The Pharmacogenomics Journal (2015), 1–7 © 2015 Macmillan Publishers Limited All rights reserved 1470-269X/15 www.nature.com/tpj

ORIGINAL ARTICLE

CYP2C9, CYP2C19, ABCB1 genetic polymorphisms and phenytoin plasma concentrations in Mexican-Mestizo patients with epilepsy A Ortega-Vázquez1, P Dorado2,3, I Fricke-Galindo1, H Jung-Cook4, N Monroy-Jaramillo5, IE Martínez-Juárez6, I Familiar-López7, E Peñas-Lledó3, A LLerena3 and M López-López8 We aimed to explore the possible influence of CYP2C9 (*2, *3 and IVS8-109 A4T), CYP2C19 (*2, *3 and *17) and ABCB1 (1236C4 T, 2677G4A/T and 3435C4T) on phenytoin (PHT) plasma concentrations in 64 Mexican Mestizo (MM) patients with epilepsy currently treated with PHT in mono- (n = 25) and polytherapy (n = 39). Genotype and allele frequencies of these variants were also estimated in 300 MM healthy volunteers. Linear regression models were used to assess associations between the dependent variables (PHT plasma concentration and dose-corrected PHT concentration) with independent variables (CYP2C9, CYP2C19 and ABCB1 genotypes, ABCB1 haplotypes, age, sex, weight, and polytherapy). In multivariate models, CYP2C9 IVS8-109 T was significantly associated with higher PHT plasma concentrations (t(64) = 2.27; P = 0.03). Moreover, this allele was more frequent in the supratherapeutic group as compared with the subtherapeutic group (0.13 versus 0.03, respectively; P = 0.05, Fisher's exact test). Results suggest that CYP2C9 IVS8-109 T allele may decrease CYP2C9 enzymatic activity on PHT. More research is needed to confirm findings. The Pharmacogenomics Journal advance online publication, 30 June 2015; doi:10.1038/tpj.2015.45

INTRODUCTION Epilepsy is a neurological disorder relevant to public health given the global prevalence of the disease, currently estimated at 70 million people.1 In Mexico, epilepsy affects approximately 1.5–2 million individuals,2 most of them younger than 40 years of age.3 Consequently, epilepsy is the primary cause of outpatient visits at the National Institute of Neurology and Neurosurgery Manuel Velasco Suárez (INNN) in Mexico City, and phenytoin (PHT) is one of the three most prescribed antiepileptic drugs at this hospital.4 PHT has a narrow therapeutic index (10–20 μg ml − 1) and nonlinear pharmacokinetics with a wide inter-individual variability in clearance, making therapeutic drug monitoring an important adjunct to the treatment of epilepsy.5 Several environmental and genetic factors can affect the metabolism of PHT leading to plasma concentrations outside the therapeutic range.6 PHT is metabolized by the polymorphic enzymes CYP2C9 (90%) and CYP2C19 (10%).7 Several population-based pharmacogenetic studies have shown that both CYP2C genes have an important role in the PHT pharmacokinetic variability, including PHT plasma concentrations and clearance.8–10 It has been reported that patients carrying at least one of the most common CYP2C9 reduced function alleles, CYP2C9*2 or CYP2C9*3, require a lower PHT dose (30–40%) to achieve therapeutic plasma concentrations than those homozygous for the wild-type CYP2C9*1 allele.11 On the other hand, the CYP2C9 intronic single nucleotide polymorphism (SNP) IVS81

109 A4T has been associated with a lower activity of CYP2C9 on losartan in healthy Swedes.12 In contrast, this variant showed an increased enzymatic activity on losartan in healthy volunteers from an Ecuadorian population.13 For CYP2C19, more than 30 reduced or null function allelic variants have been identified (http://www. cypalleles.ki.se/cyp2c19.htm). Among them, CYP2C19*2 is the most common allele in East Asians, Africans and Caucasians, while CYP2C19*3 is common in Asians but virtually absent in Africans and Caucasians.14 These two functional defective alleles may result in a poor metabolizer phenotype.7,15 The CYP2C19*17 variant has been reported with an increased enzymatic activity,16,17 although other studies have failed to support that this allele is associated with an ultra-rapid metabolism.18,19 PHT pharmacokinetic variability has also been related to the activity of P-glycoprotein, an efflux pump of the ATP-binding cassette family encoded by the ABCB1 gene.20 The ABCB1 1236C4T, 2677G4A/T and 3435C4T polymorphisms have been associated with differences in PHT transport and refractory epilepsy.21,22 These three ABCB1 variants are in linkage disequilibrium.23 Several pharmacogenetic studies have shown the influence of CYP2C9, CYP2C19 and ABCB1 genetic polymorphisms on PHT plasma concentrations and its association with PHT-induced central nervous system toxicities.24–26 However, the influence of CYP2C9 IVS8-109 A4T and CYP2C19*17 variants on PHT plasma concentrations has not been explored. The aim of this study was to assess the potential influence of CYP2C9 (*2, *3 and IVS8-109

