Differences in the inhibition of cytochromes P450 3A4 and 3A5 by metabolite-inhibitor complex-forming drugs

The objectives of this study were to characterize and compare the reversible inhibition and time-dependent inactivation of cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) by erythromycin, diltiazem, and nicardipine. In the following experiments, we used cDNA-expressed CYP3A Supersomes and CYP3A-phenotyped human liver microsomes. We estimated the apparent constants for reversible inhibition (K-i(app) and IC50) and the irreversible kinetic constants (K-I and k(inact)) for time-dependent inhibition. Based on an aggregate of K-i(app) and IC50 measurements, all inhibitors showed a greater inhibitory potency for CYP3A4 compared with CYP3A5. In addition, for each inhibitor, the k(inact) for CYP3A4 was approximately 4-fold higher than that for CYP3A5, indicating a greater propensity for time-dependent loss of CYP3A4 activity than of CYP3A5. Difference spectra experiments revealed an NADPH-dependent peak at similar to455 nm [metabolite-inhibitor (MI) complex] following incubation of all three drugs with CYP3A4. There was no discernable MI complex formation following CYP3A5 incubation with any of the inhibitors. However, when CYP3A4 and CYP3A5 were incubated simultaneously with erythromycin, both enzymes appeared to contribute to the formation of a MI complex. Additional experiments revealed that erythromycin caused a comparable type I spectral change when bound to CYP3A5 and CYP3A4 (K-s=48 muM and 52 muM, respectively). Moreover, CYP3A5 exhibited only a moderately slower rate for the initial N-demethylation than did CYP3A4 (intrinsic clearance=41 versus 99 mul/min/nmol, respectively). In conclusion, erythromycin, diltiazem, and nicardipine were weaker inhibitors of CYP3A5 and inactivated the enzyme at a slower rate than their respective effects on CYP3A4. With respect to erythromycin, the failure of CYP3A5 to form a MI complex appears to be the result of slowed or impaired metabolic events downstream from the initial catalytic step, possibly due to a different orientation of the substrate molecule in the active site.