Multisite kinetic models for CYP3A4: Simultaneous activation and inhibition of diazepam and testosterone metabolism

Some substrates of cytochrome P450 (CYP) 3A4, the most abundant CYP in the human liver responsible for the metabolism of many structurally diverse the rapeutic agents, do not obey classical Michaelis-Menten kinetics and demons trate homotropic and/or heterotropic cooperativity. The unusual kinetics an d differential effects observed between substrates of this enzyme confound the prediction of drug clearance and drug-drug interactions from in vitro d ata. We have investigated the hypothesis that CYP3A4 may bind multiple mole cules simultaneously using diazepam (DZ) and testosterone (TS). Both substr ates showed sigmoidal kinetics in B-lymphoblastoid microsomes containing a recombinant human CYP3A4 and reductase. When analyzed in combination, TS ac tivated the formation of 3-hydroxydiazepam (3HDZ) and N-desmethyldiazepam ( NDZ) (maximal activation 374 and 205%, respectively). For 3HDZ, V-max value s remained constant with increasing TS, whereas the S50 and Hill values dec reased, tending to make the data less sigmoidal. Similar trends were observ ed for the NDZ pathway. DZ inhibited the formation 6 beta -hydroxytestoster one (maximal inhibition, 45% of control), causing a decrease in V-max but n o significant change to the S50 and Hill values, suggesting that DZ may inh ibit via a separate effector site. Multisite rate equation models have been derived to explore the analysis of such complex kinetic data and to allow accurate determination of the kinetic parameters for activation and inhibit ion. The data and models presented are consistent with proposals that CYP3A 4 can bind and metabolize multiple substrate molecules simultaneously; they also provide a generic solution for the interpretation of the complex kine tic data derived from CYP3A4 substrates.