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Bioavailability and Bioequivalane

Bioavailability is a phamacokinetic term that describes the rate and extent to which the
active drug ingredient is absorbed from a drug product and becomes available at the site
of drug action. Since pharmacologic response is generally related to the concentration of
a drug at the receptor sites, the availability of a drug from a dosage form is a critical
element of a drug product’s clinical efficacy. However, drug concentrations usually
cannot be readily measured directly at the site of action. Therefore, most bioavailability
studies involve the determination of drug concentration in the blood or urine or batches of
the same drug products.
Bioequivalence- Bioequivalence refers to the comparison of bioavailability of products
or batches of the same drug products.
Bioequivalenc indicates that two or more of the drug products (pharmaceutical
equivalents) which when administered in the same dosage forms and containing the same
dose level revealed the same bioavailability.
Bioequivalence requirement - A requirement imposed by the FDA for in-vitro and/or
in-vivo testing of specified drug products, which must be satisfied as a condition for
Bioequivalent drug products:
This term describes pharmaceutical equivalent or pharmaceutical alternative products
that display comparable bioavailability when studied under similar experimental
conditions. For systemically absorbed drugs, the test (generic) and reference listed drug
(brand-name) shall be considered bioequivalent if: (1) the rate and extent of absorption of
the test drug do not show a significant difference from the rate and extent of absorption of
the reference drug when administered at the same molar dose of the therapeutic
ingredient under similar experimental conditions in either a single dose or multiple doses;
or (2) the extent of absorption of the test drug does not show a significant difference from
the extent of absorption of the reference drug when administered at the same molar dose
of the therapeutic ingredient under similar experimental conditions in either a single dose
or multiple doses and the difference from the reference drug in the rate of absorption of
the drug is intentional, is reflected in its proposed labeling, is not essential to the
attainment of effective body drug concentrations on chronic use, and is considered
medically insignificant for the drug.

When the above methods are not applicable (eg, for drug products that are not intended to
be absorbed into the bloodstream), other in-vivo or in-vitro test methods to demonstrate
bioequivalence may be appropriate. 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 comparative clinical trials or pharmacodynamic

Bioequivalent drug products may contain different inactive ingredients, provided the
manufacturer identifies the differences and provides information that the differences do
not affect the safety or efficacy of the product.
Brand name. The trade name of the drug. This name is privately owned by the
manufacturer or distributor and is used to distinguish the specific drug product from
competitor's products (eg, Tylenol, McNeil Laboratories).

Chemical name: The name used by organic chemists to indicate the chemical structure
of the drug (eg, N-acetyl-p-aminophenol).

Abbreviated New Drug Application (ANDA): Drug manufacturers must file an ANDA
for approval to market a generic drug product. The generic manufacturer is not required
to perform clinical efficacy studies or nonclinical toxicology studies for the ANDA.

Drug product: The finished dosage form (eg, tablet, capsule, or solution) that contains
the active drug ingredient, generally, but not necessarily, in association with inactive

Drug product selection: The process of choosing or selecting the drug product in a
specified dosage form.

Drug substance: A drug substance is the active pharmaceutical ingredient (API) or

component in the drug product that furnishes the pharmacodynamic activity.

Equivalence: Relationship in terms of bioavailability, therapeutic response, or a set of

established standards of one drug product to another.
Generic name: The established, nonproprietary, or common name of the active drug in a
drug product (eg, acetaminophen).

Generic substitution: The process of dispensing a different brand or an unbranded drug

product in place of the prescribed drug product. The substituted drug product contains the
same active ingredient or therapeutic moiety as the same salt or ester in the same dosage
form but is made by a different manufacturer. For example, a prescription for Motrin
brand of ibuprofen might be dispensed by the pharmacist as Advil brand of ibuprofen or
as a nonbranded generic ibuprofen if generic substitution is permitted and desired by the

Pharmaceutical alternatives: Drug products that contain the same therapeutic moiety
but as different salts, esters, or complexes. For example, tetracycline phosphate or
tetracycline hydrochloride equivalent to 250 mg tetracycline base are considered
pharmaceutical alternatives. Different dosage forms and strengths within a product line
by a single manufacturer are pharmaceutical alternatives (eg, an extended-release dosage
form and a standard immediate-release dosage form of the same active ingredient). The
FDA currently considers a tablet and capsule containing the same active ingredient in the
same dosage strength as pharmaceutical alternatives.

