The document outlines the key elements of a bioequivalence study protocol, including: 1. The objective is to show that the test and reference drug products have similar bioavailability when administered at the same dose. 2. The study design typically involves both fasting and fed conditions, uses a crossover or parallel group design, and involves pharmacokinetic analysis of blood samples taken over time. 3. Subject selection criteria aim to minimize variability through screening and exclusion of those with health issues that could impact drug absorption.

1. DELIBRATED BY: UNDER THE GUIDANCE OF
J.RAJESH Dr.SATYABRATA BHANJA M.Pharm,Ph.D
M.PHARM(CEUTICS) DEPARTMENT OF PHARMACEUTICS
256213886012 MALLARAEDDY COLLEGE OF PHARMACY

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

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

4. d.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 withdrawl
e.Adverse reaction and emergency procedures

5. 7.Facilities
8.Data Analysis
a.Analytical validation procedure
b.Statistical treatment of data
9.Drug Accountability
10.Appendix

6. Study Objective:
 The objective for a bioequivalence study is
that the drug bio availability from test and
reference products are not statiscally
different when administered to patients are
subjects at same molar dose under similar
experimental conditions.

7. Study Designs:
 For many drug products,the FDA, division
of bio equivalence, office of generic drugs
provides guidances for the performances of
in-vitro dissolution and in-vivo
bioequivalence studies
 Generally two bioequivalence studies
required for solid oral dosage forms
including

8. 1.A fasting study
2.A food intervention study
1.Cross Over Study Design:
 - Two formulations, even number of
subjects
 - Randomly divided into two equal groups

9. First Period:
 Each number of one group receive a single
dose of the test formulation and each
member of the other group receive the
standard formulations.
Subject Period 1 Period 2
1-8 Test Standard
9-16 Standard Test

10. 2.Latin Square Design:
- More than two formulations
Eg: A group of volunteers will receive
formulations in the sequence.
Volunteer
Number
Period 1 Period 2 Period 3
1 A B C
2 B C A
3 C A B

11. 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 many blood samples
 If each volunteer expected to receive atleast
2 formulations then such study can be
carried out using BIBD.

12. Volunteer
Number
Period 1 Period 2
1 A B
2 A C
3 B D
4 B C
5 C D
6 B D
7 C A
8 D A
9 C B
10 D B
11 D C
12 A B

13. 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
Treatment A Treatment B
1 2
3 4
5 6
7 8
9 10
11 12

14. 5.Replicate Cross Over Study Design:
 For highly variable drugs
 Allows comparisions of with in subject
variances
 Reduces the number of subjects needed
 4-periods, 2-sequences, 2 formulations
design(recommended)
 3-sequences, 3-periods, single dose
partially replicated

15. Period 1 2 3 4
Group 1 Test Reference Test Reference
Group 2 Reference Test Reference Test
Peeriod 1 Period 2 Period 3
A B C
B C A
C A B
A C B
B A C
C B A

16. Difference Between Parallel Group And
Cross Over Study Design:
Parallel Group Cross Over
Groups assigned different
treatments
Each patient receives both
treatments
Shorter duration Longer duration
Larger sample size Smaller sample size
No carry over effect Carry over effect

17. 6.Pilot Study:
 If the sponsor chooses in a small number of
subjects
 To access a variability, optimise the sampla
collection time intervals, and provide other
information
 Eg:Immediate release products – careful timing
of intial samples-avoid a sub sequent finding that
the first samole collection, occured after the
plasma concentration peak.
 Modified released products: To determine
sampling shedule- Assess log time and dose
dumping

18. Analytical Methods:
 Analytical methods used in an in-vivo bio
availability, bio equivalence, or pharmacodynamic
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.

19. 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

20.  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 disfunction
 H/o jaundice in past 6 months
 Chronic diseases Eg: asthma, arrthiritis
 Psychiatric illness

21. 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 ay 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.

22.  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.

23. 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.

24.  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.

25.  For first-order process , the time necessary for a
complete elimination would be infinity.
 A rule of thumb sampling in a blood level study is
to sample for 3-5 half lifes of the drugs.
 If half life is not known , sampling should proceed
untill 1/10 or 1/20 of the peak levels are reached.
 In the case of urinary excretion studies, the same
principles apply.
 The advantage of urinary excretion studies are
1.it involes non-invasive method of sampling.
2.the drug concentration in the urine is
greaterthan blood/serum allowing easy estimation
of the drug.

26.  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.

27. 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,

28. 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.

29.  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

30. 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)

31.  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

32. 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

33. 5.Pharmacokinetic Parameters and Tests
Definitions and calculaton
Statistical tests
Drug levels at each sampling time and pharmacokinetic
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

34. 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%.

35.  7. appendices
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
potency determination


36.  9. batch size and formulations
batch record
quantitative formulations

37. Applications of pharmacokinetics in drug
development,NDDS:
Drug research:
 New Drug
 i.Discovery
 ii.Development
 Drug Discovery :- Hypothesis of target enzyme on receptor for
particular disease
 Suitable models
 Screening of new drug
 Screening of in vitro/in vivo biological activity
 Drug development :
 Empirical Dosage regimen
 Semi empirical Dosage regimen
 Structaral activity
 These are development of chemical structures.

38.  Emprical Dosage Regimen :-
 It is designated by the physician based on emprical
clinical data,personal experience and clinical
observations.This approach is however,not very
accurate
 Semi emprical Dosage regimen :
 It is most accurate approach and is based on the
pharmacokinetics of drug in the individual patient.The
approch is suitable for hospitalised patients but is
quite expensive.
 Pharmacokinetics-AUC,CMAX,TMAX.
 Pharmacodynamics-On set of action, On set of
time,MSC,MEC,MTC.

39.  Applications:
 Design of new drug development:
 Design a level of optimum formalation for better use of drug
 Design a level of control and sustained released
formulation.
 Selection of appropriate new drug administration then the
selection of right drug for the particular disease
 Design and development of drug design,processing dosage
regimen
 Study of in vitro, in vivo studies
 Study of bioequivalant studies.
 The study of pharmacokinetics and pharmacodynamic
relationship
 Development of rational drug design,development rational
dose frequency and duration.

40.  Determaine the drug drug interactions
 Design of appropriate multiple dosage regimen
 Therapeutic dose of indiviual drug
 Pharmacokinetics charactarization of drug selection of the suitable novel
drug delivary system.
 NDDS parameters are the
 i.T1/2
 ii.T90
 iii.Elimination rate constant
 iv. Area under curve
 v.Volume of distibution
 vi.Steady state concentration
 vii.Mean residance time
 ix.Dosage form index
 x.Relative area
 xi.Absorption rate

41.  References:
 BIOPHARMACEUTICS AND
PHARMACOKINETICS
 second edition
o V.Venkateswarlu
o APPLIED BIOPHARMACEUTICS &
PHARMACOKINETICS
o sixth edition
o Leon Shargel
o Susanna wu-Pong
o Andrew yu