Major Role for the Cytochrome P4503A (CYP3A) Subfamily
Abstract
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 andN-desmethyl-CLAR, was confirmed by MS. Over the CLAR concentration range of 1.0–140 μM, the rate of CLAR 14-(R)-hydroxylation (KM = 48.7 ± 17.7 μM; Vmax = 206 ± 76 pmol/min/mg protein;Vmax/KM = 4.2 ± 0.21 μl/min/mg; mean ± SD, N = 3 livers) andN-demethylation (KM = 59.1 ± 24.0 μM; Vmax = 189 ± 52.0 pmol/min/mg protein; Vmax/Km = 3.3 ± 0.53 μl/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 (≤2.0 μM) or troleandomycin, CYP3A-selective inhibitors, markedly decreased (≥99%) the formation of both metabolites, whereas inhibitors selective of other CYP forms were relatively ineffective (≤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 μM) 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.
Footnotes
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Send reprint requests to: Dr. A. David Rodrigues, Drug Metabolism I, Merck Research Laboratories, Sumneytown Pike, P.O. Box 4, WP26A 2044, West Point, PA 19486-0004.
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↵2 Fractional inhibition (i) of 14-(R)-HC formation in vivo estimated using the equation: i = {AUCc − AUCi}/AUCc. AUCc and AUCi represent AUC of 14-(R)-HC in the absence and presence of RIT, respectively.
- Abbreviations used are::
- CLAR
- clarithromycin
- MAC
- Mycobacterium avium complex
- AIDS
- acquired immune deficiency syndrome
- ERN
- erythromycin
- CYP
- cytochrome P450
- MI
- metabolic intermediate
- N-desmethyl-CLAR
- N-desmethyl-clarithromycin
- 14-(R)-HC
- 14-(R)-hydroxy-clarithromycin
- 14-(S)-HC
- 14-(S)-hydroxy-clarithromycin
- QND
- quinidine
- COU
- coumarin
- TAO
- troleandomycin
- 4-MP
- 4-methylpyrazole
- KTZ
- ketoconazole
- FURA
- furafylline, SLF, sulfaphenazole
- RIT
- ritonavir
- IIAM
- Institute for the Advancement of Medicine
- KM
- apparent Michaelis constant
- Vmax
- apparent maximal initial reaction velocity
- ERODase
- 7-ethoxyresorufin O-deethylase
- COHase
- coumarin hydroxylase
- TOLase
- tolbutamide methyl hydroxylase
- DEXase
- [O-methyl-14C]dextromethorphanO-demethylase
- DMNase
- N,N-dimethylnitrosamine N-demethylase
- ERNDase
- erythromycin N-demethylase
- MEPHase
- (S)-(+)-mephenytoin 4′-hydroxylase
- MEPH
- (S)-(+)-mephenytoin
- Ki
- inhibition constant
- i
- fractional inhibition
- [I]
- inhibitor concentration
- TEA
- triethylamine
- [S]
- substrate concentration
- Cmax
- maximal concentration of drug in plasma
- AUC
- area under the plasma concentration-time curve. AUCc, area under the plasma concentration vs. time curve in the absence of inhibitor
- AUCi
- area under the plasma concentration vs. time curve in the presence of inhibitor
- Received November 5, 1996.
- Accepted February 10, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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