INFLUENCE OF THE SCN1A IVS5N + 5 G>A POLYMORPHISM ON THERAPY WITH CARBAMAZEPINE FOR EPILEPSY
Sterjev Z1,*, Kiteva G2, Cvetkovska E2, Petrov I2, Kuzmanovski I2, Ribarska TJ3, Nestorovska KA1, Matevska N1,Trajkovik-Jolevska S3, Dimovski AJ1, Suturkova, Lj1
*Corresponding Author: Zoran Sterjev, M. Sei. Pharm Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, University “St. Cyril and Methodius,” str. Vodnjanska 17, 1000 Skopje, Republic of Macedonia; Tel./Fax:+38-923-120-229; E-mail: zost@ff.ukim.edu.mk
page: 19

MATERIALS AND METHODS

We studied 147 adult Macedonian patients (63 male and 84 female, mean age 53.0  15.5) on CBZ therapy (dosage interval 200-1200 mg/day) for more than a month, had normal renal and hepatic functions, and were free of other diseases or treatments at the time of blood sample collection. The type of seizures and epileptic syndrome were classifi ed according to the International League Against Epilepsy (ILAE) classifi cation (Table 2). The control group consisted of 137 Macedonian subjects (67 newborns and 70 adults without any history of epilepsy). Participation was voluntary and could be canceled by any individual at any time during the study (according to the Helsinki II declaration). The Ethics Committees of the Faculty of Pharmacy and of the Faculty of Medicine, Sts. Cyril and Methodius University, Skopje, Republic of Macedonia, approved the research protocol and all participants signed the study informed consent form. Genomic DNA was extracted from whole blood, procedure recommended by the manufacturer (Qiagen AS, Oslo, Norway). The SCN1A IVS5N + 5 G> polymorphism (rs3812718) was analyzed by allelic discrimination TaqMan assay (MxPro 3005P; Strategene, La Jolla, CA, USA) using the TagMan SNP genotyping assay according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Plasma CBZ concentration was measured using the fl uorescence polarization immunoassay (TDx/ FLx system; Abbott Laboratories, Irving, TX, USA) and a high performance liquid chromatography (HPLC) method in which the separation was carried out on a Waters HPLC system with a reversed-phase column (Zorbax Extend C18, 150  4.6 mm, 5 m; Waters Corporation, Milford, MA, USA) using isocratic elution with acetonitrile and water (35:65 v/v as a mobile phase at 30C) with UV detection set at 220 nm. The HPLC method validation followed the recommendations of European Medicinal Agency (EMA) guideline. Prior to analysis, the samples were pre-treated by solid-phase extraction procedure. In particular, plasma samples from each patient were spiked with 100 L internal standard, vortex-mixed for 30s and loaded into Oasis hydrophilic-lipophilicbalanced (HLB) cartridges (Waters Corporation) that were pre-conditioned with 1 mL methanol/water. This was followed by washing with 1 mL 5% methanol and elution with 1 mL of absolute methanol. We defi ned drug responsiveness as a complete seizure-free history for at least 1 year of treatment with CBZ, and drug resistance as the occurrence of at least four seizures over 1 year of the treatment with CBZ. For the purpose of our investigation, individual therapeutic dose is a dose of a given drug that has not been changed for two or more consecutive visits in the history of the patient’s treatment. The individual therapeutic doses defi ned as above, were compared with individual patient’s genotypes. For normalization of doses, we calculated the dose ratio [dose ratio = prescribed daily dose (PDD)/defi ned daily dose (DDD) according to the Anatomical Therapeutical Chemical Classifi cation System (ATC) classifi cation. The index of comparison (CBZ daily dose/CBZ plasma level) was calculated for comparison of CBZ dosages and plasma levels of individual patients. The Hardy-Weinberg equilibrium for the SNP was determined using an online calculator (http:// ihg2. helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Evaluation of the association between categorical variables (CBZ doses, CBZ index of comparison and patient genotype) was performed using Chisquare. Student’s t-test (p <0.05) and z-test (for large samples >30) were used to compare the CBZ responsiveness doses between patients with different genotypes (AA/AG; AA/GG and AG/GG).



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