Bioavailability and

Bioequivalence study

Dr. Muhammad Usman

Assistant Professor
IPS, UVAS, Lahore
[email protected]
 Drug Product Performance
The release of the drug substance from the drug product
leading to bioavailability of the drug substance.
The assessment of drug product performance is important
since bioavailability is related both to the
pharmacodynamic response and to adverse events.
Drug product performance studies are used in the
development of new and generic drug products

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 Bioavailability
• Bioavailability is defined as the rate and extent at
which active drug reaches to the site of action from
it’s dosage form.
• It provides the estimation of fraction of drug
absorbed from formulation
• BA depends on
1. Pharmaceutical factors
Absolute
2. Patient related factors
Bioavailability
3. Rout of administration
Types of
Bioavailability Relative
Dr. Muhammad Usman Bioavailability 3
 Absolute Bioavailability
(F)
When the bioavailability of a drug administered orally is
determined by its comparison to IV administration is
called Absolute Bioavailability.
It is determined to characterize the drug’s inherent
absorption properties from extra-vascular rout.
[AUC]oral × Dose{IV
Absolute Bioavailability = × 100
[AUC]IV × Dose Oral

The value is always ≤ 1 or ≤ 100%

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 Relative Bioavailability
(Fr)
When the systemic absorption of one drug product is compared
with other formulation is called as Relative Bioavailability.
It is determined to compare the relative absorption properties of
two different formulations.
Bioequivalence is a specialized for of Relative BA.

[AUC]A × Dose{B
Relative Bioavailability = × 100
[AUC]B × Dose A

Relative Bioavailability may be less than or greater than 100

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Pharmaceutical Equivalents:
Two drug products are deemed to be pharmaceutical
equivalents if they have the same active ingredient(s),
strength or concentration, dosage form, and route of
administration.
Bioequivalents:
Bioequivalence is defined as the absence of a significant
difference in the rate and extent to which the active
ingredient becomes available at the site of drug action
when administered at the same molar dose under similar
conditions in an appropriately designed study.
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Therapeutic Equivalents:
Drugs products are considered to be therapeutic
equivalent only if they are pharmaceutical equivalent
and have same clinical effect and safety profile when
administered to the patients under the conditions
specified in the labeling

Pharmaceutical Therapeutic
Bioquivalent
Equivalent Equivalent

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 Bioequivalence Studies during drug
development
• In New Drug Development (NDA) BE studies are used
to;
1. Early and late clinical trial formulations
2. Formulations used in clinical trials and stability
studies
3. Clinical trial formulation and to be marketed as drug
product (Post approval changes)
SUPAC (Scale-up and post approval changes)
4. Product strength equivalent
5. To support new formulation of previously approved
product

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Bioequivalence Studies in New Drug
Development (NDA)

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 Bioequivalence Studies in Generic Drug
Development (ANDA)

• Comparative drug product performance studies are

important in the development of generic drug products
A generic drug product is a multisource drug product
that has been approved by the FDA as a therapeutic
equivalent to the reference listed drug product.
• Clinical safety and efficacy studies are not generally
performed on generic drug products
• The generic drug manufacturer must demonstrate that
the generic drug product is pharmaceutically equivalent,
bioequivalent, and therapeutically equivalent to the
comparator brand-name drug product.

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 Bioequivalence Studies in Generic Drug
Development (ANDA)

Sharjel 7th Edition Page No. 471

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 Bioequivalence study
Cross Over study
Product A Product B

Wash out
AUC AUC
AUC AUC
Cmax
AUC Cmax
AUC
CmaxAUC CmaxAUC
TmaxCmax
AUC TmaxCmax
AUC
TmaxCmax TmaxCmax
T1/2TmaxCmax T1/2TmaxCmax
T Tmax T Tmax
CL1/2
T Tmax CL1/2
T Tmax
CL1/2
T CL1/2
T
CL1/2
T
CL1/2 Difference CL1/2
T
CL1/2
CL CL

