The document discusses various aspects of bioequivalence studies including study designs and evaluation. It describes four main study designs - pilot studies, replicate designs, non-replicate designs, and food-effect studies. It also discusses evaluating bioequivalence through comparative pharmacokinetic studies, pharmacodynamic studies, clinical trials, in vitro dissolution testing, and other approaches. The key aspects covered are parameters for determining bioequivalence like AUC and Cmax, study considerations for highly variable drugs, and the role of in vitro tests in bioequivalence assessments.

1. Bioequivalence studies, design and evaluation of
Bioequivalence studies, study designs
Presented By
Selim Akhtar
Pharmaceutical Analysis
20HMPA04

2. Contents
Introduction
Design of Bioequivalence studies
Evaluation of Bioequivalence studies
References
2

3. Bioequivalence studies
3
 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".

4. Understanding the terms:
4
 Pharmaceutical equivalent
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.

5. Equivalence Studies needed For Marketing
5
 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.

6. Bioequivalence study designs
6
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

7. 7
2. Replicate Study Designs-
1. When the no. of study subjects <80, it is difficult to achieve with highly variable drugs
and drug product (%CV> 30).
2. These drugs have wide therapeutic window and despite high variability, have been
demonstrated to be safe and effective.
3. Replicate designs for these drugs require smaller no. of subject and a void exposure of
large no. of healthy subjects.
4. Used for determining individual BE, to estimate within subject variance for both test and
reference.
5. Provide an estimate of the subject- by- formulation interaction variance.

8. 8
Period 1 Period 2 Period 3 Period 4
Sequence 1 T R T R
Sequence 2 R T R T
 A four period, two-sequence, two-formulation design is recommended by the FDA.
where R = reference and T= treatment.
The same reference and the same test are each given twice to the same subject. Other
Sequences are possible.
In this design, Reference-to-Reference and Test-to-Test comparisons may also be made.

9. 9
 The advantages of replicate study designs are that they:
 Allow comparisons of within-subject variance for the test and reference products.
 Indicate whether a test product exhibits higher or lower within-subject variability in the
bioavailability measures when compared to the reference product.
 Suggest whether a subject-by-formulation interaction may be present.
 Provide more information about factors underlying formulation performance.
 Reduce the number of subjects needed in the bioequivalence study.
.

10. 10
3. Non-replicate Study Designs-
1. For the drugs having long elimination half life or depot injection in which the drug is slowly released
over weeks and month.
2. Two separate groups of volunteers are used.
3. One group will have the test product while the other will have the reference product.
4. Blood sample collection time should be adequate to ensure completion of GI transit(2-3days).
5. Cmax and AUC, 72 hrs. after dose admn. can be used to characterize peak and total drug exposure.
6. This design is not for drugs that have high intrasubject variability in distribution and clearance.

11. 11
4. Food-Effect Studies-
 Food-effect bioequivalence studies focus on demonstrating comparable bioavailability between test
and reference products when administered 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.

12. 12
 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)

13. Evaluation of bioequivalence
13
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

14. Pharmacokinetic studies:
14
 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 (AUC) and/or to the last quantifiable drug concentration (AUC)
 AUC, may be calculated using the simple trapezoidal rule while AUC, can be estimated by summing up
AUC, and C/Az where Ct is the last quantifiable concentration and Az is the terminal rate constant.

15. 15
 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 %.o
 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

16. Pharmacodynamic studies:
16
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 determined 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.

17. 17
 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= Eo +
𝐄𝐦𝐚𝐱∗𝐃
𝐄𝐃𝟓𝟎+𝟓𝟎
 Where E is the estimated (fitted) value of pharmacodynamic response, Eo is
the baseline pharmacodynamic effect, Emax is the maximum pharmacodynamic effect, and ED5o is the
dose where the pharmacodynamic effect is half-maximal.

18. Comparative Clinical Trials
18
 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

19. 19
 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 e

20. 20
 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.

21. In- vitro dissolution testing:
21
 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).

22. 22
 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 formulation
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.

23. References
23
1. Bioavailability and Bioequivalence: An FDA Regulatory Overview; Mei-Ling Chen; Vinod Shah;
Pharmaceutical Research, Vol. 18, No. 12,December 2001
2. 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.
3. Meng Li Chan; Fundamentals of Bioequivalence.

24. 24