Intractable epilepsy and the P-glycoprotein hypothesis.

Just Accepted by International Journal of Neuroscience

Intractable epilepsy and the P-glycoprotein hypothesis Guang-Xin Wang, Da-Wei Wang, Yong Liu, Yan-Hui Ma doi:10.3109/00207454.2015.1038710

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ABSTRACT Epilepsy is a serious neurological disorder that affects more than 60 million people worldwide. Intractable epilepsy (IE) refers to approximately 20-30% of epileptic patients that fail to achieve seizure control with antiepileptic drug (AED) treatment. Although the mechanisms underlying IE are not well understood, it has been hypothesized that multidrug transporters such as P-glycoprotein (P-gp) play a major role in drug efflux at the blood-brain barrier, and may be underlying factor in the variable responses of patients to AEDs. The main goal of the present review is to show evidence from different areas that support the idea that the overexpression of P-gp is associated with IE. We discuss here evidences from animal studies, pharmacology, clinical cases and genetic studies.

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Wang et al. G.-X. Wang et al. Intractable epilepsy and P-glycoprotein

Intractable epilepsy and the P-glycoprotein hypothesis

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Guang-Xin Wang1*, Da-Wei Wang2*, Yong Liu1 and Yan-Hui Ma1 Medical Institute of Paediatrics, Qilu Children’s Hospital of Shandong

University, No.23976 Jingshi Road, Jinan 250022, P.R.China

Department of Biochemistry and Molecular Biology, School of Medicine,

Shandong University, Jinan 250012, P.R.China

Corresponding

Author:

Yan-hui

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*These authors contribute equally to this work Ma,

Tel:

(86)531-81309026,

Fax:

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(86)531-87964257; E-mail: [email protected] Received: 2014-05-03

ABSTRACT

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Accepted: 2015-04-04

Epilepsy is a serious neurological disorder that affects more than 60 million people worldwide. Intractable epilepsy (IE) refers to approximately 20-30% of

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epileptic patients that fail to achieve seizure control with antiepileptic drug (AED) treatment. Although the mechanisms underlying IE are not well understood, it has been hypothesized that multidrug transporters such as P-glycoprotein (P-gp) play a major role in drug efflux at the blood-brain barrier,

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and may be underlying factor in the variable responses of patients to AEDs. The main goal of the present review is to show evidence from different areas that support the idea that the overexpression of P-gp is associated with IE. We discuss here evidences from animal studies, pharmacology, clinical cases and genetic studies. Key words: intractable epilepsy, P-glycoprotein, hypothesis 1

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INTRODUCTION

Epilepsy is a serious neurological disorder that affects more than 60 million

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antiepileptic drugs (AEDs), but up to 20%-30% of patients, or more than 20 million people including 2 million children under the age of 15, do not respond

well to pharmacotherapy [1-3]. This is known as medically intractable epilepsy

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(IE) or drug-resistant epilepsy. The International League against Epilepsy proposed a definition of IE as the failure of adequate trials of 2 tolerated and

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appropriately chosen and used AED schedules [4,5]. The mechanisms underlying IE are not yet completely understood. Several explanations for IE are as follows: (a) an overexpression of the P-glycoprotein

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(P-gp) encoded by the multidrug resistance 1 (MDR1) gene and other efflux transporters,

such

as

multidrug

resistance

protein

(MRP),

in

the

cerebrovascular endothelium in or around the region of the epileptic focus may lead to drug resistance in epilepsy; (b) the loss of AED sensitivity at certain

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people worldwide. The majority of epileptic patients can be treated with

target sites in the brain, including the sodium ion channel and the gamma-aminobutyric acid (GABA) A receptor; and (c) seizures beget seizures through a cascade of events that include various types of neuronal damage, the sprouting of neuronal axons and the formation of new synapses that establish aberrant glutamatergic synapses [6]. An important characteristic of IE is that most patients with IE are resistant to several, if not all, AEDs, though

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these drugs act by different mechanisms [3]. This multidrug resistance argues against epilepsy-induced alterations in specific drug targets as a major cause of IE and instead points to nonspecific and possibly adaptive mechanisms,

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such as decreased drug uptake into the brain via the intrinsic or acquired overexpression of multidrug transporters in the blood-brain barrier (BBB) [7].