Not Statistically Statistically

Bioequivalent
Bioequivalent Significant Inignificant
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 Product A Product B

Group A Group B

Parallel study

AUC AUC
AUC AUC
Cmax
AUC Cmax
AUC
CmaxAUC CmaxAUC
TmaxCmax
AUC TmaxCmax
AUC
TmaxCmax TmaxCmax
T1/2TmaxCmax T1/2TmaxCmax
T Tmax
CL1/2
T Tmax
Difference T Tmax
CL1/2
T Tmax
CL1/2
T CL1/2
T
CL1/2
T CL1/2
T
CL1/2 CL1/2
CL CL
Not Statistically Statistically
Bioequivalent
Bioequivalent Significant Inignificant
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 Drug Product

Patient type 2 Patient type 1

Parallel study

AUC AUC
AUC AUC
Cmax
AUC Cmax
AUC
CmaxAUC CmaxAUC
TmaxCmax
AUC TmaxCmax
AUC
TmaxCmax TmaxCmax
T1/2TmaxCmax
T Tmax
Difference T1/2TmaxCmax
T Tmax
CL1/2
T Tmax CL1/2
T Tmax
CL1/2
T CL1/2
T
CL1/2
T CL1/2
T
CL1/2 CL1/2
CL Statistically Statistically CL
Significant Inignificant
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 Methods for assessing BA and BE
The FDA’s regulations (US-FDA, CDER, 2014a) list the
following approaches to determining bioequivalence.
1. In vivo measurement of active moiety in biological fluids.
2. In vivo pharmacodynamics comparison
3. In vivo clinical comparison
4. In vitro comparison
For drug products that are not intended to absorbed into
the blood stream, bioavailability may be assessed by rate
and extent to which drug becomes available at the site of
action

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 In vivo measurement of active
moiety in biological fluids.

• This is most sensitive, accurate and reproducible

approach.
• For all systemically active drugs (with few
exceptions), BE should be demonstrated by in
vivo study based on pharmacokinetic endpoints
• Two approaches
1) Plasma drug concentration
2) Urinary excretion data

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 In vivo measurement of active
moiety in biological fluids.

• Plasma Drug Concentration

This is most direct and appropriate way to
measure the systemic bioavailability
Parameters:
❑ tmax (h)
Measure of rate of drug absorption
At tmax the rate of drug absorption becomes equal to
the rate of elimination

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 In vivo measurement of active
moiety in biological fluids.

❑Cmax (mg/L or µg/mL or ng/mL)

• Relates with the pharmacodynamics (therapeutic and toxic)
effects of drugs
❑AUC (mg.h/L)
• Measure of extent of drug absorption
• Independent of rout of administration and process of drug
elimination

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 In vivo measurement of active
moiety in biological fluids.
• For many drugs, AUC is directly proportional to dose

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 In vivo measurement of active
moiety in biological fluids.

• In some cases, AUC is not directly proportional to dose e-g

Salicylates, Phenytoin

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 In vivo measurement of active
moiety in biological fluids.

• Urinary Drug Excretion Data

Indirect estimation of bioavailability
Du is the cumulative amount of drug excreted in urine

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 In vivo Pharmacodynamic
comparison
PD comparison is used in the cases where quantitative measurement
of plasma drug concentration is not available or in vitro approaches
are not applicable.
• A dose response relationship is demonstrated.
• The PD effect should be at rising phase of dose response curve
• Sufficient measurements should be taken to assure an appropriate
PD response.
• All PD measurement assays should be validated for specificity,
accuracy, sensitivity and precision.
• Two approaches
1. Demonstration of dose response curve
2. Acute pharmacodynamics effect-time curve

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 Dose-Response Curve

Pharmacodynamics effect-time curve

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 In vivo comparison based on
clinical end point
• A parallel BE study
• Least accurate, least sensitive to bioavailability difference
• A predetermined clinical endpoint is used to evaluate the
clinical effect in the patients
• Requires the use of large number of patients which increases
cost and duration of study.
• Placebo arm is usually included
• Recommended for the products which;
1. Have negligible systemic absorption
2. For which there is no identified PD measure
3. For which site of action is local

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 In vitro studies
• Dissolution studies
• Should Correlate in vivo bioavailability
• Comparative dissolution studies are often
performed on several test formulations of
the same drug during drug development.
• Under certain conditions, comparative dissolution
profiles of higher and lower dose strengths of a
solid oral drug product such as an immediate-
release tablet are used to obtain a waiver
(biowaiver) of performing additional in vivo
bioequivalence studies.

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 Other approaches
• The use of in vitro biomarkers and in vitro binding studies
• Examples
1. The bioequivalence of cholestyramine resin is performed
by equilibrium and kinetic binding studies of the resin to bile
acid salts
2. For calcium acetate tablets, the FDA recommends a
relatively simple in vitro binding assay based on the
test/reference binding ratio over a range of phosphate
concentrations.
3. Comparative in vitro release testing and physicochemical
characterization for Acyclovir topical ointment

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BE studies based on multiple
endpoints

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 Cross over study
Two sequence, two period study

Replicated Cross over study

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NTI and TDM

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Criteria for BE

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 Criteria for BE
90% Confidence Interval for AUC and Cmax
For Non NTI
For NTI

R Reference
G1
NOT BE
G2
NOT BE
BE (NTI)
NOT BE
BE (NTI)
G3
BE
G4
BE
G5

80% 90% 100% 110% 125%

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Gozzo et al. 2022
 Multiple-Dose (Stady-State) Study Design
• A bioequivalence study may be performed using a multiple-dose
study design.
• A steady-state, randomized, two-treatment, two-way, crossover
study comparing equal doses of the test and reference products
in healthy adult subjects.
• Three consecutive trough concentrations (Cmin) are
determined to ascertain that the subjects are at steady state.
• The last morning dose is given to the subject after an overnight
fast, with continual fasting for at least 2 hours following dose
administration.
• Blood sampling is then performed over one dosing interval

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Multiple-Dose (Stady-State) Study Design

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 Multiple-Dose (Stady-State) Study Design
Pharmacokinetic analyses for multiple-dose
studies include calculation of the following
parameters for each subject:
AUC 0-tau (Area under the curve during a dosing interval)
tmax (Time to Cmax during a dosing interval)
Cmax (Maximum drug concentration during dose interval)
Cmin (Concentration at the end of a dose interval)
Cav During dose interval

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 Multiple-Dose (Stady-State) Study Design
❑Advantages:
Determination of bioavailability using multiple doses reveals changes that
are normally not detected in a single-dose study. e.g Non-linear
Pharmacokinetics, may be observed by rising Cmin and AUC after each
dose interval.
❑Disadvantages:
1. Time consuming and may lead to high cost and possibility of dropping
out of volunteers
2. More blood samples
3. Small differences in the rate of drug absorption may not be observed
with steady-state study comparisons Because Cav ∞ depends primarily
on the dose of the drug and the time interval between doses, the
extent of absorption is more important than rate.

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 Clinical End-Point BE Study
• A randomized, double-blind, placebo controlled, parallel-
designed study comparing test product, reference product,
and placebo product in patients.
• A placebo arm is usually included to demonstrate that the
treatments are active and the study is sufficiently sensitive
to identify the clinical effect in the patient population
enrolled in the study.
• In some cases, the use of a placebo may not be included for
safety reasons.
• The primary analysis for bioequivalence is determined by
evaluating the difference between the proportion of
patients in the test and reference treatment groups who
are considered a “therapeutic cure” at the end of study

