BIOAVAILABILITY TYPES, PARAMETERS,

SIGNIFICANCE, STUDY PROTOCOL AND

METHODS OF ASSESSMENT OF
BIOAVAILABILITY
Introduction
Regulatory agencies such as the FDA require submission of bioavailability data
in applications to market new drug products.
A drug product’s bioavailability provides an estimate of the relative fraction of
the administered dose that is absorbed into the systemic circulation.
Determining the fraction (f) of administered dose absorbed involves comparing
the drug product’s systemic exposure (represented by the concentration-versus-
time or pharmacokinetic profile) with that of a suitable reference product.
For systemically available drug products, bioavailability is most often assessed
by determining the area under the drug plasma concentration-versus-time
profile (AUC).
The AUC is considered the most reliable measure of a drug’s bioavailability, as
it is directly proportional to the total amount of unchanged drug that reaches the
systemic circulation.
Absolute Bioavailability
Absolute bioavailability compares the bioavailability of the active drug in the systemic
circulation following extra-vascular administration with the bioavailability of the same
drug following intravenous administration (Fig. 16-4).
Intravenous drug administration is considered 100% absorbed.
The route of extra-vascular administration can be inhaled, intramuscular, oral, rectal,
subcutaneous, sublingual, topical, transdermal, etc.
The absolute bioavailability is the dose-corrected AUC of the extra-vascularly
administered drug product divided by the AUC of the drug product given intravenously.
Thus, for an oral formulation, the absolute bioavailability is calculated as follows:

where
F
abs is the fraction of the dose absorbed, expressed as a percentage;

AUC
po is the AUC following oral administration;

D is the dose administered intravenously;

iv

AUC
iv is the AUC following intravenous administration; and
D is the dose administered orally.
Absolute availability, Fabs, may be expressed as a fraction or as a percent by multiplying
Fabs × 100.
A drug given by the intravenous route will have an absolute bioavailability of 100% (f =
1).
A drug given by an extra-vascular route may have an F abs = 0 (no systemic absorption)
and Fabs = 1.0 (100% systemic absorption).
Relative Bioavailability
Another type of comparative bioavailability assessment is provided by a relative
bioavailability study.
In a relative bioavailability study, the systemic exposure of a drug in a designated
formulation (generally referred to as treatment A or reference formulation) is compared
with that of the same drug administered in a reference formulation (generally referred to
as treatment B or test formulation).
In a relative bioavailability study, the AUCs of the two formulations are compared as
follows:

