Abstract

The Michaelis constant (K_M) for cytochrome P450-mediated drug biotransformation reactions can be an important parameter in understanding the potential for a drug to exhibit saturable metabolism in vivo and nonlinear dose-exposure relationships. K_M values were measured for several drug biotransformation reactions using recombinant heterologously expressed human enzymes. These determinations were made using an approach of monitoring substrate loss (“in vitrot_1/2” method) at multiple substrate concentrations, with the objective of comparingK_M values determined by this approach withK_M values determined using the conventional approach of measuring product formation rates at several substrate concentrations. The reactions examined were CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2D6-catalyzed dextromethorphanO-demethylation and thioridazineS-oxidation, CYP2C19-catalyzed imipramineN-demethylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, and CYP1A2-catalyzed tacrine 1-hydroxylation.K_M values spanned an 80-fold range from 0.12 μM (CYP2D6-catalyzed thioridazine S-oxidation) to 9.8 μM (CYP2C19-catalyzed imipramine N-demethylation). On average, K_M values determined by the substrate depletion approach were within 1.54-fold of those determined by measuring product formation. Thus, K_Mvalues can be determined for drug metabolism reactions without requiring knowledge of metabolite structures or requiring authentic standards of metabolites for use in construction of standard curves for quantitative bioanalysis. The in vitro t_1/2approach of determining K_M values should be useful in early drug discovery efforts to identify those compounds with low K_M values and, hence, a greater probability of exhibiting supraproportional dose-exposure relationships.