Abstract
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.
Footnotes
- Abbreviations used are::
- P450
- cytochrome P450
- HPLC-MS
- high-performance liquid chromatography-mass spectometry
- MTBE
- methyl tertiary butyl ether
- kdep
- depletion rate constant
- [S]
- substrate concentration
- Received February 21, 2002.
- Accepted April 11, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|