Drug Product Performance, In Vivo: Bioavailability and

Bioequivalence: drug product performance, purpose of bioavailability

studies, relative and absolute availability. methods for assessing

bioavailability, bioequivalence studies, design and evaluation of

bioequivalence studies, study designs, crossover study designs,

evaluation of the data, bioequivalence example, study submission

and drug review process.

Drug Product Performance,
In Vivo: Bioavailability and
Bioequivalence
Bioavailability is defined in § 320.1 as:
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.
The United States Food and Drug administration(1989)

The rate and extent to which the active drug ingredient or

therapeutic moiety is absorbed from a drug product and becomes

available at the site of drug action.

 Purpose of bioavailability studies
Bioavailability studies are performed for drug product containing
both approved API and API not yet approved for marketing.
1. API not yet approved for marketing 2. Approved API
• FDA ensures that the drug product • They have full NDA approval and
is safe and effective for its labeled BA studies are useful to define
indications for use through BA the effects of physicochemical
studies. changes of the drug
• FDA requires BA/ pharmacokinetic substance(API) and the effect of
study to have full NDA approval as drug product on the
an evidence the drug product pharmacokinetics of the drug.
meets all applicable standards of These are useful during SUPAC to
identity, strength, quality and prove bioequivalence of changed
purity. dosage form with already
approved drug.
3. For approval of multisource drug product(Generic) through BA/ BE studies with
reference listed drug (RLD) on which an applicant relies when seeking approval of an
ANDA.
Types of bioavailability
Measures of bioavailability/ Methods
for measurement of bioavailability
From plasma data
From urinary data: These measures are preferred
when,
Bioequivalence is defined in § 320.1 as:
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.
Bioavailability studies are performed as a part of
Bioequivalence study.

This data supports INDs, NDAs, ANDAs, and their

supplements like post approval changes.

In BE studies, an applicant compares the systemic

exposure profile of a test drug product to that of a
reference drug product (RLD).
For two orally administered drug products to be
bioequivalent, the active drug ingredient or active
moiety in the test product must exhibit the same rate
and extent of absorption as the reference drug
product.
Both BA and BE studies are required by
regulations, depending on the type of
application being submitted.

BE information is required to ensure

therapeutic equivalence between a
pharmaceutically equivalent test drug product
and a reference listed drug.

Regulatory requirements for documentation of

BA and BE are provided in part 320 of CFR.
Bioequivalence study
Objectives of BA/BE studies
Elements of Bioequivalence Study Protocol

1. Title
a. Principal investigator
b. Project number and date
2. Study Objective
3. Study design
a. Design
b. Drug Products
I. Test product(s)
II. Reference product
c. Dosage regimen
d. Sample collection schedule
e. Housing
f. Fasting/meals schedule
g. Analytical methods
4. Study Population
a. Subjects
b. Subject selection
- Medical history
-Physical examination
-Laboratory tests
c. Inclusion/exclusion criteria
Inclusion criteria
Exclusion criteria
d. Restrictions/prohibition
5. Clinical procedures
a. Dosage and drug administration
b. Biological sampling schedule
c. Activity of subjects
6. Ethical considerations
a. Basic principles
b. Institutional review board
c. Informed conset
d. Indications for subjects withdrawal
 e. Adverse reactions and emergency procedures
7. facilities
8. Data analysis
a. Analytical validation procedure
b. Statistical treatment of data
9.Drug accountability
10.Appendix
 Human Volunteers

Healthy Subjects versus Patients

Ideally, the bioavailability should be

carried out in patients for whom the drug
is intended to be used because of
apparent advantages:-
1. The patient will be benefitted from the study.
2. Reflects the therapeutic efficacy of the drug.
3. Drug absorption pattern in disease states can
be evaluated.
4. Avoids the ethical quandary of administering
drugs to healthy subjects
 SINGLE DOSE VERSUS MULTIPLE DOSE
Advantages:- STUDIES
1. More accurately reflects the manner in which
the drug will be used clinically.
2. Allows blood level to be measured at the same
concentrations encountered therapeutically.
3. Easy to predict the peak and valley
characteristics of the drug since the bioavailability
is determined at steady state.
4. Requires collection of fewer blood samples.
5. The drug blood levels are higher due to
cumulative effect which makes its determination
possible even by the less sensitive analytical
methods.
6. Can be ethically performed in patients
because of the therapeutic benefit to the
patient.
7. Small inter-subject variability is observed in
such a study which allows use of fewer subjects.
8. Better evaluation of the performance of a
controlled-release formulation is possible.
9. Nonlinearity in pharmacokinetics, if present,
can be easily detected.
10. Eliminates the need for out period between
doses.
Moreover, the switch-over from one formulation to
the other is possible at steady state.

