Bioavailability and Bioequivalence

391
ratoy tests.
laboratory Finally,
tests. Fin the FDA requires bioavailability/phamacokinetic studies and where
cessary bioequivalence
necessary bioequiva studies to ensure the safety and efficacy of the drug product.
hineauivalence studies, test drug product is compared with a reference standard (generally
aEDA approved drug product). Test product is called bioequivalent if it produces a
availability equivalent to that of the reference standard.

In Certain situations 505(6)(2) is also applied by innovator.

Section 505(b)(2): This provision expressly permits FDA to rely, for approval an NDA, on
data not developed by the applicant.

It permits approval of applications

other than those for duplicate
products and permits
reliance for such approvals on literature or on an Agency finding of safety and/or
effectiveness for an approved drug product.

Some of the examples of 505(b)(2) applications include

.Change in dosage form, strength, route of administration, dosing regimen,

Substitution of an active ingredient in a combination product,
.Change in the formulation beyond those considered limited confirmatory studies
appropriate to a 5050) application,
An application for a not previously approved indication for listed
a drug.
.Rx/OTCswitch.
11.3 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,
ume of administration, subjects and dosage form. The aim of bioavailability study is to
ind out the dosage form influence on the biological performance of the drug. Therefore
e Dioavailability study protocol used should be of sufficient sensitivity to detect differences
n the rate and extent of
dnd
absorption that are attributable only to dosage form variability
should avoid variabilities due to other factors. Table 11.1
lists the elements of the
bioavailability study protocol.
ometimes, due to inherent properties of the drug, analytical difficulties are encountered
at will preclude the measurement of drug or metabolite levels in body fluids, so other
u e s are used to assess bioavailability. The drug may be labeled with a radioactive
pnarmacological or clinical response may be measured, or studies may be pertormed
on animals.
 Biopharmaceutics and Pharm

392
Bioavailability
Study
Protocol
nacokinetes
Table 11.1

A Shudy objective
B Study design
Experimental design

Wash out perod

standard
Drug products Recognized
3 and (b)
product(s)
(a) Test
Route of administration

Dosage regimen
of sampling
and duration
Frequency administration

Randomizatron
of drug
multiple-dose study design
Single-versus

Subjects
versus patients
subjects
(8) Healthy
(b) Subject selection

medical history
() Physical examination,

(i) Laboratory tests

(c) Study conditions

fluids
10 Analysis of biological
Bioavailablility
C Methods of A s s e s s m e n t of
1 Plasma data
Urine data
effect
Acute pharmacological
3
4 Clinical response
Presentation of Data
D Analysis and variance (ANOVA)
Statistical treatment of data-Analysis of
1
Format of data
2
11.3.1 Study Objective
study decides the study protocol. study design
A meant
The objective of the bioavailability
for estimating essential pharmacokinetic parameters differs significantly from a bioequivalence
formulation with reference to a standard.
study meant for comparing the test

11.3.2 Study Design

the rae
Various factors have to be considered in conducting a bioavailability study since
elimina
and extent of absorption of a drug into the systemic circulation, its distribution and
are influenced by a variety of factors. Subject factors such as age, sex, disease sa
food habits, general health condition, body weight of subjects, experimental design,
used
of administration, time of sampling, analytical method used and compartment mo0
blood

in estimating pharmacokinetic parameters/bioavailability contribute to the observed

 Bioavalilabilitya n d B i o e q u i v a l e n c e
393

concentration time profile. Therefore. it is necessary to consider all these important factors
t i o n time

design.
study
in a
is based on
of study design
Selection
formulations to be compared
Number of
of the drug and its disposition
Characteristics
Study objectives
Availability of subjects
variabilities
Inter- nad intra-subject

.Duration of the study o rthe number ofperiods allowed

. Cost of adding a subject relative to that of adding one period

Dropout rates

For example: f the intra-subject variabilty is the same as or larger than the inter-subject

variability, the inference on the difference

in average bioavailability would be the same

used.
regardless of which design is
Actually, a crossover design in this situation is a poor choice, because blocking results in
the loss ofsome degrees of freedom and will actually lead toa wider confidence interval on
the difference between formulations.

In the following sections various factors are discussed keeping the bioequivalence study
also in mind. However, they are valid for simple bioavailability studies also.

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 to two groups of volunteers.
aOd a bias, formulations may be administered randomly to the volunteers. The major
disadvantage of this design is that the inter-subject variation is not being corrected. It has
Deen proved beyond doubt that most of the
times(inter-subject variation is greater than
ne variation between any formulations, Therefore, a cross-over design is preferred in
Evalability/bioequivalence trials to avoid influence of a inter-subject variation.
draliel design may be considered as an alternative to a crossover design it

Ersubject variability is relatively small compared with intrasubject variability.

