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Bioavailability and bioequivalence studies
BIOAVIALABILITY STUDY
 BIOAVAILABILITY STUDIES FOR NEW DRUGS


In vivo Bioavailability studies are performed for new
drugs to establish essential pharmacokinetic parameters
including the rate of absorption, extent of absorption, rates of
excretion and metabolism, and elimination half life after a
single and multiple doses administration.
Bioavailability studies are also conducted to determine the
influence of excipients, manufacturing procedures,
packaging materials and patient related factors on the
biological performance of a new drug formulation.
BIOAVAILABILITY STUDIES FOR APPROVED DRUGS
Bioavailability
studies for approved drugs are performed to develop a new
dosage form to improve on existing dosage form.
In
approving a drug product for marketing, the FDA must ensure that the
drug product is safe and effective for its labeled indications of use.
In
addition, the drug product must meet all applicable standards of
identity, strength, quality and purity…hence a drug product is fisrt
subjected to all applicable official laboratory tests.
Finally,
the FDA requires bioavailability / pharmacokinetic
studies and where necessary bioequivalence studies to ensure
the safety and efficacy of the drug product.
In
bioequivalence studies, test drug product is compared with
a reference standard ( FDA approved drug product).
Test
product is called bioequivalent if it produces a
bioavailability equivalent to that of the reference standard.
BIOAVAILABILITY STUDY PROTOCOL
 The assessment of bioavailability of several drug products most often
requires the measurement of drug and/or metabolite levels in either the
blood or the urine.
 The bioavailability of a drug from a dosage form depends on the dose and
route of administration, time of administration, subjects and dosage form.
 The aim of bioavailability study is to find out the dosage form influence
on the biological performance of the drug….hence the bioavailability
study protocol used should be of sufficient sensitivity to detect differences
in the rate and extent of absorption that are attributable only to dosage
form variability and should avoid variabilities due to other factors
 Sometimes, due to inherent properties of the drug, analytical difficulties
are encountered that will preclude the measurement of drug or
metabolite levels in body fluids, so other techniques are used to assess
bioavailability.
 The drug may be labeled with a radioactive tag, a pharmacological or
clinical response may be measured, or studies may be performed on
animals.
BIOAVAILABILITY STUDY PROTOCOL
STUDY OBJECTIVE
STUDY DESIGN
•Experimental Design
•Wash out period
•Drug Products
1.Test Products
2.Recognized Standards
•Route of administration
•Dosage Regimen
•Frequency and duration of sampling
•Randomization of drug administration
•Single- versus multiple dose study
•Subjects
1.Healthy subjects versus patients
2.Subject selection
a.Medical History
b.Physical examination
c.Laboratory tests
3.Study conditions
•Analysis of biological fluids
METHODS OF ASSESSMENT OF
BIOAVAILABILITY
•Plasma data
•Urine data
•Acute pharmacological effect
•Clinical response
ANALYSIS AND PRESENTATION OF DATA
•Statistical treatment of data analysis
of variance [ANOVA]
•Format of data.
STUDY OBJECTIVE
The objective of bioavailability study decides the study protocol
A study meant for estimating essential pharmacokinetic parameters differs
significantly from a bioequivalence study meant for comparing the test
formulation with reference of standard.
STUDY DESIGN
Various factors to be considered in conducting a bioavailability study
since the rate and extent of absorption of a drug into the systemic
circulation, its distribution and elimination are influenced by a variety of
factors.
Subject factors such as age, sex, disease states, food habits, general health
condition, body weight, experimental design, time of administration, time
of sampling, analytical method used and compartment model used in
estimating pharmacokinetic parameters contribute to the observed blood
concentration time profile.
It is necessary to consider all these important factors in a study design.
PARALLEL DESIGN
The aim of experimental designs is to minimize the experimental variables
and to avoid a bias.
In a parallel design, two formulations are administered randomly to the
volunteers.
Disadvantage – Intersubject variation
CROSS- OVER DESIGN
Generally, a substantial inter subject variability exists in the drug levels
achieved from any given dose of medication in a panel of subjects.
The cross- over design mininmizes the effect of inter- subject variability in
the study by using each subject as his or her own control

Types of Cross- over designs
•Latin square Cross- over design
•Balanced Incomplete Bock Design [ BIBD]
LATIN SQUARE CROSS- OVER DESIGN
A standard approach for conducting a comparative bioavailability study is to
use a randomized, balanced, cross-over design called a Latin Square or
Complete Cross-over design.
