ToxSci Advance Access originally published online on August 31, 2006
Toxicological Sciences 2006 94(2):261-271; doi:10.1093/toxsci/kfl096
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Contribution of CYP2C9, CYP2A6, and CYP2B6 to Valproic Acid Metabolism in Hepatic Microsomes from Individuals with the CYP2C9*1/*1 Genotype
Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
4 To whom correspondence should be addressed at Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, British Columbia, V6T 1Z3, Canada. Fax: 1-604-822-3035. E-mail: tchang{at}interchange.ubc.ca.
Received June 1, 2006; accepted August 25, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1–8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75–80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by
50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation. Key Words: cytochrome P450; CYP2A6; CYP2B6; CYP2C9; valproic acid.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Valproic acid (VPA, 2-propylpentanoic acid or dipropylacetic acid) is an antiepileptic drug. Its other indications include bipolar disorder, neuropathic pain, and migraine (Loscher, 1999
). Current interest in VPA is based on its inhibition of histone deacetylation (Phiel et al., 2001) and its potential therapeutic application in cancer (Minucci and Pelicci, 2006) and human immunodeficiency virus infection (Lehrman et al., 2005). The clinical use of VPA is associated with a rare, but potentially fatal, hepatotoxicity (Loscher, 1999). Based on retrospective studies (Bryant and Dreifuss, 1996), this idiosyncratic toxicity has been linked to several risk factors, including the concurrent administration of a cytochrome P450 (CYP)–inducing drug (e.g., phenobarbital), especially in patients younger than 2 years of age. The mechanism of VPA-associated hepatotoxicity is still not well understood, but it may be associated with reactive metabolites of VPA, such as 4-ene-VPA (Baillie, 1988) and its ß-oxidation product, 2,4-diene-VPA (Kassahun et al., 1991; Tang et al., 1995).
VPA undergoes terminal desaturation, 
-2 hydroxylation, -1 hydroxylation, and -hydroxylation to form 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA, respectively (Prickett and Baillie, 1984; Rettie et al., 1987). Studies conducted with individual cDNA-expressed human CYP enzymes indicated that CYP2A6, CYP2B6, and CYP2C9 catalyzed VPA terminal desaturation, whereas little or no metabolite was formed by several other CYP enzymes, including CYP2E1 and CYP3A4 (Anari et al., 2000; Sadeque et al., 1997). Very little information is available on the role of human hepatic microsomal CYP enzymes in VPA terminal desaturation. Hepatic microsomal CYP2A6 and CYP2C9 have been implicated in the formation of 4-ene-VPA, based on a study with microsome samples from two individual livers, indicating that 4-ene-VPA formation was decreased by coumarin and sulfaphenazole (Sadeque et al., 1997), which were employed to inhibit the catalytic activity of CYP2A6 (Messina et al., 1997) and CYP2C9 (Newton et al., 1995), respectively. However, the contribution of these and other CYP enzymes to the formation of 4-ene-VPA in individual hepatic microsomes remains to be determined. Even less is known about the role of human CYP enzymes in VPA hydroxylation reactions. According to experiments with cDNA-expressed enzymes, CYP2C9 catalyzes VPA 4-hydroxylation and VPA 5-hydroxylation (Ho et al., 2003). However, it is not known whether other human CYP enzymes are capable of catalyzing the formation of 3-OH-VPA, 4-OH-VPA, or 5-OH-VPA or which specific CYP enzymes are responsible for these enzymatic reactions in human hepatic microsomes.
In the present study, we conducted a detailed, systematic investigation on the role of specific human CYP enzymes in the in vitro oxidative metabolism of VPA. A complementary approach was employed that involved (1) individual cDNA-expressed human CYP enzymes, (2) chemical inhibitors of specific CYP enzymes, (3) CYP-specific inhibitory monoclonal antibodies (MAbs), (4) individual human hepatic microsomes, and (5) correlational analysis. Our primary aim was to determine the contribution of specific CYP enzymes to VPA metabolism in human hepatic microsomes. Similar to the findings of many of the other CYP2C9 substrates (Tracy et al., 2002), our previous study showed that the CYP2C9*2 and CYP2C9*3 allelic variants catalyzed VPA terminal desaturation and hydroxylation reactions with substantially reduced capacity, when compared to the wild-type CYP2C9*1 (Ho et al., 2003). Furthermore, chemical inhibition of CYP2C9 may be allele dependent (Melet et al., 2003). Therefore, the present study was conducted with microsomes obtained only from donors with the CYP2C9*1/*1 genotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and reagents.
VPA (the acid form, 99% pure) was supplied by Arcos Organics (Morris Plains, NJ), and the sodium salt of VPA was prepared (Olson et al., 1986
). NADPH, sulfaphenazole, triethylenethiophosphoramide (thio-TEPA), coumarin, N,N'-diisopropylethylamine, and tert-butyldimethylsilyl chloride were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). Dimethylformamide, pentafluorobenzyl bromide, and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide were purchased from Pierce (Rockford, IL). Authentic VPA metabolites (4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA) and heptadeuterated internal standards (4-ene-[2H7]VPA, 3-OH-[2H7]VPA, 4-OH-[2H7]VPA, and 5-OH-[2H7]VPA) were synthesized in our laboratory (Zheng, 1993).
cDNA-expressed enzymes, hepatic microsomes, and MAbs.
Microsomes isolated from baculovirus-infected cells coexpressing NADPH-CYP reductase and CYP1A1 (cat. no. 456211), CYP1A2 (cat. no. 456203), CYP1B1 (cat. no. 456220), CYP2A6 (cat. no. 456254), CYP2B6 (cat. no. 456255), CYP2C8 (cat. no. 456212), CYP2C9*1 (cat. no. 456218), CYP2C19 (cat. no. 456219), CYP2D6 (cat. no. 456217), CYP2E1 (cat. no. 456206), CYP3A4 (cat. no. 456202), CYP3A5 (cat. no. 456235), CYP4A11 (cat. no. 456221), CYP4F2 (cat. no. 456272), CYP4F3A (cat. no. 456273), or CYP4F3B (cat. no. 456274), control insect cell microsomes (cat. no. 456201), and individual human hepatic microsomes (HG24, HG30, HG88, HG95, HH13, HH18, HH47, HH64, HH74, HH91, and HK37) with the CYP2C9*1/*1 genotype were purchased from BD GENTEST Corp. (Woburn, MA). Among the 11 donors, eight were female and three were male. The mean ± SD age of the donors was 51 ± 17 years (the youngest was 28 years old and the oldest was 78 years old). MAb against CYP2A6 (MAb-2A6, clone 151-45-4) (Sai et al., 1999), CYP2B6 (MAb-2B6, clone 49-10-20) (Yang et al., 1998), and CYP2C9 (MAb-2C9, clone 763-15-5) (Krausz et al., 2001) and control MAb against lysozyme (Hy-Hel-9) (Krausz et al., 2001) were provided by Dr H. V. Gelboin and his colleagues at the National Cancer Institute, National Institutes of Health (Bethesda, MD).
VPA metabolism assay.
Each standard 200 µl incubation mixture contained 60mM Tris buffer (pH 7.4), 1.8mM MgCl2, 1mM sodium VPA, cDNA-expressed human CYP enzyme (40 pmol) or human hepatic microsomes (45 pmol total CYP), and 1mM NADPH. The complete incubation mixture (but without NADPH) was prewarmed to 37°C for 5 min in a water bath with gentle shaking. The enzymatic reaction was initiated by the addition of NADPH and terminated 40 min later (unless indicated otherwise) by the addition of ice-cold 0.1M phosphoric acid (75 µl). The substrate concentration (1mM) was chosen to approximate steady-state plasma VPA concentrations achieved in humans administered with maintenance doses of the drug (Loscher, 1999). Preliminary experiments were performed to delineate the incubation conditions whereby the VPA metabolism assay was linear with respect to incubation time and amount of enzyme. Standard curves were constructed with samples containing known amounts of authentic 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA.
VPA metabolite analysis by gas chromatography-mass spectrometry.
