Hg within
2 to 3 days and suggests that rise in IOP is due to predominant changes in trabecular meshwork
caused by α-chymotrypsin. The proteolytic action of α-chymotrypsin on zonular material and
the debris of the tissue thus formed blocks the aqueous outflow channels and elevates the IOP.
Alpha chymotrypsin induces sustained rise in intraocular pressure for several weeks.6-8
282 Drug Screening Methods

Procedure: Sears and Sears established this chronic model of glaucoma.7 Rabbits of 1.5-2 kg
body weight under nembutal anesthesia are used. One of the eyes is anesthetized with local
anesthetic 2% xylocaine. The eyeball is fixed by forceps, and a 30 G sterile needle, connected
with a tubing to a 1 ml Hamilton syringe, is inserted carefully for intravitreal injection (via
limbus). Two minutes after injection, the needle is removed and 2-3 drops of any antibiotic
solution are instilled into the eyes to prevent infection. One hundred and fifty units of alpha
chymotrypsin dissolved in sterile saline (0.5 ml) are carefully injected into the posterior
chamber so that the needle does not damage the lens. Care is taken to prevent the contact of
alpha chymotrypsin with the corneal stroma. After two days, the IOP is measured at regular
intervals. Stable rise in IOP is achieved within 15 days. A sustained pressure elevation, ranging
from 28-45 mm Hg, is observed till 50 weeks. Rabbits showing IOP less than 30 mm of Hg, are
excluded from the experiment.
To evaluate antiglaucoma activity, the test agent is administered topically or orally in a
suitable formulation once the glaucoma is established (stable IOP). The topical formulations
or vehicle are instilled onto the cul de sac of one eye (treated eye) and the contralateral eye
(control) respectively. The IOP is measured prior to drug instillation and at regular time interval
after instilling the eye drops. Change in IOP between the two eyes can be compared to see the
potential of the agent. IOP changes can also be calculated.9

Chronic Rat Model of Glaucoma

Morrison, et al. have developed a chronic rat model of glaucoma.10 The aqueous humor escapes
the eye through trabecular meshwork, Schlemm’s canal and into a vascular plexus circling the
limbus. This plexus is connected to the general circulation through several episcleral veins.
Injecting mild sclerosants (hypertonic saline) through one of these veins results in the scarring
of trabecular meshwork by which decrease in the outflow and increase in the IOP occur.
Procedure: Male Brown Norway rats (Rattus norvegicus) of 300 to 400 g weight range are kept
in light for minimum 3 days. Rats are anesthetized by intraperitoneal injection of 1.0 ml/kg
solution containing 5 ml ketamine (100 mg/ml), 2.5 ml xylazine (20 mg/ml), 1 ml acepromazine
(10 mg/ml) and 0.5 ml sterile water. A small propylene ring (5.5 mm diameter) is placed
around the globe, covering the equator. The ring has a gap of 1.00 mm in its circumference
that is adjusted in such a manner that the passage of one radial aqueous vein in the superior
quadrant remains unobstructed. The ring occludes the other aqueous veins and the sclerosing
agent (hypertonic saline) is infused to the limbus. The aqueous vein is exposed by incising
the conjunctiva. A volume of 50 µl microfiltered 1.75 M hypertonic saline solution is injected
into the limbal vascular plexus using a fabricated microneedle assembly. A force sufficient
enough to blanch the limbal artery is used for injection. The ring is removed after injecting
the saline. Polysporin ointment is applied to the eye and the animals are observed till they
recover from anesthesia. Baseline measurements of IOP are taken prior to the injection
of saline. Animals have been routinely handled in order to get a reproductive baseline IOP.
Following injections of hypertonic saline the IOP is measured twice a week. Elevation of IOP
is determined by calculating the difference between the IOP of injected and noninjected eye.
Mean IOP elevation ranged from 7-28 mm Hg.10
The model is anatomically relevant to the primate model. Prominent globes of the rats and
their docile nature helps in measuring the IOP while the animals are awake.
 Antiglaucoma Agents 283

Methylcellulose Induced Glaucoma

Glaucoma can be induced by injecting 0.5-4% methylcellulose in the anterior chamber of
animals like minipigs and rabbits.11-13 Methylcellulose (0.5%) made in 0.9% saline, free of air
bubbles is injected into the anterior chamber of rabbit eyes following evacuation of the aqueous
humor. The volume of the methylcellulose is equal to the volume of aqueous humor removed
(150 µl to 190 µl). Same needle of 0.032 cm diameter is used for both evacuation and refilling
along with a three-way stopcock. The needle is inserted into the anterior chamber just above
the aperture of iris and aqueous humor is evacuated slowly. In this model the IOP increases
from 20 to approximately 30 mm Hg in next 2 hours.12 Zhu and Cai produced a reliable and
about 8 weeks of intraocular hypertension by a series of four intra-anterior chamber injections
of 1 or 2% methylcellulose in rabbits.11

Autologous Ghost Red Blood Cells Induced Glaucoma

Fixed red blood cells or fixed ghost red blood cells (GBCs) when injected in the anterior
chamber of rabbits and monkeys produce chronic glaucoma. GBCs have a relatively rigid
membrane and do not possess the flexibility by which the normal red blood cells exit the
meshwork. Upon infusion, the GBCs cause trabecular obstruction in eyes with intraocular
hemorrhage resulting in secondary glaucoma. IOP elevation lasts in rabbits from 7 to 36
days and in monkeys 2 to 42 days. The model produces increase in IOP easily and without
intraocular inflammation.14

Hyaluronic Acid Induced Glaucoma

Chronic intracameral administration of hyaluronic acid (HA) in rats induced significant
histologic alterations and decreased electroretinographic activity in the retina and optic nerve
showing similarity with some characteristics of open angle glaucoma. Injection of hyaluronic
acid in the anterior chamber of rabbits and owls monkeys significantly raised the IOP though
the effect was short lived being 70 and 24 hours post injection in rabbits and owl monkeys
respectively.15
Single administration of HA injection induced and maintained the rise in IOP for 8 days.
However, injections administered every week induced a sustained elevation of intra ocular
pressure for 10 weeks.
Procedure: Male Wistar rats of about 200 + 40 g weight are kept in controlled humidity and
21°C temperature, and 12 h day: 12 h night cycle. Rats are anesthetized with intraperitoneally
administered mixture of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (0.5
mg/kg). Hyaluronic acid (25 µl) is injected into one eye of anesthetized rats and an equal
volume of vehicle is injected in the contralateral eye once a week.

Reduced Serum Osmolarity

The animals are subjected to forced ingestion of water or intravenous injections of 5% glucose
solution or hypertonic saline.8,16-18 This reduces the serum osmolarity and a temporary change
in the IOP is observed.
284 Drug Screening Methods

Water-loaded Rabbit Model of Glaucoma

This acute model of glaucoma is widely used and established by Sugiyama, et al.16 Changes
in IOP are temporary. This model minimizes the mechanical trauma that can alter the blood
aqueous barrier.
Procedure: Albino rabbits of 1.5-2 kg body weight of either sex are required for this model.
They are kept fasting overnight and on the day of experiment are anesthetized using 30 mg/
kg b.w. of sodium pentobarbitone, 45 minutes before the experiment. Ten minutes before
the water loading of the animals, the anesthesia maintenance doses of 4 mg/kg sodium
pentobarbitone is administered via the marginal ear vein. The baseline IOP is measured under
corneal anesthesia by instilling 2 drops of xylocaine (4%) three times at two minutes interval.
Tap water 100 ml/kg is administered orally through an intragastric infant feeding tube within
30 sec. The IOP is measured at baseline 15, 30, 45 and 60 minutes after the water loading till
the IOP reaches the baseline values. For evaluating the antiglaucoma activity of an agent, it is
administered in the form of eyedrops to one of the eyes of the water-loaded animal, while the
vehicle is instilled in the contralateral eye (control). The difference in IOP is observed in the
two eyes at various time intervals. Any significant modulation shows the potential of the drug
for lowering IOP.
Santafe et al. measured the effect of topical diltiazem in rabbits loaded with 60 ml/kg water.17

IOP Recovery Model

This rabbit model for experimental glaucoma has been used by several researchers.9,19 The
intravenous injection of hypertonic saline in the marginal ear vein results in the fall of IOP,
which comes back to normal within 2 h. If the drug used is a potential antiglaucoma agent, it
will extend the time required for the recovery of normotensive pressure.
Procedure: New Zealand white normotensive rabbits of 2.5-3.0 kg weight range are used for
this method. By using an infusion pump, 10% sodium chloride solution (10 ml) is infused
through the ear marginal vein, with a flow rate of 1 ml/min. The drug is instilled in the form of
eyedrops on to the cul de sac of the right eye (treated eye) and vehicle on to the left eye (control
eye), immediately after the infusion of hypertonic saline. IOP is measured at 40 and 20 minutes
prior to instilling the eyedrops as baseline and then at 0, 20, 40, 60, and 80 minutes thereafter
at 20 minutes interval till the baseline values are obtained.
The relative percents of IOP (IOPt%) can be calculated by the following equations:9
IOPt%= (IOPt/IOP–40) × 100%
IOPt%: Relative percent of IOP at time t
∆IOPt%: Difference of IOPt% between treated and controlled eyes

Perfused Excised Eye Model System

Novel glaucoma therapy includes modulation of cytoskeleton protein actin and tubulin in the
trabecular meshwork. Due to induced changes in cell shape and attachment, cytoskeleton of
aqueous outflow pathway cells can influence aqueous humor outflow function.
Outflow facility measurement: Freshly excised and chilled porcine eyes are perfused within
4 h. Standard constant pressure perfusion technique is followed using a Grant stainless steel
 Antiglaucoma Agents 285

corneal fitting. For the prevention of artificial deepening of the anterior chamber, radial
iridotomy is done. Dulbecco’s phosphate-buffered saline (DPBS) solution with 0.68 mM CaCl2
supplemented with 5.5 mM D-Glucose is used as perfusion medium. All the solutions are
filtered through 0.2 mm filter. The eyes are perfused at 15 mm Hg and 25°C for 1 hour to achieve
a steady state flow value and the baseline facility is recorded. The corneal fitting is removed
and the anterior chamber is emptied with a cellulose sponge and refilled with the similar
perfusion medium containing the test drug. Controls are kept as sham manipulated and
receive only the medium for perfusion and drug vehicle. Drug and control solutions are then
perfused for the remainder of the experiment.20 Outflow facilities of experimental and control
eyes are measured hourly for 5 hours.21 Drug effects are expressed as the percentage change
in the outflow facility from the baseline value in the experimental eye minus the percentage
change in the control eye.

Application of Lasers
Chronic experimental models are produced using laser treatment. Researchers have
successfully induced glaucoma in monkeys, rats, mice, etc.

Laser-induced Glaucoma in Monkeys

Normal adult cynomologus monkeys (Macaca irus) of 3-4 kg body weight are used for laser-
induced glaucoma. Monkeys with normal anterior segments, normal intraocular pressure
(15-21 mm Hg) and normal optic nerve heads during baseline examination are selected for
the study. The monkeys are anesthetized by intramuscular injection of 9 mg/kg ketamine
hydrochloride and intravenous injection of 11 mg/kg sodium pentobarbital. After anesthesia,
they are placed in front of the slit lamp of the argon laser delivery system. The eyes are
treated with 0.4% oxybuprocaine hydrochloride and approximately 200-400 circumferential
laser burns are made with a small gonioscopic lens, aiming at the middle of the trabecular
meshwork, with a beam diameter of 50-100 µm for 0.1-0.5 sec at 600-800 mW. The laser
treatment is repeated every week for 3-5 weeks. Slit lamp examination, IOP measurement, and
fundus photography with a stereofundus camera is performed every 1-2 weeks. The IOP of
laser treated eyes increase to 25-45 mm Hg after repeated laser treatment and that of normal
eyes remains at 15-21 mm Hg.22,23 Once the stable IOP is obtained, the anti­glaucoma agents are
instilled in the test group and the IOPs are compared with the control.
Serle et al. carried out a comparative study of latanoprost (Xylatan) and isopropyl unoprostone
(Rescula) in normal and glaucomatous monkeys.24

Laser-induced Glaucoma in Rats

WoldeMussie et al. 2001, produced laser induced hypertension in male Wistar rats weighing
between 350 to 450 g.25 Rats are anesthetized with a mixture of ketamine (50 mg/kg),
acepromazine (1 mg/kg), and xylazine (25 mg/kg). A blue-green argon laser treatment is given
for the elevation of IOP. Laser treatment is performed on the episcleral veins within 0.5 to 0.8
mm from the limbus and on the veins in the limbus. Treatment is given in two parts with a gap
of 1 week. The amount of energy used is 1 W for 0.2 seconds for delivering a total of 130 to 150
spots (50–100 µm spot size) with both laser treatments. IOP is measured by tonometer (Tono-
Pen) before and after laser treatment. Intramuscular injection of 3.0 mg/kg acepromazine
286 Drug Screening Methods

is administered to the rats in order to keep them calm but not to sedate them. Cornea is
anesthetized using proparacaine (0.5%) topically. IOP is measured three times for the first
2 weeks and then once a week for the remaining experimental period. IOP values increased
twofold from the baseline by the laser treatment, which is maintained for two months. The
drug treatment can be given either at the time of or 10 days after IOP elevation.25
Unilateral experimental glaucoma in Wistar rats is also induced using a diode laser (532 nm
wavelength) aimed at the trabecular meshwork and episcleral veins or only at the trabecular
meshwork through the external limbus. 26
Laser-induced experimental glaucoma is induced in rats after intracameral injection of
India ink.27,28

Laser-induced Glaucoma in Mouse

Aihara, et al. established this mouse model of laser-induced glaucoma.29 Laser photocoagulation
of the corneal limbus obstructed the outflow of aqueous. They induced mydriasis and flattened
the anterior chamber prior to photocoagulation. The procedure used by them is briefly
described below.
Thirty minutes before the initiation of laser treatment one eye of each mouse is dilated
by topical administration of 4 µl of a mixture of atropine, tropicamide, phenylephrine, and
cyclopentolate, 0.25% each. The mouse is anesthetized and placed under a stereomicroscope.
In order to prevent the drying of cornea a drop of phosphate-buffered saline is placed on
it. A fabricated microneedle is connected to a 1-µl syringe that was mounted on a micro­
manipulator. The aqueous fluid is aspirated by the microneedle. Once the anterior chamber
is flattened, the needle is withdrawn. The anesthetized mouse is kept on the platform of a
biomicroscope with a diode laser system. Laser beams of 532 nm wavelengths are then applied
to the corneal limbus. The laser power being 100 mW, duration 0.05 second and spot size 200
µm. Laser photocoagulation with flattening of the anterior chamber successfully induces 30%
elevation of IOP for at least 6 weeks in mouse eyes without any severe complications.29

Steroid Glaucoma
Elevation in intraocular pressure in humans due to the administration of corticosteroids is
already reported. Experimental glaucoma induced by steroids is reported in young rabbits and
cats.30,31 An adult feline model is developed by Zhan et al.31
Procedure: Adult cats of mixed breeds and of either sex (2.2-4.5 kg) are maintained and trained
to accept tonometery. Normal intraocular pressure is in the range of 24 ± 0.5 mm Hg (mean
± SEM). Ocular hypotensive cats show consistently lower IOP 17 ± 0.4 mm Hg for at least 1
month prior to the corticosteroid treatment without any medication.31 During the baseline
period before the first corticosteroid treatment, there is no significant difference between the
IOP of the left and right eyes. No signs of ocular irritation or inflammation are present. IOP is
measured prior to and during all treatments after corneal anesthesia by topical application
of proparacaine hydrochloride 0.5%. IOP is measured preferably at the same time and same
interval during the experimental period. During long-term treatments, IOP is measured at 0,
1, 3, and 6 h; thereafter, at least once or twice a week, and daily for a few days after a treatment
regimen is started, changed or terminated.
 Antiglaucoma Agents 287

Drug administration: A solution of dexamethasone sodium phosphate (Dex) injection

is diluted to the desired concentration of 0.5% or 1% with normal saline. The Dex solution
is applied to the corneal surface in a volume of 10 µl (to minimize systemic effect) with an
automatic micropipette either unilaterally or bilaterally one, two or three times daily. A gradual
intraocular pressure increase is observed which becomes significant within 2 to 3 weeks.
Prednisolone acetate 1% solution can also be used instead of dexamethasone.30
An ocular hypertension model in rats is generated by instillation of topical dexamethasone
to rat eyes 4 times daily for 1, 2, and 4 weeks.32 Galassi et al. (2006) induced glaucoma by
administering dexamethasone phosphate 1% drops topically in rabbit eyes.33
Bonomi, et al. administered weekly subconjunctival injections of 4 mg repository
betamethasone in rabbits for three weeks.34 This produced a sustained increase in the
intraocular pressure in 96% of the treated rabbits. The steroid treatment was well tolerated
and systemic adverse effects were seen in few animals. The rise in IOP was constant, well
reproducible and sensitive to antiglaucoma drugs.34
Hester, et al. studied the effect of three subconjunctivally injected steroids namely
betamethasone, cortisone, and triamcinolone in rabbits. All the three drugs produced
elevations in IOP but the most consistent elevation was with triamcinolone.35
Gerometta, et al. also produced steroid-induced ocular hypertension in normal cattle using
prednisolone acetate topically in one eye 3 times a day for a period of 49 days.36

OTHER MODELS
Autoimmune Glaucoma
Glaucoma usually is associated with elevated intraocular pressure, but often occurs or may
progress with intraocular pressure in the normal range. It has been suggested that autoimmune
response possibly has a role in RGC degeneration in normal pressure glaucoma. Serum samples
of glaucoma patients were found to have increased levels of heat shock protein 27 (HSP27) and
heat shock protein 60 (HSP60). An in vivo rat model was established to elicit the autoimmune
response through immunization with HSPs. HSP27 and HSP60 immunization in the Lewis rat
induced RGC degeneration and axonal loss 1–4 months later in a pattern similar to human
glaucoma. The model is a valuable tool for examining the various roles of immune system in
glaucoma. It may assist in finding the treatment strategies to prevent pressure –independent
RGC degeneration.37-40

Genetic Models
Family history and genetic factors have an important role in glaucoma as it is a progressive
disease and affects the elderly. Hereditary models are useful in understanding the genetic and
mechanistic basis of the disease.

DBA/2J Mouse Model

This is a genetic mouse model of glaucoma characterized by Chang, et al. (1999) in which IOP
increases with age due to pigment dispersion from the iris and obstruction of the trabecular
meshwork.41,42 DBA/2J strain mice spontaneously develop complex ocular abnormalities,
288 Drug Screening Methods

such as glaucomatous loss of retinal ganglion cells. Three to 11-month-old DBA/2J mice show
retinal degeneration which somewhat resembles human pigment dispersion syndrome and
pigmentary glaucoma exhibiting characteristic anterior segment changes and elevated IOP.
Neovasculogenesis and myelin-like bodies are observed during aging hence it is recommended
that the DBA/2J model requires judicious interpretation as a glaucoma model.43

Buphthalmic Rabbits, JWHR bu/bu

Inoue, et al. (2001) evaluated the effect of topical CS-088, an angiotensin AT1 receptor
antagonist, on intraocular pressure and aqueous humor dynamics in hereditary ocular
hypertensive rabbits (buphthalmic rabbits, JWHR bu/bu).8

Human Trabecular Meshwork (HTM) Culture

The trabecular meshwork of the anterior chamber is a circular zone of reticular tissue lying
between the Schlemm and the anterior chamber. It is separated into corneoscleral and uveal
meshwork. Intertrabecular spaces are present between the trabecular sheets. The aqueous
percolates through the trabecular meshwork. Major volume of the aqueous passes through the
trabecular spaces into the canal of Schlemm and eventually is emptied into the venous system.
Any obstruction in the outflow of aqueous leads to the elevation in IOP. This may be due to the
debris of the cells in the intertrabecular spaces, collapse of Schlemm’s canal or intrascleral
blockade.
Procedure: Human donor eyes enucleated within 48-96 hours of death are used. Whole globe
is soaked in DPBS containing 100 U/ml penicillin G sodium, 100 U/ml streptomycin sulfate
and 0.25 µg/ml amphotericin B for 15 minutes at room temperature. An incision is made to
bifurcate the globes at the equator. The lens, ciliary body, and iris are gently removed from the
anterior segment to expose the trabecular meshwork. Preserved anterior segments are rinsed
in Dulbecco’s modified Eagles Medium with 10% fetal bovine serum, 2 mM L-glutamine,
100 U/ml penicillin, G sodium and 100 µg/ml streptomycin sulfate. Trabecular meshwork is
separated from the anterior segment with a 0.5 mm foreign body curette and explanted onto
a culture plate. The cells are cultured at 37°C in a 7% CO2 atmosphere. Upon confluency, the
cells are passaged with 25% trypsin 1.0 mM EDTA solution in Hank’s balanced salt solution.
Subsequent passages are used in the experiments.20,44
Drug treatment on cells: Cells are placed onto glass cover slips coated with 2% sterile gelatin
solution. On reaching the confluence, the cells are treated with the test drug supposedly
interfering with actomyosin function. Sham-treated control samples are performed for each
treatment group.21
Fixation and cytoskeletal staining: Cells are rinsed to remove the medium and drug after
the drug exposure. Cells are washed with buffer. Fixation and cytoskeletal staining is done.
Cell viability is assessed by flouresceindiacetate and propidium iodide. The drug effective
in inducing a loss of cell-cell attachment and a loss of filamentous actin staining may have a
potential in increasing the outflow.
 Antiglaucoma Agents 289

CONCLUSION
Apart from surgery, therapeutic intervention is required for the treatment of glaucoma. A
large number of antiglaucoma agents are being discovered; but to screen a potential agent,
initially, a suitable animal model is required. Out of the above models, the most commonly
used are alpha chymotrypsin-induced, water-loaded and laser-induced models of glaucoma.
The alpha chymotrypsin produces irreversible glaucoma. It can permanently damage the eyes
of the animal. The changes in the IOP in water-loaded or IOP recovery models are for a short
duration. The animal recovers after few hours. Laser-induced glaucoma model in rodents is
gradually becoming the most acceptable model. Availability and maintenance of rodents is
comparatively easier. In the present circumstances when there is difficulty in the availability of
animals one can opt for the in vitro models. The cell culture techniques are preferred though
they require precision and expertise.

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14. Quigley HA, Addicks EM. Chronic experimental glaucoma in primates. 1. Production of elevated
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15. Moreno MC, Marcos HJA, Croxatto JO, Sande PH, Campanelli J, Jaliffa CO, et al. A new experimental
model of glaucoma in rats through intracameral injections of hyaluronic acid. Exp Eye Res
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16. Sugiyama K, Enya T, Kitazawa Y. Ocular hypotensive effect of 8-hydroxycarteolol, a metabolite of
cateolol. Int Ophthalmol 1989;13:85.
17. Santafe J, Martinez de Ibaretta MJ, Segarra J, et al. The effect of topical diltiazem on ocular
hypertension-induced by water loading in rabbits. Gen Pharmacol 1999;32:201-5.
18. Shah GB, Sharma S, Mehta AA, et al. Oculohypotensive effect of angiotensin converting enzyme
inhibitors in acute and chronic models of glaucoma. J Cardiovasc Pharmacol 2000;36:169-75.
19. Byron HP Li, Chiou GCY. Effects of new clonidine derivatives on rabbit intraocular pressure. Drug
Dev Res 1992;26:431-8.
20. Epstein DL, Rowlette LL, Roberts BC. Acto-myocin drug effects and aqueous outflow function.
Invest Ophthalmol Vis Sci 1999;40:74-81.
21. Epstein DL, Roberts BC, Skinner LL. Non sulfhydryl reactive phenoxyacetic acids increase aqueous
humor outflow facility. Invest Ophthalmol Vis Sci 1997;38:1526-34.
22. Jonas JB, Hayreh SS. Localised retinal nerve fibre layer defects in chronic experimental high-
pressure glaucoma in rhesus monkeys. Br J Ophthalmol 1999;83:1291-5.
23. Gherezghiher T, March WF, Nordquist RE, et al. Laser-induced glaucoma in rabbits. Exp Eye Res
1986;43:885-94.
24. Serle JB, Podos SM, Kitazawa Y, et al. A comparative study of latanoprost (xylatan) and isopropyl
unoprostone (Rescula) in normal and glaucomatous monkey eyes. Jpn J Ophthalmol 1998;42:95-100.
25. WoldeMussie E, Ruiz G, Wijono M, Wheeler LA. Neuroprotection of retinal ganglion cells by
brimonidine in rats with laser-induced chronic ocular hypertension. Invest Ophthalmol Vis Sci
2001;42:2849-55.
26. Levkovitch-Verbin H, Quigley HA, Martin KRG, Valenta D, Baumrind LA, Pease ME. Translimbal
laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Invest
Ophthalmol Vis Sci 2002;43:402-10.
27. Ueda J, Sawaguchi S, Hanyu T, et al. Experimental glaucoma model in rat-induced by laser trabecular
photocoagulation after an intracameral injection of India ink. Jpn J Ophthalmol 1998;42:337-44.
28. GuZ, Yamamoto T, Kawase C, et al. Neuroprotective effect of N-methyl-D-aspartate receptor
antagonists in an experimental glaucoma model in the rat. Nippon Ganka Gakkai Zasshi
2000;104:11-6.
29. Aihara M, Lindsey JD, Weinreb RN. Experimental Mouse Ocular Hypertension: Establishment of
the Model. Invest Ophthalmol Vis Sci 2003;44:4314-20.
30. Knepper PA, Breen M, Weinstein HG, Blacik JL. Intraocular pressure and glycosaminoglycan
distribution in the rabbit eye: effect of age and dexamethasone. Exp Eye Res 1978;27:567-75.
31. Zhan GL, Miranda OC, Bito LZ. Steroid glaucoma: corticosteroid-induced ocular hypertension in
cats. Exp Eye Res 1992;54:211-8.
32. Sawaguchi Keiko, Nakamura Yoshimi, Nakamura Yuko, Sakai Hiroshi, Sawaguchi Shoichi. Myocilin
gene expression in the trabecular meshwork of rats in a steroid-induced ocular hypertension
model. Ophthal Res 2005;37:235-42.
33. Galassi F, Masini E, Giambene B, Fabrizi F, Uliva C, Bolla M, et al. A topical nitric oxide-releasing
dexamethasone derivative: effects on intraocular pressure and ocular haemodynamics in a rabbit
glaucoma model. Br J Ophthalmol 2006;90:1414-9.
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34. Bonomi L, Perfetti S, Noya E, Bellucci R, Tomazzoli L. Experimental corticosteroid ocular

hypertension in the rabbit. Albrecht Von Graefes Arch Klin Exp Ophthalmol 1978;209:73-82.
35. Hester DE, Trites PN, Peiffer RL, Petrow V. Steroid-induced ocular hypertension in the rabbit: a
model using subconjunctival injections. J Ocul Pharmacol 1987;3:185-9.
36. Gerometta R, Podos SM, Candia OA, Wu B, Malgor LA, Mittag T, et al. Steroid-induced ocular
hypertension in normal cattle. Arch Ophthalmol 2004;122:1492-7.
37. Bouhenni RA, Dunmire J, Sewell A, Edward DP. Animal models of glaucoma. J Biomed Biotechnol
2012;2012:11 pages. Article ID 692609.
38. Wax MB, Tezel G, Yang J, et al. Induced autoimmunity to heat shock proteins elicits glaucomatous
loss of retinal ganglion cell neurons via activated T-cell-derived fas-ligand. J Neurosci
2008;28(46):12085–96.
39. Joachim SC, et al. Complex antibody profile changes in an experimental autoimmune glaucoma
animal model. Invest Ophthalmol Vis Sci 2009;50(10):4734-42.
40. Joachim SC, Mondon C, Gramlich OW, Grus FH, Dick HB. Apoptotic retinal ganglion cell death in
an autoimmune glaucoma model is accompanied by antibody depositions. J Mol Neurosci 2014;
52(2):216-24.
41. Chang Bo, Smith RS, Hawes NL, Anderson MG, Zabaleta A, Savinova O. Interacting loci cause severe
iris atrophy and glaucoma in DBA/2J mice. Nature Genetics 1999;21:405-9.
42. Anderson MG, Libby RT, Gould DB, Smith RS, John SWM. High-dose radiation with bone
marrow transfer prevents neurodegeneration in an inherited glaucoma. Proc Natl Acad Sci USA
2005;102:4566-71.
43. Schuettauf F, Rejdak R, Walski M, Frontczak-Baniewicz M, Voelker M, Blatsios G, et al. Acta
Neuropathol 2004;107(4):352-8.
44. Stamer WD, Roberts BC, Howell DN, et al. Isolation, culture and characterization of endothelial
cells from Schlemm’s canal. Invest Ophthalmol Vis Sci 1998;39:1804-12.
 cHAPTER

18
Antiarrhythmic Agents
INTRODUCTION
Arrhythmias remain among the most challenging human disorders to diagnose and to treat.
The complex pathophysiology of human arrhythmias has proven difficult to model. Direct
correlations between the traditional arrhythmia mechanisms, including abnormal excitability,
conduction, or repolarization and underlying molecular or cellular biology are poorly defined,
as the primary etiologies of many human arrhythmias remain unknown. Since the causes
of several arrhythmic syndromes have been identified, genetic models reproducing the
mechanisms of these arrhythmias have become feasible. Initial murine modeling has revealed
that in many cases the pathophysiology of the respective human disease is more complex
than had been suspected. Insights from human genetic studies and animal models strongly
suggest that the primary molecular defects may contribute at many stages in the causal chain
leading to arrhythmia. The comprehensive analysis of each arrhythmia will require knowledge
not only of the membrane effects of the primary defects, but also downstream intracellular
signals, the developmental results of these perturbations, and the integration of compensatory
responses and environmental factors. Precise modeling will require not only the mutation of
specific residues in known disease genes, but also the systematic study of each of the many
steps in arrhythmogenesis. Ultimately, such models will enable unbiased screening for disease
mechanisms and novel therapies.
Although, no animal model can accurately resemble with human disease condition and
species differences also exist, close similarities with humans suffering from or threatened
by arrhythmias can be developed by selecting appropriate model and species. Though, an
animal is not the same as a human patient, arrhythmogenic mechanisms derived from animal
experiments have tremendously helped us to diagnose and adapt therapeutic strategies.
Following are the standard models useful for the screening of antiarrythmic drugs:

CELL CULTURE TECHNIQUE

1. Studies on Isolated Ventricular Myocytes
Ventricular arrhythmias, specially torsades de pointes, can be evaluated using isolated
ventricular myocytes. Analysis of action potential and patch clamp techniques in isolated
ventricular myocytes helps us to clarify the mechanisms underlying the development of
torsades de pointes.
 Antiarrhythmic Agents 293

Guinea pigs (250–350 g) are sacrificed by decapitation and their hearts are removed and
perfused retrogradely through the aorta at a rate of 10 ml/min with oxygenated calcium free
HEPES buffered saline at 37oC for 5 min. It is then again perfused with the same solution
containing 300U/ml type II collagenase and 0.5 to 1.0 U/ml type XIV protease for 8 min and
finally with free HEPES buffered saline containing 0.2 mM calcium chloride for additional 5
min. The heart is digested and cut into small pieces, placed in a 20 ml HEPES buffered saline
containing calcium chloride and shaken until single cells are dissociated. The cells are then
resuspended in HEPES buffered saline and stored at 24oC. Transmembrane potential is
recorded using conventional glass electrodes connected to the headstage of an Axoclamp 2A
amplifier. Cells are superfused with HEPES buffered saline at a rate of 2 ml/min at 37oC. Passing
brief current pulses (1 ms, 1.2 times threshold), through the recording electrode using an active
bridge current, evokes action potentials. Cells are stimulated at a frequency of 1 Hz during
the stabilization period and at frequencies of 1 and 3 Hz during control and at 10 min after
superfusion with test drugs at cumulatively increasing drug concentration. Four individual
action potentials are digitally averaged and measured for each condition. For voltage clamp
studies, microelectrodes made from square bore, borosilicate capillary tubing are filled with
0.5 M K+ gluconate, 25 mM KCl, 5 mM K2 ATP. A List EPC-7 clamp amplifier is used to voltage
clamp the isolated cells. Voltage clamp is performed using whole cell recording mode and cell
perfusion is minimized by maintaining constant negative pressure on the electrode using a
1 ml syringe. Outward K+ currents are measured during superfusion of the cells at a rate of
2 ml/min with calcium free HEPES buffered saline. Concentration response relations are
determined by measuring action potentials of currents in each cell during control conditions
and during superfusion with two successively increasing concentrations of a given drug.1
Action potentials are assessed using a three-way ANOVA to determine significance within
treatment variations. Dunett’s t test is used to determine significance of individual treatment
means compared with control mean values.

IN VITRO MODELS
1. Isolated Guinea Pig Papillary Muscle
A simple and accurate, non-microelectrode method is available to identify and classify potential
anti-arrhythmic drugs into class I, II, III and IV. In right ventricular guinea pig papillary muscle
developed tension (DT), excitability (EX), and effective refractory period (ERP) are measured.
Na+ channel blockade decreases excitability, K+ channel blockade lengthens refractory period
and Ca2+ blockade decreases tension of cardiac muscle.
Guinea pigs (200-400 g) are stunned and their carotid artery is severed. The thoracic cage
is opened immediately and the heart is removed. The myocardium is placed into a container
filled with pre-oxygenated and pre-warmed physiological solution. The pericardium, atria and
other tissues are removed and the heart is pinned to a dissection tray. The right ventricle is
opened and tendinous end of papillary muscle is ligated with a silk thread, making sure that
the chordae tendinae are freed from the ventricle, while the other end is clamped into a tissue
holder, at the end of which is a platinum wire field electrodes. The preparation is transferred
to a tissue bath containing physiological salt solution maintained at constant temperature and
pressure. The silk thread is used to connect the muscle to a force transducer. Muscles are field
294 Drug Screening Methods

stimulated to contract isometrically at stimulus duration of 1 ms, frequency of 1 Hz. Pulses

are delivered using a Grass constant voltage stimulator and the developed tension is recorded
using a polygraph recorder. The force frequency curve is obtained by measuring developed
tension over a range of stimulation frequencies (0.3, 0.5,0.8, 1.0,1.2 Hz). The percent change in
post treatment (versus pretreatment) developed tension at 1 Hz is used to quantitate an agent’s
inotropic effect.2,3
The changes in effective refractory period (post treatment minus pretreatment), the degree
of shift in the strength duration curve (geometrical area between pre & post treatment curves)
and the percent changes in post treatment developed tension at 1 Hz are calculated. The results
of this calculation are used to classify the compound as a class I, II, III or IV anti-arrhythmic
agent on the basis of its effect on developed tension, excitability and effective refractory period.

2. Action Potential and Refractory Period in Isolated Guinea

Pig Papillary Muscle
Following the electrical stimulation, intracellular action potential in the left ventricular guinea
pig papillary muscle is recorded. To determine the refractory period, the stimulation frequency
is varied. Compounds that affect the duration of the effective refractory period may have anti-
arrhythmic or pro-arrhythmic effects. In addition, the inotropic effect of the test compound is
determined.
Guinea pigs of Marioth strain (250-300 g) are sacrificed by stunning, carotid artery are
severed, thoracic cage is opened, heart is removed and placed on a container of pre-warmed,
pre-oxygenated Ringer's solution. The left ventricle is opened and the two strongest papillary
muscles removed. A standard micro-electrode technique is applied to measure action
potential. The papillary muscle is stimulated with rectangular pulses of 1 V and 1ms duration
at an interval of 500 ms. To estimate refractory period, the second stimuli are set in decemental
intervals until contraction ceases.4
Contractile force and relative refractory period are determined before and after drug
administration. ED25ms and ED50ms values are determined. ED50 values are calculated from log
probit analysis and scored.

3. Langendorff Technique
The basic principle involved is that heart is perfused in a retrograde direction from the aorta
either at constant pressure or constant flow with oxygenated saline solutions. Retrograde
perfusion closes the aortic valves, just as in the in-situ heart during diastole. The perfusate is
displaced through the coronary arteries flowing off the coronary sinus and the opened right
atrium.
Guinea pigs of either sex weighing 300–500 g are sacrificed by stunning. The heart is
removed as quickly as possible and placed in a dish containing Ringer’s solution at 37oC.
Associated pericardial and lung tissues are removed. The aorta is located and cut below the
point of division. A cannula is inserted into the aorta and tied and the heart is perfused with
oxygenated Ringer’s solution. The heart is transferred to a double wall plexiglass perfusion
apparatus maintained at 37oC. Oxygenated Ringer’s solution is perfused at a constant pressure
of 40 mm Hg at a temperature of 37°C from a reservoir. Ligature is placed around the LAD
coronary artery and occlusion is maintained for 10 min followed by reperfusion. Test compound
 Antiarrhythmic Agents 295

is administered through perfusion medium either before or after occlusion. An epicardial ECG
electrode is used for pulsatile stimulation and induction of arrhythmias (rectangular pulses of
0.75 msec duration, usually of 10 V; frequency 400–1800 shocks per min). A small steel hook
with a string is attached to the apex of the heart. Contractile force is measured isometrically by
a force transducer and recorded on a polygraph. Heart rate is measured through a chronometer
coupled to the polygraph. Drugs are injected into the perfusion medium. Incidence and
duration of ventricular fibrillation or ventricular tachycardia is recorded in the control as well
as test group.5

4. Acetylcholine and Potassium-induced Arrhythmia

New Zealand white rabbits of the weight range 0.5–3 kg are used for the study. The animals
are sacrificed and hearts removed immediately. The atria are dissected from other tissue in
Ringer solution. The atria are attached to an electrode in the lower part of the bath and are
suspended. Fibrillation is produced when the atria are exposed to acetylcholine (3 × 10 –4 g/
ml) or (0.10 g) potassium chloride.6,7 After 5 min of exposure to acetylcholine or potassium, the
atria are stimulated with rectangular pulses of 0.75 ms duration, usually of 10 V (Frequency
400–1800 shocks per min). A mechanical record is taken on kymograph. Control arrhythmias
are produced and allowed to continue for upto 6–10 minutes. After a 30 minute rest period,
fibrillation is again induced and after allowing the arrhythmia to proceed for 3 minutes, a test
compound is added to the bath.
If the atria do not cease to fibrillate within 8–10 minutes following the addition of the test
compound, the preparation is washed and allowed to return to normal contraction. Test
compound is found to be effective if fibrillation disappears immediately or within 5 min
following test drug supplementation to the organ bath.

In vivo models
In vivo models used to screen antiarrhythmic drugs can be divided into five groups:

A. Chemically-induced Arrhythmia
A large number of agents alone or in combination are capable of inducing arrhythmias.
Administration of anesthetics likes chloroform, ether, halothane (sensitizing agents)
followed by a precipitating stimulus, such as intravenous adrenaline, ouabain alkaloids cause
arrhythmia. The sensitivity of these arrhythmogenic substances differ among various species.

Aconitine Antagonism in Rats

Aconitine, a plant alkaloid from aconitine root, acts persistently on sodium channels and
activates it resulting in ventricular arrhythmias. Drugs considered to have anti-arrhythmic
properties can be tested in aconitine intoxicated rats.
Males Ivanovas rats (300–400 g) are anesthetized intraperitoneally with urethane (1.25g/
kg). Aconitine (5 µg/kg) is dissolved in 0.1 N HNO3 and continuously infused into the rat’s
saphenous vein at a rate of 0.1 ml/min. Lead II ECG is recorded every 30 sec. Test compound is
injected orally or intravenously 5 min before the aconitine infusion.
296 Drug Screening Methods

A higher dose of aconitine in the test group compared to untreated group gives an index
of antiarrhythmic activity. The antiarrhythmic effect of test compound is measured by the
amount of aconitine/100 g animal (infusion duration) and includes ventricular extrasystoles,
tachycardia, fibrillation and death.8,9

Digoxin-induced Arrhythmia in Guinea Pigs

Digoxin overdose induces ventricular extrasystoles, fibrillation and death. Antiarrhthymic
drugs prolong the occurrence of these symptoms.
Male Marioth guinea pigs (350–500 g) are anesthetized with pentobarbitone sodium (35
mg/kg) intraperitoneally. Trachea, jugular vein and one carotid artery are catheterized and
the animal is maintained on artificial respiration (45 breaths/min). Through the jugular vein
digoxin is infused using a perfusion pump at a rate of 85 μg/kg in 0.266 ml/min until cardiac
arrest. ECG is recorded with steel needle electrodes during the whole experiment duration.
Blood pressure is recorded through the carotid artery. Test drug is administered either orally
1 hour or intravenously 1 min prior to the infusion. The period until the onset of ventricular
extrasystoles, fibrillation and cardiac arrest is recorded.10
The total amount of infused digoxin (μg/kg) to induce ventricular fibrillation extrasystoles,
ventricular fibrillation, cardiac arrest after treatment with the test drug are compared
statistically with controls receiving digoxin only.

Strophanthin/Ouabain-induced Arrhythmia
Ventricular tachycardia and multifocal ventricular arrhythmia are induced with acute
intoxication with cardiac glycoside (Strophanthin K).
Dogs (20 kg) of either sex are anesthetized with pentobarbitone sodium (30–40 mg/kg)
intraperitoneally. Two peripheral veins are cannulated for administration of the arrhythmia
inducing substance (V. brachialis) and the test compound (V. Cephalica antebrachialis). ECG
at different time intervals is registered with needle electrodes from lead II. Strophanthin K is
infused at a rate of 3μg/kg/min through the brachialis vein. 30–40 min later when ventricular
tachycardia or multifocal ventricular arrhythmia occurs, strophanthin infusion is terminated.
Test compound is administered after 10 min of stabilization of arrhythmias.11,12
A test compound is considered to have antiarrhythmic effect if the extrasystoles disappear
immediately after drug administration. If the test compound does not show a positive effect,
increasing doses are administered at 15 min intervals. If the test substance does reverse
arrhythmias, the next dose is administered after the reappearance of stable arrhythmia.

Adrenaline-induced Arrhythmia
Adrenaline at high dose may precipitate arrhythmia. Dogs (10–11 kg) are anesthetized with
pentobarbitone sodium (30–40 mg/kg) intraperitoneally. The femoral vein is cannulated.
Adrenaline is infused at a rate of 2–2.5 μg/kg through femoral vein. Lead II ECG and atrial ECG
are recorded. Test drug is administered 3 min after adrenaline infusion.13
A test compound is considered to have antiarrhythmic effect if the extrasystoles disappear
immediately after drug administration.
 Antiarrhythmic Agents 297

Calcium-induced Arrhythmia
Wistar albino rats (60–130 g) are anesthetized with Nembutal (60 mg/kg) intraperitoneally.
Ventricular flutter and fibrillation are induced by administration of 2 ml/kg 10% aqueous
calcium chloride through the femoral vein. During the injection and for 2 min thereafter, the
cardiac rhythm and behavior are studied by means of a cardioscope connected to the animal
with 2 percutaneous, precordial, clamp electrodes. Test drug is administered two minutes
prior to calcium infusion.14
Results are graded as isolated ventricular premature systoles, frequent ventricular premature
systoles, short and long run of ventricular flutter or fibrillation. The latter two are considered
positive. Comparison between test and control is made.

B. Electrically-induced Arrhythmia
Serial electrical stimulation results in flutter and fibrillation and it is possible to reproduce
some of the main types of arrhythmias of clinical importance. The flutter threshold or the
ventricular multiple response thresholds, may be determined in anesthetized dogs before or
after administration of test drug.

Ventricular Fibrillation Electrical Threshold

The maximum frequency at which atria would follow a stimulus can be used to compare
antifibrillatory compounds. Several electrical stimulation techniques have been used to
measure ventricular threshold such as single pulse stimulation, train of pulse stimulation,
continuous 50 Hz stimulation and sequential pulse stimulation.
Dogs (8–12 kg) are anesthetized with sodium pentobarbital (35 mg/kg) intraperitoneally
and maintained on artificial respiration. Blood pressure and temperature recordings are
monitored, chest opened and the heart is suspended in a pericardial cradle. The SA (sinoatrial)
node is crushed and a Ag-AgCl stimulating electrode is embedded in a Teflon disc sutured to
the anterior surface of the left ventricle.15 Anodal constant current (3 ms square) for 400 ms
is applied through the driving electrode. Electrical stimulation is programmed through a
digital stimulator. A recording electrode is placed on the surface of each ventricle. Lead II
of the body surface electrocardiogram is monitored.16 To determine ventricular fibrillation
threshold (VFT), a 0.2 to 1.8 s train of 50 Hz pulses are delivered 100 ms after every 18th basic
driving stimulus. The current intensity of pulse train required to induce sustained ventricular
fibrillation is defined as the VFT. When ventricular fibrillation occurs heart is immediately
defibrillated and allowed to recover to control condition for 15–20 min. Drug is administered
through the femoral vein.
VFT is determined before and after administration of test drug and compared using
student’s t test.

Programmed Electrical Stimulation-induced Arrhythmia

Dogs (8–12 kg) are anesthetized with 30 mg/kg pentobarbital sodium intravenously and
maintained on artificial respiration. A cannula is inserted in the left external jugular vein. Left
thoracotomy is performed between the 4th and 5th ribs, and the heart is exposed. The left
anterior descending coronary artery (LAD) is isolated. After a 20 guage hypodermic needle
has been placed on the LAD, a ligature is tied around the artery and the needle. The needle is
298 Drug Screening Methods

then removed resulting in critical stenosis of the vessel. LAD is perfused for 5 min. Ischemic
injury to the myocardium is achieved by 2 h occlusion of the LAD by a silicon rubber snare.
The vessel is then reperfused for 2 h in the presence of the critical stenosis. During the period
of LAD reperfusion, an epicardial bipolar electrode is sutured on the interventricular septum,
adjacent to the occlusion site. Silver disc electrodes are implanted subcutaneously for ECG
monitoring. After 6-9 days, chest is re-opened and programmed electrical stimulation is
performed through the electrode implanted on noninfarcted zone. The pacing stimuli is set
at 200 ms. After 15 pacing stimulation, an extra stimulus is delivered. Animals with sustained
ventricular tachycardia and ventricular fibrillation are used for the study. Heart rate, ECG
intervals are determined before programmed electrical stimulation is started. Test drug is
administered 30 min after the stimulus.17
The minimum current intensity of pulse required to induce sustained ventricular fibrillation
is recorded before and after administration of test drugs and mean values of 10 experiments
are compared using students t test.

C. Exercise-induced Ventricular Fibrillation

Tests combining coronary constriction with physical exercise, may resemble most closely the
situation in coronary patients. This model is suited to evaluate antiarrhythmic drugs for their
activity in cardiovascular parameters in an exercise-plus ischemia test.
Mongrel dogs (15–19 kg) are anesthetized with sodium pentobarbitone (10 mg/kg,
intravenously), chest cavity is opened, hearts exposed and supported by a pericardial cradle.
Around the left circumflex artery, a 20 MHz pulsed Doppler flow transducer and a hydraulic
occluder are placed. A pair of insulated silver coated wires are sutured to the epicardial surface
of both the left and right ventricular electrogram, from which heart rate is determined using
a Gould Biotachometer. A pre-calibrated solid state pressure transducer is inserted into the
left ventricle and finally, a two stage occlusion of the left anterior descending coronary artery
is performed (partially occluded for 20 min and then tied off ). Leads from the cardiovascular
instrumentation are tunneled under skin to exist on the back of the animal’s neck. Analgesics
and antibiotics are administered to the animals to minimize discomfort. Three to four weeks
after the production of myocardial ischemia, the animals are walked on a motor driven
treadmill and trained to lie quitely without restraint on a laboratory table during this recovery
period. Susceptibility to ventricular fibrillation is then tested on the motor driven treadmill.
Protocol starts with a 3 min warm up period during which the animals run at 6.4 km/h (0%
grade). The grade is increased every 3 min as follows (0%, 4%, 8%, 12% & 16%). During the last
minute of exercise, the left circumflex coronary artery is occluded, the treadmill is stopped
and the occlusion maintained for one additional min (total occlusion time, 2 min). Electrical
defibrillator is used if the animal becomes unconscious. The occlusion is released if ventricular
fibrillation occurs. The exercise plus ischemia test is repeated after pretreatment with the test
drug and compared to control (saline) group readings. On subsequent day, effective refractory
period is determined using Medtronic model 5325 programmable stimulator both at rest and
during myocardial ischemia. The effect of test drug on coronary blood flow is also studied
using flowmeter.18
All hemodynamic data (rate of change of left ventricular pressure) are recorded on to a
Gould model 2800 S eight channel recorder. The refractory period data, reactive hyperemia
 Antiarrhythmic Agents 299

response to each occlusion is averaged and the data analyzed using analysis of variance. The
effects of the drug intervention on arrhythmia formation are determined using chi-square test
with Yate’s correction.

Sudden Coronary Death Model in Dogs

Sudden coronary death is one of the leading causes of death in developed countries. This model
in dogs is used to test the protection offered against sudden coronary death. Male mongrel
dogs (14–22 kg) are anesthetized with pentobarbital sodium (30 mg/kg) intravenously. The
trachea is cannulated and the animals are maintained on artificial respiration. The jugular vein
is cannulated for the administration of test drug/saline. The chest cavity is opened and the
heart is exposed, then the left anterior descending coronary artery (LAD) is isolated and a
20-guage needle is placed on the LAD. A ligature is then tied across the artery and the needle
and subsequently, the needle is removed resulting in critical stenosis of the vessel. The LAD
is occluded for 2 h using a silicon rubber snare and then reperfused for 2 h in the presence of
critical stenosis. An epicardial bipolar electrode is sutured on the left atrial appendage for artrial
pacing and another bipolar plunge stainless steel electrode is sutured on the interventricular
septum. Two similar stainless steel electrode are sutured on the left ventricular wall, one at
the distribution of the LAD distal to the occlusion and the second in the distribution of the left
circumflex coronary artery (LCX). A silver-coated electrode is passed through the wall and into
the lumen of the LCX and sutured to the adjacent surface of the heart. For ECG monitoring
silver disc electrodes are implanted subcutaneously. Then the surgical incision is closed and
animals are allowed to recover. After the animals recover, they are treated with the test drug.
A direct anodal 15µA current from a 9-V nickel-cadmium battery is passed through a 250
ohm resistor and applied to the electrode in the lumen of LCX. The cathode of the battery is
connected to a subcutaneously implanted disc electrode and lead II ECG is recorded for 30
sec every 15 min on a cardiocasette recorder. The animals are sacrificed after 24 h of constant
anodal current or development of ventricular fibrillation. The hearts are removed and the
thrombus mass in the LCX is removed and weighed. The heart is sectioned and stained with
tetrazolium triphenyl chloride (TTC stain) to study the area of infarction. Time of onset of
ventricular ectopy and lethal arrhythmia is studied using recordings of the cardiocassette.
Non-sustained and sustained tachyarrhythmias are evaluated.19

D. Mechanically-induced Arrhythmia
Arrhythmias can be induced directly by ischemia or by reperfusion. By ischemia induced
infarction or by coronary ligation several phases of arrhythmia can be studied. The two
stage coronary ligation technique focuses on late arrhythmia. The influence on reperfusion
arrhythmia can be tested in various species.

Reperfusion Arrhythmia in Rats

Ligation of the left main coronary artery results in ventricular arrhythmia and myocardial
infarction. Electrocardiogram is recorded during ligation and subsequent reperfusion. The
amount of infarcted tissue is measured by means of p-nitro-blue-tetrazolium chloride staining
in myocardial sections.
300 Drug Screening Methods

Sprague Dawley rats (350–400 g) are anesthetized with pentobarbitone sodium (60 mg/
kg) intraperitoneally. The animal is maintained on artificial respiration, jugular vein is
cannulated for the administration of test drugs. Blood pressure is recorded from the carotid
artery using a pressure transducer connected to a polygraph. Chest is opened and heart is
exposed. The left coronary artery is located and ligated for 15–90 min (in case of infarct size
studies) and subsequently reperfused for 30 min. Test drug is administered 5 minutes before
the ligation. Peripheral blood pressure and ECG lead II are recorded continuously during the
whole experiment. The number of ventricular premature beats, ventricular tachycardia and
fibrillation are counted in the occlusion and reperfusion periods.20,21
At the end of the reperfusion period the animal is sacrificed and TTC (p-nitro blue
tetrazolium trichloride) staining is done to quantify the infarct size. The heart is dissected
and cut into transverse sections (1 mm thick) and stained with TTC prepared in Sorensen
phosphate buffer containing 100 mM, L-maleate in order to visualize the infarct tissue (blue/
violet stained healthy tissue, unstained necrotic tissue). Slices are photographed and infarct
area is measured by planimetry from projections of all slices. Changes in hemodynamic
parameters and infarct size in drug treated animals are compared to control values.

Reperfusion Arrhythmia in Dogs

Coronary artery ligation in dogs may result in increased heart rate, heart contractility, left
ventricular end diastolic pressure, blood pressure and ventricular arrhythmias, especially in
the reperfusion duration.
Dogs (20–25 kg) are anesthetized with thio-butobarbital sodium (30 mg/kg) intraperitoneally
and maintained on intravenous chloralose (20 mg/kg) and 250 mg/kg urethane intravenously
followed by subcutaneous administration of 2 mg/kg morphine. Animal is subsequently
maintained on artificial respiration. A peripheral vein (saphenous vein) is cannulated for the
administration of test compound. ECG is recorded continuously in lead II. Femoral artery is
cannulated to measure blood pressure and connected to a pressure transducer. Left ventricular
end diastolic pressure and heart rate are determined from the left ventricular pressure curves.
Myocardial contractility is measured as a rise of left ventricular pressure. The experimental
procedure followed is similar to that in rats. Coronary artery is ligated for 90 minutes. Twenty
minutes prior to ligation the test compound is administered. Animal is reperfused for 30
min.22,23 All the above mentioned parameters are recorded during the whole experiment.
Changes in parameters (mortality, hemodynamic and arrhythmia) in drug treated animals are
compared to vehicle controls.

Two Stage Coronary Ligation in Dogs

Mortality in dogs after coronary occlusion with a two stage ligation procedure is lower than
one stage ligation method.
Dogs (8–11 kg) are anesthetized by intravenous injection of methohexitone sodium (10 mg/
kg) and maintained on artificial respiration. Chest is opened and the heart is exposed. Left
coronary artery is located and coronary ligation is performed in two stages. Two ligatures are
placed around the artery and a 21 gauge needle. The first ligature is tied around the artery
and the needle, which is then removed. Thirty min later, the 2nd ligature is tied tightly around
 Antiarrhythmic Agents 301

the artery. Chest is closed in layers, 30 min after the 2nd ligature has been tied and the dog is
allowed to recover. After 24 and 48 h of ligation, arrhythmias develop and abate within 3–5
days. Lead II ECG, atrial electrogram and mean blood pressure are measured. Test drugs are
given as infusion for 10 min after coronary artery ligation.24 Changes in parameters (mortality,
hemodynamics and arrhythmia) in drug treated animals are compared to vehicle controls.
The canine model developed by Boyden and Hoffman, in which right atrial enlargement
is produced by banding of the pulmonary artery and by producing tricuspid regurgitation,
may also have a clinical counterpart in patients with chronic obstructive pulmonary disease
and tricuspid regurgitation.25 In these dogs, a functional zone of blockade and area of slow
conduction sets the stage for reentry, rather than an anatomical obstacle. Functional reentry is
also observed in the sterile pericarditis model of canine atrial flutter, first descried by Page et
al.26 This model was developed because of the fact that atrial flutter frequently occurs following
cardiac surgery in patients and this may be related to postoperative sterile pericarditis.

E. Genetically-induced Arrhythmia
Genetic Arrhythmia
A colony of German shepherd dogs has been described with inherited ventricular arrhythmias
and a predisposition for sudden death that most often occurs during sleep or at rest after
exercise or excitement. These dogs can be used to screen potential anti arrhythmic drugs.
The electrocardiogram does not show a prolonged QT interval, but frequently there is marked
notching of the T wave. The arrhythmias are rapid polymorphic ventricular tachycardia,
following long R-R intervals and are most likely due to triggered activity induced by early
depolarizations in the Purkinje system. In the epicardial myocytes, the density of the transient
outward current (Io) and the time constant of inactivation are reduced.27 Deficiencies in
cardiac sympathetic denervations also occur. At first glance, this dog model bears resemblance
to the congenital long QT syndrome in which bradycardia induced polymorphic ventricular
tachycardia and sudden death occurs and in which genetic defects in ion channels regulating
repolarization have been described. However, the dogs have no prolonged QT interval.
In patients with the long QT syndrome, no deficiencies in Io have been described.28,29 Still,
this animal model might have a counterpart because patients have been described with
polymorphous ventricular tachycardia (Torsade de pointes) that has a normal QT interval.

Discussion and Conclusion

Generally speaking, antiarrhythmic drugs exert their effects largely by modulating conduction
velocity, or refractory period duration, or both. Conduction velocity on one hand depends,
on the passive electrical properties of cardiac tissue, and on the other, the characteristics of
the Na+ and Ca2+ channels. In contrast, there are marked differences among species in the K+
currents that largely determine repolarization, so that action potential duration and duration
of refractory period differ widely in various species.
It is clear that species differences do exist with respect to factors that determine
arrhythmogenesis and it is also clear that no animal model will accurately mimic the human
suffering from or threatened by an arrhythmia. Nevertheless, the knowledge gathered from
302 Drug Screening Methods

animal studies, undoubtedly, has been instrumental in devising diagnostic and therapeutic
strategies both in supraventricular and ventricular arrhythmias. It is our conviction that in
the future, new knowledge will be obtained from experiments performed at many levels: in
systems expressing and testing the functions of molecules involved in electrical excitation,
in single cells, cell cultures, excised cardiac preparations, isolated whole hearts, whole
hearts in anesthetized animals and in conscious animals. It will be the combination of such
investigations rather than a single model or experimental technique, which will lead to
novel strategies for diagnosis and treatment. Finally, electrophysiological studies should be
encouraged in animals with ‘naturally’ occurring cardiovascular disease.
Animal models have been central to the advances in our understanding of the mechanisms
of human arrhythmia, but have also highlighted issues fundamental to all forms of disease
modeling. In any complex process, it is preferable to recapitulate as much of the causal pathway
as possible, rather than to empirically model individual components. The mechanistic insights
that have been gained over the last few decades, emphasize the complexity of the pathogenesis
of clinical dysrhythmia.30 Models capable of integrating the effects of both genetic and
epigenetic modifiers will be required to dissect the multi-step pathways involved, which
include myocyte heterogeneity, channel processing, and downstream signaling, to name but a
few.

Summary

In vitro models In vivo models

Chemically Electrically Exercise induced Mechanically Genetically
induced induced induced induced
Studies on Aconitine Ventricular Sudden coronary Reperfusion Genetic
isolated antagonism in fibrillation death model in arrhythmia in arrhythmia
ventricular rats electrical dogs rats
myocytes threshold
Isolated guinea Digoxin induced Programmed Reperfusion
pig papillary arrhythmia in electrical arrhythmia in
muscle guinea pigs stimulation dogs
induced
arrhythmia
Action potential Strophanthin Two stage
and refractory induced coronary ligation
period in isolated arrhythmia in dogs
guinea pig
papillary muscle
Langendorff Adrenaline
technique induced
arrhythmia
Acetylcholine Calcium induced
and potassium arrhythmia
induced
arrhythmia
 Antiarrhythmic Agents 303

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 cHAPTER

19
Cardiotonic Agents
INTRODUCTION
Congestive heart failure (CHF) is a constellation of symptoms, with hallmarks of fatigue and
dyspnea, which continues to be a highly prevalent and morbid clinical syndrome. Because of
the growing burden of CHF as the population ages, the need to develop new pharmacological
treatments and therapeutic interventions is of paramount importance. Common patho­
physiologic features of CHF include changes in left ventricle structure, function, and neuro-
hormonal activation.1,2
The progress made in our understanding of the pathophysiology and treatment of CHF would
not have been possible without a number of animal models of heart failure and hypertrophy,
each having unique advantages as well as disadvantages. The species and interventions used
to create CHF depend on such factors as ethical and economical considerations, accessibility
and reproducibility of the model.3,4
The use of small-animal models to study complex cardiovascular pathophysiology has
proven to be invaluable during the past decades. As a direct result of basic and translational
studies in murine models, our understanding of pathophysiology of heart failure and its
treatment has advanced considerably. Rat models have been used primarily to assess the
efficacy of specific pharmacological or molecular therapies. The ability to manipulate the
mouse genome has facilitated a particularly elegant approach to identify novel therapeutic
targets, offering a “proof of principle” approach to explore the mechanisms underlying heart
failure and its progression. Moving forward, these small animal models of heart failure will
continue to be critical tools in the identification of new therapeutic targets and evaluation of
specific therapies for heart failure.4
The recapitulation of the CHF phenotype in large animal models can allow for the translation
of basic science discoveries into clinical therapies. Models of myocardial infarction/ischemia,
ischemic cardiomyopathy, ventricular pressure and volume overload, and pacing-induced
dilated cardiomyopathy have been created in dogs, pigs, and sheep for the investigation of
CHF and potential therapies. Large animal models, recapitulating the clinical CHF phenotype
and translating basic science to clinical applications, have successfully traveled the journey
from bench to bedside. Undoubtedly, large animal models of CHF will continue to play a
crucial role in the elucidation of biological pathways involved in CHF and the development
and refinement of CHF therapies.5
306 Drug Screening Methods

IN VITRO METHOD
1. Isolated Hamster Cardiomyopathic Heart
Isolated Syrian hamster hearts can be used for evaluation of cardiotonic drugs. Hamsters
with cardiomyopathy of the age group (50 weeks) are used for the study. Normal Syrian
hamsters of the same age are used as controls. The animals are pretreated with heparin
(5 mg/kg) intraperitoneally and 20 min later the heart is prepared according to the method of
Langendorff and perfused with Ringer's solution and allowed to equilibrate in the isolated state
for 60 min at 32oC with a preload of 1.5 g. The force of contractions is recorded isometrically by
a force transducer connected to a polygraph. The heart rate is measured using a chronometer.
The coronary flow is measured using an electroflowmeter. Test compounds are injected via the
aortic cannula into the inflowing heart–Ringer's solution.
The contractile force and coronary flow in hearts of the treated and the sham control group
are compared using student’s t test. Percentage improvement is calculated and the efficacy of
the drug evaluated in increasing the coronary flow and contractile force.6

2. Isolated Cat Papillary Muscle

This method using rat capillary muscle has been described by Catell and Gold.7 Prolonged
electrical stimulation of isolated cardiac tissue results in decrease in performance. Cardiac
glycosides restore the force of contraction.
Cats (2.5–3 kg) of either sex are anesthetized with ether. Through a left thoracotomy, heart
is exposed. Papillary muscle from the right ventricle is isolated and fixed in an organ bath
containing Ringer's solution maintained at 37oC. One end of the papillary muscle is tied to a
strain guage and other end to the muscle. Electrical stimulus of 4–6 V is applied to the muscle
at a rate of 30/min and the contractions are recorded on a polygraph. On electrical stimulation
for 1 h, the muscle contractions start diminishing. The cardiac glycosides are added to the bath
at this point to restore the contractile force. Ouabain is the standard glycoside that is added at
a dose of 300 ng/ml.
Evaluation is based on increase in contractile force on adding the glycoside. Contractile
force is calculated as percentage of the predose level and comparisons between different
groups are made.

3. Ouabain Binding
The binding kinetics, i.e association process, equilibrium binding and dissociation process on
the ouabain receptor, is characteristic of the cardiac glycosides.
Rat hearts are submitted to coronary perfusion and subsequently myocytes are isolated
by collagenase digestion. From these isolated membrane fractions, myocyte sarcolemma is
obtained. Radioactive ouabain [3H] with specific radioactivity of 20 Ci/mmol is incubated
with ligands in 10 ml of binding medium kept at 37oC for 10 min. The composition of binding
medium (pH 7.4) is 1 mM inorganic phosphate, 1mM MgCl2 and 50 mM Tris HCl.
Association process: After temperature equilibration in the presence of either 10 or 100 nM
[3H]ouabain, 200 μg of membrane preparation are added to initiate the reaction. At various
times, 4.5 ml are removed and rapidly filtered.
 Cardiotonic Agents 307

Equilibrium binding: At the end of the temperature equilibration carried out in the presence
of increasing concentrations of [3H]ouabain ranging from 10 nM to 3 μM, 40 μg of membranes
are added. After 30 min, duplicate aliquots of 4.5 ml are removed and filtered.
Dissociation process: Once equilibrium has been achieved under the experimental conditions
used to study association, 10 ml of prewarmed Mg2+ plus Pi Tris-HCl solution supplemented
with 0.2 mM unlabeled ouabain are added to initiate dissociation of [3H]ouabain. At various
times, aliquots of 0.9 ml are removed and rapidly filtered.
The radioactivity bound to the filters and the specific binding measurements are determined.
Kinetic parameters for the association and the dissociation process are calculated. The results
of equilibrium binding are analyzed by Scatchard plots.

In vivo models
Rat Models of Heart Failure
Rat models are relatively inexpensive and because of short gestation period, a large sample
size can be produced in a short period of time. Therefore, rat models have been extensively
used to study the long-term pharmacological interventions including long term survival
studies.7,8 However, there are several limitations to the use of rat models regarding differences
in myocardial function compared to human heart:
1. Rat myocardium exhibits a very short action potential, which normally lacks a plateau phase.
2. Calcium removal from the cytosol is predominated by the activity of sarcoplasmic reticulum
calcium pump whereas Na+/Ca2+ exchanger activity is less relevant.
3. In normal rat myocardium, α- myosin heavy chain isoform predominates and a shift towards
the β-myosin isoform occurs and hemodynamic load or hormonal changes take place.
4. Resting heart rate is five times that of humans and the force-frequency relation is inverse.9

1. Rat Coronary Ligation Model

Myocardial infarction following coronary artery ligation in Sprague-Dawley rats is a widely
used rat model of heart failure. If the left coronary artery is not completely ligated, heart failure
may occur as a consequence of chronic myocardial ischemia.10 Complete occlusion of the left
coronary results in myocardial infarction of variable sizes with occurrence of overt heart failure
after 3–6 weeks in a subset of animals with large infarcts. The impairment of left ventricular
function is related to the loss of myocardium. Failure is associated with left ventricular dilation,
reduced systolic function and increased filling pressures.
Males Sprague Dawley rats (250–300 g) are anesthetized with 200 mg/kg hexobarbital. The
trachea is cannulated and the animal is maintained on artificial respiration. The chest cavity is
exposed and the left anterior descending carotid artery (LAD) is isolated. A ligature is placed
around the LAD and the chest cavity is sutured back and the animal maintained on food
and water ad libitum. After 4 weeks, the chest cavity is opened and carotid artery as well as
jugular vein is cannulated for measurement of blood pressure as well as administration of test
compounds. Filling pressure, systolic, diastolic and mean blood pressure are measured. After
measuring the hemodynamic parameters, the animals are sacrificed and the isolated hearts
are used for studying calcium channel, sarcoplasmic reticulum ATPase and protein levels.
308 Drug Screening Methods

It is observed that in the control group the progression of left ventricular dysfunction and
myocardial failure is associated with neurohumoral activation similar to that seen in patients
with CHF. Depressed myocardial function is associated with altered calcium transients. The
density of L-type calcium channels, SR-Ca2+ - ATPase and protein levels decrease continuously
with increasing severity of congestive heart failure. Comparison between test group and
control group are made on the basis of the above mentioned parameters.
Although a high initial mortality and induction of mild heart failure in most cases may be a
disadvantage of this model, it seems to be very useful for long term studies of pharmacological
interventions on the neurohormonal activation.11

2. Rat Aortic Banding

Restriction of blood flow to the aorta in rats induces not only hypertension but also congestive
heart failure within several weeks. After a period of several weeks, ventricular ACE activity may
decrease again to normal values, which may be related to normalization of wall stress with
increasing cardiac hypertrophy.12 Furthermore, after several months, a subset of animals goes
into cardiac failure.
Sprague–Dawley rats (250–280 g) are fasted for 12 h before surgery. Animals are anesthetized
with 200 mg/kg hexobarbitone. The abdomen is shaved, moistened with a disinfectant and
opened by a cut parallel to the linea alba. The intestine is moistened with saline and placed
in a plastic cover to prevent desiccation. The aorta is prepared free from connective tissue
above the left renal artery and underlaid with a silk thread. Then, a cannula no. 1 (0.9 × 40
mm) is placed longitudinally to the aorta and both aorta and cannula are tied. The cannula is
removed, leaving the aortic lumen determined by the diameter of the cannula. The intestine is
placed back into the abdominal cavity with the application of 5.0 mg rolitetracycline. In sham-
operated controls no banding is performed while in the test group animals are administered
drugs for 6 weeks. The skin is closed by clipping. It is observed that after 4–6 weeks heart failure
develops in these animals.
Total cardiac mass, weight of left and right ventricle of treated rats are compared with
operated controls and sham-operated controls. Heart failure is associated with increased
myosin heavy chain mRNA and atrial natriuretic factor mRNA. During compensated
hypertrophy, while catecholamine levels are normal, there is activation of local myocardial
renin-angiotensin system, which may be important for the development of heart failure.13 The
above mentioned parameters are studied in both the test group and the sham control group.
At the end of the experiment, blood pressure and heart rate are recorded via the left coronary
artery. Moreover, total cardiac mass, weight of left and right ventricle of treated rats is also
compared with sham operated controls.
This model seems to be well suited for studying the transition from hypertrophy to failure at
the level of myocardium.

3. Dahl Salt Sensitive Rats

This model is well suited to study the transition from compensated hypertrophy to failure.14
This strain of rats develop systemic hypertension after receiving a high-salt diet.
Sprague-Dawley rats (250–300 g) are selected for this study. Drinking water is replaced with
1% NaCl saline solution. High Dahl salt diet is prepared in the laboratory by mixing salt with the
 Cardiotonic Agents 309

regular diet. Animals are fed the prepared diet and 1% NaCl solution ad libitum. The test drug
rats are administered the drug orally for 1 month. After the completion of the experimental
duration, the animals of both groups (test and sham control) are sacrificed. Their hearts are
removed and total cardiac mass, weight of left and right ventricle are weighed and compared.
It is observed that the animals in the sham control group develop concentric left ventricular
hypertrophy at 8 weeks, followed by marked left ventricular dilation and overt clinical heart
failure at 15–20 weeks. Failing heart dies within a short period of time. The ability of the test
drug to reverse these changes is studied.15

4. Spontaneous Hypertensive Rat

The spontaneous hypertensive rat is a well-established model of genetic hypertension in
which cardiac pump function is preserved at 1 year of age.16 At 18-24 months, cardiac failure
develops, which includes reduced myocardial function and increased fibrosis. In this model,
although altered calcium cycling is observed, no decrease in mRNA of the sarcoplasmic
reticulum calcium pump is found during transition from compensated hypertrophy to failure.
The transition to failure is associated with significant alterations in the expression of genes
encoding extracellular matrix.17 Furthermore, an increased number of apoptotic myocytes are
observed and it is suggested that apoptosis might be a mechanism involved in the reduction
of myocyte mass that accompanies the transition from stable compensation to heart failure.
The animals are divided into two groups. To test group animals, drug is administered orally
for 1 month while to sham control group animals, no drug treatment is given. After completion
of the experimental protocol, the animals are sacrificed and their hearts are processed for the
estimation of number of apoptotic cells, sarcoplasmic reticulum calcium pump mRNA levels
and expression of genes encoding for extracellular matrix and results compared.

5. Spontaneous Hypertensive-Heart Failure Rats (SH-HF)

Spontaneous hypertensive rats, which develop failure before 18 months of age, have been
selectively bred. Development of heart failure occurs earlier in rats which carry the facp
gene (corpulent gene) that encodes a defective leptin receptor (SH-HF/Mcc-facp). In these
animals, renin plasma activity, atrial natriuretic peptide (ANP) and aldosterone levels
progressively increase with age and renin plasma activity is independently correlated to
cardiac hypertrophy. Interestingly, hearts from the SH-HF rats exhibits a more negative force
frequency relationship than control rats. In a recent study trial in SH-HF rats, it was observed
that calcium current density and function of ryanodine receptors, and sarcoplasmic reticulum
calcium uptake were normal. However, it was also observed that the relationship between
calcium current density and the probability of evoking a spark was reduced indicating that
the calcium influx was less effective at inducing SR calcium release. It was speculated that
these changes might be related to spatial remodeling between L-type calcium channels and
ryanodine receptors.18
Animals are divided into two groups. Group 1 serves as the test drug group (administered
drug for one month, orally) while group 2 serves as the sham control group (untreated). After
completion of the experiment, comparisons are made between the two groups based on their
plamsa renin activity, ANP and aldosterone levels, rynodine receptor density, sarcoplasmic
reticulum calcium uptake and endothelial nitric oxide synthase (NOS) activity.
310 Drug Screening Methods

Dog Models of Heart Failure

Generally, dog and other large animal models of heart failure may allow the study of left
ventricular function and volumes more accurately than rodent models. In particular, they
allow better chronic instrumentation. Furthermore, in dog like human myocardium the
β-myosin heavy chain isoform predominates and excitation contraction coupling processes
seem to be similar to the human myocardium.19 The force frequency relationship, the slope
of the end-systolic pressure-volume relation, is positive in automatically intact awake dogs as
well as during autonomic blockade. On the other hand, dog models are costly and require
substantial resources with respect to housing and care.

1. Chronic Rapid Pacing

Chronic rapid pacing at heart rates above 200 beats per minute in previously healthy dogs
within several weeks produces the syndrome of CHF.
Adult male dogs (18–25 kg) are anesthetized with pentobarbital (30 mg/kg) intraperitoneally.
The animal is maintained on artificial respiration (20–24 strokes/min). The chest cavity is
opened through a 3–4 cm long thoracotomy and the heart is exposed. A ventricular pacing
lead is attached to the left ventricular apex. The pacemaker is programmed to pace at 240–260
beats/min for 2–4 weeks. After the surgical procedure, the heart is placed back in the chest
cavity and the costal ribs closed and the musculus pectoralis placed over the wound. Air from
the thorax is removed by applying pressure on both sides of the thorax. After application of
an antibiotic emulsion the skin wound is closed. Significant heart failure develops by 4 weeks
and continues for upto 10 weeks. In the majority of studies, chronic rapid tachycardia results
in progressive biventricular chamber dilatation over a 3–4 week period. The test drugs are
administered subcutaneously or intramuscularly over a period of 14 days.
Heart failure is associated with a significant decrease in ejection fraction and diastolic
dysfunction, followed by decreased cardiac output and increased systemic vascular
resistance.20 It is important to note that heart failure is reversible with respect to clinical
hemodynamic and neurohumoral abnormalities when electrical pacing is stopped. The
exact pathogenesis in this model is still unclear. Similar to human heart failure there are time
dependent changes in neurohumoral activation and an early sympathetic activation, increase
in plasma catecholamine levels and attenuation of parasympathetic tone. In addition, plasma
ANP levels are elevated early in the development of left ventricular dysfunction. Systemic
activation of renin-angiotensin system is seen with progressive pump failure. Further, more
endothelial dysfunction with decreased nitric oxide mediated coronary vasodilation has been
observed similar to the patients with heart failure.
Comparison between test group and sham control group is made on the basis of changes
in parameter like ejection fraction, cardiac output and systemic vascular resistance. Further,
plasma catecholamine, ANP levels and renin acitivty are also evaluated to assess the
cardioprotective potential of test drugs.
The technique of tachycardia pacing has also been used in pigs and sheeps, and
findings similar to those in dogs have been observed with respect to clinical hemodynamic
and neurohumoral changes. This model seems very valuable for studying neurohumoral
mechanisms and peripheral circulatory alterations, both of which closely resemble that
observed in human heart failure.21
 Cardiotonic Agents 311

In a similar model of CHF, a number of transmyocardial direct current shocks applied

through a catheter into the left ventricle chamber in anesthetized dogs, result in left ventricle
hypertrophy and dilation, decreased ejection fraction and decreased cardiac output over a 4
months period. This is associated with increased plasma catecholamines but with no change
in plasma renin activity.22

2. Volume Overload
Prolonged volume overload may lead to development of heart failure. In dogs, volume
overload has been produced either by creation of an arteriovenous fistula, where an end to side
anastomosis is made between the femoral vein and artery in order to increase venous flow or by
destruction of the mitral valve in a closed chest dog by an arterially placed grasping forceps.23
Dogs (12–15 kg) are anesthetized with pentobarbital (30 mg/kg) intraperitoneally and
maintained on artificial respiration (20–24 strokes/min). Thoracotomy is performed and the
heart is exposed. Chronic experimental mitral regurgitation is produced in closed chest dogs
by disruption of mitral chordae or leaflets using an arterially placed foreceps. Within 3 months,
left ventricular hypertrophy, dilation and development of overt clinical heart failure occurs
in this model. After the surgical procedure, the heart is placed back in the chest cavity and
the costal ribs closed. By applying pressure on both sides of the thorax, air from the thorax is
removed. After application of an antibiotic emulsion the skin wound is closed. Significant heart
failure develops by 4 weeks and continues for upto 10 weeks. The test drugs are administered
subcutaneously or intramuscularly over a period of 14 days.
Neurohumoral activation including local activation of the Renin Angiotensin System is
observed in CHF dogs, which is generally associated with depressed myocardial function.
Comparisons between test group and sham control group (untreated animals) are made. The
model has been used to study the influence of chronic β-adrenoceptors blockade on myocytes
and left ventricular function, both of which significantly improve with treatment.24

3. Coronary Artery Ligation and Microembolization

Coronary artery ligation and microembolization have been used to produce myocardial
infarction and CHF in dogs.
Dogs of either sex (30 kg ) are anesthetized with intravenous bolus injection of 35–40 mg/kg
pentobarbitone. Animals are maintained on artificial respiration. A transducer is connected
to the right femoral artery for recording peripheral systolic, diastolic and mean blood
pressure. A microtip catheter is inserted via the left carotid artery for determination of left
ventricular pressure. Systolic, diastolic and mean pulmonary capillary pressure and cardiac
output are measured by thermodilution technique using a cardiac index computer. Heart is
exposed through a left thoractomy between 4th and 5th intercostal space and pericardium
is opened. Polystearyl microsphere is injected through the angiogram catheter into the
left atrium. Initially, a 10 ml (1mg/ml) microsphere is injected and later a 5 ml bolus about
5 min apart. The microsphere injection produces stepwise elevation of LVEDP. Embolism is
terminated when LVEDP has increased to 16–18 mm Hg or heart rate reaches to 200 beats/min.
Intravenous bolus injection or continuous infusion administers the test substance. Recordings
are obtained before and after embolization and administration of test compound at various
time intervals.25,26
312 Drug Screening Methods

The model has several disadvantages. Because of extensive collateral circulation, there are
important differences in the pattern of infarction between human and dog. The model is time
consuming, technically demanding and expensive. The model is associated with high mortality
and a high incidence of arrhythmias.

Rabbit Models of Heart Failure

Rabbit models are less expensive than dog models. In addition, non failing rabbit myocardium
exhibits interesting similarities to human heart.
1. The β-myosin heavy chain isoform predominates in adult animals.
2. The sarcoplasmic reticulum contributes by about 70% and the Na+/Ca2+ exchanger
contributes by about 30% calcium estimation.
3. The force-frequency relation is positive.

1. Volume and Pressure Overload

Volume overload, pressure overload and the combination of both are used to induce heart failure
in rabbits. Chronic severe aortic regurgitation in rabbits, created by aortic valve perforation with
a catheter, produces left ventricular hypertrophy, followed by systolic dysfunction and heart
failure after a period of months.
Rabbits are anesthetized with pentobarbitone sodium (35 mg/kg) intraperitoneally. Their
trachea is cannulated and the animals maintained on artificial respiration. The carotid artery
is cannulated. The chest cavity is opened and the heart is exposed. Aortic insufficiency is
produced by destroying the aortic valve with the catheter, introduced through the carotid
artery. The chest cavity is sutured back and antibiotic is applied to prevent any infection.
After 14 days, aortic constriction is performed just below the diaphragm using a PVC clamp.
Occurrence of heart failure is more consistent and rapidly observed when aortic regurgitation
is combined with aortic constriction. Test drugs are administered for 2 weeks (subcutaneously
or intraperitoneally). Heart failure occurs about 4 weeks after the initial procedure. It is
associated with alterations in the β-adrenoceptors system similar to those in humans.
Furthermore, in this model there is inversion of the force frequency relation and alteration
of the post rest potentiation, which closely resembles the situation in the human heart.
Interestingly, protein and mRNA levels of the Na+/Ca2+ exchanger are significantly increased
in failing compared to nonfailing animals, whereas sarcoplasmic reticulum Ca2+ ATPase is not
significantly altered. After completion of the experimental protocol, the animals are sacrificed
and the above mentioned neurohumoral parameters are studied in the drug treated and sham
control groups. The ability of the test group to reverse these changes is studied.27
As this model closely mimics alteration of myocardial function observed in the end
stage failing human myocardium, this model is well suited to study alteration in excitation
contraction coupling during the transition from compensated hypertrophy to failure.

2. Tachycardia Pacing
Recently, chronic rapid pacing at rates between 350–400 beats/min over a period of several
weeks in rabbits was shown to produce myocardial depression as well as, hemodynamic and
neurohumoral signs of heart failure.28
 Cardiotonic Agents 313

Rabbits are anesthetized with pentobarbitone sodium (35 mg/kg) intraperitoneally.

Their trachea is cannulated and the animals maintained on artificial respiration. The chest
cavity is opened through a 3–4 cm long thoracotomy and the heart is exposed. A ventricular
pacing lead is attached to the left ventricular apex and the pacemaker is programmed to
pace at 350–400 beats/min for 2–4 weeks. After the surgical procedure, the heart is placed
back in the chest cavity and the intercostal ribs closed and air from the thorax is removed by
applying pressure on both sides of the thorax. An antibiotic emulsion is applied and the skin
wound is closed. After 4–6 weeks the animals develop heart failure. In one experimental
group animals are administered test compound either subcutaneously or intraperitoneally
for 2 weeks. After completion of the experimental period, the animals are further subjected
to surgery. The carotid artery is cannulated for measuring blood pressure. Hemodynamic
parameters like systolic, diastolic, mean blood pressure and heart rate are measured.
The animals are then sacrificed and their hearts weighed and processed for estimation of
plasma renin activity. Comparisons between test group and sham control group are made
on the basis of hemodynamic parameters, plasma renin activity and heart weights.
It is observed that in the sham control group (untreated group) the force frequency relation
is severely depressed and inverted at higher stimulation rates. This is similar to the alteration
of the force frequency relation observed in failing human hearts. As was observed in the
tachycardia-pacing dog failure model, no left ventricular hypertrophy is developed in the
rabbit model.

3. Doxorubicin Cardiomyopathy
Doxorubicin exhibits acute and chronic cardiotoxicity and has been used to induce failure
in various animal species. Several different mechanisms involved in the pathophysiology of
doxorubicin heart failure have been suggested, including free radical generation and lipid
peroxidation, reactive sulphydryl groups, binding to channel regulatory sites, or inhibition of
mRNA and protein synthesis.29
Rabbits (5–6 kg) of both sexes of various strains can be used in this model. Doxorubicin
(1 mg/kg intravenously, twice weekly) is given for 6–9 weeks in the sham control group. In
the test group the animals are administered test drug for 4–6 weeks either subcutaneously
or through the intraperitoneal route. After the experimental duration, the animals are
anesthetized with pentobarbitone sodium (35 mg/kg) intraperitoneally and their carotid
artery is cannulated for measuring blood pressure. The heart is exposed and cannula is
inserted into the left ventricle to measure left ventricular end diastolic pressure (LVEDP)
and dP/dt. The animals are sacrificed and the hearts processed for immunohistochemical
studies through Western Blot studies. Chronic anthracycline administration to rabbits causes
impairment of cardiac contractility and decreased gene expression of the calcium-induced
calcium release channel of sarcoplasmic reticulum (SR), the ryanodine receptor (RYR2).
The C-13 hydroxy metabolite (doxorubicinol), formed in the heart, has been hypothesized
to contribute to anthracycline cardiotoxicity. Left ventricular fractional shortening (LVFS)
is decreased by chronic treatment with doxorubicin compared to age-matched pair-fed
controls. Doxorubicin, causes a significant reduction in the ratio of RYR2/Ca-Mg ATPase
(SERCA2) mRNA levels in the left ventricle. This suggests that doxorubicin may contribute
314 Drug Screening Methods

to the downregulation of cardiac RYR2 expression in chronic doxorubicin cardiotoxicity. The

above mentioned parameters are also studied in the test drug group and comparisons are
made with the sham control group. The ability of the test drug to reverse or reduce these
changes is studied. These findings may suggest that this model is suited to study functional
consequences of altered ryanodine receptor expression.

Guinea Pig Models

1. Cardiac Insufficiency
CHF in man is characterized by cardiac hypertrophy, peripheral edema, lung and liver
congestion, dyspnea, hydrothorax and ascites. Based on these symptoms, CHF has been
induced in guinea pigs with symptoms very close to human pathology. Following 8 weeks
of banding of the descending thoracic aorta in guinea pigs, overt heart failure develops in a
subgroup of animals. Alteration of myocardial function in this guinea pig model has some
similarity to end-stage failing human myocardium.
Male guinea pigs (250-400 g) are anesthetized with ether. The chest cavity is opened,
pericardium removed and heart is exposed. The beating heart is extruded from the thorax and
a ring shaped clamp covered with a thin rubber tube is placed around the basis of the heart,
keeping the heart outside of the thorax without closing off the blood circulation. A thread
soaked with diluted disinfectant solution is placed as a loop around the heart and tightened
so that the apical third of both ventricles is tied off. The degree of tightening of the loop is
essential. Complete interruption of blood supply to the apical third resulting in necrosis has to
be avoided as well as the loops slipping off. After removal of the clamp, the heart is placed back,
the incision between the 4th and 5th costal ribs closed and the musculus pectoralis placed
over the wound. Air is removed from the thorax and after application of an antibiotic emulsion,
the skin wound is closed. The test drugs are administered subcutaneously or intramuscularly
for 14 days.
The animals develop symptoms of CHF with death rate of 80% within 1 day. Lung weight,
relative heart weight are significantly increased. Exudate volume in the thorax cavity and
ascites is found between 3.5–7.5 ml. Lung edema and liver congestion are found histologically.
Peripheral edema, preterminal dyspnoea and tachycardia are observed. The ability of the test
drug to reverse these changes is studied. For survival rate ED50 of test drug is calculated.
Also a decrease in SR-Ca2+ ATPase protein levels and phospholamban protein levels is
observed in failing guinea pig heart following 8 weeks of banding of the descending thoracic
aorta as compared to an age matched banded group without clinical signs of heart failure.
Regarding myosin isoforms, guinea pig myocardium, like the human ventricular myocardium,
contains predominantly the β-myosin without any α-myosin heavy chain present in
hypertrophied and failing hearts.30
The guinea pig model, thus has similarities to human heart failure with respect to calcium
cycling, myosin isoforms and myocardial function. This model may be suited to study the
transition from cardiac hypertrophy to failure with respect to alterations in excitation-
contraction coupling systems.
 Cardiotonic Agents 315

Syrian Hamster
1. Cardiomyopathic Hamster
Cardiomyopathic strains of the syrian hamsters have been widely used as a model for
cardiac hypertrophy and heart failure.31 The model exhibits an autosomal recessive mode of
inheritance, which leads to degenerative lesions in all striated muscles and in particular in
the myocardium. The animals develop overt heart failure after 7–10 months. Histologically,
necrotic, calcified myocardial lesions are observed initially in the development of the disease.
Furthermore, a time dependent change in myosin isoform expression has been observed. The
evolution of cardiomyopathic disease is characterized by five distinct phases: A prenecrotic
stage, in which no pathology is evident, a phase of fibrosis and calcium deposition, an
overlapping period of reactive hypertrophy of the remaining viable myocytes and a final stage
of depressed myocardial performance and failure.
The test drugs are administered subcutaneously or intramuscularly for 14 days. The ability
of the test drug to reverse or delay the above-mentioned changes is studied.
In summary, the advantages of this model are absence of surgical manipulations, low cost
and the ease with which large number of animals can be studied.
It is important to state that there are differences among the strains, in the time course of
the pathologic changes, therefore, the time point at which measurements are performed are
critically important in this model. Furthermore, sub cellular alterations underlying myocardial
failure seem to be different from those in failing human hearts.

Transgenic Mice
Recent developments of techniques to alter specifically the expression of genes have greatly
improved the understanding of the pathophysiology of heart failure. Moreover, several
genetic models of heart failure by addition or deletion of genes in mice have been developed
and miniaturized physiological techniques to evaluate the resulting cardiac phenotypes
have been established.32 These models allow the identification of genes that are causative
for heart failure and to evaluate the molecular mechanisms responsible for the development
and progression of the disease. Gene targeted disruption of the muscle LIM protein (MLP) in
mice is a new model of heart failure. MLP is a regulator of myogenic differentiation. Mice who
were homozygous for the MLP knockout develop dilated cardiac myopathy associated with
myocardial hypertrophy. Adult mice show clinical and hemodynamic signs of heart failure
similar to those in humans.
Development of cardiomyopathy was also observed in mice with knockout of myogenic
factor 5. Transgenic mice overexpressing either β-adrenergic receptor kinase or G-protein
coupled receptor kinase 5, resulting in uncoupling of the β-adrenergic receptor, also exhibit
reduced contractility, but without clinical signs of overt CHF. A recent model of transgenic
overexpression of tropomodulin, exhibited dilated cardiomyopathy 2–4 weeks after birth
with reduced contractile function and heart failure. This was associated with the loss of
myofibrillar organization. One group of animals is administered drug orally, subcutaneously
or intraperitoneally for 15 days. At the end of the experimental protocol, the animals in the
test drug group are compared to sham control group on the basis of the above mentioned
parameters.33
316 Drug Screening Methods

Developing New Therapeutic Targets in CHF

Gene Therapy
As the morbidity and cost of CHF have continued to increase, so has the understanding of
cellular and molecular derangements which take place in CHF. This increasing knowledge,
combined with the urgency to discover and develop novel and improved therapeutic strategies
for CHF, has led to targeting molecular entities involved in CHF pathogenesis through gene
therapy. Efforts to create CHF therapies targeting molecular causes of myocardial failure have
focused on areas such as regulation of cardiomyocyte calcium handling. Kaye et al. used a
sheep model of rapid pacing-induced DCM to develop a percutaneous means of myocardial
gene delivery of a mutant form of the regulatory protein phospho- lamban, reporting improved
cardiac function compared with controls.34 In a different study, Kawase et al. examined the
therapeutic potential of gene delivery in a pig model of volume overload HF. The study observed
improved LV contractile performance and myocardial remodeling 2 months after SERCA2a,
the cardiac isoform of a family of calcium ATPases, gene delivery administered by antegrade
epicardial coronary artery infusion. The first human CHF gene therapy trials have now been
initiated on patients with CHF receiving SERCA2a via myocardial gene delivery, providing an
example of how large animal models serve a crucial step in the translation of basic science into
clinical application.35,36

Stem Cells
Advances in the field of cellular therapies have created a new avenue of potential CHF
treatments, specifically in area of stem cell research. Stem cells derived from various tissues
have been introduced into the post-MI myocardium in efforts to attenuate post-MI LV
remodeling. Initial studies using murine models reported dramatic results of stem cells having
the ability to localize to the heart and purported to yield new myocardium. These initial small
animal studies suggested boundless promises with respect to stem cells and myocardial
remodeling. However, more recent clinical studies using mesenchymal stem cells have failed
to demonstrate significant effects on post-MI LV remodeling and function.37 The reasons for
the failure of translation from basic stem cell studies to the clinical context are multifactorial,
but likely include a lack of consensus regarding optimal stem cell type and preparation,
delivery method, delivery location, and cell concentration. Large animal models will play a
critical role in defining these factors. For example, the relative efficacy of mesenchymal stem
cells has been evaluated in the pig MI model, with reports of improved LV EF after myocardial
delivery of mesenchymal stem cells at the time of MI. Other studies have reported decreased
MI expansion in pig and sheep MI models with mesenchymal stem cells delivered into the
myocardium within 72 hours of MI and attenuation of EF deterioration with delivery 1 month
post-MI. Alternatively, intracoronary and systemic delivery of mesenchymal stem cells have
also been examined, further exemplifying the utility of large animal models in clarifying
variables in stem cell therapies. It is likely that these translational studies in large animal
models will be necessary if the tremendous potential suggested by rodent studies is to be
realized clinically.38
 Cardiotonic Agents 317

Devices and Mechanical Support

A number of innovative devices aimed at improving LV systolic function and/or cardiac
output in patients with HF have been created and refined through large animal models.
From comparisons of early LV assist devices with the development of a totally implantable
biventricular assist system, large animals have served an integral role in groundbreaking
advances in cardiac destination therapies. The adaptation of such devices for neonatal and
pediatric use has also been possible through large animal models. The development of
minimally invasive devices to unload pressure from the failing LV have also used large animal
models. For example, Haithcock et al. used a canine microembolization model of HF to
demonstrate the benefits of LV unloading, establishing the basis for a percutaneously placed
continuous aortic flow augmentation device, which is now in clinical trial.39 Other work has
been directed toward cardiac restraint devices, which are surgically placed around the heart
itself to arrest post-MI LV remodeling. For example, Chakir et al. used a canine rapid-pacing
model to demonstrate reduced myocyte apoptosis and improved stress-response molecular
signaling with cardiac resynchronization therapy. Further advances and improvements in HF
device therapies will surely depend on large animal models to ensure the safety and efficacy of
these therapeutic options.40

Discussion and Conclusions

In the past, a large number of models have been established in animals with overt clinical
heart failure to evaluate pathophysiology from the level of the intact instrumented animal to
the tissue homogenate. These kind of studies provide a lot of information on hemodynamics,
neurohumoral activation, myocardial infarction and sub-cellular and molecular alterations
in the failing heart. There are great differences between species and models and only some
models mimic human heart failure in some aspects. Such studies seem to be currently less
important because with recent invasive and non-invasive technologies, hemodynamics
can be studied in-patients. Furthermore, with cardiac transplantation surgery, end stage
failing human myocardium became available for functional, biochemical and molecular
biology studies allowing the evaluation of alterations, which are present in end-stage failure
in the human heart itself. However, it is rather difficult or impossible to study myocardial
changes associated during compensated, less severe stages of CHF, during the transition
from hypertrophy to failure or during the process of remodeling. Therefore, in order to
study transition processes occurring in heart failure, animal models are very important.
Furthermore, animal models of heart failure may be relevant to study the effects of new
pharmacological strategies on hemodynamics, neurohumoral activation and survival. At
present, transgenic animal models of heart failure are critically important to understand
the molecular alterations underlying the development of the disease. Addition or deletion
of genes in transgenic mice together with miniaturized physiological techniques to evaluate
the resulting cardiac phenotypes allow the identification of genes that are causative for
heart failure and to evaluate molecular mechanisms responsible for the development and
progression of the disease.
318 Drug Screening Methods

In vitro models In vivo models

1. Isolated hamster cardiomyopathic heart Rat models
2. Isolated cat papillary muscle • Rat coronary ligation model
3. Ouabain binding • Rat aortic banding
• Dahl salt sensitive rats
• Spontaneous hypertensive rat
• Spontaneous hypertensive-heart failure rats (SH-HF)
Dog models
• Chronic rapid pacing
• Volume overload
• Coronary artery ligation and microembolization
Rabbit models of heart failure
• Volume and pressure overload
• Tachycardia pacing
• Doxorubicin cardiomyopathy
Guinea pig model
• Cardiac insufficiency
Syrian hamster
• Cardiomyopathic hamster
Genetic model
• Transgenic mice
New therapeutic targets in CHF
• Gene therapy
• Stem cells
• Devices and mechanical support

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28. Magid NM, Opio G, Wallerson DC, et al. Heart failure due to chronic experimental aortic
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KR, Power JM. Percutaneous cardiac recirculation-mediated gene transfer of an inhibitory
phospholamban peptide reverses advanced heart failure in large animals. J Am Coll Cardiol
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 cHAPTER

20
Antianginal Agents
Among the cardiovascular pathologies, ischemic heart disease is the leading cause of morbidity,
mortality as well as permanent premature disabilities. Ischemic heart disease occurs when
coronary blood flow is inadequate to supply the oxygen required by the heart. By far the most
frequent cause is atheromatous obstruction of the large coronary vessels (atherosclerosis
angina, classical angina). However, transient spasm of localized portions of these vessels,
which is usually associated with underlying atheromas, can also cause significant myocardial
ischemia and pain (angioplastic or variant angina).
Reperfusion of a previously ischemic heart is a standard clinical procedure. Even if
beneficial, reperfusion triggers an inflammatory response that contributes to the acute
extension of ischemic injury and later participates in the reparative processes of the damaged
myocardium.1 Occlusion of a major coronary artery in small rodents, followed or not followed
by reperfusion, has proven to be a good model to assess the relevance of pathophysiological
processes and drug effects in the setting of myocardial ischemia. Models involving reperfusion
appear to be particularly suitable to study the inflammatory response, which is much more
marked than with permanent ischemia. Ischemia/reperfusion of the myocardium in wild-
type and transgenic animals (mostly mice) allows the possibility of testing the vast array of
mediators that orchestrate the sequelae of oxidative stress and inflammation. Moreover, the
experimental models allow testing of the protective effects of drugs in experimental ischemia
and reperfusion injury.
Various animal models are available to screen anti-anginal drugs. Some of the widely
used models are discussed in this chapter. These animal models are useful for studying the
consequence of a myocardial ischemia and reperfusion on cardiac pathophysiological and
physiological functions.

In vitro models

1. Langendorff Heart Preparation

Langendorff is a highly reproducible preparation which can be studied quickly in large number
at relatively low cost. It allows measurement of broad spectrum of biochemical, physiological
and morphological indices. Measurements are made in absence of the confounding effects
of the organs. Both global and regional ischemia can be studied using this model. It allows
322 Drug Screening Methods

experiments to be continued in face of events (MI, arrhythmias) which would normally

jeopardize the survival of an in vivo experiment. However, it is a deteriorating preparation
though capable of study for several hours. The basic principle involved is that heart is
perfused in a retrograde direction from the aorta either at constant pressure or constant flow
with oxygenated saline solutions. Retrograde perfusion closes the aortic valves, just as in the
in situ heart during diastole. The perfusate is displaced through the coronary arteries flowing off
the coronary sinus and the opened right atrium. Parameters usually measured are contractile
force, coronary flow and cardiac rhythm.
Guinea pigs of either sex weighing 300 to 500 g are used for the study. They are sacrificed
by stunning. Diaphragm is assessed by transabdominal incision and cut carefully to expose
the thoracic cavity. Thorax is opened by bilateral incision along the lower margins of the last
to first ribs. Thoracic cage is reflected over the animal’s head exposing the heart. The heart is
cradled between fingers and lifted before incising the aorta, vena cava and pulmonary veins.
Immediately after excision, heart is dipped in cold perfusion solution (4oC to limit ischemic
injury during period between excision and restoration of vascular perfusion). The aorta is
located and cut below the point of division. A cannula is inserted into the aorta and tied and the
heart is perfused with oxygenated Ringer’s solution. The heart is transferred to a double wall
plexiglass perfusion apparatus maintained at 37oC.2 Oxygenated Ringer’s solution is perfused
at a constant pressure of 40 mmHg at a temperature of 37oC from a reservoir. A small steel hook
with a string is attached to the apex of the heart. Contractile force is measured isometrically by
a force transducer and recorded on a polygraph. Heart rate is measured through a chronometer
coupled to the polygraph. Drugs are injected into the perfusion medium.3 The anti-anginal
effect of the test drug is indicated by an increase in coronary blood flow. The incidence and
duration of ventricular fibrillation, coronary flow, inotropic state and K+ levels after treatment
with drug are compared with control.
This method is very useful for testing coronary vasodilator drugs. It has wide applications
in the fields of pharmacology and physiology. It is useful to study positive inotropic effects,
negative inotropic effects, coronary vasodilatory effect, calcium antagonism, effect on
potassium outflow induced by glycosides and determination of hypoxic damage. Metabolic
studies, arrhythmogenic, antiarrhythmic and antifibrillatory effects can also be assessed using
Langendorff method. Recently this model has been also used to study EDRF release from the
coronary vascular bed and electrophysiological evaluation of cardiovascular agents.

2. Isolated Rabbit Aorta Preparation

Aortic rings are used to evaluate the smooth muscle relaxant/contractile activity in this
method. Adding potassium chloride or norepinephrine to the organ bath containing slightly
modified Kreb’s bicarbonate buffer induces contraction of aorta rings.
Using an overdose of pentobarbital sodium, rabbits of either sex weighing 3 to 4 kg are
sacrificed. Thorax is opened by bilateral incision. The descending thoracic aorta is rapidly
removed and placed in Kreb’s bicarbonate buffer maintained at 37oC. Tissue is cleaned, fat
and connective tissue is carefully removed. Eight rings of 4-5 mm width are obtained and
each is mounted in 20 ml tissue bath containing Kreb’s solution. A stabilization period of 2 h is
allowed wherein the Kreb’s solution is frequently changed followed by stabilization period of
1 h. A tension of 1 g is maintained during these times. A sustained contraction is generated by
 Antianginal Agents 323

addition of KCl. Twenty minutes after addition of agonist, the test drug is added. The percent
relaxation reading is taken every 30 min after addition of the test drug. There is a 30 min time
interval between additions of different test drugs.4
Active tension is calculated for the tissue at time point just prior to the addition of the test
compound and also at the point 30 min after the addition of each concentration of the test
compound. ID50 and percentage relaxation caused by the test drug from the precontracted
level is calculated.5 Test drug with calcium channel blocking activity have a relaxing effect and
can be evaluated using this method.

3. Calcium Antagonism in Pitched Rat

This model can differentiate calcium entry blockers from other agents that do not directly
block entry of calcium.
Sprague Dawley rats (250 to 350 g) are anesthetized intraperitoneally with methohexitone
sodium (50 mg/kg). The trachea is cannulated. Thereafter the rats are pitched through one
orbit and immediately maintained on artificial respiration. The pithing rod is used as a
stimulating electrode and continuous electrical stimulation of the thoracic spinal cord with
squarewave pulses at supramaximal voltage (frequency 0.5 Hz and duration 0.5 ms) produces
a cardioaccelerator response. Only rats with a resulting tachycardia (100 beats/min) are
included for the study. The jugular vein is cannulated for administration of drugs and blood
pressure is recorded via carotid artery using a pressure transducer. In the femoral region, an
indifferent electrode is inserted subcutaneously.
When cardioaccelerator response is established for 3-5 min, calcium channel blockers and
β-blockers are administered. These test compounds dose dependently block tachycardia.6 The
level of tachycardia immediately prior to drug administration is taken as 100% and response to
drugs is expressed as percentage of predose tachycardia. ID50 is calculated and compared.

4. Relaxation of Bovine Coronary Artery

The relaxation caused by test compounds can be assayed using spiral strips from bovine
coronary artery. The tonus of coronary arteries can be regulated by eicosanoids. Prostacyclin
induces relaxation whereas thromboxane A2 causes contraction.
Beef hearts are obtained immediately after slaughtering. They are immersed in cold
oxygenated Kreb’s solution and immediately transported to the laboratory. The left descending
coronary artery is cut into spiral strips and suspended in a 4 ml organ bath under an initial
tension of 2 g and immersed in oxygenated Kreb’s bicarbonate solution at 37oC. The Kreb’s
solution contains a mixture of antagonists to inhibit actions from endogenous acetylcholine,
5-hydroxy-tryptamine, histamine or catecholamines. The strips are superfused with a solution
of test compound with oxygenated Kreb’s solution. Isometric contractions are recorded with
force-displacement transducers on a Grass polygraph. The strips are superfused with Kreb’s
solution three hours prior to the experiment. Standard compounds are 100 ng/ml PGE2
inducing contraction and 100 ng/ml PGI2 inducing pronounced relaxation.7,8
The maximal response to 100 ng/ml PGE2 or 100 ng/ml PGI2 is calculated and the relaxation
caused by the test compound is expressed as its percentage.
324 Drug Screening Methods

5. Coronary Artery Ligation in Isolated Rat Heart

Langendorff technique can also be used to produce regional ischemia by clamping the left
coronary artery close to its origin. After removal of the clip, changes in the reperfusion period
can be observed. Prevention of these symptoms is an indicator of the efficacy of the coronary
drugs.
Wistar rats of either sex weighing 280 to 300 g are sacrificed by decapitation. The hearts
are removed and dissected free from the epicardium and surrounding connective tissue.4
A cannula is introduced into the aorta from where the coronary vessels are perfused with
the non-circulated perfusion medium according to the Langendorff technique. In the
left ventricle a balloon closely fitting the ventricular cavity is placed and connected to an
artificial systemic circulation. The balloon is made of silicone material using a Teflon form.
The dimensions of the Teflon form are basically derived from casts of left ventricle of K+
arrested casts by injection of dental cement. During each heart beat the fluid volume pressed
from the balloon corresponding to the stroke volume of the heart, can be recorded by means
of a flowmeter probe and an integrator connected in series. The preload and afterload
are adjusted separately and the perfusate flow is recorded separately. For coronary artery
occlusion experiment, the isolated working hearts are perfused for 20 min with Kreb’s buffer
at 65 mmHg. Acute myocardial ischemia is produced by clamping the left coronary artery
close to its origin for 15 min. The clip is then opened and the changes during reperfusion
period are monitored for 30 min. Haemodynamic parameters like left ventricular pressure,
heart rate, cardiac output and coronary flow are measured.9 From the coronary effluent,
samples are taken for lactate dehydrogenase (LDH), creatine kinase (CK), glycogen, ATP
and lactate determinations. The test drug is given into the perfusion medium either before
occlusion or 5 min before reperfusion. The incidence and duration of ventricular fibrillation
after treatment with test drugs is compared with controls. Left ventricular pressure, left
ventricular dP/dt max, coronary flow and myocardial LDH, CPK, glycogen, ATP and lactate
are also measured.

6. Isolated Heart-Lung Preparation

The isolated heart–lung preparation of the dog has been used to study various physiological
and pharmacological processes. Now this model has also been established in rats.
Wistar rats (300 to 350 g) are anesthetized intraperitoneally with pentobarbitone sodium
(50 mg/kg). The trachea is cannulated and the animal is maintained on artificial respiration.
The chest cavity is opened and ice-cold saline is injected to arrest the heart. The aorta,
superior and inferior vena cava are cannulated. The heart-lung preparation is perfused
with Kreb’s-Ringer bicarbonate buffer (pH 7.4) containing rat RBC (hematocrit 25%). The
perfusate is pumped from the aorta and is passed through the pneumatic resistance and
collected in a reservoir maintained at 37oC. It is then returned to the inferior vena cava thus
perfusing only the heart and the lung. Test drug is administered into the perfusate 5 min after
start of experiment. Cardiac output is recorded with an electromagnetic blood flowmeter
and mean arterial pressure from the pneumatic resistance. With the help of a bioelectrical
amplifier heart rate is recorded. Hemodynamic data and recovery time of the test drug group
and control group (without any treatment) is compared using ANOVA and Kruskal-Wallis test
respectively.10
 Antianginal Agents 325

7. Plastic Casts Technique in Dogs

Coronary drug when administered for prolonged duration leads to increase in the number
and size of interarterial collaterals especially in pigs and dogs. Acute or gradual occlusion of
one of the major coronary branches may also stimulate development of collaterals. In order to
quantify the collaterals, the arterial coronary bed is filled with plastic. This provides with the
possibility to make the collaterals visible.
Dogs (10 to 15 kg) are intravenously anesthetized with pentobarbital sodium (30 mg/kg).
They are maintained on artificial respiration, chest cavity is opened and the heart is exposed.
The pericardium is removed and Ameroid cuffs are placed around the major coronary branches.
The plastic materials gradually swell and occlude the lumen within 3 to 4 weeks. The animals
are administered test drugs or placebo for 6 weeks and then sacrificed. After a recovery period
of 1 week, their hearts are removed and coronary bed flushed with saline. Liquid araldite is
filled in the bulbus aortae, the coronary, arterial and venous tree. Care is taken to maintain
the uniformity of the filling pressure, the filling time and viscosity of the filling material. After
polymerization is complete, the tissue is digested with 35 % KOH. Plastic casts from the drug
treated animals are compared with casts from the sham group (dogs subjected to the same
procedure without drug treatment). The ability of the test drug to increase the number and size
of collateral’s is evaluated.11

In vivo models
1. Occlusion of Coronary Artery
Compounds that reduce infarct size are studied using this model. Infarct size is studied
after proximal occlusion of the left anterior descending coronary artery in open chest dogs.
Nitro-blue tetrazolium chloride stain in myocardial sections are used to visualize infarct size
in coronary arteriograms made after injection of BaSO4 gelatin mass into the left coronary
ostium.
Dogs of either sex (30 kg) are used in this model. The animals are anesthetized with
pentobarbitone sodium (35 mg/kg, intraperitoneally) which is followed by its continuous
infusion at 4 mg/kg/h. Trachea is cannulated and the animal is maintained on artificial
respiration. Peripheral vein (saphenous vein) is cannulated for administration of test
compound. ECG is recorded continuously. Femoral vein is cannulated and connected to a
pressure transducer for measuring peripheral systolic and diastolic pressure. Left ventricular
end diastolic pressure, left ventricular pressure and heart rate are also measured using a Millar
microtip catheter (PC 350) inserted via the left coronary artery. Heart is exposed through a left
thoracotomy between 4th and 5th intercostal space. The pericardium is opened and the left
anterior descending coronary artery is exposed and then ligated for 360 min. Test substance
or vehicle is administered by intravenous bolus injection. Haemodynamic parameters are
monitored and at the end of the experiment, animals are sacrificed with an overdose of
pentobarbital sodium. Area at risk of infarction is measured using coronary arteriograms.
The left ventricle is cut into transverse sections. From each slice angiograms are made with
X-ray tube at 40 kv to assess the area at risk of infarction by defect opacity : reduction of
BaSO4 filled vessels in infarct tissue. The slices are then incubated in p-nitro-blue-tetrazolium
326 Drug Screening Methods

(0.25 g/l) in order to visualize the infarct tissue (blue/violet stained healthy tissue, unstained
necrotic tissue). The slices are photographed for determination of infarct area. Mortality,
haemodynamic parameters and infarct size are determined. Changes in parameters in drug
treated animals are compared to vehicle controls.12

2. Microspheres-induced Acute Ischemia

This model can be useful in evaluating the effect of test drugs on myocardial performance
during acute ischemic left ventricular failure. Microspheres (50 μm) when injected repeatedly
into the left main coronary artery may induce left ventricular failure in anesthetized dogs.
Hemodynamic parameters can be recorded and drugs tested on the basis of their improvement
of cardiac performance.
Dogs (30 kg) are anesthetized with pentobarbitone sodium intravenously (40 mg/kg) and
additionally administered a supplementary dose of 4 mg/kg/h. The trachea is cannulated
and the animal maintained on artificial respiration. The brachial vein is cannulated for
administration of analgesic and saphenous vein for administration of test compounds. ECG
is recorded continuously. The femoral artery is also cannulated and connected to a pressure
transducer for the measurement of systolic and diastolic pressure. A Millar microtip catheter
is inserted via the left coronary artery for the determination of left ventricular pressure while
left ventricular end diastolic pressure (LVEDP) is measured on a high sensitivity scale. From
the pressure curve, dP/dt and heart rate are calculated. Mean pulmonary capillary pressure,
mean pulmonary artery pressure (PAP) and cardiac output are measured using Cardiac
Index Computer and a balloon tip triple lumen catheter with the thermistor positioned in
the pulmonary artery via the jugular vein. Through a left thoracotomy, the heart is exposed.
Microspheres are injected through the angiogram catheter into the left ostium initially as
10 ml and later as 5 ml boluses about 5 min apart. Embolization is terminated when the LVEDP
increases to 16 to 18 mmHg and PAP to 20 mmHg and heart rate 200 beats/min. Test compound
is then administered by intravenous route and the above mentioned parameters recorded. In
addition to the directly measured hemodynamic parameters, stroke volume, tension index,
coronary vascular resistance, total peripheral resistance, pulmonary artery resistance can
also be measured. Changes of parameters in drug treated animals are compared to vehicle
controls. Also mean embolization time, doses of microspheres and numbers of microspheres
are evaluated.13

3. Isoproterenol-induced Myocardial Necrosis

Synthetic catecholamines like isoproterenol when injected at high dose produce cardiac
necrosis. Rona et al. have studied the infarct like lesions in the rat myocardium.14 Several drugs
such as sympatholytics or calcium antagonists can totally or partially prevent these lesions.
Wistar rats (150 to 200 g) are pretreated with test drug or standard drug orally or
subcutaneously for at least a week. These rats are then injected with 85 mg/kg isoproterenol
subcutaneously on two consecutive days. Mortality as well as symptoms are recorded in
each group and compared to group injected with isoproterenol only. After 48 h of first dose
of isoproterenol the animals are sacrificed. The heart is removed, weighed and preserved for
histological evaluation or processed for estimation of various biochemical parameters. Before
 Antianginal Agents 327

sacrificing, the animal’s hemodynamic parameters such as systolic/diastolic blood pressure

and heart rate can be recorded by cannulating the carotid artery and connecting it to a pressure
transducer. By inserting a cannula in the left ventricle, parameters such as left ventricular
end diastolic pressure (LVEDP) and dP/dt can be measured. The degree of histopathological
changes can be graded as follows:
Grade 0: No change
Grade 1: Focal areas of necrosis
Grade 2: Focal areas of necrosis and muscle fiber fragmentation
Grade 3: Confluent areas of necrosis, edema and inflammation and muscle fiber fragmentation
Grade 4: Massive areas of necrosis, edema and inflammation and mural thrombi.
Changes of parameters (histological, biochemical and hemodynamic) of drug treated
animals are compared to isoproterenol controls.15

4. Stenosis-induced Coronary Thrombosis Model

Thrombosis can be induced by stenosis in dogs. This model is characterized by alterations in
coronary blood flow with transient platelet aggregation at the site of coronary constriction.
Dogs (15 to 20 kg) are anesthetized with pentobarbitone sodium (30 to 40 mg/kg,
intraperitoneally) and then maintained on artificial respiration through a tracheal tube using
a positive pressure respirator. Through a left thoracotomy the heart is exposed at the 4th and
5th intercostal space and the pericardium is removed. An electromagnetic flowprobe is placed
on the proximal part of the left coronary artery to measure coronary blood flow. Distal to the
flowmeter, the vessel is clamped for 5 sec. A small plastic constrictor is placed around the artery
at the site of damage. The constrictor is changed several times until the required narrowing of
the coronary artery is achieved. In case the artery is occluded, the coronary artery is lifted
to induce reflow. Dogs with regular repeated cyclic flow variations of same intensity within a
pretreatment phase of 60 min are used for experimental purpose. Hemodynamic parameters
are recorded. Test compound is administered intravenously and the cyclic flow variations are
registered for 2 to 5 h and compared to pre-treatment values.
In case simple clamping of the coronary artery does not produce cyclic flow variations,
additionally adrenaline (0.2 µg/kg) is infused into the peripheral vein, 30 min before and
30 min following drug administration. Also platelet activating factor (PAF; 0.2 nmol/kg/min)
when infused for a similar duration as adrenaline into the cannulated lateral branch of the
coronary artery may produce cyclic flow variations. Cyclic flow variations are registered and
compared to the drug treated group.16

5. Electrical Stimulation-induced Coronary Thrombosis

Electrical stimulation can induce thrombosis in the coronary artery in pigs. An alteration in
coronary blood flow with transient platelet aggregation at the site of coronary constriction is
assessed using this model.
German landrace pigs (20 to 40 kg) are anesthetized with ketamine (2 mg/kg, intra–
muscularly), metomidate (10 mg/kg intraperitoneally) and xylazine (1-2 mg/kg intramuscularly)
and then maintained on artificial respiration through a tracheal tube using a positive pressure
respirator. Through a left thoracotomy the heart is exposed at the 4th and 5th intercostal space
328 Drug Screening Methods

and the pericardium is removed. A electromagnetic flowmeter is placed on the proximal part
of the left coronary artery to measure the coronary blood flow. A vanadium steel electrode
is placed in the vessel with the intimal lining and connected with the Teflon coated wire of
9-Volt battery, a potentiometer and an amperometer.17 To complete the electric circuit, a disc
electrode is placed on the thoracic muscle layer. The intima is stimulated with 150 µA for 6
h during which time an occluding thrombosis occurs. The test drug is administered either
subcutaneously with the electrical stimulation or 30 min following the electrical stimulation.
Hemodynamic parameters - systolic, diastolic, mean blood pressure and heart rate are
measured by cannulating the femoral artery and connecting it to a pressure transducer. Left
ventricular pressure, left ventricular end diastolic pressure, dP/dt are measured by inserting
a micro-tip catheter via the carotid artery retrogradely. ECG is also recorded using lead II.
The time interval until the thrombotic occlusion of the vessel occurs and the thrombus size
are determined. At the end of the experiment the animals are sacrificed with an overdose
of anesthesia. Percent change in mean values for occlusion time and thrombus size in drug
treated groups is compared to the control group. Also changes in hemodynamic parameters,
cyclic number and cycle area after drug treatment is compared to pre-treatment values.

6. Myocardial Ischemic Preconditioning Model

Myocardial preconditioning (brief duration of ischemia and reperfusion) can reduce the
damage produced by prolonged ischemia and reperfusion. Preliminary preconditioning of the
myocardium reduces infarct size, reduces leakage of cellular proteins indicative of myocyte
death, improves post-ischemic ventricular function, as well as attenuates cardiac arrhythmia
associated with frequent ischemia/reperfusion.
Rabbits (New Zealand, weighing 3 to 4 kg) are anesthetized with ketamine (50 mg/
ml)/xylazine (10 mg/ml) at a dose of 0.6 ml/kg. The trachea is cannulated and the animal
maintained on artificial respiration (30 inflations per min). The right femoral artery and
vein are catheterized for measurement of arterial pressure and administration of drugs
respectively. Hemodynamic parameters like systolic, diastolic, mean blood pressure, heart
rate, left ventricular pressure, left ventricular end diastolic pressure and dP/dt are measured.
A 4-0 suture is looped loosely around the marginal branch of left coronary artery to facilitate
coronary occlusion during the experiment. Ischemic preconditioning is induced by tightening
the loop around the coronary artery for 5 min and then loosening to reperfuse the myocardium
for 10 min prior to a subsequent 30 min occlusion. After 30 min ischemia, ligature is released for
120 min of reperfusion. Prior to 30 min of occlusion the rabbits are selected to receive ischemic
preconditioning, no preconditioning or preconditioning along with the administration of test
compound. The animals are sacrificed after the reperfusion duration. Comparisons between
systemic hemodynamic data and infarct size studies are analyzed by ANOVA using statistical
software.18

7. Models of Coronary Flow Measurement

These models are based on measurement of coronary outflow in open and closed chest animal
preparations. Various drugs can be screened for their anti-anginal potential, on the basis of
their coronary artery dilating properties.
 Antianginal Agents 329

I. Coronary Inflow Measurement in Anesthetized Dogs

Dogs (15 to 20 kg) are anesthetized with pentobarbitone sodium (30 to 40 mg/kg)
intraperitoneally and then maintained on artificial respiration using a positive pressure
respirator. Through a left thoracotomy, the heart is exposed at the 4th and 5th intercostal
space and the pericardium is removed. Through the jugular vein a catheter is inserted
to cannulate the coronary sinus. In the in-vitro studies cannula is inserted through an
opening in the atrial appendage into the coronary sinus and drained to measure coronary
flow. Haemodynamic parameters - systolic, diastolic, mean blood pressure and heart rate
are measured by cannulating the femoral artery and connecting it to a pressure transducer.
The test drug is administered through the other jugular vein. Changes in coronary flow and
haemodynamic parameters after test drug administration is compared to values before test
drug administration.
Advantage of this method is that approximately 95 % of the total coronary venous flow can be
measured.19 Disadvantages of this method are that only 60% of coronary flow returns through
coronary sinus. No constant proportion between coronary venous outflow and coronary sinus
flow is present.20 Another method with slight modification in the above mentioned model
could also be used to measure coronary blood inflow. In this method blood from superior vena
cava is diverted to pulmonary artery and flow from right ventricle is measured.

II. Coronary Outflow Measurement in Anesthetized Dogs

Various devices have been used through ages for measuring coronary flow, of which in-vogue
is electromagnetic flowmeter. It is used to measure:
A. Phasic flow: An additional probe is placed around the aorta to record changes in coronary
flow with changes in aortic pressure.
B. Mean flow: Average flow through coronary arteries per cardiac cycle.18
a. Electromagnetic Flowmeter
Dogs (10 to 12 kg) are anesthetized with pentobarbitone sodium (30 to 40 mg/kg,
intraperitoneally). Their trachea is cannulated and animals are maintained on artificial
respiration using a positive pressure respirator. Through the left thoracotomy, heart is
exposed at the 4th and 5th intercostal space and the pericardium is removed. Two poles of
electromagnet are placed in opposite sides of the coronary vessel. Distal to the electromagnets,
two chromium-vanadium electrodes are placed adhering to the coronary artery. A magnetic
field perpendicular to blood flow generates voltage in the conductor (blood stream). It is
picked up by electrodes, amplified and recorded. This method mostly records phasic flow.
Mean flow is recorded by electrical damping. Jugular vein is cannulated for the administration
of test compound and carotid artery for measurement of blood pressure. Changes in
coronary outflow and hemodynamic parameters before and after test drug administration
are compared.
To avoid polarization at pickup electrodes, magnetic current is reversed by oscillator either
of square wave or sine wave type. Initially probes were big but with advanced technology,
nowadays, small sized probes are available. They are used mainly in chronic unanesthetized
whole animal experiments by running lead wire through the skin.21, 22
330 Drug Screening Methods

III. Other Models to Measure Coronary Flow

The following methods can be employed to measure coronary flow:
a. Inert gas technique
Mainly helium or nitrous oxide is used. A mixture of room air and inert gas is inhaled (known
quantity). A series of blood samples are withdrawn simultaneously from a peripheral artery
(using needle) and coronary sinus/cardiac vein (using catheter). A-V difference is calculated.
A-V difference is the difference between the integrals of arterial and coronary sinus.20 Blood
flow through the organ/time is calculated as:
Amount of substance taken up in unit time
A-V difference
It can measure only mean flow but not regional flow.23 It takes around 10 min for one
determination.
b. Radioactive technique
The radioisotopes mainly used are 121I, 3H and rubidium. Isotopes are inhaled/injected and
change in rate over chest wall is measured using giega counter. By appropriate calculations
a measure of coronary flow can be determined. It has a close co-relation with the above-
described method. It is a fast and simple technique.24
c. Radioactive microsphere technique
This method determines regional blood flow including distribution of coronary flow across
the ventricular wall. A batch of radioactive microspheres (9-15 µDM) is suspended in a
saline detergent solution and injected into the left atrium. Microspheres lodge in only a few
capillaries, so no damage/effect on flow is observed.
The number of spheres trapped/unit of myocardial tissue is directly proportional to
myocardial blood flow.25
d. Thermodilution technique
A catheter having an end hole is passed to the beginning of the coronary sinus. A temperature
sensor (thermometer) is placed further down the coronary sinus. Cold saline of known
temperature is injected continuously through catheter-diluted by coronary sinus blood flow.
Modified temperature is measured by thermometer. The temperature difference obtained is
proportional to the blood flow.26,27
e. Coronary arteriography
Radio-opaque solutions are injected by a catheter into a coronary artery at the root of aorta.
With high speed cinematography clear visualization of coronary circulation before and after
drug administration is done. This technique is the most direct, reliable and advanced method.28
Angina is extremely variable pain syndrome with no anatomical/pathophysiological entity.
There is no direct relation between degree of pain and coronary insufficiency. Small areas of
ischemia can produce discomfort equal to that of large area ischemia.29 Also, all antianginal
drugs must not be assumed to be coronary vasodilators nor all coronary vasodilators always
relieve angina. So, any compound screened positively for angina has to be tested cautiously in
a well-planned and controlled clinical trial.
 Antianginal Agents 331

Conclusion
In the study of cardiovascular biology, both in healthy and disease conditions, there is a vast
spectrum of measurable indices of function and injury. This is particularly so in the case of
myocardial ischemia, a disease, which contributes to the majority of deaths in both the
developing and developed countries. Each experimental model, each species and each end
point has its own inherent advantages and disadvantages and appreciating these will help the
investigator select the most appropriate study system for the particular investigation under
question.
The key feature of the in vitro isolated animal hearts is that global or regional ischemia
and reperfusion can be imposed at will and the contractile, biochemical, physiological
and morphological consequences can be easily assessed. Furthermore, various degrees of
ischemia from zero flow to low flow can be induced and the rate and nature of reperfusion
can be manipulated. On the negative side, in vitro preparations have a limited laboratory life
span that rarely exceeds a few hours; they deteriorate progressively with time and cannot
be used for chronic studies. Furthermore, they are deprived of their normal central neural
connections; they are isolated from the systemic circulation and are no longer exposed to the
host of peripheral neurohormonal factors. The in vivo preparations allow measurement of
hemodynamic functions such as ECG, ventricular wall motion, and ejection fraction that are
of major diagnostic importance. They also provide scope for biochemical, pharmacological,
morphological and physiological study. Markers of cardiovascular injury such as lactate,
cytokines, catecholamines, creatine kinase or troponin–T from the peripheral circulation can
be analyzed.
Nowadays, techniques such as Nuclear Magnetic Resonance (NMR) and Positron Emission
Tomography (PET) have increased our ability to study in a non-invasive manner, some
aspects of cardiac metabolism, function and coronary flow. However, invasive catheterization
procedures will allow the collection of cardiac biopsies, measurement of arteriovenous
difference and more sophisticated electrophysiological recording.
Summary
In vitro models In vivo models
1. Langendorff heart preparation 1. Occlusion of coronary artery
2. Isolated rabbit aorta preparation 2. Microspheres induced acute ischemia
3. Calcium antagonism in pitched rat 3. Isoproterenol induced myocardial necrosis
4. Relaxation of bovine coronary artery 4. Stenosis induced coronary thrombosis model
5. Coronary artery ligation in isolated rat heart 5. Electrical stimulation induced coronary thrombosis
6. Isolated heart-lung preparation 6. Myocardial ischemic preconditioning model
7. Plastic casts technique in dogs 7. Models of coronary flow measurement
• Coronary inflow measurement in anesthetized dogs
• Coronary outflow measurement in anesthetized dogs
• Other models to measure coronary flow:
– Inert gas technique
– Radioactive technique
– Radioactive microsphere technique
– Thermodilution technique
– Coronary arteriography
332 Drug Screening Methods

References
1. Chimenti S, Carlo E, Masson S, Bai A, Latini R. Myocardial infarction: animal models. Methods Mol
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2. Balderston SM, Johnson KE, Reiter MJ. Electrophysiological evaluation of cardiovascular agents in
the isolated intact rabbit heart. J Pharmacol Methods 1991;25:205-13.
3. Burn JH, Hukovic S. Anoxia and ventricular fibrillation: With a summary of evidence on the cause of
fibrillation. Br J Pharmacol 1960;15:67-70.
4. Hof RP, Vuorela HJ. Assessing calcium antagonism on vascular smooth muscle: comparison of three
methods. J Pharmacol Methods 1983;9:41-52.
5. Matsuo K, Morita S, Uchida MK, et al. Simple and specific assessment of Ca2+ entry blocking
activities of drugs by measurement of Ca reversal. J Pharmacol Methods 1989;22:265-75.
6. Clapham JC. A method for in vivo assessment of calcium slow channel blocking drugs. J Cardiovasc
Pharmacol 1988;11:56-60.
7. Dusting GJ, Moncada S, Vane JR. Prostacyclin (PGX) is the endogenous metabolite responsible for
relaxation of coronary arteries induced by arachidonic acid. Prostaglandins 1977;13:3-15.
8. Gilmore N, Vane JR, Wyllie JH. Prostaglandins released by the spleen. Nature 1968;218:1135-40.
9. Linz W, Scholkens BA, Manwen J, et al. The heart as a target for converting enzyme inhibitors:
Studies in ischemic isolated working hearts. J Hypertension 1986;4:477-79.
10. Carpi A, Oliverio A. Effect of reserpine on the heart lung preparation of guinea pig. Arch Int
Pharmacodyn Ther 1965;157:470-86.
11. Boor PJ, Reynolds ES. A simple planimetric method for determination of left ventricular mass and
necrotic myocardial mass in postmortem hearts. Am J Clin Pathol 1977;68:387-92.
12. Gomoll AW, Lekich RF. Use of ferret for a myocardial ischemia/salvage model. J Pharmacol Methods
1990;23:213-23.
13. Smiseth OA, Mjos OD. A reproducible and stable model of acute ischemic left ventricular failure in
dogs. Clin Physiol 1982;2:225-39.
14. Rona G, Chappel CI, Balazs T. An infarct like myocardial lesion and other toxic manifestations
produced by isoproterenol in rat. Arch Path 1959;67:443-55.
15. Kannengiesser GJ, Lubbe WF, Opie LH. Experimental myocardial infarction with left ventricular
failure in the isolated perfused rat heart. Effects of isoproterenol and pacing. J Mol Cell Cardiol
1975;7:135-51.
16. Al-Wathiqui MH, Hartman JC, Brooks HL, et al. Induction of cyclic flow reduction in the coronary,
carotid and femoral arteries of conscious, chronically instrumented dogs. A model for investigating
the role of platelets in severely constricted arteries. J Pharmacol Methods 1988;20:85-92.
17. Kingaby RO, Lab MJ, Cole AW, et al. Relation between monophasic action potential duration,
ST segment elevation and regional myocardial blood flow after coronary occlusion in the pig.
Cardiovasc Res 1986;20:740-51.
18. Mickelson JK, Simpson PJ, Lucchesi BR. Streptokinase improves reperfusion blood flow after
coronary artery occlusion. Int J Cardiol 1989;23:373-84.
19. Matsuo H, Watanabe S, Kadosaki T, et al. Validation of collateral fractional flow reserve by myocardial
perfusion imaging. Circulation 2002;105:1060-65.
20. Antoniucci D, Valenti R, Moschi G, et al. Relation between preintervention angiographic evidence
of coronary collateral circulation and clinical and angiographic outcomes after primary angioplasty
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21. Lu TM, Hsu NW, Chen YH, et al. Pulsatility of ascending aorta and restenosis after coronary
angioplasty in patients >60 years of age with stable angina pectoris. Am J Cardiol 2001;88:964-68.
22. Carlier SG, Cespedes EI, Li W, et al. Blood flow assessment with intravascular ultrasound catheters:
the ideal tool for simultaneous assessment of the coronary haemodynamics and vessel wall? Semin
Interv Cardiol 1998;3:21-29.
23. Ohte N, Nakano S, Hashimoto T, et al. Noninvasive evaluation of left ventricular function using
new systolic time intervals obtained from continuous-wave Doppler echocardiography. J Cardiol
1990;20:457-64.
24. Bregman D, Kaskel P. Advances in percutaneous intra-aortic balloon pumping.
Crit Care Clin 1986;2:221-36.
25. Parker JO, West RO, Di Giorgi S. The effect of nitroglycerine on coronary blood flow and the
hemodynamic response to exercise in coronary artery disease. Am J Cardiol 1971;27:59-65.
26. Santomauro M, Cuocolo A, Celentano L, et al. Diagnosis of coronary artery disease with Tc99m-
methoxy isobutyl isonitrile and transesophageal pacing. Angiology 1992;43:818-25.
27. Parodi O, Marzullo P, Neglia D, et al. Clinical application of monitoring techniques: radioisotopic
methods. Can J Cardiol 1986; Suppl A: 155A-62A.
28. Biagioli B, Borrelli E, Maccherini M, et al. Reduction of oxidative stress does not affect recovery
of myocardial function: warm continuous versus cold intermittent blood cardioplegia. Heart
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a thermodilution technique. Cardiovasc Res 1990;24:418-22.
 cHAPTER

21
Antiplatelet Agents
Introduction
Platelets were discovered by G. Bizzozero in 1882, but drug industry did not recognize them
as viable drug targets till 1960s, after many decades of oblivion. This happened due to the
progressive recognition of the role of platelets in physiopathologic and clinical conditions such
as inflammation, cancer growth and dissemination, and organ transplant rejection. Initially,
the interest of many experts was in the role of platelets in the process of blood coagulation and
they were faced with a serious unresolved problem of the normal clotting time even in severe
thrombocytopenia. However, during the 1960s, focus shifted to the interaction of platelets with
the vascular wall (adhesion) and each other (aggregation). Platelet adhesion and aggregation
are central events in hemostasis and the pathophysiology of thrombosis. In 1968, aspirin
found that inhibits platelet aggregation and recognized by the FDA (USA) in 1988. Projects for
developing antiplatelet drugs are initiated worldwide.

