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The influence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic acid in epileptic patients
Clinical Neurology and Neurosurgery 112 (2010) 320–323
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
The influence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic acid in epileptic patients Lan Tan a,b,∗ , Jin-Tai Yu a,b , Yan-Ping Sun c , Jiang-Rong Ou a,b , Jing-Hui Song c , Yang Yu c a
Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, Shandong Province 266071, China Department of Neurology, Qingdao Municipal Hospital, School of Medicine and Pharmaceutics, Ocean University of China, 266071, China c Department of Neurology, the Affiliated Hospital of the Medical College of Qingdao University, Qingdao 266003, Shandong Province, China b
a r t i c l e
i n f o
Article history: Received 15 April 2009 Received in revised form 10 November 2009 Accepted 5 January 2010 Available online 20 January 2010 Keywords: Cytochrome P450 Genetic polymorphism Valproic acid Plasma concentration Epilepsy
a b s t r a c t Objectives: To investigate influences of the functional polymorphisms of Cytochrome P450 isozymes 2A6 (CYP2A6), 2B6 (CYP2B6), and 2C9 (CYP2C9) on pharmacokinetics of VPA in vivo. Patients and methods: In the study, we analyzed the genotypes of CYP2A6, CYP2B6, and CYP2C9 and their contribution to the steady-state standardized plasma VPA concentrations in 179 subjects with epilepsy of a Northern Han Chinese population. The genotypes were detected by the polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Results: The subjects with one or two variant CYP2A6*4 alleles showed higher mean plasma VPA concentrations compared with non-*4 alleles [(3.4 ± 0.4) g kg ml−1 mg−1 vs. (3.6 ± 0.4) g kg ml−1 mg−1 , p = 0.0055]. A significant difference [one-way ANOVA (p = 0.0203)] was also found between mean plasma VPA concentrations and the CYP2B6 genotypes. In addition, subjects with the heterozygous genotype CYP2C9*3 had higher mean plasma VPA concentrations than did those subjects with the wild-type genotype [(3.9 ± 0.4) g kg ml−1 mg−1 vs. (3.4 ± 0.4) g kg ml−1 mg−1 , p = 0.0001]. Conclusion: The presently evaluated variant alleles in the CYP2A6, CYP2B6, and CYP2C9 genes may explain part of the substantial variability in VPA pharmacokinetics between different subjects. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Valproic acid (VPA) is one of the major antiepileptic drugs (AEDs) for treatment of generalized or partial epilepsy. Moreover, its use is also increasing in the treatment of other brain diseases, including migraine and bipolar disorders [1]. Following its introduction in 1960, many studies showed that there was a poor correlation between serum VPA concentration and dosage, which could be partially attributed to pharmacogenetics [2,3]. Valproic acid is eliminated via extensive hepatic metabolism to several metabolites [4]. Studies conducted in vitro indicated that Cytochrome P450 isozymes 2A6 (CYP2A6), 2B6 (CYP2B6), and 2C9 (CYP2C9) catalyzed VPA terminal desaturation and hydroxylation, but little or no metabolite was formed by several other CYP enzymes [5,6]. CYP2A6, CYP2B6 and CYP2C9 are members of the human Cytochrome P-450 enzyme (CYPs) super-family. The three
∗ Corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: +86 532 8890 5659; fax: +86 532 85968434. E-mail address: [email protected] (L. Tan). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.01.002
enzymes are known to be genetically polymorphic [7]. Several studies have indicated that some mutations in the three enzymes are determinants of significantly impaired metabolism in many of their substrates [8–12]. In addition, Ho et al. reported that the CYP2C9*2, CYP2C9*3 alleles were associated with substantially decreased VPA oxidative metabolism in human liver microsomes [13]. However, there have been few studies, in vivo, investigating influences of the functional status of CYP2A6, CYP2B6 and CYP2C9 on pharmacokinetics of VPA. In the present study, we analyzed genotypes of CYP2A6, CYP2B6, CYP2C9 and their influence on the steady-state plasma VPA concentrations in subjects with epilepsy. 2. Patients and methods 2.1. Patients and sampling procedures This study was carried out between July, 2007 and February, 2008 in 179 epileptic outpatients who manifest no abnormal findings for hepatic and renal function tests at the Department of Neurology at Qingdao Municipal Hospital and the Department of Neurology at the Affiliated Hospital of the Medical College of Qingdao University. All of the subjects were Northern Han Chinese in
L. Tan et al. / Clinical Neurology and Neurosurgery 112 (2010) 320–323 Table 1 CYP2A6, CYP2B6 and CYP2C9 genotypes frequencies of study population.
