Abstracts

Rationale: The mechanisms underlying drug resistance in epilepsy are unclear. One of the candidate mechanisms that has attracted growing interest is the limitation of antiepileptic drug (AED) access to epileptogenic brain region(s) by overexpression of drug efflux transporters such as P-glycoprotein (P-gp). A prerequisite for this transporter hypothesis of drug resistance is that AEDs are substrates of human P-gp. However, several recent reports have indicated that, in contrast to rodent P-gp, human P-gp may not transport AEDs to any relevant extent. These recent reports have used conventional transport assays with intestinal (Caco-2) or kidney (MDCKII, LLC) cell lines expressing the human multidrug resistance-1 (MDR1) gene that encodes P-gp. The transport assays were performed in a conventional manner with the Transwell® system, applying the AED to either the apical or basolateral chamber. However, because AEDs are very lipophilic, passive diffusion could form a bias in such assays by concealing active transport. This prompted us to modify the transport assay in a way that allows evaluating active transport independently of the passive permeability component.Methods: MDCKII cells and LLC cells that were transfected with human MDR1-gene were used. Wildtype cell lines were used for comparison. Transport assays with AEDs were either used in a conventional fashion by adding the AED to either the apical or basolateral chamber (concentration gradient condition) or in a modified fashion by adding the AED to both chambers (concentration equilibrium condition). Since passive diffusion from the apical to basolateral side is equal to that in the opposite direction, determination of active efflux components should be eased under equilibrium conditions. The P-gp substrate vinblastin was used as positive standard. In case of active transport, the P-gp inhibitor tariquidar was used to substantiate the involvement of P-gp in the transport.Results: By using transport assays with MDR1-transfected cells under concentration equilibrium conditions, a clear basolateral to apical transport was observed for several AEDs, including phenytoin and phenobarbital. This transport was inhibited by tariquidar, substantiating that these AEDs were transported by human P-gp.Conclusions: Our data demonstrate that human P-gp transports several AEDs, which supports the transporter hypothesis of drug resistance in epilepsy. This work is funded by the Deutsche Forschungsgemeinschaft (Bonn, Germany)