Pre-Clinical Development

Contents
1. Introduction to Pre-clinical Studies
2. High Throughput Screening (HTS)
3. Toxicology studies
4. Pharmacological screening methods
5. Calculation of First Human Dose
Drug Development Process
 Introduction
• Pre-Clinical Trials is a study to test a drug, a procedure, or another medical
treatment in animals.
• In drug development, pre-clinical development, also named preclinical
studies and nonclinical studies, is a stage of research that begins before
clinical trials (testing in humans) can begin, and during which important
feasibility, iterative testing and drug safety data is collected.
• It also means in vivo or in vitro experiments in which test articles are
studied prospectively in test systems under laboratory conditions to
determine their safety.
 Pre-clinical Review by FDA
• Under FDA requirements, a sponsor must first submit data showing that the drug is
reasonably safe for use in initial, small-scale clinical studies. Depending on whether the
compound has been studied or marketed previously, the sponsor may have several options
for fulfilling this requirement:
– Compiling existing nonclinical data from past in vitro laboratory or animal studies on the compound
– Compiling data from previous clinical testing or marketing of the drug
– Undertaking new preclinical studies designed to provide the evidence necessary to support the
safety of administering the compound to humans.

• At the preclinical stage, the FDA will generally ask

– Develop a pharmacological profile of the drug
– Determine the acute toxicity of the drug in at least two species of animals
– Conduct short-term toxicity studies ranging from 2 weeks to 3 months, depending on the proposed
duration of use of the substance in the proposed clinical studies.
Objectives of Pre-clinical Study
• Objective is to develop adequate data to decide that
it is reasonably safe to proceed with human trials of
the drug,
• Means, a laboratory test of a new drug or a new
medical device, usually done on animal subjects, to
see if the treatment really works and if it is safe to
test on humans.
• However the main objective is to collect the data to
submit to the FDA for IND filing.
 Importance of Pre-clinical Studies

 Determination of dose, toxic dose, pharmacological action, etc.

 Necessary to check safety of drug on animals before starting to check on

human being.

 Check for kinetic profile of drug and on this basis, selection of route of
administration

 Requirement of regulatory body

 Goals of Pre-clinical Study

Identify initial safe dose Identify the target

and dose escalation organs for toxicity
schemes in humans

Goals

Identify safety Study of toxicity

parameters for clinical whether reversible
monitoring
 Steps involved in Pre-clinical studies

Lead Selection Drug

and Candidate Pre-Clinical Drug
Optimisation Confirmation Characterisation
 Get an Idea of Drug Target
• Drugs usually act on either cellular or genetic
chemicals in the targets, which are believed to be
associated with disease.
• Scientists use a variety of techniques to identify and
isolate learn more about their functions and how
they influence disease.
• Compounds are then identified that have various
interactions with targets that might be helpful in
treatment of a specific disease.
 Lead Identification
• A chemical lead is defined as a synthetically stable,
feasible, and drug like molecule active in primary
and secondary assays with acceptable specificity,
affinity and selectivity for the target receptor.
• For a compound to be considered druggable it
should have the potential to bind to a specific
target; however, also important is the compound‘s
pharmacokinetic profile regarding absorption,
distribution, metabolism, and excretion.
 Lead Optimization
• Lead optimization is the process by which a drug candidate is
designed after an initial lead compound is identified. The process
involves iterative series of synthesis and characterization of a
potential drug to build up a representation of in what way
chemical structure and activity are related in terms of interactions
with its targets and its metabolism.
• Potential leads are evaluated for a range of properties, including
selectivity and binding mechanisms during lead optimization, as
the final step in early stage drug discovery.
 Product Characterization
• When any new drug molecule shows a promising
therapeutic activity, then the molecule is characterized by
its size, shape, strength, weakness, use, toxicity, and
biological activity.
• Early stages of pharmacological studies are helpful to
characterize the mechanism of action of the compound.
 Formulation Development
• Also called as Preformulation studies.
• Pharmaceutical formulation is a stage of drug
development during which the physicochemical
properties of active pharmaceutical ingredients (APIs) are
characterized to produce a bioavailable, stable and
optimal dosage form for a specific administration route.
 Preclinical Testing
• Pre-clinical research in drug development process involves
evaluation of drug‘s safety and efficacy in animal species that
conclude to prospective human outcome.
• The pre-clinical trials also have to acquire approval by
corresponding regulatory authorities.
• ICH has established a basic guideline for technical necessities of
acceptable preclinical drug development.
• The pre-clinical trials can be conducted in two ways: General
pharmacology and Toxicology. Pharmacology deals with the
pharmacokinetic and pharmacodynamic parameters of drug.
• It is essential to explore unwanted pharmacological effects in
suitable animal models and monitoring them in toxicological
studies.
Investigational New Drug Process
(IND)
• Drug developers must file an Investigational New Drug application
to FDA before commencement clinical research.

