Elucidation of individual cytochrome P450 enzymes involved in the metabolism of clozapine

Abstract

The atypical antipsychotic clozapine has been reported to be metabolised mainly to its N-oxide and N-demethylated products. In the present study, individual recombinant cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) enzymes were used to elucidate which enzymes are responsible for these metabolic conversions. In vitro metabolism of clozapine was investigated using human CYP1A1, CYP1A2, CYP2C8, CYP2E1, CYP2C9-arg144, CYP2C9-cys144, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and FMO3 supplemented with an NADPH generating system. Clozapine and its N-oxidation and N-demethylation metabolites were determined by an HPLC method with a Hypersil CN column and a UV detector. Of the enzymes investigated, CYP1A2, CYP3A4, CYP2D6, CYP2C8, CYP2C19 and, to a lesser extent, CYP2C9-cys, CYP2C9-arg and CYP3A5 were apparently involved in N-demethylation, while CYP1A2, CYP3A4, FMO3 and, to a lesser extent, CYP2C8, CYP2C19 and CYP3A5 were found to catalyse the formation of clozapine N-oxide. A bank of 16 human liver microsome preparations was investigated for ability to catalyze the production of clozapine N-oxide and N-desmethylclozapine. Attempts were made to correlate the rates of formation of these metabolites of clozapine to previously determined catalytic activities of CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. At a clozapine concentration of 20 microM, the rate of formation of clozapine N-oxide showed significant correlations with activities of CYP3A4 (P<0.01) and CYP1A2 (P<0.05). The formation of N-desmethylclozapine exhibited significant correlations with CYP1A2 (P<0.01) and CYP3A4 (P<0.01). Similar correlations were observed when the clozapine concentration was increased to 300 microM except that the formation of clozapine N-oxide no longer correlated with CYP1A2 activity. It can be seen from these results that although some recombinant enzymes individually are capable of metabolising clozapine, the activities of several of these enzymes did not correlate with clozapine metabolism when mixtures of the enzymes are used. By combining the results of the current study and those reported in the literature, it is proposed that CYP3A4 and FMO3 are primarily responsible for the production of clozapine N-oxide, and CYP3A4 and CYP1A2 are primarily responsible for the formation of N-desmethylclozapine. The present study demonstrates the importance of the use of multiple techniques for the elucidation of the enzymes involved in the metabolism of certain drugs.

Characterization of Human Cytochrome P450s Involved in the Bioactivation of Clozapine

“…Incubations with recombinant human P450 were performed at CLZ concentrations of 10 and 100 mM, as was done previously Eiermann et al, 1997;Linnet and Olesen, 1997;Fang et al, 1998;Tugnait et al, 1999;Olesen and Linnet, 2001;Zhang et al, 2008;Dragovic et al, 2010). Duplicate incubations were performed in 100 mM potassium phosphate buffer (pH 7.4) at a final volume of 200 ml.…”

“…CYP1A2) and N-oxidation (CYP3A4 . CYP1A2, CYP2D6) of CLZ, whereas the other P450s showed only low or no activity Eiermann et al, 1997;Linnet and Olesen, 1997;Fang et al, 1998;Tugnait et al, 1999;Olesen and Linnet, 2001;Zhang et al, 2008). Figure 4 shows the relative activities of individual recombinant human P450s in bioactivation of CLZ using GSH and potassium cyanide as trapping agents.…”

“…Several studies have been performed to identify the role of individual P450s in the oxidative metabolism of CLZ to its major metabolites, N-desmethylclozapine (DMCLZ; C-2) and clozapine N-oxide (CLZ-NO; C-1) ( Fig. 1) Eiermann et al, 1997;Linnet and Olesen, 1997;Fang et al, 1998;Tugnait et al, 1999;Olesen and Linnet, 2001;Zhang et al, 2008). The combined results of these in vitro studies show that both CYP1A2 and CYP3A4 are playing major roles in the biotransformation of CLZ to these two metabolites.…”

The Aggravation of Clozapine-Induced Hepatotoxicity by Glycyrrhetinic Acid in Rats

“…Pharmacokinetic data showed that coadministration of GA significantly lowered concentrations and exposure of norCLZ and CLZ Noxide, indicating that formations of norCLZ and CLZ Noxide were inhibited by GA, which were verified by data on hepatic microsomes and hepatocytes of rats. CLZ metabolism in human liver was reported to be mainly mediated by CYP1A2, CYP3A4, CYP2C19, CYP2C9, and CYP2D6 (5), although FMO also contri buted to formation of CLZ Noxide (6,7). Activities of CYP450s in hepatic microsomes of rats were measured using corresponding substrates.…”

A therapeutic interaction between cimetidine and clozapine: case study and review of the literature

“…Nonetheless, other CYP isoenzymes such as 3A4, 2D6, 2C9, 2C19, 2E1 have also been implicated in the drug's metabolism, Troostwijk et al 2003;Fang et al 1998;Ozdemir et al 2001;Taylor 1997;Wetzel et al 1998]. In vitro data suggest that other CYPs which are also involved in the formation of norclozapine are 2C19 and 3A4, with the latter also being the main enzyme mediating the formation of clozapine N-oxide [Eiermann et al 1997;Olesen and Linnet 2001].The importance of 2D6 has been questioned by many as no difference in clozapine's metabolite generation has been seen in 2D6 ultra-rapid metabolizers compared with slow metabolizers [Dahl et al 1994].…”