Doctorate in Biological and Health Sciences, Metropolitan Autonomous University, Campus Xochimilco, Mexico City, Mexico; 2Department of Medical and Surgical Therapeutics, University of Extremadura, Campus Plasencia, Plasencia, Spain; 3CICAB Clinical Research Centre, Extremadura University Hospital and Medical School Servicio Extremeño de Salud, Badajoz, Spain; 4Department of Pharmacy, Chemistry Faculty, National Autonomous University of Mexico and Department of Neuropharmacology, National Institute of Neurology and Neurosurgery Manuel Velasco Suárez, Mexico City, Mexico; 5Department of Neurogenetics and Molecular Biology, National Institute of Neurology and Neurosurgery Manuel Velasco Suárez, Mexico City, Mexico; 6Epilepsy Clinic, National Institute of Neurology and Neurosurgery Manuel Velasco Suárez, Mexico City, Mexico; 7Department of Psychiatry, Michigan State University, Lansing, Michigan, USA and 8Department of Biological Systems, Metropolitan Autonomous University, Campus Xochimilco, Mexico City, Mexico. Correspondence: Dr M López López, Department of Biological Systems, Metropolitan Autonomous University, Campus Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, Coyoacán, Mexico City 04960, Mexico. E-mail: [email protected] Received 8 December 2014; revised 14 May 2015; accepted 21 May 2015

CYP2C9, CYP2C19 and ABCB1 effects on phenytoin concentration A Ortega-Vázquez et al

2 Table 1.

Demographic and pharmacologic characteristics of 64 patients with epilepsy treated with phenytoin

Characteristics Age, years mean (range) Weight, kg mean (range) PHT daily dose, mg mean (range) PHT plasma concentration, μg ml − 1, mean (range) Dose-corrected PHT concentration, (μg ml − 1)/(mg kg − 1), mean (range)

Total (n = 64) 32.2 67 306 12.7 3.0

(16-65) (39-129) (100-600) (1.8-37.4) (0.5-25.0)

Male (n = 44) 30.8 71.6 318.2 12.8 3.3

(16-60) (46-129) (100-600) (2.6-37.4) (0.6-25)

Female (n = 20) 35.1 56.8 280 11.1 2.3

(18-65) (39-92) (100-400) (1.8-25.7) (0.5-4.3)

PHT therapeutic group Subtherapeutic ( o10 μg ml − 1) Therapeutic (10–20 μg ml − 1) Supratherapeutic (420 μg ml − 1)

26 28 10

16 22 6

10 6 4

Co-treatment status PHT as monotherapy PHT PHT in polytherapya

25 39

16 28

9 11

Co-treatment drugs CYP2C9 inducer CYP2C9 inhibitor CYP2C9 inhibitor/inducer plus CYP2C19 inhibitor No CYP2C9 inhibitor/ inducer or no CYP2C19 inducer

19 9 7 4

14 7 3 4

5 2 4 0

Abbreviation: PHT, phenytoin. aCo-treatment drugs used in polytherapy with PHT included: CYP2C9 inducers (carbamazepine and phenobarbital), CYP2C9 inhibitor (valproate), CYP2C19 inhibitors (omeprazole, topiramate and oxcarbazepine) and drugs not considered to affect the metabolism of CYP2C9 or CYP2C19 (lamotrigine, levetiracetam and benzodiazepines).

