Mechanism-Based Inactivation of CYP3A by HIV Protease Inhibitors

Abstract

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 6␤-hydroxylation as a marker for CYP3A activity with recombinant CYP3A4(ϩb 5 ), recombinant CYP3A5, and pooled human liver microsomes (HLMs). All the PIs, except indinavir, displayed inactivation with CYP3A4(ϩb 5 ) and HLMs. Ritonavir was the most potent (K I ϭ 0.10 and 0.17 M) and demonstrated high k inact values (0.32 and 0.40 min Ϫ1 ) with both CYP3A4(ϩb 5 ) and HLMs. Ritonavir was not significantly depleted by high-affinity binding with CYP3A4(ϩb 5 ) and confirmed that estimation of reversible inhibition was confounded with irreversible inhibition. For CYP3A5, nelfinavir exhibited the highest k inact (0.47 min Ϫ1 ), but ritonavir was the most potent (K I ϭ 0.12 M). 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(ϩb 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(ϩb 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.

Structure and mechanism of the complex between cytochrome P4503A4 and ritonavir

“…The K s values obtained in this study agree well with the inhibition constant (K i ) of ritonavir for the CYP3A4-dependent metabolism of methadone, buprenorphine, and testosterone in human liver microsomes (20-50 nM (12, 13)). For other reactions catalyzed by microsomal CYP3A4, the K i for ritonavir varies from 0.10 to 0.38 μM (11,14,18).…”

“…The reactive intermediate(s) was proposed to involve the isopropyl-thiazole end-group (7), strictly required for potent CYP3A4 inhibition (9). Further, a noncovalent inhibitory complex, also known as a metabolic intermediate complex (MIC) (10), was reported to form during incubation of ritonavir with insect microsomes containing recombinantly expressed human CYP3A4 and cytochrome b 5 (11). Other studies indicated, however, that ritonavir acts as a competitive (12) or mixed competitive-noncompetitive CYP3A4 inactivator (5,13,14).…”

Effect of ritonavir on the pharmacokinetics of the benzimidazoles albendazole and mebendazole: an interaction study in healthy volunteers

“…The protease inhibitor ritonavir is among the most potent inhibitors of the CYP3A system [13]. Consequently, interaction of protease inhibitors with drugs that are cleared predominantly by CYP3A enzymes are profound and clinically significant [14].…”

Precursors for cytochrome P450 profiling breath tests from an in silico screening approach

“…CYP3A4 is most probably the one responsible enzyme in the degradation of indinavir as reviewed by Lin [57]. An aspect hampering the applicability of indinavir as precursor for a CYP3A4-specific breath test is the mechanism-based inhibition of CYP3A4 by indinavir [58]. Thus, in presence of indinavir the activity profiling for CYP3A4 is self-limited as indinavir actively decreases the CYP3A4 activity.…”