Evaluation of In Vitro Cytochrome P450 Inhibition and In Vitro Fate of Structurally Diverse N-Oxide Metabolites: Case Studies with Clozapine, Levofloxacin, Roflumilast, Voriconazole and Zopiclone.

Abstract

The role of metabolite(s) to elicit potential clinical drug-drug interaction (DDI) via cytochrome P450 enzymes (CYP) is gaining momentum. In this context, the role of N-oxides for in vitro CYP inhibition has not been evaluated. The objectives of this study were: (a) to examine in vitro CYP inhibition of N-oxides of clozapine, levofloxacin, roflumilast, voriconazole and zopiclone in a tiered approach and (b) evaluate in vitro fate of aforementioned N-oxides examined in recombinant CYPs, human microsomes and hepatocytes. CYP enzymes evaluated in the work included: CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 using standard procedures for incubation with appropriate probe substrates. The initial cutoff for CYP inhibition was ≥50% using 2 and 10µM concentrations of various N-oxide metabolites (Tier 1). IC50 values were constructed for the CYP pathway(s) that showed ≥50% inhibition (Tier 2). In addition, co-incubation of N-oxides with parent was performed to evaluate potentiation of CYP inhibition (Tier 3). N-oxides of clozapine (CYP2B6/2C19) and voriconazole (CYP2C9/3A4) showed CYP inhibition ≥50%. Clozapine-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 8.3 and 8.7µM, respectively. Voriconazole-N-oxide inhibited CYP2B6 and CYP2C19 pathways with IC50 of 10.5 and 11.2µM, respectively. Co-incubation of clozapine-N-oxide with clozapine potentiated CYP2B6/2C19 pathways; however, incubation of voriconazole-N-oxide with voriconazole did not appear to potentiate the CYP pathways because parent caused an inhibition of almost 80%. None of the N-oxides appeared to further undergo biotransformation as judged by the in vitro metabolic fate experiments (stage 2). Clinical DDI potential of specific CYP enzymes needs to be considered arising due to circulatory concentrations of certain N-oxides depending on the dose size and/or frequency of dosing of the respective parent drugs.