At the termination of the VPA metabolism assay, 50 µl of an internal standard mixture, which contained 2 µg/ml of 4-ene-[2H7]VPA, 3-OH-[2H7]VPA, 4-OH-[2H7]VPA, and 5-OH-[2H7]VPA, and 675 µl of distilled water were added to each incubation mixture. To acidify the samples, 1 ml of 1M KH2PO4 (pH 3.0) was added. Samples were extracted with ethyl acetate (8 ml) by gentle rotation for 30 min and then centrifuged at 3000 x g for 10 min at room temperature. The organic layer was transferred to a clean borosilicate glass screw-top test tube, dried with anhydrous sodium sulfate, rotated for 30 min, and centrifuged at 3000 x g for 10 min at room temperature. The organic layer was transferred to a new test tube, and the ethyl acetate was evaporated to a volume of 100–200 µl under a gentle stream of nitrogen (0.5 psi at 25°C; Zymark Turbo Vap LV evaporator, Zymark, Hopkinton, MA). To conduct the pentafluorobenzyl ester derivatization of the carboxylic acid groups, N,N'-diisopropylethylamine (30 µl) and pentafluorobenzyl bromide (10 µl) were added to each of the extracted samples followed by vortex mixing and heating at 45°C for 60 min. Subsequently, the mixture was cooled to room temperature. To perform the tert-butyldimethylsilylation of hydroxyl groups, 20 µl of dimethylformamide and 40 µl of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide containing 2% tert-butyldimethylsilyl chloride were added to the sample mixture and heated at 65°C for 2 h. The samples were cooled to room temperature and then concentrated by applying a gentle stream of nitrogen (0.5 psi at 25°C for 30 min). The residue was mixed with 200 µl of n-hexane (gas chromatography grade) by vortexing for 20 s, and the mixture was centrifuged at 3000 x g for 10 min at room temperature. Subsequently, 1 µl of the hexane layer was analyzed for VPA metabolites. The analytical system consisted of an HP6890 gas chromatograph, an HP5973 mass selective detector, and an HP7683 autosampler (Hewlett-Packard, Avondale, PA). The HP Enhanced Chemstation Software G1701BA (V. B.01.00) was used. The operating conditions and the specifications of the chromatographic columns were the same as those described previously (Ho et al., 2003).
Chemical inhibition experiments.
Sulfaphenazole (20µM), coumarin (50µM), or methanol (0.5% vol/vol final concentration; vehicle control) was added to each incubation mixture (without preincubating the inhibitor with microsomes and NADPH), and the VPA metabolism assay was performed as described above. Thio-TEPA (50µM) or distilled water (vehicle control) was preincubated with human hepatic microsomes (67.5 pmol total CYP) and NADPH (1mM) for 15 min at 37°C in a volume of 30 µl. Subsequently, a 20-µl aliquot was transferred to a tube containing 60mM Tris (pH 7.4), 1.8mM MgCl2, 1mM sodium VPA, and 1mM NADPH. The reaction was stopped 40 min later by the addition of ice-cold 0.1M phosphoric acid (75 µl). VPA metabolite analysis was performed as described above.
Immunoinhibition experiments.
The general steps in the immunoinhibition experiments were performed according to a published protocol (Krausz et al., 2001). Briefly, individual human hepatic microsomes (45 pmol total CYP) was preincubated with MAb-2A6, MAb-2B6, MAb-2C9, or the corresponding level of control MAb (as indicated in each figure legend) in 60mM Tris buffer (pH 7.4) containing 1.8mM MgCl2 for 5 min at 37°C prior to the addition of sodium VPA (1mM) and NADPH (1mM). The reaction was terminated 40 min later (unless indicated otherwise) by the addition of ice-cold 0.1M phosphoric acid (75 µl). VPA metabolite analysis was performed as described above.
Statistical analysis.
The difference between the means of the groups was analyzed by the one-tailed, paired t-test. The level of significance was set a priori at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VPA Metabolism by Individual cDNA-Expressed Human CYP Enzymes
To identify individual human CYP enzymes competent in catalyzing VPA terminal desaturation and hydroxylation reactions, the VPA metabolism assay was performed with individual cDNA-expressed CYP enzymes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9*1, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B). Among these enzymes, only CYP2C9*1, CYP2A6, and CYP2B6 catalyzed the formation of 4-ene-VPA (Fig. 1A). These three enzymes also catalyzed VPA 4-hydroxylation (Fig. 1B) and VPA 5-hydroxylation (Fig. 1C). Minimal levels of 4-OH-VPA (Fig. 1B) and 5-OH-VPA (Fig. 1C) were formed by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B (only 1–8% of the levels by CYP2C9*1). Only CYP1A1, CYP2A6, CYP2B6, CYP4F2, and CYP4F3B catalyzed VPA 3-hydroxylation (Fig. 1D).