• The regulatory authorities must ensure that trials are conducted in

safe and ethical way and would give approval for only those drugs
which are confirm to be safe and effective.

• In the IND application, developers must include:

 Preclinical and toxicity study data
 Drug manufacturing information
 Clinical research protocols for studies to be conducted
 Previous clinical research data (if any)
 Information about the investigator/ developer
 Types of Pre-clinical Study
In-vitro studies In-vivo studies
• Pharmacokinetics • Screening
• Pharmacodynamics • Isolated Organ
• Bacteria Culture
• Animal Models
• General Observation
• Confirmatory
• Mechanism of Action
• Systemic Pharmacology
• Quantitively Test
• Toxicity Test
 Bioinformatics

• Bioinformatics is an interdisciplinary subject, where three

subjects Biology, Computer science and Information
technology merge together to form the new discipline that is
Bioinformatics.
• Without the usage of bioinformatics tools it is merely
impossible to capture, manage process, analyze and interpret
the huge amounts data that is available especially after whole
genome sequencing projects.
 Why is bioinformatics important?

• Bioinformatics has help to understand the code of life that is

DNA, the massive DNA sequencing projects have evolved and
added in the growth of the science of bioinformatics.

• The ultimate goal of bioinformatics is to uncover the wealth of

biological information hidden in the mass of sequence,
structure, literature and other biological data.
 What is done in bioinformatics?

• The development of new algorithms and statistics with which

to assess relationships among members of large data sets.

• The analysis and interpretation of various types of data

including nucleotide and amino acid sequences, protein
domains, and protein structures.

• The development and implementation of tools that enable

efficient access and management of different types of
information
 How do we use Bioinformatics?

1. Store/retrieve biological information (databases)

2. Retrieve/compare gene sequences
3. Predict function of unknown genes/proteins
4. Search for previously known functions of a gene
5. Compare data with other researchers
6. Compile/distribute data for other researchers
 BLAST

• Basic Local Alignment Search Tool.

• It is an algorithm for comparing biological sequences
information, such as amino acid sequence of different
proteins or the nucleotides of DNA sequences.
• BLAST is used to identify library sequences that resembles
the query sequences.
• BLAST is a tool for alignment of sequences.
• E.g. To identify the unknown gene (query sequences) in the
mouse, the scientist will perform a BLAST search of the
human genome (library sequences) to see whether the
human carrying the similar gene or not.
 High Throughput Screening
• High Throughput Screening (HTS) is identification of one or more
positive candidates extracted from a pool of possible candidates
based on specific criteria
• It is a drug-discovery process widely used in the pharmaceutical
industry
• It allows automation to quickly assay the biological or
biochemical activity of a large number of compounds
• It is a useful for discovering ligands for receptors, enzymes, ion-
channels or other pharmacological targets, or pharmacologically
profiling a cellular or biochemical pathway of interest
 Steps in HTS
• 1st stage screening
 Test optical clarity, abrasion resistance, and adhesion
 Eliminates ~ 90% of samples

• 2nd stage screening

 Test weather ability, integrity, gloss, and surface smoothness
 ~10% of the samples