A4T), CYP2C19 (*2, *3 and *17) and ABCB1 (1236C4T, 2677G4 A/T and 3435C4T) polymorphisms and concomitant treatment on PHT plasma concentrations in Mexican Mestizo (MM) patients with epilepsy. We also determined the genotypic and allelic frequencies of the above genetic variants in a sample of MM unrelated healthy volunteers without epilepsy. SUBJECTS AND METHODS Subjects A total of 64 adults (416 years of age) patients with epilepsy currently treated with PHT at standard daily dose (100–600 mg) were consecutively recruited from the Epilepsy Clinic at the INNN. Distributions of the demographic characteristics of patients, the PHT dose and concomitant treatment drugs were extracted from medical records and summarized. PHT was given orally at the same dose for at least 4 weeks prior to blood sampling to reach steady state concentrations. Venous blood samples were obtained just before the morning dose. Samples were collected in heparinized tubes, centrifuged at room temperature and quantified using an in vitro chemiluminescent microparticle immunoassay (Abbott ARCHITECT iPhenytoin assay, Abbott Laboratories, Abbott Park, IL, USA). The calibration curve ranged from 2.5 to 40.0 μg ml − 1. The quantification limit was 0.05 μg ml − 1. The inter- and intra-assay coefficients of variation were less than 5.5% for the different calibrators. The individual percent recovery ranged from 88.1 to 97.8%. Three hundred healthy MM unrelated volunteers (mean age = 26.9 years; 64.3% females) were included in the study to determine the allele and genotype frequencies of the polymorphisms analyzed. Patients and volunteers originated from Mexico City and various Mexican states and were defined as MM if at least three generations of first degree relatives were born in Mexico. Subjects were informed about the aims of the study and gave their written informed consent prior their participation. The study protocol was reviewed and approved by the INNN ethics review board.

Genotyping analysis Genomic DNA was obtained from peripheral blood leukocytes by standard procedures. Genotyping for CYP2C9*2 (C430T; R144C; rs1799853 (C_25625805_10)), CYP2C9 IVS8-109 A4T (rs1934969; (C_27104791_10)), CYP2C19*2 (G681A; P227P; rs4244285 (C_25986767_70)) CYP2C19*3 (G636A; *212 W; rs4986893(C_27861809_10), CYP2C19*17 (-806 C4T; rs12248560 (C_469857_10)), ABCB1 1236 C4T (G412G; rs1128503 (C_7586662_10)), ABCB1 2677 G4A/T (T893P; rs2032582 (C_11711720D_40 and C_11711720C_30)) and ABCB1 3435 C4T (rs1045642 (C_7586657_20)) The Pharmacogenomics Journal (2015), 1 – 7

alleles was performed by RT-PCR with commercially available TaqMan validated SNP assays (Applied Biosystems, Foster City, CA, USA). The CYP2C9*3 (1075 A4C; I359L; rs1057910) allele was determined by PCR-restriction fragment length polymorphism with the enzyme NsiI using published primers.27

Statistical analysis Data for categorical variables are presented as numbers and frequencies, and as the mean ± standard deviation for continuous variables. Genotype and allele frequencies of patients and volunteers were assessed and Hardy– Weinberg equilibrium determined using χ2. Patients were classified into three different therapeutic groups (subtherapeutic, therapeutic and supratherapeutic), according to PHT plasma concentrations below (o10 μg ml − 1), within (10–20 μg ml − 1) or above (420 μg ml − 1) the therapeutic range, respectively. Fisher’s exact test was used to compare the genotypic frequencies of CYP2C9, CYP2C19 and ABCB1 within each therapeutic group. Dose-corrected PHT concentration was calculated dividing PHT plasma concentration (μg ml − 1) by PHT daily dose/weight (mg kg − 1). Linear regression models were used to assess univariate associations between the dependent variables (PHT plasma concentration and dosecorrected PHT concentration) and independent variables (CYP2C9, CYP2C19 and ABCB1 genotypes, ABCB1 haplotypes, age, sex, weight, and polytherapy). Genotypes with categories (for example, alleles) with less than five individuals were collapsed, using the largest group as reference category). Patients were classified into three ABCB1 haplotypes (1236C4T, 2677G4A/T, 3435C4T): haplotype 1 comprised patients homozygous for the wild-type haplotype (CGC/CGC); haplotype 2 was integrated by patients carriers of one copy of wild-type haplotype and a variant haplotype (CGC/TTT, CGC/ TGT, CGC/CAC, CGC/CGT, CGC/TAC, CGC/CTC, CGC/TGC, and CGC/TTC); and haplotype 3 included patients with both copies of variant haplotypes (TTT/ TTT, TTT/CAT, TGC/TTT, CAC/TTT, CGT/TTT, CTC/TTC, CTT/TGT, TGT/TGT and CAT/TAT). ABCB1 haplotype analysis was performed using Arlequin v. 3.5.1.3.28 Variables that were associated with each outcome at 0.1 level of significance were entered into a multivariable model to determine their independent effects on PHT plasma concentrations. Factors that remained associated with the outcomes were retained in the final model. All statistical tests were two-sided and performed in STATA version 12 (StataCorp LP, 2012, College Station, TX, USA). Statistical significance was set at Po0.05.