The main goal of the present review is to demonstrate that the evidence from

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transporters such as P-gp underlies IE. We briefly review the structure, expression and function of P-gp in an attempt to facilitate this discussion, and we then pursue the following four distinct lines of evidence that have the

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strongest support for the role of P-gp in IE:

Evidences from animal studies: a local decrease in the brain access of

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AEDs mediated by the overexpression of P-gp may be involved in pharmacoresistance in epilepsy.

Evidences from pharmacology: various major AEDs are substrates for

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P-gp.

Evidences from clinical cases: there is a significant difference in the

expression

of

P-gp

between

the

drug-resistant

patients

and

the

drug-responsive patients.

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different areas supports the hypothesis that the overexpression of multidrug

Evidences from genetic studies: the gene encoding P-gp is associated

with IE.

P-GP STRUCTURE, EXPRESSION AND FUNCTION

P-gp, which is encoded by MDR1 gene, is a 1280-amino acid plasma

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membrane protein that consists of 2 halves that share a high degree of similarity. Each homologous half contains 6 hydrophobic transmembrane domains and a relatively hydrophilic intracytoplasmic loop encoding an

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ATP-binding site. P-gp transport activity is strictly dependent on energy metabolism and ATP formation [8]. Despite sequence identity between the 2 halves, the lack of a homologous placing of introns suggests that the 2 halves

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movement after a duplication event [9]. In mice, the protein is composed of 1276 amino acids, has a molecular mass of 141 kDa in the absence of glycosylation and comprises two homologous halves. Hydrophobicity analysis

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indicates that P-gp is composed of four domains: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). The NBDs bind

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and hydrolyze two ATP molecules, whereas each TMD consists of six transmembrane helices (TM1-TM6 in TMD1 and TM7-TM12 in TMD2) that together form the drug-binding pocket (DBP) and the pathway for substrate

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transport [10].

P-gp has been found in several normal human tissues, including the liver, kidney, large and small intestines and pancreas [11]. Similarly, elevated mouse mdr1, mdr2 and mdr3 mRNA levels have been observed in the kidney,

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of P-gp have evolved independently or have undergone major intron

liver and intestine, respectively. Moreover, P-gp is present in the placenta, in secretory glands of the endometrium in the pregnant mouse and in the adrenal cortex, which argues for a role of P-gp in physiological steroid secretion [12,13]. P-gp is also expressed by endothelial cells at blood-tissue barrier sites, such as the BBB [14]. Most of the published data have demonstrated that P-gp is principally expressed at the luminal membrane of brain capillary endothelial

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cells in mammals, including humans, suggesting that P-gp may serve as a general mechanism in the mammalian BBB, which protects the brain from intoxication by potentially harmful lipophilic compounds from natural sources

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and other lipophilic xenobiotics, including anticancer drugs and AEDs, that otherwise could penetrate the BBB through simple diffusion without any limitation [14,15]. Some peripheral blood mononuclear cells, such as cytotoxic

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may be involved in cell-mediated cytotoxicity [16,17]. In addition, P-gp has

been found to colocalize and interact with caveolin-1, one of the structural proteins of caveolae, which are involved in the transport of macromolecules

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across capillary endothelial cells by transcytosis [18,19].

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EVIDENCE FROM ANIMAL STUDIES: A LOCAL DECREASE IN THE BRAIN ACCESS OF AEDs MEDIATED BY THE OVEREXPRESSION OF

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P-GP MAY BE INVOLVED IN PHARMACORESISTANCE IN EPILEPSY

Animal models have several advantages over clinical cases: the results may be easier to interpret because groups are genetically more homogenous; the environment can be controlled; and a variety of interventions is possible.

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T lymphocytes and nature killer cells, also express P-gp, suggesting that P-gp

The overexpression of multidrug transporters, primarily P-gp, is an attractive and plausible hypothesis to explain multidrug resistance in epilepsy. First and foremost, we need to determine whether the expression of P-gp differs in drug-resistant and drug-responsive individuals. The present data indicate that mammalian neurons, which normally do not express P-gp to any detectable extent, can overexpress P-gp in a pathological situation [20,21]. The low

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mRNA expression levels in the neurons from control rats are in line with the finding that P-gp is below the detection level of immunohistochemical methods in the neurons of the controls. Volk and Loscher used a rat model of temporal

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lobe epilepsy and grouped the rats into responders and non-responders based on their individual response to phenobarbital (PB) [22]. Then, they detected the P-gp

expression

in

the

brain,

including

the

hippocampus,

via

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microvessel endothelial cells that form the BBB. PB-resistant rats exhibited a marked overexpression of P-gp in brain capillary endothelial cells, whereas PB-responsive rats had a significantly lower level of P-gp expression. In a

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report by Bankstahl et al., immunohistochemical staining of Pgp did not indicate any increase of Pgp expression in brain capillary endothelial cells

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during status epilepticus (SE), whereas significant overexpression was determined in animal models 48 h after SE, so that the authors concluded involvement of Pgp in the time-dependent development of resistance of SE to

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PHT and PB [23].

