The Effect of Ritonavir on Human CYP2B6 Catalytic Activity: Heme Modification Contributes to the Mechanism-Based Inactivation of CYP2B6 and CYP3A4 by Ritonavir

Abstract

The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a K I of 0.9 mM, a k inact of 0.05 min 21, and a partition ratio of approximately 3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the protonated molecular ion of RTV exhibits an m/z at 721 and its two major metabolites are an oxidation product with MH + at m/z 737 and a deacylated product with MH + at m/z 580. Inactivation of CYP2B6 by incubation with 10 mM RTV for 10 min resulted in an approximately 50% loss of catalytic activity and native heme, but no modification of the apoprotein was observed. RTV was found to be a potent mixed-type reversible inhibitor (K i = 0.33 mM) and a type II ligand (spectral dissociation constant-K s = 0.85 mM) of CYP2B6. Although previous studies have demonstrated that RTV is a potent mechanism-based inactivator of CYP3A4, the molecular mechanism responsible for the inactivation has not been determined. Here, we provide evidence that RTV inactivation of CYP3A4 is due to heme destruction with the formation of a heme-protein adduct. Similar to CYP2B6, there is no significant modification of the apoprotein. Furthermore, LC-MS/MS analysis revealed that both CYP3A4 and human liver microsomes form an RTV-glutathione conjugate having a MH + at m/z 858 during metabolism of RTV, suggesting the formation of an isocyanate intermediate leading to formation of the conjugate.

Characterization of Ritonavir-Mediated Inactivation of Cytochrome P450 3A4

“…The integrity of the CYP3A4 heme chromophore after incubations with ritonavir suggest that inactivation occurs through a mechanism independent of heme destruction. This is contrary to the recent article by Lin et al, (2013) where the authors report finding that ritonavir inactivation of CYP3A4 was attributable to heme destruction with the formation of a heme-protein adduct, and no significant modification of the apoprotein. The report of Inactivation of CYP3A4 by Ritonavir heme destruction and a heme-protein adduct were performed with reconstituted CYP3A4 and P450 reductase, in comparison with the studies performed here in both CYP3A4 Supersomes and reconstituted 3A4 enzyme with reductase and cytochrome b 5 .…”

“…Ritonavir is also reported to be a tight, irreversible binding inhibitor of CYP3A4 via coordination to the heme iron (Sevrioukova and Poulos, 2010). More recently, ritonavir has been reported to cause heme destruction in a reconstituted CYP3A4 system (Lin et al, 2013). Together, these reports suggest that multiple mechanisms of bioactivation and inhibition may occur with ritonavir and CYP3A4.…”

Nanoflow-Nanospray Mass Spectrometry Metabolomics Reveals Disruption of the Urinary Metabolite Profiles of HIV-Positive Patients on Combination Antiretroviral Therapy

“…19,20 In agreement with previous in vitro studies two metabolites of ritonavir were detected in urine extracts (the oxidised and deacylated products), however the glutathione conjugate was not detected and this metabolite may have preferentially been excreted by the biliary route. 15 The detection of a number of metabolites of atazanavir and darunavir in this study agreed with previous work showing that these pharmaceuticals are extensively metabolised in humans via oxidation, carbamate hydrolysis, deacylation (atazanavir) and glucuronidation (darunavir). 16,17 The ability to detect the parent cART drugs and a wide range of their transformation products in urine extracts suggest that in future studies, links between pharmacogenetics, metabolism and drug efficacy could be investigated, which may lead to a more personalised approach to cART intervention.…”

“…Identification of the metabolites was supported by fragmentation patterns which were compared to those of metabolite structures that had already been detected in rodent urine, human blood and hepatocyte studies. [15][16][17][18] Analysis of the OPLS-DA loadings plot for each cART parent compound confirmed that all but one urine sample contained cART drugs at the time of sample donation. As none of the parent drugs nor any of their metabolites were detected in urine extracts from this patient in the darunavir group, this sample was excluded from further statistical analyses.…”

Cytochrome P4503A Does Not Mediate the Interaction between Methadone and Ritonavir-Lopinavir

“…This may relate to effects of ritonavir or lopinavir on nuclear receptors and their regulation of CYPs 2B6 and 3A, and P-gp (Svärd et al, 2010). Relationships of these clinical observations to mechanism-based inactivation of CYPs by ritonavir are presently unknown (Lin et al, 2013).…”