The document outlines the key elements that should be included in a bioequivalence study protocol. It discusses the study objective to show that the drug bioavailability from test and reference products is statistically equivalent. The study design may include crossover, parallel, or replicate designs. Important aspects covered include the study population, drug administration, sampling schedule, analytical methods, data analysis plan, and statistical evaluation of pharmacokinetic parameters to determine bioequivalence within the 80-125% range. In vitro dissolution and content uniformity testing should also be included to support the in vivo bioequivalence results.

1. BIOEQUIVALENCE PROTOCOL
BY
T.M.RASALA
Assistant Professor
Pharmaceutics Department
Gururnanak college of pharmacy
4/6/2022 1

2. Elements of Bioequivalence
Study Protocol
1.Title
a. Principal investigator
b. Project number and date
2. Study objective
3. Study design
a. Design
b. Drug Products
i. Test products(s)
ii. Reference product
c. Dosage regimen
d. Sample collection schedule
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3. e. Housing
f. Fasting /Meals schedule
g. Analytical methods
4. Study population
a. Subjects
b. Subject selection
i. Medical history
ii. Physical examination
iii. Laboratory tests
c. Inclusion/exclusion criteria
i. Inclusion criteria
ii. Exclusion criteria
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4. • Restrictions/prohibitions
5.Clinical procedures
a. Dosage and Drug Administration
b. Biological sampling schedule
c. Activity of subjects
6.Ethical considerations
a. Basic principles
b. Institutional review board
c. Informed consent
d. Indication for subject withdrawal
e. Adverse reaction and emergency procedures
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5. Facilities
8. Data Analysis
a. Analytical validation procedure
b. Statistical treatment of data
9. Drug Accountability
10.Appendix
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6. Study Objective:
The objective for a bioequivalence study
is that the drug bio availability from test
and reference products are not statistically
different when administered to patients
are subjects at same molar dose under
similar experimental conditions.
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7. Study Designs:
For many drug products, the FDA, division of
bio equivalence, office of generic drugs
provides guidance for the performances of in-
vitro dissolution and in-vivo bioequivalence
studies
 Generally three bioequivalence studies
required for solid oral dosage forms including-
1. A fasting study
2. A food intervention study 3. Multi dose study
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8. 1.Cross Over Study Design:
 Two formulations, even number of subjects
 Randomly divided into two equal groups
 Each number of one group receive a single dose
of the test formulation and each member of the
other group receive the standard formulations.
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Subject Period 1 Period 2
1-8 Test Standard
9-16 Standard Test

9. 2.Latin Square Design:
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More than two formulations
Eg: A group of volunteers will receive formulations
in the sequence.

10. 3.Balance Incomplete Block Design:
 More than 3 formulations
 Latin square design will not be used because
each volunteer may required drawing of too
many blood samples.
 If each volunteer expected to receive at least 2
formulations then such study can be carried out
using BIBD.
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11. 4/6/2022 11

12. 4.Parallel Group Design:
 Even number of subjects in 2 groups
 Each receive a different formulation
 No wash out necessary
 For drugs with long half life
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13. 5.Replicate Cross Over Study Design:
 For highly variable drugs
 Allows comparisons of within subject variances
 Reduces the number of subjects needed
 4-periods, 2-sequences, 2 formulations
design(recommended)
 3-periods, 3-sequences, single dose
partially replicated
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14. 4/6/2022 14

