Polymorphisms in the ABCB1 Gene in Phenobarbital Responsive and Resistant Idiopathic Epileptic Border Collies

Expression and cellular distribution of multidrug resistance-related proteins in patients with focal cortical dysplasia

Multidrug resistance is one of the most serious problems in the treatment of epilepsy that is likely to have a complex genetic and acquired basis. Various experimental data support the hypothesis that over-expression of antiepileptic drug (AED) transporters may play a pivotal role in drug resistance. Hyyt 6however, key questions concerning their functionality remain unanswered. The idea that P-glycoprotein, encoded by the ABCB1 gene, might mediate at least part of the drug resistance was met with both enthusiasm and skepticism. As in oncology, initial optimism has been clouded subsequently by conflicting results. The first study reporting a positive association between genetic variation in the P-glycoprotein and multidrug-resistant epilepsy was published in 2003. Since then, several other genetic association studies have attempted to verify this result. However, taken overall, the role of P-glycoprotein in drug resistance in epilepsy still remains uncertain. We intend to critically review the inherent problems associated with epilepsy pharmacogenetic studies in general and with ABCB1 polymorphisms studies in particular. The lessons learnt from the ABCB1 studies can help us to guide future association genetics studies to investigate AED resistance, and thereby taking us closer to the cherished dream of personalized AED therapy.

Despite considerable progress in the pharmacotherapy of epilepsy, more than 30% of patients are reported to be resistant to antiepileptic drugs. Multidrug resistance 1 (MDR1) gene could play a role in drug resistance in epilepsy. In this study, the authors investigated the association between the MDR1 gene polymorphisms, C3435T and G2677AT, and drug resistance epilepsy by using polymerase chain reaction/restriction fragment length polymorphism and pyrosequencing methods in a group of 39 patients with drug-resistant epilepsy and 92 controls. No associations were found between the polymorphisms of the MDR1 gene and drug-resistant epilepsy. Haplotype analysis showed no significant association. Compound genotype analysis showed that CC3435/GG2677 was significantly higher in the control group compared to the patient group. In conclusion, MDR1 polymorphisms investigated in this study are not associated with antiepileptic drug resistance, but the CC3435/GG2677 compound genotype might have an effect on antiepileptic drug response.

In recent years, outcomes for pediatric cardiac transplantation (PCTx) have steadily improved, with current 5-year overall survival rates estimated at 83%.1 This progress may be attributed to improvements in pretransplantation management, selection of donor hearts, surgical technique, prevention and treatment of rejection, and minimization of treatment-related adverse events. Despite these advances, children who receive cardiac transplants experience significant morbidity and mortality, and there is considerable variability in outcomes, much of which cannot be explained by known clinical risk factors. One source of interindividual variability is genetic variation in the host. There is growing evidence that genomic variation leads to differences in immune response, response to therapies, and susceptibility to adverse outcomes such as malignancy and infection.2–4 This review will focus on the effect of variation in genes that encode enzymes, transporters, and drug target molecules that influence drug response. After review of the foundational principles of pharmacogenomics, evidence for the pharmacogenomic effects on cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil (MMF), and the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus will be reviewed and clinical implications of these findings discussed. Given the limited nature of evidence from studies of PCTx patients, support will also be drawn from non-PCTx research and pediatric renal transplantation. Genetic studies of nonpharmacogenes such as those in immunomodulatory pathways and variation in the donor genotype may contribute to risk prediction but are beyond the scope of this review. ### Genetic Variation DNA variation can alter biological function through several mechanisms (Figure 1). Variation within the coding sequence or in intron/exon borders can change the protein product through changes in start codons, stop codons, splice sites, or within the coding sequence, leading to nonsense-mediated decay or altered protein function. Other variants associated with pharmacogenomic outcomes are not predicted to change the encoded protein. These synonymous and …

