Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin(9-39)

The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has rec ently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major d ifficulty with the delivery of peptide-based-agents is their short plasma h alf-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39 ), we investigated whether the conjugation of a carbohydrate structure to e xendin(9-39) would generate a peptide with intact biological activity and i mproved survival in circulation. The C-terminal portion of exendin(9-39) wa s reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alo ne or in conjunction with a recombinant GalNAc alpha 2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharid e GalNAc or the disaccharide NeuAc alpha 2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of c ontrol) with the binding of [I-125]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally e ffective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administer ed GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were m ore than twice as effective as the nonglycosylated peptide for inhibiting G LP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in viv o studies with H-3-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation. Copyright (C) 1 999 by W.B. Saunders Company.