CYP-catalysed cycling of clozapine and clozapine-N-oxide promotes the generation of reactive oxygen species in vitro

Abstract

Clozapine is an effective atypical antipsychotic indicated for treatment-resistant schizophrenia, but is under-prescribed due to the risk of severe adverse drug reactions such as myocarditis.A mechanistic understanding of clozapine cardiotoxicity remains elusive.This study aimed to investigate the contribution of selected CYP isoforms to cycling between clozapine and its major circulating metabolites, <i>N</i>-desmethylclozapine and clozapine-<i>N</i>-oxide, with the potential for reactive species production.CYP supersome™-based <i>in vitro</i> techniques were utilised to quantify specific enzyme activity associated with clozapine, clozapine-<i>N</i>-oxide and <i>N</i>-desmethylclozapine metabolism.The formation of reactive species within each incubation were quantified, and known intermediates detected.CYP3A4 predominately catalysed clozapine-<i>N</i>-oxide formation from clozapine and was associated with concentration-dependent reactive species production, whereas isoforms favouring the <i>N</i>-desmethylclozapine pathway (CYP2C19 and CYP1A2) did not produce reactive species.Extrahepatic isoforms CYP2J2 and CYP1B1 were also associated with the formation of clozapine-<i>N</i>-oxide and <i>N</i>-desmethylclozapine but did not favour one metabolic pathway over another.Unique to this investigation is that various CYP isoforms catalyse clozapine<i>-N</i>-oxide reduction to clozapine.This process was associated with the concentration-dependent formation of reactive species with CYP3A4, CYP1B1 and CYP1A1 that did not correlate with known reactive intermediates, implicating metabolite cycling and reactive oxygen species in the mechanism of clozapine-induced toxicity.