Pharmaceutical equivalents: Drug products in identical dosage forms that contain the
same active ingredient(s), ie, the same salt or ester, are of the same dosage form, use the
same route of administration, and are identical in strength or concentration (eg,
chlordiazepoxide hydrochloride, 5-mg capsules). Pharmaceutically equivalent drug
products are formulated to contain the same amount of active ingredient in the same
dosage form and to meet the same or compendial or other applicable standards (ie,
strength, quality, purity, and identity), but they may differ in characteristics such as
shape, scoring configuration, release mechanisms, packaging, excipients (including
colors, flavors, preservatives), expiration time, and, within certain limits, labeling. When
applicable, pharmaceutical equivalents must meet the same content uniformity,
disintegration times, and/or dissolution rates. Modified-release dosage forms that require
a reservoir or overage or certain dosage forms such as prefilled syringes in which residual
volume may vary must deliver identical amounts of active drug ingredient over an
identical dosing period.

Pharmaceutical substitution: The process of dispensing a pharmaceutical alternative

for the prescribed drug product. For example, ampicillin suspension is dispensed in place
of ampicillin capsules, or tetracycline hydrochloride is dispensed in place of tetracycline
phosphate. Pharmaceutical substitution generally requires the physician's approval.

Reference listed drug: The reference listed drug (RLD) is identified by the FDA as the
drug product on which an applicant relies when seeking approval of an Abbreviated New
Drug Application (ANDA). The RLD is generally the brand-name drug that has a full
New Drug Application (NDA). The FDA designates a single reference listed drug as the
standard to which all generic versions must be shown to be bioequivalent. The FDA
hopes to avoid possible significant variations among generic drugs and their brand-name
counterparts. Such variations could result if generic drugs were compared to different
reference listed drugs.

Therapeutic substitution; The process of dispensing a therapeutic alternative in place of

the prescribed drug product. For example, amoxicillin is dispensed instead of ampicillin
or ibuprofen is dispensed instead of naproxen. Therapeutic substitution can also occur
when one NDA-approved drug is substituted for the same drug which has been approved
by a different NDA, eg, the substitution of Nicoderm (nicotine transdermal system) for
Nicotrol (nicotine transdermal system).

Purpose of Bioavailability Studies

Bioavailability studies are performed for both approved active drug ingredients and
therapeutic moieties not yet approved for marketing by the FDA. New formulations of
active drug ingredients must be approved by the FDA before marketing. In approving a
drug product for marketing, the FDA ensures that the drug product is safe and effective
for its labeled indications for use. Moreover, the drug product must meet all applicable
standards of identity, strength, quality, and purity. To ensure that these standards are met,
the FDA requires bioavailability/pharmacokinetic studies and, where necessary,
bioequivalence studies for all drug products (FDA Guidance for Industry, 2003).
Bioavailability may be considered as one aspect of drug product quality that links in-vivo
performance of the drug product used in clinical trials to studies demonstrating evidence
of safety and efficacy.
For unmarketed drugs that do not have full NDA approval by the FDA, in-vitro and/or
in-vivo bioequivalence studies must be performed on the drug formulation proposed for
marketing as a generic drug product. Furthermore, the essential pharmacokinetics of the
active drug ingredient or therapeutic moiety must be characterized. Essential
pharmacokinetic parameters, including the rate and extent of systemic absorption,
elimination half-life, and rates of excretion and metabolism, should be established after
single- and multiple-dose administration. Data from these in-vivo bioavailability studies
are important to establish recommended dosage regimens and to support drug labeling.

In-vivo bioavailability studies are also performed for new formulations of active drug
ingredients or therapeutic moieties that have full NDA approval and are approved for
marketing. The purpose of these studies is to determine the bioavailability and to
characterize the pharmacokinetics of the new formulation, new dosage form, or new salt
or ester relative to a reference formulation.

