Evaluation of therapeutic potentials of site-specific PEGylated glucagon-like peptide-1 isomers as a type 2 anti-diabetic treatment: Insulinotropic activity, glucose-stabilizing capability, and proteolytic stability

PEGylation has been considered to be a good biotechnique for improving the therapeutic value of glucagon-like peptide-1 (GLP-1) analogs for the treatment of type 2 diabetes. Despite the attractive anti-diabetic potentials, GLP-1 does not exert its full biological action because of its extremely short life-time in vivo due to rapid proteolytic degradation. Here, the enzyme-resistant mono-PEGylated GLP-1 isomers substituted at Lys26- or Lys34-amine were prepared through a newly devised site-specific PEGylation process using a maleic anhydride-protection/deprotection method. The therapeutic potentials of these site-specific PEGylated GLP-1 isomers (Lys26- or Lys34-PEG-GLP-1) along with His7-(N-terminus) PEG-GLP-1 were evaluated by examining their insulinotropic activity, glucose-stabilizing capability, and proteolytic stability. Lys34-PEG-GLP-1 was found to have the well-preserved insulinotropic activity (93% efficacy versus GLP-1) in isolated rat pancreatic islets. Furthermore, Lys34-PEG-GLP-1 showed the most prominent glucose-stabilizing capability, evaluated via an oral glucose tolerance test in db/db mice by considering the following three crucial factors: (i) maximum blood glucose level (BGL), (ii) required time to lower the BGL below 100 mg/dl, and (iii) total hypoglycemic degree. Additionally, Lys34-PEG-GLP-1 had longer half-lives than the other PEGylated GLP-1s in the dipeptidyl peptidase IV (DPP IV) inhibitor-treated liver or kidney homogenate, and its stability against DPP IV was also comparable to that of Lys26-PEG-GLP-1. Taken together, Lys34-PEG-GLP-1 displayed the promising characteristics in all evaluations versus His7- or Lys26-PEG-GLP-1. This site-specific PEGylated GLP-1 analog would have therapeutic usefulness for treating type 2 diabetes on account of the well-preserved insulinotropic activity, the increased proteolytic stability, and thereby the improved glucose-stabilizing capability.