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VPA Metabolism by Human Hepatic Microsomes
The results from the experiment with cDNA-expressed enzymes indicated that CYP2A6, CYP2B6, and CYP2C9 were the major catalysts of VPA metabolism and suggested a role by the corresponding hepatic microsomal enzyme. Our initial experiment with hepatic microsomes was to determine VPA metabolism in samples characterized for their CYP2A6, CYP2B6, and CYP2C9 catalytic activities, as assessed by coumarin 7-hydroxylation, (S)-mephenytoin N-demethylation, and diclofenac 4'-hydroxylation, respectively (Table 1). Each individual hepatic microsome sample catalyzed VPA terminal desaturation, VPA 4-hydroxylation, VPA 5-hydroxylation, and VPA 3-hydroxylation. The ratio was 1:10:10:0.6 for the formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA. As shown by correlational analyses (Table 2), a positive correlation was obtained between 4-ene-VPA formation and CYP2C9-mediated diclofenac 4'-hydroxylation (r2 = 0.55, p = 0.009) and between 3-OH-VPA formation and CYP2A6-mediated coumarin 7-hydroxylation (r2 = 0.47, p = 0.02).
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Effect of Chemical Inhibitors of CYP2A6, CYP2B6, and CYP2C9 on VPA Metabolism by Human Hepatic Microsomes
To determine whether human hepatic microsomal CYP2A6, CYP2B6, and CYP2C9 played a role in catalyzing VPA terminal desaturation and hydroxylation reactions, the VPA metabolism assay was conducted with individual hepatic microsome samples in the presence of coumarin (to inhibit CYP2A6) (Messina et al., 1997
), thio-TEPA (to inhibit CYP2B6) (Rae et al., 2002), sulfaphenazole (to inhibit CYP2C9) (Newton et al., 1995), or the respective vehicle (control). Sulfaphenazole decreased the group mean formation of 4-ene-VPA (by 54 ± 4%, mean ± SEM, Fig. 2A), 4-OH-VPA (by 62 ± 4%, Fig. 2B), and 5-OH-VPA (by 66 ± 6%, Fig. 2C) but not 3-OH-VPA (Fig. 2D). In contrast, coumarin did not reduce the group mean formation of 4-ene-VPA (Fig. 2A), 4-OH-VPA (Fig. 2B), or 5-OH-VPA (Fig. 2C), but it decreased 3-OH-VPA by 57 ± 9% (Fig. 3D). By comparison, thio-TEPA did not affect the group mean formation of any of these metabolites (Figs. 3A–3D). However, in one microsomal sample (HH74), which had the greatest CYP2B6-mediated (S)-mephenytoin N-demethylation activity in our panel of microsome samples, thio-TEPA reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 26%, 15%, and 29%, respectively. Control experiments verified the inhibition of CYP2A6, CYP2B6, and CYP2C9 by coumarin, thio-TEPA, and sulfaphenazole, respectively, as evaluated by incubations containing VPA and the corresponding cDNA-expressed enzyme (data not shown).
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Effect of MAb-2A6, MAb-2B6, and MAb-2C9 on VPA Metabolism by Human Hepatic Microsomes
To assess the extent by which CYP2A6, CYP2B6, and CYP2C9 contributed to VPA terminal desaturation and hydroxylation reactions in human hepatic microsomes, we used inhibitory MAbs (i.e., MAb-2A6, MAb-2B6, and MAb-2C9) of known specificity toward CYP2A6 (Sai et al., 1999
), CYP2B6 (Yang et al., 1998), and CYP2C9 (Krausz et al., 2001). Initial experiments verified the inhibitory effect (> 90%) of these antibodies and determined the level that yielded maximal inhibition of human hepatic microsomal VPA metabolism (data not shown). MAb-2C9 decreased the group mean formation of 4-ene-VPA by 77 ± 1% (Fig. 3A), 4-OH-VPA by 75 ± 2% (Fig. 3B), and 5-OH-VPA by 80 ± 3% (Fig. 3C), but it did not affect 3-OH-VPA (Fig. 3D). In contrast, MAb-2A6 did not affect the group mean formation of 4-ene-VPA (Fig. 3A), 4-OH-VPA (Fig. 3B), or 5-OH-VPA (Fig. 3C), whereas it reduced 3-OH-VPA by 55 ± 6% (Fig. 3D). By comparison, MAb-2B6 did not affect the group mean formation of any of these metabolites (Figs. 3A–3D). However, in one sample (HH74) with high CYP2B6-associated enzyme activity, it reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 26, 18, and 42%, respectively.
