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-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 microM, respectively. Significant correlations were observed between the maximum rates of formation (Vmax) 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.
Tugnait
M
M
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
Hawes
E M
EM
McKay
G
G
Eichelbaum
M
M
Midha
K K
KK
D000906:Antibodies / Q000494:pharmacology; D014150:Antipsychotic Agents / Q000378:metabolism; D002460:Cell Line; D003024:Clozapine / Q000031:analogs & derivatives / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015394:Molecular Structure
D000906:Antibodies; D014150:Antipsychotic Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; C058272:norclozapine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003024:Clozapine; C079149:clozapine N-oxide