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.
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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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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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