PT  - JOURNAL ARTICLE
AU  - Obach, R. Scott
AU  - Reed-Hagen, Anne E.
TI  - Measurement of Michaelis Constants for Cytochrome P450-Mediated Biotransformation Reactions Using a Substrate Depletion Approach
AID  - 10.1124/dmd.30.7.831
DP  - 2002 Jul 01
TA  - Drug Metabolism and Disposition
PG  - 831--837
VI  - 30
IP  - 7
4099  - http://dmd.aspetjournals.org/content/30/7/831.short
4100  - http://dmd.aspetjournals.org/content/30/7/831.full
SO  - Drug Metab Dispos2002 Jul 01; 30
AB  - 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 vitrot1/2” method) at multiple substrate concentrations, with the objective of comparingKM values determined by this approach withKM 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.KM 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, KM values determined by the substrate depletion approach were within 1.54-fold of those determined by measuring product formation. Thus, KMvalues 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 t1/2approach 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. The American Society for Pharmacology and Experimental Therapeutics