Enzyme kinetics

Enzyme phenotyping provides information on the enzymes involved in the metabolism of a new chemical entity, which is needed to evaluate the drug interaction vulnerability of the compound as a “victim-drug”. The phenotyping of metabolizing enzymes is also needed to evaluate the risk for highly variable clearance between individuals or populations due to the involvement of enzymes with high degree of polymorphism. A thorough evaluation of metabolizing enzymes also requires metabolite identification. The most common approach for enzyme identification is the use of recombinant enzymes and extrapolation of the results based on the in vivo protein levels, and the use of liver microsomal incubations together with cytochrome P450 (CYP)-selective inhibitors under linear metabolite formation kinetics. In both methods, monitoring of metabolite formation is clearly more reliable than monitoring substrate depletion only, although the latter is often used for high throughput evaluation. The clever use of HR-MS technology allows metabolite monitoring later on from the very same LC/MS data, even if only substrate depletion was analyzed in the first place. If CYP-focused enzyme phenotyping assays do not show clear results, non-CYP mediated metabolism should be studied and the enzymes involved in the metabolic processes identified. This can be done by using recombinant enzymes, such as UDP glucuronosyltransferases (UGT), sulfotransferases (SULT), N-acetyltransferases (NAT), flavin containing mono-oxygenases (FMO), monoamine-oxidases (MAO), carboxylesterases (CE), or aldehyde dehydrogenase (ALDH). Metabolism via aldehyde oxidase (AOX), xanthine oxidase (XO), aldo-keto reductase (AKR) and carbonyl reductase (CR) can be investigated by using human liver cytosol with enzyme-selective inhibitors. For more thorough evaluation of in vivo role of each enzyme, evaluation of kinetic parameters Vmax and Km with different enzymes would be advisable.