Preparation, Characterization, and Application of Biotinylated and Biotin−PEGylated Glucagon-Like Peptide-1 Analogues for Enhanced Oral Delivery

Glucagon-like peptide-1 (GLP-1) (7–36) is a type of incretin hormone with unique antidiabetic potential. The introduction of orally active GLP-1 offers substantial benefits in the treatment of type 2 diabetes over conventional injection-based therapies. Because the intestinal absorption of GLP-1 is restricted by its natural characteristics, we developed a series of GLP-1 analogues via the site-specific conjugation of biotin−NHS and/or of biotin−poly(ethylene glycol)−NHS at Lys26 and Lys34 of GLP-1 (7–36), respectively, in order to improve oral delivery. The resultant GLP-1 analogues, Lys26,34-DiBiotin-GLP-1 (DB-GLP-1) and Lys26-Biotin-Lys34-(Biotin-PEG)-GLP-1 (DBP-GLP-1), were prepared and studied in terms of their chemical, structural, and biological properties. DBP-GLP-1 demonstrated superior proteolytic stability against trypsin, intestinal fluid, and the major GLP-1 inactivation enzyme (dipeptidyl peptidase-IV (DPP-IV)) to native GLP-1 or DB-GLP-1 (p < 0.001). The in vitro insulinotropic effects of DB-GLP-1 and DBP-GLP-1 showed potent biological activity in a dose-dependent manner, which resembled that of native GLP-1 in terms of stimulating insulin secretion in isolated rat islets of Langerhans. Intraperitoneal glucose tolerance tests (IPGTT) after the oral administration of GLP-1 analogues in diabetic db/db mice demonstrated that DB-GLP-1 and DBP-GLP-1 significantly reduced the AUC0–180 min of glucose for 3 h by 14.9% and 24.5% compared to that of native GLP-1, respectively (p < 0.01). In particular, DBP-GLP-1 concentration in plasma rapidly increased 30 min after oral administration in rats, presumably due to improved intestinal absorption. These findings revealed that site-specific biotinylated and biotin−PEGylated GLP-1 is absorbed by intestine and that it has biological activity in vivo. Therefore, we propose that this orally active bioconjugated GLP-1 might be considered as a potential oral antidiabetic agent for type 2 diabetes mellitus.