Characterization of the efflux transporter(s) repsonsible for restricting intestinal mucosa permeation of the coumarinic acid-based cyclic prodrug of the opioid peptide DADLE

Purpose. To elucidate the efflux transporter(s) responsible for restricting the permeation of a coumarinic acid-based cyclic prodrug of the opioid peptide DADLE (CD) thorough Caco-2 cell monolayers. Methods. The cellular permeability characteristics of CD 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 human MRP2 gene (MDCK-MRP2). These cells were grown as monolayers onto microporous membranes. The disappearance from the donor side and appearance on the receiver side of CD were monitored by HPLC. The substrate activity of CD for P-gp was determined by using GF120918, a known P-gp specific inhibitor. The substrate activity of CD for MRP2 was determined by using cyclosporin A (CsA), a known MRP2 and P-gp inhibitor. Results. In Caco-2 cells, the ratio of the apparent permeability coefficients (P-app) of CD flux 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 71. In the presence of GF120918 (2 muM), the Papp BL-to-AP/Papp AP-to-BL ratio was decreased to 16. In the presence of CsA (25 muM), the ratio was decreased to 5.6. In MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells, the Papp BL-to-AP/Papp AP-to-BL ratios of CD were 13, 35, and 22, respectively. CsA (25 muM) greatly decreased the Papp BL-to-AP/Papp AP-to-BL ratios in MDCK-WT and MDCK-MDR1 cells to 1.5 and 3.2, respectively. However, in MDCK-MRP2 cells, CsA (25 muM) decreased the ratio only to 11. A mixture of GF120918 (2 muM) and CsA (25 muM) decreased the Papp BL-to-AP/Papp AP-to-BL ratios of CD in MDCK-WT, MDCK-MDR1, and MDCK-MRP2 cells to 1.4, 2.7, and 5.4, respectively. Conclusions. These data suggest that CD is a good substrate for both P-gp and MRP2 and that the restricted permeation of this cyclic prodrug in Caco-2 cells and in the intestinal mucosa is probably due to its substrate activities for both of these efflux transporters.