Intensive insulin therapy-assisted glycemic control is essential for type 1 diabetes management. Recent advances in diabetes technology have successfully launched automated insulin delivery (AID) systems, providing great benefits for tight glycemic control. Despite significant progress has been made on AID, several limitations still exist. First, there is an inherent delay in insulin action due to the hexameric nature of insulin, which must dissociate into monomers to reach the bloodstream. The slow absorption of insulin from subcutaneous administration may continue for several hours, leading to post-meal hypoglycemia and making timely glycemic control difficult. Second, due to the aggregation propensity of insulin, concentrated insulin formulations resilient to room temperature and agitation stress are not available, which is essential for the sustained usage and miniaturization of wearable insulin delivery devices such as pumps and patches.
To address these challenges, novel ultra-concentrated insulin (UCI) and ultrafast-acting insulin (UFI) formulations are developed in this study. The dual role of the C-terminal region of the insulin B chain, including being critical for both insulin receptor binding and insulin dimerization, poses a significant challenge in the development of stable and effective monomeric insulin analogs. Inspired by the discovery of monomeric insulin variants in the venom of predatory snails, a potent insulin analog HALQ with low dimerization propensity has been developed. Extension of the insulin A-chain C-terminal region with the HALQ peptide sequence has been shown to compensate for the loss of receptor-binding affinity caused by truncation of the B-chain C-terminal region. In addition, a novel class of excipients based on fructan-type polysaccharides has been successfully engineered to stabilize HALQ and facilitate ultra-concentrated insulin formulations (1000 units/ml, 10-fold higher than conventional U-100 insulin products) with enhanced solubility and stability. These excipients stabilized insulin without direct interactions, ensuring its structural and functional integrity under stress aging conditions (37°C, continuous agitation). Specifically, stable UFI that exhibits 1-month stability with full bioactivity has been developed. Pharmacodynamic characterization of the UFI formulation in a diabetic swine model demonstrated a fast-on and fast-off activity, exhibiting an average duration of action of 2.5 hours (n=8), a much shorter duration than Fiasp®, the currently best available UFI in the clinic (4.1 hours, n=14). Superior stability for 1000 units/ml UCI (hInU-1000) has also been confirmed with structural and functional integrity for up to 6 months under stress aging conditions. Such novel protein design and excipient development approach successfully overcomes key challenges associated with protein therapeutics, including poor stability and suboptimal pharmacodynamics. It paves a new avenue for enhancing therapeutic strategies for patients requiring insulin therapy.
In contrast to the agonistic activity of insulin on the insulin receptor (IR), IR antagonists have attracted considerable interest due to their potential to counteract hypoglycemia and treat rare disorders such as congenital hyperinsulinism. Based on the previous reported IR binding peptides and the discovery of adding another IR binder to insulin can lead to a different receptor activation mode (partial agonism), my research focused on converting insulin from IR agonist into a fully IR antagonist. In this study, insulin modifications were investigated at the C-terminus of A chain by incorporating IR binders, which led to the development of a fully antagonistic insulin analog. The modification was enabled by designing insulin precursors with a Sortase A (SrtA) recognition sequence (LPETGG) at the A-chain C-terminus, while preserving full IR agonist potency comparable to native human insulin. Such SrtA-mediated ligation method enabled the exploration of insulin derivatives with linear IR binding peptides at the A-chain C-terminus. One such derivative, Ins-AC-S2, demonstrated high potency (IC50 = 3.0 nM) against IR phosphorylation and can effectively mitigate insulin-induced hypoglycemia in a streptozotocin-induced diabetic rat model, offers a new direction toward insulin receptor antagonist development. Moreover, the availability of a fully functional insulin receptor antagonist opens new avenues for the development of therapeutics aimed at treating insulin- and glibenclamide-induced hypoglycemia, one of the most common complications of diabetes management, as well as the rare disorder of congenital hyperinsulinism.