TABLE 7 

Enzyme-selective inhibitors reactions used in enzymology studies in human hepatocytes to determine relative contributions of human P450 enzymes to the rates of MMB metabolism and M21 formation (mean ± S.D., n = 6–8)

No turnover was observed for MMB when incubated with CYP2B6, 2D6, 2E1, or 3A5. In parallel incubations, ABT (1 mM) reduced midazolam oxidation by 95%, and hydralazine (50 μM) reduced zoniporide oxidation by 87%, confirming the effectiveness of the inhibitors. Addition of ABT (1 mM) greatly attenuated the loss of MMB, with an 87% reduction in the rate of loss. Hydralazine (50 μM) had little effect on MMB metabolism (rate reduced by 13%). ABT and hydralazine reduced the rate of M21 formation by 95% and 94%, respectively, indicating that both P450 and AOX enzymes were necessary for its formation. Simultaneous addition of ABT and hydralazine yielded a result similar to that observed with ABT alone. Additionally, the combination of furafylline, montelukast, sulfaphenazole, benzylnirvanol, and ritonavir resulted in 84% inhibition, which is close to the effect observed with ABT, indicating that the majority of P450 enzymes generating M21 had been identified. Fraction of metabolism was calculated by normalizing each percent inhibition to the total reduction in rate for all inhibitors exhibiting an inhibitory effect.