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 BE of drug products in patients
• Due to safety concern, some drugs such as antipsychotic drugs, anti-
cancer drugs etc. can not be given to healthy individuals.
• BE should be performed in patients who have been stabilized on
highest strength using multiple dose BE study.
• No wash out period can be given.
• The patient is maintained on previous dose of medication and blood
sampling is performed during a dose interval.
• Without washout, the patient takes equal dose of test or reference
product and previous drug product is discontinued.
• Dosing with each drug product is continued till steady state is
attained.
• The plasma level time curve for second drug product is obtained
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 BE of drug products in patients
• Bioequivalence or absence of bioequivalence is
determined by comparison of both plasma level
time curve data.
• The patient then continues with original drug
product.
• Products are given in a random order e.g “A” then
“B” and “B” then “A”.
• The reference product is provided by the
investigator from a known lot (not from patient’s
prescription)
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 Clinical Examples
Levothyroxine Sodium Oral Tablets
• A multiple dose relative bioavailability study of two synthetic branded
levothyroxine sodium oral tablets, product A and product B, were evaluated
in 20 euthyroid patients.
• The investigation was designed as a two-way crossover study in which the
patients who had been diagnosed as hypothyroid by their primary-care
physician were given a single 100-mg daily dose of either product A or
product B levothyroxine sodium tablets for 50 days and then switched over
immediately to the other treatment for 50 days.
• Pre-dose blood samples were taken on days 1, 25, 48, 49, and 50 of each
phase, and, on day 50, a complete blood sampling was performed.
• The serum from each blood sample was analyzed for total and free thyroxine
(T4), total and free triiodothyronine (T3), the major metabolite of T4, and
thyrotropin (TSH).

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 Levothyroxine Sodium Oral Tablets
a) Why were hypothyroid patients used in this study?
Answer: Normal healthy euthyroid subjects would be at risk if they were to take
levothyroxine sodium for an extended period of time.
b) Why were the subjects dosed for 50 days with each thyroid product?
Answer: The long (50-day) daily dosing for each product was required to obtain
steady-state drug levels because of the long elimination half-life of levothyroxine.
c) Why were blood samples obtained on days 48, 49, and 50?
Answer: Serum from blood samples was taken on days 48, 49, and 50 to obtain
three consecutive Cmin drug levels.
d) Why was T3 measured?
Answer: T3 is the active metabolite of T4.
e) Why was TSH measured?
Answer: The serum TSH concentration is inversely proportional to the free
serum T4 concentrations and gives an indication of the pharmacodynamic activity
of the active drug.
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 Clinical Examples
Mercaptopurine (Purinethol) Oral Tablets
• The FDA recommends BE steady-state studies in patients receiving
therapeutic oral doses.
• Patients should be on a stable regimen using the same dosage unit
• Plasma drug concentration–time profiles are obtained in these
patients at steady state with the brand product.
• The proposed generic drug product is then given to these patients
at the same dosage regimen until steady state is reached.
• Plasma drug concentration–time profiles are obtained for the
generic drug product;
• Then the patients return to the original brand medication

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 Design and evaluation of
Bioequivalence Studies
Team

Pharmacokinetists Clinicians Bio-Analysts Statistician Others

Nurses Phlebotomist

Technicians Helpers

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 Design and evaluation of
Bioequivalence Studies
I. Title
A. Principal investigator (Study Director)
B. Project Protocol number and date
II. Study Objective
• Should be clearly stated.
• Considerations
1. The scientific questions and objectives to be answered,
2. The nature of the reference material and the dosage form to be tested,
3. The availability of analytical methods,
4. The pharmacokinetics and pharmacodynamics of the drug substance,
5. The route of drug administration, and
6. Benefit–risk and ethical considerations with regard to testing in humans.
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 Design and evaluation of
Bioequivalence Studies
III. Study Design
A. Design (Cross over, Parallel etc)
B. Drug Products
1. Test Product(s)
2. Reference Product (RLD)
C. Dosage Regimen
D. Sample Collection Schedule
E. Housing and Confinement
F. Fasting and Meal Schedule
G. Analytical methods

