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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

The Grapefruit Juice Effect Is Not Limited to Cytochrome P450 (P450) 3A4: Evidence for Bergamottin-Dependent Inactivation, Heme Destruction, and Covalent Binding to Protein in P450s 2B6 and 3A5

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K_I, 5 µM; k_inact 0.09 min^–1; and t_1/2, 8 min. The kinetic constants obtained for the inactivation of the testosterone 6-hydroxylation activity of P450 3A5 were: K_I, 20 µM; k_inact 0.045 min^–1; and t_1/2, 15 min. Incubations of P450s 2B6 and 3A5 with 20 µM BG at 37°C for 20 min resulted in an 60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were 2 and 20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [¹⁴C]BG was irreversibly bound to the apoprotein in the BG-inactivated samples. The stoichiometry of binding was 0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6',7'-dihydroxybergamottin and bergaptol.

Address correspondence to: Dr. Paul F. Hollenberg, Department of Pharmacology, 2301 MSRBIII, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0632. E-mail: phollen{at}umich.edu

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