Measurement of Michaelis constants for cytochrome P450-mediated biotransformation reactions using a substrate depletion approach.

The Michaelis constant (KM) 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. KM 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 vitro t1/2" method) at multiple substrate concentrations, with the objective of comparing KM values determined by this approach with KM 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 dextromethorphan O-demethylation and thioridazine S-oxidation, CYP2C19-catalyzed imipramine N-demethylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, and CYP1A2-catalyzed tacrine 1-hydroxylation. KM values spanned an 80-fold range from 0.12 microM (CYP2D6-catalyzed thioridazine S-oxidation) to 9.8 microM (CYP2C19-catalyzed imipramine N-demethylation). On average, KM values determined by the substrate depletion approach were within 1.54-fold of those determined by measuring product formation. Thus, KM values 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 t1/2 approach of determining KM values should be useful in early drug discovery efforts to identify those compounds with low KM values and, hence, a greater probability of exhibiting supraproportional dose-exposure relationships.