Ultrastable Insulin-Glucagon Fusion Protein Exploits an Endogenous Hepatic Switch to Mitigate Hypoglycemic Risk

The risk of hypoglycemia and its serious medical sequelae restrict insulin replacement therapy for diabetes mellitus. Such adverse clinical impact has motivated development of diverse glucose-responsive technologies, including algorithm-controlled insulin pumps linked to continuous glucose monitors ("closed-loop systems") and glucose-sensing ("smart") insulins. These technologies seek to optimize glycemic control while minimizing hypoglycemic risk. Here, we describe an alternative approach that exploits an endogenous glucose-dependent switch in hepatic physiology: preferential insulin signaling (under hyperglycemic conditions) <i>versus</i> preferential counter-regulatory glucagon signaling (during hypoglycemia). Motivated by prior reports of glucagon-insulin coinfusion, we designed and tested an ultrastable glucagon-insulin fusion protein whose relative hormonal activities were calibrated by respective modifications; physical stability was concurrently augmented to facilitate formulation, enhance shelf life and expand access. An N-terminal glucagon moiety was stabilized by an α-helix-compatible Lys13-Glu17 lactam bridge; a C-terminal insulin moiety was stabilized as a single chain with foreshortened C domain. Studies <i>in vitro</i> demonstrated (a) resistance to fibrillation on prolonged agitation at 37 °C and (b) dual hormonal signaling with balanced activity and cross-talk. Glucodynamic responses were monitored in rats relative to control fusion proteins lacking one or the other hormonal activity, and continuous intravenous infusion emulated basal subcutaneous therapy. Whereas efficacy in mitigating hyperglycemia was unaffected by the glucagon moiety, the fusion protein enhanced endogenous glucose production under hypoglycemic conditions. These findings provide proof of principle toward a basal glucose-responsive insulin biotechnology of striking simplicity. The fusion protein's augmented stability promises to circumvent the costly cold chain presently constraining global insulin access.