Application of the relative activity factor approach in scaling from heterologously expressed cytochromes P450 to human liver microsomes: Studies on amitriptyline as a model substrate

The relative activity factor (RAF) approach is being increasingly used in t he quantitative phenotyping of multienzyme drug biotransformations. Using l ymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressa nt amitriptyline as a model substrate, we have tested the hypothesis that t he human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the bio transformation catalyzed by individual recombinant CYPs using the RAF appro ach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurement s in heterologous expression systems. In addition, we have compared the res ults of two widely used methods of quantitative reaction phenotyping, namel y, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-d emethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicte d biotransformation rates in microsomes from a panel of 12 human livers det ermined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo phar macokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correl ated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhib itors used were specific to their target CYP isoforms.