By the end of this session, students will be able to:

• Describe the concept and characteristics of

• Appreciate the importance of bioavailability studies

• Describe the different methods for the assessment of

• Discuss on the various considerations in bioavailability

• Justify the role of bioavailability studies in drug
approval process

• Identification of right plan for conducting bioequivalence

• Analyze the data produced from the bioequivalence studies



Rate and Extent of absorption of unchanged drug from its
dosage form is known as bioavailability

Bioavailable Dose

The dose available to patient.

Systemic Availability

Amount of drug that reaches systemic circulation (i.e.
extent of bioavailability).

Bioavailable fraction

The fraction of administered dose that enters systemic

F = Bioavailable Dose / Administered Dose

Why Bioavailability Studies??

Bioavailability studies performed for both approved active
drug ingredients and not yet approved for marketing by FDA

As standards like identity, strength, quality and purity
should be met

Un-marketed drugs which do not have full NDA approval by
FDA, in vitro / in vivo bioequivalence studies must be performed

In vivo bioavailability studies are also performed for new
formulations of active ingredients that have full NDA approval

These studies are useful in determining safety and efficacy
of drug products.

Objectives of
Bioavailability studies

Development of suitable dosage form for new drug entity

Development of new formulations of existing drugs

Determination of influence of excipients, patient related
factors and possible interactions with other drugs on efficiency of absorption.

Control of quality of drug product during early stages of

Considerations in
bioavailability studies

Bioavailability absolute v/s relative

Single dose vs. multiple dose

Human volunteer – healthy subject vs. patients

Types of

Single dose vs.
multiple dose

Multiple dose study has several advantages like:

1. More accurately reflects the manner in which the drug
should be used

2. Drugs levels are higher due to cumulative effect which
makes its determination possible even by less sensitive analytical methods

3. Better evaluation of the performance of controlled release
formulation is possible

4. Small inter-subject variability is observed which allows
use of fewer subjects

5. Nonlinearity in pharmacokinetics, if present, can be
easily detected.

Single dose

• Very common & easy

• Less exposure to drug & less tedious

• Difficult to predict steady state

Multiple dose

• Difficult to control

• More exposure to drug & tedious

• Time consuming

volunteer – healthy subject vs. patients

Female volunteers are used only ideally, the bioavailability
study should be carried out in patients for whom the drug is intended to be


Patient is benefited from the study

Reflects better therapeutic efficacy of drug

Drug absorption pattern in disease state can be evaluated

Avoids the ethical quandary of administering drug to healthy
subjects when oral contraceptives are to be tested

No. of subject- extent of inter- subject variability –
minimum required to obtain reliable data

They must be informed about the importance of study,
conditions to be followed and possible hazards if any. Medical examination
should be performed

Drug washout period for minimum of ten biological half-lives
must be allowed for between two studies in same subject.

Measurement of Bioavailability

methods (Indirect)

Plasma level time studies

Urinary excretion studies

methods (Direct)

Acute pharmacological response

Therapeutic response

level time studies

• Plasma level time studies or the plasma concentration – time
curve or blood level curve.

• A direct relationship exists concentration of drug at the
site of action & concentration of drug in the plasma.

• Serial blood samples are taken after drug administration
& analyzed for drug concentration.

• A typical blood level curve obtained after oral administration
of drug.

• Three parameters of plasma level-time studies considered
important for determining bioavailability are:

Cmax – gives an
indication whether drug is sufficiently absorbed systematically to provide
therapeutic response.

tmax – gives
indication of rate of absorption.

AUC – gives
indication of extent of absorption.

• Extent of
Bioavailability determined by

F = [AUC]oral Div / [AUC]iv Doral

Fr = [AUC]test   Dstd
/ [AUC]std    Dtest

F= Absolute bioavailability

Fr= Relative bioavailability

With multiple dose study, extent of bioavailability is given

excretion studies

• Based on principle that the urinary excretion of unchanged
drug is directly proportional to plasma concentration of drug

• Used for drugs extensively excreted unchanged in the urine
e.g. thiazide diuretics and drugs that have urine as site of action (nitrofurantoin)

• Three major parameters examined in urinary excretion data
obtained with single dose study are:

1) (dXu/dt)maxMaximum
urinary excretion rate. It is analogous to Cmax derived from
plasma level time studies since rate of appearance of drug in urine is
proportional to its concentration in systemic circulation.

2) (tu)max
Time for maximum excretion rate. It is analogous to tmax
of plasma level time data.

3) Xu
Cumulative amount of drug excreted in urine. It is related to AUC
of plasma level time data.