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 Design and evaluation of
Bioequivalence Studies
IV. Study Population
A. Subjects (healthy Volunteers, Patients etc)
B. Subject selection
1. Medical history
2. Physical examination
3. Laboratory tests
C. Inclusion/Exclusion criteria
D. Restrictions/Prohibitions

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 Design and evaluation of
Bioequivalence Studies
V. Clinical Procedures
A. Dosage and drug administration
B. Biological sampling schedule and handling procedures
1. Vials coding
2. Sample collection
3. Plasma separation
4. Precautions
5. Record keeping
C. Activity of subjects

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 Design and evaluation of
Bioequivalence Studies
VI. Ethical Procedures
A. Basic principles
B. Institutional Review Board
C. Informed Consent
D. Indications for subject withdrawal
C. Adverse reaction and emergency procedures
VII. Facilities
VIII. Data analysis
A. Analytical validation procedures
B. Statistical treatment of data

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 Study submission and
review process
The submission for an NDA must contain safety
and efficacy studies as provided by
❑ Animal toxicology studies,
❑ Clinical efficacy studies, and
❑ Pharmacokinetic/bioavailability studies.
For submission of ANDA (generic drug
manufacture), the BE study replaces the Animal,
Clinical and PK studies.
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 Study submission and
review process
• The investigator should be sure that the study has
been properly designed, the objectives are clearly
defined, and the method of analysis has been
validated.
• These results, along with case reports and various
data supporting the validity of the analytical
method, are included in the submission

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 Study submission and
review process
• The FDA reviews the study in details based on the
following outline;
1. Introduction
2. Study design
3. Study objectives
4. Assay description and validation
5. Summary and analysis of data
6. Comments
7. Deficiencies
8. Recommendations
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 Applicant

ANDA

Applicable No Refused to file and

and
letter issued
complete?
Yes
Review by OGD/CDER
Request for plant inspection Chemistry/Micro Review

Bioequivalence Review Labeling review

Chemistry,
BE review Yes
Labeling review
acceptable?
No acceptable No

Bioequivalence deficiency Preapproval

inspection Not approvable letter
letter
Applicable?
No

Yes Approval deferred pending

ANDA Approved satisfactory Results
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 Candidates for TDM

Drug NTI BCS Class ROA BE

Amikacin Yes III IV No
Capreomycin Yes IV IM No
Chloramphenicol Yes II Oral Yes
Colistin Yes IV IM, IV No
Gentamicin Yes III IV No
Kanamycin Yes III IV No
Streptomycin Yes I IM No
Vancomycin yes I IV No

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 Candidates for BE
Sr. No Drug BCS Class ROA
1 Ampicillin II Oral, IV
2 Azithromycin II Oral
3 Bedaquiline II Oral
4 Cefixime II Oral
5 Cefuroxime II Oral, IV
6 Ciprofloxacin III Oral
7 Clarithromycin II Oral
8 Clofazimine II Oral
9 Cloxacillin III Oral
10 Cycloserine II Oral
11 Dapsone II Oral
12 Delamanid IV Oral
13 Ethionamide II Oral
14 Nitrofurantoin II Oral
15 Rifampicin II Oral
16 Rifapentine II Oral
17 Chloramphenicol II Oral
18 Ethionamide II Oral

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 Candidates for Mandatory BE
Sr. No Drug Remarks
1 ethambutol + isoniazid FDC
2 ethambutol + isoniazid + pyrazinamide + rifampicin FDC
3 ethambutol + isoniazid + rifampicin FDC
4 isoniazid + pyrazinamide + rifampicin FDC
5 isoniazid + rifampicin FDC
6 amoxicillin + clavulanic acid FDC
7 cloxacillin + ampicillin FDC
8 sulfamethoxazole + trimethoprim FDC
9 chloramphenicol NTI

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