METABOLISM AND BIOACTIVATION OF CLOZAPINE BY HUMAN LIVER IN-VITRO

The metabolism of clozapine by human liver has been investigated in vi tro. Irreversible protein-binding and conjugation with model nucleophi les have been used as markers for bioactivation of clozapine, white st able metabolite formation has been assessed using radiometric HPLC. In all nine liver microsomal preparations investigated, clozapine was ex tensively metabolized to the stable products desmethylclozapine (range 19%-27.2%), N-oxide (1.5-20.5%) and three polar metabolites (0-20.8%) , and was bioactivated to a protein-reactive metabolite (0.6-2.1%). Th e CYP2D6 genotype did not influence the capacity of the livers to form these metabolites. All metabolic pathways were inhibited by ketoconaz ole, indicating the involvement of the cytochrome P450 enzymes, Isozym e-selective inhibitor studies demonstrated that whereas demethylation was performed by CYP1A2, N-oxidation and chemically reactive metabolit e formation were dependent upon multiple forms of P450. The N-oxide wa s readily reduced back to clozapine in the presence of NADPH, this con version being inhibited by ascorbic acid. Glutathione (1 mM) decreased covalent binding by 70%. The amount of putative adduct formed in the presence of glutathione (13.4 +/- 0.9%) was much greater than the cova lent binding (mean 1.1 +/- 0.2%). The bioactivation of clozapine was, like the N-oxidation of clozapine, a reversible process. In summary, o ur results indicate clozapine undergoes extensive metabolism by human liver to both stable and chemically reactive metabolites, the formatio n of which is catalyzed by the cytochrome P450 enzymes, The role of th e reactive metabolite, which may be a free radical, in the pathogenesi s of clozapine agranulocytosis and hepatotoxicity requires further stu dy.