u g Is potentially toxic or has a very long elimination half-life. For example
aOXilen, triptorelin, toremifene,
goserilin acetate, nevirapine.
 394
Biopharmaceutics and Pharmar
The population of interest consisits of very ill patients, and acokine
The cost for increasing the number of
subjects is much less than that
additional treatment period of addig
Other examples include
bioequivalence study of topical antifungals like ketocon
clotrimazole conazole
Cross-over Design
Usually, a substantial inter-subject variability exists in the
given dose of medication in
drug levels achieved from.
of
a panel of subjects. The cross-OVer
design minimizes the ot.any
inter-subject variability in the study by using each
Generally. two types of cross-over designs are used insubject as his or her ownCaneffed
Latin bioavailability trails. To
Square cross-over design and 2. Balanced They are,1
Incomplete Block Design (BIBDI
Latin Square Cross-Over Design
A standard
approach for conducting a comparative
balanced, cross-over design called a Latin bioavailability study is to use a randomiz
design, 1. each subject receives just
square or
complete cross-over design. In this
administered just once in each once each formulation, and 2. each formulation is
in Table 11.2,
study period. The basic elements of this design are
where the first design is a shown
two-way
way cross-over designs shown in the table 11.2 cross-over design. The three- and four
combinations. represent one of the several possible
In a two-way cross-over study,12 subjects are used to
two formulations, treatment A and treatment B.
study the bioequivalenciesof
to 6 receive treatment A, During the first study period, subjects 1
while subjects 7 to 12 receive
treatment B. A second
is initiated after the washout
period, during which a complete elimination of study period
its major metabolites takes place. In the second the drug and
treatment B and study period, subjects 1 to 6 now receive
subjects 7 to 12 receive treatment A. Therefore, each
his own control. This design has several subject acts as
advantages, listed under,
1. It minimizes the effect of
inter-subject variability in the
as his or her own control.
study by using each subjedt
2. It minimizes the carry-over
efføcts which could occur when a given dosage form
influences the bioavailability of a
formulation
subsequently administered dosage form since eaci Bala
is
preceded and succeeded by other formulations or
3. It minimizes
dosage forms. A bar
the time effect on
bioavailabilty since each dosage form is administereu with
in each study
period.
4. It requires less number of
subjects to get meaningful results
Bioavailab and Bioequivalence

Table 11.2 Latin 395

Square Designs.
Two-Way Crossover
Group No.
Subjects in Group
Treatment for Period No.
2.
1,2.3,4,5,6 A
B
7,8,9,10,11,12 B
Three-Way Crossover A

Group
No. Subjects in Group
Treatment for Period No.
. I
1,2.3,4,5,6 A C
2. B
7,8,9,10,11,12 B A
3. C
13,14,15,16,17,18 C B A
Four-Way Crossover -

Group No. Subjects in Group Treatment for Period No.

1. I1IV
1,2,3,4,5,6 A B C D
2 7,8,9,10,11,12 B D A C
3 13,14,15,16,17,18 CA D B
19,20,21,22,23,24 D CB A

The major disadvantages of the Latin-square design (Table 11.2) the

are
following
1.It requires longer time to complete the study since
a washout period exists between
two study_periods. The higher the biological half-life of drug, the longer willbe the
timecequired for completing the stugy
2. The time to complete the trialdepends on the number formulations evaluated in the
study. It takes a longer time to complete the study as the number of formulations
increases.
3. Increased number of study periods leads to high subject dropouts and the study
becormes difficult
4. Medical ethics does not allow too
many trials on asubject continuously for a longer
time
Ihese disadvantages be of balanced incomplete block design.
can overcome by use a

Balanced Incomplete Block Design

08lanced
wn tne
Incomplete Block Design (BIBD) eliminates many of the difficulties encountered
Latin square design (Table 11.3 &
11.4). The salient features of this design are
Each
subject receives not more than two formulations.
Cach tormulation is administered the same number of times.
3 Each pair
of formulations occurs together in the same number of jects
396 Biopharmaceutics and
Pharmaccokinelcs
In this des
Table 113 shows BIBD for four formulations A, B, C and D. design, as disc.
above, each Subject
su receives two formulations,
each formulation is administ
and each pair occurs together in twO SubjecS
(the pairs are S
of formulations
BC, BD and CD)
Block Design (BIBD) for Four Formulati
Table 11.3 Balanced Incomplete ions.
Treatment for Period No.
Subject
B
A
B
C

C
A D
D A

B C
C ,B
D
D B
10
11 C D
D C

Table 11.4 Randomization Scheme Used in a Bioavailability Study

Group No. Subjects in Group Treatment for Period No.

1. 1,2,3,4,5,6 A C B
2. 7,8,9,10,11,12 B A C
3. 13,14,15,16,17,18 C B

Replicated Crossover design

Replicated crossover designs are critical when an individual BE approach is used to alo
estimation of within-subject variances for the T and R measures and the subjec-D}
formulation interaction variance
component
Following is the four-period, two-sequence, two-formulation design recommended
replicated BE studies

Period
2 3 4
Sequence T R T R

2R T R T
 availability and Bioequivalence
397
dosian, the same lots of
de the I and R
formulations should
this
Forthis Each period should be separated by be used for the
replicated
an
administra
adequate washout period.
roolicated crossover designs
Other replicated crossove are possible. For example, a
shown below,
could be used. three-period design,
Period
2 3
Sequence 1 T R T
2 R
T R