321
2.3. Plasma VPA concentration determination
Genotype
N (%)
95% CI (of %)
CYP2A6*4 Nil Heterozygous Homozygous
133 (74.3) 44 (24.6) 2 (1.1)
67.4–80.5 18.5–31.6 0.1–3.9
CYP2B6*6 Nil Hetero Homo
119 (66.5) 51 (28.5) 9 (5.0)
59.1–73.3 22.0–35.7 2.3–9.3
CYP2C9*3 Nil Heterozygous Homozygous
164 (91.6) 15 (8.4) 0
86.6–95.2 4.8–13.4 –
Steady-state plasma valproate ion concentration (C) was measured in duplicate by the fluorescence polarization immunoassay (FPIA) method (TDX® , Abbott Laboratories Ltd, USA) and the average value employed. 2.4. Statistical analysis
Note: 95%CI, 95% confidence intervals.
origin. They had all been treated with the anticonvulsant drug VPA as mono-therapy for at least 6 months and all were on a stable dosage regimen [after approximately 100 h period continuous dose of a constant regimen (5 half-life periods)]. Blood samples (5 ml) for drug assays were usually obtained about 12 h after the evening dose. The blood was transferred immediately to a tube containing ethylenediaminetetraacetic acid (EDTA) and the plasma was separated within 3 h and stored at −20 ◦ C until assay. A 3 ml blood sample for CYP2A6, CYP2B6, and CYP2C9 genotyping was collected at the same time and stored with EDTA at −20 ◦ C. Each subject or his representative gave their informed consent to participate in this study, and the protocol of this study was approved by the Ethics Committee for the Study of Human Genome in each hospital. 2.2. Genotyping Genomic DNA was isolated from peripheral blood leukocytes using standard procedures [14]. The CYP2A6*4 allele associated with the poor metabolizer phenotype were determined by nestedprimer polymerase chain reaction (PCR) as described by Oscarson et al. [15]. The CYP2B6*6 allele was determined by polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) tests as described by Lang et al. [16]. Amplification products were digested with BsrI for G516T and StyI for A785G. In addition, genotyping for CYP2C9*3 was also performed by PCR-RFLP analysis according to Yasar et al. [17]. And, the amplification product was digested with NsiI for A1075C. Because CYP2C9*2 polymorphism has not been identified in a Chinese population, we did not undertake genetic testing for CYP2C9*2 [16].
Statistical analysis was performed using SPSS statistical software, version 11.5 for windows (SPSS Inc., Chicago, IL, USA). To eliminate weight and dose influence on the C, standardization of valproate ion concentration (Cs) was performed [Cs = C/(dairy dose/weight)]. Differences in the characteristics of the study population between various genotypes were examined using the Student’s t-test or the Chi-square test. Sex difference was compared using the Chi-square test, and age difference was compared using the Student’s t-test. Differences in values between various genotypes were estimated statistically by using the Student’s t-test or one-way ANOVA. If the one-way analysis of variance (ANOVA) was significant (p < 0.05), post hoc Scheffe’s F test was applied. p < 0.05 was considered statistically significant. 3. Results 3.1. Frequency of genotypic variants of CYP2A6, CYP2B6, and CYP2C9 To reduce the effect of racial and ethnic bases of stratification, we restricted the analyses to a group of 179 subjects with epilepsy from a Northern Han Chinese population. The CYP2A6, CYP2B6, and CYP2C9 genotype distributions in the population are presented in Table 1. These data correspond to allele frequencies of 13.4% (95% confidence intervals/CI = 10.1–17.4%) for CYP2A6*4, 19.0% (95%CI = 15.3–23.7%) for CYP2B6*6 and 4.7% (95%CI = 2.4–6.8%) for CYP2C9*3, which is comparable to the frequencies reported in other studies of Chinese populations [15,18,19]. In our sample, no subjects homozygous for the CYP2C9*3 allele were present. The frequencies of the genotypes of CYP2A6, CYP2B6, and CYP2C9 alleles were within the 95% confidence interval estimated by the HardyWeinberg equation [20]. 3.2. Correlation between the genotypic variants and VPA concentrations As shown in Table 2, the differences between the characteristics of subjects in each genotype were not significant, indicating
Table 2 Demographics of the study population and steady-state normalized plasma VPA concentrations as a function of CYP2A6, CYP2B6, and CYP2C9 genotypes. Genotype CYP2A6*4 Nil Carriers P values CYP2B6*6 Nil Heterozygous Homozygous P values CYP2C9*3 Nil Heterozygous P values
Age (years) (mean ± SD)
Cs (g kg ml−1 mg−1 ) (mean ± SD)
5–49 4–57
23.6 ± 3.3 23.0 ± 2.2 NS
3.4 ± 0.4 3.6 ± 0.4 0.0055
61(51.3) 29(56.9) 5 (55.6) NS
4–47 5–57 10–45
20.2 ± 3.4 23.1 ± 2.2 21.5 ± 4.1 NS
3.4 ± 0.5 3.5 ± 0.3 3.7 ± 0.4 0.0203
86(52.4) 9(60.0) NS
5–57 4–52
24.2 ± 2.3 21.5 ± 3.2 NS
3.4 ± 0.4 3.9 ± 0.4 0.0001
No. (%)
Sex, male (%)
133(74.3) 46 (25.7)
73(54.9) 22(47.8) NS
119(66.5) 51 (28.5) 9 (5.0)
164(91.6) 15 (8.4)
Age range (years)
Notes: Carriers, those of either heterozygous (CYP2A6*4) or homozygous (CYP2A6*4) genotypes. NS, not significant.