Limitations:-
1. Tedious, requires more time to complete.
2. More difficult and costly to conduct
(requires prolonged monitoring of subjects).
3. Poor compliance by subjects.
4. Greater exposure of subjects to the test
drug, increasing the potential for adverse
reactions.
Inclusion Criteria
Exclusion criteria
Blood sampling points/schedule
BA/BE studY DESIGNS
 Pilot study
A pilot study in a small number of subjects can be carried out before
proceeding with a full bioequivalence study. The study can be used to
validate analytical methodology, assess variability, optimize sample collection
time intervals, and provide other information. For example, for conventional
immediate-release products, careful timing of initial samples may avoid a
subsequent finding in a full-scale study that the first sample collection occurs
after the plasma concentration peak. For modified-release products, a pilot
study can help determine the sampling schedule to assess lag time and dose
dumping. A pilot study that documents bioequivalence may be acceptable,
provided that its design and execution are suitable and a sufficient number of
subjects (e.g., 12) have completed the study.
Currently three different studies are required for solid oral dosage forms-
1. A fasting study
2. A food intervention study
3. A multiple dose(steady state) study

Fasting study
This study is required for all immediate release and Modified release dosage forms.
1. Male and female both are included.
2. 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.
3. Subjects should be in fasting state (overnight fast for at least 10 hr) before drug
administration and should continue to fast for up to 4 hrs after dosing.
4. No other medication is normally given to the subject for at least 1 week prior to the
study.
 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.
1. The test meal is high fat and high calorie meal.
2. For bioequivalence, drug bioavailability from both the test and reference products
should be affected similarly by food.
3. The study design uses a single dose, randomized, two treatment , two period
crossover study comparing equal doses of the test and reference products.
4. All the volunteers receive identical diet and should follow identical protocol while
under study.
5. This study is required for all MR dosage forms and may be required for IR if the
bioavailability of the API is known to be affected by food.
6. Studies might also examine the effects of other food such as apple juice.
 Multiple dose (steady state ) study
In few cases a multiple dose, steady state, randomized, two treatment two way

crossover study comparing equal doses of the test and refernce products may be

performed in adult, healthy subjects. For these studies, three consecutive trough

concentrations on three consecutive days should be determined to ascertain 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 two hours following dose

administration. Blood sampling is performed similarly to the single dose study.

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 receives only B.
Crossover study design
Advantages of cross over design
Latin square design
 Balanced Incomplete Block Designs (BIBD)
BIBD is preferred when comparing three or more formulations of a
drug product, and a complete cross over design may not be of
practical interest for the following reasons
1. If the number of formulations to be compared is large the study
may be too time consuming since t formulations require t -1
washout periods.
2. It may not be desirable to draw many blood samples for each
subject owing to medical concerns.
3. Moreover a subject is more likely to drop out when he or she is
required to return frequently for tests.
If there are t formulations to be compared and each subject can only
receive exactly p formulations (t > p). A BIBD may be constructed by
taking C(t, p), the combination of p out of t formulations, and
assigning a different combination of formulations to each subject.
 Evaluation of the data
• The bio-analytical method used in the study should be validated
and same for test as well as reference product.
• Cmax, tmax, and AUC 0-t and AUC 0-∞ are to be compared.
• Statistical Interpretation of Bioequivalence Data
A) Confidence interval approach – Also called as two one-sided
tests procedure
It is used to demonstrate if the bioavailability of the test product is too
low or high in comparison to the reference product. The 90%
confidence limits are estimated for the sample means based on
Students t distribution of data. A 90% confidence interval about the
ratio of means of the two drug products must be within ± 20% for
bioavailability parameters such as AUC or Cmax i.e. the difference
between the bioavailabilities of the test product should not be greater
than ±20% of the average of reference product (between 80 and
120%).
Many statistical approaches that are used to compare assume that the
data are distributed according to a normal distribution. The distribution
of many biological parameters (Cmax, AUC) have longer right tail than
would be observed in normal distribution. Moreover the true
distribution of these biological parameters may be difficult to ascertain
because of small number of subjects used in BE study.
The distribution data that has been transformed to log values resembles
more closely a normal distribution. Therefore log transformation of BA
data is performed before statistical data evaluation for BE. When log
transformed data are used, the 90% confidence interval is set at 80-
125%. These confidence limits are also termed as bioequivalence
interval.
B) Analysis of variance (ANOVA)
Analysis of variance (ANOVA) is a statistical procedure used to test
the data for differences within and between treatment and control
groups.