ANTIPLATELET AGENTS
An antiplatelet drug is a member of a class of pharmaceuticals that decreases platelet
aggregation and inhibits thrombus formation (Table 21.1). They are effective in the arterial
circulation, where anticoagulants have little effect. They are widely used in primary and
secondary prevention of thrombotic cerebrovascular or cardiovascular diseases.

Table 21.1: Classification of antiplatelet drugs

Categories Drugs
Cyclooxygenase inhibitors Aspirin
Adenosine diphosphate (ADP) receptor inhibitor Clopidogrel, Ticlopidine
Phosphodiesterase inhibitors Cilostazol
Glycoprotein IIB/IIIA inhibitors Abciximab, Eptifibatide, Tirofiban
Adenosine reuptake inhibitors Dipyridamole
Flavonoids Rutin
 Antiplatelet Agents 335

Importance of Antiplatelet Drugs

Uncontrolled deposition of platelets on thrombogenic surfaces may lead to the occlusion of
vessels, a condition associated with numerous pathophysiological changes, such as acute
myocardial infarction and unstable angina, or the ischemic complications of coronary
intervention and stroke.1,2 The contribution of platelets to these disease processes stems from
their ability to form aggregates or platelet thrombi, as a consequence of arterial wall injury. Injury
of blood vessel walls could occur either acutely or chronically by various pathophysiological
processes. Platelets are then activated by a number of activators or agonists that are released
from the platelets or from the injured arterial walls, with subsequent adherence, aggregation
to the disrupted vessel surface, and resultant formation of an occlusive thrombus in the lumen
of the vessel. Platelet aggregation may be stimulated by collagen, thrombin, thromboxane
A2, serotonin, adenosine diphosphate (ADP), platelet activating factor (PAF), epinephrine,
or most probably by a combination of these factors. These agents are used to induce platelet
aggregation in the drug screening methods that are discussed below. Each antiplatelet drug
has its own mechanism of antiplatelet action.
Studies have shown that antiplatelet agents can prevent development of thrombotic
disorders such as myocardial infarction and peripheral vascular diseases.3 Inhibitors of
aggregation can provide protection against these symptoms that affect millions of people
worldwide. Acetylsalicylic acid (aspirin) is one such inhibitor. The chances of a second heart
attack can be reduced by as much as 40% by taking aspirin daily.4 Increasing the level of natural
platelet inhibitors in the diet may also reduce the risk of developing cardiovascular disorders
mediated by platelet aggregation.

Targets for Newer Drugs

Understanding the mechanisms underlying activation, adhesion and aggregation of platelets, is
the key to develop newer antiplatelet drugs. This fact is beautifully illustrated by the widespread
clinical use of cyclooxygenase inhibitors, most notably aspirin, as antiplatelet drugs. This was
only possible after arachidonic acid metabolism in platelets was fully characterized, which
has been shown to be responsible for platelet aggregation. The explosion of cell and molecular
biology approaches in the last two decades have led to the discovery and characterization of
platelet receptors such as the adenosine diphosphate (ADP) receptor, glycoprotein IIb/IIIa,
glycoprotein Ib/IX receptors and prostanoid receptors.5
During the last decade, vast information regarding the function of the platelet and about
the key role of von Willebrand factor (vWF) interaction with platelet glycoprotein (GP) Ib in
the initial contact adhesion of platelets to exposed subendothelium, has been gathered. This
interaction leads to the final common pathway for all agonist-induced platelet aggregate
formation, initiated by activation of the platelet glycoprotein GP IIb/IIIa and linking of
adjacent platelets by fibrinogen bound to the receptor GP IIb/IIIa.6,7 This binding of fibrinogen
is mediated in part by the RGD recognition sequence, which is common to other adhesive
proteins that bind to GP IIb/IIIa receptors.8
The glycoprotein GP IIb/IIIa belongs to the integrin superfamily of adhesive proteins and
is the most abundant platelet cell surface protein. A normal platelet contains approximately
336 Drug Screening Methods

50,000 receptor complexes, which bind like several other integrins to an Arg-Gly-Asp-Ser
(RGDS) tetrapeptide recognition sequence.9 Despite the common motif, integrins are quite
specific in their interaction with different adhesive proteins such as fibrinogen, vitronectin,
fibronectin, laminin, collagen, or vWF.
Agonist activation causes a morphological change in platelets, placing the GP IIb/IIIa
receptors in a conformation having a high affinity for the binding of fibrinogen. The binding
of fibrinogen to the activated form of GP IIb/IIIa is both a necessary and sufficient event that
can mediate the process of platelet aggregation.7 vWF is a disulfide-linked multimeric protein
composed of identical 275 kDa subunits and mediates the adhesion of platelets to the injured
vessel walls by binding both GP Ib and subendotheliums exposed on the injured vessel wall.
It is known that some conformational changes in the structure of vWF are necessary for the
binding of vWF to GP Ib, and it is likely that in vivo these conformational changes are achieved
by its binding to exposed subendotheliums, particularly under conditions of high shear
stress.10 Therefore, blocking of GP IIb/IIIa offers a superior approach in effectively preventing
arterial thrombosis. Further studies in the platelet signaling pathway and the receptor pathway,
coupled with the development of new techniques for studying platelet function, can lead to
many new classes of platelet-inhibiting drugs.
Current antiplatelet drugs are mainly effective against one of the many platelet activators.
Drugs, such as aspirin and ticlopidine, inhibit only one agonistic pathway and are therefore of
limited efficacy. Since, the specific inhibition of a single agonistic pathway leaves alternative
routes to platelet activation unaffected and is thus of limited effectiveness in preventing
thrombus formation. Antagonism of GP IIb/IIIa with an orally acting agent represents an
attractive therapy for chronic treatment of arterial thrombosis. In addition, because no oral GP
IIb/IIIa inhibitor exists, there is room for developing oral regimens. Efficacy of new drugs can
be screened in various in vitro and in vivo models of platelet aggregation.

In vitro Screening Methods

Selection of Volunteer Blood Donors
It is axiomatic that only normal healthy volunteers should be used for preparing platelets or
suspension for drug testing. The healthy volunteers selected should not have clinical signal
of von Willebrand‘s disease or symptoms compatible with primary hemostasis disorders or
blood dyscrasias; they are advised not to take aspirin or other nonsteroidal anti-inflammatory
drugs (NSAIDs) for 2 weeks before the day of the experiment.
Blood samples are mixed with 1/10 volume of anticoagulant 3.8% sodium citrate during the
screening of antiplatelet drugs. Other anticoagulants, such as heparin, EDTA cannot be used
as they affect the second wave of aggregation caused by the response to ADP and adrenaline.

Preparation of Platelets and Platelet Aggregation Assays

A typical aggregometer is basically a simple photometer consisting of a light source; usually
white light from a low voltage, DC tungsten filament and a photoelectric cell to receive the
light beam from passage through the sample. Fresh blood from male rabbit is collected into
plastic tubes containing anticoagulant dextrose solution and subsequently centrifuged at 250 g
 Antiplatelet Agents 337

for 10 min to obtain platelet-rich plasma (PRP). PRP samples are stirred at a sufficient speed
to maintain platelets in an even suspension and to provide the platelet-to-platelet contact
necessary for aggregation to proceed. High speed stirring may traumatize platelets sufficiently
to induce release and spontaneous aggregation. Maximal interference with light transmission
occurs when platelets are evenly distributed throughout the suspending medium. The levels
of light transmission are calibrated as 0% for a platelet suspension and 100% for the Tyrode/
HEPES solution. PRP is obtained by plateletpheresis of plasma centrifuged at 120 g for 15 min.
Platelet aggregation is performed according to the turbidometric method of Born.11 Test
drug at concentrations ranging from 1 to 100 µM is incubated with 0.25 ml of PRP for 1 min,
followed by activation of the platelet with various agonists. Citrated human PRP is warmed for
5 min at 37°C and then aggregation induced by ADP (100 µM), collagen or thrombin (0.8-6.4
unit/ml). The inhibition of aggregation is expressed as the percentage of the maximal rate of
aggregation observed in the absence of antagonists.10

Washed Platelets Method

Washed platelets are prepared by the method of Rho, et al.12 and platelet aggregation is
determined by a standard turbidometric method10 using an aggregometer. Platelets (300 µl)
are added to tyrode-BSA (0-35% albumin and apyrase, pH 7.35) in the aggregometer and
stirred at 1200 rpm at 37°C for 1 min before addition of the aggregating agents. The platelet
count is adjusted to 2 × 108 cells/ml. Platelet aggregation is expressed as an increase in light
transmission. Platelet suspension (0.3 ml) in the aggregometer cuvette is preincubated for 5
min at 37°C under continuous stirring at 100 rpm, and then CaCl2 or EDTA is added to a final
concentration of 1 mM. After 5 min, aggregating agent (ADP or collagen) was added and platelet
aggregation monitored for 10 min. Various concentrations of test drug were preincubated for
5 min before addition of aggregating agent (ADP or collagen) and platelet aggregation was
monitored for 20 min.
The effective concentration (EC 100 g/ml) which produces a 100% inhibition of platelet
aggregation (rabbit platelet-rich plasma) induced by arachidonic acid is considered as
pharmacological response by the test drug.4 This relatively simple method for determining the
platelet aggregation inhibitory activity and compounds giving positive results in this assay also
shows interesting properties ex vivo.

Whole Blood Aggregometry

In vitro platelet aggregation is measured with a whole blood electrical impedance aggregometer
(Chrono-log Corp.). There is a change in the impedance of whole blood, which is proportional
to the amount of platelet aggregation occurring in the cuvette. Vascular injury shown necessary
for platelet aggregation in the above discussion is achieved by exposing the blood to high shear
flow and subendothelial components.
Procedure: Blood is withdrawn from healthy human donors by venipuncture by a 21-gauge
needle into a syringe containing the anticoagulant sodium citrate (1 volume of 3.8% sodium
citrate to 9 volumes of blood) and is mixed gently. A minimum of two measurements is
performed using the blood from the same donor for each condition. An equal volume of Tris-
buffered saline (TBS; 10 mM Tris, pH 7.4, and 150 mM NaCl) is added to the blood and mixed
338 Drug Screening Methods

by inversion. The blood is stored at 20-22°C during the experiment and used within 4 hr of
venipuncture.
One ml of blood/TBS is transferred to cuvettes containing a stirbar. After incubation at
37°C for 3 min, the potential new drug is added (final concentration in whole blood is used).
Aspirin is used as positive control. The platelet inhibitory effect of acetylsalicylic acid (aspirin)
is measured at 0.36 mM, which is the approximate concentration in the blood of a 70 kg human
following a dosage of two 325 mg tablets of aspirin, assuming complete absorption into the
circulation. Aspirin is dissolved in 95% ethyl alcohol prior to dilution and then diluted in TBS so
that the final level of ethyl alcohol in whole blood is 0.0475%. Platelet aggregation is compared
to nonaspirin controls containing an equal amount of ethyl alcohol. Cuvettes are incubated at
37°C for further 4 min. The aggregometer electrodes are then inserted into the blood mixture.
Platelet aggregation is induced by adding 5 g/ml collagen. Change in electrical impedance
between the electrodes is then recorded over 7 min with stirring. The change in impedance at
6 min is used for all calculations. The test drug and the control drug aspirin both are evaluated
four times with each donor’s blood.

Preparation of Gel-filtered Platelets

Human blood samples are obtained by venipuncture from drug-free, normal male volunteers
and treated with 0.1 ml volume of 3.8% trisodium citrate. PRP is prepared by centrifugation of
the blood at 150 g for 10 min at room temperature. Gel-filtered platelets are prepared from PRP
by chromatography on a Sepharose 2B (Pharmacia, Sweden) column using modified Tyrode’s
buffer (138 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 12 mM NaHCO3, 2 mM MgCl2, 10 mM
HEPES, pH 7.4) containing 3.5% bovine serum albumin and 2% glucose for elution. Platelets
are counted electronically with a cell counter (CELLTAC MEK-5158, Nihon Kohden, Japan).
The final platelet concentration is adjusted to about 3 × 108/ml.

Preparation of Fixed Platelets

Gel-filtered platelets can be stimulated by 20 µM ADP for 10 min. They are then fixed by
treatment with 0.8% paraformaldehyde for 30 min at room temperature. The platelets are
washed three times with modified Tyrode’s buffer.10

Platelet Adhesion
Six hundred µl of test drug is added to 2.4 ml of blood. Tris buffer used as control. The drugs are
incubated with the blood at room temperature for 5-10 min. After the incubation period, the
blood is gently swirled and collected in a 5 ml syringe. The blood is then passed through a bead
column at a rate of 1 ml/min. The bead columns are composed of a 2.5 ml disposable plastic
syringe filled with 5 g silicone-coated glass beads of diameter 0.45-0.50 mm. Platelet counts are
performed in triplicate, after the passage through beads and percent adhesion is calculated by
comparing the average of these counts with those performed before bead passage. The results
with drugs are compared to those obtained with Tris buffer alone.
Cazenave’s group devised a method for evaluating platelet adhesion to glass tubes
coated with acid-soluble collagen.13 The technique utilizes washed, radiolabeled platelets
in suspension and a platelet count of 700 × 103/min. 50 µl of drug solution in Tris buffer is
 Antiplatelet Agents 339

added to 1 ml of PRP, mixed, allowed to stand for 5-10 min at 20°C. The tubes are covered with
parafilm and rotated end-over-end at 15 rpm for 10 min at room temperature. Adherence is
assessed by computing the loss of radioactivity to the collagen surface. If negligible release of
labels to the suspending medium occurs, it indicates little or no aggregation.
Platelet Function Analyzer (PFA): Through a 21-gauge cannula inserted into an antecubital
vein, a 3 ml blood sample is drawn into a plastic syringe (monovette) containing a buffered
citrate solution. Blood samples are stored at room temperature. The PFA-100® (Dade Behring,
Düdingen, Switzerland) is composed of a microprocessor-controlled device and single-use
test cartridges. An 800 µl sample containing saline or test drug (3 µM) is placed into a test
cartridge system for in vitro quantitative assessment of platelet function.11 The test cartridges
simulate an injured blood vessel, which consist of a sample reservoir, a capillary and a
membrane coated with 2 mg equine type I collagen and either 10 mg epinephrine bitartrate
(EPI cartridge) or 50 mg adenosine 5’-diphosphate (ADP cartridge). Blood is pipetted into the
reservoir and aspirated through a capillary with a diameter of 200 µm with constant negative
pressure thereby simulating high physiological shear forces (5000-6000 s-1). The capillary ends
in a membrane aperture with a diameter of 150 µm. Platelets adhere at the aperture where
they are activated by the collagen and then aggregate. The two agonist’s epinephrine and
ADP enhance aggregation. Finally, a platelet plug occludes the aperture and blood flow stops.
The time measured in seconds from the beginning of the test until formation of an occluding
platelet plug is called closure time (CT), which is a measure of the overall function of the
platelets. If an occluding platelet plug does not form after 300 sec, the analysis is stopped.

Microchannel Array Flow Analyzer (MC-FAN)

It is an in vitro screening system for platelet aggregation by monitoring aggregation and
micro-thrombi formation in the microchannels.14 In this method, whole blood is collected
from the ante-cubital vein of healthy volunteers with no known past medical history including
thrombotic disorders or hyperlipidemia. One ml of citrated whole blood is incubated with 10
μl of the indicated concentrations of platelet activation agents or phosphate-buffered saline
(PBS) as a control for adjusting the hematocrit. The final concentrations of platelet aggregation
triggers in the experiments are as follows: ADP (0–2 μM), collagen (0–100 μg/ml), and ristocetin
(0–0.75 mg/ml). Siliconized microchannels are used to evaluate blood coagulation which
consists of arrays of microgrooves formed on the surface of a single-crystal silicon substrate
and were converted to peak-proof micro-channels by covering them with an optically flat glass
plate.
The sample to be tested is transferred into a cylinder graduated in 10 μl units connected to
the inlet of the chip holder from which it flowed through the micro-channels when a solenoid
valve connecting the outlet of the holder was opened by a negative pressure is 20 cm H2O.
To determine the effect of anti-platelet agents or anticoagulants on whole blood flow rate in
the MC-FAN assay, 1 ml of citrated whole blood was incubated at 37°C and treated with 40 μl of
250 mM CaCl2, platelet activation agent and anti-platelet agent or anticoagulants. Immediately
following mixing of 500 μl of the whole blood, a sample is transferred into the cylinder of the
MC-FAN. The transit time through the cylinder of successive 10 μl aliquots of the total sample
of 100 μl, varying from 30 sec for the control to ~5 min for the highest activated samples is
340 Drug Screening Methods

determined. The average transit time through the chamber is used to determine the mean
blood flow rate.
To determine the effects of anti-platelet agents and anticoagulants on the blood flow
rate of ADP stimulated whole blood, 10 μl of the agents were added to 1 ml of whole blood,
incubated for 15 min, stimulated with 2 μM ADP, and the flow rate immediately determined
in the MC-FAN. The final concentrations of anti-platelet agents or anticoagulants used in the
experiments were as follows: FK633: 0.1–10 ng/ml, cilostazol, dilazep and sarpogrelate: 1–100
μg/ml, heparin and hirudin: 0.1–10 U/ml, APC and sTM: 0.1–1 μg/ml.
A chronolog platelet aggregometer is used to measure the decrease in optical density
accompanying aggregation.
After the induction of thrombus formation in the presence or absence of anti-platelet agents
or anticoagulants, the siliconized MC-FAN chip was fixed with 2.5% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4. The microchip is washed with phosphate buffer and re-fixed with
1% OsO4 in 0.1 M phosphate buffer. Adherent cells were dehydrated and dried, mounted on
scanning electron microscopy stubs and coated with platinum/palladium. Stubs were stored
under desiccation and analyzed with a scanning electron microscope at 15 kV accelerating
voltage.

Ex Vivo
Acid soluble collagen (100 µg/kg) is injected into the aortic arch of rabbits via the carotid
artery and serial platelet counts performed on blood withdrawn subsequent to collagen
administration. The test drug is administered to the animal before the collagen injection.

In vivo Screening Methods

Investigation of new drugs in the in vivo situation to be used in human disease states,
which have a protracted natural history and/or are multifactorial, is more complex and a
very frustrating affair. Nowhere is this brutal fact better appreciated than in the search for
antiplatelet drugs. Various artificial means are used to induce the platelet activity in vivo. It
usually involves application to an exposed portion of blood vessels to some form of insult
(mechanical, surgical, electrical, photochemical). In all cases, the experiments are either acute
or subacute in nature and, therefore, vulnerable to criticism from various fronts. Apart from the
obvious species differences, these animal models may not properly reproduce the sequence
of events of the disease as it occurs in the clinical situation. Nevertheless, there appears to be
sufficient universality in the response of blood platelets to damaged vascular endothelium,
which supports these models as useful experimental tools.

Model of Carotid Artery Occlusion in Dogs

A model of electrolytic injury-induced carotid artery occlusive thrombus formation is used.15
The experimental procedure results in the formation of a platelet-rich intravascular thrombus
along with a few erythrocytes and a rough fibrous coating at the site of an endothelial lesion
induced by electrolytic injury in proximity to distal arterial stenosis. The carotid artery response
to electrolytic injury is similar to that observed in the canine coronary artery. In the case of
 Antiplatelet Agents 341

evaluating intravenous antithrombotic efficacy of a test agent, we can use the right (control)
and left (intravenous treatment) carotid arteries or vice versa to establish time to occlusion
(min) and thrombus weight (milligrams) before and after the intravenous administration of a
test agent (i.e., the same animal can serve as control).

Surgical Preparation
Male, mongrel dogs (weight, 15 to 17 kg) are anesthetized (n = 12 for control, n = 5 to 6 for
the different oral dose levels of test substance) with sodium pentobarbital (30 mg/kg IV),
intubated, and allowed to breathe room air. Both common carotid arteries and the right
internal jugular vein are exposed. A catheter is inserted into the jugular vein for blood
sampling and administration of the test drug. Arterial blood pressure is monitored from the
cannulated femoral artery with the use of a blood pressure transducer. Standard limb lead II
of the electrocardiogram is recorded continuously. A Doppler flow probe is placed on each
common carotid artery proximal to both the point of insertion of the intra-arterial electrode
and the mechanical constrictor. The mechanical constrictor is constructed of stainless steel
in a C shape with a polytetrafluoroethylene (Teflon) screw (2-mm diameter) that could be
adjusted to control vessel circumference and produce a regional stenosis. The constrictor is
adjusted until the pulsatile flow pattern is reduced by 50% without altering the mean blood
flow. Blood flow in the carotid vessels is monitored continuously.
Electrolytic injury to the intimal surface of each carotid vessel is accomplished with the
use of an intravascular electrode composed of a Teflon-insulated, silver-coated copper wire.
Penetration of the vessel wall by the electrode is facilitated by attaching the tip of a 25-gauge
hypodermic needle to the uninsulated part of the electrode. Each intra-arterial electrode is
connected to the positive pole (anode) of a dual-channel stimulator. The cathode is connected
to a distant subcutaneous site. The current delivered to each vessel is monitored continuously
on a separate ammeter and maintained at 300 micro A. The anodal electrode is positioned to
have the uninsulated portion in intimate contact with the endothelial surface of the vessel.
Positioning of the electrodes in each of the carotid arteries is confirmed by visual inspection at
the end of each experiment.

Protocol: Prevention of Thrombus Formation

The anodal current is applied for a maximum period of 3 h or is terminated 30 min after
blood flow in the involved vessel remains stable at zero flow velocity to verify the formation
of a stable occlusive thrombus. Arterial thrombosis and thrombotic occlusion occurs in
response to intimal damage, after which the vessel segment is ligated, both proximal and
distal to the point of injury, and removed without disturbing the intravascular thrombus.
The vessel segment is opened along its length, and the intact thrombus mass is lifted off
the intimal surface of the vessel. The weight of the thrombus mass is determined with
an analytical balance. Thrombus is formed only at the injury site. Scanning electron
micrographic studies demonstrated the presence of a rough surface with fibrous coating
containing platelets and a few erythrocytes. Dogs are anesthetized 120 min after ingestion
of the capsule, the right carotid artery is cannulated, and electrolytic arterial injury is
initiated as discussed before.
342 Drug Screening Methods

Hematological Measurements
Blood is withdrawn for platelet studies from the jugular cannula into a plastic syringe
containing 3.2% sodium citrate as the anticoagulant [(1:10 citrate to blood (vol/vol)]. Blood
is taken for platelet aggregation and whole blood cell counts at baseline, 60 min, and 240 min
after the administration of test drug. Platelet count is determined with an H-10 cell counter.
PRP, the supernatant present after centrifugation of anticoagulated whole blood at 1000 rpm
for 5 min (140 g), is diluted with platelet-poor plasma (PPP) to achieve a platelet count of
200,000/mm3. PPP is prepared after the PRP is removed by centrifuging the remaining blood
at 12,000 g for 10 min and discarding the bottom cellular layer. Ex vivo platelet aggregation is
measured by established spectrophotometric methods with a four-channel aggregometer by
recording the increase in light transmission through a stirred suspension of PRP maintained
at 37°C.14 Aggregation is induced with ADP (100 µmol/l). Values are expressed as percentages
of aggregation, representing the percentage of light transmission standardized to PRP and PPP
samples yielding 0% and 100% light transmission, respectively. Bleeding time is measured in
anesthetized dogs with a simple device by making incisions on the tongue and blotting the
wound with filter paper at 30 sec intervals until blood is no longer transferred to the filter paper.

Inclusion Criteria
Animals included in the final protocol were to satisfy the following pre-established criteria:
1. A circulating platelet count of not less than 1,00,000/mm.3
2. Demonstration of ability of platelets to aggregate in response to arachidonic acid before
administration of test compound.
3. Thrombotic occlusion of the right carotid artery (control vessel) within 4 h from the onset of
vessel wall injury with a 300 micro A direct anodal current.
4. Absence of heart worms on final postmortem examination.

Photochemically Induced Thrombosis Model in Rats

Male Wistar rats weighing 230-320 g are anesthetized with sodium pentobarbital injected
into the femoral muscle (60 mg/kg). The right jugular vein and artery are cannulated for the
injection of dye and the monitoring of arterial blood pressure and heart rate, respectively. The
small intestine is exteriorized via a midline incision in the abdominal wall into a bath and
perfused with saline kept at 37°C. The mesentery around the ileum is spread out carefully on a
small circular glass stage and the other parts of the intestine are covered with moistened gauze.
Microvessels in the mesentery are observed under transillumination with a halogen lamp for
selection of venules of 41-62 µm diameter in which microthrombi are produced. Thrombus
formation is induced in microvessels by a filtered light of wavelength 420-490 nm passed
through an objective lens.16 Using a field stop, the area of irradiation around the microvessels
is adjusted on the focal plane to a diameter of about 130 µm. The light intensity is controlled at
13.8 mW/mm2.

Procedure
The test agents are administered by i.v. bolus injection into the jugular vein of a rat. One min
after the injection, irradiation with filtered light is started. One min after the start of irradiation,
 Antiplatelet Agents 343

a solution of sodium fluorescein (2.5% w/v) is injected through the jugular vein (1 ml/kg body
weight). Whole process is monitored with TV camera after injection of sodium fluorescein.
The time when the thrombus begins to form (time of initiation) and the time when blood flow
completely stops (time of occlusion) are used as indices of antithrombotic activity. Time is
measured by replay of the videotape. If the blood flow does not stop within 30 min, the result is
calculated as 30 min. Rats are sacrificed at the end of experiment by overdose of KCl.

Studies of Antiplatelet Efficacy in Rhesus Monkeys

Sixteen rhesus monkeys of either sex, 8-15 years of age, weighing 6-10 kg, are administered test
drug as a solution in 0.9% saline (total dosing volume 0.5 ml/kg) at oral doses of 0.1, 0.3, and
1.0 mg/kg. Each dose is given to at least two animals/sex. Oral dosing is achieved by passing
a 16 Fr feeding tube through the oral cavity into the stomach. Administration of test drug is
followed by a 10-20 ml water flush. Blood sampling at various time points is accomplished by
accessing the appropriate sample site (femoral or saphenous vein). The total volume of blood
sampled during the study did not exceed 1% of body weight. Blood samples are withdrawn in
citrate containing Vacutainer tubes for an assessment of the ex vivo inhibition of ADP (100 µM)
mediated platelet aggregation.17

Antiplatelet Efficacy in Baboons

Six baboons (three per group, each made up of two females and one male) weighing 15-32 kg
are fasted overnight, administered atropine (0.04 mg/kg) followed by ketamine (10 mg/kg) and
xylazine (2 mg/kg), and restrained on a treatment table. Either the femoral or saphenous vein
is cannulated for blood sampling at various time points. Using a nasogastric tube, test drug is
administered as a 0.2 or 0.6 mg/ml solution in 5% EtOH-95% saline (0.9%) at oral doses of 0.1,
0.3, 1.0, and 3.0 mg/kg. Blood samples are withdrawn in citrate containing Vacutainer tubes
for the assessment of the ex vivo inhibition of ADP (100 µM)-mediated platelet aggregation. In
vitro assessment of platelet function after test drug administration should abolish arachidonic
acid-induced platelet aggregation or prolong the lag time between exposure to collagen and
aggregation, and decreased plasma thromboxane B2 levels.18

Transgenic Rat Model

The method is developed by Sudo, et al.19 The genetically modified rat strains are housed at the
temperature 23.2°C for two weeks before the experiment. The animals are allowed free access
to standard rat chow and water. All rates for platelet aggregation are 12-week-old adult males.
Blood (10 ml) is sampled from the inferior vena cava of each rat, under ether anesthesia, with
21G needles into plastic syringes containing 0.1 volume of 3.18% trisodium citrate solution.
Platelet, leukocyte and erythrocyte counts in the citrated blood and hematocrit values are
obtained with an automatic counter. Blood is stored at room temperature until measurement.
Platelet aggregation should be measured within 60-90 min after blood collection.

Examination of PRP Aggregation Using the Turbidimetric Method

Platelet aggregation with PRP, obtained by centrifuging citrated blood, is investigated by
using a novel whole blood aggregometer, the WBA analyzer, with a screen filtration pressure
344 Drug Screening Methods

(SFP) method. The measurements for each strain are performed before noon in separate
two days. Platelet number is assessed with an automatic counter as described above, and
platelets are prepared to 5 × 108 cells/ml with autologous platelet-poor plasma (PPP). To
measure changes in the light transmission rate, PRP samples of 200 µl are incubated with
stirring for 2 min at 37°C, then with 22.2 ml Adenosine 5’-diphosphate or collagen solution
for 5 min at 37°C. The intensity of light transmission over 5 min is then measured using an
aggregometer. The baseline is set with PRP and the maximum possible increase in light
transmission (Platelet aggregation rate: 100%) is set with PPP. The concentration of ADP
and collagen causing a 50% aggregation rate are subsequently calculated as the platelet
aggregation threshold index (PATI). Area under the curve of platelet aggregation (AUC)
induced by 1µM ADP, 8µg/ml collagen or 50 µM thrombin receptor-activating peptide
(TRAP) is calculated (platelet aggregation rate [%] × seconds [sec]). AUC is depending on
the lag phase and maximum aggregation rate in collagen, and on maximum aggregation rate
and disaggregation in ADP and TRAP.

FeCl3-induced Thrombosis Model

The method of Sudo, et al. and Kurz, et al.19,20 is applied. Rats are anesthetized with sodium
pentobarbital (50 mg/kg ip). The left common carotid artery is surgically exposed and a
Doppler flow probe is placed on the surface of the artery. Baseline blood flow is recorded
using a Transonic Model flowmeter. Thereafter, filter paper is (± 10 mm) saturated with
40% FeCl3 and applied to the adventitial surface carotid artery, immediately proximal to
the probe. Time to thrombotic occlusion after initiation of artery injury is defined as the
time required for blood flow to decline to 0.0 ml/min. The times to occlusion beyond
60 min are regarded as 60 min for the purpose of statistical analysis.

References
1. Davies MJ, Thomas A. Thrombosis and acute coronary-artery lesions in sudden cardiac ischemic
death. N Engl J Med 1984;310:1137-40.
2. Fuster VF, Badimon L, Badimon JJ, et al. The pathogenesis of coronary artery disease and the acute
coronary syndromes. N Engl J Med 1992; 326:242-50.
3. Patrono C. Aspirin as an antiplatelet drug. N Engl J Med 1994; 330:1287-94.
4. Dinerman JL, Mehta JL. Endothelial, platelet and leukocyte interactions in ischemic heart disease:
insights into potential mechanisms and their clinical relevance. J Am Coll Cardiol 1990;16:207-22.
5. Armstrong RA. Platelet prostanoid receptors. Pharmacol Ther 1996;72:171-91.
6. Vorchheimer DA, Badimon JJ, Fuster V. Platelet glycoprotein IIb/IIIa receptor antagonists in
cardiovascular disease. JAMA 1999;281:1407-14.
7. Lefkovits J, Plow EF, Topol EJ. Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N
Engl J Med 1995;332:1553-9.
8. Philips DR, Kieffer N. Platelet membrane glycoproteins: function in cellular interactions. In: Palade
GE (Ed). Annu Rev Cell Biol 1990;6:329-57.
9. Mousa SA. Antiplatelet therapies: from aspirin to GP IIb/IIIa-receptor antagonists and beyond.
Drug Discov Today 1999;4:552-61.
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10. Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg MH. The effect of arg-gly-asp-
containing peptides on fibrinogen and von Willebrand binding to platelets. Proc Natl Acad Sci USA
1985;82:8057-61.
11. Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature
1962;194:927-9.
12. Rho MC, Nakahata N, Nakamura H, et al. Activation of rabbit platelets by Ca2+ influx and
thromboxane A2 release in an external Ca2+ dependent manner by Zooxanthellatoxin-A, a novel
polyol. Br J Pharmacol 1995;115:433-40.
13. Cazenave JP, Packham MA, Guccione MA, et al. Inhibition of platelet adherence to a collagen-coated
surface by nonsteroidal anti-inflammatory drugs, pyrimido-pyrimidine and tricyclic compounds
and lidocaine. J Lab Clin Med 1974;83:797-806.
14. Kamada H, Okamoto T, Hayashi T, Suzuki K. An in vitro method for screening anti-platelet agents
using a microchannel array flow analyzer. Biorheology 2010;47:153-61.
15. Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in
the ambulatory canine: a model for in vivo evaluation of antithrombotic agents. Thromb Res
1980;17:841-53.
16. Sato M, Ohshima N. Platelet thrombus induced in vivo by filtered light and fluorescent dye in
mesenteric microvessels of the rat. Thromb Res. 1984;35:319-34.
17. Kaku S, Yano S, Kawasaki T, et al. Comparison of the antiplatelet agent potential of the whole
molecule F(ab)2 Fab fragments of humanized anti-GP IIb/IIIa monoclonal antibody in monkeys.
Gen Pharmacol 1996;27:435-9.
18. Shoenfeld NA, Yeager A, Connolly R, et al. A new primate model for the study of intravenous
thrombotic potential and its modification. J Vasc Surg 1988;8:49-54.
19. Sudo T, Ito H, Hayashi H, Nagamura Y, Toga K, Yamada Y. Genetic strain differences in platelet
aggregation and thrombus formation of laboratory rats. Thromb Haemost 2007;97:665-72.
20. Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride.
Throm Res 1990;60:269-80.
 cHAPTER

22
Drugs Acting on Sympathetic
Nervous System
INTRODUCTION
Autonomic nervous system is largely involuntary and is responsible for maintaining the internal
homeostasis. Two subdivisions of autonomic nervous system include the sympathetic nervous
system and parasympathetic nervous system. Under physiological conditions, stimulation or
inhibition of sympathetic activity is in balance with that of parasympathetic activity so as to
maintain the internal homeostasis.
Autonomic nervous system consists of two groups of neurons (pre- and postganglionic
neurons) to maintain communication between CNS and peripheral organs. The neuro­
transmitters provide the means of communication between pre and postganglionic neurons.
The neurotransmitter released at the postganglionic sympathetic neurons is noradrenaline.
The adrenaline secreted by adrenal medulla provides generalized sympathetic stimulation.
The sympathetic neurotransmitters largely act through adrenergic receptors, which are
present in heart, bronchi, blood vessels, uterus, gastrointestinal tract, eye, etc. Adrenergic
receptors are of two types—α and β, which are further classified into α1, α2 and β1, β2, β3
respectively. Stimulation of these adrenergic receptors causes various responses as shown in
Table 22.1.
Sympathetic nervous system activation occurs in response to stressful stimuli including
physical activity, psychological stress, blood loss, etc. and prepares the body for emergencies.
Sympathetic nervous system stimulation leads to increased heart rate and blood pressure,
dilatation of pupils, dilatation of trachea and bronchi, stimulation of the conversion of liver
glycogen into glucose, shunting of blood away from the skin and viscera to the skeletal
muscles, brain, and heart, inhibition of the peristalsis in the gastrointestinal (GI) tract,
inhibition of contraction of the bladder and rectum stimulation of uterus and constriction of
spleen capsule.
Sympathomimetic drugs are agents with activity that mimics the responses of
adrenaline or stimulation of the sympathetic nervous system. The sympatholytic drugs
can antagonize the effects of sympathetic stimulation or the effects of sympathomimetic
agents. A summary of sympathomimetics and sympatholytics is given in Figure 22.1. The
drugs with potential sympathomimetic or sympatholytic activity can be screened using
various animal models.
 Drugs Acting on Sympathetic Nervous System 347

Table 22.1: Adrenergic receptors and their important characteristics

α1 α2 β1 β2 β3
Smooth muscles
Blood vessels Constriction Dilatation
Bronchi Dilatation
GI tract Relaxation
GI sphincters Constriction
Bladder Relaxation
Bladder Constriction
sphincter
Seminal vesicle Constriction
Eye–radial Constriction
muscle
Ciliary muscle Relaxation
Heart rate Increases
Force of Increases
contraction

Figure 22.1: Sympathetic agonists and antagonists

In vivo Methods
Cat Spleen Model
This is one of the useful preparations for the evaluation of substances affecting the release
and subsequent fate of the adrenergic transmitter from the sympathetic nervous system.
After the injection of sympathomimetic amines or electrical stimulation of the pre- and
postganglionic sympathetic nerves, spleen contracts. Different doses of the test compound
that alter the release of transmitter output of spleen are compared with the known standards.
348 Drug Screening Methods

Released noradrenaline can be measured by collecting spleen venous effluent and analyzing
its noradrenaline content. This is achieved after labeling of neuronal stores with radioactive
noradrenaline, which is taken up from the splenic arterial circulation and subsequently
released after nerve stimulation. The spleen can be used either in vivo or in vitro.

Procedure
Cats are anesthetized using chloroform. Abdomen is cut open by giving a midline incision
and the intestine is removed from the mid-duodenum to the terminal colon. The vascular
connections between the spleen and omentum are being cut. The splenic nerves are also cut
to avoid liberation of catecholamines from other sites. The adrenal glands are also removed
which is helpful to avoid the artifacts due to the release of pressor amines. A ligature is placed
around the portal vein just beyond the junction of splenic and superior mesenteric veins
and close to the adjoining gastric vein. Heparin is injected intravenously to prevent clotting
of blood. The abdominal cavity is filled with warm paraffin and aerated with a mixture of
O2 and CO2. Venous blood samples are collected by diverting effluent through a cannula by
applying increased tension to the ligature placed around the portal vein. Blood is collected in
chilled, silicone coated, calibrated centrifuge tubes during the period of stimulation together
with a subsequent 20 sec period immediately after cessation of the stimulus. This is enough to
capture more than 80% of noradrenaline released into the blood during electrical stimulation
and at the same time avoids the excessive loss and unwanted dilution of activity in the plasma.
The amount of the noradrenaline present in the blood is estimated by scintillation counter for
radioactivity measurement.1

The Rat Blood Pressure—Invasive Model

The sympathomimetics like adrenaline induce increase in heart rate and blood pressure
while the sympatholytics induce a fall in heart rate and blood pressure. These changes can be
recorded in a rat model to evaluate the activity of test drug.2

Procedure
The Sprague Dawley rat weighing 250-350 g are used. The animals are anesthetized with
urethane 1 g/kg subcutaneously. Polyethylene catheters are placed in femoral artery and
femoral vein. The catheter in femoral artery is used to record blood pressure and heart rate
while the femoral vein catheter is used for injecting drugs/vehicle. Alternatively, a catheter
placed in carotid artery can also be used for recording blood pressure and heart rate. After
cannulation the catheters are flushed with 0.2 ml heparinized (20 U/ml) saline solution. The
femoral artery catheter is connected with blood pressure transducer and heart rate and blood
pressure are recorded. After equilibrium period of 30 min 0.1-0.5 ml of various concentrations
of drug or vehicle are injected through the catheter placed in femoral vein and changes in blood
pressure and heart rate are recorded to evaluate sympathomimetic/sympatholytic activity. The
same volume of adrenaline (2 µg/kg) can be used as standard drug to compare the potential
sympathomimetic activity of test drug. To evaluate for the specific receptor agonist activity
drug induced changes in BP and HR can be recorded after treatment with specific receptor
blockers such as prazosin, an alpha1 blocker (25 µg/kg) or atenolol, a beta1 blocker (1 mg/kg).
 Drugs Acting on Sympathetic Nervous System 349

The Rat Blood Pressure—Noninvasive Model

Sympathomimetic and sympatholytic drug induced changes in blood pressure and heart rate
can also be recorded in a noninvasive model.3

Procedure
Adult male Wistar rats (250-300 g) are used. The jugular vein is cannulated in anesthetized
rats using a curved polyethylene catheter. The vinyl tubing is led under the skin of neck and
exposed on the surface of back to allow for infusion of drugs. The catheter is flushed with
heparin (100 units) to prevent clotting. Rats are allowed to recover for at least 24 hours before
starting the experiment.
On the day of experiment rats are first placed on heating pads (35-37°C) for 20-30 min. Now
the rats are transferred to a restrainer maintained at 37°C. The tail cuff and pulse sensor is
placed at the proximal end of the tail and is inflated. The heart rate is determined by manually
counting the number of beats per unit time. The systolic blood pressure is indicated at the
point where the reappearance of pulsations is detected by pulse sensor. The value for each
parameter is taken as average of at least 4 measurements. The measurements are done on
three successive days to determine the baseline values. After baseline measurements the
drug/vehicle/standard drug are injected through the catheter in jugular vein and changes in
heart rate and blood pressure are measured to evaluate for sympathomimetic/sympatholytic
activities as in invasive model.

Cat Model of Nictitating Membrane Prolapse

The nictitating membrane of cats relaxes in response to sympatholytic drugs. This activity in
cats is used to evaluate sympatholytic activity of a test drug.

Procedure
Unanesthetized cats are used and test compounds are administered orally in the animals.
Sympatholytic agents exert relaxant effect on the nictitating membrane of the cat. The extent
of relaxation of membrane is measured along the lower lid margin with ruler. Measurement
should be done carefully without disturbing the animal. For each dose of the test drug, at least
5-10 animals are used. The relative activity of the different compounds is calculated by dividing
the mean duration of the membrane prolapse of a group in hours by the dose in mg/kg.4,5

Mouse Eye Model

Norepinephrine, epinephrine and isoproterenol have the property of inducing mydriasis, this
effect is blocked by α or β blockers. α-blockers block the mydriatic effect of norepinephrine,
β-blockers block the effect of isoproterenol and α and α-blockers block the effect of epinephrine.

Procedure
Mice of weight range 15-20 g are used. Animals are divided into different groups. Vehicle
is administered subcutaneously that serves as control. Different doses of test compound
are administered subcutaneously. After 30 min, 0.1 mg/kg norepinephrine or 0.05 mg/
350 Drug Screening Methods

kg epinephrine or 20 mg/kg isoproterenol are given intravenously. The pupil diameters are
measured before and after vehicle/drug administration. The mean values of pupil diameters
are compared between the groups.6

Pithed Rat Model

In this model stimulation of the complete sympathetic outflow is performed in the intact
animal and agents affecting sympathetic nervous system are evaluated.

Procedure
Rats weighing 250-350 g are used. Animals are anesthetized with either ether or choloroform.
The trachea is cannulated. Animals are then pithed by passing a stainless steel rod through the
orbit and down the spinal column. Artificial respiration is maintained using respirator. Blood
pressure is recorded from the cannulated left carotid artery using a transducer and the arterial
pulse is used to trigger a Neilson instantaneous rate meter for the measurement of heart rate.
Drugs are administered into the cannulated left femoral vein.
Pithing rod is used as an electrode to stimulate the spinal sympathetic outflow to initiate
increase of both the blood pressure and the heart rate. To stimulate thoracolumbar region,
rod is insulated with an epoxy resin adhesive except the part in close touch with the thorax
and lumbar regions of the spinal cord. Parasympathetic or motor nerve fiber stimulation by
spread of the stimulating current is reduced by administration of atropine. The increase in the
blood pressure and heart rate can be elicited by the electrical stimulation of supramaximal
rectangular pulses of 1.0 ms duration. The magnitudes of the responses are proportional to the
frequency of the stimulus usually over the range of 0.5 to 8.0 Hz. Changes in the blood pressure
and heart rate are observed after drug (standard/test) administration and dose response
curves are plotted for comparison to assess agonistic and antagonistic activity.7
To determine α1 and α2 antagonismin pithed rats, phenylephrine (selective α1 antagonist)
in the doses of 0.1-30 mg/kg and BHT920 (selective α2-agonist) in the doses of 1 to 1000 mg/
kg are administered intravenously. Their effects on blood pressure and heart rate are observed
and dose-response curves are plotted. After that, test drug is administered intravenously and
the agonist dose response curves are repeated after 15-30 min to record any shift in the curves
of blood pressure response. Finally, potency ratios are calculated using log dose response
curves.8

Modification
Above method is modified by allowing stimulation of individual pathways of the sympathetic
and parasympathetic nerve pathways in pithed rats.8

Rat Heart and Uterus Model

β1 and β2 receptors are located in the heart and in the uterus respectively. In this method, both
heart rate and uterine relaxation are observed in the same animal after drug administration, to
determine the β1 and β2 activity of the test compound.
 Drugs Acting on Sympathetic Nervous System 351

Procedure
Female Sprague-Dawley rat is anesthetized with pentobarbital (60 mg/kg ip). After that pithing
of the animal is done.8 Artificial respiration is given to the animal using an animal ventilator.
Body temperature of the animal is kept around 36-37°C. For blood pressure monitoring, left
carotid artery is cannulated and an instantaneous rate meter is used to monitor heart rate
continuously. Drugs are administered intravenously. After that, uterine horns are exposed by
giving a midline incision in the abdomen. The ovarian artery is identified and ligated. Uterine
horn is dissected free from the ovary. At the free end of the horn a thread is tied and tissue is
mounted in an organ bath containing Krebs-Henseleit solution. Temperature is maintained at
37°C and solution is constantly bubbled with 95% O2 and 5% CO2.
Isoprenaline (nonselective β-agonist), salbutamol (β2-agonist) and norepinephrine (β1
selective agonist) are administered intravenously and their effects on both heart rate and
uterine relaxation are recorded. After that, test agent is given intravenously, their effects are
observed and compared with the effects of isoprenaline, salbutamol and norepinephrine. A
test drug is considered as an agonist when it causes an increase in heart rate and decrease
in the height of uterine contractions; whereas if test drug inhibits the effects of isoprenaline
on both heart rate and uterine relaxation, it is taken as an antagonist. Dose response curves
obtained with isoprenaline in the absence or presence of the β antagonist are compared to
assess the antagonistic activity.9

In vitro Methods
Cat Spleen Model
The contractile response of splenic capsule in response to norepinephrine can also be
determined in vitro.

Procedure
The method for the removal of spleen is similar as described above. The removed spleen is
placed in a plethysmograph filled with liquid paraffin. The organ is perfused at a rate of 7-16
ml/min with a modified Krebs solution at 37°C gassed with carbogen. Dextran (3%) is added
to raise the osmotic pressure of the perfusion fluid. Ascorbic acid at 25 µg/ml is also added to
prevent oxidation of noradrenaline. The perfusion is started and samples are collected and
estimated for noradrenaline.1

Rabbit Pulmonary Artery Model

The pulmonary artery is highly sensitive to sympathomimetic agents. The artery mainly consists
of vascular smooth muscle innervated by postganglionic adrenergic sympathetic fibers.
The noradrenaline is released in response to the nerve stimulation. The released adrenergic
transmitter is measured by labeling the neuronal stores with radioactive noradrenaline and
with the use of superfusion technique to reduce the dilution of amines.
352 Drug Screening Methods

Procedure
The rabbit of weight range 1 to 2 kg is sacrificed by exsanguination followed by the removal
of main pulmonary artery. The artery is cut transversely and spirally into vertical strip of
approximately 4 mm by 30 mm in length. The strip is suspended vertically in an organ bath
maintained at 37°C and its lower end is tied to glass support, whereas the other end is connected
by thread to an isometric strain gauge transducer. The resting tension is 2 g and the artery is
superfused with Krebs bicarbonate solution, which is allowed to flow down the connecting
thread and tissue at the rate of 6 ml/min. The tissue is then loaded with 3[H] noradrenaline
by submerging it in 20 ml of Krebs bicarbonate solution at 37°C and gassed with 95% O2 and
5% CO2. Following incubation of 60 min, the artery strip is superfused with Krebs solution
at 37°C for 30 min to wash out extraneuronal 3[H] noradrenaline. During the experiment,
superfusates are collected in vials every 2 min. Aliquots of collected samples are then assayed
for noradrenaline using scintillation spectrophotometer to count the radioactivity after adding
scintillation fluid.
The electrical stimulation is given using bipolar platinum electrodes of 30 mm length and
0.5 mm diameter, placed in parallel or either side of the strip. The gap between the tissue and
the electrode allows the strip to develop increased tension without any hindrance. The nerves
can be excited by transmural stimulation using strains by supramaximal biphasic pulses of 0.3
ms duration. Responses to successive 2 min period of electrical stimulation are reproducible
when applied at 16 min intervals. The tension produced by the tissue increases rapidly after
commencement of stimulation and decreases back to original basal value after stimulation has
ceased. The response produced is accompanied by increased release of tritium (250% above
basal level) onto the superfusate. A further reduction in basal radioactivity in the superfusion
fluid occurs after each stimulus period as the tissue gradually loses its stored radioactivity.
The total tritium detected in collected superfusates unchanged noradre­naline comprised of
approximately 30% during rest period and at least 50% during stimulation.10,11

Rat Vas Deferens Model

Vas deferens preparation has exclusive autonomic innervations and it gives constant contractile
responses to alpha-adrenergic agonists. It possess both α1 and α2 adrenoceptors at post and
presynaptic sites, respectively. Norepinephrine gives a contractile response superimposed on
the twitch response because of postsynaptic α1 receptor stimulation.

Procedure
Male Wistar rats weighing 275 to 300 g are used. Animals are killed by stunning. A midline
abdominal incision is performed to dissect out vas deferens. Tissue is suspended in an organ
bath containing Tyrode solution gassed with O2 and CO2 at 35°C. Contractions are recorded
using a lever transducer. After 30 min, norepinephrine is administered repeatedly in the
concentrations of 0.5, 1.0, 2.0 or 4.0 mg/ml. Test drug is then added into the bath and after 3
min norepinephrine is administered. Phentolamine is used as standard.
To evaluate α antagonistic activity, norepinephrine-induced contractions after test drug
administrations are compared with the contractions induced by norepinephrine alone.12
 Drugs Acting on Sympathetic Nervous System 353

Modification
Isolated hypogastric nerve—vas deferens of the guinea pig is also used to evaluate
α-sympatholytic activity.13,14

Rat Seminal Vesicle Model

Seminal vesicle of rat is used to evaluate a antagonistic activity.

Procedure
Male Wistar rats (40-50 days old) and weighing 125-150 g are used. Animals are killed by
stunning. Seminal vesicles are prepared and suspended in an organ bath containing modified
Krebs solution, provided as a continuous flow with run rate of 15 ml/min. Resting tension is
around 350 mg and solution is bubbled with 5% CO2 in O2 at 32°C.
After 30 min of equilibration period norepinephrine is administered repeatedly in the
concentrations of 0.5, 1.0, 2.0 or 4.0 mg/ml. Contractions are recorded using lever transducer.
Test drug is then added into the bath and norepinephrine is added after 3 min. Phentolamine
is used as standard. If a test drug inhibits the contractions induced by norepinephrine, it
suggests a sympatholytic activity. To evaluate α antagonistic activity, norepinephrine-induced
contractions after test drug administrations are compared with the contractions induced by
norepinephrine alone.
The preparation is quite sensitive to a few selected agonists and remains viable for over
4-6 hr. Adrenaline, noradrenaline, dopamine, and acetylcholine all produce concentration-
dependent and reproducible contractions. However, histaminergic, serotoninergic, purinergic,
and opioid agonists, prostaglandins of the E and F series and the polypeptides angiotensin,
vasopressin, and oxytocin are inactive.15

Cat Splenic Strip Model

The splenic tissue contracts in response to sympathomimetic agents and therefore can be used
for screening of drugs with potential sympathomimtic/sympatholytic activity.

Procedure
Cats of either sex weighing around 1.0-2.8 kg are anesthetized by an intraperitoneal injection
of 45 mg/kg sodium pentobarbitone. The spleen is then removed and 25 to 30 mm long and 2
to 3 mm wide strips of the spleen are prepared. The strip is then suspended in an organ bath
containing 10 ml of the Krebs-Ringer solution, which at 38°C is aerated with the bubbles of
95% oxygen and 5% carbon dioxide. Isotonic contractions are recorded on a kymograph at 0.5 g
tension with magnification 5 to 6 times. To induce contractions, norepinephrine (106 g/ml)
or epinephrine (10-6 g/ml) is administered after 30 min. Test drug is then added into the bath
followed by the administration of α agonists after 3 min. Phentolamine is used as standard.
To evaluate α sympatholytic activity, the percent inhibition of epinephrine or norepinephrine
induced contractions is determined.16,17
354 Drug Screening Methods

Guinea Pig Tracheal Chain Model

Guinea pig tracheal chain preparation contracts in response to muscarinic receptor agonist,
carbachol. The carbacholprecontracted tracheal chain preparation when exposed to a
beta agonist like isoprenaline shows complete relaxation. Antagonism of relaxant effect of
isoprenaline in presence of a beta-blocker can be demonstrated in guinea pig tracheal muscle.

Procedure
Albino guinea pig of either sex weighing 300-550 g is sacrificed by stunning and exsanguination.
The trachea is removed and cut into 10-12 rings of the same width. Six rings are connected in
series by means of short loops of silk thread and are kept in tyrode solution. The tracheal chain
is suspended in an organ bath containing tyrode solution. Solution is aerated with 95% O2 and
5% CO2 and temperature is maintained at 37°C. Tension on the lever is around 0.5 g. To block
α receptors phentolamine (0.1 mg/ml) and to induce spasm, carbachol (80 ng/ml) are added
in the bath. After a period of 30 min, spasm is relieved by adding isoprenaline (β-agonist) in
cumulative doses into the bath. After getting complete relaxation with isoprenaline, tissue is
washed out at least 2-3 times. Test compound is then added in the bath and isoprenaline is
again administered in cumulative doses. Tissue is thoroughly washed out and 10 min later,
increased dose of test drug is administered.
β2 antagonisitic activity would be considered as positive if test drug inhibits the spasmolytic
action of isoprenaline.
To evaluate β2 sympatholytic activity, percent inhibition of isoprenaline-induced relaxation
after test drug administration is compared to maximal relaxation induced by isoprenaline.
Above preparation is also used to detect β2 agonistic activity. For this, spasm is induced by
carbachol and test drug is added. After 5 min, propranolol (β blocker) is administered in the
bath. Spasmolytic effect of the test compound is decreased after propranolol administration.
Percent inhibition of spasmolytic effect of the test compound after propranolol administration
is measured to assess β2 sympathomimetic activity.18,19

Guinea Pig Isolated Heart Model

The beta adrenergic drugs are potent cardiotonic agents and the effect of these drugs can be
evaluated in isolated heart preparation.

Procedure
Guinea pigs of either sex weighing 600-800 g are used. The animals are injected with 100 units
of heparin in the marginal ear vein to avoid damage to heart due to clot formation. The animals
are now killed by a sharp blow to head, thorax is opened and heart is removed carefully
and transferred to a Petridish containing Tyrode’s solution. All the blood from the tissue is
removed by gently squeezing it. The heart and aorta are dissected free from surrounding
fascia and connective tissue. The Lagendroff’s method can be used to screen for cardiotonic
effects. The aorta is cut just below the point of its division and then the heart is transferred to
perfusion apparatus containing Tyrode’s solution at 37°C and oxygenated continuously. The
aorta is tied to the glass cannula with the precaution that no air bubble enters the aorta. The
heart is suspended by a thread through a hook into the ventricle. The thread is connected to
 Drugs Acting on Sympathetic Nervous System 355

spring levers to record contractions. After 20 min stabilization period baseline contractions
are recorded to observe for amplitude and frequency of contractions indicating baseline force
and rate of heart contractions. Now 0.1 ml of drug/adrenaline (100 µg/ml) is injected into the
perfusion fluid and recordings are done to evaluate changes in force and rate of contractions.
Beta-adrenergic drugs increase the force and rate of contraction; however they fail to do so in
presence of a beta-blocker.20

Isolated Rat Aorta Model

The isolated rat aorta model evaluates the aortic smooth muscle contractions in response to
alpha1 adrenergic stimulation.21

Procedure
Wistar rats weighing 200-250 g are killed by cervical dislocation. The thoracic aorta is
separated and gently cleared of fat and connective tissue. The separated aorta is cut into 3
mm ring segments and mounted on steel wires in 20 ml organ bath containing Kreb’s solution
(NaCl 119 mM; KC1 4.7 mM; CaCl2 2.5 mM; MgSO4 1.2 mM; NaHCO3 25 mM; KH2PO4 1.2
mM; D-glucose 11.1 mM) at 37°C and gassed with O2 + CO2 (95:5). The tissue is placed under
1.5 gram tension and a 60 min period is allowed for equilibrium with repeated washings
with Kreb’s solution at 15 min interval. Isometric muscle tension can be recorded using forced
transducer. After equilibrium period the dose response curve is prepared for phenylephrine
(alpha agonist) in the concentration range of 10-10 to 10-2 M. After washing, the aortic rings are
incubated with the test drug and dose response curve (DRC) of phenylephrine is repeated. The
shift of DRC to left or right will indicate the agonist or antagonist activity of the test drug. The
contractile response to test drug can be repeated after preincubation with prazosin (an alpha
blocker) and an inhibitory effect will indicate alpha1 agonistic activity of test drug.

Rat Submaxillary Tissue Model

Adrenergic agonists increase the utilization of glucose in presence of Ca2+ by tissues with
adrenergic receptors. Since, the rat submaxillary glands contain both the alpha and beta-
adrenergic receptors, this property can be utilized for screening of adrenergic agonists.

Procedure
Male Wistar rats weighing 150-200 g are used. The rats are killed by cervical dislocation and
submaxillary tissue slices are prepared.22 The tissue slices (200 mg wet weight) are incubated
in 50 ml flasks with 5 ml of Krebs-Henseleit bicarbonate saline containing 5 mM of glucose
and gassed with a mixture of O2 + CO2 (95:5). The estimated quantities of drug/isoproterenol
(10 µg/ml) with or without propranolol (20 µg/ml) are added to the flasks. For the control, all
additions are made as above except for the tissue. The flasks are sealed and incubated at 37ºC
for 1 hour in a shaking water bath. At the end of incubation period the medium is removed and
the quantity of glucose is estimated. Glucose removal by the tissue can be calculated from the
difference between the amount of glucose in control and that remaining in the experimental
356 Drug Screening Methods

flasks. Isoprenaline significantly increases the glucose removal but addition of propranolol
significantly inhibits the glucose removal by isoprenaline.23

Mice Metabolic Stimulation Model

Exogenous administration as well as endogenous release of catecholamines stimulates the
conversion of liver glycogen into glucose and further metabolism of glucose. Alteration in these
metabolic parameters in response to catecholamine stimulation can be used for screening of
investigational adrenergic drugs.

Procedure
Male mice used in the experiment are anesthetized with sodium pentobarbital (60 mg/kg).
The standard treatment group receives intraperitoneal injection of adrenaline (1.25 mg/g)
after priming with intravenous adrenaline (0.37 mg/kg). The control animals receive the same
volume of saline while the test group receives the test drug. The blood samples are collected
from inferior vena cava at 20, 40 and 60 min after injection. Plasma is separated by centrifugation
and is used for estimation of glucose, glycerol, nonesterified fatty acids and β-hydroxybutyrate.
The plasma concentration of all metabolites increases in response to adrenergic stimulation.24

Rat jejunal Longitudinal Muscle Model

Catecholamines can produce relaxation of non-sphincteric gastrointestinal muscle by an
action on either postjunctional α- or β-adrenoceptors or a combination of both. Hence,
adrenoreceptor-mediated changes in the contractility by experimental drugs can be
demonstrated using this model.25

Procedure
Male Wistar rats (200-300 g) are killed by a blow to the head and cervical dislocation. The
jejunum is removed and immediately placed in cold and oxygenated Krebs physiological
saline solution, at room temperature. Longitudinal muscle strips are dissected from the
intestinal segments by gently peeling the muscle in longitudinal direction. Longitudinal
smooth muscle strips (3 cm) of jejunum are then suspended in organ baths containing Krebs
solution, at 37°C, for isotonic recording. The Krebs solution is gassed with 95% O2 and 5% CO2
and consists of (mM): NaCl 118, CaCl2 2.5, KCl 4.7, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2 and
glucose 11.1. Strips are pre-contracted with potassium chloride (40 mM) before proceeding
with cumulative concentration-response curves for agonists and adrenaline can be used as a
reference drug. Alternatively, isoprenaline can be used as the intestinal responses are largely
β receptor mediated with only a small contribution from α1 receptors. Agonists produce
relaxation and the responses are expressed as % relaxation. Response to agonists is repeated
in the presence or absence of propranolol (1 µM). Tissues are allowed to equilibrate for 45 min
before addition of agonists. The antagonists are added from the beginning of the equilibration
period. In the absence of propranolol, agonists produce relaxation of the potassium chloride-
contracted strips. Presence of propranolol causes shift of dose-response curve to right by
causing β receptor blockade. 25
 Drugs Acting on Sympathetic Nervous System 357

Gravid Rat Uterus Model

Stimulation of sympathetic α1 receptors causes uterine contractions and stimulation of β2
receptors causes uterine relaxation. The gravid uterus relaxes in response to epinephrine
administration due to β2> α1. Blockade of β receptors abolishes the uterine relaxant effect of
epinephrine.

Procedure
Uteri are excised from term pregnant rats. Cross-sectional rings of excised uteri are mounted
for isometric force recording. Log dose-response curve is prepared for epinephrine in the
concentration range of 10-12 to 10-6 M. Epinephrine causes dose-dependent reduction in
uterine muscle activity. Similarly, agonists produce relaxation and the responses are expressed
as % relaxation. Response to agonists is repeated in the presence or absence of propranolol
(1 µM). In the absence of propranolol, agonists produce relaxation of the gravid rat uterus
muscle. Presence of propranolol causes shift of dose-response curve to right due to β receptor
blockade. Careful interpretation is necessary and oxytocin as well as washout of epinephrine
also antagonize catecholamine-induced tocolysis.26

Murine Macrophage Model

Catecholamines are known to play a protective role during endotoxemia by downregulating the
inflammatory response. One of the mechanisms of downregulation of inflammatory response
is through suppression of expression of macrophage inducible nitric oxide synthase (iNOS).
This model evaluates the effects of catecholamines on lipopolysaccharide (LPS) induced
macropahge NO production in vitro.27

Procedure
The male mice 8-12 week old are used. The peritoneal cavities of mice are lavaged with
ice-cold RPMI-1640 medium supplemented with 1% heat inactivated foetal calf serum,
100 U/ml penicillin, 100 µg/ml streptomycin, 25 mMN-2-hydroxyethylpiperazine-N’-2-
ethane-sulphonic acid (HEPES) and 10 mM L-glutamine. The fluid now containing resident
peritoneal exudates cells is centrifuged at 4° for 7 min at 1100 rpm. The cells are now suspended
in the same fluid as above but containing 10% foetal calf serum. The cells in the solution are
now counted and viability assessed using trypan blue dye. 100 µl of the cell suspension is now
plated on the sterile flat-bottomed culture plates to produce a final concentration of 3 × 105
cells in each well. The plates are incubated at 37° for 2 hr in a 5% CO2 incubator, followed by
removal of non-adherent cell by washing thrice with above medium with 10% calf serum. Cells
are further incubated with medium alone, LPS (10 µg/ml) alone, LPS plus drug in presence and
absence of propranolol (10-2 M) and LPS plus adrenaline (10-5 M) in presence and absence of
propranolol at 37° for 48 hr. After incubation the supernatant is removed and NO is estimated
by Griess reaction. The amount of NO production can be compared among various groups.
Adrenergic drugs suppress the NO production by murine macrophages in response to LPS
but this suppression is abolished by propranolol. Corticosterone, a steroid hormone known to
suppress NO production can be used as negative control in a concentration of 105 M.
358 Drug Screening Methods

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2. Keenan D, Romani A, Scarpa A. Differential Regulation of Circulating Mg2+ in the Rat by β1- and β2-
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3. Zhang YC, Bui JD, Shen L, Phillips MI. Antisense Inhibition of β1-Adrenergic Receptor mRNA in a
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8. Shipley RE, Tilden JH. A pithed rat preparation suitable for assaying pressor substances. Proc Soc
Exp Biol Med 1947;64:453-5.
9. Piercy V. Method for assessing the activity of drugs at β1 and β2 adrenoceptors in the same animal. J
Pharmacol Methods 1988;20:125-33.
10. Su C, Bevan JA. The release of 3H-norepinephrine in arterial strips studied by the technique of
superfusion and transmural stimulation. J Pharmacol Exp Ther 1970;172:62-8.
11. McCulloch MW, Bevan JA, Su C. Effects of phenoxybenzamine and norepinephrine on transmitter
release in the pulmonary artery of the rabbit. Blood Vessels 1975;12:122-3.
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rat deferens. J Pharmacol ExpTher 1983;224:40-5.
13. Holman M. Nerve muscle preparation of vas deferens. In Daniel EE, Paton DM (Eds): Methods in
Pharmacology. New York: Plenum Press, 1975;3:403-17.
14. Hukovic S. Responses of the isolated sympathetic nerve-ductus deferens preparation of the guinea
pig. Br J Pharmacol 1961;16:188-94.
15. Sharif SI, Gokhale SD. Pharmacological evaluation of the isolated rat seminal vesicle preparation. J
Pharmacol Methods. 1986;15(1):65-75.
16. Innes IR, Kohli JD. An action of 5-hydroxytryptamine on adrenaline receptors. Br J Pharmacol
Chemother 1962;19:427-41.
17. De Geus JP, Bernards JA, Verduyn WH. On the rhythmic activity of the cat spleen. Arch Intern
PharmacodynTher 1956;106:113-21.
18. Castillo JC, de Beer EJ. The tracheal chain: A preparation for the study of antispasmodics with
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19. Foster RW. The nature of the adrenergic receptors of the trachea of the guinea-pig. J Pharm
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20. Adome RO, Gachihi JW, Onegi B, Tamale J, Apio SO. The cardiotonic effect of the crude ethanolic
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 Drugs Acting on Sympathetic Nervous System 359

22. Thompson MP, Williamson DH. Metabolic effects of α- and β- adrenergic stimulation of rat
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 cHAPTER

23
Drugs Acting on Parasympathetic
Nervous System
INTRODUCTION
The parasympathetic nervous system performs maintenance activities and conserves
body energy. Acetylcholine is both preganglionic and postganglionic neurotransmitter of
parasympathetic nervous system. Acetylcholine, released at the cholinergic synapses and
neuroeffector junctions, mediates its pharmacological actions through cholinergic receptors
(nicotinic and muscarinic). Nicotinic receptors are found in autonomic ganglion and
neuromuscular junction. Muscarinic receptors are found primarily on the autonomic effector
cells that are innervated by postganglionic cholinergic nerves and are present in the heart,
eyes, glands, smooth muscles and blood vessels. Muscarinic receptors are of five types—M1,
M2, M3, M4, and M5. Out of these, the first three are functionally well characterized. Stimulation
of these receptors gives rise to various responses as shown in Table 23.1.
Table 23.1: Muscarinic receptors and their important characteristics
M1 M2 M3
Smooth muscles:
Blood vessels (endothelium) Dilatation
Bronchi Constriction
GI tract Constriction
GI sphincters Relaxation
Bladder Constriction
Bladder sphincter Relaxation
Eye –Circular muscle Constriction
Ciliary muscle Constriction
Heart Rate Decreases
Force of contraction Decreases

Parasympathomimetic/cholinergic agents mimic the effects of acetylcholine. The

parasympatholytic/anticholinergic drugs can antagonize the effects of parasympathetic
stimulation or the effects of parasympathomimetic agents. A summary of parasymathomimetics
(parasympathetic agonists) and parasympatholytics (parasympathetic antagonists) is given in
Figure 23.1. The drugs with potential of cholinergic or anticholinergic activity can be screened
using various animal models.
 Drugs Acting on Parasympathetic Nervous System 361

Figure 23.1: Parasympathetic agonists and antagonists

In vivo Methods
Cat Model for Anticholinesterase Activity
Cat is anesthetized using chloralose or pentobarbitone sodium. The common carotid artery
is cannulated for recording the blood pressure and the substance to be tested is injected
intravenously. The blood pressure and respiration are recorded in the anesthetized animal at
different doses of an anticholinesterase agent. For instance, at dose x, no detectable effect is
observed. At dose 2x, slight bradycardia followed by fall in blood pressure by 10-20 mm of Hg
developed over a period of 5 min. At dose 4x, blood pressure reduces by 50-100 mm of Hg
with pronounced bradycardia. Salivary and bronchial secretions are seen with fasciculation
of skeletal muscles. Initially, respiration is increased but later on it is depressed. Defecation
and urination are also observed. At dose 8x, all effects mentioned are much more prominent
as compared to dose 4x. Presence of all these effects indicates that test drug possesses
anticholinesterase activity.1

Rat Cardiorespiratory Model

The cholinergic and anticholinergic effects of test drug can be evaluated by observing the effects
of drug administration on cardiorespiratory functions in rats. Administration of cholinergic
drugs produces fall in blood pressure and reduction in heart rate. These cardiac effects are
antagonized by muscarinic blocker atropine.2 Muscarinic receptor stimulation in respiratory
tract stimulates secretions and smooth muscle contraction. Atropine in smaller doses causes
mild stimulation of medullary centers and in larger doses causes central excitation.

Procedure
Sprague Dawley rats weighing 200-350 g are used. Animals are anesthetized using a 50:50
mixture of 25% urethane and 1% alpha chloralose at a dose 5 ml/kg per body weight. Trachea is
362 Drug Screening Methods

cannulated and connected to a pneumotachograph that can record inspiration and expiration.
Femoral artery and vein are cannulated using polythene catheters. The catheter placed in
femoral artery is connected to pressure transducer for recording blood pressure and femoral
vein catheter is used for administration of drugs. Alternatively, right jugular vein may be
cannulated for intravenous administration of dose and left carotid artery can be cannulated
with a thin polypropylene tube leading to a pressure transducer for blood pressure recording.
Heart rate derived from the pulsatile pressure signal can be recorded via tachographic beat to
beat conversion with a tachograph preamplifier. Body temperature is monitored with a rectal
probe and is maintained at 37.0 ± 0.5ºC with heating lamps. Immediately after cannulation,
the animals are injected with heparin 600 u/kg so as to avoid clotting. After 30 minutes of
equilibrium period rats are injected with normal saline/test drug and changes in blood
pressure are recorded. Blood pressure is allowed to return to normal between injections. To
evaluate for cholinergic activity the drug is injected after administration of muscarinic blocker
atropine (1-6 mg/kg). To evaluate for anticholinergic activity acetylcholine (2 μg/kg) is injected
after incubation with the test drug.

Mydriasis Test
Mice of either sex, of weight range 25 to 35 g are used. Test drug and the vehicle are injected
intraperitoneally. The pupillary diameters (expressed in mm units) are measured at every 10
minutes interval for the first 60 min of drug administration, and then after 90 and 120 minutes
by a dissecting microscope having a graduated scale. If the pupil diameter exceeds 30/25 mm,
it is called positive quantalmydriatic effect.3

Modification
Rabbits are used instead of mice. At least 3 rabbits per group are taken. The left eyes of the three
rabbits are instilled with 2 drops of 0.1% aqueous solution of atropine sulfate, whereas the left
eyes of other group of three rabbits are instilled with 2 drops (0.2 ml) of 1% aqueous solution
of test compound. The right eye of the used rabbit serves as a control; and after equal intervals,
the pupillary diameters of both the eyes are measured. Average mydriasis is expressed as the
percent increase of the diameter of the test pupil as compared with that of the control pupil.4

Intestinal Spasmolytic Activity in Mice

Fasted Swiss Webster mice of either sex of weight range 25 to 35 g are used. The mice are
administered different doses of test drug orally. The aqueous solutions of soluble salts are used
for administration and resin complexes are used as suspension in 0.5% carboxymethylcellulose.
Test drug solutions are prepared in such a way that the administered dose volume is not
allowed to exceed 50 ml/kg of body weight. After 90 min of administration of the soluble salts
and 2 h after administration of the resin-complex suspension, the animals are anesthetized by
intraperitoneal injection of pentobarbitone. Abdomen is cut open and intestines are exposed.
Methacholine (0.25 µg) is applied locally to the intestines and intestines are observed for a
period of 3 sec. Data are calculated as points: if methacholine induced contractions are
completely prevented, it is scored as 2 points, whereas 1 point is scored if the contractions are
only in a limited area or reduced in intensity.5 The scores obtained are converted to the percent
 Drugs Acting on Parasympathetic Nervous System 363

of the ideal mean score and Litchfield and Wilcoxon method is used to calculate the median
protective dose.6

Continuous Cystometry in Rats

Micturition in rats is mediated mainly by the actions of acetylcholine and ATP, with later
controlling the initiation of micturition process and the former maintaining the sustained
bladder pressure during micturition. It has been reported that the anticholinergics like
atropine reduce the contractility of detrusor and this property can be utilized for evaluation of
anticholinergic activity of test drugs in rats.7

Procedure
Sprague Dawley rat weighing 230-250 g are used. The animals are anesthetized with
pentobarbital sodium (50 mg/kg). A tracheostomy tube is placed to help the respiration and
femoral artery is cannulated for administration of drugs. The urinary bladder is exposed after
midline incision and a double lumen catheter is inserted at the dome of the bladder. One
lumen of catheter is used for infusion while the other is connected to pressure transducer for
recording the bladder pressure. At first the bladder is emptied and covered with saline soaked
cotton swabs. After an equilibrium period the bladder is emptied and then continuously infused
with warm saline (25-30°C) at a rate of 0.1-0.2 ml/min. At least three micturition cycles are
recorded to obtain baseline values of urodynamic variables such as basal pressure, micturition
pressure (maximum bladder pressure during micturition), bladder capacity (residual volume
+ volume of infused saline voided) and spontaneous contractile activity (mean amplitude and
frequency of bladder contractions before micturition). After the control cystometry, the animal
is administered with the test drug and 10 min later the micturition cycles are recoded again
to calculate the changes in urodynamic variables. Following administration of muscarinic
antagonists like atropine bladder capacity and residual volume increase, the amplitude and
frequency of spontaneous bladder contractions decrease and micturition pressure decreases.
Atropine can be used as standard in a dose of 1 mg/kg.

Guinea Pig Bronchospasm Model

Parasympatholytic activity of test drug can be evaluated by monitoring the effects on bronchial
smooth muscles in guinea pigs.8

Procedure
Adult guinea pigs of either sex weighing 400-600 g are used. The animals are placed in a
chamber with inbuilt nebuliser and are exposed to acetylcholine chloride (10%) aerosol
under a constant pressure of 40 mm Hg. The time interval between the time at exposure and
beginning of dyspnea is noted. As soon as the dyspnea appears the animals are removed
from the chamber and placed in fresh air. The average time required for dyspnea to appear in
response to acetylcholine is calculated.
The experiment is repeated after pretreatment of animals with the vehicle/test drug.
Anticholinergic drugs increase the time required for dyspnea to appear following exposure
to acetylcholine as they have a relaxant effect on bronchial smooth muscles. Atropine sulfate
2 mg/kg can be used as standard.
364 Drug Screening Methods

Mice Tremor and Salivation Model

Stimulation of central muscarinic receptors elicits several responses including tremors and
hence the tremors evoked by intravenous, subcutaneous, intraperitoneal or intraventricular
administration of cholinergic agonists have been used to characterize central muscarinic
receptor stimulation in vivo. Peripheral muscarinic receptor stimulation causes increased
salivation and blockade of these receptors results in dry mouth. Therefore, these effects have
also been used to investigate cholinergic agonists and antagonists in vivo.

Procedure
Mice of either sex weighing 20-25 g are used. Test substances are administered intraperitoneally
before subcutaneous injection of oxotremorine (muscarinic agonist; 50-75 µg/kg) at the
back of the neck. Tremor intensity and salivation are assessed over a period of 60 min post-
oxotremorine injection. Tremor intensity can be scored based on the following scale: 0 =
no tremor, 1 = slight tremor (moderate, discontinuous tremor), 2 = strong tremor (intense
continuous tremor involving the whole body). Similarly, degree of salivation is scored as
follows: 0 = baseline salivation checked by placing an absorbent tissue under the mouth,
1 = slight salivation (weak and continuous salivation), 2 = excessive salivation (profuse and
continuous salivation). Alternatively, salivation can be quantified by determining the weight
of a preweighed, absorbant foam cube after it has been used to swab the oral cavity. The
cholinergic agonists potentiate and cholinergic blockers inhibit the effects of oxotremorine on
tremors and salivation.9

Modification
Rats weighing 150-200 g may also be used. The procedure used is the same as described for
mice, however, the dose of oxotremorine is 150-200 µg/kg. Alternatively, acetylcholine (875
micrograms/kg) may also be used.9,10

In Vitro Models

Guinea Pig Ileum

Guinea pig ileum is the most commonly used preparation for screening of parasympatho­
mimetic as well as parasympatholytic agents.

Procedure
Guinea pig of either sex (weight range 250 to 550 g) is used. The animal is sacrificed by stunning.
The abdomen is cut open by a midline incision. A cord is tied around the intestine just distal
to the pylorus. The intestine is gradually removed, mesentery being cut away as necessary.
When the colon is reached, the intestine is cut half-way through, so that the glass tube can
be inserted. Tyrode solution is passed through the tube and the intestine until the effluent
solution becomes clear. Mesentery around the colon is removed. A tissue clamp is attached
to the distal end. Pieces of intestine (2-3 cm in length) are cut. One piece is fixed with a tissue
clamp, suspended in a 15-20 ml tissue bath and the clamp is tied to a writing lever. Writing
 Drugs Acting on Parasympathetic Nervous System 365

lever, which magnifies the contractions 5-10 times, is used. When the spontaneous movements
of the muscle subside, acetylcholine (0.01 mg/ml) is added to the bath. Subsequently, doses
are increased as per requirement. The procedure is repeated at 10 min interval and 2-3
submaximal contractions are recorded. After getting a response, tissue is washed out every
time. When acetylcholine produces a response of 70-90% of maximal, the test substance is
added; and if contractions are observed, then it is considered as a stimulant of smooth muscles
(parasympathomimetic). If contractions do not occur and addition of acetylcholine produces
diminished response of acetylcholine, it is taken as an antagonist of acetylcholine.11,12

Modification
Apart from guinea pig ileum, the jejunum of the rat can also be used. Rest of the procedure
is same as above. This test has the advantage over the guinea pig ileum as it gives only feeble
responses to ganglion stimulants such as nicotine.13

Isolated Eye of Rodents

Rats, mice or guinea pigs with pigmented iris are used for this experiment. Immediately after
sacrificing the animals, the eyeballs are enucleated. The retrobulbar structures are removed
from the eyeball. If corneal removal is necessary, a through-and-through stab incision is given
with the help of wheeler knife. The cornea is then cut free along the limbal margin. The eyes are
washed in Krebs-Ringer solution and mounted in appropriately sized, hemispherical sockets
set in black Lucite trays. The trays are submerged in a bicarbonate buffered Krebs-Ringer
solution of approximately 20 ml. The chamber of clear Lucite is designed for this experiment,
so that it gets fitted to the standard mechanical stage of a microscope. The pH of the mixture
is maintained at 7.4 by continuously bubbling with 95% oxygen and 5% carbon dioxide. The
temperature is kept at 37°C by means of a 75 watt heating lamp. The microscope containing
ocular micrometer disk with a magnification of approximately 30 times is used to read the
pupillary diameter.
The eyes are placed in the chamber and pupillary diameter is measured as control after a
period of 30 min. The used Krebs solution is then replaced with fresh Krebs solution, which
contains known concentration of the test substance. The pupillary diameter is then measured
after 30 min and the response is expressed as a ratio of this diameter to the control diameter.
The above method is also used to determine parasympatholytic activity of a test drug. By
antagonism of miosis (induced by cholinomimetic drug), anticholinergic drugs are evaluated.14

In Vitro Assay for Anticholinesterase Activity

Acetylcholinesterase activity is detected by the assay described by Ellman et al.15 This assay
is based on measurement of the change in absorbance at 412 nm. In this assay, thiol ester
acetylthiocholine was used as a substrate. To detect inhibition of enzyme activity, 2.89
ml phosphate buffer, 0.1 ml of DTNB and 10 ml of sample (serum, plasma, blood or brain
homogenate) are mixed and incubated for 10 min. After addition of substrate, absorbance
is recorded using spectrophotometer. The rate of change of absorbance is determined and
enzyme activity is calculated. Different concentrations of inhibitors of enzymes are used and
again rate of reaction is recorded. The percent inhibition as compared to standard activity is
calculated.
366 Drug Screening Methods

Isolated Frog Rectus Muscle

The rectus muscle of frog is isolated and suspended in a bath containing 7 ml of frog ringer
solution. The fluid is replaced every 5 min by frog ringer solution containing acetylcholine. The
effect of acetylcholine is recorded for 90 sec on a kymograph. After that, tissue is washed out.
This response is recorded for at least three times. Now to detect the test compound activity,
ninety seconds prior to addition of acetylcholine, test drug is added in the bath. Response is
observed for 90 seconds. Parasympatholytic drug will decrease the effect of acetylcholine.3

Rat Isolated Aorta

The smooth muscles of aorta show a relaxation response when treated with cholinergic drugs.
The rat aorta can be used to evaluate this activity.

Procedure
Male Wistar rats of either sex weighing 250-350 g are used. The animals are killed by
intraperitoneal sodium pentobarbital and thoracic aorta is dissected clear of the connective
tissue and is transferred to a plate containing cold Krebs’ solution. Three mm ring segment from
the aorta is suspended in a 20 ml organ bath containing Krebs’ solution at 37°C and aerated
with O2 + CO2 (95:5), under 2.0 gram tension with the help of a steel wire. The preparation
is allowed to equilibrate for 60 min before starting the experiment. The relaxant response
to cholinergic drugs can be observed after precontracting the aortic rings with phenylephrine
(10-7-10-6 M). Acetylcholine in a concentration range of 10-12-10-5 M can be used as standard. The
relaxations are expressed as a percentage of the precontraction levels with phenylephrine.16

Guinea Pig Trachea

Tracheal smooth muscles contract in response to parasympathomimetic drugs like
acetylcholine and relax in response to parasympatholytic drugs. The evaluation of test drugs
can be done in guinea pig tracheal smooth muscle preparation.

Procedure
Guinea pigs of either sex weighing 300-350 g are used. The animals are sacrificed by stunning
and exsanguination. The trachea is dissected out and transferred to a dish containing Kreb,s
solution. The trachea is cut in to 2-3 mm wide rings and 6 such rings are connected to each other
with the help of a silk thread. The tracheal ring preparation is now suspended under 1 gram
tension in a 10 ml organ bath containing Krebs’ solution is 37°C and continuously aerated with
O2 + CO2 (95:5). The composition of Krebs’ solution was as follows (mM): NaCl (118), KCl (4.7),
NaHCO3 (25), KH2PO4 (1.2), MgSO4 (2.5), CaCl2 (2.5), and glucose (11.1). Carbachol (1 µM) is
used as standard. A dose response curve for cumulative concentrations of carbachol is first
obtained. The preparation is washed thoroughly with Kreb’s solution and the dose response
curve for carbachol is repeated after incubation with different doses of the test drug for 10 min.
The shift of dose response curve of carbachol to right indicates the parasympatholytic activity
of the test drug. 17
 Drugs Acting on Parasympathetic Nervous System 367

Guinea Pig Isolated Heart

The rate and force of contraction of heart is altered upon exposure to cholinergic and
anticholinergic drugs. Guinea pig isolated heart preparation can be used for evaluation of test
drugs for cholinergic/anticholinergic activity.18

Procedure
Guinea pigs of either sex weighing 600-800 g are used. Guinea pigs are injected with heparin
(1000 units) in the ear vein to avoid damage to the heart muscles by clots and then animals
are killed by a sharp blow to head. The heart is removed along with the aorta, which is cut
at just below the point of its division and is transferred quickly to a dish containing Tyrode’s
solution. Preparation is gently squeezed to remove all the blood and is then transferred to
the perfusion apparatus containing Tyrode’s solution at 37°C and continuously aerated with
O2 + CO2 (95:5). The aorta is tied to the glass cannula with the precaution that no air bubble
enters the aorta. The heart is suspended by a thread through a hook into the ventricle. The
thread is connected to spring levers to record contractions. After 30 min of stabilization the
baseline recordings are done for amplitude and rate of contractions. Subsequently, the test
drug in various doses is added to the perfusion chamber and changes in amplitude and rate
of contractions is recorded. To evaluate for cholinergic activity, the drug is injected after
incubation with muscarinic blocker atropine (1-6 mg/kg). To evaluate for anticholinergic
activity acetylcholine (2 μg/kg) is injected after incubation with the test drug.

Rabbit Isolated Corpus Cavernosum

Parasympathetic stimulation elicits erection by enhancing the coordinated relaxation of the
cavernous vessels and trabecular smooth muscles via the release of ACh and nitric oxide
leading to increased blood supply to the sinusoids.19

Procedure
Male New Zealand white rabbits weighing 1.5–2.5 kg are used. Animals are anesthetized
with pentobarbitone sodium and exsanguinated via the carotid artery. After performing
penectomy, corpus cavernosum is dissected in the Krebs solution and cleared of the tunica
albuginea and surrounding tissues. Strips of rabbit isolated corpus cavernosum are mounted
in an organ bath with warmed (37°C) and oxygenated (95% O2 + 5% CO2) Krebs solution
at a flow rate of 5 ml/min. Contractions of corpus cavernosum strips are recorded using
isotonic or isometric transducer. Tissue strips are, at first, allowed to equilibrate for 90 min
and then precontracted with noradrenaline (10−6 M). The tissues may also be continuously
infused with indomethacin (5.6 mM) to inhibit the generation of cyclo-oxygenase products.
Test substance or acetylcholine (0.3–30 nMol) are administered as single bolus injections
(10-50 µl). Atropine (1 µM), is infused over the isolated tissues 20 min before and during a
bolus injection of the agonists and significantly inhibits the acetylcholine-induced relaxation
of rabbit isolated corpus cavernosum tissue.20
368 Drug Screening Methods

References
1. Hobbiger F. Anticholinesterases. In Laurence DR, Bacharach AL (Eds). Evaluation of Drug Activities:
PharmacometricsVol –II. London and New York: Academic Press 1964:467.
2. Salahdeen HM, Yemitan OK, Alada ARA. Effect of aqueous leaf extract of tridaxprocumbens on
blood pressure and heart rate in rats. Afr J Biomed Res 2004;7:27- 9.
3. De Elio FJ. Acetylcholine antagonists: a comparison of their action in different tissues. Br J
Pharmacol Chemother 1948;3:108-12.
4. Edwards BK, Golberg AA, Wragg AH. Spasmolytic esters of N-sustituted alpha aminophenylacetic
acids. J Pharm Pharmacol 1960;12:179-86.
5. Becker BA, McCarthy LE. A comparison of the antispasmodic activities of atropine and scopolamine
and their N-methyl derivatives in mice by an in vivo technique. Arch Intern Pharmacodyn Ther
1960;126:307-14.
6. Litchfield JT, Wilcoxon F. A simplified method of evaluation of dose-effect experiments. J Pharmacol
Exp Ther 1949;96:99-113.
7. Kwak TI, Lee JG. Inhibitory effects of propiverine, atropine and oxybutynin on bladder instability in
rats with infravesical outlet obstruction. Br J Urol 1998;82:272-7.
8. Kumar DA, Ramu P. Effect of methanolic extract of benincasa hispida against histamine and
acetylcholine induced bronchospasm in guinea pigs. Indian J Pharmacol 2002;34:365-6.
9. Ogren SO, Carlsson S, Bartfai T. Serotonergic potentiation of muscarinic agonist evoked tremor and
salivation in rat and mouse. Psychopharmacology (Berl) 1985;86(3):258-64.
10. Murray CW, Cowan A, Wright DL, Vaught JL, Jacoby HI. Neurokinin-induced salivation in the
anesthetized rat: a three receptor hypothesis. J Pharmacol Exp Ther 1987;242(2):500-6.
11. Munro AF. The effect of adrenaline on the guinea pig intestine. J Physiol 1951;112:84-94.
12. Paton WD, Zar MA. The origin of acetylcholine released from guinea pig intestine and longitudinal
muscle strips. J Physiol 1968;194:13-33.
13. Van Rossum JM, Ariens EJ. Pharmacodynamics of parasympathetic drugs, structure-action
relationships in homologous series of quaternary ammonium salts. Arch Int Pharmacodyn Ther
1959;118:418-44.
14. Beaver WT, Riker WF. The quantitative evaluation of autonomic drugs on the isolated eye. J
Pharmacol Exp Ther 1962;138:48-56.
15. Ellman GL, Courtney KD, Andres V Jr, et al. A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.
16. Brahmadevara N, Shaw AM, MacDonald A. Alpha, 1-adrenoceptor antagonist properties of CGP
12177A and other beta-adrenoceptor ligands: evidence against beta3 or atypical beta-adrenoceptors
in rat aorta. Br J Pharmacol 2004;142:781-7.
17. Castillo JC, de Beer EJ. The tracheal chain-I. A preparation for the study of antispasmodics with
particular reference to bronchodilator drugs. J Pharmacol Exp Ther 1947;90:104-9.
18. Adome RO, Gachihi JW, Onegi B, Tamale J, Apio SO. The cardiotonic effect of the crude ethanolic
extract of Nerium oleander in the isolated guinea pig heart. S African Health Sci 2003;3:77-82.
19. Simonsen U, Garcia-Sacristan A, Prieto D. Penile arteries and erection. J Vasc Res 2002;39: 283-303.
20. Teixeira CE, Bento AC, Lopes-Martins RA, et al. Effect of Tityus serrulatus scorpion venom on
the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres.Br J
Pharmacol 1998;123(3):435-42.
 cHAPTER

24
Neuromuscular Blocking Agents

INTRODUCTION
Neuromuscular blocking drugs act at myoneural junctions to block the neuromuscular
transmission causing paralysis of the affected skeletal muscle. The neuromuscular blockers can
act presynaptically at receptor sites so as to inhibit the synthesis and release of acetylcholine
or postsynaptically at acetylcholine receptors. Although, some drugs like botulinum toxin and
tetradotoxin can act presynaptically, the clinically relevant drugs act postsynaptically. The
neuromuscular blocking agents fall in two categories:
a. Depolarizing agents, e.g. succinylcholine
b. Nondepolarizing agents, also known as competitive blockers, e.g. tubocurarine.
During evaluation of neuromuscular blocking agents, the following parameters should
be studied:1 (a) potency (initial intravenous mg/kg paralysing dose); (b) time required for
development of maximal effect after rapid intravenous administration; (c) duration of action
of a single paralysing dose; (d) type of block produced by the initial dose; (e) cumulative effect
or tachyphylaxis on repeated administration; (f ) change in characteristics of the block after
repeated or prolonged administration; (g) effect of tetanic stimulation or exercise on the
course of the block; (h) side effects, e.g. autonomic, histamine-releasing and (i) reversibility
by antagonists. Numerous experimental methods have been devised to obtain these data;
some can be used only in experimental animals; others are suitable for clinical testing in man
(Tables 24.1 and 24.2).

Table 24.1: Models used for evaluation of neuromuscular blockers in animals

Unanesthetized Anesthetized In vitro

• Production of contractures in birds • Nerve-muscle preparations of • Isolated nerve-muscle preparations
• Testing of righting reflex lower limbs • Frog nerve-muscle preparations
• Inclined screen and inverted grid • Phrenic nerve-diaphragm • Phrenic nerve-diaphragm
methods preparations preparation
• Rotating drum method • Close intra-arterial injection • Lumbrical nerve-muscle preparation
• Head drop method • Determination of dose causing in rabbits
• Injection into lymph sac of frog cardiac arrest • Ionophoretic micro-application
• Facial nerve stimulation technique
• Reversal of apnea in mouse • Electrical activity of single muscle
fiber
370 Drug Screening Methods

Table 24.2: Models used for evaluation of neuromuscular blockers in human

Unanesthetized Anesthetized In vitro

• Test of grip strength • Measurement of respiratory • Fetal phrenic nerve-diaphragm
• Test of exercise capacity of hand parameters preparation
muscles • Measurement of twitch response • Intercostal nerve-muscle
• Measurement of voluntary activity to indirect stimulation preparation
of other muscles
• Assessment of twitch response
following indirect stimulation
• Measurement of respiratory
parameters
• Electromyographic study
• Intra-arterial injection

Neuromuscular blocking agents show different effects in different species. Variation

in effects may also be observed among different members of the same species and even in
different muscles of the same individual.2 Neuromuscular blockers may show different effects
in in vitro preparations and in intact animals.3 For this reason, observations made on the
neuromuscular effects of an agent in one species in one set of experimental conditions cannot
be extrapolated to another species in other experimental conditions. Results obtained from
testing in animals cannot be directly applied to man and pharmacologic investigations of each
compound in human subjects must be carried out before clinical use.4

EVALUATION IN LABORATORY ANIMALS

The marked species differences in sensitivity to neuromuscular blockers5 emphasize the
importance of parallel studies of these compounds in different species.

Intact Unanesthetized Animals

Induction of Contractures in Birds
Administration of a depolarizing blocker in adult fowls, chicks or in pigeons causes a rigid
extension of the limbs and retraction of the head.6 If the dose is lethal, the animal dies in this
rigid condition; if the dose is below the lethal level, the recovery is abrupt. Curare, on the other
hand, causes flaccid paralysis in birds. The advantage of using this test on avian muscle is the
ease with which the difference in the action of these two groups of drugs can be strikingly
illustrated.

Testing of Righting Reflex

This reflex may be checked by turning the animal on its back and watching to see if the animal
rolls back over onto its sternum. Disappearance of righting reflex in mice, rats and rabbits may
be used to determine potency of neuromuscular blocking agents.7
 Neuromuscular Blocking Agents 371

Inclined Screen and Inverted Grid Methods

Doses graduated at 0.1 logarithmic intervals are given subcutaneously to groups of 10 mice
each. Mice at each dose level are placed on a screen inclined at 50° from the horizontal. Those
mice developing typical skeletal muscle paralysis and abruptly sliding off the screen within half
an hour after injection are considered positive reactors. This method is used to estimate ED50.8
Mice or rats can also be tested for their ability to stay on an inverted grid after administration
of neuromuscular blocking agents.

Rotating Drum Method

This tests the ability of small laboratory animals to remain upright in a revolving drum after
treatment with the drug.9 In mice the drug is administered subcutaneously. Immediately after
injection, the mice are placed in a rotating cylinder and mice falling away from the cylinder
during the first twenty minutes are considered as reactors.

Head Drop Method

This method measures the minimal intravenous dose of the drug required to produce head
drop. It is applicable to rabbit, mouse, guinea pig, rat, dog and monkey. The end point of
the assay, head drop is the precise relaxation state when the animal’s head drops forward to
the supporting surface and cannot be raised in response to a light tap on the back.10 Doses
graduated at 0.1 logarithmic intervals are given by intravenous injection at the rate of 1 ml/5
seconds to groups of ten rabbits each. After injection, the rabbits are placed in a large enclosure
on the floor where they can be observed for the occurrence or absence of head drop. The dose
producing head drop in 50% of the rabbits (HD50) is then calculated.

Injection into Lymph Sac of Frog

This method was first described by Bernard.11 Drugs administered via lateral or dorsal lymph
sacs in frogs are rapidly absorbed into the blood. Profound curarisation can be induced in
frogs without the need for artificial respiration as respiration occurs primarily through skin.

Anesthetized Animals
Nerve-muscle Preparations of Lower Limbs
Neuromuscular blocking agents can be tested using nerve-muscle preparations of the lower
limbs in rats, cats or dogs.7 Animals are anesthetized and artificially ventilated. The sciatic
nerve is isolated and cut. Distal end of the sciatic nerve is stimulated supramaximally and the
twitch response of the gastrocnemius, soleus or tibialis muscle is recorded.12 Diaphragmatic
and intercostal respiration is recorded. An induced current is used to stimulate the peripheral
end of the sectioned nerve once every 10 sec. After a suitable period, during which a series
of contractions of uniform height are obtained, a dose of the test drug is injected rapidly into
the femoral vein. Observation is continued until complete recovery occurs. An initial dose
of the test drug is selected which produces a partial inhibition of the muscle twitch. When
the amplitude of the muscle contractions and respiration return to the preinjection level,
subsequent doses of the test drug are increased by 0.1 logarithmic intervals, until complete
372 Drug Screening Methods

arrest of nerve impulse transmission is obtained. A crossover comparison may be obtained

in the same animal, by administering a graduated series of a second test drug in a similar
manner.8
Alternatively, the twitch response of tibialis anterior can be observed after stimulation by
supramaximal square wave stimuli 0.2 min duration applied to common peroneal nerve at 0.1
Hz. The parameters which can be used to compare the drug treated group with control include
the onset time (time interval between the end of injection and maximal block), maximal block
(twitch height depression as percentage of control twitch height), recovery time (time between
recovery from 25 to 75% of control value) and duration of action (time interval between end of
injection and 90% recovery).12
Tetanic stimulation of nerve-muscle preparations may be used to differentiate depolarizing
and nondepolarizing blocks.

Phrenic Nerve-Diaphragm Preparations

The central stump of the phrenic nerve is isolated in the neck and the nerve action potential
is recorded. Simultaneously, electrodes are placed on the ventral surface of the diaphragm to
obtain an electromyographic recording.13

Facial Nerve Stimulation

New Zealand white rabbits weighing 3.5-4.5 kg are used. Rabbits are sedated by intramuscular
injection of ketamine (20 mg/kg) and xylazine (0.05 mg/kg). Rabbits are now intubated and
placed under general anesthesia with halothane. The EMG system is set up and subdermal
electrode needles are placed in orbicularis oculi and paranasal tissue. The tympanic portion
of facial nerve is exposed through middle ear and baseline stimulations are carried out and
measured. An induced current (0.05-1.00 mA) is used to stimulate the facial nerve. Once a
series of contractions of uniform height is obtained, various doses of the test drug/vehicle
are now administered intravenously. These doses are preceded and followed by facial nerve
stimulation and monitoring. Vecuronium at a dose of 0.025 mg/kg intravenously can be used
as standard to compare the effects of test drug.14

Close Intra-arterial Injection

Close intra-arterial injection of neuromuscular blocking agents allows accurate estimation of
potency and time required for onset of action and also eliminates the influence of distribution
or breakdown en route to the muscle.15

Determination of Dose Causing Cardiac Arrest

The dose of neuromuscular blocker producing cardiac arrest is determined in artificially
ventilated animals.8

Reversal of Apnea in Mouse

Succinylcholine, a depolarizing neuromuscular blocker, has a very short duration of action
as it is hydrolyzed by butrylcholinesterase. Patients who carry genetic or acquired deficiency
 Neuromuscular Blocking Agents 373

of butyrylcholinesterase are susceptible to succinylcholine-induced apnea. Geyer et al. have

shown that a purified recombinant human BChE serves as an ideal antidote for succinylcholine
apnea.16
Mice are anesthetized with ketamine/xylazine cocktail. Respiratory rate is counted and SpO2
is recorded in anesthetized animals. Mice are then injected intravenously (tail vein) with 1 mg/
kg succinylcholine/experimental drug/vehicle. Respiratory rate and SpO2 are monitored every
2-3 min. Three minutes after injection, butrylcholine esterase/vehicle is injected to reverse
the succinylocholine-induced apnea. Since, this dose of succinylcholine is higher than LD50,
all animals injected with succinylcholine and 3 min later with vehicle die of apnea but those
receiving butylcholinesterase survive at the end of 15 min. Succinylcholine like experimental
drugs with properties of depolarizing neuromuscular blockade show a similar response.16

In Vitro Studies
Although, experiments on intact animals may reveal valuable information, in vitro experiments
allow accurate control of more variables and are likely to be useful in the analysis of the mode
of action of drugs. These methods eliminate the influence of circulation, distribution and
metabolic transport.

Isolated Nerve-Muscle Preparations

The neuromuscular blocking agent to be studied is added to the bathing fluid into which the
amphibian or mammalian muscle is immersed. Electrodes, stimulator and recording system are
used. The muscle may be stimulated directly or through the proximal end of the nerve, which is
kept outside the bathing fluid. The stimulus rate is 5 per min and working temperature is 40°C.
The bathing fluid used is a modified Krebs solution. Glucose is added immediately before use.
A gas mixture containing 5% CO2 and 95% O2 is equilibrated with the bathing fluid before use.
When first set up, the nerve-muscle preparation often shows a partial neuromuscular block,
which rapidly recovers in the presence of adequate oxygenation. The twitch tension increases
for about an hour and so some time has to be allowed for equilibration of the preparation.

Frog Nerve-Muscle Preparations

Isolated frog gastrocnemius muscle preparation was first used in the classical Claude Bernard11
experiment in which two nerve-muscle preparations were arranged so that the test drug was
applied exclusively to the nerve of one and the muscle of the other. Isolated sartorius and
rectus abdominis muscle may be used.17

Phrenic Nerve-Diaphragm Preparation

The effects of neuromuscular blocking agents on isolated respiratory muscles may be studied
in phrenic nerve-diaphragm preparations from rats18 and guinea pigs.19 Rat diaphragm
preparation is relatively insensitive to decamethonium and similar agents.5 Diaphragms from
other animals must be from very young animals or from fetuses so that they are thin enough
for oxygen diffusion.
A parallel-sided slip of diaphragm is removed with the phrenic nerve. Stimuli are applied
alternately to the muscle directly, via electrodes at each end, and to the phrenic nerve. A running
374 Drug Screening Methods

control is thus obtained against effects on the muscle as opposed to effects on neuromuscular
transmission.
Alternatively, nerve-evoked maximal twitches (T1, T2, T3, T4) of the rat hemidiaphragm
to train of four (TOF) stimulation (2 Hz for 2 s every 20 s) can be recorded continuously in
the presence and absence of vehicle/test drug. The T1 and T4 response-time profiles can
be compared with respect to amplitude depression and the TOF ratio (T4/T1) during the
development of and recovery from neuromuscular blockade.20

Lumbrical Nerve-Muscle Preparation in Rabbits

The rabbit lumbrical muscle is sensitive to depolarizing blockers. However, it is expensive
and difficult to work with; it is vestigial in some rabbits and sometimes has an aberrant nerve
supply. Three lumbrical muscles are present in the foot of the rabbit but only the medial
of these is generally thin enough to allow adequate oxygen diffusion and at the same time
strong enough to operate a lever to record its contractions. The lever bearing and writing tip
must be chosen for minimal friction. The lever must be weight-loaded to stretch the muscle
optimally and ensure a linear relation between the height of the recording and the work
done on the lever by the muscle. Depending upon the time taken during dissection, a partial
neuromuscular block may be seen when the preparation is set up. This block is due to anoxia
and recovers completely in a few minutes. The preparation is very sensitive to changes in ionic
concentration. A fall in the concentration of hydrogen, calcium or magnesium ions, especially
calcium, induces spontaneous activity, repetitive response to stimulation and irregularity of
behavior, while altering the sensitivity to all types of blocking agents.21

Ionophoretic Microapplication Technique using Isolated Tenuissimus

Nerve-Muscle Preparation in Cats
The technique has been described by Thesleff.22 Tenuissimus nerve-muscle has the advantage
of being covered by only a thin layer of connective tissue and has generally a number of
superficially located end plates, which suit the microapplication of drugs. Furthermore,
the tenuissimus muscle can be maintained for a long time in oxygenated Ringer solution
without showing signs of deterioration. In these experiments, twin micropipettes with a tip
diameter less than 1µ are used. One micropipette contains acetylcholine, while the other
contains the drug to be tested. A micromanipulator is used to move the drug pipette to an
effective position at a superficial end plate. When the tip of the pipette is close to the receptor
structure, a brief positive current pulse applied to the barrel containing acetylcholine releases
sufficient amount of drug to produce, in the end plate, a transient depolarization of a few
millivolts amplitude and a rapid time course. This potential change is recorded by inserting
a microelectrode into the muscle fiber at a distance of about 100 µ from the point of drug
application. The position of the drug pipette is adjusted so that stable and maximal responses
are obtained when acetylcholine is released by current impulses of constant intensity and
duration. With the tip of the pipette in this position, the test drug contained in the second barrel
is released in a similar way or by a constant current. Since, the tips of the two drug pipettes
are not more than 2 µ apart, both drugs affect the same receptor structure, and it is possible to
compare their effects or study their interaction. The current passing through the drug pipette
 Neuromuscular Blocking Agents 375

is recorded using an oscilloscope. The advantages of this method are that it is rapid, diffusion
times are reduced to a minimum, and much faster events can be studied. Furthermore, the
removal of the drug is automatic; there is no need for long periods of washing and many
different applications can be made to the same receptor area. The drawback, however, is the
uncertainty as to the local drug concentration obtained due to the spatial distribution of drug
receptors.

Electrical Activity of Single Muscle Fiber

Frog sartorius muscle is used. The nerve is cut close to its point of entry into the muscle. The
muscle is mounted in a bath of Ringer solution on an illuminated stage and observed from
above with a binocular microscope. The bath is divided into two compartments by means of
a partition and the muscle is drawn through a gap in this partition. Stimulating electrodes
are placed in the two compartments. The microelectrode assembly is mounted on the arm
of a micromanipulator and attached to a probe. The recording equipment consists of a DC
amplifier and oscilloscope. A large amount of information can be obtained in a relatively short
time since, the process of mounting and recording from a muscle fiber takes only a few seconds.
In this way, average values for a large number of fibers can be determined with reasonable
accuracy and the effect of environment can be assessed by comparing the properties of groups
of fibers from the same muscle.23

Evaluation in Man

In Unanesthetized Subjects
Test of Grip Strength
Onset, duration of action, relative potency and tachyphylactic or cumulative properties of
neuromuscular blocking agents can be assessed using this test.24 Grip strength, measured
by a dynamometer, is determined immediately before and after the exercise, consisting of
the squeezing of the bulb of an ergograph apparatus for 1 min with the maximum effort of
which the subject is capable, before and at 3, 5 and 10 min after the start of the injection of the
neuromuscular blockers and at 5 min intervals thereafter. In preliminary studies, the dose of
each drug, which produces a 90 to 95% decrease of grip strength, is determined.25

Test of Exercise Capacity of Hand Muscles

Information about fatigability of the hand muscles during partial depolarization block can be
obtained by this method. Ability of subjects to squeeze the bulb of an ergograph at variable
rates is recorded. 25

Measurement of Voluntary Activity of Other Muscles

Assessment of neuromuscular blockers may be made by recording the muscular strength of
finger, foot and abdomen.26
376 Drug Screening Methods

Assessment of Twitch Response Following Indirect Stimulation

In the above-mentioned methods, emotional factors may influence the action of neuromuscular
blocking agents. To eliminate this, contraction of voluntary muscles caused by indirect
stimulation of the corresponding nerves may be made.27

Measurement of Respiratory Parameters

Effect of neuromuscular blocking agents on respiratory muscles can be assessed by measuring
vital capacity and maximal expiratory pressure.21 In the preliminary experiments, the dose of
each drug which produces a 50-55% decrease of vital capacity is determined.25

Electromyographic Study
Response of individual muscles to indirect stimulation during partial depolarization or
nondepolarization block can be assessed using this method.28

Intra-arterial Injection
Close intra-arterial injections can be given in conscious subjects and the effect of neuromuscular
blocking agents on the electromyogram can be studied.

In Anesthetized Subjects
Measurement of Respiratory Parameters
In anesthetized subjects, maximal inspiratory pressure can be measured to assess the degree
of paralysis of respiratory muscles.29 The dose of neuromuscular blocking agent, which causes
paralysis of all respiratory muscles, can be measured by monitoring tidal volume.30

Measurement of Twitch Response to Indirect Stimulation

In anesthetized subjects, muscle contractions can be measured after indirect stimulation of
the corresponding nerve to assess the action of neuromuscular blocking agents.31

In Vitro Studies
Fetal Phrenic Nerve-diaphragm Preparation
This isolated nerve-muscle preparation is also used to study the effect of neuromuscular
blocking agents in vitro.32

Intercostal Nerve-Muscle Preparation

This material may be obtained by biopsy under regional anesthesia and may be used to study
the effect in vitro of neuromuscular blocking agents.33

Effect of Anticholinesterases
Neuromuscular blocking agents can be characterized further by assessing the effect
of anticholinesterases on their action in conscious and anesthetized human subjects.
 Neuromuscular Blocking Agents 377

Anticholinesterases antagonize the nondepolarizing block and prolong the depolarization

block.34

CHARACTERIZATIONS OF DEPOLARIZING AND NONDEPOLARIZING

BLOCKERS
In laboratory animals, if intra-arterial injection is followed by a spontaneous muscle twitch,
tetanus is well maintained during partial neuromuscular block, and there is no post-tetanic
stimulation, the compound is a depolarizing agent.28 On the other hand, absence of a twitch
response after intra-arterial injection, poorly maintained tetanus during partial neuromuscular
block, and presence of post-tetanic facilitation are characteristics of nondepolarizing agents.35
In birds depolarizing agents produce contracture, while nondepolarizing agents produce
flaccid paralysis.6
In human subjects, if intravenous administration of the compound produces relatively less
effect on vital capacity as compared to effect on grip strength and produces easy fatigue of
the hand muscles after rapid rate of exercise, this indicates that the agent being tested is a
nondepolarizing agent. On the other hand, depolarizing compounds affect both vital capacity
and grip strength, but exercise at a rapid rate during partial neuromuscular block caused by
them does not produce fatigue.25
The type of block produced by the compound tested may be determined by intravenous
administration of edrophonium chloride, which will rapidly antagonize the nondepolarization
block.36

Discussion and Conclusion

There is considerable disagreement regarding the applicability of various methods used for
testing neuromuscular blocking agents. To obtain complete information about neuromuscular
blocking activity of any substance, a variety of in vitro and in vivo tests must be applied to
different species including amphibians, birds and mammals. The species of test animal used
and all experimental conditions must be specified when potency, onset, duration of action,
etc. are recorded.1
Preliminary information of the pharmacological actions of neuromuscular blocking agents
can be obtained by relatively simple tests. Depolarizing and nondepolarizing compounds can
be differentiated by the production of contracture or paralysis after intravenous injection into
birds.6 The head-drop method can be used to compare relative potency, onset and duration of
action of neuromuscular blockers, without elaborate equipment.10
For more detailed information, experiments using nerve-muscle preparations in intact
animals,15 isolated nerve-muscle preparations22 and observations on single muscle fiber
with intracellular electrodes37 should be used. Close intra-arterial injection,15 observation of
the effect of tetanic stimulation on the twitch13 and electromyogram28 will provide additional
information.
Following testing of a neuromuscular blocking agent in animals, it must be tested in human
subjects before it can be introduced into clinical practice.1 For preliminary testing conscious
subjects are used. After intravenous administration, observation of the magnitude and time
378 Drug Screening Methods

course of changes produced in grip strength and vital capacity should be made. Unexpected
hypersensitivity to both depolarizing and nondepolarizing agents can be seen in apparently
normal subjects; and for this reason, during testing on human subjects, equipment and
personnel required for respiratory resuscitation must be available.38 Information regarding
cumulation, tachyphylaxis and changes in characteristics of the block can be obtained in
anesthetized subjects.39 Side effects of neuromuscular blocking agents including ganglionic
blockade, histamine release and excessive tracheobronchial secretions should also be studied
thoroughly before its introduction into clinical practice.4

References
1. Foldes FF. Animal and clinical techniques for evaluating neuromuscular blocking agents, In Nodine
JH, Siegler PE (Eds): Animal and Clinical Techniques in Drug Evaluation. Chicago: Year Book
Medical Publishers Inc 1964:383-91.
2. Foldes FF. Factors which alter the effects of muscle relaxants. Anesthesiology 1959;20:464-504.
3. Zaimis EJ. Factors influencing the action of neuromuscular blocking substances. In: Lectures on the
scientific basis of medicine. New York: John de Graar 1957:208-18.
4. Foldes FF. The pharmacology of neuromuscular blocking agents in man. Clin Pharmacol Ther
1960;1:345-95.
5. Paton WD, Zaimis E. The methonium compounds. Pharmacol Rev 1952;4:219-53.
6. Buttle GAH, Zaimis EJ. Action of decamethonium iodide in birds. J Pharm Pharmacol 1949; 1: 991-2.
7. Hoppe JO. Observations on potency of neuromuscular blocking agents with particular reference to
succinylcholine. Anesthesiology 1955;16:91-124.
8. Hoppe JO. A pharmacological investigation of 2,5-bis-(3-diethyl-aminopropylamino) benzoquinone
-bis-benzylchloride (Win 2747): a new curarimimetic drug. J Pharmacol Exp Ther 1950;100:333-45.
9. Collier HO, Hall RA, Fieller EC. Use of rotating drum in assessing activities of paralysant, convulsant
and anesthetic drugs. Analyst 1949;74:592-6.
10. Varney RF, Linegar CR, Holaday HA. Assay of curare by rabbit head drop method. J Pharmacol Exp
Ther 1949;97:72-83.
11. Bernard C. Lessons on the effects of toxic and medicinal substances: 1857. Cah Anesthesiol
1991;39:55-60.
12. Proost JH, Wierda JM, Houwertjes MC, Roggeveveld J, Meijer DK. Structure-pharmacokinetics
relationship of series of aminosteroidal neuromuscular blocking agents in the cat. J Pharmacol Exp
Ther 2000;292:861–9.
13. Paton WD, Zaimis EJ. Action of D-tubocurarine and of decamethonium on respiratory and other
muscles in the cat. J Physiol 1951;112:311-31.
14. Hester TO, Hasan A, McDonnell F, Valentino J, Jones R. Facial Nerve Monitoring under
Neuromuscular Blockade. Skull Base Surgery 1995;5:69-72.
15. Brown GL. Preparation of tibialis anterior (cat) for close intra-arterial injection. J Physiol
1938;92:22P-23P.
16. Geyer BC, Larrimore KE, Kilbourne J, Kannan L, Mor TS. Reversal of succinylcholine induced
apnea with an organophosphate scavenging recombinant butyrylcholinesterase. PLoS One.
2013;8(3):e59159.
17. Swanson EE, Gibson WR, Powell CE. Comparative potency of D-tubocurarine chloride USP and
dimethyl ether of D-tubocurine iodide. J Am Pharm Assoc 1952;41:487-97.
 Neuromuscular Blocking Agents 379

18. Bulbring E. Observations on the isolated phrenic nerve-diaphragm preparation of the rat. Br J
Pharmacol Chemother 1946;1:38-61.
19. Jenden DJ. The effect of drugs upon neuromuscular transmission in the isolated guinea pig
diaphragm. J Pharmacol Exp Ther 1955;114:398-408.
20. Cheah LS, Gwee MCE. Train of four fade during neuromuscular blockade induced by tubocurarine,
succinylcholine or alpha–bungarotoxin in the rat isolated hemidiaphragm. Clin Exp Pharmacol
Physiol 1988;15:937–43.
21. Jenden DJ, Kamijo K, Taylor DB. The action of decamethonium on the isolated rabbit lumbrical
muscle. J Pharmacol Exp Ther 1954;111:229-40.
22. Thesleff S. A study of interaction between neuromuscular blocking agents and acetylcholine at
mammalian motor endplate. Acta Anaesthesiol Scand 1958;2:69-79.
23. Nastuk WL, Hodgkin AL. Electrical activity of single muscle fiber. J Cell Comp Physiol 1950;35:39-
73.
24. Unna KR, Pelikan EW. Evaluation of curarizing drugs in man: VI Critique of experiments on
unanesthetized subjects. Ann N Y Acad Sci 1951;54:480-92.
25. Foldes FF, Monte AP, Brunn HM Jr, et al. Studies with muscle relaxants in unanesthetized subjects.
Anesthesiology 1961;22:230-6.
26. Poulsen H, Hougs W. Effect of some curarising drugs in unanesthetised man. Acta Anaesthesiol
Scand 1957;1:15-39.
27. Botelho SY. Comparison of simultaneously recorded electrical and mechanical activity in
myasthenia gravis patients and in partially curarised normal humans. Am J Med 1955;19:693-6.
28. Churchill-Davidson HC, Richardson AT. Decamethonium iodide (C10): some observations on its
action using electromyography. Proc R Soc Med 1952;45:179-86.
29. Churchill-Davidson HC, Christie TH. The diagnosis of neuromuscular block in man. Br J Anaesth
1959;31:290-301.
30. Foldes FF, Wolfson MB, Torres KM, et al. The neuromuscular activity of hexamethylene 1, 6-bis
carbaminoylcholine bromide (Imbretil) in man. Anesthesiology 1959;20:767-75.
31. Christie TH, Churchill-Davidson HC. The St. Thomas’s hospital nerve stimulator in diagnosis of
prolonged apnea. Lancet 1958;1:776.
32. Buller AJ, Young IM. Action of D-tubocurarine chloride on foetal neuromuscular transmission and
placental transfer of this drug in the rabbit. J Physiol 1949;109:412-20.
33. Dillon J, Fields J, Gumas T, et al. An isolated human voluntary muscle preparation. Proc Soc Exp Biol
Med 1955;90:409.
34. Koppanyi T, Vivino AE. Prevention and treatment of D-tubocurarine poisoning. Science
1944;100:474-5.
35. Hutter OF. Post-tetanic restoration of neuromuscular transmission blocked by D-tubocurarine. J
Physiol 1952;118:216-27.
36. Randall LO. Anticurare action of phenolic quarternary ammonium salts. J Pharmacol Exp Ther
1950;100:83-93.
37. Graham J, Gerard RW. Membrane potentials and excitation of impaled single muscle fibers. J Cell
Physiol 1946;28:99-117.
38. Pelikan EW, Tether JE, Unna KR. Sensitivity of myasthenia gravis patients to D-tubocurarine and
decamethonium. Neurology 1953;3:284-96.
39. Artusio JF Jr, Marbury DE, Crews MA. Quantitative study of D-tubocurarine, tri-(diethylamino­
ethoxy)-1,2,3-benzene (Flaxedil) and series of trimethyl and dimethylethyl-ammonium compounds
in anesthetized man. Ann N Y Acad Sci 1951;54:512-29.
 cHAPTER

25
Antiviral Drugs
INTRODUCTION
There are more than hundred viruses known to cause diseases in humans and the need for
antiviral drugs is growing rapidly as more and more new viral pathogens are discovered.
More than 40 antiviral drugs are approved for clinical use currently and about 20 of them are
used for the treatment of HIV infection and the rest for the treatment of other viral diseases.1
Currently, antiviral drug development strategy is focused to address two main issues: (i) further
improvement of existing antiviral therapy such as for hepatitis B (HBV) and C (HCV) viruses,
herpes simplex virus (HSV) and influenza viruses. (ii) development of new antiviral drugs for
the infections that do not have approved therapy yet, but their prevalence is significantly high
and they are associated with considerable morbidity, mortality and high social and economic
implications (hemorrhagic fever viruses, human papilloma viruses, severe acute respiratory
syndrome (SARS) coronavirus).2

Viruses and Viral Life Cycle

Viruses are ultra-microscopic, acellular infectious agents that are obligatory intracellular
parasites. Viruses consist of: (i) the genetic material which is either DNA or RNA, (ii) a protein
coat (capsid) and in some cases, (iii) an envelope of lipoproteins surrounding the protein coat
that is acquired from the host cell membrane. Viruses do not have their own organelles and
metabolic processes and hence they can replicate only inside the living host cells.
Virus life cycle depends on the type of virus, but generally includes 6 phases, each of which
could be a potential target for new antiviral drugs. The phases in the virus life cycle include the
following:
1. Attachment on the surface of host cell
2. Penetration/entry into the host cell
3. Uncoating
4. Gene expression and replication viral DNA or RNA
5. Virion assembly
6. Release of new virions.
 Antiviral Drugs 381

Challenges in Development of New Antiviral Drugs

The research in the field of antiviral drug development has dramatically increased over past
few decades, however, very few new drugs have been approved for clinical use. Besides high
cost, the discovery of new antiviral drugs is associated with some challenges and limitations:
i. Lack of unique targets: As stated above, viruses consist of only the genetic material and
protein coat and they completely lack their own metabolic system. It leads to insufficient
viral-specific targets for antiviral drugs and hence the possibility of their low efficacy and
high toxicity.
ii. Limited availability of in vitro and in vivo models: Some of viruses do not naturally infect
animals and cannot replicate in nonhuman cells. Hence, the availability of animal models
and in vitro systems is limited making it difficult to investigate newer drugs using multiple
models.
iii. High specificity of antiviral drugs. Most of the compounds can target only a single infectious
agent and their use for more than one viral infections remains limited.
iv. Emergence of antiviral drug resistance. Viruses are generally characterized by fast
replication that results in increased genetic variability and, therefore, greater possibility
of development of drug resistance. This further complicates the development of new
antiviral drugs.

IN VITRO MODELS
In vitro models are generally used prior to in vivo testing for the screening of potential
compounds in terms of their cytotoxicity and antiviral efficacy. Usage of in vitro models is less
time consuming and more cost effective compared to the in vivo models. Moreover, for some
of the viral infections in vivo models are not available or require a particular animal species, for
example chimpanzees or other primates, and could be limited by the ethical issues.
Most of the in vitro antiviral drug studies are based on virus-induced cytopathic effects and
include the detection of median cytotoxic concentration (CC50), median effective concentration
(EC50) and selectivity index (SI).
The selection of the type of cells for viral propagation and experimental drug treatment
depends on the virus itself. Some viruses, for example HSV, influenza viruses can replicate in
different cell lines while for others such as HVB and HVC, replication in cell lines or primary
cell cultures is very slow and variable and requires development of the replicon cell culture
systems.3

Replicon Cell Culture Model for HCV

Discovery of potential therapeutics against HCV was significantly hampered because of
lack of the ability of HCV to replicate in cell culture. The situation has been changed by the
development of HCV replicon culture system.4
Generally, replicon is defined as DNA or RNA molecule that is able to replicate. Subgenomic
replicon is derived from the Con1 strain of HCV and recapitulates the intracellular steps of the
viral replication in cultured human hepatoma Huh-7 cells.5 HCV replicon has some advantages
compared to wild-type HCV:
382 Drug Screening Methods

i. Subgenomic replicon is able to replicate at high level in cell culture system that allows its
easy detection and quantitative analysis.
ii. Subgenomic replicon is avirulent and does not support virus production, thus can be used
in a standard cell culture laboratory without particular biosafety concerns.
iii. Persistence of replicon in cell culture causes emergence of drug resistance that could be
tested as well.
The HCV replicon system has some disadvantages as well. It is not a complete viral particle
and cannot represent entire viral life cycle and some of key steps in the virus life cycle, such as
cell entry, uncoating, RNA packaging, virion assembly and release are not represented.4

Cell Culture Systems

Vero cells
Vero cells are a continuous cell line originally isolated from kidney epithelial cells of African
green monkey (Cercopithecus aethiops) by Yasumura and Kawakita. Vero cells are very easily
maintained and are extensively used in virology studies as host cells for viral propagation.
They are also used for the screening of cytotoxic and therapeutic effects of drugs. One of the
most important characteristic of Vero cells making them extremely attractive for virology
and screening of antiviral compound is their inability to produce interferon.6,7 However,
Vero cells do have the interferon-alpha/beta receptors and are able to respond to interferon
treatment.
Vero cells have been shown to support replication of different viruses including rabies virus,
reovirus, Japanese encephalitis virus, dengue fever virus, influenza A and B viruses.8,9
There are several lines of Vero cells that are available (Vero, Vero 76, Vero E6, Vero F6) but
all of them are derived from the same source and follow the same protocol.10 Vero cells are
cultured in Dulbecco’s modification of Eagle medium (DMEM), supplemented with 10% heat-
inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells grow on standard
culture plates and should be incubated at 37°C in the presence of 5% CO2. Vero cells are derived
from normal kidney cells so they have not lost their contact inhibition. Actively growing Vero
cell culture doubles approximately every 24 hours and when cells reach confluence, they stop
growing and die. To avoid that and keep cells in monolayer the cells need to be passaged 2-3
times per week depending on the number of cells seeded and the flask size.11

Huh-7 Cells
The classical cell culture system used for HCV is the human hepatoma cell line Huh-7. Huh-
7 is a well-differentiated hepatocyte derived cellular carcinoma cell line that was originally
obtained from a liver tumor in a 57-year-old Japanese male.
Currently Huh-7 subclones (Huh-7.5, Huh-7.5.1 and Huh-7-Lunet) support more efficient
viral replication and production compared to original cell line.12
While Huh-7 cells support high level of HCV replication and virus production, they are not
normal hepatocytes and have lost some essential properties and characteristics of normal cells.
Thus, there are some limitations in the assessment of antiviral effects of the investigational
drugs such as their effect on immune response, expression of certain markers etc.3 However,
Huh-7 cells and their subclones are still considered the best available in vitro model for HCV.
 Antiviral Drugs 383

Other cell culture systems used for HCV

Recently, other non-Huh-7 cell lines (Huh-6, HepG2, IMY-N9, LH86 etc.) and primary human,
mouse and chimpanzee hepatocytes have shown the ability to maintain HCV replication.
However, viral replication in these cell culture systems was found to be lower compared to that
in Huh-7 cells.3
Huh-6 cell line is derived from hepatoblastoma and is characterized by low production
of Claudin-1, an integral membrane protein responsible for the formation of tight junction
strands between cells. Initially, Huh-6 cells were not susceptible for HCV but addition of
ectopic Claudin-1 made them support HCV replicon. Huh-6 cells are also highly resistant to
interferon-γ treatment making them potentially attractive for antiviral drug screening.13

Cell Culture Systems used for HBV

HBV could be reproduced in primary hepatocytes. However, this cell culture model has
limitations due to inadequate viral replication, low viral production and poor reproducibility.
HepG2 is an immortal cell line derived from the well-differentiated hepatocellular carcinoma
of a 15-year-old Caucasian American male. Since, HepG2 cells are well-differentiated they
secrete a large range of plasma proteins, such as albumin, transferrin, and the acute-phase
proteins like fibrinogen, alpha 1-antitrypsin, transferrin, plasminogen etc. They have been
grown successfully in different cultivation systems and well support the replication of HBV
and HCV replicon. 14 HepG2 cells especially 2.2.15 line better support replication of HBV and
are widely used to evaluate potential active anti-hepatitis B virus compounds.15,16
The other cell culture systems used for HBV include HepAD38, HepAD79, YMDD, PDH.

