BIOAVAILABILITY AND

BIOEQUIVALENCE STUDIES
CONTENTS
• BIOAVAILABILITY AND ITS TYPES
• METHODS FOR DETERMINATION OF
BIOAVAILABILITY
• BIOEQUIVALENCE
• TYPES OF BIOEQUIVALENCE STUDIES
BIOAVAILABILITY STUDIES
BIOAVAILABILITY AND ITS TYPES
BIOAVAILABILITY AND ITS
TYPES
INTRODUCTION:
DRUG PRODUCT PERFORMANCE:
In vivo, it may be defined as the release of drug substance from
the drug product leading to bioavailability of the drug
substance.
Thus, performance tests relate the quality of a drug product to
clinical safety and efficacy.

Bioavailability studies are the drug product performance

studies used to define the effect of changes in the
physiochemical properties of the drug substance, the
formulation of the drug , and the manufacture process of the
drug(dosage form).
BIOAVAILABILITY AND BIOEQUIVALENCE
STUDIES:
The drug product must meet all applicable standards of identity,
strength, quality, and purity. To ensure that these standards
are met, the FDA requires bioavailability/pharmacokinetic studies
and, where necessary, bioequivalence studies for all drug products.
Bioavailability studies are performed for both approved active
drug ingredients and therapeutic moieties not yet approved for
marketing by the FDA.
• Bioequivalence studies are used to compare the
bioavailability of the same drug (same salt or ester) from
various drug products.
• Bioavailability and bioequivalence can also be considered as
performance measures of the drug product in vivo.
DEFINITION:
Bioavailability means the rate and extent to
which the active ingredient or active moiety is
absorbed from a drug product and becomes available
at the site of action.

For drug products that are not intended to be

absorbed into the bloodstream, bioavailability may
be assessed by measurements intended to reflect the
rate and extent to which the active ingredient or
active moiety becomes available at the site of action.
DRUG CONCENTRATION TIME CURVE (AUC)
• The area under the drug concentration time
curve (AUC) is used as a measure of the total
amount of unaltered drug that reaches the
systemic circulation.
• The AUC is dependent on the total quantity of
available drug, divided by the elimination rate
constant, and the apparent volume of
distribution. F is the fraction of the dose
absorbed.
TYPES OF BIOAVAILABILITY:
Two types of bioavailability:
1) ABSOLUTE BIOAVAILABILITY
The absolute availability of drug is the systemic
availability of a drug after extravascular administration
(eg, oral, rectal, transdermal, subcutaneous) compared
to IV dosing. The absolute availability of a drug is
generally measured by comparing the respective AUCs
after extravascular and IV administration.
This measurement may be performed as long as V D
and k are independent of the route of administration.
Continue……..
• Absolute Bioavailability after oral drug administration using plasma
data can be determined as follows:

Absolute bioavailability= F=[AUC]PO/[Dose]PO

[AUC]IV/[Dose]IV

Absolute availability, F, may be expressed as a fraction or as a percent

by multiplying F x 100.
Absolute availability is sometimes expressed as a percent, ie, F = 1, or
100%.
For drugs given intravascularly, such as by IV bolus injection, F = 1
because all of the drug is completely absorbed.
For all extravascular routes of administration,such as the oral
route (PO), the absolute bioavailability F may not exceed 100% (F >
1).
TYPES OF BIOAVAILABILITY:
2) RELATIVE BIOAVAILABILITY:
Relative (apparent) availability is the availability of the drug
from a drug product as compared to a recognized
standard.

The fraction of dose systemically available from an oral drug

product is difficult to ascertain.

The availability of drug in the formulation is compared to the

availability of drug in a standard dosage formulation, usually
a solution of the pure drug evaluated in a crossover study.
Continue…..
• The relative availability of two drug products given
at the same dosage level and by the same route of
administration can be obtained using the following
equation:

Relative bioavailability= [AUC]A

[AUC]B

where drug product B is the recognized reference standard. This fraction may be

multiplied by 100 to give percent relative availability.