• Rapidly identify coating samples with desired properties

• Candidates for scale up

 Test according to the specification
 Detection Methods in HTS
• Spectroscopy
• Mass Spectrometry
• Chromatography
• Calorimetry
• X-ray diffraction
• Microscopy
• Radioactive methods
 Methodology
• The heart of the HTS system is a plate, or tray, which consists of
tiny wells where assay reagents and samples are deposited,
and their reactions monitored
• The configuration of the plate has changed from 96 wells (in a
matrix of 8 rows by 12 columns) to 384, and now to a high -
density 1536 - well format, which enables large – scale
screening.
• Assay reagents may be coated onto the plates or deposited in
liquid form together with test samples into the wells.
• Both samples and assay reagents may be incubated, and those
that interact show signals, which can be detected.
• The aim of HTS is cost effectiveness and speed of compound
scanning.
 Cell Based Assays
• Cell-based assays refer to any of a number of different
experiments based on the use of live cells
• This is a general definition and can include a variety of
assays that measure cell proliferation, toxicity, motility,
production of a measurable product and morphology
• Cell-based assays offer a more accurate representation of
the real-life model since live cells are used.
• A Cell-based Assay is: one where the fundamental unit of
expression is the cell, either cell populations or single cell.
 Four Key Elements of Cell Based Assay:

1. A cellular component e.g. a cell line or a primary cell

population
2. A target (substrate) molecule that records the cellular
response
3. An instrument to conduct and monitor the assay an
informatics component to manage and analyze data from
the assay
4. Cell-based reporter assays are used where human receptors
are transfected into null cell lines either alone, (luciferin-
luciferase) or light transmission (melanophore), that can be
measured independently of radioactivity within minutes.
 Advantages/ Disadvantages
Advantages
 Assays do not require purification of the target protein
 Can immediately select against compounds / potential drugs that are generally
cytotoxic, or that cannot permeate cellular membranes to reach intracellular
sites
 Hit/lead compounds identified by cell based assays have passed important
validation steps, saving time and costs in drug development
 Cell-based assays visualize all possible drug-target interactions e.g. activators,
target interactions
 High sensitivity of assays & Automation

Disadvantages
 High Cost
 Low data quality
 Local contamination
 Need of relatively pure product
 What is an Animal Model?

• An animal model is a living organism in which normative

biology or behavior can be studied, or in which a spontaneous
or induced pathological process can be investigated, and in
which the phenomenon in one or more respects resembles
the same phenomenon in humans or other species of animal.

• An animal model is defined as a specific combination of an

animal species, challenge agent and route of exposure that
produces a disease process or pathological condition that in
multiple important aspects corresponds to the human disease
or condition of interest.
 Classification of Animal Models

1. EXPERIMENTAL : A model in which an experimenting induced

conditions mimic a human disease

2. NEGATIVE : A model in which particular condition cant be

produced , & is therefore studied to better understand the
reason for the protective or resistant affects

3. SPONTANEOUS : A model in which the animal naturally develops

a disease or some other conditions of interest

4. ALTERNATIVE MODEL: is defined as technique that reduces or

eliminates the need for live animals & there by prevents
potential pain & distress in animals
 What is Animal Testing?

• The term "animal testing" refers to procedures performed on living

animals for purposes of research into basic biology and diseases,
assessing the effectiveness of new medicinal products, and testing
the human health and/or environmental safety of consumer and
industry products such as cosmetics, household cleaners, food
additives, pharmaceuticals and industrial/agro-chemicals.

• All procedures, even those classified as “mild,” have the potential to

cause the animals physical as well as psychological distress and
suffering.

• Often the procedures can cause a great deal of suffering. Most

animals are killed at the end of an experiment, but some may be re-
used in subsequent experiments.
Animals used in clinical trials are
classified as
 The Mouse

• The mouse is a small mammal that belongs to the order Rodentia, Mus musculus, is
the species most commonly used for biomedical research.
• Mice and rats make up approximately 95% of all laboratory animals, with mice the
most commonly used animal in biomedical research.
• Mice are a commonly selected animal model for a variety of reasons, including
• small size (facilitating housing and maintenance);
• short reproductive cycle and lifespan;
• generally mild-tempered and docile;
• wealth of information regarding their anatomy, genetics, biology, and
physiology; and the
• possibility for breeding genetically manipulated mice and mice that have
spontaneous mutations.
• Some of the diseases they model include: hypertension, diabetes, cataracts, obesity,
seizures, respiratory problems, deafness, Parkinson’s disease, Alzheimer’s disease,
various cancers, cystic fibrosis, and acquired immunodeficiency syndrome (AIDS),
heart disease, muscular dystrophy, and spinal cord injuries. Mice are also used in
behavioural, sensory, aging, nutrition, and genetic studies.
 The Rat

