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

It was aimed to identify the cytochrome(s) P450 (CYPs) involved in the N-de methylation 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 o f 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 mu M, respectively. Significant correlations were observed betw een 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 an d 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-demethy lation was inhibited by 32.2 +/- 15.4% and 33.6 +/- 7.4%, respectively. Tro leandomycin (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-ox idation 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 CYP7D6, CYP1A2 and CYP3A4 in CLZ N-deme thylation. This apparent involvement of CYP7D6 in the N-demethylation of CL Z did not corroborate with the findings of other experiments. In conclusion , these data indicate that while both CYP isoforms readily catalyze both me tabolic routes in vitro, CYP1A2 and CYP3A4 are more important in N-demethyl ation and N-oxidation, respectively. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.