Abstracts

Rationale: The ABC transporter P-glycoprotein (Pgp) is one of the most important multidrug transporters at the blood brain barrier, where it restricts the brain uptake of various drugs by active efflux transport and thereby protects the brain against potential intoxication. An overexpression of Pgp has been implicated in pharmacoresistant epileptic patients. One explanation for increased Pgp expression observed in these patients may be Pgp induction by treatment with antiepileptic drugs (AEDs). However, in a recent study in the rat brain endothelial cell line GPNT, we did not determine Pgp induction by AEDs. In the present study, different brain endothelial cell lines from rats and humans were exposed to AEDs and drug uptake assays was used to determine Pgp functionality.Methods: Human brain endothelial cells hCMEC/D3 and two rat brain endothelial cell lines (RBE4 and GPNT) were treated with the widely used AEDs carbamazepine, levetiracetam, phenobarbital, phenytoin, topiramate, and valproate at different therapeutic concentrations for three days. Moreover, cells were treated with dexamethasone, puromycin, and rifampicine, which served as known inducers of Pgp activity. Medium was replaced every day with fresh substances. After treatment, cells were incubated with the known specific Pgp substrate rhodamine 123 and with or without the selective Pgp inhibitor tariquidar for 2 hours. The amount of accumulated rhodamine 123 was quantified and compared to non treated cells. In addition to functionality the expression of Pgp was determined by Western blot.Results: In all three cell lines, Pgp was functionally active, but the expression and extent of functionality varied widely. Cell lines also differed markedly in alterations of Pgp functionality after treatment with AEDs and known Pgp inducers. In all cell lines, dexamethasone and puromycin induced Pgp activity and therefore diminished accumulation of rhodamine 123 up to 30% compared to non treated cells. Rifampicine showed inducing effects on Pgp functionality only in GPNT cells and, at higher concentrations, in the human cell line. Tariquidar was always able to block the efflux by Pgp, so that accumulation of rhodamine 123 increased in all cell lines. Among the six tested AEDs, carbamazepine and phenytoin showed moderate but significant inducing effects on efflux transport in all cell lines, but only at relatively high concentrations in GPNT and hCMEC/D3 cells. With the other tested AEDs, effects were less consistent, with phenobarbital inducing Pgp in RBE4 and hCMEC/D3 but not GPNT, levetiracetam and valproate in GPNT and hCMEC/D3 but not RBE4, and topiramate in GPNT and RBE4 but not hCMEC/D3.Conclusions: Pgp induction by treatment with AEDs varied markedly between cell lines, which may be a consequence of different Pgp expression and functionality between these lines and possibly also species differences. In the human cell line, all AEDs except topiramate induced Pgp, indicating that induction of this efflux transporter by AEDs may be an important factor in the mechanisms of pharmacoresistance in epilepsy treatment.