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 P45 0 (CYP) protein(s) involved in the oxidative metabolism of [C-14]clari thromycin (CLAR) in the presence of native human liver microsomes. The identity of the two major CLAR metabolites present in microsome incub ates, 14-(R)-hydroxy-CLAR and N-des-methyl-CLAR, was confirmed by MS. Over the CLAR concentration range of 1.0-140 mu M, the rate of CLAR 14 -(R)-hydroxylation (K-M = 48.7 +/- 17.7 mu M; V-max = 206 +/- 76 pmol/ min/mg protein; V-max/K-M = 4.2 +/- 0.21 mu l/min/mg; mean +/- SD, N = 3 livers) and N-demethylation (K-M = 59.1 +/- 24.0 mu M; V-max = 189 +/- 52.0 pmol/min/mg protein; V-max/K-M = 3.3 +/- 0.53 mu l/min/mg) co nformed to monophasic (saturable) Michaelis-Menten kinetics and was hi ghly correlated (r = 0.90-0.92; p < 0.001; N = 11) with CYP3A-selectiv e erythromycin N-demethylase activity. Ketoconazole (less than or equa l to 2.0 mu M) or troleandomycin, CYP3A-selective inhibitors, markedly decreased (greater than or equal to 99%) the formation of both metabo lites, whereas inhibitors selective of other CYP forms were relatively ineffective (less than or equal to 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, th e apparent K-M characterizing the 14-(R)-hydroxylation and N-demethyla tion of CLAR in the presence of insect cell microsomes containing cDNA -expressed CYP3A4 (K-M = 18-63 mu M) 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 CLA R is primarily mediated by one or more members of the human liver CYP3 A subfamily.