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L. Tan et al. / Clinical Neurology and Neurosurgery 112 (2010) 320–323
that these characteristics did not influence the variations of VPA concentrations between the genotypes. The statistical analysis revealed significant associations between the steady-state standardized plasma concentrations of VPA with the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles tested in the study. For the CYP2A6*4 allele, subjects with one or two variant CYP2A6*4 alleles showed higher mean plasma VPA concentrations compared with non-*4 alleles [(3.3835 ± 0.4358) g kg ml−1 mg−1 vs. (3.5897 ± 0.4074) g kg ml−1 mg−1 , p = 0.0055]. A significant difference [one-way ANOVA (p = 0.0203)] was also found between mean plasma VPA concentrations and the CYP2B6 genotypes. The post hoc Scheffe’s F test showed a statistically significant difference in mean plasma VPA concentration between subjects carrying the *6/*6 genotype and non-*6 alleles (p = 0.0321), while the difference was not statistically significant between subjects with CYP2B6 *6/*6 genotype and subjects with *6 heterozygous genotype (p = 0.0833). Moreover, the difference was also not statistically significant between subjects with *6 heterozygous genotype (p = 0.0612). Similarly, we found that subjects with the heterozygous genotype CYP2C9*3 had higher mean plasma VPA concentrations than did those subjects with the wild-type genotype [(3.9 ± 0.4) g kg ml−1 mg−1 vs. (3.4 ± 0.4) g kg ml−1 mg−1 , p = 0.0001]. The subjects with CYP2A6*4 and CYP2C9*3 alleles showed 6.4% and 13.5% higher mean dose-corrected plasma VPA concentrations compared with these with non-carriers of them, respectively. And, there were 10.3% differences about the mean dose-corrected plasma VPA concentrations between subjects with the *6/*6 genotype and subjects with non-*6 alleles.
way and cytochrome P-450 (CYP) way, accounting for 50%, 40%, and 10% respectively of a dose [26]. This study suggested that CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles influenced the steadystate standardized plasma concentrations of VPA. Subjects with the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles showed higher steadystate standardized plasma concentrations of VPA than those of subjects without them. Previous studies have documented that CYP2C9 accounts for 75–80% of the formation of 4-ene-VPA, 4-OHVPA, and 5-OH-VPA, with CYP2A6 and CYP2B6 together accounting for 20–25% in vitro [6]. In addition, in an individual with a CYP2C9 poor metabolizer phenotype, CYP2A6 and CYP2B6 may account for a greater proportion of the enzymatic activity for the 4-hydroxylation and 5-hydroxylation terminal desaturation of VPA. Further studies investigating other polymorphisms, such as UGT1A6, UGT1A8, UBG2B7, are needed. To our knowledge, the present study is the first attempt to establish the influence of CYP2A6, CYP2B6, and CYP2C9 polymorphism on VPA metabolism in a Chinese population. In summary, our major findings indicate that the presently evaluated variant alleles in the CYP2A6, CYP2B6, and CYP2C9 genes may explain a part of the statistically substantial interindividual variability in VPA pharmacokinetics (i.e. 6–14%), and subjects harboring the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles may have enhanced exposure to VPA. Further studies in larger or other ethnic populations are needed to replicate our findings regarding this association. In addition, it is also urgent need to do a larger study that quantifies the impact of a host of factors on the inter- and intraindividual variability of VPA concentrations in the future. Acknowledgements
