BIOAVAILABILITY STUDY DESIGNS,

COMPONENTS & APPLICATIONS,

REPORT FORMAT
STUDY DESIGNS
 For many drug products, the FDA, Division of Bioequivalence, Office of
Generic Drugs, provides guidance for the performance of in vitro dissolution
and in vivo bioequivalence studies (US-FDA, CDER, 2010a).
 Generally, two bioequivalence studies are required for solid oral dosage

forms, including
(1) a fasting study and
(2) a food intervention study.
 For extended-release capsules containing beads (pellets) that might be poured
on a semisolid food such as applesauce, an additional “sprinkle”
bioequivalence study is recommended.
 Other study designs such as parallel design, replicate design, and multiple-

dose (steady-state) bioequivalence studies have been proposed by the FDA.

 Proper study design and statistical evaluation are important considerations for

the determination of bioequivalence.

 Some of the designs listed above are summarized here.
A. Fasting Study
 Bioequivalence studies are usually evaluated by a single-dose, two-period, two-
treatment, two-sequence, open-label, randomized crossover design comparing equal
doses of the test and reference products in fasted, adult, healthy subjects.
 This study is requested for all immediate-release and modified-release oral dosage
forms.
 Both male and female subjects may be used in the study.
 Blood sampling is performed just before (zero time) the dose and at appropriate
intervals after the dose to obtain an adequate description of the plasma drug
concentration–time profile.
 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 replicate design may be used for a drug product containing a drug that has high
intra-subject variability.
B. Food Intervention Study
 Co-administration of food with an oral drug product may affect the bioavailability of
the drug.
 Food intervention or food effect studies are generally conducted using meal conditions
that are expected to provide the greatest effects on GI physiology so that systemic drug
availability is maximally affected.
 Food effects on bioavailability are generally greatest when the drug product is
administered shortly after a meal is ingested.
 The nutrient and caloric contents of the meal, the meal volume, and the meal
temperature can cause physiological changes in the GI tract in a way that affects drug
product transit time, luminal dissolution, drug permeability, and systemic availability.
 In addition, the high fat meal can have a significant effect on certain modified-release
drug products causing them to dose dump.
C. CROSSOVER STUDY DESIGNS
 Subjects who meet the inclusion and exclusion study criteria and have given
informed consent are selected at random.
 A complete crossover design is usually employed, in which each subject
receives the test drug product and the reference product.
 Examples of Latin-square crossover designs for a bioequivalence study in
human volunteers, comparing three different drug formulations (A, B, C) or
four different drug formulations (A, B, C, D), are described in Tables 16-7
and 16-8.
 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.
 Moreover, variations due to sequence, period, and treatment (formulation) are
reduced, so that all patients do not receive the same drug product on the same
day and in the same order.
 The order in which the drug treatments are given should not stay the same in order to
prevent any bias in the data due to a residual effect from the previous treatment.
 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 (Table 16-8).
 After each subject receives a drug product, blood samples are collected at appropriate
time intervals so that a valid blood drug level–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.
 Period refers to the time period in which a study is performed.
 A two-period study is a study that is performed on two different days (time periods)
separated by a washout period during which most of the drug is eliminated from the
body—generally about 10 elimination half-lives.
 A sequence refers to the number of different orders in the treatment groups in a study.
For example, a two-sequence, two-period study would be designed as follows:
D. Replicated Crossover Study Designs
 The standard bioequivalence criterion using the two-way crossover design does not
give an estimate of within-subject (intra-subject) variability.
 By giving the same drug product twice to the same subject, the replicate design
provides a measure for within-subject variability.
 Replicate design studies may be used for highly variable drugs and for narrow
therapeutic index drugs.
 In the case of highly variable drugs (%CV greater than 30), a large number of subjects
(>80) would be needed to demonstrate bioequivalence using the standard two-way
crossover design.
 Drugs with high within-subject variability generally have a wide therapeutic window
and despite high variability, these products have been demonstrated to be both safe and
effective.
 Replicate designs for highly variable drugs/products require a smaller number of
subjects and, therefore, do not unnecessarily expose a large number of healthy subjects
to a drug when this large number of subjects is not needed for assurance of
bioequivalence (Haidar et al, 2008).
 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. A four-period, two-sequence, two-formulation design is shown below:

 In this design, the same reference and the same test are each given twice to the same
subject.
 Other sequences are possible. In this design, reference-to-reference and test-to-test
comparisons may also be made.
E. Narrow Therapeutic Index Drugs
 Narrow therapeutic index (NTI) drugs, also referred to as critical dose drugs, are drugs
in which small changes in dose or concentration may lead to serious therapeutic
failures or serious adverse drug reactions in patients.
 Narrow therapeutic index drugs consistently display the following characteristics:
(a) Sub-therapeutic concentrations may lead to serious therapeutic failure;
(b) there is little separation between therapeutic and toxic doses (or the associated
plasma concentrations);
(c) they are subject to therapeutic monitoring based on pharmacokinetic or
pharmacodynamic measures;
(d) they possess low-to-moderate within-subject variability (<30%);

