Bioavailability

and
Bioequivalence

Important definitions
Bioavailability (BA)
The relative amount of an administered dose that reaches
the general circulation and the rate at which this occurs
(American Pharmaceutical Association, 1972). OR
The rate and extent to which the active ingredient or
therapeutic moiety is absorbed from a product and becomes
available at the site of drug action (US Food and Drug
Administration, 1977)
Bioequivalence (BE): defined as the absence of a
significant difference in bioavailability between two
pharmaceutically equivalent products or pharmaceutical
alternatives under similar conditions in an appropriately
designed study.

 Pharmaceutical alternatives: drug products are considered

pharmaceutical alternatives if they contain the same
therapeutic moiety, but are different salts, esters, or
complexes of that moiety, or are different dosage forms or
strengths.
Example:-Tetracycline phosphate or Tetracycline
hydrochloride equivalent to 250 mg Tetracycline base.
Pharmaceutical Equivalents :
FDA considers drug products to be pharmaceutical equivalents
if they meet these three criteria:
1- They contain the same active ingredient(s).
2- They are of the same dosage form and route of
administration.
3- They are identical in strength or concentration.

 Pharmaceutically equivalent drug products may differ in

characteristics such as shape, release mechanism, labeling
(to some extent) and excipients (including colors, flavors,
preservatives).
Therapeutic Equivalents: drug products are considered
to be therapeutic equivalents only if they are
pharmaceutical equivalents and if they can be expected to
have the same clinical effect and safety profile when
administered to patients under the conditions specified in
the labeling.

 FDA classifies as therapeutically equivalent those products

that meet the following general criteria:
1- They are approved as safe and effective;
2-They are pharmaceutical equivalents in that they (a) contain
identical amounts of the same active drug ingredient in the
same dosage form and route of administration, and (b) meet
applicable standards of strength, quality, purity, and identity;
3- They are bioequivalent;
4- They are adequately labeled;
5- They are manufactured in compliance with Current Good
Manufacturing Practice regulations.

 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.
Therapeutic substitution: the process of dispensing a
therapeutic alternative in place of the prescribed drug
product.
Drug products containing different active ingredients that
are indicated for the same therapeutic or clinical
objectives. For example, amoxicillin is dispensed instead
of ampicillin or ibuprofen is dispensed instead of
naproxen.

 The innovator product is the original product that was

discovered and developed by a pharmaceutical company.
An pharmaceutical company must submit data about the
efficacy and safety of the new drug.
A generic product is compared to an innovator (reference
or brand) product in dosage form, strength, route of
administration, quality, performance characteristics and
intended use.
Generic drugs do not require preclinical and clinical
studies to ensure safety and efficacy, only bioequivalent
study is needed.

 Generic a formulation of drug prepared by a company that

did not innovate the drug; the company will present
bioequivalency data to the FDA for its approval, with a
view to marketing the generic formulation when the
innovator drug formulation comes off patent
Crossover study: a study design where the same subjects
receive both formulations (with a washout period in
between). This design minimizes error owing to differences
between subjects, since a given subject is used as his/her
control. Therefore, differences between formulations will
not be confounded by intersubject variability.
Washout period: this is the time for drug to be eliminated
from the body, after which the second period of the study
may proceed.

Objectives of bioavailability studies

1. Primary stages of development of a suitable dosage form
for a new drug entity.
2. Determination of influence of excipients, possible
interaction with other drugs on the efficiency of absorption.
3. Development of new formulations of the existing drugs.
4. Control of quality of a drug product during the early stages
of marketing in order to determine the influence of
processing factors, storage and stability on drug
absorption.
5. Comparison of availability of a drug substance from
different dosage forms or form the same dosage form
produced by different manufacturers (Bioequivalence).

Types of bioavailability:
1- Absolute BA
2- Comparative (or relative) BA.

 Absolute bioavailability is assessed by comparing

the values of
and/or cumulative mass of
drug excreted in the urine (Xu), obtained following the
administration of a drug in an extravascular dosage form
and an equal dose of the same
drug intravenously
From area under the plasma concentrationtime curve
data:

AUC extravascular Doseint ravenous

F

AUCint ravenous Doseextravascular

 From urinary data

The amount of drug excreted in urine can be determined

from urinary data, requiring collection of urine samples up
to at least 7t1/2 (elimination half lives) of the drug. At this
point, 99% of the administered dose of a drug is eliminated
and, therefore this procedure provides a fairly accurate
estimate of bioavailability.