Using high-resolution micro-PET scans, scientists have been able to visualize the movement of C11-labeled P-gp substrates through the BBBs of both wild-type and mdr1-knockout mice. In wild-type mice, verapamil (a substrate of

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immunohistochemistry. P-gp overexpression was observed exclusively in the

P-gp) does not penetrate into the brain to any significant extent because it binds to P-gp and is immediately transported out of the capillary endothelial cells of the BBB. However, verapamil passes freely through the BBB in mdr1-knockout mice because these mutants lack the P-gp transporter [24]. These results suggest that P-gp may transport the substrate out of the capillary endothelial cells of the BBB and reduce the penetration of the

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substrates. Some researchers have shown that AEDs, including carbamazepine (CBZ), phenytoin (PHT), PB and valproic acid, could increase the expression of P-gp

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in the rat brain and rat brain microvascular endothelial cells [25]. Jing et al chronically treated kindled rats with PB. Continuous treatment with PB had a

gradually reduced antiepileptic effect, and on day 40, PB exhibited no

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expression and a decreased PB concentration in the hippocampus. These results indicate that P-gp plays an important role in reducing the antiepileptic effect of PB and that chronic treatment with PB may induce P-gp

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overexpression. Jing et al also demonstrated that the overexpression of P-gp in the brains of subjects with pharmacoresistant epilepsy is due to a

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combination of drug effects and epileptic seizures [26]. Moerman et al investigated the concentration of C11-desmethylloperamide (11C-dLop, a radiolabeled substrate of P-gp) in mice that were pretreated with AEDs at

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doses that are therapeutic in humans [27]. These investigators found that pretreatment with AEDs at therapeutic doses significantly decreased the intracerebral

concentration

of

11C-dLop,

which

indicates

that

the

administration of AEDs at human therapeutic doses has an inducing effect on

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antiepileptic effect; this was accompanied by both an enhancement of P-gp

the P-gp transport at the BBB. These findings imply that the administration of therapeutic doses of AEDs to epileptic patients may result in an increased efflux of AEDs by P-gp, which can lead to a decrease in the therapeutic effects of these AEDs. Brandt et al selected a group of PB-resistant rats and treated them with combinations of PB and the selective P-gp inhibitor tariquidar [28]. They found

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that the coadministration of tariquidar fully restored the anticonvulsant activity of PB without altering the plasma pharmacokinetics or neurotoxicity of the antiepileptic drug. The experiment demonstrates that inhibiting P-gp in

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epileptic rats with proven drug resistance counteracts this resistance and that P-gp is likely to restrict the anticonvulsant effects of AEDs. Studies performed

in mdr1a/b (-/-) mice lacking P-gp showed that the brain distribution of select

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wide-type counterparts [29]. Furthermore, Cox et al found that the volume of

distribution for enaminone anticonvulsants was significantly higher in mdr1a/b (-/-) mice than it was in their mdr1 a/b (+/+) counterparts [30]. Experiments

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performed in mice lacking P-gp expression, including genetic knockouts and naturally occurring P-gp-deficient animals, have provided voluminous evidence

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that P-gp can significantly impede drug translocation across the blood-brain interface [31,32]. Furthermore, a blockade of the BBB P-gp via the cerebral application of P-gp inhibitors significantly increases the brain concentration of

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various drugs, which is again consistent with the function of P-gp as an efflux transporter in the BBB.

EVIDENCE FROM PHARMACOLOGY: VARIOUS MAJOR AEDs ARE

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enaminones was significantly higher compared with their mdr1a/b (+/+)

SUBSTRATES FOR P-GP

The first indication that AEDs are substrates for P-gp was obtained from in vitro experiments performed by Tishler et al [33]. They found that the intracellular PHT levels in MDR1-expressing cell lines were only one-fourth of those in MDR1-negative cells. In vitro studies involving drug-selective MDR

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cell lines expressing P-gp revealed that cellular drug accumulation was greatly reduced compared with the drug-sensitive cell line, and similar observations were also made for cells transfected with the MDR1 gene [34].