Combinatorial Immunoinhibition of VPA Metabolism by Human Hepatic Microsomes
To further illustrate that CYP2A6 and CYP2B6 contributed to the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human hepatic microsomes, we employed a combinatorial approach with MAbs (Gelboin and Krausz, 2006) in a human hepatic microsome sample (HG24) from our panel that had a large CYP2A6 and CYP2B6 catalytic capacity. The combination of MAb-2A6 and MAb-2B6 yielded greater inhibition of 4-ene-VPA (21%, Fig. 4A), 4-OH-VPA (24%, Fig. 4B), and 5-OH-VPA formation (24%, Fig. 4C), when compared to the effect by each antibody when added alone. In contrast, the effect on 3-OH-VPA formation by the combination of MAb-2A6 and MAb-2B6 was similar to that by MAb-2A6 alone, in accord with the lack of inhibition by MAb-2B6 (Fig. 4D). The combination of MAb-2A6, MAb-2B6, and MAb-2C9 decreased the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 92 (Fig. 4A), 85 (Fig. 4B), and 88% (Fig. 4C), respectively.
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Association Between the Extent of Immunoinhibition of VPA Metabolism by MAb-2B6 and MAb-2A6 and the Levels of CYP2B6- and CYP2A6-Mediated Enzyme Activities
We determined whether the extent of contribution by hepatic microsomal CYP2B6 and CYP2A6 to VPA metabolism was associated with the inherent catalytic capacity of these enzymes in microsomes. Positive correlation was obtained between MAb-2B6 inhibition of 4-OH-VPA formation and CYP2B6-mediated (S)-mephenytoin N-demethylation activity (r2 = 0.79, p = 0.001, Fig. 5A), MAb-2B6 inhibition of 5-OH-VPA formation and CYP2B6-mediated (S)-mephenytoin N-demethylation activity (r2 = 0.79, p = 0.002, Fig. 5B), and MAb-2A6 inhibition of 3-OH-VPA formation and CYP2A6-mediated coumarin 7-hydroxylation activity (r2 = 0.58, p = 0.02, Fig. 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies with cDNA-expressed enzymes showed that CYP2C9 catalyzed the formation of 4-ene-VPA (Anari et al., 2000
; Ho et al., 2003; Sadeque et al., 1997). The present study confirms this finding and further demonstrates that cDNA-expressed CYP2C9 catalyzes VPA 4-hydroxylation and VPA 5-hydroxylation. In the only study with human hepatic microsomes reported to date, it was concluded that CYP2C9 played a role in the formation of 4-ene-VPA (Sadeque et al., 1997), based on experiments with sulfaphenazole, a CYP2C9-selective inhibitor (Newton et al., 1995). However, that study was performed with microsome samples from two individuals, and their CYP2C9 genotype was not known (Sadeque et al., 1997). The percentage decrease in 4-ene-VPA formation by sulfaphenazole was 43% in one microsome sample but 15% in the other sample. The present study was conducted with hepatic microsomes obtained from donors with the CYP2C9*1/*1 genotype. A novel finding is that CYP2C9*1 is the principal human hepatic microsomal CYP catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA. This conclusion was based on our immunoinhibition data showing that MAb-2C9 decreased the group mean formation of each of these VPA metabolites by 75–80%, suggesting that CYP2C9*1 was largely (i.e., 75–80%) responsible for hepatic microsomal formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA. Consistent with this conclusion are the findings that (1) a CYP2C9-selective inhibitor, sulfaphenazole, reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA (Figs. 2A–2C); (2) CYP2C9-mediated diclofenac 4'-hydroxylation activity correlated with 4-ene-VPA formation (Table 2); and (3) VPA competitively inhibited CYP2C9-mediated tolbutamide hydroxylation activity in human hepatic microsomes (apparent Ki = 0.6mM) (Wen et al., 2001).
