| Chapter 1 Determination of Antiepileptic Drugs in Human Plasma Using Isocratic Reversed-Phase HPLCObjectiveTo establish a simple HPLC method for the simultaneous determination of zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), carbamazepine(CBZ) and two active metabolites monohydroxycar-bazepine (MHD), carbamazepine 10,11-epoxide(CBZE) in human plasma.MethodsThe analytes were pretreated by protein precipitation, using propranolol hydrochloride as internal standard. The extracts were injected onto an Agilent RX-C8 column, and the mobile phase consisted of methanol-acetonitrile-0.1% triflouroacetic acid (235:120:645, v/v/v) and pumped at 1.5 mL-min-1. PRI, LTG, MHD, PB, PHT and CBZE were detected at the wavelength of 215 nm, while ZNS, OXC and CBZ were detected at 235 nm.ResultsThe limits of quantification for ZNS, PRI, LTG, MHD, PB, CBZE, OXC, PHT and CBZ were 1.0,5.0,1.0,1.0,5.0,1.0,0.5,1.0,1.0 μg·mL-1, The method was found to be linear over the concentration ranges investigated,1～80μg·mL-1 for ZNS,5～50 μg·mL-1 for PRI, 1～25 μg·mL-1 for LTG,1～50 μg·ml-1 for MHD,5～100 μg·mL-1 for PB,1～10 μg·mL-1 for CBZE,0.5～25 μg·mL-1 for OXC,1～50 μg·mL-1 for PHT and 1～25 μg·mL-1 for CBZ. The recoveries of ZNS, PRI, LTG, MHD, PB, CBZE, OXC, PHT and CBZ were 101.26%, 101.33%,101.31%,105.08%,100.08%,100.04%,101.64%,103.27% 和 102.09%, respectively. The intra-and inter-day reproducibility was adequate with the coefficients of variation of 11.58% or below.ConclusionThis method was proved to be accurate, sensitive and convenient. It is suitable for therapeutic drug monitoring of the nine analytes concentrations in human plasma.Chapter 2 Association of gene polymorphisms with the pharmacokinetics of carbamazepineBackground and ObjectiveCarbamazepine (CBZ) is one of the most widely prescribed antiepileptic drugs. However, because of its narrow therapeutic index and large interindividual variability in dosages and concentrations, therapeutic drug monitoring (TDM) for CBZ is performed and dose is adjusted according to the result of TDM and clinical efficacy. The genes encoding sodium channels, the targets of CBZ in the brain, have genetic polymorphisms. In addition, CBZ is a substrate of Cytochromes P450 3A (CYP3A), microsomal epoxide hydrolase(mEH), UDP-glucuronosyhransferase (UGT), and multidrug resistance-associated protein 2(MRP2, or ABCC2). Those genes encoding CYP3A, mEH, UGT and ABCC2 are polymorphic, too. In that case, we hypothesized these polymorphisms as the genetic cofactors may contribute to the pharmacokinetics of CBZ and interindividual variance. The purpose of this study is to investigate the effects of SCN1A/2A, CYP3A4/5, EPHX1, UGT2B7 and ABCC2 genetic polymorphisms on CBZ pharmacokinetics in Chinese Han patients with epilepsy.MethodsA total of 166 Chinese epilepsy patients of Han ethnicity were enrolled, who were on CBZ monotherapy more than eight weeks and plasma concentration were at steady state. The peripheral venous blood was collected predose in the morning 7:00 to 9:00. SCN1A c.3184A>G, SCN2A c.56G>A and IVS7-32A>G, CYP3A4*1G, CYP3A5*3, ABCC2 3972C＞T and c.1249 G>A, UGT2B7 c.802T>C genetypes were detected by using high resolution melting curve method. A TaqMan probe technique and direct sequencing method was performed to genotype SCN1A IVS5-91G>A and EPHX1 c.337T>C, respectively. CBZ plasma concentration at steady state were determined by HPLC as mentioned in the first part of the article. The Concentration-Dose ratios (CDRs) were calculated by dividing the steady-state CBZ plasma concentration by the CBZ daily dose (mg/kg/day). The CDRs were taken natural logarithm transformation before analysis. The relationships of maintenance dose and InCDRs with corresponding genotypes and haplotypes were investigated using ANOVA and multiple linear regression. The