This document discusses bioavailability and bioequivalence studies. It defines bioavailability as the amount of drug that reaches systemic circulation in an unchanged form. Bioequivalence studies compare the test and reference formulations to show they have the same rate and extent of absorption. The document outlines study design considerations and parameters assessed, including Cmax, Tmax, and AUC from plasma concentration data. It also discusses single and multiple dose studies, as well as parallel and crossover study designs used in bioequivalence testing.

2.  INTRODUCTION
 DEFINITION OF BIOAVAILABILITY
 OBJECTIVE OF BIOAVAILABILITY
 CONSIDERATION OF BIOAVAILABILITY
STUDY DESIGN
 METHOD FOR ACCESSING BIOAVAILABILITY
 BIOEQUIVALENCE STUDIES
 METHOD OF STUDYING
BIOEQUIVALENCE
 IMPORTANT QUESTION

3.  There are two different types of bio-availability studies.
The First Type :- of study involve in bioavailability of
new drug formulation .
 New drug are obtained after administration by different
route.
 And utilized optimum dosage regimen.
The second type :- of study involve in comparison of
test formulation with standard formulation. This type of
study is known as bio-equivalence(BE) Study.
Multisource drug product is a drug product that contains
 same active drug substance in same dosage form.
 and marketed by more than one pharmaceutical
manufacture.
Single source drug product is a drug product patent has not
yet expired so that only one manufacturer can develop it.
Continue….. 1

4.  single source drug product are usually brand Name drug
product.
 Formulation and manufacture of drug product can affect
Bio Availability and Stability of drug.
 Therefore generic drug product is therapeutically equivalent
to the brand name drug product.
 Drug product selection are major Responsibility.
 For Physician, Pharmacist and other prescriber.
 For such decision US-FDA (United State – Food and Drug
Administration) published Orange Book in print.
 That contain approved drug product with their therapeutic
equivalence.
2

5.  Amount of drug that reaches the systemic circulation in the
unchanged form and available at the site of drug action.
Bioavailable fraction (F), refers to the fraction of
administered dose that enters the systemic circulation
BIOEQUIVALENCE :-
 It is a relative term that compare the test formulation with
standard formulation .
 This is known as bioequivalence.
3

6.  Bioavailability studies are important in the
1) Development of suitable dosage form for new drug entity.
2) Determination of excipient,patient related factor and
possible interaction with other drug on the efficiency of
absorption.
3) Development of new formulation of drug.
4) Quality of a drug product during the early stage of
marketing to determine the influence of processing factor,
storage, and stability on drug absorption.
4

7. 1) BIOAVAILABILITY ABSOLUTE VS. RELATIVE:-
 ABSOLUTE BIOAVAILABILITY :-
 When the systemic availability of drug administered orally is
determined in comparison to it’s intravenous administration is called
as absolute bioavailability.
 It is denoted by symbol F.
 AUC = area under the curve
 Intravenous dose is selected as a standard due to it’s 100%
bioavailability.
 If the drug is poorly water soluble, intramuscular dose can be taken
as a standard.
 It’s determination is used to characterized a drug’s inherent
absorption properties from extravascular site.
Continue….. 5

8.  RELATIVE BIOAVAILABILITY :-
 Relative bioavailability is the availability of drug, drug product
as compare to recognized standard.
 It is denoted by symbol (Fr ).
2) SINGLE DOSE VS MULTIPLE DOSE STUDY:-
SINGLE DOSE:-
6
Continue…..
 Single dose study are very
common, easy, less exposure,
and less tedious.
 It is useful for measurement
of plasma or serum
concentration of drug at
different time interval.

9.  In single dose study such determination are the peak drug
concentration, time for peak , half life, duration of drug
effect, AUC.
 Dose of drug is increased the peak effect and duration of drug
action are increased.
 However time for peak effect is unchanged.
MULTIPLE DOSE:
 Multiple dose study exposed to the more drug.
 And highly tedious and time consuming method.
 More accurately reflect the manner in which drug should be
used.
 Easy to predict the peak and valley characteristic of drug .
 Require collection of fewer blood sample.
 In multiple does study steady state has been reached.
 Drug should be administered for 5 to 6 elimination half – lives
before collecting the blood sample.
7

10.  The method is summarized in following table.
METHOD PARAMETER
(A) Pharmacokinetic
(1) Plasma drug
concentration
Time for peak plasma concentration.(t max)
Peak plasma drug concentration. (C max)
Area under the plasma drug concentration
time curve(AUC)
(2) Urinary drug
excretion
 Cumulative amount of drug excreted in the
urine. (Du)
Rate of drug excretion in the urine. (dDu/dt)
Time for maximum urinary excretion.
8
Continue…..

