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 therapeutic agents, do not obey classical Michaelis-Menten kinetics and demonstrate homotropic and/or heterotropic cooperativity. The unusual kinetics and differential effects observed between substrates of this enzyme confound the prediction of drug clearance and drug-drug interactions from in vitro data. We have investigated the hypothesis that CYP3A4 may bind multiple molecules simultaneously using diazepam (DZ) and testosterone (TS). Both substrates showed sigmoidal kinetics in B-lymphoblastoid microsomes containing a recombinant human CYP3A4 and reductase. When analyzed in combination, TS activated the formation of 3-hydroxydiazepam (3HDZ) and N-desmethyldiazepam (NDZ) (maximal activation 374 and 205%, respectively). For 3HDZ, V(max) values remained constant with increasing TS, whereas the S(50) and Hill values decreased, tending to make the data less sigmoidal. Similar trends were observed for the NDZ pathway. DZ inhibited the formation 6beta-hydroxytestosterone (maximal inhibition, 45% of control), causing a decrease in V(max) but no significant change to the S(50) and Hill values, suggesting that DZ may inhibit 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 inhibition. The data and models presented are consistent with proposals that CYP3A4 can bind and metabolize multiple substrate molecules simultaneously; they also provide a generic solution for the interpretation of the complex kinetic data derived from CYP3A4 substrates.
Kenworthy
K E
KE
Department of Mechanism and Extrapolation Technologies, GlaxoSmithKline, The Frythe, Welwyn, Herts, United Kingdom.
Clarke
S E
SE
Andrews
J
J
Houston
J B
JB
D000465:Algorithms; D014151:Anti-Anxiety Agents / Q000493:pharmacokinetics*; D001665:Binding Sites; D002460:Cell Line; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003975:Diazepam / Q000493:pharmacokinetics*; D004347:Drug Interactions; D006801:Humans; D007945:Leukemia, Lymphoid / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008954:Models, Biological; D013379:Substrate Specificity; D013739:Testosterone / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured
D014151:Anti-Anxiety Agents; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003975:Diazepam