A modified coumarinic acid-based cyclic prodrug of an opioid peptide: its enzymatic and chemical stability and cell permeation characteristics.
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66049, USA.
Hui
H
Ouyang
Fuxing
F
Tang
Teruna J
TJ
Siahaan
Ronald T
RT
Borchardt
D000143:Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D018938:Caco-2 Cells; D002460:Cell Line; D003374:Coumarins / Q000737:chemistry / Q000493:pharmacokinetics; D004285:Dogs; D016308:Enkephalin, Leucine-2-Alanine / Q000737:chemistry* / Q000493:pharmacokinetics; D004795:Enzyme Stability / Q000187:drug effects / Q000502:physiology; D019450:Genes, MDR / Q000502:physiology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D010539:Permeability / Q000187:drug effects; D011355:Prodrugs / Q000737:chemistry* / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012269:Ribosomal Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C000715087:ABCC2 protein, human; D000143:Acids; D003374:Coumarins; C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D011355:Prodrugs; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D016308:Enkephalin, Leucine-2-Alanine; C030123:coumarin