11. METHOD PARAMETER
(B) Pharmacodynamic
(1) Acute
pharmacodynamic
effect
Maximum pharmacodynamic effect (E max )
Time for maximum pharmacodynamic
effect.
(2) Clinical observation Well controlled clinical trials.
(C) In vitro Drug dissolution.
9
Continue…..

12. [1] PLASMA DRUG CONCENTRATION DATA:-
 Measurement of drug concentrations in blood, plasma or
serum after drug administration is the most direct and
objective way to determine systemic drug bioavailability.
tmax:- The time of peak plasma concentration tmax .
 Time required to maximum drug concentration after drug
administration.
 At tmax peak drug absorption occur and rate of drug
absorption is equal to rate of drug elimination.
 Drug absorption still continues after tmax is reached at slower
rate.
10
Continue…..

13. Cmax:- The peak plasma drug concentration Cmax .
 The maximum plasma drug concentration obtain after oral
administration of drug.
 Cmax indicate the drug is systemically absorbed to provide
therapeutically response.
 The unit of Cmax are concentration unit.(mg/mL)
AUC:-The area under the plasma level time curve.
 AUC is a measurement of drug bioavailability.
 Total amount of drug that reaches the systemic circulation.
 AUC is the area under the drug plasma level time curve from
t=0 to t= ∞ and is equal to the amount of unchanged drug and
divided by clearance .
 AUC is independent of the route of administration and process
of drug elimination.
 The unit of AUC are concentration time.(g hr/mL)
11
Continue…..
F= Fraction of dose
D0= Dose

14. [2] URINARY DRUG EXCRETION DATA:-
 Urinary drug excretion data is an indirect method for
estimating bioavailability.
 In addition, timely urine sample must be collected and total
amount of urinary drug excretion must be obtained.
 Du:- The cumulative amount of drug excreted in the urine,
Du is directly related to the total amount of drug absorbed.
 Urine sample are collected after administration of drug
product.
 Each urine sample is analysed for free drug using specific
assay.
12
Continue…..
The relationship between cumulative
amount of drug excreted in urine and
plasma level – time curve is shown in
figure.
When drug is completely eliminated at
point C, the plasma concentration
approaches zero, and maximum amount
of drug excreted in urine, Du is
obtained.

15.  dDu/dt:- The rate of drug excretion.
 Most drug are eliminated by first-order rate process.
 The rate of drug excretion is dependent on the first-order
elimination rate constant k, and plasma drug concentration
Cp.
 In the maximum rate of drug excretion, (dDu/dt) is at point B.
 Whereas the minimum rate of drug excretion is at point A and
C .
 Thus a graph comparing the rate of drug excretion with
respect to time should be similar in shape as the plasma level-
time curve for that drug.
 t∞ :- The total time for the drug to be excreted.
13
Continue…..
In fig. A-B is related to the rate of
drug absorption, whereas point C is
related to the total time required after
drug administration for the drug to be
absorbed and completely excreted.
The t is a useful parameter in
bioequivalence study.

16. [3] ACUTE PHARMACODYNAMIC EFFECT:-
When bioavailability measurement by pharmacodynamic method
is difficult, inaccurate or non reproducible.
Acute pharmacologic effect such as change in ECG or EEG
reading etc. is related to the time course of given drug.
An acute pharmacodynamic effect such as effect on forced
expiratory volume[FEV] or skin blanching can be use as an index of
drug bioavailability.
The acute pharmacodynamic effect is measured over period of
time after administration of drug product.
USE:
Acute pharmacodynamic effect determine bioavailability
generally requires demonstration of dose response curve.
Bioavailability is determined by characterization of dose response
curve.
The use of pharmacodynamic endpoint for the determination of
bioavailability and bioequivalence is much more variable than the
measurement of plasma or urine drug concentration.
Continue….. 14

