RF04 | PSUN233 Design of an Ultra-Stable Insulin/Glucagon Fusion Protein

Abstract

Abstract Insulin replacement therapy is lifesaving for insulin-dependent diabetic patients, but it comes with an ever-present threat of hypoglycemia, which can eventually cause ischemic events, arrhythmias, neurological damage, coma, or even death. Consequently, the risk of hypoglycemia remains the most significant obstacle that prevents insulin-dependent patients from attaining adequate metabolic control. One approach to solve this issue involves the co-injection of insulin and glucagon at a fixed ratio, which has been shown to improve glycemic control with prevention of hypoglycemia. The co-administration of both hormones is based on two known physiological processes: first, insulin is a potent inhibitor of glucagon activity, especially under hyperglycemic conditions, and second, glucagon overpowers insulin and stimulates hepatic glucose production under hypoglycemic circumstances. In this work, we took this concept further into developing an insulin/glucagon fusion protein that will provide glycemic control (by the insulin portion) while preventing hypoglycemia (through the glucagon portion). Because both hormones are known to be unstable and form amyloid-like fibrils, our approach consists of using a fibrillation-resistant single-chain insulin (SCI) analog fused to a novel fibrillation-resistant glucagon analog. These molecules were independently synthesized by Solid-Phase Peptide Synthesis (SPPS) and linked together from the C-Terminus of the glucagon analog to the N-Terminus of the SCI analog by trypsin-mediated ligation. By themselves or when fused, these ultra-stable analogs didn't form fibrils after one month of agitation at 37°C in PBS (pH 7.4). By contrast, native insulin started fibrillation at 12.14 ± 3.65 hours, and native glucagon at time 0. The glucagon analog was designed to be less potent than the native molecule to allow unopposed insulin activity while in hyperglycemia but maintaining full agonism to prevent hypoglycemia. The in vitro glucagon activity was measured by cAMP production in HEK293 expressing the glucagon receptor. The glucagon analog was determined to be a full agonist with an EC50 of 125.7 nM, a ∼58-fold potency difference to the native molecule (2.175 nM). The fusion protein was tested in vitro and in vivo to determine the activities of both moieties when fused. The in vitro insulin activity of the fusion protein measured by traditional and in-cell western blot was comparable to the SCI alone. The in vitro glucagon activity of the fusion protein was also similar to the glucagon analog alone (EC50 151.2 nM), therefore validating our fusion approach. The in vivo insulin and glucagon activities are currently being tested by subcutaneous injection in diabetic (STZ) and normal rats. So far, both insulin and glucagon moieties have shown to be fully active, and hypoglycemia has not been observed in rats. Increasing doses studies are underway. We believe that this approach will allow the design of next-generation insulin treatments protected against hypoglycemia, ultra-stable, and amenable for multiplatform delivery. Presentation: Saturday, June 11, 2022 1:54 p.m. - 1:59 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.