Oxidative metabolism of clarithromycin in the presence of human liver microsomes. Major role for the cytochrome P4503A (CYP3A) subfamily.

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) protein(s) involved in the oxidative metabolism of [14C]clarithromycin (CLAR) in the presence of native human liver microsomes. The identity of the two major CLAR metabolites present in microsome incubates, 14-(R)-hydroxy-CLAR and N-desmethyl-CLAR, was confirmed by MS. Over the CLAR concentration range of 1.0-140 microM, the rate of CLAR 14-(R)-hydroxylation (KM = 48 +/- 17.7 microM; Vmax = 206 +/- 76 pmol/min/mg protein; Vmax/KM = 4.2 +/- 0.21 microliters/min/mg; mean +/- SD, N = 3 livers) and N-demethylation (KM = 59.1 +/- 24.0 microM; Vmax = 189 +/- 52.0 pmol/min/mg protein; Vmax/KM = 3.3 +/- 0.53 microliters/min/mg) conformed to monophasic (saturable) Michaelis-Menten kinetics and was highly correlated (r = 0.90-0.92; p < 0.001; N = 11) with CYP3A-selective erythromycin N-demethylase activity. Ketoconazole (< or = 2.0 microM) or troleadomycin, CYP3A-selective inhibitors, markedly decreased (> or = 99%) the formation of both metabolites, whereas inhibitors selective of other CYP forms were relatively ineffective (< or = 10% inhibition). In agreement with chemical inhibitor studies, CLAR metabolism was only detectable with human B-lymphoblastoid microsomes containing cDNA-expressed CYP3A4 (vs. CYP2C19, CYP2C9, CYP2D6, CYP1A2, CYP2E1, or CYP2A6). Furthermore, the apparent KM characterizing the 14-(R)-hydroxylation and N-demethylation of CLAR in the presence of insect cell microsomes containing cDNA-expressed CYP3A4 (KM = 18-63 microM) was similar to that obtained with native human liver microsomes. Based on the results of this study, it is concluded that the 14-(R)-hydroxylation and N-demethylation of CLAR is primarily mediated by one or more members of the human liver CYP3A subfamily.