Synthesis and Evaluation of Human Serum Albumin-Modified Exendin-4 Conjugate via Heterobifunctional Polyethylene Glycol Linkage with Protracted Hypoglycemic Efficacy

Abstract

Albumin conjugation is considered to be one of the most effective means of protracting the short in vivo lifespans of peptides and proteins. Here, we present a new long-acting antidiabetic exendin-4 conjugate linked with human serum albumin (HSA) via polyethylene glycol (PEG). As a first step toward synthesizing this conjugate, three artificial sulfhydryl groups were introduced in HSA using 2-iminothiolane at pH 8.0. This thiolated HSA was further reacted with the monomer fraction of exendin-4 (6 equiv) conjugated with maleimide-PEG(5k)-N- hydroxysuccinimide (MAL-PEG(5k)-NHS) for 3 h. Because of the presence of PEG molecules, the resulting conjugate (HSA-PEG-Ex4) was found to have a greater apparent molecular weight and a larger particle size (ca. 195 kDa and 9.48 +/- 0.74 nm) than those of HSA-exendin-4 without the PEG linker (HSA-Ex4, ca. 84.3 kDa and 7.77 +/- 0.98 nm). Although the receptor binding affinity of HSA-PEG-Ex4 on RIN-m5F cells was significantly lower than that of Ex4, its antihyperglycemic efficacy was slightly higher than that of Ex-4 and HSA-Ex4 in type 2 diabetic db/db mice. Furthermore, HSA-PEG-Ex4 had greater circulating t(1/2) and AUC(inf) values than HSA-Ex and native exendin-4 by 2.1- and 10.3-fold, respectively. Accordingly, its hypoglycemic duration was greatly increased to 31.0 h at a dose of 250 nmol/kg vs that of native Ex4 (7.0 h). Results show that the HSA-PEG-Ex4 conjugate produced has distinct advantages over HSA-Ex4 without PEG. We believe that this exendin-4 derivative, which has the merits of albumin conjugation and PEGylation, has considerable potential as a novel type 2 antidiabetic agent.

Low Molecular Weight (1 kDa) Polyethylene Glycol Conjugation Markedly Enhances the Hypoglycemic Effects of Intranasally Administered Exendin-4 in Type 2 Diabetic <i>db</i>/<i>db</i> Mice

“…25,26) In brief, 100 µL of water-soluble Bolton-Hunter reagent (Nsulfosuccinimidyl-3-4-hydroxyphenyl propionate; 0.13 mg/mL, 36 nmol) in 10 mM PBS (pH 7.0) was mixed with 150 µL of the PEG-Ex4 analogs (0.5 mg/mL, 18 nmol) at room temperature for 90 min, and then briefly dialyzed to remove the reagent. Separately, a 100 µL aliquot of an IODO-GEN (Pierce, Rockford, IL, U.S.A.) solution in methylene chloride (0.5 mg/ mL) was dispensed into a fresh tube, and evaporated under a nitrogen stream.…”

Using albumin to improve the therapeutic properties of diabetes treatments

“…In another approach, Kim et al [88] linked exendin‐4 to HSA via polyethylene glycol (PEG) to create an HSA‐PEG‐exendin‐4 bioconjugate. Preclinical studies show that this extensively prolongs the half‐life of exendin‐4.…”

Development of a neuromedin U–human serum albumin conjugate as a long‐acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide

“…In addition to having a molecular size above the renal clearance threshold, the long half‐life is attributed to the efficient receptor‐mediated recycling pathway involving the neonatal Fc receptor (FcRn) . Conjugation of peptides to HSA has been successfully pursued, including advanced clinical candidates .…”