Characterization of the human hepatic cytochromes P450 involved in the in vitro oxidation of clozapine

Abstract

It was aimed to identify the cytochrome(s) P450 (CYPs) involved in the N-demethylation and N-oxidation of clozapine (CLZ) by various approaches using human liver microsomes or microsomes from human B-lymphoblastoid cell lines. The maximum rates of formation were measured in the microsomal fraction of human livers and the Michaelis–Menten kinetics one enzyme model was found to best fit the data with mean K_M for CLZ N-oxide and N-desmethyl-CLZ of 336 and 120 μM, respectively. Significant correlations were observed between the maximum rates of formation (V_max) for CLZ N-oxide and N-desmethyl-CLZ with the microsomal immunoreactive contents of CYP1A2 (r=0.92, P<0.009 and r=0.77, P<0.077; respectively) and CYP3A (r=0.89, P<0.02 and r=0.82, P<0.05; respectively). Antibodies directed against CYP1A2 and CYP3A inhibited formation of CLZ N-oxide in human liver microsomes by 10.7±6.1% and 37.2±6.9% of control, respectively, whereas CLZ N-demethylation was inhibited by 32.2±15.4% and 33.6±7.4%, respectively. Troleandomycin (CYP3A inhibitor) and furafylline (CYP1A2 inhibitor) inhibited CLZ N-oxidation in human liver microsomes by 23.2±12.1% and 7.8±4.3%, respectively, whereas CLZ N-demethylation was inhibited by 17.5±13.9% and 25.6±16.5%, respectively. While ketoconazole did not inhibit N-oxidation of CLZ, the N-demethylation pathway was inhibited by 34.1±10.0%. Formation in stable expressed enzymes indicated involvement of CYP3A and CYP1A2 in CLZ N-oxide formation and CYP2D6, CYP1A2 and CYP3A4 in CLZ N-demethylation. This apparent involvement of CYP2D6 in the N-demethylation of CLZ did not corroborate with the findings of other experiments. In conclusion, these data indicate that while both CYP isoforms readily catalyze both metabolic routes in vitro, CYP1A2 and CYP3A4 are more important in N-demethylation and N-oxidation, respectively.

Introduction

Clozapine (CLZ) lacks the extrapyramidal side effects of conventional antipsychotic agents and, therefore has been described as ‘atypical’. Although efficacious in patients resistant to, or intolerant of, conventional neuroleptic treatment [1], the clinical use of CLZ in psychiatry is restricted by the associated risk of agranulocytosis [2], the etiology of which remains unknown. Two mechanisms; immunological and non-immunological have been proposed [3] and further ongoing research has demonstrated that the conversion of CLZ to a chemically reactive species could interfere with the essential cellular processes [4], [5], [6], [7]. CLZ undergoes extensive hepatic metabolism in humans [8], [9], [10], in part via oxidation of the tertiary amine N-methyl group to produce the major metabolites CLZ N-oxide and N-desmethyl-CLZ [11] (Fig. 1). Many other metabolites resulting from phase I and phase II reactions have been identified in human and rat [10], [12], [13], [14].

The involvement of the various cytochromes P450 (CYPs) in the metabolism of CLZ requires clarification. Studies involving recombinant cells [15] indicated the involvement of CYP2D6 in CLZ metabolism, while more recent studies [13], [16] implicated the involvement of CYP1A2, CYP3A4, CYP2E1 and CYP2C9/10 in the metabolism of CLZ to both the N-oxide and N-desmethyl metabolites. Recent studies by Tugnait et al. [17] have demonstrated the involvement of the flavin-containing monooxygenase (FMO), specifically FMO3, in the N-oxygenation of CLZ in vitro. These studies included demonstration for a human liver microsomal preparation that N-oxidation was markedly reduced by methimazole, an alternate-substrate competitive inhibitor of FMO, and heat-mediated inactivation that involved preincubation at 45°C for 5 min in the absence of NADPH. In vivo, in human the disposition of CLZ is not associated with debrisoquine and S-mephenytoin hydroxylation polymorphisms [18], [19], indicating at most minor involvement of CYP2D6 and CYP2C19, respectively. On the other hand the major involvement of CYP1A2 in the metabolism of CLZ has been indicated by two different in vivo studies. Fluvoxamine a potent inhibitor of CYP1A2 [20], affected marked increase in CLZ plasma concentrations [21], [22] and a good correlation was found between CLZ metabolism in vivo and CYP1A2 activity as determined by the caffeine test [23]. Also the enhanced clearance of CLZ in the presence of carbamazepine [22], [24] indicated involvement of CYP3A4 in CLZ metabolism. The objective of the current investigation was to examine the involvement of CYP in the metabolism of CLZ in vitro with particular reference to the N-oxidation and N-demethylation metabolic pathways. The isozymes involved in the biotransformation of CLZ were identified and characterized using an experimental strategy combining:

• Determination of formation rates of CLZ N-oxide and N-desmethyl-CLZ in incubations with human liver microsomes characterized for the presence of various CYPs and determination of Michaelis–Menten parameters of enzyme kinetics followed by regression analysis.

• Inhibition of metabolite formation by specific antibodies and isoform selective chemical inhibitors.

• Formation of metabolites in microsomes from cell lines that were genetically engineered for stable expression of the individual CYPs.