Cell Culture System used for Influenza Viruses

Influenza viruses A and B are not as demanding as HCV and HBV and may be cultivated in
many cell culture systems including Madin Darby canine kidney (MDCK), chick embryo, chick
kidney, calf kidney, Vero, mink lung, and human respiratory epithelial cells.17

Determination of the Toxicity of the Drug

Assessment of the toxicity of the antiviral drug is based on detection of median cytotoxic
concentration (CC50). CC50 is the concentration of the drug required to reduce the cell number
by 50% compared to untreated control. CC50 could be identified using a number of cell viability
tests (Fig. 25.1).
There are a number of tests available to assess the cytotoxicity of the investigational
compound. The cell viability tests are based on various cell functions such as enzyme activity,
cell membrane permeability, ATP production, nucleotide uptake activity, etc. Among the other
tests enzyme-based calorimetric tetrazolium salts tests are the most commonly used because
of their reproducibility, safety and easy methodology.

MTT assay
The reduction of tetrazolium dyes such as 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium
bromide (MTT) by NAD(P)H-dependent oxidoreductase leads to appearance of formazan
384 Drug Screening Methods

Figure 25.1: Determination of CC50

products that have intense blue or purple color.18 Oxidoreductase is largely present in the
mitochondria and cytoplasm of actively metabolizing cells. Therefore, non-viable or dead
cells have low level of oxidoreductase and lose the ability to convert MTT into formazan
and hence the color formation. The colored formazan crystals are water insoluble and have
to be dissolved in DMSO or any other solubilizer. The absorbance of the dye is measured
spectrophotometrically at a certain wavelength (usually 570 nm) using an automated
microplate reader. The absorbance corresponding to that of untreated control cells is assumed
as 100% cell viability.
The percentage of viable cells in the treated group can be calculated as follows:

Absorbance of treated cell

% Cell viability = × 100
Absorbance of untreated cells

MTS, XTT and WST Assays

More recently developed MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide) and WST (water soluble tetrazolium salts) assays contain
tetrazolium reagents that undergoes reduction by viable cells to water soluble formazan
products. This improves the assay procedure because a second addition of solubilizing reagent
is not required. Measurement of absorbance is usually done at 490 nm.

Determination of Antiviral Activity of the Drug

Antiviral activity of the drug is expressed by the median effective concentration (EC50) which
is the concentration of the drug required to inhibit virus replication by 50 percent (Fig. 25.2).
The antiviral activity can be tested by a number of methods, including reduction assays,
cytopathic effect inhibition assays, binding and fusion assays, etc.
Some of the most commonly used antiviral assays are based on virus-induced cytopathic
effects (CPE), plaque formation or hemagglutination. These methods have disadvantages of
 Antiviral Drugs 385

Figure 25.2: Determination of EC50

being time-consuming and labor-intensive, which limit their use for screening. Further more,
these methods are less acceptable because different virus strains may differ in their ability to
cause cytopathic effects or hemagglutination.
In contrast to cytopathic effect based viral assay, test of polymerase chain reaction (PCR) or
quantitative real-time PCR (qRT-PCR) are characterized by much greater sensitivity, specificity
and reproducibility. They are currently used for detection of viral DNA or RNA (PCR) and
measurement of viral load (qRT-PCR).

PCR
Principle of PCR is based on amplification of a single copy or a few copies of a particular
DNA sequence across several cycles and finally generating thousands to millions of copies
of primal DNA sequence. Heat-stable DNA polymerase, such as Taq polymerase, is used as
an amplifier assembling new DNA strands from single-stranded DNA sample (DNA target).
DNA oligonucleotides (DNA primers) that are complementary to the particular DNA template
are used for the initiation of DNA synthesis. PCR consists of a series of repeated temperature
cycles. Each of them usually includes few steps with different temperature set up as required
for initiation and synthesis of new DNA copies. The number, level and time of the temperature
steps depend on a wide range of parameters, such as: the type of DNA-polymerase, the
concentration of ions, desoxyribonucleotides, etc.
PCR is sensitive, specific and reproducible method however it does not allow quantitation
of DNA or RNA and detection of viral load that is essential for determining the efficacy of
antiviral drug.

Quantitative Real-Time PCR

qRT-PCR defers from the standard PCR as it allows detection of the amount of DNA or RNA
formed after the each cycle with fluorescent dyes or fluorescently-tagged oligonucleotide
probes. An increase in DNA/RNA products during PCR, therefore, leads to an increase in
386 Drug Screening Methods

fluorescence intensity allowing DNA concentrations to be quantified. Each sample is assigned

a specific value in real-time PCR, defined as cycle threshold (Ct), which is the point or cycle
number when the fluorescence curve for that sample exceeds background fluorescence and
measurements become meaningful. Samples with the highest starting target amount will also
have the highest values of amplified target in a given PCR cycle number. The values obtained
from qRT-PCR do not have absolute units associated with them but express the amount of
measured DNA/RNA in the sample as a fraction of the standard.
The relative DNA or RNA level of each target nucleic acid is expressed as change by number
of folds relative to the value of corresponding control. Thereby qRT-PCR allows quantitation of
nucleic acids and detection of viral load to identify EC50.

Determination of selective index (SI)

The relative effectiveness of the investigational drug in inhibiting viral replication compared to
its cytotoxicity is defined as the therapeutic or selectivity index (SI)
CC50
SI =
EC50

SI allows selecting the compound with highest antiviral activity and minimal cell toxicity.

IN VIVO MODELS
In vivo studies are essential part for antiviral drug discovery. A number of parameters may be
used to determine the effectiveness of antiviral drug in animal models. The choice of these
parameters depends on the type of viral infection and the animal model used. However, the
most commonly used parameters include viral infection associated death, mean time to death,
change in water and food intake, change in weight, hyper or hypothermia, blood cell count,
coagulation parameters, biochemical parameters, viral titer and histopathological changes in
relevant organs and tissues etc.
Unfortunately in vivo models available for antiviral drug development studies are very
limited (Table 25.1). This can be explained by few reasons: (i) some viruses cannot replicate in
nonhuman species or require adaptation by multiple passage through the animal model, (ii)
most of the human viruses cannot produce typical clinical picture in nonhuman species, (iii)
high cost and ethical issues associated with particular animal models.
All animal models used in the in vivo studies could be divided in 3 groups:
1. Chimpanzee and other great apes
2. Nonhuman primates
3. Other animal models.

Chimpanzees (Pan troglodytes)

Chimpanzees are the closest living relative to humans sharing more than 98% genetic
identity.19 This explains the greater suitability of chimpanzees compared to all other great apes
as an animal model for vaccine development and antiviral drug discovery. Chimpanzees are
 Antiviral Drugs 387

Table 25.1: Common animal models for various viral infections

Type of virus Animal model

Cytomegalovirus Primates, transgenic immunocompromised mice
Dengue virus Nonhuman primates, transgenic immunocompromised and humanized mice
Ebola and Marburg viruses Nonhuman primates (rhesus and cynomolgus macaques), mice, guinea pigs, syrian
golden hamsters
Hepatitis B and C Primates, tree shrews, transgenic and humanized mice
HSV Mice, rabbits
Influenza viruses Ferrets, guinea pigs, transgenic mice
Japanese encephalitis virus Syrian golden hamsters
SARS corona virus Syrian golden hamster, mice
West Nile virus Syrian golden hamsters

naturally susceptible to most of the human viral photogenes and have the same pathogenic
mechanisms, similar clinical symptoms and course of illness as humans. In addition large
size of animals provides enough volume of biological materials and facilitates appropriate
investigations. Chimpanzees are used as models to study pathogenesis, vaccination and
pharmacokinetics and pharmacodynamics of new candidates for the treatment for some viral
infections such as norovirus,20 hepatitis B21-23 and hepatitis C.24,25
However, the use of chimpanzees as an animal model is very limited mostly because of
ethical considerations, low availability, and extremely high cost.

Other Nonhuman Primate Models

The genetic proximity of nonhuman primates (NHP) to humans makes them a very attractive
animal model for antiviral drug discovery. NHP such as Cynomolgus macaques, Rhesus
macaques, Green monkeys, Chacma Baboons, Cottontop tamarins and marmosets are more
commonly used models for the development of antiviral drugs and vaccines compered to
chimpanzees. For example, different macaque species are used as a model for West Nile virus
fever, Japanese encephalitis viral infection, avian influenza (H5N1), cytomegalovirus (CMV)
infection, dengue, ebola, marburg and some other virus infections.26-28
However, the high cost and ethical issues still limit the use of nonhuman primate as an in
vivo model for antiviral drug development.

Tree Shrews (Tupaia Belangeri)

The tree shrews are squirrel-like mammals belonging to Primate order and sharing with them
some similarities in anatomy (including the brain anatomy) and phylogenesis. Apparently due
to their proximity to primates, they are one of the main animal models used for the studies of
various aspects of hepatitis B and C infection including the investigations for efficacy of new
antiviral drugs.15 Many studies have demonstrated that both HBV and HCV enter and replicate
in tree shrew primary hepatocytes and infected animals develop hepatitis.29-32 Closeness to
primates, small size and uncommonness make tree shrews currently a very useful model for
HBV and HCV research.
388 Drug Screening Methods

Other Animal Models

Mice
Mice and other rodents have traditionally been used as a lab model because of many advantages
such as low cost, ease of maintenance and care, small size, which significantly reduces the
amount of drugs and other chemicals needed for experiments. Another important advantage
of these models is the availability of the large range of reagents, quantification assays,
microarray proteome expression and luminex technology-based quantification assays, etc.
required for evaluation of disease progression, immune response, histopathological changes
and viral titer.
Mice are the most widely used animal model for influenza virus and HSV studies.33-35
However, most of wild-type of human viruses do not replicate and transmit efficiently and/or
do not produce typical clinical signs of viral infections in normal immunocompetent murine
lines, requiring to be adopted by the serial passage in suckling, aged or immunocompromised
mice to show clinical features and high levels of viral titer.36 Hence, models currently used for
antiviral drug screening studies are mainly transgenic and humanized mice.

Transgenic Mice
Transgenic mice are genetically modified mice with altered genome. Currently, several
thousand strains of transgenic mice are available. Usually they are named for the gene which
has been disrupted. The genetic engineering technology allows creating the strains of mice
with different properties and increases their sensitivity to human viruses.
Knockout mice are lines of genetically modified mice where the activity of a single
gene is removed or disrupted. Currently, a large range of knockout mice strains is available
for research including pathology, immunology of viral infections and antiviral drug
development.
Knockout AG129 mice lacks interferon α, β and γ receptor genes and is more susceptible to
virus compared to wild-type mice.37 This model has been proven useful as a model for dengue
virus infection and in vivo testing of some antiviral compounds.38 IKK epsilon mutant mice
have loss of the endogenous kinase function in lung, spleen, and embryonic fibroblasts. These
mice have increased susceptibility to viral infection due to defective interferon (IFN) signaling
and are used as in vivo models for influenza virus infection.
UPA mice carry the mouse urokinase-type plasminogen activator (uPA) gene. The
overexpression of the uPA gene in the liver results in extensive liver toxicity leading to chronic
hepatic insufficiency. It also causes high plasma uPA levels and hypofibrinogenemia, which
could result in severe bleeding.

Genetically Humanized Mice

Genetically humanized mice are the mice that express human host factors and usually have
inactivation of the production of murine proteins in parallel.
MUP-uPA/SCID/Bg transgenic mice is constructed by the backcross of two murine lines:
Severe combined immunodeficiency mice subclone (SCID/Bg) and MUP-uPA. This line
expresses the secreted form of human uPA and is used as a model for HCV infection.
 Antiviral Drugs 389

Xenotransplantation Mice
Transplantation of human cells or tissues to mice may be very helpful in terms of
increasing replication of particular viruses and production of clinical signs of the infection.
Immunodeficient mice such as SCID and their subclones are usually used to produce
xenotransplantation mice. SCID mice have genetically impaired both the humoral and cellular
immunity and thus are able to sustain xenografts.
Human liver-uPA/SCID mice are produced by the cross breeding of uPA and SCID mice
followed by intrasplenical injection of human hepatocytes. Transplantation of donor cells
should be done within the 2nd week of life. Intrasplenically injected donor cells rapidly migrate
through the portal venous system into the liver and support higher level of HCV replication
compared to murine hepatocytes.39
Hu-PBL-SCID mice are constructed by the injection of peripheral blood lymphocytes to
SCID mice and are currently widely used for studies of HIV pathogenesis and anti-HIV drugs.
They were first used as a dengue model in 1995.40

Rats
Rats are not commonly used as in vivo models for antiviral drug research however, some of
them such as cotton rats are used for the study of influenza A viruses.41,42

Guinea pigs (Cavia Porcellus)

Guinea pigs are very commonly used as an alternative model to mice in viral research.
Guinea pigs are susceptible to several viruses and are used as animal models for human viral
hemorrhagic fevers, human influenza viruses, HSV, filovirus infection (Ebola and Marburg
virus infections) and others. 26,43,44 However, limitations are there due to lack of available
reagents and specific assays (ELISA, Western Blot, PCR, qRT-PCR, etc.) to monitor host
responses, including immune responses, pathological changes and viral load.

Syrian Golden Hamster (Mesocricetus Auratus)

Syrian golden hamster is widely used as animal model for human infectious diseases caused
by bunyaviruses (Crimean-Congo hemorrhagic fever virus), flaviviruses (West Nile virus,
dengue virus, Japanese encephalitis virus, yellow fever virus), henipaviruses, and SARS-
coronavirus.45,46 The main drawback of this model is the same as for guinea pig model, i.e. lack
of reagents and specific assays.

Ferrets (Mustela Putorius Furo)

The ferrets are attractive and well-established animal model for numerous viruses, such as
coronavirus, nipah virus, morbillivirus, influenza viruses and others.47 Ferrets and humans
share similar lung physiology, and human and avian influenza viruses exhibit similar
mode of interactions with cellular receptors present in respiratory tract.47 Moreover, ferrets
are very suitable for antiviral drug research because of their relatively small size and ability
to exhibit many of the typical clinical signs associated with human disease, especially with
regard to influenza virus such as nasal discharge, loss of appetite, congested eyes, otologic
manifestations and fever.48
390 Drug Screening Methods

CONCLUSION
In conclusion, at the present time, the amount of research for the screening and investigation
of new antiviral drugs is increasing due to multiple reasons. Recent advances in the techniques
of cultivation and detection of viruses on one hand has contributed to identification of new
pathogens and pathogen characteristics while on the other hand there is increasing need
for antiviral drugs because of significant outbreaks of well-known viral infections as well as
emergence of new viral infections. However, the discovery of new effective antiviral drugs faces
enormous limitations due to lack of available in vitro and in vivo models.

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 cHAPTER

26
Antipsychotic Agents
Introduction
The word psychosis was first used by Ernst von Feuchtersleben in 1845 and stems from the
Greek words psyche (mind) and -osis (diseased or abnormal condition). According to Adolf
Meyer (1866–1950), psychosis or schizophrenia is not a discrete disorder with a specific
etiology but, rather, as a reaction to a wide variety of biopsychosocial factors. Factors such as
gene mutations, brain injury, drug use (cocaine, amphetamine, marijuana, phencyclidine, and
steroids), prenatal infection and malnutrition, social isolation and marginalization, have been
implicated in the manifestation of the signs and symptoms of schizophrenia. The standardized
mortality rate in schizophrenia is about 2.5, with life expectancy between 15 and 20 years.
Mortality due to CVD is major contributor and causal factors such as lifestyle, poor diet, lack
of physical activity, smoking, and substance abuse, have been identified.1 The etiopathology
of psychosis is complex and genetic basis has been understood to play a definitive role in its
genesis with as much as 40-50% risk of developing schizophrenia in population of identical
twins. Consequently, pharmacogenetics plays pivotal role in management of schizophrenia,
and is known to affect dosage, treatment response, and occurrence of adverse effects. 2
Overactivity of dopamine neurotransmission and supersensitivity to dopamine has been
understood to be the biochemical basis of schizophrenia and the condition is successfully
alleviated by drugs that block dopamine D2 receptors.3 Currently, a number of effective
palliative antipsychotic agents including tricyclics, phenothiazenes, thioxanthenes,
dibenzepines, butyrophenones and other congeners are available. Besides the lacunae such
as delayed onset of action (2 to 3 weeks after initiation of therapy), severe extrapyramidal
neurological effects, sedation, hypotension and autonomic side effects, the pharmacological
agents used for treating psychotic disorders also have side effects that augment CVD
risk.1 This is owing to their wide array of action which includes not only blockade of D2
dopamine receptors in forebrain but also D1 dopaminergic, 5-HT2 serotonergic and
α-adrenergic receptors.
In the past, number of novel antipsychotic drugs were developed with the aim of obtaining
agents displaying therapeutic advantages over the first drug generation, often designated
“typical” antipsychotic drugs. These include a lower propensity to elicit extrapyramidal
side-effects, an improved therapeutic efficacy on negative and affective symptomatology
of schizophrenia as well as on refractory forms of the disease. These drugs are designated
394 Drug Screening Methods

as “atypical” antipsychotics”. Most of the drugs in this new class have been modeled after
the prototype drug clozapine. Although, atypical antipsychotic agents possess numerous
advantages, they are not devoid of side-effects, creating a need to develop newer agents.
Understanding of underlying neurobiology, neurotransmission anomalies and genetic
basis of schizophrenia has made it possible to design and develop animal models which
simulate the biological phenomena better. The available models can be divided into three sets,
namely, animal models with predictive validity, face validity and construct validity.1 Most of the
currently available models are valid only to make predictions with respect to pharmacotherapy
and show pharmacologic isomorphism. These models provide least insight into the processes
underlying the disease.4
Animal models with face validity are based on symptom similarity and exhibit behavioral
isomorphism like catatonia, stereotypy, impaired performance and social with­drawal.5 These
models are few in number and difficult to design, interpret and replicate. Models that mimic
the psychopathologic disturbances underlying the disease are classified as animal models
with construct validity.6 Genetic models of schizophrenia fall in this category.

IN VITRO and EX VIVO MODELS

H-Prazosin Competition Binding for α-1 Adrenoceptors
3

Direct interaction between a compound and α-1 adrenergic receptor is determined by

measuring the inhibition of binding of a radioactive ligand (3H-prazosin) to the receptor. Rats
(Male Wistar, 200 to 250 g) are housed in a controlled environment with a 12 h day-night cycle
and free access to food and water. The animals are sacrificed; their brains quickly removed,
cerebral cortex is taken out and frozen. The tissues are stored at –70°C.
The membrane preparation of rat frontal cortex is prepared and the protein content
is measured according to the method of Lowry, et al.7 Briefly, the tissue is homogenized in
20 volumes of 50 mM Tris-HCl buffer, pH 7.6. The supernatant after centrifugation of the
homogenate (1,000 g, 10 min) is recentrifuged at 25,000 g for 30 min and the resulting pellet
is stored at –20°C until incubation. Immediately before the assay, the pellet is reconstituted in
50 mM Tris-HCl buffer, pH 7.6 to obtain the final concentration of protein of approximately
1.5 mg/ml. The final incubation mixture contains 450 µl of membrane suspension, 50 µl of the
solution of the radioligand and 50 µl of the Tris-HCl buffer or of solution of displacer. Nine
concentrations of the test drug are incubated in the presence of one concentration (0.146 nM)
of 3H-prazosin. The reference standards used are phentolamine or prazosin. The incubation
is carried out at 25°C for 30 min and is terminated by vacuum-assisted filtration through
Whatman filters. Filters are washed with ice-cold Tris-HCl buffer and placed in scintillation
cocktail. The radioactivity is measured in a liquid scintillation counter and the binding is
corrected for the protein content.
Ki [nM] value is calculated from the formula Ki=IC50/(1+L/KD), where L is the concentration
of the radioligand and KD is its dissociation constant. Ki value for each compound studied is
the mean ± SEM from at least three independent competition-binding studies.
Although, this test is used mostly for in vitro studies, it can also be applied for ex vivo
experiments to determine the effects of a single dose of acutely (or chronically) given test
compound on the in vitro interaction of reference compound with α-1 adrenergic receptors.
 Antipsychotic Agents 395

The above-mentioned procedure can be adopted to study the competitive binding between
other receptors and their ligands (Table 26.1).
Table 26.1: Radioligand competitive binding with the receptor sub-type
Receptor sub-type Radioligand Reference standard
α-2 adrenoceptors 3
H-Clonidine Clonidine
α-1 adrenoceptors 3
H-Prazosin Phentolamine or Prazosin
β-adrenoceptors 3
H-CGP 12177 Propranolol
Benzodiazepine 3
H-Flunitrazepam Clonazepam or Diazepam
D1 dopamine 3
H-SCH 23390 SCH23390
5HT-1A 3
H-8-OH-DPAT 8-OH-DPAT
5HT-2 3
H-Ketanserin Ketanserin
µ opioid 3
H-Dihydromorphine Morphine
Ionophore of the NMDA-ionotropic 3
H-MK-801 Phencyclidine
glutamate receptor
NMDA ionotropic glutamate 3
H-CGP39653 L-glutamate
receptors
Glycine sites of the NMDA ionotropic 3
H-5,7dichlorokynurenic acid DCKA
glutamate receptors (DCKA)
GABA 3
H-Muscimol Muscimol

IN VIVO MODELS
Open Field Test
Automatic recording open field working station is now widely used to record locomotor
activities and stereotypic behaviors of the animals. The test field (43 × 43 × 30 cm) is divided
into 16 identical squares with a grid of infrared photocells around the arena and illuminated
with a dim light (20 lux). On the test day, the animals are individually released into the center
of the box and allowed to explore the field freely for 60 min. The general locomotor activities
and stereotypic behaviors is recorded by light beam interruptions and data are collected
over 30 min by automatic computerized system. Various useful parameters like- ambulatory
distance, ambulatory time, resting time, time spent in central area/peripheral area, time spent
in stereotypic behaviors (small movements such as scratching, grooming, or digging that
repeatedly interrupt only a single optical beam), can be recorded and compared between
different groups.8

Prepulse Inhibition
Acoustic startle and prepulse response is measured using a startle chamber (ventilate plexiglas
cylinder on a plexiglas platform). The mice are subjected to different trial types
i . PULSE ALONE—broadband 120 decibel burst for 40-ms duration
ii. PREPULSE+PULSE—either long 3 decibel for 20 ms duration or long 6 decibel for 120 ms
duration or 12 decibel for 20 ms duration prepulse stimuli are given above a 65 decibel
background and
iii. NO STIMULUS trial, in which only the background noise is presented.
396 Drug Screening Methods

The animal is allowed to acclimatize in trials presented in a pseudo-random order. Whole-

body startle responses of the animal is recorded for each stimulus.9

Sucrose Preference Test

Sucrose preference is assessed using a home cage two-bottle choice test. Animals are singly
housed with ad libitum access to standard chow and two identical water bottles. One bottle is
filled with water, while the other bottle contained sucrose solution (up to 15%). The habituation
phase (0% sucrose, water only) spans over six days and over next four days sucrose solution is
provided. The positions of the bottles are rotated to avoid position preferences. Preference can
be calculated as a ratio of the amount consumed from the sucrose bottle to the total liquid
volume consumed from both bottles.10

Catalepsy in Rodents
Catalepsy in rats is defined as a failure to correct an externally imposed, unusual posture over
a prolonged period of time. This is a typical effect of all agents, which inhibit dopaminergic
system in the nigrostriatum.
Adult Wistar rats of either sex, weighing between 180 to 220 g each are randomly divided into
two groups. One group is dosed with test drug and the other with standard drug (haloperidol
0.5 mg/kg, ip). Catalepsy is evaluated according to the slightly modified method of Delini-Stula
and Morpurgo.11 After an appropriate pretreatment time of the drug, each rat is tested with
respect to its right and left front paws, which are first put on columns 3 cm and then 9 cm high.
The cataleptic state is scored as 1 and 2, respectively (maximum 6 points for the right and left
paws) if a rat maintains an abnormal body posture for more than 10 sec. Catalepsy is scored for
2 or 3 h at 30 min intervals. Three trials are conducted for each animal.
This model tests for the potential of antipsychotic drugs to cause extrapyramidal symptoms
in man. Neuroleptics that cause extrapyramidal symptoms in man produce a cataleptic state
in rats characterized by immobility, body stiffness and inability to initiate movement.
An increase in catalepsy score predicts that the drug may cause extrapyramidal side effects
in humans. If the drug is able to decrease neuroleptic-induced catalepsy, it may have potential
antiparkinsonian activity. L-DOPA (+benserazide) or amantadine is used as a reference
compound.

Inhibition of Amphetamine-induced Stereotypy in Rats

Amphetamine is an indirect sympathomimetic agent. It induces a characteristic stereotypic
behavior (lip smacking, grooming, catalepsy, gnawing) in rats, which can be successfully
prevented by classical neuroleptic agents. The test is predictive for antipsychotic drugs with
D2-receptor antagonism.
Two groups (n = 6) of adult Wistar rats of either sex, weighing between 180 to 220 g are
treated with either test or standard drug and then placed in individual wire cages (21 cm × 21
cm × 23 cm). They are injected with d-amphetamine (5 mg/kg, ip) after 30 min. The onset of
stereotypic behavior, its duration and intensity is evaluated at 30 min intervals for 3 hours. An
animal is protected if the behavior is reduced or abolished. The intensity of stereotype activity
is assessed on an arbitrary rating scale from 0-4 for normal, periodic sniffing, continuous
 Antipsychotic Agents 397

sniffing, licking, gnawing and biting behaviors, respectively. A reduction in mean stereotypy
score is indicative for antipsychotic effect.

Inhibition of Apomorphine-induced Stereotypy in Rats

Apomorphine stimulates dopamine autoreceptors and induces stereotypic behavior
in rodents like pole climbing, licking, sniffing, gnawing and yawning.12 Apomorphine-
induced climbing behavior can be inhibited by antipsychotic drugs and is predictive for the
development of extrapyramidal side effects and tardive dyskinesia. This is a standard test used
for the screening of antipsychotic drugs and has good predictive validity for classical as well as
atypical neuroleptics.
The mice are placed in individual cylindrical cages (12 cm in diameter, 14 cm in width) with
walls made of vertical metal bars (2 mm in diameter, 1 cm apart). After an adaptation period
of 30 min, the mice are treated with either test or standard drug (clozapine 10 mg/kg, ip), and,
1 hour later apomorphine (0.75 mg/kg, sc). Immediately afterwards, the behavior is assessed
for climbing behavior namely all the paws on the floor, forefeet or all the paws held on the wall,
which is rated on an arbitrary scale of 0-2, respectively.
Apomorphine increases the behavioral score by causing climbing behavior in the mice.
Antipsychotic drugs can inhibit this behavior.

Phencyclidine-induced Bizarre Pattern of Locomotor Activity and Stereotypy

Phencyclidine (PCP) is a psychotomimetic compound that can induce a schizophrenia-like
psychosis in man. In rats, PCP produces locomotor hyperactivity and stereotyped behavior
that can be inhibited by antipsychotic drugs.
Male Wistar rats weighing between 200 to 250 g are housed in a controlled environment
with a temperature of 22 ± 2ºC and a 12 h light/dark cycle. The animals have free access to
food and water. Locomotor and stereotypy-like activities of rats are recorded individually for
each animal in an activity-monitor. Each cage is equipped with infrared emitters, located on
the X and Y-axes, and with an equivalent amount of receivers on the opposite walls of the
cage. The locomotor activity is defined as a trespass of three consecutive photo-beams, while
other movements observed as repeated interruption of the same photo-beam are regarded
as stereotypy-like movements. The PCP-induced locomotor and stereotypy-like activities of
rats are measured during a session lasting 150 min. The test may be indicative of possible
antipsychotic activity of tested drug.

Phencyclidine-induced Social Withdrawal Measured in the Social

Interaction Test
This test helps to show the effectiveness of potential antipsychotic drugs against negative
symptoms of schizophrenia. Phencyclidine (PCP) induces stereotyped behavior, hyperactivity
and social withdrawal, in rats. All of these behaviors can be evaluated in the social interaction
test. The test appears to be specific for antipsychotic drugs and can distinguish between effects
on the positive and negative symptoms.13
Male Wistar rats of 250 to 280 g are housed in a controlled environment with a temperature
of 22 ± 2°C and a 12 h light/dark cycle with free access to food and water. In the PCP-induced
398 Drug Screening Methods

social withdrawal test, naive rats are housed in pairs for 10 days prior to the start of the test.
During the test one cagemate (familiar rat) is removed and new one—intruder is placed in
the cage for 10 min. The amount of the social interaction and locomotor activity both for the
resident and intruder is recorded for 10 min. Social interaction is measured as the total amount
of time spent on various elements of the interaction, i.e. sniffing, grooming of partner, genital
investigation and following the partner. Aggressive behavior is not included (i.e. aggressive
posture, sideways threat, biting, boxing, etc.).
Apart from the locomotor stimulant effects, PCP, in a dose of 1-2 mg/kg, decreases the
time of social interaction. The doses used in this test are slightly lower than doses, which are
required to induce clear locomotor stimulant effects.

Conditioned Avoidance Reflex in Rats

In this model, the rodents are conditioned to avoid a noxious stimuli (electric shock) reflexly,
by moving from one chamber to another in response to a cue (light cue or sound buzzer).
Antipsychotic drugs decrease the number of conditioned avoidance responses (CAR) in
response to cue and prolong the total waiting time (TWT).14 Adult male Wistar rats weighing
250 to 300 g are housed in a controlled environment with the temperature at 22 ± 2°C and a 12
h light/dark cycle and free access to food and water.
Rats are trained to avoid an electric shock in an automatically controlled, two-compartment
shuttle box by moving from one compartment to the other during 3 sec of a light conditional
stimulus. If no crossings take place, the light is followed by a 3 sec shock (2 mA during the
training and 2.5 mA during the experimental session). The shock is provided through the grid
floor of the box. The rats are subjected to daily session of 10 trials for 8 consecutive days (with
30 sec intertrial intervals), which lasts for 5 min. Only rats trained for the criterion (a minimum
of 8, out of 10 possible) are used in the experiment. The compounds are administered 30 min
before the test on day 9. Two parameters CAR and TWT are recorded.
The standard drugs used in this method are haloperidol (0.2 mg/kg, ip) and clozapine (10
mg/kg, ip). CNS depressants may be delineated from antipsychotics in that the former affects
both avoidance and escape but the latter at lower doses affects only avoidance with no effect
on escape (i.e. the animal ignores the warning and does not attempt to avoid the noxious
stimulus, but escapes once stimulus is applied). At higher doses, due to ataxia and hypnosis
both avoidance and escape are affected.

Neurodevelopmental Models
From embryology, it is known that minor physical anomalies and dermatoglyphic deviations
are caused by intrauterine disturbances of the first and second trimester, respectively. The
second trimester is the critical period for the migration of neural cells to the cortex and of
dermal cells to the fingertips.15 This is the basis of the neurodevelopmental hypothesis of
schizophrenia and can explain winter peak of birth, obstetric complications, minor physical
anomalies, soft neurologic signs, reduced intelligence, dermatoglyphic differences and
structural brain abnormalities. Based on the epidemiological and clinical correlation studies,
animal models have been designed to test the etiological theories.
 Antipsychotic Agents 399

Gestational Malnutrition Model

This is a model of prenatal protein deprivation, which may induce permanent developmental
brain deficits. Malnutrition affects neurogenesis, cell migration and differentiation causing
disrupted neural circuits and neurotransmitter systems. Thus, deficits in cognition, learning
behavior are apparent. However, these are nonspecific, inconsistent and variable and therefore
of limited validity.16

Viral Infection
Prenatal exposure to virus, like influenza, utero borna disease virus (BDV), and lymphocytic
choriomeningitis virus (LCMV), has been implicated in the genesis of schizophrenia. It is
hypothesized that these viruses act either by inducing pyramidal cell disarray, defective
corticogenesis, hippocampus damage or disruption of the integrity of GABAergic neurons
and excitatory amino acid systems. Although, these are attractive models as they simulate the
etiology at the cellular level, they do not have concrete relevance to schizophrenia.17

Obstetrical and Birth Complications

It is difficult to explore the plausibility of obstetrical and birth complications as a cause for
genesis of schizophrenia in animals. However, there are a few reports indicating that in rat
models of cesarean section and anoxia at birth, there are changes in limbic dopamine function
in adult animals.18,19 As the trauma and risk factors of C-section in humans are far lesser,
obstetrical complications have not been conclusively linked with schizophrenia.

Early Stressful Experience

It has been hypothesized that long-lasting stress in early life affects brain development and
shapes adult behavioral responses. In “Two Hit” model of schizophrenia, the rodents are
subjected to dual insults, namely, aberrant genetic trait and stressful experience (like maternal
separation or social isolation).20 There are concomitant hormonal, neurochemical and
behavioral changes obvious in adult life, which respond to antipsychotic therapy.

Genetic Models
Schizophrenia is a hereditary disorder that involves anomaly of many genes. Targeted
gene deletions or gene transfer techniques have been used to set-up the animal models of
schizophrenia. These models show construct validity. However, the behavior exhibited in
these models does not mimic the disease. These models only serve as tools to study molecular
mechanisms underlying the pathogenesis of the disease.21

G72/G30 transgenic (Tg) mouse model

Bacterial artificial chromosome (BAC) containing over 117,000 bp of genomic region and
encompassing the entire G72/G30 gene complex has to be created. The BAC clone is then
microinjected into the fertilized ova of embryos from a female B6CBAF1/J mouse. The male
Tg founder mouse is mated with female B6CBAF1/J wild-type (WT) mice to keep the line. The
Tg and WT mice of both sexes (2-4 months old) are used in behavioral experiments as test
400 Drug Screening Methods

and control group, respectively. The blind experimenter subjects all animals to behavioral tests
(viz. open field test, prepulse inhibition, Sucrose preference test, Morris water maze, Forced
swimming test etc.) in soundproof behavioral room.22

Single Unit Recording of A9 and A10 Midbrain Dopaminergic Neurons

Dopamine D3 receptors are richly located in the limbic areas. D3 receptors are believed to be
involved in the pathogenesis of schizophrenia. Therefore, antipsychotic agents are targeted
at these sites. In vivo electrophysiological recording can be utilized to screen for compounds
with potential antipsychotic activity. Briefly, the number of spontaneously active midbrain
DA neurons are recorded and studied in anesthetized rats. A decrease in the number of
spontaneously active ventral tegmental area (VTA) (A10) and substantia nigra compacta (SNC)
(A9) DA neurons produced by the repeated administration of compounds may be correlated
with their therapeutic and neurological side effects, respectively.23 Alternatively, extracellular
single unit activity is also recorded to assess the clinical antipsychotic efficacy and side effects
of DA receptor blockers on A9 and A10 DA neurons.24
Male Albino Sprague Dawley rats (200 to 225 g) are anesthetized (chloral hydrate 400 mg/kg,
ip) and mounted in a stereotaxic instrument. A hole is drilled over the A9 and A10 according to
the atlas25 and the dura retracted. Single-barrel microelectrodes are used for recording single-
cell activity. Glass micropipettes are pulled with an electrode puller and the tip broken back
under a light microscope. They are filled with a solution of NaCl (2 M) saturated with fast green
dye (1%). The impedance of the electrodes is usually 0.8 to 1.2 mΩ measured at 135 Hz in vitro
and 1.5 to 2.0 mΩ in vivo. A dopaminergic neuron is identified in the A9-A10 area on the basis
of the following criterion: (1) action potential (>2.5 msec, having a distinct initial segment and
late positive component; (2) a characteristic low-pitch sound, (3) a slow, regular, or bursting
firing pattern; and (4) a spontaneous firing rate of 2 to 9 Hz.26 The number of spontaneously
active DA neurons is determined in 10 different electrode tracks separated from each other
by 200 µm, whose sequence is constant in each experiment.27 Each electrode descent is made
in a slow (1-3 µm/sec), uniform speed with a hydraulic microdrive. The number of DA cells
encountered in these descents are counted. The DA neuronal spikes obtained after the acute
and chronic administration of vehicle, haloperidol, or test drug are analyzed and the following
parameters are calculated: spikes per burst, percentage of events in bursts, mean interspike
interval (interval between successive spikes), coefficient of variation (ratio of standard
deviation and mean interspike interval × 100), and percentage of cells exhibiting burst firing
(defined as DA neurons that show two or more bursts of at least three spikes of a series of
500 spikes). These values are determined over a period of 500 intervals between successive
DA neuron spikes. The onset of a burst is defined by an interval less than 80 msec and the
termination of a burst as an interval exceeding 160 msec.

Extrapyramidal Side Effects Primed Monkey Model

The extrapyramidal side effects (EPS) are one of the most common side effects of anti-psychotic
therapy and includes perioral tremors, tardive dyskinesia etc. Monkeys have been used to set
up a sensitive model that helps to predict the liability of test drug in generating the side-effect
of EPS.28 The monkeys are sensitized to extrapyramidal side effects (EPS) by prior long-term
 Antipsychotic Agents 401

treatment with classical dopamine D2 antagonists and the EPS observed in these monkeys are
very similar to EPS induced by antipsychotics in humans.29 Other potential side effects, e.g.
gastrointestinal side effects, can also be investigated.
Six male Cebus monkeys are used for evaluation. The monkeys were housed in separate
cages in a temperature-regulated environment at a 12 h light/dark cycle The monkeys receive
haloperidol daily for 2 years and are sensitized to dystonia. The drug is given subcutaneously,
in increasing doses until two animals developed dystonia. The EPS can be rated on an arbitrary
scale ranging from 0 (not present) to 6 (extreme presence). Other behavioral paradigms that
are also recorded are arousal, unrest, stereotypy, locomotion, sedation, bradykinesia and
dystonia.
In EPS primed monkey, the test drug is administered behavior paradigms are evaluated
at time intervals (t = 30, 60, 120 and 180 min). During each experiment, data recording of the
animals can be conducted by videotaping.30
This is a useful model to predict whether a new drugs will/will not produce EPS at
antipsychotic doses.

Computational models of dopamine function and psychosis

The temporal difference (TD) algorithm is a prediction method that is gaining attention from
neuroscientists for behavioral research. It has been used to study schizophrenia and the
consequences of pharmacological manipulations of dopamine on schizophrenia. As in any
prediction method, TD takes into account the fact that predictions are often correlated and a
pattern can be developed from actually observed value. The firing rate of dopaminergic neurons
in brain, dopaminergic receptor manipulation, and the manifested behavior are correlated
into a formal framework or algorithm. This algorithm is validated with actually observed data
and after validation, TD proves useful as it offers predictive insights into clinical set-up.31 In
context of pre-clinical research, TD has been utilized to explain behavioral phenomena such
as the dose-dependent disruption of conditioned avoidance response by anti-psychotic drugs,
effect of drugs via receptor modulation and associated behavior paradigms, disruption of
latent inhibition by amphetamine, etc.32

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 cHAPTER

27
Antidepressant Agents
Introduction
Depression is a major affective disorder. It belongs to the heterogeneous group of mental
disorders characterized by extreme exaggerations and disturbances of mood, which adversely
affect cognition and psychomotor functions. It is a psychobiologic phenomena resulting from
abnormal brain mechanisms. An imbalance in the central cholinergic and adrenergic tone is
the critical pathophysiologic mechanism in affective disorders. The Biogenic Amine Hypothesis
proposes an increase in norepinephrine (NE) along with reduction in level of serotonin
(5-HT), dopamine (DA) and γ-aminobutyric acid (GABA) as the etiologic mechanism for
genesis of depression. Recently, zinc, the essential trace element that is part of regulatory
and catalytic protein, has been associated with significant role in the brain development.
Imbalance in zinc levels can lead to impaired neuronal activity, initiate neurodegenerative
processes leading to depression.1
The current therapy includes monoamine oxidase inhibitors (tranylcypromine, clorgiline,
moclobemide), tricyclics and related compounds (imipramine, amitryptyline, desipramine,
fluoxetine, fluvoxamine). Besides, the limitations of treatment lag, suboptimal efficacy
and residual cognitive dysfunction, the typical antidepressants are some of the most toxic
psychopharmacological agents and induce sedation, hypotension, and arrhythmias along with
anticholinergic symptoms. These factors limit their use. Compounds targeting glutamatergic
neurotransmission can be combined with drugs that target 5-HT or inhibit its transport and
present new opportunities for antidepressant treatment. Newer multimodal compounds
vortioxetine and vilazodone act via such diverse mechanisms in the treatment of depression
and associated cognitive dysfunction.2
A major problem in the search for new antidepressant drugs is the lack of animal models
that resemble depressive illness in humans and are selectively sensitive to simulate effective
antidepressant treatments. Most of the available screening methods are based mainly on
empirically established relationships between the clinical efficacy of known antidepressants
and their effects on various pharmacological test models. In combination with the study of
motor activity these tests allow assessment of the specificity of antidepressant activity by
establishing a ratio between the “antidepressant” dose and the “stimulant” or “sedative”
dose. It can be predicted that a substance will be antidepressant and sedative or stimulant
at the same dose if the ratio is close to 1.3 However, most of classical screening methods are
inadequate for detecting novel antidepressant drugs.
 Antidepressant Agents 405

IN VIVO METHODS
Chronic Social Defeat Stress (CSDS)
Chronic social defeat can act as stress, especially for adolescents. In order to simulate the
condition, the adolescent-age animal is exposed for 2 weeks to daily aggression in agonistic
interactions with unfamiliar adult males (hostile environment) and assessed for behavioral
changes.
Adult male rodents (potential aggressors) are placed for 5 days into one of the two equal
compartments of experimental cages separated by a transparent perforated partition. On
the sixth day, single 4-week-old male adolescents are placed into the vacant compartments
of common cages. Daily the partitions are removed and it is observed that the adult males
demonstrate aggression toward adolescents by way of attacking and chasing the young
males, who on the other hand demonstrate defensive behavior. The interaction is allowed for
5 min or for less than 3 min, depending on the intensity of the attack, after which animals
are separated. Such exposure is continued for 2 weeks, wherein the adolescent animal faces
repeated defeat and social stress. The age-matched animals of the control group are also
individually transferred daily to partitioned cage next to an unfamiliar adult but not allowed to
communicate physically. At the end of the two weeks, all animals are tested on behavioral and
biochemical parameters.4

Water Wheel Model

This model demonstrates the antidepressant property of the test drug by use of the ‘Behavioral
Despair Activity’. The animal is forced to swim without any escape in a water tank. A rotating
wheel in the water tank poses as an option for escape but adds-on to the despair as it turns
under the weight of the animal and the animal has to keep rotating the wheel in order to stay
afloat. The juncture when the animal is immobile and ceases to struggle and remains floating
motionless in the water, making only those movements necessary to keep its head above water
is denoted as endpoint. This corresponds to behavioral despair.5
The apparatus consists of a plexiglas water tank (20 cm × 8 cm × 18 cm) with a water wheel
in its center. The water wheel is made of plexiglas shaft (diameter 3 cm, length 6 cm) on which
six paddles (0.5 cm width), move when loads of more than 5 g are attached and the number
of rotations of the water wheel are counted. The tank is filled up to a height of 9 cm with water
at 25°C, such that paddles just touch the surface of the water. When placed into the apparatus
for the first time, the mice swim vigorously to find a way of escaping from the water. On
discovering the water wheel, they climb onto it and begin turning it due to their weight. After
a few minutes of attempted escape, they cling to the wheel and just float in the water showing
complete immobility.
For this method, mice of either sex and in the weight range of 20 to 25 g are selected. The
animals are randomly divided into three groups namely control, standard and test groups.
First, the animals are put through a preliminary experiment to determine the typical number
of rotations of the water wheel prior to the onset of behavioral despair. The animals are treated
either with vehicle (control group), standard drug like imipramine (5-20 mg/kg, ip) or test drug
in various doses (test group) and rechallenged on the water wheel. A potential antidepressant
will increase the number of counts of water wheel turns, indicating increased effort at escape
behavior.
406 Drug Screening Methods

The classical tricyclic antidepressants reduce immobility time in this model. However,
antidepressants acting selectively on the 5-HT system are inactive in this test and false positive
are induced by opiates and antihistaminics.
Lucki and co-workers have enhanced the sensitivity of the traditional Porsolt paradigm and
the accuracy of its scoring.6,7 This enables to better detect selective serotonin reuptake inhibitors
(SSRIs) antidepressant activity. The water depth has been increased to 30 cm from the traditional
depth of 15-18 cm, and the animal’s behavior during consecutive intervals of 5 sec measuring
climbing, swimming and immobility activity during each interval has been rated.
Another model on the principles of behavioral despair is the Forced Swim Test. The adult
male rats are forced to swim in a cylinder (40 cm × 18 cm) with no escape. The animals become
immobile after an initial struggling phase. The total duration of immobility is measured
throughout the trial. Immobility has been equated to a despair reaction, and when rats
are placed back in the water container 24 h later, they remain immobile for a significantly
longer time than naïve animals. Antidepressants decrease the immobility time.8 The forced
swimming test is a widely used behavioral model used in rodents. It is both sensitive and
selective for clinically effective antidepressant drugs. The test has been validated by most of
the current antidepressants. However, the model has the limitation that antidepressant drugs
show paradigm shift within 24 hr of treatment initiation, in contrast to weeks required for
the recovery from clinical depression. Moreover, high doses of drugs are required to produce
effects in most animal tests. 9

Learned Helplessness Test

In these models, a helpless situation is created for the animal, which results in performance
deficits in subsequent learning tasks. The rodents are exposed to chronic stress and not allowed
to escape from it. Chronic mild stress is a naturalistic paradigm of a hostile environment,
which models anhedonia, a major symptom of depression. This model reproduces a condition
of decreased sensitivity to usually pleasurable stimuli, like drinking sucrose, etc.10 In the
classical model “Inescapable Shock Treatment” the chronic stress is provided by foot shock to
the animal.11 Adult Wistar rats of either sex and in the weight range 200 to 250 g are placed in a
compartment with steel mesh grid floor. Repeated shocks (15 sec duration, 0.8 mA every min)
are applied and this serves as stress to the animals. Rats are exposed for 1 h without any escape
route. Control animals are placed in the chamber for 1 h without shocks. This forms the first
phase of the model where animal is exposed to ‘inescapable shock treatment’.
In the second phase there is ‘conditioned avoidance training’ where after chronic exposure,
the animal is trained. A cue (buzzer or light signal) precedes the shock and simultaneously a door
opened for ‘safe’ chamber, which is unelectrified, and the animal is allowed to escape towards
it and avoid the noxious stimulus (electric shock). This is termed as the ‘escape response’.
Failure to exhibit escape response by an animal is said to be an indicative of its ‘depressive
state’.12 Antidepressants reduce ‘escape failure’. This test can be successfully used to screen
potential antidepressants and determine their mode of action.13 This classic paradigm has a
variable score of 30-85%, in terms of the percentage of rats, which become hyporeactive after
training phase and as a modification, some authors use adrenalectomized rats in order to
obtain a consistent high yield.14
 Antidepressant Agents 407

Isolation-induced Hyperactivity
It is observed that rats when socially deprived for a period of 15 days, exhibit depressive
behavior. There is a reduction in spontaneous locomotor activity, exploratory behavior, rearing,
and stereotypy.
Adult Wistar rats of either sex weighing 200 to 250 g are housed singly in cages (38
cm × 26 cm × 20 cm) without any visual or auditory contact with their normally housed
counter-parts for 10-15 days. The animals are subjected to behavior testing on an arbitrary
scale for sleep, reduced response to external stimuli, ambulatory behavior, stereotype and
posture.15
Both classical and newer antidepressants reduce isolation-induced depressive behavior.

Tail Suspension Test

This model is a modification of the ‘behavior despair’ test. Mice are rendered immobile by
suspending from tail to induce behavioral despair. An animal in that situation alternates
between two kinds of behavior: agitation (mobility) and immobility. The cumulative
immobility time is a measure of the animal’s degree of helplessness (“depression”). Treatment
with antidepressant drugs reduces immobility time. It has been observed that mice exhibit
better reproducibility of results than rats.16 In a typical experiment, the mouse (20 to 30 g,
either sex, housed under standard laboratory condition with food and water ad libitum) is
hung on a wire in an upside down posture such that its nostril touches the water surface in a
container. Initially, the animal tries to escape by making vigorous movements, but is unable
to escape and becomes immobile. The period of immobility during 5 min observation period
is noted.
This test is a reliable and rapid screening method for potential antidepressants. Agents
acting via the serotonergic pathway can be screened using this method. In contrast, MAO
inhibitors fail to answer this test.16
A computerized system with 16 channels is available as a single-channel basic configuration
for measuring- time of activity, time of immobility, of the animal in real time. The tail suspension
monitor is a device for screening antidepressants in mice (Fig. 27.1).

Figure 27.1: Computerized tail suspension monitoring system for screening antidepressant activity in mice
408 Drug Screening Methods

Reserpine-induced Hypothermia
The test measures the ability of compounds to inhibit reserpine-induced hypothermia in mice.
The test is used for an early screening of potential antidepressant drugs with the mechanism of
action similar to tricyclic antidepressants.
Mice (Male Albino-Swiss, 25 to 30 g) are housed in a controlled environment with a
temperature at 22 ± 2°C, 12 h day-night cycle and free access to food and water. Reserpine in a
dose of 2.5-5.0 mg/kg, sc induces ptosis, hypothermia, catalepsy. In the experimental model,
reserpine (2.5 mg/kg, sc) is given 20 h before the test. Rectal body temperature is measured
every 30 min for 3 h after the drug injection. The experiment is preceded by two preliminary
measurements and their mean is taken as the initial temperature.17
Any pharmacological agent which reverses this typical effect of reserpine may have
antidepressant activity. It is a simple and reliable method, which can effectively screen
the tricyclics, MAO inhibitors. However, false negatives (mianserin) and false-positives
(methyldopa, antihistaminics) are also known to occur.

Amphetamine Potentiation
Amphetamine is a sympathomimetic agent, which promotes neuroexocytosis or displacement
of transmitter from axonal terminal. This test is used as a screening method to detect
adaptive changes in dopaminergic and noradrenergic systems after repeated treatment with
antidepressant drugs. Repeated treatment with antidepressants enhances the amphetamine-
induced locomotor hyperactivity.
Rats (male Wistar, 250 to 300 g) are housed in a controlled environment with temperature
22 ± 2­°C, 12 h light/dark cycle and free access to food and water. The rats receive the test drug
in their home cage usually for two weeks. Ninety minutes after the last dose of test drug,
D-amphetamine (5-10 mg/kg, ip) is injected and 30 min later they are placed singly into cages
with photocells to record their activity. The locomotor activity of single animal in the activity
cage is recorded for 1 hour.
Typical stereotypic effects (sniffing, exploratory activity, body and head movements,
gnawing, biting, licking), hyperthermia and increased locomotor activity are seen.18 Most
antidepressants including TCA, MAO inhibitors potentiate amphetamine effects. This model
effectively differentiates between neuroleptics and antidepressants.

Apomorphine Antagonism
Apomorphine is a dopamine agonist and in a dose of 16 mg/kg, ip, induces hypothermia,
stereotypy and climbing behavior. This method is simple and useful for rapid in vivo screening
of potential antidepressants. Potential antidepressants antagonize the apomorphine-induced
hypothermia in mice. This model helps to differentiate between neuroleptics and anti­
depressants as the former blocks stereotypy and climbing behavior but not hypothermia.19

Resident-intruder Paradigm in Rats

This model is designed to examine the effect of acute treatment with psychotropic drugs on the
social behavior of resident rats during encounters with unfamiliar, intruder rat. Antidepressant
drugs commonly reduce the level of aggressive behavior exhibited by the resident rats without
 Antidepressant Agents 409

modifying basal activity levels. This selective effect on rodent agonistic behavior has been
observed for all types of antidepressant drugs regardless of their pharmacology.
Male rats are housed under reverse-daylight conditions from weaning, for at least 5
weeks prior to the experiment to allow full entrainment to the phase-shifted light-dark cycle.
Standard laboratory chow and water are made available ad libitum and all subjects are
housed in standard laboratory polypropylene cages (32 cm × 50 cm × 16 cm) throughout each
experiment. In all studies, the resident and intruder rats are obtained from different sources
to ensure that resident animals have never been in contact with animals in the corresponding
intruder group. All age-matched animals within a particular group are housed in closed social
groups for at least 3 weeks to allow the development of stable social hierarchies.
Resident rats are treated with test, standard or vehicle and returned to their home cages
prior to each social encounter. Resident animals are separated 3 days before each test day and
housed individually. All social encounters are performed during the dark phase of the light-
dark cycle. At the end of the 10 min encounter, both resident and intruder rats are returned to
their respective group cages.
The nonsocial, social and agonistic behavior of the resident rats exhibited during each
social encounter is analyzed.20

Muricidal Behavior in Rats

Female rats of Holtzman strain exhibit compulsive mouse killing behavior, irrespective of their
satiety status. Nonmuricidal rats can be rendered muricidal by pretreatment with pilocarpine
(2.5-5.0 mg/kg, ip).21 It is understood to be a paradigm for depressive state. Agents reducing
muricidal behavior exhibit antidepressant action.
Antidepressants attenuate muricidal behavior at doses below that dose which induces
motor incoordination while psychotropic agents block at doses inducing motor deficits.15

Miscellaneous
Geriatric depression represents an unique set of physiological and biochemical problems due
to the regulatory differences in the hypothalamus-pituitary-adrenal (HPA) axis.22 Male Sprague
Dawley rats are obtained when they are 9 weeks or 19 months old and are housed individually
with free access to food and water and a 12 h light/dark cycle. After acclimatization, animals are
anesthetized and the top of the skull is shaved and a midline frontal incision made in the scalp
and the skin retracted bilaterally. Burr holes (2-3 mm) are drilled into the skull 2 mm lateral
to the bregma suture, after which the olfactory bulbs are severed from the frontal cortex and
aspirated according to established protocols.23 The cavity is packed with surgical foam, the skin
is closed with surgical clips and animal is allowed to recover. Sham-operated animals undergo
the same procedure except for excision and aspiration of the olfactory bulbs. After surgery,
animals are handled and weighed daily.24 Aged olfactory bulbectomy (OBX) animals show a
loss of passive avoidance. They exhibit locomotor stimulation along with decreased grooming
and suppressed feeding.24 The behavioral changes are visible 2 to 5 weeks after surgery and are
attenuated by all classes of antidepressants.
Postpartum depression (PPD) is a form of major depressive disorders that can exert
negative effects on both mothers and infants. PPD typically occurs for short durations within
the postpartum period and has moderate symptoms. Postpartum adult female cynomolgus
410 Drug Screening Methods

monkeys (Macaca fascicularis) are observed for typical huddle posture behavior as marker
of PPD. The animals are analyzed for locomotive activity, stressful events, hair cortisol levels
and for maternal interactive behaviors. This model provides translational efficiency for
systematically investigating the etiology, treatment, prevention of PPD.25

References
1. Tyszka-Czochara M, Grzywacz A, Gdula-Argasinska J, Librowski T, Wiliński B, Opoka W. The role of
zinc in the pathogenesis and treatment of central nervous system (CNS) diseases. Implications of
zinc homeostasis for proper CNS function. Acta Pol Pharm 2014;71(3):369-77.
2. Pehrson AL, Sanchez C. Serotonergic modulation of glutamate neurotransmission as a strategy for
treating depression and cognitive dysfunction. CNS Spectr 2014;19(2):121-33.
3. Bourin M. Is it possible to predict the activity of a new antidepressant in animals with simple
psychopharmacological tests? Fundam Clin Pharmacol 1990;4:49-64.
4. Iio W, Takagi H, Ogawa Y, Tsukahara T, Chohnan S, Toyoda A. Effects of chronic social defeat stress
on peripheral leptin and its hypothalamic actions. BMC Neurosci 2014;15:72.
5. Porsolt RD, Bertin A, Blavet N, et al. Immobility induced by forced swimming in rats: effects of agents
which modify central catecholamine and serotonin activity. Eur J Pharmacol 1979;57:201-10.
6. Detke MJ, Lucki I. Detection of serotonergic and noradrenergic antidepressants in the rat forced
swimming test: the effects of water depth. Behav Brain Res 1996;73:43-6.
7. Lucki I. The forced swimming test as a model for core and competent behavioral effects of
antidepressant drugs. Behav Pharmacol 1997;8:523-32.
8. Porsolt RD, Anton G, Blavet N, et al. Behavioural despair in rats: a new model sensitive to
antidepressant treatments. Eur J Pharmacol 1978;47:379-91.
9. Detke MJ, Johnson J, Lucki I. Acute and chronic antidepressant drug treatment in the rat forced
swimming test model of depression. Exp Clin Psychopharmacol 1997;5:107-12.
10. Gambarana C, Scheggi S, Tagliamonte A, et al. Animal models for the study of antidepressant
activity. Brain Res Protocol 2001;7:11-20.
11. Overmier JB, Seligman MEP. Effects of inescapable shock upon subsequent escape and avoidance
learning. J Comp Physiol Psychol 1967;63:23-33.
12. Martin P, Soubrie P, Simon P. The effect of monoamine oxidase inhibitors compared with classical
tricyclic antidepressants on learned helplessness paradigm. Prog Neuropsychopharmacol Biol
Psychiatry 1987;11:1-7.
13. Sherman AD, Sacquitne JL, Petty F. Specificity of the learned helplessness model of depression.
Pharmacol Biochem Behav 1982;16:449-54.
14. Edwards E, Harkins K, Wright G, et al. Effects of bilateral adrenalectomy on the induction of learned
helplessness behavior. Neuropsychopharmacology 1990;3:109-14.
15. Einon D, Morgan MJ, Sahakian BJ. The development of intersession habituation and emergence in
socially reared and isolated rats. Dev Psychobiol 1975;8:553-9.
16. Chermat R, Thierry B, Mico JA, et al. Adaptation of the tail suspension test to the rat. J Pharmacol
1986;17:348-50.
17. Willner P. The validity of animal models of depression. Psychopharmacology 1984;83:1-16.
18. Delina-Stula A. Psychotropic agents. In. F Hoffmeister, G Still (Eds): Antipsychotics and
Antidepressants: Part I., New York, Springer-Verlag 1980:505.
 Antidepressant Agents 411

19. Puech AJ, Chermat R, Poncelet M, et al. Antagonism of hypothermia and behavioral response
to apomorphine: a simple, rapid and discriminating test for screening antidepressants and
neuroleptics. Psychopharmacology 1981;75:84-91.
20. Grant EC. An analysis of the social behavior of the male laboratory rat. Behavior 1963;21:260-81.
21. Bhattacharya SK, Satayan KS, Ramanathan M. Experimental methods for evaluation of psychotropic
agents in rodents:II-Antidepressants. Indian J Exp Biol 1999;37:117-23.
22. Ritchie JC, Scotch RL, Nemeroff CB, et al. The effect of age on DST status and plasma dexamethasone
concentration in depressed patients. Bio Psychiatry 1990;27:A45-6.
23. Kelly JP, Wrynn AS, Leonard BE. The olfactory bulbectomized rat as a model of depression: an
update. Pharmacol Ther 1997;74:299-316.
24. Slotkin TA, Miller DB, Fumagalli F, et al. Modeling geriatric depression in animals: biochemical
and behavioral effects of olfactory bulbectomy in young versus aged rats. J Pharmacol Exp Ther
1999;289:334-45.
25. Xun-Xun CHU, Joshua Dominic Rizak, Shang-Chuan YANG. A natural model of behavioral
depression in postpartum adult female cynomolgus monkeys (Macaca fascicularis). Zoological Res
2014;35(3):174-81.
 cHAPTER

28
Antiepileptics
INTRODUCTION
Epilepsy is a common disorder with an incidence of approximately 0.3-0.5% in different
populations throughout the world and a prevalence of 5-10 persons per 1000. The characteristic
event in epilepsy is the seizure, which is a paradoxical event due to abnormal, excessive,
hypersynchronous discharges from an aggregate of central nervous system neurons.1 Epilepsy,
in contradistinction to seizures, is a chronic disorder characterized by recurrent seizures.
Pathophysiology of epilepsy involves alterations in voltage-dependent ion channels: reduction
in inhibitory, i.e. GABA-mediated or increase in excitatory, i.e. glutamate-mediated inputs.
Epileptic seizures have been classified 2 into:
•• Partial seizures: Begin focally in a cortical site and may or may not generalize. These include
simple partial seizures and complex partial seizures.
•• Generalized seizures: Involve both the cerebral hemispheres from the onset. Generalized
seizures have been classified into absence seizures (Petit mal), generalized tonic-clonic
seizures (grand mal), myoclonic seizures and atonic seizures.
Currently available antiepileptic drugs act by modulating GABA or glutamate transmission
or by modulating sodium and calcium ion channels. However, drug therapy of epilepsy is
empirical therapy and does not address the underlying pathology. Also, inspite of addition
of a large number of efficacious antiepileptic drugs during the past decade, they provide
relief in only up to 75% patients with absence seizures and in 85% patients with generalized
tonic-clonic seizures. Keeping these problems in antiepileptic pharmacotherapy, it has
been suggested that the current drug discovery process for antiepileptic drugs should be
re-evaluated. In furtherance of the objective of identifying useful models for therapy of
pharmacoresistant epilepsy, NIH held a NIH/NINDS/AES models workshop in September
2002.3 Discovery of newer genetic models of epilepsy and use of models of injury-induced
epilepsy based on electrically or chemically induced status epilepticus was recommended at
the workshop.
In order to study the antiepileptic effect of drugs and discovering their mechanism of action,
various in vitro and in vivo models of epilepsy have been devised. Some of them, which are
used most often, are described below.
 Antiepileptics 413

In vitro Methods
Hippocampal Slices
In vitro brain slice systems are being increasingly used for study of neurophysiological
mechanisms of epilepsies. In vitro hippocampal slices have been especially useful due to the
involvement of hippocampus in generation of complex partial seizures.4
Procedure: A rodent (rat, mouse, guinea pig) is used. The animal is decapitated, its brain
is removed and hippocampus is dissected out. Using a vibrotome, slices of about 0.5 mm
thickness are made. Cutting approximately perpendicular to the long axis of hippocampus
preserves the three-neuron synaptic circuit and associated recurrent circuitry. After cutting,
the slices are preincubated for 2 h in a holding chamber in which they are kept moist in 28°C
warm saline equilibrated with 95% O2 and 5% CO2. Slices can be kept healthy for more than
18 h if handled properly. For recording, the slices are transferred to a Perspex chamber (1.5 × 4
cm) and attached to its bottom. Slices are either kept in 3 mm thick layer of 32°C warm saline or
submerged in liquid artificial cerebrospinal fluid. Intracellular recordings from the pyramidal
neurons in the slice are done by passing micropipettes (tip diameter <0.5 mm) into the stratum
pyramidale under microscopic control.4-6
Evaluation: Readings are taken by adding drug to the slice medium and recording the
spontaneous or shock evoked repetitive firing of neurons.
In vitro hippocampal slices offer the advantage of mechanical stability, absence of a blood-
brain barrier for applied drugs and absence of anesthetics. It is a very useful model for studying
the neurophysiological mechanisms of convulsant and antiepileptic drugs and for screening of
putative antiepileptic drugs.

Electrical Recording from Isolated Brain Cells

Isolated brain cells are used for testing action of drugs on ion channels in excitable cell
membranes using the patch clamp techniques.7,8 In patch clamp techniques, glass pipettes are
directly opposed to membranes in order to record currents through membrane in response
to voltage, ionic or chemical change.9 The isolated brain cells are prepared by either growing
cells in tissue culture or by mechanical or enzymatic dissociation of brain slices into Petri
dishes.7 Cells are either obtained from hippocampus or from hypothalamus.
Procedure: The isolated neurons are put in a bath containing 140 mMNaCl, 5 mMKCl, 0.5 mM
CaCl2, 1 mM MgCl2, 5 mM HEPES, at pH 7.3. Patch pipettes, filled with same solution as in
bath, are attached. Drugs are added to bath solution and recording of capacitative currents is
done. Recording is done at room temperature (21-24°C). 6
Evaluation: Effect of drugs on capacitative component of current Ic is seen.
Ic = C dv/dt
where C = specific membrane capacitance
dv/dt = rate of change of membrane potential
Using this technique, neurophysiologists have explored voltage sensitive calcium and
potassium channels, membrane response to neurotransmitters and basic mechanisms of
antiepileptic drugs.5
414 Drug Screening Methods

In Vitro Assays for GABAergic Compounds

Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central
nervous system. It exerts its actions by acting on two distinct types of receptors; GABAA
receptor, which is a ligand-gated Cl- ion channel or ‘ionotropic’ receptor and GABAB, which is
a member of G-protein-coupled receptor family or ‘metabotropic’ receptor.10 Abnormalities
in the function of GABA system have been implicated in many diseases of the CNS including
epilepsy.
In relation to epilepsy, it has been suggested that enhancing GABA-mediated synaptic
inhibition would reduce neuronal excitability and raise the seizure threshold. A number of
antiepileptic drugs have been shown to act by enhancing the GABAergic inhibition, e.g.
benzodiazepines, barbiturates, vigabatrin and tiagabine.

[3H] GABA Receptor-binding Assay

[3H] GABA-binding assay is a simple and sensitive method to evaluate compounds with
GABAergic properties.
Procedure: Rats are used for the experiment. Male rats weighing about 100-150 g are decapitated
and their brains removed rapidly. The brains are homogenized in 15 volumes of ice-cold 0.32
M sucrose and centrifuged for 10 min at 1,000 g. The supernatant is then recentrifuged for 20
min at 20,000 g. After discarding the supernatant, the pellet obtained is homogenized in 15
volumes of distilled water and centrifuged for 20 min at 8,000 g. The upper, buffy layer of the
pellet is resuspended, by gentle squirling, in the supernatant and then centrifuged for 20 min
at 48,000 g. The pellet (synaptic membrane pellet) so obtained is resuspended in 15 volumes
of distilled water and centrifuged for 20 min at 48,000 g. After discarding the supernatant, the
centrifuge tubes, containing the pellets, are capped with paraffin and stored at –70°C.6
Homogenize a frozen membrane pellet in 15 volumes of 0.05 M Tris-maleate buffer (pH
7.1) at 4°C. Add Triton X-100 to a final concentration of 0.05% (enhances the specific GABA
receptor binding while lowering the nonspecific binding) and incubate the suspension for
30 min at 37°C. This is now centrifuged for 10 min at 48,000 g. Discard the supernatant and
resuspend the pellet by homogenization in 15 volumes of the buffer at 4°C.
The assay tubes are prepared in triplicate. The tissue homogenate is incubated with [3H]
GABA (15 nM) in the tris maleate buffer (0.05 M), alone or along with either the test drug or
with isoguvacine (0.1 mM) or muscimol (0.1 mM) at 4°C for 5 min and then centrifuged for 15
min at 5,000 rpm. After washing the pellet with the buffer twice, radioactivity is quantified with
liquiscint using liquid scintillation photometry.6
Evaluation: Specific [3H] GABA binding, i.e. the radioactivity that can be displaced by a
high concentration of unlabeled GABA is calculated. Difference between the total bound
radioactivity and radioactivity bound in the presence of 0.1 mM of isoguvacine i.e. nonspecific
binding gives the specific binding.
Percentage of specifically bound [3H] GABA displaced by a given concentration of the test
compound is calculated and its IC50 value with 95% confidence limits obtained using computer-
derived linear regression analysis.
 Antiepileptics 415

GABAA Receptor-binding Assay

GABAA receptor mediates the bulk of postsynaptic inhibitory actions of GABA. It is a ligand-
gated Cl- channel. It exists as pentamer, composed of 3 different subunits (α, β, γ). The receptor
subunits each exist in several subtypes giving heterogeneity to the GABAA receptors.11 Muscimol
is a powerful GABAA agonist, whereas bicuculline, picrotoxin and SR 95531 are antagonists.
Various centrally acting drugs like benzodiazepines, barbiturates and neurosteroids also
modulate GABAA receptor function. To examine the GABAA binding sites, [3H] muscimol
(agonist) and [3H] SR 95531 (antagonist) are used as radioligands.12‑15

[3H] Muscimol-binding Assay

[3H] muscimol binding is determined using a concentration of radioligand ranging from
0.5-50 nM. Prior washing of the membrane pellet three times with ice cold buffer followed
by centrifugation removes the endogenous GABA from the tissue used for assay. Membrane
samples are then incubated with the radioligand in binding buffer (Tris-citrate, pH 7.1) for
60 min at 0-4°C. Nonspecific GABA binding is determined in the presence of 100 mM GABA.
The incubation is terminated by centrifugation at 13,000 g for 10 min at 0-4°C. The resulting
membrane pellet is rinsed once with 1 ml of ice-cold buffer, and the bottom of the tube
containing the pellet is cut into a scintillation vial. Pellet material is dissolved in Protosol
(NEN) for 45 min at 40°C and left overnight at room temperature. Scintillation fluid is then
added and ligand binding is determined by scintillation counting.

[3H] SR 95531 Binding Assay

The tissue sample is incubated with [3H] SR 95531in buffer (50 mMTris-citrate supplemented
with 200 mMNaCl, pH 7.1) for 60 min at 0-4°C. Concentrations of [3H] SR 95531ranging from
1-100 nM are used for the assay and nonspecific binding is determined in the presence of 100
mM of unlabeled compound. The binding assay is terminated by filtration over Whatman
GF/B filters on a Brandel filtration manifold. The filters are washed 2 times with ice-cold buffer,
dried, and bound radioactivity quantified by liquid scintillation counting.

GABAB Receptor-binding Assay

GABAB is a metabotropic receptor, which acts by inhibiting adenylyl cyclase, K+ channel opening
or Ca2+ channel blockade.10 It mediates both presynaptic and postsynaptic inhibition in the
central nervous system. Baclofen is an agonist at the GABAB receptor. GABAB receptor-binding
assay, using [3H] baclofen, allows the screening of drugs with affinity for GABAB receptors.
Procedure: The tissue homogenate is incubated in buffer along with [3H] baclofen
(1.8-2 × 10-8 M) and either baclofen (0-1.2 × 10-6 M) or GABA or the test drug for 60 min at 4°C.
The binding is terminated by rapid vacuum filtration over glass fiber filters. Filters are washed
with ice-cold buffer and radioactivity counted after addition of ethylene glycol monomethyl
ether and liquiscint.6
Evaluation: Difference of binding of [3H] baclofen in the presence or absence of baclofen gives
the specific binding. Dissociation constant (Ki) of the test drug, i.e. the concentration of the test
drug at which 50% of the receptors are occupied, is calculated.
416 Drug Screening Methods

[3H] GABA Uptake in Rat Cerebral Cortex

GABA action is terminated by uptake of GABA into neurons and glia via the GAT-1 transporter.
Increasing the concentration of GABA by blocking the transporter offers a useful mechanism
for anticonvulsant drugs. Tiagabine, a recently introduced antiepileptic drug, acts by inhibition
of GAT-1.16 Various other uptake inhibitors such as nipecotic acid, guvacine and THPO also
exhibit anticonvulsant effects. This assay is useful in screening of potential anticonvulsants
that act by GABA uptake inhibition.
Procedure: Rats are decapitated and their brains removed rapidly. The cerebral cortices are
homogenized in 9 volumes of ice-cold 0.32 M sucrose and centrifuged for 10 min at 1,000 g.
The supernatant is recentrifuged for 10 min at 1,000 g. Supernatant is discarded and the pellet
is suspended in 9 volumes of 0.32 M sucrose and centrifuged for 10 min at 24,000 g. The pellet is
resuspended in 15 volumes of depolarizing Ringer’s solution and incubated for 10 min at 25°C.
It is then centrifuged for 10 min at 3,000 g. The final pellet is then resuspended in 15 volumes of
Ringer’s solution.
The assay tubes are prepared in triplicate. The tissue suspension is incubated in Ringer’s
solution along with either vehicle or the test drug. The nonspecific controls are incubated at
0°C while totals at 25°C for 10 min. Now [3H] GABA is added to each tube and the solution
reincubated for 10 min. Centrifuge the tubes for 1 min at 13,000 g. Dissolve the pellet by mixing
in Triton X-100+EtOH (1:4, v/v). Incubate for 3 min at 90°C and then recentrifuge for 15 min at
13,000 g. Radioactivity is quantified in 10 ml of liquiscint scintillation cocktail.6
Evaluation: Difference between cpm at 25°C and 0°C gives the value for active uptake of [3H]
GABA and percentage inhibition of uptake at each drug concentration is calculated. IC50 values
are calculated using log-probit analysis.

[35S] TBPS-binding Assay

TBPS (t-butylbicyclophosphorothionate) is a convulsant that acts by blocking the GABAergic
neurotransmission by interacting with picrotoxin sensitive site of GABA-benzodiazepine-
Cl¯channel complex.17 Inhibition of binding of [35S] TBPS to the rat cortical membranes by test
compounds is used for screening of anticonvulsant drugs.
Procedure: Assay tubes are prepared in triplicate. The tissue homogenate is incubated in
buffer (0.05 M Tris with 2 M KCl, pH 7.4) and distilled water along with [35S] TBPS (2 nM) and
picrotoxin (10-5 M) or distilled water or the test compound for 150 min at 25°C, with agitation.
The binding is terminated by rapid filtration over Whatman GF/B filters, which are presoaked
in buffer. Radioactivity is quantified by counting with 10 ml liquiscint.6
Evaluation: Difference between binding in the absence or presence of picrotoxin gives the
specific binding which is typically 85-90% of total binding. Percentage inhibition of [35S] TBPS
at each drug concentration is measured and IC50 value calculated using log-probit analysis.

Excitatory Amino Acid Receptor-binding Assays

Glutamate, and possibly aspartate, function as the principal fast excitatory neurotransmitters
in the brain. Glutamate receptors are classified functionally as either ligand-gated ion
 Antiepileptics 417

channels (ionotropic) or G-protein-coupled receptors (metabotropic). The ligand-gated ion

channel receptors are further classified, according to the identity of selective agonists, into
AMPA, NMDA and kainate receptors.18 A number of antagonists are now available for these
receptors. In case of NMDA receptors, the antagonists may act at various sites on the receptor
and antagonize glutamate actions, e.g. phencyclidine, ketamine, MK-801 are open channel
blockers, 5,7-dichlorokynurenic acid is an antagonist at the modulatory glycine site, ifenprodil
is a closed channel blocker while CPP [3-(2-carboxypirazin-4-yl)-propyl-1-phosphonic acid],
AP-7 and AP-5 are competitive antagonists at the glutamate-binding site.19 Excessive excitatory
amino acid neurotransmission has been implicated in the neuropathogenesis of epilepsy,
stroke, schizophrenia and various neurodegenerative diseases.20 Antagonists of NMDA
receptors have been shown to act as anticonvulsants and neuroprotective agents.21

[3H] CPP-binding Assay

[3H] CPP-binding assay is used to assess the affinity of compounds for the glutamate-binding
site on the NMDA receptor complex.
Procedure: Rats are decapitated and their brains removed rapidly. The cerebral cortices are
homogenized in 15 volumes of 0.32 M sucrose with a Tissumizer and then centrifuged for 10
min at 1000 g, 4°C temperature. The supernatant is recentrifuged for 20 min at 20,000 g at 4°C.
After discarding the supernatant, the pellet obtained is homogenized in 15 volumes of ice-cold
distilled water and centrifuged for 20 min at 7,600 g at 4°C. The upper buffy layer of the pellet is
resuspended in supernatant and centrifuged for 20 min at 48,000 g at 4°C. The pellet obtained
is resuspended in 15 volumes of cold distilled water and centrifuged. Discard the supernatant
and store the pellet at –70°C.6
Suspend the membrane pellet in 15 volumes of ice-cold 50 mMTris buffer (pH 7.6). Add
Triton X-100 to a final concentration of 0.05% and incubate for 15 min at 37°C, with agitation.
Now centrifuge it for 20 min at 48,000 g at 4°C. The pellet is washed thrice by resuspending
in ice-cold Tris buffer and subsequently centrifuged. Resuspend the final pellet in buffer in a
volume 20 times the original wet weight.
Assay tubes are set in triplicate. The tissue homogenate is incubated with [3H] CPP (10 mM)
in buffer (0.5 M TrisHCl, pH 7.6), distilled water along with l-glutamic acid (10-4 M) or distilled
water or the test compound (in appropriate concentration) for 20 min at 25°C with agitation.
Tubes are placed in ice bath after incubation. Binding is terminated by centrifugation for 15
min at 7,000 rpm at 4°C. Return the tubes to ice. Discard supernatant and rinse the pellets three
times with ice-cold buffer. Radioactivity is quantified by liquid scintillation counting.6
Evaluation: Difference of binding in the absence or presence of 10-4 M l-glutamic acid gives
the specific binding which is typically 60-70% of the total binding. IC50 values for the test
compound are obtained using log-probit analysis.

[3H] TCP-binding Assay

This assay is used for determining the binding affinity of noncompetitive NMDA receptor
antagonists at the phencyclidine (PCP), binding site that lies within or near the NMDA-regulated
ion channel. TCP, i.e. 1-[1-(2-thienyl)cyclohexyl]-piperidine is thienyl derivative of PCP.
418 Drug Screening Methods

Procedure: Assay tubes are prepared in triplicate. The tissue homogenate is incubated with
[3H] TCP (2.5 nM) in buffer (0.1 M HEPES, pH 7.5) and distilled water along with phencyclidine
or vehicle or the test compound and with (for stimulated binding) or without (for basal binding)
l-glutamic acid (10-4 M) and glyine (10-5 M) for 120 min at 25°C with agitation. The binding is
terminated by rapid filtration, under reduced pressure, over Whatman GF/B filters, presoaked
in 0.05% polyethylene-imine and buffer, using Brandell cell harvesters. Filters are rinsed twice
with buffer and then counted for radioactivity with 10 ml of liquiscint.6
Evaluation: Difference of [3H] TCP binding in the absence or presence of PCP gives the specific
binding. For each test compound, inhibition of [3H] TCP binding is measured both in the
absence (basal) and presence (stimulated) of l-glutamic acid and glycine. IC50 values for the
test compound are obtained using log-probit analysis.

[3H] Glycine-binding Assay

Glycine is a modulator of the NMDA receptor function, which acts at a distinct modulatory
site on the NMDA receptor complex. A strychnine insensitive [3H] glycine-binding site is
associated with NMDA receptor22 and has been shown, by autoradiographic studies, to have
similar distribution as that of [3H] TCP-binding sites.23 Binding of both glutamate and glycine is
necessary for the NMDA receptor activation. Compounds with affinity for this glycine-binding
site provide novel substances for modulation of NMDA receptor function. Such compounds
are assayed using the [3H] glycine-binding assay.
Procedure: The assay tubes are prepared in quadruplicate. The tissue homogenate is incubated
with [3H] glycine (10 nM) in buffer (0.5 M Tris maleate, pH 7.4) along with glycine (10-3 M) or
distilled water or the test drug for 20 min in an ice bath at 0-4°C. The binding is terminated by
centrifugation for 20 min at 7000 rpm at 4°C. The pellet obtained is rinsed with ice-cold buffer
and the radioactivity is quantified by counting with 10 ml of liquiscint.6
Evaluation: Difference of binding in the absence or presence of unlabeled glycine gives the
specific binding, which is typically 60-70% of the total binding. Inhibition of binding of [3H]
glycine by the test compound is measured and IC50 value calculated using log-probit analysis.
Disadvantages of in vitro models: Although, in vitro models provide insight into the
mechanism of action of putative antiepileptic drugs and serve as initial screen for drug
discovery, they do not give any indication of pharmacokinetics, pharmacodynamics or PK-PD
interactions of the compound when introduced into a living animal or human being. Further,
it is not possible to study the compensatory changes that occur in body when a drug is given.

In vivo Methods
Electrically Induced Seizures
There are three major types of electrically induced seizure models:24-26
1. Threshold models
2. Maximal electroshock seizure (MES) test
3. Focal electrical stimulation such as kindling
 Antiepileptics 419

These models are used for screening of drugs with efficacy against generalized tonic-clonic
and focal seizures.

Threshold for Maximal (Tonic Extension) Electroconvulsions

This test is done to determine the ability of a drug to alter the seizure threshold for tonic limb
extension.26 Drugs effective against generalized tonic-clonic seizures increase this threshold.
Used in parallel with MES test, it is a good test for screening of drugs effective against generalized
tonic-clonic (grand mal) seizures.
Procedure: Mice are used for the experiment. For each stimulus intensity, male mice (18 to
30 g), in groups of 8-10, are used. Corneal or ear electrodes are used to provide electrical
stimulation from a stimulator that either delivers constant current or constant voltage at
a frequency of 50-60/sec for 0.2 sec duration. Rectangular pulses are better than sinusoidal
pulses in inducing seizures, though either of the two can be used for stimulation.27 Threshold
is usually determined as the current or voltage inducing hind limb extension in 50% of the
animals, i.e. CC50 and CV50 or EV50 respectively. Control thresholds in mice are about 6-9
mA (CC50) or 90-140 V (CV50 or EV50) depending on strain, age and method of stimulation
(thresholds determined via ear electrodes are lower than via corneal electrodes).
Evaluation: Elevation of threshold by the test drug is taken as a measure of its efficacy.
Comparison of drug effects requires calculation of dose that elevates the threshold by 20%.
Control threshold determination should be undertaken on each day parallel to threshold
determinations in drug treated animals. Use of an animal more than once a day is not
recommended as post-ictal rise in seizure threshold has been noted.27

Maximal Electroshock Seizure (MES) Test

Merritt and Putnam (1938) developed the MES test and discovered the anticonvulsive effect of
diphenylhydantoin using this test.28 This model is useful for screening of drugs effective against
primary and secondary generalized tonic-clonic seizures.29
Procedure: Groups of 8 to 10 animals (rats or mice) are used per dose of a drug. Electrical
stimulation is applied via corneal or ear electrodes with a stimulator that either delivers constant
current or constant voltage at a frequency of 50-60/sec. The electrodes are moistened with
saline solution before application. All animals are stimulated with the same supra-maximal
current strength that is usually 2-5 times the threshold current strength. With constant current
stimulators, typical stimulation parameters include 50 mA in mice and 150 mA in rats, 50-60/
sec current delivered via corneal electrodes for 0.2 sec.26,27 With constant voltage stimulators,
250 V is used for mice and 750 V for rats. The resultant seizure passes through various phases:
phase of tonic limb flexion of about 1.5 sec duration followed by phase of tonic limb extension
lasting about 10 sec and finally followed by a variable short clonic interval which may lead to
asphyxial death in some animals.30
Evaluation: Suppression of tonic hind limb extension is taken as a measure of efficacy in this
test. Anticonvulsant potency is determined by calculation of ED50 for suppression of tonic hind
limb extension. Drugs effective against generalized tonic-clonic seizures such as phenytoin,
420 Drug Screening Methods

carbamazepine, phenobarbitone and primidone are effective while anti-absence seizure

drugs like ethosuximide are ineffective in this test.
Disadvantage of electrically induced seizure models: These models, though useful in
determining the anti-seizure potential of a compound, do not give any clue regarding the
mechanism of action of the compound.

Kindled Rat Seizure Model

Kindling is an animal model of epilepsy that refers to a process whereby repeated administration
of an initially subconvulsive electrical stimulus results in progressive intensification of stimulus-
induced seizure activity, culminating in a generalized seizure.31 The effect was discovered by
Delgado and colleagues32 and elaborated by Goddard et al.24 The kindling phenomenon is a
manifestation of the fact that ‘epilepsy induces epilepsy’.
Procedure: Adult female Sprague Dawley rats weighing 270 to 400 g are used for the experiment.
An electrode is implanted in the right amygdala for electrical stimulation.6 Although, amygdala
is most commonly chosen for the experiment, other regions of the forebrain can also be
kindled.24,33 Animal is allowed to recover from surgery for a minimum of 1-2 weeks, otherwise
the sensitivity of the animals to kindling is lowered. Then daily electrical stimulus trains are
applied via the electrode using either a fixed current strength (400-500 µA, 1 msec monophasic
square wave pulses for 1 sec with 50 or 60/sec frequency) or using the individual threshold
current to induce after discharges at the site of stimulation.27
During the daily electrical stimulation of amygdala, seizures develop which, as classified by
Racine,34 generally evolve through the following five stages:
Class – 1: Immobility, eye closure, twitching of vibrissae, stereotypic sniffing
Class – 2: Facial clonus and head nodding
Class – 3: Facial clonus, head nodding and forelimb clonus (contralateral to focus)
Class – 4: Rearing, often accompanied by bilateral forelimb clonus
Class – 5: Rearing with loss of balance and falling accompanied by generalized clonic seizures.
Rats are said to be fully kindled when enhanced sensitivity, as evidenced by class five seizures,
has developed.27 If the stimulation is continued for a few weeks, rats develop ‘spontaneous’
epileptic seizures that persist for as long as 7 months following termination of the stimulation.
Evaluation: The animals to be tested are given the test compound either orally or ip and tested
on the day before and after the treatment. Readings obtained after administration of test drug
and the control are compared keeping in mind four different measures for drug efficacy which
can be recorded in a kindled animal:
1. Seizure latency, i.e. time from stimulation to the first sign of seizure activity.
2. Seizure severity
3. Seizure duration
4. After discharge duration.
Alternatively, drug efficacy can be measured by determining separate ED50 values for total
suppression of:
1. Generalized seizures (class 4 and 5)
2. Focal seizures (class 1-3)
3. Amygdaloid after discharges.
 Antiepileptics 421

Kindling model has the merit that the efficacy of a drug against the process of epileptogenesis
as well as against the fully kindled state can be measured. Efficacy against generalized seizures
provides a valid model for drugs effective in secondary generalized seizures of partial epilepsy,
while efficacy against the focal components of kindled seizures provides a valid model for
drugs effective in complex partial seizures.35 Of the anticonvulsant drugs available at present,
phenobarbitone, diazepam and valproic acid block the kindled seizures as well as the kindling
process while phenytoin and carbamazepine block seizures once kindling has occurred, but
do not reliably block the establishment of kindled seizures. Every species so far studied is
subject to kindling,33 from frogs to animals like baboons, rhesus monkeys, cats and dogs.36-38

Other Methods of Kindling

1. Corneal electroshock kindling: Kindling can be done in rats and mice by giving
electroshocks via corneal electrodes. Mice can be kindled by once daily application of 3mA
current of 60 Hz frequency for 2 sec while rats can be kindled with twice daily application of
8 mA current of 60 Hz frequency for 4 sec via corneal electrodes. Occurrence of Racine stage
5 seizures (as above) indicates that the animal is kindled. Putative antiepileptic drugs can
be tested after the animals have had stage 5 seizures for 10 consecutive times.39,40
2. Kindling by stimulation of other brain areas: Kindled seizures can also be produced
by stimulation of other brain areas like neocortex or hippocampus in rats. Lothman et al.
(1985)41 have described the development of rapidly recurring hippocampal seizure (RRHS)
model of kindling in rats. Briefly, bipolar electrodes are implanted stereotactically in either
right or left hippocampus of adult albino rats. The animals are allowed to recover for one
week. Subsequently via a constant current source stimulator, hippocampus is stimulated
with suprathreshold tetanic electrical stimuli in trains of 10 sec, with each stimuli consisting
of biphasic square wave pulses of 1ms duration and 10 Hz frequency. The stimulation
is repeated every 5 min. Behavioral seizures (Class 1 to class 5 as described above for
amygdala kindling) and after discharges on EEG are recorded after every stimulus. Severe
limbic seizures (i.e. class 4 or class 5 behavioral seizures) are observed on the first day itself,
which intensify on the second day and then remain stable. Effect of putative antiepileptic
drugs against the stimulation induced behavioral or EEG seizures and the after discharge
duration can be studied. Using the RRHS model of kindling, Lothman et al (1988 a,b)42,43
have demonstrated that the response pattern of antiepileptic drug against kindling in this
model is similar to that with the amygdala kindling.
3. Chemical induced kindling: Pentylenetetrazol (PTZ), a proconvulsant chemical acting
via antagonism of GABAA function, can lead to long lasting kindling in rats when given
repeatedly in subconvulsive doses. Briefly, adult Sprague Dawley rats are administered
30 mg/kg of PTZ ip 3 times a week for 9 weeks. After every injection, seizures are recorded
and scoring done as below:
0 = no response
1 = ear and facial twitching
2 = one to 20 myoclonic jerks in 10 min
3 = more than 20 body jerks in 10 min
4 = clonic forelimb convulsions
5 = generalized clonic convulsions with rearing and falling down episodes
6 = generalized convulsions with tonic extension episodes and status epilepticus.
422 Drug Screening Methods

Beginning in second week of PTZ administration, the seizure score shows a progressive
increase over the period of 9 weeks such that by the end of 9 weeks, about 90% animals are
kindled i.e. have seizure score of more than or equal to 3. Efficacy of drugs in preventing the
development of PTZ induced kindling can be studied by injecting the putative antiepileptic
drug before each PTZ injection.44,45

Chemically Induced Convulsions

Numerous chemical compounds can produce seizures. Some, which are used as tools for
epilepsy research, include:
1. Chemoconvulsants inducing generalized seizures after systemic administration, e.g.
pentylenetetrazol, bicuculline, picrotoxin, penicillin, isoniazid, thiosemicarbazide,
allylglycine, DMCM, β-CCM, strychnine, pilocarpine, NMDA, kainic acid, gamma-
hydroxybutyric acid, DDT and methionine sulfoximine.
2. Chemoconvulsants inducing focal seizure after central administration, e.g. penicillin,
kainic acid, quinolinic acid, and pentylenetetrazol.27

Pentylenetetrazol (PTZ) Test

Pentylenetetrazol is a tetrazol derivative with consistent convulsive effect in a large number
of animal species like mice, rats, cats, primates, etc. It is believed to act by antagonizing the
inhibitory GABAergic neurotransmission.46 PTZ test is used for screening of drugs effective in
petit mal epilepsy or absence seizures.
i. Threshold for Clonic Seizures after iv Infusion of PTZ
Procedure: Eight to 10 mice are used per threshold determination. One percent solution of
PTZ is administered by continuous i.v. infusion at the rate of 0.3 ml/min.27 The animal develops
seizures in the following order: one or more isolated jerks (first twitch) followed immediately
by a generalized clonic seizure with loss of righting reflexes, followed by maximal tonic clonic
seizures after a certain time lag. Dose for the production of generalized clonic seizures with
loss of righting reflex is preferably taken as an endpoint. Threshold is calculated as the mean
dose (± SD) of PTZ that induces seizures in the group tested and is about 50 mg/kg for clonic
seizures and 90 mg/kg for maximal tonic-clonic seizures in mice.
ii. Subcutaneous PTZ Test
Procedure: Mice or rats are used for the experiment. 8 to 10 animals are used per dose of
test drug. Prior to drug efficacy experiments, it is better to determine the subcutaneous CD97
(convulsive dose in 97% of the animals) of PTZ. CD97 is usually about 70 mg/kg in rats and
80-100 mg/kg in mice.27 Mice are given 1% solution of PTZ, 80-100 mg/kg sc in the scruff of
neck. Control mice within 30 min develop a sequence of excitement, myoclonic jerks, clonic
seizures, one or more maximal tonic seizures and death.30 As endpoint, either the first episode
of clonic jerking lasting for 5 sec (a threshold seizure) or the first clonic seizure with loss of
righting reflex is taken.
Evaluation: Efficacy of a test drug as an anticonvulsant is measured by determining its ED50
for suppression of clonic seizure (either threshold seizure or clonic seizure with loss of righting
reflex). Drugs effective in petit mal epilepsy like ethosuximide, valproic acid are effective while
phenytoin, carbamazepine are not effective in the two models using PTZ.
 Antiepileptics 423

Systemic Penicillin Test

Cats are used for the experiment. Animals are injected with 3,00,000 units/kg of penicillin
G by intramuscular route. Seizure activity begins about 1 h after injection and continues
intermittently for 6-8 h and is characterized by recurrent episodes of arrested activity, staring,
myoclonus, facial-oral twitching and occasional progression to generalized tonic-clonic
seizures.47 This model is useful for screening of drugs useful in petit mal epilepsy. Ethosuximide
and valproate are effective in this model.48,49
Modification: Rats can be used for the penicillin model of epilepsy.50

Other Chemical Convulsants

Numerous other chemicals have been used systemically to study the anticonvulsant activity
of drugs, e.g. bicuculline (a GABAA antagonist used in a dose of 2.7 mg/kg sc in mice or
in a dose of 0.3 mg i.v. in monkeys to induce seizures), picrotoxin, strychnine, NMDA,
homocysteine, allylglycine, methionine sulfoximine, fluorothyl (by inhalational route),
gamma-hydroxybutyrate and pilocarpine.29,51-59

Seizures Induced by Focal Lesions

Topical or intracerebral application of certain metals and chemicals can lead to simple partial
seizures. These models are thus useful for screening of potential antiepileptic drugs effective
against these seizures.

Cortically Implanted Metals

A state of spontaneously recurrent simple partial seizures can be induced by topical application
of certain metals such as alumina cream (aluminium hydroxide), cobalt and tungstic acid
onto (or into) the cerebral cortex60 or by injection of iron into the brain cortex.61 Aluminium
hydroxide gel model is most commonly used.

Aluminium Hydroxide Gel Model

This model was discovered by Kopeloff et al.62 Four percent aluminium hydroxide is injected
into surgically exposed monkey neocortex at few adjacent sites. One to two months after the
injection, spontaneous and recurrent seizures begin. These seizures persist for several years.
Seizures consist of rhythmic jerking of an extremity or face contralateral to the lesion with
occasional progression to secondarily generalized tonic-clonic seizures. Response to standard
anticonvulsants parallels that of patients with focal epilepsy.

Miscellaneous Chemicals Used for Producing Focal Lesions

Various other chemicals have been used as convulsants by topical route such as
intrahippocampal injections of kainic acid or tetanus toxin, topical application of penicillin,
cholinergics, anticholinergics, picrotoxin, bicuculline, strychnine, ganglioside antibody
injections, zinc.63-72
Focal lesions have also been produced by cryogenic injury to areas of brain using liquid
nitrogen probe or ethylchloride, which can lead to highly epileptogenic lesions.73
424 Drug Screening Methods

Systemic Focal Epileptogenesis

This model combines the features of focal and generalized epilepsy. Rats are given radiation to
a volume of cerebrum equivalent to 0.25 ml. About 3-6 months later, bicuculline methiodide,
in a dose of 2 mg/kg, is injected systemically. A seizure focus is produced with recurrent EEG
spikes and focal seizures.74 Seizures last for several weeks after a single injection. Phenytoin,
phenobarbital, chlordiazepoxide and valproic acid are effective.

Models of Status Epilepticus

There are a number of animal models that can be used to screen drugs effective in
pharmacotherapy of status epilepticus. Some of these are described below in brief:
1. Pilocarpine-induced status epilepticus: Pilocarpine, a cholinomimetic agent, can produce
behavioral and electroencephalographic seizures suggestive of motor limbic seizures and
status epilepticus in rats when given in a dose of 380-400 mg/kg i.p. The seizures can be
scored according to the classification of Golarai et al. as follows:
Stage I - hypoactivity
Stage II - monoclonic jerks of the head, head bobbing and facial automatism
Stage III - whole body bilateral activity resembling wet dog shakes
Stage IV - rearing of forelimbs
Stage V - generalized clonic-tonic activity and loss of posture. 75-77
2. Lithium-pilocarpine induced status epilepticus: Status epilepticus can be induced in rats
by giving pilocarpine (30-40 mg/kg, i.p.) 24 h after pretreating with lithium (3 meq/kg i.p.).
Racine classification is used to score the seizures.78-82
3. Lithium-methomyl induced seizures in rats: Methomyl, a carbamate anticholinesterase,
in a dose of 5.2 mg/kg s.c., can induce long lasting status epilepticus in lithium pretreated
rats. Diazepam is effective in this model.83
4. Electrical stimulation of hippocampal perforant pathway: A week after implanting a
bipolar stimulating electrode in right angular bundle and a unipolar recording electrode in
right hippocampal dentate granule, the perforant pathway is stimulated by 2mA monopolar
pulses of 50 microsecond duration and 20 Hz frequency for 2 h. The animals develop self-
sustaining limbic status epilepticus when the stimulation is stopped.84
5. D, L-homocysteine induced status epilepticus: Status epilepticus can be induced by
D, L-homocysteinethiolactone in rats with actively epileptogenic cobalt lesions of motor
cortex. Diazepam, lorazepam, phenobarbitone and phenytoin are effective against these
seizures.85
6. Generalized myoclonic seizures in baboons: In Papio cynocephalus baboons, generalized
myoclonic jerks can be produced by hourly stimulation by stroboscope (15-35 flashes/sec for
test periods of upto 5 min), 2-8 h after injecting allylglycine (200 mg/kg i.v.). Phenobarbitone
and diazepam are effective against these seizures.86

Model of Infantile Spasms

Infantile spasms, which occur in early childhood, are insensitive to most of the available
antiepileptic drugs. Velisek et al. (2007) 87 have developed a model of infantile spasms, which
includes injecting pregnant Sprague Dawley rats with 2 doses of betamethasone (0.4 mg/
 Antiepileptics 425

kg i.p.) at 8 AM and 6 PM on gestational day 15. Seizures are produced in the pups on post-
natal day 15 by injecting NMDA (15 mg/kg i.p.). The sequence of seizures includes: twisting
movements of tail followed by arching for several seconds and finally loss of righting reflex and
flexion spasms lasting tens of seconds with multiple recurrences. ACTH, which is the only drug
effective against infantile spasms, can delay the onset of NMDA induced seizures in these rats.
Betamethasone has been proposed to sensitize the brain to onset of NMDA induced seizures.
Although, it is a step forward, this model required validation.

Genetic Animal Models of Epilepsy

Most of the animal models for screening of antiepileptic drugs are basically models of seizures
rather than of epilepsy, which is a condition of chronically recurrent spontaneous seizures.
Genetic animal models more closely approximate human epilepsy and give opportunity to
study genetic and biochemical basis of epilepsy.

Photosensitive Baboons
Baboons, Papiopapio, from the Casamance region of Senegal were first reported to suffer
from photomyoclonic syndrome by Killiam et al. in 1966.88 Intermittent light stimulation at
frequencies close to 25 flashes per second leads to seizures characterized by eyelid, then
face and body clonus and subsequently tonic spasms or full tonic-clonic convulsions.89
Drugs useful against clinical tonic-clonic and myoclonic epilepsy inhibit photosensitive
seizures in Papiopapio. Valproic acid, benzodiazepines and phenobarbital are effective as
anticonvulsants in them, while phenytoin, carbamazepine and trimethadione provide less
favorable therapeutic effects.5

Seizure-prone Mice Strains

Various seizure-prone mice strains have been developed. These include the following:
i. Audiogenic Seizure Susceptible Mice: DBA/2J mice, an inbred strain of the house mouse
(Musmusculus), is the most studied strain of audiogenic seizure susceptible mice and
has been known since 1947.90 Between the ages of 2-4 weeks, these mice exhibit sound-
induced seizures, after which susceptibility gradually declines,91 such that by 8 weeks of
age they are totally free of audiogenic seizures.
Susceptible mice are exposed to sudden, loud sound which contains frequency
components in the 12-16 kHz range.5 The seizure pattern involves a wild running phase,
followed by clonic convulsions and a tonic extension ultimately leading to respiratory
arrest (in about 60%) or full recovery.27 Anticonvulsant effect of drugs is evaluated by
efficacy against clonic seizures. Audiogenic seizures can be prevented by phenytoin
or phenobarbital92 or valproic acid. Audiogenic seizure susceptible mice are useful as
sensitive gross screening model for potential anticonvulsant drugs.
ii. Totterer Mice: The homozygous (tg/tg) strain totterer mice are prone to spontaneous
epileptic seizures. These mice are recognized by a broad-based ataxic gait. By 3 to 4
weeks of age, they develop frequent partial and absence seizures.93 Spontaneous focal
motor seizures occur a few times a day, manifested as unilateral clonic jerks of limbs with
secondary generalization. Ninety-three percent of the seizures last for 15 min or longer.94
These seizures can be suppressed by diazepam.
426 Drug Screening Methods

Totterer mice also exhibit spontaneous petit mal seizures with synchronous 6-7 per second
spike-wave discharges in EEG.95 These spike-wave discharges last 0.3 to 10 sec and occur
hundreds of times per day and are accompanied by a behavioral petit mal seizure. These
seizures are blocked by ethosuximide, diazepam and phenobarbital while phenytoin is
not effective.96
iii. E1 Mice: Imaizumi and colleagues first discovered E1 mice in 1959.97 E1 mice exhibit
seizures in response to vestibular stimulation like tossing or spinning. Manifestations
of seizure include limb and face automatism like chewing and salivation. There may
be secondary generalization to tonic-clonic seizures. EEG recordings indicate onset of
electrical discharges in deep limbic structures.98 Thus, these mice can serve as model for
complex partial epilepsy with secondary generalization. Phenytoin and phenobarbitone
are effective in this model.
iv. Quaking Mice: These are C57BL/6J mutants with myelin defects, tremors, spontaneous
or stimulus-induced myoclonic and generalized tonic-clonic seizures. Handling induces
seizures in quaking mice. These seizures are blocked by phenytoin, phenobarbitone,
carbamazepine and valproic acid. Quaking mice are useful for assessment of potential
new anticonvulsant drugs effective against focal motor seizures in humans.99
v. Lethargic (lh/lh) mice: Hosford et al. (1992) have validated lethargic (lh/lh) mouse model
as a genetic model of absence seizures. Lethargic (lh/lh) mice have behavioral, EEG and
anticonvulsant drug profiles similar to those in absence seizures in humans. Lethargic mice
are recognized by ataxic gate by 3 weeks of age. EEG electrodes are placed intracerebrally
in frontal neocortex. Subsequently, 180 min EEG recording sessions consisting of 30 min
predrug and 150 min post drug recordings are done to see the effect of drug on frequency
of epileptiform bursts. Number of seizures during 150 min period after administration
of drug is noted and IC50, i.e. concentration that reduces number of seizures by 50% is
calculated. Total duration of seizures during the 150 min recording sessions can give an
indication of pro-absence effect of drugs.100
vi. Other mice strains showing spontaneous seizures (AE mice) or induced seizures (SJL/J
strain mice with noise-induced seizures) are also being used for anticonvulsant drug
testing.

Seizure-prone Rat Strains

i. Genetically Epilepsy-prone Rats (GEPRs): Seizures can be induced in these animals by
various stimuli like sound, hyperthermia, chemical and electrical.101 When stimulated by
sound, these GEPRs run about wildly, fall from clonic jerks and may also suffer from tonic
extensor convulsions. For anticonvulsant drug evaluation, the tonic-clonic component of
the seizure is commonly used.94 Drugs effective in MES test are effective in this model.
ii. Rats with Spontaneously Occurring Petit Mal Epilepsy: About 15-30% of Sprague
Dawley and Wistar rats, both males and females, 14 to 18 weeks of age and above exhibit
spontaneous spike-wave discharges (7-11/sec) with associated behavioral components
like behavioral arrest and myoclonic twitchings mostly limited to the facial musculature.102
Spontaneous discharges last for 0.5-40 sec and occur hundreds of times a day. Drugs
effective in absence seizures in humans suppress these seizures. These rats can serve as a
good model for chronic drug efficacy studies.
 Antiepileptics 427

Mongolian Gerbils
Seizure tendency in Mongolian gerbils was first described by Thiessen et al.103 in 1968.
Seizures in these animals can be precipitated by various stimuli like placing the animal in
a new environment, onset of bright light, audiogenic stimuli, vigorous shaking of cage and
different handling techniques.104 Seizures in gerbils can be facial myoclonic (minor) seizures,
seen mostly in young animals of 7 to 10 weeks age or generalized myoclonic and tonic-clonic
(major) seizures seen in older animals. Young gerbils with minor seizures can serve as model
for petit mal epilepsy. Anti-absence seizures drugs are effective in these. While older animals
with major seizures can be used to identify drugs with efficacy against generalized tonic-clonic
seizures.

Miscellaneous Genetically Seizure-prone Animals

Various other animals can be used for studying the anticonvulsive effect of potential
antiepileptic drugs. These include Syrian golden hamsters, photosensitive epileptic chickens
and dogs.105-107

Morphological Approaches
In many animal SE models, cell necrosis and neurodegeneration are hallmark features. Acute
neuronal injury and neurodegeneration are routinely assessed to estimate the extent of
protection provided by the test compounds. The following methods are used to determine the
neuroprotective potential of test compounds.

Cell Necrosis and Apoptosis

Nissl Staining
Histological assessment is made using Nissl (Cresyl violet, CV) staining techniques, as described
previously.108 CV stains all neurophil components and any changes in cytoarchitecture of the
hippocampal cell layers like loss of cells is determined. Cresyl Violet Acetate solution is used
to stain Nissl substance in the cytoplasm of neurons. It stains both neurons and glia and is very
useful to identify the overall cell loss and neuronal damage.
TUNEL Assay
To measure apoptotic cell death, terminal deoxynucleotidyl transferase and digoxigenin-
11-dUTP nick end labeling (TUNEL) staining is mostly widely employed using the Apoptag
peroxidase in situ apoptosis detection kit.108 For quantification of cell death, photomicrographs
(30-μm) of three sequential sections are taken at dorsal hippocampus level of each animal. The
representative sections from different animals (n = 4) are permeabilized by treating with 0.25%
trypsin solution in 0.01 N HCl for 30 min at 37°C and then washed in PBS, and nonspecific
sites are blocked by using 0.1 M Tris buffer containing 3% bovine serum albumin and 20%
normal bovine serum for 30 min at 37°C. After this, sections are washed in PBS and incubated
in TUNEL reaction mixture for 90 min at 37°C. The TUNEL reaction mixture comprised
enzyme solution and fluorescein label solution at a ratio of 1:9. The sections designated as
negative controls were incubated in fluorescein label solution only. The nucleus of apoptotic
cells in sections treated with the TUNEL mixture exhibits a clear green fluorescence. TUNEL-
428 Drug Screening Methods

positive cells within a square millimeter area are counted by an observer blind to the treatment
conditions.109
Fluoro-Jade B Staining
For detecting degenerating neurons and their processes, Fluoro-Jade B (FJB) staining is
commonly performed in brain sections from rodent models. This staining procedure is a
sensitive and reliable marker for neuronal degeneration that results from SE or brain injury. The
extent of neurodegeneration after the SE is determined with FJB staining in dentate hilus (DH),
CA3 and CA1 sub-regions of the hippocampus. The rats are perfused with 4% paraformaldehyde
solution and the brains collected and processed in 30% sucrose. Consequently, 30 μm thick
sections of these brains are cut with cryostat at the dorsal hippocampus level and collected
in phosphate buffer solution (PBS) and later stored at −20°C in cryo-buffer. To visualize
neurons undergoing degeneration after SE, three sequential sections each 450 μm apart
through the dorsal hippocampi are collected in each animal, mounted on gelatin-coated
slides and air dried at room temperature overnight. Slides were then washed sequentially
in 100% ethanol, 70% ethanol and deionized water. The slides were then incubated in 0.06%
potassium permanganate solution for 15 min (with slow shaking), washed in deionized water,
and incubated in 0.004% FJB (Histo-Chem Inc., Jefferson, AK ) dissolved in deionized water
with 0.1% acetic acid for 30 min. Then slides were washed three times for 1-min in deionized
water, dried on a slide warmer at 55°C, briefly immersed in xylene and cover slipped using
DPX mounting media. The sections are analyzed by confocal microscope using FITC filter.
Cells labeled by FJB were detected as individual green shiny pyramidal shaped spots clearly
identifiable from background. The number of FJB positive cells (dying neurons) per image field
in the hippocampal DH, CA3 and CA1 are counted in each of the three sections per animal.116

Neurodegeneration, Neurogenesis and Mossy Fiber Sprouting

Neurodegeneration
The extent of neurodegeneration after the SE is determined by the immunostaining of sections
for NeuN (principal neurons) and parvalbumin- or neuropeptides-Y-positive (interneurons)
cells. The neuronal nuclei antigen (NeuN) is a very specific protein that is highly expressed in
nucleus and less expressed in cell body in differentiated neurons. NeuN is not expressed in
glial cells, oligodendrocytes, astrocytes, or microglial cells, and cerebellar Purkinje cells. Thus,
NeuN immunohistochemistry is commonly used to identify neuronal loss and to quantify total
number of neurons in various rat brain regions. To analyze the overall neurodegeneration
after the SE, the brains are removed, postfixed in 4% paraformaldehyde and cryoprotected
in PBS containing 30% sucrose. Cryostat sections are cut at 30-μm coronally through the
entire anteroposterior axis of the hippocampus and collected serially in PBS. Every 20th
section through the entire hippocampus is selected in each of the animals and processed
for Nissl staining. Nissl staining demonstrates the hippocampal cytoarchitecture of principal
cell layers and confirmed the presence of bilateral hippocampal injury in rats after SE. The
extent of neurodegeneration within different regions of the hippocampus is further assessed
by NeuN immunohistochemistry and quantified from 24 to 72 h post-SE. Because the overall
neurodegeneration in different regions of the hippocampus appeared mostly symmetrical
between the two sides, quantification is performed on only one side. The number of cells
 Antiepileptics 429

in hippocampal subregions such as the dentate hilus (DH), CA3 and CA1 are counted and
compared with controls or between various treatment groups. Reduction or lack of neuronal
loss is indicative of neuroprotective potential of the test drug.111
Neurogenesis
There is strong evidence of dramatic changes in neurogenesis in the hippocampus dentate
gyrus subgranular zone following SE and neuronal injury. Quantification of the extent
of neurogenesis and type of cells that are born after injury would be helpful to study the
pathophysiological role of neurogenesis following SE and acute neuronal injury. Interestingly,
hippocampal neurogenesis is very sensitive to physiological and pathological stimuli. Certain
pathological stimuli such as seizures alter both the amount and the pattern of neurogenesis.
Therefore, it is helpful to identify whether SE-induced changes in neurogenesis contribute
to vulnerability to neurological conditions such as cognitive dysfunction, depression and
epilepsy. Neurogenesis within the adult central nervous system is demonstrated using an
exogenous cell tracer, 5'-bromo-2'-deoxyuridine (BrdU), in combination with endogenous
neuronal markers.111 Specific primary antibodies raised against these markers are widely
available and their visualization is possible with the use of fluorescently tagged secondary
antibodies. BrdU is a thymidine analog that incorporates into dividing cells during DNA
synthesis. Once incorporated into the new DNA, BrdU will remain in place and be passed down
to daughter cells following division. Typically, BrdU is injected intraperitoneally. Different
survival times required by the desired experimental time-line will yield data on specific phases
of neurogenesis: proliferation, differentiation and maturation. One limitation of using BrdU
is uncertain penetration of the targeted cells with a uniform concentration of the compound.
Thus, for experiments requiring measurements of cell proliferation, Ki67 can be used as an
acceptable alternative. The protocol takes 3–5 days, allowing for sectioning and staining.112
Mossy Fiber (MF) Sprouting
Sprouting of neuronal axons including MF that contain zinc is generally visualized with Timm
staining.113 In the chronic epilepsy model, MF sprouting is indicative of epileptogenesis and is
commonly identified by Timm staining of the brain section at various intervals after induction
of SE. There is little or subtle change in Timm staining in acute models of SE; MF sprouting is
mostly observed in chronic post-SE models of TLE.
Neuroinflammation Markers
Neuroinflammation is a common consequence of seizures and other neuronal injury events.
Acute seizures and SE causes neuroinflammation by activating microglia, astrocytes, and
induction and enhancement of inflammatory cytokines such as IL-1β, IL-6 and TNF in key
brain regions such as the hippocampus. Further seizure related expression of inflammatory
cytokines occurs in brain regions that may undergo neuronal damage A recent report suggests
that seizure-induced release of inflammatory cytokines like IL-1β from astrocytes may
cause brain inflammation and damage the blood brain barrier. Neuroinflammation and its
secondary consequences may partly contribute to generation of recurrence of seizures.111
Immunohistochemistry of brain sections for specific glial markers such as glial fibrillary
acidic protein (GFAP, astrocytes) and Iba-1 (microglia) is helpful to identify the pattern
of neuroinflammation and relevant damage. There is a marked increase in the number
of astrocytes and microglia and their processes due to inflammation. It is shown that the
430 Drug Screening Methods

neurosteroid allopregnanolone can reduce inflammation in traumatic brain injury models.

Quantification of biomarkers of neuroinflammation is also very helpful to assess the course of
inflammation following seizures and neuronal injury events.

Conclusion
A large number of in vitro and in vivo models for screening of antiepileptic drugs are available.
However, there are many more models of seizures than of epilepsies. An ideal model of epilepsy
should show the following characteristics:
•• Development of spontaneously occurring seizures
•• Type of seizure similar to that seen in human epilepsy
•• EEG correlates of epileptic-like activity
•• Age-dependency in the onset of epilepsy as is seen in many epileptic syndromes
At present, there are no models that satisfy all these criteria. Only the genetic animal models
of epilepsy come closest to being called ideal, as they resemble idiopathic epilepsy in humans
more closely than any other experimental model. These models can prove to be very valuable
for screening of antiepileptic drugs, especially for drug-resistant epilepsies.
The Antiepileptic Drug Development Program of the National Institute of Neurological and
Communicative Disorders and Stroke (NINCDS) of the National Institute of Health (NIH), USA
is primarily based on two seizure models, the MES test and the s.c. PTZ test,108 which predict
drug efficacy against generalized tonic-clonic and absence seizures, respectively. These
models have proved to be very useful for screening of putative antiepileptic drugs.
In vitro models are also very important for gaining insight into the pathophysiology of
epilepsies and understanding the mechanism of action of drugs. They are useful in screening
of drugs having specific mechanism of action, e.g. the use of [3H] GABA uptake assay for
screening of drugs that act by inhibiting GABA uptake.
To conclude, it must be emphasized that use of a single method for screening of antiepileptic
drugs cannot predict the full pharmacological profile of the drug. Thus, for successful
development of a potential antiepileptic drug, effect of drug in various in vitro and in vivo
models must be studied.

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109. Rao MS, Hattiangady B, Reddy DS, Shetty AK. Hippocampal neurodegeneration, spontaneous
seizures, and mossy fiber sprouting in the F344 rat model of temporal lobe epilepsy. J. Neurosci.
Res 2006;83:1088-105.
110. Zeng LH, Rensing NR, Wong M. The mammalian target of rapamycin signaling pathway mediates
epileptogenesis in a model of temporal lobe epilepsy. J Neurosci 2009;29:6964–72.
111. Rao MS, Hattiangady B, Reddy DS, Shetty AK. Hippocampal neurodegeneration, spontaneous
seizures, and mossy fiber sprouting in the F344 rat model of temporal lobe epilepsy. J Neurosci.
Res 2006;83:1088–105.
112. Kuruba R, Shetty AK. Could hippocampal neurogenesis be a future drug target for treating temporal
lobe epilepsy? CNS Neurol Disord Drug Targets 2007;6:342–57.
113. Sloviter RS. A simplified Timm stain procedure compatible with formaldehyde fixation and routine
paraffin embedding of rat brain. Brain Res Bull 1982;8:771–4.
114. Librizzi L, Noè F, Vezzani A, de Curtis M, Ravizza T. Seizure-induced brain-borne inflammation
sustains seizure recurrence and blood-brain barrier damage. Ann Neurol 2012;72:82-90.
 cHAPTER

29
Anti-Alzheimer Agents
Introduction
Alzheimer’s disease (AD, the most common form of age-related dementia), is a progressive
irreversible neurodegenerative disorder that was first identified and written by Dr. Alois
Alzheimer in early 1900s. It occurs gradually affecting the short-term memory at the beginning
of the disease, followed by long-term memory loss and results in cognitive impairment,
unusual behavior, personality changes, and ultimately death. It is the most common form
of adult onset dementia.1 With the expected aging of the human population, the estimated
morbidity of AD suggests a critical upcoming health problem (Juan et al, 2014).2 Presently, it
is the 4th leading cause of death in Western countries, preceded only by heart disease, cancer
and stroke.3 The total number of people with dementia worldwide in 2010 is estimated at 35.6
million and is projected to nearly double every 20 years, to 65.7 million in 2030 and 115.4
million in 2050. The total number of new cases of dementia each year worldwide is nearly 7.7
million, implying one new case every four seconds (Duthey, 2013).4 Age is by far the main risk
factor for AD; its prevalence illustrates an exponential rise with age.3 Much of the increase is
in developing countries, especially India, and their south Asian and western Pacific neighbors
(Duthey, 2013).4
Approximately 5% of all AD cases have an early onset and are familial, based on mutations
of presenilin 1, presenilin 2 or the beta amyloid precursor protein (APP).5 By contrast,
approximately 95% of all AD cases represent the sporadic or late onset form of disease showing
no mutations of presenilin 1, presenilin 2 or APP.6
AD involves neuronal degeneration with impaired cholinergic transmission in the cerebral
cortex and hippocampus in areas of the brain particularly associated with memory and higher
intellectual functioning. The primary pathology in AD occurs in the basal forebrain that
provides the major cholinergic innervations to the neocortex, hippocampus and amygdala.7
There is a dramatic loss of neurons and synapses in these areas and are characterized by a
number of important pathological changes. The pathophysiology is complex and involves
multiple interconnected pathways.9 It is thought to arise from an interaction of several risk
factors, including genetic susceptibility, increased age, female gender, and perhaps a lower
premorbid intelligence level.
The cardinal histopathological manifestations of AD are the neurofibrillary tangles (NFT)
and amyloid plaques (AP). The NFT is present in the neurons as paired helical filaments
 Anti-Alzheimer Agents 437

and composed of hyperphosphorylated tau and the AP which is composed of a cluster of

dystrophic neurites and a few abnormal synapses surrounding a core of beta amyloid are
present extracellularly.8
The Aβ hypothesis of AD stipulates that increased brain levels of Aβ appear to be a critical
event in triggering a wide range of molecular alterations leading to AD (Fig. 29.1). It has
been proposed that exposure to sub-toxic concentrations of metals, such as copper, affects
secondary structures and acts as a seeding or nucleation core that facilitates Aβ aggregation.
Aβ aggregates to form amyloid plaques that are neurotoxic, leading to neurodegeneration
accompanied by dementia. The strongest evidence for this hypothesis comes from molecular
genetic studies of APP and presenilins in familial AD showing that all mutations increased the
propensity for Aβ to aggregate in vitro. Studies of the modifier gene product, apolipoprotein
E, showing enhanced susceptibility conferred by the E4 allele also found that the E4 isoform
increased the rate of Aβ aggregation.
Recently newer hypotheses have been proposed regarding pathogenesis of AD. For one, it is
hypothesized that Peroxisome Proliferator-Activated Receptors (PPAR) are nuclear receptors
that have potential role in exerting neuroprotective effects in various neurodegenerative
disorders, especially AD. This nuclear receptor subgroup is implicated to play a key role
in Aβ clearance and shows potential use in AD therapy (Cramer et al, 2012).10 Another
hypothesis proposes that cerebral glucose metabolism is perturbed in the pathogenesis of AD
(Fig. 29.2). Abnormality of glucose transportation contributes to intracellular glucose
catabolism dysfunction resulting in neuronal degeneration and consequently cognitive
deficits in AD patients. Thus, induction of multiple pathogenic factors such as oxidative stress,

Figure 29.1: Aβ hypothesis of AD

438 Drug Screening Methods

Figure 29.2: Cerebral glucose metabolism hypothesis of AD

mitochondrial dysfunction, and cerebral hypometabolism, etc. are understood to be both

causes and consequences of AD (Chen and Zhong, 2013).11
Effective treatment and prevention strategies and management of attendant behavioral
disturbances are paramount to the management of this illness. The efficacy of any given
intervention will ultimately be judged upon its ability to prevent memory loss or restore memory
ability. New therapeutic measures against dementia in AD are being aimed at a variety of targets
deemed instrumental in the pathogenesis of the disease. The two main targets are amyloid
plaques and neurofibrillary tangles and their principal molecular components, Aβ and tau.
The management includes inhibitors of cholinesterase (tacrine, donepezil). However, these
drugs are not able to contain the progression of the disease. Moreover, the improvement is
modest and a high morbidity exists. Side effects like abdominal cramps, nausea, vomiting and
diarrhea are dose limiting. For the development of therapeutic drugs, valid animal models
are needed that mimic the pathophysiological change in brain functions and the concomitant
behavioral deterioration seen in AD patients.
The ideal model should exhibit the same biochemical, histopathologic and behavioral
abnormalities as the human disease state. Behavioral models for studying memory storage,
recall and its manipulation by pharmacological agents do not present the typical patho­
physiology of AD, i.e. extracellular deposits of beta-amyloid in senile plaques, intracellular
accumulations of NFT.12
Most of the models currently in use are at best ‘Isomorphic Models’. Thus, for the induction
and simulation of the pathophysiology underlying AD in experimental animals, amnesia-
inducing agents like scopolamine and colchicine are available. Their dosages, mechanisms
along with comparative merits and demerits have been tabulated (Table 29.1). The effect of
test drug can then be compared to the standard drug in such amnesic animals on the basis of
behavioral tests.
The paradigms used for learning and memory can be discussed under two behavioral
tasks: behavior in avoidance chambers (active and passive avoidance) and behavior on mazes
(elevated plus maze, water maze, etc).
Table 29.1: Comparison of the various amnesic agents

Amnesic agent Mechanism of action Dose, route and animal Manifestation Remarks
33
Scopolamine Musacarinic 0.3-1 mg/kg, ip, mice Cognitive impairment. Effect similar to normal elderly but not patients of AD.32
antagonist Unlike the disease the effects are reversible with no
compromise of presynaptic cholinergic neurons.28 Does
not produce pathological hallmarks of AD (AP and NFT).
Iboteic acid Excitotoxicity Ibotenic acid: 12 g/ml Cognitive impairment Kainic acid is also a potent anticonvulsant and can
Bilateral lesion in basal Fall in choline acetyl produce distant damage (ibotenic acid is referred).24
forebrain, rats transferase activity (ChAT).34 They do not produce the histological characteristics of
AD.
Kainic acid Kainic acid: 1.5-2.5 Decreases ChAT by 70% on 5th Since these excititoxins are not specific to cholinergic
nM, Nucleus basalis day.35 cells, they provide limited information on the specific role
Meynert (nbM) of cholinergic system in learning and memory deficits.
Streptozotocin Depletes insulin 3 mg/kg, ICV, rat Damage to myelinated neurons Mimics the abnormalities of glucose and energy
receptors in in fornix and corpus callosum, metabolism found in the brains of patients of AD.38
brain leading to microgliosis),36 cognitive sporadic
abnormal impairment, increase in oxidative
glucose stress 21st day post-lesion.37, 39
metabolism, and
cognitive deficit
Colchicine Microtubule 7.5-14 µg, ICV, rat Depletion of ACh, ChAT and Selectively lesions cholinergic input into the
inhibitor causing decrease in muscarinic receptor hippocampus
cell death binding in frontal cortex and Significant impairment of learning and memory
hippocampus,39 produces Decrease ChAT activity in the hippocampus
oxidative stress in brain post- Do not produce pathological hallmarks of AD
lesion40 (AP and NFT).
Scopolamine Musacarinic 0.3-1 mg/kg, ip, Mice Cognitive impairment Effect similar to normal elderly but not patients of AD.32
antagonist Unlike the disease the effects are reversible with no
compromise of presynaptic cholinergic neurons.28 Does
not produce pathological hallmarks of AD (AP and NFT).
Anti-Alzheimer Agents

Contd...
439
Contd...
Amnesic agent Mechanism of action Dose, route and animal Manifestation Remarks
440
Iboteic acid Excitotoxicity Ibotenic acid: 12 g/ml Cognitive impairment Kainic acid is also a potent anticonvulsant and can pro-
Bilateral lesion in basal Fall in choline acetyl duce distant damage (ibotenic acid is referred).24
forebrain, rats transferase activity (ChAT).34 They do not produce the histological characteristics of AD.
Kainic acid Kainic acid: 1.5-2.5 nM, Decreases ChAT by 70% on 5th day.35 Since these excititoxins are not specific to cholinergic
Nucleus basalis Meynert cells, they provide limited information on the specific role
(nbM) of cholinergic system in learning and memory deficits.
Streptozotocin Depletes insulin 3 mg/kg, ICV, rat Damage to myelinated neurons in Mimics the abnormalities of glucose and energy me-
receptors in fornix and corpus callosum, micro- tabolism found in the brains of patients of AD.38
Drug Screening Methods

brain leading to gliosis),36 cognitive sporadic

abnormal glucose impairment, increase in oxidative
metabolism, and stress 21st day post-lesion.39
cognitive deficit
Colchicine Microtubule inhibitor 7.5-14 µg, ICV, rat Depletion of ACh, ChAT and Selectively lesions cholinergic input in to the hip-
causing cell death decrease in muscarinic receptor pocampus
binding in frontal cortex and hippocampus
hippocampus,39 produces Significant impairment of learning and memory
oxidative stress in brain post- Decrease ChAT activity in the hippocampus
lesion40 Do not produce pathological hallmarks of AD (AP and
NFT).
Ethylcholine Structural analogue 1-3 nmol/side, ICV, rat Cognitive impairment, fall in Ach41 Selectively targets cholinergic system and decreases
mustard of choline and 4 nmol/side/ and ChAT activity.42 ACh, without altering other neurotransmitters, while
aziridinum- selectively cytotoxic straitum, rat AD is multineurotransmitter disease. Does not produce
AF64A against cholinergic- 2.5 nmol/nbM, rat pathological hallmarks of AD (AP and NFT).
neurons.21
192-IgG- Antineuronal im- 0.22-1 µg, medial sep- Cognitive impairment, fall in ACh Does not produce pathological hallmarks of AD (AP and
saporin munotoxin, targets tum, rat38 and ChAT activity.45 NFT).
selectively choliner- 2 µg, ICV, rat43
gic nerve terminals in
basal forebrain
Intracerebral Beta amyloid is the Aβ[25-35], 20 µg, Produces behavioral, neurochemical Throws light on the molecular mechanism of Aβ toxicity
beta amyloid primary constituent of hippocampus, rat46 and immunohistochemical and gives support to the pivotal role of Aβ in AD patho-
(Aβ) injections the amyloid plaques Aβ [1-40], 10-20 µg × 3 changes similar to AD genesis.
days, ICV, rat47
 Anti-Alzheimer Agents 441

Intracerebroventricular (ICV) route is the favored route of drug administration as it allows

the drug to be directly injected to the appointed site and bypasses the blood-brain barrier.
However, the technique requires good expertise in surgical manipulations and postoperative
care of the animals. In contrast, systemic injections are easier to administer, but require higher
dose of the test drug, which may inadvertently lead to unwanted side effects. The techniques of
brain microdialysis and osmotic minipumps provide a suitable alternative.