The contribution of CYP2A6 and CYP2B6 to the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA was minimal in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype, as judged by the results from the immunoinhibition experiments with MAb-2A6 and MAb-2B6 and the chemical inhibition experiments with coumarin (to inhibit CYP2A6) (Messina et al., 1997) and thio-TEPA (to inhibit CYP2B6) (Rae et al., 2002). According to our combinatorial immunoinhibition analysis, CYP2A6 and CYP2B6 together accounted for 20–25% of the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in the hepatic microsome sample that was analyzed. However, interindividual variability existed in the contribution of CYP2B6 to these catalytic reactions, as illustrated by the positive correlation between CYP2B6-mediated (S)-mephenytoin N-demethylation activity and the percentage inhibition of VPA 4-hydroxylation (or VPA 5-hydroxylation) by MAb-2B6 in hepatic microsomes. The basis for this variability is the substantial interindividual differences in hepatic CYP2B6 expression (Code et al., 1997), which is due, in part, to the inducibility of this enzyme by drugs and other chemicals (Chang et al., 1997). Pharmacogenetics is another factor that may influence the relative contribution of specific CYP enzymes to a drug metabolism reaction. As an example, the CYP2C9*2 and CYP2C9*3 alleles are associated with substantially decreased VPA oxidative metabolism (Ho et al., 2003). Therefore, in an individual with a CYP2C9 poor metabolizer phenotype, CYP2A6 and CYP2B6 may account for a greater proportion of the enzymatic activity for the terminal desaturation, 4-hydroxylation, and 5-hydroxylation of VPA.
Our experiment with individual cDNA-expressed enzymes also identified other human CYP catalysts of VPA 4-hydroxylation and VPA 5-hydroxylation. However, the extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1 CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1–8% of the levels by CYP2C9*1. Among these enzymes, CYP1A2, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, and CYP4F3B are expressed in human liver (Christmas et al., 2001; Donato and Castell, 2003). However, the contribution of these enzymes to 4-OH-VPA and 5-OH-VPA formation in human hepatic microsomes was likely minimal or negligible because as shown by our combinatorial immunoinhibition analysis, CYP2C9, CYP2A6, and CYP2B6 were responsible for virtually all the VPA 4-hydroxylation and VPA 5-hydroxylation activities in human hepatic microsomes. Similarly, CYP1A1, CYP1B1, and CYP4F3A should not have accounted for any of the hepatic microsomal VPA 4-hydroxylation and VPA 5-hydroxylation activities because these enzymes are not expressed in human liver (Chang et al., 2003; Christmas et al., 2001).
An earlier study implicated CYP enzymes in the metabolism of VPA to form 3-OH-VPA (Prickett and Baillie, 1984). Another novel finding in the present study is that cDNA-expressed human CYP1A1, CYP2A6, CYP2B6, CYP4F2, and CYP4F3B are active catalysts of VPA 3-hydroxylation. CYP2A6 contributed approximately 50% to VPA 3-hydroxylation activity in human hepatic microsomes, based on the immunoinhibition experiment with MAb-2A6. The extent of contribution by CYP2A6 was associated with the inherent catalytic activity in each microsome sample, as illustrated by the positive correlation between the percentage inhibition of 3-OH-VPA formation by MAb-2A6 and CYP2A6-mediated coumarin 7-hydroxylation activity. A role for hepatic microsomal CYP2A6 in VPA 3-hydroxylation is supported by our findings that (1) an inhibitor of CYP2A6 activity, coumarin (Messina et al., 1997), reduced the formation of 3-OH-VPA in human hepatic microsomes (Fig. 2D) and (2) CYP2A6-mediated coumarin 7-hydroxylation activity correlated with the formation of 3-OH-VPA in human hepatic microsomes (r2 = 0.47, p = 0.02, Table 2). Consistent with these results is the finding that VPA is a mechanism-based inactivator of CYP2A6 (Kinact = 0.9mM) (Wen et al., 2001). The remainder of the VPA 3-hydroxylation activity in human hepatic microsomes was not due to CYP2B6 because the formation of 3-OH-VPA was not decreased by MAb-2B6 or a CYP2B6-selective chemical inhibitor (thio-TEPA). A role for CYP1A1 can also be ruled out because this enzyme is not expressed in human liver (Chang et al., 2003). Candidate enzymes include CYP4F2 and CYP4F3B, which catalyze the -hydroxylation of arachidonic acid (Powell et al., 1998) and leukotriene B4 (Christmas et al., 2001), respectively, and are expressed in human liver. The contribution of these two enzymes to VPA 3-hydroxylation in human hepatic microsomes was not determined because inhibitory antibody specific for CYP4F2 or CYP4F3B was not available.
