Elsevier

Neuropharmacology

Volume 52, Issue 2, February 2007, Pages 333-346
Neuropharmacology

Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein

https://doi.org/10.1016/j.neuropharm.2006.07.038Get rights and content

Abstract

In view of the important role of P-glycoprotein (Pgp) and other drug efflux transporters for drug distribution and resistance, the identification of compounds as substrates of Pgp-mediated transport is one of the key issues in drug discovery and development, particularly for compounds acting on the central nervous system. In vitro transport assays with Pgp-transfected kidney cell lines are widely used to evaluate the potential of compounds to act as Pgp substrates or inhibitors. Furthermore, such cell lines are also frequently utilized as a substitute for more labor-intensive in vitro or in vivo models of the blood–brain barrier (BBB). Overexpression of Pgp or members of the multidrug resistance protein (MRP) family at the BBB has been implicated in the mechanisms underlying resistance to antiepileptic drugs (AEDs) in patients with epilepsy. Therefore, it is important to know which AEDs are substrates for Pgp or MRPs. In the present study, we used monolayers of polarized MDCKII dog kidney or LLC-PK1 pig kidney cells transfected with cDNA containing either human MDR1, MRP2 or mouse mdr1a and mdr1b sequences to measure the directional transport of AEDs. Cyclosporin A (CsA) and vinblastine were used as reference standards for Pgp and MRP2, respectively. The AEDs phenytoin and levetiracetam were directionally transported by mouse but not human Pgp, whereas CsA was transported by both types of Pgp. Carbamazepine was not transported by any type of Pgp and did not inhibit the transport of CsA. In contrast to vinblastine, none of the AEDs was transported by MRP2 in transfected kidney cells. The data indicate that substrate recognition or transport efficacy by Pgp differs between human and mouse for certain AEDs. Such species differences, which are certainly not restricted to human and mouse, may explain, at least in part, the controversial data which have been previously reported for AED transport by Pgp in preparations from different species. However, because transport efficacy of efflux transporters such as Pgp or MRP2 may not only differ between species but also between tissues, the present data do not exclude that the AEDs examined are weak substrates of Pgp or MRP2 at the human BBB.

Introduction

ATP-dependent drug efflux transporters, such as P-glycoprotein (Pgp; ABCB1) or members of the multidrug resistance protein (MRP; ABCC) family, have been localized in several tissues, including the liver, kidney, gastrointestinal tract, and blood–brain barrier (BBB), where they perform excretory or protective roles, affecting the absorption, distribution, and excretion of various clinically important drugs (Schinkel and Jonker, 2003, Fromm, 2004). Overexpression of such transporters is a well-characterized mechanism of chemoresistance known as multidrug resistance (Ling, 1997, Lage, 2003, Polgar and Bates, 2005). However, such overexpression not only occurs in chemotherapy-resistant cancers but also in normal tissues, such as the BBB (Löscher and Potschka, 2005a, Löscher and Potschka, 2005b). In medically refractory epilepsy, up-regulation of drug efflux transporters in the BBB is currently discussed as one important mechanism to explain resistance to antiepileptic drugs (AEDs) by assuming that this up-regulation restricts brain access of AEDs (Schmidt and Löscher, 2005). Pgp and MRP2 are located in the apical (luminal) membrane of brain capillary endothelial cells (that form the BBB) and are thought to export drugs back into the blood before these drugs enter the brain parenchyma (Begley, 2004). Thus, overexpression of such transporters, as found in epileptogenic brain tissue from patients with pharmacoresistant epilepsy, would limit the entry of AEDs into the brain, provided that AEDs are substrates of these transporters (Löscher and Potschka, 2005a). It is therefore important to determine which AEDs are substrates for drug efflux transporters such as Pgp or MRP2.

When studying whether AEDs are substrates for efflux transporters, it is essential to note that these drugs, if at all, can only be relatively weak substrates, because strong substrates of the BBB efflux transporters are almost completely excluded from the brain (Schinkel, 1999). Over the last years, we have used a microdialysis rat model to study whether inhibition of Pgp or MRPs affects the brain entry of various AEDs (see review by Löscher and Potschka, 2005a). In this model, either Pgp or MRP inhibition (by application of inhibitors via the microdialysis probe into the brain) significantly increased the brain penetration of many major AEDs, including phenytoin (PHT), carbamazepine (CBZ), and lamotrigine, indicating that these drugs are weak substrates for Pgp or MRPs in the BBB. The only exception was the novel AED levetiracetam (LEV; Potschka et al., 2004). If true, the lack of multidrug transporters to affect the penetration of LEV into the brain would be a striking advantage compared to all other major AEDs and one important factor to explain the therapeutic efficacy of LEV as adjunctive treatment in patients with medically refractory partial seizures (Marson et al., 2001, Betts et al., 2003, Pinto and Sander, 2003).

