Role of Human Glutathione S-Transferases in the Inactivation of Reactive Metabolites of Clozapine

Abstract

The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.

Role of Residue 87 in the Activity and Regioselectivity of Clozapine Metabolism by Drug-Metabolizing CYP102A1 M11H: Application for Structural Characterization of Clozapine GSH Conjugates

“…Three of the four tested hGSTs showed strongly increased total GSH conjugation and also resulted in formation of different regioisomeric GSH conjugates of CLZ ( Fig. 1) (Dragovic et al, 2010).…”

“…However, we recently demonstrated that these GSH conjugates, CG-5 and CG-6 in Fig. 1, are formed at high levels when CLZ incubations with purified CYP102A1 M11H and human liver microsomes were supplemented with human glutathione transferases (hGSTs) (Dragovic et al, 2010). Three of the four tested hGSTs showed strongly increased total GSH conjugation and also resulted in formation of different regioisomeric GSH conjugates of CLZ ( Fig.…”

“…Recently, the highly active drug-metabolizing mutant CYP102A1 M11H (cytochrome P450 102A1 mutant M11 His-tagged) was used to investigate the role of human glutathione transferase (GST) in the inactivation of CLZ (Dragovic et al, 2010). CLZ is an atypical antipsychotic drug showing a low incidence of extrapyramidal side effects combined with excellent antipsychotic efficacy in schizophrenic and manic treatment-resistant patients (Safferman et al, 1991;Buchanan, 1995;Wagstaff and Perry, 2003).…”

“…All conjugates can be explained by direct conjugation of GSH at different positions of a reactive nitrenium ion and by chlorosubstitution of the nitrenium ion followed by reduction (see Fig. 1) (Maggs et al, 1995;Dragovic et al, 2010). Unequivocal structure determination by 1 H NMR has been published for only two of the GSH conjugates of CLZ (Fischer et al, 1991;Liu and Uetrecht, 1995;Madsen et al, 2007).…”

“…The aim of the present study was to identify the structures of these GSH conjugates by 1 H NMR by performing large-scale incubations of CLZ with selective CYP102A1 M11H mutants in the presence of glutathione transferase. Because GST P1-1 appeared to be the most active hGST in the formation of enzyme-dependent GSH conjugates (Dragovic et al, 2010), this enzyme was selected for largescale production of GSH conjugates.…”