Bioequivalence

studies
Equivalence.
Relationship in terms of bioavailability, therapeutic
response, or a set of established standards of one drug
product to another.
Bioequivalent drug products.

This term describes pharmaceutical equivalent or

pharmaceutical alternative products that display
comparable bioavailability when studied under similar
experimental conditions.
 Bases for Bioequivalence
comparison
1- A Bioequivalence is said to be established if,
the in-vivo bioavailability of a test drug product (usually the
generic product) does not differ significantly in the
product's rate and extent of drug absorption , from that of
the reference listed drug (usually the brand-name product)
when administered at the same molar dose of the active
moiety under similar experimental conditions, either single
dose or multiple dose.
2- The comparison of measured parameters are
concentration of the active drug ingredient in the blood , Cp
urinary excretion rates, or pharmacodynamic effects.
 When difference in rate of
absorption
In a few cases, a drug product that differs from the
reference listed drug in its rate of absorption, but not
in its extent of absorption, may be considered bioequivalent
if
1- The difference in the rate of absorption is intentional and
appropriately reflected in the labeling
2- The rate of absorption is not injurious to the safety and
effectiveness of the drug product.
Demonstration through In vitro
data
Bioequivalence may sometimes be demonstrated using an
in-vitro bioequivalence standard, especially when such an
in-vitro test has been correlated with human in-vivo
bioavailability data.
 In other situations, bioequivalence may sometimes be
demonstrated through
i) comparative clinical trials or
ii) pharmacodynamic studies.
 Presence of inactive
ingredients

Bioequivalent drug products may contain different

inactive ingredients,
1- provided the manufacturer identifies the differences
2- provides information that the differences do not affect
the safety or efficacy of the product.
 Differences in the predicted clinical
response
Differences in the predicted clinical response or an adverse
event may be due to
1- differences in the pharmacokinetic and/or
pharmacodynamic behavior of the drug among individuals
or
2- differences in the bioavailability of the drug from the
drug product.
Bioequivalent drug products that have the same systemic
drug bioavailability will have the same predictable drug
response.
However, variable clinical responses among individuals that
are unrelated to bioavailability may be due to differences in
the pharmacodynamics of the drug.
 Various factors affecting
pharmacodynamic drug behavior

Differences in pharmacodynamics, ie, the relationship

between the drug and the receptor site, may be due to
differences in receptor sensitivity to the drug.
Various factors affecting pharmacodynamic drug behavior
may include
◦ 1- age,
◦ 2- drug tolerance,
◦ 3- drug interactions, and
◦ 4- unknown pathophysiologic factors.
◦ 5- Fasted individuals and fed state.
◦ 6- lifestyle may affect the plasma drug levels because of
variable absorption in the presence of food ,even a
change in the metabolic clearance of the drug.
It is reported that patients on a high carbohydrate diet have
a much longer elimination half-life of theophylline,
due to the reduced metabolic clearance of the drug (t 1/2,
18.1 hours), compared to patients on normal diets (t 1/2 =
6.76 hours).
Drugs with Possible Bioavailability and Bioequivalence
Problems

When evidence from well-controlled clinical trials or

controlled observations in patients of various marketed
drug products
do not give comparable therapeutic effects.
These drug products need to be evaluated either in vitro
(eg, drug dissolution/release test) or in vivo (eg,
bioequivalence study) to determine if the drug product has
a bioavailability problem.
In addition, during the development of a drug product,
certain biopharmaceutical properties of the active drug
substance or the formulation of the drug product may
indicate that the drug may have variable bioavailability
and/or a bioequivalence problem.
Some of the biopharmaceutics
problems include
1- The active drug ingredient has low solubility in water
(eg, less than 5 mg/mL).
2- The dissolution rate of one or more such products is
slow (eg, less than 50% in 30 min).
3- The particle size and/or surface area of the active drug
ingredient is critical in determining its bioavailability.
4- Certain structural forms of the active drug ingredient
(eg, polymorphic forms, solvates, complexes, and crystal
modifications) dissolve poorly, thus affecting absorption.
5- Drug products that have a high ratio of excipients to
active ingredients (eg, greater than 5:1).
6- Specific inactive ingredients (eg, hydrophilic or
hydrophobic excipients and lubricants) either may be
required for absorption of the active drug ingredient or
therapeutic moiety or may interfere with such absorption.
7- The degree of absorption of the active drug
ingredient, or its precursor is poor (eg, less than 50%,
ordinarily in comparison to an intravenous dose), even
when it is administered in pure form (eg, in solution).
DESIGN AND EVALUATION OF
BIOEQUIVALENCE STUDIES