A statistical difference between the pharmacokinetic parameters

obtained from two or more drug products is compared by
calculating F value (using ANOVA table). The difference is
considered statistically significant when

F cal > F table at a probability of p ≤ 0.05 (less then 1 in 20 or 0.05).

The probability p is used to indicate the level of statistical

significance. If p ≤ 0.05, the differences between the two drug
products are not considered statistically significant.
Study submission and Drug review
process
 Regulatory objectives of BA/BE studies-
[Ref:(21 CFR 320.25(d)(1)]

1. To assess, through appropriately designed BA

studies, the performance of the formulations
used in the clinical trials that provide evidence
of safety and efficacy.
2. To focus on the release of a drug substance
from a drug product and subsequent
absorption into the systemic circulation.
Biopharmaceutics classification system, methods.

Permeability: In–vitro, in–situ and In–vivo methods.

Generic biologics (biosimilar drug products),clinical

significance of bioequivalence studies, special concerns

in bioavailability and bioequivalence studies, generic

substitution.
Biopharmaceutics Classification
System
 Class I Class II

High solubility- High Low solubility- High

permeability permeability
IR solid dosage forms IR solid dosage forms with improvement in
dissolution of drugs

Class III Class IV

High solubility- Low Low solubility- Low

permeability permeability
IR solid dosage forms with improvement in IR solid dosage forms with improvement in
permeability of drugs dissolution/permeability of drugs
Highly soluble –A drug substance is considered
highly soluble when the highest strength is soluble
in 250 mL or less of aqueous media within the pH
range of 1 to 6.8-USFDA (1 to 7.5-WHO) at 37±1oC.

Highly permeable- A drug substance is considered

highly permeable when the systemic BA or the
extent of absorption in humans is determined to be
≥85%-USFDA (≥90%-WHO) or more of an
administered dose or in comparison to an
intravenous reference dose.
A drug substance is considered highly soluble when the highest
strength is soluble in 250 mL or less of aqueous media within the pH
range of 1 - 6.8 (USFDA) at 37 ± 1°C.

The decrease in pH from 7.5 in the FDA Guidance's to 6.8 reflects the
need to dissolve the drug before mid jejunum to ensure enough
reserve length for absorption from the GI tract.

The volume estimate of 250 mL is derived from typical BE study

protocols that prescribe administration of a drug product to fasting
human volunteers with an 8 fluid ounce glass of water.
A drug substance is considered to be highly permeable when the
systemic BA or the extent of absorption in humans is determined to be
85 percent or more of an administered dose based on a mass balance
determination (along with evidence showing stability of the drug in
the GI tract) or in comparison to an intravenous reference dose.
The permeability criterion was relaxed from 90% in the WHO
multisource document to the FDA Guidance's to 85%. Some examples
of APIs now included in BCS class I that were previously considered to
be Class III are: paracetamol, acetylsalicylic acid, allopurinol,
lamivudine, promethazine.
 To understand the definitions
Drug A with high permeability Drug B with high permeability
Dose – 500 mg Dose-- 500mg

Solubility: 2.1 to 2.3 mg/ml Solubility: 0.21 to 0.23 mg/ml

250 ml Volume solubilises 525 250 ml Volume solubilises 52.5

to 575 mg. to 57.5 mg.

