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Treatments should be separated by adequate wash-out periods. The interval between study days should be long enough to permit elimination of essentially all of the previous dose from the body.
Preferably the interval should not be less than 5 terminal elimination half-lives of the active compound or metabolite, if the latter is measured. Normally the interval between study days should not exceed 3-4 weeks. If a longer wash-out period is required a parallel design can be considered.
6.6.3 Sample collection
Under normal circumstances blood should be the biological fluid sampled to measure the concentrations of the active pharmaceutical ingredient. In most cases the API or metabolites are measured in serum or plasma. If the active pharmaceutical ingredient is excreted predominantly unchanged in the urine, urine can be sampled. When urine is collected at the study centre the volume of each sample must be measured immediately after collection and included in the report. The number of samples should be sufficient to allow the estimation of pharmacokinetic parameters.
However, in most cases the exclusive use of urine excretion data should be avoided as this does not allow estimation of the tmax and the maximum concentration.
Blood samples should be processed and stored under conditions that have shown not to cause degradation of the analytes. This can be proven by analysing duplicate quality control (QC) samples during the analytical period. Quality control samples must be prepared in the fluid of interest (e.g.
plasma), including concentrations at least at the low, middle and high segments of the calibration range. The quality control samples must be stored with the study samples and analysed with each set of study samples for each analytical run.
Sample collection methodology should be specified in the study protocol.
6.6.4 Parameters to be assessed In bioavailability studies the shape of and the area under the plasma concentration versus time curves are mostly used to assess extent (AUC) and rate (Cmax, tmax)of absorption. Sampling points or periods should be chosen such that the concentration versus time profile is adequately defined to allow calculation of relevant parameters. For single dose studies the following parameters should be
measured or calculated:
Working document QAS/04.093/Rev.4 page 20
• Area under the plasma/serum/blood concentration-time curve from time zero to time t (AUC0-t), where t is the last time point with measurable concentration for individual formulation. The method of calculating AUC-values should be specified. In general AUC should be calculated using the linear/log trapezoidal integration method. The exclusive use of compartmental based parameters is not recommended.
• Cmax is the maximum or peak concentration observed representing peak exposure of active pharmaceutical ingredient (or metabolite) in plasma, serum or whole blood.
AUC0-t and Cmax are considered to be the most relevant parameters for assessment of bioequivalence.
In addition it is recommended that the following parameters be estimated:
• Area under the plasma/serum/blood concentration-time curve from time zero to time infinity (AUC0-∝) representing total exposure, where AUC0-∝ = AUC0-t + Clast /β; Clast is the last measurable drug concentration and β is the terminal or elimination rate constant calculated according to an appropriate method.
• tmax – the time after administration of the drug at which Cmax is observed.
For additional information the elimination parameters can be calculated:
• T1/2 – the plasma (serum, whole blood) half-life.
For steady-state studies the following parameters can be calculated:
• AUCτ - AUC over one dosing interval (τ) at steady-state.
• Cmin – concentration at the end of a dosing interval.
• peak trough fluctuation – % difference between Cmax and Cmin.
When urine samples are used cumulative urinary recovery (Ae) and maximum urinary excretion rate are employed instead of AUC and Cmax.
6.6.5 Studies of metabolites Generally evaluation of pharmacokinetic bioequivalence will be based upon the measured concentrations of the parent drug released from the dosage form rather than the metabolite.
Concentration-time profile of the parent drug is more sensitive to changes in formulation performance than a metabolite, which is more reflective of metabolite formation, distribution and elimination. It is important to state a priori in the study protocol which chemical entities (pro-drug, drug (active pharmaceutical ingredient), metabolite) will be analyzed in the samples.
In some situations measurements of metabolite concentrations may be necessary instead of parent
• If an active metabolite is formed as a result of gut wall or other presystemic metabolic process(es) and the metabolite contributes meaningfully to safety and/or efficacy, it is recommended that both the metabolite and the parent drug concentrations be measured.
• Measurement of a metabolite may be preferred when parent drug levels are too low to allow reliable analytical measurement in blood, plasma or serum for an adequate length of time or when the parent compound is unstable in the biological matrix.
It is important to note that measurement of one analyte, active pharmaceutical ingredient or metabolite, allows the risk of making a Type-I error (the consumer risk) to remain at the 5% level.
However, if more than one of several analytes is selected retrospectively as the bioequivalence determinant, then the consumer and producer risks change (5).
When measuring the active metabolites wash-out period and sampling times may need to be adjusted in order to adequately characterize the pharmacokinetic profile of the metabolite.