• Rattus norvegicus constitutes one of the most commonly used laboratory species
• Rats possess a number of qualities which make them a highly suitable and much
preferred animal model. Like mice, these traits include
• relatively small size;
• known genetic background;
• short generation time;
• similarities to human disease conditions; and
• known microbial status.
• Their tractable nature makes them easier to handle in a laboratory setting
than many other rodents.
• Rats rarely bite their handlers unless extremely stressed or in pain.
• Rats have been used as animal models in numerous areas of research from space
exploration to answering more basic scientific questions regarding nutrition,
genetics, immunology, neurology, infectious disease, metabolic disease, and
behavior. Perhaps their largest use is in drug discovery, efficacy, and toxicity studies
 The Rabbit

• The ancestral home of the European rabbit (Oryctolagus cuniculus) is the Iberian
Peninsula
• European rabbits have been used in research since the middle of the 19th century.
Early work with the species was concentrated on the comparative anatomy of the
rabbit with other species, such as the frog, and the unique features of the rabbit’s
heart and circulatory system
• The New Zealand White (NZW) rabbit is the most frequent breed of used in
research. The California and Dutch-belted rabbit breeds are also occasionally used.
• Rabbits have been used as a model of human pregnancy and for the production of
polyclonal antibodies for use in immunology research. Rabbits routinely used in
atherosclerosis, osteoporosis, ocular, and immunology research
• Rabbits are very easily heat stressed and thus must be kept at significantly lower
temperatures than other laboratory animals like rats and mice. Noise is another
significant stressor to rabbit
 Guinea pigs

• Guinea pigs (Cavia porcellus) are rodents, related to porcupines and chinchillas in
the suborder Hystricomorpha
• The guinea pig has been used as a model for infectious diseases such as
tuberculosis, Legionnaires disease, sexually transmitted diseases such as chlamydia
and syphilis, and one of the more common causes of nosocomial infections in
people, Staphylococcus aureus.
• Guinea pigs have also been useful tools in researching cholesterol metabolism,
asthma, fetus and placental development and aspects of childbirth, as well as
Alzheimer’s disease.
• Guinea pigs have many similarities to humans hormonally, immunologically, and
physiologically. Unlike other rodents, and more like primates (including people),
guinea pigs are prone to scurvy if they do not receive adequate vitamin C, typically
in their diet.
• Guinea pigs are housed similarly to other rodents, although they require more room
than the smaller rodents.
• Guinea pigs have also been used as models for infectious disease associated with
bacteria, parasites, and viruses, such as leptospirosis, leishmaniasis, and severe
acute respiratory syndrome (SARS) and Ebola viruses.
 Hamsters

• Hamsters are of the Rodentia order, suborder Myomorpha along with the
mouse and the rat. There are over 24 species of hamsters described in the
literature, with the most common hamster used in research being the
Golden or Syrian hamster, Mesocricetus auratus.
• Specific anatomical and physiological features including their
susceptibility to disease and infection make them a useful model for
study.
• Hamsters are still used in many areas of research, including investigations
into metabolic diseases like diabetes mellitus, cardiovascular disease,
reproductive endocrinology, and oncology.
 Monkeys

• Human biology and disease can be studied in monkeys because they are very similar
to humans; biologically anatomically and physiologically.
• Most often used monkeys are; Cynomolgus macaques (Macaca fascicularis), Rhesus
macaques (macaca mulatta), Marmosets (Callithrix jacchus).
• Drug testing-
• Determination of serum concentrations of penicillin ,doxycycline and
ciprofloxacin in anthrax infected monkeys.
• Studying of efficacy of psychostimulants like cocaine.
• More relevant data about the metabolites of cyclophosphamide and
ifosamide from CSF of monkeys.
• Genetical similarity of monkeys to humans make them particularly suitable
candidates for testing the safety of new drugs and studying of variable diseases .
• But those similarities to humans also raise specific ethical questions about their use
for scientific experiments.
• However they play an indispensable role in the process of medical research and
development.
 Birds