15. Difference between parallel group and
cross over study design
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16. 6. Pilot Study:
 If the sponsor chooses in a small number of
subjects
 To access a variability, optimize the sample
collection time intervals, and provide other
Information
Eg: Immediate release products: careful timing
of initial samples-avoid a sub sequent finding that
the first sample collection, occurred after the
plasma concentration peak.
Modified released products: To determine
sampling schedule- Assess log time and dose
dumping
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17. 7. Analytical Methods:
 Analytical methods used in an in-vivo bio availability,
bio equivalence, or Pharmacodynamics studies must be
validated for accuracy and sufficient sensitivity.
 The analytical method for measurement of drug must be
validated for accuracy, precision, sensitivity, specificity,
and robustness. The use of more than one analytical
method during a bio equivalence study may not be valid
because different methods may yield different values.
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18. Subject Selection:
 Healthy adult volunteers
 Age 18-45years
 Age/sex representation corresponding to
therapeutic and safety profile weight with in
normal limits
 women-pregnency test period to first and last
 Dose of study
Selection Of Number Of Subjects:
 Sample size estimated by
 Pilot Experiments
 Previous Studies
 Published Data
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19.  Significance level desired usually 0.05
 Power of study normally 80% or more
 Minimum 16 subjects unless ethical justification
 Allow for drop outs
Exclusion Criteria:
 H/o allergy to test drug
 H/o liver or kidney dysfunction
 H/o jaundice in past 6 months
 Chronic diseases Eg: Asthma, Arthritis
 Psychiatric illness
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20. Administration of drug products:
 Administration of drug products to the should be
based on randamization. After the administration
of drug products, biood samples are withdrawn
from the subjects at fixed time points.
 It takes some to take a sample from each subject,
and the total time difference between first and last
subject range from 10 to 20 minutes depending
upon the number of subjects and technicians in
the study.
 This 10 to 20 minutes difference would represent
a substantial change in the drug concentrations
observed in the blood.
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21. If under these conditions treatments are
administered to the the subjects in a
seqential manner (such as teatment A to the
first 6 volunteers, teatment B to volunteers
7 to 12, and teatment C to Volunteers 13
to18), the error between the time of
administration and sampling will gradually
increase from treatment group to treatment
group. This is because of sequential
administration of drug products to different
treatments.
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22. Sampling:
 The biological sample to be used in the study as to
be decided before the commencement of a
bioavailability study.
 If the bioavailability of a given dosage form is to
be evaluated by a blood level study, some estimate
of the area under the serum concentration v/s
time curve, peak serum concentration, time of
peak concentration must be obtained from the
study.
 These factors can markedly influence the
‘apparent’ results obtained in a given study.
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23.  The sampling scheme should frequent enough to
define the absorption phase, the peak, and the
elimination phase during a drugs time course in the
body.
 The absorption rate, volume of distribution,
elimination rate, all influence the apparent drug
concentration one obtains in a given sample.
 It is necessary to see that all these factors influence
each dosage form equally. To estimate the AUC
from the data, sampling as to be carried out till the
concentration of the drug reaches the linear
elimination phase.
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24. The amount of drug excreted in urine is
obtained directly. In the case of a blood level
study, the amount of drug in the body is
estimated using pharmacokinetic parameters.
The urinary excretion method has several
disadvantages
1. Urinary excretion studies are not useful in
estimating the drug absorption rate.
2. In some cases, the metabolites of the drug are
also concentrated in the sample that interferes
with the estimation of unchanged drug in the
urine sample.
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25. Evaluation of data:
Pharmacokinetic evaluation of the data for single
dose studies, including a fasting study or a food
intervention study, the pharmacokinetic analyses
include calculation for each subject of the area
under the curve to the last quantifiable
concentration (AUC0 ) and to infinity (AUC0),
tmax and Cmax .Additionally ,the elimination rate
constant,k, the elimination half-life,t1/2,
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26. Statistical evaluation of the data:
 Bioequivalance is generally determined using a
comparision of population averages of a
bioequivalance metric, such as AUC and Cmax.
 This approach, termed average bioequivalence,
involves for the ratio of averages of the test and
reference drug products.
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27. Statistical Analysis For Average
Bio
equivalence:
 Based on log transformed data
 Point estimates of the mean ratios
Test / reference for AUC and Cmax are between
80% -125%
 AUC and Cmax
90% confident intervals must fit between 80%-
125%
 Statistical model typically includes factors
accounting for following sources of variations:
 Sequence, subjects, nested in sequences, period
in treatment 4/6/2022 27

28. Proposed And Contents Of An In vivo Bio
equivalence Study Submission And Accompaning In
vitro Data:
 Title Page
 Study Title
 Name of sponsor
 Name and Address of clinical laboratory
 Name of Principal Investigator(S)
 Name of Clinical Investigator
 Name of Analytical Laboratory
 Dates of Clinical Study
 Signature of principal investigator(and date)
 Signature of Clinical Investigator(and date)
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29.  Table Of Contents
1.Study Resume
 Product Information
 Summary of Bio equivalence study
 Summary of Bio equivalence data
 Plasma
 Urinary Excreation
 Figure of mean plasma concentration-time profile
 Figure of mean cumulative urinary excreation
 Figure of mean urinary excreation rates
2.Protocol And Approvals
 Protocol
 Letter of acceptance of protocol from fda
 Informed consent form
 Letter of approval of institutional review board
3.Clinical study
 Summary of Study
 Details of study
 Demographic characteristics of the subjects
 Subject assignement in the study
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30.  Mean physical characteristics of subjects arranged by sequence
 Details of clinical activity
 Deviation from protocol
 Vital science of subjects
 Adverse reactions report
4.Assay Methodology And Validation
 Assay method discription
 Validation procedure
 Summary of validation
 Data on linearity of standard samples
 Data on interday precision and accuracy
 Data on intraday precision and accauracy
 Figure for standard curve for low/high ranges
 Chromatograms of standard and quality control samples
 Sample calculation
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31. 4/6/2022 31
5. Pharmacokinetic Parameters and Tests
• Definitions and calculaton
• Statistical tests
• Drug levels at each sampling time and harmacokinetic
parameters
• Figure of mean plasma concentration-time profile
• Figure of individual subjects plasma concentrations-
time profiles
• Figure of mean cumulative urinary excreation
• Figures of individual subject urinary excreation rates
• Tables of individual subject data arranged by drug,
drug/period, drug/sequence

32.  6.statistical analyses
 statistical considerations
 summary of statistical significance
 summary of statistical parameters
 analysis of variance,least squares estimates and least
 squares means
 assessment of sequence, period, and treatment
effects
 90% confidence intervals for the differences between
 test and reference products for the log-normal-
transformed
 parameters of AUC0-t, AUC0-infinty, CMAX should
be 80%-125
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33.  7. Appendices
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• Randamization schedulule
• sample identification codes
• Analytical raw data
• Chromatograms of at least 20% of subjects
• Medical records and clinical reports
• Clinical facilities discription
• Analytical facilities discription
• Curricula vitae of investigators
8. Invitro testing
• Dissolution testing
• Dissolution assay methadology
• Content uniformity testing
• Cotency determination
9. Batch size and formulations
• Batch record
• Quantitative formulations

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