A areater number ofsubjects would be encouraged for the

recommended four-period design to achieve the samethree-period
statistical
design compared
power to conclude
BE
Other Designs
Eor the highly variable drugs(HVD) andhighly variable drug products(HVDP) havingwithin
Subiect variability of more than 30o, FDA has recommended
bioequivalence approach) where the bioequivalence limits are notfreference scaled average
size. Rather, they are scaled based on the within-subject determined by the sample
For drugs with an expected within-subject variability of variability of the reference product.
30%
three-period, reference- replicated, crossover design with or greater, a BE study with
RRT is proposed. Specifically, sequences of TRR, RTR, and
subjects receive a single dose of
reference product twice on separate occasions with random the test product once and
sequences of product administration. This partial replicate assignment to the three possible
of withinsubject design allows for the estimation
variability for the reference product. Treatments should be separated by a
washout period of adequate duration such that the drug of
in plasma. that would be acceptable is 24. interest can no longer be detected
The three-peried design was selected over a
four-period design because of efficiency. The only advantage of the four period design is
that it allows the
IS not used
calculation of the variability of the test product. The test product variability
in the
proposed statistical method,
11.3.3 Washout Period
a Latin
Square cross-over study design each subject
ven BlBD in
each subject receives two formulations at different receives each formulation and
een the two treatments is called "washout occasions. The time interval
nauonof administered dose _of a drua period".Washout
the
so as
period is required for the
nas Come into use for a crossover design for to avoid the carryover. A guideline,
of -llves should be allowed between treatments.most drugs is that a period of at least
99.9%
first treatoo the ministered dose apd a
This should ensure an elimination

washout period
maximum_carryover of less than 0.1% from the
is a function of the half-life and the dose of the drug
398 Biopharmaceutics and Pharmaco
administered The number of washout periods in a study depends upon the
macokinet
type ofe
design used and the number of formulations to be evaluated. In the case of diaitovi CIOSSO
ha[ a half-life of 6 to 9 days, the total study period exceeds one , whie
year if four formi
have to be evaluated using the Latin Square
design. Because a very larqe ni
drugs have been found to have a half-life between 1 and 10
hours, a washot
mber
of 1 week was usually found suitable in most of the
reported studies. peks
It should be
noted that the metabolites of the drug should also be
body before the commencement of next treatment. Unfortunately, the exacteliminated fromom te
of all drugs is not known. Whether all the
metabolic sche
metabolites of the drug are eliminated or
is unknown. However, as most of the
metabolites are more
drug and have a shorter residence water-soluble than the.pa
is assumed that their
time in_the body than the parent drug molecule
elimination.ocCurS well before the elimination- efthe parent compount
11.3.4 Drug Products
Test Products: Test product(s)
new drug formulations developed by:
may be
pharmaceutical technologist or new dosage forms of an
be
compared t o a reference existing drug. A test product me
standard recognized by the Food and Drug
for getting
approval for Administa
for following reasons. marketing the drug product. Test products are generally evalua
1. To select best
dosage form of a new drug or existing drug
forms (e.g. Tablet, among different dosa
capsule, emulsion, and
2. To select the best suspension)
formulation
formulations that have
of a new
drug or existing drug among
shown equal
performance in dne
3. To
compare biological performance of a in vitro tests.
test product to that of a
e. bioequivalence studies). recognized stalu
Reference Standard
A chemical
or
verify generic product has to be
performance, In general, thecompared
its in-vivo with form
some
dosag standard
any innovator's drug Food and
Drug
product
received approval from as a
reference standard. The (F Administration
manufacturers may hold
the FDA to
market the product in the is the innovator
one wno
mes, seve

drug approval for certain drugs. country. Some

products
can used as a
be Therefore, any one of u permile

In many of these reference standard.

be used as a instances, the FDA would
reference product in request that only one
order to obtain e
ot tn
produc"

Generally highest.dose is the a more

easily comparao al
where the safety of the
volunteer
reference
is
listed drug. There are some exceptions
For example, atomoxetine HCI
a concern. excepu
(60 mg),
(2 mg), Lamotrigine (2 *
25 mg), glimepiride (1 mg), risperidone (1 lerazo

olanzepine (5 m mg
 Bioavailab, and Bioequivalence

Route of Administration

of the Orall
c
the times, oral
of.
administered dosage forms are 399
Most

e
However, dosage forms
formsadministered
intramascuk should
should alsob administered
also be
evaluated
by other
tor
Or
subjected for
routes
t h e i O u t e s c e d