• Birds have been used as research models of human disease and are important in
evaluation of aging, memory, parasitology, atherosclerosis, reproduction, and
infectious disease among other topics.
• Historically, chickens (Gallus domesticus) are the most common bird species studied
in biomedical and agricultural research and are a classic model in areas such as
immunology, virology, infectious disease, embryology, and toxicology
• Chickens are also studied to evaluate reproductive development and retinal disease.
Embryonated chicken eggs have been used to commercially produce vaccines (such
as for human influenza), studied for developmental analysis, and are now being
treated with viral vectors like lentivirus to produce transgenic embryos.
• Pigeons (Columba livia) have been evaluated in areas such as comparative
psychology, neuroanatomy, neuroendocrinology, and atherosclerosis. They are
studied to understand their navigational skills and memory which allow homing,
vision and discrimination ability.
• Also Zebra Finch, Owls, Japanese Quails, Amazon Parrots are used for research.
 Toxicological Studies
• Toxicology is the scientific study of adverse effects that occur in living
organisms due to chemicals.
• It involves observing and reporting symptoms, mechanisms, detection and
treatments of toxic substances, in particular relation to the poisoning of
humans.
• Adverse drug effects – Any undesirable &/ or unintended effects of drug
– Predictable (Type A reactions)
– Non-predictable (Type B reactions)
• Side Effects: Unwanted but often unavoidable effects at therapeutic doses.
• Secondary Effects: Indirect consequences of primary action of drug.
• Toxic Effects: Are results of excessive pharmacological effect of drug due to
over dosage or prolonged use.
 Types of Toxicological Studies
• Acute Toxicity:
– Single dose of substance or
– multiple doses given within 24 hrs or
– an inhalation exposure of 4 hrs
– Observed for 14 days
• Subacute Toxicity:
– Repeated doses
– Observed for28 days
• Sub-chronic Toxicity:
– Repeated doses
– Observed for 1-3 months
• Chronic Toxicity:
– Exposure either repeated or continuous
– Observed for 6 months - 2 years
• Special Toxicity:
– Carcinogenicity, Mutagenicity, Teratogenicity etc.
 Acute Toxicity
• 14 days study.
• Study on at least two species.
• One rodent –mice/rat.
• One non rodent –usually rabbit.
• Dose administered orally & parenterally.
• Various dose levels to groups of both sexes.
• Dose selection such that causing less than 50% but not
• 0% and more than 50% but not 100% mortality.
• Establish the:
– Maximum tolerated dose (MTD)
– Minimum lethal dose (MLD)
– Target organ of toxicity (if possible)
• Other parameters :
– Symptoms, signs and mode of death, signs of intoxication, effect on body weight
– Gross and histopathological changes
– LD 10 and LD 50 values, (preferably with 95 % CI)
– Genetic effects if any
 Acute Toxicity
• LD50
– LD stands for "Lethal Dose“
– LD50 is the amount of a drug, given all at once, which causes the death of 50%
(one half) of a group of test animals. The LD50 is one way to measure the short-
term poisoning potential (acute toxicity) of a material.

• LC50
– LC stands for "Lethal Concentration“
– LC values usually refer to the concentration of a chemical in air but in
environmental studies it can also mean the concentration of a chemical in
water.
– The concentrations of the chemical in air that kills 50% of the test animals
during the observation period is the LC50 value.

• Minimum lethal dose (MLD): The least amount of drug that can produce death in a
given animal species under controlled conditions.
Acute Toxicity
 Maximum tolerated dose (MTD)
• Maximum tolerated dose, is defined as the highest dose of a drug that does not cause
unacceptable side effects or overt toxicity in a specific period of time.
• These side effects can range from mild effects such as reduced weight gain, moderate
effects such as weight loss up to 20% or substantial effects such as unresponsiveness.
• The MTD can be determined by acute toxicity studies, short duration dose escalation
studies and dose ranging studies.
• These studies are designed with a minimum number of animals and include
toxicological endpoints such as clinical observations and clinical pathology, for
example blood tests for liver function.
• This maximum tolerated dose is then used for longer-term safety assessments.
• The rationale for using the MTD in long term studies is to maximize the likelihood of
detecting any chronic disease effects or other hazards of a drug candidate.
• It is also more humane to determine the MTD before conducting any PK or ADME
studies to minimize animal morbidity.
• Maximum tolerated dose studies are not designed to cause mortality, therefore death
is not an appropriate end point.
 Repeated dose toxicity studies
(Sub acute/ sub chronic toxicity)
• Aim:
– To identify Target Organ Toxicity
– To establish MTD for subsequent studies
• Animal- at least 2 mammalian species(1 rodent & 1 non- rodent)
– Rodent- must be between 6-8 weeks
– Non-rodent should be between 4-9 months
– Younger and still growing animals are preferred in initiation of sub-chronic
studies
• Drug is given on 14, 28, 90 & 180 days.
• 3 other groups are formed :
– Highest dose Observable toxicity [MTD]
– Lowest dose No observable toxicity [NOAEL] should be comparable to intended
therapeutic dose or multiple of it
– Intermediate dose Some symptoms ; not gross toxicity or death placed
logarithmically between two doses
 Repeated dose toxicity studies
(Sub acute/ sub chronic toxicity)
 Chronic Toxicity
• Objectives
1. Chronic Toxicity Test: To determine the toxicity observing changes in the function,
shape, of a living organism.
2. On Reproductive Potential and Future Generations
3. Teratogenicity Test: To determine damage to the birth of the fetus.
4. Mutagenicity Test: To test carcinogenicity and genetic impact on the next
generation.