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Researchers have demonstrated that fourteen AEDs (PHT, PB, CBZ, lamotrigine, gabapentine, felbamate, topiramate,levetiracetam et al.) are

substrates for P-gp (see Table 1). Moreover, Zhang et al demonstrated that

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not be observed if concentrations below or above the therapeutic range are used [49]. Xu et al examined the inhibitory effect of a P-gp inhibitor (1416) on

P-gp both in vitro and in vivo and observed that 1416 successfully increased

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the concentration of the drugs that are P-gp substrates in vitro and enhanced the activities of P-gp substrates in multidrug-resistant mice, effectively

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reversing the multidrug resistance [50]. These results verified the reversal of the multidrug-resistance phenotype by 1416 through the inhibition of the drug efflux function of P-gp both in vitro and in vivo. C11-Phenytoin was found to be

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a moderate P-gp substrate because the brain-to-plasma concentration ratio increased by 45% after complete P-gp inhibition with tariquidar, which indicates that inhibiting P-gp could increase the concentrations of AEDs [51]. Extracellular brain levels of PHT were significantly increased by the local

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P-gp-mediated transport highly depends on the AED concentration and may

administration of P-gp inhibitors, providing evidence that P-gp may reduce the concentration of PHT in the brain [52]. These studies indicate that the drugs that can inhibit P-gp might inhibit multidrug resistance. Marchi et al measured the brain-to-plasma partition of the convert of oxcarbazepine (OXC) in epilepsy patients undergoing surgery [53]. They found a

significant

inverse

linear

correlation between

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the

brain-to-plasma

concentration ratio of OXC and the level of MDR1 mRNA. The in vitro uptake studies demonstrated lower intracellular 10-OH CBZ levels in cells with higher P-gp expression levels, and the intracellular drug concentration was increased

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with a P-gp blocker. These two findings combined suggest that the level of expression of P-gp in an epileptogenic area might affect the brain concentrations of AEDs.

References

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Evidence for AEDs as AEDs substrates of P-gp Patients Animals Cell lines Acetazolamide – – Y Carbamazepine Y Y N Carbamazepine-10,11-e – – Y poxide Eslicarbazepine acetate – – Y Felbamate – Y N Gabapentin – ? ? Lacosamide – – Y Lamotrigine Y Y Y Levetiracetam Y N Y Oxcarbazepine Y – Y Phenobarbital – Y Y Phenytoin – Y Y S-licarbazepine – – Y Sodium valproate – – Y Tiagabine – Y – Topiramate – Y ?

[35] [36,37,38,39,40] [36] [36] [39, 41] [35,39,42] [43] [38,39,40,41,44] [38,39,40,44,45] [36,40] [38,39,41,42,44] [37,38,42,44,46,47] [36] [42] [47] [39,47,48]

Y: yes; N: no; –: not reported; ?: conflicting evidence

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Table 1. AEDs as substrates of P-gp in in vitro and in vivo models

EVIDENCE FROM CLINICAL CASES: THERE IS A SIGNIFICANT DIFFERENCE

IN

THE

EXPRESSION

OF

P-GP

BETWEEN

THE

DRUG-RESISTANT PATIENTS AND THE DRUG-RESPONSIVE PATIENTS

The P-glycoprotein hypothesis is well supported in animal models of IE.

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However, relevant in vivo human data are sparse [54]. Tishler et al were the first to evaluate specimens of brain tissue from patients undergoing surgical producers to control intractable seizures [33]. In this study, eleven of 19 brain

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specimens had MDR1 mRNA levels 10 times greater than normal tissue, and 14 of 19 specimens had increased expression of P-gp in the capillary

endothelium. Tishler et al proposed that P-gp may play a clinically significant

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expression may thus contribute to the refractoriness of seizures in patients

with IE. Liu et al quantified P-gp immunoreactivity in the epileptogenic, sclerotic hippocampus and the non-epileptogenic undamaged brain regions; a

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comparison of the two regions indicated that a higher percentage of P-gp immunopositive labeling was present in the sclerotic hippocampus than in the

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undamaged brain regions [55]. Lazarowski et al examined the intractable seizures of a patient treated with AEDs including PHT, PB and lorazepam. Despite several consecutive intravenous loading doses, the PHT blood levels invariably

subtherapeutic.