such as
fo
bioavailability studies
se dosage forms
ormsdepen
depe their buccal,
form depends on the biological performance. Thetransdermal
form dosage forms
these dosage fate and extent
absorption oftherapeulio
ormtnese of
Ingeneral, orally administ
istered dosage forms show a the
drug
because of nter-subject and
tra-subject variations. Allmuchthevariation in their
by an extravascular
route do require a dosage forms performance
bioavailability assessment.
3.5 Single- Versus Multiple- Dose administered
Whet cinale dose studies
Study Design
Wne nt are better or
of the
assessment of
bioavailability of a drug multiple-dose studies
always difficult tobetter
are
answer However, it is possible to product?
discuss the issue
It is for the
give a
single
concepts involved in decidin the
dosage thoroughly so as to
regimen for a bioavailability understand
are to be evaluated only for the
cient. This is because the bioequivalence purposes, single-dosestudy. the dosage
If
relative
tarmined on a single-dose basis, andbioavailability studies
of most tablets and capsules are usually
Dasane forms meant for a single usually, this is predictive of can be
multiple-dose levels.
analaesics for the relief of headachedose administration for a therapeutic
need only single-dose studies. However,benefit such as
formsdesigned to achieve special release profiles of certain
For example,
drugs may require multiple-dose dosage
time-release products, studies.
iniections. Even the drugs that undergoenteric-coated
the
preparations, some tntramustutar-
first-pass
study. Use of an improper study design leads to the metabolism do need a multiple-dose
inappropriate data. Further, if special dosage regimens collection of insufficient and/or
such as loading dose or
a-day dosing are to be used, a multiple-dose twice-
study design may also be necessary.
11.3.6 Administration of Drug Products
Administration of drug products to the
subjects should be based on randomization.
the
administration of the drug products, blood samples are witharawn from the After
TDXed time points. It takes subjects
ime
some time to take a sample from each subject, and the total
diference between first subject and the last subject may range from 10 to 20
minutes
depending upon the number of subjects and technicians in the study. If the sampling schedue
s
not followed rigorously in the same sequentiat manner, significant
Concevably exist in the actual duration of the drug in the body and the diierences can
stated sampling
ue given for each subject. This 10 to 20 minutes' difference would represent a substantial
n
the drug concentrations observed in the blood. If under these conditions treatments
6 siered to_the subjects in a sequential manner (such as treatment Ato the Tirst
volunteers, treatment B to volunteers 7 to 12, and treatment C to volunteers 13 to 18,
n in table 11.4), the error between the time of administration and sampling w
yCrease from treatment group to treatment group. This is because of sequenua
 Biopharmaceutics and Pharmaco:
400
treatments. To avoid this type
cokinetics
n
products to different ef
s t r a t i o n of drug The order is not
of dosing seec
randomized adm dministration of drug products is used. in
a part of each treatment is given first,
a part
the middle and a part in
in sequentia
the las
u
Multiple-dose study
determine the bioavailability of drug
a Drocd.
required to
Multiple-dose study may be in
the following circumstances. of absornti.
absorption but not in the extent tion.
There is a difference in the rate of
from subject to subject.
There is excessive variability in bioavailability
concentration of the active drug
ingredient or therapeutic moiety
The acc
from a singie dose is too low for accurate
metabolite(s), in the blood resulting
method.
determination by analytical
an extended
release dosage form.
The drug product is
for which multiple-dose study
is performed are rivastiami
mine
Examples for the drugs
fumerate (tablets oral).
tartarate( oral capsules), quatiapine
vsT

11.3.7 Sampling
The biological sample to be used study has
in the to be decided before the commencemeni
to be evaluated
of a bioavailability study. If the bioavailability of a given dosage form is
s o m e estimate of the area under the serum
concentration versie
by a blood leyel study, and time of peak serum Concentration must ha
time curve, peak serum concentration, and the duration of samplina
obtained from the study. Therefore, the frequency of sampling
factors can markedly influence the "apparent"' results obtainer
are very important since these
in a given study. While the blood sampling Schedule
requiredHo evatuate bioavailablitv
will vary with the drug, it is possible to list a few factors that
ensure a satisfactory
will
be frequent enough
determination of the blood-level profile. The sampling scheme should
to define the absorption phase,
the peak, and the elimination phase during a drug's time
course in the body.
In order to estimate the rate of absorption it is necessary to have enough data points
in the absorption phase and hence, the frequency of sampling is more in this
phase. Even
it not be possible to get
though the frequency of colection of samples is monitored, may
the time of peak serum concentration directly from the data and is generally_estimae
from the data. Since the relative amount of the drug absorbed is determined from the
AUC parameter, there must be sufficient sampling points to allow for proper evaluation
of the area under the blood-level curve. The absorption rate, volume of distribution, a
elimination rate, all influence the apparent drug concentration one obtains in a given sam
It is necessary to see that all these factors influence each dosage form equally. To estma
the AUC from the data, sampling has to be carried out till the concentration of the a
reaches the linear elimination phase. For first-order process, the time necessaed
complete elimination would be infinity. However, for all practical purposes it is ap
of 10 half-lives for any given drug. A rule of thumb sampling in a blood-level stuay
sample for three to five half-lives of the drug. If the half-life is not known, sampling s
proceed until 1/10 or 1/20 of the peak levels are reached.
Boavala
ailability and Bioequivalence

In the cas of urinary excretie

retion studies, the
ies are used whe hen it is either same 401
exCreton s t u d i

s e r u m or
when
or whe
ethical
not principles
possible to apply. Generally,
or
h0od, plasma, me (e g. subjects such as considerations measure
do not allow a
given drug
urinary
patients, children). Thethe collection of in the
o v e ra perio

tudies are: 1. it inv volves non-invasive

excreton studies

ine is often method of advantages samples

of
greater than that in urinary
sampling, 2. the concentration
of
drug,
a n d 3. the amount of
the drug blood/serum
of the
caseofa
blood-level stud udy, the amount ofexcreted in urine is allowing easy estimation
haacokinetic parameters. O narameters.
On the other
the drug in obtained
the body is directly. In the
pharm