RESULTS Demographic and pharmacologic characteristics of patients are shown in Table 1. Overall, patients in this study were young (mean © 2015 Macmillan Publishers Limited

CYP2C9, CYP2C19 and ABCB1 effects on phenytoin concentration A Ortega-Vázquez et al

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Figure 1. Frequencies of CYP2C9, CYP2C19 and ABCB1 genotypes and phenytoin plasma concentrations by therapeutic group: subtherapeutic ( o10 μg ml − 1), therapeutic (10–20 μg ml − 1) or supratherapeutic range ( o20 μg ml − 1). Numbers above bars indicate the number of individuals. *CYP2C9 IVS8-109T allele frequency was higher in the supratherapeutic group than in the subtherapeutic group (0.13 versus 0.03, respectively; P = 0.05, Fisher's exact test).

age = 32.2 years) and predominantly male (69%). Mean PHT plasma concentrations were within the therapeutic range (10–20 μg ml − 1) and PHT was used as monotherapy in 25 (39%) patients. For the remaining 39 patients in polytherapy (for example, at least one more drug used to control seizures in addition to PHT), cotreatment drugs included CYP2C9 inducers (carbamazepine and phenobarbital), CYP2C9 inhibitor (valproate), CYP2C19 inhibitors (omeprazole, topiramate and oxcarbazepine) and drugs not considered to affect the metabolism of CYP2C9 or CYP2C19 (lamotrigine, levetiracetam and benzodiazepines). None of the patients were treated with CYP2C19 inducers. Of the 64 patients with epilepsy studied, 44% had therapeutic, 41% sub-therapeutic and 15% had supratherapeutic PHT plasma concentrations (Table 1). The frequencies of CYP2C9, CYP2C19 and ABCB1 genotypes and PHT plasma concentrations by therapeutic group are shown in Figure 1. Fifty percent of patients with supratherapeutic PHT plasma concentrations carried at least one copy of the CYP2C9 IVS8-109 T allele as compared with 27% and 36% of patients in subtherapeutic and therapeutic group, respectively. Moreover, homozygous carriers for this variant were only observed in patients with PHT plasma concentrations in supratherapeutic range © 2015 Macmillan Publishers Limited

(Figure 1a). CYP2C9 IVS8-109 T allele frequency was higher in the supratherapeutic group than in the subtherapeutic group (0.13 versus 0.03, respectively; P = 0.05, Fisher's exact test). The remaining CYP2C9, CYP2C19 or ABCB1 genotypic and allelic frequencies were not significantly different between the three therapeutic groups (Figures 1b and f). Genotype and allele frequencies of the CYP2C9, CYP2C19 and ABCB1 polymorphisms in patients and volunteers are shown in Table 2. Frequencies of genetic variants in patients and volunteers were in agreement with Hardy–Weinberg equilibrium (P40.05). Allelic frequencies were significantly different only for CYP2C9 IVS8109 T and CYP2C19*1 alleles; with 19% and 91% in patients, while in volunteers were 29% and 82%, respectively (P = 0.02, in both cases; Fisher's exact test). However, the disparity may be due to the difference in the sample size of these groups (n = 64 versus n = 300). The CYP2C19*3 allele was not identified in patients or volunteers. Table 3 shows the PHT plasma concentrations and dosecorrected PHT concentrations in the 64 patients with epilepsy studied according to CYP2C9, CYP2C19 and ABCB1 genotypes, and ABCB1 haplotypes. From this table, it seems that patients with CYP2C9 IVS8-109 TT or AT genotypes may have slightly higher dose-corrected values than those with CYP2C9 IVS8-109 AA The Pharmacogenomics Journal (2015), 1 – 7

CYP2C9, CYP2C19 and ABCB1 effects on phenytoin concentration A Ortega-Vázquez et al

