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Bioequivalence is established by undertaking a single or in certain circumstances a number of bioequivalence studies.
Most bioequivalence studies employ a randomised crossover design in healthy volunteers, in which each individual acts
as his/her own control. Clearance, volume of distribution, and physiological variables that might affect absorption (e.g.
gastric emptying, motility, pH), distribution and elimination will normally have lower within-patient than between-patient
variability. Therefore, a crossover design will usually have more statistical power for a given number of subjects than
a parallel-group design.
The test (generic) and reference (innovator) products should be administered with a standard quantity of water under
fasting conditions or following a standard meal.
So that each subject returns to a ‘baseline’ state prior to each treatment period, a ‘washout’ period
of no treatment should be employed between each treatment period. The washout period should be at least 5 half lives of
the substances to be measured and the absence of carryover of plasma concentrations into the second period should be confirmed
by pre-dose plasma assay. If significant carryover is present in the data, the trial results will be considered void.
Sampling times should be appropriate to describe the absorption, distribution, and elimination phases of the drug. Sampling
frequency around Tmax should be sufficient to provide an accurate estimation of Cmax. Sampling duration
should be sufficient to provide an accurate estimation of AUC extrapolated to infinity (measured AUC0-t should be at least
80% of extrapolated AUC0-∞). At least three to four samples can be obtained during the terminal log-linear phase
of the elimination period in order to calculate terminal elimination rate constant accurately.
Usually drug or metabolites are measured in serum or plasma. Where plasma measurement is not possible total urine collection
may be more appropriate for analysis.
The number of subjects required for a bioequivalence study is determined statistically and is typically about 20, although
smaller numbers can be used if sufficient statistical power has been determined.
Plasma concentration curves for the test and the reference product are derived from the data obtained in the study (see
1, What is Bioavailability and Bioequivalence?). As it is very difficult to test whether two curves are sufficiently
similar to each other the internationally accepted indices of area under the curve (AUC), peak and timing of the peak
drug concentration (Cmax) are used to characterise the curves. If differences are apparent between either
the values for the Cmax of the two products or those for the AUC then the two curves will have different shapes.
The data from each individual patient is used to calculate the mean, standard deviation and confidence intervals of
the pharmacokinetic variables (Cmax and AUC).
Statistically the assessment of bioequivalence is based upon 90% confidence intervals for the ratio of the population
geometric means (test/reference) for the variables under consideration. This method is equivalent to two one-sided tests
with the null hypothesis of bioinequivalence at the 5% significance level.