advantages as well. 1. hand, urinary

urinary excretion studies estimated using
are not
excretion method has several
absorbing
drug. This useful in
rate ofa
the
rapidly
absorption phase to
is because it estimating
is difficult to
define the absorption rate the
obtain manyabsorption
in emptying the rinary bladder
duning
frequently accurately. samples
some cases, the
and it leads to a
Subjects feel difficulty
nple. 2. In metabolites of the drug are also carryover effect on the next
that interferes with the estimatio of the concentrated in
unchanged drug in the urine
the sanmple
However, in order:toobtain useful data, it is sample.
If possible, urine necessary to plan the
frequency and duration
of sampling carefully. samples should be collected
the drug to ensure a 9999.9% of elimination of the drug in urine. If the for 10 half-lives of
af known, a plot of the cumulative amount of drug excreted over time elimination half-life
will reach a plateau
a $Ome point in time consistent with a complete elimination of the drug.
11.3.8 Selection of Subjects
Healthy Subjects Versus Patients: Bioavailability studies are designed to find out the
dosageform biological performance. Hence, it is necessary to minimize all possible variations
f t is not possible to eliminate them. Use of healthy volunteers avoids much of variations
that are possible with patients. However, it is true to saythat the conditions should mimic
as much as possiblethe actual conditions of usage for patients. Thevariables associated
with most disease states make it impossible to design a meaningful bioequivalencetest
Some of the specific problems associated with testing in patients are given below
. t is difficult to obtain many patients in a given place
2. The severity of a disease varies from one patient to another

Ethicalconsiderations do not allow withdrawal of many blood samples from the patients
tor a longer time.
4. IU 1S not ethical to administer a dosage form to a patient whose therapeutic efficacy
is unknown.
5 the effect
Treatment of a disease involves use of several drugs simultaneously,
oE known before
the bioavailability of the drug to be tested should be
nese drugs on
the
interpretation of the bioavailability test results
he,normal subjects are preferred in the bioavailability studies over patients
studies include
which patients are used in bioavailabil.ty
Es or the drugs for
leuprolide
Ar temozolomide, capecitabine,
drugs like azacitidine, nabilone,
acelat
acetate.
402 Biopharmaceutics and Pharm.
nacoki
Selection of Subjects BIG
I it is accepted that healthy volunteers should be used in bioavailability st
volunteers? Healthy me, the
studies
is, what is meant by "healthy"
OVe

question one faces

health. It is ascertained by vital o nen
aa person
having an overall good state of physical
me

temperature, pulse, respiration, blood pressure,

and laboratory tests on blood SuCh no

WBC count, hemoglobin, blood sugar, etc.), urine (pH, albumin, sugar etc.) RBC COun th
liver function tests such as serum alkaline phosphatase and serum glutamin nd also b te
transaminase. Depending upon the drug products used in the study, certainl0aces for

be included or excluded.
in
tests acelmayic se

Age, sex and body weight also influence the blood level profile of a drun sh
general 21-year old, male subjects weighing 150-Ib are ideal to act as volunteer prod In
study. It is difficult to obtain a sufficient number of subjects with these specificati he
hence acceptable normal ranges are 20-50 years of age ana 120 to 200 Ib of body
Males are preferred over females because menstrual cycle, pregnancy, lactati
menopause stages that occur in females may affect the blood level profiles of SI
the
However, females are also included in the bioavailability study taking above points drug
consideration. into CL

p
Medical history of the subjects has to reviewed critically by a panel of experts. Forexa W
a person who has undergone gastrectomy shows normal values for the general screa
tests used, but he may not absorb certain drugs like normal volunteers because of
in the gastric pH. ence difer
d
The selected subjects should be distributed randomly to different groups in
order th
achieve a uniform distribution of the available volunteers with respect to
ndage, sex
body weight and to avoid bias.

11.3.9 Study Conditions

The selected subjects should be maintained on a uniform diet and none of them
should
have taken any drug at least one week prior to the study. Before the
commencement o
the study it is necessary to define the study conditions such as the
the administration, time period after
fasting period before
drug product administration during which fasting s
continued, standard diet to be given after fasting, fluid intake and volume to be
etc. The quality and quantity of food intake allowed
drastically affects the bioavailability of some
drugs. In general, bioavailability trails are conducted on
subjected that has fasted ovemign
11.3.10 Analysis of Biological Samples
Ideally, the biological samples collected as per the
sampling procedure have to be anay.
immediately after the study. But most of the times, the samples are stored for severai ua
before they are subjected to a chemical
an important aspect in a
analysis. The storage of biological sam
bioavailability study, since, during storage the sample may uicaidergo
a chemical
degradation, adsorption on to the walls of the container, etc. ytical
method used for the estimation of the active ne atic
must ingredient responsible for the tneid
efficacy be selective and sensitive.
of the unchanged drug and metabolitesNonspecific analytical methods measuringrnsS
are less desirable even in
ro:

well-controlle
 Cnta
Jate
IAd

Bloave
vailablity
and Bioequivalence
that undergo the
ruaS 403
verstucies.

metabolite ratio deper

first-pass
on the rate of effect exhibil different
mncific, then
the results of the study may not reveal If the analytical unchanged
absorption. drug/
method used is
the difference in therapeutic
exists between drug products. Most of the manufacturers
prode
efficacy
that
for the analysis of the samples. use the latest
a Only one analytical analytical
There may be moremethod should be used
t e c h n i q u e

of all the samples

analysi es of a
study.
the
forsitive m e t hQds
o d s for the analysis of the than one selective and
given chemical
for the analysis
but the results obtained with moiety. Any one of them may
one
those obtained in a other analytical method in one study
used
be
not be compared
alytical method used must bestudy with a different
should

dudt. nethod. In
summary, the analytical
specific to the active chemical
and should exhibit high sensitivity.
moiety