4 Table 2.

CYP2C9, CYP2C19 and ABCB1 genotypic and allelic frequencies in 64 patients with epilepsy and 300 healthy volunteers from Mexico Patients (n = 64)

Gene CYP2C9

CYP2C19

ABCB1

Volunteers (n = 300)

Genotype/allele

n

Frequency

95% CI

n

Frequency

95% CI

*1/*1 *1/*2 *1/*3 *1 *2 *3 IVS8-109 AA IVS8-109AT IVS8-109 TT IVS8-109A IVS8-109T *1/*1 *1/*2 *1/*17 *2/*2 *2/*17 *17/*17 *1 *2 *3 *17 1236CC 1236CT 1236 TT 1236C 1236T 2677GG 2677GA 2677GT 2677 AA 2677AT 2677 TT 2677G 2677A 2677T 3435CC 3435CT 3435 TT 3435C 3435T

61 3 0 125 3 0 42 20 2 104 24 52 7 5 0 0 0 116 7 0 5 15 33 16 63 65 18 5 25 1 4 11 66 11 51 23 29 12 75 53

0.95 0.05 0.00 1.00 0.02 0.00 0.66 0.31 0.03 0.81 0.19 0.89 0.11 0.08 0.00 0.00 0.00 0.91 0.05 0.00 0.04 0.23 0.51 0.25 0.49 0.51 0.28 0.08 0.39 0.02 0.06 0.17 0.52 0.09 0.40 0.36 0.45 0.19 0.59 0.41

0.87–0.99 0.01–0.13 0.00–0.07 0.93–0.10 0.01–0.07 0.00–0.04 0.53–0.76 0.21–0.43 0.00–0.11 0.74–0.87 0.13–0.26 0.79–0.95 0.05–0.21 0.03–0.17 0.00–0.07 0.00–0.07 0.00–0.07 0.84–0.95 0.02–0.11 0.00–0.04 0.01–0.09 0.15–0.35 0.40–0.63 0.16–0.37 0.41–0.58 0.42–0.59 0.19–0.40 0.03–0.17 0.28–0.51 0.00–0.09 0.02–0.15 0.10–0.28 0.43–0.60 0.05–0.15 0.32–0.49 0.25–0.48 0.34–0.57 0.11–0.30 0.50–0.67 0.33–0.50

271 27 2 571 27 2 150 128 22 428 172 197 51 47 1 1 3 492 54 0 54 71 155 74 297 303 69 28 137 0 17 49 303 45 252 70 155 75 295 305

0.90 0.09 0.01 0.95 0.04 0.00 0.50 0.43 0.07 0.71 0.29 0.66 0.17 0.16 0.00 0.00 0.01 0.82 0.09 0.00 0.09 0.24 0.52 0.25 0.49 0.50 0.23 0.09 0.46 0.00 0.06 0.16 0.50 0.07 0.42 0.23 0.52 0.25 0.49 0.51

0.87–0.93 0.06–0.13 0.00–0.03 0.93–0.97 0.03–0.06 0.00–0.01 0.44–0.56 0.37–0.48 0.05–0.11 0.68–0.75 0.25–0.32 0.60–0.71 0.13–0.22 0.12–0.20 0.00–0.02 0.00–0.02 0.00–0.03 0.79–0.85 0.07–0.12 0.00–0.01 0.07–0.12 0.19–0.29 0.46–0.57 0.20–0.30 0.46–0.53 0.47–0.54 0.19–0.28 0.07–0.13 0.40–0.51 0.00–0.02 0.04–0.09 0.13–0.21 0.47–0.55 0.33–0.41 0.08–0.13 0.19–0.29 0.46–0.57 0.20–0.30 0.45–0.53 0.47–0.55

Abbreviations: CI, confidence interval; n, number of subjects; Hardy–Weinberg equilibrium (P40.05).

