BIOAVAILABILITY AND

BIOEQUIVALANCE STUDIES :
PROTOCOL AND REGULATORY
REQUIREMENT

Prepared By: Md. Tayfuzzaman

DEFINITION
Bioavailability
Regulatory Definition (21 CFR 320.1(a)):

“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.”
Bioequivalence
Regulatory Definition (21 CFR 320.1(e)):

“Bioequivalence means the absence of a significant difference in the rate and

extent to which the active ingredient or active moiety in pharmaceutical
equivalents or pharmaceutical alternatives becomes available at the site of drug
action when administered at the same molar dose under similar conditions in
an appropriately designed study.”
 PHARMACEUTICAL EQUIVALENTS
Pharmaceutical equivalents are drug products that contain identical amounts of
the identical active drug ingredient, i.e., the same salt or ester of the same
therapeutic moiety, in identical dosage forms, but not necessarily containing
the same inactive ingredients.

 PHARMACEUTICAL ALTERNATIVES

Pharmaceutical alternatives are drug products that contain the identical

therapeutic moiety, or its precursor, but not necessarily in the same
amount or dosage form or as the same salt or ester.

 THERAPEUTIC EQUIVALENTS
Therapeutic equivalents are drug products that contain the same active
substance or therapeutic moiety and, clinically show the same efficacy and
safety.
REFERENCE PRODUCT
Identified by the Regulatory Authorities as
“Designated Reference Product”

 Usually the Global Innovator’s Product

 Protected by a patent

 Marketed under manufacturers brand name

 Clinical efficacy & safety profile is well documented in extensive trials

 All generics must be Bioequivalent to it

TEST PRODUCT
For Oral solid forms for systemic action:
a) The test product should usually originate from a batch of at least 1/10 of production scale
or 100,000 units, whichever is greater, unless otherwise justified.
b) The production of batches used should provide a high level of assurance that the product
and process will be feasible on an industrial scale. In case of a production batch smaller than
100,000 units, a full production batch will be required.
c) The characterisation and specification of critical quality attributes of the drug product, such
as dissolution, should be established from the test batch, i.e. the clinical batch for which
Bioequivalence has been demonstrated.
d) Samples of the product from additional pilot and/or full scale production batches, submitted
to support the application, should be compared with those of the BE study test batch, and
show similar in vitro dissolution profiles when employing suitable dissolution test condition.
e) Comparative dissolution profile testing should be undertaken on the first three production
batches.
f) If full scale production batches are not available at the time of submission, the applicant
should not market a batch until comparative dissolution profile testing has been completed.
For other immediate release pharmaceutical forms for systemic action, justification of the
representative nature of the test batch should be similarly established.
REQUIREMENT OF BA & BE STUDIES

 For IND/NDAs:
To establish equivalence between:
• Early & late clinical trial formulations
• Formulations used in clinical trial & stability studies, if different
• Clinical trial formulations & to-be-marketed drug product
• Any other comparisons, if appropriate

 ANDA for a generic drug product

 Change in components, composition, &/or manufacturing process

 Change in dosage form (capsules to tablet)

NDA VS. ANDA REVIEW PROCESS
NDA Requirements ANDA Requirements

1. Chemistry 1. Chemistry
2. Manufacturing 2. Manufacturing
3. Controls 3. Controls
4. Labeling 4. Labeling
5. Testing 5. Testing
6. Animal Studies
7. Clinical Studies 6. Bioequivalence
8. Bioavailability
DESIGN AND CONDUCT OF STUDIES

I. Pharmacokinetic Studies

II. Pharmacodynamic Studies

III. Comparative Clinical Studies

IV. Dissolution Studies

PHARMACOKINETIC STUDY

Pharmacokinetic Study

a) Study b) Study c) Selection d) Study e) Statistical

Design Population Criteria Condition evaluation
STUDY DESIGN

The basic design of an in-vivo bioavailability study is

determined by the following:
• What is the scientific question(s) to be answered.
• The nature of the reference material and the
dosage form to be tested.
• The availability of analytical methods.

• Benefit-risk ratio considerations in regard to

testing in humans.
STUDY POPULATION
 The number of subjects to be included in the study based on an
appropriate Sample size which is estimated by:
• Pilot experiment
• Previous studies
• Published data
 The number of subjects recruited should be sufficient to allow for
possible withdrawals or removals (dropouts) from the study.
 Significance level desired, usually 0.05
 Power of the study, normally 80% or more
 Minimum 12 subjects, unless ethical justification
 if the drug product is intended for use in both sexes, the sponsor
attempt to include similar proportions of males and females in the
study.
SELECTION OF SUBJECTS
 Healthy adult volunteers

 Age: 18years or older

 Age/Sex representation corresponding to therapeutic & safety profile

 Non-smokers/without a history of alcohol or drug abuse Medical history/Clinical Lab test values
must be within normal ranges

 Weight within normal limits→ BMI (18.5-30 kg/m2)

 Women:Women should be required to give assurance that they are neither pregnant, nor likely to
become pregnant until after the study.

 Drug use intended in Elders, the sponsor attempt to include as many subjects of 60yrs of age or
older as possible.