Continue…..
• When different doses are administered, a
correction for the size of the dose is made, as in
the following equation:
Relative bioavailability=[AUC]A/DOSEA
• [AUC]B/DOSEB
Relative bioavailability may exceed the value of 1 or
100% as compared to the reference drug product.
DETERMINATION OF
BIOAVAILABILTY
INTRODUCTION

Bioavailability testing is a means of

predicting the clinical efficacy of a drug
the estimation of the bioavailability of
a drug in a given dosage form is direct
evidence of the efficiency with which a
dosage form performs its intended
therapeutic function
OBJECTIVES OF THE STUDY

To compare
The ability to bioavailability of a
analyze the drug The The route of drug drug substance
pharmacodynamics administration, from same or
(and of the drug and the nature of different dosage
metabolites) in substance the drug product. forms produced
biological fluids by different
manufacturers
METHODS FOR ASSESSING BIOAVAILABILITY AND
BIOEQUIVALENCE
• Direct and indirect methods may be used
INDIREC
DIRECT
T
METHOD
METHOD
Plasma drug Urinary drug
concentration excretion

Clinical
observations

Acute
pharmacodyna
mic effect

In-vitro
studies
PLASMA DRUG CONCENTRATION
• Most direct and objective way to determine
systemic drug bioavailability.
• Measurement of drug concentrations in blood,
plasma, or serum after drug administration.
IMPORTANT PHARMACOKINETIC
PARAMETERS
• AUC: area under the concentration-time curve
 measure of the extent of bioavailability
• Cmax: the observed maximum concentration of
drug  measure of both the rate of absorption
and the extent of bioavailability
• tmax: the time after administration of drug at
which Cmax is observed  measure of the rate of
absorption
PLASMA CONCENTRATION TIME PROFILE
concentration

Cmax
AUC

Tmax time
DEFINITE INTEGRAL METHOD FOR
DETERMINING AUC
• The area between time intervals is the area of a
trapezoid and can be calculated with following
formula
• [AUC]tn-1tn = Cn-1 + Cn / 2 (tn – tn-1 ) Where [AUC]= area
under curve, tn = time of observation of drug conc ,
Cn and tn-1 is the time of prior observation of drug
conc corresponding to Cn-1
• The AUC between any two time intervals can be
calculated by
[AUC]t1t4 = [AUC]t1t2 +[AUC]t2t3 + [AUC]t3t4
Cmax
• The concentration of drug in the blood is the net
difference between drug input and drug output.
• Cmaxprovides warning of possibly toxic levels of
drug.
• The units of Cmax are concentration units (e.g.,
mg/mL, ng/mL).
tmax
• For a given dose and bioavailability fraction tmax
is inversely dependent on absorption rate .
• Units for tmax are units of time (e.g., hours,
minutes).
URINARY DRUG EXCRETION
INTRODUCTION
• An alternative and indirect method for assessment
of bioavailability.
• METHOD INVOLVES:

Determination of the total

Collection of urine samples quantity of drug excreted in
• IMPORTANCE: urine as a function of time

Urinary drug excretion is directly proportional to the plasma concentration

of total drug, thus the quantity of drug excreted in urine is a reflection of the
quantity of drug absorbed from the GIT.
EXAMPLE

EXAMPLE
80 mg drug
Two products, A recovered in urine This indicates that
and B (100 mg from Product A twice as much
drug each) drug was
administered 40 mg drug absorbed from A
orally recovered from as from B.
Product B
IMPORTANT CONSIDERATIONS:
Atleast 20% of a dose must be excreted
unchanged in urine

Fraction of drug entering the bloodstream

and being excreted intact by the kidneys must
remain constant.

