Bioavailability, Bioequivalence & Dissolution

 This chapter provides general information about the conduct of bioequivalence (BE) studies
as a surrogate measure of in-vivo drug product performance and dissolution profile
comparisons as a measure of in vitro drug product performance.
 It also discusses conditions when an in Vivo BE requirement may be waived (biowaiver) for
certain drug products and shows how the Biopharmaceutics Classification System (BCS) can
be used to predict a drug product's performance.
 Definition - A bioavailability study measures the extent and rate at which the active
ingredient or active moiety of a drug is absorbed and becomes available at the site of action
in the body. This is crucial for determining the correct dosage and ensuring the drug’s
effectiveness.
 BA and BE generally can be obtained by serially measuring drug and/or metabolite
concentrations in the systemic circulation over a prescribed period. BE studies can use other
approaches when systemic drug concentrations cannot be measured or are not appropriate.
 BA (Bioavailability) and BE (Bioequivalence) information are crucial for regulatory
submissions.BA primarily addresses the absorption, distribution, metabolism, and excretion
of the API (Active Pharmaceutical Ingredient).
 BE studies are conducted to establish the performance of a generic drug compared to an
innovator product. The goal of BE studies is to demonstrate that a generic drug product is
equivalent to the reference-listed drug (RLD) product, typically the brand or innovator drugs.
 The ICH document titled Guidance on Q6A Specifications: Test Procedures and Acceptance
Criteria for New Drug Substances and New Drug Products by the Committee for Medicinal
Products for Human Use describes the regulatory approach towards setting acceptance
criteria for drug product performance.
 This approach relies on dissolution or disintegration based on clinically acceptable batches,
as does FDA's BE studies focus on the performance of the drug product. and usually involve
comparisons of two drug products: the test (T) and reference (R) or comparator product.
 The required studies and determination of BE are the purview of regulatory agencies. In the
United States, R is termed the reference listed drug (RLD) and is so noted in FDA's Approved
Drug Products with Therapeutic Equivalence Ratings (Orange Book) (2008)
(http://www.fda.gov/oc/orange_book). The ICH document is termed the Common Technical
Document (CTD), which is a summary document that clearly defines the set of CPP (Critical
Process Parameters)

Bioequivalence
A generic product must be pharmaceutically equivalent (PE). The WHO document allows generic
products interchangeable if they are therapeutically equivalent and must be shown to be BE for
their generic products' BE to be considered PE. For a product to be considered PE, it must have
the same active ingredient, same strength, same dosage form, same route of administration,
and same labeling as the comparator product. Several methods exist to assess and document BE.
These include

1. Comparative Pharmacokinetic Studies: In humans, these studies assess the active drug
and/or its metabolite(s) are measured as a function of time-inaccessible biological fluid
such as blood, plasma, serum, or urine to obtain pharmacokinetic measures such as area
under the plasma drug concentration vs. time curve (AUC) and maximum concentration
(Cmax) that are reflective of systemic exposure.
 2. BE study Design to Compare the In Vivo Performance of a Generic Product with an R-
Product: Generally, the design is a two-period, two-sequence, single-dose crossover
randomization strategy in 18 to 36 subjects. The number of subjects should be
statistically justified and not less than 12. During the study, blood samples are collected
at sufficient intervals for assessing AUC, Cmax, and other parameters. Valid samples are
analysed using appropriately validated bioanalytical methodology with standard
pharmacokinetic measures and statistical approaches. The statistical method for testing
pharmacokinetic BE is based on the determination of the 90% confidence interval
around the geometric mean ratio of the log-transformed population means(generic/R)
for AUC and Cmax by carrying out two one-sided test at the 5% level of significance.
3. In Vitro Dissolution Studies (BCS): The Biopharmaceutics Classification System (BCS) can
sometimes be used to waive in vivo studies by comparing the dissolution profiles of the
test and reference products. If the dissolution characteristics are similar, it can indicate
bioequivalence without the need for in vivo testing, particularly for drugs that fall under
certain BCS classes.

Objective

The objective of BE study is to measure and compare formulation performance between two or
more pharmaceutically equivalent drug product. Drug availability of the test and reference drug
should not be statistically difference when administered a patient.

