Bioequivalence and Bioavailability Forum

Ciao Paola!

❝ The bioanalytical results from a BE study (crossover, 3 periods, 36 healthy volunteers) shown the concentrations at the pre-dose sampling time above the LLOQ.

❝ These results occur in 20% of the subjects, and for each subject happen in more than one period, and always during the period 1, before the starting of the drug administration.

I’m not sure whether I do understand you correctly. In those 20% of subjects do you get pre-dose concentrations in all periods? Please clarify.

❝ Following several bioanalytical check, the results seem to be correlated to an eventual contamination of the pre-dose samples during the clinical phase, rather than to a bioanalytical bias.

I know the term ‘contamination’ is sometimes used – but how should that practically occur? Somebody spiking vacutainers at BD’s factory (sabotage)? The drug leaking from the formulation, sneeking downstairs to the room where the centrifuges are, and slipping into sample vials? I would rather say it’s an analytical issue.

❝ All these pre-dose values (except for one subject) are lower than 5% of the respective Cmax.

At least we are talking about small concentrations. Are you using LC/MS-MS or a ligand-binding assay? Guidelines call for checking of interferences and the matrix-effect in ≥6 sources of matrix. Even if you were successful in validation it might be possible to be hit in a larger study by pure chance. In the future consider checking more than six sources of blank matrix if you plan for a large study.

❝ Since the EMA guideline on BE reports that, if the pre-dose concentration is greater than 5 % of the Cmax value, the subject being removed from the analysis, we cannot exclude these data from the statistical analysis.

❝ Which is in your opinion the most suitable approach to treat our bioanalytical results?

According to the GL you should report all data and remove subject’s data of all periods where pre-dose concentrations are >5% of C_max. This procedure should have been stated in the protocol. Was it?

❝ What is the position of the regulatory authorities in these cases?

I’m not a regulator. Personal experience close to nil. Messed up a 36 subject 3 treatment study (two tests, one reference) ten years ago where the wash-out was too short (stupidly we improved the LLOQ by a factor of five compared to a pilot study) and found pre-dose concentrations in the second period in 21 subjects and in the third period in 18 subjects (but all of them <5% C_max). We presented:

1. Evaluation as planned (ignoring pre-dose values).
   
2. Evaluation based on the first period only (i.e., as a parallel design).
   
3. Estimating λ_z including the pre-dose values of the respective next period. Correcting all concentrations in higher period(s) based on the estimated time-course of earlier period(s).

Both test formulations were BE based on all methods. Study was accepted (mainly based on [2] – study was large enough). Nowadays [1] would be sufficient. [2] is statistically flawed and should not be done according to the GL [“A test for carry-over is not considered relevant and no decisions regarding the analysis (e.g. analysis of the first period only) should be made on the basis of such a test.”]. [3] only for very adventurous people.

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