Mechanism-based inactivation of CYP3A by HIV protease inhibitors.
Human immunodeficiency virus (HIV) protease inhibitors (PIs) are inhibitors of CYP3A enzymes, but the mechanism is poorly defined. In this study, time- and concentration-dependent decreases in activity as defined by maximum rate of inactivation (k(inact)) and inhibitor concentration that gives 50% maximal inactivation (K(I)) of CYP3A by amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir were quantified using testosterone 6beta-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(+b(5)), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(+b(5)) and HLMs. Ritonavir was the most potent (K(I) = 0.10 and 0.17 microM) and demonstrated high k(inact) values (0.32 and 0.40 min(-1)) with both CYP3A4(+b(5)) and HLMs. Ritonavir was not significantly depleted by high-affinity binding with CYP3A4(+b(5)) and confirmed that estimation of reversible inhibition was confounded with irreversible inhibition. For CYP3A5, nelfinavir exhibited the highest k(inact) (0.47 min(-1)), but ritonavir was the most potent (K(I) = 0.12 microM). Saquinavir and indinavir did not show time- and concentration-dependent decreases in activity with CYP3A5. Spectrophototmetrically determined metabolic intermediate complex formation was observed for all of the PIs with CYP3A4(+b(5)), except for lopinavir and saquinavir. The addition of nucleophilic and free aldehyde trapping agents and free iron and reactive oxygen species scavengers did not prevent inactivation of CYP3A4(+b(5)) by ritonavir, amprenavir, or nelfinavir, but glutathione decreased the inactivation by saquinavir (17%) and catalase decreased the inactivation by lopinavir (39%). In conclusion, all the PIs exhibited mechanism-based inactivation, and predictions of the extent and time course of drug interactions with PIs could be underestimated if based solely on reversible inhibition.
Ernest
C Steven
CS
Division of Clinical Pharmacology, Wishard Memorial Hospital, 1001 W. 10th St., WD Myers Bldg., W7123, Indianapolis, IN 46202, USA.
Hall
Stephen D
SD
Jones
David R
DR
D000447:Aldehydes / Q000737:chemistry; D000465:Algorithms; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D051544:Cytochrome P-450 CYP3A; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors*; D017382:Reactive Oxygen Species; D013056:Spectrophotometry, Ultraviolet
D000447:Aldehydes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D017320:HIV Protease Inhibitors; D017382:Reactive Oxygen Species; D007501:Iron; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating