Bioavailability & Bioequivalence Testing Protocol

Bioavailability

Bioavailability is a measurement of the rate and extent of active drugs that reach systemic circulation and are available at the site of action.

Bioequivalence

It refers to a procedure that compares the bioavailability of a drug from different formulations.

• The aim of the bioavailability study is to find out the dosage form influence on the biological performance of the drug.
• The bioavailability study protocol is used to detect differences in the rate and extent of absorption that are attributable only to dosage form variability and should avoid variabilities due to other factors.

The bioavailability study protocol is divided into

A. Study objective

B. Study design

• Experimental design
• Wash out period
• Drug products

1. Test product
2. Recognized standard

• Route of administration
• Dosage regimen
• Frequency and duration of sampling
• Randomization of drug administration
• Single-versus multiple-dose study design
• Subjects

1. Healthy subjects versus patients
2. Subject selection: Medical history, Physical examination, Laboratory tests
3. Study conditions

• Analysis of biological fluids

C. Methods of Assessment of Bioavailability

• Plasma data
• Urine data
• Acute pharmacological effect
• Clinical response

D. Analysis and Presentation of Data

• Statistical treatment of data-Analysis of variance (ANOVA)
• Format of data

Study objective

• Bioavailability studies are performed for new drugs to establish pharmacokinetic parameters including

1. Rate of absorption
2. Rate of excretion
3. Metabolism
4. Elimination
5. Half-life of single and multiple dose administration

Study design

The study design has two types

1. Parallel design
2. Cross over design

• Latin square cross-over design
• Balanced incomplete block design

1. Parallel design

• The aim of experimental design is to minimize the experimental variables and to avoid a bias.
• In parallel design, two formulations are administered to two groups of volunteers

Disadvantage

The intersubject variation needs to be corrected.

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2. Cross over design

Minimizes the effect of intersubject variability in the study.

Latin square cross-over design

In this design

• Each subject receives just once each formulation.
• Each formulation is administered just once in each study period.

Advantages

• Minimize the effect of intersubject variability.
• It minimizes the carry-over effects.
• It minimizes the time effect on bioavailability.

Disadvantages

• It requires a longer time to complete the study.
• Increase number of study periods leads to high subject dropouts and the study becomes difficult.

Balanced incomplete block design

The salient features of this design are

• Each subject receives not more than two formulations
• Each formulation is administered the same number of times

Washout period

The time interval between the two treatments is called the washout period.

• Washout period is required for the elimination of the administered dose of a drug.
• The washout period is a function of the half and dose of the drug administered.

E.g. digitoxin has a half-life of 6-9 days washout period.

Drug products

Test products:

Test products are generally evaluated for the following reasons.

• To compare the biological performance of a test product to that of a recognized standard i.e. bioequivalence studies.
• To select the best dosage form of a new drug or existing drug among different dosage forms. E.g. tablet, capsule, emulsion, and suspension.

Route of administration

• Most often, orally administered dosage forms are subjected to bioavailability studies.
• Dosage forms administered by other routes (buccal, transdermal, and intramuscular) should also be evaluated for their biological performance.

Single dose vs multiple-dose study design

• Useful to know whether single-dose studies are better or multiple-dose studies are better for assessing the bioavailability of a drug product.
• If dosage forms are to be evaluated only for bioequivalence purposes, single-dose studies are usually sufficient. E. g. analgesics for the relief of headaches need only single-dose studies.
• However certain dosage forms (time-release products, enteric-coated preparations) require multiple dose studies.

Administration of drug products

• Administration of drug products to the subjects should be based on randomization.
• After administering the drug products, blood samples are withdrawn from the subjects at fixed time points. It takes some time to take a sample from each subject, and the total time difference between the first subject and the last subject may range from 10 to 20 minutes.
• This 10 to 20-minute difference would represent a substantial change in the drug concentrations observed in the blood.

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Sampling

• The sampling scheme should be frequent enough to define the absorption phase, the peak, and the elimination phase during a drug’s time course in the body.
• To estimate the AUC from the data, sampling has to be carried out till the concentration of the drug reaches the linear elimination phase.

Selection of subjects

Healthy subjects vs patients:

• Use of healthy volunteers avoids many of the variations that are possible with patients.
• Some of the special problems associated with testing in patients are given below

1. It is difficult to obtain many patients in a given place.
2. The severity of a disease varies from one patient to another.

Study conditions

The selected subject should be

1. Maintained on a uniform diet.
2. None of them should have taken any drug at least one week before the study.

In general bioavailability trials are conducted on subjects that have fasted overnight.

Analysis of biological samples

• The biological samples collected as per the sampling procedure have to be analyzed immediately after the study.
• Analysis of biological samples is carried out by

1. Analytical method
2. Non-specific analytical method

Methods of assessment of bioavailability

Two types

1. Pharmacokinetic methods (indirect methods)
2. Pharmacodynamic methods (direct methods)

Pharmacokinetic methods

The parameters that are useful in determining the bioavailability of a drug from a drug product based on indirect methods are

1. Plasma data

• Time of peak plasma concentration (tp)
• Peak plasma concentration (Cmax)
• Area under the plasma concentration-time curve (AUC)

2. Urine data

• The rate of drug excretion in the urine (dXu/dt)
• The cumulative amount of drug excreted in the urine (Xu∞)
• The time for maximum urinary excretion (tu∞)

Pharmacodynamic methods

Two methods used for the estimation of bioavailability are based on the measurement of

1. Acute pharmacological effect
2. Clinical response

Statistical analysis of the data

• The purpose of a bioavailability test is to find out whether the test formulation gives a blood level profile identical to that observed for a reference standard product or not.
• Statistical methods are used to evaluate the data in order to identify the different sources of variation and if possible, to measure the contribution of each identified variable and isolate the specific observation of primary interest.
• The analysis of variance (ANOVA), a statistical procedure used for a cross over design, is used widely in bioavailability testing and is the procedure that will be encountered most frequently by the health scientist.

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