The design of be study depends on the objective study, the ability to analyse the drug in biological
fluids, route of administration and pharmacodynamics of the drug substance done by observing the
pharmacokinetic, pharmacodynamic, clinical trials and invitro studies is used to determine the drug
BE

Some be design includes

 Single-dose, two-way crossover study under fasted conditions.

 Single-dose, two-way crossover study under fed conditions.
 Single-dose, parallel study under fasted conditions.
 Single-dose, replicate design.
 Single-dose, partial replicate design.
 Multiple-dose, two-way crossover study, fasted conditions.
 Pharmacodynamic or clinical endpoint study.
 In vitro dissolution profile comparisons.

The standard bioequivalence (BE) study employs a crossover design, typically a Latin square format,
where each subject receives both a test drug product and a reference product in separate periods.
The studies are usually structured as single-dose, two-period, two-treatment, two-sequence, open-
label, randomized crossover designs, comparing equal doses in fasted or fed healthy adult subjects.
For certain extended-release formulations, a multiple-dose study may be necessary. A washout
period is scheduled between doses to ensure complete elimination of the first dose before
administering the second.

In fasting studies, subjects fast for at least 10 hours, followed by a pre-dose blood sample. The drug
is administered with 240 mL of water, and no food is allowed for at least 4 hours post-dose, with
blood sampling conducted periodically thereafter. For food effect studies, a standard meal designed
to maximally affect drug absorption is used, typically comprising a high-fat (approximately 50% of
total calories) and high-calorie (800 to 1000 calories) meal. This meal should provide around 150,
250, and 500-600 calories from protein, carbohydrates, and fats, respectively. The drug is given after
the meal with 240 mL of water, and subjects consume identical meals at the same time during the
testing period.

Analysis of sample- samples usually plasma are analysed for the active drug and on occasion, active
metabolites concentrations by a validated bioanalytical method.

Pharmacokinetic parameters- are obtained from the resulting concentration time curves. Two major
parameters are used to assess the rate and extent of systemic drug absorption, and the peak drug
concentration reflects the rate of drug absorption. Other pharmacokinetic parameters may includes
the time to peak drug concentration, the elimination rate constant, elimination half-life, lag time,
and others.

Statical analysis: The main points regarding the statistical analysis of pharmacokinetic parameters in
bioequivalence (BE) studies can be summarized as follows:

1. Objective: The analysis aims to determine whether test (T) and reference products yield
comparable pharmacokinetic values.

2. Data Transformation: Log transformation of data is often employed to normalize the

frequency distribution, allowing for parametric statistical analyses.

3. Statistical Model: For a typical two-treatment, two-period, two-sequence (2x2) crossover

study, an Analysis of Variance (ANOVA) is recommended. The model accounts for factors
such as sequence, subjects, period, and treatment.

4. Testing Variance: The sequence effect is assessed using a specific mean square from ANOVA,
while all other effects are tested against the residual error.

5. Least Squares Means: The LSMEANS statement is used to calculate adjusted treatment
means and their differences, along with standard errors.

6. Assumptions for ANOVA:

o Randomization of subjects

o Homogeneity of variances

o Additivity of effects (no interactions)

o Independence and normality of residuals

7. Response to Assumption Violations: If assumptions are not met, data transformation or

nonparametric tests may be considered. However, ANOVA is robust to small to moderate
deviations from normality and variance homogeneity. The rationale for using log-
transformed data is supported by FDA guidance, and justification is needed for using
untransformed data

The Two One-Sided Tests Procedure (TOST) is utilized to assess the comparability of geometric mean
values for pharmacokinetic parameters following the administration of test (T) and reference (R)
products. This procedure determines whether T is not importantly less than R and vice versa, with an
important difference typically defined as 20%. The statistical analysis involves calculating a
confidence interval for the ratio (or difference) between the pharmacokinetic variable averages of T
and R. For bioequivalence (BE), the point estimate mean ratios derived from log-transformed area
under the curve (AUC) and maximum concentration (Cmax) must fall between 80% and 125%.
Consequently, the 90% confidence intervals for the geometric mean ratios (T/R) for AUC and Cmax
should also lie within this range, though regulatory requirements may vary in different countries.