NOTE:
The reference standard in absolute bioavailability of a
drug must always be an intravenous solution since the
drug administered intravenously is presumed to be always
completely bioavailable.
Furthermore, the value of (AUC) or the value of Xu=7t
for the intravenous solution, (the reference standard) must
always be in the denominator of the respective equations.
It is important to recognize that the absolute
bioavailability or fraction of the administered dose of a
drug that reaches the general circulation can equal 1 or a
number less than 1; however, it cannot be greater than 1.

 Comparative (relative) bioavailability: is assessed by

comparing the bioavailability parameters derived from
plasma drug concentrationtime plot data and/or urinary
excretion data following the administration of a drug in
two different dosage forms (i.e. tablet and syrup, capsule
and suspension, etc.) and/or two different extravascular
routes of administration (i.e. oral and intramuscular).

AUC extravascular1 Doseextravascular 2

Frel

AUC extravascular 2 Doseextravascular1

 From plasma concentration versus time data:

 From urinary data

Bioequivalence
Bioequivalence is a type of comparative or relative
bioavailability study.
However, in a bioequivalence study, (AUC) , peak plasma
concentration and peak time are determined for two or
more pharmaceutically equivalent products (identical
dosage forms) where at least one of them is an innovator
product (also known as the Brand Name or Reference
Standard).

 The difference between a bioequivalence study and a

comparative bioavailability study is that a
bioequivalence study compares a drug formulation with
a reference standard that is the innovator product.
Moreover both formulations must be identical dosage
forms.
The parameters evaluated in a bioequivalence study are
(AUC) , peak plasma concentrations and peak time.

 Factors affecting bioavailability of drug:

1- Formulation factors .
2- Physiological factors.
3- Drug interactions.
4- Food.

1-Formulation factors:
There are many formulation factors effecting on BA of
drug such as:
A- Excipients (type and concentration) used in the formulation
of a dosage form.
B- Physiochemical properties of drug substance.
C- Route of administration and dosage form.

A- Excipient:
Any change in excipient may cause increase or decrease
of BA of drug which could lead into intoxication or
therapeutic failure.
Examples:
Outbreak of phenytoin intoxication among epileptic patients
due to change of excipient in phenytoin capsules, elevated
in blood phenytoin concentration after administration of
phenytoin capsules with calcium sulphate excipient instead
of phenytoin with lactose excipient.

 Cimetidine bioavailability was decreased when mannitol

was used instead of sucrose in formulation. The mannitol
reduce small intestinal transit time, so the contact time
between cimetidine and small intestine become shorter that
led to decreased the amount of drug absorbed
B- Physiochemical properties of drug substance
Physiochemical properties of drug substance such as particle
size, surface area, salt form, polymorphism, solubility,
crystalline or amorphous nature of drug.

 Most drugs are either weak acids or weak bases. One of the
easiest approach to enhance the solubility and dissolution
rate of such drugs is to convert them into their salt form.
Decreasing the particle size, increasing the surface area
improve the solubility and dissolution of drug substance
that lead to increase drug absorption and bioavailability.
For example, the bioavailability was at highest value
when a phenytoin suspension prepared by micronized
particles and lowest was when tablet prepared by large
particles were administered.

 Polymorphism referred to existence the drug substance

in more than one crystalline forms, which lead to
difference in solubility, dissolution, melting point and
bioavailability.
For example, chloramphenicol palmitate has three
polymorphic form: A, B, C, the B form shows best
bioavailability. The B form is more soluble and better
absorbed.

C. Route of administration and dosage form

The IV route of drug administration is the only route that
guarantees that the entire dose reaches the systemic
circulation, that is, 100% BA. Most of the drugs
administered by the extravascular routes have incomplete
BA.
After oral intake of drug several steps should be passed
in order to be absorbed. The number of steps different
between oral dosage forms.

 Following tablet administration, firstly it will be

disintegrated into small particles followed by dissolution
and then absorption. While solution dosage form, there are
no disintegration or dissolution steps and the drug will be
absorbed rapidly. The bioavailability of solution dosage
form is higher and has less problem than tablet.