genotype. Moreover, in univariate models, only CYP2C9 IVS8109 A4T was significantly associated with PHT plasma concentrations. Patients with CYP2C9 IVS8-109 TT or AT genotypes had on average 1.94 higher dose-corrected PHT concentration (t(64) = 2.46; P = 0.02), compared with patients with AA (Table 4). Sex and age explained 1% (F(60) 0.88; P = 0.46) and 12% (F(60) 3.78; P = 0.02) of the variance observed in multivariate models with PHT plasma concentration and dose-corrected PHT concentrations as outcome, respectively. After adjusting for sex and age, CYP2C9 IVS8-109 A4T was significantly associated with higher dosecorrected PHT concentrations. Patients with AT and TT genotypes had on average 1.7 higher dose-corrected PHT concentration than patients with the AA genotype (t(64) = 2.27; P = 0.03) (Table 4). DISCUSSION To the best of our knowledge, this is the first study to explore possible associations between CYP2C9, CYP2C19 and ABCB1 polymorphisms, and concomitant treatment on PHT plasma concentrations in MM patients with epilepsy, and to describe the frequencies of CYP2C9 IVS8-109 A4T, and ABCB1 1236 T4C and 2677 G4A/T variants in a MM sample. The Pharmacogenomics Journal (2015), 1 – 7

We found low CYP2C9*2 and *3 allele frequencies in MM patients and in healthy volunteers, which is in accordance with previous studies in Mexican Americans, MM and Mexican Tepehuanos.29–32 In both our patient and volunteer MM samples, we observed a predominance of the wild-type CYP2C19*1 allele, a low frequency of CYP2C19*2 and an absence of CYP2C19*3 allele, as it has been previously reported.33–38 The frequency of CYP2C19*17 allele found in the present study (MM from Central Mexico) shows a minor nonsignificant difference from that found in a recent report in MM from the state of Jalisco (0.09 and 0.14, respectively).38 The frequency of ABCB1 3435C4T in the MM population studied here is similar to that found in previous reports.39,40 Also comparable with our results were the frequencies of the three ABCB1 polymorphisms reported among individuals with Mexican ancestry living in Los Angeles, California (ABCB1 1236C = 0.540, 2677G = 0.570 and 3435C = 0.540; data from HapMap project consulted in NCBI dbSNP).41 All patients included in this study were currently treated with PHT and 61% had co-administration of at least one more drug, including antiepileptics reported to reduce or enhance PHT plasma concentrations.42 It is well known that PHT is a cytochrome P450 inducer and several interactions with other antiepileptic © 2015 Macmillan Publishers Limited

CYP2C9, CYP2C19 and ABCB1 effects on phenytoin concentration A Ortega-Vázquez et al

Table 3. PHT plasma concentrations and dose-corrected PHT concentrations in 64 patients with epilepsy by CYP2C9, CYP2C19 and ABCB1 genotypes, and ABCB1 haplotypes Gene

Genotype

CYP2C9

*1/*1 *1/*2 IVS8-109AA IVS8-109AT IVS8-109TT CYP2C19 *1/*1 *1/*2 *1/*17 ABCB1 1236CC 1236CT 1236TT 2677GG 2677GA/T 2677AA/AT/ TT 3435CC 3435CT 3435TT Haplotype 1 Haplotype 2 Haplotype 3

n

PHT plasma concentration (μg ml − 1)a

Dose-corrected PHT concentration (μg ml − 1)/(mg kg − 1)a

61 3 42 20 2 52 7 5 15 33 16 18 30 16

12.9 ± 7.1 10.4 ± 3.1 12 ± 6.5 13.7 ± 7.5 21.8, 21.6 12.5 ± 8.9 12.3 ± 8 14.9 ± 7.4 13.9 ± 9.2 12.2 ± 6.5 12.6 ± 5.6 12.8 ± 7.5 13.9 ± 6.9 10.4v6.0

3.0 ± 3.2 2.8 ± 1.2 2.4 ± 1.1 4.3 ± 5.2 3.7, 4.5 3.0 ± 3.4 2.7 ± 1.6 3.7 ± 2.1 4.5 ± 5.9 2.5 ± 1.2 2.8 ± 1.4 2.9 ± 1.7 3.6 ± 4.2 2.2 ± 1.4

23 29 12 10 25 29

12.5v8.1 13.7v1.3 10.8 ± 5.0 10.3 ± 3.0 13.7 ± 7.3 12.7 ± 7.5

3.6 ± 4.9 3.0v1.3 2.1 ± 0.8 2.4 ± 0.7 3.2 ± 1.8 3.1 ± 4.3

Abbreviation: PHT, phenytoin. aMean values ± standard deviation.

