The INVOLVEMENT OF CYP3A4 AND CYP2C9 IN THE METABOLISM OF 17α-Ethinylestradiol

Abstract

ABSTRACT:The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 M). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (V max /K m ) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoformspecific monoclonal antibodies, in which the inhibition of formation was ϳ54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.Oral contraceptives (OCs) have been used widely by 60 to 70 million women worldwide since the 1970s. Ethinylestradiol (EE) and norethindrone are two components in OCs. Interactions with OCs in clinical studies have been reported for several compounds, including rifampicin (Bolt et al., 1977), rifabutin (LeBel et al., 1998), and ritonavir (Ouellet et al., 1998), and the subsequent failure to inhibit contraception was attributed to the increased metabolism of EE.The metabolism of EE has been studied extensively. It undergoes hydroxylation at the 2 (major), 4, 6, and 16␣ positions of the steroid nucleus (Fig. 1). Metabolism can occur by way of glucuronidation at 17 and 3 positions, methylation, or sulfation at the 3 position. Hydroxylation of EE is catalyzed primarily by CYP3A4 (Guengerich, 1988), 3-O-glucuronidation by uridine diphosphate glucuronosyltransferase 1A1 (Ebner et al., 1993), and 3-O-sulfation by sulfotransferase 1E1 (Forbes-Bamforth and Coughtrie, 1994). Changes in the activities of these metabolizing enzymes have been implicated in the observed OC interactions (Guengerich, 1990a(Guengerich, ,b, 1997. More recently, estradiol and estrone have been shown to be oxidized at several positions by several P450 isoforms, namely, CYP1A2, CYP3A4, CYP1B1, and CYP2C9 (Hayes et al., 1996;Shou et al., 1997;Yamazaki et al., 1998;Badawi et al., 2001;Lee et al., 2001Lee et al., , 2002. However, the involvement of P450 isoforms, other than CYP3A4, in the metabolism of EE has not been established clearly, although a previous report suggested that isoforms from the CYP2C and CYP2E families also may be involved (Ball et al., 1990). The objective of this present study was to investigate systematically the human P450 isoforms involved in the ox...

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

“…Hepatic as well as extra-hepatic P450s such as P450s 3A4, 2C9, 2C19, 1A1 and 1A2 have been found to metabolize 17EE primarily to 2-hydroxy 17EE and 4-hydroxy 17EE (16,31). The metabolism of 17EE by human P450 enzymes such as P450s 3A4 and 2B6 results in the production of reactive intermediates that are able to alkylate the P450 heme or modify the apoprotein (16)(17)(18).…”

“…Similar molecular ions and fragmentation patterns would be expected if the GSH conjugate arose from conjugation with an ortho quinone on the A ring (subsequent to hydroxylation and further oxidation). Metabolites consistent with 2-and 4-hydroxylation of the A ring of 17EE aromatic ring have been observed in humans (16,31,40). Previous studies in microsomes from phenobarbital-treated rats resulted in the identification of 2-and 4-hydroxylation products on the A ring of 17EE.…”

A Review of the Toxicity of HIV Medications II: Interactions with Drugs and Complementary and Alternative Medicine Products

“…Ethinyl estradiol undergoes oxidative metabolism by CYP3A4, CYP2C9, and other CYP isoforms [19] in addition to glucuronosyltransferases (UGTs) and pgp, which may also play a role [20]. Cytochrome p450 3A4 is also involved in the metabolism of the parent compound or active metabolites of t h e p r o g e s t i n s : e t o n o g e s t r e l , l e v o n o r g e s t r e l , medroxyprogesterone acetate, norelgestromin, norethindrone, n o r g e s t i m a t e , a n d n o r g e s t r e l .…”

Potent Inhibition of Human Sulfotransferase 1A1 by 17α-Ethinylestradiol: Role of 3′-Phosphoadenosine 5′-Phosphosulfate Binding and Structural Rearrangements in Regulating Inhibition and Activity

“…Because of the extensive usage of EE2 in women of child-bearing age and its extensive metabolism, several drug-drug interactions have been described. Many of these interactions involve the cytochromes P450 (P450), resulting in the inhibition or the induction of the P450s, leading to increased EE2 metabolism and clearance (Barditch-Crovo et al, 1999;Wang et al, 2004;Zhang et al, 2007). Although EE2 is readily sulfated in humans, less is known concerning the interactions and inhibition of the sulfotransferase (SULT) isoforms by EE2 that may result in clinically relevant drug-drug interactions (Sinofsky and Pasquale, 1998;Christensen et al, 2007).…”

“…1). The SULT isoforms responsible for intestinal EE2 sulfation have not been well described; however, SULT1E1 and P450s have been reported to be the major metabolizers of EE2 in human liver (Schrag et al, 2004;Wang et al, 2004). With a relatively high K m for EE2, SULT1A1 probably has only a small role in EE2 sulfation in liver; however, in tissues such as skin where SULT1A1 is highly expressed, it may have a greater role, especially in situations in which estrogens, including EE2, are applied topically.…”