In this design…
Each subject receives just once each formulation
Each formulation is administered just once each study period
TWO WAY CROSSOVER
GROUP NUMBER
SUBJECTS IN GROUP
1
2
TREATMENT FOR PERIOD
I
II
1,2,3,4,5,6
A
B
7,8,9,10,11,12
B
A
THREE WAY CROSSOVER
GROUP NUMBER
SUBJECTS IN GROUP
1
TREATMENT FOR PERIOD
I
II
III
1,2,3,4,5,6
A
C
B
2
7,8,9,10,11,12
B
A
C
3
12,13,14,15,16,17,18
C
B
A
FOUR WAY CROSSOVER
GROUP NUMBER
SUBJECTS IN GROUP
1
TREATMENT FOR PERIOD
I
II
III
IV
1,2,3,4,5,6
A
B
C
D
2
7,8,9,10,11,12
B
D
A
C
3
12,13,14,15,16,17,18
C
A
D
B
4
19,20,21,22,23,24
D
C
B
A
LATIN SQUARE CROSS- OVER DESIGN FOR BIOEQUIVALENCE STUDY
OF THREE DRUG PRODUCTS IN SIX HUMAN VOLUNTEERS
SUBJECTS
TREATMENT FOR PERIOD
I
II
III
1
A
B
C
2
B
C
A
3
A
A
B
4
A
C
B
5
C
B
A
6
B
A
C
LATIN SQUARE CROSS- OVER DESIGN FOR BIOEQUIVALENCE STUDY
OF FOUR DRUG PRODUCTS IN SIXTEEN HUMAN VOLUNTEERS
SUBJECTS
TREATMENT FOR PERIOD
I
II
III
IV
1
A
B
C
D
2
B
C
D
A
3
C
D
A
B
4
D
A
B
C
5
A
B
D
C
6
B
D
C
A
7
D
C
A
B
8
C
A
B
D
9
A
C
B
D
10
C
B
D
A
11
B
D
A
C
12
D
A
C
B
13
A
C
D
B
14
C
D
B
A
15
D
B
A
C
16
B
A
C
D
ADVATAGES OF LATIN SQUARE CROSS- OVER DESIGN
•It minimizes the effect of inter subject variability in the study by using each subject as
his or her own control.
•It minimizes the carry over effects
•It minimizes the time effect on bioavailability since each dosage form is administered in
each study period.
•It requires less number of subjects to get meaningful results.
DISADVATAGES OF LATIN SQUARE CROSS- OVER DESIGN
•It requires longer time to complete the study since a washout period exists between two
study periods….t1/2
•The time to complete the trails depends on the number of formulations to be evaluated
in the study…more no…..more time
•Increased number of study periods leads to high subject dropouts and the study
becomes difficult
•Medical ethics does not allow too many trails on a subject continuously for a longer
time
BALANCED INCOMPLETE BLOCK DESIGN [BIBD]
BIBD eliminates many of the difficulties encountered with the Latin square
design.
Salient aspects….
Each subject receives not more than two formulations
Each formulation is administered the same number of times
Each pair of formulations occurs together in the same number of
subjects.
In this design, each subject receives two formulations, each formulation is
administered six times and each pair of formulations occurs together in two
subjects (AB, AC, AD, BC, BD & CD)
BALANCED INCOMPLETE BLOCK DESIGN [BIBD]
SUBJECTS
TREATMENT FOR PERIOD
I
II
1
A
B
2
B
A
3
A
C
4
C
A
5
A
D
6
D
A
7
B
C
8
C
B
9
B
D
10
D
B
11
C
D
12
D
C
WASHOUT PERIOD
In a Latin Square cross- over study design each subject receives each
formulation and even in BIBD each subject receives two formulations at
different occasions.
The time interval between the two treatments is called WASHOUT PERIOD
Washout period is required for the elimination of the administered dose of
a drug so as to avoid carryover.
Official guidelines recommend 10 half lives should be allowed between
successive treatments.
Washout period is a function of the half life and the
dose of the drug administered.
The no. of wash out periods depends upon the type of
crossover design used and the no. of formulations to be
evaluated.
Metabolites of the drug should also be eliminated from
the body before the next treatment…..more water
soluble…rapid elimination.
DRUG PRODUCTS
TEST PRODUCTS
Test products may be new drug formulations developed by a pharmaceutical
technologist or new dosage forms of an existing drug.
A test product may be compared to a reference standard recognized by the FDA
for getting approval for marketing the drug product.
Test products are evaluated for the following reasons….
To select best dosage form of a new drug or existing drug among different dosage
forms
To select best dosage form of a new drug or existing drug among different
formulations that have shown equal performance that have shown equal performance
in in-vitro tests.
To compare biological performance of a test product to that of a recognized
standards ( Bioequivalence studies)
REFERENCE STANDARD
A chemical or generic product has to be compared with some standard
dosage form to verify its in-vivo performance.
FDA accepts any innovator drug product as a reference standard.