IN VITRO and EX VIVO MODELS

Transfected Cell Lines
HEK293 cells are transfected with human APP695 and N2a cells with human APP695 cDNAs.
APP695-HEK293 transfectants are grown in Dulbecco’s modified Eagle’s medium plus 10%
fetal bovine serum, penicillin and streptomycin, whereas, APP695-N2a cells are maintained in
Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, penicillin
and streptomycin. For drug treatments, cells are treated at confluence as per indicated
concentrations and for desired incubation time. Medium is changed regularly, and treatments
are continued. For siRNA-directed silencing, 200 pmol of purified siRNA directed against the
proteasome subunit β5 are transfected with 10 μl of Lipofectamine 2000 in APP695-HEK293
cells plated in 35 mm dishes. At 48 h post-transfection, cells are incubated in the absence
or presence of test drug for another 24 h. Cells and conditioned medium are harvested and
analyzed (Marambaud P, Zhao H, Davies P).13

Study of Field Excitatory Postsynaptic Potentials

Long-term potentiation (LTP) is a form of synaptic plasticity, which exhibits several features
that make it an attractive candidate as the neural basis of vertebrate learning and memory.14
Study of the amplitude of field excitatory postsynaptic potentials (fEPSPs) evoked in the
hippocampal slices, may help to understand the underlying mechanisms involved.
Adult female Wistar rats weighing between 150 g and 200 g are housed under standard
laboratory conditions of light, temperature, food and water. The animals are singularly housed
in clean cages. On the day of the experiment, they are anesthetized using halothane and
decapitated. Transverse hippocampal slices, 400 µm thick, are prepared. The CA3 region is
removed before the slices are transferred to a recording chamber at the interface between
a warmed (29-31°C) artificial cerebrospinal fluid and oxygen enriched (95% O2/5% CO2)
humidified atmosphere. The standard perfusion medium comprising of (mM) NaCl 124, KCl
3, NaHCO3 26, NaH2PO4 1.24, CaCl2 2, MgSO4 1,D-glucose 10, is bubbled with 95% O2/5% CO2
and perfused at a rate of 1 ml/min. Extracellular recordings of fEPSP are obtained from stratum
radiatum of the CA1 region using 3 M NaCl filled glass electrodes (resistance 2-10 MΩ). The
Schaffer collateral-commissural fibers are stimulated every 30 sec using a bipolar stimulating
electrode placed in stratum radiatum towards the fimbrial end of the slice. The stimulus
strength is adjusted to evoke a fEPSP half the maximum amplitude and a stable baseline of
at least 15-20 min is recorded before any drugs are added. Test drug is administered either by
addition to the perfusion fluid for a period of 5 min, or by iontophoresis into the CA1 recording
region.
442 Drug Screening Methods

For iontophoretic experiments, triple-barrelled electrodes can be used in which one barrel
is used for recording, one contains drug solution and one contains 200 mM NaCl for current
balancing. The resistance of the drug and balance barrels is 40-80 MΩ and a holding current of
25-40 nA is applied to the drug barrel to limit leakage.15

Cultures of Rat Cerebral Cortical and Hippocampal Neurons

Cultures of rat cerebral cortical and hippocampal neurons have been used to evaluate various
intracellular mechanisms involved in synaptic plasticity, neuronal development and learning
and memory.16
Timed pregnant Sprague-Dawley rats are procured and housed individually in a climate-
controlled environment on a 12 h light/dark cycle with free access to food and water. Newborn
rat pups are anesthetized with pentobarbitone and their cerebral cortices are dissected and
placed in an isotonic salt solution containing 100 units of penicillin G, 100 µg of streptomycin,
and 25 µg of amphotericin B per milliliter, pH 7.4. The pia mater and the blood vessels are
cleaned from the cortices, which are then chopped into pieces and incubated in 4 ml of an
isotonic salt solution containing 0.25% trypsin (w/v) in a shaking water bath at 37°C for 10
min. The reaction is stopped by the addition of 4 ml of 0.016% DNase and further incubation
is carried out in a water bath for 5 min. After incubation, regular DMEM (with 10% PDHS) is
added and the cells are triturated to dissociate them. The cell suspension is then centrifuged at
600 g for 10 min at 24°C. The supernatant is aspirated and the pellet is resuspended in regular
DMEM and plated onto poly-L-lysine-coated culture dishes at a density of 3 × 106 cells/35
mm dish. The dishes are then incubated at 37°C in a 5% CO2, 95% O2 atmosphere. On day 3,
the media is replaced with fresh DMEM containing 10µ M β-cytosine arabinoside to diminish
glial cell proliferation. On day 5, the β-cytosine arabinoside-containing media is aspirated
and regular DMEM is added to the cells. The cells are grown for seven more days. The cells
express receptors belonging to glutamate family and the cellular mechanism for memory can
be studied using these cells in the presence of various agonists and antagonists.16
Similarly, hippocampal cultures can be prepared and utilized for determining in vitro effects
of test drugs. In brief, the hippocampus is dissected, chopped into pieces, and incubated with
2 ml of 0.25% trypsin (w/v) in a 37°C water bath for 10 min. The reaction is stopped by addition
of 2 ml of 0.016% DNase, followed by four to five times trituration with a fire-polished pipette
and a 5 min incubation at 37°C. The supernatant is transferred to a tube containing 20 ml of
DMEM/PDHS, and the trypsin treatment is repeated two more times. The procedure for the
cortical cultures is then followed and the cells are plated at a density of 1 × 106 cells/35 mm
dish. The neurons are maintained similarly to the cortical cultures. The cells are rinsed and
incubated for 50 min at 37°C in HEPES buffer (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,
100 nM glycine, 15 mM glucose, and 25 mM HEPES, pH 7.4) containing 1 µM tetrodotoxin,
TTX. Drug treatment is carried out.

Motor-based Intracellular Transport

Motor-based intracellular transport involves directed movement of cargos along microtubules
and forms a key component of transport within a cell. Its regulation is crucial to the functioning
of a cell, as disruption of transport is linked to Alzheimer’s and other neurodegenerative
 Anti-Alzheimer Agents 443

diseases.17 A model of two-motor arrangement driven cargo transport is one of the simplest
case of intracellular motor transport. Motors frequently detach and reattach; so as to transport
cargo to the microtubules. Involvement of at least one motor is essential for the transport. In
the presence of tau protein, rebinding is suppressed. Therefore, once the first motor disengages
from the microtubules, it is unlikely to reattach before the second motor also detaches. The
processive motion ceases and is diffused away. Tau protein assumes importance as it reduces
cargo travel distances. By blocking rebinding, tau reduces the number of motors that on
average drive the cargo. Therefore, tau reduces the total force that the motors can apply to
move the cargo as also the force needed to detach the cargoes from microtubules.
This model suggests that it might be possible to regulate the number of engaged motors
by controlling motor on rate, i.e. how long it takes motors to bind to the microtubules. The
more quickly the motors can reattach to a microtubule, the more time it will spend actively
participating in transport. Conversely, if the motors’ on rates were lowered, on average fewer
of the geometrically available active motors would be bound at any given time. Tau critically
controls the number of engaged motors and thereby cargo transport. Thus, this model depicts
a potentially important mechanism of local regulation of intracellular transport mechanism.

Hypoxia-induced Memory Deficit

Oxygen homeostasis is essential for the development and physiology of an organism. Hypoxia-
inducible factor-1 (HIF-1) (is the principal molecule regulating oxygen homeostasis. The
hypoxia) signal transduction pathway plays a major role in vascular development and ischemia,
as well as in neurodegeneration.
APP23 transgenic mice carry the human APP751 cDNA with the Swedish double mutation
at positions 670/671 (KM→NL) under control of the murine Thy-1.2 expression cassette.
Littermates of APP23 mice carrying no human Swedish mutant APP751 cDNA are used as
controls. APP23 mice (8 months of age, n = 20, 50% female) are assigned randomly to hypoxia
and control groups. The hypoxia group was treated in a hypoxia chamber at 8% O2 for 16 h/day
for 1 month. The oxygen level is regulated by infusing nitrogen into a semisealable chamber
controlled by a controller.
This model is useful for evaluating the hypoxia facilitated enzyme cleavage of APP and
deposition of Aβ.18

Acetylcholinesterase Activity
The procedure was first described by Ellman (1961) and is also known as Ellman Esterase
Assay.19 The method aims to determine the rate of hydrolysis of acetylthiocholine (ATCh)
by acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) in tissues taken from the
laboratory rat or dog.
This assay is a spectrophotometric method, which involves two linked reactions to
produce a colored compound. The production of the compound is monitored by measuring
the absorbance of light by the reaction mixture over time. ATCh is hydrolyzed enzymatically
to give acetate and thiocholine. Thiocholine reacts with 5,5’-dithiobis-2-nitrobenzoic acid
(DTNB) producing the yellow colored 5-thio-2-thionitrobenzoic acid anion (TNB). TNB has
absorbance maxima at a wavelength of 412 nm.
444 Drug Screening Methods

Rat striata are homogenized in 10 volumes of 0.1 M phosphate buffer (pH 8.0). Five
minutes after the addition of 400 µl of homogenate, 30 µl of test compound solution at various
concentrations, and 100 µl of DTNB (10 mM) to 2.45 ml of 0.1 M phosphate buffer (pH 8.0) are
added and AChE activity is determined at 25°C over 1 min in a photocell after the addition of
20 µl of acetylthiocholine iodide (75 mM) as substrate.
A substrate blank (i.e., no tissue, only substrate and buffer and DTNB) should be run with
each group of assays; this measures nonenzymatic substrate hydrolysis. A tissue blank (i.e.,
only tissue, buffer and DTNB but no substrate) should be run for each tissue to determine
the degree of binding of DTNB to sulfhydryls in the tissue sample. If this is minimal after the
preincubation period, then the tissue blank will not be needed on a regular basis for that tissue.

IN VIVO MODELS
The development of animal models mimicking the complex pathogenesis and clinical
symptoms of AD still represents a challenge for the preclinical investigators. There is no ideal
model of AD, but there are several models, which may help in the screening of potential
therapeutic agents so as to accomplish an effective armamentarium to combat AD.

Aging Animals (Rodents and Monkeys)

Impairment of learning and memory in aging animals are severe and consistent with behavioral
impairments in the elderly. Since AD is a neurodegenerative disorder associated with aging,
aged animals are the most common for investigating drugs potentially active on AD. This model
has several advantages namely; (a) old mice and rats are used most frequently since they are
easy to obtain and relatively cheap, (b) aged rodents and monkeys show cognitive impairment.
Old monkeys have been used as the last preclinical step before clinical trials, and (c) amyloid
beta plaques, one of the classical pathological hallmarks, have also been found in aged (23 to
31 years) rhesus monkeys. Therefore, testing in nonhuman primates is an important model.20,21
The limitations of this model being: (a) since aging and AD are distinct, aged rodents and
monkeys are not ideal animals for this disease, (b) old monkeys are expensive for drug screening,
and (c) the poor health of aged animals and individual pharmacokinetic variabilities in drug
absorption, metabolism, and distribution may sometimes yield statistically inconclusive
data.22,23

Transgenic Mice
Identification of AD-related genes (four genes located on chromosomes 21, 19, 14 and
1) has made it possible to rationally design transgenic animal models and test some of the
hypothetical mechanisms of amyloidogenesis. The mutations of these genes cause changes in
processing of APP, leading to amyloid formation in the brain.24
There are now a number of genetically engineered Alzheimer’s mouse models. Transgenic
mice expressing familial AD mutations or fragments of normal APP: the Minnesota mouse
and exemplar/Athena mouse have been developed. Both these models are characterized by
accumulation and deposition of amyloid, Minnesota mouse at 7 to 12 months and Athena
mouse at 4 to 8 months of age. However, data published for these two models seem to indicate
 Anti-Alzheimer Agents 445

that several key features of AD are established; but in either model, there is no change in the
number of neurons in those areas of the brain in which amyloid deposits are found. Moreover,
there are no reports showing degeneration of the cholinergic cells of the basal forebrain in APP
transgenic mice.25-27
Of other transgenic AD animal models exhibiting senile plaques and Abeta-associated
neuropathology, different types of transgenic mice expressing human APP, Abeta, the
C-terminal fragment of APP and the APP genes carrying familial AD mutations have been
created. Transgenic mice over-expressing human APP751, which develop early AD like
histopathology with diffuse deposits of Abeta and aberrant tau protein immunoreactivity
in some, exhibit age-dependent deficits in spatial learning in a water maze task and in
spontaneous alteration behavior in a Y-maze. Transgenic mice models have been reported
by number of research groups. The most commonly used models express variants of amyloid
precursor protein (app), presenilin-1 (ps1) or presenilin-2, tau or apolipoprotein E.
The development of transgenic (Tg) mice that over express the Swedish mutation of human
APP has been developed. Amyloid plaques in hAPPs mouse brain, like those seen in human
AD patients, lead to activated microglia, oxidative stress and alterations in neurotransmitter
systems. Distribution of amyloid plaques is not uniform and densely concentrated in the
cortex, hippocampus, and amygdala throughout the brains of Tg2576 mice. This is similar to
the nature and pattern of the neuropathology seen in human AD patients.
One of the main challenges in cognitive studies of APP transgenic mice has been determining
the onset of memory deficits.28 Although, transgenic mice develop a wide variety of behavioral,
biochemical, pathological, and physiological traits, not all traits have been represented within
an individual mouse line.

Bigenic Mice
All of the transgenic mice used to evaluate potential therapeutic interventions have been app
transgenic mice or app/ps1 bigenic mice, because they are the only transgenic mouse models
in which both amyloid deposition and progressive memory loss have been shown. The lack of
neurofibrillary tangles or significant neuronal loss in app or app/ps1 mice limits the utility of
the model. It is possible to produce both amyloid plaques and neurofibrillary tangles within
individual mice by crossing app to tau transgenic mice, the resultant mice are not suitable for
learning or memory assessments because they become paralyzed.29

Triple Transgenic Mouse Model

3xTg-AD mouse is a Triple Transgenic Mouse Model of AD mouse, which shows etiopathology
similar to the progression of the disease in humans. An age-dependent increase in soluble
amyloid β (Aβ), its neuronal accumulation and extracellular deposition is recorded by the age
of seven months. The relative abundance is seen in the following order: subiculum > amygdale
> CA1 region of hippocampus. Concomitantly, changes in behavioral paradigms are recorded
from three months onwards. 30

Femtosecond Laser Ablation for Inducing Vascular Dementia in Transgenic Mice

Vascular dementia (VaD) accounts for roughly 15-20% of all types of dementia making it the
second leading cause of dementia behind only Alzheimer’s disease. The pathological features
446 Drug Screening Methods

commonly associated with VaD include large artery infarctions, small artery subcortical
infarctions, cerebral amyloid angiopathy, hippocampal sclerosis, chronic subcortical ischemia
leading to selective loss of neurons, glial cells and endothelial cells. Depression, apathy and
mood changes leading to personality changes accompany this disease and are an important
behavioral component of VaD. The risk factors for vascular dementia are stroke, microbleeds,
hypertension, type 2 diabetes and atherosclerosis.
Femtosecond laser ablation can be used to injure the endothelium of a targeted vessel
and trigger clotting in transgenic mice that express fluorescent proteins in neurons. Blood
flow changes can then be determined by tracking red blood cell motion in 2-photon images.
Occlusions in the penetrating arterioles cause a severe drop in flow in the downstream
capillaries and lead to a region of neuronal death. Dendrite degeneration, can be observed
within minutes of vascular occlusion in real time using. In a stepwise manner, degeneration
progresses to cellular death in the ischemic region created due to small vessel occlusions, and
lastly measurable behavioral and functional impairment. These lesions are accompanied by
activation of inflammatory cells, such as microglia and leukocyte invasion.31

BEHAVIORAL PARADIGMS TO ASSESS LEARNING AND MEMORY

Passive Avoidance
Step-down Model
This is a classical model for the assessment of cognitive performance after inducing brain
lesions. The term “passive avoidance” is usually employed to describe experiments in which
the animal learns to avoid a noxious event by suppressing a particular behavior. The step-down
type of passive avoidance task is used to examine the long-term memory based on negative
reinforcement.
The apparatus consists of a transparent acrylic cage (30 cm × 30 cm × 40 cm) with a grid
floor, inserted in a semi-soundproof outer box (35 cm × 35 cm × 90 cm). The cage is illuminated
with a 15 W lamp during the experimental period. A wooden platform (4 cm × 4 cm × 4 cm) is
fixed in the center of the grid floor (35 cm above the floor) and electric shocks (1 Hz, 500 msec,
40 V DC) are delivered using an isolated pulse stimulator.
The training is carried out in two sessions. The animal (mouse or rat) is placed on the
elevated platform. Due to its innate exploratory behavior, the animal steps down on to the
grid. The step-down latency (SDL) is noted. As soon as the animal steps on the grid it is given
an electric shock (for duration of 15 sec). SDL, number of flinching reactions and vocalizations
are measured.
Animals showing a SDL of 3 to 30 sec during the first training session are selected for
second (60 to 90 min after first) and retention trials. Animals staying 60 sec on the platform are
considered as remembering the task and do not receive electric shocks any more. The retention
test is carried out 24 h after training, in a similar manner, except that the electric shocks are not
applied to the grid floor. Each animal is again placed on the platform and SDL is recorded,
with an upper cut-off time of 300 sec. Both saline-treated and amnestic-drug treated animals
show the same SDL during the first training session. However, amnestic drugs are expected to
increase the SDL in the subsequent trials indicating the impairment of learning and retention
tasks. The failure should be ameliorated by test drug.48
 Anti-Alzheimer Agents 447

Active Avoidance
Step-through Model
Active avoidance is induced by a sequence of conditioned and unconditioned stimuli to
the animal. In response, the animal should actively step through to relocate to an adjoining
compartment within a preset time, in order to avert the mild electric shock.49 The latency from
stimuli onset to escape of subject, after the pretraining, is related to the retention of memory
task. This model is based on the innate behavior of the animal to seek dark areas and avoid
brightly lit area. The trial can be divided into three phases:
1. Familiarization
2. Learning
3. Retention.
After training the animal on this system, it is treated with amnesia-inducing agent and
sufficient time is allowed to elapse for the manifestation of the signs and symptoms. The animal
is again tested for its step-through latency before and after administering test or standard drug.
A typical shuttle box apparatus consists of two identical opaque dark compartments
(31 cm × 18 cm × 29 cm) separated by a wall with a rectangular (7 cm × 10 cm) opening with a
sill situated on the grid floor level. Each compartment is illuminated by a 5 W lamp mounted
centrally on the top of the apparatus. The floor in each compartment is made of stainless steel
bars.
i. Familiarization: The animal is exposed to a conditioned stimulus (darkness). The conditioned
stimulus lasts up to 5 sec or until the conditioned reaction emitted, whichever comes first.
ii. Learning: The animal receives a foot shock (Mice—1 mA, 1 sec; Rat—1.5 mA, 2 sec). The
animal has 3 to 5 sec to avoid the unconditioned stimulus.
iii. Retention: After learning, the animal takes the cue and avoids step-through thereby
increasing latency.
A trained animal is then subjected to scopolamine or colchicine; and after recovery, the
animal is again exposed to test. The effect of test drug is compared with standard drug.

Maze
Mazes are traditional tools for assessing learning and memory performance in laboratory
animals. Conventionally, maze consists of open and enclosed arms. The rodents have a natural
inclination towards enclosed area and spend more time there in comparison to open area. On
the basis of this, the transfer latency of the animal is recorded. The animal learns to avoid open
arms and shortens transfer latency to enclosed area. Nootropic agents are effectively screened
using this paradigm in scopolamine-induced dementia. Elevated Plus Maze, Radial Maze, Y
Maze, and Figure 8 Maze are based on this phenomenon.
The plus maze consists of two opposite open arms (50 cm × 10 cm), crossed with two closed
arms of the same dimensions with 40 cm high walls. The arms were connected with a central
square (10 cm × 10 cm). Rats are placed individually at one end of an open arm, facing away
from the central square. Time taken for the rat to move from the open arm and enter into one of
the closed arms is recorded and termed as “initial transfer latency” (ITL). Animals are allowed
to explore the maze for 30 sec after recording transfer latency. Retention transfer latency (RTL)
is recorded by placing the rats similarly on the open arm at specified intervals.50
448 Drug Screening Methods

Morris Water Maze

The Morris water maze was developed by Richard Morris in 1984. It is the most popular task in
behavioral neuroscience; and in its most basic form, it assesses spatial learning and memory
along with nonspatial discrimination learning.51 Performance in the Morris water maze is
acutely sensitive to manipulations of the hippocampus.
The rodents are placed in a large circular pool of water from where they can escape onto
a hidden platform to avoid swimming. In this way, the animal learns the spatial location
of the platform. The latency to escape from water onto hidden platform is measured. The
neuroendocrine mechanisms involved in this case may be responsible for the rapid learning
capability of animals. It is useful for studying working and reference memory processes also.
Motor, motivational or sensory factors can be easily eliminated. The animals are trained faster,
easily and with reliability using water maze in comparison to radial arm maze.
The water maze consists of a large, circular, galvanized steel pool (1.8 m in diameter, 0.6 m in
height). A white platform (10 cm in diameter) is placed inside, and the tank is filled with water
(22°C) until the top of the platform is submerged 1 cm below the water surface. A sufficient
amount of white paint is added to make the water opaque and render the platform virtually
invisible. In addition to the visual cues on the walls of the laboratory (shapes), five sheets
of paper with black-and-white geometric designs attached to the sides of the tank serve as
additional cues. An automated tracking system can be used to analyze the swim path of each
subject and calculate escape latencies (the time between being placed in the water and finding
the hidden platform), total path lengths, average swim speed and thigmotaxia (percentage of
time spent in periphery).
The protocol for reference memory is as follows. Before beginning acquisition training,
mice are given a pretraining acclimatization session during which they are allowed to swim
in the pool for 5 min without the platform present. On the following day, mice are given seven
acquisition sessions that consist of four trials per day with an intertrial interval of 10 min.
Throughout the course of this acquisition period, the hidden platform remains in the same
fixed position for all mice. Four points along the perimeter of the maze arbitrarily designated
as N, S, E, and W, serve as starting points where the mice are released, facing the wall of the
tank, at the beginning of each trial (the order of the starting points is determined randomly,
except that each starting point is used only once in each session). Once a mouse locates the
platform, it is allowed to remain there for 30 sec before being removed from the tank. If a mouse
fails to locate the platform within 120 sec, it is manually guided to it. After seven sessions of
acquisition training, mice are subjected to a reversal test in which the platform is moved to the
opposite side of the tank. Other task parameters remain identical to the acquisition procedures
(i.e. 10 min intertrial interval, each trial begins from a different release point).
The training for working memory task is described briefly. The platform is located in one of
the 24 possible positions, with the exact platform position on any given day being randomly
determined (positions along the perimeter of the tank and in the exact middle are excluded).
As in the reference memory procedure, if a mouse fails to locate the platform in 120 sec, it is
manually guided to it. The second trial begins after a period of 30 sec on the platform, when
the mouse is again released into the water from the same position as the first trial (first trial
start positions are still randomly determined). To be eligible for testing with sample or vehicle,
the subjects are required to locate the platform in less than 30 sec on two of the three trials
 Anti-Alzheimer Agents 449

subsequent to the first, and are required to meet this criterion on three of their four most recent
training sessions.
Sample tests are conducted once or twice per week, with at least 72 h and one training session
between tests to ensure sample clearance. In addition, the tests are conducted identically to
training sessions except that only two trials are run.
Experiments using a cued procedure can also be conducted. The location of the platform is
made known to the mice by placing a black rubber stopper (height, 3 cm; radius, 1.5 cm) on the
platform that extends about 2 cm above the surface of the water. The platform, which remains
submerged 1 cm below the surface of the water, is moved to a new location each day in the
same manner as in the working memory procedure. Test sessions consist of four trials, each
starting from one of the four release points. Mice are allowed to rest on the platform for 30 sec
in between trials.52

Conditioned Avoidance Response (CAR)

This test characterizes the influence of the compound on both learning ability and memory
(retention of the avoidance behavior after extinction period, i.e. the rest period without training
and drug treatment). The method is valid and considered as essential in vivo procedure for
testing the cognition-enhancers, nootropics and neuroprotective agents (e.g. piracetam,
nimodipine, bilobalide, memantine). This test shows particularly convincing results in
neurodeficit-animal models in which memory processing is impaired by scopolamine,
lesions or aging. The test is also often used to identify drugs with antipsychotic activity where
antipsychotics disrupt learning.
Test is carried out in a shuttle-box, which is divided into two equal compartments, which
are connected. The floor of each compartment contains a steel grid with the help of which the
unconditioned stimulus, a foot-shock of 2.0 mA is delivered. The protocol for the experiment is
described in the chapter “Antipsychotics”.
A shortening of avoidance latency and an increase in the number of correct responses
relative to the vehicle-treated group indicate that the test drug improves avoidance behavior
and enhances memory processing.53

Microdialysis of Acetylcholine Release

In vivo microdialysis techniques can be used to estimate the cholinergic neuronal activity in
the hippocampus of freely moving rats.
Adult Wistar rats (200 to 250 g, either sex) are anesthetized with ketamine (100 mg/kg,
ip) and a 3 mm concentric guide cannula stereotaxically implanted into the hippocampus
(5.08 mm posterior, 4.8 mm lateral to the bregma, 4.0 mm ventral to the dura). Two days
after surgery, a dialysis probe is inserted through the guide cannula and perfused at a
flow rate of 1 µl/min with Ringer’s solution (KCl 2.7, NaCl 147, CaCl2 2.3 mM) containing
10 µM physostigmine. Perfusion is performed to obtain the stable baseline for 120 to 180 min,
and successive 20 µl samples are collected at 20 min intervals.
Test samples can be administered into the hippocampus via the dialysis probe. The
extracellular levels of ACh are measured using high performance liquid chromatography
450 Drug Screening Methods

(HPLC) with an electrochemical detector. ACh and choline are separated using an AC-Gel
column attached to enzyme reactor containing acetyl cholinesterase and choline oxidase.
ACh can be assayed using the HPLC-electrochemical detector following its conversion to
hydrogen peroxide, which is electrochemically detected with a platinum working electrode
at 450 mV versus the Ag/AgCl reference electrode. The mobile phase contains 0.1 M sodium
phosphate buffer (pH 8.5), 0.6 mM tetramethylammonium chloride and 0.8 mM sodium
1-decanesulfonate. The HPLC separation and enzymatic reactions are to be performed at
33°C.54

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 cHAPTER

30
Antiparkinsonian Agents
Introduction
Parkinson’s disease (PD), a neurological syndrome is characterized by bradykinesia, postural
instability, rigidity and involuntary tremors. These motor symptoms result from progressive
neurodegeneration marked by selective and extensive loss of dopaminergic neurons of
substantia nigra. It is understood that interplay of genetic and environmental factors may
disrupt normal intrinsic physiology and contribute to neuronal death along with possible
initiation of repair mechanisms. Biochemically, there is depletion of dopamine (DA) and
increment of acetylcholine (Ach) in the affected area.
In India, with an aging population and increased life expectancy, it is expected that the
disease burden due to PD will be enormous, but there is no prospective study to estimate its
incidence and mortality. The incidence rates (IRs) in different countries vary from 1.5 to 20 per
100,000 per year.1
Recently, manganese has also been held responsible for neurotoxicity in the central
nervous system and the basal ganglia, and produces neurological symptoms that are similar
to that of Parkinson’s disease.2 Chronic exposure to manganese initiates a cascade of events
that leads to selective dopaminergic dysfunction, neuronal loss, gliosis in basal ganglia
structures, astrocytic changes ultimately leading to a disruption of oxidative phosphorylation.
In addition, manganese causes a number of other functional changes in astrocytes that lead to
compromised energy metabolism and neurotoxicity.
Advances in understanding of pathogenesis of PD indicate role of Renin-angiotensin system
(RAS). Angiotensin, via type 1 receptors, activates NADPH-oxidase complex, to mediate key
events leading to oxidative stress. Hyperactivation of RAS and its components in the basal
ganglia and nigrostriatal system exacerbates OS and the microglial inflammatory response
and contributes to progression of dopaminergic degeneration. In addition counter-regulatory
interactions between angiotensin and dopamine have also been observed in several peripheral
tissues.3
Belladonna alkaloids, antispasmodics and antihistaminics are some of the class of drugs,
which have been traditionally used for the management of parkinsonism. However, their
introduction into the physician’s itinerary has been incidental and not as a result of systematic
search. Recent studies using adenosine receptor agonists have also shown neuroprotective
effect in PD through the reduction of excitatory neurotransmitter release, apoptosis and
454 Drug Screening Methods

inflammatory responses.4 The current drug therapy includes dopamine precursor (l-dopa),
dopamine agonists and anticholinergics. L-dopa is decarboxylated to dopamine in the
dopaminergic neurons which helps to maintain adequate motor functions. L-dopa provides
therapeutic benefit for most early-stage PD patients. However, from the available line of drugs,
no single agent is capable of controlling neuronal degeneration. They only provide symptomatic
relief. In addition, there are reports that l-dopa is itself neurotoxic and an active participant
in the oxidative stress cascade.5,6 Further debilitating conditions like neurogenic orthostatic
hypotension are part of PD and no pharmacotherapy exists. Recently, short-term clinical
trials have shown the potential of Droxidopa, an oral prodrug converted by decarboxylation
to norepinephrine, for symptomatic relief of neurogenic orthostatic hypotension in PD.
Improvement in daily activities, falls, and standing systolic blood pressure has been reported.7
It is, therefore, essential to have an alternative line of therapy, which not only provides
symptomatic relief but also has a neuroprotective activity. Since to date no such wonder drug
is available, it is imperative that the research in this field be intensified.
The molecular players in genesis of PD have been identified as inflammatory cytokines
(tumor necrosis factor-α, interleukin [IL]-6, and IL-1 β), nitric oxide, prostaglandin E2, and
reactive oxygen and nitrogen species (Fig. 30.1). Uncontrolled activation of microglia, the
resident innate immune cells, affects neurons by releasing the molecular mediators of
neuroinflammation leading to PD.8 Addressing these lacunae may prove useful strategy for
development of future preventive and therapeutic agents for this neurodegenerative disorder.
Appropriately designed models, which allow such investigations are essential.

Figure 30.1: Etiopathology of Parkinson’s disease

 Antiparkinsonian Agents 455

The patients suffering from idiopathic PD exhibit the three cardinal symptoms of muscular
rigidity, resting tremors and hypokinesia. The rodents or primates per se do not exhibit these
signs and symptoms. Hence, to induce the underlying pathologic changes and mimic the
condition of parkinsonism in experimental animals, certain chemical agents are available.
Compounds, which were reported to produce adverse effects resembling parkinsonism in
patients, were tested in laboratory animals for the induction of extrapyramidal disorders.
Major tranquilizers, reserpine and phenothiazine derivatives have been successfully used
to set-up models of parkinsonism in animals. The protective effect of the test drugs can then
be compared to that of standard in these animals by assessing them on various behavioral
paradigms.

IN VITRO and EX VIVO MODELS

In Vitro Primary Microglial Cultures
Primary rat microglial-enriched cultures are obtained from cerebral cortices of two-day-old
rat pups. After removing the meninges, cortices are sectioned and washed in Hank’s Balanced
Salt Solution supplemented with Hepes/Na pH 7.4 (10 mM), MgSO4 (12 mM), 50 U/ml
penicillin and 50 μg/ml streptomycin and dissociated with 2.5 mg/ml trypsin type IX in the
presence of 1 mg/ml deoxyribonuclease for 10 min at 37°C. The cell suspension obtained is
diluted in medium containing 10% horse serum and centrifuged (50 g for 15 min). The cells are
cultured in Minimum Essential Medium Eagle supplemented with 10% horse serum, 33 mM
glucose, 2 mM Glutamax, 50 U/ml penicillin, 50 μg/ml streptomycin and 20 ng/ml GM-CSF
and maintained in flasks at 37°C in a humidified 5% CO2 incubator. Microglia cells are obtained
by gentle manual shaking of the flasks two to three days after dissection. Detached cells (about
75 to 85% microglia with a 15 to 25% astrocytic contamination) are plated with fresh medium
containing GM-CSF on 12-well plates coated with poly-l-lysine (100 μg/ml). Purity of highly-
enriched microglial cells is assessed.
Stimuli (LPS, IL-1β, TNF-α and INF-γ) are administered to the culture medium and assessed
for activation of inflammatory and neurodegenerative processes in microglia.9

Experiments Using Rat Striatal Slices

Striatum is the brain region, which is primarily affected in parkinsonism. The release of the
neurotransmitters like dopamine and acetylcholine in response to test agent serves as a good
in vitro marker of its activity.
Male Sprague Dawley rats (150 to 250 g) are decapitated; the skull is opened and the right
and left striata are removed and placed in ice-cold Krebs’ solution of the following composition
(in mM): NaCl 118, KCl 4.85, CaCl2 1.3, KH2PO4 1.15, NaHCO325 and C6H12O6 11.1. The striata
is cut into 0.4 mm thick slices using a tissue chopper. The slices are kept floating for 30 min in
Krebs solution continuously gassed with 95% O2 and 5% CO2 at room temperature. The slices
are labeled by incubating for 30 min at 37°C with [3H] dopamine (5 mCi/ml) and [14C] choline
(2 mCi/ml) in the presence of 0.15 mM pargyline chloride and 0.1 mM ascorbic acid. Labeled
slices are transferred to superfusion chambers and perfused with Krebs solution at 37°C at
a flow rate of 0.5 ml/min. After washing and stabilization, 5 min fractions of superfusate are
collected. The perfusion buffer contains 1 mM nomifensine to inhibit dopamine reuptake and
456 Drug Screening Methods

10 mM hemicholinium to inhibit choline uptake. The slices are subjected to field stimulation
with rectangular pulses of alternating polarity, with a current strength of 10-15 mA/cm2 and a
pulse duration of 2 msec at a stimulation frequency of 3 Hz for 5 min. Drugs to be tested are
present in the superfusion fluid. The radioactivity in the superfusate samples and in the tissue
is determined by liquid scintillation counting.10
The radiolabelled choline method makes it possible to study ACh release in vitro without
inhibiting cholinesterase, thus minimizing auto inhibition of transmitter release caused by an
accumulation of unhydrolysed ACh.

Dopamine-stimulated Adenylyl Cyclase Activity

Male Sprague Dawley rats (150 to 250 g) are decapitated and the right and left striata are
removed. Striatal tissue is homogenized by teflon glass homogenizer in chilled buffer
containing 10 mM imidazole, 2 mM EGTA, and 10% sucrose, pH 7.3. The homogenate is
centrifuged at 1,000 g for 10 min, and the supernatant that is obtained is re-centrifuged at
27,000 g for 20 min. The pellet obtained is washed twice and suspended in 10 mM imidazole,
pH 7.3. Membrane protein is determined by Bradford’s method using bovine serum albumin
as standard. Adenylyl cyclase activity is measured by calculating the conversion rate of [32P]
ATP to [32P] cAMP. The assay is performed in 250 µl of solution containing 10 mM imidazole, pH
7.3, 2 mM MgCl2, 0.1 mM papaverine, 0.2 mM EGTA, 1 mM dithiothreitol, 1 µM GTP, 0.1 mM
ATP, 2 mM phosphocreatine, 5 units of creatine phosphokinase, and 1 µCi of [a-32P] ATP. The
reaction mixture is preincubated at 30°C for 5 min, and the reaction is initiated by adding 50 to
60 µg of membrane proteins and incubated for an additional 10 min. The reaction is terminated
by the addition of 300 µl of stopping solution (2% SDS, 25 mM ATP, and 1.3 mM cAMP). Formed
[32P] cAMP is separated from [32P] ATP by chromatography.11

Radioligand Binding Studies for D1 and D2 Dopamine Receptors

Male Sprague Dawley rats (150 to 250 g) are decapitated and the right and left striata removed.
Striatal tissue is homogenized with teflon glass homogenizer in 20 volumes of ice-cold
buffer containing 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, and 10% sucrose. The homogenate
is centrifuged for 5 min at 800 g, and the supernatant is centrifuged for 20 min at 49,000 g.
The pellet is washed and suspended in 50 mM Tris-HCl buffer, pH 7.4. Reaction mixtures
containing 50 mM Tris-HCl buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2
and [3H] SCH23390 (0.1-6.4 nM) or [3H] raclopride (0.25-16 nM), in a total volume of 250 µl
is incubated at 37°C for 30 min and terminated by vacuum filtration through Whatman filter.
Further, washing with cold 50 mM Tris-HCl buffer, pH 7.4 may be given. Nonspecific binding is
defined as binding in the presence of 10 µM cis-flupenthixol for D1 dopamine receptor binding
or 1 µM haloperidol for D2 dopamine receptor binding. In addition, 10 µM of mesulergine
is added into the D1 dopamine receptor-binding assay to prevent binding of SCH23390 to
serotonin receptors. The receptor densities and affinities are calculated by scatchard analysis.11

Dopamine Release from Synaptosomes

Mice are bred in pathogen-free colony and maintained on a 12 h light/dark cycle and housed
five in a cage with same-sex littermates, free access to food and water. Each mouse is sacrificed
 Antiparkinsonian Agents 457

by cervical dislocation and the brain removed and immediately placed on ice and chilled for
5 min. One millimeter coronal slices are taken and nucleus accumbens (NA) dissected from
slices in which the anterior commissure is visible using a 1.2 mm glass capillary tube. Frontal
cortex (FC), olfactory tubercle (OT), and striatum (ST) are dissected from the slices using
forceps. Tissue is homogenized in 0.5 ml of 0.32 M sucrose buffered with 5 mM HEPES, pH 7.5.
P2 synaptosomal pellets are then prepared from each region by centrifugation at 1000 g for
10 min, followed by centrifugation of the resulting supernatant at 12,000 g for 20 min. The P2
pellets are resuspended in perfusion buffer (in mM): NaCl 128, KCl 2.4, CaCl2 3.2, KH2PO4 1.2,
MgSO4 1.2, HEPES 25, glucose 10, ascorbic acid 1, and pargyline 0.01, pH 7.5). Synaptosomes
are diluted 5- to 15-fold with perfusion buffer to maintain uptake in a linear range (below 10%
of input counts per minute). Test and standard drugs are dissolved directly into perfusion
buffer. Aliquots of synaptosomes (90 µl) are added to appropriate concentrations of drugs
(10 µl) and incubated at 37°C for 5 min before addition of 0.5 µCi of [3H] dopamine (final
concentration 0.1 µM). After an additional 5 min of incubation at 37°C, each sample is diluted
with 0.5 ml of cold buffer and filtered. Radioactivity is determined by scintillation counting.11

In Vitro Neuroprotective Efficacy

Human Neuroblastoma cells, SH-SY5Y are cultured and maintained in Dulbecco’s modified
Eagle’s medium supplemented with 10% (v/v) fetal calf serum and a 1% (v/v) penicillin/
streptomycin antibiotics mixture. Cells were grown in an atmosphere of 95% air and 5% CO2 at
37°C for 24 h. SHSY5Y cells are seeded at a density of 4 × 104 cells/well in 96-well culture plates
and allowed to attach overnight. The cells are subjected to stress by incubating with or without
hydrogen peroxide (100 or 300 µM) for 6 h. Appropriate concentration of test drug is added to
culture plate 0.5 h before H2O2, so as to evaluate its efficacy as neuroprotective agent.
The cell survival is evaluated, by performing the MTT. Briefly, MTT is added to the cultures
at a final concentration of 0.2 mg/ml, and after incubation at 37°C for 2 h, the media is removed
carefully and the reaction is stopped by addition of isopropanol containing 0.04 N HCl. The
absorbance of each well is measured at 590 nm by using a microplate reader.
Increase in viability of the cells in test wells may indicate the efficacy of the test drug as
neuroprotective agent against a stress of H2O2.12

BEHAVIORAL MODELS OF PARKINSONISM

Reserpine-induced Parkinsonism
Reserpine is an alkaloid extracted from the dried roots of Rauwolfia serpentina belonging to
the family, Apocynaceae. The pharmacological activity of this herb is well documented in the
ancient literature and was used for the management of hypertension and psychosis. It acts by
interfering with vesicular uptake and storage of Nor-epinephrine (NE), DA and serotonin (5-
HT), both centrally and peripherally. Depletion of biogenic amines impairs the sympathetic
activity, which takes about weeks to restore after discontinuation of the drug. However, owing
to its CNS side effects, the drug has been discontinued for therapeutic use. Currently, it finds
use as an agent for inducing extrapyramidal symptoms in laboratory animals.
Both intravenous (5 mg/kg) and intraperitoneal (2.5 mg/kg) injections of reserpine in rats
produce the signs and symptoms of parkinsonism. After 20-30 min of drug administration,
458 Drug Screening Methods

motor disorders are apparent. The animals are sedated and markedly hypokinetic with poor
movement coordination. Hind limb rigidity, arched body position, fixed facial expression and
ptosis are the other typical effects of reserpine. The effects peak at 1-2 h postadministration
and subsides within 24 h. The animals may be divided into three groups, namely, test, standard
and vehicle control. About 30 min after reserpine administration, the test or standard drug
may be administered and percentage inhibition of the peak reserpine effect may be evaluated.
Various behavioral paradigms are established to quantitatively score the effect of the test drug.

Assessment of Hypokinesia
A wooden box of 88 cm × 88 cm × 60 cm is constructed and the floor is divided into 16 equal
squares. For a total duration of 120 min, the number of squares entered per 2 min by the rat is
counted. In a hypokinetic animal, the score reduces significantly.

Assessment of Muscular Rigidity

A simple grasping test helps to assess the muscular rigidity of the animal. A metal rod (id
0.5 cm) is held at a height of 50 cm above table. The animal is made to grasp the rod with its
forelimbs and the total time for which it remains on bar is noted.

Assessment of Catatonia
Appearance of catalepsy is the most obvious behavioral consequence of acute injection of a
dopamine antagonist. It is hypothesized that catatonia may be equated with Parkinson’s disease
as it also originates as a dysfunction at the level of cortico-cortical and cortico-subcortical
connectivity. Clinical similarities between Parkinson’s disease and catatonia with respect to
akinesia may be related with involvement of the basal ganglia in both disorders.13 The ability of
antiparkinson drugs to counteract this manifestation is an often studied behavioral paradigm.
An arbitrary scale is used to grade the degree of catatonia by challenging each paw to different
heights (0-9 cm).
The advantage of using reserpine model is that it helps to simulate the symptoms of
parkinsonism effectively. Thus, it helps to predict the in vivo pharmacological activity of the
test agent. However, as reserpine produces sudden depletion of biogenic amines without
producing neurodegeneration of neurons, this model cannot be used to screen potential
neuroprotective agents.

Neuroleptics-induced Parkinsonism
Neuroleptics act by blocking striatal dopamine receptors and are useful for the management
of psychosis in patients. However, phenothiazine derivatives with piperazine side chain like
trifluoperazine, thioproperazine and chlorpromazine produce typical extrapyramidal side
effects and are used as a pharmacological tool to mimic parkinsonism in laboratory animals.
Chlorpromazine in a single dose of 4 mg/kg, ip, in rats produces marked catatonia, muscular
rigidity and bradykinesia. The reversal of neuroleptic effect by test and standard drugs may
be quantitated using various behavior scoring systems (described in the previous model).
Hypokinesia may also be accompanied with tremors and rigidity, when rats are administered
chlorpromazine chronically (2 mg/kg, ip, daily for 7 days). Chlorpromazine also diminishes
 Antiparkinsonian Agents 459

exploratory behavior and conditioned avoidance response (Method for assessment described
in the chapter “Antipsychotics”).

Assessment of Tremors
The animal is lifted by tail such that its hindquarters are suspended approximately 8 cm above
table. The forelimbs are maintained in this position for 10 sec and observed for body/hindleg
tremors and scored on the basis of degree of tremors on an arbitrary scale from 0-4.
Cumulative scores of 30-60 min are analyzed in vehicle control, standard and test
groups. Test agent with antiparkinsonism effect will significantly reduce the scores.
Instruments, which allow digital recording of the tremors, have also been devised.13 There
are many pitfalls of this model. Catalepsy is the primary symptom observed in this model
of parkinsonism. L-dopa, which is the choice of drug for management of parkinsonism in
humans, partially antagonizes this symptom in rodents.14 On the other hand, anticholinergic
agents, which provide symptomatic relief to patients of parkinsonism, are quite effective in
ameliorating the signs and symptoms of neuroleptics-induced parkinsonism in rodents.15
Moreover, the neurobehavioral symptoms produced by neuroleptics are due to temporary
blockade of dopamine neurotransmission and not owing to degeneration of dopaminergic
neurons. Thus, this model does not accurately simulate the underlying pathogenesis of the
disease.

Cholinomimetics-induced Parkinsonism
In PD loss of nigrostriatal dopaminergic inhibitory neurons results in cholinergic hyperactivity.
In accordance with this hypothesis, anticholinergic agents are used for management of parkin­
sonism in patients and cholinomimetics used to set-up experimental models of parkinsonism.
Cholinomimetics like ACh, arecoline, tremorine and its potent derivative oxotremorine,
carbachol, physostigmine, nicotine, harmine and harmaline have been traditionally used, both
centrally (0.5-1.5 mg, intrastriatally) and peripherally (1 mg/kg, ip), to produce cholinergic
symptoms like tremors, excitement, limb rigidity, salivation, lacrimation, defecation, urination
and stereotypy and convulsions at higher doses (1 mg/kg, iv) in rodents. Tremorine is also
known to produce rage in cats. These are observational symptoms. The ability of the test agent
to ameliorate these symptoms indicates its anticholinergic activity.
Oxotremorine-induced tremors in mice is a sensitive, useful and convenient method for
finding antiparkinsonian drug. This model serves as a simple, precisely defined, observational
method for screening drugs with anticholinergic activity.
However, neuroleptics, antidepressants and antihistaminics can also give false positive
results in this model. In such case, the test drug may be subjected to further tests to clearly
delineate its mechanism of action:
i. In vitro antagonism of the effect of ACh on isolated tissue preparation—frog rectus
abdominus, guinea pig ileum.
ii. Peripheral in vivo effect on mouse pupil—atropine-like drugs produce dilatation, whereas
acetylcholine produces constriction.
iii. Directly testing anticholinergic effect in central nervous system—ability to antagonize
physostigmine-induced sinistrotorsion in guinea pigs.
460 Drug Screening Methods

Certain precautions ought to be taken when any drug is being centrally administered. Firstly,
its pH should be correctly balanced to avoid any drug-induced lesion in brain. Secondly, the
osmolarity of the drug solution should be checked. It is advisable to prepare all solutions in
CSF buffer. Thirdly, the volume of the drug injection should be kept to minimum (0.5-2 ml)
as large volumes can displace brain tissue. All injections should be made slowly so as to avoid
diffusion of solution into a large area.16

Surgical Induction
Direct lesion of the nigrostriatal dopaminergic neurons by injecting 6-OH-dopamine (6-OH-
DA), 1-methyl,4-phenyl,1,2,3,6 tetrahydro pyridine (MPTP) or electrolesion are selective and
reproducible ways to simulate the clinical condition in animals.

Unilateral Administration of 6-OH-DA

Unilateral administration of 6–OH-DA in the substantia nigra, ventral tegmentum or medial
forebrain bundle in rats produces degeneration of the nigrostriatal pathway.
Adult male Wistar rats weighing between 200 to 250 g are housed under standard
laboratory conditions of 12 h day/night cycle, with food and water ad libitum. The animals
are anesthetized with ketamine hydrochloride (80 mg/kg, ip) and the head region between
eye and ear of the animal is shaved. The rat is positioned on the stereotaxic apparatus with
the incisor bar at 5° below the interaural line. The animal is held in position with the help of
ear-bars. A longitudinal midline incision is made and the skin retracted. A point is marked at
the appropriate stereotaxic coordinate and a burr hole is made.17 A prefilled probe drive with a
microliter syringe mounted on top is lowered to appropriate height and a slow infusion made.
The test group is administered with 8 mg of 6-OH-DA and the control rats receive equivalent
volume of the vehicle. Minimum volume of vehicle (normal saline) is used to dissolve the
drug. Ascorbic acid (0.2 mg/ml) is added to prevent auto-oxidation. After 5 min of injection,
the infusion probe is slowly withdrawn and the wound sutured. The animal is allowed to
recuperate for 10-15 days before challenging it to battery of tests.
The neurotoxin 6-OH-DA induces permanent and selective damage of the neuronal region
where it is injected. The altered levels of cerebral monoamines, i.e. norepinephrine, 5HT
and DA induce behavioral deficits.18 In rats, inhibition of spontaneous locomotor activity,
hypokinesia, adypsia, aphagia and increase in pain sensitivity are some of the typical effects
of central administration of 6-OH-DA. Ipsilateral and contralateral turning of lesioned rats
in response to psychomotor stimulant administration is a critical behavior paradigm used to
assess the activity of test drug.
a. Spontaneous locomotor activity: A well-illuminated, sound attenuated chamber (45 cm ×
50 cm × 35 cm) containing a transparent acrylic cage (37 cm × 24 cm × 30 cm) is fabricated.
It is fitted with a photocell beam system (described in chapter “Antipsychotics”). This
system can be used to record the spontaneous locomotor activity and rearing behavior in
a variety of animals such as rats, mice and marmosets. The animal is introduced into the
acrylic cage and allowed to acclimatize for 5-10 min. The total counts of the interruptions of
the photocell beam, for a predecided duration are made. The numbers of events of rearing
behavior are also counted.
 Antiparkinsonian Agents 461

Motor activity is a good index for studying the effects of pharmacological agents.
Traditionally, investigations center around counting the number of times an animal
interrupts a photocell beam, but novel methods also use interruption of magnetic field as
a paradigm. The interruptions are significantly reduced in a hypokinetic rat. Novel sensor
systems with provision for multichannel measurements (body heat measurement, online
cannulation and monitoring) are also available.19
b. Rotational behavior in rats: The neurotoxin 6-OH-DA depends upon DA transporter for
selective presynaptic uptake, where it exerts neurotoxicity. Unilateral lesion of nigrostriatum
by 6-OH-DA in rats, induces a selective presynaptic terminal loss of dopaminergic neurons
along with postsynaptic hypersensitivity in the lesioned hemisphere.20 The rotational
behavior model, as proposed by Ungerstedt, is an important model. Systemic or central
injection of apomorphine stimulates the intact but hypersensitive postsynaptic dopamine
D2 receptors and induces contralateral rotations in rat. On the other hand, amphetamine
acts on the intact presynaptic terminals in the hemisphere contralateral to the lesion,
and induces release of DA. Thus, the rat rotates in a direction ipsilateral to the lesioned
hemisphere.
In unilaterally lesioned rats, a clear cut rotational behavior is observed in response
to very low doses of apomorphine (0.05 mg/kg, ip). The onset is within 5 min of drug
administration and lasts for 30-40 min. The total number of rotations is recorded for this
duration and its modulation by the test drug reveals the nature of activity of the test drug.
Concomitant stereotypy (gnawing, sniffing, rearing, ptosis) and cholinergic (defecation,
salivation, urination) behavior are also observed. Comparable effects of apomorphine in
normal rats are observed at a dose 10-20 times of the magnitude. Implantation of chronic
cortical and neck muscle electrodes helps to record EEG and EMG in lesioned rats and
study disease progression along with chronic effects of drug administration.21
L-dopa induces similar rotational behavior at a threshold dose of 10 mg/kg.
The cholinergic and stereotypy behavior is less pronounced. Doses between 200 and
1000 mg/kg are required to induce rotations in normal rats.22 Dexamphetamine is an
indirect dopaminergic agent which at a dose of 5 mg/kg, ip induces ipsilateral rotations.23
Unilaterally lesioned animal model is at best a representation of hemiparkinsonism. In
contrast, both the brain hemispheres are affected in humans. Moreover, these animals do
not exhibit the triad symptoms like rigidity, akinesia and tremors.

Bilateral Administration of 6-OH-DA

Bilateral administration of 6-OH-DA in the medial forebrain bundle at the level of the lateral
hypothalamic area results in a widespread depletion of regional brain catecholamine content
and are accompanied with severe neurobehavioral disturbances.24
Adult Wistar male rats weighing between 200 to 250 g are housed under standard laboratory
conditions of 12 h day/night cycle, with food and water ad libitum. They are fixed on the
stereotaxic instrument as described earlier. A bilateral infusion of 6-OH-DA (2 × 5 mg) is made
to the lateral hypothalamic site. The neurochemical and behavioral deficits are evident 48 h
later. Prominent hypokinesia, muscular rigidity, and resting tremors are accompanied with
severe adypsia, aphagia and weight loss.25 Modulation of behavior by test drug is evaluated
using paradigms described earlier.
462 Drug Screening Methods

MPTP Model
1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) is an environmental toxin, which
produces a parkinsonian syndrome after its conversion to 1-methyl-4-phenylpyridine (MPP+)
by B-form monoamine oxidase (MAO) in the brain. MPP+ perfusion into the striatum increases
extracellular DA levels. This increase may concomitantly induce the formation of reactive free
oxygen radicals (.OH), which induce lipid peroxidation of striatum membranes, as detected by
increased nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA).26 MPTP treatment
has provided best animal model of PD primarily in primates and later in mice. It produces
the same syndrome as in clinical situation and the animals respond to the same therapeutic
modalities as their human counterparts. The side effects (dyskinesia etc.) to the drugs
administered are also the same.
All primate species are sensitive to MPTP, though most studies have been conducted on
cynomolgus monkeys (Macaca fascicularis). The MPTP salt should be handled with due
precaution as it is a very potent neurotoxin. Monkeys weighing between 2 to 4 kg are used for
the experiment. For the injection, they are placed in a chair and MPTP solution is administered
intravenously in the leg vein or subcutaneously. The dosage schedule is fixed as 0.3 mg/kg/d
for 5 days. Immediately after each injection, the animals exhibit a syndrome for 10-30 min
characterized by agitation, ataxia, myoclonus, hallucinations, linguinal dyskinesia and vomiting.
The animals become akinetic progressively followed by stooped posture, adypsia and aphagia, to
an extent that it has to be intubated. After 6 to 8 weeks, the syndrome stabilizes and experiments
to test antiparkinsonian agents can be initiated. The response to test agent has to be evaluated in
terms of motor parameters. Tremors, rigidity by electromyography, and akinesia by photocells
fixed within cages. Disability scoring systems, inspired from the scoring system of human
patients, have been employed to assess the degree of disability amongst the animals.
Common marmosets (Callithrix jacchus; 300–350 g) may also be used as animals to
study parkinsonism. The animals are kept in controlled housing conditions, with constant
temperature (25°C), relative humidity (50%), and 12 h light/dark cycles and free access to food
and water. Marmosets are rendered parkinsonian by administration of MPTP-hydrochloride
(2 mg/kg s.c. for 5 consecutive days). Following stabilization of the parkin­sonian state, the
animals are treated with standard (12 mg/kg L-DOPA and 3 mg/kg benserazide) or test drug
and observed for behavioral paradigm as described above.
C57 BL/6 mice are also sensitive to systemic administration of MPTP. MPTP (2 × 40 mg/kg,
s.c., separated by a 24 h interval) is administered. After 4 to 6 weeks, the symptoms stabilize and
behavioral testing can be conducted.27 Striatal dopamine levels fall by 90%.28 There is reduction
of all three behavior parameters of spontaneous motor activity, i.e. locomotion, rearing and
total activity, during both the initial, exploratory stage (first 90 min), and later stages of the 3 or
4 h test periods.29
Primate model is an expensive and tedious method. Despite the disadvantages, it serves
as an excellent platform to test new drugs before initiating them for patients. The effect on the
various components of Parkinson’s syndrome is established along with potential to induce
side effects.
A modification in the protocol has been described by Jackson-Lewis et al. (1995) to induce
severe and mild parkinsonism in mice. In severe model, the mice receive four-dose of MPTP
(20 mg/kg, ip × 4) at 2 h intervals. Two doses of the same protocol induce milder cell injury.
 Antiparkinsonian Agents 463

FeCl3-induced Model of Parkinsonism

The cause of degeneration of nigrostriatal dopaminergic neurons in idiopathic PD is unknown.
The “oxidative stress” hypothesis suggests that there is an imbalance between the formation
of cellular oxidants and the antioxidative processes.30 Abnormalities in the metabolism of
the transition metals, iron and copper, have been demonstrated to play a crucial role in its
pathogenesis.31 Iron is central in the initiation of fenton reaction mediated cascade of events,
which ultimately ends in neuronal death.32 Based on this hypothesis in vivo rat model of
parkinsonism has been established.
Adult Wistar rats of either sex, weighing between 200 to 250 g are housed under standard
laboratory conditions of light, temperature, food and water. After acclimatization the animals
are prepared for stereotactic lesion. The animals are anesthetized using ketamine (80 mg/kg,
ip) and fixed on the stereotaxy instrument as described above. According to the coordinates
for substantia nigra the prefilled probe is lowered through the burr hole. The test group
receives 50 mg of FeCl3 dissolved in 5 ml of normal saline, whereas the control group receives
equivalent volume of vehicle. After 5 min of infusion, the probe drive is slowly withdrawn to
avoid spreading of chemical. The wound is sutured and the animal allowed recuperating for
15 to 20 days. On 21st day postlesion, the animal is subjected to apomorphine (1 mg/kg, ip)
challenge. The animals suffering from nigral lesion exhibit ipsilateral rotations. The number of
rotations executed for duration of 40 min is counted and its modulation by test and standard
drug indicates their interaction with the dopaminergic system.
In FeCl3-induced unilateral lesion of substantia nigra there is free radical mediated injury to
which both pre- and postsynaptic neurons are equally susceptible. Hence, when apomorphine
is systemically administered, it acts on the intact neurons in the contralateral hemisphere and
induces ipsilateral rotations.33
In concordance with the human patients, this model also induces permanent damage from
which the system cannot recover. In contrast, studies indicate that there are signs of recovery
from toxicity of 6-OH-DA.34
Alternatively, unilateral destruction of nigrostriatal dopaminergic neuron can also be
conducted using Ferrous citrate (2 mM in 0.9% NaCl). Rats are anesthetized (Tiletamine
hydrochloride, 125 mg/kg,ip + Zolazepam hydrochloride, 125 mg/kg, ip) and positioned
on stereotaxic instrument. The 4.0 µL of ferrous citrate solution (2 mM) is infused through
microdialysis probe with glued outlet and tear off membrane at the flow rate of 0.8 µL/min to
the right SNC using stereotaxic coordinates; AP: 3.2 mm, L: 2.1 mm and DV: 2.0 mm relative
to bregma. The injections were delivered over the period of 5 min and probes were kept in
situ for an additional 5 min after each injection to avoid the reflux along the injection tract.
The sham controls were injected with 4.0 µL of 0.9% NaCl. After surgery, animals are observed
for behavior paradigms and sacrificed at different time intervals and brain sections assessed
histologically and biochemically for DA levels.35

Ultrafine Particle Air Pollution Model of Parkinson’s Disease

Exposure to components of air pollution and agricultural pesticides has been associated
with lesions of neurons in substantia nigra and striatum and increased risk of PD in humans.
Based on this, mice are exposed to concentrated ambient ultrafine particles (CAPS; <100 nm
diameter) during the first two weeks, postnatal. Parkinson-like pathology can be further
464 Drug Screening Methods

induced during adulthood using agricultural pesticides (paraquat+maneb, 10+30 mg/kg, ip,
2 × per week × 6 weeks). Symptoms of loss in locomotor activity along with alterations in striatal
GABA inhibitory function, nigrostriatal dopamine, dopamine-glutamate function in midbrain
and excitotoxicity can be recorded.36

Monitoring Dopamine Concentration Using Microdialysis

Microdialysis has become a widely used method for the analysis of the extracellular fluid
composition in anesthetized and moving animals.
Male Wistar rats weighing between 220 to 240 g are housed individually at ambient
temperature, 12 h light/dark cycle and food and water ad libitum. The animals are allowed
to acclimatize for 4 days before initiating experiment. The animals are anesthetized using
pentobarbitone (70 to 80 mg/kg, ip) and placed on stereotaxic apparatus. The head is shaved
and the skin retracted. The exposed skull bone is cleaned and allowed to dry. A guide cannula
with a stainless steel obturator is implanted with its tip at the upper limit of the striatum.37 The
cannula is fixed into place using screws and dental cement. One week post-surgery the animal
is subjected to repeated dialysis after treating with test or standard drug. To perform dialysis,
the obturator is removed and dialysis probe inserted into the guide cannula and connected to
microinjection pump. It is perfused with artificial CSF (in mM: NaCl 147, KCl 4, CaCl2 3.4) at
a flow rate of 1 ml/min. After 30 min equilibration, 20 ml samples are collected in microtubes
containing 0.5 M perchloric acid and refrigerated at –80°C. At the end of dialysis, the probe is
gently removed and the obturator reinserted. The dialysate is separated by HPLC (flow rate-
0.3 ml/min, C18 reverse phase column, mobile phase: 100 mM KH2PO4, 0.1 mM EDTA, 0.28 ml
triethylamine, 1 mM octanesulfonic acid, 10% methanol/acetonitrile, 2/1 v/v, pH 4.4 adjusted
with citric acid). 31 This model helps to measure DA levels and concentrations repeatedly up to
4 months. It can be successfully used for kinetic monitoring of a given neurotransmitter during
chronic drug treatment.

Monitoring Dopamine Concentration Using LC-MS/MS

Liquid chromatography with triple quadrupole tandem mass spectrometer (LC-MS/MS) can
be used to detect DA in brain samples. Briefly, striatum is accurately weighed and mixed with
acetronitrile containing 200 µg/ml dithiothreitol and 2 µg/ml cimetidine (internal standard).
The homogenized mixture is vortexed and centrifuged (8,000 rpm) and supernatant is
collected, dried under nitrogen, and reconstituted in methanol (containing 0.1% formic acid).
The sample is injected into LC-MS/MS system with ESI operated in the positive ion mode
and analyzed under the multiple reaction monitoring (MRM) mode: DA (m/z 154 → 177,
CE = 10 eV), BH4 (m/z 242.2 → 166.0, CE = 17 eV), cimetidine (m/z 253.1 → 159.0, CE=14 eV).
For chromatographic separation hydrophilic interaction chromatography column can be
used. The recommended mobile phase is of 0.1% formic acid-acetonitrile/water (75:25, v/v).35

Effect of Dopamine Receptor Stimulation on Electrophysiological Output

from Rat Basal Ganglia
The basal ganglia functional model predicts that DA, by stimulating the striatonigral pathway
via D1 receptors and inhibiting the striatopallidal pathway via D2 receptors, should inhibit
 Antiparkinsonian Agents 465

output from the substantia nigra pars reticulata and internal pallidal segment (SNpr/GPi).
Inhibition of output from the SNpr/GPi should in turn disinhibit the thalamus to facilitate
movement.38
Male rats (250 to 275 g) are implanted bilaterally with 23-gauge stainless steel guide
cannulae into the VLS just before electrophysiological experiments. Activities of SNpr neurons
are recorded in rats anesthetized with chloral hydrate (400 mg/kg ip). SNpr neurons are
located within the pars reticulata region of the substantia nigra, within the following stereotaxic
coordinates: L, 2.0 to 2.4 mm; A, 2.8 to 3.2 mm; V, >7.0 mm. These neurons are distinguished
electrophysiologically by their sharp, biphasic extracellular waveforms, duration (<1 msec),
firing rates (10-40 spikes/sec), and location just ventral to the pars compacta dopamine
neurons.37 Electrodes are glass micropipettes filled with 1% pontamine sky blue dye in 2 M
NaCl. Standard methods are used for amplifying, displaying, and discriminating the single unit
activities of SNpr neurons.38 At the end of recording periods, a small amount of the blue dye is
iontophoretically deposited in the brain at the recording site. The animal is sacrificed and the
brain is removed, sectioned, and mounted on slides to verify location of the blue spot within
the pars reticulata. The effects of striatal drug infusions on SNpr firing are evaluated by first
obtaining a stable 3 to 5 min period of baseline firing then infusing bilaterally into the VLS over
2 min test or standard drugs or saline (1 µl/side). SNpr neuronal firing is monitored for 30 min
after infusions. Firing rates are averaged over 5 sec intervals and plotted as a percentage of the
preinfusion baseline firing rate of the cell. For each drug treatment, the average percentage
of change in firing of all SNpr neurons after the infusions is compared with their preinfusion
(baseline) rates.35,36

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12. Northoff G. What catatonia can tell us about “top-down modulation”: a neuropsyhiatric hypothesis.
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of a Novel Poly(ADP-Ribose) Polymerase-1 Inhibitor, 2-{3-[4-(4-Chlorophenyl)-1-piperazinyl]
propyl}-4(3H)-quinazolinone (FR255595), in an in vitro Model of Cell Death and in Mouse
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14. Dill RE, Dorman HL, Nickey WM. A simple method for recording tremors in small animals. J Appl
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15. Derkach P, Larochelle L, Bieger D. L-Dopa-chlorpromazine antagonism on running activity in
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in rats: relevance for evaluation of therapeutic and extrapyramidal side effect potential.
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17. Matthews RT, Chiou CY. Cholinergic stimulation of the caudate nucleus in rats: a model of
Parkinson’s disease. Neuropharmacology 1978;17:879-82.
18. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Sydney: Academic Press, 1982.
19. Butterworth RF, Belanger F, Barbeau A. Hypokinesia produced by anterolateral hypothalamic
6-hydroxydopamine lesions and its reversal by some antiparkinson drugs. Pharmacol Biochem
Behav 1978;8:41-5.
20. Masuo Y, Matsumoto Y, Morita S, et al. A novel method for counting spontaneous motor activity in
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lesions of the nigrostriatal dopamine system. Brain Res 1970;24:485-93.
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23. Ernst AM. Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats.
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27. Obata T. Dopamine efflux by MPTP and hydroxyl radical generation. J Neural Transm 2002;109:
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29. Fredriksson A, Palomo T, Chase T, et al. Tolerance to a suprathreshold dose of L-Dopa in MPTP
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1997;72:123-35.
 cHAPTER

31
Antimigraine Agents
Introduction
Migraine is a neurovascular disorder characterized by episodes of severe, throbbing
headache that is frequently unilateral, usually associated with nausea, vomiting or sensitivity
to light, sound or movement. When untreated, these attacks typically last for 4–72 h.1 In about
one-third of the patients, migraine attacks are usually preceded or accompanied by transient
focal neurological symptoms, which are usually visual; such patients have migraine with aura
(previously known as classical migraine),2 while majority of patients do not present with such
symptoms of aura (migraine without aura or common migraine).3 Migraine is a very common
disorder with a prevalence of 15–18% in females and 6% in males.4
The exact pathophysiology of migraine is still not known. Neurovascular hypothesis has been
proposed to explain the pathophysiology of migraine.3,5 The initiating trigger, which remains
unknown, is followed by a wave of cortical spreading depression.6 There is subsequent decrease
in regional cerebral blood flow leading to aura symptoms. This phase of oligemia is followed
by vasodilatation of cranial extracerebral large arteries and arteriovenous anastomoses in the
dura mater, base of skull and scalp during the headache phase. The pathophysiology of this
vasodilatation involves changes in the activity of the neurons innervating these blood vessels.
These neurons may release various vasodilating neurotransmitters like 5-HT, substance P,
calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP)7 and nitric oxide.8
This vasodilatation leads to stimulation of ‘stretch’ receptors in the vessel wall leading to
increase in perivascular (trigeminal) sensory nerve activity that provokes headache and other
associated symptoms. These trigeminal nerve fibers may also release neuropeptides, which
reinforce vasodilatation and perivascular sensory nerve activity.9
The mechanisms by which various antimigraine drugs are proposed to act include:
constriction of the dilated extracerebral blood vessels,10,11 reduction of neuropeptide release
and plasma protein extravasation across dural vessels12 and inhibition of impulse transmission
centrally within the trigeminovascular system.13
At present, there are no true animal models that can reproduce a complete clinical picture
of migraine. However, the constituent parts of migraine can be explored to a large extent in
experimental animals, both in vivo and in vitro. Some of the models used in the screening of
potential antimigraine drugs are described below.
 Antimigraine Agents 469

In vitro Models
[3H]5-HT-binding Assay
[3H]5-HT-binding assay is done for screening of potential antimigraine drugs, acting as
5-HT1B/1D agonists. The assay is performed using rat or bovine brain tissues in the presence of
spiroperidol, which inhibits the binding of 5-HT to 5-HT1A and 5-HT2 receptors.
Procedure: Male Wistar rats are used for the assay. The rats are sacrificed, decapitated and
their striata removed and weighed. These are homogenized in 20 volumes of 0.05 M Tris buffer,
pH 7.7 and subsequently centrifuged at 48,000 g for 10 min. The supernatant is discarded and
the pellet suspended in same volume of 0.05 M Tris buffer and incubated at 37°C for 10 min.
This is again centrifuged at 48,000 g for 10 min. The final pellet is suspended in 0.05 M Tris
buffer containing 10 mM pargyline, 4 mM calcium chloride and 0.1% ascorbic acid.
The binding assay consists of 50 µl [3H]5-HT, 50 µl of spiroperidol (final concentration 1
mM), 800 µl of tissue preparation, 80 µl of 0.05 M Tris buffer with calcium chloride, pargyline
and ascorbic acid and 20 µl of vehicle/5-HT (final concentration of 10-5 M)/the test drug.
Following incubation at 25°C for 15 min, the binding is terminated by vacuum filtration
through Whatman GF/B filters. The filters are then washed twice using 5 ml of ice-cold 0.05 M
Tris buffer. Radioactivity is determined in 10 ml of liquiscint scintillation cocktail.14
Evaluation: Specific binding is determined in the presence or absence of 10-5 M 5-HT. IC50
values are calculated from the percent specific binding at each drug concentration. The Ki
value is determined by Cheng Prusoff equation.14

Contraction of Isolated Blood Vessels

A number of isolated blood vessels from various species including the canine basilar and
coronary arteries, canine and rabbit saphenous vein and human middle meningeal artery15‑18
contract in response to acutely acting antimigraine drugs. These are used for studying the
action of potential antimigraine drugs on the 5 HT1B/1D receptors.

Contraction of Dog Isolated Saphenous Vein

Procedure: Beagle dogs of either sex, body weight 7–12 kg are used. The animals are
anaesthetized and their lateral saphenous veins removed and cut spirally into strips. Four
preparations are obtained from each vein. The strips are suspended in organ bath containing
modified Krebs solution, under resting tension of 0.5 g and continuously aerated with 95% O2
and 5% CO2 at 37°C. All tissues are equilibrated for at least 1 hour and then primed with KCl
(final bath concentration of 30 mM). Test drugs are given at least 30 min following washout
of KCl. To exclude the actions of 5-HT and the test drug on other receptor sites, blockers of
various receptors are used viz. atropine (1 µM), ketanserine (1 µM) and mepyramine (1 µM).
Cumulative concentration response curves to 5-HT are obtained on all tissues. One hour later,
a cumulative concentration response curve of the test drug is determined in one preparation
and of 5-HT in another preparation (to monitor any spontaneous changes in sensitivity to
5-HT).
470 Drug Screening Methods

To find out the effect of antagonist on the contractile effect of the test drug, concentration
response curves to the test drug are established in the presence of a single fixed antagonist
concentration in the bath, allowing 30 min of antagonist contact time.19-21
Evaluation: EC50 is calculated for 5-HT and the test drug by log concentration response curves.
Relative potency is determined by dividing EC50 for the test compound with EC50 value of 5-HT
in the same preparation. This value is then corrected for spontaneous changes in sensitivity to
5-HT by dividing it with the ratio of EC50 values for 5-HT in the control strip.21

Contraction of Rabbit Isolated Saphenous Vein

Procedure: Right and left lateral saphenous veins of male New Zealand white rabbits
(2.5–3.0 kg) are cleaned of surrounding adipose and connective tissue in situ under a binocular
microscope. The veins are then cut off and placed in cold oxygenated Krebs-bicarbonate
buffer solution, and cut into 4 rings of approximately 5 mm in length. The tissue is suspended
with wire hooks and mounted in an organ bath filled with 10 ml Krebs-bicarbonate solution
maintained at 37°C and continuously gassed with 95% O2 and 5% CO2 at 7.4 pH. Initial optimal
resting force of 4 g is applied and tissues are equilibrated for 60-90 min before the concentration
responses with repeated washings at every 15 min. Isometric contractions are recorded on a
physiological recorder with isometric force displacement transducers. The response of the test
drug is obtained in the presence of standard agonist and antagonist. 22-24

Contraction of Human Isolated Middle Meningeal Artery

Procedure: Pieces of dura mater containing segment of middle meningeal arteries are
obtained, as from the neurosurgery department. The arteries are dissected free from the dura
mater and ring segments (2–3 mm in length) are prepared. These are mounted in organ baths
containing standard physiological salt solution, under a resting tension of 4 g and aerated
with 95% O2 and 5% CO2 at 37°C. Contractile response to the reference agonist, KCl (45 mM)
is taken. After a 30 min wash-off period, cumulative concentration-effect curves to 5-HT,
5-carboxamidotryptamine (5-CT) and the test drug are obtained. Concentration-response
curves to the test drug are also obtained in the absence or presence of 5HT1B/1D antagonists like
GR125,743 or GR 127,935 (10 nM), equilibration time 30 min.18
Evaluation: The concentration-effect curves to the agonist are calculated as a percentage of KCl
evoked contractions. For the antagonist experiment, the responses are expressed relative to the
maximum response obtained in the control (in the absence of antagonist) curve (=100%). The
data are analyzed using weighted least square nonlinear regression analysis and the equation:
E = Emax/[1 + {EC50/(agonist)nH}]

Where Emaxis the maximum contraction evoked by the agonist, EC50 is the molar concentration
of an agonist eliciting half maximal response and nH is the Hill coefficient.18

Contraction of Isolated Human Coronary Artery

This model is used for screening the antimigraine compounds for their coronary side effect
potential.
 Antimigraine Agents 471

Procedure: Hearts of patients who die of noncardiac causes like cerebrovascular accidents,
trauma, hypoxia, etc. are obtained. The right epicardial coronary artery is removed and cut
into rings approximately 4 mm long. These are suspended in organ baths containing Krebs
bicarbonate solution, maintained at 37°C and aerated with 95% O2 and 5% CO2. Vessel segments
containing macroscopically visible atherosclerotic lesions are discarded. Segments are allowed
to equilibrate for at least 30 min with 2–3 washings in between and then K+ (30 mM) is added
twice. To verify the functional integrity of the endothelium, relaxation in response to substance
P (1 nM) after precontraction with prostaglandin F2α(1 µM) is observed. Any segment that
does not relax to substance P is discarded. After washout, the maximal contractile response
to K+ is determined by exposing the tissue to 100 mM of K+. The tissue is then equilibrated in
Krebs solution for 30 min.
After equilibration, cumulative concentration response curves to the test and standard drugs
are obtained. Responses are expressed as a percentage of K+ (100 mM)-induced contractions.
All curves are obtained in a paired, parallel experimental set up. The averaged data per artery
are used for further analysis.25,26
Evaluation: pD2 values [–log of molar concentration of an agonist needed to reach half of
its maximal effect (Emax), i.e. –log EC50] are obtained and averaged for the agonist. Data are
analyzed using multiple analysis of variance (MANOVA) followed by paired t-test.25

In vivo Models
Constriction of Carotid Arteriovenous Anastomoses
in Anesthetized Animals
Dilatation of carotid arteriovenous anastomoses has been implicated in the pathogenesis
of migraine.27 It has been postulated that selective constriction of carotid arteriovenous
anastomoses is responsible for the antimigraine effect of a number of drugs including the ergot
alkaloid and the triptans.28-30 This screening method is thus useful for screening of potential
antimigraine drugs acting by constriction of carotid arteriovenous anastomoses.
Procedure: Domestic pigs (Yorkshire X Landrace; 10–15 kg) are used. After an overnight fast,
the pigs are anesthetized, intubated and connected to a respirator for intermittent positive
pressure ventilation. Arterial blood gases and pH are maintained within physiological limits
(pH 7.35–7.48, pCO2 35–48 mm Hg, pO2 100–120 mm Hg) by adjusting the respiratory rate, tidal
volume and oxygen supply. Body temperature is kept at about 37°C and continuous infusion
of saline is given to maintain fluid and electrolyte balance. Inferior vena cava is catheterized
via the left femoral vein for administration of drugs and aortic arch is catheterized via the left
femoral artery for measurement of arterial blood pressure and withdrawal of arterial blood for
determining blood gases.
After cutting both vagi and the accompanying cervical sympathetic nerves, a catheter is
placed in the right external jugular vein for withdrawal of venous blood samples and a needle
is inserted into the right common carotid artery against the blood flow for the administration
of radioactive microspheres. Right carotid blood flow and heart rate are measured using a flow
probe (internal diameter: 2.5 mm) and tachograph, respectively. After a stabilization period of
1 hour, baseline heart rate, mean arterial blood pressure, carotid blood flow and its distribution
472 Drug Screening Methods

are measured. Arterial and jugular venous blood gases are monitored continuously. The test
drug is then given and its effect on the hemodynamic variables is seen at different time-points.
For determining the distribution of common carotid blood flow, a suspension of about
2,00,000 microspheres [15 ± 0.1 (SD) µm diameter labeled with either 141Ce, 113Sn, 95Nb, 103Ru or
46
Sc] is injected into the carotid artery. At the end of the experiment, the animal is sacrificed.
Heart, kidneys, lungs and the different cranial tissues are dissected, weighed, put in vials and
radioactivity counted in these vials for 5–10 min in a gamma-scintillation counter.26,31
Evaluation: Effect of the test drug on the arterial and jugular venous O2 saturation (A-V O2
difference), systemic hemodynamics (mean arterial blood pressure, heart rate, changes in
distribution of blood to other organs) and on carotid hemodynamics (total carotid blood flow
and its arteriovenous anastomotic fraction, capillary fraction of blood flow) is seen. Data are
evaluated statistically using Duncan’s new multiple range test and paired t-test.
For calculating the distribution of carotid blood flow to different tissues (by the use of
microspheres), the ratio of tissue and total radioactivities is multiplied by the total common
carotid blood flow at the time of injection of microspheres. The amount of radioactivity in the
lungs is an index of the arteriovenous anastomotic fraction of the carotid blood flow.26

Neurogenic Plasma Extravasation Model

This screening method is used for studying the ability of potential antimigraine drugs to block
the release of vasodilating peptides from trigeminal sensory nerve endings.
Procedure: Male Sprague-Dawley rats (380–450 g) are used for experiment. After anesthetizing
the animal, femoral vein is cannulated for intravenous injections. Animals are placed in a
stereotaxic frame with the incisor bar set at –1.5 mm. Burr holes are drilled symmetrically 4
mm lateral and 4 mm posterior to the bregma. Paired nonconcentric bipolar electrodes are
lowered bilaterally into the trigeminal ganglia, which are located 9.5 mm from the dura mater.
The animals are now injected with 125I radiolabeled human serum albumin (50 µCi/kg) and
Evan’s blue (20 mg/kg). Drug or vehicle is administered 5 min later. The left or right trigeminal
ganglion is stimulated electrically (5 Hz, 2 ms, 2.2 mA, 3 min duration) 10 min after drug
administration. To wash the blood from the head region, animals are perfused with 0.9% saline
via the left cardiac ventricle for 5 min at a constant pressure of 120 mm Hg immediately after
stimulation. The dura mater is then dissected, weighed and counted for radioactivity. Samples
of extracranial tissues innervated by the trigeminal nerve (eyelid, conjunctiva and lower lip)
are also removed, weighed and counted for radioactivity.32
Evaluation: 125I human serum albumin extravasation ratio is expressed as the ratio of
cpm/mg wet weight (stimulated side) to cpm/mg wet weight (unstimulated side). Maximum
inhibition for a drug is achieved when the ratio of radioactivity on the stimulated versus the
unstimulated side does not differ from 1.33 Data are analyzed using student’s unpaired t-test.

Inhibition of Neurogenic Dural Vasodilatation in Anesthetized Animals

This procedure is used for testing the ability of test drug to block the release of vasoactive
peptides like CGRP from the trigeminal sensory nerve endings and thereby inhibit the
vasodilatation of dural vessels.
 Antimigraine Agents 473

Procedure: Male Sprague-Dawley rats (300–400 g) or male Dunkin Hartley guinea pigs
(300–450 g) are used. After anesthesia, the trachea is cannulated for artificial respiration.
The left carotid artery and jugular vein or the femoral artery and vein are cannulated for
measurement of mean arterial blood pressure (MABP) and i.v. injections of drugs. The
animals are now placed in a stereotaxic frame and their skull exposed. Using a saline cooled
drill, the right parietal bone is thinned until the dural blood vessels are clearly visible
through the intact skull. Using an intravital microscope, a branch of middle meningeal
artery is viewed and a video dimensions analyzer continuously measures dural blood vessel
diameter. A bipolar stimulating electrode is placed on the surface of the cranial window
close to the vessel of interest. To evoke dilatation of dural blood vessels, the cranial window
is electrically stimulated (5 Hz, 1 ms, 250–300 µA for 10 sec). Test drugs are administered
15 min before the electrical stimulation. In guinea pigs, the dural vessels are observed
to be fully dilated following introduction of cranial window. Therefore, it is necessary to
preconstrict the vessels by giving intravenous endothelin-1 (ET-1, 3 µg/kg) 3 min before
electrical stimulation.34,35
Evaluation: Ability of the test drug to constrict the dilated blood vessels is taken as a measure
of efficacy. The effect of electrical stimulation is calculated as a percentage increase from
the prestimulation baseline diameter. The control responses are compared to the responses
obtained after drug administration. The data obtained are analyzed using ANOVA and paired
student’s t-test.35

Activation of CB2 Receptors: Potential Therapeutic Target for Migraine36

Existence of interactions between the endocannabinoid system and pain mediation in
migraine have been suggested by experimental animal models. Role of cannabinoid-1
(CB1) receptor in antinociception has been demonstrated and it has been suggested that
CB2 receptors, located outside the central nervous system, play a role in the perception of
pain. Systemic administration of nitroglycerin (NTG) consistently induces spontaneous-like
headache attacks in migraneurs; in the rat, systemic NTG induces hyperalgesia, probably
through the activation of cerebral/spinal structures involved in nociceptive transmission.
Greco et al. 2014 evaluated the role of CB2 receptors in animal models of pain that may be
relevant for migraine. 36

Knockin Mouse Models37

A group of researchers generated a knockin mouse model carrying the human pure
FHM-1 R192Q mutation and observed multiple gain-of-function effects. These include
increased Cav2.1 current density in cerebellar neurons, enhanced neurotransmission at
the neuromuscular junction, and in the intact animal, a reduced threshold and increased
velocity of cortical spreading depression (CSD; the likely mechanism for the migraine aura).
They showed that the increased susceptibility for CSD and aura in migraine may be due to
cortical hyperexcitability. The R192Q FHM-1 mouse is a promising animal model for testing
novel therapeutic strategies for migraine aimed at decreasing neuronal hyperexcitability
and/or preventing CSD.37
474 Drug Screening Methods

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36. Greco R, Mangione AS, Sandrini G, et al. Activation of CB2 receptors as a potential therapeutic
target for migraine: evaluation in an animal model. J Headache Pain 2014;15:14.
37. van den Maagdenberg AM, Pietrobon D, Pizzorusso T, et al. A Cacna1a knockin migraine mouse
model with increased susceptibility to cortical spreading depression. Neuron 2004;41(5):701-10.
 cHAPTER

32
Analgesic Agents
INTRODUCTION
Pain is an unpleasant sensory and emotional experience associated with actual and potential
tissue damage. Various types of pain are seen in humans, e.g. somatic pain (arising from the
skin, muscles, joints, ligaments and bones), visceral pain, referred pain, neuropathic pain,
cancer pain, etc.
Chemical mediators of pain are numerous. These mediators come from sources intrinsic to
the neuron, including various neurotransmitters such as 5-HT and substance-P, and extrinsic
to the nervous system, including substances from inflammatory/immune cells and red blood
cells such as prostaglandins, kinins, cytokines, chemokines and ATP that are released following
injury to the tissue.
Pain is produced by the excitation of particular receptors, the nociceptors or of their
afferent fibers. These remarkable cells respond to a broad spectrum of physical (heat, cold
and pressure) or chemical noxious stimuli. In general, perception of noxious stimuli is termed
as nociception. Nociception is not exactly same as pain, pain is a subjective experience and
includes a strong affective component, whereas nociception lacks affective component. Most
of the afferent fibers that are excited by noxious stimuli are non-myelinated C-fibers with low
conduction velocities (< 1 m/s), known as C-polymodalnociceptors (PMN) other fibers are
fine myelinated (Aδ) fibers with rapid conduction.
Pain can be classified as acute or chronic. The distinction between acute and chronic pain is
not based on its duration of sensation, but rather the nature of the pain itself. Acute pain, which
has as its source soft tissue damage, infection and/or inflammation, will be short in duration.
The primary distinction is: acute pain serves to protect one after an injury, whereas chronic
pain does not serve this or any other purpose. Acute pain is the symptom of pain. Chronic pain
was originally defined as pain that lasts 6 months or longer. It is now defined as, “the disease
of pain”. The most common causes of chronic pain include cancer pain, neuropathic pain and
arthritic pain.

MECHANISM AND MODULATION OF PAIN/NOCICEPTION

Nociception is the mechanism whereby noxious peripheral stimuli are transmitted to the
central nervous system. Nociceptive fibers terminate in the superficial layers of the dorsal
 Analgesic Agents 477

horn, forming synaptic connections with transmission neurons running to the thalamus.
PMN neurons release glutamate, substance P, etc. contributing to neurogenic inflammation.
Transmission in the dorsal horn is subjected to various modulations constituting the gate
control theory. Descending inhibitory pathways from the midbrain (Periaqueductal gray
area) and brainstem (nucleus raphe magnus) exert a strong inhibitory effect on dorsal horn
transmission. Main transmitters in this pathway are enkephalin, 5-HT and noradrenaline.
Drugs in clinical use as analgesics belong to two main groups—narcotic or morphine group
and analgesic-antipyretic (nonsteroidal anti-inflammatory drugs) group. Morphine-like drugs
produce analgesia by acting on the central nervous system, while analgesic-antipyretic drugs
act by both central and peripheral mechanisms.

ANIMAL MODELS Of ACUTE PAIN

Animal tests used in the search for new analgesics are designed as models for the treatment
of pathological pain in man; but they usually differ from the original in that the drug is given
before the noxious stimulus (thermal, electrical, chemical, and mechanical types of stimuli).
Hence, these tests only measure the power of a drug to increase the minimal stimulus required
to elicit pain or nociceptive response. The methodology to perform these tests using above
stimuli is described below.

MODELS USING THERMAL STIMULUS

Hot Plate Method

Hot plate method has been widely used to evaluate opioid analgesics.

Procedure
Mice weighing 18–22 g are used. Animals are placed on the hot plate, which consists of
electrically heated surface. Temperature of the hot plate is maintained at 55–56°C. Responses
such as jumping, withdrawal of the paws and licking of the paws are seen. The time period

Figure 32.1: Hot plate

478 Drug Screening Methods

(latency period), when animals are placed and until responses occur, is recorded by a
stopwatch.
Test compounds are administered orally or subcutaneously and latency or latency period
is recorded after 20, 60 and 90 min. These values are compared with the values before
administration of the test drug by using t-test.1,2

The Tail-Flick Test

The tail-flick test is a widely and reliably used test for revealing the potency of opioid analgesics.
In this also heat is used as the noxious stimulus. The stimulus causes a simple nociceptive
spinal reflex response in which the rat or mouse flicks its tail away from the heat source. The
dependent variable in this test is the time taken by the animal to flick its tail. It is a very useful
test for discrimination between centrally acting morphine like analgesics and non-opioid
analgesics. Another advantage of the method lies in the fact that there is minimal interanimal
variation.
There are two variants of the tail-flick test. One consists of applying radiant heat to a small
surface of the tail and the other involves immersing the tail in water at a predetermined
temperature.

Figure 32.2: Tail-Flick apparatus

The Tail-Flick Test Using Radiant Heat

Procedure
Mice weighing 18–22 g are used and placed into small cages, leaving the tail exposed. Mouse-
tail is held gently by the observer. A light beam is focused (exerting radiant heat) to the proximal
third of the tail. The mouse tries to pull the tail away and rotates the head, this reaction is
known as escape reaction. The reaction time of this movement is recorded. The reaction time
varies with the surface area stimulated.
The test drug and standard are administered either orally or subcutaneously. Same
procedure is repeated and reaction time is noted after 30, 60 and 120 min. A lengthening of the
reaction time is interpreted as an analgesic action of test drug.
At each time interval those animals that show higher reaction time than the time before drug
administration are regarded as positive. Percentages of positive animals are counted for each
time interval and each dose and ED50 values of test compounds can be calculated according to
Litchfield and Wilcoxon. Codeine, pethidine and morphine are used as standard.3,4
 Analgesic Agents 479

The Tail-Flick Test Using Immersion of the Tail

Young female Wistar rats weighing 170–210 g are used. Rats are placed into separate cages
in such a way that their tail hangs out freely. The distal 5 cm part of the tail is marked, which
is immersed (maximum upto 15 sec) in a cup filled with warm water (temperature 55°C). A
tail withdrawal reflex is seen within a few seconds. This reaction time is noted by stopwatch.
Normal reaction time is 1–5.5 sec. The test substances are given orally or subcutaneously and
reaction time is recorded after 0.5, 1, 2, 3, 4 and 6 h.
Centrally acting analgesic drugs are capable of causing prolongation of tail withdrawal
reflex, and hence, a withdrawal time of more than 6 sec in test animals denotes positive
analgesic response of the test drug.5

Modifications
Cold Tail-Flick Test
In the test, 1–2 cm of the tails of rats are immersed in a cold 1:1 mixture of water and ethylene
glycol at –10°C. The reaction time is measured as the time taken by the rats to deflect their tails.
The first reading is discarded and the reaction time is taken as the mean of the last two readings.
A test drug having potential of analgesic (centrally acting) would increase the reaction time. 6

Cold Ethanol Tail-Flick Test (CET)

Cold ethanol tail-flick test can also be used for evaluating opioid analgesics. In the test –20°C
is selected as the noxious cold stimulus. Male Sprague-Dawley rats weighing between 175 and
225 g are used and their tails submerged, approximately, halfway into the bath containing a
bath solution of water or 75% ethanol (temp –20°C). The nociceptive threshold is taken as the
latency until the rat removes or flicks its tail from the bath. The time from immersion to tail
removal is measured to the nearest tenth of a second with a timer.7

MODELS USING ELECTRICAL STIMULUS

Tooth Pulp Test
In the test electrical stimulus is given in rabbits tooth pulp.

Procedure
Rabbits weighing 2–3 kg are used and anesthesia is given with thiopental in the doses of 15 mg/
kg intravenously. Using dental drill, tooth pulp chambers are exposed close to the two front
upper incisors. Clamping electrodes are placed into the drilled holes. After 30 min, electrical
stimulus is applied by rectangular current (frequency 50 Hz) upto 1 sec. Current is started with
0.2 mA and increased until animal starts licking. After that, a threshold is determined at least 3
times in each animal. Animal serves as its own control. Test compound is administered orally
or intravenously. After 15, 30, 60 and 120 min, threshold current is measured and compared
with the threshold current prior to drug administration.8
480 Drug Screening Methods

Modifications
Electrical Stimulation of the Tail
Electrical stimuli of gradually increasing intensities can be delivered by subcutaneous
electrodes in the tail of the rat or the mouse. When such gradually increasing intensities of
electrical stimuli are applied, a reflex movement of the tail can be observed. The threshold for
this response is determined before (relative ratio: 1, 1.5, and 3, approximately) and 45 min after
the administration of test drug. The results are expressed as percentages of these thresholds on
a semilogarithmic chart. Over the range of doses used, if test drug increases the threshold for
the responses, it would have analgesic potential.9-11

Monkey Shock Titration Test

The monkey shock titration test is used for final evaluation of a new compound having analgesic
potential.

Procedure
The monkeys are seated in restraining chairs. By a Coulbourn Instrument Programmable
Shocker electrical current is delivered through electrodes, which are coupled to two test
tube clamps attached to a shaved portion of tail. The current ranges from 0–4 mA through 29
progressive steps. The monkey presses a bar to interrupt the shock. For each monkey, a stable
baseline shock level is recorded. After 24 h, drug is administered and shock titration activity
is measured according to a change in maximum level of median shock intensity for drug as
compared to control levels.12,13

MODELS USING CHEMICAL STIMULUS

The chemical tests involve administration of an irritant, algogenic chemical agent as
the nociceptive stimulus. Chemical stimulation induces a slow form of stimulation. The
characteristic features of chemical stimuli are that they are progressive and persist for a longer
duration.
Hence, chemical tests can be distinguished clearly from those mentioned above, in terms
of their physical nature, duration, and measurement of a behavioral score in units of time.
Without doubt, these experimental models are the closest in nature to clinical pain. Tests
where chemical stimulus is applied are:

Formalin Test
The formalin test uses a 10% formalin solution as a chemical noxious stimulus. By injecting
the formalin solution into the paw of a rat or a mouse, a model of persistent (chronic) pain
caused by peripheral tissue injuries and inflammation is created. This model is highly
sensitive for opioids like drugs. In formalin test, animals show two phases (biphasic) of
nociceptive behavior involving two different stimuli. The first (early) phase starts immediately
after injection of formalin and last for 3–5 min. This occurs due to chemical stimulation of
nociceptors causing C-fiber activation. Second (late) phase starts after 10–15 min of formalin
 Analgesic Agents 481

injection and lasts for 20–40 min. This phase appears due to combination of an inflammatory
reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord.
The C-fiber barrage initiates functional changes during the early phase. Opioid analgesics are
antinociceptive for both phases, although the second is more sensitive to these substances.
Non-steroidal antinflammatory drugs are effective in second phase, while the first phase
remains unaffected.14,15

Procedure
Male Wistar rats weighing 180–300 g are used. In the dorsum of front paw of the animal 0.05
ml of 10% formalin is injected subcutaneously. Each animal is placed into separate cage
for observation of pain responses in early and late phases. These responses are elevation or
favoring of the paw or excessive licking and biting of the paw. Scoring of these pain responses
is done according to a pain scale. After administration of the test drug, again scoring is done
after 30, 60 min for comparison. If both paws of the animal are allowed to rest on the floor with
no obvious favoring of the injected paw, this is taken as a positive analgesic response.16

Writhing Test
This is a model of visceral or peritoneal pain in animals, which also involves the administration
of algogenic agents. This test is used to detect peripheral analgesic activity of a test compound.
In this test, pain is induced by intraperitoneal administration of chemicals that irritate serous
membranes and provoke a stereotyped behavior in the mouse or rat known as writhing. These
behaviors are considered reflexive, and are evidence of peritoneovisceral or visceral pain
associated with visceral chemoreceptors.17

Procedure
Mice of either sex approximately 20–25 g are used. Rats can also be used in this test.
Phenylquinone (0.02%) is suspended in 1% suspension of carboxy methylcellulose. An aliquot
of 0.25 ml of this suspension is injected intraperitoneally in each animal. The animal reacts
with a characteristic stretching behavior, i.e. a series of constrictions occur that travel along the
abdominal wall, sometimes accompanied by turning movements of the body and extension of
the hind limbs. This response is called as writhing.
A group of animals (n=6) is used as test group in which prior to phenylquinone administration,
test drugs are administered orally or subcutaneously. The mice are placed individually into
glass chambers and number of writhes are recorded for 10 min in each animal.
For scoring, a writhe is indicated by stretching of the abdomen with simultaneous stretching
of at least one hind limb. Formula for computing percent inhibition is: average writhes in the
control group minus writhes in the test group divided by writhes in the control group times
100%. The time period with the greatest percent of inhibition is considered the peak time.18

Other Chemical Compounds that can Elicit Writhing

Several other chemicals can give rise to writhing behavior. For example: acetic acid,
acetylcholine, bradykinin, prostaglandin E1, aconitine, 4% NaCl, ethacrynic acid. 19-23
482 Drug Screening Methods

Distension of Hollow Organs Using Chemical Stimulus

Such tests involve injecting chemical substances directly into hollow organs and they represent
as models for true visceral pain such as:

Rat Sigmoid Colon Model

This model is a true visceral pain model.
In this model, formalin (50 µl, 5%) is administered into the rat sigmoid colon, which produces
a complex biphasic pain behavior involving an initial phase of body stretching and contraction
of either the flanks or the whole body followed by a second phase, showing abdominal licking
and nibbling. A pain score can be calculated after analysis of the formalin-induced behaviors.
The score can be dose-dependently reduced by test drug with analgesic activity.24

Inflammatory Uterine Pain Model

This model resembles closely a state of inflammatory uterine pain and also provides further
insight into the neural processes contributing to visceral nociception.
Procedure
Mustard oil is injected into one uterine horn in the rat to produce chemical inflammation.
Control rats are sham-operated. Rat behavior is observed using non-stop video-tape
recording for 7 days. Rats with uterine inflammation show abnormal behavior during the first
4 days (hunching, hump-backed position, licking of the lower abdomen, repeated waves of
contraction of the ipsilateral oblique musculature with inward turning of the ipsilateral hind
limb, stretching, squashing of the lower abdomen against the floor) suggestive of visceral pain
and evidence of flank muscle hyperalgesia over 7 days indicative of referred visceral pain.25

MODELS USING MECHANICAL STIMULUS

Haffner’s Tail Clip Method
In this method, mechanical stimulus is applied. The preferred sites for applying nociceptive
mechanical stimuli are the hind paw and the tail. This method is highly sensitive for centrally
acting drugs. Tests using constant pressure have been abandoned progressively for those
applying gradually increasing pressures.

Procedure
Male mice weighing 18–25 g are used. An artery clip is placed at the root of the tail of the mice
to apply noxious stimulus. A quick response of the animal is seen as biting the clip or tail, where
clip has been placed. The time (reaction time) between application of the clip and response
is noted by a stopwatch. For testing analgesic activity, test compounds are administered
subcutaneously or orally. After 15, 30 or 60 min, same procedure is repeated and reaction time
is measured. For evaluation of analgesic drugs cut off time is determined, i.e. average reaction
time plus 3 times the standard deviation of the combined latencies of the control mice at all
time periods.
 Analgesic Agents 483

Reaction time of the test animals, greater than the cut off time denotes a positive response
indicating analgesic activity.26,27

Randall Selitto Test

The principle of the test is that inflammation increases the sensitivity to pain, and thus,
decreases pain threshold. In Randall Sellito test, inflammation is induced by subcutaneous
administration of Brewer’s yeast and then pressure is applied, which increases the intensity of
pain. This test is used to detect peripheral analgesic activity of a test compound.

Procedure
Male Wistar rats are divided into two groups—test and control. In the test group, test agents
are administered and vehicle is administered in control group animals. After 15–30 min, 0.1
ml of Brewer’s yeast (20% suspension) in distilled water is injected subcutaneously into the
plantar surface of the left hind paw of the rat. After 3 h, using a special apparatus (Randall
Selitto apparatus) pressure is applied to the plantar surface of the rat’s foot at a constant rate
until animal struggles or squeals. In both the groups each animal is tested for its control pain
threshold and then comparison is made between the groups. Animal, which is showing control
pain threshold greater than 80 g, is eliminated.28
Colburn et al. evaluated a mechanical visceral pain model, where chronic intermittent
intestinal distension (repeatable and reversible) is produced in the rat using a chronic
indwelling intraduodenal balloon catheter.29,30
Commonly used animal models of acute pain, according to types of stimuli are shown in
Table 32.1.

Table 32.1: Common animal models of acute pain according to types of stimuli

Thermal Mechanical Chemical Electrical

Tail-flick test Haffner’s tail clip method Formalin test (intradermal injections) Tail stimulation
Hot-plate test Randall and Selitto test Writhing test (intraperitoneal injections) Tooth pulp test
Chemical stimulation of visceral organs

ANIMAL MODELS OF CHRONIC PAIN

Neuropathic Pain Models
Partial somatosensory nerve injury is the cause of causalgiform pain disorders in man. Causalgia
is characterized by spontaneous burning pain combined with hyperalgesia and allodynia. The
presence of pain in the inflammation models is inferred by an increased response to a noxious
stimulus (hyperalgesia) or a nocifensive behavior in response to an innocuous stimulus,
normally not perceived as painful allodynia. This model represents disorders of neuropathic
pain sensation like those seen in man.

Procedure
Male Sprague-Dawley rats are used and anesthetized with 4% halothane. A local incision is
given and sciatic nerves of both legs are exposed at the level of mid thigh. Four (4-0) chromic
484 Drug Screening Methods

gut sutures are tied loosely with a square knot around the right sciatic nerve. Left sciatic nerve
is just mobilized. Incisions are closed layer to layer. During the next days animals show a mild
aversion of the affected paw and foot drop.31 The thermal nociceptive threshold of hind paws
is measured in each animal. For this, rats are placed beneath a transparent plastic cage upon a
raised glass plate, such that a halogen projector lamp could be placed below it. Lamp (source
of radiant heat) is focused at the planter area of one hind paw. As soon as heat is applied a
withdrawal response of hind paw is seen. The time interval between the exposure of heat
and withdrawal response is measured in each animal.32 After 7–8 days, test drug and vehicle
are injected intrathecally in test and control group, respectively. Paw withdrawal latency
(PWL) of hind paws is recorded before and after 5, 15, 30, 60 and 90 min of drug and vehicle
administration. PWL, which was the maximum during the first 30 min after drug or vehicle
injection is called as maximum PWL. To evaluate hyperesthesia, the difference score (DS) is
calculated by subtracting the maximum PWL of the control side (left side) from the maximum
PWL of the affected side (right side). For evaluation of drug effects in hyperesthetic rats, the
dose is plotted against the change in DS (post- drug difference score minus pre-drug difference
score).

Modifications
A rat model of partial sciatic nerve injury has also been produced.33 This model produces
reproducible tactile allodynia and thermal hyperalgesia. Unilateral tight ligation of about half
of the sciatic nerve in rats rapidly produces sympathetically dependent neuropathic pain that
lasts many months and resembles causalgia in humans.
An animal model of persistent peripheral neuropathic pain has also been produced
involving spared nerve injury. This involves a lesion of two of the three terminal branches
of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve
intact. Co-mingling of distal intact axons with degenerating axons is restricted, and it permits
behavioral testing of the non-injured skin territories adjacent to the denervated areas. The
spared nerve injury model results in early (< 24 h), prolonged (> 6 months), robust (all animals
are responders) behavioral modifications.34

Vincristine-induced Neuropathy Model

Hyperalgesia induced by vincristine in the rat provides a good model for the experimental study
of painful peripheral neuropathies in patients receiving vincristine as a chemotherapeutic
agent.

Procedure
Vincristine (100 µg/kg) is administered daily for 2 weeks in rats. A decrease in mechanical
nociceptive threshold and hyperalgesia occurs after the second day of administration. Chronic
lowered threshold and increased response to stimuli (determined 24 h after each injection)
is seen during the second week of vincristine administration. Responses gradually return to
baseline following discontinuation of treatment. Thermal hyperalgesia is also produced with
vincristine in this model.35
 Analgesic Agents 485

Diabetic Neuropathy Model

Painful diabetic neuropathy is one of the most common complications of insulin-dependent
diabetes in man. The streptozotocin-induced diabetic rat has been put forward as a model
of chronic pain with signs of hyperalgesia and allodynia that may reflect signs observed in
diabetic humans.

Procedure
Streptozotocin (STZ) (75 mg/kg, ip) is administered in rats so that they develop diabetes
(hyperglycemia > or = 14 mM). The animals are subjected to various pain stimuli: mechanical,
thermal (warm and cold) and chemical. The time course of the scores was followed for 4 weeks
simultaneously with the clinical symptoms (weight, body and skin temperature, motility) and
hyperglycemia. A decrease in reaction thresholds to noxious heat stimuli and to non-painful
thermal (cold: 10­°C, and warm: 38–42°C) and mechanical stimulation (paw pressure) is
observed. This serves as evidence for hyperalgesia and allodynia, respectively. These troubles
appear after 2 weeks of establishment of diabetes. Four weeks after the induction of diabetes,
the scores can be obtained in diabetic rats injected with formalin (chemical stimuli). If score is
greater than those in normal rats, it indicates hyper­algesia.36
Kiguchi S et al. evaluated the antinociceptive effect of oxcarbazepine (OCBZ), a keto
derivative of carbamazepine in diabetic neuropathy rat model and suggested that OCBZ has
an analgesic action and is a possible therapeutic agent for the treatment of painful diabetic
neuropathic pain.37

Persistent Post-thoracotomy Pain Model

Chronic post-thoracotomy pain (CPTP) recurs or persists after a thoracotomy incision at least
2 months following the surgical procedure.38 This pain is described as a continuous dysesthesia
with burning and aching in the general area of the thoracotomy incision.
This type of pain is chronic and persistent in nature and commonly seen after thoracotomy,
although its basis and therapy have not been well characterized. In this model, the allodynic
responses (mechanical and cold) as well as the histopathologic changes after thoracotomy
and rib retraction in rats are observed to evaluate the antinociceptive potential of test drug.

Procedure
Male Sprague-Dawley rats are anesthetized and the right 4th and 5th ribs surgically exposed.
The pleura is opened between the ribs and a retractor placed under both ribs and opened 8
mm. Retraction is maintained for 5, 30, or 60 min. Control animals are given pleural incision
only.
After two days post-surgery, animals are tested for mechanical allodynia using calibrated
von Frey filaments and cold allodynia using acetone applied to the incision site. Two weeks
after surgery, animals are tested for reduction of allodynia with administration of test drugs.
In 50% of the animals with 60 min retraction, allodynia develops and when the retraction time
was 5 and 30 min allodynia is seen in 11% and 10% of animals, respectively. Control animals
do not develop allodynia. Allodynic animals show extensive axon loss in the intercostal nerves
of the retracted ribs. If a test drug reduces allodynia, it would have analgesic potential. This
486 Drug Screening Methods

model is useful for quantifying the efficacy of techniques to reduce the frequency and severity
of long-term post-thoracotomy pain.39

Rat Model of Incisional Pain

This model helps to understand mechanisms of sensitization caused by surgery and investigate
new therapies for postoperative pain in humans. In the model, it is revealed that both the
sural and tibial nerves are responsible for transmitting input from the incision that produces
hyperalgesia.
A longitudinal incision of 1 cm is given through skin, fascia and muscle of the plantar surface
of the hind paw in halothane-anesthetized rats. Withdrawal responses are measured using von
Frey filaments at different areas around the wound before surgery and for the next 6 days. The
results of tests for withdrawal responses suggest that a surgical incision of the rat foot causes
mechanical hyperalgesia lasting for several days after surgery.40

Pharmacological Characterization of Rat Model of Incisional Pain

Whiteside et al. assessed the validity and reliability of rat model of postincisional pain and
evaluated the effects of different classes of clinically effective analgesic drugs against multiple
behavioral end points, the time course of mechanical hyperalgesia, tactile allodynia using
the Randall-Selitto (paw pressure) assay and electronic von Frey, respectively. Behavioral
evaluations began 24 h following surgery, and continued for 9–14 days.41

MODELS OF CANCER PAIN

Rat Model of Bone Cancer Pain
Bone metastasis is one of the major causes of cancer-related pain, and not all bone cancer pain
can be effectively treated.
In this model, bone cancer is induced in the rat by the syngeneic MRMT-1 mammary tumor
cell line. Model is characterized by mechanical hyperalgesia and allodynia along with the
progression of the tumor in the bone marrow cavity, while the general condition of the animal
remains satisfactory.

Procedure
Sprague-Dawley rats are given intra-tibial injections of syngeneic MRMT-1 rat mammary
gland carcinoma cells. Control rats receive heat-killed cells or vehicle. Sprague-Dawley
rats, given intratibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells,
develop behavioral signs indicative of pain, including: mechanical allodynia, difference of
weight bearing between hind paws and mechanical hyperalgesia. The development of the
bone tumor and structural damage to the bone was monitored. Intra-tibial injections of
3 × 10(3) or 3 × 10(4) syngeneic MRMT-1 cells produced a rapidly expanding tumor within the
boundaries of the tibia, causing severe remodeling of the bone. Damage to the cortical bone
and the trabeculae by day 10–14 after inoculation of 3 × 10(3) MRMT-1 cells, and by day 20, the
damage was threatening the integrity of the tibial bone. A large number of polykariotic cells,
resembling those of osteoclasts within the tumor are observed with tartarate-resistant acid
phosphatase staining.
 Analgesic Agents 487

No tumor growth was observed after the injection of heat-killed MRMT-1 cells. No changes
in body weight and core temperature occurs after intra-tibial injections of 3 × 10(3) or 3 × 10(4)
MRMT-1 cells, heat-killed cells or vehicle. The general activity of animals after injec­tion with
live or heat-killed MRMT-1 cells was higher than that of the control group, howe­ver, the activity
of the MRMT-1 treated group declined during the progress of the disease.
Rats receiving intra-tibial injections of MRMT-1 cells show development of mechanical
allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb,
beginning on day 12–14 or 10–12 following injection of 3 × 10(3) or 3 × 10(4) cells, respectively.
Rats receiving heat-killed cells or vehicle do not show these symptoms.42

Modifications
In modified model, syngeneic Walker 256 mammary gland carcinoma cells are injected into the
tibia medullary cavity via intercondylar eminence in rats. The rats inoculated with carcinoma
cells show significant ambulatory pain, mechanical allodynia, and reduction in weight bearing,
as well as increased incidence of spontaneous activity in Abeta fibers in affected limb, whereas
PBS (vehicle) or heat-killed cells (sham) injected rats showed no significant difference in
comparison to normal rats.43
Zhao C et al. used sarcoma cells (NCTC 2472) that were injected into the medullary cavity of
the humerus, femur, or calcaneus.44
Lee BH et al. developed another mouse model of cancer pain, in which Murine
hepatocarcinoma cells, HCa-1, were inoculated unilaterally into the thigh or the dorsum of
the foot of male C3H/HeJ mice. Four weeks after inoculation, behavioral signs were observed
for mechanical allodynia, cold allodynia, and hyperalgesia using a von Frey filament, acetone,
and radiant heat, respectively. Bone invasion by the tumor commenced from 7 days after
inoculation of tumor cells and was evident from 14 days after inoculation. Cold allodynia,
but neither mechanical allodynia nor hyperalgesia, was observed in mice that received an
inoculation into the thigh. On the contrary, mechanical allodynia and cold allodynia, but not
hyperalgesia, were developed in mice with an inoculation into the foot. Sometimes, mirror-
image pain was developed in these animals.45

IN VITRO METHODS
Identification of several types of opioid receptors in the brain has allowed to perform
in vitro binding tests to study the action of central analgesics. Various new receptors have been
identified by using in vitro methods as therapeutic targets for the treatment of pain, especially
neuropathic and cancer pain, such as receptors for nociceptin, vasoactive intestinal peptide,
cannabinoids and vanilloid receptors. Agonists and antagonists for these receptors are being
evaluated.46-49

H-Naloxone Binding Assay

3

Opiate agonists and antagonists have ability to displace radiolabeled naloxone that is a potent
narcotic antagonist. 3H-Naloxone binding assay is developed to classify opioid analgesics as
agonists, mixed agonist-antagonists and antagonists. The basic principle of this assay is to
determine IC50 values for 3H-Naloxone in the presence or absence of Na+.
488 Drug Screening Methods

Reagents that are used in the assay are: 3H Naloxone (38–58 Ci/mmol): concentration is
5 nM in 3 test tubes.
Levorphanol tartrate: 1 mM stock solution of levorphanol is diluted 1:200 in distilled water
and in 3 tubes 20 µl is added to yield a final concentration of 0.1 µM in the assay.
Dextrorphan tartrate: 1 mM stock solution is diluted 1:200 in distilled water and in 3 tubes
20 µl is added to get a final concentration of 0.1 µM in the assay.
Test compounds: 1 mM stock solution is made in an appropriate solvents and diluted
serially to get the final concentration in between 10-5 and 10-8 M.

Procedure
Male Wistar rats are decapitated and their brains are removed. Whole brains without cerebella
are homogenized in 50 volumes of ice-cold 0.05 M tris buffer with a tissue homogenizer.
Centrifugation of homogenate is done at 40,000 g for 15 min. Pellet is resuspended in buffer
and recentrifuged at 40,000 g. After this, the final pellet is resuspended in freshly prepared
0.05 M tris buffer. Finally, tissue concentration in the assay becomes 10 mg/ml.
In test tubes a mixture is prepared, which consist of 310 µl H2O, 20 µl 5 µM dextrorphan
(total binding) or 5 µM levorphanol (non specific binding), 50 µl 2 M NaCl or H2O, 50 µl 0.05
M Tris buffer, pH 7.7, 20 µl drug or vehicle, 50 µl 3H-Naloxone and 500 µl tissue suspension.
The tubes are incubated for 30 min at 37°C. Vacuum filtration through Whatman GF/B filters
is done to stop the assay and washing is performed at least 3 times with ice-cold 0.05 M Tris
buffer, pH 7.7. The filters are then counted in 10 ml of Liquiscint liquid scintillation cocktail.
Difference between binding in the presence of 0.1 µM dextrorphan and 0.1 µM levorphanol is
known as stereospecific binding.
Specific binding is around 1% of the total added ligand and 50% of the total bound ligand
in the absence of Na+ and 2% of the total added ligand and 65% of the total bound ligand in the
presence of Na+ (100 mM). An increase in specific binding denotes an increase in binding.
To evaluate analgesic activity, data are converted into % stereospecific 3H-naloxone binding
displaced by the test drug. Determination of IC50 is done by using computer-derived log-probit
analysis. IC50 is used to calculate sodium shift. Opioids agonists show high sodium shifts,
antagonists show low shift and mixed opioids agonists-antagonists show medium shift. Data
are analyzed by a computer program.50,51

µ Opiate Receptor Binding Assay

Opioids drugs exert their analgesic effects mainly through µ opioid receptors. 3H-
Dihydromorphine is highly selective for µ receptors. The compounds that inhibit binding of
3
H-dihydromorphine in a synaptic membrane preparation from rat brain can be identified by
this assay.52
Reagents that are used in the assay are—20 nM stock solution of 3H-dihydromorphine,
0.1 mM stock solution of levallorphan tartrate and 1 mM stock solution of test compounds. All
compounds are taken in 3 test tubes. 50 µl of 3H-dihydromorphine and 20 µl of levollarphan
tartrate are added to each tube. In the assay final concentration of 3H-dihydromorphine and
levollarphan tartrate are 0.5 nM and 0.1 µM, respectively and concentration of test compounds
ranges from 10-6–10-9 M. Total volume of assay mixture is 2 ml.
 Analgesic Agents 489

Procedure
Male Wistar rats are used. Animals are sacrificed by decapitation. Whole brains without
cerebella are removed, weighed and homogenized in 30 volumes of ice-cold 0.05 M Tris
buffer, pH 7.7. Centrifugation of homogenate is performed at 48,000 g for 15 min and pellet
is resuspended in the same volume of buffer. This homogenate is incubated to remove the
endogenous opiate peptides and centrifuged again. The final pellet is resuspended in 50
volumes of 0.05 M Tris buffer.
In test tubes, a mixture consisting of 1850 µl tissue suspension, 80 µl distilled water, 20 µl
vehicle or levallorphan or appropriate concentration of drug and 50 µl 3H-dihydromorphine
is prepared. Then incubation is performed for 30 min at 25°C. The assay is stopped by vacuum
filtration through Whatman GF/B filters, which are washed twice with 5 ml of 0.05 M tris
buffer. Filters are placed into scintillation vials with 10 ml liquiscient scintillation cocktail and
counted. Specific binding is the difference between total binding and binding in the presence
of 0.1 mM levollarphan. At each drug concentration IC50 values are calculated from the percent
specific binding.53,54

Assay to Study Cannabinoids Activity

Cannabinoids such as 9-THC (tetrahydrocannabiol) are capable of inhibiting nociception, i.e.
pain transmission. Cannabinoids exert their effects by cannabinoid receptors CB1& CB2, that are
localized in the brain. These receptors have been well characterized and cloned. Cannabinoids
produce analgesia without respiratory depression that is associated with opioids analgesics.

Procedure
Male Sprague-Dawley rats of around 150–200 g are decapitated and brains are removed
immediately. The cortex is dissected free and immersed in 30 ml of ice-cold centrifugation
solution (320 mM sucrose, 2 mM Tris EDTA, 5 mM MgCl2). The process is repeated until
the cortices of five rats are combined. The cortical material is homogenized with a Potter-
Elvehjem glass-Teflon grinding system, which is then centrifuged for 15 min at 1,600 g. The
supernatant is combined with the two subsequent supernatant obtained from washing and
1,600 g centrifugation of the P1 pellet. The combined supernatants are centrifuged at 39,000 g
for 15 min. The P2 pellet resuspended in 50 ml buffer (50 mM Tris HCL, 2 mM Tris EDTA,
5 mM MgCl2, pH 7.0) and incubated for 10 min at 37°C. Then centrifuged again at 23,000 g for
10 min. The P2 membrane is resuspended in 50 ml of buffer A, incubated again and centrifuged
at 11,000 g for 15 min. Finally, obtained and wash treated P2 pellet is resuspended in assay
buffer B, (50 mM Tris HCL, 3 mM Tris EDTA, 3 mM MgCl2, pH 7.4) to a protein concentration
of approximately 2 mg/ml. Four aliquots are prepared from the preparation and freezed in dry
ice solution and 2 methylbutane and stored at –80°C.
150 mg of P2 membrane is added to test tubes that contain [3H] CP-55,940 (79 Ci/mmol),
a cannabinoid analog (for displacement studies) and a sufficient quantity of buffer C (50 mM
Tris-HCI, 1 mM Tris EDTA, 3 mM MgCl2, 5 mg/ml BSA) to get the total incubation volume to
1 ml. In displacement studies, the concentration of [3H] CP-55,940 is 400 pM and in saturation
studies it varies from 25 to 2500 pM. Nonspecific binding is measured by the addition of 1 mM
unlabeled CP-55,940. The standard CP-55,940 and other cannabinoid analogs are prepared in
suspension buffer C from a 1 mg/ml ethanolic stock, without evaporation of the alcohol.
490 Drug Screening Methods

After this, incubation is performed for 1 h at 30°C, binding is terminated by addition of 2 ml

ice-cold buffer D (50 mM Tris-HCl, 1 mg/ml BSA) and vacuum filtration. Reaction vessels are
washed once with 2 ml of ice-cold buffer D, and the filters washed twice with 4 ml of ice-cold
Buffer D. Filters are placed into 20 ml plastic scintillation vials with 1ml of distilled water and
10 ml of Budjet-Solve. After shaking for 1 h, the radioactivity is determined by liquid scintillation
photometry.55,56

ROLE OF VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) and

PITUITARY ADENYLATE CYCLASE- ACTIVATING PEPTIDE (PACAP)
AND NOCICEPTIN IN ANALGESIA
Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating peptide (PACAP),
play a role in the altered transmission of sensory information in neuropathic pain (arising
from trauma or compression injury of peripheral nerves). Both of these peptides act through
G-protein coupled receptors, named VPAC (VIP1), VPAC2 (VIP2), PAC1 (PACAP type 1) receptors.
These receptors would be used as new therapeutic targets for the treatment of neuropathic
analgesia. Various agonists and antagonists for VIP have been evaluated.
To study properties of these receptors, CHO cell lines are used, which stably express VIP-
PACAP type I & type II receptors.57,58
Nociceptin or orphanin FQ is a heptadekapeptide, which acts through a specific receptor
that has been cloned and well characterized in man and animals and named as opioid
receptor like (ORL1) receptor. Nociceptin receptor (ORL1) and opioid receptors show structural
and transductional similarities. Hence, nociceptin receptor has been included into the opioids
receptor family with the name of OP4. Nociceptin induces analgesia when given intrathecally in
rats.59-60

CONCLUSION
The word pain is applied to a wide variety of subjective phenomenon ranging from the
perception of an experimental noxious stimulus to the most severe and excruciating pain
in humans suffering from cancer, trigeminal neuralgia, etc. Animal models have been used
extensively in basic pain research based on the premise that animal models can serve as
surrogate assays that can reliably predict the potency and efficacy of the pharmacologic action
of, and, in some cases, the molecular response to, agents that work in human pain states. But
in contrast to the polymorphic nature of pain in humans, pain in animals can be estimated
only by examining their reactions to various chemical, thermal, and mechanical stimuli, with
the latency or nature of response altered in the pain state. Most commonly used methods for
evaluation of analgesic drugs are tail-flick test and hot plate test. However, formalin test has
also been widely used.
Although different pain models based on use of nociceptive stimuli (electrical, thermal,
mechanical, or chemical) have been used, none is ideal. However, test using chemical stimuli
probably most closely mimic acute clinical pain. The monitored reactions are almost always
motor responses ranging from spinal reflexes to complex behaviors. Most have the weakness
that they may be associated with, or modulated by, other physiological functions. The
 Analgesic Agents 491

weaknesses of the tests, include (i) in most tests responses are monitored around a nociceptive
threshold,whereas clinical pain is almost always more severe; (ii) differences in the fashion,
whereby responses are evoked from healthy and inflamed tissues; and (iii) problems in
assessing threshold responses to stimuli, which continue to increase in intensity.
In summary, animal models have contributed much to the understanding of the mechanisms
of pain in humans, and current clinical treatments are based, in part, on those studies. But the
future of effective strategies that go beyond palliative care will also use these models to screen
novel, safe, and useful approaches in a preclinical setting. Much resource effort and expense
can be conserved by testing novel methodologies in multiple animal models—not relying on
a single animal model, strain, or species—before clinical testing begins. As pain in humans is
chronic in nature, there is a critical need for development of such models that could provide
knowledge about mechanisms involved in chronic pain in humans. As far as neuropathic pain
is concerned, it is difficult to design this type of model in animals for both technical and ethical
reasons and much more difficult is to devise tests that could measure affective component of
pain.

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 cHAPTER

33
Anti-inflammatory Agents
INTRODUCTION
Inflammatory diseases cover a broad spectrum of conditions including autoimmune diseases
(e.g. rheumatoid arthritis), osteoarthritis, asthma, chronic obstructive pulmonary disease,
interstitial cystitis, prostatitis, inflammatory bowel disease, multiple sclerosis, allergic rhinitis,
infectious diseases, various types of cancers and cardiovascular diseases, etc.1 Therefore,
inflammation can be described as a universal host defense process involving complex cell-
cell, cell-mediator and tissue interactions. These events involved in inflammation may
appear common across various inflammatory diseases, but there are underlying differences
in paracrine signaling mechanisms which are orchestrated by differences in chemokines,
cytokines and growth factors, lipids and genetic influences.2,3
Although, inflammation is the unifying factor across a host of diseases but the treatment
approach is often unique for each of the inflammatory disease. Each disease population
has distinct therapeutic needs that are inadequately served by current prevention and
treatment strategies. Therefore, there is need for improved method to search for new drugs.
Inflammation most commonly occurs when microbial invasion or tissue injury overcomes
the body’s non-specific defense mechanisms.4 Subsequent to infection, immune system
gets activated, communication and coordination occurs between different classes as well as
actions of immune cell to produce inflammation. Normally, inflammation is tightly regulated
by the body and is the starting point of the body’s self repair process initiated by body’s defense
system to thwart pathologic assaults but occasionally it runs amok, leading to physiological
chaos and death.5
Inflammation occurs in response to a variety of harmful exogenous and endogenous stimuli.
Exogenous stimuli can be of physical, chemical, mechanical, nutritional and biological origin,
whereas endogenous stimuli can be of immunological, neurological and genetic origin. The
inflammation could be acute, subacute or chronic in nature. The acute inflammation is short
lasting, whereas chronic inflammation may persist for weeks, months or years. The classic
triad of acute inflammation is: (a) pain, fever and swelling, which is caused by increased blood
flow, (b) increased capillary permeability and (c) increased migration of leukocytes of which
neutrophils are first to ingress into the affected tissue area. Acute inflammatory response often
transitions into chronic inflammation, which is defined by tissue proliferation, granuloma,
and repair. The cardinal features of inflammation are shown in Figure 33.1.
496 Drug Screening Methods

Figure 33.1: Inflammation is a characteristic response of tissue to injury or microbial invasion. Classic triad of inflammation
at injury site is initiated by vasodilation following release of Nitric oxide (NO) from endothelium, then increased capillary
permeability facilitates extravasation (increased migration) of leukocytes especially neutrophils towards the affected
tissue area. Release of chemokines by injured cells drives the chemoattraction of other leukocytes such as monocytes,
neutrophils and lymphocytes from blood. Extravasation of leukocytes to injured tissue requires expression of molecules,
such as selectins, which supports rolling and stable arrest of leukocytes bound to platelets on activated vascular
endothelium. Adhesion molecules, such as ICAM-1 associates with receptors of the integrin family to induce a reversible
adhesion interaction

In the recent years, there has been increased focus on leukocyte migration. The first steps in
leukocyte recruitment include rolling of the leukocytes on the vessel wall mediated by selectins
and glycoproteins bearing the sialyl Lewisx moiety6 (Fig. 33.1). Adhesion between the activated
platelets and neutrophils is mediated by one of the selectins, P-selectin. Drug screen methods
based on adhesion assays evaluate the binding of thrombin-activated human platelets to
neutrophils. Adhesion molecules, vascular cell adhesion protein 1 (VCAM-1), intracellular cell
adhesion molecule (ICAM-1) also play a major role in the development and persistence of
inflammatory diseases.7
Accumulating research evidence has proven that inflammation is an orchestra involving
many players including histamine, prostaglandins (PGE2 and prostacyclins), leukotrienes
(LTB4), serotonin, bradykinin, cytokines (IL-1, IL-6, IL-8, TNF-α), growth factors,6 lysosomal
contents of neutrophils, adipokines (leptin, adiponectin, resistin), reactive oxygen species
(ROS), etc.6 Reactive oxygen species (ROS) generated in endoplasmic reticulum and
mitochondria contribute to the inflammation by participating in the process of autophagy,
which is a highly conserved housekeeping pathway that plays a critical role in the removal of
aged or damaged intracellular organelles. ROS include Superoxide (O2.-), Hydrogen peroxide
(H2O2), Hydroxyl radical (OH.), singlet oxygen. Gasotransmitters like Hydrogen sulfide (H2S)
 Anti-inflammatory Agents 497

together with Nitric oxide (NO)8 and carbon monoxide CO, are also emerging as a regulators of
inflammation.9 Efforts to develop new, safer and more effective anti-inflammatory drugs are
based on the improved understanding of the role of key mediators and the processes involved
in triad of inflammation (Fig. 33.1).
Advent of genomic era has emphasized on the role of altered gene expression as fundamental
to the etiology of inflammation and immune disorders.10 Many genes for proinflammatory
enzymes (e.g. COX-2, iNOS,)8 acute phase proteins and cytokines (e.g. TNF-α) contain binding
sites for multiple transcription factors in their regulatory elements, which are activated by a
variety of exogenous stimuli like bacterial lipopolysaccharide (LPS) and endogenous stimuli,
cytokines (IFN-γ, IL-6) and growth factors. Consensus sequences for the transcription
factors NF-κB, AP-1 and STAT1 have been found, e.g. in the promoters of COX-211 and iNOS.9
Proinflammatory agents like TNF-α and LPS activate the mitogen-activated protein (MAP)
kinase pathways resulting in the stimulation of ERK1/2, c-Jun, N-terminal kinases and p38
kinases which in turn activate transcription factors AP-1, NF-κB which participate in the
regulation of expression of immediate early genes involved in immune, acute phase and
inflammatory responses.
Cytokines have been shown to play central roles in inflammatory diseases12 such as psoriasis,
rheumatoid arthritis and septic shock, and inhibition of their action or their activation
is a proven approach to modulation of these diseases. The secretion of these diffusible
growth factors and heparin- binding chemokines by resident tissue is further amplified by
immune cells during inflammation.13 It has been demonstrated that chemokine expression
temporally precedes the inflammatory cell infiltration.14 Certain hyper inflammatory states
are linked to inflammasome activation, which facilitates caspase-1 and interleukin IL-1β
processing by autophagy, leading to amplified inflammatory response.15-17 During autophagy,
cell size increase and the presence of increased numbers of membrane vacuoles termed
autophagosomes is noticeable. Autophagy can limit inflammasome activity by lysosome
mediated destruction of inflammasomes. Autophagy can therefore be described as a double-
edged sword as destruction of inflammatory cells like macrophages and inflammasome by
autophagy is desirable in control of inflammation,18 but unchecked autophagy of differentiated
cells could have serious implications19 in aging and degenerative diseases.
Until a few years ago, inflammatory disorders were treated primarily with relatively
nonselective anti-inflammatory drugs such as corticosteroids and various nonsteroidal anti-
inflammatory drugs, however, nowadays specific mediator antagonists alone or in combination
and gene therapy are also being tried. Inhibitors, which specifically interfere with components
of different intracellular signaling pathways or inhibit the activation of transcription factors
responsible for the expression of disease, related genes might have applications as novel
therapeutic agents in inflammation.11,20-22 In order to search new inhibitors of signaling
involved in inflammation, inducible reporter gene vectors are constructed containing the
natural promoters of inflammatory genes (COX-2, iNOS, TNF-α)11,20-22 or using binding sites
for defined transcription factors (NF-κB, AP-1, glucocorticoid receptor, STATs). Present day
anti-inflammatory drug discovery is based on preliminary in vitro observations in a number
of standard anti-inflammatory assays, in which the test compound produces unusually potent
antagonism of inflammatory pathways. The effective candidate drug in in vitro tests is later
tested in whole animal models of acute, subacute and chronic inflammation.
498 Drug Screening Methods

IN VITRO METHODS
Measurement of NO Production in LPS/ IFN-γ- costimulated and unstimulated murine
macrophage RAW264.7 cell line
In this in vitro screening method, ability of test drug to inhibit NO, which is one of the
early mediators of inflammation through increased vasodilation (Fig. 33.1), is evaluated.
Murine macrophage cell line RAW264.7 is maintained at 37° C in Dulbecco’s modified eagle
medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 units per ml)
and streptomycin sulfate (100 µg/ml) in a humidified atmosphere of 5% CO2.23 The cells are
stimulated with either 1 µg/ml of LPS for 4 h or 20 ng/ml IFN-γ and then test compounds,
Griess reagent (100 μl) is added to 100 μl of each supernatant from LPS or IFN-γ stimulated
cells in triplicate. Inhibitory test compounds are dissolved in DMSO before addition to cell
line; final concentrations of DMSO is kept at 0.1% or less than that. Controls with DMSO alone
are also run alongside the test drugs. The protein determination is performed by Bradford
protein assay. The plates are read at 550 nm against a standard curve of sodium nitrite. Nitrite
accumulation is used as an indicator of NO production in the medium and is assayed by the
Griess reaction. Hydrocortisone, a well-known steroidal anti-inflammatory drug, is used
as a positive control for such experiments and potential candidates are compared against
hydrocortisone for efficacy measurement.

