Novel Exenatide Analogs with Peptidic Albumin Binding Domains: Potent Anti-Diabetic Agents with Extended Duration of Action

et al. (2014) Novel Exenatide Analogs with Peptidic Albumin Binding Domains: Potent Anti-Diabetic Agents with Extended Duration of Action. PLoS ONE 9(2): e87704. doi:10.1371/journal.pone.0087704 Novel Exenatide Analogs with Peptidic Albumin Binding Domains: Potent Anti-Diabetic Agents with Extended Duration of Action Odile E. Levy 0 Carolyn M. Jodka 0 Shijun Steven Ren 0 Lala Mamedova 0 Abhinandini Sharma 0 Manoj Samant 0 Lawrence J. D'Souza 0 Christopher J. Soares 0 Diane R. Yuskin 0 Li Jenny Jin 0 David G. Parkes 0 Krystyna Tatarkiewicz 0 Soumitra S. Ghosh 0 Nigel Irwin, University of Ulster, United Kingdom 0 Amylin Pharmaceuticals LLC , San Diego, California , United States of America The design, synthesis and pharmacology of novel long-acting exenatide analogs for the treatment of metabolic diseases are described. These molecules display enhanced pharmacokinetic profile and potent glucoregulatory and weight lowering actions compared to native exenatide. [Leu14]exenatide-ABD is an 88 residue peptide amide incorporating an Albumin Binding Domain (ABD) scaffold. [Leu14]exenatide-ABP is a 53 residue peptide incorporating a short Albumin Binding Peptide (ABP). [Leu14]exenatide-ABD and [Leu14]exenatide-ABP exhibited nanomolar functional GLP-1 receptor potency and were metabolically stable in vitro in human plasma and in a pancreatic digestive enzyme mixture. Both molecules displayed picomolar and nanomolar binding association with albumin across multiple species and circulating half lives of 16 and 11 hours, respectively, post a single IV dose in rats. Unlike exenatide, both molecules elicited robust glucose lowering when injected 1 day prior to an oral glucose tolerance test, indicative of their extended duration of action. [Leu14]exenatide-ABD was compared to exenatide in a Lep ob/ob mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu14]exenatideABD displayed superior glucose lowering and weight loss in diabetic mice when compared to continuously infused exenatide at the same total weekly dose. A single oral administration of each molecule via an enteric coated capsule to cynomolgus monkeys showed superior pharmacokinetics for [Leu14]exenatide-ABD as compared to [Leu14]exenatide-ABP with detectable exposure longer than 14 days. These studies support the potential use of these novel long acting exenatide analogs with different routes of administration for the treatment of type 2 diabetes. - Peptide hormone-based therapeutics deliver significant therapeutic benefits while displaying exquisite receptor selectivity. While insulins have long been a treatment mainstay for diabetes, the development of peptide drugs from somatostatin [1], calcitonin [2,3], parathyroid hormone (PTH) [4], vasopressin [5], and glucagon like peptide-2 (GLP-2) [6] classes have become important treatment options for metabolic indications. With the recent introduction of ByettaTM, VictozaTM, LyxumiaH and BydureonTM, the GLP-1-based drug class has emerged as an important therapeutic regimen for treating patients with type 2 diabetes. These anti-diabetic agents illustrate product differentiation based on differences in efficacy, tolerability, frequency of administration and device presentation. Long-acting injectable peptide drugs, given by injection, afford greater convenience and promote increased compliance due to less frequent administration. The design of such drugs needs to address proteolytic degradation and renal filtration which plays a significant role in their clearance from the bloodstream. Efforts to reduce renal clearance have focused on increasing molecular size beyond the 60 kDa renal threshold [7] through the covalent fusion of the bioactive molecule to molecular scaffolds such as polyethylene glycol (PEG) polymers [8,9,10,11], XTEN [12,13], polysaccharides, large natural proteins such as Fc [14,15,16] and albumin [17]. Alternate strategies involve modification of peptide and protein drugs to allow a reversible non-covalent association to serum albumin, resulting in half life extension commensurate with their albumin binding affinity [18,19,20,21]. These approaches take advantage of the high abundance of serum albumin in mammalian sera, its wide tissue distribution and extended half-life resulting from its large molecular size and neonatal Fc receptor (FcRn) mediated cell recycling [22,23,24]. The half-life of serum albumin is directly proportional to size of species, as exemplified by halflives in rabbits and humans of 4.66.2 and 19 days, respectively [25]. Thus, fatty acid acylation of peptides has emerged as a successful half-life extension approach via non-covalent association of the fatty acid alkyl moiety with albumin, and is exemplified by the once-daily anti-diabetic drugs Insulin detemir [26,27] and liraglutide [28,29]. Their protracted effect is attributed to drug self association that delays absorption, and their intermittent association with circulating albumin that also protects against enzymatic degradation. Our half-life extension strategy focused on the fusion of bioactive peptides with small peptidic motifs that display high affinity for albumins across species. Albumin Binding Peptides (ABP) and Albumin Binding Domains (ABD) have been reported in the literature. Dennis et al [19] have used phage display to identify a short albumin-binding peptide, that when recombinantly fused to an immunoglobulin fragment D3H44-Fab, improved its half life by 37 fold in rabbits and 26 fold in mice. In addition, an array of bacterial proteins showing high, and specific, affinity for various human plasma proteins have been identified and structural studies have elucidated homologous domains involved in the specific interaction with human albumin [30,31]. Linhult, Johansson and Jonsson have reported on an ABD from the bacterial protein Streptococcal G (strain 148) that can be molecularly tuned to bind with nanomolar to femtomolar affinity to albumin [32,33,34]. The binding of ABD to albumin was shown not to interfere with albumins pH-dependent interaction with FcRn that is critical for its recycling mechanism [35]. In our studies, we utilized a previously reported ABP and an ABD licensed from Affibody AB (ABD035) as fusion partners for [Leu14]exenatide to generate potent, metabolically stable and long acting exenatide analogs with prolonged glucoregulatory action. The ABP and the ABD components are 1.4 and 5 kDas, respectively, and confer extended circulation half-lives to the conjugates through tight, reversible binding to plasma albumin. Our fusion strategy is amenable to chemical or recombinant manufacturing processes, and results in significantly smaller conjugates compared to those with molecular scaffolds such as Fc, XTEN and PEG. They display comparable half-life extension with preservation of biological potency, afford the ability to screen the molecules in animal models from different species and offer flexibility to deliver the fusion molecules by alter (...truncated)