Site-Specific PEGylated Exendin-4 Modified with a High Molecular Weight Trimeric PEG Reduces Steric Hindrance and Increases Type 2 Antidiabetic Therapeutic Effects

Abstract

The purpose of this study was to optimize an Exendin-4 (Ex4-Cys) site-specific PEGylation method with a high-molecular-weight trimeric PEG. Here, we describe the preparation of C-terminal specific PEGylated Ex4-Cys (C40-tPEG-Ex4-Cys), which was performed using cysteine and amine residue specific coupling reactions between Ex4-Cys and activated trimeric PEG. The C40-PEG-Ex4-Cys was obtained at high yields (~83%) and characterized by MALDI-TOF mass spectrometry. The receptor binding affinity of C40-PEG(5K)-Ex4-Cys was 3.5-fold higher than that of N-terminal PEGylated Ex4-Cys (N(ter)-PEG(5K)-Ex4-Cys), and receptor binding by the trimeric PEG (tPEG; 23, 50 kDa) adduct was much higher than that of branched PEG (20 kDa). Furthermore, C40-tPEG(50K)-Ex4-Cys was found to have greater blood circulating t(1/2) and AUC(inf) values than native Ex4-Cys by 7.53- and 45.61-fold, respectively. Accordingly, its hypoglycemic duration was much greater at 59.2 h than that of native Ex4-Cys at 7.3 h, with a dose of 25 nM/kg. The results of this study show that C-terminal specific PEGylation using trimeric PEG is effective when applied to Ex4-Cys and suggest that C40-tPEG(50K)-Ex4-Cys has considerable potential as a type 2 antidiabetic agent.

A comparison of three 67/68Ga-labelled exendin-4 derivatives for β-cell imaging on the GLP-1 receptor: the influence of the conjugation site of NODAGA as chelator

“…Ga-Ex4NOD12 showed the highest affinity to the receptor whereas both Ga-Ex4NOD40 and Ga-Ex4NOD27 showed to be similar, albeit lower binding affinities. This is in contrast to reports in which C-terminally modified exendin-4 derivatives show similar binding as the unmodified peptide [[11],[16],[24]]. The differences to literature values can be attributed to the fact that different cell lines were used.…”

“…Jin et al were also able to show that biotinylated exendin-4 derivatives showed no significantly lower affinities compared to unmodified exendin-4 when conjugated at the same positions [[17]]. In contrast, the investigations of Kim et al on PEGylated exendin-4 derivatives led to the conclusion that conjugations at both K27 and a C-terminally added cysteine had similar affinities as exendin-4, while the K12 derivative showed reduced binding to the receptor [[16]].…”

Glycosaminoglycan Conjugation for Improving the Duration of Therapeutic Action of Glucagon-Like Peptide-1

“…32 Therefore, we hypothesize that CH is more effective even at low molecular weight ranges than PEG in preventing renal clearance. The anionic charge and lower flexibility 33 of CH may have favored the slowing of glomerular filtration.…”

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

“…However, current approaches lack the sensitivity required for clinical application[25, 41, 45], so more development is needed. Modifications such as PEGylation have shown prolonged therapeutic effects[46–47], which may benefit a slower-clearing imaging agent if rapid distribution and blood clearance result in low targeting. An imaging agent that clears from the blood slowly has more time to accumulate in the target tissue, and in the well-vascularized islets, endocytosis of GLP-1R agonists occurs rapidly[48], suggesting a slower clearing agent may theoretically result in a higher TBR in the pancreas.…”