11.3.11 Estimation of Drugs and/or its Metabolites in Biological Fluids

Weg
ion oenuivalence testing is based on the premise that equivalent blood levels
Since bioed
produce
he equivalent pha nharmacological responses, it is obvious that one must be certain that equivalent
componer are measured inthe biological fluids after administration of chemical equivalent
products. Theddevelopment of highly sensitive and specific instrumental techniques, coupled
selective extraction and separation procedures, has enabled a residue
with advances in
exampe anavsis of drugs and their metabolites in biological fluids with a high degree of precision
creenn and accuracy. In most analytical problems, the final quantitative analytical step is the least
eng diirutt Dart of the entire procedure. In the analysis of blood or urine, the most easily
the major problem is to extract quantitatively and
obtainable and useful biological fluids,
ordet then separate the intact drug
from its major metabolites or even to separate a mixture
sex, a more drugs from their metabolites.
of two or
active component should be measured in the
biological
ldeally, the pharmacologically active and should
method used must be specific to the component
sample. The analytical methods of analysis that
there are several potential
show good sensitivity. Since usually a concise
shout
m should be mentioned and also,
.are usable, the type of procedure employed other combination of
ementd drug, metabolite, or some
statement as to whether the unchanged
included with the bioavailability data.
od beéoe
fasting products measured should be
allowe
of sone
11.4 Methods of Assessment of Bioavailability in humans and the
selection of
bioavailability
Ihere are several methods of assessing and the analytical
Ovemigt of the study, nature of the dosage form,
a method on the purpose
depends pharmacokinetic
as
available are classified
of measurement. The methods
method drug
methods.
methods and pharmacodynamic
analye

veral dar methods for the assessment

ofbioavailability
biological
pharmacokinetic
level in the a
ne assumptionthatusing
in between the drug
linear relation indirect
amples products is there exist a are also known as
y undegr Ug Therefore, these
methods
measured
in
can be accurately
response.
analynia nerapeutic active drug bioavailabiny.
therapeutically information on
free or
SBecause the the most objective
Nerapeut

urine data give

I c a l tluids, plasma and
9 a
m t s
 Biopharmaceutics and
armacok
404
Methods
inetics
Pharmacokinetic
1.4.1 Indirect Methods or of aa drug fron
determining
the bioavailability a drug
The parameters that are useful in
product based on indirect methods are

1. Plasma Data
(max
concentration
(a) The time of peak plasma
concentration (Cmax
(6) The peak plasma concentration-time
curve (AUC)
The area under the plasma
(C)
2 Urine Data
excretion in the urine (dX, / dt)
(a) The rate of drug
excreted in the urine (X)
cumulative
amount of drug
(b) The
maximum urinary
excretion (t,)
The time for
(C)
11.4.1.1 Plasma
This is the Data used and accepted method for the assessment of bioavaiabi,
most widely and
tacrolimus, whole blood
some drugs like
sirolimus, indapamide,
For serum is used for assessin
of drug products.
some drugs
like triptorelin, aminosalicylic acid, sing
or
ioavailability that are bioequivalent produa
The basic assumption
in this method is that drug producis measure
bioavailability is a of
level-time curves. By definition,
superimposable plasma The parameters t, and C_
absorption of a drug from a drug product.
rate and extent of AUC is a measio
the rate of absorption of the drug while the parameter
are the
measures of
of the extent of absorption.
concentration (max): The time required to reach the
(a) The time of peak plasma
concentration in plasma after drug administration is known as the
maximum drug
time of peak plasma
concentration (tma (Fig. 11.2). It indicates the time at which
of
the rate of absorption is maximum following
drug administration. At tmay the rate
when comparing
exactly equals the rate of drug elimination. Therefore,
drug absorption absorption
an approximate indicator of the drug
drug products, ta Can be used as
rate. Small tmay value indicate that less
time is required to reach peak plasma
tor
more rapid absorption. Units
concentration. In other words, small tmay means a

rax are units of time (e.g. hours, minutes).

Mathematiacally, may iS a function of both absorption and elimination rate constain
to US
Jt is highly dependent the sampling scheme, and it is therefore difficult
on
less
it to detect diferences between two products, especially when max Values ale
than 2 hrs.
tained
In contrast, for slow-reeleasing dosage forms, plasma concentrations are na
at a plateau for long time and may
max
does not reflect the rate of bioavailabil
 bility andBioequivalence
Bixnvatab

narisions
tatistical comparisions of
max tor the 406
ndertaken for rapidd reelase nnovator and
compounds and/or generic
only
when products
some clinical relevance are