4. Discussion Pharmacogenetics is a factor that may influence the relative contribution of specific CYP enzymes to a drug metabolism reaction. It gives rise to important interindividual and interethnic variability in the metabolism and disposition and clinical response of several therapeutic agents. Many studies have reported that the CYP2A6, CYP2B6, and CYP2C9 enzymes activity were dramatically reduced in the homozygotes for CYP2A6*4, CYP2B6*6 or CYP2C9*3 or the heterozygotes with these alleles, respectively [15,21,22]. CYP2A6*4 allele deletes the whole CYP2A6 gene [15]. CYP2B6*6 allele is known to be associated with lower levels of CYP2B6 expression in the liver [21]. The CYP2C9*3 has an exchange of A1075C in exon 7 causing an Ile359Leu substitution. One study reported that the variant CYP2C9*3 coding for enzyme with approximately 5% of the activity of CYP2C9*1, which was apparently a result of the amino acid substitution altering the interaction of the enzyme with Cytochrome P450 oxidoreductase [22]. Consequently, the high mean plasma VPA concentration of subjects with CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles may be explained by the low enzymatic activity of these respective genotypes. Previous genotyping analysis had revealed significantly different frequencies of the CYP2A6, CYP2B6 and CYP2C9 mutations in Chinese when compared to other ethnic populations such as Caucasians, Afro-Americans or Japanese, especially that comparison of CYP2A6*4 allele in Chinese to that in Caucasians is 15.1% to 0.5–1.0% [15,18,19,23–25]. The frequencies of CYP2A6*4, CYP2B6*6 and CYP2C9*3 are the highest mutant of CYP2A6, CYP2B6 and CYP2C9 gene isoforms, respectively, in the Chinese population [15,18,19]. So, it has special significance to study the clinical relevance of the CYP2A6*4, CYP2B6*6 and CYP2C9*3 alleles in the Chinese population. Metabolism by CYP pathway is not a prominent route of VPA elimination. The biotransformation of VPA involves three major metabolic ways, including uridine diphosphate glucuronosyltransferases (UDPGT) mediated pathway, mitochondrial -oxidation
We are grateful to all of the subjects who kindly agreed to participate in this study. We thank Prof. John G Nicholls (SISSA, Trieste, Italy) for critically reading this manuscript. The study was supported by grants from the Shandong Province Nature Science Foundation (Y2007C123) and National Natural Science Foundation of China (30870884). References [1] Rogawski MA, Löscher W. The neurobiology of antiepileptic drugs for the treatment of nonepileptic conditions. Nat Med 2004;10:685–92. [2] Shorvon S. Handbook of epilepsy treatment. second ed. Oxford: Blackwell; 2005. [3] Klotz U. The role of pharmacogenetics in the metabolism of antiepileptic drugs: pharmacokinetic and therapeutic implications. Clin Pharmacokinet 2007;46:271–9. [4] Davis R, Peters DH, McTavish D. Valproic acid: a reappraisal of its pharmacological properties and clinical efficacy in epilepsy. Drugs 1994;47:332–72. [5] Anari MR, Burton RW, Gopaul S, Abbott FS. Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography-mass spectrometry. J Chromatogr B 2000;742:217–27. [6] Kiang TK, Ho PC, Anari MR, Tong V, Abbott FS, Chang TK. Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype. Toxicol Sci 2006;94:261–71. [7] Ingelman-Sundberg M, Sim SC, Gomez A, Rodriguez-Antona C. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacol Ther 2007;116:496–526. [8] Nakajima M, Fukami T, Yamanaka H, Higashi E, Sakai H, Yoshida R, et al. Comprehensive evaluation of variability in nicotine metabolism and CYP2A6 polymorphic alleles in four ethnic populations. Clin Pharmacol Ther 2006;80:282–97. [9] Nakajima M, Komagata S, Fujiki Y, Kanada Y, Ebi H, Itoh K, et al. Genetic polymorphisms of CYP2B6 affect the pharmacokinetics/pharmacodynamics of cyclophosphamide in Japanese cancer patients. Pharmacogenet Genomics 2007;17:431–45. [10] Crettol S, Déglon JJ, Besson J, Croquette-Krokkar M, Gothuey I, Hämmig R, et al. Methadone enantiomer plasma levels, CYP2B6, CYP2C19, and CYP2C9 genotypes, and response to treatment. Clin Pharmacol Ther 2005;78:593–604. [11] Tsuchiya K, Gatanaga H, Tachikawa N, Teruya K, Kikuchi Y, Yoshino M, et al. Homozygous CYP2B6 *6 (Q172H and K262R) correlates with high plasma efavirenz concentrations in HIV-1 patients treated with standard efavirenzcontaining regimens. Biochem Biophys Res Commun 2004;319:1322–6.
