The multidrug transporter P-glycoprotein in pharmacoresistance to antiepileptic drugs

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Review

The multidrug transporter P-glycoprotein in pharmacoresistance to antiepileptic drugs

Karolina M. Stêpieñ¹, Micha³ Tomaszewski^2,3, Joanna Tomaszewska^3,4, Stanis³aw J. Czuczwar^5,6

1Clinical Biochemistry and Metabolic Medicine Department, Central Manchester Foundation Trust, Oxford Road, M13 9WL Manchester, UK

2Department of Cardiology,³Chair of Internal Medicine and Department of Internal Medicine in Nursing, Medical University of Lublin, Jaczewskiego 8, PL 20-954 Lublin, Poland

4Department of Vitreoretinal Surgery, Medical University of Lublin, Chmielna 1, PL 20-079 Lublin, Poland

5Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland

6Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland Correspondence: Micha³ Tomaszewski, e-mail: mdtomaszewski@yahoo.com

Abstract:

This review provides an overview of the knowledge on P-glycoprotein (P-gp) and its role as a membrane transporter in drug resistance in epilepsy and drug interactions. Overexpression of P-gp, encoded by the ABCB1 gene, is involved in resistance to antiepileptic drugs (AEDs), limits gastrointestinal absorption and brain access of AEDs. Although several association studies on ABCB1 gene with drug disposition and disease susceptibility are completed to date, the data remain unclear and incongruous. Although the litera- ture describes other multidrug resistance transporters, P-gp is the main extensively studied drug efflux transporter in epilepsy.

Key words:

P-glycoprotein, multidrug transporter, pharmacoresistance, antiepileptic drugs

Abbreviations: AEDs – antiepileptic drugs, ATP – adenosine triphosphate, BBB – blood brain barrier, BCRP – breast cancer resistance protein, DRE – drug resistant epilepsy, MRPs – multidrug resistance-associated proteins, P-gp – P-glycoprotein, PKA – protein kinase A, PKC – protein kinase C, SNP – single nucleotide polymorphism, TLE – temporal lobe epilepsy, TNF – tumor necrosis factor

Introduction

Epilepsy is a chronic and often progressive brain disorder, characterized by the periodic and unpredictable occurrence

of seizures. It affects approximately 1 to 2% [35, 51]

of the population. The main treatment of choice in epilepsy is chronic administration of antiepileptic drugs (AEDs) [10]. However, seizures continue to re- cur in 30% of people with epilepsy despite drug therapy. Current treatment options are limited among these patients. Morbidity and mortality are increased as well [64]. Long periods of drug resistant epilepsy (DRE) are associated with difficulties in psychosocial functions such as dependent behavior, lifestyle re- strictions, poor academic achievement and decline in self-esteem [15, 38]. Intractable epilepsy is associated with five-fold higher mortality rate when compared to

Pharmacological Reports 2012, 64, 10111019 ISSN 1734-1140

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Drug-resistant epilepsy (DRE)

The definition of DRE is elusive. A number of plausi- ble hypotheses of DRE have been proposed. Most cli- nicians would consider an epilepsy pharmacoresistant that had not been controlled by any of two to three first-line AEDs usually used for a given epilepsy syn- drome [42]. Potential causes of apparent resistance – such as misdiagnosis, non-compliance, inappropriate treatment or interaction between AEDs must be ex- cluded [33, 64]. Failure of drug response is a major limitation in the treatment of epilepsy.

The epilepsy itself has multiple etiologies so the intractability to AEDs may also be multifactorial. It has not been conclusively determined whether pharmacoresistance develops as the result of the disease process or exists at the time of initial seizure [15].

Elger [15] suggests that drug resistance is present in the early stage of the disease. Patients who do not respond to the first AED have a chance of only 10%

or lower to be controlled by other AEDs (even those

suffer from partial seizures [56].

The pathophysiological basis of pharmacoresistance in epilepsy is still poorly understood. One of suggested mechanisms refers to reduced target sensitivity and may occur through alterations of the composition and functionality of voltage-gated ion channels or neurotransmitter receptors in epileptogenic brain tissue. Another DRE hypothesis refers to P-glycoprotein (P-gp) expressed at the blood brain barrier (BBB) as a result of seizures, induction mediated by the nuclear receptor PXR, or genetic factors.

