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SHORT COMMUNICATION

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

Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey

(Received April 9, 2004; accepted August 9, 2004)

	Abstract

Top Abstract Materials and Methods Results and Discussion References

 
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 µM). 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 isoform-specific 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.

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.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Structure of ethinylestradiol.

	Materials and Methods

Top Abstract Materials and Methods Results and Discussion References

 
Chemicals. Ethinylestradiol (EE), NADP, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO); 17-[6,7-³H(N)]-ethinylestradiol ([³H]EE, specific activity 41 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Two hydroxylated metabolites (2-hydroxy- and 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 µM [³H]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 [³H]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 V_max and K_m 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% acetic acid (10 µl, final concentration = 2%) and methanol (50 µl, 20%), and the suspensions were vortex-mixed followed by centrifugation at 3000g for 5 min. Aliquots (100 µl) 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., V_max and K_m) 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 µl/mg microsomal protein; anti-CYP1A2, anti-CYP2A6, anti-CYP2C8, 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 µM [³H]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 [³H]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 µM [³H]EE and a selective chemical inhibitor (13 µM sulfaphenazole or 1 µM 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 [³H]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 µl) 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 baculovirus-infected 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 Gene-Tools 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 (SCL-10Avp), 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 YMC-ODS-AM column (4.6 x 250 mm; 5-µm 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):

(1)

where rP450n = each recombinant P450, and mP450n = the specific content of each P450 in human liver microsomes.

	Results and Discussion

Top Abstract Materials and Methods Results and Discussion References

 
Metabolism of EE by Recombinant Human P450s. The metabolism of [³H]EE (Fig. 1) was studied in a pool of human (female) liver microsomes. The two major metabolites were identified as 2-hydroxy-EE and 4-hydroxy-EE by comparing their retention times with the synthetic standards. Since 2-hydroxy-EE accounted for >90% of the total metabolites_, only the 2-hydroxylation pathway was investigated in detail in this study. To determine the P450 reaction phenotyping of EE, microsomes expressing individual recombinant human P450 isozymes (rCYP1A1, rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6, rCYP2E1, rCYP3A4, and rCYP3A5) were incubated with [³H]EE (1 µM) in the presence of an NADPH-regenerating system at 37°C for 5 to 60 min. Formation of 2-hydroxy-EE was observed in the incubations with rCYP1A1, rCYP1A2, rCYP2C8, rCYP2C9, rCYP3A4, and rCYP3A5, whereas the formation of 4-hydroxy-EE was observed only with rCYP3A4 and rCYP3A5, at rates 5- to 10-fold lower than those for 2-hydroxy-EE formation (Fig. 2). The formation rates of 2-hydroxy-EE with rCYP1A1 and rCYP3A4 were faster than with the other P450 isozymes. Also, the highest catalytic efficiency (intrinsic clearance, V_max/K_m) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2 (Table 1).

View larger version (13K): [in this window] [in a new window]   FIG. 2. Cytochrome P450 isozymes involved in the in vitro hydroxylation of ethinylestradiol. [3H]EE (1 µM) was incubated with microsomes expressing human recombinant P450 isozymes (150 or 250 pmol) for 5 min (CYP1A1), 10 min (CYP3A4 and CYP3A5), 30 min (CYP2C9 and CYP2C19), and 60 min (all other P450 isozymes). Formation of 2- and 4-hydroxy-EE was monitored by HPLC analysis with UV and radiometric detection. Results are presented as average of duplicate incubations.

Inhibition of EE Oxidation by Monoclonal Anti-P450 Antibodies. Inhibitory P450 isoform-specific monoclonal antibodies were used to identify the human P450 isoforms involved in the 2-hydroxylation of [³H]EE in the same pool of human (female) liver microsomes in which the specific P450 isozyme contents were quantified (see below). A titration curve for each monoclonal antibody (1–10 µl) was tested (Fig. 3) and the optimal amount of antibody used in the inhibitory reaction was determined to be 5 µl of antibody/mg of microsomal protein. The metabolism of EE was inhibited by 6, 26, 24, and 52% by the anti-1A2, anti-2C8, anti-2C9, and anti-CYP3A4 antibodies at 5 µl of antibody/mg of microsomal protein, respectively. No inhibition was observed with anti-CYP2A6 or anti-CYP2D6 antibodies at 10 µl/mg microsomal protein (data not shown). The results from the inhibition study with monoclonal antibodies were compared with the relative contribution of individual P450 isozymes (Table 2).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Inhibition of 2-hydroxylation of ethinylestradiol in human liver microsomes by isozyme-specific monoclonal antibodies. A pool of female human liver microsomes (0.1 mg of protein in 200 µl of buffer) was preincubated with isozyme-specific monoclonal antibodies (anti-CYP1A2, anti-CYP2C8, anti-CYP2C9, and anti-CYP3A4) at room temperature for 30 min. [3H]EE (5 µM) was added and incubations continued at 37°C for 30 min. Formation of 2-hydroxy-EE was monitored by HPLC analysis with UV and radiometric detection. Each point represents the average of duplicate incubations.