 Teratogenic Drugs→ Male volunteers

 Highly toxic drugs: Patients with concerned disease (stable) eg. Cancer
STUDY CONDITION

Selection of Blood Sampling

Points/Schedules
The lot Numbers of both reference
listed products and the expiration date for
 For at least three elimination half-lives (cover >80% of AUC)
the reference product would be stated.

 Absorption phase : 3-4 points The drug content of the test product
cannot differ from that of reference
 Around T
max : 3-4 points product by more than ± 5%.
 The sponsor can include a statement of
 During elimination : 4 points the composition of the test product and if
possible a side-by-side comparison of the
 Intervals not longer than the half-life compositions
of the drug of the test and reference
product.
 If urine tested, collect it for at least 5  Samples of the test and reference listed
half-lives
product must be retained for 5 years.
Fasting or fed conditions
Fasting state:
• A single dose study should be conducted after an overnight fast (at least 10 hours), with subsequent
fast of 4 hours following dosing.
• For multiple dose fasting state studies, when an evening dose must be given, two hours of fasting
before and after the dose is considered acceptable .
Fed State:

Required when: In general BE Study conducted under

 Drug recommended with food
fasting condition.
1. Where the SmPC recommends intake
 Modified release product
of the reference medicinal product on
 Assessment of C
max and Tmax difficult with fasting state study
an empty stomach or irrespective of
food intake, BE study conducted under
 Requires consumption of a high fat food, 30 minutes before dosing
fasting condition.
2. Where SmPC recommends intake of
 Provide 800-1000 kcals with about 50% of calories derived from fat.
Where information is required in both FastingRMP and fed
onlystate, it is
in fed acceptable
state, to
BE Study
conduct either; conducted under fed state.
 Fat- 500-600, Proteins - 150, Carbohydrate- 250 Kcal
a. Two separate two-way cross-over OR, 3. For specific formulation both study
b. A four way cross-over study recommended, unless product taken
 Ethnic & cultural variation considered
only in fasting or fed state.
 Specified in protocol
STEADY STATE/ MULTIPLE DOSE STUDIES
Long elimination half life→ Accumulation in the body

 Toxic drugs requiring multiple dose therapy

 Some Modified-release drugs

 Combination products

 Drugs inducing own metabolism

 Drugsshowing non-linear pharmacokinetics

 Disadvantages:

• Difficult to conduct
• Costly
• Longer monitoring
• Longer exposure to drug
PARAMETERS IN MULTIPLE DOSING STUDIES
Cmaxss

AUCss

Cminss

Fluctuation:
Cmax - Cmin
 CHARACTERISTICS TO BE MEASURED

 Evaluations of BA/BE will be based upon the measured concentrations of the active drug substance(s) in
the biological matrix.
 The measurements of an active or inactive metabolite may be necessary:

a)where the concentrations of the drug(s) may be too low to accurately measure in the biological matrix, (b)
limitations of the analytical method, (c) unstable drug(s), (d) drug(s) with a very short half-life or (e) in the case
of prodrugs.
 Racemates should be measured using an achiral assay method.

 The plasma-time concentration curve is mostly used to assess the rate and extent of absorption of the study drug.
This include Cmax, Tmax, AUC0-t and AUC0-∞.
 Pharmacokinetic Parameters measured are:

• Plasma conc. and time points

• Subject, period, sequence, treatment Measurement of Individual enantiomers in BE

• Cmax, Tmax, AUC0-t ,AUC0-∞ Studies is recommended if
1. Exhibit different Pharmacodynamic
• Partial AUC only for early exposure of drug content.
characteristics
 For steady state studies: 2. Exhibit different Pharmacokinetics
• Swing characteristics
• Cav 3. Primary efficacy and safety activity resides with
• Cmin minor enantiomers
• Degree of fluctuation 4. Nonlinear absorption is present
STATISTICAL EVALUATION
 Primary concern of bioequivalence is to limit Consumer’s & Manufacturer’s risk
• Cmax & AUC analysed using ANOVA
• Tmax analysed by non-parametric methods

 Use natural log transformation of Cmax and AUC

 Calculate 90% confidence interval for this GMR for C max

 Similarly calculate GMR for AUC

Criteria for bioequivalence
• To establish Bioequivalence, the calculated 90% confidence interval for AUC and C max should

fall within the bioequivalence range, usually 80-125%.

• Tighter limits for permissible differences in bioavailability may be required for drugs that
have:
I A narrow therapeutic index.
II A serious, dose-related toxicity.
III A steep dose/effect curve, or
IV A non-linear pharmacokinetics within the therapeutic dose range.
DIFFERENT TYPES OF BE STUDY
 Two-Period Crossover Design
 Latin Square Design
 Balance Incomplete Block Design (BIBD)
 Parallel-Group Design
 Replicate Crossover-study design
VI. Pilot Study

 If the sponsor chooses, in a small number of subjects

 To assess variability, optimize sample collection time intervals

& provide other information, Validate analytical methodology.