• Extent of bioavailabilityis calculated from equation

F = (Xu∞)oral Div
/ (X
u∞)iv Doral

Fr = (Xu∞)test  Dstd
/ (X
u∞)std   Dtest


F = (Xu,
ss)test  Dstd
test / (Xu,ss )std Dtest  τ std

Thus, the bioavailability can be calculated in the steady

of Urinary Excretion Method

Useful when there is lack of sufficiently sensitive
analytical techniques to measure concentration of drug in plasma

Noninvasive method therefore better subject compliance

Convenience of collecting urine samples in comparison to
drawing of blood periodically

If any case the urine drug concentration is low, assaying of
larger sample volume is relatively more

Direct measurement of bioavailability, both absolute &
relative is possible without the necessity of fitting the data to the
mathematical model

Pharmacologic Response

• Used when bioavailability measurements by pharmacokinetic
method is difficult, inaccurate or non-reproducible

• Method requires measurement of Reponses for at least 3
biological half-lives of drug


Pharmacologic response tend to be more variable and accurate
correlation between measured response & drug available from formulation is

The observed response may be due to an active metabolite
whose concentration is not proportional


Based on observing clinical response to a drug formulation given
to patients suffering from disease for which it is intended to be used

A major drawback of this method – Quantization of observed
response is too improper to allow for reasonable assessment of relative
bioavailability between two dosage forms of same drug

Concept of


It is a term that compares drug products w.r.t. a specific
characteristic or function or to a defined set of standards

Several types of

1. Chemical
Two or more drug products contain the same labeled chemical substance

2. Pharmaceutic
Two or more drug products are identical in strength, quality,
purity, content uniformity and disintegration & dissolution characteristics

3. Bioequivalence:-
It denotes that drug substance in two or more identical dosage forms, reaches
the systemic circulation at the same relative rate and relative extent

4. Therapeutic
It indicates that two or more drug products that contain same
therapeutically active ingredient, elicit identical pharmacologic effect

and its Importance

• A drug product that differs from reference material in its
rate of absorption, but not in its extent of absorption may be considered

• Bioequivalence requirement may be imposed by FDA on the
basis of following:

1. Various drug products do not give comparable therapeutic
effect as seen from well controlled clinical trials.

2. Availability of multiple brand of same compound each with
claims for perfect bioavailability.

3. It is important for drug with narrow therapeutic index.

• For bioequivalence studies, both test and reference drug
formulations contain the pharmaceutical equivalent drug in same dose strength,
in similar dosage forms & both are given by same route of administration

• Study is performed in normal, healthy male volunteers who
have given informed consent to be in the study

• Study might exclude any volunteers who are smokers, have
known allergies to drug, are overweight or have taken any medications within a
specified period (1 week) prior to the study


1. Completely randomized design

2. Randomized block design

3. Repeated measures, cross-over & carry over design

4. Latin squire design

randomized design

• All treatments are randomly allocated among all
experimental subjects

• Method – label
all subjects with the same number of digit

Ex: If 20 subjects are available then number them from 1-20.

• Randomly select the non-repeating numbers for the first
treatment then repeated for all other treatments


1. Easy to construct

2. Can accommodate many number of subjects & treatments

3. Easy & simple to analyze


1. It is best suited for situation where few treatments are

2. All subjects must be as homogenous as possible

block design

• Subjects are first sorted into homogenous group called as
blocks then treatments are assigned at random within blocks


• Subjects having similar background characteristics are
formed as blocks, then they are randomized within each block

• Randomization for different blocks are done independent of
each other


 • It can accommodate
any number of treatments or replications

• Different treatment need not to have equal sample size

• Easy to construct & analyze

• If an entire block needs to be dropped from the analysis
for some reason, analysis is not complicated

• More precise results are obtained

• Variability in experimental units can be introduced to
widen validity


• Missing observation within a block require more complex

• The degree of freedom of experimental error are not as
large as with a completely randomized design

measures, cross-over & carry-over designs

• It is also a randomized block design in which the same
subject serves as a block

• The same subject is utilized for each of the treatments
under study, since repeated measures on each subject are done so it is called
as repeated measure design

• The study involves several treatments or a single
treatment evaluated at different point of time

• The administration of two or more treatments one after the
other in a specified or random order to the same group of patients are called
as crossover or change-over design

• The drawback of crossover studies is the potential for
distortion due to carryover that is residual effects from preceding treatments

• To prevent carryover effects a wash out period of 10
elimination half-life should be maintained

• Complete randomization is used to randomize the order of
treatments for each subject.

