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


METHODS
The 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.


RESULTS
In 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.


CONCLUSIONS
These 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.
Tang
Fuxing
F
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
Borchardt
Ronald T
RT
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D004285:Dogs; D016308:Enkephalin, Leucine-2-Alanine / Q000737:chemistry / Q000493:pharmacokinetics*; D019450:Genes, MDR / Q000502:physiology*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D018847:Opioid Peptides / Q000493:pharmacokinetics; D010539:Permeability / Q000187:drug effects; D011355:Prodrugs / Q000737:chemistry / Q000493:pharmacokinetics*; D012269:Ribosomal Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C000715087:ABCC2 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D018847:Opioid Peptides; D011355:Prodrugs; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D016308:Enkephalin, Leucine-2-Alanine