Inhibition of Oxidative Stress by Reducing Reactive Oxygen Species (ROS)

Generation
Generation of excessive ROS has been implicated in a number of diseases including
inflammatory diseases (Fig. 33.1). Cells of murine macrophage cell line RAW264.7 grown
overnight are treated with the test drugs in different concentration for 24 h, and then
incubated with fluorescent marker 5 μM carboxy-2',7'-dichlorodihydrofluorescein diacetate
at 37°C for 30 min. Drug treated cells are compared to positive control cells exposed to
hydrogen peroxide H2O2 (0.03%) for 1h prior to being stained with 5 μM of carboxy-2',7'-
dichlorodihydrofluorescein diacetate. Treated cells are washed with PBS, and fluorescent cells
are counted by flow cytometry. Decrease in percentage of fluorescent cells relative to positive
control is index of ROS generation. Another in vitro model of superoxide (O2–) generation by
polymorphonuclear cells PMNs has been used by Dianzani et al. 2006 for drug screening.24
These investigators have cultured the PMNs and challenged them with a concentration of
10-7M n-FMLP (N-formylmethionyl-leucyl-phenylalanine) to optimally generate O2–.
Superoxide production was determined spectrophotometrically by measuring the superoxide
dismutase inhibit able reduction of cytochrome C reduced/106 PMNs/min.

Blocking Inflammasome Activation

Inflammasome are cytoplasmic and multimolecular protein complexes, which activate
Caspase-1, enzyme present in an inactive pro-form in the cytoplasm.15-17 Isolated macrophages/
mast cells from mouse peritoneum or cell lines such as RAW264.7 and HMC-1 plated overnight
can be used for this assay. Cultured cells are pretreated with test drugs (0.1, 1, and 10 μg/ml)
for 1 h before stimulation with LPS (100 ng/mL)25 or proinflammatory cytokine (interleukin-
1beta, IL1β, 5 ng/ml) for 24 h. Afterwards, the cells and supernatants are harvested for analysis.
 Anti-inflammatory Agents 499

Caspase-1 activity is quantitatively measured by Western blot of pro- and processed caspase-1
in drug treated tissue homogenates.26 Efficacy of drugs in blocking inflammasome activation is
measured by reduction in the active form of caspase-1 and the increased level of pro caspase-1
form relative to active drug comparator.

Induction of Autophagy
Recent studies on autophagy frequently use RAW264.7 macrophage cells that stably or
transiently express a green fluorescent protein (GFP)-tagged LC3 protein (microtubule-
associated protein 1 light chain 3). RAW264.7 macrophage cells stably expressing GFP-LC3 are
grown in multi-well plates and exposed to test drugs and positive controls. Drugs capable of
inducing autophagy cause characteristic redistribution of GFP-LC3, which leads to a change
from diffuse fluorescence throughout the cytosol to numerous punctate with concentrated
fluorescent signal in vesicles due to vacuole formation.18 Effect of potential anti-inflammatory
drugs in causing redistribution of fluorescence is measured by confocal microscope.27 The
differences in expression levels of LC3-II and LC3-I in immunoblots can serve as a quantitative
measure of autophagy induction.18
Autophagy occurs in a stepwise fashion as cells gain in size with increase in numbers of
membrane vacuoles termed autophagosomes. The final step of autophagy involves the fusion
of the autophagosome with the lysosome to form the autophagolysosome, in which the
gathered cargo is degraded by lysosomal hydrolases. In the drug screen for autophagy, GFP-
LC3 is recruited from the cytosol to become part of the autophagosome and there it undergoes
site specific proteolysis and lipidation near the C terminus to form LC3-II. Effect of test drugs
on the expression of autophagy-related proteins such as light chain 3B (LC3B), autophagy
protein 5 ATG5, beclin 1, LAMP-2, can be used as measure of efficacy.28

Mast Cell Degranulation

Mast cells play a key part in allergic inflammation as their degranulation releases cytokines,
chemokines, proteases, and histamine after re-exposure to an allergen.29 Previous drug screen
methods relied on harvesting peritoneal mast cells to evaluate ability of test drugs to inhibit
mast cell degranulation. Recent studies used phorbol 12-myristate 13-acetate (PMA) plus
A23187-stimulated mast cell line, HMC-129 for this purpose. Cultured mast cell line is exposed to
test drugs for 30 min prior to stimulation with PMACI (20 nM of PMA plus 1 μM of A23187) and
incubated at 37°C for 6 h. The cells are separated from the released histamine by centrifugation
at 400 rpm for 5 min at 4°C. Histamine levels are then measured in supernatant by measuring
fluorescent intensity at 460 nm (excitation at 355 nm) using a spectrofluorometer. The total
content is measured after treatment of the cell suspension with Triton X-100. The percentage
release determined is considered the index of anti-inflammatory activity.

Measurement of Cytokine Expression in Murine Macrophages

Macrophages are the crucial agents for chronic inflammation and therefore serve as ideal
platform for screening agents against chronic inflammation. To obtain primary macrophages,
female CD-1 mice, 5 to 10 weeks of age are injected intraperitoneally with 2 ml of 4%
thioglycollate broth. Four days after injection, peritoneal macrophages are harvested and
processed according to Ding’s procedure.30 Cells are seeded in 96-well plates at 2 × 105 cells/
500 Drug Screening Methods

well and incubated for 48 h with 20 ng/ml IFN-γ in the presence or absence of test drugs.
Following treatment, cytokines levels in medium from drug treated and untreated cells can
be measured by ELISA or by multiplex.31 Besides protein, the effect of tested agents on the
mRNA expression of proinflammatory mediators can be assessed by harvesting the cells and
processing for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) or
quantitative PCR (qPCR).23,32

Measurement of Inflammatory Gene Expression

Murine peritoneal macrophages are isolated26 as described above and treated with test drugs
in the presence or absence of LPS (5µg/mL) for 18 h. Effect of test drugs on inducible NOS
(iNOS), COX-233 and microsomal PGE synthase-1 protein, and p38 MAPK phosphorylation34
is evaluated by Western Blot analysis. Effect of drugs on NF-κB nuclear translocation can be
visualized using the technique of immunofluorescence.

Adhesion Assays
The vascular proteins VCAM-1, ICAM-1, and E-selectin, are investigated in primary cells
derived from umbilical vein (HUVEC) and a microvascular cell line (HMEC-1) owing to
their prominent roles in regulating leukocyte extravasation. Adhesion assays using cultured
(HUVECs) or human dermal microvascular endothelial cells (HDMECs) are used to recapitulate
the in vivo setting.35 Confluent monolayers of endothelial cells are cultured overnight in 96 well
plates and then incubated with test drug at concentrations from 10-6 to 2 × 10-5 M for 20 min
prior to stimulation with TNF-α 20 ng/ml for 24 h. TNF-α upregulates intercellular adhesion
molecule (ICAM)-1 expression and 24 h stimulation with TNF-α can be replaced in this assay
with another noxious stimulus of LPS 4 mg/L for 6 h.36 Neurophills intravitally labeled with
2’, 7’- bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) are overlaid (2 × 105 cells/
well) and allowed to adhere for 20 min to TNF-α stimulated endothelial cells at 37°C.
Fluorescently labelled cells of human monocytic leukemia cell line THP-1 can also be used
instead of neutrophils. Adherent cells are rinsed with PBS and adherence is then evaluated by
lysing adherent cells with 0.5% CTAB (cetyl-trimethyl-ammonium bromide), and measuring
fluorescence at 485 nm in an ELISA reader which is a quantitative measure of the activity
present in test drug.

FMLP-induced Adhesion of PMN to HUVEC

The bacterial peptide FMLP only activates the PMN adhesive machinery, therefore this stimulus
was selected by Dianzani et al. 2006 to evaluate the anti-inflammatory activity of various
compounds on PMN adhesion to HUVEC.24 The near maximal concentration of FMLP (10-7M)
produced maximal activation of PMNs. PMNs and HUVEC can also be challenged with other
inflammatory stimuli like platelet activating factor (PAF), IL-1β, TNF-α and phorbol myristate
acetate (PMA) to observe adhesion phenomenon in inflammation. PAF (10-7) induced stronger
inhibition of PMN adhesion than other stimuli at the concentration used.
Platelet-neutrophil adhesion: Thrombin-activated human platelets are incubated with drug
(107–10-4 M) at 20­°C for 10 min, and mixed with neutrophils at a ratio of 10:1. Neutrophils with
two or more (number positives) and one or no adherent platelets (number negatives) are
counted as index of activity. The test drug blocks the adhesion with respect to controls.
 Anti-inflammatory Agents 501

Neutrophil adhesion to hypoxia-stimulated porcine aortae: Fresh porcine aortae are

stimulated by placing them into PBS gassed with N2, and fixed between a Teflon block and a
stainless steel plate with drilled holes. Neutrophils and the test drug (10-5 to 10-7 M) are added
onto the luminal side for 90 min. Adhesion is blocked by test drug, and the adherent cells are
lysed to assess the (MPO activity photometrically.

Interleukin 1beta-stimulated human articular chondrocytes

Human chondrocytes (CHs) isolated from osteoarthritic patients37 can serve as ideal screen
to test potential drugs for arthritis. Isolated cells are stimulated with a proinflammatory
cytokine (interleukin-1beta, IL1beta, 5 ng/ml) and the effect on NO and cytokine expression is
measured as described for macrophages in above sections.

Cyclooxygenase (COX) Assays

The enzyme COX catalyses the conversion of arachidonic acid to prostaglandins. COX is now
known to exist in two isoforms COX-1 and COX-2, which are constitutive and inducible in
nature, respectively. Thus, constitutive form is associated with physiological function whereas
inducible form plays a pathological role (major role in inflammation). The in vitro assays for
these enzymes may be carried out as under.
COX-1 assay: Briefly, purified recombinant human COX-1 (50 μl of 1 μg ml-1 in 100 mm Tris-
HCl, pH 8.0, 5 mm EDTA, 1 mm phenol, 1 μm hematin)38 is preincubated with 2 µl of test drug
solution for 15 min. The reaction is then initiated by the addition of 5 μl of 1 μM arachidonic
acid to obtain a final concentration of 0.1 μM. After a 7 min incubation at room temperature,
the reaction is stopped by the addition of 5 μl 1M HCl and 50 μl acetonitrile. Aliquots of 50 μl
of each reaction mixture are analyzed for substrate conversion into PGE2 by a PGE2 enzyme
immunoassay.
COX-2 assay: Drugs can be tested for COX-2 inhibitory activity spectrophotometrically by
measuring the velocity of oxidation of N, N, N’, N’-tetramethyl-p-phenylenediamine (TMPD).39
TMPD is oxidized during the reduction of PGG2 to PGH2. The assay mixture consists of 100 mM
sodium phosphate, 1 μM of hematine, 1 mg/ml gelatin, 2-5 μg/ml of purified COX-2 and 4 μl of
test compound in DMSO. The total volume of assay mixture is 180 μl. This is then preincubated
for 15 min at 22°C and then 20 μl of a solution of 1 mM arachidonic acid and 1mM TMPD in
the assay buffer is added. The assay buffer contains the assay solution except hematin and
enzyme. The absorbance at 610 nm is measured over the first 36 sec and percentage inhibition
calculated. The nonenzymatic oxidation of TMPD in the absence of COX-2 is also observed
and subtracted from the activity in the presence of COX-2.

IN VIVO METHODS
Animal models are the way in which the results from simpler in vitro research can be tested
in “intact” biological systems. The logical link of animal modeling to the clinical context can
help build a confidence in a new drug before clinical trial. Animal studies can be viewed as
hierarchical in nature as studies using rodents and small laboratory animals are relatively
simpler to perform and maintain than studies done using larger animals. Research done
with small mammals is less expensive, more accessible and less time-consuming to carry
out.
502 Drug Screening Methods

However, none of the models currently employed using variety of phlogistic agents inducing
varying degree and duration of inflammation; adequately mimic the chain of events underlying
inflammation in patients. Various models that have been used to date have different strengths
and weaknesses and findings should ideally be reproduced in more than one mammalian
species before extrapolating data to human subjects. A recent PNAS paper reported that mouse
models of inflammatory diseases correlate poorly with the human conditions,40 which makes
clinical translation of findings from certain mouse models less certain. The revised chapter lay
greater emphasis on rat models over mouse models. The use of rats also affords superior pain
behavior modeling, and their comparatively larger size facilitates surgical manipulation. It is
important that these aspects are taken into account for identifying experimental findings that
constitute general principles that are predictive of clinical efficacy from those that are unique
to a model.
It is recommended to concomitantly use several in vivo methods, which together can
mimic a broad spectrum of acute, subacute and chronic inflammatory events such as
redness, heat, plasma exudation, edema, pain, leukocyte migration, tissue proliferation
and partial necrosis. Whole animal like guinea pig, rat, mouse, rabbits or dog may be used
for this purpose. Most prominent in vivo models of inflammation are croton oil-induced
mouse ear edema, carrageenan induced edema,41 carrageenan-induced rat pleurisy,42 and
cotton pellet induced rat granuloma.43 The models inducing edema screen drugs for their
ability to halt vasodilation and edema formation, whereas models inducing pleurisy and
granuloma evaluate test compounds against exudative phases and proliferative phases of
inflammation, respectively. Various in vivo models of inflammation have been given in Table
33.1. Preliminary anti-inflammatory mechanisms, is determined in the animal models by
measuring the levels of myeloperoxidase (MPO) for assessing neutrophil migration and levels
of free radical damage by measuring superoxide dismutase (SOD) and malondialdehyde
(MDA) in tissue.

Table 33.1: Various in vivo models of inflammation

S. No Model of inflammation Animal Site of application/ injection of Reference(s)

species inflammogen
1. UV-β-induced erythema Guinea pig Depilated skin 44,45
2 Ear edema Mice Topical application on ear 41, 43
3. Carrageenan-induced paw edema Rats, mice Subplantar region 44, 46, 47, 48
4. Pleural exudation Rats Pleural space 42
5. Cotton pellet-induced granuloma Rats, mice Subcutaneously through the skin 43
incision, groin region, flanks
6. Freund’s adjuvant arthritis Rats Subplantar region 53
7. Papaya latex-induced arthritis Rats Subplantar region 54
8. Collagen/LPS-induced accelerated arthritis Mice Intradermal injection at the base 55, 56
of the tail
9. Air pouch Rats, mice Dorsal surface 51, 52
10. Cyclophosphamide-induced cystitis Rats, mice Systemic injection 8, 57
11. Prostatitis Rats, mice Intraprostatic application 33, 46
12. TNBS-induced colitis Rats, mice Rectal application 64
 Anti-inflammatory Agents 503

UV-B-induced Erythema in Guinea Pigs

Erythema (redness) is the earliest sign of inflammation, not yet accompanied by plasma
exudation and edema. Guinea pigs are the frequently used animals to study the anti-
inflammatory activity of drugs in this model.44 In albino guinea pigs, the pure erythema
reaction appears 2 h after exposure of the depilated skin to ultraviolet irradiation.28 Guinea
pigs are pretreated with the test drugs half an hour before UV-exposure from a UV lamp that
emits radiation in the wavelength of 180-200 nm. This model can be used as a pure measure
of the vasodilatory phase in the inflammatory reaction. However, the test suffers from the
drawback that shaving of skin is required before application of irritant. The test also depends
on the skin thickness and the intensity of erythema. It is difficult to quantify and requires a
skilled investigator. However, the method is easily translatable in clinic and in recent studies it
has been applied to Human volunteers.45

Ear Edema Model

Croton oil is obtained from the expression of the seeds of Croton tiglium. Apart from its role as
a promoting agent in chemical carcinogenesis, it is widely used agent to induce ear edema in
mice. Croton oil ear edema (induced by topical application) is a predictable model to detect
the activity of topical anti-inflammatory drugs. In this test, the inflammatory response is
quantified by measuring the increase in earplug weight at single time interval (peak at 6 h)
after croton oil application.41 The method can be used to test both steroidal and nonsteroidal
anti-inflammatory drugs. Both the rats and mice can be used for this test.
A total of 15 µl of an acetonic solution containing 75 µg of croton oil is applied to the outer
and inner surface of the right ear of each mouse (about 1 cm2 area). The left ear remains
untreated. Control animals receive only the irritant while indomethacin (100 µg/ear) serves as
the reference. Varying dose levels of the test drug are applied to the inner surface of the right
ear of each mouse either 1 h before drug administration or by dissolving the drug in croton
oil solution. The animals are sacrificed by cervical dislocation 6 h later and a plug (6 mm in
diameter) is removed from both the treated and untreated ear. The difference in weight between
the two plugs is taken as a measure of edematous response.43 The inhibition percentage was
calculated by the following equation:

Inhibition (%) = (Econtrol– Etreated) ÷ Econtrol × 100

where Econtrol and Etreated is the extent of edema from the control group and treated groups.
Possible mechanism of action is determined by measuring MPO activity in harvested tissue.

Carrageenan-induced Paw Edema Model

To study the acute and subacute phases of inflammation in rodents (rats and mice),
carrageenan is a widely used irritant or inflammogen or a phlogistic agent.41 Chemically, it is a
sulfated polysaccharide from seaweeds. The experimental tissue injury caused by this irritant
initiates a cascade of events leading to formation of exudates. The inflammation induced
by it is biphasic in nature.46 Injection of carrageenan induces inflammatory cell infiltration
504 Drug Screening Methods

and increased COX-2 expression, which facilitates inflammatory processes. COX-2 derived
prostaglandins, particularly prostaglandin E2, is the primary pathogenic factor of symptoms,
representing the classic triad of inflammation.11,20-22 The well recognized method of Winter
et al. 196247 is followed. A 1% w/v suspension of carrageenan is prepared freshly in normal
saline and injected into subplantar region of left hind paw (usually 0.1 ml in rats and 0.025-0.05
ml in mice).48 In control animals, only vehicle is injected. Test drug is usually administered
orally or intraperitoneally, according to body weight immediately or half an hour or one hour
before (depending on the expected peak effect) carrageenan challenge.49 A mark is made at the
ankle joint of each rodent. Paw volume up to the ankle joint is measured in drug treated and
untreated groups before and 3 h after carrageenan challenge using a plethysmograph filled
with mercury.50 However, paw edema in rats has also been measured beyond 3 h also after
carrageenan challenge.
The sophisticated electronic devices are also being used nowadays to record the paw volume
or rodents. The % reduction in edema is calculated using the following formula:
Mean edema in untreated control
group–mean edema in drug treated group
% Reduction in edema = × 100
Mean edema in control group

The method is simple, easy and short lasting as well as reproducible. However, it is non-
specific and difficult to quantify. It is also difficult to examine cells and their modification by
anti-inflammatory drugs. One must avoid injecting the irritant in both the hind paws of the
animal on account of severe pain. The carrageenan causes unalleviated pain and deformity.
Carrageenan in this model can be replaced by other irritants such as formalin, mustard
oil, snake venom, dextran and polyvinylpyrollidone, etc., which produce varying degree of
inflammation.

Carrageenan-induced Rat Pleurisy

Various irritants produce nonimmune acute inflammation when injected into natural cavities
(pleural and peritoneal cavities, knee joints). The pleural cavity of rats and guinea pigs has
been successfully utilized for screening of anti-inflammatory drugs. In this model, it is easy
to measure the volume of exudates and to determine the amounts of protein, mediators and
leukocytes in the exudates. Pleurisy can be induced in rats by an intrapleural injection of
carrageenan,42 turpentine, Evan’s blue, Arabic gum, glycogen and dextran, enzymes, antigens,
microbes, mast cell degranulators, etc. This model not only allows quantitation of the anti-
inflammatory activity, but also allows for investigating the mechanism underlying the action
of new test drugs. The method is suitable for detection of both steroidal and nonsteroidal anti-
inflammatory drugs.
Experimental pleurisy is produced by injecting 0.1 ml of turpentine oil into right pleural
space in rats under light anesthesia as described by Spector. The test drugs are injected
intraperitoneally in graded doses 1 h before turpentine injection. The rats are decapitated and
pleural exudate is collected half an hour after turpentine treatment. The exudate is removed,
preferably by washing the pleural cavity with a known volume of Hank’s solution to ensure
 Anti-inflammatory Agents 505

complete recovery of the exudate and integrity of the cells. Volume of the exudate is measured
as an index of activity of the test drug.

Cotton Pellet-induced Granuloma

This method is widely used to study the exudative and proliferative phases of inflammation.
Sterile cotton pellets (5 mg), each impregnated with 0.4 ml of 5% aqueous solution of ampicillin,
are used.43 Under ether anesthesia, pellets are inserted subcutaneously through skin incision
in the back of the animal (rats and mice). Drug treatment is started 2 h after cotton pellet
implantation and continued for 5 consecutive days. Vehicle treated animal receive normal
saline for the same duration as the test group receives drug. On 5th day, animals are sacrificed,
granulomas are removed, dried for 24 h at 60°C and the dry weights determined. The weight
of granulomatous tissue formed is calculated by subtracting initial weight from the final dry
weight of cotton pellets and % protection by the drug can be calculated. Cotton pellets of
larger size (30 mg) introduced subcutaneously into the groin region have also been used. The
proinflammatory effect of cotton pellets can be enhanced by soaking the pellets in turpentine,
carrageenan or some other irritants.

Air Pouch Model

The air pouch model of acute inflammation has been used over the last 30 years for screening
anti-inflammatory drugs as well as for other applications.51,52 Injection of air into dorsal surface
of rat or mouse followed by a suitable irritant provides a useful model of non-immune subacute
exudative inflammation. The air pouch model was originally devised by Selye, 1953 and later
on modified by several investigators. Subcutaneous dorsal pouches are created in anesthetized
mice by injecting 5 ml of air. 51,52 After 3 days, the pouches are re-injected and on day 6, 1 ml of
1% w/v carrageenan in sterile saline is injected. Control animals receive saline alone. Twenty-
four hours after carrageenan, mice are anesthetized and killed. The anti-inflammatory effect
of different doses of test compound is investigated by giving them orally 30 min before, 8 and
20 h after carrageenan. Controls receive drug vehicle. Indomethacin (5 mg/kg) orally is kept
as standard and fed according to the above schedule. The pouches are washed with 1ml of
saline, exudates are immediately cooled on ice and the volume is recorded. The total number
of leukocytes migrated into the pouch are evaluated after staining with erythrosine B and
the remaining exudate is centrifuged at 3,000 rpm for 10 min at 4°C and supernatant stored
at –20°C until use. The chemical mediators involved in this inflammation remain unknown,
protein synthesis and kinin formation are necessary for this granuloma formation.

Adjuvant Arthritis
Adjuvant arthritis in rats is considered to be an immunologically mediated and most frequently
investigated model of chronic inflammation. This model depicts the very close similarity with
the clinical rheumatoid arthritis. The strain of rat, preparation (emulsion or suspension) of the
adjuvant (emulsion induces both primary and secondary lesions in greater% of animals), site
of injection and the time of measurement of primary and secondary lesions affects the results
obtained.53 The arthritis is induced by s/c injection of either Freund’s complete adjuvant FCA
or mycobacteria suspended in oil. The subplantar injection of 50-100 µl of this suspension
506 Drug Screening Methods

results in a primary, non-immune, localized inflammatory response in the paw followed by

the secondary immune systemic disease. Mycobacterial constituents in FCA are recognized by
toll like receptors and activate Th1 immune response leading to production of TNF-α and IL-12
and arthritic changes. The local swelling begins in the injected paw within 24 h, reaches a peak
on day 4 or 5 and becomes stabilized on day 6 to 11. The systemic disease usually starts on day 7
and is characterized by the swelling of the contralateral non-injected limb. Foot thickness and
body weight changes can be monitored in the drug treatment group and compared with that
of untreated control. This model allows evaluation of chronically administered drug against
inflammation. Drug administration is done daily starting a day before injection of adjuvant.
There is delayed systemic response to the Freund’s adjuvant, which makes this model superior
to other models in assessing the efficacy of all types of potential anti-rheumatic drugs.

Papaya Latex-induced Arthritis

The slow reacting antirheumatic drugs (SARDs) such as gold, chloroquine, levamisole and
penicillamine etc. have failed to show significant activity in the conventional experimental
models. It has now been demonstrated that lysosomal enzymes play an important role in
adjuvant induced arthritis. These enzymes are known to cause tissue damage. Keeping in view
the disease etiology, papaya latex induced model of experimental rheumatoid arthritis has
been developed to test the anti-inflammatory activity of SARDs. Cartilage destruction in this
model is caused by cysteine protease called caricain found in the latex of papaya. 0.1 ml of
0.25% solution of papaya latex (prepared in 0.05 M sodium acetate buffer, pH 4.5 containing
0.01% thymol) is injected into the rat hind paw.54 The peak effect occurs at 3 h and lasts for more
than 5 h. The method is sensitive for evaluating NSAIDs like aspirin, ibuprofen and steroidal
anti-inflammatory drugs (particularly SARDs), which do not show appreciable activity in
adjuvant induced arthritis and other models of inflammation.

Collagen-induced Arthritis (CIA)

This mouse model of arthritis is induced by immunization with type II collagen in FCA, which
induces an autoimmune disease directed against the cartilage in the joints.55,56 Unlike other
models, CIA is characterized by inflammation and destruction of the joints in a T cell– and
B cell–specific manner. The model successfully predicted the clinical efficacy of (TNF-α)
antibodies in rheumatoid arthritis.
Chicken collagen type II55,56 is solubilized overnight at 10°C in 0.05 N acetic acid at a
concentration of 1 mg/ml and emulsified in FCA at a ratio of 1:1 (v/v). Using 0.1 ml emulsion
each adult female DBA/1 Lac J mice (age 12-14 weeks, weighing 22-26 g) are immunized by an
intradermal injection at the base of the tail with 100 µg of chicken type II collagen. Seventeen
days later, the immune response to collagen is boosted by a subcutaneous injection in the
back of the neck with 200 µg of LPS solubilized in 0.2 ml PBS. The thickness of each affected
hind paw is measured daily with microcalipers. Clinical severity of inflammation (redness,
swelling, joint deformity) is scored periodically on a scale of 0-3 in all 4 extremities as follows:
0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced edematous swelling, and 3
= joint rigidity. Each limb is graded, thus allowing a maximum score of 12 per mouse. This
experiment was repeated on 3 separate occasions. The incidence and frequency of arthritis is
calculated as follows:
 Anti-inflammatory Agents 507

Incidence was the number of mice having at least one affected paw divided by the number
of mice per group.

Number of affected paws
Frequency =
Total number of paws per group

The advantages of this method are:

i. This model is specifically well suited for high throughput screening to identify novel
inhibitors of integrin VLA-4, the very late antigen-4.
ii. The end point of the model is reached within 21 days whereas in other models it takes
weeks to months.
iii. Inflammation is not self-limiting as with other models and therefore mimics clinical
arthritis.
iv. It limits the amount of test compound required for the study by administration of the test
compound at appropriate time (these investigators have administered antibody to VLA-4
on day 16-22 (effector phase of the response). Therefore, the animals do not need to be
treated from the time of collagen immunization.
v. Administration of LPS causes a highly synchronous response with reduced variability.

Cyclophosphamide-induced Cystitis in Rodents

Systemic chronic administration of cyclophosphamide (CYP) in rodents has been proposed
as a relevant preclinical model of urinary bladder inflammation or cystitis.8,57 Single or repeat
systemic injections of CYP in mice or rats can induce acute or chronic model of bladder
inflammation. Cyclophosphamide is metabolized in liver and kidney into acrolein, which is
the actual agents that cause bladder injury leading to bladder inflammation. Acute cystitis
is induced by a single intraperitoneal (ip) injection of CYP at the dose of 100-150 mg/kg.
Inflammation in bladder tissue is characterized by submucosal edema (Fig. 33.2), infiltration
of inflammatory cells, telangiectasia (prominent dilated blood vessels engorged with red blood
cells) increase in proinflammatory cytokine gene expression. Increased bladder weight and
wall thickness were associated with edema and hemorrhage. Bladder tissue levels of IL-1β,
IL-6, MCP-1 and VCAM-1, and urinary levels of PGE2 were increased. Oral administration of
anti-inflammatory agents aspirin and ibuprofen reversed the increased bladder wall thickness,
macroscopic damage and levels of cytokines IL-1β, IL-6 and PGE2.58 Therefore, the model of
bladder inflammation allows noninvasive repeat assessment of the drug effect from the same
animal without the need of animal sacrifice. The two genders respond differently to CYP and
females perform better in this model.8,57 Model allows evaluation of both local59 and systemic
treatments.
Rat model of Prostatitis: Compared to cystitis which is typically observed in females, prostatits
is a quintessential male disorder as it is the inflammation of prostate. Several animal models
have been reported in last 30 years including chemical induced prostatitis, bacterial infection-
induced prostatitis models, hormone-associated prostatitis models and other miscellaneous
prostatitis models. For chemically induced prostatitis, irritant chemicals are directly injected
into the surgically exposed prostate of male rat. Chemicals used in studies so far include,
capsaicin, 5% formalin33 and 3% carrageenan46 in saline.
508 Drug Screening Methods

Figure 33.2: Cyclophosphamide induced inflammation in rat bladder. Bladder tissue harvested 24 h after injection
showing interstitial edema and infiltration of inflammatory cells and telangiectasia (prominent dilated blood vessels
engorged with red blood cells). Hematoxylin and eosin stain, original magnification 4x

For intraprostatic injection, rats are anesthetized with isoflurane (5% for induction and
3% for maintenance) and lower abdomen above the penis is shaved and the skin in this area
sterilized using 3 applications of 10% povidone-iodine solution. A small midline incision is
made in the sterile area to expose the bladder and the adjacent prostate at the bladder outlet.
With a 30-gauge needle, 50 ul injection of irritant chemical is made into both right and left
ventral lobes of the prostate gland. For the control group, commensurable sterile normal saline
is injected in similar sites. After the injection, surgical site is sutured back and animals allowed
to recover from anesthesia. At different time points (after 24 h, 7 days, 14 days and 30 days of
injection), rats are sacrificed and the prostate is harvested. For histological analysis, one part
of the prostate is fixed in buffered 10% formaldehyde for 24 h, embedded in paraffin, cut with
a microtome, and stained with hematoxylin-eosin. Under a low-power microscopy field, each
slide is evaluated randomly in 4 different areas containing inflammatory cells (Fig. 33.3).

Figure 33.3: Intraprostatic injection of 5% formalin induces a model of prostatic inflammation (prostatitis) marked by a
host of inflammatory changes including hyperplastic acini lined by tall columnar epithelium and infiltration shown by
arrows. Hematoxylin and eosin stain, image is magnified 20x
 Anti-inflammatory Agents 509

Compared to saline injected prostate, intraprostatic injection of chemicals causes prostatic

edema and increased inflammatory cell accumulation. There is inflammation of prostate with
minimal tissue necrosis and damage without any bacterial infection. Compared to capsaicin
induced prostatitis60 that lasts only for a week, the formalin induced prostatitis lasts up to a
month to test interventions.
Bacterial prostatitis in male rats is induced by injecting 105 -108 colony-forming units
(CFU)/ml of E. coli in to the urinary tract.61 Frozen bacterial isolate is cultured in tris-buffered
saline (TBS) immediately prior to the injection to a concentration of 108 CFU. Male rats are
anesthetized and a sterile polyethylene tube (0.9 mm outer diameter, 2.5 cm length) is inserted
to the urethra, and 0.2 ml of E. coli suspension instilled into prostatic urethra using insulin
syringes. Sufficient time for the bacteria to infiltrate to the inside of the prostate is maintained
by preventing excretion of urine through maintenance of anesthesia for 1 h. Model can be
validated 4 weeks after the injection of E. coli by performing McConkey culture test on collected
urine or prostate biopsy to detect bacteria. This model is suitable for assessing the effect of
drugs with chronic action.
A mice model of experimental autoimmune prostatitis also exists, which is developed by
subcutaneous injection of prostate antigen62 or spermine binding protein (p25) peptide63 which
induces immune mediated prostatic inflammation. However, these immune mediated models
of prostatitis in mouse frequently develop diabetes and other organ-specific autoimmune
diseases that may interfere in the study of drugs targeting prostatic inflammation.

Trinitrobenzene Sulfonic Acid (TNBS)-induced Experimental Colitis

Under normal conditions the intestinal mucosa functions within a delicate balance of
inflammatory cells where cytokine synthesis and cytokine-induced signal transduction
pathways are tightly regulated by intricate feedback mechanisms and regulatory T-cells. Colitis
leads to a dramatic shift/imbalance in the cytokine production profile at different stages of
the disease process. TNBS induces inflammation of the rat colon called colitis. Intracolonic
administration of 2, 4, 6-trinitrobenzene sulfonic acid (50 mg/ml) dissolved in 50% ethanol
(v/v) is administered via a transanal approach (total volume 0.5 ml) using a PE-90 catheter
whose tip will be placed approximately 6 cm proximal to the anal verge.64 Sham animals receive
0.5 ml of normal saline. Inflammation is examined in 8 cm distal part of rat colon 14 days later.
Cytokines are principal mediators of the innate and adaptive arms of the immune responses
in mucosal inflammation and TNBS induces drastic increase. TNBS instilled rats show colonic
damage, weight and adhesions (macroscopic and microscopic), diarrhea, body weight and
increased colonic levels of free radicals (nitric oxide and lipid peroxidation).

CONCLUSION
From the searched literature, it is evident that numerous experimental methods for evaluation
of anti-inflammatory drugs have been developed over the last few years. The lead compounds
may be identified using in vitro assays and may be subjected to further testing in vivo. These
methods help in understanding of inflammation process as well as in identifying a potential
drug. The findings they generate may drive medical advances and understanding, but the
information gained must be interpreted within the limitations of the model. Irrespective of
510 Drug Screening Methods

the type of animal model used, findings should be carefully validated before extrapolation to
humans. How closely the chosen model reproduces the disease in question will dictate the
extent of validation necessary for translation.
Most of the currently available antirheumatic drugs have shown anti-inflammatory
activity in carrageenan-induced edema, which utilize the transudative and exudative phases
of inflammation. One of the key steroidal anti-inflammatory drug hydrocortisone failed to
show anti-inflammatory activity in pleural exudation method. So, this method is not the most
recommended one for testing inflammatory activity. Therefore, for detecting anti-inflammatory
activity in a new compound, one may rely either on the inhibition of the proliferative phase of
inflammatory reaction viz. inhibition of granulation tissue formation or in the reduction of
exudative phase of inflammation viz. carrageenan induced edema. The former methods are
more-time consuming and require larger quantities of drugs. Carrageenan-induced edema
seems most suitable for screening anti-inflammatory drugs because it is convenient, less time-
consuming and detects activity in all the clinically useful drugs. Collagen-induced arthritis
is suitable model for new biotechnology based drugs for arthritis, considering the successful
prediction the clinical efficacy of TNF-α antibodies this model.

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 CHAPTER

34
Ocular Inflammation
INTRODUCTION
Ocular inflammation is responsible for a number of eye ailments. Ocular inflammation can
cause number of ocular disorders like blepharitis (lids), conjunctivitis (inflammation of
conjunctiva), keratitis (inflammation of cornea), scleritis (inflammation of sclera), uveitis
(inflammation of uvea), etc. These inflammations commonly occur due to bacterial or
viral infections and hypersensitivity. Surgical procedures and physical injury to the eye are
important causes of ocular inflammation.
Any type of ocular inflammation can affect the vision of an individual. The recurrent
inflammation requires immediate attention as several internal structures get adversely
affected and vision is hampered. Gram-negative bacterial infections cause severe ocular
surface inflammations. Lipopolysaccharide (LPS), a component of gram-negative bacterial
membrane has potent proinflammatory and proapoptotic effect, which is mediated through
membrane receptors expressed on host cells. Inflamed cells or tissues release inflammation
mediators (histamine, bradykinin, prostaglandin, leucotrienes, cytokines etc.) in case of acute
or chronic inflammation. The concentration of mediators suggests the degree of inflammation.
The efficacy of the anti-inflammatory drug can be evaluated by determining the levels of these
mediators.
One of the common forms of ocular inflammation is uveitis, which is the inflammation of
uveal tract comprising iris, ciliary body and choroid. Uveitis is responsible for over 2.8% of
blindness in the United States. Each year, 17.6% of active uveitis patients experience a transient
or permanent loss of vision.1
It can be categorized into:
1. Anterior uveitis: Inflammation of the anterior part of the uvea causing iritis and iridocyclitis.
2. Intermediate uveitis: Inflammation of the middle part of the uvea affecting the ciliary
muscles.
3. Posterior uveitis: Inflammation of the posterior portion of the choroid.
4. Pan uveitis: All anterior, middle and posterior zones are affected.
Ocular inflammation can be treated by corticosteroids. Instillation of steroids and pupil
dialators help in reducing the inflammation and pain. Systemic medications are supplemented
for treating appropriate cases. If the treatment gets delayed it may lead to several complications
like glaucoma, cataract and development of new blood vessels ultimately resulting in visual
loss.
 Ocular Inflammation 515

The ethical and practical limitations prevent the availability of human ocular tissues with
active inflammation at different time intervals. Therefore, the studies on animal models remain
the main source of the information. Various animal models have been established to evaluate
the anti-inflammatory activity of new therapeutic interventions. The models are mainly
categorized into non-immunogenic model and immunogenic model, besides inflammation
can be produced experimentally by inflicting injury to the cornea.

EXPERIMENTAL MODELS FOR OCULAR INFLAMMATION

Guinea Pig Model of Giant Papillary Conjunctivitis
Chronic exposure to foreign bodies like contact lenses, leads to giant papillary conjunctivitis
(GPC). Increased incidence is seen during the spring season although it is not related to any
allergan. Levels of IL-3, IL-4, and IL-5; IgG, IgE, and IgM have been found increased in tears.2
Procedure: Contact lens associated GPC, is mimicked in guinea pigs. A sterile needle is
inserted directly into the conjunctiva of unsensitized animal. Pricking results in high number
of neutrophil infiltration in the traumatized tissues.3 This model does not represent the true
histopathological picture found in human GPC.

Rat Models of Allergic Conjunctivitis

Ovalbumin Induced
Ovalbumin induced allergic conjunctivitis model is a generally used experimental model for
studying the inflammatory responses and effect of various therapeutic approaches.4
Procedure: Brown Norway rats, of 8-10 weeks age, are injected subcutaneously in the hind
footpad with 100 µg of ovalbumin emulsified with 100 µl Complete Freund’s Adjuvant (CFA).
Then, the intraperitoneal injection of the test compound or phosphate buffered saline (PBS)
treatment is given for 13 days. On 13th day all the rats are instilled with ovalbumin eye drops
(250 µg in 50 µl of PBS). After 24 h the rats are sacrificed and the eyes, blood and lymph nodes
are used for histologic studies and other related parameters.4, 5

Pollen Induced
Ragweed pollen (RW): RW has been used as an allergan for inducing conjunctivitis. Iwamoto
et al. (2000) has compared the effects of immunization with ragweed pollen in two different
adjuvants.
Procedure: Lewis or Brown Norway rats are immunized with 100 µg of RW in emulsion with
aluminum hydroxide or CFA. The rats are instilled with ragweed pollen eye drops in phosphate
buffer saline (PBS) after three weeks. Inflammatory parameters are observed after 24 h of drug
instillation in eyes, blood and lymph. The response to ragweed pollen is similar to that of
ovalbumin.6
Fukushima et al. (2006) induced allergic conjunctivitis in mice using ragweed pollen as per
the method described below:
Procedure: BALB/c mice are actively immunized with ragweed pollen. The ragweed pollen,
which is adsorbed on alum, is injected in the hind footpad and the tail base. Each injection
516 Drug Screening Methods

contains 50 µg of RW and 2 mg of alum. The test drug is administered intraperitoneally on

alternate days till day 8. RW in a dose of 2 mg in 10 µl of PBS per eye is instilled in the eyes of
mice on the tenth day. After the 24 h of ragweed challenge the conjunctivae are processed for
the evaluation of histological changes and other parameters as needed in the study.7
An experimental model of allergic conjunctivitis to ragweed was produced in guinea pigs
by Merayo-Lloves et al. in 1995.8 The model mimics human hay fever conjunctivitis and helps
in evaluating the reponse of conjunctivitis to various therapeutic approaches.8 Merayo-Lloves
et al. in 1996 exposed SWR/J mice to ragweed by topical contact with the conjunctival and
nasal mucosas. Serum IgE levels and histopathological changes, infiltration of conjunctival
eosinophils, change in number of mast cells, cytokine release is evaluated.9
Japanese cedar pollen: It has been used by Yasuda et al. (2004) for developing allergic
conjunctivitis in guinea pigs.10 The method is useful in analyzing the mechanism of allergic
conjunctivitis.
Procedure: Small gelatin sponge pieces containing pollen extracts and aluminum hydroxide
are inserted into the palpebra superior and/inferior sulci of both eyes of Male Hartley guinea
pigs for 8 h/day for 6 days. Three pieces are inserted in each eye. After six days the guinea pigs
are challenged once a week by instilling pollen suspension in each eye.10

Rabbit Model of Staphylococcus aureus Keratitis

Oguz et al. (2005) tested a broad-spectrum nonantibiotic antimicrobial agent against bacterial
keratitis.11 They used rabbit model of Staphylococcus aureus keratitis for the study and compared
the efficacy of the test compound with topical ciprofloxacin, ofloxacin, and 5% cefazolin.
Procedure: One of the corneas of the rabbits is intrastromally injected with 100 colony-forming
units of Staphylococcus aureus ATCC strain 25923. The animals are divided into different group
as per the need of the experiment. The eyes are instilled with the test drug every 30 min from
4 to 9 h post injection. At 10 h post injection signs of inflammation are scored by slit-lamp
examination. Then, the corneas are processed for further evaluation. The number of colony-
forming units per cornea in all eyes is also calculated.11

Endotoxin-induced Uveitis (EIU)

This is a model for acute anterior uveitis in human beings. Lipopolysaccharide (LPS) is
a glycolipid component of the outer membrane of Gram-negative bacteria. It induces a
generalized proinflammatory response during infection. Systemic administration of sublethal
dose of LPS produces bilateral acute ocular inflammationin rats and mice. Maximum effect of
EIU is seen in 24 h of LPS injection and wanes out in the next 48 h. Percolation of proteins from
the serum and infiltration with macrophages and neutrophils into the eye is the characteristic
feature of EIU.12

In Rabbits
LPS induced uveitis in rabbits has been used by several workers for testing anti-inflammatory
response of the drugs (Fig. 34.1).12-17 The mechanism of EIU is not clearly known, but the role
of cytokines has been clearly mentioned.18,19 Cytokines are signaling proteins released by cells
 Ocular Inflammation 517

Figure 34.1: Clinical signs of anterior uveitis in control group: (A) Just before intravitreal endotoxin injection; (B) 24 hours
post intravitreal endotoxin injection; (C) 72 hours post-intravitreal endotoxin injection.
[Courtesy: Researchers of Ocular Pharmacology Laboratory, Delhi Institute of Pharmaceutical Sciences and Research,
New Delhi.]

and act as important mediators. The upregulation of cytokines such as tumor necrosis factor
(TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and macrophage
inflammatory protein (MIP)-2 has been reported in rabbits, rat and mice models of EIU.12
Procedure: New Zealand male rabbits weighing between 1.5 and 2.0 kg are used for inducing
uveitis. Rabbits are anesthetized using proparacaine HCl topically. E. coli endotoxin (LPS) is
dissolved in sterile physiological saline solution at a concentration of 10 ng/µl. Intravitreal
injection of 10 µl (100 ng) of the LPS is given into both eyes of each rabbit using a 30 G needle
attached to a Hamilton constant range syringe. The severity of the inflammation is compared
in the treated group with their own vehicle treated control group, as the grade of uveitis is
known to vary from one experiment to another. The test drug is administered to rabbits one h
before intravitreal injection of endotoxin. Within 24 h time maximal inflammation occurs and
wanes out in 48 h. The rabbits are euthanized 24 h after the LPS injection with an overdose of
pentobarbital sodium and immediately aqueous humor is withdrawn by paracentesis using
30 G needle. To dissect the ciliary body the eyes are enucleated and dissected around the
equator. Various parameters can be evaluated in aqueous humor and ciliary body.13
Burgundy Fawn rabbits of 2.5 to 3.9 kg weight range are also used using ketamine and same
dose of LPS as mentioned above.14
Liang et al. (2006) used two new tools to assess the ocular inflammation induced by LPS.16
They used Confocal Microscopy for the evaluation of inflammatory infiltrates and conjunctival
impression cytology for assessing TNF alpha and TNF receptor-1 expression.

In Rats
In endotoxin induced uveitis in Lewis rats mainly anterior segment (iridocyclitis) is inflamed
and the inflammatory cells get into the vitreous humor and retina.12,20 Lewis eight-week-old
rats weighing between 180 to 220 g are used. LPS from Salmonella typhimurium is diluted
in sterile saline and 200 µg/0.1 ml is injected in one of the footpads of the rats to induce the
uveitis. Test drug is given immediately after LPS injection in the test group whereas vehicle is
administered in the control group.
518 Drug Screening Methods

Satofuka et al. (2006) produced EIU in Long Evans rats by a single intraperitoneal injection
of 100 µg LPS.21

In Mice
In order to induce EIU Ohta et al. injected 200 µg LPS from Salmonella typhimurium in PBS in
footpads of C3H/HeN mice of 8-10 weeks. An acute intraocular inflammation is induced that
peaks within 24 h and dissipates by 48 h.15

Autoimmune Uveitis
Experimental autoimmune uveitis (EAU) is a model of idiopathic human uveitis.1 EAU is
produced against S-antigen, a major protein on retinal photoreceptor cell, interphotoreceptor
retinoid binding protein (IRBP) like retinal proteins in susceptible strains of rats, mice and
subhuman primates.15, 22-25

Interphotoreceptor Retinoid-Binding Protein-induced Uveitis

A method for inducing experimental autoimmune uveitis by Ohta et al. is described briefly.
Procedure: B10.A mice are immunized (s.c) with 50 µg interphotoreceptor retinoid-
binding protein (IRBP) in 0.2 ml emulsion mixed in a ratio of 1:1 with complete Freund’s
adjuvant supplemented with Mycobacterium tuberculosis to a final concentration of 2.5 mg/
ml. Simultaneously, 500 ng of pertussis toxin is injected intraperitoneally to the mice as an
additional adjuvant. Aqueous humor is collected at different time points and assayed for
leukocyte content and the ability to suppress or enhance T-cell proliferation. Inflammation in
the anterior segment is detected after 10 days.15
Fox et al. (1987) compared the inflammatory response produced by IRBP and S-antigen.
They immunized Lewis rats with IRBP and found that the response was more compared
to S-antigen in lower doses (less than or equal to 4 µg/rat) however, uveitis produced by
S-antigen at higher doses (greater than or equal to 20 µg/rat) was more severe in comparison
to the inflammation produced by same dose of IRBP. They also observed that the rats differ in
susceptibility when immunized with these two proteins.22
Ke et al. (2007) induced autoimmune uveitis in C57BL/6 mice by transfer of activated T-cells
specific for the IRBP 1-20 peptide. Disease onset occurs at 10 days. The test compound can be
given at 0 or 10th day. Clinical signs, ocular histology and infiltrated inflammatory cells in the
eye are compared.26

Bovine Serum Albumin-induced Uveitis in Guinea Pigs

Ultrastructural changes and leptin expressions in the guinea pig eyes were studied by Kükner,
et al. in 2006.27 Retinas of uveitis induced group when observed under light and electron
microscope showed oedematous ganglion cells and increased thickness of inner plexiform
layer. They reported that leptin expressions are closely related to ocular inflammation. They
also observed the effect of intraperitoneal vitamin E, melatonin and aprotinin on leptin
expression.
 Ocular Inflammation 519

Procedure: Male guinea pigs are divided into control and treated groups and are injected
intravitreally with bovine serum albumin (BSA) to produce experimental uveitis. On the
third day, the test group is given the test drugs and on the sixth day, the clinical scoring of the
inflammation produced and histopathological examination is done. Leptin expressions are
evaluated in retina, choroids, sclera and episclera. 27

Melanin-induced Uveitis
A number of researchers have used Experimental melanin-protein-induced uveitis (EMIU)
also known as experimental autoimmune anterior uveitis (EAAU) model induced by melanin
granules extracted from bovine choroids, iris, hair and skin, and from human, monkey and
rabbit choroids.28-32
Procedure: Susceptible strains of rats (Lewis, Fischer 34, Porton rats) of 10 weeks are housed
at 21°C and 50% humidity in a 12 h light and 12 h dark cycle, and fed water and dried feed.
Melanin is extracted from bovine choroids as per the procedure of Broekhuyse et al. (1993).29
Susceptible Lewis rats are immunized with bovine ocular melanin with a dose of 250 µg, which
induced maximally severe disease in all injected animals in about 10 days of treatment. Rats
are given 125 µg of bovine ocular melanin in a 1:1 emulsion of sterile, non-pyrogenic normal
saline and Hunter’s TitreMax adjuvant by right hind footpad injection (60 µl). Immediately
afterwards, they are injected intraperitoneally with the same quantity of melanin mixed with
1 µg of pertussis toxin in normal saline (40 µl). Animals are examined daily through slit-lamp
biomicroscope for clinical signs of uveitis, and are scored using a clinical scoring system.33
Inflammatory cellular infiltrates into the iris, ciliary body, and the anterior chamber show
predominance of CD4+ T-cells, monocytes/macrophages, and neutrophils.34

Myelin Basic Protein-induced Anterior Uveitis

Adamus et al. (1998) induced experimental autoimmune encephalomyelitis (EAE) with myelin
basic proteins (MBP). EAE is an acute CD4+ T-cell mediated inflammatory disease of the central
nervous system. It is an animal model for multiple sclerosis (MS) and is induced in susceptible
animals by a number of myelin antigens including MBP.34-35 Lewis rats are immunized with
MBP or certain peptides of MBP with adjuvant and develop a self-limited form of AAU along
with EAE. MBP-induced EAU persists for more than 11 days post immunization even after
clinical signs of EAE subsides, with spontaneous AAU remission occurring about 30 days after
immunization. The inflammatory cellular infiltrates accumulate around anterior surface of
iris, trabecular meshwork and in some cases within the ciliary body and aqueous humor.34-36
Kuchroo et al. (1991) inducted experimental allergic encephalomyelitis by myelin
proteolipid-protein-(PLP) specific T-cell clones and synthetic peptides in SJL(H-2s) mice.37

T-Helper Lymphocytes Type 2-induced Ocular Inflammation

Ocular inflammation is often mediated by T-helper (Th) lymphocytes. These are divided into
two Th1 and Th2, and differ in their cytokine production and biological activities. Kim et al.
(2002) induced inflammation using T-helper lymphocytes Type 2. They examined the capacity
of Th1 and Th2 cells to induce ocular inflammation.38
520 Drug Screening Methods

Procedure: Transgenic (Tg) mice are used for induction of ocular inflammation. These mice
express hen egg lysozyme (HEL) in their lens, by adoptively transferring Th cells, which
transgenically express HEL-specific receptor. Th1 and Th2 populations are polarized in vitro,
and their selective cytokine production is checked by RT-PCR. Conventional histological
methods are used for monitoring the inflammation.38

Inflammation-induced by Topical Irritants

Topical irritants like carageenan, nitrogenized mustard, Freund’s adjuvant, croton oil, etc.
have been used in establishing a model of ocular inflammation. The model fulfills most of the
criteria sought by the researchers such as the induction of pathological inflammatory changes
in the eyes to study inflammatory activity that can be quantified using standard methods such
as estimation of edema, inflammation, mediators, inflammatory cells, etc. The model is easily
standardized and reproduced.37

Croton Oil-induced Uveitis

Rabbits are instilled with 3% croton oil. Croton oil is dissolved in 2-ethoxyethanol and
40 µl (single drop) is instilled in the cornea. The model probably involves the activation of
arachidonic acid pathway and breakdown of blood-aqueous barrier and permits the entry of
high molecular weight proteins in aqueous humor.39
Villena et al. also induced inflammation by applying 80 µl (40 µl at the interval of 5 min) of
0.1 and 0.2% carageenan using 1% carboxymethyl cellulose and 1% between 80 and; 120 µl (40
µl at 5 min interval) Freund’s adjuvant.39

Schistosoma mansoni Model of Ocular Inflammation

The model has an important role in screening of the chemotherapeutic agents for suppressing
the granulomatous uveitis and understanding the cellular interaction in the formation
of granuloma. The role of immunologic factors in ocular defense mechanism can also be
understood.40
Procedure: Golden Syrian hamsters are injected with 200 cercariae of Schistosoma mansoni.
After 8 weeks the eggs are recovered from the livers by trypsin digestion and sieving. Eggs are
counted and transferred to the tuberculin syringes. New Zealand rabbits of 2.0 kg weight are
anesthetized. S. mansoni eggs are injected in the eye through pars plana carefully so that the
lens and retina is not damaged. The eggs are injected in various concentrations. After 5 days
an inflammatory response involving vitreous, choroids, retina, and optic nerve is seen which
depends on the number of eggs injected. 40

Laser-induced Ocular Inflammation

Several workers have induced ocular inflammation in rabbits, rats, mice, primates using
laser. Apart from changes in the IOP the injury due to laser produces ocular inflammation.
Laser-induced ocular hypertension model can also be used as ocular inflammation model.41
Laser treatment to the iris of rabbits causes miosis, a rise in intraocular pressure and an
increase in the protein content of the aqueous humor. These effects on IOP and the blood-
 Ocular Inflammation 521

Figure 34.2: Laser treatment in pigmented rabbit eyes: (A) Normal eye (B) post-laser with hyphema (Courtesy: Researchers
of Ocular Pharmacology Laboratory, Delhi Institute of Pharmaceutical Sciences and Research, New Delhi)

aqueous barrier are to some extent due to the release of prostaglandins of the E type and
are reduced by pretreatment with prostaglandin synthetase inhibitors. The miosis and part
of the rise in intraocular pressure and breakdown of the blood-aqueous barrier may also be
due to antidromic stimulation of sensory nerves. A combination of indomethacin and local
anesthetic helps to block the miosis and elevation in IOP and greatly reduces the increased
protein content of the aqueous humor. The human eye may react in a similar way to laser
irradiation of the iris.42
Gherezghiher and Koss (1989) applied argon laser of 0.75 watts, 0.5 sec duration and 8 spots
of 500-micron size to the iris of pigmented rabbits. This resulted in acute rise in IOP and miosis
together with an increase in aqueous protein concentration. The response lasted for three
days.41
Neodymium-yttrium aluminum garnet (Nd-YAG) laser was used by Joo and Kim (1992) to
induce ocular changes like prostaglandin E, protein, pupil diameter, and intraocular pressure
by photo disruption of pigmented rabbit iris.43 Figure 34.2 shows post laser hyphema in
pigmented rabbits treated with the Nd-Yag laser for producing hypertension.
Zhou et al. (2005) induced choroidal neovascularization by laser photocoagulation in adult
male C57BL/6J mice. A brief procedure used by them is given below.44
C57BL/6 male mice between 6 to 8 weeks old are fed standard laboratory chow and
maintained in a 12 h light 12 h dark environment. The mice are anesthetized with ketamine
(80 mg/kg) and xylazine (8 mg/kg), and pupils are dilated with topical 1% tropicamide. Both
eyes of mice are photocoagulated by Diode laser (75 µm spot size, 0.1 s duration, 140 mW). They
evaluated neutrophil infiltration and showed that post-laser treatment neutrophils infiltrated
the sites of laser injury on the day 1 and peaked at day 3.44

Endophthalmitis
Inflammation of the ocular cavities and their adjacent structures is defined as endophthalmitis.
It may occur as a complication of intraocular surgeries and can lead to loss of vision or
sometimes enucleation of the eye. One of the most common causes of this disease is bacterial
or fungal infection. Trauma and foreign bodies may also result in endophthalmitis. The
522 Drug Screening Methods

symptoms usually are pain; redness of conjunctiva and episclera and sometimes hypopyon is
also present. Some of the fungi and bacteria induced experimental models, used by researchers
for evaluating the therapeutic potential of the drugs or understanding the mechanism, are
described here.

Candida albicans Induced Endophthalmitis

With candida endophthalmitis becoming common Demant and Easterbrook (1977) produced
an experimental rabbit model of endophthalmitis to understand whether a clinical lesion can
be related to the number of organisms present in it as the drugs required to treat this condition
are very toxic and need to be stopped as early as possible. The model produced by them gave
reproducible results making it a convenient model for experimental endophthalmitis.45
Procedure: A suspension of Candida albicans is injected in rabbits intravenously, which
produced typical ocular lesions in all of them. The rabbit fundus is examined by indirect
ophthalmoscopy. As per the requirement the rabbits are killed to prepare quantitative cultures
of their eyes.45
In a recent study, Kusbeci et al. (2007) injected New Zealand white rabbits by inoculation of
1 × 104 colony forming units (CFU)/ml of C. albicans. The test drug is injected in the vitreous
after seventy-two h of inoculation. Differences in the clinical scores and histopathological
scores and mean CFU/ml between the control and test group are evaluated.46

Fusarium solani-induced Endophthalmitis

Mayayo et al. (1998) induced inflammation in immunocompetent mice by Fusarium solani
to produce an experimental model, which can be used for evaluating the efficacy of various
treatments and can establish the pathogenecity of the fungus in eye. 47
Procedure: Isolates of F. solani are injected in the lateral tail vein of immunocompetent mice
to produce systemic infection along with ocular infection. Each mouse is injected with 5 × 106
conidia.
The study of Mayayo et al. showed that out of the 20 F. solani injected mice 16 developed
panophthalmitis, four had bilateral infection and 34% showed presence of fungal infection.47

Bacteria-induced Endophthalmitis
Bacterial infections lead to endophthalmitis with tissue destruction and also produce host
inflammatory response. Inflammatory cells infiltrate to the site of infection mediated by
adhesion molecules expressed on the surface of endothelial and inflammatory cells. In order
to understand the mechanism of infiltration of the inflammatory cells, the model is produced
and described by Giese et al.48-50
Procedure: Female Lewis rats of 8-10 weeks are used. One of the eyes of rats is intravitreally
injected with 25 µl of S. aureus. Controls can be given normal saline. To confirm the number of
S. aureus injected into the eye 25 µl of S. aureus suspension is added to rabbit and sheep blood
agar plates and kept under incubation at 37°C for 24 h. The maximum inflammatory response
occurs at 24 to 48 h after injection and then reduces.49
 Ocular Inflammation 523

Kowalaski et al. (2005) and Deng et al. (2006) induced inflammation using Staphylococcus
species in rabbits. The inflammation thus produced was reproducible.51,52
Meyers-Elliott and Dethlefs (1982) injected Klebsiella oxytoca in the vitreous of rabbits
to produce a model of endophthalmitis with anterior segment inflammation. They
observed polymorphonuclear leucocytes at corneal limbus, adjacent to endothelium, in
iris and ciliary body, vitreous and optic nerve within 24 h. Within 48 h retinal photoreceptor
degeneration was seen. Mononuclear cells were observed in the vitreous in 72 h.53
Davey et al. 1987 also induced endophthalmitis in rabbits using a different species of Klebsiella,
i.e. K. pneumoniae and Pseudomonas aeruginosa. Rabbits were injected intravitreally with
either of the organisms. The injection containing 500 colony-forming units of organisms in 0.1
ml of saline were given to produce unilateral inflammation.54
Experimental animal models of ocular inflammation are contributing a lot in understanding
the mechanism of the ocular inflammations in humans and the efficacy of newer therapeutic
agents though researches in this direction are still going on. There are numerous models of
inflammation of which only few have been described here. Models using different fungal or
bacterial strains have been produced to evaluate antibacterial or antifungal agents for treating
the particular disease condition. Corneal transplant models and several other eye irritants
or allergens are also used for producing the inflammatory responses in the experimental
animals.55

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Jun 28.
 cHAPTER

35
Antiulcer Agents
Introduction
The term peptic ulcer refers to a spectrum of disorders that includes gastric ulcers, duodenal
ulcers and postoperative ulcers at or near the site of surgical gastrointestinal anastomosis. There
are two types of duodenal ulcers – acute and chronic duodenal ulcers. Pathogenesis of peptic
ulcers involves disturbances in the acid-pepsin status of the gastric contents and infection with
H. pylori. Neutralization of gastric acid by antacids or inhibition of acid secretion by drugs like
H2 receptor blockers, proton pump (H+ K+-ATPase) inhibitors, cytoprotective prostaglandin
analogs and various other drugs are the main modes of pharmacological treatment of peptic
ulcers. Due to the high morbidity associated with this disease, there is a continuous need for
newer anti-ulcer drugs. Screening models of antiulcer agents have been reviewed recently by
many investigators.1,2 This chapter further updates various methods used for the screening of
potential anti-ulcer agents. Before the selection of experimental ulcer model, it is desirable
that the following requirements are met.1
1. They should be simple, reproducible and allow for easy quantification of results.
2. They should make use of a variety of animal species.
3. They should induce characteristic ulceration in specific locations (stomach and duodenum).
4. They should involve different mechanism by which ulceration is produced.
5. The ulcers induced should not spontaneously heal during the observation period.
Apart from above requirements the selected model
6. Should also be cost effective and
7. Model should not be time consuming.

IN VITRO METHODS
[125I] Gastrin Binding Assay
Gastrin is one of the major stimuli for gastric acid secretion. Its release is also triggered by
partially digested proteins, peptides, blood borne factors and by vagal stimulation.3 After
release from gastric antrum, it stimulates acid secretion by binding to its receptors on parietal
cells as well as by releasing histamine from enterochromaffin-like cells. Compounds with
gastrin receptor antagonistic activity can prove to be useful antiulcer drugs. [125I] Gastrin is
used for radioligand binding assay for gastrin receptors. Proglumide like drugs, which are a
CCKR antagonists, may also be screened using this assay.4
528 Drug Screening Methods

Procedure: The assay is done using fundic gland suspension obtained from guinea
pig stomach. For the binding and competition assays, the gland suspension is incubated
with 50 µl of [125I] gastrin in the presence of either buffer alone (for total binding) or in the
presence of unlabeled gastrin (for non-specific binding) or in the presence of test compound
for 90 min at 37°C. Subsequently, ice cold buffer, in microcentrifuge tubes, is layered with
incubated mixture and centrifuged for 5 min at 10,000 g. Radioactivity is quantified in pellet
after discarding the supernatant.
Evaluation: Total binding, non-specific binding and specific binding are determined.
Percentage of specifically bound [125I] gastrin displaced by a given concentration of the test
compound is calculated and its IC50 and dissociation constant (Ki) values are calculated.5-7

[3H] Tiotidine Binding Assay for Histamine H2 Receptors

Histamine H2 receptor blockers have been the mainstay of anti-ulcer therapy since the early
1970s due to their potent acid-suppressing properties. Tiotidine is an H2 receptor blocker that
is used for H2 receptor binding assay.
Procedure: The assay is done using cerebral cortex homogenate obtained from male White
leghorn chicks or from guinea pigs. The cerebral cortex homogenate is incubated with
[3H] tiotidine for 90 min at 4°C in the presence of Na2HPO4/KH2PO4 buffer (pH 7.4) alone to
determine total binding or in the presence of unlabeled ranitidine and buffer to determine
non-specific binding or in the presence of test compound in buffer for competition assay. 5 ml
of ice-cold phosphate buffer is added to terminate the incubation. Subsequently the reaction
mixture is filtered under vacuum through glass fiber filters that are pre-soaked with buffer.
Filters are then washed with 5 ml of ice-cold buffer twice and radioactivity measured by liquid
scintillation counting.8,9
Evaluation: Specific binding, i.e. total binding minus non-specific binding and IC50 are
determined.
In another study, the [3H]-tiotidine has also been used as a specific ligand for H2 receptors
in dispersed mucosal cells from guinea pig stomach and it was found to have limited binding
to H2 receptors and as such [3H]-tiotidine was not a suitable ligand for labelling the H2-receptor
on gastric mucosal cells.10

H+/K+- ATPase Inhibition Assay

H+/K+- ATPase or proton pump is the final step in the synthesis of acid by parietal cells. It
exchanges intracellular H+ with extracellular K+ in the canaliculi of parietal cells in response to
stimulation by all gastric acid secretagogues, i.e. histamine, acetylcholine and gastrin. Proton
pump inhibitors like omeprazole, lansoprazole, etc. are now established antiulcer drugs.
Procedure: The assay is carried out using homogenates of microsomal gastric H+/K+-
ATPase obtained from pig gastric mucosa.11 For inhibition assay, 80 ng microsomal H+/K+-
ATPase is incubated with 100 µl buffer (pH 7.4), 1 mM ATP and test compound in microtitre
plate for 30 min at 37°C. After 30 min of incubation, the reaction is stopped by adding
malachite green colorimetric reagent and then after 10 sec 15% sodium citrate is added for 45
min. Release of orthophosphate from ATP is quantified by colorimeter at 570 nm. For study
 Antiulcer Agents 529

of omeprazole and other proton pump inhibitors available at present, which produce active
metabolite at acidic pH, the microsomal homogenate is initially suspended in buffer at pH 6.1
along with the drug and incubated for 30 min. This homogenate is then transferred to buffer
at 7.4 and the procedure described above is followed. Percentage inhibition of H+/K+– ATPase
is calculated.12

Percentage of enzyme inhibition is calculated using the formula:

[Activity (control) – Activity (test)]
% Inhibition = × 100
Activity (control)

IN VIVO METHODS
Pylorus Ligation in Rats
This procedure was described by Shay et al.13 The basis for this model is that accumulation of
unbuffered gastric juice over a certain length of time leads to peptic ulceration in rats whose
pylorus has been ligated.
Procedure: Wistar rats (150 to 180 g) are used for the experiment. The animals are fasted for 48
hours before the operative procedure. However they are given free access to water ad libitum.
To prevent cannibalism and coprophagy, the animals are housed singly in cages with raised
bottoms of wide wire mesh. Under ether anesthesia, a one-inch midline abdominal incision
is given below the xiphoid process. The pylorus is carefully lifted out with minimal handling
and traction and ligated without damaging its blood supply. The stomach is now replaced and
the abdominal wall closed with sutures. The test compound is administered either orally or
subcutaneously and the animals are placed in plastic cylinders. About 17-19 h after pyloric
ligation, the animals are sacrificed and the stomach dissected out. The contents of the stomach
are drained into a graduated centrifuge tube and their acidity determined by titration with 0.1
N NaOH. The stomach is opened along its greater curvature, pinned on a cork plate and inner
surface examined for ulceration with a binocular microscope. The ulcer index is calculated
and the ulcer severity graded as mentioned below.
Ulcer severity is graded as:
0—No ulcer, 1—Superficial ulcer, 2—Deep ulcer and 3—Perforation

The ulcer index (UI) is calculated by the following equation:

UI = UN + US + UP × 10–1

where, UN = Average of number of ulcers/animal; US = Average of severity scores; UP = Percentage

of animals with ulcers.
Ulcer index and acidity of the gastric content of the treated animals are compared with the
control.
Aspirin alone at a dose of 150 mg/kg p.o. can induce ulcers in rats.14 Aspirin plus pylorus
ligation induced gastric ulcers in rats have also been successful to study the antiulcerogenic
effects of drugs.15
530 Drug Screening Methods

Stress Ulcer Models

Stress plays a significant role in the pathogenesis of gastric ulcers in human being. However,
the recent studies suggest no harmful effect of chronic stress on gastric ulceration.16 Selye
(1936), for the first time, described the use of restraint for the production of gastric ulcers.17
Various models involving various types of stress have since been developed. The involvement
of psychogenic factors and the ease of production of gastric ulcers using these models is a real
advantage over the pyloric ligation method of gastric ulcer production.

Restraint-induced Ulcers
Procedure: Albino rats, of either sex, weighing 150 to 200 g, are used for the study. After 36
hours of fasting, the test drug is administered. Thirty minutes later, the animals are subjected
to restraint by molding a special galvanized steel window screen around the animal and
tying the limbs of the animal in pair so that the animal cannot move. The animals are kept
under restraint for 24 h. The animals are then sacrificed and their stomachs dissected out. The
stomachs are opened along greater curvature and fixed to cork plate.18 Ulcer index and ulcer
severity are determined as described in the pyloric ligation method.

Cold Water Immersion-induced Ulcers

It has been observed that when the restrained animals are subjected to additional cold
water immersion, the occurrence of gastric ulcers is accelerated and this also shortens the
immobilization time.19,20
Procedure: Wistar rats (150 to 200 g) are used for the experiments. After fasting the animal
for 16 hours, the test compound is administered orally. The animals are placed individually in
restraint cages vertically and then immersed in water at 22°C for 1 h. Azovan blue (Evan’s blue),
in a dose of 30 mg/kg, is injected intravenously via the tail vein after removal of the rats from
the cage. They are sacrificed 10 min later. The stomach is removed and ligated at both ends. It
is filled with formol saline and kept overnight. On the next day, the stomach is opened along
the greater curvature, washed in warm water and examined for ulcerative lesions. Evan’s blue
helps in evaluation of the lesion score, which is calculated by adding the lengths of the longest
diameters of the lesions.20 Gastric mucosal lesions can also be induced by 2 h of cold restraint
stress in rats.21
Abdel-Sater KA et al,22 2012 have used acute cold restraint stress by fixing the four limbs of
the rat and placing it in a refrigerator at 4oC for 3 h to study the gender difference of selective
serotonin reuptake inhibitors, fluoxetine. Stressed male rats were found to be more responsive
to the antiulcer effect of fluoxetine more than stressed females.

Stress and NSAIDs-induced Ulcers

Procedure: Wistar rats (150 to 200 g) are used for the experiment. The animals are fasted for
24-36 h and then given the test agent (in 1% carboxymethyl cellulose) via gastric intubation
and an NSAID such as aspirin, indomethacin or diclofenac intraperitoneally. After placing the
rats in stress cages, they are immersed in water up to the level of xiphoid process at 23°C for
7 h. The animals are then sacrificed, their stomach removed and evaluated for ulcer index.
 Antiulcer Agents 531

The dose of NSAID required to increase gastric erosion by 100% relative to immobilization is
compared with that of NSAID required to produce 100% increase in gastric erosion under the
protective effect of test drug.23

Swimming Stress Ulcers

Procedure: Albino rats of either sex are fasted for 24 h with free access to water. The rats
are forced to swim in a deep concrete tube filled with water at 23°C for 5 h. The animals are
removed from the tube after five hours, sacrificed and their stomachs removed. The stomachs
are opened along the greater curvature and severity grading is done and ulcer index calculated.
Severity of the ulcerative lesions is graded as follow:
0—no lesions, 1—lesions with diameter less than 1 mm, 2—lesions with diameter 1-2 mm,
3—lesions with diameter 2-4 mm, and 4—lesions with diameter more than 4 mm.
Ulcer index is calculated by summation of scores for individual erosions and ulcers.24

Histamine-induced Gastric Ulcers

Histamine has been used widely for the production of gastric ulcers. Enhanced gastric
acid secretion has been implicated in the production of ulcers due to histamine.25
Procedure: Male guinea pigs, weighing 300 to 400 g, are used for the experiment. The animals
are fasted for 36 h with water available ad libitum. Histamine acid sulphate is injected in a
dose of 50 mg intraperitoneally. To prevent histamine toxicity, promethazine hydrochloride
is injected intraperitoneally 15 min before and 15 min after the histamine injection in a dose
of 5 mg. The test drug is administered 30-45 min before the histamine injection. Four hours
after the histamine injection, the animals are sacrificed and their stomachs dissected out. The
degree of ulceration grading is done as follows:
•• Type 0: No visible ulceration on gross examination
•• Type 1: 1-3 mm ulcers, ten or less in number
•• Type 2: 1-3 mm ulcers, eleven or more in number
•• Type 3: 4-6 mm ulcers, one or more in number
•• Type 4: 7 mm or bigger ulcers, one or more in number
•• Type 5: Gastric or esophageal wall perforation.26
Other routes of administration of histamine such as intraperitoneal/intramuscular have
also been found useful.27 Repeated administration of histamine by intraperitoneal route
(0.09 mg/kg) or intramuscular route 0.09 mg/kg × 8 doses) has been found to selectively induce
gastric or duodenal ulcers demonstrating differential of histamine in ulcer induction.
Modification: Duodenal ulcers were also induced by repeated ip administration of histamine
acid phosphate at a dose of 0.25 mg/kg at every 30 min interval for 4 h after 45 min of test drug
administration.28
Apart from guinea pig, other animals such as rats can also be used to induce histamine gastric
ulcers. Histamine, intraperitoneal injection at a dose of 300 mg/kg induces gastric lesions.29

Methylene Blue-induced Ulcers

Methylene blue (MB), a synthetic drug has been used for the induction of duodenal and gastric
ulcers in rats. Methylene blue uncouples the ATPase enzyme and also possesses the affinity for
532 Drug Screening Methods

muscarinic receptors. The model is used for the screening of antiulcer agents involving H+/K+
ATPase system or via anticholinergic action exerted via muscarinic receptors.
Procedure: Methylene blue produces ulceration of gastric mucosa by reduction in blood
supply to gastric mucosal region that causes oxidative stress. Rats are fasted for 24 h before the
administration of MB. Animals are administered MB at a dosage of 5-125 mg/kg body weight
p.o. followed by the administration of the test drug. After 4 h the animals are sacrificed and
ulcer index is determined.30

Serotonin-induced Gastric Ulcers

Adinortey et al. 2013 have reviewed the serotonin (5-HT)-induced gastric ulcer model.
Serotonin produced local vasoconstriction and thus reduced the gastric mucosal blood supply,
resulting in local mucosal injury.2
Procedure: Rats were administered with a single dose of serotonin creatinine sulfate (0.5 ml
of 50 mg/kg subcutaneous injection) for the induction of glandular lesions after fasting for
24–36 h and water deprivation for 2 h before the experiments. They are housed in wide mesh
wire bottom to prevent coprophagy. Serotonin was administered by intragastric intubation
with the aid of an orogastric cannula. Six hours later, the animals are sacrificed by cervical
dislocation for examination.
Endogenous serotonin is also reported to play a dual role in the pathogenesis of indo­
methacin induced small intestinal ulceration in mice - proulcerogenic action via 5-HT3
receptor and antiulcerogenic action via 5-HT4 receptors.31

Ethanol-induced Mucosal Damage

Ethanol (absolute)-induced gastric lesion is a reproducible method in experimental animal.32
Using a transmission densitometer, it is possible to quantify the extent of gastric lesions induced
by ethanol, by measuring the optical density of photographic negatives of gastric mucosa.33
Procedure: Wistar rats, weighing 250 to 300 g, are used for the experiment. The animals are
placed individually in cylindrical stainless steel cages with flat bottoms to limit their mobility
and prevent coprophagy. They are fasted for 18 h but given water ad libitum. Now the test
drug or the vehicle is given to the animals orally. Thirty minutes later, 1 ml of absolute ethanol
is given orally. The animals are sacrificed 1 hour later and their stomachs dissected out. The
stomachs are opened along the greater curvature, washed with tap water and ulcer severity
grading done. Using a cork borer, 13 mm, full thickness circular patches are cut from each lobe
of the fundus below the ridge dividing glandular from non-glandular portion of the stomach
and placed into holes of a special template. Photographs of the tissues are taken and the
negatives examined under light transmission densitometer. Damaged areas have lower optical
density values.32,33
Absolute ethanol has also been used at higher dose of 5 ml/kg to induce gastric lesions in
rats. Animals were sacrified after 1 h and stomach opened to observe gastric lesions.34
Khazaei and Salehi,35 2006 have induced gastric ulcers in male albino rats by administering
ethanol 50% (in distilled water) at a dose of 10 ml/kg. One h after ethanol administration, rats
were killed and lesions produced in glandular part of stomach were measured/counted with a
graticules under stereo microscope and effects of test drug observed. Ethanol induced severe
gastric hemorrhagic erosions. It induced both long ulcers and petechial lesions. Decrease in
 Antiulcer Agents 533

gasric mucus and increased lipid peroxidation are the mechanisms suggested for ethanol
induced gastric ulcers.

Acetic Acid-induced Gastric Ulcers

This model produces chronic ulcers that resemble human ulcers and is used to screen drugs
for their gastroprotective effect in chronic gastric and duodenal ulcers. Spontaneous relapse of
healed ulcers > 100 days after ulceration is the characteristic feature of this model.36
Procedure: Albino rats are used for the experiment. A volume of 0.05 ml of acetic acid
(1-30%) is injected into the submucosal layer of the stomach, which results in the formation of
penetrating peptic ulcers that are confined by adhesions to contiguous organs like liver. They
are typically chronic ulcers with repeated healing and re-aggravation. 100% acetic acid can
also be applied to the serosal surface of stomach or duodenum for production of ulcers. Effect
of test drug given twice daily for 10-15 days is noted.37
Rabbit gastric ulcer models namely acetic acid induced and mucosal resection-induced
models are more clinically relevant models in terms of round, deep ulcers with clear-cut
margin and well-defined healing stages that are difficult to define in rat models.38
Qin and Chen,39 2005 have used modified method for induction of gastric ulcers. Gastric
ulcers were produced in male Sprague-Dawley rats by application of round filter paper
(diameter 5 mm) immersed in a 100% acetic acid on the serosal surface of the anterior wall of
the stomach approximately at the center of the corpus for 30 sec and the process was repeated
twice. Immediate production of necrosis of the entire mucosa and submucosa (but not serosa)
within the area (20 mm2) where the acetic acid applied, was observed. Excess of acetic acid was
then removed and serosa was gently washed with saline. The abdomen was then sutured and
the animals were allowed to recover and returned to their cages with free access to food and
water. The above method was found useful to study the synergistic action of famotidine and
chlorpheniramine on acetic acid-induced gastric ulcer in rats.

Indomethacin-induced Gastric Ulcers

Gastric ulcers may be induced by oral administration of indomethacin (25 mg/kg) after
24 h fasting male albino rats. Indomethacin is dissolved in sodium bicarbonate to form a
clear solution. Gastric ulcer can be examined after 4 h of indomethacin administration by
opening the stomach along the greater curvature, washed in normal saline to remove debris
and pinned on a cork mat for ulcer screening. This can be done by locating the wounds in the
glandular region under a simple microscope. The length (mm) of all the elongated black–red
lines parallel to long axis of the stomach in the mucosa is measured. Ulcer index is calculated
by adding the lengths of all the lesions in the glandular region of the stomach. NSAIDs are
believed to cause inhibition of COX and thereby inhibiting the production of cytoprotective
prostaglandins and causing gastrointestinal side effects. Indomethacin has shown to cause
oxidative stress also leading to formation of gastric ulcers.40

Reserpine-induced Chronic Ulcers

Histamine liberation from the mast cells in stomach wall with subsequent increase in gastric acid
secretion has been implicated in reserpine-induced gastric glandular ulcer formation in rats.41
534 Drug Screening Methods

Procedure: Female Sprague-Dawley rats, weighing 130 to 180 g, are used. The animals are fasted
for 48 hours with free access to 0.8% sucrose in 0.2% NaCl w/v. They are housed in wide mesh
wire bottom cages to prevent coprophagy. One h before starting the experiment, the liquid diet
is also withdrawn. Now the animals are injected with the test drug intraperitoneally and half an
hour later reserpine (5 mg/kg) or vehicle is injected intraperitoneally. Four h later, the animal
is sacrificed and stomach removed and examined for mucosal lesions.42 Reserpine dose (5 mg/
kg intraperitoneally, 18 h before sacrifice) has also been used by other investigators,43 and it
induced marked glandular ulceration with release of free β-glucuronidase.

Cysteamine-induced Duodenal Ulcers

Selye and Szabo44 first described the production of duodenal ulcers in rats by cysteamine
HCl (β-mercaptoethylamine HCl). The pathogenesis of cysteamine-induced duodenal ulcers
involves: inhibition of alkaline mucus production, increased gastric acid secretion, increased
serum gastrin levels and delayed gastric emptying. This model is widely used to evaluate the
anti-ulcer activity of anticholinergics, antacids, prostaglandins and H2 receptor antagonists.
Procedure: Female Sprague-Dawley rats are used. Cysteamine (10% in normal saline)
is administered in dose of 28 mg/100 g body weight, 3 times at intervals of 3.5 h orally or
20 mg/100 g body weight, twice at an interval of 4 h subcutaneously. The animals are
sacrificed 28 hours after the first dose in case of orally administered cysteamine and 40 h after
subcutaneous administration of cysteamine. Perforating duodenal ulcers are produced that
are located 2-4 mm from the pylorus, mainly on the anterior wall of the duodenum. Presence
of necrotic material and acute inflammatory response on the luminal layers of the crater are
characteristics of active ulcers. The ulcer and its features in test group are compared to those
in control group.
Acute duodenal ulcers can be induced in rats by administration of a single dose of
cysteamine hydrochloride (400 mg/kg p.o.). For induction of chronic type duodenal ulcers,
rats are administered with cysteamine (400 mg/kg p.o.) twice at an interval of 4 h followed by
addition of cysteamine hydrochloride to drinking water.45

Dimaprit-induced Duodenal Ulcers

Dimaprit, an H2 receptor agonist, has been shown to induce gastric erosions in rats after a single
intravenous dose and duodenal ulcers in guinea pigs after repeated subcutaneous doses.46 This
model is especially useful for screening of H2-blockers.
Procedure: Female Sprague-Dawley rats (150 to 180 g) or female guinea pigs (250 to 300 g) are
used for the experiments. The animals are fasted for 24 h before the experiment but allowed
free access to water. In rats, dimaprit is given in a dose of 100 mg/kg intravenously, single dose.
The animal is sacrificed one hour later and the stomach dissected out and examined for gastric
erosions. The test drug or vehicle is given orally 60 min before injecting dimaprit.
In case of guinea pigs, the animals are given multiple subcutaneous injections of dimaprit
(2 mg/kg every hour for 6 h). The test drug is given either 30 min before the first dose of
dimaprit or 30 min before and then hourly along with dimaprit. The animals are sacrificed
one hour after the last dimaprit injection and stomach and duodenum are examined for
lesions.46
 Antiulcer Agents 535

Other routes of administration of dimaprit such as intramuscular injection at a dose of 0.09

or 0.18 mg/kg body weight and intraperitoneal injection at a dose of 1.81 or 3.62 mg/kg have
also been used in guinea pigs for induction of duodenal ulcers. The intramuscular injection
produced particularly severe ulcers.27

Mepirizole-induced Duodenal Ulcers

Okabe et al. (1982) have described the production of duodenal ulcers in rats by mepirizole, a
non-steroidal anti-inflammatory drug. This model is a useful model for screening of antiulcer
drugs like antacids, anticholinergics and H2 receptor antagonists as mepirizole–induced
gastric secretions are inhibited by these drugs thereby important in study of pathogenesis of
duodenal ulcers.47
Procedure: Male Sprague-Dawley rats, 200-220 g in weight, are administered 200 mg/kg
of mepirizole suspended in 1% carboxymethyl cellulose solution via gastric intubation.
Subsequently rats are kept in cages with raised mesh bottom and deprived of food and water
for 24 h. This leads to ulceration in proximal duodenum and erosions in antrum. Antiulcer
drug therapy is started after 24 h of mepirizole administration. On the 11th day, the animals are
sacrificed and their duodenum and stomach evaluated for ulcer area under microscope. Ulcer
or erosion indices are calculated from the sum of area of ulcers and erosions, respectively.48
Duodenal ulcers can also be induced by subcutaneous administration of mepirizole at
doses of 60 and 200 mg/kg to increase acid secretion in a dose dependent manner within 8
hours.49

Gastric Mucosal Injury by Local Ischemia-Reperfusion in Rats

Gastric ischemia-reperfusion is an important model for studying acute gastric mucosal injury.
Procedure: Wistar rats, 180-200 g weight, are deprived of food for 24 h before experiments, but
allowed free access to tap water. The animals are anesthetized and their abdomen opened by
a midline incision. Celiac artery is identified and clamped by a microvascular clamp, 0.5 cm
from its origin, for 30 min to induce ischemia and then reperfusion for 60 min is performed by
removal of clamp. After completion of reperfusion, the animals are sacrificed by exsanguination
and stomach removed and lesions examined macroscopically and microscopically. Ulcer
index is then calculated.50-53
Modifications: Hassan et al. (1997) produced ischemia by clamping left gastric artery for 15
min and then reperfusion for 30 min before sacrificing the animals by cervical dislocation.
The stomachs were removed and opened along the greater curvature and photographed
for assessment of macroscopic mucosal injury planimetrically. Microscopic injury was also
assessed by staining a sample of corpus with hematoxylin and eosin, and examining under a
microscope.54
Mojzis et al.55 2000 induced gastric ischemia in male Wistar rats by 30 min clamping of
the coelic artery followed by 30 min of reperfusion and the mucus content, extent of gastric
lesions and length of lesions was determined at the end of ischemia/reperfusion. The method
has been used to evaluate the effects of sucralfate, malotilate, 0.5% methylcellulose and
N-acetylcysteine.
536 Drug Screening Methods

In Silico Model Based Approach for the Development of Antiulcer Antibiotics

Mandal and Das,56 2014 have developed an in silico based approach for the development of
antiulcer antibiotics. They targeted bacterial lysine biosynthetic pathway for antibacterial drug
development using inhibitors of H. pylori DapE-encoded N-succinyl–L,L-diaminopimetic
acid desuccinylase, an essential enzyme responsible for lysine synthesis in bacterial cell wall.
Procedure: The study used 3D structured model of DapE-encoded H. pylori having two
domains, one catalytic domain and other dimerization domain developed by MODELLER
software. After conformation of the stability of the model by GROMACS, the identification of
inhibitors of DapE, drug–like small molecule screening library was developed by Tanimoto
based Pubchem Database with DapE substrate L, L- SDAP as a query molecule followed by
docking approach by using GLIDE XP to identify the potential inhibitors of DapE which can be
used further in the development of novel antibacterial antiulcer drugs.

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5-HT3 receptor and antiulcerogenic action via 5-HT4 receptors. Pharmacol Res 2012;66(3):226-34.
32. Robert A, Nezamis JE, Lancaster C, et al. Cytoprotection by prostaglandins in rats: Prevention
of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury.
Gastroenterology 1979;77:433-43.
538 Drug Screening Methods

33. Witt CG, Will PC, Gaginella TS. Quantification of ethanol induced gastric mucosal injury by
transmission densitometery. J Pharmacol Methods 1985;3:109-16.
34. Arumugam S, Selvaraj SV, Velayutham S, Natesan SK, Palaniswamy K. Evaluation of anti-ulcer
activity of Samanea saman(Jacq)merr bark on ethanol and stress induced gastric lesions in albino
rats. Indian J Pharmacol 2011;43(5):586-90.
35. Khazaei M, Salehi H. Protective effect of Falcaria vulgaris extract on ethanol-induced gastric ulcer
in rat. Iranian J Pharmacol Ther 2006;5:43-6.
36. Okabe S, Amagase K. An overview of acetic acid ulcer models – the history and state of the art of
peptic ulcer research. Biol Pharmaceutical Bull 2005;28(8):1321-41.
37. Kimura M, Saziki R, Arai I, et al. Effect of 2’- carboxy-methoxy-4,4’-bis(3-methyl-2-butenyloxy)
chalcone (sofalcone) on chronic gastric ulcers in rats. Jpn J Pharmacol 1984;35:389-96.
38. Maeng JH, Lee E, Lee DH, Yan SG. Rabbit gastric ulcer models: Comparison and evaluation of acetic
acid-induced ulcer and mucosectomy-induced ulcer. Lab Anim Res 2013;29(2):96-102.
39. Qin Z, Chen C. Synergistic action of famotidine and chlorpheniramine on acetic acid induced
chronic gastric ulcer in rats. World J Gastroenterol 2005;11(45):7203-7.
40. Ajeigbe KO, Olaleye SB, Oladejo EO, Olayanju AO. Effect of folic acid supplementation on
oxidative gastric mucosa damage and acid secretory response in the rat. Indian J Pharmacol
2011;43(5): 578-81.
41. Lau HK, Ogle CW. The influence of cimetidine, a histamine H2-receptor antagonist, on the gastric
effects of reserpine in rats. Eur J Pharmacol 1981;70:139-48.
42. Kim KS, Shore PA. Mechanism of action of reserpine and insulin on gastric amines and gastric acid
secretion and the effect of monoamine oxidase inhibition. J Pharmacol Exp Ther 1963;141:321-5.
43. Pfeiffer CJ, Cho CH, Cheema A, Saltman D. Reserpine induced gastric ulcers: Protection by
lysosomal stabilization due to zinc. Eur J Pharmacol 1980;61(4):347-53.
44. Selye H, Szabo S. Experimental model for production of perforating duodenal ulcers by cysteamine
in the rat. Nature 1973;244:458-9.
45. Szabo S. Animal model of human disease. Duodenal ulcer disease : Cysteamine induced acute and
chronic duodenal ulcer in the rat. Am J Pathol 1978;73(1):273-76.
46. Del Soldato P, Ghiorzi A, Cereda E, et al. Cimetidine, ranitidine and mifentidine in specific gastric
and duodenal ulcer models. Pharmacology 1985;30:45-51.
47. Okabe S, Ishihara Y, Inoo H, et al. Mepirizole-induced duodenal ulcers in rats and their pathogenesis.
Dig Dis Sci 1982;27:242-9.
48. Ishihara Y, Okabe S. Effects of antiulcer agents on healing of mepirizole-induced duodenal ulcers in
rats. Digestion 1983;27:29-35.
49. Ueshima K, Takeuchi K, Ohuchi T, Okabe S. Acid secretory and duodenal ulcerogenic responses
induced by mepirizole in anaetetized rats. Digest Dis Sci 1994;39(8):1625-32.
50. Brzozowski T, Konturek PCh, Konturek SJ, et al. The role of melatonin and L-tryptophan in
prevention of acute gastic lesions induced by stress, ethanol, ischemia and aspirin. J Pineal Res
1997;23:79-89.
51. Ueda S, Yoshikawa T, Takahashi S, et al. Role of free radical and lipid peroxidation in gastric mucosal
injury induced by ischemia-reperfusion in rats. Scand J Gastroenterol Suppl 1989;162:55-8.
52. De La Lastra CA, Cabeza J, Motilva V, et al. Melatonin protects against gastric ischemia-reperfusion
injury in rats. J Pineal Res 1997;23:47-52.
53. Wada K, Kamisaki Y, Kitano M, et al. Protective effect of cystathionine on acute gastric mucosal
injury induced by ischemia-reperfusion in rats. Eur J Pharmacol 1995;294:377-82.
 Antiulcer Agents 539

54. Hassan M, Kashimura H, Matsumaru K, et al. Gastric mucosal injury induced by local ischemia-
reperfusion in rats. Role of endogenous endothelin-1 and free radicals. Dig Dis Sci 1997;42:1375-80.
55. Mojzis J, Hegedusova R, Mirossay L. Role of mucus in ischemia/reperfusion-induced gastric
mucosal injury in rats. Physiol Res 2000;49:441-6.
56. Mandal RS, Das S. In silico approach towards identification of potential inhibitors of Helicobacter
pylori DapE. J Biomol Struct Dyn 2014;9:1-14.
 cHAPTER

36
Agents Affecting Gut Motility
INTRODUCTION
The change in absorptive, secretory and motor functions of the gut can undermine human
well-being. Appropriate motility along the gut plays a significant role in the absorption of
nutrients and water across the gastrointestinal tract. Drugs can stimulate or reduce intestinal
motility and thus alter the transit time of compounds across intestine and thereby hinder or
stimulate absorption. Following are some of the models most commonly employed to study
the effect of drugs on intestinal motility.

IN VITRO MODELS

[125I]CCK Receptor Binding Assay

Cholecystokinin (CCK) is an important hormone regulating gastric emptying, intestinal
motility and biliary and pancreatic secretions. Dexloxiglumide, a CCK-A receptor antagonist,
is in clinical development phase for the therapy of gastroparesis and constipation-dominant
irritable bowel syndrome.
Procedure: [125I]CCK receptor binding assay is performed using rat pancreatic membrane
suspension.1,2 The membrane suspension is incubated with 40 pM of [125I]CCK in presence of
buffer alone (for total binding) or in presence of CCK (for non specific binding) or in presence
of test compound (for competitive assay) for 40 min at 25°C. Subsequently, ice cold buffer
supplemented with 0.5% bovine serum albumin, in microcentrifuge tubes, is layered with
the incubated mixture and centrifuged for 5 min at 10,000 g. Radioactivity is determined in
γ−scintillation counter.
Evaluation: Specific binding, i.e. radioactivity that can be displaced by a high concentration of
unlabelled CCK is calculated by taking difference between the total binding and nonspecific
binding. IC50 value is further calculated by computer-derived linear regression analysis.

[3H]GR-113808 Binding Assay for 5-HT4 Receptors

5-HT is one of the major transmitters affecting gut motility. It acts via 5-HT4 receptors to
stimulate gut motility. Thus, 5-HT4 agonists will have motility stimulating activity in the
 Agents Affecting Gut Motility 541

gastrointestinal (GIT). Tegaserod, a partial agonist at 5-HT4 receptors, is approved for use in
constipation-dominant irritable bowel syndrome. Prokinetic drugs like cisapride, mosapride
and metoclopramide act as agonists at these receptors. GR-113808 is a 5-HT4 receptor
antagonist used for radioligand studies.
Procedure: Guinea pig striatal or hippocampal brain tissue homogenates are obtained as
described by Grossman et al.3 For binding and competition studies, 400 μl of [3H]GR-113808
in HEPES buffer is incubated for 30 min at 37°C in presence of either buffer alone (for total
binding) or 5-HT (for non-specific binding) or the test compound. Reaction is terminated
by rapid vacuum filtration and washing with ice-cold buffer. Radioactivity is counted by
scintillation counter after placing the filters in scintillation cocktail overnight.
Evaluation: Specific binding (total binding minus non-specific binding) and IC50 are calculated.

[3H]Zacopride Binding Assay for 5-HT3 Receptors

5-HT, acting via 5-HT3 receptors, contributes to relaxation of gut. Thus 5-HT3 antagonistic
activity can stimulate the gut motility. Cisapride, a prokinetic drug, has weak 5-HT3 antagonistic
activity in addition to its 5-HT4 agonistic activity. Zacopride is a 5-HT3 receptor antagonist used
for radioligand binding studies.
Procedure: Assays are performed using homogenates of tissue from entorhinal cortex of
male Hooded-Lister rats.4 For binding studies, 50 μl of [3H]zacopride is incubated in presence
of buffer alone for 20 min at 37°C (for total binding) or in presence of 5HT (for non-specific
binding) or test compound for 15 min at 37°C. Reaction is terminated by rapid filtration and
the filters are washed with ice-cold buffer. Radioactivity is quantified by liquid scintillation
counting.
Evaluation: Specific binding (total binding minus non-specific binding) and IC50 are
determined.

Guinea Pig Ileum

Magnus for the first time described this method.5 It is one of the most commonly used models
to study the effect of drugs on gut motility, like screening of drugs for spasmolytic activity.
Other parts of the gut, such as duodenum and colon, can also be used as isolated preparations
for gut motility studies.
Procedure: Guinea pigs of either sex are used for the experiment. The animal is sacrificed by
stunning. The abdomen is cut opened. After tying a ligature around the intestine just distal to
the pylorus, the intestine is cut above the ligature and carefully dissected free up to colon after
cutting the mesentery. The intestine is severed near the ileocolic junction. Now, a nick is made
in the intestine near the cord at the upper end. A glass tube is inserted through the cut and the
intestine is flushed thoroughly with Tyrode’s solution until the solution coming out of the other
end is clear. The intestine, preferably the lower part (more sensitive), is cut into 2.5-3.0 cm long
pieces and kept in Tyrode’s solution. A piece of intestine is selected and suspended in an organ
bath containing Tyrode’s solution, under a preload of 1 g and aerated with 95% O2 and 5% CO2
at 37°C. After a 30 min incubation period, concentration-response curve to the standard drug
542 Drug Screening Methods

is obtained. Subsequently, the concentration-response curve of the test drug (agonist) or of

the standard in the presence of test drug (antagonist) is established. The contractions can be
recorded on a kymograph under a magnification of 5-10 times. Alternatively, the contractions
can be recorded on a polygraph using isometric force transducer. Drugs acting as agonists
or antagonists on various receptors like muscarinic, nicotinic, serotonergic, histaminergic,
prostaglandin receptors as well as directly acting agents like BaCl2 and papaverine can be
assayed using guinea pig ileum. The agonists (acetylcholine, carbachol, histamine, BaCl2,
serotonin, PGE2) and antagonists (atropine, scopolamine, papaverine) can be used as
standards for the experiments.

Cascade Superfusion Technique

The technique was developed by Gaddum.6 Using rat stomach strip, guinea pig ileum, guinea
pig vas deferens and various other isolated tissues, Vane (1964) has extended the technique to
multiple tissue superfusion for identification and assay of circulating hormones in the blood
of animals.7
Procedure: Depending on the activity to be tested, tissues of various origins like guinea pig
ileum and vas deferens, rat fundus, rat duodenum, rat colon, rat bladder, and rabbit stomach
strip, etc. can be used for the cascade superfusion. Up to 5 tissues can be simultaneously used.
The tissues are suspended one above the other, on plastic platforms attached to a vertical
rectangular rod inside an organ bath consisting of double-wall glass container (20-25 cm in
height and 7-8 cm inner diameter). The plastic platforms are placed in such a way that the
effluent from the donor tissue above bathes the tissue below and so on. Depending on the
tissue used, various physiological salt solutions can be used as superfusion fluid. The tissues
are aerated with 95% O2 and 5% CO2. Warm water is circulated through the outer jacket of the
organ bath to maintain a temperature of 38°C.
For recording, the threads from various organs, separated from each other by about 5 mm,
are connected over isotonic levers to isometric tension transducers. The superfusate is passed
down the cascade of tissues at uniform flow rate and tension from each tissue is recorded on a
polygraph.

Isolated Rat Thoracic Esophageal Muscularis Mucosae

This method is useful for study of drugs acting on 5-HT4 receptors as this tissue is rich in these
receptors.
Procedure: Sprague-Dawley rats, 180-300 g weight, are used. The animal is sacrificed and
its esophagus removed. External muscularis propria is carefully separated from the tunica
muscularis mucosae of lower 2 cm of esophagus. The tissue is suspended in organ bath
containing Krebs’ Henseleit solution, under an initial tension of 1g and aerated with 95% O2
and 5% CO2. The tissue is allowed to equilibrate at 37°C for 60 min. Tissue is washed every 15
min. After 60 min equilibration, 3 μM of carbachol is added to the organ bath for contracting
the tissue and then a cumulative concentration-response curve to 5-HT is obtained. 5-HT will
relax the precontracted tissue. Tissue is then washed and allowed 60 min recovery period.
Subsequently, the antagonistic test compound is added for 45 min and again concentration-
 Agents Affecting Gut Motility 543

response curve is obtained or for agonist drug, a concentration-response curve to the agonist
is obtained on the precontracted tissue. All responses are recorded isometrically.8,9

IN VIVO MODELS
Charcoal Passage Test
After administration of a test compound, the distance traveled by charcoal meal can be used to
determine the effect of the compound on gut motility.
Procedure: Rats or mice can be used. The animals are fasted for 18 h, but with free access to
water, before experiment. After varying time intervals of oral or subcutaneous administration
of the test drug, the animals are administered a charcoal meal consisting of 10% charcoal
powder, 5% gum Arabic with or without 1% carboxymethylcellulose suspension in distilled
water. The dose of charcoal meal is 0.3 ml/mouse or 1 ml/100 g/rat. The animals are sacrificed
by cervical dislocation or asphyxiation with CO2 at various time intervals after the meal
depending on the goal of study, e.g. for gastric emptying studies, the animals are sacrificed
after 5 min while for intestinal transit, the animals can be sacrificed after 20, 40, 60 or 120 min.
Immediately after sacrifice, the GI tract is removed and distance traveled by charcoal meal
through the intestine is measured and expressed as percentage of total length of intestine
from pylorus to cecum.2,10-12

In Vivo Evaluation of Spasmolytic Activity

To assess organ selectivity of spasmolytics, Maggi and Meli (1982) devised an in vivo procedure
for determining the comparative potencies of spasmolytics against smooth muscle contractions
induced by topically applied acetylcholine on rat colon, rectum and urinary bladder.13
Procedure: Male albino rats, weighing 350 to 400 g are used. After anesthetizing the animal,
the left jugular vein is cannulated for injecting the test drugs. The abdomen is opened and
colon and rectum isolated. Pocket-like spaces are made in them by applying occluding
silk ligatures at a distance of 2 cm from each other; and into these spaces, flanged tips of
polyethylene tubing (internal diameter 1 mm and outer diameter 1.5 mm) are inserted via
a small incision. Similarly, a polyethylene tube is inserted into the urinary bladder via a
small uretheral incision. The free ends of the tubes are connected to pressure transducer
and the whole system is filled with saline. Warm saline, at 37°C, is filled into the organs so
as to obtain a resting pressure of 4-12 mm of Hg and warm saline soaked cotton-wool swabs
are used to maintain the temperature and moisture of the exteriorized organs. The organs
are allowed to stabilize for 15 min and then concentration-response curve to acetylcholine
is obtained by bathing the outer surface of organs with 0.5 ml of acetylcholine solution.
After 3 or more comparable control curves are obtained at 10 min interval from each other,
antagonist is injected intravenously and dose-response curve to acetylcholine obtained.
ED50 values are calculated, and from them, DR10 values (dose of antagonist required to
produce an acetylcholine dose ratio of 10) are calculated according to the method of Daly
et al.14
544 Drug Screening Methods

Colonic Motility Studies in Rats

The anesthetized rat is used as one of the experimental models to study the influence of
spasmolytic drugs on colon motility induced by carbachol. Bickel (1983) used this model to
study the stimulant property of an enkephalin analogue pentapeptide.15
Procedure: Male Sprague-Dawley rats, weighing 350 to 500 g are used. The animals are fasted
for 18 h with free access to water. After anesthetizing the animal, the abdomen is opened and
a latex balloon inserted into colon ascendens and recording of intraluminal pressure changes
done.15 Duration and height of colonic contractions in response to carbachol in the absence or
presence of the test drug are noted.

Gut Motility Studies in Dogs

This method includes the study of intraluminal pressure and motility of the small intestine in
unanesthetized dogs with balloon catheter systems via a duodenal Mann-Bollman fistula.
Procedure: Male beagle dogs, weighing 15 to 20 kg, are used. After anesthetizing the animal,
the abdomen is opened by a midline incision. Terminal ileum and the portion of the intestinal
tract, where the fistula is desired, are exposed and carefully packed off. A 15-20 cm loop of
ileum is isolated, clamped at both ends and cut so that the two cut ends are left in clamps. The
remaining ileum is anastomosed end-to-end or side-to-side. Without stretching the mesentery
of the isolated loop, it is anastomosed end-to-side with the intestine at the desired site of fistula.
The free end of the loop is brought to the surface through a stab wound and sutured to the skin.
The abdomen is closed in layers.16,17
Gut motility experiments are carried out in these animals after they have been fasted
for 18 h. For measurement of intraluminal pressure of intestines, air filled latex balloons
attached to air filled polyethylene catheters are placed in intestine through the fistula
and filled with air at a pressure of 10 mm of Hg. Recording is done on a polygraph after
connecting the catheters to a pressure transducer. Frequency and amplitude of pressure
waves are recorded over 10 min and comparison of predrug and postdrug readings is made.

Gastrointestinal Motor Activity in Conscious Dogs

Beagle dogs of either sex, weighing 9-13 kg, are used. The animal is anesthetized and
abdominal cavity is opened. Force transducers are implanted extraluminally in gastric
antrum (3 cm proximal to pyloric antrum) and in colon (10 and 20 cm distal to the ileo-
caecal valve). Through an incision between the scapulae, the lead wires are brought out at
the back of animal and sutured to the skin. A 6.5 F catheter is placed in superior vena cava
of the animal (for intravenous injection of drugs) and the outer end sutured to the skin. The
animal is covered with a protective jacket and allowed to recover for 2 weeks. For gut motility
experiments, the test drug is given intravenously, 2-3 hr after food and GI motor activity
recorded by connecting lead wires to polygraph system. Motor index is calculated from the
amplitude of contraction and the time for which the contraction remains in an area over
a fixed time period (30 min period is taken). Motor index with and without the drug are
compared.9,18
 Agents Affecting Gut Motility 545

REFERENCES
1. Steigerwalt RW, Williams JA. Characterization of cholecystokinin receptors on rat pancreatic
membranes. Endocrinology 1981;109:1746-53.
2. Gully D, Frehel D, Marcy C, et al. Peripheral biological activity of SR 27897: a new potent non-
peptide antagonist of CCKA receptors. Eur J Pharmacol 1993;232:13-9.
3. Grossman CJ, Kilpatrick GJ, Bunce KT. Development of a radioligand binding assay for 5HT4
receptors in guinea-pig and rat brain. Br J Pharmacol 1993;109:618-29.
4. Barnes NM, Costall B, Naylor J. [3H]zacopride: ligand for the identification of 5HT3 recognition
sites. J Pharm Pharmacol 1988;40:548-51.
5. Magnus R. Versuche am uberlebenden Dunndarm von Saugethieren. Pflugers Arch 1904;102:123-51.
6. Gaddum JH. The technique of superfusion. Br J Pharmacol 1953;8:321-6.
7. Vane JR. The use of isolated organs for detecting active substances in the circulating blood. Br J
Pharmacol 1964;23:360-73.
8. Baxter GS, Craig DA, Clarke D. 5-Hydroxytryptamine4 receptors mediate relaxation of the rat
oesophageal tunica muscularis mucosae. Naunyn-Schmiedeberg’s Arch Pharmacol 1991;343:439-46.
9. Mine Y, Yoshikawa T, Oku S, et al. Comparison of effect of mosapride citrate and existing 5-HT4
agonists on gastrointestinal motility in vivo and in vitro. J Pharmacol Exp Ther 1997;283:1000-08.
10. Vischer P, Casals-stenzel J Influence of prostacyclin and indomethacin on castor oil-induced
gastrointestinal effects in rats. J Pharm Pharmacol 1983;35:152-6.
11. Mascolo N, Izzo AA, Barbato F, et al. Inhibitors of nitric oxide synthetase prevent castor oil-induced
diarrhea in the rat. Br J Pharmacol 1993;108:861-4.
12. Izzo AA, Mascolo N, Pinto L, et al. The role of cannabinoid receptors in intestinal motility,
defaecation and diarrhea in rats. Eur J Pharmacol 1999;384:37-42.
13. Maggi CA, Meli A. An in vivo procedure for estimating spasmolytic activity in the rat by measuring
smooth muscle contractions to topically applied acetylcholine. J Pharmacol Methods 1982;8:39-46.
14. Daly MJ, Flook JJ, Levy GP. The selectivity of β-adrenoceptor antagonists on cardiovascular and
bronchodilator responses to isoprenaline in the anaesthetized dog. Br J Pharmacol 1975;53:173-81.
15. Bickel M. Stimulation of colonic motility in dogs and rats by an enkephalin analogue pentapeptide.
Life Sci 1983; 33:469-72.
16. Tasaka K, Farrar JT. Intraluminal pressure of the small intestine of the unanaesthetized dog. Pflugers
Arch 1976;364:35-44.
17. Mann FC, Bollman JL. A method for making a satisfactory fistula at any level of the gastrointestinal
tract. Ann Surg 1931;93:794-7.
18. Itoh Z, Honda R, Takeuchi S, et al. An extraluminal force transducer for recording contractile
activity of the gastrointestinal smooth muscle in conscious dogs: Its construction and implantation.
Gastroenterol Jpn 1977;12:275-83.
 cHAPTER

37
Antiemetic Agents
INTRODUCTION
Nausea and vomiting are amongst the most common and distressing symptoms observed
in a variety of conditions such as pregnancy, peptic ulcer, gastrointestinal infections,
gastrointestinal obstruction, renal disorders, hepatitis, motion sickness, following anesthesia
and surgery and as adverse effect of treatment with a wide range of drugs especially the cancer
chemotherapeutic agents. Nausea and vomiting are also an important component of defense
mechanism of the body against accidental ingestion of toxins. Nausea is a nonobservable
subjective feeling of having an urge to vomit. The unpleasant sensation is experienced at
the back of throat and epigastrium that may or may not culminate into emesis.1 The emesis
consists of retching and vomiting. The retching is characterized by attempt to vomit without
expulsion of the contents of upper gastrointestinal tract. During retching muscles of diaphragm
and abdomen contract and relax simultaneously. The vomiting consists of more sustained
abdominal contraction in coordination with intercostal muscles and muscles of larynx and
pharynx. The glottis is closed, the soft palate elevated, the gastric fundus relaxes and contents
of stomach, both solid and liquid, are forcefully expelled out through the nose or mouth. The
occurrence and frequency of retching and vomiting can be measured objectively.
Acute emesis usually occurs within a few minutes to several hours of exposure to emetogen.
It usually resolves within 24 h. Emesis that occurs before the person receives the emetic stimuli
such as the cancer chemotherapy is known as the anticipatory emesis. It is a conditioned
response due to negative past experiences. Emesis occurring despite the prophylactic
antiemetic treatment and requiring rescue antiemetic therapy is known as the breakthrough
emesis. Delayed emesis is observed more than 24 h after the institution of cancer chemotherapy.
It usually peaks at 48-72 h and can continue for 6-7 days.
Integration of the complex interplay of physiological events ultimately leading to emesis
takes place in the vomiting center located in medulla oblongata. Output neurons that control
the muscles involved in emesis are scattered throughout the medulla oblongata. Afferent
inputs to the vomiting center originate from four sources that include (Fig. 37.1):
1. Cerebrocortical pathways, stimulated by learned associations.
2. Chemoreceptor trigger zone (CTZ) in area postrema of cortex, stimulated by chemical
stimuli from blood and cerebrospinal fluid.
3. Vestibular pathway, stimulated by positional changes of the body.
4. Peripheral pathways, stimulated by neurotransmitter receptors in gastrointestinal tract.
 Antiemetic Agents 547

Figure 37.1: Pathophysiology of nausea and vomiting

To further elaborate the understanding of the pathways involved in generation of the

sensation of nausea and reflex vomiting, to identify pharmacological approaches for
development of new antiemetics and to understand the underlying problems in diseases with
nausea and vomiting as the cardinal features, use of appropriate animal models is required.

IN VIVO MODELS
Choice of Animal Models
Animals
For research in nausea, emesis and antiemetics, selection of appropriate animal species is
extremely important. In general, the vomiting reflex, although not essential for survival, is an
important defense mechanism, which helps body to get rid of ingested toxins. Many animal
species used in laboratory research such as rat, mouse, rabbit, guinea pig and hamster appear
to lack the vomiting reflex as is seen in human. There are isolated reports suggesting presence
of a degenerate vomiting reflex in rodents2,3 and presence of a neophobic character to avoid
ingestion of toxins.4 Vomiting reflex is present in other mammalian species such as dogs,
monkey, cat, ferret, pigs and vertebrates like birds, reptiles, fishes and amphibians.5-9 However,
it should be noted that all species with vomiting reflex are not sensitive to all emetic stimuli
and function of vomiting reflex in some species is to get rid of undigested material and not the
defense against the toxins.
Animal species commonly used to evaluate antiemetic activity of test drugs include dogs,
cat, monkeys, ferret, house musk shrew (Suncus murinus), least shrew (Cryptotis parva),
gerbils and pigeons. Rodents do not possess the emetic reflex, however exposure to emetic
stimuli can lead to significant increase in the intake of non-food substances, a response known
548 Drug Screening Methods

as pica. Dogs, cats and monkeys have good learning abilities and therefore cannot be used for
repeated testing. Pigeons carry viruses infectious to human and therefore they are not widely
used. Ferret is a widely accepted animal model and the emesis in ferret closely resembles that
in human. The disadvantage in using ferret is the high maintenance cost. The Suncus murinus
is an insectivore and is considered phylogenetically more closely related to primates than other
animal species like rodents. Emetic response to a wide range of stimuli is well-known in this
animal species however, sometimes the frequency of retches can be very high as compared
to other species such as cat, dog and ferret making it difficult to observe individual retches.
Therefore, the emetic response consists of both the retching and vomiting and is measured
as emetic episodes. The least shrew (Cryptotis parva) was also found to be useful as it showed
the emetic response to a wide range of emetogens.10,11 Animals are individually housed and
maintained in standard environmental conditions with free access to food and water. The
observation cages are kept in a quite room. A wide range of emetogens can be used to evaluate
antiemetic properties of test drugs.

Emetogens
The commonly employed emetic stimuli in animals include drugs such as cancer
chemotherapeutic drugs like cisplatin and methotrexate, apomorphine and copper sulfate.
The cancer chemotherapeutic drugs act by directly stimulating the enterochromaffin cells in
small intestinal mucosa to secrete serotonin. Serotonin is thought to stimulate vagal afferent
fibers through 5-HT3 receptors located at the vagal afferent terminals. Sensory signals reach
the area postrema to initiate nausea and emesis. Apomorphine is a derivative of morphine and
induces vomiting by stimulating central dopamine receptors. Copper sulfate is an irritant to
gastrointestinal mucosa and its oral administration initiates emesis by stimulating the vagal
afferents. Other drugs used as emetogens include cyclophosphamide, doxorubicin, morphine,
nicotine, etc.
Other useful emetogens used in experimental studies include motion stimulus, which
induces emesis through vestibular pathways and radiations acting through early peripheral
and late central serotonergic pathways.

Parameters
Emetic episodes are easy to study and can be overtly measured in laboratory animals showing
vomiting reflex. Observations are made for latency to first retching and vomiting and number
of vomiting episodes are counted for each animal. An interval of at least 1 min is needed to
separate one emetic episode from the next. Episodes of retching and vomiting are considered
separate when animal changes position in the observation cage or when the interval between
the retches and/or vomiting is more than 5 seconds. Exposure to emetogens can also induce
behavioral changes in animals, which are considered analogous to nausea. These behavioral
changes include increases in the incidence of swallowing, lip licking, backward walking,
burrowing, curling up, rearing or decreases in locomotor activity. It is considered relatively
straightforward to assess if a compound has an activity to reduce retching and vomiting,
but more problematic to determine a reduction of nausea, since animals are unable to
communicate directly their emotional status.
 Antiemetic Agents 549

Animals with absent vomiting reflex such as rats are also used to study behaviors associated
with nausea and vomiting. These specific behaviors include conditioned flavor avoidance (CFA)
or pica. Mitchell et al (1976, 1977) suggested that pica, i.e. eating of nonnutritive substances
such as kaolin, is an illness-response behavior in rats.12,13 These behavior responses are often
measured as indicators of emesis in rats. Kaolin is prepared using the pharmacological grade
kaolin (or hydrated aluminum silicate) and acacia mixed in a ratio of 99:1. A thick paste is
prepared by mixing with distilled water. The paste is rolled and cut into pieces resembling
regular rat chow pellets, which are then dried at room temperature for 72 h.14 Rats expressing
pica behavior show increase in the wet weight of stomach, which can be measured at the end
of the experiment. An incision is made proximal to the gastroesophageal junction and distal to
the pyloric sphincter to isolate the stomach. After isolation, the stomach is blotted dry, and the
wet weight is recorded.15
Emesis is associated with delayed gastric emptying due to reduced gastric motility. Increase
in the rate of gastric emptying in response to administration of test drugs may also indicate
their antiemetic efficacy.16 To determine the rate of gastric emptying, 200-250 g rats are orally
administered with a 1.5 ml of test meal consisting of 0.05% phenol red in a 1.5% aqueous
methylcellulose solution. Rats are sacrificed 45 min after the meal. Stomach is removed and
homogenized in 100 mL of NaOH 0.1 M. Stomach tissue proteins are precipitated with 20%
trichloroacetic acid (0.5 ml of trichloroacetic acid in 5 ml homogenate) and centrifuged. The
supernatant is mixed with 4 ml of NaOH 0.5 M and absorbance of the sample is read at a
wavelength of 560 nm. Phenol red recovered from the stomach of rats sacrificed immediately
after administration of methylcellulose meal serves as standard. The percentage gastric
emptying of each rat is calculated as follows:

Gastric emptying (%) = 1-Cphenol red (test stomach)/Cphenol red (standard stomach) × 100

Drug-induced Emesis Models

Cisplatin-induced Emesis Model
Cancer chemotherapy based on cisplatinum is known to be associated with the adverse effects
of nausea and vomiting. Emesis occurring within first 24 h of the institution of therapy is the
early phase acute vomiting and can be effectively antagonized by 5-HT3 receptor antagonists.
The emesis occurring 24-120 h after the start of therapy is delayed emesis and does not respond
well to 5-HT3 receptor antagonists.17,18 Cisplatin is popularly used as an emetogen in animal
models to evaluate antiemetic drugs especially for early phase emesis and is usually prepared
in normal saline at 70°C followed by slow cooling to 40°C. Dogs are most sensitive to cisplatin
followed by ferret, cat and Suncus murinus.

Cisplatin-induced Dog Model

Cisplatin-induced emesis in dogs has previously been described by Gylys et al. (1979)19 and can
be used to evaluate antiemetic properties of 5-HT3 receptor antagonists. Animals are divided
into test and control groups. Test group receives the test drug and control group receives the
vehicle. Ten minutes later cisplatin is administered intravenously at a dose of 3.2 mg/kg/ml.
Animals are then observed continuously for next 5 h for emetic episodes. Dogs with no obvious
toxicity are retested after an interval of 4 weeks.
550 Drug Screening Methods

Cisplatin-induced Cat Model

John et al. (2000) have described the use of Cisplatin-induced cat model.20 Cats of either sex
weighing 2-6 kg are used. On the day of experiment animals are fed with 200 g of cat food. Light
anesthesia is administered using 5% halothane. A cannula is then inserted into the cephalic
vein through which cisplatin (3-7.5 mg/kg) is infused slowly over a period of 4 min. Cisplatin
administration is immediately followed by administration of drug/vehicle. Cannula can now
be removed and animals transferred back to cages for observation of emesis as in case of dogs.
A video-monitoring system can also be used to monitor behavioral changes during prolonged
observation period if drug is being tested for effects on delayed emesis.

Cisplatin-induced Ferret Model

Ferrets weighing 1-1.5 kg are used. After overnight fasting each ferret is administered
simultaneously with the test drug/vehicle and cisplatin (10 mg/kg) intravenously. If the test
drug is administered orally, cisplatin can be administered 30 minutes after the test drug
administration. Following cisplatin administration the animals are observed for emesis for
next four hours.21

Cisplatin-induced Pigeon Model

Adult pigeons weighing 350-550 g are used. Cisplatin 4 mg/kg is administered intravenously
via the brachial wing vein. Test drug/vehicle is administered prior to cisplatin administration
and time interval between the drug and cisplatin administration depends upon the expected
time required for the drug to act. Following cisplatin administration animals are kept under
observation for emesis.22

Cisplatin-induced Suncus murinus Model

Adult Suncus murinus of either sex weighing 30-80 g are maintained under standard laboratory
conditions are allowed to acclimatize in transparent cages to make the observations easier.
Test drug/vehicle is administered usually by subcutaneous route and 30 min later the emetic
stimulus cisplatin (20 mg/kg) is administered intraperitoneally. Animals are individually
observed for next two hours for the behavioral effects to characterize nausea status, latency to
emetic episodes and frequency of emesis.23

Cisplatin-induced Rat Model

Administration of cisplatin in rats induces significant kaolin intake, which can be used as
the measure analogous to emesis in other species. Rats pretreated with drug/vehicle are
administered with cisplatin (3-10 mg/kg, intraperitoneal) 30 minutes later. Kaolin intake, food
intake, and body weight were measured every 24 h for 120 h.24

Methotrexate-induced Delayed Emesis Model

Cisplatin induced emesis in animals may fail to demonstrate delayed emesis, which occurs
24 h after the start of chemotherapy. To evaluate the antiemetic activity against delayed emesis
during chemotherapy, methotrexate (MTX) is a useful emetogen.25 It can be used in dogs,
 Antiemetic Agents 551

cats, ferrets and Suncus murinus. MTX is prepared by dissolving in 5% dextrose. Test drug/
vehicle is administered at 24, 36, 48 and 60 after MTX. Animals are kept under observation
soon after MTX administration till 72 h. It is preferable to use a video camera with automatic
night photographic system so as to provide with a continuous record of animal behavior for
72 h post MTX treatment. Animals can be retested with MTX at least 6 weeks later.

Apomorphine-induced Emesis Model

Apomorphine is an opiate that acts as a potent central dopamine agonist directly at the
area postrema via dopamine receptors.26,27 As the vestibular pathways are also involved in
apomorphine-induced emesis the active animals develop emesis readily as compared to
animals that are sedate and motionless. Dogs are the most sensitive to apomorphine followed
by ferret while Suncus murinus is unresponsive. Use of apomorphine in cat is controversial as
the administration of apomorphine can cause excitation in cats.

Apomorphine-induced Dog Model

Dogs are very sensitive to emetic response to apomorphine. Apomorphine hydrochloride is
prepared as solution in saline. Each animal receives the test drug/vehicle subcutaneously
or orally. Apomorphine is administered at a dose of 0.3 mg/kg subcutaneously. The interval
between the administration of drug and emetogen can be modified depending upon the
expected time required for drug action.20 Subsequently, animals are observed for emetic
episodes.

Apomorphine-induced Ferret Model

Ferrets have been used successfully to evaluate antiemetic drugs against apomorphine-
induced emesis. Animals are allowed to habituate in the observation cages 2 h every day for
4-5 days. On the day of experiment the animals are kept in the observation boxes for at least
30 min prior to the start of experiment. Animals are then administered with the test drug/
vehicle and 30 min later apomorphine hydrochloride (0.25 mg/kg) is injected subcutaneously.
The animals are now observed for further 60 min for behavioral changes as well as emetic
episodes.28

Apomorphine-induced Rat Model

Administration of apomorphine in rats induces pica (ingestion of non-food substances e.g.
kaolin). The antiemetic activity of test drug can be determined by observing this behavior in
animals (pretreated with drug/vehicle) before and after challenge with apomorphine (10 mg/
kg, intraperitoneal).29

Copper Sulfate-induced Emesis Model

Copper sulfate is a powerful oxidizing agent and an irritant to mucous membranes. If
administered orally causes irritation of gastric mucous membrane and leads to nausea and
vomiting. It has conveniently being used as an emetogen in experimental studies.
552 Drug Screening Methods

Copper Sulfate-induced Dog Model

After overnight fasting dogs receive the test drug or the vehicle depending upon the group
they belong to. Copper sulfate solution prepared by dissolving copper sulfate pentahydrate
in distilled water is rapidly administered at a dose of 100 mg/kg through an orogastric tube.
Animals are observed for next one hour for episodes of emesis and those showing no toxicity
can be retested two weeks later.30

Copper Sulfate-induced Cat Model

Evaluation of antiemetics can be done in adult cats after administering copper sulfate in a
dose of 40 mg/kg orally. Alternatively, the threshold dose is determined by once a week
administration of copper sulfate orally in a dose of 20, 30 and 40 mg/head. The cats with a
threshold of more than 40 mg or latency of less than 5 min or of more than 45 min should
be excluded. Cats included in the experiment are administered with the test drug/vehicle
followed by oral administration of the threshold dose of copper sulfate. The efficacy of test
drug can now be evaluated by observations for the emetic episodes.31

Copper Sulfate-induced Ferret Model

Copper sulfate-induced emesis can also be induced in ferrets. After overnight fasting and
pretreatment with drug/vehicle ferrets are administered with the solution of copper sulfate
40 mg/kg orally. Subsequently, animals are observed for latency and frequency of emetic
episodes.32

Copper Sulfate-induced Suncus murinus Model

Suncus murinus can also be used to investigate antiemetic effects against copper sulfate induced
emesis. Copper sulfate is administered 40 mg/kg (intragastric) 30 min after the adminis­tration
of test drug/vehicle. The animal is observed for further 60 min for emetic episode.23

Copper Sulfate-induced Chick Model

Antiemetic activity of experimental drugs may also be evaluated using chick emesis model.
4-day old chicks are administered with experimental drug or vehicle orally. Subsequently, after
10 min, copper sulfate is administered orally at a dose of 50 mg/kg body weight and animals
are observed for latency and frequency of emetic episodes.33

Motion-induced Emesis Model

Dog is probably as sensitive to motion-induced emesis as man. Motion-induced emesis can
also be established in cats, Suncus murinus and rats.