6.6.6 Measurement of individual enantiomers A non-stereoselective assay is currently acceptable for most pharmacokinetic bioequivalence studies.
When the enantiomers have very different pharmacological or metabolic profiles, assays that distinguish between the enantiomers of a chiral active pharmaceutical ingredient may be appropriate.
Stereoselective assay is also preferred in the case when systemic availability of different enantiomers is demonstrated to be non-linear.
6.6.7 Use of fed state studies in bioequivalence determination 18.104.22.168 Immediate release formulations Fasted state studies are generally preferred. When the product is known to cause gastrointestinal disturbances if given in the fasted state, or if labelling restricts to administration to the fed state only, then the fed state pharmacokinetic bioequivalence study becomes the preferred method. The details of the meal may depend on local diet and customs.
22.214.171.124 Modified release formulations
Food effect studies are necessary for all multisource modified release formulations to ensure the absence of "dose dumping". The latter signals a formulation failure such that the dose is released all at once rather than over an extended period of time. This results in a "spike" in plasma concentrations time profile. A high fat meal is recommended to provide maximum perturbation to challenge the robustness of release from the formulation with respect to prandial state. The composition of the meal may depend on local diet and customs. (See also Section 6.2.4) In addition to the fast state studies, see also section 9.3.3 dose proportional studies.
6.7 Active pharmaceutical ingredients' quantification All analytical test methods used to determine the active compound and/or its biotransformation product in the biological fluid must be well-characterized, fully validated and documented. The objective of the validation is to demonstrate that a particular method used for quantitative Working document QAS/04.093/Rev.4 page 22 measurement of analytes in a given biological matrix, such as blood, plasma, serum or urine, is reliable and reproducible for the intended use.
Applicable principles of GLP should be followed in the conduct of chemical analysis (6).
Bioanalytical methods should meet the requirements of specificity, sensitivity, accuracy, precision and reproducibility. Knowledge of the stability of the active pharmaceutical ingredient and/or its biotransformation product in the sample material is a prerequisite for obtaining reliable results.
The Bioanalytical Method Validation Conference held in 2000 made several recommendations for the conduct of analysis of biological samples in a pharmacokinetic study (7). Some important
• Validation comprises before-study and within-study phases. During the pre-study phase stability of the stock solution and spiked samples in the biological matrix, specificity, sensitivity, accuracy, precision and reproducibility should be provided. Within-study validation proves the stability of samples collected during clinical trial under storage conditions and confirms the accuracy and precision of the determinations.
• Validation must cover the intended use of the assay.
The calibration range must be appropriate to the study samples. A calibration curve should be • prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte. A calibration curve should consist of a blank sample, a zero sample, and six to eight non-zero samples covering the expected range. Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study.
• If an assay is to be used at different sites, it must be validated at each site, and cross-site comparability established.
• An assay which is not in regular use requires sufficient revalidation to show that its performance is according to the original validated test procedures. The revalidation study must be documented, usually as an appendix to the study report.
• Within a study, the use of two or more methods to assay samples in the same matrix over a similar calibration range is strongly discouraged.
• If different studies are to be compared and the samples from the different studies have been assayed by different methods, and the methods cover a similar concentration range and the same matrix, then the methods should be cross-validated.
• Spiked quality control samples at a minimum of three different concentrations in duplicate should be used for accepting or rejecting the analytical run.
• All the samples for one subject (all periods) should be analysed in the same analytical run, if possible.
method during the analysis of study samples will require adequate revalidation. Results of study sample determination should be reported in the analytical report together with calibration and QC sample results, repeat analyses (if any), and a representative number of sample chromatograms.
6.8 Statistical analysis
The primary concern in bioequivalence assessment is to limit the risk of a false declaration of equivalence. Statistical analysis of the bioequivalence trial should demonstrate that the clinically significant difference in bioavailability is unlikely. The statistical procedures should be specified before the data collection starts in the protocol.
The statistical method for testing pharmacokinetic bioequivalence is based upon the determination of the 90% confidence interval around the ratio of the log transformed population means (multisource/ comparator) for the pharmacokinetic parameters under consideration and by carrying out two onesided tests at the 5% level of significance (8). To establish pharmacokinetic bioequivalence the calculated confidence interval should fall within a preset bioequivalence limit. The procedures should lead to a decision scheme which is symmetrical with respect to the two formulations (i.e. leading to the same decision whether the multisource formulation is compared to the comparator product or the comparator product to the multisource formulation).