Example: bioavailability studies with pentobarbital from

various dosage forms show the absorption rate of
pentobarbital after administration in various oral dosage
forms decreased in the following order:
aqueous solution > aqueous suspension of the free acid >
capsule of the sodium salt > tablet of the free acid.

2- Physiological factors
Many physiological factors affecting bioavailability of
drug include gastrointestinal motility, gastric emptying,
intestinal transit rate and change in gastrointestinal PH.
Delay in gastric emptying could result in extensive
decomposition and reduced bioavailability of drugs that
are unstable in the acidic media of the stomach e.g.
penicillin G.

 Generally drugs are better absorbed in the small intestine

(because of the larger surface area) than in the stomach,
therefore quicker stomach emptying will increase drug
absorption.
For example, a good correlation has been found between
stomach emptying time and peak plasma concentration for
acetaminophen. The quicker the stomach emptying (shorter

stomach emptying time) the higher the plasma concentration.

Peak Acetaminophen Plasma Concentration as a Function of

Stomach Emptying Half-life

 The first-pass effect:

The fraction, f, of orally administered drug that successfully
passes through gut lumen and gut wall is then taken via the
hepatic portal vein to the liver, where metabolism of the drug
by enzymes may take place.

F = f F*

F : is the fraction of administered drug that eventually reaches

the general (systemic) circulation (this is the fraction that
would be obtained in an absolute bioavailability calculation).
f : is the fraction of dose absorbed from gut into the portal
circulation (not the systemic circulation).
F* : is the fraction of absorbed dose that survives the first pass
effect.

For example, let us assume that the orally administered dose

of a drug is 100 mg; the fraction absorbed into the portal
circulation is 0.9 and the fraction that survives the first-pass
effect is 0.5.
Then, F = f F* = 0.9 0.5 = 0.45 (or 45%). In this example,
therefore, 45 mg out of 100 mg of the administered dose will
reach the general circulation

3- Drug interactions
Interactions can occur by pharmacokinetic or
pharmacodynamic mechanisms.
Pharmacokinetic drug interaction alter the pharmacokinetic
properties of drug including absorption, distribution,
elimination and excretion of drug while pharmacodynamic
interaction occur at the site of action of drug and is not related
to change in the serum concentration of drug.

Examples
Probenecid reduces renal tubular secretion of penicillin
which decrease elimination and increase serum
concentration of penicillin.
Valproic acid increases the serum concentration of
phenytoin by displacement from protein binding sites.
Renal elimination of weak organic acids (such as
nitrofurantoin, sulfonamides, aminoglycosides, and
vancomycin) is increased with urine alkalinization and
decreased with urine acidification.

 Drugs may form insoluble complexes by chelation in the

gastrointestinal tract. The quinolone antibiotics in
combination with magnesium- and aluminum-containing
antacids, ferrous sulfate. These di- and trivalent cations
complex with the 4-oxo and 3-carboxyl groups of the
quinolones, resulting in clinically significant decreases in
the quinolone area under the concentrationtime curve
(AUC) by 30 to 50%.

 The complexation of tetracycline and iron. This interaction

leads to decrease tetracycline antibiotic AUCs by up to
80%.
Ketoconazole inhibits the metabolism of oral cyclosporine
by approximately 80%.
4- Food
Food can effect on bioavailability of drug by alteration the
absorption of drug through interaction with drug substance
or drug product, or changing gastrointestinal physiological
parameters as PH and/or delay gastric emptying.
Presence of food may either delay, reduce, increase or may
not affect absorption.

Examples:
Tetracycline, for example, forms non-absorbable complexes
with calcium and iron, and thus it is advised that patients do
not take products containing calcium or iron, such as milk,
iron preparations or indigestion remedies, at the same time
of day as the tetracycline.
High-fat meals can significantly increase the extent of
absorption of fat-soluble compounds such as griseofulvin.
The type and the amount of food effected bioavailability
of valproic acid. The plasma concentration of valproic
acid was higher when administered after high fat meal
than when administered in fasting condition

 Assessment of bioavailability :

1- Pharmacokinetic studies:
Pharmacokinetic study is the most widely method used in
assessment of BE of drug. This type depended on
determination of pharmacokinetic parameters in
biological fluids such as plasma, serum and urine, that
indicated the release and absorption of active ingredients
and became available into systemic circulation.
Typically, the pharmacokinetic study usually done by
two or more products compared to each others in
crossover design with adequate washout period to ensure
complete elimination before administration of the next
dose.

