Several major antiepileptic drugs are substrates for human P-glycoprotein
Introduction
Resistance to antiepileptic drugs (AEDs) is a major, unresolved problem in epilepsy therapy, affecting about 30–40% of all patients (Kwan and Brodie, 2000, Chang and Lowenstein, 2003). Most patients with AED-resistant epilepsy are resistant to several, if not all, AEDs, despite the fact that these drugs act by different mechanisms. The consequences of uncontrolled epilepsy can be severe, and include shortened lifespan, bodily injury, neuropsychologial and psychiatric impairment, and social disability (Sperling, 2004). Consequently, there is a pressing need to develop new and more effective treatment strategies to counteract or prevent pharmacoresistance. For this goal, we need to understand the mechanisms underlying AED resistance. One of the candidate mechanisms that has attracted growing interest is the limitation of AED access to epileptogenic brain region(s) by localized overexpression of drug efflux transporters such as P-glycoprotein (Pgp) at the blood–brain barrier (BBB) (Löscher and Potschka, 2005a). A prerequisite for this transporter hypothesis of drug resistance is that AEDs are substrates of human Pgp.
However, several recent reports, including studies by our group, have indicated that, in contrast to rodent Pgp, human Pgp may not transport AEDs to any relevant extent (Schinkel et al., 1996, Mahar Doan et al., 2002, Crowe and Teoh, 2006, Baltes et al., 2007a, Baltes et al., 2007b). These recent reports have used conventional (bi-directional) transport assays with polarized intestinal (Caco-2) or kidney (MDCKII, LLC) cell lines expressing the human multidrug resistance-1 (MDR1; ABCB1) gene that encodes Pgp. The transport assays were performed in a conventional manner with the Transwell® system that allows studying drug transport between an apical and basolateral compartment separated by a polarized cell monolayer on a polyester filter membrane, applying the AED to either the apical or basolateral chamber for studying bi-directional transport. However, because most AEDs are very lipophilic, passive transcellular diffusion could form a bias in such assays by concealing active transport. Thus, the conventional bi-directional transport assay may fail to identify highly permeable compounds as Pgp substrates, particularly if they are not high-affinity substrates for this efflux transporter (FDA, 2006). In line with this possibility, Robey et al. (2008) recently suggested that it may be that AEDs are Pgp substrates but are not so well transported that they can be detected by the model systems used in previous studies.
This prompted us to modify the transport assay in a way that allows evaluating active transport independently of the passive permeability component. For this purpose, we adapted a method recently described for measuring Pgp-mediated transport of highly permeable antibiotics in the Caco-2 model (Pachot et al., 2003). Instead of applying the drug to either the apical or basolateral chamber for studying bi-directional transport, the drug is initially added at identical concentration to both chambers, resulting in concentration equilibrium conditions (Pachot et al., 2003). This concentration equilibrium transport assay thus minimizes the problem of drug concentration gradients that is known to affect identification of highly permeable compounds as Pgp substrates. In the present study, the concentration equilibrium transport assay was used to determine whether major AEDs are transported by human Pgp, using kidney cell lines transfected with MDR1. The known high-affinity Pgp substrates vinblastine and digoxin were included in the study for comparison.
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Cell lines and cell cultures
LLC-PK1 cells transfected with human MDR1 (LLC–MDR1) and respective wildtype (Wt) LLC cells were kindly provided by Prof. P. Borst (The Netherlands Cancer Institute, Amsterdam, Netherlands). Some experiments were also performed with MDCK type II cells transfected with human MDR1 (MDCKII–MDR1) and respective wildtype cells, which were also kindly provided by Prof. P. Borst. After obtaining the cells, they were cultured as described in detail recently (Baltes et al., 2007a, Baltes et al., 2007b).
Comparison of transport of vinblastine, digoxin, phenytoin and phenobarbital by Pgp in conventional vs. concentration equilibrium transport assays
Under concentration gradient conditions as commonly used in transcellular transport assays in Pgp-overexpressing cell lines, the prototype Pgp substrates vinblastine and digoxin exhibited directional (basolateral to apical) transport with cTRs of 3.01 and 3.41, respectively, in LLC–MDR1 cells (Fig. 2). With digoxin, directional transport was also observed in LLC wildtype cells, indicating transport by endogenous pig Pgp. Substantial basolateral to apical transport in wildtype cells was also
Discussion
Our recent study (Baltes et al., 2007a) indicating species differences in the Pgp-mediated transport of AEDs with significant transport of phenytoin and levetiracetam by mouse but not human Pgp in transfected LLC cells has cast serious doubt on the hypothesis that overexpression of multidrug transporters such as Pgp may mediate resistance to AEDs in patients with epilepsy (Löscher and Sills, 2007). The transporter hypothesis of AED-resistant epilepsy is fundamentally supported by four key
Acknowledgements
We thank Dr. Astrid Volz (Boehringer Ingelheim, Department of Drug Discovery Support, Biberach, Germany) for discussions on the concentration equilibrium transport assay and Prof. Piet Borst (The Netherlands Cancer Institute) and his group for kindly providing us with the cell lines used in this study and the information that Prof. Borst and Dr. A.H. Schinkel have previously used the concentration equilibrium transport assay for identifying transport by Pgp. The skilful technical assistance of
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