Motion-induced Cat Model

Cats weighing 1.5-3 kg are placed in a Plexiglas chamber on a circular platform having holes
at the base so as to reduce air resistance during oscillations. Chambers are suspended from
the ceiling by springs. A gentle push is applied to initiate and maintain the vertical oscillations
at ~0.3 Hz through a distance of ~75 cm. The motion sickness is characterized by repetitive
 Antiemetic Agents 553

licking, salivation often dripping out of the mouth, or vomiting. The increased latency and/or
decreased frequency of motion-induced emesis in cats pretreated with the test drug indicates
antiemetic property of the test drug.34

Motion-induced Suncus murinus Model

Suncus murinus after receiving the test drug/vehicle are placed individually in transparent
cages, which are then kept on a reciprocal shaker. This is followed by a period of 5 min for
acclimatization. Shaker is then started to give a horizontal oscillation of 4 cm at a frequency of
1 Hz for 10 min. The emetic episodes are noted during motion as well as after the motion ceases.
Individual animals can be exposed only twice at an interval of 1 week as more exposures lead
to adaptation to motion stimulus.35

Motion-induced Rat Model

In rats, 60 min double rotation can induce increased kaolin intake indicating motion-induced
emesis. Pretreatment with the test drug if found to decrease the kaolin intake, indicates
antiemetic activity of the test drug.29

Radiation-induced Emesis Model

Ferrets are most sensitive to radiations followed by dogs. Cats are considerably resistant to
radiations.

Radiation-induced Dog Model

A radiation-induced emesis can be induced in dogs by using 6°Co and 8 Gy administered to
total body surface. An irradiated control group is established which is given no medication or
vehicle while the other similarly irradiated groups receive the test drug. Latency to retching/
vomiting and number of vomiting episodes are observed in each animal and a comparison
can be made to evaluate efficacy of the antiemetic drug. Alternatively, drug/vehicle pretreated
dogs are exposed to 8 Gy 6°Co gamma mid-abdominal irradiation. Subsequently, animals are
observed for latency and frequency of emetic episodes in two groups.36

Radiation-induced Ferret Model

The radiation-induced model can also be induced in ferret. The ferrets are irrradiated with
bilateral 6°Co gamma radiation at 201 cGy. An emesis incidence of 100% is reported at 201 cGy
in ferret.37 Increased latency and/or reduced frequency of emetic episodes indicates antiemetic
property of test drug.

Radiation-induced Rat Model

Exposure to radiation can induce pica in rats. Pica is a behavior characterized by ingestion
of non-nutritive substances such as kaolin and can be used as an index of radiation-induced
vomiting. Rats pretreated with drug/vehicle are exposed to 4 Gy of total-body irradiation.
The 4 Gy of abdominal irradiation is reported to be more effective to induce pica than that of
head irradiation.38 Inhibition of kaolin intake in drug treated group as compared to control is
considered a measure of antiemetic properties of test drug.
554 Drug Screening Methods

Suncus murinus Model for Anticipatory Nausea and Vomiting

Anticipatory nausea and vomiting, often associated with cancer chemotherapy, is described as
the conditioned response to cues present at the time of exposure to toxins as a result of pairing.
Conditioned retching in Suncus murinus was found to provide an effective model to evaluate
efficacy of drugs against anticipatory nausea and vomiting by Parker et al. 2006.39 The emetic
stimuli, which can be effectively used in this model, include horizontal motion (1 Hz, 4 cm, 10
min), nicotine, 4 mg/kg sc and lithium chloride 100 mg/kg ip.
Shrews of either sex weighing 20-50 g are used. All animals included in the study are weaned
at the age of 20 days. They are individually housed and maintained under standard laboratory
conditions. Animals are housed in transparent cages with a mirror fitted at the bottom so as to
allow the observation of the ventral surface of the shrews. Observation chambers are kept in a
well-illuminated room with a video camera fitted to record the activities of shrews. Animals are
then divided into test and control groups. All animals now receive three conditioning trials 72 h
apart. Within each group (study, control) half the animals receive of lithium chloride while the
other half will receive the vehicle intraperitoneally just before they are put in the observation
chambers. The episodes of vomiting and retching are observed over next 45 minutes. Lithium
treated animals develop retching and vomiting during these conditioning trials. Six days after
completing the three conditioning trials the animals receive two test trials 72 hours apart. On
the day of trial the animals are first injected with the test drug/vehicle and returned to their
home cages. Thirty minutes later all animals are injected with 60 ml/kg of physiological saline
just before putting them in observation chambers. The shrews are then observed and behavior
is video recorded for next 45 minutes. Finally four groups of animals i.e. test drug-lithium
conditioned, test drug-saline conditioned, vehicle-lithium conditioned and vehicle-saline
conditioned are compared to evaluate efficacy of test drug. The shrews display conditioned
retching upon exposure to a context associated with drug-induced vomiting in vehicle
treated lithium conditioned group.40 The frequency of retching can be more than that during
the last conditioning trial. Decreased frequency of retching episodes in drug treated lithium
conditioned group indicates efficacy of drug against anticipatory vomiting. The cannabinoids
have been found to be effective against anticipatory nausea and vomiting however, 5-HT3
antagonists are not as effective.

Rat Model for Anticipatory Nausea and Vomiting

Effectiveness of the test drugs against anticipatory nausea and vomiting can also be evaluated
in rats.41 The animals undergo 4 conditioning trials 72 h apart as described for Suncus murinus.
Seventy two hours after 4th conditioning trial, all rats are surgically implanted with intraoral
cannulae. For implantation of cannulae, first a thin-walled 15-gauge stainless steel needle is
inserted at the back of the neck, directed subcutaneously around the ear and is brought out
behind the first molar inside the mouth. A length of thin plastic tubing with an inner diameter
of 0.86 mm and an outer diameter of 1.27 mm is then passed through the needle and then
the needle is removed. Two circular elastic discs are placed over the tubing and drawn to
the exposed skin at the back of the neck to stabilize the cannulae. The tubing is secured in
the oral cavity by a ring, which is sealed behind the tubing prior to cannulation surgery. This
indwelling cannulae is connected with an infusion pump through a plastic tube for delivery
 Antiemetic Agents 555

of 0.1% saccharin solution. Cannulae are flushed daily with chlorhexidine solution to prevent
infection.
All animals are subjected to test trials after receiving drug/vehicle as described for Suncus
murinus, 72 h after implantation of cannulae. During the 30 min trial in observation chamber,
rats receive an intraoral infusion of 0.1% saccharin solution every 5 min for 1 min (at a rate
of 1 ml/min), resulting in a total of 6 infusions in 30 min. During the intraoral infusions, the
rat’s somatic and orofacial responses are video-recorded. Grading is done for gaping, rearing
and active locomotion. Gaping is rapid, large-amplitude opening of the mandible expressed
while rats are infused with 0.1% saccharin. Frequency of gaping is recorded separately for the
duration of saccharine infusion (6 min) and for inter-infusion interval (24 min). The rearing
activity is obtained by recording the frequency when both front forepaws of rat are lifted off the
floor and not touch the wall of the chamber. An overall activity duration score is obtained by
summing the frequency of instances of forward locomotion i.e. movement of the rat’s forepaws
along the floor of the chamber. These scores are converted to an activity/min score and a
comparison is made among groups.
Rats display the characteristic response of gaping when intraorally infused with a flavor
previously paired with lithium chloride. The gaping response of the taste reactivity test appears
to be a selective marker of nausea in rats. Only drugs that produce emesis in species capable of
vomiting produce conditioned gaping.

IN VITRO MODELS
The in vitro models can be used to demonstrate the pharmacological activity of the newer
drugs indicating their potential use as antiemetics. A large number of antiemetics belonging
to different classes of drugs are in therapeutic use. These include selective H-1 receptor
antagonists, anticholinergics, antidopaminergics, corticosteroids and cannabinoids. Among
all, 5-HT3 receptor antagonists are considered potent antiemetics. The experimental drugs
with possible antiemetic activity can be evaluated for 5-HT3 receptor antagonist activity using
in vitro methods.

5-HT3-Receptor Antagonists
Selective 5-HT3 receptor antagonists such as ondansetron are extremely potent antiemetics
especially in the treatment of emesis associated with cancer chemotherapy. The possible 5-HT3
receptor antagonist activity indicative of antiemetic activity can be evaluated using isolated
guinea pig colon preparation.
Male guinea pig is killed by blow on the head, abdomen opened and distal part of colon
is removed and placed in a dish containing Krebs-Henseleit solution (pH 7.3–7.5) consisting
of NaCl 118 mM, KCl 4.7 mM, CaCl2 2.5 mM, KH2PO4 1.2 mM, NaHCO3 25 mM, MgSO4
1.2 mM and glucose 10 mM. A 20 mm piece of colon (in relaxed condition) is washed with
Krebs-Henseleit solution. The piece of colon is now suspended in 10 ml organ bath containing
Krebs-Henseleit solution at 37°C and bubbled through with oxygen/carbon dioxide (95% O2
+ 5% CO2). The preparation is fixed with an isotonic transducer loaded with 1-gram weight.
After 60 min to achieve equilibrium, a selective 5-HT3 receptor agonist 2-methyl-5-HT (initial
concentration10-5 M) is used to elicit the concentration-response curve. The preparation is
556 Drug Screening Methods

washed with bath solution every 5 min until the baseline is achieved. The test compound is
now pipetted into the organ bath and 30 min time is allowed to equilibrate before challenging
with the agonist again. Selective 5-HT3 receptor antagonist competitively inhibits the
contractile responses to 2-methyl-5-HT or it can enhance the inhibitory activity of another
selective 5-HT3 antagonist like tropisetron (0.1 mmol.l–1). 5-HT3 receptor antagonist will
fail to affect the contractile response evoked by acetylcholine receptor agonist carbachol
(1 mmol/l-1).42

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17. Kris MG, Gralla RJ, Clark RA, Tyson LB, O’Connell JP, Wertheim, et al. Incidence, course, and
severity of delayed nausea and vomiting following the administration of high-dose cisplatin. J Clin
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18. Kris MG, Pisters KM, Hinkley L. Delayed emesis following anticancer chemotherapy. Support Care
Cancer 1994;2:297-300.
19. Gylys JA, Doran KM, Buyniski JP. Antagonism of cisplatin-induced vomiting in dogs. Commun
Chem Pathol Pharmacol 1979;23:61-8.
20. Rudd JA, Tse HYH, Wai MK. Cisplatin-induced emesis in the cat: effect of granisetron and
dexamethasone. Eup J Pharmacol 2000;391:145-50.
21. Yoshikawa T, Yoshida N, Oka M. The broad-spectrum anti-emetic activity of AS-8112, a novel
dopamine D2, D3 and 5-HT3 receptors antagonist. British J Pharmacol 2001;133:253-60.
22. Tanihata S, Hiroaki I, Masami S, Toshimitsu U. Cisplatin-induced early and delayed emesis in the
pigeon. British J Pharmacol 2000;130:132-8.
23. Andrews PLR, Okada F, Woods AJ, Hagiwara H, Kakaimoto S, et al. The emetic and anti-emetic
effects of the capsaicin analogue resiniferatoxin in Suncus murinus, the house musk shrew. British
J Pharmacol 2000;130:1247-54.
24. Mehendale S, Han A, Anbao W, Jun-Jie Y, Chong-Zhi W, Jing-Tian X, et al. American ginseng berry
extract and ginsenoside Re attenuate cisplatin-induced kaolin intake in rats. Cancer Chemoth
Pharmacol 2005;56:63-9.
25. Hisashi Y, Hiroe S, Yasue M, Katsunori I, Hiroyuki S, Masahiko M, et al. Probable involvement of the
5-hydroxytryptamine4 receptor in methotrexate-induced delayed emesis in dogs. Pharmacol Exp
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26. Andrews PLR, Davis CJ, Maskell L. The abdominal visceral innervation and the emetic reflex:
pathways, pharmacology, and plasticity. Can J Physiol Pharm1990; 68:325-45.
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antagonist CP-99,994 does not require the presence of the area postrema in the dog. Neurosci Lett
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28. Lau AH, Ngan MP, Rudd JA, Yew DT. Differential action of domperidone to modify emesis and
behaviour induced by apomorphine in the ferret. Eur J Pharmacol 2005;516:247-52.
29. Takeda N, Hasegawa S, Morita M, Horii A, Uno A, Yamatodani A, et al. Neuropharmacological
mechanisms of emesis. I. Effects of antiemetic drugs on motion- and apomorphine-induced pica in
rats. Methods Find Exp Clin Pharmacol 1995;17:589-90.
30. Fukui H. Yamamoto M, Sasaki S, Sato S. Possible involvement of peripheral 5-HT4 receptors in
copper sulfate-induced vomiting in dogs. Eur J Pharmacol 1994;257:47-52.
31. Kayashima N, Hyama T. Reproducibility of emesis by orally administrated copper sulfate in cats.
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33. Akita Y, Yang Y, Kawai T, Kinoshita K, Koyama K, Takahashi K, et al. New assay method for surveying
anti-emetic compounds from natural sources. Natural Product Sciences 1998;4:72-77.
34. Ivan ML, Sarna SK, Shaker R. Gastrointestinal motor and myoelectric correlates of motion sickness.
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37. King GL. Characterization of radiation-induced emesis in the ferret. Radiat Res 1988;114:599-612.
38. Yamamoto K, Noriaki T, Atsushi Y. Establishment of an animal model for radiation-induced
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39. Parker LA, Magdalena K, Raphael M. Delta-9-tetrahydrocannabinol and cannabidiol, but not
ondansetron, interfere with conditioned retching reactions elicited by a lithium-paired context
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 cHAPTER

38
Absorption and Metabolism
Introduction
Absorption and metabolism are important pharmacokinetic parameters for effective drug
therapy. Many drugs are absorbed by the small intestine. Drug absorption is affected by
multiple factors such as physiochemical factors—solubility of the drug, particle size, routes of
administration, gastrointestinal motility, permeability area of absorbing surface, etc. The small
intestinal epithelium acts as a primary barrier to control the absorption of orally administered
xenobiotics and nutrients. Apart from its absorptive property, it is also involved in the first
pass metabolism of the drugs, involving both phase I and phase II reactions. The outcome
of an orally administered drug depends on both physicochemical properties of the drug and
physiological factors like gastric emptying rate, intestinal motility, intestinal mucosal cells
and composition of the intestinal juice. Sufficient intestinal absorption of orally administered
drugs from gastrointestinal tract is one of the essential requirements for the success of oral
drug therapy.
Further, after absorption of drug from any route, it undergoes distribution and metabolism
in the body before being excreted. Liver is the main site of metabolism of most of the
xenobiotics. CYP450 superfamily of enzymes catalyzes most of the metabolism reactions. Drug
metabolism studies are very important both from the industrial perspective, where they are
used to study drugs in discovery and development as well as in the pursuit of basic research.
Importance of metabolic studies can be gauged from the recent banning of terfenadine,
astemizole and cisapride, due to their propensity to cause serious ventricular arrhythmias
when given in conjunction with CYP450 inhibitors like erythromycin, ketoconazole, etc. Some
of the commonly used methods for the screening of drugs for absorption and metabolism are
described below.

ABSORPTION STUDIES
In vitro Models
Everted Gut Sac Model
Wilson and Wiseman in 19541 first introduced everted intestinal gut sac model to study nutrient
transport. The technique was later modified to study drug transport across the intestine.2
560 Drug Screening Methods

Male Sprague-Dawley rats of 250–300 g weight range are used. Small intestine is quickly
removed from the decapitated animal and placed in Krebs-Henseleit bicarbonate buffer
solution. Using a glass rod, the intestine is inverted and tied at both the ends to prepare an
intestinal sac. The sac is then filled with the physiological solution using blunt needle syringe.
A small air bubble is also injected to oxygenate the serosal side of the intestinal segment. The
everted sac is then placed in a beaker containing the compound of interest dissolved in well-
oxygenated physiological salt solution. The amount of drug permeated through the intestine
layers to the serosal side is measured.3
This model can be used for measuring absorption of test compounds at different sites in the
intestine.4 The model is also used to study pro-drug conversion in different intestinal sites and
also to investigate efflux transporters, like ‘p’ glycoprotein mediated efflux of [3H] vinblastine,
[14C] doxorubicin and veraparmil. Thus, can be prove as an important tool for study of
p-glycoprotein mediated transport and potential p-glycoprotein substrate modifiers.5 It is also
useful for estimating the first-pass metabolism of drugs in intestinal epithelial cells. A potential
disadvantage of this approach is limited tissue viability6 and the presence of the muscularis
mucosa, which is not usually removed from everted sac preparations. Thus, the compound has
to cross all the layers (including muscles) of the small intestine. Apart from this, as the volume
of fluid on the serosal side, i.e. inside the sac is limited; there may be possibility of saturation
of transport due to accumulation of compound. This model has recently been reviewed by
Alam et al. 2011.7 This model is affected by the age of the animal, sex and species, pathological
conditions, chronic therapy and intestinal selectivity. Many experimental factors such as pH,
media, temperature, substrate related factors (time lapse in the harvesting of the intestine and
animal state—live or dead) affect active transport in the duodenal segments of the animal. This
model is very useful to study product conversion in different intestinal sites, drug interaction
and mechanism of transport, screening of excipients and formulation in transport modulation
and to study permeability of drugs in various experimental settings.

Tissue Mounted in Ussing Chamber

The technique of performing drug absorption studies, using intestinal tissue mounted in
Ussing chamber, was first introduced by Ussing and Zerahn8 for studying active transport of
sodium as a source of short circuit current in isolated frog skin. Later on, it was modified to
study the transport of drugs. In this system, the drug can be exposed at either the mucosal side
(apical side of enterocytes) or the serosal side (basolateral side of enterocytes). The procedure
for carrying out the intestinal Ussing chamber studies is described by Jezyk et al.9
Albino rats, weighing 225–275 g, are used. After an overnight fast, the animal is decapitated.
The small intestine is removed and small sections, 2.5–3 cm in length, are made. These are
opened along the mesenteric border. The tissue is mounted in Ussing chamber, which is then
placed in a 37°C heating block, aerated with 95% O2/5% CO2 and filled with 5 ml buffer at
37°C and pH 7.4. After 15 minutes of equilibration period, the original buffer is replaced with
warm fresh buffer solution. The transport studies are initiated by the addition of labeled and
unlabeled drug to either the apical or the basolateral chamber. Samples of 500 µl are taken
from the receiver chamber every 30 min for up to 120 min and replaced with fresh warm buffer
of same pH. Sample activity is then measured by liquid scintillation counter. Some researchers
have used a stirring rotor system instead of gas lift, to avoid excess foaming when surfactants
or proteins are used in buffer solution preparations.10
 Absorption and Metabolism 561

The usefulness of Ussing chambers for intestinal transport studies has long been recognized,
and they have also been used to study the intestinal metabolism of xenobiotics.11 When
properly equipped with electrodes, Ussing chambers are useful for studying the effects of
compounds on electrophysiological parameters of the intestinal barrier.3 However, to study
this effect, integrity and viability of intestinal segments is very important and this is ensured
by electrophysiological parameters, like transepithelial resistance (TEER) responsible for
tissue integrity and short circuit current (SSC) reflects the ionic flux across the membrane
epithelium.12 Molecules like mannitol and PEG 400 can be used as marker to check the integrity
of cell layers of intestinal segment.

Porcine Intestinal Tissue System

Healthy porcine intestinal tissue mounted in an Intestine TM system to predict human intestinal
absorption of compounds with different chemical characteristics and within bio relevant
matrices has been a new approach used. Feasibility of this new approach to study regional
differences (duodenum, jejunum and item) in permeability of compounds and to study the
effects of luminal factors on permeability was also investigated. The authors concluded that
this system can be applied as a reliable tool for the assessment of intestinal permeability in the
absence and presence of bio relevant samples.13

Cell Culture Models

Cell culture models are based on the assumption that intestinal epithelium, in the form of
monolayer of cells, is the main barrier for the drug molecules to reach the systemic circulation.
These models are used to perform rapid screening of compounds for their absorption across
the intestine. As enterocytes present in the intestinal epithelium play a major role in the
absorptive functions, various immortalized tumor cells having intestinal epithelial cells are
used to investigate the transport of drugs across the intestinal epithelium. One of the most
commonly used cell lines for studying the drug absorption is Caco-2 cell line.

Caco-2 Cells
Caco-2 cells are derived from human colon carcinoma cells and have similarity to the intestinal
enterocytes. Drug transport studies can be carried out, as recently described by Kim et al.14 and
Pauli-Magnus et al.15 Caco-2 cells are grown as polarized monolayers on semiporous filters.
During this period, P-glycoprotein is expressed on their apical surface. These cells allow
study of vectorial transcellular transport, i.e. basal to apical and apical to basal. Cells of passage
number 33–50 are plated on polycarbonate transwell cell culture insert plates with a cell count
of 2 × 105 cells/well. Transport experiments are performed on day 7 after plating. About 1 hour
prior to the start of the experiment, the medium in each compartment is replaced by transport
medium. Test drug is then added for the transport experiments either in the apical or the
basolateral compartment and the amount of drug appearing in the opposite compartment
(basal or apical) after 1, 2, 3 and 4 h is measured in 25 µl aliquots. Net basal to apical transport
is calculated after 4 hours by substracting the apical to basal from the basal to apical transport
rate. Permeability coefficient (Papp) is determined according to the equation:
Papp = dQ/dt * 1/(A * Co) [cm/sec]
562 Drug Screening Methods

Where, dQ/dt (µmol/sec) is the transport rate, Co (µmol/cm3) is the initial concentration
in the donor chamber and A (cm2) is the surface area of the monolayer.16 Inhibition of
P-glycoprotein-mediated transport across confluent Caco-2 cell monolayers is determined in
a similar manner after addition of the putative inhibitor to both compartments. Experiments
are conducted only in those wells that show transepithelial electrical resistance (TEER) of more
than 350 ohms. TEER is verified after each transport experiment in all the wells to determine
the effect of test substance on the monolayer integrity.
Apart from expression of P-glycoproteins, they also express transporter protein and phase
II conjugation enzymes to model a variety of transcellular pathways as well as metabolic
transformation of test substances. Caco-2 cells, in contrast to normal cells, lack expression of
cytochrome P450 isozyme particularly CYP 3A4, however, drug treatment of Caco-2 cells with
vitamin D3 can induce this enzyme.
Caco-2 cell permeability assays may be used employing LCMS and LC-MS–MS for
simultaneous measurement of multiple compounds. Caco-2 cells are useful in studying more
than one pharmacokinetic parameter.17
Permeability characteristics of HT29-18-C1 colonic epithelial cell line with Caco-2 have
been compared. It was concluded that HT29-18-C1 monolayers can be used to study drug
permeability at transepithelial electrical resistance (Rt) values similar to human intestine
without the need for Ca2+ chelation and as such they offer a useful alternative to Caco-2 for
modeling intestinal drug absorption.18

Other Cell Lines

Various other cell lines like Madin Darby Canine Kidney (MDCK) cells isolated from a dog
kidney by Madin and Darby,19 Intestinal Epithelial Cell line (IEC)20 and Rat Intestinal Epithelial
(RIE) cell line21 are also being used for drug intestinal absorption studies.

Advantages and Disadvantages of Cell Culture Models

Cell lines offer a variety of advantages:
• Easy to work with once cell culture conditions are established
• Less amount of test drug needed than in other systems
• Importance of different cell populations in absorption and metabolism of drugs can be
studied by culturing specific cell types like enterocytes, crypt cells, etc.
Some of the disadvantages associated with the use of cell culture models include:
• Some are cancerous in origin, and thus, may not mimic the exact physiological features of
normal intestinal cells
• Are not necessarily phenotypically stable and properties may differ with passage number
• Great expertise is needed in handling cell culture systems
• Molds, bacteria and fungi can infect the cultured cells and lead to erroneous results.

In Situ Model
In Situ Rat Gut Perfusion
In situ rat gut perfusion technique is an important model for the investigation of intestinal
transport, since in situ perfusion of the intestinal segments in anesthetized rats more closely
 Absorption and Metabolism 563

mimics the in vivo absorption studies. A significant advantage of the model is that biliary
excretion and enterohepatic circulation are eliminated, thus allowing the study of intestinal
events in isolation.
Male Wistar rats, weighing 225–275 g, are used. Overnight fasted animals are anes­thetized
with urethane 30 min before surgery. The small intestine is exposed through a midline
longitudinal abdominal incision. A silicon cannula is then inserted into the duodenum and an
outlet cannula is inserted just proximal to the ileocecal junction. The intestine is then flushed
using isotonic saline followed by infusion of air to remove the remaining solution. The drug
solution with or without inhibitor is then infused and the effluent samples are estimated for
drug content. The amount of the drug measured in the inlet and the outlet cannula is used to
calculate the permeability coefficient of the drug across the small intestinal epithelium.22,23

In-silico Model
For in-silico predictions of gastrointestinal drug absorption in pharmaceutical products
development, SjÖrgen E et al. 201324 successfully developed a mechanistic absorption model.
The GI-Sim deployed a compartmental gastrointestinal absorption and transit model as well
as algorithms describing permeability, dissolution rate, salt effects, partitioning into micelles,
particle and micelle drifting in aqueous boundary layer particle growth and amorphous or
crystalline precipitation. The model’s overall predictive performance was good in screening
the selected APIs.

METABOLISM STUDIES
An overview of different in vitro models such as supersomes, microsomes, cytosol, S9 fraction
cell lines, transgenic cell lines, primary hepatocytes, liver slices, isolated perfused liver with
advantages, disadvantages and future prospectives is given by Brandon et al. 2003.25 For
successful screening of drug candidate’s selection of proper models and data interpretation
are crucial. Methodologies for investigating drug metabolism at the early drug discovery stage
and prediction of hepatic drug clearance and P450 contribution are reviewed by Emoto C
et al. 2010.26 Recently, different in vitro and in vivo preclinical experimental models of drug
metabolism and drug disposition in drug discovery and development are described in detail
by Donglu et al. 2012.27

In vitro models
For drug metabolism studies, various in vitro models are used. These models involve the use of
liver slices and hepatocytes in evaluating the metabolism of the test compound. The advantages
offered by these models are the presence of drug metabolizing enzymes like CYP450s, other
microsomal and cytosolic enzymes and also cofactors that contribute to metabolism. However,
interindividual genetic variations in enzyme expression lead to differences in variable rate
of drug metabolism. Such genetic variations, leading to variable human CYP 3A-mediated
metabolism are covered by Lamba et al. 2002.28 These models can also be used for studying
564 Drug Screening Methods

the hepatotoxic potential of the compounds. However, these cellular systems also have some
inherent disadvantages, like the cells and slices cannot be easily frozen and subsequently
thawed for use in assays, cell culture of hepatocytes are primary and cannot be passaged and
these cellular systems cannot be prepared from liver tissue that has previously been frozen.
There is also an alternative approach of using a complete mixture of enzymes that involves
working with simple homogenates of liver or other tissues. Although all of the enzymes are
present in the system containing homogenates, but their cofactors are diluted.

Hepatocytes
Hepatocytes are the functional units of liver, the major site responsible for xenobiotic and drug
metabolism. Hepatocytes isolated from a large number of laboratory animal species, such as
rat, mouse, dog, monkey as well as human liver tissue, have been used. Hepatocytes can be used
to study several aspects of drug metabolism, such as metabolite profiling, biotransformation
pathway reaction phenotyping, metabolic drug-drug interactions comparison of metabolism
of xenobiotics and drugs and the detoxification of toxicants of xenobiotics derived from
metabolism. The collagenase perfusion method, first developed using rat liver, is now used
for preparation of hepatocytes using various species of animals. Collagenase perfusion is used
because the extracellular matrix of the liver comprises largely of collagen (1 mg/g of wet weight
in rats and 5 mg/g in humans), thus yielding the highest viable hepatocytes.
To carry out the isolation of hepatocytes from rat liver, liver is obtained and portal vein is
cannulated. To remove blood from the liver, it is perfused with calcium free oxygenated buffer
(Krebs-Ringer bicarbonate buffer, Hanks balanced salt solution or Dulbecco’s phosphate
buffered saline) at a flow rate of 20–40 ml/min under a hydrostatic pressure of 20–25 cm of H2O
for 5–10 min. Too high or too low perfusion rates can damage the liver tissue by causing shearing
and anoxia, respectively. EGTA (ethylene glycol bis β-aminoethyl ether N, N, N, N tetra-acetic
acid), a calcium-chelating agent, can be added to the perfusion buffer to favor cleavage of
hepatocytes. Collagenase is then added to the reservoir and the liver perfused for an additional
10-15 min or until visible softening is evident. Subsequently, disruption or removal of liver
capsule and gentle stroking of the cell mass leads to dissociation of liver cells. The resulting cell
suspension is filtered through a double layer of sterile cotton gauze or nylon mesh of 50-250
µm. The cells are washed twice by centrifugation and the cloudy supernatant, which contains
residual collagenase, nonparenchymal cells, erythrocytes, nonviable hepatocytes and cell
debris, is discarded. The hepatocytes are then centrifuged again through a density gradient of
porcell to obtain hepatocytes of the highest viability.29 Using trypan blue exclusion assay; the
hepatocytes are then tested for their viability. More than 90% viability is desirable. The cells
are then cultured in collagen-coated (95–98% type I collagen), 100-mm diameter plates at a
density of 107 cells/7 ml of culture medium. Hepatocytes are cultured for the first 4 h in 1:1
(v/v) Ham’s F12/William’s medium E supplemented with fetal calf serum, sodium bicarbonate,
penicillin, streptomycin, ethanolamine, transferin, insulin, dexamethasone, glucagon, linoleic
acid, glucose, sodium pyruvate, ascorbic acid and trace elements and subsequently cultured
in serum free medium30 in a humidified atmosphere of 5% CO2/95% air at 37°C. The drug with
and without inhibitor is put in the different wells of the plate to study the metabolism of the
drug.
 Absorption and Metabolism 565

In vitro human hepatocyte-based experimental systems for the evaluation of human drug
metabolism, drug-drug interactions, and drug toxicity are recently described by Shahi J et al.
201031 and Li, 2014.32

Precision-cut Liver Slices

Precision-cut liver slices are used frequently for in vitro metabolism studies. Liver slices from
a large variety of laboratory animals, including human beings, can be used for metabolism
studies. The liver samples are obtained and stored in ice-cold culture medium consisting of
Earle’s Balanced Salt Solution (EBSS) containing 25 mM of D-glucose, 50 µg/ml of gentamicin
and 2.5 µg/ml of fungizone till the initiation of slicing procedure. The medium is pregassed with
95% O2 and 5% CO2. Tissue is prepared for slicing in tissue cylinders with the help of a motor
driver tissue coring tool. Using a Krumdieck tissue slicer, 200-300 µm tissue slices are prepared
in well-oxygenated culture medium. The tissue slices are now floated onto steel mesh inserts
and incubated in culture medium in polystyrene vials for 30 min at 37°C in an atmosphere of
95% air and 5% CO2, using a roller system rotated at 9 rpm. Pre-incubation enables the liver
slices to restore their ATP levels. Subsequently, the tissue is incubated with culture medium
containing the test drug and the medium is analyzed for metabolites.33
Precision-cut tissue slices from various organs and different species can provide excellent
in vitro models for biotransformation and chemical-induced organ specific toxicity. They
offer the advantage of maintaining tissue architecture and avoiding damage to the cells as
may occur during cell isolation procedures. Further, the technique for preparing tissue slices
is relatively easy and can be employed for preparing tissue slices from different organs from
different species.

Microsomes
Majority of drugs administered by the human beings are metabolized in the liver by CYP3A4
enzymes. It is important to choose a proper experimental system to study the drug metabolism.
Microsomes of varied origin are used in vitro. The experimental approaches using cytochrome
P450 are reviewed by Zuber et al. 2002,34 based on drug metabolising system. Briefly, for
CYP1A mediated pathways all the commonly used experimental models are appropriate
except probably the dog; on the contrary the dogs are suggested to be suitable for modeling
of processes depending on the CYP2D. With CYP2C, which is possibly the most large and
complicated subfamily, the system based on monkey (Maccacus rhesus) is suggested to be a
good representative. The CYP3A is suggested to be well modeled by pig or minipig CYP3A29.
The use of microsomes is another in vitro approach to study the metabolism of test compound
and patterns of biotransformation. For the preparation of liver microsomes, the organs from
the sacrificed animals are immediately removed and placed in ice. Using a mechanical meat
grinder, these are grounded and then homogenized in 2 volumes of 0.25 M sucrose, 0.05 M
HEPES (pH 7.4) using a potter Elvehjem homogenizer coupled with a motor driven Teflon
pestle (2,500 rpm). The homogenate is then diluted by adding 25% w/v buffered sucrose and
centrifuged at 10,000 g for 20 min to remove the cell debris, nuclei and mitochondria. The
mitochondrial supernatant is again centrifuged for 45 min at 1,00,000 g. The microsomal pellet
so obtained is suspended in 0.15 M KCl containing 0.02 M HEPES (pH 7.4) and centrifuged at
566 Drug Screening Methods

1,92,000 g for 45 min. The washed pellet is resuspended in buffered KCl.35 The protein content
of the microsomal preparation is estimated by the method of Lowry et al.36
To perform metabolism studies, substrate is preincubated with 50 µg of microsomal
protein, 30 mM MgCl2, and 50 mM KH2PO4, pH 7.4 for 2 min in a shaking water bath at 37°C.
The reaction is started by the addition of 4.8 mM NADPH and incubated for another 5 min.
The reaction is terminated after 5 min with the addition of 1.7 ml of ice-cold ethanol. The
incubation performed in presence of different concentration of inhibitors is used to determine
the nature of the inhibition.37
The microsomal preparations can be stored in liquid N2 or at –80°C for long duration. Human
liver microsomes can be availed from industries or from other sources. The disadvantage with
human liver microsomes is the presence of different subfamilies of CYP450. Normally, liver
is considered as a main enzyme source, although depending on the specific enzyme and
xenobiotic, other organs may also play an important role in drug metabolism.38

Recombinant Systems
Due to the technical difficulties in purification of specific CYP450 enzymes from human
liver and the unavailability of tissues, various recombinant systems expressing CYP450s
have been developed for drug metabolism studies. Several cell lines expressing individual
human CYP450s, including V79 Chinese hamster39 and human lymphoblastoid cell lines,
have been developed and are commercially available. High level of CYP450 expression has
also been achieved in yeast,40 insect cells using recombinant baculovirus41 and bacteria.42 For
toxicological studies, expression of P450 in mammalian cells is most appropriate as exemplified
by activation and inactivation of chemotherapeutic drugs.43 The advantage of recombinant
systems is the availability of large amount of purified enzymes for drug metabolism studies,
but the cost of these systems has limited their use.

Acknowledgments
The encouragement from Dr GN Singh, Secretary-cum-Scientific Director, Indian
Pharmacopoeia Commission, Ghaziabad and Professor SK Gupta, Delhi Institute of
Pharmaceutical Sciences and Research, Pushp Vihar, New Delhi-110017, during the revision
of this chapter is gratefully acknowledged. Typing assistance provided by Ms Reena Tripathi is
also acknowledged.

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 cHAPTER

39 Hormones of Pituitary, Thyroid,

Parathyroid, Adrenal Cortex,
Ovary and Testes
Introduction
Functions of the body are mainly regulated by two control systems, the nervous system and
the endocrinal system. The endocrine system is a collection of glands that secrete chemical
messengers known as hormones. A hormone is a specialized molecule that is synthesized,
stored and secreted by a group of specialized cells known as the endocrine gland. These glands
are ductless gland and secret hormones directly into the blood stream to reach the target
organ possessing cells with appropriate receptors. The hormones deliver the message through
receptors to bring about changes in cellular functions.
The neural center for the control of endocrinal secretions is located in the hypothalamus.
The hypothalamus secretes releasing or inhibitory hormones and sends them to pituitary via
hypothalamic-hypophyseal portal veins. The pituitary gland often known as the ‘master gland’
is responsible for secretion of a battery of hormones that collectively influence virtually all
physiological processes of the body (Figs 39.1 and 39.2).
An integrated feedback regulation mediated by complex interactions among the
hypothalamus, pituitary and peripheral endocrine glands is present, and better understanding
of the mechanisms responsible for the interactions provides the rationale to diagnose and to
treat the endocrine disorder. Various endocrine disorders mostly arise from the endocrine
gland dysfunction, which may result in excessive or scanty secretion of hormones resulting in
clinical symptoms.
The elucidation of the structures of various hormones of different origin with the
advancement of the protein chemistry made it possible to produce synthetic peptide agonists
and antagonists that play very important role in diagnostic as well as therapeutic application.
To evaluate these drugs, various in vitro and in vivo experimental models have been developed.
This chapter deals with the different biological screening models of hormones, which are useful
to evaluate various natural and synthetic derivatives of hormones for their pharmacological
activity.

growth hormone
Growth hormone (GH) is the most abundant anterior pituitary hormone. It produces growth at
open epiphyses via stimulation of insulin-like growth factor I (IGF-I, somatomedin C). It also
570 Drug Screening Methods

Figure 39.1: Hormones of hypothalamus and anterior pituitary

causes lipolysis in adipose tissue and growth of skeletal muscle. In vitro and in vivo bioassays
used for screening GH analogues include the following models.

In vitro studies
Glucose Uptake Inhibition
The conversion of glucose to lipid in murine adipocytes is inhibited by human growth hormone
(hGH) in a dose-dependent manner. This is a very sensitive in vitro method developed by
Foster et al.1 and is used to screen growth hormone analogues for their biological activity.

Procedure
Murine fibroblasts cells are grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
containing antibiotics. These cells are plated in 60 to 100 mm plastic culture dishes at a
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 571

Figure 39.2: Hormones of hypothalamus and posterior pituitary

density of 200 cells/cm2 and are allowed to be confluent in 25 mM galactose and 10% fetal calf
serum supplemented media with 90% air and 10% CO2 at 37°C. Medium is replaced every 2
to 3 days; and once fibroblasts are confluent, they are converted to characteristic adipocytes
by incubation for 48 h in a medium supplemented with 25 mM glucose, 10% FCS, 0.5 mM
methylisobutylxanthine, 2 µg/ml insulin and 250 nM dexamethasone. The medium is then
replaced by the medium which contains 10% fetal bovine serum and 2 µg/ml of insulin every 2
days for 5 to 8 days until at least 70% of the cells have characteristics of adipocytes as assessed
by phase contrast microscopy.
For the evaluation of growth hormone activity, the DMEM medium is uniformly
radiolabeled with [14C] D-glucose. Cultures are incubated with increasing concentrations of
standard and test preparations or with medium alone for 24. The cells are treated with Doles
reagent followed by the scraping of cells and finally the contents are transferred to glass tube.
Lipids are extracted and the radioactivity is determined by the scintillation counter. Lipid
accumulation in the control cells is considered to be 100%. Decrease in lipid accumulation is
directly proportional to the increase in dose range. From dose response curve, activity of the
unknown can be calculated.
572 Drug Screening Methods

Human Growth Hormone (hGH) Bioassays

Bioassay of hGH can be assessed either by evaluating biological interaction of hGH with its
receptor through Ligand binding assay or its intracellular activity that it evokes on particular
cell. Based on affinities of binding on receptor it is possible to screen the agents having hGH
activity. hGH bioassays include radioreceptor assays, receptor modulation assays and cell
proliferation assays.

Radioreceptor Assays
The biological potency of hGH is determined by employing competitive binding between
unlabeled and radiolabelled hGH to its receptor. It has been known that radiolabeled agent
that is bound to the receptor can be replaced by the unlabeled agent in direct proportion to
their biological activity.

Procedure
The method of radioimmunoassay was proposed by Nederman and Sjodin et al.2 Human
lymphocytes cells (IM-9) are cultured in RPMI 1640 medium complemented with FCS (l0%),
L-glutamine (2 mM), streptomycin (100 mg/ml) and penicillin (100 IU/ml). These cells in
the late log or early plateau phase are harvested and washed in assay buffer (100 mM Hepes,
120 mM NaCl, 1.2 mM MgSO4, 2.5 mM KCI, 15 mM sodium acetate, 10 mM glucose, 1 mM
EDTA and 1% bovine serum albumin, pH 7.0). The cells are resuspended in assay buffer and
preincubated at the desired temperature in a shaking waterbath for 1 h before incubation was
started.
The cells (400 ul) are added to the tubes containing purified tracer [125I] hGH (about
1.0 ng/ml) and serial concentrations of unlabeled hGH or other samples to be assayed.
The incubation is carried out for 90 min at 30°C in a constant temperature water bath. To
determine binding, duplicate aliquots of the incubation mixture are layered over iced assay
buffer in polypropylene microfuge tubes. The samples are centrifuged at 90,00 g for 1min. The
resulting pellets are washed three additional times with iced assay buffer. The radioactivity in
the duplicate washed pellets and in corresponding duplicate medium samples is counted in
a gamma counter.
Total binding is calculated as percentage of radioactivity in the medium samples. Non-
specific binding, defined as the binding of [125I] hGH in the presence of unlabeled hGH, is
subtracted from the total binding to obtain the specific binding.

Cell Proliferation Bioassays

The bioactivity of hGH can be evaluated by cell proliferation assays utilizing human cell lines
or other cell types. The cell proliferation assays are based on the somatotrophic response
produced by hGH in dose dependent manner as described by Ishikawa et al.3 This assay
reflects not only receptor binding of hGH but also mimic signal induction at the intracellular
level.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 573

Procedure
Establishment of Ba/F3-hGHR
Mouse pro-B cell lymphoma cells expressing hGH receptors are developed as described
by Wada et al.4 The Ba/F3-cells are cultured in RPMI-1640 supplemented with 10% FCS,
50 mM 2-mercaptoethanol, 50 mg/ml streptomycin sulfate, 50 U/ml penicillin G, and
1 ng/ml recombinant mouse IL-3. Fifty micrograms of mammalian plasmid pCXN2-hGHR are
transfected into 1×107 Ba/F3 cells. Cells expressing hGHR are cultured in selection medium
(RPMI-1640 medium containing 1 mg/ml G418, 10% FCS, 50 mM 2-mercaptoethanol, 10 nM
22K-hGH, and antibiotics). Resultant hGH-responsive cells are examined for hGHR expression
by binding assay to [125I] 22K-hGH.
Bioassay with Ba/F3-hGHR cell line
The procedure for the bioassay was explained by Ishikawa et al.3 Approximately, 4h before the
start of experiment, cells are washed twice with assay medium (RPMI 1680, supplemented
with 5% FCS, 50 mM 2-mercaptoethanol, and antibiotics, without hGH) and are transferred to
the assay medium and incubated for 4-6 h to slow down the rate of cell replication. The cells
are collected by centrifugation (3 min at 1000 rpm) and resuspended in the assay medium
at a concentration of 1×105 cells/mL of which an aliquots (200 µl) distributed in each well of
96-well microplate. Standards hGH are diluted with 0.01 m PBS supplemented with 0.1% BSA.
Samples are incubated at 56°C for 40 min to inactivate the serum. Standards or samples (25 µl)
are added to each well. The cultures are incubated in a CO2 incubator (5% CO2, 95% air) for
48 h at 37°C. At the end of the incubation, the colorimetric end point is determined by an
eluted stain bioassay (ESTA). Briefly, 20 µl MTT solution (3-[4, 5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) (5 mg/ml in 0.01 M PBS) is added to each well and incubated
at 37°C for 4 h in a CO2 incubator. During this time, activated cells reduce the yellow MTT
salt to purple formazan. The plate is centrifuged at 800 rpm for 10 min, and the stain is eluted
into dimethyl sulfoxide (100 µl). Bioactive responses are determined with a kinetic microplate
reader reading optical densities at the test wavelength of 550 nm and a reference wavelength of
650 nm to correct for differential scattering. A control serum with known bioactivity was used
at every assay for the quality control.
The bioactivity of hGH is evaluated on the basis of cell proliferation in the dose dependent
manner which serve as a standard curve for quantification of unknown sample.

Immunoassays
These tests are the widely used, sensitive proficiencies for the determination of hGH in
biological fluids with high degree of accuracy and reproducibility. These are based on
detection of immunological epitopes present on the polypeptide human growth hormone.
Antibodies are being developed on the basis of detection of these epitopes such as monoclonal
and polyclonal antibodies. However, these techniques are having shortcomings viz. it do
not reflect the biological activity of hGH. Immunoassays include radioimmunoassays
(RIAs), immunoradiometric assays (IRMAs), enzyme-linked immunoassays (ELISAs) and
immunofunctional assays (IFAs).
574 Drug Screening Methods

Immunofunctional Assay (IFA)

hGH has been known to contain two receptor binding epitopes and induces dimerization
of receptor as an initial step of signal transduction mechanism in target cells. Immuno­
functional assay is developed to combine the speed of immunoassay and merit of bioassay.
The IFA is based on the interaction of hGH with an immobilized mAb specific for binding
site 2 epitope of the hormone and with biotin-labeled recombinant hGH (rhGH)-binding
protein (hGHBP), which represents the full length hGH receptor ecto domain (Strasburger
et al.).5

Procedure
The method of IFA was demonstrated by Strasburger et al.5 Anti-hGH mAb 7B11, which binds
to an epitope largely overlapping with binding site 2 of the hGH molecule, is adsorbed to flat
bottom 96 well microtiter plates by incubation of 500 ng mAb 7811 in 200 µl 50 mmol/l sodium
phosphate buffer, pH 9.6. The plates are sealed with a self-adhesive cover film and stored at
4°C for 12 h to 1 month. After aspiration of the coating solution, the plates are washed three
times with washing solution. Twenty-five microliters of standard or sample are pipetted into
the wells, followed by 175 µl assay buffer. The plates are incubated at ambient temperature for
3 h on a horizontal shaker. After a 3-fold wash step, 50 ng/well biotinylated rhGHBP are added
in 200 µl assay buffer, and the plates are sealed and incubated overnight (12-16 h) at 4°C.
After a 3-fold wash step, the plates are incubated with streptavidin-europium and processed
as described above. The calibration curve is produced by plotting the hGH concentration of
standards and the signal from the time-resolved fluorometric end point. Concentrations of
unknown samples are read by interpolation of the signal obtained on the standard curve.

In vivo methods
Weight Gain Model
After attaining maturity at six months of age, female rats continue to grow at a very slow rate
and they almost reach the plateau of weight gain process. However, they can be induced to
grow and gain weight by the administration of growth hormone. Though this method needs
a large amount of test materials, it is highly specific for the evaluation of growth hormone
activity.

Procedure
Ten female Wistar rats weighing between 250 to 280 g who fails to gain more than 10 g in 20
days period are used. Various doses of unknown growth hormone preparations and standard
dissolved in saline are injected subcutaneously for 20 consecutive days. During this period,
weight gain between 10 to 40 g is expected. A relationship exists between the logarithm of the
daily dose level and the increase in body weight of the rats in grams.
The mean values of the weight gain are calculated after administration of 2 doses of standard
and 2 doses of test preparations used for 2+2 point assay.6
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 575

Rat Model of Suboptimal Nutrition

In the regulation of secretion of GH and insulin like growth factor I (IGF), nutritional status and
energy intake play a critical role. Poor nutritional status and energy intake reduce the serum
GH and IGF levels leading to retarded growth. In animals with suboptimal nutrition, growth
can be stimulated by administration of growth hormone.

Procedure
The procedure was described by Carrillo et al. (1998).7 Four-month-old Sprague Dawley rats
are maintained under standard laboratory conditions with ad libitum access to water. Daily
record of body weight and estimation of food intake is obtained for each animal. Various
doses of unknown growth hormone preparation and standard dissolved in saline are injected
subcutaneously in the back. Control animals are treated with same volume of normal saline.
Within each group the rats are fed with balanced purified 1:1 carbohydrate to fat diet at three
energy levels, i.e. ad libitum and 80 and 60% of ad libitum. Protein and micronutrient levels are
maintained at constant level. Each rat from ad libitum group is paired with one rat each from
80 and 60% diet restriction groups. The amount of food to be fed to the diet restricted groups is
calculated from the amount of food intake by corresponding rat of ad libitum group according
to the following formula:
Food consumed by the ad libitum fed rat in the previous day/weight of this rat in the
previous day) × (0.8 or 0.6, depending on the restriction level) × (the current weight of the rat
for which the food is calculated).
Body weight is recorded daily and tail length measured from hairline to the tip of the tail.
For each rat weekly average is determined for body weight gain, increase in tail length, calorie
intake and food sufficiency. Calorie intake is calculated by multiplying the amount of food
intake by calorie density of diet. Food efficiency is calculated by multiplying the ration of
amount of food intake and body weight gain by 100. In addition at the end of the experiment
the rats can be sacrificed and blood is collected for estimation of insulin and insulin growth
factor using radioimmunoassay. The growth hormone treated rats in restricted diet groups
show higher growth as compared to vehicle treated groups.

Tibia Test
Cessation of epiphyseal growth occurs after hypophysectomy. Following hypophysectomy,
width of the epiphyseal cartilage is markedly reduced. Remarkable increase in the width of
the epiphyseal cartilage plate can take place by administration of the growth hormone to
hypophysectomized rats.

Procedure
Hypophysectomy is done in female Sprague Dawley rats at the age of 26 to 28 day. They are
used for the experiment 12 to 14 days after the operation. Animals are randomly divided into
different groups of standard and test preparations, each comprising of 6 to 8 animals. The
drugs are administered (i.p.) twice daily for 4 days. On fifth day, animals are sacrificed and
both tibias are dissected out and freed from soft tissue. The bones are split into half with a
576 Drug Screening Methods

sharp knife. The halves are washed in water, then immersed in acetone and again washed
in water; and thereafter, they are placed in 2% silver nitrate solution and rinsed with water.
While rinsing with water, bones are exposed to a strong light so that calcified portions of the
bone turn dark brown. The stained tibia are then processed to a microscopic stage and the
width of the uncalcified cartilage plate, which does not take stain, is measured under low
power with a calibrated micrometer eyepiece. Ten individual readings are taken across the
epiphysis.
Mean values are obtained and average is calculated for the each dose group. The potency
ratio of the test preparations is calculated using 2+2 point assay.8

Uptake of 35S
In hypophysectomized animal, radiolabeled sulfur uptake into the cartilage is greatly reduced
and is found to be restored following growth hormone application. This phenomenon can be
utilized to evaluate the growth hormone activity in the compounds.

Procedure
Hypophysectomized female Sprague Dawley rats (21 day old) are used for this experiment.
Three weeks after hypophysectomy, animals are given growth hormone together with
radiolabeled sulfur (i.p.) once daily for 4 days. Eight to ten animals are used for various doses
for standard and test preparations. The rats are sacrificed 24 h after the last injection and the
amount of radiosulfur present in seventh rib cartilage is determined. The radiosulfur uptake is
directly proportional to the dose of the growth hormone.
Mean value of each group is calculated and potency ratios of the test preparations compared
to the standard are calculated with the help of 2+2 assay.9

prolactin
Prolactin is a 198 amino acid peptide hormone produced in the anterior pituitary. It is the
principal hormone responsible for lactation. A deficiency of prolactin, which can occur in
states of pituitary deficiency, is manifested by failure to lactate or by a luteal phase defect.
Hyperprolactinemia can produce galactorrhea and hypogonadism and may be associated
with symptoms of a pituitary mass. No preparation is available for the use in prolactin deficient
patient. For patients with symptomatic hyperprolactinemia, inhibition of prolactin secretion
can be achieved with bromocriptine and other dopamine agonists.

Radioimmunoassay
Prolactin is a glycoprotein hormone with specific activity. For evaluation of gonadotropin-
releasing activity or prolactin inhibiting factor activity, radioimmunoassay is necessary.

Procedure
Standards or samples are incubated with antiserum (rabbit-anti-rat-prolactin) for 24 h at 4°C.
Now 125I-rat-prolactin is added and incubated for another 48 h. The secondary antibody (1:50),
200 ml/tube is added and incubated for 48 h at 4°C. Separation is done with 1 ml ice-cold
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 577

phosphate-buffered saline, pH 7.4. The vials are centrifuged at 13,000 g for 15 min. Thereafter,
the supernatant is discarded and the residue is counted for 1 min in a gamma counter.10

Pigeon Crop Method

Pigeons are very sensitive to lactogenic hormone. Suitable assay method has been developed
on the basis of this phenomenon.

Procedure
Pigeons of 2-3 months old of either sex are injected with various doses of standard and test
preparations once daily for 4 days intraperitoneally. On the 5th day, the birds are euthanized.
A midventral incision is made through the skin and crop wall from keel to head. The contents
of the crop and the adherent crop milk are removed. The two lateral pouches are removed,
cleaned and wet weight is recorded.
Mean values of various doses of standard and test preparations are calculated and plotted
against log dose of the corresponding group. Potency ratios with confidence limits are also
calculated.11

Lactation in Rabbits
For mammalian assays of lactogenic hormone, only rabbits and guinea pigs have shown
satisfactory results so far. There is an induction of mammary growth and milk secretion
of pseudopregnant animal following prolactin administration. This is useful to evaluate
compounds having prolactin-like activity.

Procedure
Pseudopregnancy is induced in mature estrus rabbits by 50 IU of human chorionic
gonadotropin given intravenously. On the 14th day, animals are examined for the presence
of well-developed mammary gland, which is characteristic of pregnancy. Various doses of
standard and test preparations are injected (s.c.) once daily for 6 days. Animals are euthanized
on the 7th day and the abdominal skin is incised in the midline and separated from the
mammary gland underneath. The degree of glands enlargement with secretion is rated as the
following:
– No response
+ All ducts are filled with milk
++ All ducts and most of the lobules are filled with milk
+++ Entire gland is filled with milk
++++ Mammary glands are greatly extended with milk
Mean values of 6 animals in each group of test are compared with the standard mean.12

corticotropin
Adrenocorticotropin is a peptide hormone produced by anterior pituitary. Its primary
endocrine function is to stimulate synthesis and release of cortisol by the adrenal cortex.
Corticotropin can be used therapeutically, but a synthetic derivative, e.g. cosyntropin is more
commonly and almost exclusively used to assess adrenocortical responsiveness.
578 Drug Screening Methods

In vitro studies

Receptor-binding Assay
Adrenocorticotropin hormone (ACTH) was one of the first hormones that were shown to cause
an increase in intracellular cAMP and one of the first peptides shown to bind specifically and
reversibly to a cell surface receptor.13 This receptor is primarily expressed in the adrenal cortex
and the role of ACTH is to stimulate steroid production by target cells.
Demonstration of specific-binding of ACTH to receptors is extremely difficult. Cloning of
the human ACTH receptor in 199214 permitted a detailed investigation of the ligand-binding
and agonist properties of ACTH and its analogues.

Procedure

Stable Transfection of HeLa Cells

Human HeLa cells are cultured in DMEM with 10% FBS. The mouse ACTH receptor
expression vector, in which the coding region of the gene is inserted into the pcDNA1 vector,
is co-transfected with the pTK and neomycin resistance expression vector. Cells are selected
in G418 (final conc. 800 µg/ml) and several resistant colonies are picked by ringing after 2
weeks. These cells are further cultured and are characterized for cAMP generation response
to ACTH.

Stimulation of Cells with ACTH and Analogues

HeLa cells (5×105) are seeded into 6 well tissue culture dishes 24 h before stimulation. On
the day of the experiment, cells are incubated in DMEM that include 1 mM 3-isobutyl-1-
methylxanthine and incubated for 20 min at 37°C with increasing concentrations (10-12 to 107
M) of ACTH and various analogues. After incubation, the cells and the media are placed into
Eppendorf tubes and boiled for 10 min, centrifuged at 10,000 rpm to discard the cell debris and
stored at –20°C until aliquots are assayed to determine levels of cAMP, which is measured by a
specific protein-binding assay.15

Ligand-binding Assay
The-binding assay is done by the method described by Penhoat et al.16 HeLa cells are seeded
into 12 well culture plates at a density of 106 cells/well. On the second day of the culture,
the cells are washed and incubated for 60 min at 20°C with increasing concentrations of
nonradio­active ACTH or various ACTH analogues and the reaction initiated on the addition
of (125 I-iodotyrosyl) ACTH (2000 Ci/mmol, final concentration 0.1 pmol/l) in DMEM. Post
incubation, the medium is removed and the cells washed for three times with 0.9% NaCl and
then dissolved in 0.5 M NaOH/0.4% sodium deoxycholate (w/v). Each point is determined in
triplicate and radioactivity is measured using a gamma counter. Specific-binding is determined
by subtracting from the total-binding. Binding parameters are determined using the LIGAND
programme.17
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 579

In vivo models
Thymus Involution
Corticotropin can reduce thymus weight in a dose dependent manner. Young Wistar rats are
sensitive to this effect of corticotropin.

Procedure
Sprague Dawley or Wistar rats of either sex of 7 to 10 days of age (10 to 15 g) are used and
animals from the same litter are preferred for this experiment. The rat pups are randomly
distributed into different groups of standard and test compounds. Drugs (both standard and
test agents) are injected subcutaneously at different doses in oily suspension once daily for 3
days. Animals are sacrificed after 24 h of the last injection. Thymus glands are immediately
dissected out and weighed. The response is expressed as the average of the individual weight
for each dose of standard and test drug.18
Dose response curve of average weight is plotted against log dose of the drugs and the
potency ratios are calculated by using 3+3 point assay.

Modifications
The sensitivity of the method can be enhanced by using the quotient between increase of the
weight of the adrenals and decrease of the weight of the thymus gland.

Depletion of Adrenal Ascorbic Acid

Temporary reduction in adrenal ascorbic acid is observed after the administration of pituitary
ACTH. The depletion of ascorbic acid is dose dependent. This relationship has been used for
the quantitative assay of ACTH.

Procedure
Hypophysectomy is done in male Wistar rat (100 to 200 g) one day prior to the experiment.
Three doses of test and standard are used. Test and standard preparations are dissolved in 0.5%
phenol solution and diluted with gelatin solution. The hypophysectomized rats are randomly
divided into 6 groups, each comprising of 6 to 8 animals. Each animal is injected (s.c.) with
various doses of test compounds and standard. Post-treatment animals are anesthetized
and both adrenal glands are removed, freed from external tissues, and finally their weight
is recorded. The glands are homogenized in 4% trichloroacetic acid and the ascorbic acid
determination is done according to the method of Roe and Kuether.19

Ascorbic Acid Determination

Trichloroacetic acid (4%) is added to 0.0, 0.5, 1, 2, 3, 4, 6, 8 ml of the 0.02% (w/v) ascorbic
acid solution and 1, 1.5 and 2 ml of the 0.2% ascorbic acid solution to reach final volume
of 8 ml. About 100 mg charcoal is added to each sample and is mixed by shaking for 1 min.
The solutions are filtered after 5 min and an aliquot of 0.1 ml of the 6% thiourea solution is
580 Drug Screening Methods

added to 2.0 ml of the filtrate followed by 0.5 ml dinitrophenylhydrazine solution. The whole
mixture is shaken and heated at 57°C for 45 min in a water bath. The solutions are then placed
in ice-cold water bath; and with further cooling, 2.5 ml of 85% sulfuric acid is added. At the
wavelength of 540 nm, the calibration curve is established using the solution without ascorbic
acid as a blank.

Preparations of the Adrenal Glands

The dissected adrenal glands are homogenized in 200 mg purified sand and 8 ml of 4% TCA.
The reagents are added in a similar way for the calibration curve. The potency including the
confidence limits is calculated by 3+3 assay.

Modifications
This ascorbic acid depletion test can also be performed in dexamethasone blocked rats.
However, the potency ratios of various synthetic corticotropins have been found to be different
in hypophysectomized rats.

Increased Corticosterone Level in Blood

In hypophysectomized or dexamethasone blocked rats, ACTH activity can be measured by the
increase in corticosterone level in venous blood. This phenomenon is useful to measure the
corticotropin activity of the corticotropin analogues.

Procedure
Male Sprague Dawley rats (150 to 200 g) are injected ACTH preparation or the standard
dexamethasone (5 mg/kg) subcutaneously 24 h and 1 h prior to the experiment. Eight animals
are taken for the each dose of test and standard. Rats are anesthetized with pentobarbital
(60 mg/kg, i.p.) at various time intervals after ACTH injection and blood is withdrawn by
cardiac puncture. One ml plasma is diluted with 2 ml distilled water and extracted with 5 ml
petrol ether in order to remove the lipids. The petrol ether is discarded and 2 ml of water layer
is extracted twice with 5 ml of methylene chloride by vigorous shaking for 15 minutes. The
methylene chloride phase is separated by centrifugation. Both methylene chloride extracts
are unified and shaken with 1 ml ice-cold 0.1 N NaOH. The water phase is immediately
removed and the methylene chloride extracts dried by addition of sodium sulfate. Five ml of
methylene chloride aliquot is mixed with 5 ml of fluorescence reagent. After vigorous shaking,
the methylene chloride phase is removed and fluorescence is measured with primary filters of
436 mµ and secondary filters of 530 to 545 mµ. To establish a calibration curve, concentrations
of 0, 20, 50, 100 and 250 µg/ml corticosterones are treated identically and measured in each
assay.20
Using 3 doses of test compounds and standard, activity ratios with confidence limit can be
determined with the help of 3+3 point assay.

Modifications
Corticosteroid can also be determined fluorimetrically in guinea pigs.21
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 581

GONADOTROPINS
Hypophysectomized Female Mice Model
Effect of FSH like activity can be evaluated by studying ovarian follicular development in adult
hypophysectomized mice.

Procedure
Adult female mice undergo hypophysectomy and 12 days later the animals start receiving
various doses of drug, subcutaneously daily for 4 day. The control group receives the same
volume of vehicle subcutaneously. Ovine FSH subcutaneously (4 µg/day) can be used as
standard to compare the FSH like activity of test drugs. After 4 days of treatment the ovaries are
examined for follicular development. The FSH activity leads to increased number of preantral
follicles (stage 1-3) as compared to control. The number of antral follicle, i.e. stage 4 and 5 starts
increasing after 1 day of treatment with concomitant decrease in the number of atretic follicles.
After 2 days of FSH treatment the follicles reach the stage 6 or the preovulatory size. The serum
levels of progesterone and androstenedione also increase in the drug treated groups if the test
drug possesses FSH like activity.22

Immature and Mature Male Rat Model

The male sex hormone, testosterone is secreted by the Leydig cells located in the interstitium
of testes. The functions of Leydig cells are regulated by Luteinizing Hormone (LH) however
the studies have revealed that FSH can modulate the LH response of Leydig cells indirectly
through Sertoli cells.

Procedure
Twenty-eight day old immature or 90 day old adult male rats are used. The test group of rats
receives different doses of drug by intraperitoneal route for 6 days. The control group receives
the same volume of vehicle by same route. Another group of animals treated with recombinant
human FSH (rFSH) (100 ng/day for immature rats and 300 ng/day for adult rats in 100 µl
of gelatin phosphate buffered saline (GPBS) by intraperitoneal route for 6 days is added to
compare the efficacy of test drugs. On the 7th day the animals are sacrificed and the testicular
tissue from different groups is used to isolate Leydig cells and estimate in vitro testosterone
production. The Leydig cells are isolated by collagenase dispersion of the testis and Percoll
density gradient fractionation as described by Hardy et al. (1990)23 and Sriraman et al. (2000).24
As described by Sriraman and Rao (2004)25 the Leydig cells (1×105) are cultured in 1 ml of
medium containing 0.1% BSA at 34°C for 4 h in shaking water bath with or without 100 ng
of hCG or 22-R-hydroxycholesterol (20 µM). The testosterone secreted in to the medium is
estimated by radioimmunoassay. There is a significant increase in the amount of testosterone
production in FSH treated groups.

Male Rat Model for LH Activity

The testosterone production by Leydig cells in testes is regulated by LH. LH stimulated increase
in testosterone production can be used to evaluate LH like activity of investigational drugs.
582 Drug Screening Methods

Procedure
Male Wistar rats weighing 200-250 g and 3-4 month old are used. The interstitial tissue
(containing Leydig cells) obtained from wet dissection of testes is incubated at 32°C for 1 h in
2 ml of Kreb’s – Ringer bicarbonate buffer containing 0.2% glucose at pH 7.4. The tissue
is further incubated with different concentrations of drug/vehicle/LH (100 ng/ml) in an
atmosphere of O2 + CO2 (95:5). The incubation tubes are then placed in ice and total volume
made up to 1 ml with Kreb’s-Ringer Bicarbonate glucose buffer. The tissue is now homogenized
using a sonicator. The homogenate is used for testosterone estimation by radioimmunoassay.
Addition of LH or drugs with LH like activity significantly increases the testosterone synthesis
as compared to control.26

Granulosa Cell Culture for Effects of FSH and LH

The granulosa cells from the ovarian follicles of rats when challenged with gonadotropins
in culture show increased secretions of steroidal hormones, the estradiol and progesterone.
These hormones can be estimated in culture medium to evaluate the gonadotropin like activity
of test drug.

Procedure
The rats undergo hypophysectomy at the age of 23 days. The granulosa cells are obtained
from the ovaries on day 26 using a needle and syringe. The collected cells are washed with
serum free medium thrice. After each wash cells are separated by centrifugation (200 g, 10
min). Finally, the cells are suspended in 1:1 mixture of DMEM and Ham’s F12 containing
gentamicin, glutamine and sodium bicarbonate. After checking for cell viability with trypan
blue, culture is established in multiwell plates at a concentration of 2×105 cells/ml. The culture
is maintained at 37°C under 5% CO2 atmosphere. To evaluate for the activity of drug the medium
is supplemented with vehicle/drug/FSH/LH. The medium is collected at 48 h with addition
of fresh medium. The medium is collected again after 48 h and the levels of estradiol and
progesterone are measured by radioimmunoassay. The supplementation of medium with FSH
(1-100 ng/ml) or LH (30 ng/ml) leads to significantly higher levels of estradiol, progesterone by
96 h of culture as compared to control.27

Oxytocins
Oxytocins are agents that increase the force and frequency of uterine contractions. Apart from
this, they also cause milk ejection reflex and with higher doses, they produce reduction in
blood pressure.

Isolated Uterus
Isolated uterus of virgin guinea pigs is very sensitive to oxytocin and is used to determine the
oxytocic activity of the unknown compounds. In contrast to guinea pig uterus, isolated rat
uterus is less sensitive, but it does not show spontaneous contraction in solution containing
low calcium and glucose concentrations.28
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 583

Procedure
Female Wistar rats weighing 120 to 200 g are used. Eighteen to 20 h prior to the experiment,
animals are injected (i.m.) with 100 mg of estradiol benzoate. One horn of the uterus is dissected
out and suspended in De Jalon’s solution, maintained at 32°C in tissue bath 10 ml in volume,
bubbled with 95% oxygen and 5% carbon dioxide. Suspended tissue is allowed to rest for 30-
60 min. Dose response curve of standard oxytocin and the test compounds are obtained. The
potency of the test compound is evaluated by 2+2 assay.

Modifications
Apart from rat uterus, Berde et al.29 used the rat uterus in situ, the cat uterus in vitro and the cat
uterus in situ for the evaluation of synthetic analogues of oxytocin.

Stimulation of Myometrium
An improved bioassay for the testing of oxytocic compounds by the use of myometrium
layer preparations has been developed to test the oxytocic activity of synthetic analogues in
comparison to oxytocin.

Procedure
The vitality of the tissue is found for more than 8 h. The sensitivity of the bioassay is found in
the region of 10-11mol/l oxytocin. The maximum of the stimulation of muscle strips varies from
5 × 10-5 IU/ml to 10-4 IU/ml oxytocin in the organ bath. Various doses of synthetic hormone
analogues are added to the organ bath and the contractions of the myometrium strips are
recorded. The calculation of the results is afforded planimetrically and by measuring of the
concentration-maximum during the testing period.30
Potency ratios of the test preparations are compared to standard by 2+2 assay.

Chicken Blood Pressure

Administration of oxytocin causes transient fall in blood pressure in chicken and also in other
birds in a dose dependent manner, and this response is used to evaluate oxytocic property of
unknown preparation.

Procedure
White Leghorn chickens (1.2 to 2.0 kg) are anesthetized by sodium pentobarbitone (200 mg/
kg, i.v.). Ischiadic artery is cannulated and connected to polygraph. Baseline blood pressure
is usually between 100 and 120 mm Hg. The crural vein is cannulated for injections of the test
preparations. Different doses of oxytocin used as standard are chosen which cause fall of 20
to 40 mm Hg. Two doses of standard and test are selected and result is evaluated by 4-point
assay.31

Milk Ejection Method

This is a sensitive method and shows the milk ejecting properties of oxytocic compounds.
584 Drug Screening Methods

Procedure
Female rabbits weighing 1.5 to 2 kg are anesthetized with urethene (700 mg/kg) or by pento­
barbitone (40 mg/kg). One of 6 ducts of rabbit nipples are cannulated and connected to
polygraph. Jugular vein is isolated for injecting test compound and standard. Time interval
of the doses is kept at 3 to 10 min. Two doses of standard and test drugs are taken. Oxytocic
potency ratios of the test preparations are evaluated by 2+2 assay.32

Modifications
Apart from rabbits, rats can also be used for this experiment. Female rat (300 g) in 3-21st
day post-parturition is used. They are anesthetized with pentobarbital and tip of one teat is
connected to the polygraph. Both standard and test compounds are injected through jugular/
femoral vein.33 Tindal and Yokoyama34 recommended the use of guinea pigs applying the
similar procedure but the injection of standard and test is given into the internal saphenous
artery.

vasopressin
Vasopressin is structurally related to oxytocin. It is extracted from animal posterior pituitary.
Vasopressin has both antidiuretic and vasopressor effects and is the main hormone regulating
the body fluid osmolality.

Vasopressor Activity
Vasopressor activity can be demonstrated in animal by blocking response of other pressor
substances in the body.35

Procedure
Male Wistar rats (300 g) are anesthetized with 1.75 mg/kg of urethane anesthesia (s.c.).
After half an hour, the trachea of the animal is cannulated. Simultaneously, one femoral
vein and one carotid artery are cannulated for drug injections and for the measurement of
blood pressure respectively. Heparin (2,000 U/kg) is injected through the venous cannula.
Dibenamine, which is going to block other pressor substances, is injected twice (i.v.) at
10 min interval at the concentration of 1 mg/kg. The basal blood pressure obtained is
about 50 mm Hg. Different doses of vasopressin are injected and there is a dose dependent
increase in blood pressure. Two selected doses from standard as well as test are repeatedly
administered to obtain the Latin square design and potency ratio is calculated using 2+2
point assay.

Modifications
Apart from rats, rabbits can also be used.36 Knape and van Zwieten37 used pithed rat to study
vasoconstrictor activity of vasopressin after pretreatment with various drugs.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 585

Vasopresser Activity in Guinea Pig Ileum

Procedure
Guinea pigs of either sex (0.5 to 1 kg) are sacrificed under ether anesthesia. Ileum is dissected
out and cut into 2 to 3 cm pieces. Pieces of guinea pig ileum are suspended in an organ bath.
The contractions of ileum in response to the standard and test drugs are measured by a strain
gauge transducer. Potency ratios are calculated using 2+2 point assay.38

Antidiuretic Activity
Various tests in water loaded rat model were developed by Burn39 to demonstrate antidiuretic
activity of vasopressin.

Procedure
Wistar or Sprague Dawley rats of either sex (140 to 250 g) are used for the experiment. One
preliminary test is carried out with the saline solution instead of the test preparation. 0.1 ml of
saline per 100 g body weight is injected (i.v.). Any rat showing undue excitement or frequent
micturition or stress is not taken for the main test. Food and water are not given during the
tests. Animals are divided into various groups of standard or test drug, each comprising of 6
animals. Animals are weighed and placed in separate chambers. They are given warmed water
through Ryle's tube and urine of each animal is collected. Rats are provided total water load
equivalent to 8% of the animal’s body weight.
Urine during the first 5 min after injection is discarded and the same is collected at an
interval of 15 min until the excreted urine volume becomes greater than 30% of the total water
load. The potency ratio is calculated from 2+2 point assay.

Modifications
Hydrated conscious dogs were used to test the antidiuretic activity of vasopressin by van Dyke
et al.40

Hereditary Model (Brattleboro Strain)

These animals have genetic deficiency of vasopressin synthesis. Schmale et al.41 observed a
single base deletion in the vasopressin gene, which ultimately causes diabetes insipidus in
Brattleboro rats.

Procedure
Animals are placed in metabolic cages provided with a wire mesh bottom and a funnel to
collect the urine. Stainless-steel sieves are placed in the funnel to retain the feces. Animals
are given standard diet and water ad libitum. Fifteen hours prior to the experiment, food
and water are withdrawn. For screening procedures, two groups of three animals are
used for one dose of the test compound. The test agent is given orally dissolved in 5 ml of
water/kg body weight. Two groups of 3 animals receive 1 g/kg urea orally. Additionally, 5 ml of
0.9% NaCl solution per 100 g body weight are given by gavage. Urine excretion is recorded after
5 and 24 h. Sodium content is determined by flame photometry.
586 Drug Screening Methods

thyroid hormones
Thyroid gland secretes two hormones thyroxine and tri-iodothyronine, commonly called as T4
and T3, respectively. Various bioassays are following to screen thyroid hormone analogues for
their activity.

In vitro methods
Lipogenic Enzyme Assay
Fish of body weight 20 ± 2.0 g are collected from fresh water bodies. Liver samples are collected
and are pooled as well as minced with ice-cold Hank’s balanced salt solution (HBSS). Samples
are centrifuged and a known quantity of the liver samples are transferred to separate culture
flasks containing 14C-acetate in 2 ml culture medium (HBSS) and various concentrations (10-7,
10-8 or 10-9 M) of test agents and standard (thyroxine). Medium without hormone serves as
control group. Samples are incubated at 30°C in a shaking water bath for 8 h. Post-incubation,
samples are washed in HBSS to remove unbound 14C-acetate. Samples are stored at –20°C
for analysis of 14C-acetate incorporation into lipids. Similar experiments without 14C-acetate
are done to assay lipogenic enzymes. Major lipogenic enzymes are measured in the liver
samples and the values are expressed as IU/mg protein. Absorbance is recorded by using
spectrophotometer. Lipids are extracted from the liver tissue. Extracted lipids are separated
by thin layer chromatography (TLC). Lipids of interest are collected from the TLC plate
and placed in a separate scintillation vials containing 5 ml of scintillate fluid and 0.2 g of
1, 4 bis (2-15-phenyloxazolyl) benzene/liter. Activity is counted in scintillation counter and
expressed as cpm/mg tissue.42

Tadpole Tail Culture Method

The tadpole (Xenopus laevis) is a valuable model for thyroid hormone actions as the chemical
structure of thyroxine and T3 in Xenopus are similar to mammalian thyroid hormones. The
process of metamorphosis in tadpole is dependent on the availability of thyroid hormones
from developing thyroid gland. Moreover, each tissue responds directly and independently to
thyroid hormones in organ culture.

Procedure
Tadpole tails are cultured according to the procedure described by Tata et al. (1991).43 Staged
tadpoles are first treated with Steinberg’s solution (10 mm Hepes, 60 mM NaCl, 0.67mM
KCl, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4, pH 7.4) containing gentamicin (70 µg/ml) and
streptomycin (200 µg/ml). Twenty-four hours later tadpoles are anesthetized in water
containing 0.01% amino ester benzoic acid and 6 mm of the tail is cut and transferred to 1
ml of Steinberg’s solution containing antibiotics and 5 µg/ml insulin in falcon tissue culture
dishes. The drug treatment is instituted 24 h later with change of culture medium every 48 h.
The length of each tail is measured every 24 h and a comparison between the control and test
groups is made.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 587

In vivo methods
Thyroidectomy
Pharmacological evaluation of thyroid hormones and analogues are mainly performed in
thyroidectomized rats. Bomskov et al.44 described the methods of thyroidectomy in various
animal species such as guinea pigs, rats and mice.

Procedure
Neck fur of the animal is removed with electric clippers and the area is disinfected. A median
incision of 2 cm is made upwards from the sternum. On both sides, large salivary glands and
maxillary lymph nodes are pushed to the side to make muscles covering the trachea visible.
This muscle is split. The isthmus of the thyroid glands is separated from trachea and blood
vessels are ligated. Alternatively, thyroid can be removed by electrocauterization.45

Iodine Release Inhibition

I release from thyroid gland is inhibited by thyroxine and the degree of inhibition is dose
131

dependent.46 This activity is used to compare the potency of thyroid hormone derivatives with
standard thyroxine.

Procedure
Male Sprague Dawley rats (200 to 240 g) are fed with normal diet with or without supple­
mentation of 0.03% propylthiouracil. 131I is administered (i.p.) and food is withheld for 8 h
before and 24 h after the 131I injection. Thereafter, the radioactivity over the thyroid region
is determined 40 h later under light ether anesthesia. This value is taken as a zero time and
after this reading, diet is changed to the diet containing 0.03% propylthiouracil and various
doses of standard and test preparations are injected (s.c.) at 24 h intervals for a total of four
doses.
After last 4 doses, percent zero counts of the remaining 131I is plotted against logarithm of
the dose and potency ratios of unknown are calculated from this curve.

Antigoitrogenic Activity
Administration of the exogenous goitrogenic compounds block thyroid hormone secretion
resulting in reduced thyroid hormone level in circulation. This stimulates the secretion of
thyroid stimulating hormone (TSH), which induces the enlargement of the thyroid gland. By
the administration of thyroxine or other thyroid derivatives, thyroid gland hyperplasia can be
prevented.

Procedure
Male Sprague-Dawley rats (150 to 180 g) are divided into various groups, each comprising of
8 to 10 animals. During treatment period, 0.1% thiouracil is added to food. After 2 weeks, rats
are administered test or the standard drug (thyroxine) subcutaneously at the dose of 10 to 40
588 Drug Screening Methods

mg/kg. Animals in the control group receive thiouracil diet and saline injection only and are
fed normal diet. Rats are sacrificed after 2 weeks. Thyroid gland is dissected out and weighed.
Weight of the thyroid gland is increased by 2 to 3 times by thiouracil diet and the size is
reversed to normal in dose dependent manner by thyroidal substances.46 Dose response curve
of standard and test compounds are plotted and potency ratio is calculated.

Reduction in Tensile Strength

Short-term treatment with thyroid hormone decreases tensile strength of connective tissue in a
dose dependent manner. This activity can be used to evaluate the thyroid hormone derivatives.

Procedure
Male Sprague Dawley rats (100 to 120 g) are administered various doses of thyroid hormone
subcutaneously. After 24 h, animals are sacrificed and the tensile strength of the distal femoral
epiphyseal plates, tail tendons or skin strips is measured by the following methods.
a. Measurement of tensile strength of femoral epiphyseal plates: After sacrificing the animal, the
hind legs are stretched at the hip joints and fastened at the column femoris. Longitudinal
tension results in rupture of the femoral epiphyseal cartilage. The ultimate load of the
femoral epiphyseal plate is registered by an instrument at an extension rate of 5 cm/
min.47 Single injection of thyroid hormone results in decrease in tensile strength in a dose
dependent manner.
b. Measurement of tensile strength of tail tendons: After sacrificing the animal, the tail is
amputated at the base and tail skin is removed. Single tendons are pulled out from the dorsal
and ventral bundles and kept in normal saline. Tendons of the same diameter (0.25 mm) are
selected and tendons of the same vertebral insertion are tested alternatively. The tendons
are fixed in special clamps at a distance of 2 cm and immersed in a bath with physiological
saline. Stress-strain curves and ultimate loads are determined with an Instron46 instrument
with an extension rate of 5 cm/min.48
c. Measurement of tensile strength of skin strips: The animal is sacrificed and the back is shaved
and a skin flap of about 5 × 5 cm is removed. The skin flap is placed between 2 pieces of plastic
material with known thickness and the actual thickness of the excised skin is measured by
calipers. Two dumb-bell shaped specimens are cut with a special punch in perpendicular
direction to the body axis. They are fixed between the clamps of an Instron46 instrument.
Stress-strain curves and ultimate loads are registered at a strain rate of 5 cm/min. From
stress-strain curves, the values of ultimate load and extension are registered.49
Dose response curve of different doses of test compounds and standard are established
and potency ratio is calculated.

Parathyroid Hormone
Parathyroid hormone (PTH) acts chiefly on bone and kidney by regulating calcium and
phosphate passage. Processes that are regulated by parathyroid hormone includes the
absorption of calcium from the gastrointestinal tract, the deposition and mobilization of bone
calcium and the control of excretion of calcium in urine, feces, sweat and milk.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 589

In vitro methods
Tissue Culture Assay
The tissue culture technique is based on the method described by Fell and Weiss et al.50 for
the study of PTH induced bone resorption in vitro. For 2+2 assay design, 40 mice, 6 to 9 day
old are required. The mice near the same age and size from two or three complete liters are
chosen. The calvaria are dissected out under sterile conditions. The bone samples are pooled
immediately into culture fluid at room temperature. The calvaria are cleaned and trimmed in
order to get a symmetrical bone sample consisting of two parietal bones. This is halved along
the sagittal suture and the halves are placed on wire table in each of the pair of culture vessels
inside a petridish unit. Two halved calveria per pair of culture dishes are needed when the
volume of culture fluid is 2 ml. Thereafter, petridishes are kept in incubator at 37°C with 5%
CO2 in air for 10 min. After 3 days, culture fluid is removed with a sterile Pasteur pipette and
is retained for analysis. Fresh medium containing PTH derivatives are then added to one of
the pair of culture vessels, the other receiving medium without PTH, to serve as the control.
Experiment is terminated after 3 days and the medium from each culture vessels is numbered
at the beginning of the experiment and various doses of test compounds are alternated
throughout all the dishes.
Samples of rat serum and medium are directly placed into Auto Analyzer cups and stored at
4°C until analyzed. A technicon Auto Analyser is used for estimation of calcium and phosphate.
When tissue culture media are being analyzed, a sample of medium without serum from the
batch of media, which is used for the experiment, is run at the beginning and end of each set of
samples.

In vivo methods
Increase in Serum Calcium
Administration of parathyroid extract results in increase in serum calcium level in animals.
Dogs, rabbits and rats can be used as animal models. However, healthy rats are insensitive to
parathyroid hormone but sensitivity can be increased by parathyroidectomy.

Procedure
Parathyroidectomy is performed in anesthetized male Wistar rats (200 to 250 g) by cauterization.
After a recovery period of 1 week, blood is withdrawn by retro-orbital puncture. Various doses
of test and standard preparations are given (s.c.) to different groups comprising of 6 to 8
animals. Retro-orbital puncture is performed again after 21 hours. Blood samples from all the
groups are collected and centrifuged and finally calcium is determined in the serum by flame
photometry.51
Mean values of the increase in serum calcium are plotted against log dose of the test
preparations and standard. Thereafter, potency ratio is calculated.
590 Drug Screening Methods

Modifications
Treatment with parathyroid hormone in normal rats and in rats with osteoporosis induced by
pregnancy and lactation under a low calcium diet show increase in whole body calcium and
skeletal mass was found by Hefti et al.52

Decrease in Serum Phosphate Level

This method involves the decrease in serum phosphate following the injection of parathyroid
hormone.

Procedure
Male Wistar rats (150 to 200 g) are fed normal diet for 2 weeks before the experiment. During
the experiment, animals are only allowed to have water. Blood sample (0.6 ml) is taken out
from each animal of different groups given test and standard preparations. It is centrifuged
for 10 min and 0.2 ml of sample serum of each animal is added to 6 ml of 10% trichloroacetic
acid. Again, this preparation is centrifuged and 5 ml of aliquot is used for the estimation of
inorganic phosphorus. Serum phosphorus is measured initially and 3 h after subcutaneous
administration of various doses of test and standard.53
Dose response curve of test drug and standard preparations is established to calculate the
potency ratio.

cAMP Release
Parathyroid hormone and its derivatives cause release of cAMP from adult bone and this can
be measured in a perfusion system of isolated rat femora.

Procedure
Femur is removed from five-week-old Wistar rats under anesthesia. Adhering muscles are
cleaned from the bone. A hole is made at the nutrient foramen below the femoral neck.
Thereafter, a 21-gauge needle is inserted through the hole to avoid the leakage of the
perfusate. The bone is then placed in an apparatus for liver perfusion and perfused at a
flow rate of 1 ml/5 min by a pump with Krebs-Ringer bicarbonate continuously gassed with
95% O2 and CO2 and containing 1 mg/ml glucose. Once the perfused bone is assembled,
the bone is allowed to equilibrate for 45 min. Samples are collected into a chilled tube
for determination of basal cAMP levels. Then various doses of the test preparations or the
standard are injected for 5 min. In the perfusate, cAMP in the perfusate is measured by
radioimmunoassay.54 Dose response curve of test and standard preparations are obtained
to calculate the potency.

Modifications
Activation of plasma membrane adenylatecyclase in canine renal cortex by parathyroid
hormone was measured by Nissenson et al.55 Saito et al.56 established a new biological assay
where eleven day old chick embryonic femur is labeled with 45Ca in vitro. t1/2 is calculated
from the sequential release of labeled calcium into the medium. Parathyroid hormone reduces
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 591

the t1/2 indicating enhanced bone resorption. Docherty and Heath57 used osteosarcoma cells
for an in vitro bioassay determining cAMP formation.

Cytochemical Bioassay
This method has been developed to determine agonist and antagonist activities of parathyroid
hormone analogue.58

Procedure
Renal Cytochemical Assay
Kidney segments from guinea pigs are maintained in non-proliferative organ culture for
5 h using Trowell’s T8 medium. The medium is changed before exposure to various doses of
standard and test drug for an additional 8 min. To test the antagonistic activities, each segment
is exposed to a single concentration of hPTH- (1-84) (106 fmol/l) or to hPTH-(1-34) (255 fmol/l)
in the presence or absence of the analog being tested. The segments are then stored at –70°C
in N-hexane before being sectioned at 16 µm on a cryostat. The sections are then examined
for their glucose-6-phosphate dehydrogenase activity. The precipitated formazane activity is
then quantified in the cells of the distal convoluted tubules by means of a microdensitometer
(585 nm).
For Metatarsal Cytochemical Assay
Metatarsals of young female Wistar rats (50 to 100 g) are removed and are maintained
individually in non-proliferative organ culture in 5 to 15 ml of Trowell’s T8 medium (pH 7.8)
in presence of 95% O2 and 5% CO2 at 37°C for 5 h. After the culture period, the medium is
discarded and each metatarsal is exposed to fresh medium containing a low priming dose of
PTH (0.5 fg/ml) for 8 min followed by exposure to known concentrations of standard PTH or
various concentration of PTH analogues for 8 min. The metatarsals are then briefly dipped in a
5% solution of polyvinyl alcohol and chilled immediately in N-hexane at –70°C.
Each bone is sectioned by cryostat. The sections are then examined for their glucose-6-
phosphate dehydrogenase activity in a similar way described in renal cytochemical assay. The
enzyme activity of each section is measured in 10 individual hypertrophic chondrocytes or
osteoblasts lining the metaphyseal trabeculae by scanning and integrating micro-densitometry
at a wavelength of 585 nm. Dose response curves are tested for linearity and parallelism and
potency ratios against standard is calculated.

adrenal steroids
Adrenal glands lie at the superior poles of two kidneys and are composed of two distinct parts-
adrenal medulla and adrenal cortex. The adrenal cortex releases a large number of steroids
into the circulation. The hormonal steroids may be classified as:
a. Glucocorticoids, having important effect on intermediary metabolism.
b. Mineralocorticoids, having principally salt-retaining activity.
Both natural and synthetic corticosteroids are used for diagnosis and treatment of disorders
of adrenal function. They are also used in the treatment of a variety of inflammatory and
immunologic disorders.
592 Drug Screening Methods

Glucocorticoids

In vitro methods
Receptor Binding Assay
The relative-binding affinities for the glucocorticoid receptor present in rat liver or thymus
cytosol can be measured by competitive displacement of [3H]-dexamethasone.

Procedure
Male Wistar rat (130 to 150 g) is adrenalectomized under ether anesthesia. After two days of
adrenalectomy, liver is surgically removed and homogenized in phosphate buffer containing
50 mM Tris HCl, 1 mM EDTA, 2 mM dithiothretol, 10 mM Na2MnO4 and 10% glycerol (pH
7.4). The homogenate is centrifuged for 1 h at 10.500 g at 4°C and supernatant is collected.
The cytosol is mixed with 5 nM [3H]-dexamethasone in presence or absence of competitors
and incubated for 2 h at 4°C. The reaction is terminated by the addition of hydroxyapatite to
separate the receptor steroid complex from the free [3H]-dexamethasone. The radioactivity
bound to the receptors is determined by liquid scintillation spectrometry.59 IC50 values are
estimated by probit analysis.

Modifications
Apart from the rat liver, cytosol can be prepared from other organs such as cultured hepatoma
cells, from normal human lymphocytes, thymocytes from rat thymus gland, from human
leukemic lymphoid cell line, from rat and human lung.60-65 Transient co-transfection of receptor
cDNA and suitable receptor genes were used to study human glucocorticoid receptor function
using CV-1 mammalian cell line. Various natural and synthetic steroids have been analyzed to
see whether they can activate gene expression through glucocorticoid receptors.

Transactivation Assay
Transactivation assay is useful in determining steroid agonistic and antagonistic properties.66
The transactivation assay is based on the principle that steroid receptor proteins act as ligand
regulated tanscriptional activators. After-binding of hormone, the steroid receptor interacts
with hormone responsive elements (HREs) of hormone regulated genes, thereby inducing a
cascade of transcriptional events.67
The transactivation assay determines the agonist and also the antagonistic potency of a
given compound by induction or inhibition of reporter gene activity.68

Procedure
CV-1 cells and COS-1 for transient transfection are grown in DMEM supplemented with 10%
fetal calf serum, 4 mmol/l L-glutamine, penicillin and streptomycin. Stable and transient
transfections are performed by using Lipofectin Reagent according to the procedure of Flegner
et al.69
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 593

Stable transfections are carried out as described by Fuhrmann et al.70 For transient
transfections, 1 × 106 COS-1 or CV-1 cells are harvested onto 100 mm dishes one day prior to
transfection. After 24 h, when cells become about 80% confluent, cells are washed twice with 1
ml Opti-MEM per dish to prepare for transfection. For each dish, 5 µg pHGO (hGR expression
plasmid) and 5 µg pM MTV-CAT are diluted with 1 ml of Opti-MEM. Next, the DNA and the
Lipofectin Reagent dilutions are combined in a polystyrene snap-cap tube to obtain 2 ml of
transfection solution per dish, gently mixed and incubated at room temperature for 5 min
and added to the washed cells. After 5 h, the transfection solution is replaced by 6 ml DMEM
containing 10% fetal calf serum.
After 24 h of transfection, transiently transfected cells are trypsinized, pooled and replated
onto 60 mm dishes at a density of 4.5 × 105 per dish to study the effect of glucocorticoids
analogues. Stably transfected cells are seeded onto 6-well dishes (1 × 105 cells/dish). Cells are
cultured in medium supplemented with 3% charcoal stripped FCS and appropriate hormones
for 48 h. Cells are cultured in 1% ethanol and act as negative controls for reporter gene
incubation.

CAT Assay
By freezing and thawing (37°C water bath) for at least three times, transiently transfected
cells and stably transfected cells are disrupted. Protein concentrations of the cell extracts
are determined according to the method of Bradford.71 The CAT assay is performed by the
method of Gorman et al.72 After the cells are centrifuged for 15 min at 4°C, the supernatants
are separated for enzyme assay. The assay mixture contains (final vol 180 ml) 100 ml of 0.25
M Tris-HCl, 20 ml of cell extract, 1 mCi of [14C] chloramphenicol and 20 ml of 4 mM acetyl co-
enzyme A. Controls contain CAT instead of cell extract. All the reagents except coenzyme A
are preincubated together for 5 min at 37°C. After equilibration is reached at this temperature,
the reaction is started by adding coenzyme A and is terminated by 2 ml cold ethyl acetate. The
organic mixture is dried and is taken up in 30 ml of ethyl acetate and spotted on silica gel thin
layer plate. The plate is developed in a mixture of chloroform: methanol (95:5). The spots are
cut and transferred to the counting vials. Data are expressed as the amount of chloramphenicol
acetylated by 20 ml of the extract. Concentration-response for CAT induction is established
to determine the potency of test hormone. Dexamethasone (10-10 to 10-6mol/l) is used as a
standard.

Inhibition of Cell Growth

Human fibroblasts are grown in Eagle’s Minimum Essential Medium supplemented with
10% fetal calf serum. The cultures are buffered by the addition of NaHCO3 to the medium
and are maintained on 5% CO2 and 95% air in an incubator. Cells are subcultured in 25 cm2
culture flask. Treated cultures receive test compounds dissolved in 95% ethanol while control
cultures receive ethanol alone. With the addition of 10 µl aliquots of these solutions, the final
concentration of ethanol (0.063%) does not affect cell growth in control cultures. Designating
the day of drug addition as day 0, cells are harvested and are counted on day 5 when the cells
are still in logarithmic phase growth. Cells are trypsinized (1:250 trypsin) and are scraped
from the culture flask. The results are expressed in terms of percentage of control cell growth.
594 Drug Screening Methods

This expression is obtained by dividing the difference in cell counts between day 5 and day 0
cultures, which have been treated with drug.

Tyrosine Aminotransferase Activity

Glucocorticoid induces tyrosine aminotransferase synthesis in hepatoma tissue culture cells.73

Procedure
Hepatoma tissue culture cells are grown at a density of about 8 × 105 cells/ml. Before
centrifugation, cells are washed 3 times at 0°C with buffer. After centrifugation, cell pellets
are resuspended in serum free medium containing 0.1% BSA and 0.1% NaHCO3. Standard
(dexamethasone) and test steroids of different concentrations are added to the supernatant
of cell suspension. After 16 h of incubation at 37°C, the cells are harvested and tyrosine
aminotransferase is determined. The cells are washed twice and then disrupted with an
ultrasonicator. The enzyme is assayed at 37°C by conversion of p-hydroxyphenylpyruvate to
p-hydroxybenzylaldehyde.
Enzyme specific activity is expressed in milliunit of tyrosine aminotransferase/mg of cell
protein. Standard dose response curve is drawn and potency of test preparation is calculated.

In vivo methods
Adrenalectomy in Rats
Majority of the studies regarding the evaluation of the physiological role and pathological
effect is done in adrenalectomized animal.

Procedure
Sprague Dawley or Wistar rat (120 to 150 g) of either sex is first anesthetized in a closed vessel
containing ether. The dorsal fur is shaved and the rat is placed on a wooden block on its
abdomen with fore and hind limb well extended. In this position, the spine tends to arch which
is a useful landmark for skin incision. A transverse incision of about 5 mm long is made in the
midline at the costovertebral angle. To remove the left adrenal gland, the skin is retracted to
the ventral side and the lumber muscles are incised just superior and anterior to the splenic
shadow. Now the gland is visible just below the incision. The adrenal gland along with the
periadrenal fat is removed. Any remnants of the capsule to which cortical tissue may adhere
are removed.
After surgical removal of the left gland, the animal is turned around and the right gland
is removed through skin incision. A small incision is made through the lumber muscles
above and anterior to the lumbocostal artery near the costal margin. With the help of curved
forceps, liver is elevated which covers the adrenal gland on this side; and thereafter, the gland
along with periadrenal fat and the mesentric attachments is removed. The skin is closed by
skin clip. The entire procedure is done within a short period of time so that additional ether
anesthesia is not to be given. The animals become normal within a few minutes following the
surgery.74
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 595

Adrenal and Thymus Gland Atrophy

When corticosteroid is administered repeatedly, it is going to produce both central and
peripheral effects. In immature rat, thymus gland involution is seen. Adrenal gland atrophy is
observed when ACTH secretion from pituitary is inhibited.

Procedure
Immature male Sprague Dawley rats are used for this experiment. Animals are injected different
doses of test drug subcutaneously daily for 6 days. Standard hydrocortisone is given daily at the
dose of 0.05 mg per animal. Vehicle is injected to the control group in similar frequency. On 7th
day, all animals are sacrificed and the adrenal and thymus gland weight is determined.
The degree of reduction of the weight of thymus gland represents the degree of catabolic
activity of the test compounds. Involution of the adrenal glands is a measure of the ability of
the compound to inhibit the secretion of ACTH from pituitary. From the dose response curves
of these two parameters of the standard, the potency of the test compound is compared.

Decrease in Eosinophillic Cell Count

Reduction in eosinophillic cell count occurs in animals as well as in man by glucocorticoids.
By this phenomenon, glucocorticoid effect can be quantitated in adrenalectomized mice in
vivo.

Procedure
Male albino mice (20 to 25 g) are adrenalectomized and they are maintained at 28°C with
1% NaCl in place of drinking water. Fifteen milligram pellets of deoxycorticosterone acetate
(DOCA) are implanted at the time of operation. Steroids are dissolved in benzyl alcohol or
mixed with sesame oil. Three days after the operation, the mice receive 5 µg of epinephrine
subcutaneously and after 4 h, various concentrations of test substance are given. Blood
samples are obtained from tail before and 3 h after the steroid injection.75
Potency ratio of the test compound is calculated from the dose response curve of the
standard.

Liver Glycogen Test

It is a simple and specific test for evaluation of glucocorticoid activity.

Procedure
Male Sprague Dawley rats (140 to 160 g) are adrenalectomized and given standard laboratory
diet and 1% NaCl instead of drinking water. On the 4th postoperative day, food is withdrawn;
and on the 5th day of operation, drinking fluid is withdrawn. Animals are given test compound
by a single subcutaneous injection dissolved in sesame oil. After 7 h, rats are sacrificed under
anesthesia and liver is dissected out. Liver is perfused with double distilled water to remove
blood. Following this, liver is weighed and put into the flasks containing hot 30% potassium
hydroxide and digested on a hot plate. The digest is diluted and aliquot is assayed for liver
glycogen by an anthrone procedure.76
596 Drug Screening Methods

Calibration curves are established using glucose as a standard. Three doses of test
compound and of standard are given to the animals in order to find dose response activities.
From mean values, dose response curves are established for each compound and potency
ratio is calculated from standard curve.

Mineralocorticoids

In vitro methods
Receptor-binding Assay
For mineralocorticoid receptor-binding assay, rat kidney preparations and radioactive-labeled
aldosterone are used.

Procedure
Kidney is dissected out from adrenalectomized rats and homogenized in phosphate buffer
solution containing 10 mM Tris, 0.25 M saccharose and HCl, pH 7.4. Supernatant is obtained
after centrifugation at 0°C for 10 min at 8000 g. RU 28362 is added at the concentration of
0.001 M to the aliquot in order to inhibit binding of aldosterone to the glucocorticoid receptor.
The aliquot is again centrifuged at 10.500 g for 60 min. The cytosol (supernatant) is removed
and kept for incubation at 0°C with different concentrations of test compounds and [3H]-
aldosterone (5 nM). Nonspecific binding is determined in presence of 1 mM aldosterone.
By the help of charcoal-dextran technique, free [3H]-aldosterone is separated after 1 h or 24 h
of incubation. Supernatant is centrifuged and concentration of bound ligand is determined by
liquid scintillation counter. The following parameters are calculated:
a. Total binding of [3H]-aldosterone
b. Nonspecific binding
c. Specific binding: Total binding—nonspecific binding
d. % inhibition: 100—specific binding as percentage of the control value.
Binding potency of the test compounds is evaluated as the relative-binding affinity with
respect to the standard.77

Modifications
Mineralocorticoid receptor-binding assay can also be done in the supernatant of rabbit kidney
or rat kidney slices. Arizza et al.78 cloned the transfected monkey kidney cells with plasmids
containing the human mineralocorticoid receptor.

In vivo methods
Electrolyte Excretion
Sodium retention and potassium excretion is increased by mineralocorticoids. This property
can be used to estimate mineralocorticoid activity of the unknown compound.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 597

Procedure
Male Sprague Dawley rats (140 to 160 g) are adrenalectomized and are given normal laboratory
diet and 1% NaCl instead of plain water. Food and NaCl are withdrawn on the 4th postoperative
day. Next day, each animal is given water by Ryle’s tube followed by 0.9% NaCl orally and
injected with test compounds suspended in vehicle. To collect the urine, each animal is
anesthetized by ether and the bladder is evacuated. The animals are kept in cages for 4 h and
then further they are anesthetized with the same anesthetic agent and are removed from the
cages. Urine volume is measured and diluted. Appropriate dilutions are analyzed for sodium
with a flame photometer. Deoxycorticosterone acetate is used as a standard.79
Potency ratio of the test compound is calculated by comparing the dose response curve of
the test drug with that of the standard.

Modifications
Both sodium and potassium concentration were estimated by Simpson and Tait80 in urine
and the sodium to potassium ratio is used as a index of mineralocorticoid activity of the test
compound. Nikisch et al.81 infused glucocorticoid substituted adrenalectomized rats with
saline glucose containing aldosterone. The sodium and potassium concentration of urine in 1
h fractions are calculated.

androgens
The principal circulating androgen in men is testosterone. Androgens are responsible for
male sexual differentiation in utero and male pubertal changes. The clearest indication for the
administration of testosterone is male hypogonadism.

In vitro methods
Receptor-binding Assay
Prostate, from Wistar male rats (150 to 200 g) castrated 24 h before sacrifice, are removed.
They are minced and homogenized in three volumes of Tris-HCl buffer, pH 7.4 (20 mmol/l)
containing EDTA and dithiothreitol (1.5 mmol/l) each. The homogenate is centrifuged at
1,05,000 g for 1 h to obtain the cytosol fraction.82 Cytosol (2 ml) is preincubated with [3H]-
5 alpha dihydro­testosterone (2 × 10-9 mol/l) at 0°C for 30 min to ensure equal concentration
of the label in all parallely processed samples. Aliquot portions (150 µl) of cytosol labeled with
[3H] dihydro­testosterone are labeled to the dry residue of tested steroid, mixed and incubated at
0°C under shaking for 2 h. For standard (dihydrotestosterone) and test 2.5, 5, 10, 20 × 10-9 mol/l
concentrations are taken. Separation of free and bound ligands and the determination of the-
binding characteristics are carried out by a polyacrylamide gel electrophoresis method of
Krieg et al.83

Modifications
Tezon et al.84 studied the influence of androgen and antiandrogens on androgen receptors,
which are distributed intracellularly in the rat epididymis.
598 Drug Screening Methods

The use of tritiated 7 alpha, 17 alpha-dimethyl-19-nor-testosterone for the assay of androgen

receptors was recommended by Schilling and Liao.85

Sebum Secretion Test in Rat

Androgens are known to have an effect on sebaceous gland in rat as described by Archibald
and Shuster et al.86 A bioassay for androgenic and non-androgenic steroids is developed on
the basis of this effect. The sebum secretion rate stimulated by androgenic hormones is found
parallel to increasing dose of androgenic steroids. This bioassay system can be used to evaluate
various androgenic and anti-androgenic substances for their activity.

Procedure
Measurement of sebum secretion in rat
The procedure of surface lipid extraction and measurement of sebum secretion rate were
depicted by Archibald and Shuster et al. Pre-pubertally castrated male rats are used, when
they reached a weight of 150-200 g and age of 6 to 24 weeks. They are anesthetized with ether
in glass jar. The anesthetized rats are suspended by the forepaws and immersed in 300 ml of
solvent (an ethanol-ether mixture which gave a 90-94 % extraction of surface lipid) with the
head extended so that the ears remain above the surface of the solvent. After 60 sec the rats are
gently lifted up and down 6 times and transferred to a second volume of solvent for a further 30
sec. The rats are dried with a hot air blower and returned to the cage. The solutions are filtered
into weighed aluminium cups and evaporated to dryness in a fume cupboard. The cups were
then re-weighed. The minimum 2 extractions are required for the same rat for lipid extraction.
The quantity of lipid recovered from each extraction is calculated as a percentage of total lipids
removed from each rat.
By subsequent dipping at different time intervals it is shown that up to 4 days after the first
lipid extraction the rate of accumulation of lipid is found linear; after this it tends to flatten off.
It is therefore possible to measure the sebum secretion rate from surface lipid accumulation
during the first 4 days after an initial defatting and four-day sebum collections are made in all
assays.87

Bioassay Method of Androgens

The response to increasing concentration of androgenic steroid daily is measured at intervals
for a month. The slope of the dose-response curves increases rapidly until the 17th day after
which it increased very little. In all subsequent assays a 13 to 17 day collection of sebum is
therefore made.
The sebum secretion rate is shown to increase in dose of androgenic steroids and vice versa.
This bioassay can be used to assess the androgenic potential of unknown compounds.88

In vivo methods
Castration Procedure
Castration is best performed in young male rats weighing not more than 60 g. Under ether
anesthesia, a small transverse incision is given in the skin on the ventral site over the symphysis.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 599

Testis present in the scrotum is gently pushed into the abdominal cavity. The abdomen is
then opened and with the help of forceps, the testis with the epididymis is pulled out from
the wound. The ductus deferens with the testicular vessels is separated and the testis together
with the epididymal fat pad is removed with the help of a fine scissors. The same procedure is
performed on the other side. The skin wound is closed with wound clips. The animal recovers
immediately with no bleeding.89

Chicken Comb Method

Growth of capon comb by androgen has been a popular method to evaluate androgenic
activity. This method is useful for the isolation and structural elucidation of natural androgens.

Procedure
Single comb White Leghorn chicks (2 to 3 day old) are used for the experiment. At the
beginning of the assay, the sum of the length and height of each comb is calculated. Chickens
are distributed in different groups, each comprising of 8 animals. Chickens are injected with
various doses of standard (olive oil as vehicle) and the test preparations intramuscularly daily
for 5 consecutive days. Twenty-four hours after the last injection, chickens are sacrificed and
comb size is determined by excision. Growth of the comb is expressed as the sum of length and
height in millimeters.90
The mean values of each group are calculated and plotted as dose response curve for the
test compound and the standard in order to calculate the potency ratio of the unknown.

Modifications
Newly hatched chicks of either sex can be used to study growth of the combs after systemic and
local administration.

Growth of Secondary Sex Organs

Androgens have a dose dependent effect on male secondary sex organs. The growth of ventral
prostate, seminal vesicles and musculus levatorani is dependent on androgen.

Procedure
Castrated immature male Sprague Dawley rats are used for the experiment. The rats are
administered various doses of test preparations and standard (testosterone) in 0.5 ml of 0.5%
carboxymethylcellulose orally or subcutaneously in 0.2 ml of sesame oil suspension daily
over a period of 10 days. Controls receive the vehicle only. Each group is comprised of 8 to
10 animals. On the 11th day all rats are euthanized and seminal vesicles, ventral prostate and
levato rani muscle are carefully dissected and weighed. Body weight of each animal is noted at
the beginning and the end of the experiment.91
The ratio of the organ weight/body weight is calculated for each organ and for each animal.
Mean values are calculated for each group and dose response curves are established for each
organ. Potency ratios are calculated by comparing the test with standard.
600 Drug Screening Methods

Nitrogen Retention Test

Positive nitrogen balance can be induced by anabolic agents in living organisms.

Procedure
Castrated rats (25 day old) are kept untreated till the body weight becomes 300 g with normal
laboratory chow. Thereafter, animals are given liquid-diet force feeding regime. Besides
carbohydrates and fat, it contains casein and brewer’s yeast as nitrogen source. At the
beginning of the experiment, rats are given 10 ml per day and this is increased to 26 ml per day.
This feeding is continued for 30 days with simultaneous administration of various doses of test
and standard preparations. Twenty-four hour urine specimens are collected 3 times weekly
and analyzed for total nitrogen.
Greatest daily retention of sodium and total nitrogen retention is calculated for each test
and standard group.92

Modifications
Apart from rats, monkey (Macaca mulatta) can also be used as an experimental animal for
sodium retention study.93

Change in β-glucuronidase Activity

There is an alteration in the testosterone levels in experimental animals with the change in
kidney β-glucuronidase activity. It is, therefore, suggested that β-glucuronidase activity can be
used as an assay of androgens.94

Procedure
Male Wistar rats (100 day old) are used for this study. The rats are maintained under a regulated
12:12 h light and dark schedule and are provided food and water ad libitum. Animals are
randomly divided into different groups of control, standard and treated, each comprising of 6
to 8 animals. Control is injected with normal saline (0.9% NaCl) and experimental groups are
given various doses of standard and test preparations (s.c.) for three alternate days. On the 7th
day, animals are euthanized by decapitation. Blood is collected and kidneys are dissected out.

Determination of β-glucuronidase Activity

Approximately 100 mg of kidney tissue is homogenized in 100 mM acetate buffer (pH 4.5)
and stored at –20°C until assayed. The homogenate is made up to 10 ml with the same buffer.
An aliquot of 0.3 ml homogenate is incubated at 37°C for 90 min with 0.1 ml of 0.005 M
phenolphthalein β-glucuronidase and 0.6 ml of 0.1 M acetate buffer (pH 4.5). The incubation
is terminated by placing the tubes in boiling water bath for 1 min and 1.5 ml distilled water.
Absorbance is measured at 540 nm on a synthetic spectrophotometer.

progesterone
Progesterone is produced by corpus luteum and converts the uterine epithelium from
proliferative to secretory phase. It is necessary for successful implantation of the ovum.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 601

The clinical uses of the progestational agents are ill-defined apart from contraception and
postmenopausal hormone replacement therapy. Following are the well-established models to
screen progestational agents.

In vitro methods
Receptor-Binding Assay
Preparation of Cytosol
Female New Zealand white rabbits weighing approximately 3 kg are injected (s.c.) with 50 mg
estradiol benzoate in sesame oil daily for 4 days. On day 5, the animals are anesthetized with
sodium pentobarbitone, the uteri are excised and placed immediately in ice-cold TESHMo
(10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 15 mM thioglycerol, 10 mM sodium molybdate)
buffer. All subsequent procedures are performed at 0 to 4°C. The uteri are weighed, mixed and
homogenized with a Polytron homogenizer in 4 vol of TESHMo. Following centrifugation at
10.500 g for 1 h, supernatant (cytosol) is passed over phosphocellulose prior to chromatography
on any other resin. The nonadsorbed fraction is collected and used for subsequent experiments.

Determination of Progesterone Binding to the Receptor

In some experiments, cytosol is labeled overnight with 10 mM [3H] progesterone at 4°C. Free
steroid is removed by mixing the sample with 1.33 vol. of a charcoal suspension (0.5% dextran:
0.5% charcoal, v/v) and incubating for 10 min on ice followed by centrifugation at 5,000 g for
10 min.
During chromatography of progesterone receptor without bound steroid, receptor
concentration is determined at selected point by Seat chard analysis of the binding of [3H]-
progesterone (0 to 20 mM) ± 100 M excess progesterone. At all steps in the procedure, binding
is estimated by incubation with 10 mM [3H]-progesterone ± 100 M excess progesterone,
in triplicate. Receptor concentration is expressed as [3H]-progesterone bound (p mol/mg
protein).95

Modifications
The binding of progesterone agonist and antagonist to the progesterone receptor from calf
uterus was characterized by Hurd and Moudgil et al.96 Different DNA-binding properties of
the calf uterine estrogen and progesterone receptors were explained by different dimerization
constants.97 Mutation of the progesterone receptor was found to be responsible for species
specificity and has been used for the evaluation of agonistic and antagonistic activity.98

In vivo methods
Clauberg Test in Rabbits (McPhail Test)
Clauberg et al.99 first described the histological changes of the endometrium in rabbits that
are pretreated with estrogen and after treated with progestational compounds and McPhail
systemically examined the test and introduced scores for the changes of endometrium.100
602 Drug Screening Methods

Procedure
Immature female rabbits (550 to 650 g) are administered daily injections (s.c.) of 5.0 mg of
estradiol benzoate in sesame oil solution per animal for a period of 6 days. Thereafter, rabbits
are given various doses of test compounds and the standard for the next 5 days. Control group
receives either the vehicle or estradiol benzoate only. Animals are sacrificed on the 15th day of
the experiment and both the horns of the uterus are removed and fixed in 10% formalin. For
histological examinations, sections are made from the middle part of the each horn.
Increase in uterine weight is compared in control and treated groups by the following
scoring system:
0—ramification of uterine mucosa, no proliferation
1—slight proliferation of mucosa
2—medium proliferation with slight additional ramification of uterine mucosa
3—pronounced proliferation of mucosa
4—pronounced proliferation as well as pronounced ramification.
The scores are arranged from each group, mean values are obtained and are plotted for dose
response curve in order to calculate the potency ratio.

Modifications
Direct injection of progesterone can also be given in the uterine segment. This is performed in
immature rabbits primed for 6 days with estrogen. On the 7th day, the upper middle segment of
each horn of the uterus is ligated without disturbing the circulation. Test agent of various doses
is injected into the lumen of one segment and in the opposite horn, only vehicle is injected.
After three days, animals are sacrificed and sections of horn are evaluated histologically
according to McPhail scores.101

Endometrial Carbonic Anhydrase

Progesterone enhances endometrium carbonic anhydrase activity. Measurement of carbonic
anhydrase activity in the rabbit endometrium can be used to evaluate compounds having
progesterone like activity.

Procedure
Immature female rabbits are used and are given estrogen and progestin treatment as
described in Clauberg test. After sacrificing the animals, the uteri are opened longitudinally
and endometrium is dissected, weighed and homogenized. After centrifugation, carbonic
anhydrase activity is measured by colorimetric method.
Mean values of carbonic anhydrase activity/g wet tissue are calculated and dose response
curve of test and standard are plotted to calculate the potency ratio.102

Rat Decidualization Model

This model measures the progestational activity of the compounds in the uterus. The target
cells are the endometrial stromal cells, which undergo a progestin dependent proliferation
and differentiation that is required for normal embryo implantation and maintenance.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 603

This response is very specific for progestin and is used to evaluate the compounds having
progesterone like activity.

Procedure
Adult female Sprague Dawley rats (200 to 250 g) are ovariectomized. Ovariectomies are
performed 10 days prior to treatment. Upon arrival, the rats are randomized to groups of 5 to 6
animals each. The standard and test preparations are administered once daily for 7 days orally
by gavage (0.5 ml). Approximately 24 h following the final treatment, rats are euthanized by
CO2 asphyxiation. The uteri are removed, trimmed of fat and the decidualized (D) and control
(C) horns are weighed separately. The decidual response is expressed as D/C.103

Rat Uterine C3 Model

Female Sprague Dawley rats (50 day old) are ovariectomized. The ovariectomies are
performed 8 days prior to the treatment. The rats are randomly divided into various groups,
each consisting of 6 animals. The rats are treated once daily for 2 days per orally in a volume of
0.5 ml or s.c. injection in the nape of the neck (0.2 ml) with either the standard or test
preparations or vehicle. On the second day of the treatment, the animals are also treated with
17 alpha-ethinyl estradiol (EE) (0.08 mg/kg body weight) orally by gavage. Approximately 24
h after the final treatment, the animals are euthanized by CO2 asphyxiation. The uteri are then
removed, stripped of remaining fat and mesentery, weighed and snap frozen on dry ice.
The total RNA is isolated from the uteri using the Trizol Reagent. To find the potencies of
the test preparations, northern blot analysis is performed with RNA that are separated in a 1%
agarose gel containing 1% formaldehyde. The 28S rRNA is quantified with ethidium bromide
stained gels. cDNA probes are labeled with a 32P-dCTP using the Radiprime random primer
DNA labeling kit. C3 mRNA is quantified using a phosphorimager and normalized to 28S
rRNAlization in rodents.104

Rat Ovulation Inhibition Model

Progestins function at the level of hypothalamus to block the LH surge associated with
ovulation. This activity of progestins has been utilized to develop a method to evaluate
unknown compounds having progestational activity.

Procedure
Random cycling mature female rats weighing 180 to 200 g are used for the experiment. Animals
are synchronized for estrous with 2 mg/rat of luteinizing hormone-releasing hormone (LH-
RH) (in PBS containing 0.1% BSA) s.c. at 09:00 h and again at 16:00 h. Animals are allowed to
rest for 8 days prior to the administration of the test compounds. Animals are then divided into
various treatment group, each comprising of 7 to 9 animals. From the morning of the ninth
day following LH-RH treatment, the rats are treated with standard and test preparations orally
once a day for 4 consecutive days. The animals are euthanized in the morning following the
last treatment. Oviducts are removed, placed between two glass slides and are viewed through
a dissecting microscope to count ova. The animals presenting ova in the oviduct are also
recorded.105
604 Drug Screening Methods

estrogens
The dramatic actions of estrogen in the maintenance of female reproductive tissues are well
known. Pharmacological intervention using a variety of steroidal and nonsteroidal estrogens
is widely practiced for contraception, for hormonal control of prostatic carcinoma and
for alleviation of many of the serious sequelae of menopause. This wide array of important
therapeutic actions has sustained a high level of interest in the continued development of new
estrogens.

In vitro assay
Receptor Binding Assay
Cytosol Preparation
Adult female Wistar rats (200 to 250 g) are ovariectomized and five days later uteri are removed
and washed with cold saline. All the procedures are carried out at 4°C. One gram of the tissue
is homogenized in 1 ml phosphate buffer. The homogenate is centrifuged at 10.500 g for 90 min
and the supernatant is used as estrogen receptor-binding cytosol.

Competition Experiment
Ten microlitre of [3H]-estradiol (final conc. 5 × 10-8mol/l) and 10 µl of unlabeled estradiol for
the standard curve or 10 µl of the test substances in appropriate concentrations are added to
40 µl of the cytosol. Each sample is incubated for 120 min at 4°C. Postincubation unbound
estrogens are adsorbed by incubating with 0.5 ml of a suspension of dextran coated charcoal in
Tris buffer (0.01 M, pH 7.5) containing 1.5 mM EDTA and 10% glycerol for 10 min at 4°C. After
centrifugation for 5 min at 15,000 rpm, an aliquot of the supernatant is withdrawn and counted
for radioactivity. The relative-binding affinity is evaluated according to the method of Bouton
and Raynaud.106

Recombinant Yeast and Beta-galactose Assay

The recombinant (BJ-ECZ) yeast strain contains a reported gene with two estrogen responsive
elements upstream from the yeast proximal cytochrome C1 promoter fused to the Lac Z gene.
These yeast cells are transfected with rtER or hER expression vectors. The activation of the
reporter gene is strictly dependent on the presence of estrogen receptor and estrogens and
results in the production of galactosidase (beta-Gal).
Four independent colonies are incubated at 30°C in a shaking incubator at 300 rpm for 36 h.
About 100 ml of yeast suspension (0.6 OD600) is distributed in each well of conical bottomed 96
well plates. After 4 h of incubation at 30°C in presence of test compounds, plates are centrifuged
and the media is discarded. Fifty microliter of lyticase solution is added to each well, and the
plates are incubated at room temperature for 30 min. About 100 ml of 0.1% triton × 100 is added
to each well. Plates are then centrifuged at 10.500 g for 10 min and 100 ml of supernatants
are transferred to flat bottomed 96 well microtitration plates and 20 ml of O-nitrophenyl
β-D-galactopyranoside substrate is added to each well. The enzymatic reaction is performed
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 605

at 30°C for 1 h. The reaction is stopped by adding 50 ml of 1 mol/l Na2CO3 and the OD405 is read
after 5 min equilibration. The beta-galactosidase units are defined as OD405/mg protein/min of
enzymatic reaction.107

Alkaline Phosphatase Assay

Cell Culture
The Ishikawa cell line is maintained in Eagle’s Minimum Essential Medium (MEM) containing
10% (v/v) fetal bovine serum (FBS) supplemented with antibiotics, glutamine and sodium
pyruvate. On the day of the experiment, cells are harvested and plated in 96 well plate
(2 × 104 cells/200 µl) in phenol red free DMEM/F-12 medium supplemented with 5% FBS
stripped of endogenous estrogen with dextran-coated charcoal. On the day of the experiment,
2 µl of the test compound is added. Dose response is performed in 1/5 serial dilution. After
48 h of the treatment, the plates are rinsed with 200 µl Tris/HCl 0.1 ml/l, pH 7.4 and cells are
lysed by adding 20 µl Tris/HCl 0.1 mmol/l, pH 9. Now the plates are placed at –80°C for at least
15 min followed by thawing at room temperature for 5 to 10 min. This freeze and thaw cycle
can be omitted with loss of about 15% of detectable ALP activity. The plates are placed in ice
and 50 µl of ice-cold solution containing 5 mM p-nitro phenyl phosphate, 0.24 mM MgCl2 and
1 M diethanolamine (pH 9.8) is added. Plates are warmed to room temperature and the yellow
colour from the production of p-nitrophenol is allowed to develop. Plates are monitored at 405
nm in an enzyme-linked immunosorbent assay plate reader.108
Dose response experiments are analyzed mathematically by nonlinear regression method.
The sigmoidal dose response curve is used as a model. The EC50 is calculated from log EC50.

In vivo methods
Castration of Female Rats
Ovariectomy is usually done in immature female rats weighing less than 60 g. The whole
procedure is performed under ether anesthesia. A single incision is made in the skin of the
back. A small puncture is then made over the site of the ovary, which can be seen through the
abdominal wall embedded in a pad of fat. With the help of fine forceps, fat around the ovary is
removed without rupturing the ovarian capsule. The ovary together with the fallopian tubes is
removed with a single cut by a pair of fine scissors. Usually, no bleeding is observed. The ovary
of the other side is removed in a similar way. The skin wound is closed by one or two clips. The
animals recover immediately.

Vaginal Cornification
Immature female rats (approx. 55 g) are ovariectomized and kept for about 1 week on standard
laboratory diet and water ad libitum. The test compounds are administered in 0.5% solution of
carboxymethylcellulose or in cotton-seed oil either orally or subcutaneously in various doses.
Estradiol is used as standard and each group comprises of 10-20 animals. The compounds are
given twice daily for two consecutive days at the gap of 8 h interval. Vaginal smears are taken
on third day afternoon and on fourth day morning with the help of spatula or cotton swabs
606 Drug Screening Methods

moistened with saline. Smears are transferred to a glass slide and evaluated microscopically
according to the following scores:
0—diestrous stage, mainly leukocytes and a few epithelial cells are present
1—metestrous stage, mixture of leukocytes and epithelial cells
2—proestrous stage, nucleated or nucleated plus cornified cells
3—estrous stage, cornified cells only
Animals showing score 2 or 3 are considered to be positive. The number of positive animals
in each group is recorded. ED50 value is calculated by using various doses of the test drug.109

Modifications
The sensitivity of the assay can be enhanced by local application of estrogen into the vagina of
castrated animals.

Increased Uterus Weight

Estrogen enhances the weight of the uterus when it is given repeatedly in a dose dependent
manner in castrated female rats.

Procedure
Immature female Wistar rats (55 g) are ovariectomized and divided into various groups, each
comprises of 6 to 8 animals. Animals are administered with various doses of test and standard
compounds simultaneously for seven days. The test compound is administered in 0.5%
solution of carboxymethylcellulose or in cotton-seed oil. Control animals are given vehicle
only. On the 8th day, the animals are sacrificed and uterine weight is determined.110
Dose response curve of test and standard compounds are obtained to find out the potency
ratios.

Modifications
Rubin et al.111 used albino mice who are administered the oil solution of hormone (s.c.).
Twenty-four hours after the injection, animals are sacrificed and uterine as well as body weight
are taken. The uterine ratio is calculated by dividing the uterine weight (mg) by body weight
(g), multiplied by 100.
In addition to uterus weight, Branhan et al.112 studied luminal and glandular epithelium
height in cross sections of the uterus horns of rats by histological means.

Chick Oviduct Method

Administration of natural or synthetic estrogen enhances the oviduct weight of young chicken
in a dose dependent manner.

Procedure
Seven-day-old chicks are given subcutaneous injections of various doses of test compounds
and standard twice daily for 6 days. Six to ten chicks are used for each group. On the day after
the last injection, the animals are sacrificed and body as well as oviduct weight is determined.
 Hormones of Pituitary, Thyroid, Parathyroid, Adrenal Cortex, Ovary and Testes 607

The ratio of oviduct weight/body weight is calculated for each animal. Mean values of test and
standard groups are plotted to get dose response curve in order to calculate the potency.113

References
1. Foster CM, Borondy M, Padmanabhan V, et al. Bioactivity of human growth hormone in serum:
validation of an in vitro bioassay. Endocrinology 1993;132:2073-82.
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 cHAPTER

40
Antidiabetic Agents
INTRODUCTION
The pancreas is an organ composed of (98%) exocrine and (2%) endocrine cells. Islets of
Langerhans, which form the endocrine part of pancreas, consist of four types of cells α-cells,
β-cells, δ-cells and PP-cells, which secrete glucagons, insulin, somatostatin and pancreatic
polypeptide.
Glucose stimulates the β-cells to release insulin, which then promotes glucose uptake and
storage in various tissues. Diabetes mellitus is a disease characterized by derangement in
carbohydrate, protein and fat metabolism caused by the complete or relative insufficiency of
insulin secretions and/or insulin action.
Two major types of diabetes, one associated with insulin deficiency called type-I or insulin-
dependent diabetes mellitus (IDDM) and the other associated with insulin resistance called

Figure 40.1: Pathophysiology of hyperglycemia

614 Drug Screening Methods

Figure 40.2: Factors regulating β-cell mass

type-2 or noninsulin-dependent diabetes mellitus (NIDDM). Type-1 is associated with a

specific and complete loss of pancreatic β-cells. Type-II diabetes is the most common type and
is associated with obesity, hyperinsulinemia and insulin resistance. Insulin resistance may be
due to the defect at receptor level or at the postreceptor level. This defect may be in the effector
cell or in the β-islet cell causing insulin resistance.
The β-cell mass is the overall balance of β-cell growth and β-cell loss, depending on the four
mechanisms1 as follows (Fig. 40.1):
1. Replication of existing differential β-cells.
2. Neogenesis of β-cells from precursors located in the pancreatic ductal epithelium.
3. β-cell size.
4. β-cell death.
In the 1980s, von Mering was working on the absorption of fat from the intestine after
removing the pancreas of a dog. The animal developed polyuria and polydipsia and was found
to have diabetes mellitus. Many experiments on rabbits and dogs followed, although history
has given a special place to Marjorie, one of the dogs used by Banting and Best in their seminal
experiments on the isolation and purification of insulin in the 1920s.2 Marjorie is probably the
most famous experimental animal in history.

Models for IDDM

Chemically-induced Diabetes
Chemically induced Type-I diabetes is the most commonly used animal model of diabetes.
Chemical agents which produce diabetes can be classified into three categories, and include
agents that:
•• Specifically damage β-cell
•• Cause temporary inhibition of insulin production and/or secretion
•• Diminish the metabolic efficacy of insulin in target tissues.
In general, chemicals in the first category are of interest as they reproduce lesions resembling
IDDM.