Three important parameters in blood data:

Cmax (maximum concentration of drug or peak
concentration),

Tmax ( the time require to reached the maximum

concentration or peak concentration).

AUC ( area under plasma concentration versus time

curve).
The AUC represent the extent of absorption and Cmax
and Tmaxt represent the rate of absorption.

 Trapezoidal method:
Most common method of estimating AUC.
The principle of the trapezoidal rule for calculating the
AUC is based on dividing the plasma drug concentration
time profile to a number of trapezoids.
The method requires the computation of the average
plasma concentration from two consecutive concentration
values (starting with concentrations at time 0 and time 1,
then time 1 and time 2, etc.). These average values are
multiplied by the difference between the corresponding
time (dt) values. By employing this approach, you can
compute the AUC value for each trapezoid.

 The area of each trapezoid is calculated as:

The following expression yields the AUC for the first

trapezoid of the concentration versus time plot:

The AUC for the second trapezoid of the concentration versus

time plot is:

 This procedure is followed to determine the AUC for each

trapezoid until the last observed plasma concentration.
Then the total AUC can be calculated from the sum of the
areas of these trapezoids in addition to the area under the
tail of the curve.
The area of the tail is:
OR
C last: is the last measured concentration.
k is the first-order elimination rate constant.

 The total AUC is the sum of all trapezoids plus the area
of the tail:

AUC is the sum of the area of all trapezoids and the area under the
tail of the curve.

Rectilinear plot of plasma or serum concentration versus time

following the administration of an intravenous bolus of a drug fitting a
one-compartment model.

Three important parameters in urine excretion data:

1- ( dxu / dt )max :(Maximum urinary excretion rate):
Its value increases as rate and/or extent of absorption
increases
Obtained from peak of plot between rate of excretion
versus midpoint time of urine collection period
2- ( tu ) max: Time for maximum excretion rate:
Its value decreases as absorption rate increases Analogues
of tmax of plasma level data.
3- Xu :Cumulative amount of drug excreted in urine:
It increases as the extent of absorption increases

2- Pharmacodynamic( Pharmacologic effect ) studies:

In local acting drugs as topical dermatological products or
oral inhaled drugs exhibit no systemic absorption.
Determination of BA cannot depend on measuring the drug
concentration in blood or other biological fluids.
In this situation, determination of BA depended on
assessing the pharmacological effect of a drug. The
measured pharmacological effect should be reflect the
efficacy and safety of drug.
The pharmacodynamic endpoint should be accurate,
sensitive, reproducible and reflect the BA of drug. As
example, bronchodilator drugs that used in treatment of
asthma, can determine the pharmacodynamic effect by
measuring the forced expiratory volume of lung

3- Comparative clinical studies:

It is involve comparing the outcome (involve the
therapeutic and adverse effect ) of test and reference
formulations.
It is not the first choice in bioavailability and
bioequivalence studies because need to large number of
subjects that no favorable in ethical consideration.
This type of studies usually done in inability to do
pharmacokinetic and pharmacodynamic studies, because its
less accurate and less sensitive than other methods

4- In vitro studies
In-vitro dissolution test can be used to assess the BA and BE
of drug under certain condition. Such as in drug substances
that are highly soluble and highly permeable and formulated
in rapid dissolving products needed only in-vitro dissolution
test to establish BE.

 Methods and criteria for bioavailability testing

1- The general procedure involves administering a drug to
healthy human subjects, collecting blood and/or urine samples,
analyzing the samples for drug content and tabulating and
graphing results.
2- For comparative bioavailability studies, a crossover design
must be conducted to minimize individual subject variation. A
crossover study means that each subject receives each of the
dosage forms to be tested.
3- A minimum of 12 subjects is recommended; however, 18 to
24 subjects are normally used to increase the database for
statistical analysis.

4- In addition to informed written consent from each subject,

physical examination and laboratory testing are also required
to establish them as healthy volunteers.
5- Usually the volunteers fast overnight and drug is taken in
the morning with a prescribed
amount of water.