This document discusses the design and evaluation of bioequivalence studies. It defines bioequivalence as the absence of a significant difference in the rate and extent to which the active drug becomes available at the site of action when administered under similar conditions. The document discusses various study designs including crossover, replicate, and non-replicate designs. It also covers sampling, criteria for comparisons between test and reference products, and the roles of bioequivalence studies in drug review and approval processes.

1. BIOEQUIVALENCE STUDIES, DESIGN AND EVALUATION OF
BIOEQUIVALENCE STUDIES, STUDY DESIGNS,
CROSSOVER STUDY DESIGNS, EVALUATION OF THE
DATA, BIOEQUIVALENCE EXAMPLE, STUDY SUBMISSION
AND DRUG REVIEW PROCESS
Submitted to: Prepared by:
Dr. Sanjula Baboota Dipak Kumar Gupta
Dr. Javed Ali M. Pharm, II Sem
SPER, Jamia Hamdard Pharmaceutics

2. Bioequivalence studies:
 Is defined as “the absence of a significant difference in
the rate and extent to which the active ingredient or
active moiety in pharmaceutical equivalents or
pharmaceutical alternatives becomes available at the
site of drug action when administered at the same
dose under similar conditions in an appropriately
designed study”.

3. Understanding the terms:
 Pharmaceutical equivalent
1. It refers to drug products, which contain the same active
ingredient in the same strength (concentration) and dosage form,
and is intended for the same route of administration. In general, it
has the same labelling and meets compendial and other standards
of strength, quality, purity, and identity.
2. Pharmaceutical equivalent does not necessarily imply
therapeutic equivalence as differences in the excipients and/or the
manufacturing process can lead to differences in product
performance.
 Pharmaceutical Alternatives
1. Drug products are considered pharmaceutical alternatives if
they contain the same therapeutic moiety, but are different salts,
esters, or complexes of that moiety, or are different dosage forms
or strengths. Different dosage forms and strengths within a
product line by a single manufacturer are thus pharmaceutical
alternatives, as are extended-release products when compared
with immediate or standard-release formulations of the same
active ingredients.

4. EQUIVALENCE STUDIES
NEEDED FOR MARKETING
AUTHORIZATION Pharmaceutically equivalent multi-source pharmaceutical
products must be shown to be therapeutically equivalent
to one another in order to be considered interchangeable.
 Several test methods are available to predict bio-
equivalence, including:
(a) Pharmacokinetic studies in humans in which the active
drug substance or one or more metabolites are measured in
an accessible biologic fluid such as plasma, blood or urine.
(b) Comparative pharmacodynamic studies in humans.
(c) Comparative clinical trials.
(d) In-Vitro Studies.

5. Bioequivalence study designs:
1. Pilot Study-
 A pilot study in a small number of subjects can be carried
out before proceeding with a full bioequivalence study.
 The study can be used to validate analytical methodology,
assess variability, optimize sample collection time
intervals, and provide other information. For example, for
conventional immediate-release products, careful timing
of initial samples may avoid a subsequent finding in a full-
scale study that the first sample collection occurs after
the plasma concentration peak.
 For modified-release products, a pilot study can help
determine the sampling schedule to assess lag time and
dose dumping. A pilot study that documents
bioequivalence may be acceptable, provided that its
design and execution are suitable and a sufficient number
of subjects (e.g., 12) have completed the study.

6. 2. Replicate Study Designs-
 Replicate study designs are recommended for
bioequivalence studies of modified-release dosage
forms and highly variable drug products (within-
subject coefficient of variation ≥ 30%), including
those that are immediate release, modified-release,
and other orally administered drug products.
 Replicate study designs offer several scientific
advantages compared to non- replicate designs.

7.  The advantages of replicate study designs are that they:
(i) Allow comparisons of within-subject variance for the test
and reference products.
(ii) Indicate whether a test product exhibits higher or lower
within-subject variability in the bioavailability measures
when compared to the reference product.
(iii) Suggest whether a subject-by-formulation interaction
may be present.
(iv) Provide more information about factors underlying
formulation performance.
(v) Reduce the number of subjects needed in the
bioequivalence study.
3. Non- replicate Study Designs-
 Non- replicate study designs are recommended for
bioequivalence studies of most orally administered,
immediate-release dosage forms.

8. 4. Food-Effect Studies-
 Food-effect bioequivalence studies focus on
demonstrating comparable bioavailability between
test and reference products when administered with
meals. Usually, a single-dose, two- period, two-
treatment, two-sequence crossover study is
recommended for food-effect bioequivalence study.
 Food- effect bioequivalence studies are generally
recommended for modified release products. Food-
effect bioequivalence studies are also recommended
for certain conventional release drug products.