Drug Resistance in Epilepsy: Expression of Drug-resistance Proteins in Common Causes of Refractory Epilepsy Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M. Brain 2002;125(Pt 1):22–31 Epilepsy is resistant to drug treatment in about one third of cases, but the mechanisms underlying this drug resistance are not understood. In cancer, drug resistance has been studied extensively. Among the various resistance mechanisms, overexpression of drug-resistance proteins, such as multidrug resistance gene-1 P-glycoprotein (MDR1) and multidrug resistance–associated protein 1 (MRP1), has been shown to correlate with cellular resistance to anticancer drugs. Previous studies in human epilepsy have shown that MDR1 and MRP1 also may be overexpressed in brain tissue from patients with refractory epilepsy; expression has been shown in glia and neurons, which do not normally express these proteins. We examined expression of MDR1 and MRP1 in refractory epilepsy from three common causes, dysembryoplastic neuroepithelial tumors (DNTs; eight cases), focal cortical dysplasia (FCD; 14 cases), and hippocampal sclerosis (HS; eight cases). Expression was studied immunohistochemically in lesional tissue from therapeutic resections and compared with expression in histologically normal adjacent tissue. With the most sensitive antibodies, in all eight DNT cases, reactive astrocytes within tumor nodules expressed MDR1 and MRP1. In five of eight HS cases, reactive astrocytes within the gliotic hippocampus expressed MDR1 and MRP1. Of 14 cases of FCD, MDR1 and MRP1 expression was noted in reactive astrocytes in all cases. In five FCD cases, MRP1 expression also was noted in dysplastic neurons. In FCD and DNTs, accentuation of reactivity was noted around lesional vessels. Immunoreactivity was always more frequent and intense in lesional reactive astrocytes than in glial fibrillary acidic protein–positive reactive astrocytes in adjacent histologically normal tissue. MDR1 is able to transport some antiepileptic drugs (AEDs), and MRP1 also may do so. The overexpression of these drug-resistance proteins in tissue from patients with refractory epilepsy suggests one possible mechanism for drug resistance in patients with these pathologies. We propose that overexpressed resistance proteins reduce the interstitial concentration of AEDs in the vicinity of the epileptogenic pathology and thereby render the epilepsy caused by these pathologies resistant to treatment with AEDs. P-Glycoprotein and Multidrug Resistance–associated Protein Are Involved in the Regulation of Extracellular Levels of the Major Antiepileptic Drug Carbamazepine in the Brain Potschka H, Fedrowitz M, Loscher W. Neuroreport 2001;12:3557–3560 Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epilepsy patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED also is refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, overexpression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance–associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

been satisfied for the transporter hypothesis, however, only in rodent models of epilepsy as yet [7]. Accordingly, P-gp expression is: increased in epileptogenic brain tissue of rodents; associated with lower brain levels of AEDs; higher in AED-resistant rats than in responsive animals; and most importantly, co-administration of the highly selective P-gp inhibitor, tariquidar, reverses AED resistance. Thus, it is reasonable to conclude that P-gp plays a significant role in mediating resistance to AEDs in rodent models of temporal lobe epilepsy and that inhibition of P-gp can circumvent this mechanism – however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear [7]. Increased brain expression of efflux transporters, such as P-gp, could be a result either of prolonged or frequent seizures, as demonstrated in rodent models of epilepsy, or of genetic factors, such as polymorphisms in the ABCB1 gene or, theoretically, of both [2]. Siddiqui et al. were the first to report that a SNP in the ABCB1 gene is associated with resistance to multiple AEDs, leading to the suggestion that drug resistance in epilepsy might be genetically determined [8]. In this study, patients with AED-resistant epilepsy were more likely to have the CC genotype at the ABCB1 C3435T polymorphism, which is associated with increased expression of the protein, than the TT genotype [8]. Since then, different studies of various designs have tried to confirm the association between the 3435C>T polymorphism and AED resistance in epilepsy, but most of these subsequent studies were negative [9]. A recent meta-ana lysis of 11 case–control studies involving 3371 patients (1646 patients with drug-resistant epilepsy and 1725 controls) did not identify a significant association between AED resistance and ABCB1 polymorphisms, including the 3435 SNP [10]. However, in most of these pharmacogenetic studies, patients were treated with various AEDs, ignoring the fact that not all AEDs are substrates Resistance to multiple anti-epileptic drugs (AEDs) is a significant problem in epilepsy affecting at least 30% of patients with this common and often devastating disease [1]. One prominent hypothesis to explain this multidrug resistance suggests an inadequate penetration or excess efflux of AEDs across the blood–brain barrier (BBB) as a result of overexpressed efflux transporters, such as P-glycoprotein (P-gp), the encoded product of the multidrug resistance-1 (MDR1, ABCB1) gene [2]. Drug efflux transporters such as P-gp have a major impact on the pharmacological behavior of most clinically used drugs, critically affecting drug absorption, disposition and elimination in the body [3]. The idea that enhanced BBB expression of P-gp could be involved in AED resistance was first proposed by Tishler et al. in 1995, who reported that P-gp and ABCB1 are markedly increased in brain tissue of pa tients with medically intractable partial epilepsy [4]. This finding was subsequently confirmed by several other groups and formed the basis of the transporter hypothesis of intractable epilepsy [5].