In summary, clinical studies are useful in determining the safety and efficacy of drug
products. Bioavailability studies are used to define the effect of changes in the
physicochemical properties of the drug substance and the effect of the drug product
(dosage form) on the pharmacokinetics of the drug. Bioequivalence studies are used to
compare the bioavailability of the same drug (same salt or ester) from various drug
products. Bioavailability and bioequivalence can also be considered as performance
measures of the drug product in-vivo. If the drug products are bioequivalent and
therapeutically equivalent (as defined above), then the clinical efficacy and the safety
profile of these drug products are assumed to be similar and may be substituted for each

Biovailability Studies:
1- Biovailability studies are carried out to compare the availability of a drug substance
from different dosage forms. Such comparison as:
• An immediate release tablet with sustained release tablets For this type of
tablet ka values should be slower; but F values should be similar or
• Two (or more) dosage forms made by two different manufacturers
2-To determine the rate and amount of drug absorbed from a dosage form.
3-To determine the duration of the drug present in the biological fluids or tissues when
correlation with the patient response

Relative and Absolute Bioavailability

"Relative” or “Comparative”bioavailability refers to the availability of a drug product as
compared to another dosage form or product of the same drug given in the same dose.
These measurements determine the effects of formulation differences on drug absorption.
The relative bioavailability of product A compared to product B, both products
containing the same dose of the same drug, is obtained by comparing their respective
Relative Bioavailabilty = AUCa

Where drug product B is the reference standard. When the bioavailability of a generic
product is considered, it is usually the relative bioavailability that is referred to. A more
general form of the equation results from considering the pos-stability of different doses.

"Absolute"bioavailability,F,is the fraction of an administered dose which actu-ally

reaches the systemic circulation, and ranges from F =0 (no drug absorption) to
F =1 (complete drug absorption). Since the total amount of drug reaching the sys-temic
circulation is directly proportional to the area under the plasma drug concen-tration as a
function of time curve (AUC),F is determined by comparing the
respective AUCs of the test product and the same dose of drug administered intra-
venously. The intravenous route is the reference standard since the dose is,by def-inition,
completely available.

(where AUC EV and AUC IV are,respectively,the area under the plasma concentra-tion-
time curve following the extravascular and intravenous administration of a given dose of
drug.Knowledge of F is needed to determine an appropriate oraldose of a drug relative to
an IV dose.

Absolute Bioavailability= AUCor

i AUCiv

The difference between absolute and relative bioavailability is illustrated by the

following hypothetical example. Assume that an intravenous injection (Product A)and
two oral dosage forms (Product B and Product C),all containing the same dose of the
same drug,are given to a group of subjects in a crossover study.

The F for Product B and Product C is 50%(F =0.5) and 40%(F =0.4), respectively.
However, when the two oral products are compared, the relative bioavailability of
product C as compared to product B is 80%.

Drug Product Area Under the Curve (mcg/ml)x hr

A Intravenous injection 100
B Oral dosage form, brand or reference standard 50
C Oral dosage form, generic Product 40

Study Design:
Bioavailability studies involve the administration of the tested dosage form to a panel of
subjects, after which blood and / or urine samples are collected and analyzed for drug
content. Based on drug concentration profile of the drug, a judgment is made regarding
the rate and extent of absorption of the drug.
Normally, the study is conducted in a group of healthy male subjects:
1-The healthy subjects with normal height, weight (70-90kg to avoid variations in
volume of distribution), and ages between 18-35 years should be preferred.
2-Smokers and or subjects taking other medication may cause alteration of enzymatic
activity or drug-drug interaction. Therefore, no other medication is given to the
volunteers for one week to ensure complete excretion.
3-The number of volunteers should be sufficient to see any real differences in
bioavailability. Usually, 10-20 subjects are used; at least 6 subjects in each study are
5-One assay method for all tested experiments should be done.
6- A complete cross-over design is used with which each subject receive all products with
a wash out period between each dose administration to ensure complete elimination of
the drug from the body before the next dose.

7-Other important considerations in the methodology of a bioavailability study are:

Sample size, period of trial, and sampling to get enough data to draw valid conclusion.

The bioavailability testing period should be of a sufficient period of time at least three
half-lives of the drug, five times of half-lives is preferred. Blood samples should be taken
to permit an accurate determination of tmax, Cpmax, and AUC.

Method of Assessing Bioavailability:

The bioavailability testing is a way to obtain an evidence of therapeutic utility of a drug

product. Bioavailability determinations are performed by drug manufacturers to ensure
that a drug product will get the therapeutic agent to its site of action in an adequate
concentration. Also, to compare the drug product from different dosage forms or from the
same dosage forms produced by different manufacturers.