Alloxan-induced Diabetes
Alloxan, a cyclic urea analog, was the first agent in this category, which was reported to produce
permanent diabetes in animals.
 Antidiabetic Agents 615

Mechanism of Action
The mechanism by which it induces diabetes is not very clear. Alloxan is a highly reactive
molecule that is readily reduced to diuleric acid, which is then auto-oxidized back to alloxan
resulting in the production of free radicals. These free radicals damage the DNA of β-cells and
cause cell death. Second mechanism proposed for alloxan is its ability to react with protein SH
groups, especially the membrane proteins like glucokinase on the β-cells, finally resulting in
cell necrosis. However, there are major species differences in response to alloxan.
Procedure
Rabbits weighing 2–3 kg are used. Alloxan is infused via the ear marginal vein at a dose of 150
mg/kg for 10 min. About 70% animals become hyperglycemic and uricosuric. The remaining
animals either die or are temporarily hyperglycemic.
When rats of Wistar or Sprague-Dawley strain each weighing 150–200 g are used, alloxan is
injected s.c. in the dose of 100–175 mg/kg. In male Beagle dogs weighing 15–20 kg, alloxan is
injected i.v. at a dose of 60 mg/kg. Alloxan has been given to nonhuman primates, like monkeys
and baboons in the dose of 65–200 mg/kg i.v. to induce diabetes. All the animals, which are
given alloxan, receive glucose and regular insulin for one week and food ad libitum. Thereafter,
single daily dose of 28 IU insulin is administered s.c. The blood glucose level shows triphasic
change, first a rise at 2 h, followed by hypoglycemic phase at 8 h, and finally an increase at 24 h
probably due to depletion of β-cells of insulin.
Modifications
Diabetes can be induced in neonatal Sprague-Dawley rats by intraperitoneal injection of 200
mg/kg alloxan on days 2, 4, or 6.
Drawbacks:
• High mortality in rats
• Causes ketosis in animals due to free fatty acid generation
• Diabetes induced is reversible
• Some species like guinea pigs are resistant to its diabetogenic action.
Alloxan has been almost completely replaced by Streptozotocin (STZ) for inducing diabetes
because of these drawbacks.1-4

Streptozotocin-induced Diabetes
STZ [2-deoxy-2-(3-methyl-3-nitrosourea) 1-D-glucopyranose] is a broad-spectrum antibiotic,
which is produced from Streptomyces achromogens. Rakieten et al. first described the
diabetogenic property of STZ.5
Mechanism of Causing β-cell Damage
• By process of methylation
• Free radical generation and
• Nitric oxide production.
Procedure
STZ induces diabetes in almost all species of animals. Diabetogenic dose varies with species
and the optimal doses required in various species are: rats (50–60 mg/kg, i.p. or i.v.), mice
(175–200 mg/kg i.p. or i.v.) and dogs (15 mg/kg, for 3 days). The blood glucose level shows the
same triphasic response as seen in the alloxan treated animals, with hyperglycemia at 1 h,
616 Drug Screening Methods

followed by hypoglycemia, which lasts for 6 h, and stable hyperglycemia by 24–48 h after STZ
administration.
Modifications
Multiple low dose of STZ also induces diabetes by causing immune mediated pancreatic
insulitis in rats. It has also been shown to have diabetogenic effect on the golden hamsters when
given i.p at a dose of 50 mg/kg. Cyclosporin-A when given with STZ enhances its diabetogenic
efficacy. STZ combined with complete Freund’s adjuvant: each of CFA, incomplete Freund’s
adjuvant, Mycobacterium butyricum (component of CFA), Listeria monocytogenes, or endotoxin
administered 24 h prior to STZ (25 mg/kg) and then repeated in the three subsequent weeks,
all produce hyperglycemia. Fasting for 48 h (24 h prior to and 24 h subsequent to the STZ
injection) also produces hyperglycemia. Neither four administration of CFA nor of STZ alone
result in persistent hyperglycemia.
Advantages and Disadvantages
STZ has almost completely replaced alloxan for inducing diabetes because of:
• Greater selectivity towards β-cells
• Lower mortality rate
• Longer or irreversible diabetes induction
• However, guinea pigs and rabbits are resistant to its diabetogenic action.5-9

Hormone-induced Diabetes Mellitus

Dexamethasone, a long-acting glucocorticoid, is used to produce NIDDM. NIDDM form of
diabetes is produced when dexamethasone is administered at a dose of 2–5 mg/kg i.p. twice
daily over a number of days in rats.
Besides rat models, other experimental models using guinea pigs and rabbits are also
reported for the study of diabetes using corticoids.11,12 In these models, corticotrophin is used
to stimulate adrenal cortex that results in hormonal imbalance causing steroid diabetes.13,14

Insulin Antibodies-induced Diabetes

Giving bovine insulin along with CFA to guinea pigs produces anti-insulin antibodies.
Intravenous injection of 0.25–1.0 ml guinea pig anti-insulin serum to rats induces a dose
dependent increase in blood glucose levels up to 300 mg%. This unique effect to guinea pig
anti-insulin serum is due to neutralization of endogenous insulin by the insulin antibodies.
It persists as long as the antibodies are capable of reacting with insulin remaining in the
circulation. Slow i.v. infusion or i.p. injection prolongs the effect for more than a few hours.
However, large doses and prolonged administration are accompanied by ketonemia, ketonuria,
glycosuria and acidosis and are fatal to the animals. After lower doses, the diabetic syndrome
is reversible after a few hours.15

Diabetes-induced by Viral Agents

Viruses are thought to be one of the etiologic agents for IDDM. Viruses may produce diabetes
mellitus by:
• Infecting and destroying of β-cells in pancreas
• A less infecting or cytologic variant producing a comparable damage by eliciting immune
autoreactivity to the β-cells
• Viruses producing systemic effect, not directly affecting the β-cells.
 Antidiabetic Agents 617

Various human viruses used for inducing diabetes include RNA picornoviruses, Coxsackie-
B4 (CB4), encephalomylocarditis (EMC–D and M variants), Mengo-2T, as well as two other
double stranded RNA viruses, reovirus and lymphocytic choriomeningitis virus (LMCV,
Armstrong variant) (Table 40.1). Primary isolates of these human pathogenic agents are,
generally, not pancreatotrophic or ilytic to mouse β-cells and must be adapted for growth
either by inoculation into suckling mice, or by passage in cultured mouse β-cells.16,17

Table 40.1: Susceptible mouse strain

Virus Susceptible mouse strain

EMC–D or M variant SJL/J
SWR/J
DBA/1J
DBA2J
BALB/cCUM
Mengo–2T SJL
C57BL/6J
CBA/J
C3H/HCJ
AKR/J
CE/J
CB4 SJL/J
SWR/J
Reo SJLj
Kilham rat virus BB –DR

Surgically-induced Diabetes
Induction of diabetes mellitus can be achieved through the surgical removal of all or part of
the pancreas. In partial pancreactectomy more than 90% of the organ must be removed to
produce diabetes. Depending on the amount of intact pancreatic cells, diabetes may range
in duration from a few days to several months. Total removal of the pancreas results in an
insulin-dependent form of diabetes, and insulin therapy is required to maintain experimental
animals. The portion of the pancreas usually left intact following a subtotal pancreatic resection
is typically the anterior lobe or a portion thereof.

Disadvantages
1. Surgical removal of pancreas results in loss of α- and δ-cells in addition to β-cells. This
causes loss of counter-regulatory hormones, glucagon and somatostatin.
2. There is a loss of the pancreatic enzymes necessary for proper digestion, therefore, the diet
for pancreatectomized animals must be supplemented with these pancreatic enzymes.
3. The total resection of the pancreas in rat is very difficult to achieve and the development
and severity of the diabetic state appear to be strain specific.
618 Drug Screening Methods

The use of pancreatectomy in combination with chemical agents, such as alloxan and STZ,
produces a stable form of diabetes mellitus in animals, such as cats and dogs, that does not
occur when each procedure is applied independently. The combination therapy reduces the
organ damage associated with chemical induction and minimizes the interventions, such as
enzyme supplementation, necessary to maintain a pancreatectomized animal.18,19

Genetic Models
The NOD Mouse
Non-obese Diabetic (NOD) mice are an inbred strain of albino mice developed by Makino and
coworkers, in Japan. It is derived from breeding Jcl:ICR (Swiss mice) progenitors, and NOD
mice represent the product of over 80 generations of sib matings. Over the first 20 generations
of sib matings, the strain was being maintained as a normoglycemic control line to match with
another line being selected for impaired glucose tolerance (NON strain). Once spontaneous
development of IDDM was observed in a female of the control NOD strain at F20, development
of frank hyperglycemia and glycosuria rather than normoglycemia, it became the selected
phenotype.20

The BB Rat
Spontaneous diabetes in the BB Wistar rat was initially diagnosed in 1974 by the Chapel brothers
at the Biobreeding Laboratories commercial breeding facility in Ottawa, Ontario, Canada, in
a noninbred but closed outbred colony of Wistar rats. It was decided to name this syndrome
BB after the initials of the breeding lab. The clinical presentation of diabetes in the BB rat is
similar to that of its human counterpart. Marked hyperglycemia, glycosuria, and weight loss
occur within a day of onset and are associated with decreased plasma insulin that if untreated
will result in ketoacidosis within several days. Like the NOD mouse, the BB rat is one of the few
rodent models in which significant ketosis occurs in the absence of obesity. Unlike most NOD
mouse colonies, both sexes of BB rats are equally affected.21

WBN/KOB Rat
Wistar Bonn/Kobori (WBN/KOB) rats show hyperglycemia and glucosuria by the age of 5
month. Degeneration of islet in size and number is evidenced by the age of 3 months.22 Other
changes observed are fibrinous exudation, deterioration of exocrine pancreatic tissues and
demyelination by the age of 4 months.14

Cohen Diabetic Rat

Cohen diabetic rat is an excellent hyperglycemic rat model characterized by insulin
resistance.23 In this model, high blood glucose level, high insulin level, glucosuria and other
diabetic complications are evidenced in rats on high sucrose diet.14,24

Other Diabetogenic Compounds

Other diabetogenic compounds used for the study of diabetes are dithizone25 or
goldthioglucose26 or monosodium glutamate.27 Chelators like dithizone, 8-(p-toluene-
 Antidiabetic Agents 619

sulfonylamino)-quinoline (8-TSQ), and 8- (benzenesulfanylamino)-quinoline are reported to

used in the study of diabetes in experimental animals.28 Dithizone, used in a single dose of 40-
100 mg/kg by intravenous route, produce hyperglycemia in rats, cats, rabbits, golden hamsters
and mice. In rabbits dithizone injected, produce hyperglycemia characterized by triphasic
glycemic reaction; hyperglycemia after 2 h of injection followed by a normal blood glucose
level for 8 h and permanent hyperglycemia after 24–72 h.

Models for NIDDM

Neonatal STZ Model of NIDDM (Chemically-induced Diabetes)
Neonatal rats of Wistar or Sprague-Dawley strain are treated with STZ (80–100 mg/kg i.p.) at
birth or within the first 5 days following birth. There is severe pancreatic β-cell destruction,
accompanied by a decrease in pancreatic insulin stores and a rise in plasma glucose levels.
However, in contrast to adult rats treated with STZ, the β-cells of the treated neonates partially
regenerate. Following an initial spike in plasma glucose the STZ treated neonatal rat becomes
normoglycemic by 3 weeks of age. In the next few weeks, the β-cell number increases mainly
from the proliferation of cells derived from ducts, the extent, depending upon both the age at
which the animal is treated with STZ and the species of the treated rat.29,30

Other Chemically-induced NIDDM Models

Agents used for induction of NIDDM in rabbits include adrenaline (0.1 mg/kg s.c.). The peak
hyperglycemic effect is noticed at 1 h and lasts up to 4 h. The increase in blood sugar levels is
found to be 120–150 mg/100 ml. Oral hypoglycemic agents can be screened by this method.
Diabetes can also be induced in animals with chelating agents 8-hydroxy quinoline and
biphenyl thio carbazine. EDTA has been reported to be diabetogenic in partially depancreatized
rats. Injection of an antiserum produced against ox insulin in guinea pigs or sheep causes
diabetes in mice. Diabetes is associated with acute insulin deficiency and the animals exhibit
marked hyperglycemia and ketonuria. It is, however, temporary in nature. This model does
not have serious toxic side effects like other models, but induces mild pancreatitis in rats.
Administration of thiazides, chlorthiazide, hydrochlorthiazide, diazoxide and furosemide,
produced hyperglycemia and glycosuria in experimental animals, including rabbits, rats and
mice. Diazoxide is found to be effective either alone or in combination with other drugs.15

Genetic Models of NIDDM

Monogenic Models of Obesity and NIDDM
The defining phenotypes of the monogenic rodent models include obesity, hyperinsulinemia,
transient or sustained hyperglycemia and hyperlipidemia.
Animal models used are as follows:
Yellow Mouse (The Agouti Mouse)
The agouti mouse is believed to have first appeared in China, where it was treated as a curiosity
because of its brilliant hair color. The agouti locus was identified as a result of studies on coat
color pigments, and was named after the South American rodent Dasyprocta agouti, which
620 Drug Screening Methods

has a banded pattern of hair color. Hyperinsulinemia, hyperglycemia, insulin resistance and
NIDDM in males are found around 4–5 weeks of age.31-37
Obese and Diabetic Mouse
The obese (ob) mutation was detected in noninbred mouse stock and was subsequently
maintained in the C57Bl/6J strain. The diabetes (db) mutation occurred in the C57BL/KSJ
inbred strain. Both ob and db are autosomal recessive with full penetrance. Obese phenotype
is characterized by extreme insulin resistance, glucose intolerance, and mild hyperglycemia,
and, therefore, exhibits many of the characteristics of NIDDM. At 20–28 days fasting and fed
states, both the number and size of pancreatic β-cells are increased. Hyperinsulinemia is
accompanied by decreased glucose tolerance. Serum insulin eventually reaches a peak and
then falls. Improvement and normalization of blood glucose tolerance, stabilization of serum
insulin level and decrease in body weight follow this. In the db phenotype mouse, plasma
insulin increases by 10 days and peaks at 6–10 times the normal by 2–3 months, when animals
are severely hyperglycemic. Insulin levels drop rapidly to near normal values at the time when
islets are hyperplastic and hypertrophic. Progressive degranulation and necrosis follow this,
and the islet insulin content becomes greatly reduced. Glucose-induced insulin secretion is
severely decreased and there is a rapid rise in blood glucose to over 22 mM up to 5–8 months
of age.32,38
Tubby Mouse
The tub mutation arose spontaneously in a mouse colony at the Jackson Laboratory, and the
tubby colony was bred from a single C57BL/6J male. Tubby mice are characterized by slowly
developing obesity. Although hyperinsulinemia, hyperactivity of the islet β-cells and β-cell
degranulation are conspicuous features of these animals, hyperglycemia is not observed, and
the obesity syndrome does not normally progress to severe diabetes. Both, females and males
develop mild hypoglycemia and hyperinsulinemia at 12 weeks, but then become euglycemic.
The hyperinsulinemia persists, increasing in severity with age, and ultimately, is associated
with insulin resistance.31-39
Fat Mouse
The fat (CPE fat) mutation was discovered in a HRS/J inbred mouse colony at the Jackson
Laboratories. Inheritance is autosomal recessive. Animals develop obesity at 6–8 weeks of
age. Males develop hyperglycemia by 7–8 weeks and then they return to normal. Chronic
hyperinsulinemia is present in both sexes from weaning, and is associated with hypertrophic
and hyperplastic pancreatic islet cells.31-39
Zucker Diabetic Fatty Rat (ZDF)
These arose from the inbreeding of a substrain of fa/fa rats that exhibited hyperglycemia. In
this strain all males develop obesity, insulin resistance and overt NIDDM between 7 and 10
weeks of age, by which time their average plasma glucose exceeds 22 mM. Females are also
obese and insulin resistant, but do not become diabetic.31-40
Wdf/Ta-Fa Rat
The WDF/Ta-fa rat, commonly referred to as the Wistar fatty rat, is a genetically obese,
hyperglycemic rat established by the transfer of the fatty (fa) gene from the Zucker rat to
the Wistar Kyoto rat. 41-43 The Wistar fatty rat exhibits obesity, hyperinsulinemia, glucose
 Antidiabetic Agents 621

intolerance, hyperlipidemia, and hyperphagia similar to Zucker rats being, however, more
glucose intolerant and insulin resistant than Zucker rats. Hyperglycemia is usually not observed
in females, but can be induced by addition of sucrose to the diet. Kobayashi and coworkers44
found an increase of insulin sensitivity by activation of insulin receptor kinase by pioglitazone
in Wistar fatty rats (fa/fa). Sugiyma and coworkers45 found a reduction of glucose intolerance
and hypersecretion of insulin in Wistar fatty rats after treatment with pioglitazone for 10 days.46
The WDF/Ta-fa rat is genetically modified rat model also known as the Wistar
fatty rat. It is generated by injecting fatty (fa) gene from the Zucker rat to the Wistar Kyoto
rat.41-43 It is characterized by obesity, insulin resistance, glucose intolerance, hyperphagia and
hyperlipidemia.44-46
Other spontaneous diabetic rat models reported in the literature are ESS-rat,47,48 OBESE
SHR rat49-51 and BHE rat.52
Koletsky and JCR: LA-Corpulent Rats
The obese, spontaneously hypertensive, Koletsky rat strain develops obesity, hyperlipidemia
and proteinuria with kidney disease. Several substrains have been developed from Koletsky
rats, including the SHR/N-cp, LA/N-cp, and JCR: LA-cp strains. JCR: LA–cp rat develop marked
hyperinsulinemia by 5–6 weeks of age and decreased glucose uptake. The hyperinsulinemia
effectively maintains virtual normoglycemia, but this results in marked islet hyperplasia, and
islets occupy 15–20% of the total pancreatic volume in 9-month-old cp/cp male rats.53,54

Polygenic Models of Obesity and NIDDM

No single gene has been demonstrated to be involved in the majority of patients with obesity
and/or NIDDM. The overall contribution of single gene defects to the total NIDDM population
is small and only 10% of the genetic risk factors for NIDDM are known. The more common
forms of NIDDM probably result from interaction between the environment and several gene
defects, each of which when expressed individually has little effect on glucose tolerance.
Therefore, polygenic animal models represent human condition more closely.
Animal strains used are as follows:
New Zealand Obese (NZO) Mouse
Bielschowsky and Bielschowsky first described the NZO mouse. NZO mouse is hyperphagic,
obese, hyperglycemic, hyperinsulinemic, insulin resistant, and mildly glucose intolerant. NZO
mice become overtly obese by 8–10 weeks of age and reach their maximal weight (males) of 70 g
at 12 months. Hyperinsulinemia and hyperglycemia develop early (by 4 weeks), and pancreatic
insulin stores are increased at this time. Glucose intolerance decreases continuously with age
and body weight.55
Diabetic db/db Mice
The Diabetic db/db Mice is a unique mice model derived from mice of the strain the C57BL/KsJ
strain having spontaneous recessive mutation in leptin receptor gene, i.e. C57BL/6J db/db.56 It
is characterized by high blood glucose level up to 20–25 mmol/l57obesity and nephropathy.58
Mutations on the leptin receptor result in an obese phenotype identical to that of ob mice.59,60
In the db/db mouse strain observe abnormal splicing because the Ob-Rb transcript contains
a premature stop codon.61,62 Insulin level is high and insulin receptors are significantly low.63
622 Drug Screening Methods

Japanese KK Mouse
The KK mouse belongs to the Kasukabe (K) group of mouse strains. These mice show
hyperinsulinemia, nonfasting hyperglycemia and glucose intolerance. They have insulin
resistance due to defect in both the insulin receptor and postreceptor signal transduction
systems, including glucose uptake, pentose pathways and impaired insulin sensitive
phosphodiesterase in fat cells.32
Nagoya–Shibata–Yasuda (NSY) Mouse
NSY mouse was established as an inbred strain from a JcL: ICR mouse colony by selective
breeding for glucose intolerance. Spontaneous diabetes develops in 98% of males and 30%
females by 48 weeks of age. NSY mice do not become obese, but exhibit fasting hyperinsulinemia,
and pancreatic insulin content at 36 weeks of age.64
PBB/Ld Mouse
The PBB/Ld mouse originated from pet store stock, selected on the basis of black coat color.
They develop apparent obesity by 3–4 months and obese animals exhibit hyper­lipidemia,
hyperinsulinemia, mild hyperglycemia and reduced tolerance to glucose load.65
Otsuka–Long–Evans–Tokushima Fatty Rat (OLEFT)
Kawano and co-workers established the OLEFT rat strain by selectively inbreeding members of
a normal colony of Long Evans rats, which developed polyuria, polydypsia, hyper­insulinemia,
persistent hyperglycemia, hypertriglyceridemia and mild to moderate obesity. Obesity is
evident approximately 2 weeks after weaning. There is a late onset hyperglycemia (after 18
weeks of age) associated with marked glucose intolerance. Diabetes develops in 80–100% of
males by 25 weeks of age, females do not show any disability till 40 weeks of age.66
Goto–Kakisaki Rat
The GK rat was developed by Goto and Kakisaki through the selection of 18 rats from the local
Wistar stock that were slightly glucose intolerant. These hyperglycemic animals were mated and
inbred until, after 30 generations, the diabetic state became stable in subsequent generations.
The GK rat is one of the best-characterized animal models of spontaneous nonobese NIDDM,
since it exhibits similar metabolic, hormonal and vascular disorders to the human disease.
This includes fasting hyperglycemia, pronounced glucose intolerance, peripheral and hepatic
insulin resistance, impaired glucose-induced insulin secretion and late complications, such as
neuropathy and nephropathy.67
Chinese Hamster
Mier and Yerganian first reported a diabetic syndrome in the Chinese hamster (Cricetulus
griseus). Prediabetic hamsters are hyperphagic from birth and develop hyperglycemia,
polydypsia and glycosuria early, but they do not become obese.68
Djungarian (Siberian) Hamster
An extremely high incidence of spontaneous diabetes has been reported in Djungarian
hamsters (Phodopus sungorus).69
South African Hamster
The South African Hamster (Mystromys albicandatus) also develops diabetes without obesity.
The degree of hyperglycemia and glucose intolerance varies with age of onset, incidence, and
 Antidiabetic Agents 623

degree of severity in different colonies. Diabetic animals are characterized by polydypsia,

glucosuria and ketonuria.31

Animal Models of NIDDM with Unknown Hereditary and

Environmental Component
A number of models of NIDDM have been developed, as a result of the observation that
animals taken from their natural environment developed diabetes mellitus when fed normal
laboratory diet.
Sand Rat
Psommomys obesus is a small rodent (gerbil), indigenous to the desert regions of the Middle
East where access to food and water is limited. They exhibit a genetic predisposition to the
development of NIDDM cataracts, when fed standard high calorie laboratory diet ad libitum.66
Spiny Mice
The development of diabetes has been studied in two species of spiny mouse, Acomys russates
and A. cahirinus. Both, males and females have congenital hyperplasia of pancreatic islets,
with high insulin content. When laboratory bred A. cahirinus exhibits a moderate weight gain
on a fat diet, accompanied by hyperglycemia but neither hyperlipidemia nor ketonuria. A.
russates become obese on either a regular or fatty diet, but do not exhibit hyperglycemia or
hyperlipidemia.70
Tuco-tuco
The tuco-tuco (Ctenomys talarum) were observed at 3 months of age, many of these animals
developed cataracts. The ones with cataracts were hyperglycemic and were also mildly obese.71
Polygenic animal models produced by hybrid crosses
• BSB (C57BL/6J × Mus spretus)
• AKR/J × SWR/J Model
• GK crosses: GK × Fisher 344 strain, GK × nondiabetic Brown Norway rat.72

Transgenic and Knock-out Animals

The genes controlling various aspects of metabolism and insulin secretion are manipulated to
produce animal models of diabetes mellitus.
The genes that are manipulated to cause insulin resistance correspond to:
1. Insulin receptor
2. Insulin receptor substrate 1 and 2
3. Glucose transporters
4. Hexokinase II
5. Tumor Necrosis Factor-α (TNF-α)
6. Fatty acid-binding Protein 2
7. RAS associated with diabetes (Rad).
The genes that are manipulated to cause defective insulin secretion correspond to:
1. GLUT–2
2. Glucokinase
3. Hepatic Nuclear Factors
624 Drug Screening Methods

4. Islet Amyloid polypeptide.

Genes that increase body fat:
1. Knock out of uncoupling proteins
2. Knock out of β3-adrenergic receptors.73,74

Gene Targeting and Transgenic Techniques

Gene targeting is the process by which single gene is disrupted in an embryonic stem cell and
then transmitted along the germ line. This process leads to ‘knockout animals’. The transgene
is randomly incorporated into the host genome and some offspring will, therefore, express the
modified gene.
The female mouse or rat (superovulate) is allowed to mate. The subsequent day, single cell
zygotes are collected and maintained in culture for few hours. A genetic construct (insulin
gene) is then injected into pronucleus of zygote and the construct integrates itself into the
genome of the zygote, which results into the over-express insulin in adult life. The insertion
of metallothionein promoter will enhance transgene expression when heavy metals are
administered in vivo (Fig. 40.3).

Knockout Animals
Knockout animals are produced by using a genetic construct that will disrupt normal gene.
Construct is developed, which contains DNA sequence homologues to the target gene but
that are disrupted or contain a deletion. These are injected into embryonic stem cells (ES)
and will undergo recombination with the normal gene, causing it to be ‘knocked out’. ES
cells are injected into pre-implantation mouse embryos and transferred to the oviducts of

Figure 40.3: Producing transgenic animals that over express a gene

 Antidiabetic Agents 625

pseudopregnant mice and allowed to develop to term. ES cells contribute to the germ line of
the offspring, selective breeding will allow the production of mice that are either heterozygous
or homozygous for the knockout.
This approach has been used to produce a large number of animals to understand the
pathogenesis of Type 1 and Type 2 diabetes.75,76

Miscellaneous Models
Invertebrate Animal Model
The silk worm Bombyx mori provides an excellent diabetes model alternative to conventional
mammalian diabetes models owing to its various advantages like low maintenance cost, ease
of experimentation and no ethical issues. In this model, high glucose diet, i.e. 10% glucose is
fed to the silk worm for 3 days in the presence and absence of test drug and intervention by test
drug is observed by comparing the blood sugar level, body size, weight and other parameters
between normal, control and treatment silk worm groups.14,77

Diet-induced Metabolic Dysregulation

This model mimics the diabetes induced by metabolic dysregulation due to diet. In this model,
hyperglycemia is induced in Baboon (Papio hamadryas sp.) by feeding them with high sugar,
high fat diet (73% Purina Monkey Chow 5038 (a grain-based meal), 7% lard, 4% Crisco, 4%
coconut oil, 10.5% flavored high fructose corn syrup, and 1.5% water) after 12 h fasting. High
blood glucose level is demonstrated after 2 months of high glucose and lipid diet along with
other diabetic changes like increased blood level of glycosylated hemoglobin (HbA1c), lipids
and adipokines.14,78

Normoglycemic Animal Models

Rabbit Model
Rabbits are the animals of choice for this model as they have been used for standardization of
insulin for many years and also for the ease in handling.

Procedure
Mixed breed rabbits of either sex weighing 3–4.5 kg are chosen and divided into groups of
4–5. For the evaluation of the insulin and insulin like compounds, food is stopped a day
earlier to the experiment; while for the evaluation of hypoglycemic agents, the animals have
free access to normal diet till the experiment. The test drug is given orally through gavage
in the dose of 1 ml/kg in 0.4% starch suspension. Control group receives only the vehicle.
Different doses are tried on other groups. Blood is withdrawn from the ear vein immediately
before, and 1, 2, 3, 4, 5, 24, 48, and 72 h after treatment. Blood glucose is determined in 10 µl
blood samples using the hexokinase enzyme method. Blood sugar values are plotted against
time.79
626 Drug Screening Methods

Rat Model
Procedure
Male Wistar rats of 180–240 g body weight are chosen and fed on standard diet. They are divided
into groups of 4–7. The test drug is given orally or i.p. in various doses suspended in 0.4% starch
suspension. Control group receives only the vehicle. Blood is withdrawn from the tip of the tail
immediately before, and 1, 2, 3, 5, and 24 h after administration of the test compound. Blood
glucose is determined in 10 µl blood samples with hexokinase enzyme method. Average blood
sugar values are plotted versus time or each dosage.

Modifications
Male guinea pigs (pirbright white) can be used instead of rats, each weighing 250–380 g. Blood
is withdrawn by puncturing ear veins before and 1, 3, and 5 h after administration of the test
compound or the vehicle.79

Dog Model
Procedure
Male beagle dogs weighing 15–20 kg, fed normal diet, are chosen. Food is stopped 18 h prior
to the administration of the test compound that is given orally or i.v. in various doses. Control
animals receive only vehicle. Blood is collected at various intervals up to 48 h. Blood glucose is
determined with hexokinase enzyme method and plasma insulin with a radioimmunological
method.79

Modifications
Pancreatectomized dogs up to 2–3 years prior to the study are given dry feed with 2–3 g
pancreatic enzymes. Insulin is substituted with a single daily s.c. dose of 32 IU insulin (long
acting); Vitamin D is given i.m. at a dose of 1 ml every 3 months. On the day before the
study, animals receive 32 IU of short-acting insulin. This is given along with the food and
test compound. Test compound is given as oral suspension in tap water. Blood glucose is
determined before and up to 6 h after treatment at hourly intervals. Control animals receive
only tap water.79
Diabetes is also induced in dogs by a single i.v. dose of 60 mg/kg of alloxan. Then 1000 ml
of 5% glucose with 10 IU insulin is infused via jugular vein daily for one week and standard
diet ad libitum. Then, a single dose of 28 IU of the shorter-acting insulin is given daily and the
animals are fed commercial diet. On the day before the study, the dogs receive 28 IU of the
short acting insulin. This insulin is given at the same time as food and test compound. The test
drug is given as oral suspension in tap water. Blood glucose is determined before and up to 6 h
after treatment at hourly intervals. Control animals receive tap water only.79
 Antidiabetic Agents 627

In Vitro Methods on Isolated Organs, Cells and Membranes

Isolated Pancreas of Rat
The in vitro perfusion of isolated pancreas helps us in studying the effect of the drug on insulin,
glucagon and somatostatin secretion without interference from other organ changes.

Procedure
Animals used are male Wistar rats weighing 200–250 g. The animals are fed ad libitum. The
pancreas is removed under pentobarbital (50 mg/kg i.p.) anesthesia. Once the pancreas is
removed, through a portal vein cannula, Krebs-Ringer bicarbonate buffer with 2% bovine
albumin and 5.5 mmol/l glucose is perfused at a rate of 1.75 ml/min. The temperature of the
perfusion fluid is kept at 37.5°C and the pressure at which it is perfused is about 100 mmHg. The
perfusate is collected every minute for 30 min. After the first 5 min of perfusion, test compound
is added till the 15th min (conc. of test compound being 0.05–0.5 mM). From 16th min till 30th
min, glucose of 5.5 mM and 16.6 mM is perfused. The samples collected are stored at –20°C.
Hormones insulin, glucagon and somatostatin are estimated radioimmunologically. At least
3 experiments per concentration are performed. The effect of the test compound, whether
it increases or decreases the secreted hormones of pancreas in response to elevated glucose
level, is compared with the control.80

Isolated Rat Pancreatic Islets

Procedure
Two male Wistar rats weighing 200–250 g act as donors of pancreas, which is removed under
pentobarbital anesthesia. The islets are obtained by the collagenase method and collected
under a stereomicroscope. In every test, up to 10 chambers each with 15 islets are perifused.
Cut-off microfuge tubes, sealed with Tuohy-Borst adapters, serve as perfusion chambers.
Two thick-walled, small diameter Teflon catheters are passed through the adapter into
the chamber. One of the catheters extends to the bottom of the chamber and acts as the
perifusate inlet; the other extends to the lower edge of the adapter cone and acts as outlet.
The latter is connected to a multichannel peristaltic pump, which delivers the perifusate to a
fraction collector. The chamber volume is 0.15 ml. The perifusate flow rate is 0.1 ml/min. The
perifusate consists of Krebs-Ringer bicarbonate buffer with 1.0 mmol/l glucose, 0.25% bovine
albumin and 5 mmol/l theophylline. The storage vessels for the perifusate, the chambers and
the inlet catheters are immersed in a water bath of 37°C. After a pre-perifusion phase of one
hour, the perifusate is collected every minute for 46 min. From the 2nd until the 18th min,
the test compound is added at concentrations between 0.1 and 2.5 µmol/l, and from the 34th
to the 46th min, the glucose concentration is raised to 20.0 mol/l. Insulin is determined by
radioimmunological methods. The determination is done immediately after the end of an
experiment.81

Modifications
Free cell suspensions from mouse pancreatic islets have been tried, where the response to
glucose is lower than that of intact isolated islets. Long term monolayer culture of adult rat islet
628 Drug Screening Methods

of Langerhans as an experimental model for studying chronic modulation of α-cell function

has been performed.82

Isolated Rat Liver

Procedure
Male Wistar rats weighing 200–250 g are anesthetized with 150 mg/kg hexobarbital i.p. after
which the liver is removed from the animal, it is washed with 100 ml heparinized (5 IU/ml)
physiological saline solution at 37°C for 3 min through the portal vein. The preparation is then
transferred to perfusion apparatus, where portal vein cannula is attached to tubing containing
the oxygenated medium. Perfusion is done with Krebs-Ringer bicarbonate buffer with 25%
bovine erythrocytes, 1.6% bovine serum albumin, and 22.5 mmol/L Na-L-lactate at a rate of
30 ml/min. Seventy ml are used for recirculation over 2 h. The test compound is added to the
perfusate medium in a concentration of 40–100 µmol/l. A variable CO2/O2 mixture, the ratio
of which is dependent on the pH value of the perfusate is used for gassing. The perfusate is
bubbled with 70 ml gas mixture/min. To avoid foam formation, a detergent has to be added to
the perfusate. The samples for analyses are withdrawn by catheter.83

Isolated Hepatocytes
Procedure
Male Wistar rats weighing 200–300 g act as donors. The hepatocytes are then isolated by
collagenase method from the liver. The isolated hepatocytes are suspended in 3.0 ml of Krebs-
Ringer bicarbonate buffer containing 4% bovine albumin (pH 7.4). The cell suspension is
preincubated for 15 min at 37°C in a Dubnof metabolic shaking incubator gassed with CO2.
The following substrates are added in various combinations and each sample is incubated
for 60 min:
1. Alanine, fructose, glycerol, lactate, pyruvate (10 mM), or
2. Palmitate (0.5 mM as sodium palmitate bound to albumin). Test drugs are added
in concentrations between 0.05 and 5.0 mM. At the end of the 60 min incubation period,
0.2 ml of 70% HClO4 is added into the medium to stop the reaction. The reaction mixture
is then centrifuged, and the supernatant obtained is used to determine the intermediate
metabolites. Glucose is assayed by the glucose-oxide method, lactate, pyruvate, acetoacetate
and α-hydroxybutarate by enzymatic methods.84
Modifications
Instead of isolated hepatocytes, cultures of Hep G2 cells can be used. The Hep G2 cell line is a
minimal deviation of human hepatoma that maintains the liver cell morphology and function.
Hep G2 cells express insulin receptors, display a number of metabolic responses to insulin and
insulin-like growth factor-I and have been used in various studies. Troglitazone (cs-045) was
found to increase glycogen synthase-I activity I Hep G2 and BC3H-1 muscle cell.85,86
Fructose-2,6-bisphosphate Production in Rat Hepatocytes
Glycogenesis in hepatocytes is regulated by the cytosolic level of fructose-2,6-bisphosphate.
Insulin increases fructose-2,6-bisphosphate by inhibiting fructose-2,6-biphosphatase through
phosphorylation. If the drug increases the insulin secretion or acts like insulin, it should
increase fructose-2,6-bisphosphate level.
 Antidiabetic Agents 629

Procedure
106 cells/ml suspension of hepatic cells is perfused with Hanks 10 mM HEPES buffer solution
(pH 7.5, containing 0.5% BSA, and 1 mM palmityl oleate) at 37°C for 10 min. The cells are cen­
trifuged at 40 g for 15 sec, and the supernatant is discarded. To the remainder, the same buffer
and various concentrations of test compound are added and the mixtures are then incubated
at 37°C for 10 min. The reaction is stopped by cooling on ice. The mixture is cen­tri­fuged at 170 g
at 4°C for 60 sec. The pellet is homogenized with buffer solution containing 1 mM EGTA and 10
mM MgCl2. After heating at 80°C for 20 min, the homogenate is mixed with an equal volume of
400 mM Tris-HCl buffer (pH 7.5) and the mixture is centrifuged at 17,500 g at room temperature
for 5 min. In the supernatant, fructose-2,6-bisphosphate is measured by adding a solution of
54 mM Tris-HCl, 1 mM Fructose-6-phosphate, 0.2 mM NADH, 7.5 mM dithiothreitol, 0.5 mM
EDTA, 0.01 U phosphofructokinase, 0.4 U aldolase, 1 U glycerine-3-phosphate dehydrogenase,
and 3 U trisphosphate isomerase. The concentration of fructose-2,6-bisphosphate in the
sample solution is measured by colorimetry and compared with the reaction rate of a known
concentration of fructose-2,6-bisphosphate.86-89

Isolated Target Tissue: Muscle

Perfused Hind Limb in Rats
Procedure
Female Wistar rats weighing 170–230 g are used. They are fasted for 48 h before experiment. They
are anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. After identification of
aorta, a ligature is placed around it above the origin of the renal vessels and then tied. The aorta
is incised between the left renal and iliolumbar vessels and a No. 18 polyethylene catheter,
filled with 0.85% NaCl containing 200 U of heparin/ml, is introduced and passed up to a point
midway between the iliolumbar vessels and the aortic bifurcation and after flushing with
heparin-NaCl solution, it is tied in place. The vena cava is cannulated with a No. 16 needle that
is secured in position, such that its tip is at the same level as the aortic catheter. The needle
is connected to vinyl tubing. The preparation is then transferred to a perfusion apparatus.
Aortic cannula is attached to tubing containing oxygenated medium. Perfusion medium
consists of Krebs-Ringer bicarbonate buffer with 25% bovine erythrocytes, 4% bovine serum
albumin, and 10 mmol/l D-glucose. It is perfused at a rate of 8 ml/min and 70 ml is used for
recirculation over 2 h. The test compounds are added in a concentration of 40–100 mmol/l to
the perfusate medium. The ratio of CO2/O2 bubbled, depends on the pH value of the perfusate
and is bubbled at a rate of 70 ml gas mixture/min. To avoid foam formation, a detergent (14 ml/
ml 0.1% Genapol PF-10) is added to the perfusate.90,91

Models to study insulin secretion from β cells

Insulin Secreting Cell Lines
Insulin secreting cell line culture provides an excellent ex vivo diabetic model alternative
to conventional animal models. In this model, the HIT cell line is used to assess the insulin
secretary and inhibitory activity of test drug. INS-1cells and INS-2 cells from parental RINm5f
630 Drug Screening Methods

cells is another model used to study diabetes. In this model, glucose transport activity of test
drug is observed and compared with standard anti diabetic formulations available in the
market.
Another ex vivo model reported in the literature is genetically engineered insulin-secreting
human liver cell line, i.e. betacyte (HEP G2ins/g cell), which is responsive to glucose.92-97

Muscle Cell Lines

BC3H1 Myocytes
The BC3H1 cell line is a nonfusing spontaneously and reversibly differentiating mouse muscle
cell line derived from a mouse neoplasm. BC3H1 myocytes have electron microscopic features
of both smooth and skeletal muscle cell, but they have nicotinic acetylcholine receptors and
an action potential more like that of a skeletal muscle cell.
BC3H1 myocytes are cultured to confluence in 100 mm dishes over 10–14 days in Dulbecco’s
Modified Eagle’s Medium (DMEM) added with 15% Process serum Replacement-I, 25 mM
glucose (added 18 h before the experiment). Cells are rinsed and pre incubated at 37°C for
20 min in Dulbecco’s phosphate buffered saline with 0.1 mM CaCl2 and 1 mg/ml BSA, then
treated with buffer (control) or agonists in the buffer for 30 min.
Test compound can be studied on this cell line for:
• Its direct effect on glucose transport
• Insulin-induced decrease of 5'-nucleotidase activity in skeletal muscle membranes
• Glucose transport associated with diacylglycerol-like activation of protein kinase C.98,99

L6 Muscle Cells
They are cultured myogenic cell lines, cultured from thigh of a 1-day-old rat. It has features of
skeletal muscle cell. They are grown in 75 cm2 flasks in Ham’s F-10 medium containing 15%
horse serum, 2.5% fetal calf serum, 0.87% glutamine, 0.87% penicillin-streptomycin, and 7.5%
NaHCO3 at 37°C in a 5% CO2 atmosphere. After 2–4 days, the cells are in a monolayer, confluent,
aligned, and fused, but no myotubes are present. The medium is changed and test substances
are added and are used to study glucose uptake.100

C2 C12 Cells
It is a mouse skeletal muscle cell line isolated from dystrophic mouse muscle. The cells are
seeded in a 100 mm diameter, collagen coated, tissue culture dishes at 5 × 104 cells/ml and
plated in growth medium consisting of DMEM containing 20% fetal bovine serum, 0.5%
chick embryo extract, and 1% penicillin/streptomycin. Cells are grown in this medium to
confluence between 75–85% and then induced to differentiate by transferring them to DMEM
supplemented with 2% adult horse serum and 1% penicillin/streptomycin.101

In Vitro Assays for Insulin or Insulin-like Substances on Adipocytes

Isolated Adipocytes Preparation
The uptake of (3H) glucose into fat cells and incorporation of this radiolabelled glucose in the
lipids can measure insulin or insulin-like activity.
 Antidiabetic Agents 631

Procedure
Epididymal fat pads are removed from male Wistar rats weighing 160–180 g. The fat pads are
cut into pieces and incubated for 20 min at 37°C with 1 mg/ml collagenase in KRHB (Krebs-
Ringer bicarbonate buffer), 25 mM HEPES/KOH, (pH 7.4), 0.1 mM glucose, 1% w/v BSA
(bovine serum albumin). Through 100 mm Nylon screen the cell suspension is filtered and
washed 3 times by flotation (accumulation of a thin cell layer on top of the medium after
centrifugation at 1000 g for 1 min in a swing-out rotor) with KRHB without glucose, and
finally, suspended in the same solution. The suspension is adjusted to a final titer of 4 × 105
cells/ml.102,103

Rat Adipocytes Primary Culture

Insulin resistant adipocytes are generated in vitro by incubating freshly isolated cell in the
primary culture with 25 mM glucose and 10 mM insulin, for 20 h at 37°C under slow shaking.
When assayed for insulin stimulated glucose transport, these adipocytes show decreased
insulin sensitivity and decreased insulin responsiveness.

3T3 Adipocytes
These cells were isolated from an established mouse fibroblast line, 3T3. These subclonal
sublines accumulate large amounts of triglycerides when the cells are in the resting state.
These cells have been used for the study of insulin-sensitizing agent. 3T3-L1 cells are grown
in DMEM containing 25 mm glucose, 10% FCS and antibiotics. Almost 80–90% of these cells
become confluent and differentiated (express the adipocyte phenotype). They are washed two
times with KRHB and then incubated in the same buffer with test compound.104,105

Assays for Lipid Synthesis in Isolated Adipocytes

The assays measure the insulin stimulated glucose transport and conversion into lipid
products like fatty acids, triglycerides and phospholipids. At low glucose concentration
(0.1 mM), when the glucose transport is rate limiting, majority of the radiolabelled appears in
the glycerol backbone. At higher concentrations (2 mM), when transport is not rate-limiting,
2/3rd of the label is incorporated into the fatty acids of the lipids. These lipids are then measured.

Procedure
Adipocytes are incubated with D-(3-3H) glucose (0.55 mM concentration) and the cells are
lysed. Toluene based scintillation cocktail is added to separate the lipids formed from the
water-soluble products. After phase separation, radioactivity incorporated into the lipids is
determined by liquid scintillation counting directly without removal of the lipid phase.
Incorporation of D-(3-3H) glucose into toluene-extractable acylglycerides is measured after
incubation of adipocytes with 1 ml KRPB in the absence or presence of test compound. Before
measuring the radioactivity of (3-3H), the reaction is started by adding 0.1 ml (3-3H) glucose
(2 µci/ml), 0.1 mM, 0.4 ml Krebs-Ringer bicarbonate buffer 2-fold, and 0.3 ml of substance with
test compound to 0.2 ml of adipocyte suspension to scintillation vials. The scintillation vials
are placed under a stream of CO2 for 10 sec, then closed and placed in a very slowly shaking
water bath (37°C). After incubation for 90 min, the reaction is terminated by the addition
632 Drug Screening Methods

of 10 ml of toluene-based scintillation cocktail. The vials are mixed vigorously using vortex
and subsequently left standing for 2–4 h to allow phase separation. The (3-3H) radioactivity is
determined with a liquid scintillation counter. Blank values obtained from a typical reaction
mixture containing buffer and (3-3H) glucose, but not containing adipocytes and the test
compounds, have to be included in each experiment. Since, the lipid synthesis increases in
basal state (quality of cell decreases with time), it is recommended to set up to basal incubation
for every 20–25 test mixtures. The blank values are subtracted from the values measured for the
corresponding set of test mixtures to correct for 3-3H radiation originating from the aqueous
phase.105

In Vitro Assays of Glucose Transport in Adipocytes

Adipocytes are incubated with 0.2 mM D-(V-14C) glucose for 20 min. Cells are then centrifuged
on silicon oil, removed and counted for radioactivity. This assay measures the total insulin-
stimulated glucose uptake, transport and metabolism irrespective of whether the glucose is
utilized via the oxidative or nonoxidative pathway.

Glucose Uptake by the Isolated Diaphragm from Mice and Rats

This model is useful for the study of the effects of insulin and insulin-like substances on muscle
tissue.

To Study Glycogen Synthesis

Diaphragms are obtained from male Sprague-Dawley rats weighing 70–100 g. The diaphragms
obtained are divided into two equal pieces. The hemidiaphragms are incubated in Krebs-
Henseleit buffer, gassed with 95% O2/5% CO2 and with 5 mM (U-14C) glucose (0.5 mci/ml),
insulin or the compound to be tested. After 30 min, the hemidiaphragms are blotted on tissue
and frozen in liquid nitrogen. The powdered tissue is dissolved by heating for 45 min at 100°C
in 30% KOH (1 ml/100 mg tissue) before ethanol is added to a concentration of 70%. After
freezing at –20°C, the samples are centrifuged at 2000 g for 10 min. The glycerin pellets washed
3 times with 70% ethanol before the amount of 14C labeled glycogen is determined by liquid
scintillation counting. Total glycogen is determined after hydrolysis to glucose (1 N HCL at
100°C for 3 h).106

To Study Glucose Transport

Freshly dissected rat diaphragms are incubated for 30 min at 37°C in HEPES-buffered saline
(25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 0.5 mM
sodium pyruvate, 1.5 mM KH2PO4, pH 7.4) under constant bubbling with 95% O2/5% CO2. The
diaphragms are then washed two times with the same buffer without glucose, and incubated
further for 30 min in 5 ml of the above buffer with test compound or insulin. Glucose transport
is initiated by addition of 50 ml of 10 mM 2-(1-3H) deoxyglucose (10 mci/ml) in the absence
or presence of 25 mM cytochalasin B (control). After 15 min, the diaphragms are rinsed four
times with ice-cold buffer containing 10 mM glucose, 25 mM cytochalasin B, then blotted with
filter paper and homogenized. Portions of the homogenate are used for protein determination.
Samples of the supernatant in a volume of 1 ml are centrifuged at 10,000 g for 15 min and
 Antidiabetic Agents 633

mixed with scintillation cocktail and counted for radioactivity. Glucose transport (dpm/mg of
protein) is calculated as difference between diaphragm associated radioactivity measured in
the absence (total uptake) and presence of cytochalasin (nonspecific uptake).105

To Study Glycogen Synthase Activity

Intact hemidiaphragms obtained from male Wistar rats are incubated in DMEM (10 ml/
hemidiaphragm) with test compounds or insulin and 5 mM glucose at 37°C and constantly
bubbled with O2: CO2 (95:5). The homogenate is prepared by blotting and freezing the
diaphragms in liquid nitrogen. Frozen diaphragms are grounded in a porcelain mortar and
then homogenized at 0°C in 10 vol of 25 mM tris/HCl (pH 7.4), 100 mM NaF, 5 mM EDTA,
0.1 mM PMSF. The homogenate is centrifuged (10,000 g, 20 min). The supernatant is used for
glycogen synthase assay. Ten microliters of diaphragm homogenate is added to 200 ml of 25
mM Tris/HCl (pH7.4), 50 mM NaF, 10 mM EDTA (preincubated at 30°C) containing either 0.1 or
10 mM glucose-6-phosphate. The reaction is initiated by supplementing 0.2 mM (U-14C) UDP-
glucose (10 mci) and terminated after 15 min by addition of 2.5 ml of ice-cold 66% ethanol and
filtration over pre-wetted Whatman GF/C glass fiber disks. The filters are washed, dried and
counted for radioactivity. Blanks are assayed by adding the homogenate to tubes containing
the complete reaction mixture plus ice-cold ethanol. The fractional velocity, as parameter
for the portion of glycogen synthase active in vivo (l-form) toward the total enzyme contents
(l- + d-forms) at the point of homogenization, is calculated as ratio between the activities
measured at 0.1 (l-form) and 10 mM glucose-6-phosphate (l-+ d-forms).106

Insulin Receptor-binding Assays

Subcutaneous adipose tissue is obtained from the abdomen of patients undergoing
gasteroenterological surgery. The adipose tissue is finely chopped and incubated for 90 min at
37°C in a HEPES buffer (pH 7.4), containing human serum albumin (25 g/l) and collagenase
(0.5 g/l). The isolated adipocytes are subsequently washed five times in a HEPES buffer
containing 50 g/l human albumin. The diameters, surface and volume are calculated for every
cell. Insulin receptor-binding studies with isolated human adipocytes are performed in a
300 µl cell suspension containing about 1 × 105 cells/ml in a HEPES buffer (10 mmol/l HEPES,
50 g/l human albumin (pH 7.4) at 37°C. The iodine-labeled ligand (I125 TyrA14-monoiodinated
insulin, specific activity about 350 mCi/mg) in a final concentration of 20 pmol/l is incubated
with increasing amounts of unlabeled human insulin and insulin derivative to be tested. The
reaction is stopped by adding 10 ml of chilled 0.154 mol/l NaCl and subsequent centrifugation
with silicon oil. Nonspecific-binding is measured by incubating tracer in the presence of a large
excess of unlabeled insulin. For association studies, the 125I-labeled ligand is incubated for
different period (1–240 min) and the reaction is terminated as described above. At each time
point, the nonspecific-binding is measured and substracted subsequently from the data for
total-binding. Dissociation rates are determined by first incubating isolated adipocytes at 37°C
with either (125I) TyrA14- insulin, or the test compound labeled in the same position for 90 min
to achieve steady-state-binding conditions. Each incubation mixture is then centrifuged for
60 sec. The adipocytes are rapidly washed twice, by diluting with buffer to the original volume
at 4°C and centrifugation and aspirations are repeated. After the third aspiration, the cells are
diluted to the original volume with buffer alone or native insulin or the insulin derivative to be
634 Drug Screening Methods

tested at a final concentration of 0.2 mmol/l at 22°C. The reaction is stopped at various times
between 10 and 180 min and cell-associated radioactivity is determined.107,108

Modifications
Insulin receptor-binding assays have been done on receptors prepared from the membranes
prepared from the rat liver.109 The studies have also been done on solubilized purified insulin
receptors from livers of Zucker fatty rats and Sprague-Dawley rats with dietary obesity.110
Receptor-binding and tyrosine kinase activation by insulin analogues, in the human hepatoma
HepG2 cells72 have been studied. Binding of insulin derivative to human insulin receptor
overexpressed on a transfected Chinese Hamster ovary cell line has also been studied.112
An ideal antidiabetic agent is one which normalizes all of the metabolic disturbances which
occur in the diabetic patient. Diabetes, which is due to some degree of insulin deficiency, can
be-induced experimentally by removal of source or action of insulin.
Screening of antidiabetic agents must take into consideration methods for discovering
activity as well as definitive studies. Selection of physiologic end point for screening a large
number of chemicals for preliminary activity is of prime importance. Historically and because
diabetes is diagnosed by tests of glucose tolerance, attention has been generally directed to
glucose metabolism. Although diabetes is a complicated disease involving all aspects of the
intermediary metabolism, changes in blood glucose level are a convenient and useful tool in
screening for antidiabetic agents.
The most useful screening method is based on depression of blood sugar values in intact
animals, since the intact animal theoretically possesses all the mechanisms involved in blood
regulation.

Experimental models for Diabetic complications

The uncontrolled blood glucose level affects different body organ which causes some of the
undesirable effects which include microvascular complications such as retinopathy (which
can lead to blindness), nephropathy (ultimately kidney failure) and painful neuropathy
(which can lead to amputations). In addition, macrovascular complications of diabetes
include coronary artery disease, atherosclerosis, hypertension, and stroke. The micro and
macrovascular complications observed in the diabetic population result from the actions of
various pathological factors and pathways functioning independently or in combination.113-115
Some specific experimental models commonly used for screening of diabetic
complications like cardiomyopathy, nephropathy and retinopathy are also reported. Models
for cardiomyopathy and nephropathy are given below. Models for retinopathy are explained
in detail in Chapter 41.

Diabetic Cardiomyopathy
Cardiovascular complications are leading cause of morbidity and mortality related to
uncontrolled diabetes. Initially, increased coronary artery disease was considered as major
risk for failing heart, but risk of developing heart failure remained one of the major health
problem which was independent of coronary artery disease. One of the study reveals that
 Antidiabetic Agents 635

diabetic patients with normal coronary artery suffered from heart failure suggesting that
other etiologies are involved. Similarly, other studies have shown that diabetic patients with
normal blood pressure, body weight, serum lipid profile and coronary artery developed heart
failure. This research led to coin the term ‘diabetic cardiomyopathy’, which has been defined
as ventricular dysfunction occurring in diabetes patients in the absence of coronary artery
disease and hypertension.116,117
High glucose regulates structural and functional changes of cardiomyocytes via the
activation of several signal transduction pathways, including impaired calcium homeostasis,
activation of the renin-angiotensin system and increased oxidative stress.118-123

The heterozygous Ins2+/- Akita Diabetic Mouse

The Ins2Akita/+ (Akita) mouse is a model of type 1 diabetes, characterized by a point-mutation
causing proinsulin misfolding with subsequent endoplasmic reticulum stress leading to beta-
cell apoptosis. This affects folding of proinsulin in the endoplasmic reticulum, leading to
endoplasmic reticulum stress, proteotoxicity in pancreatic β-cells, and cell loss. The Ins2WT/
C96Y mouse provides an ideal nonobese model of type 1 diabetes that is based on a mutation
described in human diabetes, while being free of potential confounding effects of STZ-
induced type 1 diabetes. Moreover, Ins2WT/C96Y mice have several advantages over inbred
mouse strains that require STZ treatment, including a better defined etiology, along with a
more pronounced and durable hyperglycemia.
In Ins2Akita/+ (Akita) mouse, cardiomyopathy is characterized by early diastolic dysfunction
in the absence of systolic dysfunction. It has been studied that the diastolic dysfunction in
Ins2Akita/+ mice could be brought about by a number of factors, including elevated levels of
β-MHC isoform and reduced SERCA2a levels, which may have contributed to the impaired
relaxation of the LV.
Lipotoxicity may arise from myocardial triacylglycerol accumulation, increased oxidation
of long-chain fatty acids, and increased production of ceramide and DAG, important markers
of lipotoxicity in the heart. In addition, mitochondrial functions are impaired and the
expressions of mitochondrial encoded genes are reduced. The elevated levels of fatty acids,
triglycerol, ceramides, DAG, as well as lipid depositions in the Ins2Akita/+ hearts strongly
suggest myocardial lipotoxicity as the dominant mechanism of the diastolic dysfunction in
Ins2Akita/+ mice.124-126

OVE26 Mouse Model

OVE26 mouse is a model of type 1 diabetes. OVE26 mouse model shows severe and consistent
diabetes, and has an early onset. It can survive for more than a year without insulin treatment,
direct damage is specific to the pancreatic β-cell and breeding is simple. Overexpression of the
Ca2+ binding protein calmodulin in pancreatic β-cells led to insulin deficient diabetes in first
week after birth producing cell damage remains to be completely understood.127
OVE26 mice show cardiac abnormalities, which include reduced cardiomyocyte
contractility, degenerating mitochondrial morphology and reduced mitochondrial glutathione
content. Freshly isolated OVE26 diabetic cardiomyocytes had reduced contractility compared
to control FVB myocytes. Several studies have shown impaired peak shortening, prolonged
636 Drug Screening Methods

time to peak shortening, prolonged time to 90% re-lengthening and reduced velocities of
shortening and re-lengthening.128
OVE26 diabetic hearts showed several distinguishable ultrastructural abnormalities like
swollen mitochondria, mottled matrices and broken mitochondrial membranes with impaired
pyruvated-supported mitochondrial state 3 respiration. Mitochondrial content is increased
in OVE26 hearts and proteomic analysis showed induction of several proteins involved n
the biogenesis of mitochondria. It is very well evident important role of oxidative stress in
OVE26 hearts. In OVE26 diabetic hearts, reduced levels GSH, increased catalase expression
and increased malondialdehyde levels are found. OVE26 diabetic hearts showed impaired
intracellular Ca2+ handling, reduced activity of sarcoendoplasmic reticulum Ca2+-ATPase
2a, impaired Ca2+ release and uptake from sarcoplasmic reticulum, and reduced Na2+-Ca2+
exchanger expression.129

Diabetic Nephropathy
Diabetic nephropathy is one of the microvascular complications after chronic diabetes and
is the major cause of morbidity and mortality in patients with type 2 diabetes. End-stage
renal disease has been increased drastically worldwide in patients suffering from type 2
diabetes during the past two decades, and diabetes is associated with worse survival rate in
patients undergoing dialysis. Despite the high prevalence of diabetic nephropathy, around
40% of all diabetic patients are at risk of developing end-stage kidney failure, and genetic
studies indicate grave risk for diabetic nephropathy.130 A large number of pool of genes have
been identified in the onset and progression, but still are weak predictors of nephropathy in
patients with type 2 diabetes. Experimental models of type 2 diabetes with nephropathy may
offer a key to a better understanding of this complication in a multifactorial disease such as
type 2 diabetes.131
Increased blood glucose level causes the nephropathy via different pathogenic mechanism
like changes in hemodynamic mechanisms, activation of protein kinase C, aldose reductase
pathway, increases level of cytokines and oxidative stress.132-137

Insulin-2 Akita Mouse

Akita mice develop type 1 diabetes because of the spontaneous mutation in Ins-2 gene.
The mutation leads to the misfolding of insulin protein, which is toxic to pancreatic β-cell.
Therefore, the ability of β-cell to secrete insulin is mainly decreased. The Ins-2 Akita mutation,
which is autosomal dominant, was originally found in C57BL/6 mice in Akita, Japan.
Heterozygous mice develop significant hyperglycemia as a result of severe insulin deficiency
at 3–4 weeks of age. A male suffers considerably worse insulin deficiency compared to female.
Mice with Ins-2 Akita mutation exhibit modest levels of albuminuria and mild-to-moderate
glomerular mesangial expansion. However, Akita mice develop higher levels of hyperglycemia,
albuminuria, blood pressure, and more consistent structural changes of kidney compared
with STZ-induced diabetic nephropathy. In one of the study, Gurley et al. 2006, have found
that renal phenotype of Akita mice depend largely on their genetic background strains, which
suggest that genetic factors might influence susceptibility to diabetic nephropathy in Akita
mice which is the same in human disease. Thus, mice having Ins-2 Akita mutations have
significant advantages as a model of type 1 diabetes.138,139
 Antidiabetic Agents 637

BTBR ob/ob Mice

When the ob/ob mutation is crossed with BTBR background, the mice are initially insulin
resistant with elevated insulin levels, pancreatic islet hypertrophy and marked hyperglycemia
by 6 weeks of age. They are largely resistant to significant lowering of blood glucose by insulin
administration. The kidney phenotype of these mice shows early loss of podocytes and onset
of proteinuria, both of which are detectable by 8 weeks of age.
Glomerular hypertrophy marked expansion of mesangial matrix, mesangiolysis, and
capillary basement membrane thickening have been identified in this model. These features
model morphologically mimics early human DN, and by 18 weeks of age, these mice model
have progressive, advanced DN with features of marked proteinuria, more extensive mesangial
expansion, mesangiolysis, persistent podocyte loss, and basement membrane thickening.
Like other mice with leptin deficiency, these mice are infertile and breeding mice in sufficient
numbers for interventional studies is labor intensive and expensive.140,141

eNOS Deficient Mice

Endothelial dysfunction is present in diabetes and is associated with impaired vascular nitric
oxide (NO) synthesis. A number of polymorphisms in the endothelial NO synthase gene
(eNOS), located on human chromosome 7, have been linked to renal vasculopathies. There are
currently two models of diabetic nephropathy dependent on genetic deletion of endothelial
nitric oxide synthase (eNOS−/−). eNOS provides the principal means by which nitric oxide
is generated in the vasculature; NO is in turn a major regulator of vascular tone. Reduction
of NO availability has been considered a major mechanism for the development of diabetic
complications involving the vasculature, including diabetic nephropathy.142
The eNOS−/−/db/db mouse is a model of type 2 diabetes generated by backcrossing of
eNOS knockout mouse on the C57/B6 background with db/db mouse on the C57BLKS/J
background. The eNOS−/−/lepr db/db double-knockout mice exhibit obesity, hyperglycemia,
hyperinsulinemia, hypertension, dramatic albuminuria, and decreased GFR. These mice develop
histopathological features of DN-like human disease such as mesangial expansion, glomerular
basement thickening, mesangiolysis, focal segmental and nodular glomerulosclerosis, nodules,
fibronectin accumulation in glomeruli, arteriolar hyalinosis, minimal tubulointerstitial
fibrosis, and microaneurysms.143,144
In second model, eNOS deficiency has been introduced in streptozotocin induced type I
diabetes in eNOS−/− mice of C57BL/6 background. Although C57BL/6 mice are resistant to
development of diabetic nephropathy; however, eNOS deficiency was found to be sufficient to
induce development of features of advanced diabetic nephropathy, including hypertension,
albuminuria, mesangial matrix expansion, mesangiolysis and mesangial nodule formation,
and renal insufficiency. Like eNOS−/−/db/db mice, this model also offers a means to study
mechanisms underlying advanced diabetic nephropathy, but several important pitfalls needs
to be considered.145

Podocyte Specific Insulin Receptor Knockout Mouse

A recent study shows that a mixed mouse strain (with C57BL/6, 129/Sv, and FVB backgrounds)
with podocyte specific deletion of insulin receptors using both podocin and nephrin promoters
638 Drug Screening Methods

to drive cre-recombinase directed excision of the receptor. Furthermore, loss of podocyte

insulin responsiveness in the intact perfused glomerulus results in glomerular pathology with
a number of features characteristic of diabetic nephropathy (DN), including increased matrix
production, glomerulosclerosis, thickening of the glomerular basement membrane, and,
with time, podocyte apoptosis. These mice develop proteinuria and were reported to develop
histologic features of DN despite maintaining normal insulin levels and normoglycemia.
Features of DN exhibited by these mice included increased podocyte apoptosis at 8 weeks of
age, detected at a time when significant proteinuria was becoming established, podocyte foot
process effacement, and increased mesangial matrix.146,147

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 cHAPTER

41
Diabetic Retinopathy
INTRODUCTION
Diabetes mellitus is a heterogeneous disorder defined by the presence of hyperglycemia.
Diagnosis criteria for diabetes mellitus include (1) a fasting plasma glucose >126 mg/dl, (2)
symptoms of diabetes plus a random plasma glucose > 200 mg/dl, or (3) a plasma glucose
level > 200 mg/dl after an oral dose of 75 g of glucose. Hyperglycemia in all cases is due to
a functional deficiency of insulin action, which may be either due to a decrease in insulin
secretion by the β-cells of the pancreas or insulin resistance. Majority of cases of diabetes are
regarded as primary processes for which individuals have a genetic predisposition and are
classified as either type I or type II. Type I diabetes mellitus is characterized by autoimmune
β-cell destruction leading to severe insulin deficiency.
In contrast to Type I diabetes mellitus, Type II diabetes mellitus is 10 times more common,
has stronger genetic component, occurs commonly in adults, increases in prevalence with age
and is associated with increased resistance to the effects of insulin.
The diabetic patient is susceptible to a series of complications that cause morbidity and
premature mortality. Diabetic retinopathy is one such complication that is associated with
long-standing diabetes. It remains as one of the leading cause of blindness. Diabetic retinopathy
has been understood as a microvascular complication, the pathogenesis of which is not
completely understood. Microscopically, the basement membrane appears to be thickened
with increased amounts of collagen and decreased amounts of proteoglycan. In conditions
of high circulating levels of sugar, glucose nonenzymatically reacts with amino groups in
proteins to form Advanced Glycation End Products (AGEs). AGEs bind to matrix components
of basement membrane, specific receptors on macrophages, and endothelial cells to initiate a
cascade of release of cytokines, which in turn affect the proliferation, and function of vascular
cells (Fig. 41.1).
Diabetic retinopathy can be classified in different ways, but there are three main types:
a. Background retinopathy: There are tiny swellings in the blood vessel walls. These blebs
(micro aneurysms) appear as small red dots on the retina. There are tiny yellow patches of
hard exudates (proteins from the blood) on the retina. Dots and blots of hemorrhage appear
on the retina (Figs 41.2A and B).
b. Maculopathy: In maculopathy, the hemorrhages, exudates and swellings of the first stage
occur in the macula. This may interfere with vision, particularly for reading and seeing fine
details (Fig. 41.3).
646 Drug Screening Methods

c. Proliferative retinopathy: The fundamental characteristics of proliferative retinopathy are

new vessel formation and scarring. The stimulus for neovascularization may be retinal
hypoxia secondary to capillary or arterial occlusion (Fig. 41.4).

Figure 41.1: Etiopathology of diabetic retinopathy

Figure 41.2A: Normal human retina Figure 41.2B: Background diabetic retinopathy with
characteristic microaneurysms and hard exudates

Figure 41.3: Diabetic maculopathy Figure 41.4: Proliferative retinopathy

 Diabetic Retinopathy 647

Diabetic retinopathy occurs in two distinct stages: non-proliferative and proliferative.

Nonproliferative retinopathy is characterized by microaneurysms of the retinal capillaries,
appearing as tiny red dots. This background retinopathy has often been documented as the
earliest clinically detectable sign of diabetic retinopathy. Further there is a loss of pericytes
that surround and support the capillary walls leading to increased vascular permeability and
leaking of fat. This progresses to formation of hard exudates in areas of macula. Macular edema
describes the condition where retinal blood vessels can develop tiny leaks. When this occurs,
blood and fluid seep from the retinal blood vessels, and fatty exudates deposit in the retina.
This causes swelling of the retina and is called diabetic macular edema leading to reduced or
blurred vision.
The above-mentioned preproliferative stage often worsens to a more severe proliferative
stage. Proliferative retinopathy describes the changes that occur when new, abnormal
blood vessels begin growing on the surface of the retina. This abnormal growth is called
neovascularization. If these abnormal blood vessels grow around the pupil, glaucoma can
result from the increasing pressure within the eye. These new blood vessels have weaker walls
that may break and bleed, or cause scar tissue to grow. These complications may cause retinal
detachment and finally severe vision loss, blurred and distorted images including blindness.

MAGNITUDE OF DISEASE
Diabetic retinopathy is emerging as one of the important causes of blindness in both developing
and developed countries. In 2013, there are more than 382 million (or 8.3% of Adults) people
suffering from diabetes, and it is being projected that the number of diabetic patients will reach
592 million, or one adult in 10, will have diabetes in 2035. This equates to approximately three
new cases every 10 sec or almost 10 million per year.1 India is also plagued by the condition
and it is expected that the country will have over 50 million cases by 2025. Diabetic retinopathy,
a complication of diabetes occurs in patients of both type 1 and type 2. Recently, it has been
shown that nearly all type 1 and 75 percent of type 2 diabetes will develop diabetic retinopathy
after 15 year duration of diabetes.2 In a clinic population of a cohort of 6792 type 2 diabetic
patients attending a diabetes center in south India, the prevalence of diabetic retinopathy was
found to be 34.1 percent. 2

PRESENT THERAPY FOR DIABETIC RETINOPATHY

Currently, there is no specific drug therapy for diabetic retinopathy. The sole management
lies in surgery. Surgical treatment is decided, after taking into account the stage of the disease
and the specific problem that requires attention. The retinal surgeon relies on several tests to
monitor the progression of the disease and to make decisions for the appropriate treatment.
These include fluorescein angiography, retinal photography and ultrasound imaging of the
eye. Focal photocoagulation (focal laser treatment for sealing specific leaking blood vessels) or
Scatter (pan-retinal) photocoagulation (laser treatment over a wide area of retina) are the two
oft-used approaches for the management. Other management approaches include intraocular
steroid injection, cryotherapy and vitrectomy. However, these manage­ment options suffer
from drawbacks as laser photocoagulation burns and destroys part of the retina that itself can
648 Drug Screening Methods

result in some permanent vision loss. Treatment may cause mild loss of central vision, reduced
night vision, and decreased ability to focus. Some people may lose their peripheral vision. It
may also lead to ocular neovascularization. Rare complications of laser photocoagulation that
may cause severe vision loss include vitreous hemorrhage, traction retinal detachment, and
accidental laser burn of the fovea.
Management of diabetic retinopathy remains a challenge with very few modalities available.
There is a paucity of pharmacological approaches towards its management. Some of the
molecules that have come into fore are tabulated (Table 41.1) and have substantial role to play
in both prevention and therapeutics. Therefore, screening of new molecules in preclinical and
clinical studies against models of diabetic retinopathy, takes prime importance.

Table 41.1: Novel molecules for prevention of diabetic retinopathy

S.No. Novel molecule Putative mechanism of action
1 Vitamin C Antioxidant, Prevents protein glycosylation
2 Vitamin E Antioxidant, Prevents protein glycosylation
3 Acetyl-L-carnitine Antioxidant
4 Magnesium Role unclear
5 Ginkgo biloba Antioxidant

IN VITRO MODELS
Retinal Endothelial Cell Culture
The capillaries of the retina constitute a barrier (Blood Retinal Barrier, BRB) between the
systemic circulation and retina that physiologically functions to regulate the passage of blood
borne solutes, compounds, amino acids, and glucose from blood to retina. Histologically, BRB
comprises of microvascular endothelial cells (also known as Inner BRB) and retinal pigment
epithelial cells (also known as Outer BRB). Chronic exposure to hyperglycemia, as in diabetes
and its associated complications such as diabetic retinopathy, has been recently reported to
alter the physiology of BRB, especially inner BRB.3
Primary cell cultures of retinal endothelial cells are being extensively used to study the
physiology, pathogenesis and putative therapeutic interventions for diabetic retinopathy.
The retinal endothelial cells isolated from human, murine or bovine vessels are seeded on
a reconstituted basement membrane extracellular matrix in the absence or in the presence
of the substance under test, and the efficacy of the test substance to inhibit formation of a
capillary-like network is observed and quantified by digital image analysis.4

Primary Cell Cultures of Bovine Retinal Endothelial Cells

Bovine retinas are the source of capillaries used to isolate cells for primary culture. Cow eyes can
be obtained from a local abattoir. Primary bovine retinal endothelial cell (BREC) cultures are
established from fresh calf eyes. Under sterile conditions, the retinas are isolated and washed
in Dulbecco’s modified Eagle’s medium (DMEM) and pieces of adherent retinal pigment
epithelial cells are removed. The retinas are transferred to an enzyme solution containing
pronase (100 µg/ml), collagenase (500 µg/ml) and DNase (70 µg/ml) and incubated with
 Diabetic Retinopathy 649

shaking at 37°C for 20 min. After incubation, the retinal digest is passed through 210 and 50 µm
nylon mesh and the microvessels trapped on top of the 50 µm mesh are collected in DMEM
by centrifugation. The fragments are resuspended in DMEM with 15% fetal calf serum (FCS),
20 µg/ml endothelial growth supplement, heparin (100 µg/ml) and antibiotic-antimycotic
solution, plated and grown on fibronectin coated dishes in low glucose DMEM, at 37°C with
5% CO2. To determine the effect of high glucose, BREC can be grown in low (5.5 mM) or high
(25 mM) D-glucose medium up to 48 h.5,6

Cell Line of Retinal Capillary Endothelial Cells

A conditionally immortalized retinal capillary endothelial cell line can be grown and
maintained in DMEM with low glucose, containing 10% fetal bovine serum (FBS), 100 U/ml
penicillin G, 100 U/ml streptomycin in humidified atmosphere composed of 95% air and 5%
CO2 at 33°C. Cells are grown to approximately 40-50% confluence and incubated in regular
DMEM containing 5.5 or 25 mM glucose at 37°C. Control experiments can be performed using
mannitol to test the effect of high osmolarity on various intracellular paradigms like GLUT1
expression.5

Retinal Neural Cells

During the event of pathogenesis of diabetic retinopathy, methylglyoxal and glyoxal are
intermediates of AGEs. These substances in conjunction with other factors such as oxidative
stress, induce apoptosis of retinal neurons. Studies conducted on retinal neural cells provide
crucial information with regard to diabetic retinopathy.7
E1A-NR3 is a rapidly proliferating rat retinal precursor cell line that has been used in several
studies to study the expression of various retinal and neuronal markers. The rat retinal cell
line E1A-NR is maintained in DMEM containing 10% FCS, 2 mM glutamine, 1% nonessential
amino acids, and 1%vitamins.
Immunocytochemistry studies can be conducted by growing the cells on glass coverslips
coated with collagen IV (5 µg collagen IV/ml 0.05 M HCl for 60 min). Monoclonal mouse
antihuman antibodies can be added directly to the cell cultures and appropriately incubated.
After washing the cells are fixed with 4% paraformaldehyde. FITC-conjugated antibodies can
be used and the cells can be viewed with a fluorescence microscope.
Apoptosis assays are conducted on retinal neural cells to demonstrate the influence of
various stresses on cell viability and functionality. The cells are seeded and when they are
subconfluent, glyoxal, methyl glyoxal, or hydrogen peroxide is added to the culture medium.
After incubation, the supernatant is removed and the detached cells are collected. Adherent
cells are collected after trypsin treatment. Adherent and detached cells are pooled, washed
with PBS containing 1% horse serum, and subjected for apoptosis assays like fluorescence
activated cell sorting (FACS), terminal deoxynucleotidyl transferase biotin-dUTP nick end
labeling (TUNEL).7

Retinal Pigmented Epithelium (RPE) Cell Line

Human Retinal pigmented epithelium (HRPE) cell line is widely used to screen drugs having
protective effect on diabetic retinopathy. High blood glucose in diabetes is associated with
650 Drug Screening Methods

increased intracellular soluble adenylyl cyclase (sAC), and intracellular cAMP levels in HRPE
cells which results in impairment of integrity of HRPE cell line barrier.
In this model, HRPE cell line is treated with high glucose concentration (25 mM) to mimic
the hyperglycemic condition in the absence or presence of the test drug. The efficacy of
test drug is assessed by measuring its ability to reverse drop in the transepithelial electrical
resistance (TEER) due to high glucose concentration.8

EX VIVO MODELs
The effect of chronic hyperglycemia on retinal vasculature can also be studied in vivo, using ex
vivo models. The colony of Goto Kakizaki (GK) rats shows persistent hyperglycemia after six
weeks of age. Six month to one year old diabetic rats are used for the study. They are fed normal
rat chow ad libitum and maintained in temperature controlled facilities with 12 h light-dark
cycles. Glucose concentrations are routinely measured on tail blood samples using a glucose
monitor.
To study changes associated with diabetic retinopathy, the rats are sacrificed by decapitation
and their eyes are quickly removed. Retinas are isolated and wrapped in aluminum foil, frozen
on liquid nitrogen and stored at –80°C until used. The study is conducted on membrane fraction
of the retina from diabetic GK rats. Total retina homogenates from diabetic rats is obtained by
tissue lysis in 10 mM Tris-HCl, 1 mM EDTA, 250 mM sucrose, protease inhibitors, pH 7.4, at 4°C
and mechanical disruption using a homogenizer (50-60 strokes). The samples are centrifuged
at 9,000x g to remove nuclei, mitochondria and unlysed cells, and recentrifuged at 1,00,000x g
for 75 min to obtain the total cell membranes. The membrane pellet is resuspended in 10
mM Tris-HCl, 1 mM EDTA, pH 7.4 containing protease inhibitors, 0.5% sodium deoxycholate
(DOC) and 1% Triton X-100. The samples are then centrifuged at 14,000x g to remove the
unsoluble fraction.
Using standard techniques of Western, Northern blot, RT-PCR, protein changes in the
basement membrane can be qualitatively and quantitatively assessed.5

IN VIVO MODELS
Diabetic Retinopathy in Streptozotocin Induced Diabetic Rats
Healthy adult Wistar rats of either sex weighing between 250-300 g are maintained on standard
laboratory chow and tap water under 12 h light: dark cycles. Diabetes is induced by single
injection of streptozotocin (50-70 mg/kg, ip).9
The control group is simultaneously adminis­tered equal volume of vehicle (3 mM citrate
buffer, pH 4.5). After 10 days, the plasma glucose of the animals is monitored and rats with
plasma glucose > 300 mg/dl are included in the study. After two weeks, diabetic retinopathy
can be studied in the rats. The following paradigms can be evaluated to assess the etiology,
pathogenesis, preventive and therapeutic interventions in this model:
a. Proliferative diabetic retinopathy is assumed to develop because vasogenic factors are
released from retinal areas that are ischemic and hypoxic secondary to occlusion of the
retinal vascular bed. Vascular occlusion in diabetic retinopathy may be due to the deposition
of periodic acid Schiff positive glycoprotein compounds in the retinal vascular walls.
 Diabetic Retinopathy 651

Periodic acid Schiff staining and immunohistochemistry helps to investigate the expression
of laminin, fibronectin, type VI collagen and VEGF in areas of retinal capillary closure.
b. Blood sugar monitoring: Blood glucose level of normal, control and experimental groups
of rats can be estimated periodically. Around 0.05 ml of blood is withdrawn from the tail
vein by tail puncture and applied to the strip. The blood glucose levels are recorded using
digital glucometer.
c. Blood HbA1c, Hb-AGE measurements: According to the method of Al-Abed et al. (1999),10
under light ether anesthesia, whole blood of rats is collected from tail vein into heparinized
tubes. Hemolysates are prepared and purified hemoglobin is prepared in the standard
method. Hemoglobin content is measured by Drabkin’s reagent and the quantity of HbA1c
is measured by using a standardized chromatography method. AGE-modified hemoglobin
(Hb-AGE) can be measured by an AGE-specific ELISA.
d. Antioxidant activity: The retina is homogenized in 50 mM PBS, under cold conditions using
hand homogenizer for the estimation of Thiobarbituric acid reacting substances (TBARS),
antioxidant enzymes (reduced glutathione, catalase and superoxide dismutase).
e. Fluorescein angiography: Clinical presentation of the pathology can be monitored by
fluorescein angiography. Fluorescent dye is injected into the leg vein of the rat. The dye
travels through the body including the eyes. With special camera, meant for the purpose of
photography of the retina, the retinal vessels are observed and photo documented as the
dye flows through the retinal vessels.11
f. Visualization of vessel leakage: Increased vascular permeability is a useful marker and can
be assessed after 2 weeks of induction of hyperglycemia. The dye Evans blue, which has the
property of plasma albumin binding, is used to study the blood trajectory and vessel leakage,
if any. Briefly, the following protocol is followed. Under deep anesthesia, rats weighing
approximately 200 g are kept on a warming plate (37°C, 20 min) before injection of 200 µl
of 2% (wt/vol) Evans blue dissolved in sterile physiological solution through the femoral
vein. The animals are returned to the warming plate for 20 min before sacrificing. Retinas
are rapidly isolated and stored in 10% formaldehyde, flat mounted,and immediately viewed
and photographed under fluorescent light (excitation filter 546 nm, barrier filter 590 nm).
Modifications of the standard protocol are also widely applied. Retinal vascular leakage can
also be measured by intravenously injecting FITC-BSA. After induction of anesthesia, the
rats receive tail vein injections of 100 mg/kg FITC-bovine serum albumin and the animals
are sacrificed 20 min later, and their eyes are removed, embedded in OCT medium, and
snap-frozen in liquid nitrogen. The plasma is simultaneously collected and assayed for
fluorescence with a fluorescence spectrophotometer based on standard curves of FITC-
BSA in normal rat plasma. Frozen retinal sections (6 µm thick) are collected and viewed
with fluorescence microscope. Quantification of FITC-BSA fluorescence intensity indicates
the vascular leakage.12

Diabetic Retinopathy in Streptozotocin-induced Diabetes in C57Bl/6 Mice

Male C57Bl/6 mice weighing 20-25 g (5-6 weeks old) are randomly assigned to nondiabetic
control or diabetic groups. Diabetes is induced by a single intraperitoneal injection of
streptozotocin at (180 mg/kg). Control animals receive an equivalent dose of the drug vehicle
(citrate buffer at pH 4.6). The mice are caged individually and allowed food and water ad
652 Drug Screening Methods

libitum. Blood glucose level has to be measured fortnightly. Diabetic animals with blood
glucose levels between 20 and 30 mM are included in the study.13
Feit-Leichman et al. (2005), induced diabetes in C57Bl/6J mice of 7 to 10 weeks with 5
consecutive injections of streptozotocin @ 55 mg/kg.14 The blood levels were maximum at 4
weeks after the STZ treatment.

Diabetic Retinopathy in Galactose Fed Rat model

Feeding with galactose is another method to induce hyperglycemia. In this model, blood
aldohexose concentration is elevated in the animal and other biochemical parameters such
as concentrations of insulin, glucose, fatty acids, and amino acids remain unaltered. The
monitoring of animals can be done up to 28 months as galactose-fed rats have a longer life
span as compared to other diabetic models.
Method: In this model, weanling Sprague-Dawley rats are used; normal control group is fed
with laboratory chow plus 50% starch, diabetic control group is fed with 50% D-galactose,
and treatment group is fed with 50% D-galactose with test drug for 4 to 8 months and then
changes by addition of test drug are measured and compared with control groups.15

Nondiabetic Models of Proliferative Retinopathy

Proliferative retinopathy can also be induced in animals by inducing neovascularization
in the ocular region. Proliferative retinopathy is induced mainly by two methods: (i) by
introduction of ischemia to the eyes, such as oxygen-induced retinopathy (OIR) or retinal
occlusion; and (ii) by direct injection or genetically induction of the angiogenic factor, VEGF,
into the ocular region.
In OIR model one week old neonatal mice are kept in a 75% oxygen chamber for five days
and then returned to room air. Vessel loss in the central area of retina, which is associated
with hypoxic challenges, is observed immediately at this time. Neovascularization extending
from the inner retina into the vitreous begins at two days after the return to room air, peak
is observed on third week, and is gradually regressed and spontaneously resolved by fourth
week.16
DR can also be induced by producing ischemia in the retina using retinal occlusion models;
by unilateral ligation of pterygopalatine artery (PPA) and external carotid artery (ECA),17 branch
retinal vein occlusion,18 and elevating the intraocular pressure (IOP).19 In these occlusion
models, reduced thickness of retinal cell layers, increased apoptotic cells, reduced a-wave,
b-wave, and OPs amplitudes of the ERG are evidenced.

Transgenic Models of Spontaneous Retinopathy

HLA-A29 Mice
Humans inheriting the class I major histocompatibility allele HLA-A29 are at a markedly
increased relative risk of developing eye diseases. The human MHC class I specificity HLA-A29
has been recorded in over 95.8% patients of chorioretinopathy. Transgenic mice expressing
HLA-A29 molecules have been developed as an animal model for retinopathy.20
 Diabetic Retinopathy 653

For constructing the transgene, HLA-A*2902 cDNA (A29c) is obtained from patient suffering
from bird shot chorioretinopathy (BSCR). The SalI–Hind III cDNA insert is cloned into
pBluescript II SK expression vector (Stratagene). After digestion, the purified insert is inserted
into pBSK-SP19 containing the H-2Kb promoter and the simian virus 40 poly(A) addition site.
The resultant recombinant gene is excised by XhoI and NotI and used to generate HLA-A29
transgenic mice. Briefly, the protocol for producing transgenic mice is described here.
Procedure: The DNA fragments are purified free of vector DNA by preparative agarose gel
electrophoresis and microinjected into fertilized (C57BL/6×SJL) × BALB/c oocytes. Embryos
surviving microinjection are reimplanted into oviducts of pseudopregnant females, and
offsprings are tested for the integration of the transgene by Southern blot analysis of tail-
derived DNA digested by ClaI and BamHI with the use of a 0.9-kb fragment containing the
simian virus 40 poly(A) addition site as a probe.20 The descended mice can be utilized to
conduct studies on retinopathy.

Spontaneous Hypertensive Rats (SHR)

Inflammation has an important role in the pathogenesis of diabetic retinopathy. The main
secondary risk factor associated with DR is hypertension. Genetic hypertension in early retinal
inflammation in experimental diabetes has been studied by Silva et al. 2007.21
They induced diabetes in both fully hypertensive rats and rats developing hypertension
along with the age matched control normotensive Wistar Kyoto (WKY) rats. They concluded
that the developing hypertension and fully developed hypertension lead to earlier development
of inflammation in diabetic retina.
Procedure: Spontaneous hypertensive rats of 12 weeks age (fully hypertensive) and 4 weeks
age (developing hypertension) are administered 50 mg/kg streptozotocin intravenously. After
20 days the rats are sacrificed to collect the retina and the changes in the retinal expression of
inflammatory parameters can be evaluated.
Spontaneous diabetic rodent models: There are some strains of rodents which are reported to
show spontaneous hyperglycemia used in the study of DR including db/db mice,22 Non-obese
diabetic (NOD) mice,23 Akita (Ins2Akita) mice,24 Zucker diabetic fatty rats,25 Otsuka long-Evans
Tokushima fatty rats,26 Goto-Kakizaki rats,27 Diabetic Torii rats.28

Transgenic Vascular Endothelial Growth Factor; Kimba Mouse Model

Role of vascular endothelial growth factor (VEGF) in the pathogenesis of DR instigated
Shen et al. 2006, to investigate whether transgenic mice with moderate VEGF expression in
photoreceptors (trVEGF029) developed changes similar to diabetic retinopathy and whether
retinopathy progressed with time.29
Evaluation of human VEGF165 (hVEGF165), serum glucose levels; changes in the density of
retinal vasculature and changes similar to diabetic retinopathy such as retinal leukostasis,
capillary endothelial cell and pericyte loss and number of acellular capillaries, suggested that
an early short-term elevation in hVEGF165 expression leads to progressive retinopathy. Mice
can be generated by the following method.
654 Drug Screening Methods

Procedure: Mice are generated through microinjection of a DNA construct containing

hVEGF165 gene (pcDNA.opsin.VEGF) driven by a truncated mouse rhodopsin promoter
confining transgene expression to the eye. Backcrossing with C57BL/6J mice produces fifth
and sixth generation heterozygous mice and age matched wild type littermates. These mice
demonstrate moderate elevation of hVEGF165, limited neovascular changes and minimal
retinal damage; and help in the examination of early pathological changes.29,30
Akimba mice model: Akimba mice is an ideal model created by crossing of Akita mice with
Kimba mice, (Ins2AkitaVEGF+/−) and has the advantage that it inherits the diabetic phenotypes
from their parental strains and show hyperglycemia and retinal neovascularization at the same
time.31
Monkey Model of DR: Monkey provides a good model in eye research, as the ocular
structures of monkeys are similar to that of humans. Some of the reported monkey models
are Hyperglycemic DR model,32 VEGF-induced proliferative retinopathy33 and Spontaneous
diabetes34,35 in obese rhesus monkeys.
In Hyperglycemic DR model, pancreatectomy or STZ injections are used to induce
hyperglycemia in monkeys32 and retinal changes due to hyperglycemia are measured. In VEGF-
induced proliferative retinopathy model, human recombinant VEGF in pellet is implanted into
the vitreous cavity of the animal to mimic the inflammatory changes associated with natural
DR.33 In Spontaneous diabetes model hyperglycemia occurs spontaneously in obese rhesus
monkeys.34,35
In monkey models morphological changes in retina like BRB break down, vascular tortuosity
are observed after 2 weeks of implantation. However these models are not used widely and not
validated due to many demerits like ethical issues, lack of proliferative retinopathy, high cost,
longer duration of study, etc. Other animal models reported for the study of DR are dog,36-38
rabbit,39 cat,40-42 and swine.43,44
Rat model of streptozotocin-induced diabetes is one of the most widely accepted models
for long-term studies. Retinal lesions seen in rats have similarity to the initial process of DR in
man.20 However, mice can be genetically manipulated and provide the opportunity to study
the molecular pathways in the development of diabetic retinopathy. Transgenic mice help in
the examination of individual factors, which contribute, to early stages of diabetic retinopathy
and to test early therapeutic interventions. The models described above vary from each other
in some aspect or the other having their own advantages and disadvantages. The selection of
the appropriate experimental model therefore has a prime importance in execution of one’s
study.

Zebrafish Model of DR
Zebrafish is an important tool to study visual development and impairments. They are proving
to play important role in the study of etiology, pathogenesis, preventive and therapeutic
interventions in DR owing to their very small size, large breeding size, easy to handle and
similarity to those seen in human. The distinctive pattern of the mammalian retinal cell layers,
ranging from ganglion cell layer to retinal pigment epithelium, is observed in zebrafish.45 They
have a short life span and a large breeding size, which in turn allow a shorter experimental
turnover time. Moreover, a number of studies showed that genes of interest can be specifically
induced, deleted, or overexpressed in zebrafish, allowing mechanistic studies of diseases.46
 Diabetic Retinopathy 655

DR can be studied in zebrafish via direct elevation of glucose in the surrounding as well as
angiogenesis without the involvement of glucose and by genetic manipulation.

Glucose-induced Diabetic Model of DR

In this model, hyperglycemia is induced in zebrafish by immersing the fish in 2% glucose
solution and water every other day for one month. After one month animal is sacrificed,
eyes are dissected and pathological changes are observed and compared between control
(untreated zebrafish) and treated zebrafish group.
Method: In this model, adult zebrafish are acclimated to standard laboratory conditions;
14 h light and 10 h dark cycle, 28°C temperature are selected for screening. The acclimated
fish are placed in 2% glucose solution and water alternate days for one month under standard
conditions of laboratory. After a period of one month fish are anesthetized by 0.04% tricaine
methanesulfonate solution environment. Anesthetized fish are decapitated by a sharp
blade and blood glucose readings are collected immediately. Whole eyes are dissected, and
incubated for fixation in 4% formaldehyde solution for 1 week. The fixed eyes are equilibrated in
phosphate-buffered saline solution containing 30% sucrose at 4°C for overnight and sectioned.
The sectioned retinal tissues are labeled with 4,6-Diaminidino-2-phenyloindole (DAPI) and
images are viewed under fluorescence microscopy. Various morphological changes like
thickness of the inner plexiform layer (IPL) and inner nuclear layer (INL) are measured and
comparison is done between normal, treated (administered test drug) and control group
(untreated zebrafish) to detect any change by the test drug in the pathogenesis of disease.47
Angiogenesis model for study of DR in Zebrafish: Two models are reported to study
angiogenesis in zebrafish environmentally and transgenic-induced models.
Hypoxia-induced retinal angiogenesis in zebrafish: In this model, retinal neovascularization
is induced by keeping the fli-EGFP-Tg zebrafish in hypoxic aquaria where the air saturation is
gradually reduced to 10% (820 bbp) over a course of 2 to 3 days. Zebrafish is exposed in this
hypoxic environment for different time points with a maximal period of 15 days. After 12 days
of hypoxic challenge, neovascularization is observed in the retina evident by increased number
of branch points, sprouts, and vascular area as well as reduced intercapillary distance.48
Transgenic Zebrafish model: Retinal angiogenesis can also be induced in zebrafish via
transgenic approach. Zebrafish carrying vhl−/− mutation displays an upregulation of hypoxia-
inducible factor, which in turns triggers VEGF production and expression of the VEGF
receptors. However, this model is not commercially available, which limits its use in the field
even though severe neovascularization and proliferative retinopathy are observed.47-50

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function in an aged monkey with spontaneous diabetes mellitus. Exp Eye Res 2005;80(1):37-42.
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with spontaneous type 2 diabetes. Invest Ophthalmol Vis Sci 2004;45(12):4543-53.
36. McLeod DS, Crone SN, Lutty GA. Vasoproliferation in the neonatal dog model of oxygen-induced
retinopathy. Invest Ophthalmol Vis Sci 1996;37(7):1322-33.
37. Kobayashi T, Kubo E, Takahashi Y, Kasahara T, Yonezawa H, Akagi Y. Retinal vessel changes in
galactose-fed dogs. Arch Ophthalmol 1998;116(6):785-89.
38. Takahashi Y, Wyman M, Ferris F 3rd, Kador PF. Diabetes like preproliferative retinal changes in
galactose-fed dogs. Arch Ophthalmol 1992;110(9):1295-302.
39. Drago F, La Manna C, Emmi I, Marino A. Effects of sulfinpyrazone on retinae damage induced by
experimental diabetes mellitus in rabbits. Pharmacol Res 1998;38 (2)97-100.
40. Hatchell DL, Toth CA, Barden CA, Saloupis P. Diabetic retinopathy in a cat. Exp Eye Res
1995;60(5):591-93.
41. Linsenmeier RA, Braun RD, McRipley MA, et al. Retinal hypoxia in long-term diabetic cats. Invest
Ophthalmol Vis Sci 1998;39(9):1647-57.
658 Drug Screening Methods

42. Mansour SZ. Reduction of basement membrane thickening in diabetic cat retina by sulindac. Invest
Ophthalmol Vis Sci 1990;31(3):457-63.
43. Umazume K, Barak Y, McDonald K, Liu L, Kaplan HJ, Tamiya S. Proliferative vitreoretinopathy in
the Swine-a new model. Invest Ophthalmol Vis Sci 2012;53 (8):4910-6.
44. Lee SE, Ma W, Rattigan EM, et al. Ultrastructural features of retinal capillary basement membrane
thickening in diabetic swine. Ultrastruct Pathol 2010;34 (1):35-41.
45. Goldsmith P, Harris WA. The zebrafish as a tool for understanding the biology of visual disorders.
Semin Cell Deve Biol 2003;14 (1):11-8.
46. Collery RF, Cederlund ML, Smyth VA, Kennedy BN. Applying transgenic zebrafish technology to
study the retina. Adva Exp Med Biol 2006;572:201-7.
47. Gleeson M, Connaughton V, Arneson LS. Induction of hyperglycaemia in zebrafish (Danio rerio)
leads to morphological changes in the retina. Acta Diabetol 2007;44 (3):157-63.
48. Cao R, Jensen LDE Söll I, Hauptmann G, Cao Y. Hypoxia-induced retinal angiogenesis in zebrafish
as a model to study retinopathy. PLoS One 2008;3 (7);e2748.
49. Rooijen E van, Voest EE, Logister I, et al. Von Hippel-Lindau tumor suppressor mutants faithfully
model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish. Dis
Models Mech 2010;3(5-6):343-53.
50. Lai AKW , Lo ACY. Animal Models of Diabetic Retinopathy: Summary and Comparison. J Diab Res
2013;2013: 29 pages. Article ID 106594, doi:10.1155/2013/106594.
 cHAPTER

42
Ocular Toxicity
INTRODUCTION
Many substances are regularly used that are capable of damaging the eyesight. The eyes
represent a valuable asset to people and the sense of vision is critical for normal functioning of
an individual. They are in danger of accidental or intentional exposure to chemical preparations.
This can occur at any step during the production, transportation, use and disposal of these
preparations. In the mid-twentieth century, the aftermath of chemical warfare research and
the hazards of unsafe cosmetics justified the need of public protection. Thus, the Draize eye
test1 became the standard method endorsed by the government of various countries to evaluate
the safety of materials meant for use in or around the eyes. The test involves a standardized
protocol for instilling agents on to the cornea and conjunctiva of rabbits. In the past few years,
the ethical concerns over and the cost involved in the use of animals in research, has led to
active development in the field of alternative testing for ocular toxicity assessment.

IN VIVO IRRITATION TESTING

An in vivo test to study the eye irritation potential of chemicals was developed in 1944 by Draize
et al,1 and became the famous Draize test. This was based on the original work of Friedenwald
et al.2 For years, the Draize test has been used as the animal test to identify human eye irritants.
For the pharmaceutical industry, eye irritation testing is performed when the material is
intended to be put into the eye as a means or route of application or for ocular therapy. The test
has been modified by regulatory agencies around the world in terms of numbers of animals
to be used, dose to be applied, scoring of the parameters and evaluation of the results, but the
basis of the test procedure remains constant.

Procedure
For the test, usually six albino rabbits are used. 0.1 ml of the liquid or 0.1 g of solid test substance
is instilled into the lower conjunctival sac of one of the eyes of each rabbit; the eyelids are held
together for a few seconds and then released. The other eye of the animal serves as control. The
test eye is not washed. Both the eyes are examined at 1, 24, 48, 72 h after treatment for the degree
or extent of opacity of the cornea, the redness on the iris, and the chemosis and discharge
on the conjunctiva. A numerical score as is shown in Table 42.1 is assigned subjectively for
660 Drug Screening Methods

Table 42.1: Eye irritation test: grading values for ocular lesions

Description of lesion Value

1. Cornea
A. Opacity, degree of density
• No opacity 0
• Scattered or diffuse areas of opacity, but details of iris clearly visible 1
• Easily discernible translucent areas, details of the iris slightly obscured 2
• Opalescent areas, no details of iris visible, size of pupil barely discernible 3
• Opaque cornea, iris invisible through opacity 4
B. Area of cornea involved
• One quarter (or less), but not zero 1
• Greater than one quarter 2
• Greater than one half 3
• Greater than three quarters, up to complete area 4
Score = Part A × Part B × 5 – Maximum score = 80
2. Iris
A. Normal 0
• Folds above normal, congestion, swelling circumcorneal injection, iris still reacting to light
(sluggish reaction, but positive) 1
• No reaction to light, hemorrhage, gross destruction 2
Score = Part A × 5 – Maximum score = 10
3. Conjunctiva
A. Redness (refers to palpebral conjunctiva)
• Blood vessels normal 0
• Blood vessels hyperemic (injected) 1
• Diffuse, crimson color, individual, vessels not easily discernible 2
• Diffuse vessels, beefy red 3
B. Chemosis (lids and nictitating membranes)
• No swelling 0
• Any swelling above normal (includes the nictitating membranes) 1
• Obvious swelling with partial eversion of lids 2
• Swelling with lids half-closed 3
• Swelling with lids more than half-closed 4
C. Discharge
• No discharge 0
• Any amount different from normal (does not include small amounts observed in
inner canthus of normal animals) 1
• Discharge with moistening of lids and hairs adjacent to lids 2
• Discharge with moistening of the lids and hairs of considerable area around eye 3
Score = (Parts A + B + C) × 2 – maximum score = 20
Source: Draize JH, Woodard G, Calvery HO. J Pharmacol Exp Ther 1944; 82: 377-90. With permission.
 Ocular Toxicity 661

each of these parameters at the different time periods of examination. Each animal is graded
separately, and the scores for the different parameters are added. The total score for each
animal does not exceed 20. The treated eye may be washed at the 24 h period if considered
appropriate to show whether washing with water results in decrease or increase in irritation.
If there is no evidence of irritation at 72 h, the study may be terminated. If residual injury is
present, examination of the eyes may be prolonged. Extended observation, e.g. at 7 and 21
days may be necessary if there is persistent corneal involvement or other ocular irritation in
order to determine the progress of the lesions and their reversibility or irreversibility. However,
the observation period normally need not exceed 21 days after instillation.
On comparing the scores for each of the rabbit, if only two or three rabbits show a positive
effect, the test is considered as inconclusive and is repeated with a new set of animals. If four
or more rabbits are positive in response, the test is considered positive.
The Draize test has been a subject of controversy both in the animal rights group and the
scientific community. The test has been criticized on the dose volume, use of rabbit as model,
number of animals, the infliction of pain to animals, and the subjective nature of assessment
giving rise to variable interpretation.
There are a few modifications, which have been proposed and adapted for the Draize test.
These modifications have been targeted at making the tests more accurate in predicting human
responses and at reducing both the use of animals and the degree of discomfort or suffering
experienced by them.
A modification of the standard Draize procedure is the Low-Volume Eye Test (LVET) in
which only 0.01 ml or 0.01 g of the test substance is applied directly to the cornea instead of
the conjunctival sac, and the eyelids are not held shut following treatment.3 Several studies
have shown that the LVET more accurately predicts human ocular exposure, good correlations
being obtained between LVET results and the human response.4,5 The LVET can be successfully
conducted with three rabbits rather than six usually used.6
Over the years, it has been proposed that topical anesthetics be administered to the eyes of
rabbits prior to their use in the test to decrease the pain inflicted to the rabbit. However, use
of anesthetics can interfere with test results and thus compromise scientific validity and/or
advantages of using anesthetic remain inconclusive. Irrigation of the eye at various times after
dosing is another measure proposed to decrease animal suffering. Alternatively, use of sodium
fluorescein dye and/or slit lamp has also been proposed for evaluation of the changes in the
eye to decrease the subjectivity of evaluations.
Humane and scientific concerns regarding the use of the animals in toxicology and the
subjective nature of the Draize test that leads to inter- and intra-laboratory variations has
prompted active research for the replacement of animal testing for toxicity evaluation. One
of the approaches to the above is the prediction of ocular toxicity from pre-existing data. The
data on ocular irritation published in the literature and/or contained in computer databases
may be evaluated for investigating the probable toxicity of a new chemical. However, these
strategies suffer from the drawbacks of limited information available, discrepancies in scoring,
variability and lack of strict reproducibility making interlaboratory comparisons very difficult.
From these databases, only a broad classification of the chemical as irritant or non-irritant can
be concluded. Computer modeling developed on the basis of computer databases is another
approach proposed. But this also suffers from the drawback similar to that of databases.
662 Drug Screening Methods

Prediction of toxicity from physical/chemical data is an alternative widely employed. It is

assumed that compounds having extreme pH (< 3 or > 12) are severe irritants and does not
require animal testing. Comparisons have been made between dermal and ocular irritation
data under the hypothesis that if a material is irritating to the skin, it will also be irritating
to the eye. It was found that the degree of correlation between the two was dependent upon
the in vivo classification scale used.7 The correlation between the two tests was better if the
compound was classified as irritant or non-irritant than when compound was classified as
non-, mild, moderate or severe irritant.

IN VITRO METHODS
The next approach is the use of in vitro methods for evaluation of toxicity. The major parameters
scored in the in vivo test are corneal opacity, inflammation and cytotoxicity. While no single
test can cover all the three parameters, individual tests can address some of these end points.
It seems probable to use a battery of these validated tests as screens for toxicity evaluation
thereby reducing animal experimentation, which may be used only for the final and crucial
premarket testing of products for regulatory requirements.

Opacity Tests
Corneal opacity is the most heavily weighted component of the Draize score. Several in vitro
tests have been developed that assess opacity.

Isolated Rabbit Eye

The method was first introduced by Burton et al.8 and modified by Price and Andrews9 with the
end points of the assay being opacity, corneal thickness and fluorescein dye retention.
Rabbits with a normal eye and corneal thickness are selected for the study and killed by
overdose of sodium pentobarbitone. The eyes are enucleated and lightly supported by clamps
within temperature-regulated (32°C) chambers and warm isotonic physiological saline is
dripped continuously over the cornea at a rate of 0.1-0.15 m1/min for 45-75 min. Slit lamp
examination is repeated for observation of any corneal changes; and if no change is found,
the eye is used for further experimentation. 0.1 ml of the test solution is dripped over the
cornea for 10 sec or 0.1 g powder is sprinkled on the corneal surface and left for 10 seconds.
The test substance is washed off with physiological saline and effects of treatment are assessed
at intervals of 1 h over a period of 4 h by slit lamp examination. The eyes are observed for:
(i) generalized or patched opacities that represent severe tissue damage, (ii) changes in the
thickness of the surface epithelium and objective measurement of any swelling of the corneal
stroma using a slit lamp with a corneal pachymeter attachment. Mild to moderate irritants
result in some loss of surface epithelium and swelling of the stroma. For scoring system, please
refer Price and Andrews,9 and (iii) changes in the epithelial layers and in penetrability that can
be detected by applying 2% fluorescein sodium to the surface of the eye for a few seconds and
then rinsing off with the isotonic salt solution. This procedure is followed when required.
The advantages of the method are that no living animal is subjected to pain. Also, two
eyes are available for test from a single animal. The eyes may be taken from rabbits used for
 Ocular Toxicity 663

other tests not relevant to the eye. Changes are observed in cornea, which is a specialized
nonvascular structure essential for the proper functioning of the eye and is undisturbed in the
whole eye organ preparation. This test has been validated with a wide range of compounds,
with good correlations being established between in vitro effects and in vivo irritancy.9 The
effects of liquid test substances were more successfully predicted in vitro as compared to solid
test agents. However, one serious drawback of this technique over assessment in a live animal
is that it cannot indicate the degree of discomfort caused by the test material entering the eye.

Isolated Chicken Eye (ICE) Test

Based on the above method, Prinsen and Köeter (1993) described a protocol for isolated
chicken eye test.10 The components of the test method were more or less same except for the
dose-volume of the test substance, which was modified for the chicken eye. The parameters
considered are corneal swelling, corneal opacity, and flourescein retention. The method
is being used since 1993 without any significant change. The method has been evaluated
by National Toxicological Program Interagency Centre for the Evaluation of Alternative
Toxicological Methods (NICEATM) and the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM).11
Intact chicken heads are transported from the slaughterhouse to the laboratory within 2 h in
the humidified boxes so as to prevent the damage due to transportation and dessication. The
eyes are excised without damaging the cornea. The eyes are mounted on a stainless steel clamp
individually with corneas in a vertical position. The eyes are then transferred to a superfusion
apparatus. The corneas are moistened with isotonic saline, which is supplied at a rate of 0.10-
0.15 ml/min through a bent stainless steel tube attached to a peristaltic pump. The corneal
integrity is again examined under slit lamp microscope to exclude damaged corneas from the
test. Once the eyes are examined and approved they are equilibrated for 45 to 60 min before
dosing. A single dose of the test substance (30 μl for liquids and 30 mg for solids) is applied
to cover the whole cornea for 10 sec. Corneas are rinsed with 20 ml isotonic saline. Mean
values for corneal swelling, corneal opacity and flourescein retention are evaluated at regular
intervals up to 4 h post treatment.11
Advantage of the test includes the availability of the test system. Chicken eyes can be obtained
easily as they are widely consumed by the people worldwide. The removal of eyes compared
to bovine and porcine eyes is much easier. Moreover, chicken cornea are comparable to rabbit
cornea structurally. The changes in the opacity are more easily observed due to black iris of the
chicken eye.
Other investigators have experimented with the technique using enucleated eyes of cattle
and pigs held by clamps in suitable chambers containing a suitable medium.10,12

Bovine Corneal Opacity-Permeability (BCO-P) Test

The method was developed by Sina and Gautheron.13 The corneas are obtained from an abattoir
as target tissue. They are dissected free of the bovine eye and mounted in plastic holders, which
allow access to both sides of the corneas. The isolated corneas are allowed to equilibrate in
culture medium containing 1% serum for 1 h. Then, the posterior chamber is filled with fresh
culture medium containing 1% serum and the anterior chamber is filled with test substance in
either medium or polyethylene glycol 400. After an appropriate exposure period, the exposure
664 Drug Screening Methods

medium is replaced with fresh medium, and the opacity of the treated cornea is measured
relative to control without removal of the corneas from the holders. This measurement is done
in an opacitometer (which is similar to a dual beam spectrophotometer), with a control cornea
in one compartment and the treated one in another. The difference in light transmission is a
measure of the chemically induced increase in opacity of the treated cornea.
It was found that the correlation between the opacity measurement and in vivo Draize
score was very good. This model also allows the assessment of recovery from injury, by simply
continuing incubation of the cornea in fresh medium after taking the initial opacity reading.
Readings taken at various times thereafter would give an indication of reversibility of the
lesion.
One modification of this technique includes filling up of the posterior chamber with
fresh medium after taking opacity readings. The anterior compartment is filled with sodium
fluorescein in buffer. After 90 min of incubation period, the amount of dye in the posterior
chamber is determined spectrophotometrically at 490 nm. Since, the epithelial layers of
the cornea form a barrier resistant to chemical penetration, the amount of dye penetrating
through the cornea to the posterior chamber is directly proportional to the degree of damage
to the epithelium.
The limitation of the method, however, is the difficulty in assessment of the compound that
is hydrophobic or insoluble, a weak irritant, as well as the agents that cause minimal in vivo
irritation initially (24 or 36 h) but induce increasing irritation at a later stage over a period of
time.

EyetexTM Assay
This is another assay for alternative measurement of the corneal opacification. It is a complete
nonbiological test. This method stems from the observation that transparency of the cornea
depends on the hydration and organization of proteins and that the presence of high molecular
weight aggregates of protein cause opacity. The test uses a “proprietary reagent” of a soluble
protein matrix from the jack bean, which turns opaque when an appropriate test substance
causes alterations in the hydration/organization of the soluble protein, thereby reducing light
transmission through a cuvette placed in a simple spectrophotometer. The change in light
transmission is equated with a standard curve provided in the kit relative to in vivo eye irritancy
scores. The correlations of Eyetex data with in vivo data are conflicting as some investigators
showed good correlations while others showed the correlations to be poor. The correlation
was not good when alcohol-containing formulations were tested. Another potential drawback
of this assay is that if a chemical exerts its irritant effects by a mechanism other than protein
precipitation or coagulation, the test would give false results.13

EpiOcularTM Assay
EpiOcularTM corneal model is a three dimensional tissue construct of normal human derived
epidermal keratinocytes, cultured to form a stratified squamous epithelium, and models
the human corneal epithelium. The EpiOcularTM tissue construct models the top epithelial
layer only and not the stromal and endothelial layers of the cornea. The test is based on the
hypothesis of Maurer and Jester (2002) that the level of ocular irritation is related to the extent
of initial injury.14
 Ocular Toxicity 665

It estimates the potential ocular irriation of a test article when exposed topically. To measure
the ocular irritation potential of any test material the time taken to reduce tissue viability by
50% [ET50] is considered. Relationship between the ET50 of a test material and nature and
extent of histological changes that occur is established by this method. As the depth and
severity of the tissue damage increases the tissue viability decreases progression and types of
changes associated with tissue damage helps in distinguishing the degrees of ocular irritation.
In vivo ocular irritation potential of a test article is directly related with its in vitro potential for
cytotoxicity and tissue penetration.15,16

Long-term Corneal Culture Assay

Raabe et al. 2005 has proposed the long-term corneal culture as an in vitro model to evaluate eye
irritation and post-treatment recovery after chemical exposures. The method is modification
of the previously published method.17,18
Porcine eyes are obtained from the abattoir. The eyes are disinfected. Corneas are dissected
out leaving a 3-5 mm circular rim of sclera intact, and cultured within 24 h of sacrifice. Corneas
are suspended in 24 well plates wells with epithelial side facing the bottom of the well filled
with Hank’s Balance Salt Solution (HBSS). An agar/gelatin/M-199 mixture is added to the
endothelial cavity to support the corneal architecture drop by drop and allowed to gel till
the cavity is completely filled and the gel solidifies. Corneas are inverted and transferred to
large deep well dishes. The M-199 culture medium is added to each dish so that the limbal
conjunctivae are covered with medium and corneal epithelium exposed to air. The dishes
are then incubated at 37°C, 5% CO2 and 90% RH for 24 h prior to dosing. The corneas are
moistened by brief immersion in the medium every 2.7 h using a modified plate rocker. The
corneas are treated either with SLS, ethanol and water (control) after 24 h of initiating the
cultures. The exposure time ranged between 2-10 min. The surface of the corneas is rinsed
with PBS till they are free from the test substance. They are again transferred to fresh wells
and cultured for varying time points. The observations both visual and microscopic are
made daily. After the incubation, the corneas are fixed, embedded, sectioned and stained for
histological examination. Corneas can be cultured upto 96-120 h with normal morphology.
SLS and ethanol-induced damage in porcine corneas exhibited histological similarities with
observations made in other ex vivo models. The cornea exhibited re-epithelialization after
exposure to ethanol. Thus, the model demonstrates the potential for further optimization of
evaluating recovery of damaged corneas.17

Cytotoxicity Tests
Majority of the alternatives to Draize test assess cellular toxicity in vitro. This assay involves
the use of immortalized cell lines of different origin and examines dye inclusion/exclusion/
leakage as indices of membrane integrity. Usually, these methods are simple, straightforward,
and relatively rapid, with defined end points, which can be reproducibly and accurately
measured.
The disadvantages are that these assays do not provide information on the mechanism by
which a chemical causes irritation and, therefore, any correlation with in vivo data for one
group of compounds may be due to chance and may not hold for another type of chemical.13
666 Drug Screening Methods

The dyes that are used include: neutral red or fluorescein diacetate which enters only
viable cells, and either gets localized in the cells or leaks out back from the damaged plasma
membrane; propidium iodide and ethidium bromide that can enter only the damaged cell
and stain the DNA but are large enough to be excluded from the viable cells; and trypan
blue that stains the proteins in nonviable cells by entering through the damaged cell
membranes.

HCE-T TEP Assay

For assessing the eye irritation potential of chemicals and formulations HCE-T TEP assay is
being evaluated by ICCVAM to be used as a Draize Alternative Test Method.19
The method is used for evaluating the human eye irritation potential of water-soluble
chemicals and formulations within the irritation range referenced by Draize. It measures
the dose-dependent efficacy of any test material on fluorescein transepithelial permeability
(TEP) in human corneal epithelial cells (HCE-T). An HCE-T cell line has been used to develop
a three dimensional in vitro model of human corneal epithelium.20 These cells are grown at
the air-liquid interface on a collagen membrane in serum free medium which stratify and
differentiate forming an epithelial barrier like in the human eye. These cells are treated with
the test material. Addition of sodium fluorescein to the culture and consequent measurement
of the concentration of fluorescein present in the medium determines damage to the epithelial
cell barrier.
Another example of a dye penetration assay is the method developed by Tchao et al.21
Madin-Darby Canine Kidney cells (MDCK) are grown on a filter and form tight junctions upon
confluency, forming two separate compartments. Fluorescein is placed in the inner chamber
and the passage of dye through the cell sheet to the other chamber is monitored. When an
intact monolayer covers the filter, the passage of fluorescein is restricted. If a chemical damages
the cells, extensive leakage of fluorescein occurs. Sina and Goutheron13 found that the assay
correctly identified only the severe irritants. Mild and moderate compounds, as well as some
severe irritants (which presumably act by a mechanism other than junction disruption),
showed no increased penetration. Besides, MTT dye reduction assay is also used as a cytotoxic
test to study cellular metabolism.22 Uridine uptake study has also been proposed as one of the
methods.23
The cytotoxicity assays can be used as a complementary assay in a battery of tests to screen
a compound in vitro for its irritation potential.

Inflammatory Tests
Inflammation is another important aspect of Draize test. Few in vitro tests have been
developed that can produce an inflammatory response. Inflammation is a complex event
and is difficult to mimic a similar response in an in vitro system. However, the right type
of cells can release chemotactic factors and/or inflammatory mediators such as histamine,
serotonin, prostaglandin, thromboxane, and leukotrienes which can be quantitated indirectly
via their chemotactic effects on neutrophils or by direct assay using high performance liquid
chromatography (HPLC).
 Ocular Toxicity 667

Bovine Corneal Cup Model

The model is developed by Elgebaly and colleagues24 on the basis that chemotactic factors are
released from a variety of tissues in response to injury. In this assay, corneas (bovine, rabbit
or human) are put into temperature-controlled plastic holders such that the epithelial surface
forms the inside of a cup. Physiological medium with or without a test substance is pipetted
onto the cup. Following an appropriate incubation period, the released chemotactic factors
associated with inflammation are removed with the medium for subsequent interaction with
isolated neutrophils. In the assay, the isolation or identification of chemotactic response of
the neutrophils is the end point of assay. In a modification of this assay by Benassi et al.25 the
inflammatory mediators released from mast cells (histamine, serotonin, leukotrienes) are
derivatised by fluorescamine prior to their quantitation by HPLC.
In isolated rat vaginal tissue, Dubin et al.26 have attempted to quantify and associate the
release of eicosanoids with inflammation, measuring the cellular release of prostaglandin
(PGE2, PGF2, 6-keto PGF1) and thromboxane B2 in the medium by using HPLC with fluorescence
or radioimmunoassay (RIA).
These techniques, however, require extensive interlaboratory validation with a broad range
of known or potential ocular irritants.

Chorioallantoic Membrane (CAM) Assay or The Hen’s Egg Test-Chorioallantoic

Membrane Test (HET-CAM)
In this assay, the vascularized respiratory membrane surrounding the embryonic chicken
within an egg is used as the site of irritant activity.
Fertile eggs, on day 0 are incubated at 37°C. On day 3, the shell is penetrated in two places.
First, near the pointed end of the egg a small opening is ground and the shell membrane is
exposed. Through the opening, a needle is inserted and 1.5 to 2 ml of albumen is removed and
discarded. A rectangular window is made on the wall of the egg. A distinct false air space is
seen below the window. The fluid content of the egg is present at the floor of the air space and
the shell dust that results from cutting of window sinks into the fluid. The window is covered
with a transparent tape. On the 14th day, the tape over the window is removed and a Teflon
ring (10 mm internal diameter) is placed on the CAM. The ring serves as a marker of the test
and as a container for holding the test substance. Forty ml of the test sample is placed in the
ring and the window is resealed with tape. The eggs are further incubated for 3 days and on the
17th day of incubation the tape is removed, the window is enlarged and the CAM is observed
for the extent of necrosis.27
HET-CAM is a similar technique.28 The eggs to be treated are first candled in order to discard
the defective eggs and only fresh, fertile eggs are used for the test. Eggs weighing less than
50 g and more than 60 g, are also rejected. For the assay, the eggs are put in incubator trays
with large ends up and put in an incubator at 37.5°C and 62.5% relative humidity where the
trays rotate automatically. The eggs are candled on the 5th day and each day thereafter to
discard the nonfertile eggs. On day 10 of incubation, the shell is scratched around the air cell
by a dentist’s rotary saw and then pared off. The vascular CAM is exposed after removal of the
inner egg membrane. The liquid test substance is dropped onto the membrane in a volume of
0.2 ml; and the solid test substance (0.1 g) is applied on the vascular chorioallantois and
668 Drug Screening Methods

irrigated after 20 sec with 5 ml of warm water. In every case, a series of four eggs are used;
two eggs treated with vehicle only, serves as controls. After application of the test substance,
the CAM, the blood vessels, including the capillary system, and the albumen are examined
and scored for irritant effects (hyperemia, hemorrhage, coagulation) at 0.5, 2 and 5 min after
treatment.
A modification of this assay, the CAM Vascular Assay (CAMVA), is carried out in the
same manner, but vascular changes constitute the lesion, a positive test being indicated
by hemorrhage, obstruction, or narrowing of blood vessels 30 min after applying the test
substance to the CAM of a 14-day-old fertilized hen’s egg.
The CAM assay has been validated, but the results are mixed. High number of false positive
results have been obtained; the scoring is again subjective in nature like the in vivo test; the
tissue under investigation has little anatomical similarity to the cornea; the test substance may
be toxic to the embryo; there is no evidence for inflammatory response; circulating neutrophils
are absent in chicken embryo; and at the embryonic stage, the immune response is not
completely developed.29 Therefore, this assay also will not be a very useful alternative for the
replacement of Draize test.
The in vitro assays described above measure different end points. Therefore, a single test
measuring a single end point cannot be used as an alternative to Draize test. Each test can
only be used together in a battery of tests to evaluate the ocular irritation potential of a new
chemical. Also, many of these tests are not validated; results in interlaboratory variations; and
good correlations do not exist with the in vivo test. It does not appear in the near future that
either single or a group of these tests can completely replace the Draize test.
The regulatory authorities worldwide consider the Draize eye irritancy test among the most
reliable methods currently available for evaluating the safety of a substance in and around
the eye; and that to-date there are no alternatives of equivalent efficacy. Continued use of
Draize test for the evaluation of ocular irritancy potential is not due to a shortage of potentially
useful alternative methods, since more efforts have probably been put into the development of
alternatives to the Draize test than in seeking replacements to all other in vivo test put together.
However, no test, combination of tests, or testing strategy has yet been developed which meets
all of the requirements of the regulatory authorities.
Active research is going on globally in the field of development of in vitro alternatives to
Draize test and also sincere efforts are underway for their validation. These tests can definitely
be used as prescreens to the final determination of irritation potential by Draize test.

REFERENCES
1. Draize JH, Woodard G, Calvery HO. Methods for the study of irritation and toxicity of substances
applied topically to the skin and mucous membranes. J Pharmacol Exp Ther 1944;82:377-90.
2. Friedenwald JS, Hughes WF, Hermann H. Acid-base tolerance of the cornea. Arch Ophthalmol
1944;31:279-83.
3. Griffith JF, Nixon GA, Bruce RD, et al. Dose-response studies with chemical irritants in the albino
rabbit eye as a basis for selecting optimum test conditions for predicting hazard to the human eye.
Toxicol Appl Pharmacol 1980;55:501-13.
4. Freeberg FE, Nixon GA, Reer PJ, et al. Human and rabbit eye responses to chemical insult. Fundam
Appl Toxicol 1986;7:626-34.
 Ocular Toxicity 669

5. Williams SJ. Changing concepts of ocular irritation evaluation: pitfalls and progress. Food Chem
Toxicol 1985;23:189-93.
6. Bruner LH, Parker RD, Bruce RD. Reducing the number of rabbits in the low-volume eye test.
Fundam Appl Toxicol 1992;19:330-5.
7. Gad SC, Walsh RD, Dunn BJ. Correlation of ocular and dermal irritancy of industrial chemicals. J
Toxicol-Cutan Ocular Toxicol 1986;5:195-213.
8. Burton ABG, York M, Lawrence RS. The in vitro assessment of severe eye irritants. Food Cosmet
Toxicol 1981;19:471-80.
9. Price JB, Andrews IJ. The in vitro assessment of eye irritancy using isolated eyes. Food Chem Toxicol
1985;23:313-5.
10. Prinsen MK, Koeter HBWM. Justification of the enucleated eye test with eyes of slaughterhouse
animals as an alternative to the Draize eye irritation test with rabbits. Food Chem Toxicol 1993;31:69-
76.
11. http://iccvam.niehs.nih.gov/methods/ocutox/ocutox.htm.
12. Whittle E, Basketter D, York M, et al. Findings of an interlaboratory trial of the enucleated eye
method as an alternative eye irritation test. Toxicol Methods 1992;2:30-41.
13. Sina JF, Gautheron PD. Ocular toxicity assessment in vitro. In: Gad SC (Ed). In vitro Toxicology. New
York: Raven Press Ltd, 1994:21-46.
14. Maurer JK, Jester JV. Extent of initial corneal injury as the mechanistic basis for ocular irritation: key
findings and recommendations for the development of alternative assays. Regul Toxicol Pharmacol
2002;36:106-17.
15. Blazka ME, Diaco M, Harbell JW, Raabe H, Sizemore A, Wilt N, et al. EpiOcularTM human cell
construct: tissue viability and histological changes following exposure to surfactants. Presented
at the 5th World Congress on Alternatives and Animal Use in the Life Sciences. Berlin, Germany,
2005;21-26.
16. Curren R, Harbell J, Trouba K. The EpiOcularTM Model Protocol, Performance and Experience.
Regul Toxicol Pharmacol 2002;36:106-17.
17. Raabe H, Bruner L, Snyder T, Wilt N, Harbell J. Optimization of an in vitro long term corneal culture
assay. Presented at the 5th World Congress on Alternatives and Animal Use in the Life Sciences,
Berlin, Germany 2005;21-6.
18. Foreman DM, Pancholi S, Jarvis-Evans J, McLeod D, Boulton ME. A single organ culture model for
assessing the effects of growth factors on corneal re-epithelialization. Exp Eys Res 1996;62:555-64.
19. http://iccvam.niehs.nih.gov/methods/ocutox/hce.htm.
20. Kruszewski FH, Walker TL, Dipasquale LC. Evaluation of a human corneal epithelial cell line as an
in vitro model for assessing ocular irritation. Toxicological Sci 1997;36:130-40.
21. Tchao P, Cotovio J, Dossou KG, et al. Assessment of surfactant cytotoxicity: comparison with the
Draize eye test. Int J Cosmet Sci 1989;11:233-48.
22. Gay R, Jadlos S, Marenus K. The living dermal equivalent (LDE) as an assay for ocular irritation
potential. Presented at the 7th Annual CAAT Symposium, 1989.
23. Shopsis C, Sathe S. Uridine uptake inhibition as a cytotoxicity test: Correlations with the Draize test.
Toxicology 1984;29:195-206.
24. Elgebaly SA, Forouhar F, Kreutzer DZ. In vitro detection of cornea-derived leukocytic chemotactic
factors as indicators of cornea inflammation. In: Goldberg AM (Ed). Alternative methods in
toxicology. New York: Mary Ann Liebert 1987;6:257-68.
670 Drug Screening Methods

25. Bennasi CA, Angi MR, Salvalaio L, et al. Ocular irritancy evaluated in vivo by conjunctival lavage
technique and in vitro by bovine eyecup model. In: Goldberg AM (Ed). Alternative methods in
toxicology. New York: Mary Ann Liebert 1987;5:235-42.
26. Dubin NH, Ghodgaonkar RB, Parmley TH. Differential response of in vitro vaginal tissue to various
test agents. In Goldberg AM (Ed). Alternative methods in toxicology. New York: Mary Ann Liebert,
1988;5:153-8.
27. Leighton J, Nassauer J, Tchao R. The chick embryo in toxicology: an alternative to the rabbit eye.
Food Chem Toxicol 1985;23:293-8.
28. Leupke NP. Hen’s egg chorioallantoic membrane test for irritation potential. Food Chem Toxicol
1985;23: 287-91.
29. Bagley DM, Waters D, Kong BM. Development of a 10-day chorioallantoic membrane vascular
assay as an alternative to the Draize rabbit eye irritation test. Food Chem Toxicol 1994;32:1155-66.
 cHAPTER

43
Phototoxicity
INTRODUCTION
Many systemic, therapeutic agents photosensitize human skin to solar or artificial sources of
UV radiation (UVR). The phototoxic potential of an agent is often only noted during clinical
trials late in product development. Withdrawal of an agent at this stage is extremely costly
to the manufacturer and although various animal skin phototoxicity models exist, there is
increasing ethical pressure to develop alternative methods.1
The solar energy has been considered essential for the development and evolution of life on
earth. It can produce photobiological effects on microorganisms, plants, animals and humans.
The portion of the solar spectrum containing the biologically most active region is from 290
to 700 nm. The UV part of the spectrum includes wavelengths from 200 to 400 nm. The most
relevant spectral regions are UVB (290-320 nm) and UVA (320-400 nm). Shorter wavelength
radiation (λ = 290 nm) is efficiently filtered by the atmosphere, mainly by the stratosphere
ozone layer and does not reach the earth’s surface. Sunlight containing radiation of longer than
400 nm has much lower biological effectiveness. The most thoroughly studied photobiological
reactions that occur in skin are induced by UVB. UVB represents approximately 1.5% of the
solar energy received at the earth’s surface; it elicits most of the known chemical phototoxic
and photoallergic reactions. The visible portion of the spectrum, representing about 50% of the
sun’s energy received at the sea level, includes wavelengths from 400 to 700 nm. Visible light
is necessary for such biological events as photosynthesis, vision, the regulation of circadian
cycles and melanogenic effects. The various models used for screening of phototoxicity are
given in Table 43.1.
Table 43.1: Various models used for screening of phototoxicity

In vivo models Photosensitization models In vitro assays

• Rabbit Dorsal Surface Method • Guinea Pig Dorsal Surface • Photohemolysis
• Guinea Pig Dorsal Surface Model • Armstrong Assay • Photosensitized Candida
• Mouse Ear Swelling Model • Photo Hen’s Egg Test (PHET) Albicans Growth Inhibition
• Rabbit Ear Model • Photodegranulation of Mast Cells
• Hairless Mouse Model • Photo-Basophil-Histamine Release Test
• Histidine Photodegradation
672 Drug Screening Methods

The intensity of irradiation used in phototoxicity testing is determined with a light meter,
which provides output as watts/m2. The shelves on which the animals are tested during the
exposure periods are normally adjustable in order to control the dose of light to the exposure
area. The irradiation from fluorescent lights will somewhat vary from day to day, depending on
temperature, variations in the current, etc. The dose the animals receive is generally represented
as joules/cm2. One joule is equal to 1 watt/sec. Therefore, the dose of light is dependent on the
period of exposure.2
Period of exposure = W sec/J

Three protocols for assessing topical phototoxicity potential in rabbit, guinea pig and mouse
are discussed here.