Resistance to antiepileptic drugs (AEDs) is one of the most serious clinical problems in epilepsy, and along with AED teratogenicity, perhaps the major concern of epilepsy pharmacogenetics. Studying the genetics of drug resistance in epilepsy is important, as it may identify or confirm key mechanisms underlying this phenomenon that have real clinical importance; it might also offer insights into its prediction and management. Drug resistance in epilepsy is likely to be multifactorial: overactivity of multi-drug transporters provides one likely underlying mechanism through lowering of AED concentration in the epileptogenic focus. Genetic association studies may provide a tool to assess this ‘transporter’ hypothesis by determining whether differences between individuals contribute to resistance phenotypes. Most of these studies have investigated one variant in the ABCB1 gene, and have provided, thus far, inconclusive evidence. This review also considers current knowledge of the role of genetic polymorphisms in multi-drug transporters in pharmacoresistant epilepsy, to highlight possible confounding factors affecting the implementation and interpretation of association studies in this field.

Drug resistance in epilepsy: Why is a simple explanation not enough?

Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

Current theories on drug resistance in epilepsy include the drug transporter hypothesis, the drug target hypothesis, and a novel approach called the inherent severity model of epilepsy, which posits that the severity of the disease determines its relative response to medication. Valuable as each of these hypotheses is, none is currently a stand-alone theory that is able to convincingly explain drug resistance in human epilepsy. As a consequence, it may be of interest to update and integrate the various hypotheses of drug resistance and to explore possible links to the severity of epilepsy. The observation that a high frequency of seizures prior to onset of treatment is a prognostic signal of increased severity and future drug failure suggests that common neurobiological factors may underlie both disease severity and pharmacoresistance. Such a link has been proposed for depression; however, the evidence for a direct mechanistic link, genetic or otherwise, between drug response and disease severity of human epilepsy is still elusive. Although emerging data from experimental studies suggest that alterations in GABA(A) receptors may present one example of a mechanistic link, clearly more work is needed to explore whether common neurobiological factors may underlie both epilepsy severity and drug failure.

There is a lack of data on idiopathic epilepsy (IE) in Border Collies (BCs) in the veterinary literature. Genetic epilepsy occurs in BCs and is frequently characterized by a severe clinical course and poor response to medical treatment. Forty-nine BCs diagnosed with IE. Medical records, seizure data, treatment data, and pedigree information of affected dogs were collected. Cases were classified phenotypically as affected or not affected; mild, moderate, or severe clinical course; active epilepsy (AE) or remission; and drug resistant or not drug resistant. Clinical manifestations were classified as having a moderate (33%) or severe clinical course (49%), characterized by a high prevalence of cluster seizures and status epilepticus. Survival time was significantly decreased in dogs < 2 years of age at seizure onset, and in dogs with a severe clinical course. Drug resistance was apparent in 71% of 24 dogs treated with > 2 antiepileptic drugs. The epilepsy remission rate was 18%. Median age at onset was significantly higher and initial seizure frequency was significantly lower in dogs with remission compared with dogs with AE. Pedigree analyses indicated a strong genetic founder effect in the appearance of epilepsy, resembling autosomal recessive inheritance. The present study confirms the occurrence of genetically mediated epilepsy with a frequent severe clinical course and drug resistance in BCs. The results provide information about the long-term prognosis of IE in BCs for veterinarians and concerned owners, and may benefit breeders as well.