9.  Selection of conventional release drug products that
require food studies is based upon certain
considerations, such as:
(i) Documented evidence of effect of food on drug
absorption (e.g., cefaclor);
(ii) The drug is recommended to be administered with
food;
(iii) The drug may produce gastric irritation under
fasting conditions, thus may be taken with food (e.g.,
NSAIDs)

10. Evaluation of bioequivalence
 a) Comparative pharmacokinetic studies
 (b) Comparative pharmacodynamic studies
 (c) Comparative clinical trials
 (d) Comparative in vitro tests
 (e) Any other approach deemed adequate by FDA

11. Pharmacokinetic studies:
 BE between a test (T) and reference (R) product can be achieved by
the conduct of comparative pharmacokinetic studies.These studies
are generally performed with a limited number of healthy
volunteers, e.g., 24–36 subjects.
 Most studies have a two-sequence, two-period, crossover design
where each subject is randomly assigned to either sequence TR or
RT with an adequate washout interval between the two treatment
periods.
 Derived from the plasma or serum concentration–time profile, the
rate of drug absorption is commonly expressed by maximum
concentration (Cmax) and time to maximum concentration (Tmax)
whereas the extent of absorption is expressed by the area-under-
the-curve from time zero after drug administration to time infinity
(AUC1) and/or to the last quantifiable drug concentration (AUCt).
 AUCt may be calculated using the simple trapezoidal rule while
AUC1 can be estimated by summing up AUCt and Ct/λz where Ct is
the last quantifiable concentration and λz is the terminal rate
constant.

12.  Both AUCs and Cmax are statistically analyzed using
the two one-sided tests procedure to determine if the
average values between the T and R products are
comparable.
 These comparisons require the calculation of a 90 %
confidence interval for the geometric mean ratios of
the T and R products. BE is generally declared if the 90
% confidence interval is within the BE limit of 80.00–
125.00 %.
 However, the BE limits for highly variable drugs and
narrow therapeutic index drugs have been scaled to
the intrasubject variability of the reference product in
the study.
 To obtain geometric means, the data of AUCs and
Cmax are log-transformed prior to conducting an
analysis of variance (ANOVA), then back-transformed
before calculating the T/R ratio.

13. Pharmacodynamic studies:
a) DOSE- RESPONSE RELATIONSHIP: Pharmacodynamic
endpoints selected for BE studies are required to have the
capacity of detecting potential differences between the
test and reference products.
 The basic pharmacodynamic study design for BE
determination may include two doses of the reference
product.
 This can be ascertained by a pilot study that demonstrates
the existence of a clear dose–response relationship, which
should be done before the conduct of pivotal BE studies.
 Depending on the drugs, the dose–response curve may be
linear, nonlinear, steep, or shallow. A shallow dose–response
curve may not allow for detection of potential formulation
differences between products. Linearity may be obtained in
some cases when the dose is expressed on logarithmic
scale.

14.  For many drugs, however, the dose–response
relationship based on a pharmacodynamic endpoint
is nonlinear and can be fitted to a hyperbolic Emax
model as follows
 E = E0 + Emax * D
ED50 + D
 where E is the estimated (fitted) value of
pharmacodynamic response, E0 is the baseline
pharmacodynamic effect, Emax is the maximum
pharmacodynamic effect, and ED50 is the dose where
the pharmacodynamic effect is half-maximal.

15. Comparative Clinical Trials
 Clinical responses are often located near or at the plateau of
the dose–response curve, thus insensitive to distinguish the
therapeutic difference between a test and reference
formulation.
 As a result, conduct of these studies for BE assessment requires
a large number of patients to detect formulation differences.
 Demonstration of dose–response relationships is not required
for clinical BE studies since they are intended only to confirm
the lack of important clinical differences between products in
comparison.
 Because of all the reasons mentioned above, BE studies using
clinical endpoints will be considered only when both
pharmacokinetic and pharmacodynamic approaches are
impossible for BE determination.
 Several FDA guidance documents for industry are available on
the application of clinical approaches to document BE for
topical drug products .

16.  Typically, a randomized, double-blind, placebo-controlled,
parallel group study is required. However, placebo treatments
are not needed for drugs treating infectious diseases.
 BE is established if the T product is equivalent to the R product
and superior to the placebo treatment. In the case of nasal
sprays for local action, the USFDA may waive the in vivo BE
studies and also for solution-based products as BA/BE is self-
evident for these products. However, such testing is required
for suspension based nasal sprays due to the lack of a suitable
method for particle size determination in suspension
formulations.
 Moreover, in vivo BE testing cannot be exempted for nasal
solutions in metered dose devices because they are drug device
combination products.
 Ex- For establishment of equivalence in local delivery of
suspension-based nasal sprays, the US FDA has recommended
clinical trials in seasonal allergic rhinitis patients. The study
design is a randomized, double-blind, placebo-controlled,
parallel group of 14-day duration. The clinical endpoints for
equivalence and efficacy analyses are patient self-rated mean
total nasal symptom scores.