L. Tan et al. / Clinical Neurology and Neurosurgery 112 (2010) 320–323 [12] Kirchheiner J, Brockmöller J. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther 2005;77:1–16. [13] Ho PC, Abbott FS, Zanger UM, Chang TK. Influence of CYP2C9 genotypes on the formation of a hepatotoxic metabolite of valproic acid in human liver microsomes. Pharmacogenomics J 2003;3:335–42. [14] Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal Biochem 1988;174:485–8. [15] Oscarson M, McLellan RA, Gullstén H, Yue QY, Lang MA, Bernal ML, et al. PCRbased detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett 1999;448:105–10. [16] Lang T, Klein K, Fischer J, Nüssler AK, Neuhaus P, Hofmann U, et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 2001;11:399–415. [17] Yasar U, Eliasson E, Dahl ML, Johansson I, Ingelman-Sundberg M, Sjöqvist F. Validation of methods for CYP2C9 genotyping: frequencies of mutant alleles in a Swedish population. Biochem Biophys Res Commun 1999;254:628–31. [18] Yu BN, Luo CH, Wang D, Wang A, Li Z, Zhang W, et al. CYP2C9 allele variants in Chinese hypertension patients and healthy controls. Clin Chim Acta 2004;348:57–61. [19] Guan S, Huang M, Li X, Chen X, Chan E, Zhou SF. Intra- and inter-ethnic differences in the allele frequencies of cytochrome P450 2B6 gene in Chinese. Pharm Res 2006;23:1983–90.
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[20] Terwilliger JD, Ott J. Handbook of human genetic linkage. New York: John Hopkins Univ. Press; 1994. [21] Xie HJ, Yasar U, Lundgren S, Griskevicius L, Terelius Y, Hassan M, et al. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation. Pharmacogenomics J 2003;3:53–61. [22] Haining RL, Hunter AP, Veronese ME, Trager WF, Rettie AE. Allele variants of human cytochrome P450 2C9: baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms. Arch Biochem Biophys 1996;333:447–58. [23] Hannu R, Arja R, Harriet G, Olavi P. Polymorphisms of CYP2A6 and its practical consequences. Br J Clin Pharmacol 2001;52:357–63. [24] Hiratsuka M, Takekuma Y, Endo N, Narahara K, Hamdy SI, Kishikawa Y, et al. Allele and genotype frequencies of CYP2B6 and CYP3A5 in the Japanese population. Eur J Clin Pharmacol 2002;58:417–21. [25] Sipeky C, Lakner L, Szabo M, Takacs I, Tamasi V, Polgar N, et al. Interethnic differences of CYP2C9 alleles in healthy Hungarian and Roma population samples: relationship to worldwide allelic frequencies. Blood Cells Mol Dis 2009;43:239–42. [26] Devane CL. Pharmacokinetics, drug interactions, and tolerability of valproate. Psychopharmacol Bull 2003;37:25–42.
Contents lists available at ScienceDirect
Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
The influence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic acid in epileptic patients Lan Tan a,b,∗ , Jin-Tai Yu a,b , Yan-Ping Sun c , Jiang-Rong Ou a,b , Jing-Hui Song c , Yang Yu c a
Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, Shandong Province 266071, China Department of Neurology, Qingdao Municipal Hospital, School of Medicine and Pharmaceutics, Ocean University of China, 266071, China c Department of Neurology, the Affiliated Hospital of the Medical College of Qingdao University, Qingdao 266003, Shandong Province, China b
a r t i c l e
i n f o
Article history: Received 15 April 2009 Received in revised form 10 November 2009 Accepted 5 January 2010 Available online 20 January 2010 Keywords: Cytochrome P450 Genetic polymorphism Valproic acid Plasma concentration Epilepsy
a b s t r a c t Objectives: To investigate influences of the functional polymorphisms of Cytochrome P450 isozymes 2A6 (CYP2A6), 2B6 (CYP2B6), and 2C9 (CYP2C9) on pharmacokinetics of VPA in vivo. Patients and methods: In the study, we analyzed the genotypes of CYP2A6, CYP2B6, and CYP2C9 and their contribution to the steady-state standardized plasma VPA concentrations in 179 subjects with epilepsy of a Northern Han Chinese population. The genotypes were detected by the polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Results: The subjects with one or two variant CYP2A6*4 alleles showed higher mean plasma VPA concentrations compared with non-*4 alleles [(3.4 ± 0.4) g kg ml−1 mg−1 vs. (3.6 ± 0.4) g kg ml−1 mg−1 , p = 0.0055]. A significant difference [one-way ANOVA (p = 0.0203)] was also found between mean plasma VPA concentrations and the CYP2B6 genotypes. In addition, subjects with the heterozygous genotype CYP2C9*3 had higher mean plasma VPA concentrations than did those subjects with the wild-type genotype [(3.9 ± 0.4) g kg ml−1 mg−1 vs. (3.4 ± 0.4) g kg ml−1 mg−1 , p = 0.0001]. Conclusion: The presently evaluated variant alleles in the CYP2A6, CYP2B6, and CYP2C9 genes may explain part of the substantial variability in VPA pharmacokinetics between different subjects. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Valproic acid (VPA) is one of the major antiepileptic drugs (AEDs) for treatment of generalized or partial epilepsy. Moreover, its use is also increasing in the treatment of other brain diseases, including migraine and bipolar disorders [1]. Following its introduction in 1960, many studies showed that there was a poor correlation between serum VPA concentration and dosage, which could be partially attributed to pharmacogenetics [2,3]. Valproic acid is eliminated via extensive hepatic metabolism to several metabolites [4]. Studies conducted in vitro indicated that Cytochrome P450 isozymes 2A6 (CYP2A6), 2B6 (CYP2B6), and 2C9 (CYP2C9) catalyzed VPA terminal desaturation and hydroxylation, but little or no metabolite was formed by several other CYP enzymes [5,6]. CYP2A6, CYP2B6 and CYP2C9 are members of the human Cytochrome P-450 enzyme (CYPs) super-family. The three
∗ Corresponding author at: Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, No.5 Donghai Middle Road, Qingdao, Shandong Province 266071, China. Tel.: +86 532 8890 5659; fax: +86 532 85968434. E-mail address: [email protected] (L. Tan). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.01.002
enzymes are known to be genetically polymorphic [7]. Several studies have indicated that some mutations in the three enzymes are determinants of significantly impaired metabolism in many of their substrates [8–12]. In addition, Ho et al. reported that the CYP2C9*2, CYP2C9*3 alleles were associated with substantially decreased VPA oxidative metabolism in human liver microsomes [13]. However, there have been few studies, in vivo, investigating influences of the functional status of CYP2A6, CYP2B6 and CYP2C9 on pharmacokinetics of VPA. In the present study, we analyzed genotypes of CYP2A6, CYP2B6, CYP2C9 and their influence on the steady-state plasma VPA concentrations in subjects with epilepsy. 2. Patients and methods 2.1. Patients and sampling procedures This study was carried out between July, 2007 and February, 2008 in 179 epileptic outpatients who manifest no abnormal findings for hepatic and renal function tests at the Department of Neurology at Qingdao Municipal Hospital and the Department of Neurology at the Affiliated Hospital of the Medical College of Qingdao University. All of the subjects were Northern Han Chinese in
L. Tan et al. / Clinical Neurology and Neurosurgery 112 (2010) 320–323 Table 1 CYP2A6, CYP2B6 and CYP2C9 genotypes frequencies of study population.
321
2.3. Plasma VPA concentration determination
Genotype
N (%)
95% CI (of %)
CYP2A6*4 Nil Heterozygous Homozygous
133 (74.3) 44 (24.6) 2 (1.1)
67.4–80.5 18.5–31.6 0.1–3.9
CYP2B6*6 Nil Hetero Homo
119 (66.5) 51 (28.5) 9 (5.0)
59.1–73.3 22.0–35.7 2.3–9.3
CYP2C9*3 Nil Heterozygous Homozygous
164 (91.6) 15 (8.4) 0
86.6–95.2 4.8–13.4 –
Steady-state plasma valproate ion concentration (C) was measured in duplicate by the fluorescence polarization immunoassay (FPIA) method (TDX® , Abbott Laboratories Ltd, USA) and the average value employed. 2.4. Statistical analysis
Note: 95%CI, 95% confidence intervals.
origin. They had all been treated with the anticonvulsant drug VPA as mono-therapy for at least 6 months and all were on a stable dosage regimen [after approximately 100 h period continuous dose of a constant regimen (5 half-life periods)]. Blood samples (5 ml) for drug assays were usually obtained about 12 h after the evening dose. The blood was transferred immediately to a tube containing ethylenediaminetetraacetic acid (EDTA) and the plasma was separated within 3 h and stored at −20 ◦ C until assay. A 3 ml blood sample for CYP2A6, CYP2B6, and CYP2C9 genotyping was collected at the same time and stored with EDTA at −20 ◦ C. Each subject or his representative gave their informed consent to participate in this study, and the protocol of this study was approved by the Ethics Committee for the Study of Human Genome in each hospital. 2.2. Genotyping Genomic DNA was isolated from peripheral blood leukocytes using standard procedures [14]. The CYP2A6*4 allele associated with the poor metabolizer phenotype were determined by nestedprimer polymerase chain reaction (PCR) as described by Oscarson et al. [15]. The CYP2B6*6 allele was determined by polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) tests as described by Lang et al. [16]. Amplification products were digested with BsrI for G516T and StyI for A785G. In addition, genotyping for CYP2C9*3 was also performed by PCR-RFLP analysis according to Yasar et al. [17]. And, the amplification product was digested with NsiI for A1075C. Because CYP2C9*2 polymorphism has not been identified in a Chinese population, we did not undertake genetic testing for CYP2C9*2 [16].
