This document provides an introduction to bioequivalence studies, including definitions of key terms, the need for and importance of bioequivalence studies, criteria for establishing a bioequivalence requirement, types of bioequivalence studies, design of bioequivalence studies, evaluation of bioequivalence study results, and clinical significance. It discusses in vivo and in vitro bioequivalence study types and designs, including factors such as single dose, multiple dose, fasting, food effect, and crossover designs. Statistical evaluation methods including ANOVA, confidence intervals, and bioequivalence limits of 80-125% are also summarized.

1. PREPARED BY: PARTH
M.PHARM SEM-I (Q . A)
GUIDED BY : Ms.TANVI PANDYA
DEPARTMENT OF Q. A
A.P.M.C.COLLEGE OF PHARMACY
HIMMATNAGAR

2. CONTENT
 Introduction
 Need for Bioequivalence studies
 Importance of Bioequivalence studies
 Criteria for Establishing a bioequivalence requirement
 Types of Bioequivalence studies
 Design of Bioequivalence studies
 Evaluation of Bioequivalence study
 Clinical significance
 References

3. INTRODUCTION
DEFINITIONS:
 Bioavailability:
Bioavailability means the rate and extent to which the active
ingredient or active moiety is absorbed from a drug product and
becomes available at the site of action.
 Equivalence:
It is a relative term that compares drug products with respect to a
specific characteristic or function or to a defined set of standards.
There are several types of equivalences:
• Chemical equivalence: It indicates that two or more drug
products contain the same labeled chemical substance as an
active ingredient in the same amount.

4.  Pharmaceutical equivalence: This term implies that two or more
drug products are identical in strength, quality, purity, content
uniformity and disintegration and dissolution characteristics. They
may, however differ in containing different excipients.
 Bioequivalence: denotes that the drug substance in two or more
identical dosage forms, reaches the systemic circulation at the same
relative rate and to the same relative extent i.e. their plasma
concentration-time profiles will be identical without significant
statistical differences. When statistically significant differences are
observed in the bioavailability of two or more drug products, bio-in
equivalence is indicated.
• Therapeutic Equivalence: This term indicates that two or more
drug products that contain the same therapeutically active
ingredient elicit identical pharmacological effects and can control
the disease to the same extent.

5. NEED FOR BIOEQUIVALENCE STUDIES
 New product is intended to be a substitute for an approved
medicinal product as a pharmaceutical equivalent or alternative
 To ensure clinical performance of such drug products
 Bioequivalence studies are conducted if there is:
 A risk of bio-in equivalence and/or
 A risk of pharmacotherapeutics failure or diminished clinical
safety
 In vivo bioavailability / bioequivalence studies and in vitro
dissolution testing recommended to applicants intending to
submit Investigational new drug application (INDs)New drug
applications (NDAs)Abbreviated new drug applications
(ANDAs) for conventional and extended release dosage forms
administered orally.

6.  In conditions where a suitable method for determining active
drug is not available, an indirect indication of bioavailability
and bioequivalence by comparing the pharmacodynamic
responses of the formulations may be possible

7. IMPORTANCE OF BIOEQUIVALANCE STUDIES
 To evaluate the absolute bioavailability of dosage form compared
with reference dosage forms.
 Dose proportionality study to determine if bioavailability
parameters are linear over proposed dosage range.
 Intra/inter subject variability
 Intervention study to examine effect of e.g. Food and concomitant
medication.
 Dosage form proportionality study to determine if equipotent drug
treatments administered at different dose strength of the market
form produce equivalent drug bioavailability.
 Bioequivalence study needed as a result of changes in the
formulation or manufacturing processes.

8. CRITERIA FOR ESTABLISHING A BIOEQUIVALENCE
REQUIREMENT
 Evidence from well-controlled clinical trials, or controlled
observations in patients and bioequivalence studies :
 Various drug products do not give comparable therapeutic
effects are not bioequivalent drug products.
 Narrow therapeutic ratio and minimum effective concentration
in the blood.
 Serious adverse effects
 Physicochemical:
 Low solubility in water.
 Dissolution rate slow
 Particle size and surface area of the active drug ingredient

9.  Structural forms dissolves poorly.
 High ratio of excipients.
 Hydrophilic or hydrophobic excipients and lubricants
• Pharmacokinetic:
 GI tract or localized site.
 Degree of absorption poor

10. TYPES OF BIOEQUIVALANCE STUDIES
 In vivo:
1. Oral immediate-release products with systemic action
 Indicated for serious conditions requiring assured response.
 Narrow therapeutic margin.
 Absorption <70%
 Unfavorable physiochemical properties.
 Bioavailability problems.
2. Non-oral immediate-release products.
3. Modified-release products with systemic action.