Background Genetic polymorphisms of drug efflux transporters as ATP-binding cassette subfamily B, member 1 (ABCB1) have been suggested to modulate antiepileptic drugs (AEDs) response. We aimed to explore the association of ABCB1 polymorphisms and AEDs resistance among epileptic patients. Methods A total of 86 Jordanian epileptic patients treated with AEDs was included in the study. DNA was extracted from blood samples and genotyping and haplotypes analyses were conducted for Nine single nucleotide polymorphisms (SNPs) on the ABCB1 gene. Results Data revealed that none of the examined SNPs were associated with resistance to AEDs neither on the level of alleles nor genotypes. However, strong association was found between rs2235048 (OR = 10.6; 95%CI = [1.89–59.8], p= 0.01), rs1045642 (OR = 14; 95%CI = [1.3–156.7], p= 0.02), rs2032582 (OR = 9.1; 95%CI = [1.4–57.3], p= 0.04) and rs1128503 (OR = 18.7; 95%CI = [1.6–222.9], p= 0.02), ABCB1 polymorphisms and resistance to AEDs among females but not males. Haplotype analysis revealed statistically significant associations. The strongest significant associations were for haplotypes containing 2677G_1236 T in two-SNPshaplotypes (OR = 4.2; 95%CI = [1.2–14.9], p = 0.024); three-SNPs-haplotypes (OR = 4.2; 95% CI = [1.2–14.9], p = 0.02); four-SNPs-haplotypes (OR = 4.1; 95%CI = [1.2–14.3], p = 0.026). Conclusion Data suggests that there is a gender dependent association between ABCB1 genetic polymorphisms and response to AEDs. Additionally, ABCB1 haplotypes influence the response to AEDs. Further investigation is needed to confirm the results of this study.

The pathogenesis underlying pharmacoresistance in epilepsy is unclear. One of the candidate mechanisms that has attracted growing interest is the limitation of antiepileptic drug (AED) access to the seizure focus by a range of efflux transporters, the prototype of which is P-glycoprotein (P-gp). P-gp is encoded by the multidrug resistance (MDR1 or ABCB1) gene. Predominantly expressed in organs with excretory functions and at blood-tissue barriers, P-gp is thought to act as a physiologic defense by extruding xenobiotics from mammalian cells and affording protection of sensitive organs. The high level of P-gp in the cerebrovascular endothelium is believed to contribute to the functionality of the blood-brain barrier. Overexpression of P-gp causes multidrug resistance in certain cancers. It has been hypothesized that overexpression of P-gp and other efflux transporters in the cerebrovascular endothelium, in the region of the epileptic focus, also may lead to drug resistance in epilepsy. This hypothesis is supported by the findings of elevated expression of efflux transporters in epileptic foci in patients with drug-resistant epilepsy, induction of expression by seizures in animal models, and experimental evidence that some commonly used AEDs are substrates. Conflicting reports suggest a possible association between variants of the MDR1 gene and medical intractability in epilepsy. Further studies to delineate the exact role, if any, of P-gp and other efflux transporters in drug-resistant epilepsy are warranted.

The multidrug transporter P-glycoprotein in pharmacoresistance to antiepileptic drugs

Drug-resistant epilepsy with uncontrolled severe seizures despite state-of-the-art medical treatment continues to be a major clinical problem for up to one in three patients with epilepsy. Although drug resistance may emerge or remit in the course of epilepsy or its treatment, in most patients, drug resistance seems to be continuous and to occur de novo. Unfortunately, current antiepileptic drugs (AEDs) do not seem to prevent or to reverse drug resistance in most patients, but add-on therapy with novel AEDs is able to exert a modest seizure reduction in as many as 50% of patients in short-term clinical trials, and a few become seizure free during the trial. It is not known why and how epilepsy becomes drug resistant, while other patients with seemingly identical seizure types can achieve seizure control with medication. Several putative mechanisms underlying drug resistance in epilepsy have been identified in recent years. Based on experimental and clinical studies, two major neurobiologic theories have been put forward: (a) removal of AEDs from the epileptogenic tissue through excessive expression of multidrug transporters, and (b) reduced drug-target sensitivity in epileptogenic brain tissue. On the clinical side, genetic and clinical features and structural brain lesions have been associated with drug resistance in epilepsy. In this article, we review the laboratory and clinical evidence to date supporting the drug-transport and the drug-target hypotheses and provide directions for future research, to define more clearly the role of these hypotheses in the clinical spectrum of drug-resistant epilepsy.