Collection of the urine has to continue until

all the drug has been completely excreted.
DRAWBACKS OF THIS METHOD
In practice, these methods are subject to a high
degree of variability, and are less reliable than
those obtained from plasma concentration-time
profiles, thus these studies can only be used in
conjunction with blood level data for
confirmatory purposes
A) CUMULATIVE AMOUNT OF DRUG
EXRETED IN URINE - Du
• The cumulative amount of drug excreted in urine is
directly related to the total amount of drug absorbed.
• Urine samples are collected periodically after
administration of a drug product.
• Each urine sample is analyzed for free drug using specific
assay procedure.
• Graph is plotted that relates cumulative drug excreted to
the collection-time interval.
• When the drug is almost completely eliminated, the
plasma concentration approaches zero and maximum
amount of drug excreted in urine is obtained.
 31
Cumulative Amount of Drug Excreted in
the Urine
Cumulative amount excreted

One needs to
collect urine
Du samples for a
minimum of 7-
10 half-lives of
the drug to
assure all the
drug is
excreted into
the urine.
Time
B) RATE OF DRUG EXCRETION IN URINE
DDU/DT
• The measured urinary excretion rate reflects the
average plasma concentration during the
collection interval.
• Minimum rate of urinary drug excretion at zero
time and maximum rate as estimated from the
graph.
C) TIME FOR MAXIMUM URINARY DRUG
EXCRETION (T)

