Synthesis and Evaluation of Exenatide Mono-PEGylates with Novel Branched Configuration for A Type II Diabetes Therapy

Abstract

K12C-PEG-K12C and K27C-PEG-K27C. The conjugation results in a lower yield and it was difficult to purify the homodimer for two kinds of analog, however homodimer K12C-PEG-K12C show the higher yield than K27C-PEG-K27C. The half-maximum effective concentration (EC50) of the homodimer PEGylate K12C-PEG-K12C was increased ca. 1.5-fold compared to the monomeric PEGylate K12C-PEG and 14-fold compared to the native exenatide. For in vivo stability test, optical imaging based on fluorescence probes was used for the power of visualization. Flamma® Vinylsulfone 675 was applied as a fluorescence label because of its superior properties such as aqueous phase solubility and high temperature reaction. This dye was conjugated to native exenatide Exn, analog K12C, PEGylates K12C-PEG 5 kD, K12C-PEG 40 kD, C40-PEG 40 kD and homodimer K12C-PEG-K12C. Each sample was subcutaneously injected once to nude mice. In vivo imaging shows that branched form K12C-PEG 40 kD has the longest retention, about 3-fold longer than the native exenatide, follow linear form C40-PEG 40kD. This prolonged duration can reduce the requirement adminstration frequency for patient. Although homodimer does not demonstrate the increased half-life, it could show better efficacy compared to mono-PEGylate. We expect that our K12C-PEG 40 kD and homodimer K12C-PEG-K12C are a promising peptide PEGylate to diabetes type 2 treatment.; Exenatide is a 39-mer peptide of synthetic Exendin-4, which is approved by US FDA as a therapeutic against type II diabetes mellitus. But its in-serum half-life is relatively short (~2.4 h). PEGylation, a widely used approach to increase the size of protein/ peptide to reduce kidney filtration, can improve both pharmacokinetic and pharmacodynamic properties; however, it often yields non-specific and multi-site binding of PEG so that the separation and purification of the desired PEGylates are difficult. Therefore, in this dissertation, we would like to introduce mono- and site-specific PEGylation. From the very first experiment, the partial separation between mono- and di-PEGylates of native exenatide by cation exchange chromatography was a challenge. Thus, point-mutation approach was used. Two point-muation or analogs, were prepared by Lys 12 to Cys (K12C) and Lys 27 to Cys (K27C). Both PEGylations are site-specific and the PEGylation yield are nearly 100%. These PEGylates are novel because of branched-form PEGylates structure, which are different from the previous C-terminal PEGylate to generate linear-form. Preparative cation exchange chromatography (HiTrap SP) showed complete separation of each mono-PEGylate from the mixture of both PEGylates by the difference in elution time. The distribution of charged amino acid surrounding the binding site of PEG and swimming action of conjugated PEG could be elution time difference. The immunobinding affinity by ELISA showed the similar binding to the native exenatide. Proteolytic stability of the analog PEGylates was improved more than 50-fold in trypsin solution. In cell model, exenatide binds to glucagon-like peptide-1 (GLP-1) receptor, activates some signals and increases cyclic adenosine monophosphate (cAMP). Role of cAMP signaling is enhanced insulin secretion. The bioactivity test shows all the PEGylated analogs demonstrate improved receptor binding but decrease the efficacy. A study of PEG chain effect indicates that elution time decreases as PEG molecular weight increaces because of “swimming action” or “shielding effect”. A set of experiments with various PEG molecular weight (5, 10, 20 and 40 kD) conjugated with the analogs was performed at various pHs (2.5, 3.0, 3.5 and 4.0). From the calculated net surface charge, we try to find the PEG’s shielding range in the terms of the number of amino acids with the hypothesis that PEG has spherical shape and exenatide has secondary structure. The experimental data were well correlated with the net charge calculation that PEG 5, 10, 20 and 40 kD would cover 5, 8, 12, and 17 amino acid residues, respectively, in each side of the conjugation point. Another analog was prepared, named C40, in which Cys was added to the C-terminal. PEGylated C40 is a linear form and it could be compared with the branched form PEGylates. Bioactivity study indicates that PEG 40 kD has better efficacy than other smaller PEGs, and all PEGylates, branched form as well as linear form, demonstrated improved receptor binding in cell culture. Dimeric PEGylation can be another effective means for enhancing receptor binding. So, a new bifunctional PEG, called MA-PEG-MA, was conjugated to K12C and K27C to generate a homodimer