• Randomization for different for subjects are independent
of each other

Repeated measures,
cross-over & carry-over designs


• Good precision for comparing treatments because experimental
errors are excluded

• If the effect of a treatment overtime need to be known
then observing the same subject at different intervals of time , then other way

• It is economic on the subjects

• There may be an order effect, which is connected with the
position in the treatment order

• There may be a carryover effect, which is connected with
the preceding treatments

square designs

• In other methods the subjects remains on treatment from
start to the end of experiment so called as continuous trials

• Each subject receive each treatment during the course of
the experiment

• It is a 2 factor design (subjects & treatments) with
one observation in each cell

• In this design 3 or more treatments are to be compared
& carry over effects are balanced

• Rows represent subjects, and columns represent treatments

• r rows & r columns such that each of the r square cell
contains one & only one of the r letters representing the treatments

• Each letter appears only once in the row & column
Latin square design is considered as standard if the first row & first
column consist of the r letter in alphabetical order

• Randomised, balanced, crossover latin square design are
commonly used for bioequivalance studies


Minimizes the inter subject variability in plasma drug

Minimizes the carry over effects which could occur (intra
subject variability)

Minimizes the variation due to time effect

Focus on the formulation variables which are key to the


Small degree of freedom for experimental errors when only a
few treatments are used

The randomization required is somewhat more complex than the
other design

Takes a long time since an appropriate wash out period b/w 2

When the number of formulation to be tested is more, the
study becomes more difficult & subject dropout rates are also high

equivalence protocol

1. Title

– Principal investigator

– Project number & date

2. Study objective

3. Study design

a) Design

b) Drug products

– test products

– Reference products

c) Dosage regimen

d) Sample collection schedule

e) Housing

f) fasting/ meals

g) Analytical method

4. Study population

a) Subjects

b) Subject selection

– Medical history

– Physical examination

– Laboratory tests

c) Inclusion /exclusion criteria

-Inclusion criteria

-Exclusion criteria

d) Restriction/ prohibition

5. Clinical

– Dosage &drug administration

– Biological sampling schedule

– Activity of the subjects

6. Ethical

a) Basic principle

b) Institutional review board

c) Informed consent

d) Indication for the subject’s withdrawal

e) Adverse reaction or emergency situation

7. Facilities

8. Data analysis

a) Analytical validation

b) Statically treatment of data

9. Drug

10. Appendix

Interpretation of Bioequivalence data

• After data has been collected, statistical methods must be
applied to determine the level of significance of any observed difference in
the rate or extent of absorption in order to establish bioequivalence between two
or more drug product.

• Typically, an analysis of variance (ANOVA) method is
applied to determine statistical difference (p). if p <  0.05 then the product is not considered
statically significant

• If relative bioavailability of test formulation is in
range of 80 to 120% of reference standard, it is considered bioequivalent.

• The difference between bioavailability of test formulation
should not be greater than +/- 20% of average reference standard.


• Bioavailability is the fraction of administered dose that
enters systemic circulation in an unchanged form

• Bioavailability studies performed for both approved active
drug ingredients and not yet approved for marketing by FDA

• Measurement of bioavailability involves both direct and
indirect method

• Bioavailability is the determination of rate and extent of
absorption of unchanged drug from its dosage form

• Bioavailability studies are performed for both the drug
and excipients as a mandatory requirement of FDA to ascertain the safety and
efficacy of drug products

• The main objective of bioavailability studies is
development of suitable dosage forms for new drug entities as well as existing

• The various considerations in bioavailability studies are:

   Absolute and
relative bioavailability

  Single dose vs.
multiple dose

   Human volunteer –
healthy subject vs. patients

• Bioavailability can be estimated by Pharmacokinetic (Blood
level studies and Urinary excretions studies) and     Pharmacodynamics methods (Acute pharmacological
response and therapeutic response

• The key pharmacokinetic parameters estimated by plasma
level studies (indirect method) are tmax, Cmax and AUC

• Urinary excretion studies are based on the principle that
the urinary excretion of unchanged drug is directly proportional to plasma
concentration of drug

• Pharmacologic response tend to be more variable and accurate
correlation between measured response & drug available from formulation is
difficult, hence indirect is preferred

• Bioequivalence is a term that compares drug products
w.r.t. a specific characteristic or function or to a defined set of standards.

• Different approaches for bioequivalence studies are
randomized block approach, completely randomized approach, repeated measures,
cross-over & carry-over design and latin square design

• Randomised and crossover latin square design are commonly
used designs for bioequivalence studies

• Analysis of variance (ANOVA) is generally is applied to
determine statistical difference (p) for bioequivalence data

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