Molecular and Cellular Pharmacology
Exploring the possible interaction between anti-epilepsy drugs and multidrug efflux pumps; in vitro observations

https://doi.org/10.1016/j.ejphar.2008.09.014Get rights and content

Abstract

Approximately one-third of patients with epilepsy display an inherent resistance to pharmacological therapy, manifest as continuing seizures despite maximal tolerated doses of anti-epileptic drugs. One hypothesis for the underlying mechanism of anti-epileptic drug pharmacoresistance is lower drug entry to the epileptic neurones due to the activity of multidrug efflux pumps from the ATP Binding Cassette (ABC) superfamily at the blood-brain barrier. There has been a steady accumulation of animal and human data supporting this theory, particularly for ABCB1 (P-glycoprotein). However, much of this evidence is indirect. In the present study, several anti-epileptic drugs (carbamazepine, valproic acid, phenytoin, lamotrigine and primidone) were examined for their ability to interact with three ABC transporters that have been implicated pharmacoresistance of anti-epileptic drugs — ABCB1, ABCC1 and ABCG2. Interaction of anti-epileptic drugs with the transporters was assessed by determining whether they could reverse the ability of multidrug ABC transporters to confer a drug resistance phenotype on cancer cell lines. None of these compounds was able to affect the phenotype, suggesting an absence of any interaction with the multidrug transporters. This finding was further investigated by examination of transporter activity; namely the ability to reduce steady-state intracellular [3H]-radiolabelled drug accumulation. None of the anti-epileptic drugs affected labelled drug accumulation by any of the triumvirate of multidrug transporters examined, indicating that they are unlikely to be substrates. The lack of direct modulation by anti-epileptic drugs of ABC transporter function suggests that these proteins do not contribute significantly to resistance in epilepsy.

Introduction

Approximately one-third of patients with epilepsy fail to achieve control of their seizures with medical treatment, despite multiple different anti-epileptic drugs being tried (Kwan and Brodie, 2004). This phenotype has been termed pharmacoresistance and represents a serious clinical obstacle to successful disease management. The emergence of a resistant phenotype following prolonged exposure to anti-epileptic drugs and the common situation where the resistance is to a wide variety of different compounds led to the hypothesis of an underlying multidrug resistance mechanism, similar to that observed in cancer (Remy and Beck, 2006, Schmidt and Loscher, 2005). Multidrug resistance in cancer is partly attributed to the ability of certain members of the ATP Binding Cassette (ABC) superfamily of transporters to prevent sufficient intracellular accumulation of cytotoxic drugs (Modok et al., 2006). The ABC proteins involved belong to a subset capable of transporting an extraordinarily large range of structurally and functionally unrelated compounds. The proteins in question include P-glycoprotein (ABCB1), MRP1 (ABCC1) and the BCRP (ABCG2). These transporters are not purely localised to cancerous tissue, with expression demonstrated in a number of secretory tissues (e.g. gastrointestinal tract) in addition to crucial barrier tissues including the blood-brain barrier (Cordon-Cardo et al., 1989, Schinkel et al., 1995, Thiebaut et al., 1987). Moreover, resected brain tissue from patients with pharamacoresistant epilepsy has revealed elevated expression of ABCB1 and ABCC1 (Aronica et al., 2004, Sisodiya et al., 2002). The blood-brain barrier localisation in the apical membranes of capillary endothelial cells represents the primary and predominant expression, although the proteins have also been detected at the astro-glial end-feet (Sisodiya et al., 2002). This expression pattern suggested that pharmacoresistance in epilepsy was due to ABC transporters mediating efflux of anti-epileptic drugs from the brain parenchyma back into the blood.

As described above, the localisation of multidrug ABC transporters at the blood-brain barrier is not specific to epilepsy patients and consequently, the correlative evidence for their role between this expression and pharmacoresistance required substantiation. A number of whole animal studies attempted to address this issue. Typically, the animal models demonstrated that inhibition of ABCB1 by intra-peritoneal or intra-cerebral administration of verapamil led to small, but consistent increases in the brain:blood plasma ratio for phenytoin (Rizzi et al., 2002). Similarly, mice with an ABCB1 knock-out genotype displayed increased uptake of phenytoin at the brain (Potschka et al., 2002). However, verapamil belongs to the first generation of ABCB1 inhibitors (McDevitt and Callaghan, 2007) and therefore, these studies were criticised for the potentially low selectivity to inhibit ABCB1. This led to the use of a third generation inhibitor, Tariquidar (XR9576), which also generated an elevation in brain levels of anti-epileptic drugs (Brandt et al., 2006, Marchi et al., 2005). The study by Marchi et al. could only generate altered oxycarbazepine accumulation in P-glycoprotein expressing cell lines with high concentrations of Tariquidar — the levels were in considerable excess of the values known to specifically inhibit ABCB1. In addition, recent evidence has demonstrated that Tariquidar also inhibits other ABC transporters, namely ABCG2 (Robey et al., 2004). Similar models have been employed to demonstrate that inhibition of ABCC1 also produces a pharmacokinetic advantage with respect to the passage of anti-epileptic drugs across the blood-brain barrier (Kubota et al., 2006, Sisodiya et al., 2006).

