Balkan Journal of Medical Genetics

THE INFLUENCE OF CYP2C8*3 ON CARBAMAZEPINE SERUM CONCENTRATION IN EPILEPTIC PEDIATRIC PATIENTS
Milovanovic DD, Milovanovic JR, Radovanovic M, Radosavljevic I, Obradovic S, Jankovic S, Milovanovic D, Djordjevic N
*Corresponding Author: M.D., Ph.D., Associate Professor, Department of Pharmacology and Toxicology, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34 000 Kragujevac, Serbia. Tel: +381-34-306-800, ext 223. Fax: +381-34-306-800. E-mail: natashadj2002@yahoo.com
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MATERIALS AND METHODS

The study was conducted at the Clinical Centre, Kragujevac, Serbia, and involved 40 epileptic pediatric patients on ongoing therapy with carbamazepine [9]. Except for four patients, who were on comedication with valproate, all others were on monotherapy. The study was approved by the relevant Ethics Committee (approval No. 01-7848) and conducted in accordance with the Declaration of Helsinki and its subsequent revisions. Blood samples for drug concentration analysis were collected twice, both times at the minimal concentration point (8-12 hours after the administration of the last dose): at the beginning of the study, and 4 weeks after dose adjustment. The steady-state carbamazepine serum concentrations were determined by validated high pressure liquid chromatography (HPLC) assay, as described by Jankovic et al. [10]. An additional blood sample was taken for CYP2C8 genotyping, and DNA was isolated using the PurelinkTM genomic DNA kit (Invitrogen, Carlsbad, CA, USA). CYP2C8*3 (416G>A, rs11572080) and CYP2C8*5 (475delA, rs72558196) were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific (AS)-PCR methods, respectively, according to Nakajima et al. [11]. Primers and restriction enzyme were from Invitrogen and New England Biolabs (Ipswich, MA, USA), respectively, while all other reagents used were made by Thermo Scientific (Waltham, MA, USA) or Qiagen GmbH (Hilden, Germany). Electrophoresis on a 1.2% agarose gel, stained with Sybr® safe DNA gel stain (Invitrogen), was used to detect the obtained PCR products and restriction fragments. To determine the factors affecting carbamazepine clearance, population pharmacokinetic modeling was employed, using the nonlinear mixed effect model (NONMEM) software, version 7.3.0 (Icon Development Solution, Hanover, MD, USA), and ADVAN 1 subroutine (within NONMEM) (one compartment model with no absorption). Carbamazepine serum concentrations, age, body weight, sex, total carbamazepine daily dose, CYP2C8 genotype and concomitant therapy with valproate, were included as covariates by the stepwise addition process in the model construction, and their significance was estimated by the likelihood ratio test. The final model was built through a backward deletion from the full model, using covariates that met criteria of the minimum objective function value difference of more than 6.6 for nominal p <0.01 and degree of freedom (df) = 1. Inter-patient varia-bility of the clearance and intrapatient (residual) variability in the concentration was estimated by exponential, additive, or proportional error model. Data distribution was assessed through the ratio of predicted (PRED) and measured the dependent variable (DV) concentrations of the drug, as well as the ratio in the weighted residuals (WRES) and PRED values of carbamazepine from the base to the final model. Bootstrapping analysis, as a preferable validation procedure for the small study sample size, was used to evaluate the predictive performance of the final model. Statistical Analyses. The haplotype analysis was done by the population genetic software program Arlequin, version 3.11 (http://cmpg.unibe. ch/software/arlequin3), and Statistica, version 7.1 (StatSoft, Tulsa, OK, USA) was used for all other statistical analyses. The observed and expected allele frequencies were compared by the χ2 test, and consistency of the data with the normal distribution was assessed by the Shapiro-Wilk test. The Spearman analysis was used to correlate doses and concentrations of carbamazepine, and the Student t-test for independent groups was used for assessment of dose requirements and carbamazepine serum concentrations in the CYP2C8*3 carrier and non carrier groups. A p value of <0.05 was considered significant.

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