1-Plasma Data or Blood level studies: Blood level studies, the most common type of
human bioavailability, Studies are based on the assumption that: there is a direct
relationship between the concentration of drug in the blood or plasma and the drug’s
concentration at the site of action, by monitoring the concentration in the blood. The
possible of an indirect measure of drug response by plotting the plasma drug conc. versus
time. The following bioavailability parameters could be calculated to assess the rate and
extent of drug absorption: 1- Cpmax, 2-tmax, and 3- AUC

AUC : is representative of, and proportional to, the total amount of drug absorbed into the
Cpmax : is proportional to the rate of absorption.
tmax : is invesely proportional to the rate of absorption. The faster the absorption of a
drug the higher the maximum concentration and the less time will take to reach the
maximum concentration.
Urinary excretion data: An alternative bioavailability studies is to measure the
cummulative amount of unchanged drug in the urine.

These Studies are based on the premise of that urinary excretion of the unchanged drug is
directly proportional to the plasma drug concentration of total drug. Thus, the total
quantity of drug excreted in the urine is a reflection of the quantity of drug absorbed from
the gastrointestinal tract.

See examples on page 8-13

These techniques of studying bioavailability are most useful for those drugs that are not
extensively metabolized prior to urinary elimination. Only at least 20% of a dose is
excreted unchanged in the urine after iv dose. This is for determination of
bioavailability using urinary excretion data. Other conditions for valid results included:

1. The fraction of drug entering the blood stream and being excreted intact by the
kidneys must remain constant.
2. Collection of the urine has to continue until all the drug has been completely
excreted (five half-lives).

Urinary excretion data are primarily useful for assessing extent of drug absorption. The
time for cumulative amount of drug excreted in the urine can also be used to estimate the
rate of absorption. Thus, urinary excretion of drug is not recommended as a substitute for
blood concentration data; rather, these studies should be used in conjunction with blood
level data for confirmatory purposes

Consider the following example:

Two products, A and B, each containing 100 mg of the same drug are administered
orally. A total of 80 mg of drug is recovered in the urine from Product A, but only
40 mg is recovered from Product B. This indicates that twice as much drug was absorbed
from Product A as from Product B. (The fact that neither product resulted in excretion of
the entire dose might be due to the existence of other routes of elimination, e.g,
metabolism). This technique of studying bioavailability is most useful for those drugs
that are not extensively metabolized prior to urinary elimination. As a rule-of-thumb,
determination of bioavailability using urinary excretion data should be conducted only if
at least 20%of a dose is excreted unchanged in the urine after an iv dose (56).

1-Disintegration test:
The early attempts to establish an indicator of drug bioavailability focused on
disintegration as the most pertinent in-vitro paramete. Solid dosage forms must
disintegrate before significant dissolution and absorption can occur. It doesnot ensure that
the rate of soluton of the drug is adequate to produce suitable blood level of the active
ingredients.Thu, the disintegration test is very useful for quality control for
2-Dissolution test:
Since a drug must go into solution before it can be absorbed, and since the rate at which a
drug dissolves from a dosage form often determines its rate and/or extent of absorption,
attention has been directed at the dissolution rate. It is currently considered to be the
most sensitive in-vitro parameter most likely to correlate with bioavailability.

Official dissolution tests -There are two official USP dissolution methods: Apparatus I,
(basket method), and Apparatus II (paddle method). For details of these dissolution tests,
the reader is recommended to consult USPXXII/NFXVII (66).
Dissolution tests are an extremely valuable tool in ensuring the quality of a drug product.
Generally, product-to-product variations are due to formulation factors, such as particle
size differences, excessive amounts of lubricant and coatings.
These factors are reactive to dissolution testing. Thus, dissolution tests are very effective
in discriminating between and within batches of drug product(s). The dissolution test, in
addition, can exclude definitively any unacceptable product.

Correlation In-Vitro /In-Vivo

Invitro methods are important in the development and optimization of dosage forms
while in vivo tests are essential in obtaining information on the behavior of medication in
living organisms.
The in-vitro dissolution studies are most useful for monitoring drug product, stability and,
manufacturing process control. Thus, dissolution testing is of immense value as a tool for
quality control. Dissolution is difficult for immediate release solid dosage forms because
of highly solubility of some compounds compared to the gastric emptying rate. On other
hand, dissolution test has been reported for modified release solid dosage forms that
exhibit slow dissolution. The correlation in-vitro/ in-vivo tests is based on comparing:
1-Dissolution rate versus absorption rate:

Dissolution time

Absorption time
2-Percent of dissolution versus percent of drug absorbed:
If a drug absorbed completely after dissolution, a linear correlation may be obtained

Absoprtion time

% Dissolved

3-Maximum plasma concentration versus percent drug dissolution:

Drug products that have a highest percentage of dissolution exhibit higher peak drug
concentration Cpmax

%Drug Dissolved
4-tmax versus percent drug dissolved:
Highly dissolved drugs exhibited the shortest time to reach peak concentration (tmax)
The tmax is depenedent on absorption rate, the fastest absorption would result in the
shortest tmax.