Bioequivalence studies are performed to compare the

bioavailability of the generic drug product to the brand
name product.
Statistical techniques should be of sufficient sensitivity to
detect differences in rate and extent of absorption that are
not attributable to subject variability.
Once bioequivalence is established, it is likely that both the
generic and brand-name dosage forms will produce the
same therapeutic effect.
 Design Requirements
The design and evaluation of well-controlled bioequivalence
studies require cooperative input from pharmacokinetics,
statisticians, clinicians, bioanalytical chemists, and others.
The basic design for a bioequivalence study is determined by
1) The scientific questions to be answered.
2) The nature of the reference material and the
dosage form to be tested.
3) The availability of analytical methods,
4) Benefit-risk and ethical considerations with regard to
testing in humans.
How a drug is Bioequivalent
with respect to standard drug
For bioequivalence studies, the test and reference drug
formulations must contain
The pharmaceutical equivalent drug
The same dose strength,
 In similar dosage forms
 The same route of administration.
The basic guiding principle in
performing studies
Do not do unnecessary human research.
Generally, the study is performed in normal, healthy male
and female volunteers who have given informed consent to
be in the study.
 Critically ill patients are not included in an in-vivo
bioavailability study unless the attending physician
determines that there is a potential benefit to the patient.
Principles for using
Reference Standard

For bioequivalence studies, one formulation of the drug is

chosen as a reference standard against which all other
formulations of the drug are compared.
1- The reference drug product should be administered
by the same route as the comparison formulations unless
an alternative route or additional route is needed to
answer specific pharmacokinetic questions.
For example, if an active drug is poorly bioavailable after
oral administration, the drug may be compared to an oral
solution or an intravenous injection.
 2- Before beginning an in-vivo bioequivalence study, the
total content of the active drug substance in the test
product (generally the generic product) must be within
5% of that of the reference product.
3-- First invitro then in-vivo bioequivalence study
For bioequivalence studies on a proposed generic drug product
the reference standard is, the reference listed drug
 Selection of subjects
1- The number of subjects in the study will depend on
the expected inter subject and intra subject variability.
2- Subject selection is made according to certain
established criteria for inclusion into, or exclusion from,
the study.
a- Who have known allergies to the drug,
b- Overweight,
c- Have taken any medication within a specified
period (often 1 week) prior to the study.
d- Smokers are often not included in these studies.
3- The subjects are generally fasted for 10 to 12 hours
(overnight) prior to drug administration and may
continue to fast for a 2 to 4 hour period after dosing.
STUDY DESIGNS

Currently, different studies may be required for solid oral

dosage forms, including
1) Fasting study,
2) Food intervention study, and/or
3) Multiple-dose (steady-state) study.
4) The using a replicate design
5) Two-way crossover food intervention study.
Bioequivalence studies are usually evaluated by a
 Single-dose,
 Two-period,
 Two-treatment,
In an A,B/B,A crossover trial, patients are randomly assigned
to receive either treatment A in the first period followed by
treatment B in the second period or
treatment B in the first period followed by treatment A in
the second period.
two-sequence, (orders)
open-label,
A clinical trial in which researchers and participants know
which drug or vaccine is being administered.
Randomized crossover design
A type of clinical trial in which all participants receive the
same two or more treatments, but the order in which they
receive them depends on the group to which they are
randomly assigned.
The crossover trial allows for a within-patient comparison
between treatments because each patient serves as his or
her own control subject, removes the interpatient variability
from the comparison between treatments, and can provide
unbiased estimates for the differences between treatments.
Fasting Study
The subjects should be in the fasting state (overnight fast
of at least 10 hours) before drug administration and should
continue to fast for up to 4 hours after dosing.
No other medication is normally given to the subject for at
least 1 week prior to the study.
In some cases, a parallel design may be more appropriate
for certain drug products, containing a drug with a very long
elimination half-life.
A parallel study is a type of clinical study where two groups
of treatments, A and B, are given so that one group receives
only A while another group 
 A replicate design may be used for a drug product
containing a drug that has high intra subject variability.
Food Intervention Study