This review will present the overview of the current knowledge on biochemical properties and mechanisms of P-gp, particularly that several AEDs frequently used in the treatment of epilepsies are substrates of P-gp both in rodents (gabapentin, lamotrigine, phenobarbital, phenytoin and topiramate) and humans (phenytoin, phenobarbital, lamotrigine and levetiracetam) [4, 41, 44] (Tab. 1). Data on carbamazepine, levetiracetam and valproic acid are not clear [44, 48, 77].

Biochemical and functional properties of P-pg

P-gp is a prototype of membrane transporters. It is a 170-kD transmembrane glycoprotein that contains 1280 amino acids. It is a subject of several post- translational modifications, such as N-glycosylation, phosphorylation and ubiquitination [79] and consists of two similar halves of 610 amino acids each, joined by a linker region consisting of 60 charged residues.

Each of the two halves forms six transmembrane spans and hydrolyzes ATP during molecular transport.

The presence of both halves is essential for the transport activity [79].

According to Schmidt and Löscher [59], P-gp meets all the criteria for the transporter hypothesis as 1) it is increased in epileptogenic brain tissue of rodents; 2) associated with lower brain levels of AEDs;

3) higher in AED-resistant rats than in responsive ani- mals and 4) coadministration of the highly selective P-gp inhibitor, tariquidar, reverses AED resistance.

Thus, P-gp plays a significant role in mediating resis-

Tab. 1. Antiepileptic drugs (AEDs) that are substrates/inhibitors of P-glycoprotein with high/moderate or low/none affinity

AEDs References

Moderate/High affinity Low/No affinity Carbamazepine [6, 63, 73, 76, 77] [4, 22, 39, 43, 44,

52, 60]

Phenytoin [6, 14, 39, 41, 43, 44, 48, 63, 73, 75, 76]

Phenobarbital [8, 39, 41, 43, 44, 73, 75, 76]

Levetiracetam [6, 39, 41, 43, 44, 48, 76]

Lamotrigine [39, 41, 43, 44, 73, 76]

Gabapentin [73, 76]

Topiramate [48] [73, 76]

Valproate [4, 5, 39, 43, 44,

48, 73, 76]

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tance to AEDs in rodent models of temporal lobe epilepsy (TLE) and inhibition of P-gp can outwit this mechanism [59].

P-gp is specifically localized to secretory surfaces of liver, pancreas, renal tubules, small intestine and bile canaliculi acting as an energy-dependent pump for liphophilic compounds, including noxious xenobi- otics [38, 57]. It is also present in the brain capillaries that form the BBB; localized in the lining endothelium where it can restrict entry of lipophilic drugs into the brain and export substances, such as b-amyloid, from neuroparenchyma [32, 34]. P-gp pumps xenobi- otics from intracellular space back to the capillary lumen, thereby maintains the integrity of BBB and reduces the cerebral accumulation of substrate drugs [32].

P-gp is the first discovered human ABC transporter in drug-resistance ovarian cells obtained from Chinese hamsters [25]. It was initially discovered in multidrug resistance of cancer cells, but was also the first drug efflux transporter in multidrug resistance that was de- tected in endothelial cells of human BBB [20, 47].

Two genes in humans encode P-gp: MDR1 (systematic name ABCB1) [67] and MDR2 (ABCB4). In ro- dents, it is encoded by three genes mdr1a, mdr1b and mdr2 that have strikingly similar sequence to several bacterial transport proteins [19]. P-gp has very chemi- cally diverse substrates and inhibitors [31].

Overexpression of P-gp

Brain capillaries restrict the penetration of hydro- philic, polar and protein-bound compounds, whereas non-polar and highly lipid-soluble drugs penetrate easily through BBB by passive diffusion [42]. To penetrate BBB drugs have to be highly liphophilic.

Multidrug transporters are believed to function as an active defense mechanism, preventing lipophilic substances from reaching the brain through either the BBB or blood-cerebrospinal fluid barrier [32, 66].

P-gp is believed to cause multidrug resistance by reducing intracellular drug accumulation through its function as an active efflux pump [31]. It prevents high accumulation of the drug in the brain tissue [69]. The active transport from the brain back into blood vessels causes lower brain accumulation of drugs than it would be expected based on their lipophilicity [69].