 The FDA currently recommends that bioequivalence studies of narrow therapeutic

index drugs should employ a four-way, fully replicated, crossover study design.
 The replicated study design permits comparison of both test and reference means
and test and reference within-subject variability (Davit et al, 2013).
F. Reference Scaled Average Bioequivalence
 Recently a three-sequence, three-period, two-treatment partially replicated crossover
design for bioequivalence studies of highly variable drugs has been recommended by
the FDA (Haidar et al, 2008).
 The partially replicated design allows the estimation of the within-subject variance and
subject-by-formulation interaction for the reference product.
 The time for completion of this study is shorter than the fully replicated four-way
crossover design.
 This design is usually used for highly variable drugs with within-subject variability
≥30%.
G. Parallel Study Designs
 A non-replicate, parallel design is used for drug products that contain drugs that have a
long elimination half-life or drug products such as depot injections in which the drug
is slowly released over weeks or months.
 In this design, two separate groups of volunteers are used.
 One group will be given the test product and the other group will be given the
reference product.
 It is important to balance the demographics of both groups of volunteers.
 Blood sample collection time should be adequate to ensure completion of
gastrointestinal transit (approximately 2–3 days) of the drug product and absorption of
the drug substance.
 Cmax and a suitably truncated AUC, generally to 72 hours after dose administration,
can be used to characterize peak and total drug exposure, respectively.
 For drugs that demonstrate low intrasubject variability in distribution and clearance,
an AUC truncated at 72 hours (AUC072 hours) can be used in place of AUC0 or AUC0.
t

 This design is not recommended for drugs that have high intra-subject variability in
distribution and clearance.
H. Multiple-Dose (Steady-State) Study Design
 Multiple doses of the same drug are given consecutively to reach steady-state plasma
drug levels.
 The multiple-dose study is designed as a steady-state, randomized, two-treatment,
two-way, crossover study comparing equal doses of the test and reference products in
healthy adult subjects.
 Each subject receives either the test or the reference product separated by a “washout”
period, which is the time needed for the drug to be completely eliminated from the
body.
 To ascertain that the subjects are at steady state, three consecutive trough
concentrations (Cmin) are determined.
 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 then performed over one dosing interval.
 The area under the curve during a dosing interval at steady state should be the same as
the area under the curve extrapolated to infinite time after a single dose.
 Pharmacokinetic analyses for multiple-dose studies include calculation of the
following parameters for each subject:
 AUC0-tau—Area under the curve during a dosing interval
 tmax—Time to Cmax during a dosing interval
 Cmax—Maximum drug concentration during dosing interval
 Cmin—Drug concentration at the end of a dosing interval
 Cav—The average drug concentration during a dosing interval
 Degree of fluctuation = (Cmax− Cmin)/Cmax
 Swing = (Cmax− Cmin)/Cmin
 The data are analyzed statistically using analysis of variance (ANOVA) on the log-
transformed AUC and Cmax.
 To establish bioequivalence, both AUC and Cmax for the test (generic) product should
be within 80%–125% of the reference product using a 90% confidence interval.
 Estimation of the absorption rate constant during multiple dosing is difficult, because
the residual drug from the previous dose superimposes on the dose that follows.
 However, the data obtained in multiple doses are useful in calculating a steady-state
plasma level.
 The extent of bioavailability, measured by assuming the [AUC] 0, is dependent on
clearance:

 Determination of bioavailability using multiple doses reveals changes that are

normally not detected in a single-dose study.
 For example, non-linear pharmacokinetics may occur after multiple drug doses due to
the higher plasma drug concentrations saturating an enzyme system involved in
absorption or elimination of the drug.
 Non-linear pharmacokinetics after multiple-dose studies may be observed by rising
Cmin drug concentrations and AUCt after each dosing interval.
 With some drugs, a drug-induced malabsorption syndrome can also alter the
percentage of drug absorbed.
 In this case, drug bioavailability may decrease after repeated doses if the fraction of
the dose absorbed (F) decreases or if the total body clearance (k.V D) increases.
 It should be noted that nonlinear PK can also be observed by high single doses of the
drug.
H. Clinical Endpoint Bioequivalence Study
 Study design for a clinical endpoint study generally consists of a randomized, double-
blind, placebo-controlled, parallel-designed study comparing test product, reference
product, and placebo product in patients.
 A placebo arm is usually included to demonstrate that the treatments are active (above
the no-effect part of the effect versus dose curve, see Fig. 16-11) and the study is
sufficiently sensitive to identify the clinical effect in the patient population enrolled in
the study.
 In some cases, the use of a placebo may not be included for safety reasons.
 The primary analysis for bioequivalence is determined by evaluating the difference
between the proportion of patients in the test and reference treatment groups who are
considered a “therapeutic cure” at the end of study.
 The superiority of the test and reference products against the placebo is also tested
using the same dichotomous endpoint of “therapeutic cure.”