11.  In vitro:
Dissolution study can be used in lieu of in vivo bioequivalence
under certain circumstance, called as biowaivers.
1. The drug product differs only in strength of the active substance
it contains, provided all of the conditions hold-
 Pharmacokinetics linear.
 Qualitative composition same.
 Ratio between active substance and the excipients are same.
 Same manufacturer and same production site.
 Bioavailability or bioequivalence study performed with the
original product.
2. Slightly reformulated or manufacturing method slightly modified
by the original manufacturer.

12. 3. Meets all of the following requirements-
 Solution or solublised form
 Active ingredient in the same concentration
 No excipients
 Topical administration
 Oral administration
 By inhalation as a gas or vapour.

13. DESIGN OF BIOEQUIVALENCE STUDIES
DESIGN:
 The design and evaluation of well-controlled bioequivalence
studies require cooperative input from
pharmacokineticists, statisticians, clinicians, bioanalytical
chemists, and others.
 The basic design for a bioequivalence study is determined by:
 The scientific questions to be answered,
 The nature of the reference material and the dosage form to be
tested,
 The availability of analytical methods, and
 Benefit–risk and ethical considerations with regard to testing
in humans. For some generic drugs, the FDA offers general
guidelines for conducting these studies.

14. Bioequivalence study protocol
1. Title c.Dosage regimen
a. Principal investigator d. Sample collection schedule
b. Project number & date e. Housing
2. Study objective f. Fasting/meals schedule.
3. Study design g. Analytical methods
a. Design 4. Subject selection
- Medical history
b.Drug product
- Physical examination
- Test products
- Laboratory tests.
- Reference product
c. Inclusion/exclusion criteria

15. 5. Clinical procedures
a. Dosage & drug e. Adverse reactions &
administration emergency procedures.
b. Biological sampling 7. Facilities
schedule 8. Data analysis
c. Activity of subjects 9.Analytical validation
6. Ethical considerations procedure
a. Basic principles a. Drug accountability
b. Institutional review board b. Statistical treatment of
c. Informed consent data
d. Indications for subject 10. Appendix
withdrawal

16. • Detailed protocol
• Same dose strength, similar dosage forms and same route of
administration.
• No unnecessary human research
• Normal, healthy male volunteers given informed consent.
• Fasted 10 to 12 hours prior to drug administration and 2 to 4 hour
after dosing.
Reference standard
 Same route
 Fully approved
 Innovator’s or original manufacturer
Brand name product
 In vivo: 5% of the reference product
 In vitro:Both test and reference

17. Study designs:
Fasting study
• Single dose, two-period, two-treatment, two sequence, open
label, randomized cross over designs.
• Fasted, adult, healthy subjects.
• All immediate release and modified release oral dosage forms
• Both male and female subjects
• Overnight fast and 4 hour after dosing
Food intervention study
• Co-administration of food with an oral drug product may affect
the bioavailability of the drug.

18. Multiple dose
• Multiple dose, steady state, randomized, two-treatment, two-
way, crossover study.
• Adult and healthy subject
• Three consecutive trough concentration on three consecutive days
Types of test designs
1.Completely randomized designs
All treatments( factor levels) are randomly allocated among all
Experimental subjects.
Method of randomization : label all subjects with the same number
digits. Randomly select non-repeating numbers.
Advantages:-
 Easy to construct.

19.  Any number of treatments & subjects.
 Easy & simple to analyses.
Disadvantages:-
 Best suited for relatively few treatments.
 All subjects must be as homogenous
2. Randomized block designs
Subjects are sorted into homogenous groups called blocks.
Method of randomization: Subjects having similar background
characteristics are formed as blocks.

20. Then treatments are randomized within each block, just like the
simple randomization.
Advantages:-
 Effective & systemic way of grouping.
 Any number of treatments or replications.
 Different treatments need not have equal sample size.
 Statistical analysis simple & easy to construct.
 Spoiled results, the design is easy to construct.
Disadvantages:-
 More complex analysis
 Degrees of freedom of experimental error are not as large as
with a completely randomised design.