It has been hypothesized that overexpression of efflux transporters at the BBB [25], which prevents AEDs from reaching sufficiently high brain concentrations despite adequate plasma levels, could be one such mechanism [31]. P-gp overexpression may be the consequence of (i) seizures themselves, (ii) PXR- mediated upregulation through exposure to certain an- ticonvulsants such as carbamazepine, or (iii) genetic variation [37].

Additionally, it has been shown that P-gp can be induced by AEDs in both endothelial and astroglial cells in culture [74]. On the contrary, Ambroziak et al.

[1] have demonstrated that exposure to major antiepileptic drugs (such as phenobarbital, phenytoin and carbamazepine) does not alter the functionality of P-gp [1, 4].

The expression of P-gp in different individuals var- ies probably due to genetic and environmental influ- ences [62]. It is crucial whether overexpression of efflux transporters in epileptic brain tissue is constitutive or acquired/induced or both mechanisms coexist.

A constitutive overexpression could be a result of genetic predisposition or it could be intrinsic to the development of the specific pathology [30, 31]. Theory that overexpression of efflux transporters may be constitutive or intrinsic, may be supported by the obser- vation of P-gp upregulation in patients’ post-mortem tissues with malformations of cortical development that had died before experiencing seizures or exposure to AEDs [67]. Kwan and Brodie [31] hypothe- size that increased expression of drug transporters is associated with refractory epilepsy and that AEDs are substrates of these transporters. The evidence for upregulation of efflux transporters such as P-gp was derived from brain tissues removed during epilepsy surgery from patients with drug resistant epilepsy.

Pathologically elevated expression of P-gp has been found in resected brain tissue of patients with refractory temporal lobe epilepsy (TLE) undergoing epilepsy surgery as well as in limbic brain regions of mouse and rat models of TLE [38, 40, 41].

Recent report by Bankstahl et al. [7] has demonstrated in experimental study on mice that increased P-gp expression and activity post status epilepticus occurs not only in primary epileptogenic regions e.g., hippocampus, but also in the thalamus and the cere- bellum [7].

In the animal models, increased expression of P-gp was associated with decreased brain concentration of AEDs such as phenytoin [40, 41].

The multidrug transporter P-glycoprotein

Karolina M. Stêpieñ et al.

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protein kinases. It has been documented that P-gp contains sites with structural features mimicking phosphorylation sites for protein kinases such as protein kinase C (PKC) or protein kinase A (PKA) [12]. P-gp is also phosphorylated in vivo and the linker region (amino acids 629–686) was identified to be the major phosphorylation domain [17]. It has been shown that substitution of PKA and PKC phosphorylatable residues in the P-gp linker region affects the stimulation of P-gp ATP-ase activity by some substrates [17].

Pharmacogenetics of P-gp

Genetic polymorphism in transporters may explain why two patients with the same type of epilepsy may have different response to AEDs [15]. Some AEDs are substrates [22] and inhibitors of P-gp (phenytoin, carbamazepine, lamotrigine, phenobarbital, valproic acid, levetiracetam, gabapentin) that can be affected by ABCB1 gene polymorphisms (Tab. 1). Because the efflux drug transporter P-gp is highly expressed at BBB, genetic variation in its expression or functionality could directly affect brain uptake and extrusion of AEDs. The overexpression of several ABC-transporters, particularly P-gp (MDR-1/ABCB1 gene) has been recognized to play a central role in the pharmacoresistant phenotype in epilepsy by limiting the drug efficacy as well as the plasmatic levels of AEDs in affected cases [36, 55].

There is still considerable controversy whether polymorphisms in the ABCB1 gene encoding P-gp could influence drug-response and seizure frequency [27, 61]. Recent genetic association studies have indicated an association of the 3435CC genotype, which is associated with increased P-gp expression, with AED resistant epilepsy [37]. Noteworthy, cerebrospinal fluid concentrations of phenobarbital were significantly lower in epilepsy patients with the 3435CC genotype [8]. Mosyagin et al. [49], however, examined the impact of polymorphisms 3435CT and 2677GT in the ABCB1 gene on the ABCB1 mRNA expression and P-gp content. Authors concluded that they cannot exclude an association of ABCB1 variants on P-gp function, but their results suggest that brain ABCB1 mRNA and protein expression is

phism (SNP), at the position C3435T in exon 26 of the MDR1 gene, is associated with differential intestinal expression of P-gp. However, the homozygous T-allele (T3435T or TT genotype) is associated with decreased MDR-1 expression by approximately 2-fold compared with patients homozygous for C-allele (C3435C or CC genotype), and heterozygous individuals exhibit an intermediate phenotype (T3435C or CT genotype) [23, 35].