Co-administration of food with an oral drug product may

affect the bioavailability of the drug.
Conducted by using meal conditions that are expected to
provide the greatest effects on GI physiology so that
systemic drug availability is maximally affected.
The test meal is a high-fat and high-calorie meal.
A typical test meal is two eggs fried in butter, two strips of
meat, two slices of toast with butter, 4 ounces of brown
potatoes, and 8 ounces of milk.
Food Intervention
Study------
Both the test and reference products should be affected
similarly by food.
The study design uses a single-dose, randomized, two-
treatment, two-period, crossover study comparing equal doses of
the test and reference products.
Following an overnight fast of at least 10 hours, subjects are
given the recommended meal 30 minutes before dosing.
The meal is consumed over 30 minutes, with administration of
the drug product immediately after the meal.
The drug product is given with 240 mL (8 fluid ounces) of water.
No food is allowed for at least 4 hours post dose
This study is required for all modified-release dosage forms
and may be required for immediate-release dosage forms if
the bioavailability of the active drug ingredient is known to
be affected by food (eg, ibuprofen, naproxen).
 For certain extended-release capsules that contain coated
beads, the capsule contents are sprinkled over soft foods
such as apple sauce, which is taken by the fasted subject
and the bioavailability of the drug is then measured.
Bioavailability studies might also examine the affects of
other foods and special vehicles such as apple juice.
Multiple-Dose (Steady-State) Study

 For these studies, three consecutive trough concentrations

(C min) on three consecutive days should be determined to
determine that the subjects are at steady state.
The last morning dose is given to the subject after an
overnight fast, with continual fasting for at least 2 hours
following dose administration.
Blood sampling is performed similarly to the single-dose
study.
Crossover Designs

Subjects are selected at random.

 Each subject receives the test drug product and the
reference product.
Latin-square crossover designs
Human volunteers, comparing three different drug
formulations (A, B, C) or four different drug formulations (A,
B, C, D)
 The Latin-square design plans the clinical trial so that each
subject receives each drug product only once, with
adequate time between medications for the elimination of
the drug from the body .
 In this design, each subject is his own control, and subject-
to-subject variation is reduced.
variation due to sequence, period, and treatment are
reduced, so that all patients do not receive the same drug
product on the same day and in the same order.
 Possible carryover effects from any particular drug
product are minimized by changing the sequence or order
in which the drug products are given to the subject.
Thus, drug product B may be followed by drug product A,
D, or C .
After each subject receives a drug product, blood samples
are collected at appropriate time intervals so that a valid
blood drug l-time curve is obtained.
The time intervals should be spaced so that the peak blood
concentration, the total area under the curve, and the
absorption and elimination phases of the curve may be well
described.
Replicated Crossover Design
four-period, two-sequence, two
formulation
Replicated crossover designs are used for the
determination of individual bioequivalence, to estimate
within subject variance for both the Test and Reference
drug products, and to provide an estimate of the subject-by
formulation interaction variance.
Generally, a four-period, two-sequence, two-formulation
design is recommended by the FDA.
 Period 1 Period 2 Period 3 Period 4
Sequence 1 T R T R
Sequence 2 R T R T

In this design, Reference-to-Reference and Test-to-Test

comparisons may also be made.
EVALUATION
OF THE DATA
Analytical Methods

The analytical method used in an in-vivo bioavailability or

bioequivalence study to measure
The concentration of the active drug ingredient ,
therapeutic moiety, or its active metabolite(s),
STUDY IN
body fluids
Excretory products,
 measure an acute pharmacological effect,
Analytical Method-------

