High-throughput fluorescence assay for cytochrome P450 mechanism-based inactivators

Abstract

The mechanism-based inactivation (MBI) of the human cytochrome P450 (P450 or CYP) drug-metabolizing enzymes may lead to adverse drug-drug interactions, especially for drugs with narrow therapeutic windows. Unlike reversible inhibitors of P450, drug-drug interactions originating from MBI may persist in patients for some time after the body eliminates the offending drug because P450 enzymatic activity can be recovered only after de novo synthesis of the P450. In a pharmaceutical setting, a substantial amount of effort is often expended to understand the potential for mechanism-based inactivation and its possible contribution to the drug-drug interaction pro file of drug candidates. Therefore, in vitro assays that identify and characterize which drug candidates are P450 MBIs are critically important in preclinical drug metabolism and pharmacokinetic studies. A detailed method is described for the adaptation of a 7-ethoxytri fluoromethyl coumarin O -deethylation fluorescence activity assay to a 96-well plate format to characterize the K I and k inact values for an MBI. The advantages of this microtiter format compared with the conventional method include a signi ficant reduction in the amount of enzyme used, a reduction in assay time, and an increase in experimental throughput. © Springer Science+Business Media New York 2013.