Importantly, the correlation between intestinal expression of cytochrome P450 enzymes and ACB transporters with dose requirement and plasma levels of carbamazepine and phenytoin, evaluated in 44 epileptic patients, indicated that differences in intestinal MDR1 and MRP2 expression may influence carbamazepine and phenytoin disposition and may account for interindividual pharmacokinetic variability [63].

Additionally, phenytoin plasma levels has been shown to be affected by polymorphisms in MDR1, CYP2C9 and CYP2C19 [26]. Results of a recent Japanese study showed an association between C3435T and low intestinal expression of cytochrome P450 (CYP3A4) that similarly to ABCB1 gene, is located at chromo- some 7q21 [18]. As a consequence, the induction of CYP3A4 is associated with MDR-1 induction [78].

The results of another recent study by Meng et al. [46]

were consistent with the published results for the Japanese patients and showed that the 3435TT genotype was associated with increased P-gp expression [46] that was converse to the results of the study on Caucasian group of patients [23]. Authors concluded that patients with the 3435-TT genotype had significantly lower plasma carbamazepine concentrations than those with the 3435-CC genotype [46], but there were no significant associations between all the studied genotypes, haplotypes and diplotypes involving SNPs of ABCB1 gene.

Several studies have been performed on patients treated with multiple AEDs. In studies performed on patients treated with monotherapy, an association between ABCB1_3435CT polymorphisms and drug- resistance was found in individuals treated with phenytoin [14] or phenobarbital [8], whereas in those with carbamazepine a reverse association [60] or non-association [52] was found.

The clinical impact of ABCB1 polymorphisms on AED resistance in patients with epilepsy has been

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assessed in clinical studies with P-gp inhibitors, such as nimodipine or cyclooxygenase-2 inhibitors that significantly improve the anticonvulsive action of AEDs [24, 71]. These drugs optimize the control of P-gp expression, improve AEDs brain penetration, help overcome the pharmacoresistance and control seizures [18]. One study examining an inhibitory interaction of several AEDs with P-gp in vitro at concentrations exceeding therapeutic plasma concentrations, has suggested that modulation of P-gp is not a major determinant of AED action in monotherapy. It is possible that additive or even synergistic inhibition of transport will occur when several AEDs that compete for P-gp binding sites are given concomitantly [73].

A meta-analysis by Nurmohamed et al. [51] has shown that although the ABCB1 gene had been highly investi- gated and plays many important roles, it is unlikely that the polymorphism at 3435 plays a major role in the development of drug resistance to AEDs [55]. Similarly, Haerian et al. [21] have failed to show an association between the C3435T polymorphism in ABCB1 gene and the risk of drug-resistance [21]. Authors of meta-analyses do not recommend testing for C3435T polymorphisms of ABCB1 gene as the investigation will not yield any infor- mation that would be useful in the diagnosis, manage- ment or prognosis of epilepsy [9, 21].

The role of ABCC2 (MRP2) transporter is not well understood yet [68]. However, ABCC2 expression was reported in brain-derived endothelial cells from patients with drug-refractory epilepsy, indicating

a potential upregulation of ABCC2 because of repeti- tive seizures. This study has demonstrated a higher risk of AED failure in ABCC2 (MRP2)-24T allele carriers, possibly though a compensatory upregulation of ABCB1 (P-gp) [68].

P-gp vs. other drug transporters

P-gp compared to other known multidrug transporters has one known isoform, whereas multidrug resistance- associated proteins (MRPs) have multiple confirmed ones such as MRP1, MRP2, MRP3, MRP4 and MRP5 that were recently isolated in brain microvessel endothelial capillaries and on the luminal side of brain capillary endothelial cells [50, 72] (Tab. 2). Both P-gp and MRP have been also found to be expressed in TLE specimens [2, 66].