21. Repeated measures, cross-over & carry-over designs:-
• Same subject serves as a block.
• Repeated measures on each subject we get the design name
“repeated measures design”
• The administration of two or more treatments one after the other in
a specified or random order to the same group of patients is called
a crossover design or change-over design
• Carry over effects
• Wash out period
Advantages:
 Good precision for comparing treatments
 Economic on subjects

22. Disadvantages:
 Order-effect.
 Carry over effect.
Latin Square designs:
• Each Subject receives each treatment during the course of the
experiment.
• A Latin square design is a two factor design with one
observation in each cell.
• Rows represent subjects & columns represent treatments.
• Standard: the first row & the first column consist r letters in
alphabetical order.

23. Latin-Square Crossover Design for a Bioequivalence Study of Three
Drug Products in Six Human Volunteers
Drug Product
Subject Study Washout Study Washout Study
Period 1 period 1 Period 2 period 2 Period 3
1 A B C
2 B C A
3 C A B
4 A C B
5 C B A
6 B A C

24. Advantages:
 Minimizes inter-subject variability, Intra-subject variability &
time effect.
 Formulation variables.
Disadvantages:-
 Degrees of freedom for experiments error larger than
necessary.
 More complex.
 Wash out period very long

25. Evaluation of bioequivalence studies
Analytical method
• Accuracy, precision and specificity.
• More than one analytical method not be valid.
• Data presented in both tabulated and graphical form
• Plasma drug concentration versus time curve for each drug
product and each subject.
Pharmacokinetic evaluation of the data
• Single dose study:AUC0-t ,AUC0-∞,Tmax and Tmax.
• Multiple dose studies:AUC0-t,Tmax,Cmax,Cmin and percent
fluctuation[100*(Cmax-Cmin)/Cmin].

26. Statistical evaluation of the data
• No statistical difference between the bioavailability of the test
product & the reference product.
• Bell- shaped curve
• Log values resembles more closely a normal distribution
Analysis of variance(ANOVA)
• No significance difference.
• AUC0-24,AUC0-∞, Tmax & Cmax
• Evaluate variability in subjects , treatment groups, study
period, formulation & other variables.
• Variability in the data is large then two drug products are
bioequivalent.
• Statistically significant, If p≤0.05
• P- level of Statistical significance.

27. • If p> 0.05 the difference between the two drug products are not
statistically significant.
• To detect small differences between the test products, a power
test is performed.
• Sample size, variability of the data & desired level of
significance.
• Power is set at 0.80 with an α=0.2 & a level of significance of
0.05.

28. Two one-sided tests procedure
• Confidence interval approach.
• Greater 20%.
• 90% confidence limits.
• Student’s t-distribution of the data.
• within 20%.
• Lower 90% confidence interval for the ratio of means cannot
be less than 0.80 & the upper 90% confidence interval for the
ratio of the means cannot be greater than 1.20.
• Log transformed data , 90% confidence interval is set at 80 to
125%.

29. • Confidence limits termed the bioequivalence interval.
• No-statistical differences between the mean AUC & Cmax
parameters.
• 90% confidence intervals for AUC & Cmax values of the test
drug product should not be less than 0.80(80%) nor greater
than 1.25(125%) of the reference product based on log-
transformed data.

30. CLINICAL SIGNIFICANCE
 Clinical interpretation is important in evaluating the results of
a bioequivalence study.
 Differences of less than 20% in AUC & Cmax between drug
products are unlikely to be clinically significant in patients.
 A small, statistically significant difference if the study well
controlled & the number of subjects is sufficiently large.
 Above MEC & do not reach the MTC.
 Elderly or patients.
 Normal healthy volunteers.
 Minimize product to product variability by different
manufactures & lot to lot variability with a single manufacture.

31. REFERENCES
 Leon Shargel; Andrew B.C Yu, Applied Bio pharmaceutics and
pharmacokinetics, Fourth edition,pp 256-271.
 D.M.Brahmankar, Sunil b. jaiswal, Biopharmaceutics and
Pharmacokinetics, A Treatise, second edition, vallabh
prakashan,pp336-344.

32. thanks