17.  In general, for drug products that BE determination is
made on the basis of pharmacodynamic or clinical
endpoints, measurement of the active ingredients, or
active moieties in an accessible biological fluid (i.e.,
pharmacokinetic approach) is necessary to ensure
comparable systemic exposure (albeit minimal)
between the T and R product.
 However, for some locally acting drug products, such
pharmacokinetic studies may be limited by the labeled
maximum dose, drug bioavailability, and sensitivity of
the bioassay used.
 In such circumstances, pharmacodynamic or clinical
studies could be used to document comparable
systemic effects of these drug products.

18. In- vitro dissolution testing:
 Dissolution/release testing is the most commonly used in vitro method
for BE assessment.
 Although in vitro dissolution/release testing has seldom been used
alone as a tool for BE demonstration, dissolution/release information
along with the in vivo study data is routinely submitted by drug
sponsors for BE documentation of orally administered drug products.
 Dissolution/release data have often been employed to substantiate BE
when there is a minor change to formulation or manufacturing. In
addition, in vitro dissolution/release data are utilized to support waiver
of BA/BE studies for lower strengths of a drug product, provided that
an acceptable in vivo study has been conducted for a higher strength
and compositions of these strengths are proportionally similar
 Together with the use of BCS, in vitro dissolution/release testing has
played an increasingly important role in the regulatory determination
as to whether the waiver off in vivo BE studies can be granted for an
immediate-release drug product (FDA 2000).

19.  To serve as an indicator for BE, an in vitro dissolution/
release test should be correlated with a predicative of
in vivo BA (FDA 1995,2003).
 In this setting, the in vitro dissolution/release
methodology should be optimized to closely mimic the
physiological environment in vivo.
 For a drug product, proper in vitro dissolution/release
behavior in the presence of different formulations with
defined in vivo absorption characteristics will be useful
to facilitate the establishment of an in vitro–in vivo
correlation (IVIVC).
 The in vitro dissolution/release method developed in
such a manner may be utilized as a surrogate for BA/BE
studies when a change occurs in manufacturing or
formulation.

20. Cross- over designs:

21. Replicated crossover design:
Sequence studies- 3- way and 4-
way design
period
1 2 3
T R T
R T R
period
1 2 3 4
T R T R
R T R T

22. Sampling:
 In a typical BE study, the T and R product are generally
administered with 8 oz (i.e., 240 mL) of water to each
participating subject under fasting conditions, unless the
study is to be conducted under fed conditions where a high-
fat meal will be given.
 For fasting studies, subjects are usually fasted overnight
before drug administration in the following day and
standardized meals will be provided to subjects no less than
4 h after dosing.
 For BE studies with pharmacokinetic measures, under
normal circumstances, a series of blood samples (rather
than urine or tissue samples) will be collected after dosing
and parent drug (and major metabolites) concentrations in
serum or plasma will be measured.
 However, depending on the drug kinetics, whole blood may
be more appropriate for analysis of some drugs, e.g.,
tacrolimus

23. Criteria for comparisons:
 It focuses on the degree of certainty needed in the
analysis of relative BA or BE studies. An equivalence
approach is generally recommended.
 The approach usually relies on
(i) a criterion to allow the comparison
(ii) a confidence interval for the criterion
(iii) a BE limit (also called the goalpost).
 Log- transformation of exposure measures is generally
recommended.
 To compare measures in these studies, data are
analyzed by using an average BE criterion with other
criteria allowed more recently .

24. Drug review processes: INDs-
NDAs:
 BE documentation may be useful during the IND-NDA period
to establish links between:
(i) early and late clinical trial formulations; (ii) formulations
used in clinical studies and stability studies, if different; and (iii)
clinical trial formulations and the to-be-marketed drug
product.
 In each comparison, the new formulation or new method of
manufacture is the test product, and the prior formulation
or method of manufacture is the reference product. It may
not be possible to conclude BE because the test product
produces higher or lower measures of rate and extent of
absorption or because the performance of the test or
reference is more variable.
 In some cases, “bioinequivalence” is observed because of
inadequate numbers of subjects entered into the BE study.

25. ANDAs:
 Sponsors of ANDAs are required to establish BE between a
pharmaceutically equivalent generic drug product and the
corresponding listed drug.
Postapproval Changes:
 Information on the types of in vivo BE studies and in vitro
dissolution needed for post- approval changes to drug products
approved as either NDAs or ANDAs are provided in FDA
guidances.
 In the presence of certain major changes in components and
composition, and/or method of manufacture after approval, in
vivo BE between pre- and post change product may need to be
re- established.
 Under such circumstances, for approved NDAs, the drug
product after change should be compared with the drug
product before change, whereas for approved ANDAs, the drug
product after change should be compared with the reference
listed drug.

26. References:
 Bioavailability and Bioequivalence: An FDA
Regulatory Overview; Mei-Ling Chen; Vinod Shah;
Pharmaceutical Research, Vol. 18, No. 12,
December 2001
 Review of methods and criteria for the evaluation
of bioequivalence studies; G. Pabst and H. Jaeger
LAB GmbH and Co.; European Journal of
Pharmacology; Springer Publications.
 Meng Li Chan; Fundamentals of Bioequivalence.

27. THANK YOU!