The effect of pharmaceutical excipients on activity and expression of P-glycoprotein in Caco-2 cells

Abstract

Background- P-glycoprotein (P-gp), encoded by the MDR1 gene in humans, is a 170 kD integral membrane protein expressed in the apical surface of epithelial cells in the intestine of human body which effluxes its substrates using ATP hydrolysis energy preventing the entry of undesirable, potentially toxic materials into systemic circulation; consequently, limits the bioavailability of orally administered drugs. Aim- The present study was designed to investigate the effect of non-ionic surfactants on activity and expression of P-gp in human colon adenocarcinoma cells (Caco-2). Methods- Non-cytotoxic concentrations of excipients were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test. Measuring the activity of P-gp was performed with Rhodamine-123, a P-gp fluorescent substrate, uptake test assay in the presence of excipients and compared to control samples and verapamil 0.3 mM, a P-gp known inhibitor, as positive control. Moreover, increase in Rhodamine-123 uptake was shown by Fluorescent microscopy qualitatively. Furthermore, the expression of P-gp was investigated by Western blotting using P-gp specific antibody supporting Rho-123 uptake data and fluorescence microscopy likewise. Results- Results demonstrated a wide range. Among all excipients, PEG 400, cremophor EL, tween 20, tween 40, PEG 6000 demonstrated P-gp functional inhibition both in activity and in the expression but other excipient's inhibition was not significant. Conclusion- Our results showed that some excipients in suitable concentrations are able to inhibit P-gp. The idea behind this study was to develop a way to improve drug absorption and introduce a new efficient concentration of excipients in drug formulations to improve drug bioavailability.