The biotransformation of clomipramine in vitro, identification of the cytochrome P450s responsible for the separate metabolic pathways.

The aim of the study was to identify the cytochrome P450s (CYPs) that catalyze the biotransformation of clomipramine in vitro. A high-performance liquid chromatography method was developed to assay N-desmethylclomipramine, 8-hydroxyclomipramine, 2-hydroxyclomipramine, 8-hydroxydesmethhylclomipramine, didesmethylclomipramine and 2-hydroxydesmethylclomipramine formed by microsomes prepared from human liver and yeast expressing human CYP1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4. There was a statistically significant correlation between the formation rate of desmethylclomipramine and the immunoquantified concentration of CYP3A4 in 12 human liver microsome preparations (rs = 0.664, P = .028). Ketoconazole was a very potent inhibitor of desmethylclomipramine formation (Ki = 0.054 microM) and microsomes from yeast expressing CYP3A4 were also active in forming the metabolite (formation rate: 25.6 nmol/nmol of CYP per hr). Thus, the results are consistent with the assumption that the N-demethylation of clomipramine is catalyzed by CYP3A4. As expected from in vivo panel studies, CYP2C19 in yeast was also very active in the N-demethylation (formation rate, 145 nmol/nmol of CYP per hr). Fluvoxamine was a potent inhibitor of desmethylclomipramine formation (Ki, 0.15 microM), suggesting that CYP1A2 is a third CYP involved in the N-demethylation. CYP2D6 in yeast microsomes catalyzed the 8-hydroxylation of clomipramine and desmethylclomipramine (formation rates, 65 and 75 nmol/nmol of CYP per hr) and quinidine was a very potent inhibitor (Ki, 0.10 and 0.16 microM). Both results confirm that CYP2D6 catalyzes the 8-hydroxylation in agreement with the results obtained in previous in vivo studies. Besides quinidine, paroxetine, fluoxetine and norfluoxetine, all were potent inhibitors of the 8-hydroxylations (Ki, 0.24-1.5 microM) and sertraline was a less potent inhibitor (Ki, 16 and 27 microM, respectively).