Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin

•1,8-Dibenzoyl-rifampicin (DiBenzRif) was obtained by dibenzoylation of rifampicin.•DiBenzRif inhibited P-glycoprotein (P-gp) activity in RBE4 cells.•DiBenzRif decreased ATP intracellular levels and inhibited P-gp ATPase activity.•DiBenzRif increased membrane fluidity and enhanced rhodamine123 accumulation in liposomes.•DiBenzRif may be useful to enhance the access of drugs to the brain.

PurposeTo assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood–brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes).MethodsP-gp expression (western blot) and activity [rhodamine 123 accumulation studies] were assessed until 72 h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin–luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes.ResultsA significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells.ConclusionDiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs.