Short Communication THE INVOLVEMENT OF CYP3A4 AND CYP2C9 IN THE METABOLISM OF 17 -ETHINYLESTRADIOL

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 (Vmax/Km) 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 54 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,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., 2001, 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 oxidative metabolism of EE to facilitate our understanding of the underlying mechanisms associated with OC interactions. Materials and Methods Chemicals. Ethinylestradiol (EE), NADP, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO); 17 -[6,7-H(N)]-ethinylestradiol ([H]EE, specific activity 41 Ci/ mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Two hydroxylated metabolites (2-hydroxyand 4-hydroxy-EE) were synthesized by Dr. Matt Braun of the Merck Labeled Compound Synthesis Group (Rahway, NJ). All chemicals were of the highest analytical purity available. Microsomes and Antibodies. Human liver microsomes (catalog number 452183, lot 2, a pool from seven female subjects), as well as microsomes from baculovirus-infected Sf21 cells expressing recombinant human P450 isoforms (rCYP1A1, rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6, rCYP2E1, rCYP3A4, and rCYP3A5) were purchased from BD Gentest (Woburn, MA). P450 isoform-specific inhibitory monoclonal antibodies for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4 were prepared by Dr. M. Shou of Merck Research Laboratories. The antibodies for CYP1A2, CYP2C8, and CYP2C9 were licensed from the National Institutes of Health (Bethesda, MD) (Krausz et al., 2001). Rabbit polyclonal antibodies for human P450 isoforms (CYP2C9, CYP2C19, CYP3A4, and CYP3A5) were purchased from BD Gentest and Oxford Biomedical Research (Oxford, MI) (CYP1A2). Horseradish peroxidase-conjugated anti-rabbit antibody was purchased from BD Gentest. Incubation Conditions. The typical reaction mixtures (0.2 ml) contained 1 M [H]EE, 100 mM phosphate buffer (pH 7.4), 6 mM MgCl2, 10 mM EDTA, an NADPH-regenerating system (5 mM glucose 6-phosphate, 1 mM NADP, and 0.7 IU/ml glucose-6-phosphate dehydrogenase), and human liver microsomes (0.5 mg of protein/ml). Stock solutions of [H]EE were made in 75% aqueous acetonitrile such that the final concentration of acetonitrile in the reaction mixture was 1.5%. The reactions were initiated by adding the NADPH-regenerating system and were allowed to proceed for 15 to 60 min at 37°C in a static water bath. Incubation conditions were optimized so that the rate of metabolism was linear with respect to incubation time and microsomal protein concentration. The Vmax and Km parameters for EE 2-hydroxylation were determined from 30-min incubations with a microsomal protein concentration of 0.5 mg/ml. The reactions were terminated by the addition of 50% Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.104.000182 . ABBREVIATIONS: OC, oral contraceptive; EE, ethinylestradiol; [H]EE, 17 -[6,7-[H(N)]]ethinylestradiol; P450, cytochrome P450; rP450, recombinant cytochrome P450; NR, normalized rate; TNR, total normalized rate; HPLC, high-performance liquid chromatography. 0090-9556/04/3211-1209–1212$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 11 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 182/1179893 DMD 32:1209–1212, 2004 Printed in U.S.A. 1209 at A PE T Jornals on M ay 2, 2017 dm d.aspurnals.org D ow nladed from acetic acid (10 l, final concentration 2%) and methanol (50 l, 20%), and the suspensions were vortex-mixed followed by centrifugation at 3000g for 5 min. Aliquots (100 l) of the supernatants were analyzed by HPLC. Incubations with microsomes from baculovirus-infected Sf21 cells expressing human P450 isoforms contained 150 or 250 pmol of P450/ml. P450 isoform-specific substrates were incubated as positive controls to validate the enzyme activity of each isozyme (data not shown). Kinetic parameters (i.e., Vmax and Km) were calculated by fitting the data to the Michaelis-Menten equation, using nonlinear least-squares regression with KaleidaGraph software, version 3.5 (Abelbeck/Synergy, Reading, PA). For immuno-inhibition studies, aliquots of human liver microsomes (final concentration, 0.5 mg/ml) were preincubated with a P450 isoform-specific antibody (1–10 l/mg microsomal protein; anti-CYP1A2, anti-CYP2A6, antiCYP2C8, anti-CYP2C9, anti-CYP2D6, or anti-CYP3A4) in 100 mM phosphate buffer (pH 7.4), without EE, at room temperature for 30 min. At that time, 5 M [H]EE and an NADPH-regenerating system were added and incubations were allowed to proceed as described previously. The percentage of inhibition was calculated from the extent of [H]EE metabolism in the presence versus absence of monoclonal antibodies. For chemical-inhibition studies, aliquots of human liver microsomes (final concentration, 0.5 mg/ml) were incubated with 1 M [H]EE and a selective chemical inhibitor (13 M sulfaphenazole or 1 M ketoconazole) in the presence of an NADPH-regenerating system. Incubations were allowed to proceed as described previously. The percentage of inhibition was calculated from the extent of [H]EE metabolism in the presence versus absence of the chemical inhibitors. Quantitative Western Blot. Microsomal proteins, suspended in Laemmli sample buffer, containing 5% 2-mercaptoethanol and 2% sodium dodecyl sulfate, were denatured by heating in a boiling water bath. The samples (20 l) were separated by SDS-polyacrylamide gel electrophoresis using 7.5% polyacrylamide gel and transferred onto nitrocellulose membranes, as described by Towbin et al. (1979). Detection of P450 proteins was performed using rabbit polyclonal antibodies for human CYP2C9, CYP2C19, CYP3A4, CYP3A5, or CYP1A2. Visualization of antibody-antigen interactions was achieved using horseradish peroxidase-conjugated anti-rabbit antibody and the Hyperfilm enhanced chemiluminescence detection system, as described by the manufacturer (Amersham Biosciences Inc., Piscataway, NJ). Serial dilutions of microsomes from baculovirusinfected Sf21 cells expressing corresponding recombinant human P450 isozymes were used as standards to define the linear range of the optical density in enhanced chemiluminescence used for the detection. Prestained SDS-polyacrylamide gel electrophoresis molecular weight standards (Bio-Rad, Hercules, CA) were also loaded onto the same gel. The optical density of the immunoblot bands was measured with a GeneGnome Chemiluminescence Imaging System using GeneTools version 3.02a software (The Synoptics Group, Cambridge, UK). The amount of each P450 isozyme in human liver microsomes was calculated against a standard curve constructed with the human rP450 standards loaded on the same gel as the human liver microsomes. HPLC Analysis. The HPLC system (Shimadzu Scientific Instruments Inc., Columbia, MD) consisted of two pumps (LC-10ADvp), a controller (SCL10Avp), an autosampler (SIL-10ADvp), and a UV detector (SPD-10AVvp). Radioactive EE and its metabolites were monitored using a UV detector at 210 nm and an on-line radiometric detector (IN/US Systems Inc., Tampa, FL) with Ultima-Flo M (PerkinElmer Life and Analytical Sciences) as scintillant at a flow rate of 3 ml/min. Chromatography of all samples was performed using a YMCODS-AM column (4.6 250 mm; 5m particle size; Waters, Milford, MA). The mobile phase consisted of solvent A (10 mM ammonium acetate in water containing 0.1% trifluoroacetic acid) and solvent B (10 mM ammonium acetate in a mixture of 10% methanol and 90% acetonitrile containing 0.1% trifluoroacetic acid). The column was eluted at 1 ml/min using a linear gradient from 25 to 65% B in 30 min, followed by a gradient from 65 to 95% B in 0.1 min, and an isocratic hold at 95% B for 5 min. The elution times for 4-hydroxy-EE, 2-hydroxy-EE, and EE under these conditions were 25.4, 26.4, and 30.4 min, respectively. Normalization of rP450 Data for Human Liver Activity. For each rP450, the reaction rate was normalized by multiplying the rate with the specific content of the corresponding P450 (pmol of P450/mg of microsomal protein) in human liver microsomes, and was expressed as a normalized rate (NR; pmol/min/mg microsomal protein) as described by Rodrigues (1999). The NR values for each P450 were summed to obtain the total normalized rate (TNR). Finally, the NR for each P450 was expressed as a percentage of the TNR (eq. 1):