After

surgical

treatment,

an

ST

were

immunohistochemical analysis of the resected brain tissues revealed high P-gp expression levels [56]. Dombrowski et al found that in endothelial cells isolated from the temporal lobe blood vessels of patients with IE, P-gp was

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role by limiting the access of AEDs to the brain parenchyma; increased MDR1

overexpressed relative to the controls [57]. Rambeck and colleagues used microdialysis probes to measure AED concentrations in the extracellular space of epileptogenic tissue, cerebrospinal fluid (CSF) and the blood plasma of patients undergoing resective surgery [58]. An analysis of the perfusates, collected over 1 h prior to tissue excision, revealed that the AED concentrations were significantly decreased in the epileptogenic zones of

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these individuals compared with the CSF concentrations. These data are the first indication that decreased drug levels in the epileptogenic zone may be involved in the drug resistance of patients with epilepsy. In view of the data

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indicating that the endothelial barrier function of the BBB is transiently disrupted during seizures, the overexpression of multidrug transporters in the

glial end feet covering the blood vessels may represent a "second barrier"

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lowers the extracellular concentration of an AED in the vicinity of an

epileptogenic pathology and thereby renders the epilepsy caused by the pathology resistant to AED treatment [59].

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In 75% of hippocampal sclerosis (HS) cases, P-gp was detected in blood vessels with prominent endothelial labeling, which indicates that P-gp is

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unregulated in concert in the hippocampus of patients with IE [60]. Lazaroski et al reported a tuberous sclerosis patient who manifested with IE [56]. An immunohistochemical analysis of the tissues resected from patients

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undergoing surgical treatment revealed high P-gp expression levels. Summers et al added verapamil (a P-gp inhibitor) to the antiepileptic drug regimen of a patient with IE [61]. They found that the addition of verapamil to CBZ therapy greatly improved the overall seizure control and subjective quality

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under these conditions. Sisodiya et al proposed that the overexpressed P-gp

of life in this AED-resistant patient; this outcome was accompanied by randomly acquired CBZ serum concentrations that were higher after the verapamil was added. Given the emerging evidence that P-gp is overexpressed in epileptogenic brain tissue, particularly in capillary endothelial cells and astrocytes contributing to BBB permeability, and the increasing evidence that various

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major AEDs are substrates for P-gp, we can conclude that the overexpression of P-gp may contribute to IE.

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EVIDENCE FROM GENETIC STUDIES: THE GENE ENCODING P-GP IS ASSOCIATED WITH IE

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tissues and organs to potentially toxic xenobiotics, it has been suspected that P-gp activity in genetically impaired patients might increase the susceptibility to disease [62]. In rats, AED-resistance and AED-responsiveness appear to be

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associated with different frequencies of several polymorphisms in the mdr1a gene that encodes P-gp in the rodent BBB [63]. Researchers have also

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attempted to obtain clinical support for the overexpression of P-gp in IE by studying the effects of polymorphisms in the genes that encode for P-gp in humans. The C1236T, G2677T/A and C3435T loci, which were reported to

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influence function and (or) expression of P-gp, are the most commonly studied genetic variants in the MDR1 gene [64]. Among these three loci, the C3435T polymorphism has received the most attention as a critical variant in the resistance to AEDs [65,66]. Siddiqui et al. were the first to report that C3435T

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Because the changes in P-gp function increase the exposure of various

is associated with IE, patients with IE were more likely to have the CC genotype at the MDR1 C3435T polymorphism, which is associated with increased expression of the protein, than the TT genotype[52]. Since then, various studies have tried to examine this association, but there is a discrepancy in the results of these studies. Ebid et al. genotyped the MDR1 gene in the epileptic patients and found that the patients with drug-resistant

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epilepsy were more likely to have the CC than the TT genotype compared with either the responsive patients or the control subjects [67]. Taur et al. has also reported that drug resistant patients

were more

likely to have the ‘C’

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allele than the ‘T’ allele of C3435T polymorphism in the MDR1 gene in a Indian population [68]. However, in another study carried out by Shaheen et al. in a South Indian

population, it was reported that patients with drug resistant to have TT genotype when compared to drug

