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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. [Leu(14)]exenatide-ABD is an 88 residue peptide amide incorporating an Albumin Binding Domain (ABD) scaffold. [Leu(14)]exenatide-ABP is a 53 residue peptide incorporating a short Albumin Binding Peptide (ABP). [Leu(14)]exenatide-ABD and [Leu(14)]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. [Leu(14)]exenatide-ABD was compared to exenatide in a Lep (ob/ob) mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu(14)]exenatide-ABD 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 [Leu(14)]exenatide-ABD as compared to [Leu(14)]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.

Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lep(ob)/Lep (ob) mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (HbA1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1–3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 ± 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10–20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an IP glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 ± 0.08 mg 1 week after transplantation to 4.34 ± 0.27 mg in mice sacrificed at 27–35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.