BCS Class I BCS Class II

 To understand the definitions
Drug C with low permeability Drug D with low permeability
Dose – 5 mg Dose– 40 mg

Solubility: 0.1 to 0.3 mg/ml Solubility: 0.1 to 0.3 mg/ml

250 ml Volume solubilises 25 250 ml Volume solubilises 25

to 75 mg. to 75 mg.

BCS Class III BCS Class IV

Why should we be acquainted with
BCS?
1. Development of dosage form based on BCS
 2. Bio waivers based on BCS

Generic product if drug product contains BCS Class I drug ; may be approved

without BA studies ( bio-waived) If-

1. It is IR product.

2.It exhibits > 85% dissolution in 30 minutes.

3.Similarity factor (f2) >50 i.e. similarity in dissolution with innovator product.

4. Drug with wide therapeutic index.

5. Excipient used in product is approved for IR.

6. Drug stable in GIT.

 Biowaivers based on BCS
The USFDA may waive off BA/BE studies for certain IR solid dosage forms That

meet specific criterion. The guidance is applicable for BA/BE waivers (biowaivers)

based on BCS, for BCS class 1 and class 3 IR solid oral dosage forms.

For BCS class 1 drug products, the following should be demonstrated:

• the drug substance is highly soluble.

• the drug substance is highly permeable.

• the drug product (test and reference) is rapidly dissolving, and

• the product does not contain any excipients that will affect the rate or extent of

absorption of the drug.

For BCS class 3 drug products, the following should be demonstrated:

• the drug substance is highly soluble

• the drug product (test and reference) is very rapidly dissolving and

• the test product formulation is qualitatively the same and quantitatively very

similar (should contain same excipients).

For certain drug products, the in vivo bioavailability or bioequivalence of the drug
product may be self-evident. FDA (CFR 320.22) shall waive the requirement for the
submission of evidence obtained in vivo measuring the bioavailability or
demonstrating the bioequivalence of these drug products. A drug product's in vivo
bioavailability or bioequivalence may be considered self-evident based on other data
in the application if the product meets one of the following criteria:
(1) The drug product:
(i) Is a parenteral solution intended solely for administration by injection, or an
ophthalmic or otic solution; and
(ii) Contains the same active and inactive ingredients in the same concentration as a
drug product that is the subject of an approved full new drug application or
abbreviated new drug application.
(2) The drug product:
(i) Is administered by inhalation as a gas, e.g., a medicinal or an inhalation anesthetic; and
(ii) Contains an active ingredient in the same dosage form as a drug product that is the subject
of an approved full new drug application or abbreviated new drug application.
(3) The drug product:
(i) Is a solution for application to the skin, an oral solution, elixir, syrup, tincture, a solution for
aerosolization or nebulization, a nasal solution, or similar other solubilized form; and
(ii) Contains an active drug ingredient in the same concentration and dosage form as a drug
product that is the subject of an approved full new drug application or abbreviated new drug
application; and
(iii) Contains no inactive ingredient or other change in formulation from the drug product that
is the subject of the approved full new drug application or abbreviated new drug application
that may significantly affect absorption of the active drug ingredient or active moiety for
products that are systemically absorbed, or that may significantly affect systemic or local
availability for products intended to act locally.
(4) The drug product is in the same dosage form, but in a different strength, and is

proportionally similar in its active and inactive ingredients to another drug product for

which the same manufacturer has obtained approval

(i) The bioavailability of this other drug product has been measured;

(ii) Both drug products meet an appropriate in vitro test approved by FDA; and

(iii) The applicant submits evidence showing that both drug products are proportionally

similar in their active and inactive ingredients.

(iv) Paragraph of this section does not apply to delayed release or extended release

products.
Points to remember-

1.BCS classification.
2.Definitions of high solubility and high
permeability.
3.Applications of BCS
Permeability: In–vitro, in–situ and In–vivo
methods.
 Determination of Permeability
In situ intestinal perfusion in rats is the best option for correctly
classifying the permeability of compounds according to human data.