ROUTE OF ADMINSTRATION
Generally orally administered dosage forms are subjected for bioavailability
studies.
Others such as buccal, transdermal, intramuscular should also be
evaluated for their biological performance.
Orally administered dosage forms show a much variation in their
performance because of inter- subject and intra- subject variations.
All the dosage forms administered by an extra vascular route do require a
bioavailability assessment.
SINGLE- VERSUS MULTIPLE- DOSE STUDY DESIGN
Single dose studies? …..Multiple dose studies?.....Better??????
If the dosage forms are to be evaluated only for
bioequivalence purposes, single dose studies are usually
sufficient….since relative BA of most tablets and capsules can
be determined on a single dose basis…which is a predictive of
multiple doses levels.
Certain dosage forms designed to achieve special release
profiles of drugs may require multiple studies.
Ex: Time release products, enteric coated tablets,
intramuscular injections.
Drugs that undergo first pass metabolism do require
multiple dose study.
Use of an improper study design leads to the collection of
insufficient or inappropriate data
If special dosage regimens such a s loading dose or twicea-day dosings are to be used, a multiple dose study design
may also be necessary.
ADMINISTRATION OF DRUG PRODUCTS
•Adminstration of drug products to the subjects should be based on
randomization.
•After the administration of the drug products, blood samples are
withdrawn from the subjects at fixed time points.
•It takes some time to take a sample from each subject, and the total
time difference between first and last subject may range from 10 to 20
minutes depending upon the no. of subjects and technicians.
This 10-20 minutes difference would represent a substantial change
in the drug concentrations observed in the blood.
If so, error between the time of administration and sampling will
gradually increase from treatment group to treatment group.
To avoid this type of effect, randomized administration of drug
products is used.
The order of dosing is not sequential but a part of each treatment is
given first, a part in the middle and a part in the last.
SAPMLING
The biological sample to be used in the study has to be decided before the
commencement of a BA study.
To have as estimate of the parameters, frequency and duration of sampling are
very important since they can markedly influence the apparent results obtained
in the study.
The sampling scheme should be frequent enough to define the absorption
phase, the peak, and the elimination phase during a drugs time course in the
body.
Estimate of Ka….more data points in absorption phase
Estimate of relative amount of absorption….AUC curve…more points
To estimate the AUC from the data, sampling has to be carried out till the
concentration of the drug reaches the linear elimination phase
In case of Urinary excretion studies….bit difficult during absorption phase
Inorder to obtain useful data, it is necessary to plan the frequency and
duration of sampling carefully
If possible, urine samples should be collected for 10 half lives of the drug to
ensure 99.9% of elimination
SELECTION OF SUBJECTS
HEALTHY SUBJECTS VERSUS PATIENTS
BA studies are designed to find out the dosage form biological performance, hence it is
necessary to minimize all possible variations if it is not possible to eliminate them
Use of healthy volunteers avoids much of variations that are possible with patients.
The variables associated with most diseases states make it impossible to design a
meaningful BE test.
Some problems associated with testing in patients are…
It is difficult to obtain many patients in a given place
The severity of a disease varies from one patient to another
Ethical considerations do not allow withdrawl of many blood samples from the
patients for a longer time.
It is not ethical to administer a dosage form to a patient whose therapeutic
efficacy is unknown.
 Treatment of a disease involves use of several drugs simultaneously , the effect
of these drugs on the bioavailability of the drug to be tested should be known
before the interpretation of the bioavailability test results.
Normal subjects are preferred in the bioavailability studies over patients.
SELECTION OF SUBJECTS
It is accepted that healthy volunteers should be used in bioavailability
studies.
Healthy volunteers ?
Healthy means a person having an overall good state of physical health,
which is asceratained by vital signs such as temperature, pulse, respiration,
B.P and laboratory tests on blood ( RBC count, WBC count, haemoglobin,
blood sugar), urine(pH, albumin, sugar) and also liver function tests (
serum alkaline phosphatase and serum glutamic oxaloacetictransaminase)
Age, sex and body weight also influence the blood level profile of a drug
product.
In general, 21 year old, male subjects weighing 150 ld are ideal to act as
volunteers in the study.
Difficult to obtain sufficient number…hence acceptable normal ranges
are 20-50 years of age and 102 to 220 lb body weight.
Males are preferred over females because menstrual cycle, pregnancy,
lactation and menopause stages that occur in females may affect the
blood level profiles of the drug.
Medical history of the subjects has to be reviewed critically by a panel
of experts.
The selected subjects should be distributed randomly to different groups
in order to achieve a uniform distribution of the available volunteers with
respect of age, sex, and body weight and to avoid bias.
STUDY CONDITIONS
The selected subjects should be maintained on a uniform diet and none of
them should have taken any drug at least one weak prior to study.