• Observations:
• Morbidity or mortality.
• Specific signs of toxicity in particular for neurofunctional and
neurobehavioural sign.
• Ophthalmological examination, using an ophthalmoscope or other suitable
equipment.
• Body weight
• Haematology and clinical biochemistry: Various blood count level. Eg.
Platelet count, etc.
• Glucose, urea (urea nitrogen), creatinine, total protein, albumin, calcium,
sodium, potassium, total.
Chronic Toxicity
 Reproductive Toxicity
1. Male Fertility
2. Female Reproduction & Developmental toxicity
 Female Fertility
 Teratogenicity
 Perinatal development
Reproductive Toxicity
 Male Reproductive Toxicity

• One rodent species (preferably rat)

• 3 dose groups and a control group :
Dose selection : results of previous 14 or 28-day toxicity study
Highest dose : showing minimal toxicity in systemic
studies
• 6 adult male animals per group
 Female Reproductive Toxicity

For all drugs proposed for women of child bearing age

• Segment I : Female fertility
• Segment II : Teratogenicity
• Segment III : Perinatal development
Segment I, II and III studies in albino mice or rats, and
Segment II study also in albino rabbits as a second test species
 Female Reproductive Toxicity

For all drugs proposed for women of child bearing age

• Segment I : Female fertility
• Segment II : Teratogenicity
• Segment III : Perinatal development
Segment I, II and III studies in albino mice or rats, and
Segment II study also in albino rabbits as a second test species
Female Fertility Study (Segment I)
 Teratogenicity Study (Segment II)
• Rodent (preferably rat) and Non-rodent (rabbit)
• Drug administered throughout organogenesis
• 3 dose levels and a Control group:
• Highest dose : minimum maternal toxicity
• Lowest dose : as proposed clinical dose or its multiple
• Route of administration : same as intended for humans
• At least 20 pregnant rats (or mice) and 12 rabbits, for each dose
 Perinatal Study (Segment III)
• Specially if
→Drug to pregnant / nursing mothers for long periods
→Indications of possible adverse effects on foetus
• One rodent species (preferably rat)
• Dosing comparable to multiples of human dose and route
• At least 4 groups (including control), 15 females / group
• Drug throughout last trimester (from day 15 of gestation)
• Dose causing low fetal loss continued throughout weaning
• Second generation (F2) selected at weaning and studied.
• Observed parameters are same as teratogenicity study
 LOAEL/NOAEL
Lowest-observed-adverse-effect level (LOAEL) Lowest concentration
or amount of a substance, found by experiment or observation,
which causes an adverse alteration of morphology, functional
capacity, growth, development or life span of the target organism
distinguishable from normal (control) organisms of the same species
and strain under the same defined conditions of exposure.

No-observed-adverse-effect level (NOAEL) Greatest concentration or

amount of a substance, found by experiment or observation, which
causes no detectable adverse alteration of morphology, functional
capacity, growth, development or life span of the target organism
under defined conditions of exposure. Alterations of morphology,
functional capacity, growth, development or life span of the target
may be detected which are judged not to be adverse.
Chronic toxicity
Genotoxicity
Mutagenicity
Teratogenicity
Carcinogenicity
Reproductive toxicity
In vitro studies
In vivo studies
In silico methods
Ex vivo methods
Calculation of first human dose
NOAEL
MABEL