DEFINITION

Bioavailability is defined as the rate and the

absorption of drug that reaches the biological
system in an active form, capable of exerting the
desired pharmacological effect, including its
onset, intensity and duration of its action.
 Objectives of bioavailability studies:

 Development of new formulation.

 Determination of influence of excipients, patient related
factors and possible interaction with other drugs on the
efficiency of absorption.
 Control of quality of a drug product during the early stages
of marketing in order to determine the influence of
processing factors, storage, stability on drug absorption.
 Primary stages of the development of a suitable dosage form
for a new drug entity.
 CONSIDERATIONS IN IN-VIVO BIOAVAILABILITY STUDY
DESIGN:

 Bioavailability—Absolute versus Relative

 ABSOLUTE BIOAVAILABILITY:
 The systemic availability of a drug administered orally is determined in
comparison to its iv administration.
 Characterization of a drug's absorption properties from the e.v. site.
 F = AUCev
AUCiv

 RELATIVE BIOAVAILABILITY:
 The availability of a drug product as compared to another dosage form or product
of the same drug given in the same dose.
 Characterization of absorption of a drug from its formulation.
 Fr=AUCA
 Route Bioavailability (%) Characteristics

Intravenous 100 (by definition) Most rapid onset

(IV)

Intramuscular 75 to ≤ 100 Large volumes often possible; may be

(IM) painful

Subcutaneous 75 to ≤ 100 Smaller volumes than IM; may be painful

(SC)

Oral (PO) 5 to < 100 Most convenient; first pass effects may be
significant

Rectal (PR) 30 to < 100 Less first-pass effects than oral

Inhalation 5 to < 100 Often very rapid onset

Transdermal 80 to ≤ 100 Usually very slow absorption; used for

lack of first-pass effects; prolonged duration
of action
 Single Dose versus Multiple Dose Studies
 The dose to be administered for a bioavailability study is determined from
preliminary clinical experiments.
 Single dose bioavailability studies are very common. They are easy, offer less
exposure to drugs and are less tedious.
Limitation: Difficult to predict the steady-state characteristics of a drug and inter-
subject variability.
• multiple dose study :
• the bioavailability is determined at steady-state.
• Better evaluation of a controlled-release formulation is possible.
• Small inter-subject variability is observed,
• Requires collection of fewer blood samples.
• Limitations:
◦ Poor compliance by subjects.
◦ Greater exposure of subjects to the test drug, increasing the potential for adverse
reactions.
In multiple dose study the drug should be administered for 5 to 6 elimination half-lives
until the steady-state has been reached, before collecting the blood samples.
 ASSESSMENT OF BIOAVAILABILITY
 Pharmacokinetics method – This method is more practical and
discriminative. Pharmacokinetic methods are of two types.
a) Determination of whole blood, plasma or serum concentration
b) Urinary excretion method
 Pharmacodynamic methods:
 Acute Pharmacologic Response Method : When bioavailability measurement by
pharmacokinetic method is difficult, an acute pharmacologic effect such as effect
on pupil diameter, heart rate or BP can be useful as an index of drug bioavailability.
 Disadvantage: It tends to be complex, expensive, time-consuming and require a
sensitive and quantitative measure of the desired response.
 Therapeutic Response Method: Clinical response of the drug for which it is
intended to be used is measured.
 E.g.: heart rate, body temperature, blood sugar levels, and for anti-inflammatory
drugs, reduction in inflammation is determined.
 Drawbacks: quantification of observed response is too improper to allow for
reasonable assessment of relative bioavailability between two dosage forms of the
same drug.
A) The blood (or serum or plasma) concentration-
time curve -
 These are based on the assumption that there is a direct relationship between
the concentration of drug in blood or plasma and the concentration of drug at
the site of action.
 If the drugs are given to the volunteers through iv dose, the blood samples
should be withdraw after 5min. And the frequency of sampling should be
15min.
 In case of one compartment open model – at elimination phase 3 points are
required to describe he kE value.
 In case of two compartmental model – 5 to 6 points are required to describe kE
value.
 If Oral dose is given – 3 points are required to describe ka value.
The key parameters for determining bioavailability
AUC: The AUC is proportional to the total amount of drug reaching the systemic
circulation, and thus characterizes the extent of absorption.

Cmax: Gives indication whether drug is sufficiently absorbed systemically to provide a

therapeutic response.

Tmax: The Tmax reflects the rate of drug absorption, and decreases as the absorption
rate increases.

MEC: The minimum plasma concentration of the drug required to achieve a given
pharmacological or therapeutic response

MSC: plasma concentration of the drug beyond which adverse effects are likely to
happen.
 THERAPEUTIC RANGE-The range of plasma drug concentration in which the
desired response is achieved yet avoiding adverse effect. The aim is clinical practice
is to maintain plasma drug concentration within the therapeutic range.

 ONSET OF ACTION-On set of action is the time required to achieve the minimum
effective plasma concentration following administration of drug formulation.