• The total time for drug to be excreted is t 

• It is related to the total time required for the
drug to be absorbed and completely excreted
after its administration.
• Useful parameter in bioequivalence studies that
compare several drug products.
BIOEQUIVALENCE STUDIES
ACUTE PHARMACODYNAMIC OR
PHARMACOLOGIC EFFECT
• In some cases, the quantitative measurement of a drug is
not available, or it lacks sufficient accuracy and/or
reproducibility. In such cases an acute pharmacodynamic
effect, such as effect on pupil diameter, heart rate, or blood
pressure, can be used as an index of drug bioavailability.
• This method is based on the assumption that a given
intensity of response is associated with a particular drug
concentration at the site of action,e.g..variation of meiotic
response intensity can be directly related to the oral dose of
chlorpromazine. In this case, an acute pharmacodynamic
effect-time curve is constructed.
• Monitoring of pharmacologic data is often difficult, precision
and reprocubility are difficult to establish, and there are only
a limited number of pharmacologic effects,e.g.. Heart rate,
body temperature, blood sugar levels..that are applicable to
this method.
• Measurement of the pharmacodynamic effect should be made
with sufficient frequency to permit a reasonable estimate of
the total AUC for time period at least three times the half-life
of the drug. Pharmacodynamic parameters that are obtained
include maximum pharmacodynamics effect(Emax), time for
maximum pharmacodynamics effect,area under the
pharmacodynamic effect-time curve and onset time for
 Pharmacodynamic effect.
• CLINICAL OBSERVATIONS
• One method for assessing the bioavailability of a drug
product is through the demonstration of a clinically
significant effect. Well controlled clinical trials in
humans can establish the safety and effectiveness of the
drug product. However, this approach is the least
accurate, least sensitive, and least reproducible of the
general approaches for determining in vivo
bioavailability. Moreover, such clinical studies are
complex,expensive,and time consuming.
• They also require a sensitive and quantitative measure of the
desired response. Furthermore, response is often quite
variable, requiring a large test population. Practical
considerations,therefore,preclude the use of this method
except in initial stages of development while proving the
efficacy of ea new chemical entity.
• SINGLE-DOSE VERSUS MULTIPLE-DOSE..
• Most bioavailability evaluations are made on the basis of
single-dose administration. The argument has been made
that single doses are not representative of the actual
clinical situation, since in most instances, patients require
repeated administration of a drug.
• When a drug is administered repeatedly at fixed intervals,
with the dosing frequency less than five half-lives, drug will
accumulate in the body and eventually reach a pleateau,or a
steady-state. At steady-state, the amount of drug eliminated
from the body during one dosing interval is equal to the
available dose(rate in=rate out),therefore, the area under
the curve obtained when a single dose is administered. This
AUC can therefore be used to assess the extent of absorption
of the drug, as well as its absolute and relative
bioavailability.
• Multiple-dose administration has several advantages over
single-dose bioavailability studies, as well as some
 Limitations.
• ADVANTAGES..
• Eliminates the need to extrapolate the plasma concentration
profiles to obtain the total AUC after a single dose.
• Eliminates the need for a long wash-out period between doses.
• More closely reflects the actual clinical use of the drug.
• Allows blood levels to be measured at the same concentration
s encountered therapeutically.
• Because blood levels tend to be higher than in the single-dose
method, quantitative determination is easier and more
reliable.
• Saturated pharmocokinetics,if present, can be
more readily detected at steady-state.
• Limitations..
• Requires more time to complete.
• More difficult and costly to conduct(requiring
prolonged monitoring of subjects)
• Greater problems with compliance control.
• Greater exposure of subjects to the test drug,
increasing the potential for adverse reactions.
INVITRO DISSOLUTION STUDIES
AND BIOAVAILABILITY
• Drug dissolution studies may under certain
conditions give an indication of drug
bioavailability. Ideally,the in-vitro drug
dissolution rate should correlate with the 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 drug dissolution in-
vitro will generally have the most rapid rate of
drug bioavailability in-vivo.
• Pharmaceutical scientists have for many years been
attempting to establish a correlation between some
physiochemical property of a dosage form and the biological
availability of the drug from that dosage form. The term
commonly used to describe this relationship is “in-vitro/in-
vivo correlation”. Specifically,it is felt that if such a
correlation could be established,it would be possible to use
in-vitro data to predict a drug;s in-vivo bioavailability. This
would drastically reduce,or in some cases,completely
eliminate the need for bioavailability tests. The desirability
for this becomes clear when one considers the cost and time
involved in bioavailability studies as
• well as the safety issues involved in
administration drugs to healthy subjects or
patients. It would certainly be preferable to be
able to subsitute a quick,inexpensive in-vitro
tests could reliably and accurately predict drug
absorption and reflect the in-vivo performance
of a drug in humans.
DISINTEGRATION TEST
• The early attempts to establish an indicator of
drug bioavailability focused on disintegration as
the most pertinent in vitro parameter. The first
official disintegration test appeared in USP in
1950.
• However it is true that solid dosage form must
disintegrate before significant and dissolution
may occur, meeting disintegration test
requirements only ensures that dosage form will
break up into small particles in sufficient length
of time.
• It does not ensure that rate of solution of drug is
adequate to produce suitable blood level of
active ingredient.
• Therefore, while the test for tablet disintegration
is very useful for quality control purposes in
manufacturing, it is poor index of bioavailability.
DISSOLUTION TESTS
• Since a drug must go into solution before it can
be absorbed and since the rate at which a drug
dissolves from dosage form often determines its
rate and extent of absorption(dissolution rate).
• It is currently most sensitive in vitro parameter
most likely to correlate with in vitro
bioavailability. Dissolution tests are valuable tool
in ensuring quality of drug product.
• Generally, product to product variations are due
to formulation factors such as particle size
differences, excessive amount of lubricant and
coating. These factors are reactive to dissolution
testing. Thus, dissolution test are effective in
discriminating between and within batches of
drug products. The dissolution test in addition
can exclude definitely any unacceptable product.
PROBLEMS WITH IN VITRO
DISSOLUTION TESTING
• These are those problems which make correlation
with in vivo availability difficult. The first is related
to instrument variance and absence of standard
method. The tests described in USP are but a few of
large number of dissolution methods proposed to
product bioavailability. Since dissolution rate of
dosage form is dependent on methodology used in
dissolution test, changes in apparatus, dissolution
medium can dramatically modify results.
• Another problem is related to difference between in
vitro and in vivo environments in which dissolution
occurs. In vitro studies are carried out under
controlled conditions in one or perhaps two,
standardized solvents. The in vivo ,on other hand
changes continuously.
Factors that effect dissolution rate in GIT are:
• pH
• enzyme secretion, surfacetension,motility,presence
of other substances.
• Adding to complexity of correlating dissolution
with in vivo absorption are factors such as drug-
drug interactions,age,food effect, health and
physical activity. these effects rate and extent of
absorption of drug. Thus ,in vivo environment is
far more complex than in vitro test environment
making in vitro/in vivo correlation difficult.
SELECTION OF PARAMETERS FOR IN VITRO/IN
VIVO STUDIES.
• The in vitro parameter should be selected that has
greatest effect on absorption characteristics of
drug. The in vitro/in vivo correlative methods
used most often are of single point where
dissolution rate (expressed as percent of drug
dissolved in single time) is correlated to certain
parameter of bioavailability. Examples of in vivo
parameters used are Cmax. ,AUC,time to reach
half maximal plasma conc., the average plasma
conc. After 0.5 or 1 hr,max. urinary excretion rate.
• According to Wagner,
“The best in vitro variable to use is the time for
50 percent of drug to dissolve and best variable
for in vivo data to use is time for 50 percent of
drug to be absorbed.”
IN VITRO/IN VIVO CORRELATION STUDIES
• There have been many attempts to establish in vitro/in
vivo correlations for variety of drugs.
• While there are many published examples of satisfactory
correlations between absorption parameters and in vitro
dissolution test most studies resulted in poor
correlations. Moreover, positive correlation that have
been found generally applied only to specific formulation
studies. There have been instances where dissolution
rate or various formulations of same drug have been
different yet little or no difference was observed in
bioavailability parameters.
• There have been cases where drug has failed to meet
dissolution standards but has demonstrated adequate
bioavailability. Welling states that, To the writers knowledge
there has been no studies that have accurately correlated in
vitro/in vivo data to point that use of upper or lower limits for
in vitro dissolution parameters can be confidently used to
predict in vivo behavior and therefore to replace in vivo testing.
• Even if in vitro test could be designed that would accurately
reflect dissolution process in GIT ,dissolution is only one of
many factors that effects drug bioavailability. Dissolution
studies also would not predict poor bioavailability due to
instability in gastric fluid or complication with another drug .
• Thus, the ultimate evaluation a drug product’s
performance under conditions expected in
clinical therapy must be in vivo test ;a dissolution
test is unlikely to entirely replace bioavailability
testing. In vitro methods are important in
development and optimization of dosage forms
while in vivo tests are essential in obtaining
information on the behavior of medication in
living organisms. One cannot be substituted for
the other.
BIOEQUIVALENCE:
• Two medicinal products are bioequivalent if they are
pharmaceutical equivalents or alternatives and if
their bioavailabilities (rate and extent) after
administration in the same molar dose are similar to
such degree that their effects, with respect to both
efficacy and safety, will be essential the same.