where
Frel is the relative bioavailability of treatment (formulation) A, expressed as a percentage;
AUCA is the AUC following administration of treatment (formulation) A;
DA is the dose of formulation A;
AUCB is the AUC of formulation B; and
DB is the dose of formulation B.
Relative bioavailability studies are frequently included in regulatory submissions.
For example, the FDA recommends that new drug developers routinely use an oral
solution as the reference for a new oral formulation, for the purpose of assessing how
formulation impacts bioavailability.
Other types of relative bioavailability studies used in drug development include studies
to characterize food effects and drug–drug interactions.
In a food-effect bioavailability study, oral bioavailability of the drug product given with
food (usually a high-fat, high-calorie meal) is compared to oral bioavailability of the
drug product given under fasting conditions.
The drug product given under fasting conditions is treated as the reference treatment.
The goal of a drug–drug interaction study is to determine whether there is an increase or
decrease in bioavailability in the presence of the interacting drug.
As such, the general drug–drug interaction study design compares drug relative
bioavailability with and without (reference treatment) the interacting drug.
Relative bioavailability studies are used in developing new formulations of existing
immediate-release drug products, such as new modified-release versions or new fixed-
dose combination formulations.
In the case of a new modified-release version, the reference product is the approved
immediate-release product.
In the case of a new fixed-dose combination, the reference product can be the single-
entity drug products administered either separately (i.e, three treatments for a fixed-dose
combination doublet) or concurrently according to an approved combination regimen
(i.e, two treatments).
Relative bioavailability study designs are also commonly used for bridging formulations
during drug development, for example, to evaluate how drug systemic availability from
a new pre-market formulation compares with that from an existing premarket
formulation.
STUDY PROTOCOL
Reference Listed Drug (RLD)
For bioequivalence studies of generic products, one formulation of the drug is chosen as
a reference standard against which all other formulations of the drug are compared.
The FDA designates a single reference listed drug as the standard drug product 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.
The reference drug product should be administered by the same route as the comparison
formulations unless an alternative route or additional route is needed to answer specific
pharmacokinetic questions.
For example, if an active drug is poorly bioavailable after oral administration, the drug
may be compared to an oral solution or an intravenous injection.
For bioequivalence studies on a proposed generic drug product, the reference standard is
the reference listed drug (RLD), which is listed in the FDA’s Approved Drug Products
with Therapeutic Equivalence Evaluations—the Orange Book and the proposed generic
drug product is often referred to as the “test” drug product.
The RLD is generally a formulation currently marketed with a fully approved
NDA for which there are valid scientific safety and efficacy data.
The RLD is usually the innovator’s or original manufacturer’s brand-name
product and is administered according to the dosage recommendations in the
labeling.
Before beginning an in vivo bioequivalence study, the total content of the
active drug substance in the test product (generally the generic product) must
be within 5% of that of the reference product.
Moreover, in vitro comparative dissolution or drug-release studies under
various specified conditions are usually performed for both test and reference
products before performing the in vivo bioequivalence study.
METHODS FOR ASSESSING BIOAVAILABILITY AND
BIOEQUIVALENCE
Methods For Assessing Bioavailability And Bioequivalence
Direct and indirect methods may be used to assess drug bioavailability.
Bioequivalence of a drug product is demonstrated by the rate and extent of drug
absorption, as determined by comparison of measured parameters.
The FDA’s regulations (US-FDA, CDER, 2014a) list the following approaches to
determining bioequivalence, in descending order of accuracy, sensitivity, and
reproducibility:
In vivo measurement of active moiety or moieties in biological fluid (i.e, a
pharmacokinetic study)
In vivo pharmacodynamic (PD) comparison
In vivo limited clinical comparison
In vitro comparison
Any other approach deemed acceptable (by the FDA)
A. IN VIVO MEASUREMENT OF ACTIVE MOIETY OR MOIETIES IN
BIOLOGICAL FLUIDS
1. Plasma Drug Concentration
Measurement of drug concentrations in blood, plasma, or serum after drug administration
is the most direct and objective way to determine systemic drug bioavailability.
By appropriate blood sampling, an accurate description of the plasma drug
concentration–time profile of the therapeutically active drug substance(s) can be obtained
using a validated drug assay.
tmax: The time of peak plasma concentration, tmax, corresponds to the time required to
reach maximum drug concentration after drug administration.
At tmax, peak drug absorption occurs and the rate of drug absorption exactly equals the
rate of drug elimination (Fig. 16-3).
Drug absorption still continues after tmax is reached, but at a slower rate.
When comparing drug products, tmax can be used as an approximate indication of
drug absorption rate.
The value for tmax will become smaller (indicating less time required to reach peak
plasma concentration) as the absorption rate for the drug becomes more rapid.
Units for tmax are units of time (eg, hours, minutes).
For many systemically absorbed drugs, small differences in tmax may have little
clinical effect on overall drug product performance.
However, for some drugs, such as delayed action drug products, large differences in
tmax may have clinical impact.
Cmax: The peak plasma drug concentration, Cmax, represents the maximum plasma drug
concentration obtained after oral administration of drug.
For many drugs, a relationship is found between the pharmacodynamic drug effect and
the plasma drug concentration.
Cmax provides indications that the drug is sufficiently systemically absorbed to provide a
therapeutic response.
In addition, Cmax provides warning of possibly toxic levels of drug.
The units of Cmax are concentration units (e.g, mg/mL, ng/mL).
Although not a unit for rate, Cmax is often used in bioequivalence studies as a surrogate
measure for the rate of drug bioavailability. So, the expectation is that as the rate of drug
absorption goes up, the peak or Cmax will also be larger. If the rate of drug absorption
goes down, then the peak or Cmax is smaller.
AUC: The area under the plasma level–time curve, AUC, is a measurement of the
extent of drug bioavailability (see Fig. 16-3).
The AUC reflects the total amount of active drug that reaches the systemic circulation.
The AUC is the area under the drug plasma level–time curve from t = 0 to t = ∞, and is
equal to the amount of unchanged drug reaching the general circulation divided by the
clearance.

