Received for publication October 22, 2004.
Revised December 9, 2004.
Accepted for publication December 15, 2004.

THE "GRAPEFRUIT JUICE EFFECT" IS NOT LIMITED TO P450 3A4: EVIDENCE FOR BERGAMOTTIN-DEPENDENT INACTIVATION, HEME DESTRUCTION AND COVALENT BINDING TO PROTEIN IN P450s 2B6 AND 3A5

Abstract

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochrome P450s 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 a ~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. LC-MS 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-PAGE analysis demonstrated that [¹⁴C]BG was irreversibly bound to the apoprotein in the BG-inactivated samples. The stoichiometry of binding was ~ 0.5 mol of a BG metabolite bound per mol of each P450 inactivated. HPLC 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.

Key words: Drug-drug interaction, Mechanism-based inactivation, P450 2B6, P450 3A5, bergamottin, protein adduct

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