Statistical analysis was performed using SPSS statistical software, version 11.5 for windows (SPSS Inc., Chicago, IL, USA). To eliminate weight and dose influence on the C, standardization of valproate ion concentration (Cs) was performed [Cs = C/(dairy dose/weight)]. Differences in the characteristics of the study population between various genotypes were examined using the Student’s t-test or the Chi-square test. Sex difference was compared using the Chi-square test, and age difference was compared using the Student’s t-test. Differences in values between various genotypes were estimated statistically by using the Student’s t-test or one-way ANOVA. If the one-way analysis of variance (ANOVA) was significant (p < 0.05), post hoc Scheffe’s F test was applied. p < 0.05 was considered statistically significant. 3. Results 3.1. Frequency of genotypic variants of CYP2A6, CYP2B6, and CYP2C9 To reduce the effect of racial and ethnic bases of stratification, we restricted the analyses to a group of 179 subjects with epilepsy from a Northern Han Chinese population. The CYP2A6, CYP2B6, and CYP2C9 genotype distributions in the population are presented in Table 1. These data correspond to allele frequencies of 13.4% (95% confidence intervals/CI = 10.1–17.4%) for CYP2A6*4, 19.0% (95%CI = 15.3–23.7%) for CYP2B6*6 and 4.7% (95%CI = 2.4–6.8%) for CYP2C9*3, which is comparable to the frequencies reported in other studies of Chinese populations [15,18,19]. In our sample, no subjects homozygous for the CYP2C9*3 allele were present. The frequencies of the genotypes of CYP2A6, CYP2B6, and CYP2C9 alleles were within the 95% confidence interval estimated by the HardyWeinberg equation [20]. 3.2. Correlation between the genotypic variants and VPA concentrations As shown in Table 2, the differences between the characteristics of subjects in each genotype were not significant, indicating
Table 2 Demographics of the study population and steady-state normalized plasma VPA concentrations as a function of CYP2A6, CYP2B6, and CYP2C9 genotypes. Genotype CYP2A6*4 Nil Carriers P values CYP2B6*6 Nil Heterozygous Homozygous P values CYP2C9*3 Nil Heterozygous P values
Age (years) (mean ± SD)
Cs (g kg ml−1 mg−1 ) (mean ± SD)
5–49 4–57
23.6 ± 3.3 23.0 ± 2.2 NS
3.4 ± 0.4 3.6 ± 0.4 0.0055
61(51.3) 29(56.9) 5 (55.6) NS
4–47 5–57 10–45
20.2 ± 3.4 23.1 ± 2.2 21.5 ± 4.1 NS
3.4 ± 0.5 3.5 ± 0.3 3.7 ± 0.4 0.0203
86(52.4) 9(60.0) NS
5–57 4–52
24.2 ± 2.3 21.5 ± 3.2 NS
3.4 ± 0.4 3.9 ± 0.4 0.0001
No. (%)
Sex, male (%)
133(74.3) 46 (25.7)
73(54.9) 22(47.8) NS
119(66.5) 51 (28.5) 9 (5.0)
164(91.6) 15 (8.4)
Age range (years)
Notes: Carriers, those of either heterozygous (CYP2A6*4) or homozygous (CYP2A6*4) genotypes. NS, not significant.
