Abstracts

Rationale: The mechanisms for drug resistance in epilepsy are unclear. One possible mechanism is overexpression of P-glycoprotein (Pgp, also called ABCB1 or MDR1) in cerebral capillary endothelial cells which form the blood-brain barrier. Some studies indicated that Pgp is upregulated in epileptic foci in drug-resistant patients and that several antiepileptic drugs (AEDs) are Pgp substrates, although others revealed conflicting evidence. There is still no consensus on whether (or which) AEDs are substrates of Pgp. In vitro transport assays with MDR1 transfected cells are widely used to evaluate whether drugs may be Pgp substrates. But since most AEDs are lipophilic and have high passive diffusion, conventional bi-directional transport assays may not be sensitive enough to identify AEDs as Pgp substrates, and concentration equilibrium transport assays (CETA) may be needed. Methods: MDCKII and LLC-PK1 cells transfected with the human MDR1 gene and respective wildtype cells (kindly provided by Prof. P. Borst, The Netherlands Cancer Institute) were grown in monolayers in Transwells^®. Integrity of monolayers was verified by measuring transepithelial electrical resistance and by using atenolol and propranolol as paracellular and transcellular markers, respectively. Transport assays were performed in triplicate for three AEDs: phenytoin (PHT, 10μg/mL), phenobarbital (PB, 10μg/mL), and ethosuximide (ESM, 5.6μg/mL). Potential cytotoxicity of AEDs was tested by MTT (3-[4,5 dimethyl thiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. Drug concentrations were measured by HPLC/UV. Bi-directional transport assays were initiated by adding drugs to either the apical side or basolateral side of the monolayer. The apparent permeability (P_app) of drug across the monolayer was calculated and compared between MDR1 transfected and wildtype cells. In CETA, drugs were initially added to both sides of the monolayer at identical concentration. Drug concentrations were measured at various time points, and changes from baseline were compared between the apical and basolateral sides. Financial support was provided by CUHK Direct Grant 2008.1.078. Results: MTT assays indicated that the drugs were safe for cells at the tested concentrations. In bi-directional transport assays, the P_app values (basolateral to apical direction) of PB and ESM were similar between wildtype cells and MDR1 transfected cells, while the P_app value of PHT in MDCK-MDR1 cells was marginally significantly higher than that of wildtype cells (Figure 1). In the CETA, concentrations of PHT and PB, but not ESM, in the apical side, from 60 minutes onward, were significantly higher than those in the basolateral side of MDCK-MDR1 and LLC-MDR1 cells but not wildtype cells, indicating transport from the basolateral to the apical side by Pgp (Figure 2). Conclusions: Using MDR1-transfected monolayer cell models, we demonstrated that PHT and PB, but not ESM, are substrates of human Pgp. The CETA is a more sensitive model than the conventional bi-directional transport assay to detect Pgp transport of AEDs.