title: Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.
abstract: The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.
authors.affiliation: Center of Pharmacy, Institute of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria. andreas.bernkop-schnuerch@univie.ac.at
authors.forename: A
authors.initials: A
authors.lastname: Bernkop-Schnürch
authors.forename: H
authors.initials: H
authors.lastname: Zarti
authors.forename: G F
authors.initials: GF
authors.lastname: Walker
mesh_terms: D000180:Acrylic Resins / Q000138:chemical synthesis / Q000494:pharmacology*; D000818:Animals; D000930:Antidiarrheals / Q000138:chemical synthesis / Q000494:pharmacology*; D018826:CD13 Antigens / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003545:Cysteine / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D013439:Sulfhydryl Reagents / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D013552:Swine
chemical_list: D000180:Acrylic Resins; D000930:Antidiarrheals; D013439:Sulfhydryl Reagents; C030312:calcium polycarbophil; D018826:CD13 Antigens; D003545:Cysteine