Table 4.

drugs have been reported.43 However, our results showed no relation between polytherapy and PHT plasma concentrations; furthermore, PHT daily doses of patients were within standard recommendation.44 Several studies have reported the influence of CYP2C9*2, CYP2C9*3, CYP2C19 *2 and CYP2C19*3 variants on PHT pharmacokinetics.5,8,9,24,45–47 In contrast, we did not find an association between CYP2C9 (*2, *3) and CYP2C19 (*2, *3) alleles and PHT plasma concentration, supporting the report by Taguchi and colleagues.10 However, our observation could be owing to the low frequency of these alleles found among MM patients coupled with our small sample size. More research is needed to explore this potential association. The potential effect of IVS8-109 A4T on CYP2C9 activity could be due to linkage with other SNPs of CYP2C9, because the SNP IVS8-109 A4T has been found to be linked with other SNPs in the 5'-UTR, exon 1 and intron 1 of the CYP2C9 gene.48 In this study, approximately a twofold higher dose-corrected PHT concentration (P o 0.05) was found between patients carriers of at least one copy of CYP2C9 IVS8-109 T allele as compared with patients with AA genotype. Although the number of individuals with supratherapeutic PHT plasma concentrations (420 μg ml − 1) is low, a higher frequency of this intronic variant was observed in patients within this group as compared with patients in the subtherapeutic group (0.13 versus 0.03, respectively; P = 0.05, Fisher's exact test). These preliminary results suggest that the CYP2C9 IVS8-109 T allele could be related to a reduced CYP2C9 enzymatic activity on PHT. Our findings are supported by previous data reported by Hatta et al.,12 who showed that the CYP2C9 IVS8-109 T allele was associated with

Results from linear regressions (univariate and multivariate associations) Dose-corrected PHT concentration (μg ml − 1)/(mg kg − 1)

PHT plasma concentration (μg ml − 1) Covariates Univariate associations Females Age (years) Weight (kg) Co-treatment drugsa CYP2C9 inducers CYP2C9 inhibitors CYP2C9 inhibitor/inducer plus CYP2C19 inhibitor Genotypes CYP2C9*1/*1 and *1/*2b CYP2C9 IVS8-109 A4T A/T and T/Tc CYP2C19*1/*2 and *1/*17d ABCB1 1236C4T C/T and T/Te ABCB1 2677G4A/T G/A/T and A/A, A/T or T/Tf ABCB1 3435C4T C/T and T/Tg ABCB1 haplotype 2h ABCB1 haplotype 3i Multivariate associations Females Age (years) CYP2C9 IVS8-109 A/T and T/T

b ( 95% CI)

P-value

b (95% CI)

P-value

− 0.59 (−4.3–3.2) 0.06 (−0.1–0.2) − 0.06 (−0.2–0.04)

0.75 0.40 0.23

− 1.0 (2.7–0.68) 0.06 (−0.005–0.1) 0.01 (−0.04–0.05)

0.24 0.07 0.81

− 3.0 (−7.1–1.1) − 1.5 (−6.8–3.8)

0.15 0.54

− 1.0 (−2.8–0.9) 0.05 (−2.3–2.4)

0.30 0.97

− 3.1 (−8.9–2.8)

0.30

− 1.2 (−3.8–1.5)

0.38

− 2.4 2.55 0.08 − 0.06 − 1.2 − 0.05 3.3 2.5, 0.18

(−10.6–5.8) (−1.1–6.2) (−3.6–5.3) (−3.1–1.9) (−3.5–1.2) (−2.9–1.9) (−6.8–3.8) (−7.1–1.1)

− 0.69 (−4.5–3.4) 0.06 (−0.09-0.20) 2.4 (−1.3-6.1)

0.56 0.16 0.71 0.63 0.33 0.67 0.22 0.32 0.72 0.43 0.20

− 0.24 1.94 0.12 − 0.85 0.33 − 0.73 0.77 0.70

(−3.9–3.5) (0.36–3.5) (−1.9–2.1) (−2.0–0.2) (−1.4–0.7) (−1.8–0.3) (−1.6–3.1) (−1.6–3.0)

0.90 0.02 0.91 0.13 0.54 0.18 0.52 0.54

− 1.1 (−2.7–0.5) 0.06 (0.001-0.1) 1.7 (0.20-3.3)

0.16 0.05 0.03

Abbreviations: CI, confidence interval; PHT, phenytoin. Bold type indicates P-value o 0.05. aNo CYP2C9 inhibitor/ inducer or no CYP2C19 inducer is reference category. bCYP2C9*1/*1 is reference category. cCYP2C9 IVS8-109 genotype AA is reference category. dCYP2C19 genotype *1/*1/ is reference category. eABCB1 1236 genotype CC is reference category. fABCB1 2677 genotype GG is reference category. gABCB1 3435 genotype CC is reference category. hABCB1 haplotype 1 is reference category. iABCB1 haplotype 1 is reference category.

