LAUSR

&NA; Riluzole is currently one of two approved medications for the treatment of amyotrophic lateral sclerosis (ALS). However, brain disposition of riluzole, as a substrate of P‐glycoprotein (P‐gp), is limited by the efflux transporters at the blood‐brain barrier (BBB). We propose to develop a liposomal co‐delivery system that could effectively transport riluzole to brain cells by reducing efflux pumps with a P‐gp inhibitor, verapamil. Riluzole and verapamil cocktail liposomes were prepared by lipid film hydration. The average particle size of cocktail liposomes was 194.3 ± 6.0 nm and their polydispersity index (PDI) was 0.272 ± 0.017. The encapsulation efficiencies of verapamil and riluzole in the cocktail liposomes were 86.0 ± 1.4% and 85.6 ± 1.1%, respectively. The drug release from cocktail liposomes after 8 h in PBS at 37 °C was 78.4 ± 6.2% of riluzole and 76.7 ± 3.8% of verapamil. The average particle size of liposomes did not show significant changes at 4 °C after three months. Verapamil cocktail liposomes inhibited P‐gp levels measured by western blotting in dose and time‐dependent manners in brain endothelial bEND.3 cells. Increased drug efflux transporters were detected in bEND.3 and astrocytes C8D1A cells, promoted by tumor necrosis factor (TNF‐&agr;) or hydrogen peroxide (H2O2). Restored accumulations of riluzole and fluorescent dye rhodamine 123 were observed in bEND.3 cells after treatments with cocktail liposomes. It indicated that inhibitory potential of co‐delivery liposome system towards P‐gp could mediate the transport of both P‐gp substrates. Verapamil and riluzole co‐loaded liposomes may be used to overcome pharmacoresistance of riluzole for improving ALS therapy. Graphical abstract Figure. No Caption available.