Limited information is available on the role of other mammalian CYP enzymes in VPA metabolism. Immunologically purified rat CYP2B1 catalyzes the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA with a ratio of 1:37:5 (Rettie et al., 1995). This pronounced preference for VPA 4-hydroxylation by rat CYP2B1 was not apparent in our experiment with cDNA-expressed human CYP2B6, which yielded a ratio of 1:47:51. In contrast to rat CYP2B1, purified rabbit CYP4B1 has a greater preference for VPA 5-hydroxylation (a ratio of 1:2:117 for the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA) (Rettie et al., 1995). However, among the human CYP enzymes investigated in the present study, none of them showed a preference for VPA 5-hydroxylation over VPA 4-hydroxylation. Rat CYP3A enzymes account for the majority of VPA 3-hydroxylation activity in hepatic microsomes from rats treated with pregnenolone 16
-carbonitrile (an inducer of CYP3A) (Fisher et al., 1998). However, as shown in the present study, human CYP3A4 and CYP3A5 did not metabolize VPA, in accord with previous findings (Anari et al., 2000; Sadeque et al., 1997). Collectively, these results suggest species-dependent effects of CYP enzymes on VPA metabolism.
The biotransformation of VPA involves glucuronidation, ß-oxidation, and CYP-catalyzed terminal desaturation and hydroxylation (Abbott and Anari, 1999). Although the CYP-catalyzed metabolism of VPA is quantitatively minor relative to the other two pathways, it is of toxicological interest because of the formation of 4-ene-VPA. As shown in previous studies conducted in cultured rat hepatocytes (Jurima-Romet et al., 1996; Kingsley et al., 1983) and in rats in vivo (Kesterson et al., 1984; Loscher et al., 1993), signs and symptoms of hepatic injury could be demonstrated under specific experimental conditions by the direct administration of relatively high doses of 4-ene-VPA. However, it is not clear from human studies whether the in situ concentrations of the enzymatically formed 4-ene-VPA can account for the hepatotoxicity in patients administered with VPA (Nau et al., 1991; Siemes et al., 1993). This may relate to the notion that the hepatotoxicity of 4-ene-VPA is due to reactive metabolites produced by the ß-oxidation of 4-ene-VPA (Tang et al., 1995). Future studies are needed to investigate directly whether modulation of CYP-catalyzed formation of 4-ene-VPA and the subsequent alteration in the levels of ß-oxidation–derived reactive metabolites would influence the extent of toxicity in cultured human hepatocytes treated with VPA.
In summary, based on the in vitro VPA (1mM) metabolism assay conducted with human hepatic microsomes from individuals with the CYP2C9*1/*1 genotype, (1) CYP2C9*1 was responsible for the majority (75–80%) of VPA terminal desaturation, VPA 4-hydroxylation, and VPA 5-hydroxylation activities, whereas CYP2A6 and CYP2B6 contributed to the remainder of these reactions; (2) CYP2A6 accounted for approximately 50% of VPA 3-hydroxylation activity; and (3) the extent by which CYP2A6 and CYP2B6 contributed to VPA oxidative metabolism was associated with the inherent catalytic capacity of these enzymes in each microsome sample.
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NOTES |
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1 Present address: School of Pharmacy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
2 Present address: Genelabs Technologies, Inc., Redwood City, CA 94063-4737.
3 Present address: Department of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065.
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ACKNOWLEDGMENTS |
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We thank Dr H. V. Gelboin and his colleagues at the National Cancer Institute, National Institutes of Health for the generous provision of the MAbs. This research was supported by grant MOP-13744 (to F.S.A. and T.K.H.C.) from the Canadian Institutes of Health Research (CIHR). T.K.L.K. received a CIHR Canada Graduate Scholarship Award and a Michael Smith Foundation for Health Research Incentive Award. V.T. received a CIHR Doctoral Research Award.
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