• i.e. their plasma concentration-time profiles will be

• identical without significant statistical differences.
ADVANTAGES AND DISADVANTAGES
• Advantages:-
• Minimizes the effect of inter subject variability.
• It minimizes the carry over effect.
• Requires less number of subjects to get meaningful results.
• Disadvantages:-
• Requires longer time to complete the studies.
• Completion of studies depends on number of formulations evaluated
in the studies.
• Increase in study period leads to high subjectdropouts.
• Medical ethics does not allow too many trials on a subject continuously
for a longer time.
REQUIRMENTS / OBJECTIVES
• If a new product is intended to be a substitute for an
approved medicinal product as a pharmaceutical
equivalent or alternative, the equivalence with this
product should be shown or justified.
• In order to ensure clinical performance of such drug
products, bioequivalence studies should be performed.
• Bioequivalence studies are conducted if there is:
• A risk of bio - in equivalence and/or
• A risk of pharmacotherapeutic failure or diminished
clinical safety.
• Some of the important terms relevant in this context will be
defined.
• EQUIVALENCE :-
It is a relative term that compare drug products with respect
to a specific characteristic or function or toa defined set of
standards.
• There are several types of equivalences.
• Chemical Equivalence
• Pharmaceutical Equivalence
• Bioequivalence
• Therapeutic Equivalence
TYPES OF EQUIVALENCE
A. CHEMICAL EQUIVALENCE :- It indicates that two or
more drug products contain the same labelled chemical
substance as an active ingredient in the same amount.