relationship between investigated polymorphisms and other clinical cofactors (such as age, gender, age of onset epilepsy, and course of disease) and maintenance dose and InCDRs were determined by multiple linear regression models.Results1. The frequencies of allele, genotypes and haplotypes analysisThe frequencies of SCN1A IVS5-91G>A, SCN1A c.3184A>G, SCN2A c.56G>A and SCN2A IVS7-32A>G in 166 epilepsy patients were 54.8%,12.4%,13.5% and 11.7%, respectively. The frequencies of CYP3A4*1G, CYP3A5*3, EPHX1 c.337T>C, UGT2B7 c.802T>C were 26.5%,70.5%,44.9% and 64.8% respectively. The frequencies of ABCC2 3972OC＞T和 c.1249 G>A were 16.9% and 10.5%. There was a strong linkage disequilibrium between SCN1A IVS5-91G>A and c.3184A>G (D’=0.79), SCN2A c.56G>A and IVS7-32A>G (D’=0.99), CYP3A4*1G and CYP3A5*3 (D’=0.71), repectively. Construction of haplotypes (SCN1A/2A 4SNPs) via expectation maximization resulted in five haplotypes of the frequencies more than 3%.2. The association analysis between gene polymorphism and CBZ maintenance dose and InCDRsStatistical analysis demonstrated that carriers of the variant SCN1A IVS5-91G>A and EPHX1 c.337T>C allele tended to require higher CBZ dosages and lower In (concentration-dose ratios) than noncarriers. There is significantly different among genotypes. The patients with SCN1A IVS5-91 AA and GA genotypes require higher maintenance CBZ dosages than wild-types (8.48±1.89 vs 10.35±3.27,8.48±1.89 vs 10.44±3.28, P＜0.05). Patients with EPHX1 c.337 CT genotype were more likely to require higher maintenance dosage of CBZ than patients with wild-types (9.65±3.04vs 10.98±4.08, P＜0.05)The patients with SCN1A IVS5-91 AA and GA genotypes have significantly lower InCDRs (day/ml) than wild-types (2.38±0.14 vs 2.56±0.14,2.41±0.13 vs 2.56±0.14, P＜0.001). And the patients with EPHX1 c.337 CC genotype have significantly lower In CDRs (day/ml) than wild-types (2.36±0.17 vs 2.46±0.12, P<0.05), there is no significantly different between CT and TT on CBZ InCDRs. The carriers of the variant ABCC2 3972 allele T also seemed to require higher CBZ dosages than allele C (9.89±3.08 vs 10.80±3.35, p=0.047). But the maintance dose and InCDRs were no significantly different among the three genotypes of ABCC2 3972C>T. In addition, the multiple regression model also revealed that genetic variants in SCN1A, and EPHX1 genes interactively affect the the InCDRs of CBZ (p<0.05). We did not find the other SNPs affect OXC maintenance dose and InCDRs.ConclusionThe present study identified that SCNIA and EPHX1 gene polymorphisms are the genetic factors associated with CBZ therapy optimization and provided useful information for individualized CBZ therapy in epileptic patients. Further studies in larger populations are needed to confirm our results.Chapter 3 Association of gene polymorphisms with the pharmacokinetics of OxcarbazepineBackground and ObjectiveOxcarbazepine (OXC) as second-generation AED is a keto-substituted analog of carbamazepine. Although OXC is structurally related to carbamazepine and has a common target-sodium ion channel, they are still different in the pharmacokinetics, efficacy and adverse reaction. Once OXC absorbed from the gastrointestinal tract, OXC is almost immediately reduced by a hepatic cytosolic arylketone reductase to form the major active metabolite monohydroxy carbamazepine (MHD). MHD is the major compound with antiepileptic effect. The major pathway for elimination of OXC and MHD is the formation of glucuronic acid conjugates by the UDP-glucuronosyhransferase (UGT) in the urine. In addition, ATP-binding cassette (ABC) transporter is also involved in the process of OXC in vivo. Multidrug resistance-associated protein 2 (MRP2), also known as ABCC2, is one of ABC transporter subfamily. The genes encoding SCN1A/2A, UGT and ABCC2 are