Unfortunately, a polyclonal anti-CYP2C8-specific antibody was unavailable for quantitative Western blot analysis; thus, the role of CYP2C8 in the metabolism of EE is inconclusive. However, based on the low abundance of CYP2C8 in human liver microsomes (Lasker et al., 1998), it can be concluded that its contribution to EE hydroxylation is minor (<10%). Although our inhibition study with the monoclonal anti-CYP2C8 antibody suggested a large contribution of CYP2C8 (26% inhibition of turnover), the value may be overestimated due to crossover inhibition with CYP2C9 (2C9^*1), as demonstrated by Krausz et al. (2001), using diazepam as the substrate.

Inhibition of EE Oxidation by Selective Chemical Inhibitors. Sulfaphenazole and ketoconazole were used to further confirm the involvement of the human CYP2C9 and CYP3A4 in the 2-hydroxylation of [³H]EE in the aforementioned pool of human liver microsomes. The metabolism of EE was inhibited by 31 and 75% by sulfaphenazole (13 µM) and ketoconazole (1 µM), respectively.

Relative Contribution of Different P450 Isozymes to the 2-Hydroxylation of EE in Human Liver Microsomes. To calculate the relative contribution of P450 isozymes to the 2-hydroxylation of EE, the specific P450 contents in a pool of liver microsomes obtained from seven female donors were determined by quantitative Western blot using cDNA-expressed enzymes as standards (Table 2). The rates of formation of 2-hydroxy-EE from EE (1 µM) by different rP450 isozymes were normalized with respect to the specific content of each P450 isozyme in the aforementioned pool of human liver microsomes (Table 2). The relative contribution, or %TNR, of each P450 isozyme to the 2-hydroxylation pathway was calculated. Thus, CYP3A4 and CYP2C9 contributed 61% and 23%, respectively, to the formation of 2-hydroxy-EE, whereas CYP1A2, CYP2C19, and CYP3A5, combined, contributed less than 20%.

The typical human dose of EE is 50 µg or less. The peak plasma concentration of EE is also very low and ranges from 60 to 160 pg/ml after a single oral dose of 30 µg (Orme et al., 1989). Previous reports indicated that CYP3A4 was the principal enzyme involved in the oxidative metabolism of EE (Guengerich, 1988). However, relatively high concentrations of EE (50–100 µM) were used in these incubations, which may not be clinically relevant. Our studies showed that using a low concentration of EE (1 µM), CYP2C9, as well as CYP3A4, catalyzed the 2-hydroxylation of EE. Thus, the decrease in the 0- to 24-h area under the curve of EE caused by rifampicin as previously reported (Chen et al., 2004) may be attributed to the increased activity of not only CYP3A4 but also CYP2C9. Therefore, the potential drug interaction of EE with CYP3A4 and CYP2C9 inducers or inhibitors should be evaluated clinically. Although EE oxidation appears to represent a minor pathway in the metabolism of EE in human (Abdel-Aziz and Williams, 1970; Williams et al., 1975; Williams and Goldzieher, 1980), CYP3A4 and CYP2C9 inducers are known to cause significant changes in the pharmacokinetics of EE (Barditch-Crovo et al., 1999; Finch et al., 2002; Hall et al., 2003). In addition, the production of EE sulfate conjugates was also induced in rifampicin-treated primary human hepatocytes. However, the clinical relevance of this finding is still unclear (Li et al., 1999).

The present studies showed that rCYP1A1, a predominantly extra-hepatic P450 isozyme, exhibited higher intrinsic catalytic activity than rCYP3A4 and rCYP2C9 for EE 2-hydroxylation. However, the role of CYP1A1 in the first-pass metabolism of EE is unclear due to the large interindividual variation in CYP1A1 expression in human small intestine (Paine et al., 1999; Zhang et al., 1999). In addition, it has been reported that CYP1A1 is inducible in human intestine by omeprazole and smoking (Buchthal et al., 1995; Kashfi et al., 1995). Therefore, increased EE elimination (even attenuated pharmacological effect of EE) may be seen in omeprazole users and smokers. Our attempts to demonstrate metabolism of EE in several preparations of human intestinal microsomes (n = 4) were not successful. The CYP3A4 activity in these preparations was measurable but very low when testosterone was used as a probe substrate, whereas CYP1A1 activity was not detected when ethoxyresorufin was used as a substrate.

In conclusion, CYP3A4 and CYP2C9 were shown to be the major isozymes contributing to the 2-hydroxylation of EE in human liver microsomes, whereas CYP2C8, CYP2C19, and CYP1A2 also contributed to a lesser extent.

	Acknowledgments

 
We are grateful to Dr. M. Braun of Radiolabeled Compound Synthesis at the Merck Research Laboratories for providing synthetic reference compounds. We gratefully acknowledge the critical review and helpful discussions of R. Wang and Drs. S. Vincent and R. Evers of Drug Metabolism at Merck Research Laboratories.

	Footnotes

 
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; [³H]EE, 17-[6,7-[³H(N)]]ethinylestradiol; P450, cytochrome P450; rP450, recombinant cytochrome P450; NR, normalized rate; TNR, total normalized rate; HPLC, high-performance liquid chromatography.

Address correspondence to: Ms. Bonnie Wang, Dept. of Pharmaceutical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, NJ 08540. E-mail: bonnie.wang{at}bms.com

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.104.000182v1 32/11/1209    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, B. Articles by Huskey, S.-E. W. Search for Related Content PubMed PubMed Citation Articles by Wang, B. Articles by Huskey, S.-E. W.