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responsive epileptic patients [69]. Hoffmeyer et al analyzed the MDR1

sequence in 21 volunteers, along with P-gp expression and function, to evaluate whether alterations in the MDR1 gene correlate with intestinal P-gp

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expression and the uptake of orally administered P-gp substrates, the results showed a significant correlation of the C3435T polymorphism in MDR1 gene

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with the expression level and function of MDR1, individuals homozygous for this polymorphism had significantly lower duodenal MDR1 expression and the highest substrate plasma levels [70]. Subsequent to this report, the results

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from at least four more studies with adequate statistical power that included patients with IE of similar ethnic backgrounds have been published, but the studies obtained conflicting results. In the study of Basic et al., PB levels were analyzed in plasma and cerebrospinal fluid (CSF), demonstrating that while

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epilepsy were more likely

the C3435T polymorphism did not affect plasma levels, the CC genotype of C3435T was associated with significantly lower PB levels in CSF and CSF: plasma ratio than the CT or TT genotypes, these results indicated that PB levels in the CSF of epileptics with the CC genotype of C3435T were lower because of increased brain expression of P-gp [71]. However, in the study of Kwan et al., no significant difference in ABCB1 mRNA level was found

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between brain tissues excised from drug-resistant epileptics with different genotypes of C3435T [72]. In this respect, it is important to note that the C3435T polymorphism has been reported to result in P-gp with altered drug

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and inhibitor interaction sites [73]. So that lack of altered P-gp expression does not exclude that the C3435T polymorphism affects transport of AEDs by P-gp,

as indicated by the report of Basic et al. [71]. Maleki et al found that the risk of

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CT than in those with the TT genotype, which indicates that T1236C polymorphism is associated with drug resistance in Iranian female epileptic patients [74]. Other polymorphisms, such as G2677T/A, have been reported to

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be related to IE. Nevertheless, a recent randomized controlled trial involving 152 patients (69 patients with IE and 83 patients with drug-responsive epilepsy)

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and a meta-analysis of 23 studies involving 7067 epileptics did not identify a significant association between IE and ABCB1 polymorphisms [75,76].

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CONCLUSIONS

In the present review, we summarized four distinct lines of evidence that corroborate the role of P-gp in the pathogenesis of IE. The overexpression of

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drug resistance was higher in Iranian female epileptic patients with 1236 CC or

P-gp in the BBB can reduce the penetration of AEDs into the brain, which leads to decreased drug concentrations around epileptic foci and contributes to IE.

Nevertheless, it is necessary to note that a single theory most likely cannot explain the complexity of IE. In relation to the first line of evidence from the animal studies described here, it can be concluded that, although the role of

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P-gp in the mechanisms underlying IE is unambiguous, focusing on P-gp alone is an oversimplification; other factors may be involved in epileptogenesis [77,78]. Regarding the evidence from pharmacology, there is no doubt that

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several AEDs are P-gp substrates. However, some AEDs, such as ethosuximide, that are not P-gp substrates could also be effective in treating

epilepsy or IE. Evidence from clinical cases included those whose tissues

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tissue. However, there are limitations in study designs to the experiments, such as species differences, and the findings will require confirmation in a larger patient cohort. Evidence from genetic studies suggests that the MDR-1

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gene encoding P-gp is important to IE. Whether specific P-gp polymorphisms contribute to IE remains controversial; more conclusive evidence that

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polymorphisms in P-gp influence antiepileptic drug resistance is required. In addition, other genes have also been studied regarding the genetics of IE. Knowledge of P-gp in the pathogenesis of IE will allow us to develop novel

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drug therapies for IE. The modulation of P-gp expression is a growing area of epilepsy research [79]. Novel AEDs that target the transcriptional activators of P-gp

expression,

including

the

N-methyl-d-aspartate

receptor,

the

inflammatory enzyme cyclooxygenase-2 and the prostaglandin E2 EP1

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exhibited a significantly higher expression of P-gp compared with normal brain

receptor, are under investigation.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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ACKNOWLEDGEMENTS

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This work was supported by the JNSTI (201003120) and NSFC (81071042). The authors are deeply grateful for Guang-fang Ren for rewriting the

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Intractable epilepsy, hemispheric malformation, and generalized electroencephalography abnormalities.

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"Subtotal" hemispherectomy in children with intractable focal epilepsy.

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