If BE is not demonstrated, it may be due to a performance failure of the T product or an inadequate

study design, often related to improper sampling techniques or insufficient subject numbers,
particularly with highly variable drugs. In reporting the results, untransformed drug concentrations
in biological fluids at each sampling time should be provided for all subjects, along with derived
pharmacokinetic parameters. The final report should include means, standard deviations, and
coefficients of variation (CV) for each variable. To facilitate comparisons, pharmacokinetic
parameters for each individual should be displayed side by side for both formulations tested,
showing differences (T-R), ratios (T/R), and log ratios (log T/R) for AUC and Cmax. Additionally,
summary tables should indicate the sequence in which products were administered to each subject.
Visual representations, such as histograms of the frequency distribution for differences and ratios,
are also encouraged in submissions. All calculated means, including arithmetic means, geometric
means, and means of the logs, along with their standard deviations and CVs, should be
comprehensively reported.

Dissolution and in vitro product performance

As noted for an official preparation, USP monographs pro- vide a public specification that includes a
list of tests, references to analytical procedures, and acceptance criteria. Most solid oral dosage
forms, including oral suspensions, require a dissolution or a drug release test. Drug dissolution and
drug release testing are described in USP general chapters Dissolution (711) and Drug Release (724).
These public specifications are used for quality control tests and for market approval. The USP
dissolution test in the monograph is related to BA and BE only when closely allied with a sound
regulatory determination. Without this link it should be re carded solely as a quality control test for
batch release. FDA Guidance’s are:

1. Guidance for Industry Dissolution Testing of Immediate Release Solid Oral Dosage Forms
(1977)
2. Guidance for Industry-Extended-Release Oral Dosage Forms: Development, Evaluation, and
Application of in Vitro/In Vivo Correlation (1977).

Dissolution and in vitro bioavailability

Dissolution and in vitro bioavailability testing play a crucial role in drug product
development, helping to identify critical manufacturing attributes that affect product
performance. These tests are essential for developing optimal dissolution conditions that
can differentiate between drug product formulations and manufacturing process changes.
After a drug product receives marketing approval, dissolution and release tests continue to
be valuable in predicting performance changes resulting from scale-up and post approval
changes (SUPAC).
The FDA has issued several guidance’s that provide frameworks for these processes,
including "Immediate Release Solid Oral Dosage Forms: Scale-Up and Post approval
Changes" (1995) and "SUPAC-MR: Modified-Release Solid Oral Dosage Forms" (1977). For
certain oral drug products, in vitro dissolution can be correlated with in vivo performance,
such as bioavailability and systemic drug exposure. The USP General Information Chapter
1088 outlines various methods for establishing in vitro-in vivo correlation (IVIVC),
emphasizing its importance in ensuring consistent drug performance throughout the
product lifecycle.
Dissolution and in vitro equivalence
The dissolution test is a critical in vitro physicochemical assessment that evaluates the
quality and performance of various dosage forms, including solid oral, transdermal,
suspensions, and certain semisolid forms. The United States Pharmacopeia (USP)
categorizes tests for finished dosage forms into two main types: drug product quality tests,
which assess attributes like assay and content uniformity, and drug product performance
tests, which focus on product efficacy, often relating directly to dissolution.
Initially developed as a quality control measure to ensure product quality and batch-to-
batch consistency, the dissolution test procedures are outlined in USP general chapters 711
and 724. The introduction of the Biopharmaceutics Classification System (BCS) has enhanced
the understanding of dissolution tests by classifying drug substances based on their
solubility and permeability through biological membranes, such as intestinal mucosal cells.
The dissolution rate is particularly important in supporting biowaivers under the BCS
framework, facilitating regulatory approvals by demonstrating that similar formulations can
be expected to perform equivalently in vivo.
Dissolution profile comparisons
In vitro drug dissolution and release testing are increasingly recognized for their relevance
to in vivo drug performance, particularly in establishing bioavailability (BA) and
bioequivalence (BE). The comparison of dissolution profiles has become a critical
component of documenting comparative BA studies. A biowaiver allows for the substitution
of in vivo BE studies with in vitro tests, streamlining the approval process.
A model-independent mathematical approach is employed to compare the dissolution
profiles of two products: (1) between the test (generic, multisource) product and the
reference (comparator) product for biowaiver considerations; (2) between different
strengths of products from the same manufacturer; and (3) for post-approval changes under
SUPAC guidelines. To compare dissolution profiles, the similarity factor fff is calculated using
the following equation:

Where:
 Rt is the cumulative percentage of drug dissolved from the reference product at time
t.
 Tt is the cumulative percentage of drug dissolved from the test product at time t.
  n is the number of selected time points.
An f value of 50 or greater (ranging from 50 to 100) indicates similarity in the dissolution
profiles, suggesting that the two products are equivalent in performance. For profile
comparisons, a minimum of three time points should be used, with no more than one point
exceeding 85% dissolution. If a product dissolves very rapidly (85% dissolution in 15
minutes), profile comparison may not be necessary.

Biowaiver
Biowaiver is applied to a regulatory approval process when the application is approved on the basis
of evidence of equivalence other than an in vivo BE test.

Biowaiver based on pharmaceutical dosage form

A drug products in vivo comparative BA or BE study requirement may be waived if the
products compared contain the same API(s) in the same concentration, contain the same
excipients in comparable concentrations, and meet one of the following criteria:
 Aqueous solutions to be administered parenterally.
 Solutions for oral use that do not contain an excipient that is known or is suspected
to affect gastro-intestinal transit or absorption of the active substance.
 Gases
 Powders for reconstitution as a solution
 Optic or ophthalmic products prepared as aqueous solutions.
 Topical products prepared as aqueous solutions.
 Inhalation products or nasal sprays tested to be administered with essentially the
same device. Special in vitro performance testing should be required to document
comparable device performance.
Biowaiver based on dosage form proportionality
When conducting a single-dose fasting bioequivalence (BE) study on the highest strength of
a drug product, additional in vivo BE studies for lower strengths may be waived under
certain conditions:
1. Dosage Form: The lower strength must be in the same dosage form as the highest
strength.
2. Proportional Similarity: The lower strength must be proportionally similar in both
active and inactive ingredients to the higher strength.
3. Drug Release Mechanism: For extended-release products, the lower strength must
have the same drug release mechanism as the higher strength.
 dissolution profile comparison criterion (typically a similarity factor 𝑓≥50).\
4. In Vitro Dissolution Profile: The lower strength must meet an appropriate in vitro

5. Linear Pharmacokinetic Range: Both the lower and higher strengths must be within
the linear pharmacokinetic range.
Biowaiver based on the Biopharmaceutics classification system
The Biopharmaceutics Classification System (BCS) categorizes drug substances based on
their aqueous solubility and intestinal permeability, influencing the rate and extent of drug
absorption from immediate-release dosage forms. The BCS divides drugs into four classes:
Class 1: High solubility, high permeability
Class 2: Low solubility, high permeability
Class 3: High solubility, low permeability
Class 4: Low solubility, low permeability
The BCS can facilitate the documentation of bioequivalence (BE) without the need for in
vivo studies, as outlined in the FDA guidance from 2000 regarding waivers for immediate-
release solid oral dosage forms.
In vitro dissolution studies are typically conducted using either the basket method (100 rpm)
or the paddle method (50 or 75 rpm) in 900 ml of medium at pH levels of 1.2, 4.5, and 6.8.
Pharmaceutical dosage forms are classified based on their dissolution rates as follows:
Very rapidly dissolving: 85% or more dissolves in 15 minutes or less
Rapidly dissolving: 85% or more dissolves in 30 minutes
Slowly dissolving: More than 30 minutes required for 85% dissolution
For a biowaiver to be granted, both the generic and reference products must undergo
dissolution tests under the same conditions, with the reference product belonging to the
same BCS class and meeting specific dissolution profile comparison criteria. Regulatory
authorities may consider biowaivers based on BCS classification and adherence to these
criteria.