 Example:

• Immediate-release products: careful timing of initial samples→ avoid a

subsequent finding that the first sample collection, occurred after the plasma
concentration peak

 Can be appropriate, provided its design & execution are suitable & sufficient
number of subjects have completed the study
BIOWAIVERS–
In vitro studies ,i.e. dissolution studies can be used in place of in
vivo bioequivalence under certain conditions ,called
BIOWAVIERS.
1. The drug product differs only in strength of active substance,
provided the following condition hold ;
a) Pharmacokinetics are linear.
b) The qualitative composition is same.
c) The ratio between active substance and excipient is same.
d) Both product are produced by same manufacturer at same site
with same manufacturing process.
2. The drug has been slightly reformulated or manufacturing
method has been slightly modifies by same manufacturer in
ways that can be argues irrelevant for BA.
3. The product meets following requirement :
a) The product is in form of solution or solublised form ( elixir,
syrup) etc.
b) The product contain active ingredient in same conc. as approved
drug.
c) The product contain no excipient known to significantly affect
absorption of active ingredient.
d) The product is administered by inhalation as gas or vapor.
e) The product is for oral administration but not intended for
absorption ( antacid or radio opaque medium ).
f) The product is intended for topical administration
(ointment, creams, gels etc,) for local effect.
PHARMACODYNAMIC STUDY
 Measurement of effect on a Patho-physiological process as a function of time, after
administration of 2 different products

Necessity:

1. Quantitative analysis in plasma or urine not possible with sufficient accuracy &
sensitivity

2. Drug concentrations are not surrogate endpoints e.g. Topical formulations without
systemic absorption

3. In situations of ‘Superiority Claims’

 Incase only Pharmacodynamic data is collected→ other methods tried & why they were
unsuitable should be mentioned.
COMPARATIVE CLINICAL STUDIES
 Necessity:

• Both pharmacokinetic & pharmacodynamic parameters not properly

measurable or not feasible
• Mention which methods were tried & found unsuitable

 Statistical principles to be considered:

• No. of patients→ Variability of assessed parameters & acceptance

range Much higher than BE studies
FOLLOWING CRITICAL POINTS NEED TO BE DEFINED IN ADVANCE, ON CASE TO CASE BASIS:

 Clinical end points (Target parameters)→ intensity & onset of response

 Size of equivalence range→ case-to-case basis (dependings on natural

course of disease, efficacy of available treatments, target parameter)

 Statistical confidence interval

 Placebo included when appropriate

 Safety end-points in some cases

 DISSOLUTION STUDIES

1. Suitable to confirm unchanged product quality with minor changes in formulation /

manufacturing after approval→ SUPAC ( Scale-Up & Post-Approval Changes)

2.Different strengths of drug manufactured by same manufacturer

where:
• Qualitative composition is same

• Ratio of active ingredients & excipients is same

• Method of manufacture is same

• BE study has been performed on 1 strength

• Linear pharmacokinetics

3. Assess batch-to-batch quality

 More than 1 batch of each formulation tested

Design should include:

 Individually testing of at least 12 dosage units of each batch →Mean

& Individual results with Sd or SE

 Measurement of percentage of content released at suitably spaced time

points(eg. At 10, 20 & 30 mins or appropriate for complete dissolution)

 Dissolution profile in atleast 3 aqueous media with pH range of 1.0-

6.8 Or 1.0-8.0 wherever necessary

 Conduct tests on each batch with same apparatus & on same or

consecutive days
Dissolution testing should be carried out in:

 USP Apparatus I at 100 rpm or Apparatus II at 50 rpm

using 900 ml of the following dissolution media:

• 0.1N HCl or Simulated Gastric Fluid USP without enzymes

• a pH 4.5 buffer
• a pH 6.8 buffer or Simulated Intestinal Fluid USP without enzymes

 For capsules and tablets with gelatin coating

• Simulated Gastric and Intestinal Fluids USP (with enzymes) can be

used
DOCUMENTATION
• With respect to the conduct of bioequivalence/bioavailability studies
following important documents must be maintained:
A) Clinical Data :
• All relevant documents as required to be maintained for compliance with
GCP Guidelines .
B) Details of the analytical method validation including the following:
a. System suitability test
b. Linearity range
c. Lowest limit of quantization
d. QC sample analysis
e. Stability sample analysis
f. Recovery experiment result
C) Analytical data of volunteer plasma samples which should include
the following:
a. Validation data of analytical methods used,
b. Chromatograms of all volunteers,
c. Inter-day and intra-day variation of assay results,
d. Details including chromatograms of any repeat analysis performed,
e. Calibration status of the instruments .
D) Raw data
E) All comments of the chief investigator regarding the data of the
study submitted for review.
F) A copy of the final report
DIFFICULTIES DURING BE REPORT REVIEW
 No practical knowledge regarding overall BE Study design
 Lack of knowledge on Bioanalysis

 No knowledge of Biostatistics

 Little knowledge on Good Clinical Practice (GCP)

 No knowledge on different Clinical investigation

(Hematology, Serology), Pharmacokinetics parameter
(Cmax, AUC0-t, AUC0-inf etc.) and Pharmacodynamic study.
 No knowledge regarding statistical analysis software
(SAS), WinNonlin and methods (ANOVA)