Mono-PEGylates of exenatide in branched and dimeric structures can improve in vivo stability and hypoglycemic bioactivity

Exenatide, a synthetic version of exendin-4, is a glucagon-like peptide-1 receptor agonist (GLP-1RA) used for treating diabetes, but its relatively short half-life is a major disadvantage. In this study, we attempted residue-specific mono-PEGylation to the middle of the amino acid backbone to extend its in vivo half-life. Exenatide was point-mutated from Lys to Cys at the 12th residue to yield a variant (K12C), and PEG-maleimide of varying molecular weights (MW) (5, 10, 20, 40 kD) was site-specifically conjugated to yield a mono-PEGylate with branched T-shape molecular structure. In another approach, we conjugated a bis-maleimide PEG (10 kD) to the middle of two K12Cs to yield an H-shape homodimer PEGylate In vitro bioactivity assays indicated that: (1) PEGylates conjugated with higher MW PEG lead to stronger receptor binding, (2) the branched form was superior to the linear configuration in the binding, and (3) both T-shape and H-shape mono-PEGylates demonstrated better potency than the native exenatide, evidenced by lower EC50. Db/db mouse experiments to evaluate in vivo hypoglycemic activity indicated that: (1) all mono-PEGylates resulted in improved glucose tolerance compared to the native exenatide, (2) the homodimer PEGylate demonstrated much stronger hypoglycemic activity, especially during the initial period, and (3) the H-shape and T-shape mono-PEGylates (40 kD) maintained hypoglycemia for up to ca. 168 and 140 h, representing approximately 12- and 14-fold increase, respectively, compared with the native exenatide. Our findings suggest that the exenatide mono-PEGylates in unclassical molecular structures can improve in vivo pharmacokinetics properties.