Before the commencement of the study it is necessary to define the study
conditions such as the fasting period before the administration, time period
after drug product administration during which fasting is continued, standard
diet to be given after fasting, fluid intake and volume to be allowed.
The quality and quantity of food intake drastically affects the bioavailability
of some drugs
Generally bioavailability trails are conducted on subjects that have fasted
overnight.
ANALYSIS OF BIOLOGICAL SAMPLES
The biological samples collected as per the sampling procedures have
to be analyzed immediately after the study.
Most of the times, the samples are stored for several days before they
are subjected to a chemical analysis.
The storage of biological samples is an important aspect in a
bioavailability study, since during storage the sample may undergo a
chemical degradation, adsorption on the walls of the container.
The analytical method used for the estimation of the active ingredient
responcible for the therapeutic efficacy must be selective and sensitive.
If the analytical method used in nonspecific, then results of
the study may not reveal the differences in therapeutic efficacy
that exists between drug products.
Only one analytical method should be used for the analysis of
all the samples of study.
Analytical method used must be specific to the active chemical
moiety and should exhibit high sensitivity.
ESTIMATION OF DRUGS &/or METABOLITES
IN BIOLOGICAL FLUIDS
Since BE testing is based on the premise that equivalent blood levels
produce equivalent pharmacological responses, it is obvious that one must
be certain that equivalent components are measured in the biological fluids
after administration of chemical equivalent products.
The development of highly sensitive and specific instrumental techniques,
coupled with advances in selective extraction and separation procedures
has enabled a residue analysis of drugs and their metabolites in biological
fluids with a high degree of precision and accuracy.
In the analysis of blood and urine samples, the most easily obtainable and
useful biological fluids, the major problem is to extract quantitatively and
then separate the intact drug from its major metabolites or even to separate
a mixture of two or more drugs from their metabolites.
METHODS OF ASSESSMENT OF BIOAVAILABILITY
The assumption in using pharmacokinetic methods for the assessment
of bioavalilability of drug products is that there exists a linear
relationship between the drug level in the biological fluid and
therapeutic response….Indirect methods.
INDIRECT METHODS or PHARMACOKINETIC METHODS
The parameters that are useful in determining the BA of a drug from
drug product based on indirect methods are….
PLASMA DATA
URINE DATA
1. Tmax
1.
dXu / dt
2. Cmax
2.
Xuα
3. AUC
3.
tuα
DIRECT METHODS or PHARMACOKINETIC METHODS
The pharmacokinetic methods are based on the assumption that the drug
concentration in blood or the drug excretion in the urine are related to the
observed therapeutic effect.
Pharmacodynamic methods are used when the assessment of bioavailability by
pharmacokinetic methods is not possible due to non availability of a sensitive
analytical method for the measurement of the drug or the analytical methods
lacks sufficient accuracy or precision.
Two Pharmacodynamic methods used for the estimation of bioavailability are
based on the measurement of ….
Acute Pharmacological Effect
Clinical Response
Measurement of Acute Pharmacological Effect
In order to estimate the bioavilaibility of a drug product accurately by this
method, the following criteria should be met….
An established dose related response curve
An easily measurable pharmacological response such as heart rate, ECG,
blood pressure, pupil diameter.
Experimentally, the pharmacological effect is measured at different time intervals
following the admi9nistration of drug product.
A plot of observed pharmacological effect versus time is made inorder to get a
smooth curve
is difficult to obtain.
The AUC is the measure of the performance of the drug product
and is used for the estimation of the bioavailability.
The study should be conducted for at least three times the half
life of the drug and a measurement of the pharmacological
response should be made with sufficient frequency to permit a
reasonable estimate of the total area under the curve
Main drawback of this method is that an accurate linear
relationship between the drug level and observed
pharmacological response
CLINICAL RESPONSE
Theoretically best among all….practically it is not.
The drug may be available to the systemic circulation from a drug
product at a sufficient rate and extent, but may not elicit a clinical
response in an individual because his receptors are less sensitive to the
drug compared to others.
STATISTICAL ANALYSIS OF THE DATA
The purpose of the BA test is to find out whether the test formulation gives a blood level
profile identical to that observed for a reference standard product or not.
Due to biological and experimental variations, some differences always exist and it is
necessary to ascertain whether these differences are simply chance occurrences or due
to actual differences in treatment administered to subjects.
Statistical methods are used to used to evaluate the data in order to identify the different
sources of variation and, if possible, to measure the contribution of each identified
variable and isolate the specific observation of primary interest.
The Analysis of Variance [ANOVA], a statistical procedure used for cross over design, is
used widely in bioavailability testing and is the procedure that will be encountered most
frequently by health scientists.