 DURATION OF ACTION-Duration of action of the therapeutic effect of the drug

is defined as the time period during which the plasma concentration of the drug
exceeds the minimum effective level.

 INTENSITY OF ACTION-In general, the difference between the peak plasma

concentration and the minimum effective plasma concentration provides a relative
measure of the intensity of the therapeutic response of the drug.
B) Urinary Excretion Data:
 These studies are based on the principle that urinary excretion
of the unchanged drug is directly proportional to the plasma
concentration of total drug.

 This technique of studying bioavailability is most useful for

those drugs that are not extensively metabolized prior to
urinary elimination.
 The three major parameters examined in urinary excretion data
are as follow:
1.(dXu/dt)max : maximum urinary excretion rate, gives the
rate of appearance of drug in the urine is proportional to its
concentration in systemic circulation. Its value increases as the
rate of and/or extent of absorption increases
2. (tu)max : time for maximum excretion rate, is analogous to
the of plasma level data, its value decreases as the absorption
rate increases.
3. Xu : cumulative amount of drug excreted in the urine is
related to the AUC of plasma level data and increases as the
extent of absorption increases.

 These studies used for certain thiazide diuretics and

sulfonamides and for drugs that have urine as the site of action-
for example, urinary antiseptics.
BIOEQUIVALENCE STUDIES

 DEFINITIONS
 Equivalence – Equivalence is more relative term that compares
one drug product with another or with a set of established
standards. Equivalence may be defined in several ways:

 Chemical equivalence indicates that two or more dosage forms

contain the labeled quantities of drug.

 Clinicalequivalence occurs when the same drug from two or

more dosage forms gives identical in vivo effects as measured
by a pharmacological response or by control of a symptom or a
disease.
 Therapeutic equivalence implies that one structurally different
chemical can yield the same clinical result as another chemical.
DESIGN AND CONDUCT OF STUDIES:
 The design and conduct of the study should follow ICH regulations
on Good Clinical Practice, including reference to an Ethics
Committee.

 A bioequivalence study is basically a comparative bioavailability

study designed to establish equivalence between test and reference
products.

1. Design:
The various types of test designs that are usually employed in
bioequivalence stdies;
i. Completely randomised designs
ii. Randomised block designs
iii. Repeated measures, cross-over and carry-over designs
iv. Latin square designs
i. Completely randomised designs
 Method of randomisation
 Label all subjects with the same number of digits, for e.g., if there are 20 subjects,
number them from 1 to 20.
 Randomly select non-repeating random numbers (like simple randomisation) with
among these labels for the first treatment, and then repeat for all other treatments.
Advantages
 The design is extremely easy to construct.
 It can accommodate any number of treatments and subjects.
 Disadvantages
 Although the design can be used for any number of treatments, it is best suited for
situations in which there are relatively few treatments.
 Any unrelated sources of variability will tend to increase the random error term,
making it difficult to detect differences among the treatment (or factor level) mean
responses.
ii. Randomised block designs
 First, subjects are sorted into homogeneous groups, called blocks and the treatments
are then assigned at random within the blocks.
Method of Randomisation
 Subjects having similar background characteristics are formed as blocks.
 Then treatments are randomised within each block, just like the simple
randomisation.
 Randomisations for different blocks are done independent of each other.
Advantages
 It can accommodate any number of treatments or replications.
 Different treatments need not have equal sample size.
 The statistical analysis is relatively simple. The design is easy to construct.
 Disadvantages
 Missing observations within a block require more complex analysis.
 The degrees of freedom of experimental error are not as large as with a completely
randomised design.
iii. Repeated measures, cross-over and carry-over designs
 This is essentially a randomised block design in which the same subject serves as a
block.
 The administration of two or more treatments one after the other in a specified or
random order to the same group of patients is called a crossover design or change-
over design
 Method of Randomisation
 Complete randomisation is used to randomise the order of treatments for each subject.
Randomisations for different subjects are independent of each other.
 Advantages
 They provide good precision for comparing treatments because all sources of
variability between subjects are excluded from the experimental error.
 It is economic on subjects. This is particularly important when only a few subjects can
be utilized for the experiments.
 Disadvantages
 There may be an order effect, which is connected with the position in the treatment
order.
 There may be a carry-over effect, which is connected with the preceding treatment or
treatments.
iv. Latin square designs
 A Latin square design is a two-factor design (subjects and treatments are the two
factors) with one observation in each cell.
 Such a design is useful to compare the earlier ones when three or more treatments
are to be compared and carry-over effects are balanced.
 In a Latin square design, rows represent subjects, and columns represent
treatments.
 Advantages
 It minimizes the inter-subject variability and carry-over effect in plasma drug
levels.
 Minimizes the variations due to time effect.
 Treatment effects can be studied from a small-scale experiment. This is particularly
helpful in preliminary or pilot studies.
 Disadvantages:
 The randomisation required is somewhat more complex than that for earlier designs
considered.
 The study takes a long time since an appropriate washout period between two
administrations is essential which may be very long if the drug has a long t½.
WASHOUT PERIOD
The time interval between the 2 treatments is
called as WASHOUT PERIOD
It is required for the elimination of the
administered dose to avoid the carry over effect

