Abstracts

Despite the launch of several new antiepileptic drugs (AED) during the last two decades pharmacoresistance of epilepsy remains an unsolved problem. It is hypothesized that local overexpression of multidrug transporters (MDT) in the epileptic tissue of pharmacoresistant individuals results in limited access of AED to the epileptic focus region. Downregulation of MDT by RNA interference (RNAi) would be a possible approach to overcome pharmacoresistance. Aim of the present study was to test if specific delivery of siRNA targeting the mRNA sequence of the major MDT P-glycoprotein (Pgp) to brain-capillary endothelial cells is possible., Liposomes were coupled with ApoE4 peptides which are discussed to mediate an uptake of liposomes into brain-capillary endothelial cells via LDL receptors. ApoE4-liposomes loaded with fluorescence-labelled siRNA targeting the rat Pgp mRNA sequence were administered i.v. to female Wistar rats. Distribution of siRNA was investigated at different time points (4, 10, 24 h) post administration by confocal microscopy in rat brain sections. Based on a fluorescence resonance energy transfer (FRET) effect the integrity of siRNA was controlled (siRNA was labelled with two fluorescent dyes). The influence of siRNA molecules on Pgp expression was evaluated 24 h post administration by immunhistochemistry and compared with control groups., Fluorescence microscopy of rat brain sections after siRNA administration demonstrated a successful targeting to brain-capillary endothelial cells. An accumulation of labelled siRNA in the cells was obvious at all time points. The intensity of fluorescence increased from 4 to 24 h post administration. FRET analysis indicated that the siRNA was still intact 24 h following administration. Preliminary results of the immunhistochemical investigations gave evidence for a reduced Pgp expression in rats treated with siRNA in comparison with control groups., It was demonstrated that specific delivery of siRNA encapsulated in immunoliposomes to brain-capillary endothelial cells can be achieved. Thus, the study substantiates the suitability of this novel approach for siRNA delivery. Reduction of Pgp expression in brain-capillary endothelial cells will be investigated in further experiments to ensure that an RNAi-based downregulation of Pgp takes place and enhances AED efficacy in the amygdala kindling model of temporal lobe epilepsy., (Supported by CURE (Citizens United for Research in Epilepsy).)