Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of an acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE.

purposeTo elucidate the efflux transporter(s) responsible for restricting the permeation of an acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE (AD) through Caco-2 cell monolayers.

methodsThe cellular permeation characteristics of AD were investigated using Caco-2 cells, Madin-Darby canine kidney wild-type II cells (MDCK-WT), MDCK cells transfected with the human MDR1 gene (MDCK-MDR1), and MDCK cells transfected with the human MRP2 gene (MDCK-MRP2). These cells were grown as monolayers onto microporous membranes. The disappearance of AD from the donor side and its appearance on the receiver side were monitored by high-performance liquid chromatography. The substrate activity of AD for P-glycoprotein (P-gp) was determined using GF120918, a known P-gp specific inhibitor. The substrate activity of AD for MRP2 was determined by using cyclosporin A, a known MRP2 and P-gp inhibitor.

resultsIn Caco-2 cells, the ratio of the apparent permeability coefficients (Papp) of AD flux measured in the basolateral (BL) to apical (AP) direction vs. the flux in the AP-to-BL direction (Papp BL-to-AP/ Papp AP-to-BL) was 99. In the presence of 2 microM GF120918 or 25 microM cyclosporin A. the Papp BL-to-AP/Papp AP-to-BL ratio was decreased to 11. In MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells, the Papp BL-to-AP/Papp AP-to-BL ratios of AD were 4.7, 10, and 5.8, respectively. A mixture of GF120918 (2 microM) and cyclosporin A (25 microM) decreased the Papp BL-to-AP/Papp AP-to-BL ratios of AD in MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells to 1.2,1.8, and 2.3, respectively.

conclusionsThese data suggest that AD is a much better substrate for P-gp than MRP2 and that the restricted permeation of this cyclic prodrug in Caco-2 cells and in the intestinal mucosa probably is due primarily to its substrate activity for P-gp.