Because C rallects extent of

1ay Is known tousually
have
absorption, there is
and max strong correlation
lasma concentratlon botwoon AUC
b) Thepeak
ximum plasma drug (maxhe peak plasma
concentrat
concentration
the maation
administratiof the drug
(19 11.2) max
concentration
obtained following an oxtrrepresents
sents
sufficiently ahsorbed
abso. to elicit a provides
des an an ina gan extravascular
is within
within therapeiui response. It also indicates
sponse" indication that the drug is
in plasma is
in plasma
Is

ug/ml or ng/ml
therapeutic levels or
reaching toxic levels.whetheris the drug level
Cay expressed as
alse Cmax reflects extent ot
Becaus

and max
absorplion, there is strong
correlation between AUC
chould be recognized tnat maxIS
hsarotion and is only an
influenced by extent indirect measure of
the rate of drug
of
cad its clinical
absorption.
on

concentration) applicability
reflects the [it
may relate to patients highest
in the body and drug exposure (e.g.
strona case can be made that risk for
(although Cmax is a poor toxicity and/or efficacV
clinically appropriate one for
assessing rate of measure, it is the most
absorption
13

Time (hrs)

Fig.11.2 Plasma drug level versus time curves

following oral administration.
ne area under the plasma concentration-time
plasma concentration-time curve, AUC, is a
curve (AUC):
The area under
O a
dosage form or
of the
measure
extent
the fraction of the dose that reaches the
of drug absorption
systemic circulation.
AUC is the sum of [AUCI, and [AUC|I:, where 't' is the last time
point of
aSma sample collection. [AUCI is calculated by the Trapezoidal rule and the
IS
obtained from the [AUCR
Equation 11. 65.
AUC), - [AUCI, +[AUC] (11.5)

AUC)= C/K (11.6)

 Biopharmaceutics and Pha.
6
Where, C' is the concentration of the drug n the last plasma samnof
ar
mple
macrinhe
independent of the routo otnd and Y
Overall elimination
rate constant AUC is
as long a s the
ess ddo as
eliminalion process adrnniste
"di

chare
eliminaton
of drug ug-hr/ml)
and
processes
c o n c e n t r a t i o n - t i m e r v o l u m e

(e 9
h e units for AUC are
the administered doen 0se
proportional to
For many drugs AUC directly

administered
is

dose and AU,

In general, indicales the avi Nonilinea
between the VWhen the AUC
absorption
and/or elimination. 1s notr
saturable kinetics
of
the drug is difficult to au
proportional
dose. bioavailability
to the
of aluate
for one dosing interval at th
dose studies, AUC
In the case of multipie
absorption from the dosage form T -
level is a measure of
the extent of drug The folowit
fraction of the dose absorbed
equations are used to estimate the .e., te
bIoavailability of the drug product).

(AUC)ora[Dose],
F 11.7
AUC,
AUC),[Doseoral
If doses are equal, then

F AUCloral 11.8
AUC
Where F = Absolute bioavailability

AUCHDoselsandard
Fr[AUCI tandard x[Dose}lest 11.9

If doses are equal, then

Fr =
11.10
Where, Fr Relative bioavailability.
Truncated AUC
The comnon measures used in
bioequivalence study are AUC and C
Estimation of AUC requires
frequent blood samples. For long half-life drugs, samplu
for long periods of time become
may cumbersome. To resolve this issue some investiga
have suggested the use of truncated AUC in ie
drugs. bioequivalence studies for long na
For example, pimozide, sirolimus, one
apriprazole, anastrazole. entacavir, salefivacin succinate, atavay

Partial AUC
Here AUC is truncated at tma, for the innovalor compound.
 vailabilityand Bioequivalence
Bioavailabi

Fororally a d m eninistered immediate release 407

be sufficient. in products,
situations in which measures of peak and total
may

pha
nharmacodynamic
relation may be
studies indicate clinical safety and/or
that
exposure
efficacy trials or
warranted, assessment ofbettet control of drug
ystemic c i r c

early exposure via useabsorption

into
be indicated
AUCs may of partial
vample zolpidem extended release tablets
11.4.1.2 Urine Data

of the unchar
iry e x c r e t i o n
anged drug versus time data
ilability of a drug product. This can be used to
method is based on estimate the
on of
urinary excretion the
the rate of
a
drug is
directly proportional to general observation that
drug in
the bl hlood. In other words, the same fraction of the concentration
of the
the urine unchanged. Therefore, the
bioavailability can absorbed drug always
totalamount of the unchanged drug recovered in urine
be calculated as the ratioreaches
of the
and standard formulations
ns. Example for which urine following the administration of test
data is used to
is alendronate.
assess
bioavalability
la obtain valid estimates, the drug must
arder to
be excreted in
nurine and urine samples must be colected for at least significant quantities
7 half-lives. This
l l the drugs that are excreted in urine method is useful
data is not used for theunchanged
in
otaholite excretion significant quantities. Urinary
estimation of bioavailability
can undergo metabolism at different Sites including the since the drug
metabolism may vary because of various factors. gut and liver and the rates
of
lal The average rate of drug excretion in urine
of the unchanged drug in urine generally follows first(AX, I At): The rate of excretion
order process, since, it
on the blood concentration of the
drug. Therefore, a plot of AX,/At versusdepends
time at
the mid point of urine collection (t) is a mirror
versus time curve (compare
image of the plasma drug concentration
Figures 11.2 and 11.3). In Fig. 11.3, the maximum rate
of drug excretion would be at
point B, (AX,/ At)max, and it corresponds to the time
at which
Cmax OcCurs. Hence, (AX,/ At}max is used to estimate the
of drug products bioavailability
(b) The cumulative amount of drug excreted in the urine in infinite time
B
(X,):

Time (hrs)

Fig. 11.3 Rate of urinary excretion of drug versus time plot.

