Bioequivalence and Bioavailability Forum

Dear Devisha,

you are raising a terribly complicated issue

I would go for a biowaiver, but still it may be difficult to obtain the permeability data for classification.

Do you have a validated bioanalytical method--which is not a trivial task?

Unfortunately with both enzymes you will not only see a circadian rhythm of excretion, but also highly variable levels dependent on nutrition.
I would suggest to standardize food/water not only on treatment days, but also perform a ‘run-in period’ for a couple of days prior to each treatment.
Since there is already a variable blank profile, you should collect bloodsamples at the same time points as intended on treatment days.
Then you should subtract this blank profile from both tretament profiles.
You should also prepare a decision tree describing how you will deal with negative values (which are not uncommon especially if you are subtracting low values). Generally such values should be set to zero.
Below is an example for an LOQ of 1 Unit/ml: corrected values have a 'true zero'--which is not the lower limit of quantification of the method.
1. Blank = 1.1 U/ml, measured = 1.2 U/ml, corrected concentration = 0.1 U/ml
2. Blank = 1.2 U/ml, measured = 1.1 U/ml, corrected concentration = 0.0 U/ml

In any case (Biowaiver or in-vivo study) I strongly would suggest talking to the regulator(s) first.
If you decide to go for an in-vivo study, I would also suggest to start with a small pilot study first (not to estimate CV, but mainly to get an impression whether the run-in, the sampling times, and the analytical method ‘works’ and you are obtaining a plausible pharmacokinetic profile from the subtraction procedure).