where
F = fraction of dose absorbed,
D0= dose, k= elimination rate constant, and V D= volume of distribution.
The AUC is independent of the route of administration and processes of drug elimination as long as
the elimination processes do not change.
The AUC can be determined by a numerical integration procedure, such as the
trapezoidal rule method.
The units for AUC are concentration × time (eg, mg·h/mL).
For many drugs, the AUC is directly proportional to dose. For example, if a single dose
of a drug is increased from 250 to 1000 mg, the AUC will also show a fourfold increase
(Figs. 16-5 and 16-6).
In some cases, the AUC is not directly proportional to the administered dose for all
dosage levels.
For example, as the dosage of drug is increased, one of the pathways for drug
elimination may become saturated (Fig. 16-7).
Drug elimination includes the processes of metabolism and excretion.
Drug metabolism is an enzyme-dependent process. For drugs such as salicylate and
phenytoin, continued increase of the dose causes saturation of one of the enzyme
pathways for drug metabolism and consequent prolongation of the elimination half-life.
The AUC thus increases disproportionally to the increase in dose, because a smaller
amount of drug is being eliminated (i.e, more drug is retained).
When the AUC is not directly proportional to the dose, bioavailability of the drug is
difficult to evaluate because drug kinetics may be dose dependent.
Conversely, absorption may also become saturated resulting in lower-than-expected
changes in AUC.
2. Urinary Drug Excretion Data
Urinary drug excretion data is an indirect method for estimating bioavailability.
The drug must be excreted in significant quantities as unchanged drug in the urine.
In addition, timely urine samples must be collected and the total amount of urinary drug
excretion must be obtained.

The cumulative amount of drug excreted in the urine, Du, is related directly to the total
amount of drug absorbed.
Experimentally, urine samples are collected periodically after administration of a drug
product.
Each urine specimen is analyzed for free drug using a specific assay.
A graph is constructed that relates the cumulative drug excreted to the collection-time
interval (Fig. 16-8).
The relationship between the cumulative amount of drug excreted in the urine and the
plasma level– time curve is shown in Fig. 16-8. When the drug is almost completely
eliminated (point C), the plasma concentration approaches zero and the maximum
amount of drug excreted in the urine, Du, is obtained.