Rabbit Dorsal Surface Method

The traditional method uses rabbit for phototoxicity testing.3 A brief procedure has been
described here. Eight adult female New Zealand white rabbits, 6 for each test and 2 for positive
control are used. Animals are acclimatized at least for 7 days prior to the study. Animals are
given free access to commercial laboratory feed and water. A dose of 0.5 ml of test drug in liquid
form or 500 mg in solid form or semisolid dosage is applied to each test site. A liquid substance
is used undiluted. For solids the test article is moistened with water (500 mg test article/0.5 ml
water or another suitable vehicle) to ensure good contact with the skin. The positive control
material is a lotion containing 1% 8-methoxypsoralen. The animals are weighed on the first day
of dosing. The fur of the test animal is clipped from the dorsal area of the trunk using a small
animal clipper, then shaved to clean the dorsal trunk with a fine blade clipper one day prior to
the dosing. On the day of dosing, the animals are placed in a restrainer to prevent licking. One
pair of patch (approximately 2.5 × 2.5 cm) per test article is applied to the skin of the back, with
one patch on each side of the backbone. A maximum of two pairs of patches, at least 2 inches
apart, are applied to each animal. The patches are secured to the skin by means of an occlusive
tape for the 2 h exposure period. Following exposure the occlusive tape as well as the patches on
the right side of the animal, are removed. The left side of the animal is covered with an opaque
material. The animal is then exposed to approximately 5 J/cm2 of UVA (320-400 nm). After
exposure to the UVA light, the patches on the right side of the animal as well as the occlusive
tape are replaced. The tape is again removed approximately 23 h after the initial application
of the test article. Residual test article is carefully removed, using water or another suitable
vehicle. Animals are examined for signs of erythema and edema, and the responses scored at
24, 48 and 72 h, after the initial test article application, according to the Draize reaction grading
system. Any unusual observation and mortality is recorded. The data from the irradiated and
nonirradiated sites are evaluated separately. The scores from erythema, scar formation and
edema at 24, 48 and 72 h, are added for each animal. The values are then divided by 3, yielding
six individual score. The mean of the six individual animal irritation score represents the mean
primary irritation score (maximum score = 8, as in the primary dermal irritation study).
 Phototoxicity 673

Guinea Pig Dorsal Surface Model

This protocol uses guinea pig for phototoxicity testing.4 Ten young adult male Hartley guinea
pigs (300 to 500 g) are divided into two groups. Four animals are used as irradiation controls
and 6 animals for material treatment. All the animals are acclimatized at least for 5 days prior
to the study. Animals are given free access to food and water. The test assumes that material
is in solution form. The most volatile, nonirritating organic solvent such as ethanol, acetone,
dimethylacetamide, or some combination is used. There can be upto four sites per animal,
each measuring 1.5 × 1.5 cm (2.25 cm2). In general, one side is selected for a vehicle control,
and another for positive control [8-methoxypsoralen (8-MOP), 0.005% in ethanol]. A dose of
0.025-0.05 ml is applied using a micropipette or HPLC Hamilton syringe to each site. Animals
are weighed on the first day of dosing. Approximately 48 h prior to the treatment, the hair is
removed from a 6 × 8 cm area on the back with a fine clipper. The animals are dosed as described
above. Test areas are separated to prevent mixing of test solutions after application. No patches
or wraps are used. Immediately after dose application, the animals are placed in a restrainer
while keeping the sites uncovered. Prior to irradiation, the heads of the animals are covered to
prevent eye damage from the light exposure. Thirty minutes after dosing, animals are exposed
to a nonerythmogenic dose of light in the UVA band (peak intensity between 335 and 365 nm).
The dose of light should be 9 or 10 J/cm2 for UVA and 0.1-0.3 J/cm2 for UVB. Immediately after
the light exposure, the animals are wiped clean if necessary and returned to the respective
cages. Animals are inspected and scored at 24 h and 48 h post-exposure according to the
following scoring scale: 0, no reaction; 1, slight erythema; 2, moderate erythema; 3, severe
erythema, with or without edema.
This scoring scheme is similar to that used for dermal sensitization scoring, whereas
the scoring method for the rabbit model discussed previously is that for dermal irritation
studies. Any unusual clinical sign during exposure should be noted. The following descriptive
parameters are calculated from the data.
Number of positive sites × 100
Phototoxicity irritation index =
Number of exposure sites

Total of scores
Phototoxicity severity index =
Total of observations

Mouse Ear Swelling Model

The method is used for topical phototoxicity testing.5 In this female BALB/c mice are used and
divided into two or three groups if positive control is included. Five mice each for solvent/
irradiation controls and for test material treatment are used. Five animals are used for positive
control (8-MOP). Animals should be juvenile, weighing 20 to 25 g. Animals are acclimatized at
least 5 days prior to the experiment. Animals are given free access to food and water. This test
assumes that material is in solution form. Generally, the most volatile and non-irritating organic
solvent like ethanol, acetone, dimethylacetamide or some combination is used. For treatment
each ear in each mouse in a group is treated with the same solution. A dosage of 8-10 μl is
applied on each side of the ear using a micropipette. Animals are weighed on the first day of
dosing. Approximately 48 h prior to treatment, the ears are inspected and animals assigned
674 Drug Screening Methods

to treatment groups. Animals with swollen or damaged ears are excluded from the test.
Preliminary ear thickness measurements are taken by vernier caliper. On the day of dosing,
animals are placed in specially designed restrainers and the animals are dosed as described
previously. No patches or wraps are used. Thirty to 60 min after dose application, the animals
are exposed to a nonerythmogenic dose of light in the UVA band (150 W short-arc UV solar
simulator with internal 1 mm UG-11 and WG 320 filters with peak intensity between 290 and
410 nm). Irradiation in W/cm2 is determined using a radiometer with an appropriate UVA or
UVB detector. Light is between 5 and 7 J/cm2 for UVA and 0.2-0.4 J/cm2 for UVB. Only the
right ear is exposed. The left ear is protected from light by the researcher’s hand. Immediately
after light exposure, the animals are wiped clean if necessary and returned to respective cages.
Ear thickness measurements are taken at 24, 48 and 72 h post-dosing using a handheld dial
micrometer. The ear is positioned so that the contact plates of the micrometer can be placed
towards the outer edge of the ear. The spring-loaded lever of the micrometer is slowly released
until the plates come in contact with the surface of the ear as judged visually. Care is taken
so that the plates do not close too quickly or tightly on the ear, causing compression of any
edema that might be present. Two measurements are taken at adjacent sites on each ear and
the average ear thickness is determined. Changes in ear thickness from the pretreatment
baseline are calculated and expressed as mm × 10–2. A statistically significant (paired t-test)
increase in ear thickness in the irradiated ears vs. the nonirradiated ears within the test article
treated groups is considered sufficient evidence for phototoxicity, provided that there is not a
statistically significant change in the control group.
This method has obvious advantages over the guinea pig and rabbit dorsal surface models
already described. The animals are less expensive to obtain and maintain, and the test is very
objective and reliable.

ALTERNATIVE DESIGNS IN THE IN VIVO METHODS

Rabbit Ear Model

There are at least two in vivo alternatives to this traditional test. The first uses just the ear of the
rabbit. The rabbit ear provides a site, which is of sufficient size with uniform thin skin without a
thick, hairy coat, thus, offering advantages over the whole body rabbit and mouse models. The
external surface of the pinna is shaved with clippers and radiation is delivered using a xenon
arc solar simulator with multiple liquid light guides (Solar Light Co, Philadelphia, PA) adjusted
to provide intensity steps varying by 40%. Depending on the test situation, exposures range
from 0.5 to 12 min. In the absence of phototoxic agents or sunscreens, a 1 minute exposure
generally produces detectable erythema from the midrange light guides. The intensity of the
erythema is graded and corresponds with the radiation flux.6

Hairless Mouse Model

In this method, the dorsal skin thickness is measured using vernier skinfold calipers. In this
way, the degree of dermal edema induced by phototoxic agents is measured readily and is not
subjected to the problems of visual assessment of erythema as in rodent skin.7
 Phototoxicity 675

Advantages and Disadvantages

The classic and alternative methods share common advantages and disadvantages. They have
no appreciable false negative results. These are easy to perform and relatively inexpensive. On
the other hand, the disadvantages of the method are that the photometers must be carefully
calibrated to control UV exposure, results are subjective rather than objective, and significant
amount of test material is required.

PHOTOSENSITIZATION MODELS
Guinea Pig Dorsal Surface
This method uses dermal exposure without any adjuvant to increase the response.8 Young
adult female Hartley guinea pigs, weighing between 300 to 400 g are used. The females are
preferred because the aggressive social behavior of males may result in considerable skin
damage that might interfere with the interpretation of the challenge reactions. Animals that
show poor growth or illness in any way are not used because illness markedly decreases the
response. Animals with skin marked or scarred from fighting are avoided. The guinea pigs
are quarantined and observed for at least 2 weeks to detect any illness prior to the study. The
guinea pigs are randomly assigned to a test group of 10 animals and negative control group
of 6 animals. If a pretest group is necessary then animals, as needed for that group, are also
randomized. The test and control group guinea pigs are weighed 1 week prior to dosing (day-
7), on the day of dosing (day-0) and weekly thereafter.
If pre-test is necessary, then several animals are exposed to different concentrations of
test substance dissolved in acetone. This is used to determine the topical dermal irritation
threshold concentration on the skin that is exposed to UVB and UVA irradiation sequentially
and on the skin that is exposed to UVA irradiation alone. The hair of these animals is clipped
over the whole dorsal region. A volume of 0.2 ml of each test concentration is applied twice
to each guinea pig: (i) at the nuchal region and (ii) the dorsal lumbar region. Thirty minutes
after application, the treated nuchal sites are irradiated with sunlamp [fluorescent “sunlamp”
of tubes (UVB irradiation): type of tubes Westinghouse FS40; skin distance, 15 cm; dose,
measured and calculated at time of exposure; (exposure time, 30 min; emission, 285-350 nm)
emissions (UVB for 30 min)], while the lumbar sites are shielded with elastoplast tape. After
the UVB exposure the tape is removed from the lumbar region, and both the treated nuchal
sites and lumbar sites are irradiated with black light [fluorescent ‘black light’ tubes (UVA
irradiation): type of tubes, GE F40 BL; skin distance, 10 cm; dose measured and calculated
at time of exposure; exposure time, 30 min; emission, 320-450 nm; a pane of window glass
(3 mm thick) is used to eliminate passage of radiation of lower than 320 nm] emissions (UVA)
for 30 minute. Thereafter the animals are returned to their respective cages after the UVA
exposure. Twenty-four hours after the initial exposure to the test substance, the nuchal and
lumbar skin sites are scored for erythema formation. A concentration is chosen for induction
application that causes a mild or weak erythema response at the nuchal sites. If the substance
does not cause erythema response then the highest concentration level that is practical should
be used for the erythema induction. The highest concentration of the test substance that is
nonirritating to the lumbar sites is used for challenge application. Two lower concentrations
676 Drug Screening Methods

of the test substance, prepared by serial dilution from the highest concentration are also used
for the challenge application.
Days 0, 2, 4, 7, 9, and 11 are used for induction stage. The hair in an area of approximately
3 × 3 cm is clipped from the nuchal region of each test and control group guinea pig. A volume
of 0.2 ml of a relatively high concentration of the test substance in either acetone or ethanol is
applied to the shaved nuchal region of each test group guinea pig. The concentration will be
the highest level that can be well tolerated locally by the guinea pig, as determined by a pretest
for dermal irritation. Then a volume of 0.2 ml of solvent (acetone or ethanol) is applied to the
shaved nuchal region of each control group guinea pig. Thirty minutes after application, the
treated nuchal sites of test and control guinea pigs are irradiated with sunlamp emissions for
30 min and black light emissions for 30 min, successively. The lumbar region of the back is
shielded from the light sources during the irradiation procedures with elastic bandage, which
is wrapped around the torso of each animal. The clipping, topical exposures to test substance
and irradiation procedures are repeated six times during a 12-day period (induction days are
0, 2, 4, 7, 9, and 11). The day 32 is a challenge day. The elicitation of contact photosensitivity
is performed on 21 day from the last sensitizing (induction) exposure. The hair of the dorsal
lumbar region of each of 10 test group and 3 of 6 control group guinea pigs is clipped for the first
time. Three different concentrations of the test substance using the solvent used for induction,
as determined from the pre-test, are applied topically to this region; test and control animals
are treated alike. Each concentration is applied to the right and left side of the dorsal midline.
The torso of each test and control guinea pig is wrapped in Saran Wrap (1 layer thick) after the
test chemical is applied. The Saran Wrap is held in place at the ends with athletic adhesive
tape. The same tape is used to shield the left side of each animal from the UVA light source.
Thirty minutes after application, the right side of each animal is exposed to nonerythmogenic
(> 320 nm) UVA emissions for 30 min. The radiation is passed through a pane of window
glass 3 mm thick in order to eliminate passage of radiation lower than 320 nm. After black
light exposure, all animals are unwrapped, returned to their respective cages, and placed in a
darkened room for 24 h. If the test substance leaves a colored residue the excess test material
is removed by washing with a suitable solvent at 24 h so that the area of challenge skin can
be evaluated accurately. All the test sites, both irradiated and nonirradiated, are scored and
interpreted 24 and 48 h after the initial test substance application and subsequent exposure
to black light irradiation. The erythema is scored as follows: 0, no erythema; 1, minimal, but
definite erythema; 2, moderate erythema; 3, considerable erythema; 4, maximal erythema.
If the test substance is judged a nonphotosensitizing agent after the challenge application,
a second and final challenge application is performed on each test animal 7 days after
the initiation of the first challenge dose. Controls from the first challenge application are
rechallenged because they have been exposed to the test substance and are no longer true
negative (naive) controls. The three remaining naive control group animals (not used for the
first challenge) are challenged for comparison to the re-challenge of test group animals. The
procedure used for the first challenge application is used for the second challenge application.
Either the same procedure will be used or new concentrations of test substance, including
reshaving, the same patching method and the same duration of exposure is used. Observations
are again made 24 h and 48 h after the second challenge application and skin reactions are
recorded. The negative control group of animals, having received no previous photosensitive
 Phototoxicity 677

(induction) exposure, serves to identify any phototoxic or primary irritant (nonphototoxic)

substances. An erythema score of one or more is considered a positive response.

Armstrong Assay
This method originally published by Ichikawa et al9 introduced the use of adjuvant in a
photosensitization test system in guinea pig. The assay has been recommended by the
Cosmetic, Toiletries, and Fragrances Association (CTFA). This assay uses UVA light (320-400
nm) in the induction and challenge phase. The UVA lights are commonly known as “black
lights” (“BLB” fluorescence-type bulbs). However, the selection of the light source is critical
because the range of wavelengths emitted by the bulb is controlled by the phosphor coating
and different manufacturers use different phosphors to produce BLB lights. There may
even be different phosphors used by the same manufacturer with no code on the bulbs to
indicate the type of phosphor being used. The General Electric BLB emits effective energy
only at wavelength longer than 350 nm, whereas the entire spectrum between 250 and 350
nm is covered by Sylvania BLB bulb. Less than 2% of the energy emitted by General electric
BLB light is between 250 and 350 nm, whereas 42% of the energy from the Sylvania BLB light
falls in this range. There are known photoallergens that require the energy contained in the
spectrum below 345 nm for activation, and thus give false negative results if the incorrect light
source is used. The best precaution is to determine the emission spectrum of the lights, which
are to be used in the assay.10
It is necessary to determine the total energy being emitted by the lights in order to calculate
the proper J/cm2 exposure. An International Light Model 700 provides a relatively inexpensive
means of measuring the light energy when fitted with a cosine-corrected UVA detector (W150s
quartz diffuser, UVA-pass filter SEL015 detector). The device has a peak sensitivity of 360 nm
and width of 50 nm. A bank of eight bulbs is readily prepared by bolting together two industrial
four-bulb (48” long) reflectors. Two sets of these will allow 40 animals to be treated at one
time. The lights are allowed to warm 30 min before use. They are turned off just before the
animals are placed under them and then turned back on. The light intensity is measured at
several locations at the level of the top of the backs of the animal and the correct exposure
time is then calculated. The lights are adjusted between 4 and 6 inches above the back and
10 J/cm2 is the proper exposure. The Hill Top Chamber provides a good patching system in this
assay. A volume of 0.3 ml is used. Suitable animal restrainers for holding the animals during
the patching and the exposure to the light as well as in providing excellent occlusion should
be used. The majority of hair is removed from the intended patching site with a small animal
clipper fitted with a No. 40 blade. The assay frequently requires the complete removal of hair
using a depilatory cream, which is applied and left in contact with the skin for not more than
15 min. It must be washed away completely with a stream of warm running water. The animals
are dried with a towel and the inside of the cages wiped clean of any depilatory cream before
putting the guinea pigs. When required, the epidermis is partially removed by tape stripping.
The skin must be completely dry otherwise the stripping will be ineffective. An approximately
8 inches long tape strip is used. Starting at one end of the rope, it is placed against the skin and
rubbed with the finger to adhere firmly. It is then pealed away, taking with it some dry epidermis
cells. A new section of the tape is then applied to the skin and the procedure repeated four
or five times. The skin will have a shiny appearance due to the leakage of moisture from the
678 Drug Screening Methods

dermis. The tape should not be jerked away from the skin because this can cause the rupture
of dermal capillaries.
The potential of the animal to respond to a sensitizer is enhanced by the injection of
Freund’s complete adjuvant (Calbio-chem-Behring, San Diego, CA, or Difco, Detroit, MI). The
adjuvant is diluted 1:1 with sterile water before use. The injections must be given by intradermal
route. In the Armstrong assay, a pattern of four 0.1 ml injections is given just prior to the first
induction patching in the nuchal area. All four injections should fit under the edge of the area
to be covered by the Hill Top chamber. It is advisable to perform the skin stripping operation
before the injections as the adjuvant can leak onto the skin and prevent effective removal of
the epidermis. The test site(s) is exposed to the UVA light after 2 h of occlusion. The animal is
left in the restrainer and the dental dam above the test site to be exposed is cut and the patch
removed. Excess material is wiped from the site to be exposed and the remaining parts of the
animals are covered with aluminum foil. All patches are removed after the light exposure step,
the patched areas wiped free of excess material, and the animals are returned to the cage. The
grading is similar to as described earlier.
With the exception of water it is desirable to use a vehicle for the induction, which is
different from the one used at the challenge. Because the controlled animals in the Armstrong
assay are sham treated (including any vehicle), one can patch the test and controlled animals
with vehicle at the challenge if the same vehicle is to be used for both the induction and the
challenge. It is advantageous to use a vehicle, which dissolves the test compound and is inert.

Assay Procedure
For Irritation/Toxicity Pretest (8 animals)
Day 0: The hair from the lumbar region is removed by clipping and depilation. Two
concentrations are applied on each animal on adjacent left side/right side locations for a
total of four dose concentrations. The patches are occluded for 2 h (+ 15 min). The right side is
exposed to 10 J/cm2 of UVA light after removing the patches on the right side. The remaining
patches and excess material are removed after the exposure to light. Day 1: All test sites are
graded 24 h (± 1 h) after removal of all patches (24 h grade). Day 2: The grading is repeated
48 h (± 2 h) after removing the patches (48 h grade).
For Induction (20 tests + 10 sham controls + any rechallenge controls)
Day 0: All test and control animals are weighed. The hair from the nuchal area is removed
with clippers and depilatory cream. The epidermis is removed by stripping four or five times
with tape. Four 0.1 ml id. injections of a 1 : 1 dilution of Freund’s complete adjuvant are made
in an area to be covered by the patch. This area is covered on the test animals with a Hill Top
Chamber, which has 0.3 ml of test material preparation. The sham controls are patched with
water or solvent on the patch. Occlusion with dental dam is done, and animals are restrained
in a holder for 2 h (± 15 min). The patches are removed and the non-patched areas are covered
with foil, and are exposed to 10 J/cm2 of UVA light for 30 min. On day 2, 4, 7, 9, and 11 the
activities of Day 0 are repeated with the following exceptions. Animals are not weighed and
not injected with adjuvant. The patch is removed when the original induction site becomes
too damaged but it is kept in the nuchal area. Depilation may not be needed at each induction.
For challenge, 20 tests + 10 sham control animals (9-13 days after last induction exposure) are
 Phototoxicity 679

required. On day 0: All the animals are weighed, the lumbar region is clipped free of hair, and
depilated. The skin is not stripped. Each animal is patched with a pair of adjacent patches (one
on the left side and one on the right side) containing 0.3 ml of a nonirritating concentration
of test material on a Hill Top Chamber. The patches are removed from the right side and the
remaining animal is covered with foil. The right side is exposed to 10 J/cm2 of a UVA light. The
remaining patches are removed and any excess material is cleaned. On day 1, all challenge
sites either exposed or unexposed to light (24 h ± 1 h) are graded after removal of the patches
(24 h grade) and recorded separately. On day 2, the grading is repeated at 48 h (± 2 h) after
removal of the patches (48 h grade). All or selected animals are rechallenged with the same
or a different test material 7-12 days after the challenge. Ten new sham treated controls and
naive test sites on all animals are used following the same procedure as used in the challenge.
The number of positive responders is determined (number of animals with a score >1 at either
the 24 or 48 h grading or with a score 1 unit higher than the highest score in the control). The
average score is determined at 24 and 48 h for the test and control groups using face values. The
data for the sites exposed to light are kept separate from the data unexposed to light.
The Armstrong assay has been found to give responses similar to human in the guinea pig:
positive responses for 6-methyl coumarin and musk ambrette. A major disadvantage is that
the procedure is time consuming with six induction exposures. The procedure is very stressful
on the animals because of the injection of adjuvant and the multiple skin strippings and
depilation.
The recommended positive control is musk ambrette. Tetrachlorosalicylanilide (TCSA) also
gives positive results, but it should be noted that this material is reported to be an allergen
without UV light activation. Animals are induced with a 10% w/v solution of musk ambrette in
acetone and sham controls are patched with acetone alone. The Hill Top Chamber is used for
all patch studies.

The Photo Hen’s Egg Test (PHET)

As an alternative to the rabbit eye test (Draize test) hen’s egg test (HET), was introduced by
toxicologists as a screening model for mucocutaneous toxicity.11-13 Neumann et al employed
the yolk sac blood vessel system (YS) of the incubated hen’s egg in combination with UV
irradiation as a new valid and inexpensive screening model for phototoxicity.14-18
Fertile white Leghorn eggs are incubated in horizontal position at 37.5°C and relative
humidity 65%. At 3 days of incubation all eggs are candled in order and defective eggs are
discarded. A hole is made into the eggshell and 5 ml of egg white is removed to lower the
embryo and its surrounding yolk substance. Afterwards a 1.5 × 2.5 cm window is sawed out
of the shell. Eggs are covered by a wax sheet and replaced back in the incubator. At day 4 of
incubation, only eggs with normally developed embryos and YS are used for testing. The PHET
has a 2 × 2 factorial test designs with the factors ‘‘irradiation’’ and ‘‘substance application’’ and
the levels ‘‘yes’’ and ‘‘no’’. PHET is established to investigate phototoxic reactions; the yolk sac
blood vessel system of the incubated hen’s eggs is exposed only to nontoxic concentrations of
the test substances and to a nontoxic UVA dose (5 J/cm2) concurrently. At day 4 of the incubation
period, test group is exposed to a test substance, immediately followed by an irradiation with
5 J/cm2 UVA.
680 Drug Screening Methods

Physiological salt solution (PSS) may be used as vehicle. Serving as controls, three additional
test groups exposed only to PSS and 5 J/cm2 UVA or to PSS or to a test substance alone.
Readings performed 24 h after irradiation. During this observation period, the morphological
parameters such as membrane discoloration (MD), hemorrhage (HR) were monitored via a
macroscope and graded following a four point scale:
Level 0: No visible MD or HR
Level 1: Just visible MD or HR
Level 2: Visible MD or HR, structures are covered partially
Level 3: Visible MD or HR, structures are covered totally
The test parameters MD and HR as well as embryo lethality are summarized in morphology
and a lethality index (Table 43.2). Using these indices, the relative phototoxic potential of an
assumed photosensitizer compared to other well-known photosensitizers can be calculated.

Table 43.2: Photo hen’s egg test

Relative lethality: Lethality rate of the interaction group (Li) minus the average lethality rate of controls LC = (lc1 + lc2
+ lc3)/3
Relative lethality L = Li _ Lc
Relative lethality L (%) = L × 100
Relative hemorrhage I and II: Sums of the hemorrhage (HR) levels of the interaction group HRG1i and HRG2i:
Frequencies of level 0 HR + level 1 HR = HRG1i
Frequencies of level 2 HR + level 3 HR = HRG2i
Sums of the hemorrhage (HR) levels of the controls:
Frequencies of (level 0 HR + level 1 HR = HRG1c
Frequencies of level 2 HR + level 3 HR = HRG2c
Relative hemorrhage I (HR I) = HRG1i–[(HRG1c)/3]
Relative hemorrhage II (HRII) = HRG2i–[(HRG2c)/3]
Sums of the membrane discoloration (MD) levels of the interaction group MDG1i and MDG2i:
Frequencies of level 0 MD + level 1 MD = MDG1i
Frequencies of level 2 MD + level 3 MD = MDG2i
Sums of the membrane discoloration (MD) levels of the controls:
Frequencies of level 0 MD + level 1 MD = MDG1c
Frequencies of level 2 MD + level 3 MD = MDG2c
Membrane discoloration: Relative membrane discoloration I (MD I) = MDG1i–[(MDG1c)/3]
Relative membrane discoloration II (MD II) = MDG2i–[(MDG2c)/3]

IN VITRO ASSAYS
Photohemolysis
Photohemolysis is quantified in two ways: (a) by the release of hemoglobin, through the
absorbance of the supernatants (after centrifugation) at 540 nm; (b) by the decreasing number
of intact red blood cells, which is proportional to the optical density at 650 nm. An intrinsic
limitation of this test system is that it detects photosensitization only at the cell membrane level.
Hence, false negative predictions must be expected for compounds acting via photodamage to
 Phototoxicity 681

DNA or other biological targets different from the membrane components. This assay is rapid
and easy to perform.19

Photosensitized Candida albicans Growth Inhibition

Candida albicans growth inhibition is evaluated as the diameter of the yeast free zone.20,21 Such
inhibition is assumed to indicate injury to DNA.
The test compound is dissolved in ethanol, DMSO or acetone. A known volume of a 0.05-
10% solution is applied to 7.5 mm filter discs. The latter is transferred to Sabouraud agar
plates where a suspension of a fresh culture of C. albicans has been evenly spread. The plates
(duplicate samples) are exposed to UVA radiation (1 mW/cm2). The controls are kept in the
dark and the vehicle alone. After exposure is complete, the diameter of the yeast free zone is
measured. The pathogenicity of this species is a drawback of this assay.

Photodegranulation of Mast Cells

Phototoxicity is frequently associated with urticaria and histamine release from the skin
mast cells. The release of histamine is measured by means of spectrophotometric methods22
or by radioactive mediator (3H-serotonin) resulting from mast cell degranulation, in the
supernatant.23

Photo-Basophil-Histamine Release Test

Suspensions of human leucocytes are used in this case as the model system, and photosensitized
release of histamine from the cells to the supernatant (measured spectrofluorimetrically after
derivatization) serves as an end-point for identifying phototoxic drugs.24

Histidine Photodegradation
Solutions of tiaprofenic acid in 10% propylene glycol in PBS (0.01 M, pH 7.4) are mixed with
the same amount of L-histidine monochloride solution (0.61 mM) in PBS. These mixtures
are bubbled with oxygen and irradiated with the light of psoralens and ultraviolet A radiation
(PUVA) unit (365 nm, 16 mW/cm2). When irradiation is complete, the samples are incubated in
the dark at room temperature for 30 min. Histidine is determined by a modified Pauly reaction.
For this, 200 μl of test solution is made up to 2 ml with PBS; 200 μl of 1% sulfanilic acid in 0.87
N HCl and 200 μl of 5% sodium nitrite are added and the mixture is left for 10 min; 0.6 ml of
20% sodium carbonate is then added, followed by the addition of 2 ml of ethyl alcohol after
2 min. The OD of the final solution is read at 530 nm in a spectrophotometer against a control;
the remaining histidine is measured from a standard curve.25

REFERENCES
1. Ferguson J. Fluoroquinolone photosensitization: A review of clinical and laboratory studies.
Photochem Photobiol 1995;62:954-8.
2. Lambert J, Warmer W, Kornhauser A. Animal models for phototoxicity testing. Toxicol Methods
1996;2:99-114.
3. Marzulli FM, Maibach HI. Perfume phototoxicity. J Soc Cosmet Chem 1970;21:685-715.
682 Drug Screening Methods

4. Nilsson R, Maurer T, Redmond N. A standard protocol for phototoxicity testing: Results from an
interlaboratory study. Contact Dermatitis 1993;28:285-90.
5. Gerberick GF, Ryan CA. A predictive mouse ear swelling model for investigating topical phototoxicity.
Food Chem Toxicol 1989;12:813-9.
6. Marks R, Gabriel KL, Hershman RJ, et al. The rabbit ear as an animal model for phototoxicity and
photobiology studies. J Am College Toxicol 1986;5:606.
7. Lowe NJ. Cutaneous phototoxicity reactions. Br J Dermatol 1986;115:86-92.
8. Harber LC, Shalita AR. The guinea pig as an effective model for the demonstration of immunologically
mediated contact photosensitivity. In: Maibach H (Ed). Animal Models in Dermatology. Edinburgh,
UK:Churchill-Livingstone, 1975:90-102.
9. Ichikawa H, Armstrong RB, Harber LC. Photoallergic contact dermatitis in guinea pigs: Improve
induction technique using Freund’s complete adjuvant. J Invest Dematol 1981;76:498-501.
10. Cole CA, Forbes PD, Davies RE. Different biological effectiveness of blacklight fluorescent lamps
available for therapy with psoralens plus ultraviolet. J Am Acad Dermatol 1984;11:599-606.
11. Draize HJ. Intracutaneous sensitization test on guinea pigs. In Apraisal of the Safety of Chemicals
in Food, Drugs and Cosmetics, Association of Food and Drug Officials of the United States, Austin,
Texas, 1959.
12. Duffy PA. Irritancy testing-a cultured approach. Toxicol In Vitro 1989;3:157-8.
13. Rosenbruch M. Toxizita¨tsuntersuchungen am bebru¨teten Hu¨hnerei, Derm Beruf Umwelt
1990;38:5-11.
14. Freeman RG, Murtishaw W, Knox JM. Tissue culture techniques in the study of cell photobiology
and phototoxicity. Invest Dermatol 1970;54:164-9.
15. Maier K, Schmitt-Landgraf R, Siegmund B. Development of an in vitro test system with human skin
cells for evaluation of phototoxicity. Toxicol In Vitro 1991;5/6:457–61.
16. Neumann NJ, Hö lzle E, Lehmann P, Rosenbruch M, Klaucic P, Plewig G. Photo hen’s egg test: a
model for phototoxicity. Br J Dermatol 1997;136:326-30.
17. Neumann NJ, Klaucic A, Hö lzle E, Lehmann P. Evaluation of the phototoxic potential of ciprofloxacin
in the photo hen’s egg test. Arch Dermatol Res 1995;287:384.
18. Neumann NJ, Hö lzle E, Wallerand M, Vierbaum S, Ruzicka S, Lehmann P. The photoprotective
effect of ascorbic acid, acetylsalicylic acid, and indomethacin evaluated by the photo hen’s egg test.
Photodermatol Photoimmunol Photomed 1999;15:166-70.
19. Kahn G. Fleischaker B. Red blood cell hemolysis by photosensitizing compounds. J Invest Dermatol
1971;56:85-90.
20. Daniels F. A simple microbiological method for demonstrating phototoxic compounds. J Invest
Dermatol 1965;44:259-63.
21. Kavli G, Volden G. The Candida test for phototoxicity. Photodermatology 1984;1:204-7.
22. Sik RH, Paschall CS, Chignell CF. The phototoxic effect of benoxaprofen and its analogs on human
erythrocytes and rat peritoneal mast cells. Photochem Photobiol 1983;38:411-5.
23. Gendimenico GJ, Kochevar IE. Degranulation of mast cells and inhibition of the response to
secretory agents by phototoxic compounds and ultraviolet radiation. Toxicol Appl Pharmacol
1984;76:374-82.
24. Przybilla B, Schwab-Przybilla U, Ruzicka T, et al. Phototoxicity of nonsteroidal anti-inflammatory
drugs demonstrated in vitro by a photo-basophil-histamine-release test. Photodermatol 1987;4:73-8.
25. Figueiredo A, Fontes Ribeiro CA, Goncalo M, et al. Experimental studies on the mechanisms of
tiaprofenic acid photosensitization. J Photochem Photobiol B1993;18:161-8.
 cHAPTER

44
Anti-asthmatic Agents
INTRODUCTION
Asthma is a complex inflammatory airway disease characterized by airflow obstruction
that remains leading cause of hospitalization and death worldwide. In Asthma the airway
occasionally constricts, becomes inflamed, and is lined with excessive amounts of mucus,
often in response to one or more triggers. These episodes may be triggered by such things as
exposure to an environmental stimulant (or allergen), cold air, warm air, moist air, exercise
or exertion, or emotional stress. In children, the most common triggers are viral illnesses
such as those that cause the common cold. This airway narrowing causes symptoms such as
wheezing, shortness of breath, chest tightness and coughing. Between episodes, most patients
feel well but can have mild symptoms and they may remain short of breath after exercise for
longer periods of time than the unaffected individual. The symptoms of asthma, which can
range from mild to life threatening, can usually be controlled with a combination of drugs and
environmental changes. Patients usually have reduced forced expiratory volume in one second
(FEV1) as well as reduced airflow. Other features, characteristic of asthma but not unique to the
disease are airway inflammation and bronchial hyperresponsiveness.1
Short-term relief is most effectively achieved with bronchodilators, agents that increase
airway caliber by relaxing airway smooth muscle and of these the α-adrenoreceptor
stimulants (α2-agonists) are the most widely used. Theophylline, a methyl xanthine drug,
and antimuscarinic agents (e.g. ipratropium bromide) are also used for reversal of airway
constriction. Long term control is most often achieved with an anti-inflammatory agent such
as an inhaled corticosteroid (e.g. budesonide) or an inhibitor of mast cell degranulation, e.g.
cromolyn or nedocromil sodium. Of the several drugs currently available neither clinicians
nor patients are completely satisfied with their effects. There is no doubt that there is an urgent
need of new and effective drugs, which are able to treat or even possibly cure the allergic
inflammation.
To mimic bronchial asthma in animals, a wide variety of animal models have been
developed. The use of an appropriate model could help us to develop new chemical entities
for the treatment of human allergic disorders in a more predictable way. There have been
many attempts to develop and characterize animal models that approximate human allergy or
asthma. Each model has its own inherent advantages and shortcomings and unfortunately, no
model is identical to the conditions found in human disease. Nonetheless, the development
of relevant animal models continues in anticipation that they will aid in the examination of
684 Drug Screening Methods

underlying processes that contribute to asthma or allergy, or will assist in the identification of
novel therapies that can be used to treat these diseases.
Animal systems that accurately reflect disease pathophysiology continue to be essential to
the development of new therapies for asthma. In this review, the pre-clinical in vitro and in
vivo models that recapitulate many of the features of asthma are described. Specifically, the
pro’s and con’s of the standard models are discussed and recently developed systems designed
to more accurately reflect the complexity of both diseases are reflected. The new models of
asthma described herein demonstrate that improved clinical understanding of the diseases
and better preclinical models is an iterative process that will hopefully lead to therapies that
can effectively manage asthma.

In Vitro models
Binding Assays
Histamine Receptor Assay
This method evaluates the affinity of test compound to histamine H1 receptor. This is done by
measuring their inhibitory activities on the binding of 3H pyrilamine (H1 antagonist) to guinea
pig brain plasma membrane preparation.
Male guinea pig weighing 300-600 g is sacrificed by CO2 necrosis. The brain is homogenized
in ice-cold Tris buffer (pH 7.5, 1 g in 30 ml buffer) and homogenate is centrifuged for 10 min
at 4°C at 50,000 g. Supernatant is discarded and pellet is resuspended in buffer, centrifuged
again. The pellet obtained after centrifugation is re-suspended in Tris buffer (1 g/5 ml) and
aliquots of 1 ml are frozen at -70°C. In a shaking bath maintained at 25°C 50 ul3H pyrilamine
(2 ×10-9 M), 50 ul test compound (10-5-10-10 M) and 100 ul membrane suspension from guinea
pig whole brain (10 mg/ml) per sample are incubated for 30 min. Incubation buffer used is
TrisHCl buffer (50 mM, pH 7.5). With 11 conc. of 3H pyrilamine (0.1-50 × 10-9 M) saturation
experiments are performed. Total binding is determined in the presence of incubation buffer,
non-specific binding in presence of mepyramine (10-5 M). By rapid vacuum filtration through
glass fiber filters reaction is stopped. Subsequently the membrane bound is separated from
the radioactivity. The retained membrane bound radioactivity on the filter is measured after
addition of 3 ml scintillation cocktail/sample in liquid scintillation counter.3
The parameters calculated are total binding of 3H pyrilamine, non-specific binding and
specific binding (total binding–non-specific binding) and % inhibition of 3H pyrilamine
binding (100- specific binding as % of control value).
The dissociation constant (Ki) and IC50 value of test compound are determined from
experiment of 3H pyrilamine Vs non-labeled drug by computer-supported analysis of the
binding data.

Cell Culture Method

CULTEX Technique
The CULTEX technology is a new experimental system for cultivation and exposure of cells
intermittently at the air/liquid interface with ultrafine particles, gases, or mixtures of both
 Anti-asthmatic Agents 685

which fixedly flows. Studies on cytotoxicity of air contaminants such as gaseous or particulate
compounds and complex mixtures have traditionally used animal experiments because of the
difficulties in exposing cell cultures directly to these substances. This technique has enhanced
the efficiency of in vitro studies, and allows direct exposure of the bronchial epithelial cells.
CULTEX technique uses a transwell membrane technique for direct exposure of complex
mixtures like sidestream cigarette smoke at the air/liquid interface. Before exposure, the
bronchial epithelial cells are washed with PBS and then transferred from the companion
plate to the cell exposure unit. The factors influencing the susceptibility of human bronchial
epithelial cells (e.g. gas flow rate or duration of exposure) are studied and the cells are finally
exposed for one hour to clean air or different concentrations of sidestream smoke. To achieve
homogeneous aerosol distribution above the cell cultures, a specially designed exposure
top can be adapted to the CULEX unit to allow direct exposure. Incubation intervals and cell
preparation for analysis are the same for cells exposed to the test atmosphere and the air/
liquid controls. The test group bronchial epithelial cells are incubated with the test drug for 24
hr and then exposed for 1 h to different concentrations of sidestream smoke.4 The biological
parameters that can be estimated in the control and treated groups are number of cells,
metabolic activity and glutathione concentration. Also cell viability measurements in the
control and test groups can be carried out by using the WST assay and electronic cell counting.5

WST Assay
Briefly, in the WST assay the cells are transferred from the cell exposure vessels to conventional
companion plates containing 2 ml of fresh RPMI medium per well. Five hundred microliters
of medium with 100 ml of WST-1 dye is layered on the attached cells and removed after 1 hr
of incubation. Aliquots of 100 µl are transferred into a 96 well microplate for measuring their
absorbance at 450 nm/630 nm using a microplate reader. Additional cells from the same
membrane are trypsinized by adding 500 µl trypsin/EDTA solution on top of the monolayer.
After 4 min of incubation at 37°C, the enzymatic activity is stopped after adding 25 µl of
trypsin inhibitor (10,000 BAEE units/mg protein). The cells are gently suspended and 100 µl
of the suspension diluted in 9.9 ml CASYton. Aliquots are analyzed with an electronic cell
counter.
Thus, CULTEX technique enables treatment of bronchial epithelial cells with sample
atmospheres for subsequent in vitro assays. The introduction of these cultivation and exposure
techniques offers new testing strategies for the toxicological evaluation of a broad range of
airborne and inhaled compounds.

Tests in Isolated Organs

Spasmolytic Activity in Guinea Pig Lungs
Several autacoids such as histamine and leukotrienes induce bronchoconstriction. Histamine
causes bronchoconstriction by activating H1 receptors. It is an important mediator of
immediate allergic and inflammatory reactions. Calcium ionophores induce the release of
leukotrienes via the 5-lipoxygenase pathway that cause potent bronchoconstriction. Using this
method, drugs are tested for their capability of inhibiting bronchospasm induced by histamine
or calcium ionophore.
686 Drug Screening Methods

Albino guinea pigs of either sex, weighing 300 to 450 g, are sacrificed with an overdose of
ether. The chest is opened and the lungs are removed and cut into strips of 5 cm each and
placed into a physiological saline solution. Thereafter, the lungs are mounted in an organ bath
containing a nutritive solution. The nutritive solution has following components in percent of
anhydrous salts: NaCl 0.0659, NaHCO3 0.0252, KCl 0.046, CaCl2 0.005, MgCl2 0.0135, NaH2PO4
0.01, Na2HPO4 0.008, glucose 5%, pH 8. The bath is bubbled with carbogen and maintained
at 37°C. Under a preload of 0.5 to 3 g, the tissue is left to equilibrate for 30 to 60 min. Prior to
the testing, carbachol is added to the bath to test the lung strip’s ability to contract. Twenty
minutes later two pre-values are obtained by adding the spasmogen to the bath and recording
contractile force at its maximal level. Following a 20 min equilibration period the spasmogen
is administered again. The spasmogens commonly used are histamine dihydrochloride
(10-6 g/ml for 5 min), Ca-inophore (10-6 g/ml for 5 min), leukotine C4 and D4 (10-8 g/ml for
10 min). Five minutes thereafter the test compound is administered. The contractile dose is
determined isometrically. The percentage inhibition of spasmogen-induced contraction by
the test drug is calculated.6

Vascular and Airway Responses to the Isolated Lung

The model provides the opportunity of simultaneous registration of pulmonary vascular and
airway responses to drugs.
Sprague Dawley rats (300 to 350 g) are anesthetized intraperitoneally with pentobarbitone
sodium (50 mg/kg). The trachea is cannulated and the animal is maintained on artificial
respiration. Rat is heparinized with 1,000 units of heparin and rapidly exsanguinated by
withdrawing blood from the carotid artery. Lung is exposed by median sternotomy and a
ligature is placed around the aorta to prevent systemic loss of blood. Lung is removed and
suspended in a warmed (39°C), humidified (100%) water-jacketed chamber. The pulmonary
artery is also catheterized. An external heat exchanger is used to maintain temperature of
the perfusate solution (Krebs-Henseleit), which is placed in a reservoir and mixed constantly
by a magnetic stirrer. Lungs are perfused using a peristaltic roller pump at a flow rate of 8 to
14 ml/min to maintain pulmonary arterial pressure of 15 mmHg. Pulmonary arterial pefusion
pressure, airway pressure and reservoir blood level are continuously monitored, electronically
averaged and recorded with the help of a polygraph. Changes in pulmonary arterial pressure
and airway pressure after injection of test compounds are measured in mmHg and compared
with baseline values.7

Reactivity of the Isolated Perfused Guinea Pig Trachea

This model is best suited to study the mechanism by which the epithelium affects the reactivity
of tracheal musculature. The effect of histamine, calcium ionophores, bradykinin, leukotrienes
and potassium channel openers can be studied using this method. Contractile agonists can be
added either to the extraluminal (serosal) or the intraluminal (mucosal) surface.
Albino guinea pigs of either sex (300 to 550 g) are sacrificed by CO2 narcosis. The entire
trachea is dissected out and cut into individual rings, 12 to 15 rings are tied together with silk
threads and mounted in an organ bath containing Krebs-Henseleit buffer solution. The solution
is pumped at a rate of 30 ml/min through the lumen. The tissue is maintained at 37°C under a
tension of 0.5 g and gassed with carbogen. Responses of the tracheal musculature are obtained
 Anti-asthmatic Agents 687

by changes in inlet-outlet pressure between the side holes of indwelling catheters. Both the
catheters (inlet and outlet) are connected to the positive and negative sides respectively of a
differential transducer. Isometric contractions are recorded via a transducer connected to a
polygraph. After 45 min equilibration, spasmogens are added. The spasmogens commonly used
are carbachol (2 × 10-7 g/ml) histamine dihydrochloride (10-7 g/ml), Ca-inophore (10-6 g/ml),
leukotine C4 and D4 (10-8 g/ml). When the contraction has reached maximum (initial spasm)
the standard drug [(isoprenaline (1 ng/ml), aminophylline (10 ng/ml)] is administered. The
bronchial responses are allowed to plateau and are recorded. The tissue is washed thoroughly
and control contractions are induced again after adding spasmogen. After obtaining the initial
contraction again, the test drug is added and the contractile force is recorded to its maximal
level. A 15 min washing time is observed during the experiment.
Responses are quantified as change in pressure in cm of water. EC50 values are determined
from least square analysis of a logrit model and presented using 95% confidence intervals.7
From dose-response curves ED50 values can be calculated and the percent inhibition of
spasmogen-induced contractions is calculated.8

IN VIVO MODELS
Bronchospasmolytic Activity in Anesthetized Guinea Pigs
Changes in air volume of a living animal in a closed system consisting of the respiratory
pump, the trachea and the bronchi are registered using this method. Also reservoir permitting
measurement of volume or pressure of excess air can be measured. A decrease in the volume
of inspired air and increase in the volume of excess air is induced by bronchoconstriction.
Contraction of bronchial smooth muscles results on administration of spasmogen. The method
permits the evaluation of bronchospasmolytic effect by measuring the volume of air, which is
not taken up by the lungs after bronchospasm.
Guinea pigs of either sex (250 to 500 g) are anesthetized with 1.25 g/kg urethane intra­
peritoneally and it is ensured that there is no spontaneous respiration. The trachea is cannu­
lated, one arm is connected to a respiratory pump and the other to a Statham P23 Db trans­
ducer. The animal is artificially ventilated at a frequency of 60 strokes/min. Excess air, not
taken up by the lungs, is measured and recorded in a polygraph. The jugular vein is cannulated
for test drug administration and carotid artery for blood pressure measurement. Each animal
is placed in a plastic container of 15 l volume. An aerosol of 0.25% histamine solution at
180 mmHg pressure is sprayed. The exposure time is 5 min. The test drug is administered orally
1 h before the exposure. The spasmogen challenge is repeated. Unprotected animals fall on
their sides, asphyxiated. ED50 is calculated and the results are expressed as percent inhibition
of induced bronchospasm over the control agonistic responses.9

Arachidonic Acid or PAF-induced Respiratory and Vascular

Dysfunction in Guinea Pigs
Thromboxane and prostacyclin are the products of arachidonic acid metabolism. Thromboxane
leads to brochoconstriction and thrombocytopenia whereas prostacyclin leads to reduction in
systolic and diastolic pressure.
688 Drug Screening Methods

Male guinea pigs (300 to 600 g) are anesthetized with 60 mg/kg pentobarbitone sodium
(intraperitoneally). Jugular vein is cannulated for administration of a spasmogen/test
compound. Both carotid arteries are cannulated and one is connected to a pressure
transducer for measuring blood pressure and the other is used for blood withdrawal. The
trachea is connected to a respirator (70 to 75 strokes/min). Excess air, not taken up by the
lungs, is conducted to a transducer with bronchotimer, which translates changes in airflow to
an electric signal. Changes in airflow and arterial blood pressure are recorded continuously.
Animals receive multiple intravenous injections of the same dose of arachidonic acid
(60 µg/kg) until two bronchospasms of equal intensity are obtained. The test compounds are
administered intravenously and the spasmogen is given again. Percent inhibition or increase
of bronchospasm, reduction of blood pressure, thrombocytopenia and hematocrit following
test drug administration are calculated in comparison to control values before drug treatment.
For the reduction of blood pressure both the magnitude and the duration are determined.10

Anaphylactic Microshock in Guinea Pigs

In the anaphylactic response, histamine is released from various sites. Antihistamines are
useful in various anaphylactic manifestations such as bronchial asthma and serum sickness.
Introduction of a foreign protein in the body can produce microshock. A microshock is
apparently one that is interrupted before death, and is repeatable.
Guinea pigs (200 to 300 g) are sensitized with subcutaneous injection of egg albumin. After
three weeks the animals are exposed to an aerosol of 5% albumin in an exposure chamber.
They are removed from the chamber as soon as they become dyspneic. If not removed at that
moment, they die. The time from the commencement of exposure to severe dyspnea, the
preconvulsion time, is noted which serves as a measure of the severity of the shock. If no signs
of shock are seen after 6 min, the animal is regarded as protected and the preconvulsion time
is taken as infinite. The degree of protection (p) is calculated from the formula:
p = [1 – (C/T )] × 100

C and T are preconvulsion time of the control and treated animals respectively.
After a control exposure the guinea pig is treated with a drug. After another 4 to 7 days,
it is again given a control exposure.11,12 Animals with C below 40 sec and above 165 sec are
excluded.

Serotonin Aerosol-induced Asphyxia in Guinea Pig

Guinea pigs when exposed to an aerosol containing serotonin, develop constriction of the
bronchi, which, if sufficiently large causes asphyxia.
Guinea pigs (200 to 300 g) are placed in an anesthetic box and 2% serotonin aerosol
is introduced by means of a compressor. Animals exposed to the aerosol behave in a
characteristic manner and show progressive signs of difficulty in breathing, convulsion and
death. By observation, experience is gained so that the pre-convulsion time can be judged
accurately, and is quiet constant if the guinea pigs are not used frequently. As soon as the
preconvulsive breathing commences, the animals are placed in fresh air. An infusion pump
injects the test drug within 1 min. Alternatively, the animal is treated orally or subcutaneously
with the test drug. The percent protection afforded by the drug is calculated from the formula
 Anti-asthmatic Agents 689

(1-T1/T2) × 100 where T1 is the mean of the control preconvulsion time 2 days before, and 2
days after the administration of the drug and T2 is the preconvulsion time determined with the
administration of the drug.13,14

Histamine-induced Bronchoconstriction in Anesthetized Guinea Pigs

Respiratory parameters such as respiratory frequency and respiratory amplitude can be
measured in guinea pigs by plethysmograph. Bronchodilatory drugs attenuate the decrease
in respiratory amplitude and the reflectory increase of respiratory frequency after histamine
inhalation. Additional respiratory parameters can be recorded using Fleisch tube and a
catheter inserted into the pleural cavity. In addition, using this method antagonism against
bradykinin-induced bronchoconstriction or the bronchodilator effects of potassium channel
openers can also be evaluated.
Guinea pigs of either sex weighing 400 to 600 g are anesthetized with 70 mg/kg pentobarbital
intraperitoneally and the trachea, jugular vein and carotid artery are cannulated. Animal
is maintained on artificial respiration (60 strokes/min). The guinea pigs are placed inside a
whole body plethysmograph box and tracheal, venous and arterial catheters are connected
to onset ports in the wall of the plethysmograph box. The tracheal port is connected to the
respirator. Airflow rate into and out of the plethysmograph is measured using a differential
pressure transducer. Tidal volume and transpulmonary pressure are measured. Signals from
the airflow, tidal volume and transpulmonary pressure are fed into an online computer system
for calculation of pulmonary resistance (PR) and dynamic lung compliance (LC). Systemic
arterial pressure is measured using a Statham pressure transducer. Heart rate is computed from
pressure pulses. Intravenous injection of histamine (0.5 to 2.0 µg/kg) leads to decrease in LC
and increase in PR by 200% compared to baseline values. After 5 min interval, challenges are
repeated yielding the same increase in pulmonary resistance during the whole experimental
duration. After three reproducible responses the test compound is administered intravenously
1 min before histamine injection. Inhibition of histamine-induced bronchoconstriction by
test compound is recorded and ED50 is calculated. Further, the time required for histamine
antagonism is evaluated.12

Pneumotachography in Guinea Pigs

Pneumotachograph allows simultaneous measurements of several respiratory and circulatory
parameters in anesthetized guinea pigs. Its use is based on the principle of the Fleisch tube.
Guinea pigs (300 to 400 g) are anesthetized intraperitoneally with 1.5 g/kg urethane. The
animals are fixed at the upper extremities on a heated operating table. The trachea is cannulated.
A thin plastic catheter is inserted into the esophagus and the tip is located inside the thorax
to register intrathoracic pressure. Further, the cephalic vein of one side and carotid artery of
the other side are cannulated. The tracheal cannula is connected to the pneumotachograph.
The pneumotachograph is connected to a differential pressure transducer. One side of another
pressure differential transducer is connected with the esophageal catheter and the other side
remains open to room air. For recording of arterial pressure a Gould pressure transducer is
used. The signals of airflow and esophageal pressure are monitored using an oscilloscope.
Using an analog computer various respiratory and circulatory parameters are measured.
690 Drug Screening Methods

Pulmonary mechanics like airway resistance, dynamic compliance and end respiratory work
can be measured.
Each animal serves as its own control. For each individual experiment the data of the last
5 min before the first substance application are averaged and used as controls. The response
values after substance application are then expressed as percentages of the control.6

Microshock in Rabbits
Although the guinea pig has been favored for screening antihistaminic agents because of its
sensi­tivity to histamine, the rabbit may serve as a suitable model when a second species is
desired.
Rabbits (200 to 300 g) are placed in a glass aquarium 61 × 30.5 × 30.5 cm with its open side
down on a smooth rubber surface, having a hole connected to a nebulizer. Each rabbit is
placed in a separate chamber and subjected to an aerosol of 0.2% histamine aerosol. In small
doses rabbit exhibits shock symptoms in gradual succession. The first sign is the drawing in of
the abdominal walls to assist in breathing. The movements become stronger, the respiration
becomes slower and deeper or more rapid and shallow. In the latter case the animal’s head
moves rapidly to and fro. At this stage, the animal has to be removed from the chamber.
Otherwise the sequelae in quick succession are seen, e.g. gasping for air, convulsions, urination,
cyanosis and possibly death. In order to prevent desensitization, an interval of 8 days or more
is interposed between tests. The test drug is administered intraperitoneally 30 min before the
experiment. The time at which the animal has to be removed from the chamber is recorded.15‑17
The percent protection offered by the drug is calculated using the formula
(1-T1/T2) × 100
where
T1 = control preconvulsion time
T2 = preconvulsion time after administration of the drug.

Bronchial Hyperactivity in Guinea Pigs

Inhalation of histamine or other spasmogens can induce symptoms like asphyctic convulsions
resembling bronchial asthma in guinea pigs. The challenging agents are applied as aerosols
produced by an ultrasound nebulizer. Early symptoms are increased breathing frequency,
forced inspiration and finally anaphylactic convulsions. Antagonistic drugs can delay the
occurrence of these symptoms. Preconvulsion time can be measured.
Male albino guinea pigs (300 to 400 g) are used. The inhalation cages consist of three boxes
each ventilated with airflow of 1.5 l/min. The animal is placed into box A to which the test drug
or the standard is applied using an ultrasound nebulizer which provides an aerosol of 0.2 ml
solution of the test drug injected in an infusion pump within 1 min. Alternately, the animal
is treated orally or subcutaneously with the test drug or the standard. Box B serves as sluice
through which the animal is passed into Box C. There, the guinea pig is exposed to an aerosol
of 0.1% solution of histamine hydrochloride provided by an ultrasound nebulizer. Time until
appearance of asphyctic convulsions is measured. Then, the animal is immediately removed
from the inhalation box. Percent increase of preconvulsion time is calculated versus controls.
ED50 (50% increase in preconvulsive time) is also calculated.18
 Anti-asthmatic Agents 691

Airway Microvascular Leakage in Guinea Pigs

Evans Blue dye can be used to study plasma exudation in guinea pig airways in vivo. The
antagonism against bradykinin and platelet-activating factor (PAF)-induced microvascular
leakage and vagal stimulation-induced airway responses can be studied using this method.
Guinea pigs (380 to 600 g) are anesthetized with urethane (1.5 g/kg, intraperitoneally). The
trachea, jugular vein and carotid arteries are cannulated. Jugular vein is cannulated for the
administration of test compounds and carotid artery for the measurement of blood pressure.
The animal is maintained on artificial respiration (60 strokes/min). Lung resistance is measured
as an index of airway function and monitored throughout the experiment. Transpulmonary
pressure is measured using a pressure transducer with one side attached to the catheter inserted
into the right pleural cavity and the other side attached to the intratracheal cannula. Airflow is
measured using a pneumotachograph. The test compound is given intravenously. Ten minutes
later, Evans Blue dye is injected intravenously for 1 min. After 1 min, bronchoconstriction and
microvascular leakage is induced by injection of bradykinin or PAF or vagal stimulation. Six
minutes after induction of leakage the thoracic cavity is opened and a cannula is inserted
into the aorta through a ventriculotomy. Perfusion is performed with 100 ml (0.9%) saline
at a pressure of 100-120 mmHg in order to remove the intravascular dye from the systemic
circulation. The right ventricle is opened and perfused with 30 ml saline.
The lungs are also removed. The tissues are blotted dry and weighed. Evans Blue dye is
extracted in 2 ml of formamide and measured in a spectrophotometer at 620 nm. Evans Blue
dye concentration, expressed as ng/mg tissue as well as lung resistance are compared by
statistical means between treated groups and controls receiving the challenge only.19‑21

Airway Inflammation in Mice

Balb/c mice sensitized with ovalbumin and challenged by repeated exposure to ovalbumin
yields marked eosinophilia in bronchoalveolar lavage (BAL) fluid. It has also been seen that
eosinophil influx varies dramatically in mice of the different stains. Strains such as 129/SV,
CBA belong to non- or low-responder. Strains such as SWR, FVB, C57BL/6 respond to antigen
challenge with a marked increase of eosinophils both in the BAL and in the lung tissue.22,23
Balb/c mice, used for this study are subcutaneously implanted with heat-coagulated egg
white. Fourteen days later the mice are challenged intratracheally with heat-aggregated
ovalbumin. Test drug is administered subcutaneously, intraperitoneally or orally. Forty-eight
hours after antigen challenge, bronchoalveolar lavage fluid is collected from animals of both
groups and total number of eosinophils, neutrophils and eosinophil peroxidase activity are
assessed. The animals are then sacrificed and histopathological evaluation is carried out.
Based on the results of histopathological study and BAL fluid examination, protection that is
offered by the test drug is evaluated.24,25

Innovation in Model Development

The lack of translation from preclinical to clinical studies of new asthma compounds and
biologics is of particular importance to the severe and/or therapy-resistant asthma group,
where new effective therapies for improved asthma control are crucial. The challenge is
to develop models that more accurately recapitulate the asthmatic airway for mechanistic
692 Drug Screening Methods

studies, target identification and validation, and efficacy and safety testing. Addressing this
will require a multidisciplinary, collaborative and innovative approach.

Biomimetic Models
The opportunities afforded by tissue engineering, coupled with advances in bioreactor
and scaffold design, have resulted in several tissue-engineered human airway equivalents
becoming available. Although these have added valuable insight into the pathophysiology
of the disease, they are still too simplistic to mimic important in vivo features, such as fully
functioning immune and/or circulatory system.26
Microfluidics
Tissue engineering is a rapidly evolving science and tissue engineers have embraced the
challenge of building complexity into their models. Although there might still be some way to go
in recapitulating in vitro entire immune or circulatory systems, steps are being made. Advances
in the emerging field of microfluidic lab-on-a-chip technologies, combined with tissue
engineering, offers great opportunity in this regard. Microfluidics provide many advantages
over current macroscopic techniques and have been used to microfabricate successfully blood
vessels, muscles, brain, kidney and liver for basic research and drug discovery.27.
In silico modelling
In silico modelling has the potential to change the way drugs develop, and accelerate the
drug discovery process. These approaches are forming the newest wave of modelling tools
in asthma research and drug development. Numerous models have been developed to study
basic events, such as molecular interactions (ligand–receptor), whole-organ functions (e.g.
bronchoconstriction and aerosolized drug deposition in the lung), and even virtual patients.28
Precision cut lung slices
Ex vivo precision cut lung slices (PCLS) from human lung also offer exciting opportunities for
increasing understanding of asthma development and progression, and bridge the gap between
cell culture and isolated tissue preparations and animal studies. Despite this, however, PCLS
approaches are rarely adopted. Originally developed for toxicological purposes, PCLS have
been successfully adapted to study airways disease and have several advantages over current
in vivo approaches. The main focus of their use has been to assess airway smooth muscle
responses, including hyperresponsiveness, remodeling and bronchoconstriction in response
to several stimuli, such as allergens, pharmacological challenge and infection. Recently, PCLS
has been used to investigate the response of airways to drugs used in the treatment of asthma,
where these models are already identifying alternative treatment strategies as potential future
asthma therapies; however, these are yet to be tested clinically.29

Transgenic Approaches
Genetically modified animals, especially mice, are considered important models in basic
research to define specific pathways for drug discovery, mechanistic studies and target
identification and/or validation. They have been extensively used in asthma, with numerous
‘off-the-shelf’ transgenic mice and species-specific probes and reagents commercially
available, which enable researchers to suppress, switch off or upregulate a single molecular
 Anti-asthmatic Agents 693

signalling pathway specifically. However, it is questionable how useful these models are for
studying a disease that is associated with several molecular and cellular pathways that function
synergistically or independently of each other.30

DISCUSSION
Animal models of asthma are necessary and irreplaceable for the further understanding of
the pathophysiological mechanisms and preclinical evaluation of new treatments. Since all
allergic diseases are characterized by early and late phase symptom’s models of both phases
are available. Furthermore, it is also necessary to investigate mediators and cells involved in
early and late phase of an allergic reaction. Additionally, the increased airway responsiveness
should also be modeled. In animals, inflammatory changes should be induced that result in
pathological changes including remodeling.
A murine model for asthma presents numerous advantages when compared with the use
of other animal species. This model offers the opportunity to explore mechanisms of allergic
reactions because of the existence of numerous immunological reagents specific for murine
cytokines, adhesion molecules, etc. Murine models have provided fundamental information
regarding certain features of asthma such as involvement of lymphocytes. Because mice are
extremely useful for immunological studies, a suitable murine model of allergic pulmonary
inflammation can be invaluable to study drug effect on it. There are certain transgenic or knock
out strains available. On the other hand, because of considerable physiological dissimilarities
with primates, the ability to extrapolate murine findings to humans will be difficult.
The rat has received considerable attention during recent decades. Experimental data
suggest that the most characteristic features of human asthma including pathological changes
can be duplicated in rats. Certain strains such as Brown Norway rats produce IgE as the major
anaphylactic antibody. Since a large number of corresponding immunological reagents are
available the role of cytokines and chemokines in allergic inflammation can also be studied
in a rat model. In contrast to guinea pigs, allergic sensitization requires use of adjuvant
such as alum or Bordetella pertussis. Another disadvantage of this species is that early phase
bronchoconstriction cannot be induced via inhalation of the antigen. It should be given
intravenously. The rat also offers economic advantage.
The guinea pig was or perhaps is still the most popular animal model of allergic reactions.
The benefits are the pragmatic benefits of economy and ease of animal handling to the
features, which allergen-induced bronchoconstriction and human bronchial asthma have in
common. Such features include the bronchoconstrictor response to antigen contact, the hyper-
responsiveness of the airway to mediators and the eosinophilic nature of allergic bronchial
inflammation. However, the scarcity of inbred strains is a disadvantage with regard to studying
genetic influence.
The hamster is seldom used in asthma research. Its oral mucosa is well suited for intravital
microscopy and therefore for studying microcirculation. It is sometimes employed for testing
in vitro effect of kinins. The rabbit provides an interesting animal model. This species can
demonstrate early and late phase responses with accumulation of eosinophils, BHR is also
observed. A further advantage of this species is the production of IgE. Rabbits are small, docile
animals and relatively without danger of desensitization. The sheep represents a species, in
694 Drug Screening Methods

which sensitization to Ascaris occurs naturally. The antigen response consists of an early and
a late phase bronchoconstriction. The latter is accompanied with cell infiltration. Dogs can be
sensitized by natural exposure to Ascaris, but other antigens can also immunize them.
In asthma research, little attention has been paid to domestic pigs or mini- or micropigs.
From an anatomical point of view, pigs have several similarities to humans. The real handicap
with pigs is their size. Monkeys demonstrate an IgE mediated early and late phase respiratory
response to antigen. They have shown BHR. Monkeys are considered to have an immune
system very similar to that of humans in comparison to mice, rats and guinea pigs.

CONCLUSION
In summary, it should be emphasized that any animal model will have both strengths and
weaknesses and its value in answering particular research questions must be evaluated with
respect to its relevance to human diseases. Unfortunately, this relevance becomes more
difficult to determine when the cause of the underlying disease is unclear, or potentially
complex as in asthma. Thus many species have been utilized in the development of animal
model of asthma including mice, rats, guinea pigs, ferrets, hamsters, rabbits, dogs, sheep, pig,
horses and nonhuman primates. Each possesses certain advantages and disadvantages as a
model of asthma. Therefore, certain caveats must be recognized in using animal systems. It
must however, be appreciated that animals are only surrogates. Results from such studies may
be compared with information obtained from experiments performed with human materials
in order to minimize or even to avoid faulty extrapolations. It is also important to recognize
that no single model is sufficient to draw conclusions on the therapeutic value of new
chemical entities. Prudent employment of well-designed animal models can provide valuable
information on drug effects. Advances in new technologies, including tissue engineering,
imaging and in silico modelling, can provide researchers with new opportunities for innovative
model development. Integrating these technologies in existing preclinical testing programs is
crucial to accelerating the development of more predictive but fewer animal models and a
fundamental shift in the way that asthma research and drug development is carried out.

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1. Tai A, Ranganathan S. Optimizing medications for poorly controlled asthma. Am J Respir Crit Care
Med 2007;176:520-1.
2. Martin BL, Caruana-Montaldo B, Craig T. Comprehensive management of asthma. Drugs of Today
1997;33:149-60.
3. Hill SJ, Emson PC, Young JM. The binding of 3H mepyramine to histamine H1 receptor in guinea pig
brain. J Neurochem 1978;31:997-1004.
4. Knebel JW, Ritter D, Aufderheide M. Exposure of human lung cells to native diesel motor exhaust:
Development of an optimized in vitro test strategy. Toxicol In Vitro 2001;16:185-92.
5. Piperi C, Pouli AE, Katerelos NA, et al. Study of the mechanisms of cigarette smoke gas phase
cytotoxicity. Anticancer Res 2003;23:2185-90.
6. Kleinstiver PW, Eyre P. Evaluation of lung parenchyma strip preparation to measure bronchoactivity.
J Pharmacol Methods 1979;2:175-85.
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7. Allen DA, Schertel ER, Bailey JE. Reflex cardiovascular effects of continuous prostacyclin
administration into an isolated in situ lung in the dog. J Appl Physiol 1993;74:2928-34.
8. Baersch G, Frolich JC. A new bioassay to study contractile and relaxant effects of PGE2 on perfused
guinea pig trachea. J Pharmacol Toxicol Methods 1996;36:63-8.
9. Miura M, Belvisi MG, Barnes PJ. Modulation of nonadrenergic noncholinergic neural broncho­
constriction by bradykinin in anesthetized guinea pigs in vivo. J Pharmacol Exp Ther 1994;268:482-6.
10. Lefort J, Vargaftig BB. Role of platelets in aspirin-sensitive bronchoconstriction in the guinea pig:
Interactions with salicylic acid. Br J Pharmacol 1978;63:35-42.
11. Chand N, Diamantis W, Nolan K, et al. Azelastine inhibits acute allergic dyspnoea in a conscious
guinea pig asthma model. Res Commun Mol Pathol Pharmacol 1994;85:209-16.
12. Vitkun SA, Foster WM, Bergofsky EH, et al. Large and small airway responses to bronchoconstrictors
in the guinea pig and the ferret. Lung 1990;168:249-57.
13. De Bie JJ, Henricks PA, Cruikshank WW, et al. Modulation of airway hyperresponsiveness and
eosinophilia by selective histamine and 5-HT receptor antagonists in a mouse model of allergic
asthma. Br J Pharmacol 1998;124:857-64.
14. Konno S, Adachi M, Matsuura T. Bronchial reactivity to methacholine and serotonin in six inbred
mouse strains. Arerugi 1993;42:42-7.
15. Ali S, Mustafa SJ, Metzger WJ. Adenosine receptor mediated bronchoconstriction and bronchial
hyperresponsiveness in allergic rabbit model. Am J Physiol 1994;266:L271-7.
16. Ali S, Mustafa SJ, Metzger WJ. Modification of allergen-induced airway obstruction and bronchial
hyperresponsiveness in the allergic rabbit by theophylline aerosol. Agents Actions 1992;37:168-70.
17. Harris JO, Bice D, Salvaggio JE. Cellular and humoral bronchopulmonary immune response of
rabbits immunized with thermophilic actinomyces antigen. Am Rev Respir Dis 1976;114:29-43.
18. Rogers DF, Boschetto P, Barnes PJ. Plasma exudation: Correlation between Evans Blue dye and
radiolabelled albumin in guinea pig airways in vivo. J Pharmacol Methods 1989;21:309-15.
19. Aoki S, Bouberkeur K, Kristersson A, et al. Is allergic airway hypersensitivity of the guinea pig
dependent upon eosinophil accumulation in the lung. Br J Pharmacol 1988;94:365-71.
20. Chand N, Nolan K, Diamantis W, et al. Repeated aeroallergen challenge induces lung dysfunction
but not bronchial hyperresponsiveness in conscious guinea pigs. Agents Actions 1992;37:184-7.
21. Perretti F, Manzini S. Activation of capsaicin sensitive sensory fibers modulates PAF-induced
bronchial hyperresponsiveness in anesthetized guinea pigs. Am Rev Respir Dis 1993;148:927-31.
22. Mitchell HW, Turner DJ, Gray PR, et al. Compliance and stability of the bronchial wall in a model of
allergen-induced lung inflammation. J Appl Physiol 1999;86:932-7.
23. Waserman S, Xu LJ, Olivenstein R, et al. Association between late allergic bronchoconstriction
in the rat and allergen stimulated lymphocyte proliferation in vitro. Am J Respir Crit Care Med
1995;151:470-4.
24. Hessel EM, Van Oosterhout AJ, Hofstra CL, et al. Bronchoconstriction and airway hyper
responsiveness after ovalbumin inhalation in sensitized mice. Eur J Pharmacol 1995;293:401-12.
25. Temann UA, Geba GP, Rankin JA, et al. Expression of IL-9 in the lungs of transgenic mice causes
airway inflammation, mast cell hyperplasia and bronchial hyper responsiveness. J Exp Med
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27. Song JW, et al. Microfluidic endothelium for studying the intravascular adhesion of metastatic
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28. Van de Waterbeemd H, Gifford E. ADMET in silico modelling: towards prediction paradise? Nat Rev
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 CHAPTER

45
Cell-Based Assays in High-
Throughput Screening of Drugs
ABSTRACT
Drug discovery is a complex process which is aimed to develop drug candidates with all
desired pharmacological and pharmacokinetic properties with minimal side effects. With
the advancement of combinatorial chemistry, genomics, proteomics and bioinformatics
which necessitate a very rapid screening strategy for lead identification and optimization.
Conventional animal models become obsolete due to its high cost, low throughput and moral
issues. Over the past two decades, high-throughput screening (HTS) assays has emerged and
matured as a platform in the early stage of drug discovery in the pharmaceutical industry.
More than half of the HTS assays are being replaced with cell-based assays for target validation
and ADMET (absorption, distribution, metabolism, elimination and toxicity) in the early
drug discovery processes. In this review, different types of cells, culture systems (2D, 3D and
microfluidic cell culture), detection methods and its recent advancements in cell based HTS
are discussed with successful examples in commercial development of new drugs.

BACKGROUND
Cell-based high-throughput screening (HTS) assays have been developed to provide more
relevant in vivo biological information for drug discovery processes. Historically, drug
screening greatly relies on animal models as proxies for human beings in drug target validation
and ADMET (absorption, distribution, metabolism, elimination and toxicity) but because of
its low throughput and moral issues, the animal models are being replaced with cell-based
HTS assays to accelerate early-phase drug discovery.1

TYPES OF CELL-BASED ASSAYS

Cell-based assays for HTS mainly include three types:
•• Second messenger assays: This is based on the principle that it monitors signal transduction
following activation of cell surface receptors. In this assay, fluorescent molecules that
respond to intracellular Ca2+ concentrations, membrane potential, pH, etc. are being used
to assay receptor/ion channel activation.2,3
698 Drug Screening Methods

•• Reporter gene assays: This assay monitor cellular responses at the transcription or
translational level, e.g. Quantification of G-protein coupled receptor (GPCR) internalization
using GPCR-green fluorescent protein hybrids.4
•• Cell proliferation/cytotoxicity assays: The overall cell growth or death in response to external
stimuli or stress is evaluated using this assay, e.g. Virus–induced cytopathic effects on
cell proliferation monitored by following the reduction of tetrazolium salt to formazan
quantified by measuring absorbance at 410 nm.5

COMPONENTS OF CELL-BASED HTS ASSAYS

The components of cell-based HTS assays include:
•• Cells – Sources and types
•• Devices for culturing cells
•• Detection system to quantify cell/cellular activities.

Cells—Sources and Types

Primary cells: Primary cells are the cells that have been freshly isolated from a living organism
and maintained for growth in vitro. These cells closely mimic the physiological state of cells
in vivo and generate more relevant data representing living systems. The cell types most
frequently found in primary cell culture are epithelial cells, fibroblasts, keratinocytes,
melanocytes, endothelial cells, muscle cells, hematopoietic and mesenchymal stem cells.
Disadvantages: They have a limited life span in culture, difficult to grow and transfect.6
Immortalized cell lines: Immortalized cells are the cells whose growth characteristics have
been altered to grow continuously and divide indefinitely in vitro, e.g. HEK293 cells derived
from human kidney, HeLa cells are human epithelial cells from a fatal cervical carcinoma
transformed by human papilloma virus 18 (HPV18).7 They are cheap, easy to grow, reliable
and reproducible, and hence widely used for drug screening assays.
Disadvantages: Significant mutations and altered biological characteristics in the
immortalization process will make them different from those of the native/normal cells.6
Human cancer cell lines: These cells are derived from human cancers and they are widely used
for anticancer drug screening in pharmaceutical research, e. g. NC160, a panel of 60 human
tumor cell lines (NC160) representing 9 tissue types for screening potential new anti-cancer
agents.8 The presence of mutations in these cell lines may affect the experimental outcome.
Cancer stem cells: Cancer stem cells may be genetically raised from oncogenic transformation
of either stem cells or progenitor cells. They can be isolated from tumors and have the capability
to self renew, differentiate and regenerate a phenotype of the original tumor,9 e.g. Phase II
screening of new drugs in ovarian cancers and malignant melanoma.10
Mesenchymal stem cells (MSCs): MSCs (MSCs, also known as bone marrow stromal cells or
skeletal stem cells) are multipotent stem cells that can differentiate into chondrocytes (cartilage
cells), osteoblasts (bone cells) or adipocytes (fat cells)—making them ideal candidates for
tissue engineering. MSCs can contribute to the regeneration of bone, cartilage, muscle and
tendons. It has also been shown that—when transplanted systemically into animals—they
 Cell-Based Assays in High-Throughput Screening of Drugs 699

are able to migrate to the sites of the injury, e.g. Human MSCs derived osteoblasts for testing
purmorphamine.11
Embryonic stem cells (ESCs): These cells exhibit an almost unlimited proliferative capacity in
culture and maintain their pluripotent potential to differentiate into all cell lineages in the
body.These cells can serve as better cell models for both drug efficacy and toxicity screening,
e.g. Human embryonic stem cell derived cardiomyocytes for electrophysiological drug
screening.12
Induced pluripotent stem cells (iPSCs): iPSCs are pluripotent cells artificially derived from
somatic cells (Fibroblasts and other adult cell types) by inducing a small set of powerful
pluripotency genes. Their previous somatic cell properties are lost and are similar to human
ESCs in terms of morphology, growth properties, gene-expression profiles and differentiation
potential. iPSCs derived from patients with specific diseases have been considered as a new
tool in drug discovery.

Devices for Culturing Cells

The majority of cells cultured in vitro grow as monolayers on an artificial substrate. Hence, the
substrate must be correctly charged to allow cell adhesion, or to allow the adhesion of cell-
derived attachment factors that will allow cell adhesion and spreading. Therefore, the normal
cells need to be spread out on a substrate to proliferate and inadequate spreading due to poor
adhesion or overcrowding will inhibit proliferation.13,14 However, the hemopoietic cell lines,
rodent ascites, tumors and a few other selected cell lines such as small-cell lung cancer,15 many
transformed cell lines grow in suspension and can be independent of surface charge on the
substrate.
The factors which govern the choice of culture vessel include:
1. The cell mass required,
2. Whether the cells grow in suspension or as a monolayer,
3. Whether the culture should be vented to the atmosphere or sealed,
4. The frequency of sampling,
5. The type of analysis required, and
6. The cost.
The vessels used for adherent cultures and suspension cultures are given in Figure 45.1.

TYPES OF CELL CULTURE SYSTEMS

Conventional 2D Culture Systems

It is well documented that the cells grown on 2D surfaces do not mimic true in vivo physiology.
Though 2D cell-based assays in multiwall plates together with automated operation are widely
used in drug screening, 2D assays may result in errors in predicting tissue-specific responses
due to the loss of native morphology and limited cell-cell and cell-matrix interaction. This is
overcome by the development of 3-dimensional (3D) cell cultures.
700 Drug Screening Methods

Figure 45.1: Types of vessels for human cell cultures: (A) Adherent culture system; (B) Suspension
culture system (Ref. www.sigmaaldrich.com)16
 Cell-Based Assays in High-Throughput Screening of Drugs 701

3D Culture Systems
The cells grown on 3D scaffolds are demonstrated to show similar in vivo morphology with
intimate cell-cell and cell-ECM (extracellular matrix) interaction.1 The 3D scaffold provides
another direction for cell-cell interactions, cell migration and cell morphogenesis which are
critical in regulating cell cycle and tissue functions.1 In addition, 3D cell cultures provide not
only the templates for cells to adhere and grow but also allow the distribution of nutrients and
metabolites thus enabling long-term cell culture in vitro.17 It holds promise for being more
predictive of in vivo responses to drug treatments.
Numerous studies have documented that cell response to drugs in 3D cultures are distinct
from those in 2D cultures, which highlights the advantages of using 3D-based models.18-20
Therefore, it is having a greater potential to become a superior platform for drug development
to bridge the 2D monolayer cell culture systems and the animal models.

Figure 45.2: Schematic representation of 2D vs 3D cultures

3D Microfluidic Cell Cultures

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-
scale cell based research and applications in drug screening. Various natural and synthetic
hydrogels have been incorporated into microfluidic cell culture systems to support cells in
3D.1 A variety of 3D microfluidic cell culture models have been developed.21,22 Vickerman et al.
(2008) developed a microfluidic platform capable of mimicking the in vivo microenvironments
by integrating fluidic microenvironments and 3D microenvironments using microinjection of
gel solution containing cells. An open lumen-like structure was created when human adult
dermal microvascular endothelial cells were cultured on this microfluidic platform for up to
7 days.

DETECTION METHODS
Detection methods used in cell-based HTS assay fall into two groups:
1. Electrochemical methods
2. Optical methods
702 Drug Screening Methods

Figure 45.3: Schematic representation of 3D microfluidic culture system. Microfluidic cell culture ar-
ray showing concentration gradient generator and microchambers On 2×2 cm device (Adapted from
Hung et al 200523 & Zang et al, 20121)

Electrochemical Methods
The detection method by electrochemical technique is based on:
A. Cellular activity and function,
B. Cellular barrier behaviour, and
C. Recording/stimulation of electric potential of electrogenic cells.
Methods based on Cellular Activity and Function
The cell viability of a living cell can be measured as a function of electron generation and charge
transfer caused by redox reaction and changes of ionic composition, e.g. the tumor cells when
attached to a specialized electrode exhibit an irreversible voltammetric response, which is
related to the oxidation of guanine and thus the oxidation peak can be used to investigate the
exogenous effect to study anti-tumor drug sensitivity.
In addition, the cellular activities can be measured using conventional potentiometry and
amperometry methods. The potentiometric method utilizes either ion-selective electrode
(ISE) or gas-sensing electrode (GSE) coated with a layer of cells to monitor metabolic products
during cell growth,24 e.g. screening of toxins by ISE. This is achieved by integrating cells with a
K+ selective film wherein a change in potential caused by the ion accumulation or depletion
on the electrode can be measured. This method requires a very stable reference electrode
which limits the application of potentiometry sensors.
Amperometric methods are widely used for the determination of pH, dissolved oxygen or
glucose as a measure of cellular biochemical changes. The application of this approach in HTS
cell based assays is limited due to many uncontrollable environmental factors.
 Cell-Based Assays in High-Throughput Screening of Drugs 703

Method based on Cellular Barrier

In general, cells with insulating properties would significantly increase the electrode
impedance.25,26 This has been exploited to study the biological status of cells including cellular
viability, morphology, cell number, cell apoptosis and cell adhesion using electrochemical
impedance spectroscopic techniques, e.g. A novel electrical impedance sensor array integrated
into the bottom of a microtiter plate has been specially designed for the quantitative detection
of living cells. Real-time assessment of cytotoxicity and acute toxicity can be achieved using
this device.26
Method based on the Recording/Stimulating of cellular electrical potential
Electrogenic cells and tissues such as heart, muscle, pancreas beta and nerve cells are capable
of generating bioelectrical signals when they are grown in culture on microelectrode arrays
or on field effect transistors in response to any neuroactive compounds added to the culture
medium. Such signals can be used to test drugs against critical diseases such as cardiac
arrhythmia, hypertension, Parkinson’s disease, diabetes, depression and neuropathic pain.27 A
nanoelectronic biosensor was developed based on single-wall carbon nanotubes (SWCNTs) to
measure non-invasive detection of cellular activities of electrogenic cells with high throughput,
high sensitivity, easy handling and the capacity of long-term cell culture.
Disadvantages of Electrochemical Methods of Detection
•• No specific information can be obtained on cellular activities directly related to certain cell
functions, biomarkers or signalling pathways which are essential for better understanding
of mechanisms of cytotoxicity and action of drugs.
•• Not suitable for 3D cell cultures which requires cell direct contact with the electrode.

Optical Methods
The optical methods of detection include, colorimetric, luminescent or fluorescent methods.

Colorimetric Method
The colorimetric method of detection relies on the color change of the growth medium after
cell metabolites react with chemical agents, e.g. Assays using ruthenium dye28 and alamar
blue have been developed.29 These types of assays are basically employed to study the effect of
test drugs on cell proliferation or cellular viability. However, ruthenium dye and alamar blue
(Resazurin) (Fig. 45.4A) are not being used in HTS measurements because of its low sensitivity
and reproducibility.

Figure 45.4A: Principle of cell viability/toxicity assay using alamar blue

704 Drug Screening Methods

A spectrum of assays using tetrazolium salts such as MTT [3-(4,5-Dimethylthiazole-2-yl)-2,5-

diphenyltetrazolium bromide] and the variants of this assay using MTS [3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and XTT [2,3-bis-(2-
methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) are also available commercially
(Fig. 45.4B).

Figure 45.4B: Principle of cell viability/toxicity assay using tetrazolium salt (MTT)

Limitations
•• Multiple additions of chemicals at pre-scheduled time points which interfere or disrupt the
targeted cells
•• Time consuming and laborious
•• Insufficient to obtain dynamic data which can provide more information about the effects
of drugs on cells
•• Requires expensive robotic arms for HTS application
•• Low sensitivity.

Luminescent Method
This assay is based on the principle that the oxidation of luciferin catalyzed by luciferase
produces light that is detected by an illuminometer or optical microscope, allowing observation
of biological processes.30 This reaction may be mediated by ATP or calcium ions, e.g. Luciferase
assays for kinase activity in which luciferase is widely used as a reporter in cells expressing a
luciferase gene under the control of a promoter if interest to assess its transcriptional activity.31
Limitations
•• Its application is limited to end-point assay
•• Requires cell lysis and addition of luciferase substrate
•• Luminescent intensity is affected by many factors, such as luciferin absorption, availability
of co-factors, pH and transparency of culture media or buffer.

Fluorescent Method
The fluorescent assays are very sensitive as compared to luminescent assays, and very easy to be
miniaturized for HTS applications and thus enable the measurement of cell activities, pathway
 Cell-Based Assays in High-Throughput Screening of Drugs 705

activation, toxicity and phenotypic cellular responses of exogenous stimuli. The fluorescent
methods for cell based assays were initially developed using small, highly-fluorescent, organic
molecules to monitor ion concentrations, membrane potential and as intracellular substrates
for reporter genes. These conventional fluorescent molecules have been replaced with photo-
unstable quantum dots (QDs). QDs are semiconductor nanocrystals and are photo-chemically
stable, provide narrow and adjustable emission. These QDs can be excited by light of any
wavelength shorter than that of the emission peak.32
For example, two fluorescent proteins fused with a peptide linker comprising a caspase-3-
cleavage site is being used to study the activation of caspase-3 or apoptosis in live cells.33

RECENT DEVELOPMENTS IN 3D CELL-BASED HTS ASSAYS FOR DRUG

DISCOVERY
3D Cell-Based Fluorescence Assays
This assay enables the real-time analysis of cell proliferation based on fluorescence read-
outs from a fluorometer and the fluorescence signals are generated from cells cultured on
conventional 96-well plates. The lack of sensitivity and accuracy, high fluctuating background
signals mediated by change in pH, culture environment and other autofluorescent components
in the medium render it unreliable for assessing cytotoxicity or cell proliferation. These
limitations can be overcome by culturing GFP-expressing cells in a PET scaffold in a modified
well which significantly reduces background noises and increases the total cell number per
unit area. Such a 3D culture significantly improves a background noise and provides a 20-
fold higher cellular fluorescence. This kind of assays has been successfully employed to study
cytotoxicity effects of chemicals, cancer drugs and Chinese herbal medicines in early-stage
drug discovery process.1

Microfluidic Cell-Based Assays

Microfluidics refers to the science and technology that allows one to manipulate tiny amounts
(10-9 to 10-6 liter) of fluids using microstructures with characteristic dimensions on the order
of tens to hundreds of micrometers. This technology has been emerged as a promising tool. Its
unique design paves the way to create a more in vivo–like cellular microenvironment in vitro. It
can be fully automated for HTS assays with improved data quality, reduced assay time and cost.
A digital microfluidics (DMF) has emerged as an alternative to conventional format of
enclosed micro-channels in which nano-liter sized droplets are manipulated on an open
surface of an array of electrodes, e.g. the first time lab-on-a-chip platform has been developed
by Barbulovic-Nad et al. (2010)34 which is capable of implementing all steps required for
complete mammalian cell culture. This is a very promising technique for improvising HTS via
well-controlled fluid handling without the need for complex robotics.

CELL-BASED HTS IN COMMERCIAL DRUG DEVELOPMENT

The global market for cell-based assays in drug discovery was estimated as $6.2 billion in
2010 and it is expected to increase at an annual growth rate of 11.6% to nearly $10.2 billion
in 2015.1 There has been a growing interest in drug discovery to use cell-based assays for lead
706 Drug Screening Methods

identification and optimization since they provide more relevant physiological information
than biochemical assays. Hence, now it becomes an integral part of successful drug discovery
processes. The successful examples of cell based HTS in commercial drug discovery is given
in Table 45.1.
Table 45.1: Examples of cell-based HTS in commercial drug discovery

Name of the drug Indication Target class Type of assay Year FDA Reference
(US trade name; employed approval
Company)
Eltrombopag Thrombocytopenia Cytokine Cell-Based 2008 Duffy et al.,
(Promacta; receptor Luciferase 200135
GlaxoSmithKline) reporter assay
BMS-790052 Hepatitis C HCV-NS5A Cell-based Phase III Lee C, 201136
(Daclastavir;Bristol- replicon
Myers Squibb) screening
method
Bortezomib Myeloma Protease Cell-Based assays 2003 Paull
(Velcade; Millinium using NC160 KD,198937
Pharmaceuticals)

CONCLUSION AND PERSPECTIVES

The advancement of combinatorial chemistry, genomics, proteomics and bioinformatics
necessitate the development of a very rapid screening strategy for lead identification
and optimization. Conventional animal models become obscure due to its high cost, low
throughput and moral issues. Hence, in order to meet the high screening attrition rate in lead
identification and optimization, HTS assays have been emerged as a potential platform in early
drug development program. Today, more than half of the HTS assays are replaced with cell-
based assays. However, the conventional 2D static cell culture systems have many limitations
which restrict its use in cell proliferation and cytotoxicity studies. This can be overcome by the
development of cell-based 3D culture with fluorescent detection. Such system is fast, sensitive
and physiologically more relevant which can be used to bridge the gap between biochemical
assays and animal tests. The recent advances in 3D cell culture with microfluidic technology
coupled with stable fluorescent probes offer a great advantage of long-term study of drugs in
an in vivo like 3D environment and flow fields. This would enable more effective screening and
exploration of new drugs for their health benefits.

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index

Page numbers followed by f refer to figure and t refer to table

A Aluminium hydroxide gel model 423

Abciximab 334 Alzheimer’s disease 25, 29, 39, 45, 48, 436, 445
Acetic acid-induced gastric ulcers 533 Alzheimer’s mouse models 444
Amnesic agents 439t
Acetylcholine-induced arrhythmia 295
Amphetamine potentiation 408
Aconitine antagonism in rats 295
Amphetamine-induced stereotypy in rats 396
Acquired immunodeficiency syndrome 88
Amyloid precursor protein 436
Acrosomal status 148
Amyotrophic lateral sclerosis 50, 260
Adenocarcinoma 179
Anaphylactic microshock in guinea pigs 688
Adenoma 179
Androgens 597
Adenosine diphosphate receptor 335
method of 598
Adherent culture system 700f
Angiogenesis 104, 114, 184
Adhesion assays 500
Animal models for
Adipocytes (fat cells) 631, 698 acute pain 477
Adipose tissue 42 chronic pain 483
cell 161 female sexual dysfunction 128
size and number 153 study of antiobesity drugs 152
Adjuvant arthritis 505 various viral infections 387t
Adrenal ascorbic acid, depletion of 579 Animal models, choice of 547
Adrenal gland atrophy 595 Anti-Alzheimer agents 436
Adrenal gland, preparations of 580 Anti-androgenic activity 149
Adrenal steroids 591 Antianginal agents 321
Adrenalectomy in rats 594 Antiarrhythmic agents 292
Adrenaline-induced arrhythmia 296 Anti-asthmatic agents 683
Adriamycin 193 Anticancer agents 170
Adriamycin-induced nephropathy 195 Anticataract agents 226
Adult neural stem/progenitor cells 42, 44, 56f Anticholinesterase, activity 365
Agarose gel electrophoresis 213 Anticholinesterases, effect of 376
Air pouch model 505 Antidepressant activity in mice 407f
Air-jet stimulation-induced hypertension 271 Antidepressant agents 404
Airway inflammation in mice 691 Antidiabetic agents 613
Akimba mice model 654 Antidiuretic activity 585
Alkaline phosphatase assay 605 Antiemetic agents 546
Alkaloid morphine 34 Antiepileptics 412
Allergic rhinitis 495 Anti-estrogenic activity 139
Alloxan-induced Antifertility agents 132
cataract 234 Antiglaucoma agents 278
diabetes 614 Antigoitrogenic activity 587
Alpha chymotrypsin-induced experimental Anti-HIV agents 88
glaucoma 281 Antihypertensive agents 266
Alpha-smooth muscle actin 194 Anti-implantation activity 144
710 Drug Screening Methods

Anti-inflammatory agents 495 Benzopyrene-induced forestomach tumors in

Antimigraine agents 468 mouse 183
Antiobesity agents 152 Beta-cell mass, factors regulating 614f
Antiparkinsonian agents 453 Beta-glucuronidase activity
Antiplatelet agents 334 change in 600
Antiplatelet drugs 335 determination of 600
classification of 334t Bile duct ligation 201
Antiplatelet efficacy in Bilobalide 449
rhesus monkeys 343 Biogenic amine hypothesis 404
Anti-progestational activity 142 Blepharitis 514
in immature rabbits 142 Blood brain barrier models 98
Antipsychotic agents 393 Blood pressure in
Antisense oligoneucleotides 220 conscious rats 273
Antistroke agents 71 pithed rats 269
Antiulcer agents 527 Blood retinal barrier 648
Antiulcer antibiotics, development of 536 Blood sugar monitoring 651
Antiviral activity of drug 384 Bone marrow derived stem cells 56
Antiviral drugs 380 Borderline hypertensive rats 271
Apnea in mouse, reversal of 372 Borna disease virus 155
Apomorphine antagonism 408 Bovine corneal cup model 667
Apomorphine-induced Bovine corneal opacity-permeability test 663
dog model 551 Bovine coronary artery 331
emesis model 551 Bovine retinal endothelial cells 648
primary cell cultures of 648
ferret model 551
Brain cells 413
rat model 551
Brain derived neurotrophic factors 49, 56
stereotypy in rats 397
Brain glutathione level 80
Apoptosis, detection of 209, 210, 221
Brain injury 393
Arachidonic acid 687
Brattleboro strain 585
Aromatase inhibition 140
Bronchial hyperresponsiveness 683
Arteriogenic impotence 124
Brown adipose tissue 162
Ascorbic acid determination 579
Budesonide 683
Aspartate transaminase levels 199
Aspirin 334 C
Asthma 256, 495, 683
Atherosclerotic impotence 124 C2C12 cells 630
Caenorhabditis elegans 30
Atomic absorption spectroscopy 9
Calcitonin gene-related peptide 468
Audiogenic seizure susceptible mice 425
Calcium-chelating agent 564
Autoimmune glaucoma 287
Calcium-induced arrhythmia 297
Autoimmune uveitis 518
Callithrix jacchus 462
Autonomic nervous system 346
Camp release 590
Azoxymethane-induced aberrant crypt
Cancer 39
foci in rat 181
pain 486
stem cells 698
B
tissue arrays 106
Ba/F3-hGHR cell line 573 types of 170
Bacteria-induced endophthalmitis 522 Candida albicans 522
Basic fibroblast growth factor 56 induced endophthalmitis 522
BC3H1 myocytes 630 Canine distemper virus 155
Benzenesulfanylamino 619 Cannabinoids activity 489
 Index 711

Carbon tetrachloride Cell transfection 7

acute models with 199 Cell viability/toxicity assay alamar blue 703f
chronic models with 199 Cell viability/toxicity assay tetrazolium salt 704f
Carboxymethylcellulose 605 Cell-based assays, types of 697
suspension 543 Cell-based HTS assays, components of 698
Cardiac arrest, dose causing 372 Cells, primary 698
Cardiac insufficiency 314 Cells, types of 698
Cardiomyocytes 54 Cellular barrier 703
Cardiomyopathic disease 315 CEM cells 96
Cardiotonic agents 305 Central nervous system 29
Carotid arteriovenous anastomoses in Cerebral cortex homogenate 528
anesthetized animals 471 Cerebral ischemia 55t, 82
Carotid artery 77
damage 83
occlusion in dogs 340
Cerebral stroke 54
Carrageenan-induced paw edema model 503
Cerebrovascular diseases 334
Carrageenan-induced rat pleurisy 504
Charcoal passage test 543
Cartilage cells 698
Cat model for anticholinesterase activity 361 Chemically-induced arrhythmia 295
Cat papillary muscle 306 Chemically-induced hypothalamic obesity 155
Cat spleen model 347, 351 Chemoreceptor trigger zone 546
Cat splenic strip model 353 Chick
Catalepsy in rodents 396 chorioallantoic membrane 113
Cataract oviduct method 606
enzymatically-induced 231 Chicken blood pressure 583
experimental models of 226 Chicken comb method 148, 149, 599
induction of 235 Chicken eye (ice) test, isolated 663
mechanical stimulation and 239 Cholinergic agonists 279
surgery complication 228 Cholinomimetics-induced parkinsonism 459
Cataractogenesis 227f Cilostazol 334
Catatonia, assessment of 458 Cisplatin-induced
Cavernous nerve injury model of erectile cat model 550
dysfunction 126 dog model 549
Cavia porcellus 389 emesis model 549
Cell counting assay 175 ferret model 550
Cell culture 605 pigeon model 550
method 684 rat model 550
models 561, 562 suncus murinus model 550
systems 382 Clauberg McPhail test 140, 601
for HBV 383 Clonogenic assays 175
for HCV 383
Clonogenic properties 172
for influenza viruses 383
Clopidogrel 334
types of 699
Cohabitation test 145
technique 54, 292
Cohen diabetic rat 618
Cell division 172
Cell fractionation 214 Colchicine 439, 440
Cell free assays 11 Cold ethanol tail-flick test 479
Cell growth, inhibition of 593 Cold water immersion-induced ulcers 530
Cell line of retinal capillary endothelial cells 649 Collagen-induced arthritis 506
Cell necrosis and apoptosis 427 Colonic motility studies in rats 544
Cell proliferation bioassays 572 Congestive heart failure 53, 305
Cell proliferation/cytotoxicity assays 698 Conjunctiva 660
712 Drug Screening Methods

Copper sulfate-induced Degenerative disease, chronic 43

cat model 552 Dendritic cells 30
chick model 552 Dengue virus 387
dog model 552 Dexamethasone 594, 616
emesis model 551 Dextran-coated charcoal adsorption technique
ferret model 552 138
suncus murinus model 552 Diabetes 39, 618
Cornea 660 chemically-induced 614, 619
Corneal culture assay 665 induced by viral agents 616
Corneal electroshock kindling 421 mellitus 121, 127, 254, 645
Corneal micropocket model 111 mouse 158
Cornified epithelial cells 136f, 137f types of 613
and leukocytes 136f
Diabetic cardiomyopathy 634
Cornified epithelial sperm 137f
Diabetic complications 634
Coronary artery disease 634
Diabetic DB/DB mice 621
Coronary artery ligation 311, 324
Diabetic macular edema 647
Coronary artery, occlusion of 325
Coronary flow measurement 328 Diabetic maculopathy 646f
Coronary ligation in dogs 300 Diabetic nephropathy 636, 638
Coronary thrombosis 327 Diabetic neuropathy model 485
Corpus cavernosum 123 Diabetic rats, care of 235
Cortically implanted metals 423 Diabetic retinopathy 645, 646f
Corticosterone level, in blood 580 in galactose fed rat model 652
Corticotropin 577 diabetic rats 650
Corticotropin-releasing hormone 152 prevention of 648t
Cotton pellet-induced granuloma 505 therapy for 647
Coulter counting 9 Diabetogenic compounds 618
Cricetulus griseus 622 Dialysis probe 15
Croton oil-induced uveitis 520 Diet-induced metabolic dysregulation 625
Croton tiglium 503 Diet-induced obesity 154
Cryopreserved embryos 40 Dietary hypertension 269
Cryptotis parva 547, 548 Digoxin-induced
Ctenomys talarum 623 arrhythmia in guinea pigs 296
Cultex technique 684 duodenal ulcers 534
Cultured brain microvascular endothelial cells 98 Dimethylnitrosamine 202
Culturing cells, devices for 699 Dipyridamole 334
Cupric acetate-induced ovulation in rabbits 133 Dithizone 618
Cyclooxygenase assays 501 Djungarian (siberian) hamster 622
Cyclophosphamide-induced
DMBA sustained release suture technique 182
cystitis in rodents 507
DMBA-induced
inflammation in rat bladder 508f
mouse skin papillomas 178
Cysteamine-induced duodenal ulcers 534
Cystometry in rats 363 oral cancer in hamster 181
Cytochemical bioassay 591 rat mammary gland carcinogenesis 179
Cytokine expression in murine macrophages 499 DNA fragmentation, detection of 213, 214
Cytomegalovirus 387 DNA staining 217
Cytosol, preparation of 601, 604 DNA technology 261
Cytotoxicity tests 665 Dog model 626
of heart failure 310
D of hypertension 273
Dahl salt sensitive rats 308 Dopamine function and psychosis 401
Deciduoma reaction, in rats 141 Dopamine stimulated adenylyl cyclase activity 456
 Index 713

Dose response curve 173f Eye irritation test 660t

Doxorubicin cardiomyopathy 313 Eye of rodents 365
Drug discovery program 3 Eye test, low-volume 661
Drug screening, target for 77 EyetexTM assay 664
Drug treatment on cells 288
Drug-induced emesis models 549 F
Drugs acting on Facial nerve stimulation 372
parasympathetic nervous system 360 Fat mouse 158, 620
sympathetic nervous system 346 Fatty liver disease 198
Dulbecco’s modified eagle’s medium 570 Female sexual dysfunction 122
Dulbecco’s phosphate buffered saline 285, 564 Female Sprague Dawley rats 534, 603
Dye exclusion tests 174 Femtosecond laser ablation 445
Fertility test 145
E Fetal bovine serum 498, 605
Eagle’s minimum essential medium 605 Fetal calf serum 98
Earle’s balanced salt solution 565 Fetal neural stem/progenitor cells 41, 44
Ebola virus 387 Fetal phrenic nerve-diaphragm preparation 376
Electric shock 406 FGF-2-fibroblast growth factor-2 47
Electrically-induced arrhythmia 297 Fibroadenoma 179
Electrolyte excretion 596 Field-inversion gel electrophoresis 215
Electrospray ionization 25 First strand cDNA preparations 6
Embolic models of focal cerebral ischemia 76 Fixation and cytoskeletal staining 288
Embryonic carcinoma cells 44 Fixed platelets, preparation of 338
Embryonic stem cells 40, 44, 49, 699 Flow cytometry 24, 216
Endocrine cells 613 Fluorescein angiography 651
Endocrine hypertension 270 Fluorescence activated cell sorting 9, 10
Endometrial carbonic anhydrase 602 Fluorescence microscopy 211
Endophthalmitis 521 Fluorescence-type bulbs 677
Endothelial cells 121f, 192 Fluorescent techniques 23t
Endothelial dysfunction 637 Fluoro-Jade B staining 428
Endotoxin-induced uveitis 516 Focal ischemia 55, 75
Enos deficient mice 637 Forkhead transcription factor 170
Enterochromaffin-like cells 527 Formalin test 480
Enzyme aldose reductase 231 Freund’s adjuvant 515
Enzyme assays 220 Fructose-induced hypertension in rats 269
Enzyme-linked immunosorbent assays (ELISAs) Fructose-2,6-bisphosphate production in rat
262, 573 hepatocytes 628
Eosinophillic cell 595 Fusarium solani-induced endophthalmitis 522
count 595
Epilepsy, pathophysiology of 412 G
EpiOcularTM assay 664 GABA receptor-binding assay 414
Epithelial cells 135 Gabaergic compounds 414
Epithelial mesenchymal transition 192 Galactose-induced cataract 233
Eptifibatide 334 Gallus gallus domesticus 30
Erectile dysfunction, complication of 128 Gamma rays-induced cataract 239
Estrogens 604 Gastric mucosal injury 535
Estrus cycle in rats 135 Gastrin binding assay 527
Ethacrynic acid 481 Gastrointestinal motor activity in conscious dogs
Ethanol-induced mucosal damage 532 544
Excitatory amino acid receptor-binding assays 416 Gel-filtered platelets, preparation of 338
Exercise-induced ventricular fibrillation 298 Gene targeting 624
Gene therapy 316 H
Genetic animal models of epilepsy 425 Haffner’s tail clip method 482
Genetic arrhythmia 301
Hairless mouse model 674
Genetic hypertension 271
advantages 675
Genetic models 115, 287, 399, 618
disadvantages 675
of NIDDM 619
Hamster cardiomyopathic heart 306
of obesity 156
Hamster cheek pouch model 111
Genetically epilepsy-prone rats 426
Hand muscles, capacity of 375
Genetically humanized mice 388
Genetically induced arrhythmia 301 Heart attack 53
Genetically modified mice 128 Heart diseases 39
Genetically seizure-prone animals 427 Heart-lung preparation 324
Gestational malnutrition model 399 Hematopoietic stem cells 39
Glaucoma 278 Hen’s egg test 679, 680t
experimental models of 280 chorioallantoic membrane test 667
Global ischemia 55 Hepatic cell 203
Glomerulonephritis 191 types 204
anti-Thy1 antibody-induced 197 Hepatic stellate cells, role of 192
Glucocorticoid-induced cataract in developing Hepatitis B and C 387
chick embryo 237 viruses 192, 380
Glucocorticoids 592 Hepatitis B virus 258
Glucose transport 632 Hepatitis C virus 259
in adipocytes 632 Hepatocellular carcinoma 183
Glucose uptake inhibition 570 Hepatocytes 564
Glucose, conversion of 570 Herbal medicines 246
Glucose-induced diabetic model 655 Hereditary cataract model 239
Glutathione 80 Hereditary model, in glucoma 585
Glycine-binding assay 418 Herpes simplex virus 380
Glycogen synthase activity 633 High-fat diet-induced metabolic syndrome 128
Glycogen synthesis 632 Hind limb in rats 629
Gold fish 29 Hippocampal perforant pathway 424
Goldblatt hypertension 267 Histamine receptor assay 684
Goldman applanation tonometer 281f Histamine-induced
Graft-versus-host disease 91 bronchoconstriction in anesthetized guinea
Granulocyte colony stimulating factor 56 pigs 689
Granulosa cell culture for effects of FSH and LH 582 gastric ulcers 531
Gravid rat uterus model 357 Histidine photodegradation 681
Green fluorescent protein 26, 112 Histocompatibility complex 91
Growth hormone 569 HIV in CNS, systems for 98
GTPCH-1(guanidine triphosphate cyclohydro­
HIV transmission 94
lase-1) 47
HLA-A29 mice 652
Guinea pig
H-naloxone binding assay 487
bronchospasm model 363
Hollow-fiber solid tumor model 110
dorsal surface 673, 675
Hollow-fiber technique 185
ileum 364, 541, 585
Hormone responsive elements 592
isolated heart 354, 367
lungs, spasmolytic activity in 685 Hormone-induced diabetes mellitus 616
model 314 Hormones of
of giant papillary conjunctivitis 515 adrenal cortex 569
papillary muscle 293, 294 hypothalamus and
trachea 366, 686 anterior pituitary 570f
tracheal chain model 354 posterior pituitary 571f
 Index 715

ovary 569 Hypoxia-induced memory deficit 443

parathyroid 569 Hypoxia-stimulated porcine aortae 501
pituitary 569
testes 569 I
thyroid 569 Iboteic acid 439, 440
Hot plate method 477, 477f Immortalized cell lines 698
HTS Immune-based disorders 254
absorption studies 31 Immune-mediated diseases 193
hepatotoxicity 33 Immunoassays 573
metabolism studies 32 Immunoelectrophoresis 261
pharmacokinetic studies 31 Immunofunctional assay 573, 574
roles of 35 Immunoinflammatory disorders of central
HTS-cytochrome inhibition 32 nervous system 257
HTS-scintillation methods in drug screening 28 Indomethacin-induced gastric ulcers 533
HUH-7 cells 382 Inert gas technique 330
Human adenovirus 155 Infantile spasms 424
Human adipogenesis 165 Inflammation of cornea 514
Human cancer cell lines 698 Inflammation, models of 502t
Human capsular bag model 228 Inflammatory bowel disease 495
Human chorionic gonadotropin 132 Inflammatory gene expression 500
coronary artery 470 Inflammatory tests 666
embryonic stem cells 98 Inflammatory uterine pain model 482
Human growth hormone bioassays 572 Influenza viruses 387
heart failure 310 Insulin antibodies-induced diabetes 616
immune system 254 Insulin-dependent diabetes mellitus 613
Human immunodeficiency virus 259 Insulin-dependent diabetes mellitus, models for
isolated middle meningeal artery 470 614
Insulin or insulin-like substances on adipocytes
Human lens epithelial cell 228
630
culture 227
Insulin receptor-binding assays 633
Human leukocyte antigen 259
Insulin secreting cell lines 629
Human lymphocytes 96
Intercostal nerve-muscle preparation 376
Human mesenchymal stem cells 165
Interphotoreceptor retinoid-binding protein-
Human PBMC cultures 97 induced uveitis 518
Human retina 646f Interstitial cystitis 495
Human retinal pigmented epithelium cell 649 Intestinal spasmolytic activity in mice 362
Human T cell leukemia virus 97, 260 Intracellular cell adhesion molecule 496
Human trabecular meshwork culture 288 Invertebrate animal model 625
Human-cord blood leukocyte-neonatal 93 Iodine release inhibition 587
Humanized transgenic mice 94 Ipratropium bromide 683
Humoral cells 24 Iris 660
Huntington’s disease 42, 48, 49f Ischemic hind limb model 107
Hyaluronic acid 283 Isoprenaline 351
induced glaucoma 283 Isoproterenol-induced myocardial necrosis 326
Hydrated aluminum silicate 549
Hydrogen peroxide-induced cataract 231 J
Hyperbaric oxygen-induced cataract 237 Japanese cedar pollen 516
Hyperglycemia, induction of 651 Japanese encephalitis virus 387
Hyperglycemia, pathophysiology of 613f
Hypokinesia, assessment of 458 K
Hypo-osmotic swelling test 147 Kainic acid 439, 440
Hypothalamic obesity 154 Keratitis 514
716 Drug Screening Methods

Kidney failure 634 Luteinizing hormone-releasing hormone 603

Kimba mouse model 653 Lymphoblastoid leukemia cell line 96
Kindled rat seizure model 420 Lymphocytic choriomeningitis virus 617
Klebsiella oxytoca 523 Lymphocytic leukemia, chronic 174
Knockin mouse models 473
Koletsky rat strain 621 M
Kreb’s bicarbonate 322 Macaca fascicularis 410, 462
Kreb’s buffer 324 Macaca nemestrina 90
Kreb’s solution 322 Maccacus rhesus 565
Kreb’s-Ringer bicarbonate 582 Madin-Darby canine kidney cells 666
Krebs-Henseleit bicarbonate saline 355 Male erectile dysfunction 121, 122
Krebs-Henseleit solution 266 causes of 122f
Krebs-Ringer bicarbonate buffer 564 Male guinea pigs 314
Kruskal-Wallis test 324 Male rat model
Kupffer cells 192 for LH activity 581
immature 581
L mature 581
L6 muscle cells 630 Male Sprague-Dawley rats 533, 544, 595
Lactation, in rabbits 577 Male wistar rats 353, 356, 469
Langendorff heart preparation 321, 331 Marburg viruses 387
Langendorff technique 294, 324 Mast cell 667
Laparotomy, procedure for 144 degranulation 499
Laser treatment in pigmented rabbit eyes 521f Matrigel implant model 109f
Laser-induced glaucoma in Matrigel plug 108
monkeys 285 Maximal electroshock seizure test 419
mouse 286 Maze 447
rats 285 McGinty test 142
Laser-induced ocular inflammation 520 Melanin-induced uveitis 519
Lasers, application of 285 Memantine 449
Left ventricular end diastolic pressure 313, 326 Mepirizole-induced duodenal ulcers 535
Leptin in obesity, role of 163 Mesenchymal stem cells 42, 56f, 698
Levator ani 148 Mesocricetus auratus 389
Ligand-binding assay 578 Metabolic syndrome 122
Lipid peroxidation 509 Metabolism studies 563
Lipid proton MR spectroscopy 221 Metatarsal cytochemical assay 591
Lipid synthesis in isolated adipocytes 631 Methotrexate-induced delayed emesis model 550
Lipogenic enzyme assay 586 Methylcellulose induced glaucoma 283
Lithium-methomyl induced seizures in rats 424 Methylene blue-induced ulcers 531
Lithium-pilocarpine induced status epilepticus Mice metabolic stimulation model 356
424 Mice tremor and salivation model 364
Liver cirrhosis, pathogenesis of 192 Microbe-based screening assays 11
Liver disease 198 Microchannel array flow analyzer 339
Liver fibrosis 198, 202f Microculture tetrazolium test or MTT 172
Liver glycogen test 595 Microdialysis of acetylcholine release 449
Liver slice system 204 Microdialysis technique 14, 17
local ischemia-reperfusion in rats 535 Microfluidic cell-based assays 705
Local toxicity test 250 Microspheres-induced acute ischemia 326
Long-term toxicity test 249 Microvascular endothelial cells 99
Lou Gehrig’s disease 50, 260 Microwave-induced cataract 239
Lumbrical nerve-muscle preparation in rabbits Midbrain dopaminergic neurons 400
374 Middle cerebral artery occlusion 76
 Index 717

Migraine 468 Neural progenitor cells 45

Milk ejection method 583 Neurodegenerative diseases 442
Mineralocorticoids 596 Neurodegenerative disorders 209
Mongolian gerbils 427 Neurodevelopmental models 398
Monkey shock titration test 480 Neurogenesis 429
Monoamine oxidase 462 Neurogenic dural vasodilatation in anesthetized
Monocrotaline-induced pulmonary hypertension animals 472
267 Neurogenic hypertension 269, 274
Monogenic and polygenic models of obesity 156t Neurogenic plasma extravasation model 472
Monogenic models of obesity and NIDDM 619 Neuroinflammation markers 429
Monosodium glutamate-induced hypothalamic Neuroleptics-induced parkinsonism 458
obesity in mice 155 Neuromuscular blockers in
Morris water maze 448 animals 369t
Mossy fiber sprouting 429 human 370t
Motion-induced Neuromuscular blocking agents 369
cat model 552 Neurons of substantia nigra 453
emesis model 552 Neurons, groups of 346
rat model 553 Neuropathic pain models 483
suncus murinus model 553 Neuroprotective efficacy 457
Motor-based intracellular transport 442 Neutrophil adhesion 501
Mouse ear swelling model 673 New drug development 20
Mouse eye model 349 Newborn rat model 186
Mucosal cells 559 Newer drugs, targets for 335
Multiple drug resistance 177 NIDDM models, chemically-induced 619
Multiple fluorochrome comet assay 7 Nimodipine 449
Multiple sclerosis 495 Nitric oxide 122, 509
Multiprobe ribonuclease protection assay 8 Nitrogen retention 149
Murine fibroblasts cells 570 test 600
Murine macrophage model 357 Nociceptin receptor 490
Muscarinic receptors 360t Noncontact tonometer 281f
Muscimol-binding assay 415 Nondiabetic models of proliferative retinopathy
Muscle 629 652
cell lines 630 Nonhuman primate models 88, 387
Muscular rigidity, assessment of 458 Noninsulin-dependent diabetes mellitus 614
Mycoplasma 256 Non-mammalian models 113
Mydriasis test 362 Normoglycemic animal models 625
Myelin basic protein 77 NPC (neural progenitor cells) 47
induced anterior uveitis 519 Nucleated epithelial cells 136
Myocardial infarction 53 Nucleus basalis of Meynert lesion 48
Mystromys albicandatus 622
O
N Obesity 152
Nagoya-Shibata-Yasuda mouse 622 mutations in rodents and mutant proteins 156t
Nano screens 12 Obstructive pulmonary disease, chronic 495
Naphthalene-induced cataract 232 Ocular inflammation 514, 515
Nausea 546 Ocular injections 280
and vomiting, pathophysiology of 547f Ocular lesions, values for 660t
Neodymium-yttrium aluminum garnet laser 521 Ocular toxicity 659
Neonatal STZ model of NIDDM 619 Oedematous ganglion cells 518
Nerve growth factor 49 Olfactory bulbectomy 409
Nerve-muscle preparations of lower limbs 371 Opacity tests 662
718 Drug Screening Methods

Organ culture 229 Phrenic nerve-diaphragm preparation 372, 373

Osteoarthritis 495 Pigeon crop method 577
Osteoblasts (bone cells) 698 Pigtailed macaques 90
Otsuka-long-evans-tokushima fatty rats 160, 622 Pilocarpine-induced status epilepticus 424
Oxidative stress induced experimental cataract Piracetam 449
230 Pithed rat model 350
Oxidative stress measurement 79 Pituitary adenylate cyclase-activating peptide 490
Oxygen species, reactive 269, 498 Pituitary tumor-transforming gene-1 170
Oxytocins 582 Plasma leptin 164
Plasma membrane integrity, assessment of 147
P Plastic casts technique in dogs 325
PAF-induced respiratory dysfunction 687 Platelet adhesion 338
Pan troglodytes 89, 386 Platelet function analyzer 339
Pan uveitis 514 Platelet neutrophil adhesion 500
Pancreas 613 Platelet poor plasma 344
of rat, isolated 627 Platelet rich plasma 337
Pancreatic cancer models 184 Pluripotent stem cells 699
Papaya latex-induced arthritis 506 Pneumotachography in guinea pigs 689
Papio cynocephalus 90 Podocyte specific insulin receptor knockout
Parallel artificial membrane permeability assay 31 mouse 637
Parasympathetic agonists and antagonists 361f Polygenic models 159
Parasympathetic nervous system 360 Polygenic models of obesity and NIDDM 621
Parathyroid hormone 588 Polymerase chain reaction 262
Parkinson’s disease 42, 43, 453, 463 Porcine intestinal tissue system 561
etiopathology of 454
Posterior uveitis 514
Passive avoidance 446
Post-traumatic arteriogenic erectile dysfunction
Patch clamp technique 12
125
advances in 14
Potassium chloride 356
Patch electrode on cell membrane 13f
Potassium-induced arrhythmia 295
PBB/LD mouse 622
Potential therapeutic target for migraine 473
Pelvic nerve electrical stimulation in female dogs
129 Precision-cut liver slices 565
Pentylenetetrazol 421, 422 Proapoptotic and antiapoptotic agents 209
Peptic ulcer 527 Progesterone binding to receptor, determi­nation
Perfused excised eye model system 284 of 601
Perfused heart 53 Progesterone 600
Peripheral vein 325 receptor-binding assay 141
Permanent loss of vision 514 Programmed cell death 209
Peroxisome proliferator-activated receptors 437 Programmed electrical stimulation-induced
Persistent post-thoracotomy pain model 485 arrhythmia 297
Petit mal epilepsy 426 Prolactin 576
Pharmacovigilance of herbal drugs 252 Proliferative retinopathy 646f
Phencyclidine 397 diabetic 650
Phodopus sungorus 622 Prostate cancer in gerbils 180
Photo-basophil-histamine release test 681 Prostatitis 495
Photochemically-induced cataract 231 Protein overload rat model 196
Photodegranulation of mast cells 681 Pseudomonas aeruginosa 523
Photosensitive baboons 425 Psychical model of erectile dysfunction 127
Photosensitized Candida albicans growth Psychogenic hypertension 271
inhibition 681 Pulmonary resistance, calculation of 689
Phototoxicity 671, 671t Pylorus ligation in rats 529
 Index 719

Q of heart failure 307

Quantitation of DNA fragments 214 of hypertension 267
of incisional pain 486
of prostatitis 507
R
of suboptimal nutrition 575
Rabbit aorta preparation 322, 331 ovulation inhibition model 603
Rabbit dorsal surface method 672 pancreatic islets, isolated 627
Rabbit ear model 674 preadipocytes 164
Rabbit eye, isolated 662 primary cultured mature adipocytes 164
Rabbit isolated corpus cavernosum 367 rotational behavior in 461
Rabbit isolated saphenous vein 470 seminal vesicle model 353
Rabbit model 625 sigmoid colon model 482
of heart failure 312 striatal slices 455
of Staphylococcus aureus keratitis 516 submaxillary tissue model 355
Rabbit pulmonary artery model 351 thoracic esophageal muscularis mucosae 542
Rabbit-anti-rat-prolactin 576 uterine C3 model 603
Radiation-induced vaginal smear 136f, 137f
dog model 553 of mated animal 137f
emesis model 553 vas deferens model 352
ferret model 553 Receptor screens 12
rat model 553 Receptor-binding assay 578, 596, 597, 601
Radioimmunoassay 262 Renal and liver fibrosis 191
Radioreceptor assays 572 Renal cytochemical assay 591
Randall Selitto test 483 Renal fibrosis 193
Rapid pacing at heart rates, chronic 310 pathogenesis of 191
Rat Renal hypertension, chronic 273
adipocytes primary culture 631 rats 268
Renal ischemia/reperfusion-induced fibrosis 196
aorta model 355
Renin inhibition in monkeys 274
aortic banding 308
Renin-angiotensin system 267
basal ganglia 464
Reno-vascular hypertension 267
blood pressure 348, 349
Reperfusion arrhythmia in
cardiorespiratory model 361
dogs 300
cerebral cortex 416
rats 299
cerebral cortical and hippocampal neurons
Replicon cell culture model for HCV 381
442 Reporter gene assays 698
coronary ligation model 307 Reserpine-induced
decidualization model 602 chronic ulcers 533
dorsal air sac models 112 hypothermia 408
fundus 542 parkinsonism 457
gut perfusion 562 Resident-intruder paradigm in rats 408
heart 331 Respiratory parameters 376
and uterus model 350 Restraint-induced ulcers 530
isolated aorta 366 Retinal endothelial cell culture 648
jejunal longitudinal muscle model 356 Retinal neural cells 649
liver, isolated 628 Retinal pigmented epithelium cell line 649
model 626 Rheumatoid arthritis 254, 259, 495
of allergic conjunctivitis 515 RNA extraction 6
of bone cancer pain 486 Rous-associated virus-7 155
of glaucoma, chronic 282 Rutin 334
720 Drug Screening Methods

S Sponge implant model 107

Saccharomyces 11 Spontaneous diabetic rodent models 653
Salt-sensitive DAHL rats 271 Spontaneous locomotor activity 460
Sand rat 623 SR 95531 binding assay 415
Sandwich ELISA 262 Stable transfection of hela cells 578
Saphenous vein 325 Status epilepticus 424
SARS corona virus 387 Stem cells 39, 316
Scdia (stromal cell-derived inducing activity) 47 factor 56
Schistosoma mansoni model of ocular in various animal models of neurological
inflammation 520 disorders 44t
Scid-hu mice (THY/LIV model) 91 therapy 39
Scintillation proximity assay 28 for cardio- and cerebrovascular disorders
Sclera, inflammation of 514 53
Scleritis 514 for neurological disorders 43
Scopolamine 439 types of 39
Sebum secretion in rat 598 viz embryonic stem cells 46f
Seizure-prone mice strains 425 Stenosis-induced coronary thrombosis model 327
Seizure-prone rat strains 426 Steroid glaucoma 286
Seizures induced by focal lesions 423 Steroid-induced cataract 232
Selenite-induced cataract 230, 236 Stimulation of cells with ACTH and analogues 578
Seminal vesicles 148 Stimulation of myometrium 583
Serotonin aerosol-induced asphyxia in guinea pig Stimuli, types of 483t
688 Streptozotocin 439, 440
Serotonin receptor type-2C null mutant mouse cataract 235
161 Streptozotocin-induced diabetes 615
Serotonin-induced gastric ulcers 532 Stress and NSAIDs-induced ulcers 530
Serum calcium 589 Stress ulcer models 530
Serum osmolarity 283 Stroke or brain attack 71
Serum phosphate level 590 Stroke, types of 72, 73f
Severe combined immunodeficiency Strophanthin/ouabain-induced arrhythmia 296
disease 256 Subcutaneous PTZ test 422
mice models 91 Subtotal nephrectomy 194
Severe immune disease 94 Sucrose preference test 396
Sex organs, growth of secondary 599 Sudden coronary death model in dogs 299
Sexual dysfunction 121 Sugar cataract models 233
SFM (serumfree medium) 47 Sugar-induced cataract 231
Sheep choroids plexus 96 Sulphorhodamine B assay 173
Shiotz tonometer 281f Suncus murinus 547, 548, 554
Single muscle fiber 375 and rats 552
Single photon emission computed tomography 26 model for anticipatory nausea and vomiting
Sinusoidal endothelial cells 192 554
Slow reacting antirheumatic drugs 506 Superoxide dismutase 80
Smoke-induced cataract 238 Surgically-induced diabetes 617
Smooth muscle of corpora cavernosa 121f disadvantages 617
Social defeat stress, chronic 405 Surgically-induced hypothalamic obesity 154
Sodium deoxycholate 578 Susceptible mouse strain 617, 617t
Sodium sarkosyl 214 Swimming stress ulcers 531
South African hamster 622 Sympathetic agonists and antagonists 347f
Spasmolytic activity 543 Sympathetic nervous system 346
Sperm revival test 147 Syngeneic grafts 106
Spermicidal activity 146 Syngeneic mouse-non-obese diabetic mouse 255f
 Index 721

Systemic drugs 280t Transfected cell lines 441

Systemic focal epileptogenesis 424 Transfections of cells 13
Systemic lupus erythematosus 254 Transforming growth factor β-induced cataract
Systemic penicillin test 423 238
Systolic blood pressure 349 Transgenic and knock-out animals 623
Transgenic animal 48
T models 94
Tachycardia pacing 312 Transgenic mice 94, 315, 388, 444
Tadpole tail culture method 586 Transgenic models of spontaneous retinopathy
Tail cuff method in rats 272 652
Tail-flick apparatus 478f heart failure 309
Tail-flick test 478 spontaneous hypertensive rat 309, 653
using immersion of tail 479 Transgenic mouse model 186
using radiant heat 478 Transgenic or knockout animal models 160
TBARs reactive substances 80 Transgenic rabbits 95
TBPs-binding assay 416 Transgenic rat 95
TCP-binding assay 417 model 343
Tensile strength, reduction in 588 Transgenic zebrafish model 655
Tenuissimus nerve-muscle preparation in cats 374 Translucent models 110
Tetrazolium salt assay 172 Tri-iodothyronine 586
T-helper lymphocytes type 2-induced ocular Trinitrobenzene sulfonic acid-induced
infla­mma­tion 519 experi­mental colitis 509
Thermal cataract 239 Triple fluorochrome analysis 8
Thermal stimulus 477 Triple transgenic mouse model 445
Throat and epigastrium 546 Tubby mouse 158, 158f, 620
Thrombosis model in rats 342 Tubocurarine 369
TH (tyrosine hydroxylase) 47 Tumor angiogenesis 104
Thymus gland atrophy 595 Tumor models 178
Thymus involution 579 Tumor necrosis factor receptor 72
Thyroid hormones 586 Tumor-cell cytotoxicity 175
Thyroidectomy 587 Tunel staining 81
Thyroxine 586 Tyrosine aminotransferase activity 594
Tibia test 575
Ticlopidine 334 U
Tiotidine binding assay for histamine H2 receptors U-937-scid mice 93
528 Ultraviolet-induced cataract 233
Tirofiban 334 Umbilical cord blood stem cells 42
Tissue culture assay 589 Uteri of immature rabbits 143f
Tissue excision models 105 Uterine endometrium in estrogen-primed rabbits
Tissues for counting 139 140
Tissues of endodermal 40f Uterine weight assay 134
Tonic extension 419 Utero borna disease virus 399
Tooth pulp test 479 Uterus of virgin guinea pigs 582
Totterer mice 425 Uterus weight 606
Toxicity of drug 383 Uterus, isolated 582
Toxicity test UV-B-induced erythema in guinea pigs 503
acute 248 Uvea, inflammation of 514
special 250 Uveitis 514
Toxicity testing models 29 anterior 514
Transactivation assay 592 in control group, signs of anterior 517f
Transcriptional polymerase chain reaction 5 in lewis rats 517
722 Drug Screening Methods

in rabbits 516 Visna virus model 96

intermediate 514 Vital dyes 220
UV-induced cataract 238 Volume and pressure overload 312
Volunteer blood donors 336
V Vomiting 546
V. brachialis 296
V. cephalica antebrachialis 296 W
Vagal stimulation 527 Washed platelets method 337
Vaginal cornification 135, 605 Water wheel model 405
Vaginal opening 133 Water-loaded rabbit model of glaucoma 284
Vaginal or clitoral smooth muscle preparations WBN/KOB rat 618
128 WDF/TA-FA rat 620
Vaginal smears, preparation of 138 West nile virus 387
Vascular dysfunction in guinea pigs 687 Whole blood aggregometry 337
Vascular endothelial growth factor 170 Willebrand‘s disease or symptoms 336
Vasculogenic impotence, model for 123 Wistar fatty rat 620
Vasoactive intestinal Writhing test 481
peptide 468
polypeptide, role of 490 X
Vasopressin 584 Xenobiotics and nutrients 559
Vasopressor activity 584 Xenografts 105
Venous incompetence induced impotence 126 Xenopus laevis 114
Ventricular arrhythmias 292 Xenotransplantation mice 389
Ventricular fibrillation electrical threshold 297 Yeast 11
Ventricular myocytes 292 and beta-galactose assay 604
Vero cells 382
Vessel leakage, visualization of 651 Y
Vessels for human cell cultures, types of 700f Yellow obese mice 156, 157f
Vincristine-induced neuropathy model 484
Viral diseases 88, 258, 380
Viral infection 399
Z
Virtual screening 5 Zacopride binding assay for 5-HT3 receptors 541
Viruses and viral life cycle 380 Zebrafish (danio rerio) 29
Viruses causing obesity in animals 155t Zebrafish model of DR 654
Virus-induced obesity 155 Zucker diabetic fatty rat 620