enkephalin--leucine-2-alanine and coumarin

A coumarin-based prodrug system has been developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural features of the drug moiety. The effect of the phenyl ring substitutions on the release kinetics of such prodrugs of model amines was examined recently and it was found that appropriately positioned alkyl substituents on the phenyl ring could help to facilitate the release. Aimed at further understanding the structure-release rate relationship of the coumarin-based cyclic prodrugs, we synthesized and examined a series of substituted coumarinic acid derivatives of opioid peptides, DADLE, and [Leu(5)]-enkephalin.

Topics: Coumarins; Enkephalin, Leucine-2-Alanine; Enkephalins; Esterases; Kinetics; Molecular Structure; Prodrugs

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.. 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.. In Caco-2 cells, the ratio of the apparent permeability coefficients (Papp) 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 microM), the Papp BL-to-AP/Papp AP-to-BL ratio was decreased to 16. In the presence of CsA (25 microM), the ratio was decreased to 5.6. In MDCK-WT. MDCK-MDR1, and MDCK-MRP2 cells, the Papp BL-AP/Papp AP-to-BL ratios of CD were 13, 35, and 22, respectively. CsA (25 microM) greatly decreased the Papp BL-P-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 microM) decreased the ratio only to 11. A mixture of GF120918 (2 microM) and CsA (25 microM) 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.. 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.

Topics: Acids; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caco-2 Cells; Cell Line; Coumarins; Dogs; Enkephalin, Leucine-2-Alanine; Genes, MDR; Humans; Intestinal Mucosa; Mitochondrial Proteins; Multidrug Resistance-Associated Protein 2; Opioid Peptides; Permeability; Prodrugs; Ribosomal Proteins; Saccharomyces cerevisiae Proteins

To evaluate the chemical/enzymatic stability and the cell permeation characteristics of the modified coumarinic acid-based cyclic prodrug 2 of DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), which has an aldehyde equivalent (oxymethyl) inserted between the phenolic group of the promoiety and the carboxylic acid group of the peptide.. The rates of the chemical/enzymatic conversion of the oxymethyl-modified prodrug 2 to DADLE were measured by HPLC. The cellular permeation characteristics of DADLE and its oxymethyl-modified prodrug 2 were measured by HPLC using Caco-2 cells, wild type Madin-Darby Canine Kidney cells (MDCK-WT), MDCK cells transfected with human MDR1 gene (MDCK-MDR1), and MDCK cells transfected with human MRP2 gene (MDCK-MRP2) grown onto microporous membranes.. The oxymethyl-modified coumarinic acid-based cyclic prodrug 2 degraded chemically to DADLE in a pH-dependent manner, i.e., rates of conversion increased with increasing pH. The prodrug 2 degraded rapidly in rat plasma (t1/2 = 39 min) and rat liver homogenate (t1/2 = 59.2 min), but much slower in Caco-2 cell homogenate (t1/2 = 678.7 min) and human plasma (t1/2 = 264.3 min). In all four cell lines used for transport studies, the flux rates of the oxymethyl prodrug 2 in the basolateral (BL)-to-apical (AP) direction (Papp BL-to-AP) were significantly greater than the flux rates in the AP-to-BL direction (Papp AP-to-BL). The Papp BB-to-AP/Papp AP-to-BL ratios were >116, 35.1, 21.2, and 12.6 in Caco-2, MDCK-MDR1, MDCK-MRP2, and MDCK-WT cells, respectively. The efflux of the modified prodrug could be inhibited by GF120918 (an inhibitor for P-gp) and cyclosporin A (an inhibitor for P-gp and MRP2).. The oxymethyl-modified coumarinic acid-based cyclic prodrug 2 of DADLE could be converted to DADLE in both chemical and enzymatic media. However, the prodrug was a good substrate for both P-gp and MRP2 suggesting that its permeation across intestinal mucosa and blood-brain barrier would be significantly restricted.

Topics: Acids; Animals; Caco-2 Cells; Cell Line; Coumarins; Dogs; Enkephalin, Leucine-2-Alanine; Enzyme Stability; Genes, MDR; Humans; Liver; Male; Mitochondrial Proteins; Multidrug Resistance-Associated Protein 2; Permeability; Prodrugs; Rats; Rats, Sprague-Dawley; Ribosomal Proteins; Saccharomyces cerevisiae Proteins