Breast cancer resistance protein (BCRP) also known as ABCG2/MXR/ABCP, is another ABC transporter family and expresses significant overlap in substrate specificity profile with P-gp [53]. BCRP seems to have a low affinity for AEDs so that its role in drug resistance epilepsy is not as important as it is for P-gp [13]. BCRP has been expressed in capillary endothelial cells, but in contrast to P-gp and MRP, BCRP levels are not changed in the brain tissue from patients with hippocampal sclerosis [2, 3]. Addition- ally, all three types of ABC transporters are induced

Tab. 2. Comparison of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP)

P-gp MRP References

Number of isoforms One More than one [50, 53, 72]

Hippocampal sclerosis expression Yes Yes [2, 3]

Seizure induced Yes Yes [70]

Transporter for AEDs Yes No [39, 43, 44, 77]

Effect on expression TNFa

IL-1b IL-6

Increased Increased Decreased

Increased No effect No effect

[29, 58]

[58]

Blood cells expressing transporter:

Erythrocytes Platelets Lymphocytes

Yes Yes Yes

Yes Yes No

[28]

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frequency [70].

Interestingly, P-gp has been shown to be a transporter for major AEDs such as carbamazepine [77], phenytoin, lamotrigine, levetiracetam and phenobarbital, however, MRP1, 2 and 5 has not been demonstrated to have such properties [39, 43, 44]. Zhang et al. [75] have exhibited that human P-gp is involved in concentration dependent transport of phenytoin and phenobarbital (but not ethosuximide) [75]. It is not known yet if BCRP is a transporter for AEDs.

Some studies examined the effect of inflammatory mediators on expression of multidrug transporters and have shown that the expression of P-gp can be in- creased by TNFa, but not by IL-1b or IL6 [58]. Simi- larly, MRP1 expression can be increased by TNFa and polyinosinic-cytidylic acid a ligand for Toll-like receptor-3 [29]. These results suggest that P-gp- expressing astrocytes in the close proximity to blood vessels form a glial barrier that may alter the ability of AEDs to access the epileptogenic focus. In addition, this barrier remains under the control of pro- inflammatory pathways [29]. Currently, there are no similar studies on BCRP and inflammatory mediators.

Multidrug transporters have been found to be present in glial cells in the brain, hippocampus, frontal and peripheral cortex [4, 65], but there is some evidence that MRP 1–5 may be expressed in blood cells such as erythocytes, platelets, and P-gp additionally in lymphocytes [28] (Tab. 2). The fact is important in the aspect of the transport of AEDs such as valproate. It has been suggested that valproate accumulates in erythrocytes when, as a result of drug transporters inhibition at the erythrocyte membrane, its plasma concentration significantly decreases during co-medica- tion with carbapenem antibiotics [45]. The reduction of valproate plasma levels was associated with the occurrence of seizures in patients with epilepsy [16].

The transporter for valproate has not been identified as yet as there is no evidence that valproate is trans- ported by P-gp or MRPs [39, 43, 44].

Conclusion

In conclusion, the knowledge on pharmacoresistance mechanisms to AEDs becomes important in drug de-

nation with AEDs [18]. Finally, the identification of genetic polymorphisms, in either transporter or AEDs genes, would increase the chance of the antiepileptic therapy. However, the most recent data suggest that ABCB1 variants provide no evidence of an association of drug responsiveness to AEDs.

In view of current controversial knowledge about associations of polymorphisms and AEDs therapeutic efficacy, but also which AEDs are substrates for P-gp, further research is necessary to determine the substrate characteristics of different AEDs. It would be desirable to design an AED that would have low or medium affinity for multidrug transporter proteins.

AEDs are highly lipophilic agents and pass mem- branes by rapid diffusion. In addition, they generally pass the blood-brain barrier, and are only affected when pathophysiologic mechanisms upregulate P-gp- expression brain-penetration rates [54]. Modulating P-gp is likely to overcome resistance to some of AEDs, but definitely will not solve the issue of multidrug resistance. Some advancement in the form of P-gp modulators such as cyclooxygenase-2 has been exhibited and requires further evaluation of its clinical relevance.

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Received: January 30, 2012; in the revised form: May 27, 2012;

accepted: June 8, 2012.