However, because of lack of selectivity of the Pgp inhibitors used in most of our previous experiments and some inherent problems of the model used (Löscher and Potschka, 2005a), our findings from the microdialysis rat model need to be substantiated by other models used to study whether drugs are substrates for Pgp or MRPs. One strategy here is the use of cells lines which have been transfected with transporter genes. Such cell lines are widely used to study whether drugs are substrates for Pgp (Polli et al., 2001, Schwab et al., 2003, Löscher and Potschka, 2005a). The drug-transporting Pgp is encoded by the MDR1 gene in humans, while rodents have two drug-transporting Pgp genes, mdr1a and mdr1b (Fromm, 2004). The primary mdr1 isoform detected in brain microvessels in rodents is mdr1a, whereas mdr1b mRNA is the main isoform detected in brain parenchyma, indicating that Pgp in these different locations may confer different functions (Löscher and Potschka, 2005a). Using monolayers of a polarized pig-kidney epithelial cell line (LLC-PK1), transfected with complementary DNA containing either MDR1 or mdr1a sequences, Schinkel et al. (1996) reported transport of PHT by Pgp, which could be blocked by the Pgp inhibitor PSC 833 (valspodar). Using MDR1-transfected Madin Darby canine kidney (MDCK type II) cells as a substitute for more labor-intensive BBB models, Mahar Doan et al. (2002) found no evidence that CBZ is a substrate for Pgp. However, Weiss et al. (2003) reported that CBZ, at high concentrations, inhibits the transport of other Pgp substrates, indicating that CBZ might be a weak Pgp substrate itself. To our knowledge, PHT and CBZ are the only major AEDs which have been evaluated as yet for transport by Pgp in such cell monolayer efflux systems. Furthermore, although MDCK type II cells transfected with MRP2 are available (Evers et al., 1998, Tang et al., 2002), they have not been used to study whether AEDs are transported by MRP2 in such cell lines.

This prompted us to study the transport of AEDs in cell lines overexpressing either Pgp or MRP2. For our experiments, we used polarized kidney cell lines (MDCK type II and LLC-PK1) which had been transfected with either human or mouse cDNAs for the genes encoding Pgp (human MDR1 or mouse mdr1a and mdr1b) or MRP-2, respectively. Wildtype cells lines were used for comparison. In addition to comparing transport between transfected and wildtype cell monolayers, we used the Pgp inhibitor PSC833 to study whether the observed drug transport was specific for Pgp. Three AEDs, i.e., PHT, CBZ, and LEV, were included in the study. Cyclosporin A (CsA), which is a well-known substrate of Pgp (Schinkel et al., 1995, Fromm, 2004), was included as a positive control in Pgp-overexpressing cell lines. The questions that we aimed to answer by our study were as follows: (1) Which AEDs are transported by Pgp in monolayer efflux assays and how do their transport ratios compare with CsA? (2) Are there species differences in transport of AEDs or CsA by Pgp in MDR1-, mdr1a- and mdr1b-transfected cells? (3) Do MDR1-transfected MDCKII and LLC-PK1 cell lines yield comparable results for AEDs and CsA? (4) Are AEDs transported by human MRP2 in a monolayer efflux assay? The vinca alkaloid vinblastine was used as a reference substrate for MRP2 (Evers et al., 1998).

Section snippets

Cell lines

MDCK type II cells transfected with either human MDR1 (MDCKII-MDR1) or human MRP2 (MDCKII-MRP2) and respective wildtype cells (MDCKII/wt) were kindly provided by Prof. P. Borst (National Cancer Institute, Amsterdam, Netherlands). Furthermore, Prof. Borst kindly provided LLC-PK1 cells transfected with either human MDR1 (LLC-MDR1), mouse mdr1a (LLC-mdr1a), or mouse mdr1b (LLC-mdr1b) and respective wildtype LLC cells (LLC/wt). After obtaining the cells from Prof. Borst, they were grown in the

Transport experiments with MDCKII cells

In general, no significant directional transport was seen with MDCKII wildtype cells for CsA, vinblastine or any of the AEDs examined, but TRs were usually around 1.0 (Fig. 1, Table 1). Fig. 1 illustrates that CsA, which was used as a positive standard for Pgp-mediated transport, is transported in a directional fashion by MDR1-transfected MDCKII cells. This is shown by the increased permeability in the b-A direction and the decreased permeability in the a-B direction in comparison to the

Discussion

Monolayer efflux assays using transfected cell lines are widely used to test whether drugs are substrates for transport by Pgp or other drug-efflux transporters such as MRPs (Polli et al., 2001, Schwab et al., 2003, Löscher and Potschka, 2005a). In addition to identification of Pgp or MRP substrates, inhibitors of these transporters can be characterized by such assays. Various Pgp substrates, including CsA and verapamil, have been demonstrated to competitively inhibit the transport of other Pgp

Acknowledgements

We thank Prof. P. Borst and his group (National Cancer Institute, Amsterdam, Netherlands) for kindly providing us with the cell lines used in this study and advice during establishment of the assays. The skilful technical assistance of M. Weissing, M. Hausknecht and M. Gramer is gratefully acknowledged. We thank Carlos Luna Tortós for help with the vincristine and vinblastine experiments. The study was supported by a grant (Lo 274/9-3) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).

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    Present address: International Neuroscience Institute (INI), Hannover, Germany.

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