B. PHARMACEUTICAL EQUIVALENCE :- This term

implies that two or more drug products are identical in
strength, quality, purity, content uniformity and
disintegration and dissolution characteristics. They may,
however, differ in containing different excipients.
 C. BIOEQUIVALENCE :- It is a relative term which denotes that the
drug substance in two or more identical dosage forms, reaches the
systemic circulation at the same relative rate and to the same relative
extent i.e. their plasma concentration-time profiles will be identical
without significant statistical differences. When statistically
significant differences are observed in the bioavailability of two or
more drug products, bio-inequivalence is indicated.

D. THERAPEUTIC EQUIVALENCE :- This term indicates that two

or more drug products that contain the same therapeutically active
ingredient elicit identical pharmacological effects and can control
the disease to the same extent.
TYPES OF BIOEQUIVALENCE STUDIES
Bioequivalence can be demonstrated either –

• In vivo, or
• In vitro.
IN VIVO BIOEQUIVALENCE STUDIES
• The following sequence of criteria is useful in assessing the need for in
vivo studies:
1. Oral immediate-release products with systemic action-
• Indicated for serious conditions requiring assured response.
• Narrow therapeutic margin.
 Pharmacokinetics complicated by absorption
• < 70 % or absorption window, nonlinear kinetics,
• presystemic elimination > 70 %.
• Unfavorable physiochemical properties, e.g. low solubility, metastable
modification, instability, etc.
• Documented evidence for bioavailability problems.
• No relevant data available, unless justification by applicant that in vivo
study is not necessary.
2. Non-oral immediate-release products.

3. Modified-release products with systemic action.

 In vivo bioequivalence studies are conducted in the
• usual manner as discussed for bioavailability
• studies, i.e. the pharmacokinetic and the
• pharmacodynamic methods.
• 1. Pharmacokinetic Methods
• a) Plasma level-time studies
• b) Urinary Excretion studies

• 2. Pharmacodynamic Methods
• a) Acute pharmacological response
• b) Therapeutic response
IN VITRO BIOEQUIVALENCE STUDIES
• If none of the above criteria is applicable,
• comparative in vitro dissolution studies will suffice.
• In vitro studies, i.e. dissolution studies can be used
• in lieu of in vivo bioequivalence under certain
• circumstances, called as biowaivers(exemptions)-
1.The drug product differs only in strength of the active substance It contains,
provided all the following conditions hold Pharmacokinetics are linear.
 The qualitative composition is the same.
• The ratio between active substance and the
• excipients is the same, or (in the case of small
• strengths) the ratio between the excipients is the
• same.
 Both products are produced by the same manufacturer at the same
production site.
 A bioavailability or bioequivalence study has been performed with a
original product.
 Under the same test conditions, the in vitro dissolution rate is the same.
2. The drug product has been slightly reformulated or the manufacturing
method has been slightly modified by the original manufacturer in ways
that can convincingly be argued to be irrelevant for the bioavailability.
3. The drug product meets all of the following requirements –
 The product is in the form of solution or solubilised form (elixir, syrup,
tincture, etc).
 The product contains active ingredient in the
 same concentration as the approved drug product.
 The product contains no excipients known to significantly affect
absorption of the active ingredient.
• The product is intended for topical administration
• (cream, ointment, gel, etc.) for local effect.
• The product is for oral administration but not intended to be
absorbed (antacid or radio-opaque medium).
 The product is administered by inhalation as a gas
• or vapour.
• The criteria for drug products listed above indicate
• that bioavailability and bioequivalence are self-
• evident.