General study design

The study should be designed so as to set test conditions which reduce intra- and inter-subject variability and avoid biased results. Standardization (exercise, diet, fluid intake, posture, restriction of the intake of alcohol, caffeine, certain fruit juices, and concomitant drugs in the time period before and during the study) is important to minimize the magnitude of variability other than in the pharmaceutical products.

A cross-over design with randomized allocation of volunteers to each leg is the first choice for bioequivalence studies. The design of studies should, however, depend on the type of drug, and other designs may be more appropriate for specific cases, for example, highly variable drugs and those with a long half-life. In cross-over studies a wash-out period between administration of the test product and the reference product of more than five times the dominant and/or terminal drug half-life is usual, but special consideration will need to be given to extending this period if active metabolites with longer half-lives are produced and under other circumstances.

The administration of the product should be standardized with a defined time of day for ingestion, volume of fluid (150 ml is usual) and usually in the fasting state.

Parameters to be assessed

In bioavailability studies the shape of, and the area under, the plasma concentration curve, or the profile of cumulative renal excretion and excretion rate are mostly used to assess extent and rate of absorption. Sampling points or periods should be chosen such that the time versus concentration profile is adequately defined to allow calculation of relevant parameters. From the primary results the bioavailability parameters desired are derived, such as AUC₄, AUC_t, C_max, t_max, Ae₄, Ae_t, dAe/dt, or any other justifiable parameters (see Appendix 2). The method of calculating AUC-values should be specified. AUC₄ and C_max are considered to be the most relevant parameters for assessment of bioequivalence. In case of use of urine excretion data this corresponds to Ae₄ and dAe/dt_max. For additional information t_½ and MRT can be calculated. For steady-state studies AUC_J, and % peak trough fluctuation can be calculated. The exclusive use of modelled parameters is not recommended unless the pharmacokinetic model has been validated for the active substance and the products.

Additional considerations for complicated drugs

Drugs which would show unacceptable pharmacological effects in volunteers (e.g., serious adverse events, or where the drug is toxic or particularly potent or the trial necessitates a high dose) may require crossover studies in patients or sometimes parallel group design studies in patients.

Drugs with long half-lives may require a parallel design or the use of truncated Area Under Curve (AUC_t) data or a multi-dose study. The truncated area should cover the absorption phase.

Drugs for which the rate of input into the systemic circulation is important may require the collection of more samples around the time of the t_max.

Multi-dose studies may be helpful to assess bioequivalence for:

- drugs with non-linear kinetics (including those with saturable plasma protein binding);

- cases where the assay sensitivity is too low to cover a large enough portion of the AUC₄;

- drug substance combinations, if the ratio of plasma concentrations of the individual drug substances is important;

- controlled-release dosage forms;

- highly variable drugs.

Number of subjects

The number of subjects required for a sound bioequivalence study is determined by the error variance associated with the primary parameters to be studied (as estimated from a pilot experiment, from previous studies or from published data), by the significance level desired, and by the deviation from the reference product compatible with bioequivalence and with safety and efficacy. It should be calculated by appropriate methods (see page 124) and should not normally be smaller than 12. In most of the cases 18-24 subjects may be needed (see Diletti E, Hauschke D, Steinijans VW,: Sample size determination for bioequivalence assessment by means of confidence intervals. Int J Clin Pharmacol Ther and Toxicol, 1991, 29:1-8; Hauschke D, Steinijans VW, Diletti E, Burke M: Sample size determination for bioequivalence assessment using a multiplicative model. J Pharmacokin Biopharm, 1992, 20:559-563; and Phillips KE:

Power of the two one-sided tests procedure in bioequivalence. J Pharmacokin Biopharm, 1990, 18:137-144). The number of recruited subjects should always be justified.

Investigational products

Test products (samples) used in the bioequivalence studies for registration purposes should be identical to the projected commercial pharmaceutical product. Therefore not only the composition and quality characteristics (including stability) but also manufacturing methods should copy those in the future routine production runs.

Samples ideally should be taken from batches of industrial scale. When this is not feasible, pilot or small-scale production batches may be used provided that they are not smaller than one tenth (10%) of expected full production batches.

It is recommended that potency and in vitro dissolution characteristics of the test and reference pharmaceutical products be ascertained prior to performance of an equivalence study. Contents of the active drug substance(s) between the two products should not differ by more than +/-5%. If the potency of the reference material deviates from the declared content of 100% by more than 5%, this difference may be used subsequently to dose-normalize certain bioavailability metrics in order to facilitate comparisons between the test and reference pharmaceutical products.