17. 15
[4] IN VITRO STUDIES:-
Drug dissolution studies may under certain conditions give an
indication of drug bioavailability.
Ideally, the in vitro drug dissolution rate should correlate with in
vivo drug bioavailability.
Dissolution studies are often performed on several test
formulations of the same drug.
The test formulation that demonstrates the most rapid rate of
drug dissolution.
In vitro will generally have the most rapid rate of drug
bioavailability in vivo.
The FDA may also use other in vitro approaches for establishing
bioequivalence.
For example, cholestyramine resin is a basic quaternary
ammonium action-exchange resin that is hydrophilic, insoluble in
water, and not absorbed in the gastrointestinal tract.
The bioequivalence of cholestyramine resin is performed by
equilibrium and kinetic binding studies of the resin to bile acid
salts.

18.  Bioequivalence is commonly observed that there are several
formulation of the same drug, in the same dose, in a similar
dosage form and given by same route.
 There are four types of equivalence.
►TYPES OF EQUIVALENCE :
 Chemical Equivalence
 Pharmaceutical Equivalence
 Bioequivalence
 Therapeutic Equivalence
16
Continue…..

19. 1) Chemical Equivalence:-
 When two or more drug products contain the same labeled
chemical substance as an active ingredient in the same
amount.
2) Pharmaceutical Equivalence:-
 Drug product that contain same active drug ingredient and
are identical in strength, concentration, dosage form and
ROA are called pharmaceutical equivalence.
 Pharmaceutical equivalent drug product must the identical
standard of strength, quality, purity and identity but differ
in colour, flavor, shape, packaging, excipient, preservative,
expiry date, and labeling within certain limit.
3) Bioequivalence:-
 It is relative term drug substance in two or more identical
dosage forms, reaches the systemic circulation at the same
relative rate and to the same relative extent.
Continue….. 17

20. 4) Therapeutic Equivalence:-
 When two or more drug product that contain same
therapeutically active ingredient, identical pharmacological
effect and control the disease to the same extend.
18

21.  There are four different type of methods.
1. In vivo bioequivalence study
2. In vitro bioequivalence study
3. Pharmacodynamic study
4. Comparative clinical trial
Continue….. 19

22.  Currently three different studies may be require for solid oral
dosage form.
1) Fasting study
2) Food intervation study
3) Multiple dose study
▲TYPES OF DESIGN:
1) Parallel group design:-
 In parallel group design, subjects are divided randomly into
groups, each group receiving one treatment randomly.
 Here number of groups is same as number of treatments to be
compared.
 Each subject receives only one treatment.
Continue….. 20

23. 2) Cross over design:-
SUBJECTS
PERIOD
1 2
1 A B
2 B A
3
4
Each subject receives two or more different treatments on
successive occasions, are known as cross over design.
In this design, the number of treatments is same as the number
of periods.
Continue….. 21

24. CROSS OVER DESIGN PARALLEL GROUP DESIGN
Subject variability does not enter
into the error.
Subject variability is very much
and is included in error.
Number of subjects required are
less.
Number of subjects required are
more.
Due to long wash out periods,
subjects may drop out.
No fear of drop out.
If observations are missing,
design loses efficiency.
The statistical analysis is simple
even if some results are missing.
The trial will be too consuming. Investigated drug and/or time
metabolite have long half life, this
design can be used.
22

25.  There are two types of design
1) LATIN square or completed cross-over design.
2) Replicated cross-over design.
1)Latin Square or Complete Cross-Over Design:-
 In this design, each subject receives each formulation just
once and each formulation is administered just once in each
study period.
 The basic element of this design are shown in table.
 TWO-WAY CROSS-OVER
Group No. Subjects in group Treatment for period No.
I II
(1) 1,2,3,4,5,6 A B
(2) 7,8,9,10,11,12 B A
Continue….. 23

26.  THREE-WAY CROSS-OVER
Continue….. 24
Group No. Subjects in group Treatment for period No.
I II III
(1) 1,2,3,4,5,6 A B C
(2) 7,8,9,10,11,12 B C A
(3) 13,14,15,16,17,18 C A B
 In a two-way cross-over study, 12 subjects are used to study the
BE of two formulation, treatment A and treatment B.
During the first study period, subjects 1to 6 receive treatment A,
while subjects 7 to 12 receive treatment B.
A second study period is initiated after the washout period.
In the second study period, subjects 1 to 6 now receive treatment
B and subjects 7 to 12 receive treatment A.