polymorphic and we hypothesized these polymorphisms may contribute to the pharmacokinetics of OXC, and interindividual variance. The purpose of this study is to investigate the effects of SCN1A/2A, UGT2B7 and ABCC2 genetic polymorphisms on OXC pharmacokinetics in Chinese Han patients with epilepsy.MethodsA total of 184 Chinese epilepsy patients of Han ethnicity were enrolled, who were on OXC monotherapy more than eight weeks and plasma concentration were at steady state. The peripheral venous blood was collected predose in the morning 7:00 to 9:00. SCN1A c.3184A>G, SCN2A c.56G>A and IVS7-32A>G, ABCC2 3972C＞T and c.1249 G>A, UGT2B7 c.802T>C genetypes were detected by using high resolution melting curve method. A TaqMan probe technique was performed to genotype SCN1A IVS5-91G>A. MHD plasma concentration at steady state were determined by HPLC as mentioned in the first part of the article. The Concentration-Dose ratios (CDRs) were calculated by dividing the mean steady-state MHD plasma concentration by the OXC daily dose (mg/kg/day). The CDRs were taken natural logarithm transformation before analysis. The relationships of maintenance dose and InCDRs with corresponding genotypes and haplotypes were investigated using ANOVA and multiple linear regression. The influence of investigated polymorphisms and other clinical cofactors, such as age, gender, Age of onset epilepsy, and course of disease on maintenance dose and InCDRs was determined by multiple linear regression models.Results1. Correlation analysis of MHD plasma concentration at steady-state and the maintenance dose (Dose/Weight)The correlation analysis of MHD plasma concentration at steady-state and the maintenance dose (Dose/Weight) revealed that MHD plasma concentration at steady-state and the maintenance dose is positively correlated, r= 0.583, p ＜0.001.2. The frequencies of allele, genotypes and haplotypes analysisThe frequencies of SCN1A IVS5-91G>A, SCN1A c.3184A>G, SCN2A c.56G>A and SCN2A IVS7-32A>G in 184 epilepsy patients were 43.2%,9.2%,13.9% and 10.9% respectively. The frequencies of UGT2B7 c.802T>C, ABCC23972C>T and c.1249 G>A were 66.0%,12.0% and 10.5% respectively. There was a strong linkage disequilibrium between SCN1A IVS5-91G>A and c.3184A>G (D’=0.95), SCN2A c.56G>A and IVS7-32A>G (D’=0.99), repectively. Construction of haplotypes (SCN1A/2A 4SNPs) via expectation maximization resulted in seven haplotypes of the frequencies more than 3%.3. The association analysis between gene polymorphism and OXC maintenance dose and lnCDRsStatistical analysis demonstrated that carriers of the variant SCN1A IVS5-91G>A, UGT2B7 c.802T>C and ABCC2 c.1249 G>A allele tended to require higher OXC dosages than noncarriers. The patients with SCN1A IVS5-91 AA genotypes require higher maintenance OXC dosages than GG and GA genotypes (16.95±6.56 vs14.54±4.74, p=0.007, 14.42±5.18 vs 16.95±6.56, P=0.046). The patients with UGT2B7 c.802 CC and CT genotypes require higher maintenance OXC dosages than TT (12.14±3.49 vs 16.15±6.10, P=0.007; 12.14±3.49 vs 15.11±5.48, P=0.042, respectively). The patients with ABCC2 c.1249 AA and GA genotypes require higher maintenance OXC dosages than GG genotype (14.63±5.25 vs 25.66±10.17, P=0.006,14.63±5.25vs 16.98±6.27, P=0.018, respectively). lnCDRs (day/ml) in the patients with SCN1A IVS5-91 differs by genotypes in order GG> GA> AA. and there was a significant difference (2.90±0.29 vs 2.88±0.34 vs 2.75±0.21, P<0.05). In addition, the multiple regression model of lnCDRs also revealed that genetic variants in SCN1A IVS5-91G>A affected the concentration-dose ratio of OXC (p<0.05). We did not find the other SNPs affected OXC maintenance dose and InCDRs.Conclusion:The present study identified that SCN1A, UGT2B7 and ABCC2 gene polymorphisms are the genetic factors associated with OXC therapy