%Drug Dissolved

Limitation of In-vitro Dissolution test

There are, however, problems with in-vitro dissolution testing which should be noted
problems which make correlation with in-vivo availability difficult.
1-The first is related to instrument variance and the absence of a standard method.
The tests described in the USP but a few of the large number of dissolution methods
proposed to predict bioavailability. Since the dissolution rate of a dosage form is
dependent on the methodology used in the dissolution test, changes in the apparatus
dissolution medium, temperature …etc.
Factors Affecting dissolution rate of the drug: degree of agitation, size and shape of
container, composition of dissolution medium, pH, ionic strength, viscosity, surface
tension, temperature, volume of dissolution medium, evaporation, and flow rate
2-The difference between the in vitro and invivo environments in which dissolution
Invitro studies are carried out under controlled condition in one or in two standard
The invivo environment (The GIT) is a continuously changing, complex environment.
Many factors can affect the dissolution rate of a drug in the GIT, including: pH, enzymes
secretion, surface tension, motility presence of other substances and absorption surfaces.
3-The invivo environment is far complex, variables and unpredictable than in vitro
environment, making invitro/invivo correlation very difficult. A simple dissolution can’t
reflect the invivo absorption of a drug across population.
Single Dose Versus Multiple Dose
Most Bioavailability evaluations are made on the bases of single dose administration.
Repeated dose administration at fixed time intervals with the dosing frequency less than
five half-lives, drugs will accumulate in the body and eventually reach a plateau or steady
At steady state the amount of drug-eliminated equals the available dose (rate in = rate
out), therefore the AUC during a dosing interval at steady state is equal to the total AUC
obtained when a single dose is administered. The AUC can be used to assess the extent of
absorption of the drug, as well as its absolute and relative bioavailability.

Multiple dose administration (Steady-State)

Multiple-Dose (Steady-State) Study P-10
In a few cases, a multiple-dose, steady state, randomized, two-treatment, two-way
crossover study comparing equal doses of the test and reference products may be
performed in adult, healthy subjects. For these studies, three consecutive trough
concentrations (C min) 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 2 hours following dose administration.
Blood sampling is performed similarly to the single-dose study.

Advantages of a multiple-dose over a single dose bioavailbility studies:

• Eliminate the need to extrapolate the plasma concentration profiles to obtain the
total AUC after a single dose.
• Eliminate the need for a long washout period between doses.
• More closely reflects the actual clinical use of the drug.
• Allows blood levels to be measured at the same concentrations encountered
• Because blood levels to be higher than in the single dose method, quantitative
determination is easier and more reliable.
• Saturable pharmacokinetics, if present, can be more readily detected at steady
• Requires more time to complete
• More difficult and costly to conduct (requiring prolonged monitoring of subjects)
• Greater problems with compliance control
• Greater exposure of subjects to the test drug, increasing the potential for adverse

Crossover 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 below. 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, variation 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. 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 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.

Table 15.3 Latin-Square Crossover Design for a Bioequivalence Study of Three Drug
Products in Six Human Volunteers

Drug Product
Subject Study Period 1 Study Period 2 Study Period 3
1 A B C
2 B C A
3 C A B
4 A C B
5 C B A
6 B A C

Table 15.4 Latin-Square Crossover Design for a Bioequivalency Study of Four

Drug Products in 16 Human Volunteers

Subject Study Period 1 Study Period 2 Study Period 3 Study Period 4
1 A B C D
2 B C D A
3 C D A B
4 D A B C
5 A B D C
6 B D C A
7 D C A B
8 C A B D
Subject Study Period 1 Study Period 2 Study Period 3 Study Period 4
9 A C B D
10 C B D A
11 B D A C
12 D A C B
13 A C D B
14 C D B A
15 D B A C
16 B A C D

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:

Period 1
Period 2
Sequence 1
Sequence 2

Where R = reference and T = treatment.

shows a design for three different drug treatment groups given in a three-period study
with six different sequences. 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.