The investigations above provide indirect evidence to support the proposition that ABC transporters contribute to pharmacoresistance to anti-epileptic drugs. This has been supported further by studies in epilepsy animal models which have provided evidence that the inhibition of ABC transporters in vivo affects the response to therapy with anti-epileptic drugs (Hocht et al., 2007). In one such study drug resistance to the anti-epileptic drug oxycarbazepine in spontaneously seizing post-pilocarpine treated rats was reversed by coadministration with intra-hippocampal delivery of verapamil (Clinckers et al., 2005). Additionally there are two clinical case reports of verapamil reversing pharmacoresistance in patients (Iannetti et al., 2005, Summers et al., 2004).

Thus, the in vivo and clinical observations suggest, but not unequivocally, that pharmacoresistance in epilepsy is mediated by ABC transporters. However, the majority of the data supporting a role for ABC transporters pharmacoresistance to anti-epileptic drugs is rather indirect and the magnitude of the alteration in brain:blood plasma ratios produced by non-specific inhibitors is not large. Recent, direct transport studies in cultured cells appear to cast doubt on the hypothesis for a small number of anti-epileptic drugs. For example, valproic acid transport across an epithelial monolayer was unaffected by the expression of P-glycoprotein or the MRP1 and 2 isoforms (Baltes et al., 2007a). These authors have also verified the result for phenytoin, carbamazepine and levetiracetam (Baltes et al., 2007b). This disagreement with the transporter hypothesis as a basis for pharmacoresistance has been reconciled by the suggestion that the anti-epileptic drugs belong to a class of “weak substrates” of the ABC transporters.

Provision of direct evidence for an interaction between the anti-epileptic drugs and the multidrug ABC transporters is critical in absolutely defining the role of these transporters in the resistant phenotype. This issue is at the heart of the present manuscript and we provide a systematic and comprehensive evaluation of the potential for a series of commonly used anti-epileptic drugs to interact with ABCB1, ABCC1 and ABCG2 using specific cell based assays.

Section snippets

Materials

RPMI1640, Foetal Calf Serum and penicillin/streptomycin were obtained from Invitrogen (Paisley, UK). Methylthiazoletetrazolium (MTT), vinblastine sulphate, vincristine sulphate, doxorubicin hydrochloride, daunomycin hydrochloride, etoposide, mitoxantrone, verapamil, nicardipine, cyclosporin A, carbamazepine, valproic acid, phenytoin, lamotrigine and primidone were purchased from Sigma Aldrich (Poole, UK). The MCF7/FLV1000 and COR/L23 cell lines were kindly provided by Prof Susan Bates (NCI,

Characterisation of cell lines

The breast and lung cell lines chosen for the present investigation have been well characterised for their sensitivity to anti-cancer drugs and the resistant cells were generated through long-term selection with anti-cancer drugs as described (Aronica et al., 2004, Rhodes et al., 1994, Robey et al., 2001). The relative expression of the ABC transporters ABCB1, ABCC1 and ABCG2 has been verified in the present investigation as shown in Fig. 1. The BXP21 monoclonal antibody reveals that ABCG2

Discussion

The present investigation provides a systematic characterisation of the interactions of anti-epileptic drugs and the multidrug ABC transporters known to be expressed at the blood-brain barrier. Two assays were used to test the hypothesis that multidrug ABC transporters contribute to the pharmacoresistance observed in epilepsy by preventing the passage of anti-epileptic drugs across the blood-brain barrier. Data produced demonstrated that the drugs were not capable of interacting with ABCB1, ABC

Acknowledgements

The authors kindly acknowledge the provision of the COR/L23Adr and MCF7/FLV1000 cell lines by Prof M Barrand (Cambridge University) and Prof S Bates (NCI, USA) respectively. The research was supported by a Cancer Research UK Program Grant (SP1861/0401) to R Callaghan. The authors would like to thank J. Ryland and D. Mullins their patience and inspiration in the early stages.

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