Metabolism of Bergamottin by Cytochromes P450 2B6 and 3A5

Abstract

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 5Ј-OH-BG, and M4 was a mixture of 6Ј-and 7Ј-OH-BG. Because 6Ј-and 7Ј-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 (5Ј-OH-BG), and M5 (2Ј-OH-BG)], and two minor metabolites [M2 (6Ј,7Ј-dihydroxy-BG) and M4 (6Ј-and 7Ј-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 2Ј-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.The polymorphic cytochromes P450 (P450) 3A5 and 2B6 are involved in the metabolism of many clinically important drugs used in chemotherapy, hormone therapy, suppression of immune responses, or as inhibitors of human immunodeficiency virus proteases (Ingelman-Sundberg, 2004). P450 3A5 is found primarily in liver, intestine, and lung and shares 85% amino acid sequence homology with the major human drug-metabolizing enzyme P450 3A4 (Wrighton et al., 1990;Ding and Kaminsky, 2003). The P450 3A family of enzymes is involved in the metabolism of at least 60% of drugs that are currently in clinical use (Wrighton et al., 1990). Recent reports indicate important clinical consequences for individuals expressing the polymorphic P450 3A5 in the metabolism of immune suppressants and statins (Hesselink et al

Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

“…The molecular ions at m/z 662, 678, and 696 are from the reaction of GSH with the furanoepoxide of BG, furanoepoxide of monohydroxy BG, and furanoepoxide of DHBG, respectively. The two GSH conjugates with MH ϩ values at m/z 662 and 678 are similar to those previously reported for the GSH conjugates formed by CYP2B6 and 3A5 (Lin et al, 2005;Kent et al, 2006). The MS/MS spectrum of the GSH conjugate with MH ϩ at m/z 696 is shown in Fig.…”

“…6). As reported previously (Kent et al, 2006), the WT 3A4 generated four major metabolites (M1, M2, M3, and M5), but the Q273V mutant only generated two major metabolites (M3 and M5). On the basis of our previous reports on the metabolism of BG by CYP3A5 and CYP2B6, M1 is bergaptol, M2 is DHBG, M3 is 5Ј-OH-BG, and M5 is 2Ј-OH-BG (Kent et al, 2006).…”

“…ϩ2 for this modified peptide can be identified in the MS/MS spectrum. The mass increase of 388 Da observed in the b2 to b10 ions, whereas the lack of any mass increase in the y3 through y9 ions observed in the modified peptide, indicates that Gln273 is the amino acid residue modified by a reactive intermediate of DHBG, which is a metabolite of BG (Goosen et al, 2004;Kent et al, 2006).…”

“…The organic phase was dried under N 2 and dissolved in 0.1% acetic acid/50% acetonitrile for HPLC analysis. The metabolites and BG were separated on a C8 column and monitored at 310 nm according to previous methods (Lin et al, 2005;Kent et al, 2006).…”

“…These findings have led us to hypothesize that BG and DHBG, which both contain the intact furanocoumarin core structure, are activated via a similar mechanism to form a reactive intermediate capable of covalently binding with GSH or with amino acid residues in the 3A4 apoprotein (Lin et al, 2005;Kent et al, 2006).…”