title: Mono-PEGylated dimeric exendin-4 as high receptor binding and long-acting conjugates for type 2 anti-diabetes therapeutics.
abstract: Dimerization is viewed as the most effective means of increasing receptor binding affinity, and both dimerization and PEGylation effectively prolong the life spans of short-lived peptides and proteins in vivo by delaying excretion via the renal route. Here, we describe the high binding affinities of two long-acting exendin-4 (Ex4) conjugates, dimerized Ex4 (Di-Ex4) and PEGylated Di-Ex-4 (PEG-Di-Ex4). Di-Ex4 and PEG-Di-Ex4 were prepared using cysteine and amine residue specific coupling reactions using Ex4-Cys, bisMal-NH(2), and activated PEG. The Ex4 conjugates produced were of high purity (>98.5%), as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. The receptor binding affinity of Di-Ex4 on RIN-m5F cells was 3.5-fold higher than that of Ex4, and the in vivo antihyperglycemic efficacy of Di-Ex4 was also greater than that of native Ex4 in type 2 diabetic db/db mice. Furthermore, Di-Ex4 and PEG-Di-Ex4 were found to have greater blood circulating t(1/2) and AUC(inf) values than native Ex4 by 2.7- and 13.7-fold, and by 4.0- and 17.3-fold, respectively. Accordingly, hypoglycemic durations were greatly increased to 15.0 and 40.1 h, respectively, at a dose of 25 nmol/kg (native Ex4 7.3 h). The results of this study show that combined dimerization and PEGylation are effective when applied to Ex4, and suggest that PEG-Di-Ex4 has considerable potential as a type 2 anti-diabetic agent.
authors.affiliation: College of Pharmacy, SungKyunKwan University , 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea.
authors.forename: Tae Hyung
authors.initials: TH
authors.lastname: Kim
authors.forename: Hai Hua
authors.initials: HH
authors.lastname: Jiang
authors.forename: Seulki
authors.initials: S
authors.lastname: Lee
authors.forename: Yu Seok
authors.initials: YS
authors.lastname: Youn
authors.forename: Chan Woong
authors.initials: CW
authors.lastname: Park
authors.forename: Youngro
authors.initials: Y
authors.lastname: Byun
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
authors.forename: Kang Choon
authors.initials: KC
authors.lastname: Lee
mesh_terms: D000818:Animals; D001665:Binding Sites; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy*; D019281:Dimerization; D004195:Disease Models, Animal; D000077270:Exenatide; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D011092:Polyethylene Glycols / Q000737:chemistry*; D014018:Tissue Distribution; D014688:Venoms / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*
chemical_list: D007004:Hypoglycemic Agents; D010455:Peptides; D014688:Venoms; D011092:Polyethylene Glycols; D000077270:Exenatide