Metabolism of bergamottin by cytochromes P4502B6 and 3A5

Cytochromes P450 (P450) 2B6 and 3A5 are inactivated by bergamottin (BG). P450 2B6 metabolized BG primarily to M3 and M4 and one minor metabolite (M1). The metabolites were analyzed, and the data indicated that M1 was bergaptol, M3 was 5'-OH-BG, and M4 was a mixture of 6'- and 7' -OH-BG. Because 6'- and 7'- OH-BG were the primary metabolites, it suggested that P450 2B6 preferentially oxidized the geranyloxy chain of BG. Metabolism of BG by P450 3A5 resulted in three major metabolites: [ bergaptol, M3 (5'-OH-BG), and M5 (2'- OH- BG)], and two minor metabolites [M2 (6', 7'- dihydroxyBG) and M4 (6'- and 7'-OH-BG)]. Because bergaptol was the most abundant metabolite formed, it suggested that P450 3A5 metabolized BG mainly by cleaving the geranyl-oxy chain. Molecular modeling studies confirmed that docking of BG in the P450 2B6 active site favors oxidation in the terminal region of the geranyl-oxy chain, whereas positioning the 2'-carbon of BG nearest the heme iron is preferred by P450 3A5. Glutathione (GSH)-BG conjugates were formed by both P450. Each enzyme predominantly formed conjugates with m/z values of 662. Tandem mass spectrometry analysis of the GSH conjugates indicated that the oxidation forming a reactive intermediate occurred on the furan moiety of BG, presumably through the initial formation of an epoxide at the furan double bond. The data indicate that oxidation of the geranyl-oxy chain resulted in the formation of stable metabolites of BG, whereas oxidation of the furan ring produced reactive intermediates that may be responsible for binding to and inactivating P450 2B6 and 3A4.