dDu/dt: The rate of drug excretion. Because most drugs are eliminated by a first-
order rate process, the rate of drug excretion is dependent on the first-order elimination
rate constant, k, and the concentration of drug in the plasma, Cp.
In Fig. 16-9, the maximum rate of drug excretion, (dD u/dt)max, is at point B, whereas the
minimum rate of drug excretion is at points A and C.
Thus, a graph comparing the rate of drug excretion with respect to time should be
similar in shape to the plasma level–time curve for that drug (Fig. 16-10).
t∞: The total time for the drug to be excreted. In Figs. 16-9 and 16-10, the slope of the
curve segment A–B is related to the rate of drug absorption, whereas point C is related to
the total time required after drug administration for the drug to be absorbed and
completely excreted, t = ∞.
The t∞ is a useful parameter in bioequivalence studies that compare several drug
products.
B. Bioequivalence Studies Based On Pharmacodynamic End points—in Vivo
Pharmacodynamic (PD) Comparison
In some cases, the quantitative measurement of a drug in plasma is not available or in
vitro approaches are not applicable. The following criteria for a PD endpoint study are
important:
A dose–response relationship is demonstrated.
The PD effect of the selected dose should be at the rising phase of the dose–response curve,
as shown in Fig. 16-11.
Sufficient measurements should be taken to assure an appropriate PD response profile.
All PD measurement assays should be validated for specificity, accuracy, sensitivity, and
precision.
For locally acting, non-systemically absorbed drug products, such as topical
corticosteroids, plasma drug concentrations may not reflect the bioavailability of the
drug at the site of action.
An acute pharmacodynamic effect, such as an effect on forced expiratory volume,
FEV1 (inhaled bronchodilators), or skin blanching (topical corticosteroids) can be used
as an index of drug bioavailability.
In this case, the acute pharmacodynamic effect is measured over a period of time after
administration of the drug product.
Measurements of the pharmacodynamic effect should be made with sufficient frequency
to permit a reasonable estimate for a time period at least three times the half-life of the
drug (Gardner, 1977).
The use of an acute pharmacodynamic effect to determine bioavailability generally
requires demonstration of a dose–response curve (Fig. 16-11 and Chapter 21).
Bioavailability is determined by characterization of the dose–response curve.
For bioequivalence determination, pharmacodynamic parameters including the total area
under the acute pharmacodynamic effect–time curve, peak pharmacodynamic effect, and
time for peak pharmacodynamic effect are obtained from the pharmacodynamic effect–
time curve (Fig. 16-12).
The onset time and duration of the pharmacokinetic effect may also be included in the
analysis of the data.
The use of pharmacodynamic endpoints for the determination of bioavailability and
bioequivalence is much more variable than the measurement of plasma or urine drug
concentrations.
Some examples of drug products for which bioequivalence PD endpoints are
recommended are listed on Table 16-2.
C. Bioequivalence Studies Based On Clinical Endpoints—clinical Endpoint
Study
The clinical endpoint study is the least accurate, least sensitive to bioavailability
differences, and most variable.
A predetermined clinical endpoint is used to evaluate comparative clinical effect in the
chosen patient population.
Highly variable clinical responses require the use of a large number of patient study
subjects, which increases study costs and requires a longer time to complete compared to
the other approaches for determination of bioequivalence.
A placebo arm is usually included to demonstrate that the study is sufficiently sensitive
to identify the clinical effect in the patient population enrolled in the study.
The FDA considers this approach only when analytical methods and pharmacodynamic
methods are not available to permit use of one of the approaches described above.
The clinical study is usually a limited, comparative, parallel clinical study using
predetermined clinical endpoint(s).
Clinical endpoint BE studies are recommended for those products that have negligible
systemic uptake, for which there is no identified PD measure, and for which the site of
action is local.
Comparative clinical studies have been used to establish bioequivalence for topical
antifungal drug products (eg, ketoconazole) and for topical acne preparations.
For dosage forms intended to deliver the active moiety to the bloodstream for systemic
distribution, this approach may be considered acceptable only when analytical methods
cannot be developed to permit use of one of the other approaches.
Some examples of drug products where a clinical endpoint bioequivalence study is
recommended (Davit and Conner, 2015) are listed in Table 16-3.
D. In Vitro Studies
Comparative drug release/dissolution studies under certain conditions may give an
indication of drug bioavailability and bioequivalence.
Ideally, the in vitro drug dissolution rate should correlate with in vivo drug
bioavailability (see Chapter 15 on in vivo–in vitro correlation, IVIVC).
The test and reference products for which in vitro release rates form the basis of the
bioequivalence usually demonstrate Q1/Q2 sameness (qualitatively same inactive
ingredients in the quantitative same amounts).
Comparative dissolution studies are often performed on several test formulations of the
same drug during drug development.
Comparative dissolution profiles may be considered similar if the similarity factor (f2)
is greater than 50 (see Chapter 15).
For drugs whose dissolution rate is related to the rate of systemic absorption, the test
formulation that demonstrates the most rapid rate of drug dissolution in vitro will
generally have the most rapid rate of drug bioavailability in vivo.
Under certain conditions, comparative dissolution profiles of higher and lower dose
strengths of a solid oral drug product such as an immediate-release tablet are used to
obtain a waiver (biowaiver) of performing additional in vivo bioequivalence studies (see
section on biowaivers).
E. Other Approaches Deemed Acceptable (By The FDA)
The FDA may also use in vitro approaches other than comparative dissolution for
establishing bioequivalence.
The use of in vitro biomarkers and in vitro binding studies has been proposed to
establish bioequivalence.
For example, cholestyramine resin is a basic quaternary ammonium anion-exchange
resin that is hydrophilic, insoluble in water, and not absorbed in the gastrointestinal tract.
The bioequivalence of cholestyramine resin is performed by equilibrium and kinetic
binding studies of the resin to bile acid salts (US-FDA, CDER, 2012a).
7. Bioequivalence Studies Based On Multiple Endpoints
The FDA may recommend two or more bioequivalence studies, each based on
a different approach, for some drug products with complex delivery systems or
mechanisms of action.
Some examples of drug products that FDA requires multiple bioequivalence
studies (Davit and Conner, 2015) are listed in Table 16-4.
Reference & Keys

Applied Biopharmaceutics & pharmacokinetics by LEON

SHARGEL Sixth Edition, Page # 406-413.