 Biopharmaceutics and
408
Pharmacokine
excreted in the
urine IS directly relate
d to t (C
of the drug conti
ntinues till the
The cumulative amount excretion into urine
amount of the drug
absorbed. The drug
samples must be collected dry
for a lone
for
zero. Therefore, urine
level in blood falls to
a better
estimate of X: Aar.
time (at least for 7
half-lives) in order
to get graph
excreted into the urine versus timeic.
the unchanged drug
cumulative amount of excreted up to point C coae
amount of the drug
The cumulative
11.4.
in Fig. of bioavailability.
the estimation
This value is used for
to X
Absolute bioavailability.

Tine (hrs)

Fig. 11.4 Cumulative amount of unchanged drug excreted in urine versus time following oral
administration.

F Xral[Dose), .(11.1
K, *[Doseornal
Relative bi0availability,

Fr
X JsDoselslandard
Xatandard x [Dosels .(11.12

When doses administered are equal,

Absolute bioavailability.

F
...(11.19

and Relative bioavailability

Fr
ndard ..
(11.14
 abilityand Bioequivalend
me
for aximum urinary excretion 409
The time

(t,): The time at

point C in Fig. 114
nothing
but
,

t. tis
It is tthe time required for the absorption and a
s

following complete elimination

t h e drug
dministration
lowing the adm of
drug product. t is a useful
a
inbioequiva
comparing several drug products,
studies parameter
of
both he rate and extent of drug
absorption from a drug since, it is a measure
Steadystate: For studies at teady state, the
product.
following characteristics should be
during one dosing interval or cycle, O.at
calculaek-trough fluctuation, PTF
/culated
steady state: AUC( 0-T):
Cmin
(CmaxCminCav, where Cav AUC (0-T/T)
=

and
denotes the average steady state concentration.
For
firmative bioequivalence assessment. Primary characteristics for
the rate and
extent of absorption
should be stipulated
prospectively in the study protocol
114.2 Direct Methods or Pharmacodynamic Methods

acokinetic methods are

The p h a r m a c o k i n e t
based on the assumption that the
drug concentration
n blood or the drug excretion in the urine are related to the observed therapeutic effect.
namic methods
Pharmacodynamic methc used when the assessment of
are
bioavailability by
armacokinetic methods is not possible due to non-availability of a sensitive analytical
method for the measurement of the drug or the analytical method lacks sufficient accuracy
andlor reproducibility. The fwo pharmcodynamic methods used for the estimation of
are based on the measurement of
bioavailabilty
effect
1.Acute pharmacological
2. Clinical response

11.4.2.1 Measurement of Acute Pharmacological Effect

In order to estimate the bioavailability of a drug product accurately by this method, the
folowing criteria should be met.
1. An established dose-related response curve
2. An easily measurable pharmacological response such as heart rate, ECG, blood

pressure, pupil diameter, etc.

different time intervals following
Ementally, the pharmacological effect is measured at effect versus
Oministration of a drug product. A plot of observed pharmacological a measure
under this curve is
nf 0e in order to get a smooth curve. The area estimation of the bioavailability
performance of the drug product and is used for the
Theffrequency of of the pharmacological effect and
the total duration of
the study affect theneasurement
he results. The study should be conducted for at least three times the
halfi-ife of the drug and a measurement of the pharmacological response should be made
uff estimate of the total
area under the curve
Theemain
main ency to permit a reasonable
the drug
drawback of an accurate linear
relationship between
level and this method is that
observed pnarmacological response is difficult to obtain.
 Biopharmaceutics and Pharmacokineti
410

11.4.2.2 Clinical Response discussed

best among the methods ta
Theoretically, this method s e e m s to be the
observed differences in therapeut
Dut,practically, it is not. This is because of the fact that
not be attributed only to t i e 1ofmulation
sponse following different formulations can 1acokinetir
differences both in
the pharmnar
Differences in the
clinical response may be due to
and pharmacodynamic behavior of the drug among individuals. The drug may be availatl
avallable
to the systemic circulation from a drug product at a sufficient rate and extent, but may
are less sesitive t
clinical
a in individual because his receptors
the elicit compared to others. This is because of differences in pharmacodynamicsehavior
O drug response an
of the
of
drug in a particular patient. Various factors affecting a pharmacodynamic drug behavior
may include age, drug tolerance, drug interactions and unknown pathophysiological tactors
In addition, quantification of clinical response useful in the assessnent
is too inaccurate to be
of bioavailability of drug products. Examples mesalamine, ketoconazole, clotrimazole.
In vitro studies: In some cases in vitro studies are recommended for bioequivalence studies
Binding studies: in case of sevelamer carbonate and sevelamer HCL tablets two in vitro
teste are suggested by FDA.
i n vitro equilibrium binding which is considered as a pivotal bioequivalence study and
() in vitro kinetic binding which is used to support the pivotal equilibrium binding study
The bioequivalence is based on Langmuir binding consatant k, from the equilibrium
binding study.
Similarly in vitro studies are also recommended for calcium acetate ( in vitro phosphate
binding), colesevelam hydrochloride and cholestyramine powder.