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that these characteristics did not influence the variations of VPA concentrations between the genotypes. The statistical analysis revealed significant associations between the steady-state standardized plasma concentrations of VPA with the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles tested in the study. For the CYP2A6*4 allele, subjects with one or two variant CYP2A6*4 alleles showed higher mean plasma VPA concentrations compared with non-*4 alleles [(3.3835 ± 0.4358) g kg ml−1 mg−1 vs. (3.5897 ± 0.4074) g kg ml−1 mg−1 , p = 0.0055]. A significant difference [one-way ANOVA (p = 0.0203)] was also found between mean plasma VPA concentrations and the CYP2B6 genotypes. The post hoc Scheffe’s F test showed a statistically significant difference in mean plasma VPA concentration between subjects carrying the *6/*6 genotype and non-*6 alleles (p = 0.0321), while the difference was not statistically significant between subjects with CYP2B6 *6/*6 genotype and subjects with *6 heterozygous genotype (p = 0.0833). Moreover, the difference was also not statistically significant between subjects with *6 heterozygous genotype (p = 0.0612). Similarly, we found that subjects with the heterozygous genotype CYP2C9*3 had higher mean plasma VPA concentrations than did those subjects with the wild-type genotype [(3.9 ± 0.4) g kg ml−1 mg−1 vs. (3.4 ± 0.4) g kg ml−1 mg−1 , p = 0.0001]. The subjects with CYP2A6*4 and CYP2C9*3 alleles showed 6.4% and 13.5% higher mean dose-corrected plasma VPA concentrations compared with these with non-carriers of them, respectively. And, there were 10.3% differences about the mean dose-corrected plasma VPA concentrations between subjects with the *6/*6 genotype and subjects with non-*6 alleles.
way and cytochrome P-450 (CYP) way, accounting for 50%, 40%, and 10% respectively of a dose [26]. This study suggested that CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles influenced the steadystate standardized plasma concentrations of VPA. Subjects with the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles showed higher steadystate standardized plasma concentrations of VPA than those of subjects without them. Previous studies have documented that CYP2C9 accounts for 75–80% of the formation of 4-ene-VPA, 4-OHVPA, and 5-OH-VPA, with CYP2A6 and CYP2B6 together accounting for 20–25% in vitro [6]. In addition, in an individual with a CYP2C9 poor metabolizer phenotype, CYP2A6 and CYP2B6 may account for a greater proportion of the enzymatic activity for the 4-hydroxylation and 5-hydroxylation terminal desaturation of VPA. Further studies investigating other polymorphisms, such as UGT1A6, UGT1A8, UBG2B7, are needed. To our knowledge, the present study is the first attempt to establish the influence of CYP2A6, CYP2B6, and CYP2C9 polymorphism on VPA metabolism in a Chinese population. In summary, our major findings indicate that the presently evaluated variant alleles in the CYP2A6, CYP2B6, and CYP2C9 genes may explain a part of the statistically substantial interindividual variability in VPA pharmacokinetics (i.e. 6–14%), and subjects harboring the CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles may have enhanced exposure to VPA. Further studies in larger or other ethnic populations are needed to replicate our findings regarding this association. In addition, it is also urgent need to do a larger study that quantifies the impact of a host of factors on the inter- and intraindividual variability of VPA concentrations in the future. Acknowledgements
4. Discussion Pharmacogenetics is a factor that may influence the relative contribution of specific CYP enzymes to a drug metabolism reaction. It gives rise to important interindividual and interethnic variability in the metabolism and disposition and clinical response of several therapeutic agents. Many studies have reported that the CYP2A6, CYP2B6, and CYP2C9 enzymes activity were dramatically reduced in the homozygotes for CYP2A6*4, CYP2B6*6 or CYP2C9*3 or the heterozygotes with these alleles, respectively [15,21,22]. CYP2A6*4 allele deletes the whole CYP2A6 gene [15]. CYP2B6*6 allele is known to be associated with lower levels of CYP2B6 expression in the liver [21]. The CYP2C9*3 has an exchange of A1075C in exon 7 causing an Ile359Leu substitution. One study reported that the variant CYP2C9*3 coding for enzyme with approximately 5% of the activity of CYP2C9*1, which was apparently a result of the amino acid substitution altering the interaction of the enzyme with Cytochrome P450 oxidoreductase [22]. Consequently, the high mean plasma VPA concentration of subjects with CYP2A6*4, CYP2B6*6 or CYP2C9*3 alleles may be explained by the low enzymatic activity of these respective genotypes. Previous genotyping analysis had revealed significantly different frequencies of the CYP2A6, CYP2B6 and CYP2C9 mutations in Chinese when compared to other ethnic populations such as Caucasians, Afro-Americans or Japanese, especially that comparison of CYP2A6*4 allele in Chinese to that in Caucasians is 15.1% to 0.5–1.0% [15,18,19,23–25]. The frequencies of CYP2A6*4, CYP2B6*6 and CYP2C9*3 are the highest mutant of CYP2A6, CYP2B6 and CYP2C9 gene isoforms, respectively, in the Chinese population [15,18,19]. So, it has special significance to study the clinical relevance of the CYP2A6*4, CYP2B6*6 and CYP2C9*3 alleles in the Chinese population. Metabolism by CYP pathway is not a prominent route of VPA elimination. The biotransformation of VPA involves three major metabolic ways, including uridine diphosphate glucuronosyltransferases (UDPGT) mediated pathway, mitochondrial -oxidation
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