Large no. of drugs have been found to have half

–life between 1-10 hrs, awashout period of 1
week was usually found suitable in most cases.
2. Subjects:
A. Selection of subjects:
 Aim to minimize variability and permit detection of differences
between pharmaceutical products.
 The studies should normally be performed with healthy
volunteers.
 They should be screened for suitability by means of clinical
laboratory tests, review of medical history, and medical
examination.
B. Standardization of the study:
 The test conditions should be standardized in order to minimize
the variability of all factors involved.
 Standardization of the diet, fluid intake and exercise is
recommended.
 The subjects should not take other medicines during a suitable
period before and during the study.
C. Inclusion of patients:
 If the investigated active substance is known to have adverse effects
and the pharmacological effects or risks are considered unacceptable
for healthy volunteers it may be necessary to use patients instead,
under suitable precautions and supervision.
D. Genetic pheno-typing:
 Pheno-typing of subjects should be considered as well in crossover
studies.
 If a drug is known to be subject to major genetic polymorphism,
studies could be performed in panels of subjects of known phenotype
or genotype.
3. Characteristics to be investigated:
 In most cases evaluation of bioavailability and bioequivalence will be
based upon the measured concentrations of the parent compound.
 In some situations, measurements of an active or inactive metabolite
is carried out.
 The plasma concentration versus time curves are mostly used to
assess extent and rate of absorption.
 The use of urine excretion data may be advantageous in determining
the extent of drug input in case of products predominately excreted
renally.
 Specificity, accuracy and reproducibility of the methods should be
sufficient.
4.Chemical analysis:
 It is conducted according to the applicable principles of Good
Laboratory Practice (GLP).
 Determination of the active moiety and/or its biotransformation
product(s) in plasma, urine or any other suitable matrix must be well
characterized, fully validated and documented to yield reliable results
that can be satisfactorily interpreted.
5 Reference and test product:
 The choice of reference product should be justified
by the applicant and agreed upon by the regulatory
authority.
 The test products used in the biostudy must be
prepared in accordance with GMP-regulations.
6.Data analysis:
 To quantify the difference in bioavailability
between the reference and test products and to
demonstrate that any clinically important
difference.
A.Statistical analysis:
The statistical analysis (e.g. ANOVA) should
take into account sources of variation that can
be reasonably assumed to have an effect on the
response variable.
Pharmacokinetic parameters derived from
measures of concentration, e.g. AUC, Cmax
should be analyzed using ANOVA.
B. Acceptance range for pharmacokinetic parameters:
1.AUC-ratio:
 It should lie within an acceptance interval of 0.80-1.25.
2.Cmax-ratio:
 It should lie within an acceptance interval of 0.80-1.25.
 The wider interval must be 0.75-1.33.
 For tmax if there is a clinically relevant claim for rapid release or
action or signs related to adverse effects. The interval should lie
within a clinically determined range.
C. Handling deviations from the study plan:
 The protocol should also specify methods for handling drop-outs.
D.A remark on individual and population bioequivalence:
 Bioequivalence studies are designed for population and individual
is limited.
7.In vitro dissolution for bioequivalence study:
 The term commonly used to describe is "in-vitro/in-vivo
correlation".
 The specifications for the in vitro dissolution of the product should
be derived from the dissolution profile of the batch.
A. Official dissolution tests:
 Apparatus 1, (basket method).
 Apparatus 2 (paddle method).

B.Parameters used:
1. Degree of agitation
2. Size and shape of container
3. Composition of dissolution medium
• pH, ionic strength, viscosity
4. Temperature of dissolution medium
5. Volume of dissolution medium
8.Reporting of results:
 The report of a bioequivalence study should give the complete
documentation of its protocol, conduct and evaluation
complying with GCP-rules and ICH guideline.
 Drop-out and withdrawal of subjects should be fully
documented.
 The method used to derive the pharmacokinetic parameters
should be specified.
 The analytical report should include the results for all standard
and quality control samples.
APPLICATIONS FOR PRODUCTS CONTAINING
APPROVED ACTIVE SUBSTANCES:
A. Bioequivalence studies:
 In vivo bioequivalence studies are needed when there is
a risk that possible differences in bioavailability may
result in therapeutic in equivalence.

 oral immediate release drug formulations with systemic

action
 Non-oral and non-parenteral drug formulation designed
to act by systemic absorption.
 Sustained or otherwise modified release drug
formulation.
 Fixed-dose combination products.
REFERENCES:

 Brahmankar.D.M,Sunil.B.Jaiswal,VallabhPrakashan
Biopharmaceutics and Pharmacokinetics-A Treatise ,page
no.282-305.
LeonShargel & Andrew Yu Applied Biopharmaceutics &
pharmacokinetics, page no 247-260.
Madan.PL Biopharmaceutics & pharmacokinetics page no
118-127.

TERIMA KASIH