However, considering the throughput limitations of in situ methods in

screening a large number of compounds, a combined application of
permeability models was proposed to improve the accuracy of the
BCS permeability classification.
1. The ex vivo rat intestinal tissue in Ussing chamber. Ex vivo method
2. The MDCK (Madin–Darby canine kidney ) In vitro cell line
3. Caco-2 (Human colon adenocarcinoma cell line) cell monolayers.
In vitro cell line
4. The parallel artificial membrane (PAMPA-parallel artificial
membrane permeability assay ). In vitro method
A variety of approaches were developed to predict permeability of NCEs in early
drug discovery.
The PAMPA (parallel artificial membrane permeability assay) using a chemical
membrane offers an avenue to quickly estimate permeability for NCEs with passive
diffusion mechanisms and small molecular weights (e.g., <500). PAMPA is artificial
membrane lipid layer made up of mixtures of lecithin or membrane phospholipids
and inert organic solvents on a permeable support .
The Madin–Darby canine kidney (MDCK) cell model that originates from dog kidney,
the expression of transporters is quite different from that of human intestine. As a
result, the MDCK monolayer is commonly used for permeability evaluation of NCEs
transported by passive diffusion mechanisms, as does the PAMPA model. The latest
development has extended the permeability studies using a P-glycoprotein (P-gp)-
transferred MDCK model to estimate the contributions of efflux transporters.
The human colon adenocarcinoma (Caco-2) cell permeability model,
exhibiting morphological (e.g., tight junction and brush-border) as well
as functional (e.g., multiple transport mechanisms) similarities to
human intestinal enterocytes, has been widely received applicability
and utility in drug discovery and development. Caco-2 cells
extensively express a variety of transport systems beyond P-gp
normally found in small intestinal enterocytes, which made it
possible to investigate the interplay among different transport
systems and differentiate the relative contributions from passive and
active transport mechanisms to the overall permeability across the
human gastrointestinal (GI) tract.
 Limitation of Caco 2 cell line

Caco2 cells seem to underestimate the transport than rat

duodenal cell line 2/4/A1 cells or the excised intestinal
segments or perfusions. If the paracellular route is to be
utilized, data generated using relevant models to be used to
train the model. Also, paracellular transport seems to be
species dependent; and these differences need to be taken
into account for data interpretation.
Despite the extensive insights offered by the Caco-2 model
over other in vitro permeability models, its lengthy and
complicated cell culturing has led to high cost for assay
maintenance and daily operation. It is preferable to
use PAMPA as a fast and HT prescreening tool for
permeability ranking and apply Caco-2 assays only to the
challenging compounds that failed in the PAMPA assay.
Permeability assays employing cultured Caco-2 epithelial cell
monolayers derived from a human colon adenocarcinoma cell line are
widely used to estimate intestinal drug absorption in humans. Caco-2
cells undergo spontaneous morphological and biochemical
enterocytic differentiation, express cell polarity with an apical brush
border, tight intercellular junctions, and several active transporters as
in the small intestine. Due to a potential for low or absent expression
of efflux (e.g., P-gp, BCRP, MRP2) and uptake (e.g., PepT1, OATP2B1,
MCT1) transporters, the use of Caco-2 cell assays as the sole data in
support of high permeability for BCS classification is limited to
passively transported drugs.
Generic biologics(Biosimilars)
 BA data for a given formulation provide
1. An estimate of the relative fraction of the orally
administered dose that is absorbed into the systemic
circulation when compared to the BA data for a solution,
suspension, or intravenous dosage form.
(21 CFR 320.25(d)(2) and (3)).

2. Other useful pharmacokinetic information related to

distribution, elimination, the effects of nutrients on
absorption of the drug, dose proportionality, linearity in
pharmacokinetics of the active moieties and, where
appropriate, inactive moieties.

3. Information indirectly about the properties of a drug

substance before entry into the systemic circulation, such
as permeability and the influence of presystemic enzymes
and/or transporters (e.g., p-glycoprotein).
4. Confirmation of dose proportionality and
linearity in kinetics.
5. Estimation of food/nutrients effect on the
absorption.
WHEN INVIVO BIOAVAILABILITY STUDIES ARE NECESSARY
A) Oral IR formulations with systemic action
.Indicated for serious conditions
.Narrow therapeutic window
.Complicated pharmacokinetics (variable and incomplete absorption)
.Unfavorable physiochemical properties
.Documented evidence for bioavailability problems
.High excipients/active ingredients ratio

B) Non oral and non Parenterals formulations

C) SR or MR acting by systemic absorption

D) Fixed dose combination products with systemic action

E) Non solution for non systemic use