The analytical method for measurement of the drug must

be validated for accuracy, precision, sensitivity, and
specificity.
The use of more than one analytical method during a
bioequivalence study may not be valid, because different
methods may yield different values.
Data should be presented in both tabulated and graphic
form for evaluation.
The plasma drug concentration-time curve for each drug
product and each subject should be available.
Pharmacokn
ieticEvaluationofthe Data

For single-dose studies, including a fasting study or a food

intervention study, the pharmacokinetic analyses include
calculation for each subject of the AUC to the last
quantifiable concentration (AUC-t) , Tmax, and Cmax.
Additionally, the elimination rate constant, k, the
elimination half-life, t1/2, and other parameters may be
estimated.
Pharmacokinetic evaluation of the data for multiple dose regimen

 For multiple-dose studies, pharmacokinetic analysis

includes calculation for each subject of the steady-state
AUC, (AUC-t), Tmax, Cmin, Cmax, and
the percent fluctuation [100 x (Cmax - Cmin)/C min].
Proper statistical evaluation should be performed on the
estimated pharmacokinetic parameters.
Statistical Evaluation of the Data
Average Bioequivalence
Bioequivalence is generally determined using a
comparison of population averages of a bioequivalence
metric, such as AUC and C max.
This involves the calculation of a 90% confidence interval
for the ratio of averages (population geometric means) of
the bioequivalence metrics for the Test and Reference drug
products.
To establish bioequivalence, the calculated confidence
interval should fall within a prescribed bioequivalence limit,
usually, 80-125% for the ratio of the product averages.
Individual bioequivalence
This approach is proposed by the FDA and others.
Individual bioequivalence requires a replicate
crossover design, and estimates within-subject
variability for the Test and Reference drug products,
as well as subject-by formulation interaction.
 Presently, only average bioequivalence estimates
are used to establish bioequivalence of generic
drug products.
USE OF LOG VALUE
The true distribution of these biological
parameters may be difficult to establish because of
the small number of subjects used in a
bioequivalence study.
The distribution of data that has been
transformed to log values resembles more closely a
normal distribution compared to the distribution of
non-log-transformed data.
Therefore, log transformation of the bioavailability
data (eg, Cmax, AUC) is performed before statistical
data evaluation for bioequivalence determination.
To prove bioequivalence, there must be no statistical
difference between the bioavailability of the Test product
and the Reference product.
Several statistical approaches are used to compare the
bioavailability of drug from the test dosage form to the
bioavailability of the drug from the reference dosage form.
Many statistical approaches (parametric tests) assume that
the data are distributed according to a normal distribution
or "bell-shaped curve" .
CLINICAL SIGNIFICANCE OF
BIOEQUIVALENCE STUDIES

Bioequivalence of different formulations of the same drug

substance involves equivalence with respect to rate and
extent of systemic drug absorption.
Clinical interpretation is important in evaluating the results
of a bioequivalence study.
A small difference between drug products, even if
statistically significant, may produce very little difference in
therapeutic response.
 Generally, two formulations whose rate and extent of
absorption differ by 20% or less are considered
bioequivalent.
The considered that differences of less than 20% in AUC
and C max between drug products are "unlikely to be
clinically significant in patients."
Therefore, normal variation is observed in medical practice
and plasma drug levels may vary among individuals greater
than 20%.
A small, statistically significant difference in drug
bioavailability from two or more dosage forms may be
detected if the study is well controlled and the number of
subjects is sufficiently large.
 When the therapeutic objectives of the drug are
considered, an equivalent clinical response should be
obtained from the comparison dosage forms if the plasma
drug concentrations remain above the (MEC) for an
appropriate interval and do not reach the (MTC).
Therefore, the investigator must consider whether any
statistical difference in bioavailability would alter clinical
efficiency.
Why the healthy volunteers
are preferred ?
Special populations, such as the elderly or patients on drug
therapy, are generally not used for bioequivalence studies.
 Normal, healthy volunteers are preferred for
bioequivalence studies, because these subjects are less at
risk and may more easily endure the discomforts of the
study, such as blood sampling.
The objective of these studies is to evaluate the
bioavailability of the drug from the dosage form, and use of
healthy subjects should minimize both inter- and
intrasubject variability.