optimization and provided useful information for individualized OXC therapy in epileptic patients. Further studies in larger populations are needed to confirm our results.Chapter 4 Association between ABCC2, SCN1A/2A Polymorphisms and drug-resistant EpilepsyBackground and ObjectiveAround a third of patients with epilepsy will not have their seizures controlled despite treatment with appropriate antiepileptic drugs (AEDs). And the patients with multidrug resistance will typically not respond to a variety of AEDs with differing modes of action, the reason and mechanism of multidrug resistance is still unclear. Previous studies have shown that the overexpression of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs), the drug targets sodium ion channels, and GABA-receptor subunits may be the main mechanism of drug-resistance. MRP2 and sodium ion channel is encoded by the highly polymorphic ABCC2 and SCN1A/2A gene. The aim of this study is to evaluate the association between the genetic polymorphisms of SCN1A/2A and ABCC2 gene with its haplotype and the drug-resistance of AEDsMethodsWe recruited patients with epilepsy who were treated by optimal AEDs. Demographic and clinical information were collected for all patients. Depending on their response to AEDs patients were divided into two groups, drug-resistant and drug-responsive group. Drug resistance was defined as four or more seizures a year despite two AEDs (monotherapy or polytherapy) at maximally tolerated doses. While drug response was defined as seizure free for the previous year or freedom from seizures for a minimum of three times the longest preintervention interseizure interval. Venous blood sample was cellected and DNA was extracted. SCN1A c.3184A>G, SCN2A c.56G>A and IVS7-32A>G, ABCC2 3972C＞T and c.1249 G>A genetypes were detected by using high resolution melting curve method. A TaqMan probe technique was performed to genotype SCN1A IVS5-91G>A. Haplotypes were reconstructed by SHEsis programs, which based on the maximum likelihood expectation maximization algorithm. Binary logistic regression was employed to compare the genotypes and haplotypes between two groups, and calculate the odds ratios (ORs) and 95% confidence interval(CI)ResultsA total of 453 Chinese epilepsy patients of Han ethnicity (258 male and 195 female) were recruited, of whom 246 were drug-resistant and 207 were drug-responsive. SCN1A IVS5-91 AA genotype was significantly associated with drug resistant epilepsy OR= 3.41 (1.73-6.70), p ＜0.001. SCN1A IVS5-91 allele A was significantly associated with drug resistant epilepsy, OR= 1.52 (1.17-1.98), p= 0.002. ABCC2 c.1249 GA genotype was significantly associated with drug resistant epilepsy OR= 2.14 (1.23-3.71), p= 0.007, allele A was significantly associated with drug resistance, OR= 2.05 (1.31-3.19), p= 0.001. SCN1A c.3184 A> G, SCN2A c.56 G> A and IVS7-32A> G and ABCC2 3972C＞ T polymorphisms were not associated with drug resistant epilepsy. Linkage disequilibrium analysis showed that SCN1A IVS5-91G> A was in strong LD with SCN1A c.3184A>G (D’ = 0.899), and SCN2A c.56G> A was in strong LD with SCN2A IVS7-32A> G (D’= 0.991). Haplotype AAGA (SCN1A IVS5-91G> A/SCN1A c.3184A> G/SCN2A c.56G> A/SCN2A IVS7-32A> G) in the resistant group was significantly higher than the control group (43.3% vs 34.3%, p= 0.006). Haplotype AAGG in the resistant group was also significantly higher than the control group (8.2% vs 4.7%, p= 0.036).Conclusion:SCN1AIVS5-91G> A and ABCC2 c.1249 G> A polymorphism and haplotypes AAGA and AAGG (SCN1A IVS5-91G>A/SCN1A c.3184A>G/SCN2Ac.56G>A/SCN2A IVS7-32A>G) were significantly associated with AEDs resistance. SCN1A c.3184 A>G, SCN2A c.56G>A, and IVS7-32A>G polymorphism were not associated with AEDs resistance. |
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