Site-specific PEGylation of exenatide analogues markedly improved their glucoregulatory activity.
OBJECTIVE
Exenatide is a 39-amino-acid peptide widely used to manage type 2 diabetes mellitus. However, it has a short plasma half-life and requires a twice daily injection regime. To overcome these drawbacks we used maleimide-polyethylene glycol to induce site-specific PEGylation.


METHODS
The analogue PB-105 (ExC39) was produced by replacing cysteine at position 39 of exenatide to provide a free thiol group. PB-105 showed the same glucoregulatory activity as exenatide in mice. Site-specific PEGylation of PB-105 was performed to produce PB-110 (ExC39PEG5kDa), PB-106 (ExC39PEG20kDa), PB-107 (ExC39PEG30kDa) and PB-108 (ExC39PEG40kDa). Their effects on intracellular cAMP, acute glucoregulatory activity and pharmacokinetic profile were compared in mice and rats.


RESULTS
PEGylation shifted the concentration-response curve of PB-105 to the right in a parallel, polyethylene glycol mass-dependent manner but with an inflexion point of at least 20 kDa. The activities of PB-107 and PB-108 but not PB-106 were reduced by 90% and 99%. PEGylation affected in vivo glucoregulatory activity in the same 'Inflexion-Shift' fashion at least at 20 kDa, but linearly increased plasma duration and systemic exposure without inflexion. PB-106 had a plasma t(1/2) approximately 10-fold that of PB-105, and exhibited superior glucoregulatory activity compared with PB-105 in normal and diabetic mice.


CONCLUSIONS
Site-specific PEGylation of exenatide with a permanent amide linkage affects its activity in a new type of 'Inflexion-Shift' fashion. PB-106 is a putative new analogue for treating diabetes; it possesses no loss of in vitro activity, prolonged plasma duration and superior, improved in vivo glucoregulatory activity compared with exenatide.
King's Lab, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Nian
N
Gong
Ai-Niu
AN
Ma
Li-Jie
LJ
Zhang
Xiao-Su
XS
Luo
Yin-Hui
YH
Zhang
Michael
M
Xu
Yong-Xiang
YX
Wang
D000595:Amino Acid Sequence; D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D000242:Cyclic AMP / Q000096:biosynthesis; D003545:Cysteine / Q000737:chemistry; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000188:drug therapy; D000077270:Exenatide; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry; D014688:Venoms / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*
D001786:Blood Glucose; D007004:Hypoglycemic Agents; D010455:Peptides; D014688:Venoms; D011092:Polyethylene Glycols; D014443:Tyrosine; D000077270:Exenatide; D000242:Cyclic AMP; D003545:Cysteine
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