© 2015 Macmillan Publishers Limited

The Pharmacogenomics Journal (2015), 1 – 7

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CYP2C9, CYP2C19 and ABCB1 effects on phenytoin concentration A Ortega-Vázquez et al

6 a higher losartan metabolic ratio among 85 Swedish healthy volunteers. However, the same researchers were not able to replicate this result in 128 Koreans part of the same study. In contrast, in a recent study in 187 Ecuadorians, the CYP2C9 IVS8109 T variant was associated with an increased CYP2C9 hydroxylation capacity of losartan.13 The discrepancy on CYP2C9 enzymatic activity across studies could be due to differences in the genetic background of the populations analyzed. Particularly, there might be differences in linkage disequilibrium structures between them, suggesting that alterations in CYP2C9 activity are not caused by CYP2C9 IVS8-109 T intronic variant itself but by the alleles linked to it. Another reason for the variable results in the literature could partly be attributed to the different CYP2C9 drug substrates used in studies. Overall, inconsistencies across studies highlight the importance of adding new data from different populations that can help to elucidate the influence and mechanism of CYP2C9 IVS8-109 T intronic variant on commonly used drugs. Traditionally, CYP2C19*1/*17 individuals have been classified as extensive metabolizers49,50 and/or ultra-rapid metabolizers.17,19 At least some of these discrepancies may be due to alternate routes of clearance between different CYP2C19 drug substrates.51 We did not observe a significant PHT plasma concentration difference between patients with CYP2C19*1/*1 and CYP2C19*1/*17 genotypes, and none of our patients were homozygous for CYP2C19*17 (Table 4), thus the role of this variant on PHT plasma concentrations could not be concluded. Although some studies have associated ABCB1 1236C4T, 2677G4A/T and 3435C4T polymorphisms with PHT disposition,52–54 they could not explain the high plasma levels of PHT in reports of PHT intoxication.25,55 In this study, we did not observe a significant difference on PHT plasma concentrations among patients’ carriers of different ABCB1 genotypes and haplotypes. It has been reported that the discrepancy in the association studies of ABCB1 polymorphisms in epilepsy treatment could be due to confounders such as ethnicity and polytherapy.56 Results presented here have to be viewed in the light of some limitations. Sample size was restricted to patients attending the Epilepsy Clinic at INNN and treated with PHT, limiting generalizability of results. However, the INNN is a large reference hospital in Mexico receiving epilepsy cases from several parts of the country. Because of the small sample size accrued, it is possible that we were underpowered to detect associations in rare variants (for example, CYP2C9*3 and CYP2C19*3). More studies with larger sample sizes are required to determine the inclusion of CYP2C9 IVS8-109 A4T variant as a biomarker of PHT plasma concentrations in MM patients with epilepsy. However, the preliminary results presented here are an important contribution to our understanding on the potential influence of CYP2C9 IVS8-109 A4T polymorphism on PHT plasma concentrations. Replications of our results in a larger sample could add knowledge of the reasons for variability in PHT plasma concentrations per dose in MM patients with epilepsy. In conclusion, CYP2C9 IVS8-109 T carriers showed significantly higher dose-corrected PHT concentrations and this allele was found in a higher frequency in patients that exhibited supratherapeutic PHT plasma concentrations. Our finding suggests that this variant decreases enzymatic CYP2C9 activity on PHT as compared with the wild-type variant in MM patients with epilepsy. Although additional research is warranted to confirm the influence of CYP2C9 IVS8-109 T on PHT plasma concentrations and other pharmacokinetic parameters, this preliminary study represents a valuable first step in PHT pharmacogenetics research in Mexico. CONFLICT OF INTEREST The authors declare no conflict of interest.

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ACKNOWLEDGMENTS This research was supported by a grant from Consejo Nacional de Ciencia y Tecnología de México (CONACyT) (#167261). AOV was supported by a scholarship (Doctor’s degree) from CONACyT (#328295).

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