27. ADVANTAGES OF LATIN SQUARE DESIGN:-
 In this design, each subjects is his own control, and subject-to-
subject (inter-subject) variation is reduced.
 Variation due to sequence, period, and treatment (formulation) are
reduced, so that all patients do not receive the same drug product
on the same day in the same order.
 It requires less number of subjects to get meaningful results.
DISADVANTAGES OF LATIN SQUARE DESIGN:-
 It requires longer time to complete the study.
 The higher the biological half life of drug, the longer will be the
time required for completing the study.
 It takes a longer time to complete the study as the number of
formulations increases.
 Increased number of study periods leads to high subject dropouts
and the study becomes difficult.
 Medical ethics does not allow too many trials on a subject
continuously for a longer time.
Continue….. 25

28.  A two-period study is a study that is performed on two
different days (time periods) separated by a washout period.
 A sequence refers to the number of different orders in the
treatment groups in a study.
 For example, a two-sequence, two-period study would be
designed as follows:
Continue….. 26
Period 1 Period 2
Sequence 1 T R
Sequence 2 R T
Where R= reference and T= treatment

29. 2)Replicated Cross-Over Design:-
 Replicated cross-over designs are used for the determination
of individual bioequivalence, to estimate within- subject
variance for both the test and reference drug products, and to
provide an estimate of the subject- by formulation interaction
variance.
 Generally, a four period, two-sequence, two-formulation
design is recommended by the FDA.
Period 1 Period 2 Period 3 Period 4
Sequence 1 T R T R
Sequence 2 R T R T
Where R= reference and T= treatment
The same reference and the same test are each given twice
to the same subject.
In this design, reference-to-reference and test-to-test
comparisons may also be made.
Continue….. 27

30. ADVANTAGES OF CROSS-OVER DESIGN:-
 Minimize intersubject variability in plasma drug level.
 Minimize intersubject variability affecting bioavailability of a
subsequently administered product.
 Minimize variation due to time effect.
DISADVANTAGES OF CROSS-OVER DESIGN:-
 Time may be longer.
 The study becomes more difficult and subject dropout rate may
increase.
28

31.  In following circumstances equivalence may be assessed by
the use of in vitro dissolution testing.
 (B.1) Drugs for which the applicant provide data to
substantiate all of the following:
1) Highest dose strength is soluble in 250 ml of an aqueous
media over the pH range of 1-7.5 at 37°C.
2) Atleast 90% of the administered oral dose is absorbed on
mass balance determination or comparison to intravenous
reference dose.
3) Speed of dissolution demonstrated by more than 80%
dissolution within 15 minute at 37°C using IP apparatus 1 at
50rpm or IP apparatus 2 at 100rpm
Continue….. 29

32.  (B.2) Different strength of drug manufacture by the
same manufacture where all the following criteria
are fulfilled:
1) The qualitative composition between the strength is
essentially the same.
2) The ratio of active ingredient and excipient between the
strength is essentially the same.
3) The method of manufacture is essentially the same.
4) An appropriate equivalence study has been performed on
atleast one of the strength of the formulation.
30

33.  Studies in healthy volunteers of patients using
pharmacodynamic parameters may be used for establishing
equivalence between two pharmaceutical products.
 These studies may become necessary.
1) If quantitative analysis of the drug and/or metabolite in
plasma or urine cannot be made with sufficient accuracy
and sensitivity.
2) If measurement of drug concentrations cannot be used as
surrogate endpoints for the demonstration of efficacy and
safety of the particular pharmaceutical product. Eg. Topical
products without an intended absorption of the drug into the
systemic circulation.
31

34.  It is carried out when
1) The plasma concentration time-profile date may not be
suitable to assess equivalence between two formulations.
2) Pharmacodynamic studies cannot be performed because of
lack of meaningful pharmacodynamic parameters, which
can be measured.
3) Pharmacodynamic and pharmacokinetic studies are not
feasible.
32

35. Q.-1: Define bioavailability and bioequivalence and enlist
objective of bioavailability?
Q.-2: Write note on bioavailability absolute vs. relative ?
Q.-3: Enlist methods for assessing bioavailability and
describe any one.
Q.-4: Describe layout of Latin square design with
advantage and disadvantage ?
Q.-5: Type of bio-equivalence?
33