Preparation and Evaluation of a 4-Branched Polyethylene Glycol Derivative Modified with Exendin-4 and Stearylamine for Extended Hypoglycemic Action

Abstract

Albumin-modification has been viewed as one of the most effective ways of extending the short in vivo lifetimes of peptide drugs by delaying glomerular filtration. In this study, we describe a new type 2 anti-diabetic exendin-4 (Ex4) peptide derivative with significant binding ability to human serum albumin (HSA). This exendin-4 derivative consists of a 4-branched polyethylene glycol (PEG)5k (Mw: 20 kDa) modified with three stearylamines (C18-NH2) and one exendin-4 on its branches. PEG and stearylamine were selected to provide functionality to increase molecular size and bind to albumin, respectively. This derivative (3C18-4PEG5k-Ex4) was shown to have larger molecular size (Ca. 152 kDa) than actual (25.0 kDa) when subjected to size-exclusion chromatography, and the fluorescein-tagged 3C18-4PEG5k-Ex4 displayed significant binding to the HSA-immobilized Sepharose CL-4B resin using confocal laser scanning microscopy. Furthermore, 3C18-4PEG5k-Ex4 was found to have acceptable anti-hyperglycemic efficacy via three consecutive oral glucose tolerance testings (OGTT) in fasted type 2 diabetic db/db mice. The HDtotal value (57.6±12.3%) of 3C18-4PEG5k-Ex4 at a 50 nmol/kg dose was 2-fold greater than that (31.0±8.7%) of native exendin-4 in non-fasted db/db mice. Especially, the blood glucose levels in the mice group treated with 3C18-4PEG5k-Ex4 did not rebound to ~150 mg/dL until 24 h after the injection, which obviously shows the extended hypoglycemia. We believe that this derivative has great pharmaceutical potential as a novel long-acting type 2 anti-diabetic injection treatment.