Prolonged activity of exenatide: Detailed comparison of Site-specific linear polyglycerol- and poly(ethylene glycol)-conjugates

Abstract

Exenatide is a small therapeutic peptide being currently used in clinic for the treatment of diabetes mellitus type II, however, displaying a short blood circulation time which makes two daily injections necessary. Covalent polymer modification of a protein is a well-known approach to overcome this limitation, resulting in steric shielding, an increased size and therefore a longer circulation half-life. In this study, we employed site-selective C-terminal polymer ligation of exenatide via copper-catalyzed azide-alkyne-cycloaddition (CuAAC) to yield 1:1-conjugates of either poly(ethylene glycol) (PEG) or linear polyglycerol (LPG) of different molecular weights. Our goal was to compare the impact of the two polymers on size, structure and activity of exenatide on the in vitro and in vivo level. Both polymers did not alter the secondary structure of exenatide and expectedly increased its hydrodynamic size, where the LPG-versions of exenatide showed slightly smaller values than their PEG-analogs of same molecular weight. Upon conjugation, GLP-1 receptor activation was diminished, however, still enabled maximum receptor response at slightly higher concentrations. Exenatide modified with a 40 kDa LPG (Ex-40-LPG) showed significant reduction of the blood glucose level in diabetic mice for up to 72 h, which was comparable to its PEG-analog, but 9-fold longer than native exenatide (8 h).

Introduction

Exenatide is a small biotherapeutic drug of 4.2 kDa and is used as a treatment of diabetes mellitus type II since its approval in 2005 (Byetta™) [1]. Originally derived from the natural peptide Exendin-4 found in saliva of Heloderma suspectum, exenatide works as an agonist for the GLP-1 receptor (GLP-1R) leading to a glucose-dependent release of insulin, among other effects [2]. Despite of its higher stability against peptidases compared to its physiological analog glucagon-like peptide-1 (GLP-1), exenatide displays only a short half-life of 2.4 h in humans after subcutaneous administration [3], making twice-daily injections necessary. Many other biotherapeutics are facing a similar problem which mostly originates from their small molecular weights and sizes being below the renal threshold of around 50–60 kDa [4]. To overcome these limitations, several approaches for long-acting preparations of exenatide have been followed, with the poly(lactide-co-glycolide) (PLGA) microsphere-formulated and approved Bydureon^TM entering the clinical field [5]. Exenatide embedded in PLGA-microspheres however shows some drawbacks like peptide degradation [6], [7] and an initial lag-phase in its release profile [8], leaving still room for other techniques to enhance exenatide’s therapeutic performance.

Besides incorporation into microparticles, other promising strategies are the covalent modification of the biotherapeutic, ranging from conjugation of albumin-binding motifs [9], [10], [11] and polypeptides [12] to modification with hyaluronic acid [13]. Among these, the most prominent technique surely is PEGylation, the covalent modification of a protein or peptide with polyethylene glycol (PEG), leading to steric shielding of immunogenic epitopes and an increase in molecular weight and size, which results in an extended circulation time. Branched [14], trimeric [15] or linear [16], [17] architectures of PEG as well as PEG-derived polymers bearing a brush-like architecture on the polymer backbone [18] have already demonstrated an extension of therapeutic activity for exenatide. However, within the last years, PEG’s image as a safe and well-tolerated excipient got questioned by emerging reports on antibody-induction and other immunogenic reactions in clinic linked to PEGylated protein drugs [19], [20], [21]. While the mechanism and rationale behind PEG-immunogenicity is still unclear, a large variety of alternative polymers for half-life extension has been investigated over the years [22], [23]. Linear polyglycerol (LPG) is a highly hydrophilic polymer that shows similar structure to PEG however bearing an additional hydroxymethyl group per repeating unit. It displays an excellent biocompatibility profile even at high concentrations (10 mg/mL), with no significant effects on complement activation or coagulation observed [24], [25]. LPG was able to prevent accelerated blood clearance of liposomes [26], displayed a very low intrinsic viscosity and a longer blood circulation time than PEG of similar molecular weight [25]. Moreover, oligoglycerol esters of up to 10 repeating units are approved by regulatory authorities (American Food and Drug Administration, FDA) and have been used as pharma and food additives for several decades [27]. Therefore, it represents a logical candidate as PEG alternative to extend the activity of therapeutic biomolecules.

To synthesize site-specific Exenatide-LPG and -PEG conjugates we used copper-catalyzed azide-alkyne-cycloaddition (CuAAC), which enables conjugates with one polymer chain only. Several techniques for modification of biomolecules exist but they mostly lack specificity, as they often target –NH₂ or –COOH groups which are abundant in every protein therefore hampering specific coupling, purification and bioactivity of the conjugates. The CuAAC reaction on the other hand, allows specific peptide modification via unnatural amino acids and has already been successfully applied to conjugation of several proteins [28], [29], [30] therefore making this the coupling chemistry of our choice.

In this study we aim to demonstrate the suitability of LPG to extend the therapeutic activity of exenatide in vivo in a similar manner than PEG, being the gold standard in this field. The site-specific exenatide-polymer conjugates were directly compared and characterized in terms of hydrodynamic size, structural stability and activity in vitro followed by a therapeutic model in diabetic mice.