Ultra-fast insulin-pramlintide co-formulation for improved glucose management in diabetic rats

Dual-hormone replacement therapy with insulin and amylin in patients with type 1 diabetes has the potential to improve glucose management. Unfortunately, currently available formulations require burdensome separate injections at mealtimes and have disparate pharmacokinetics that do not mimic endogenous co-secretion. Here, we use amphiphilic acrylamide copolymers to create a stable co-formulation of monomeric insulin and amylin analogues (lispro and pramlintide) with synchronous pharmacokinetics and ultra-rapid action. The co-formulation is stable for over 16 hours under stressed aging conditions that cause a commercial "fast-acting" insulin formulation, Humalog, to aggregate in only 8 hours. The faster insulin pharmacokinetics achieved by delivery of monomeric insulin alongside pramlintide in this new co-formulation resulted in an increased overlap of 75 (s.e. = 6)% compared to only 47 (s.e. = 7)% for separate injections. Pramlintide delivered in the co-formulation resulted in similar delay in gastric emptying compared to pramlintide delivered separately, indicating pramlintide efficacy is maintained in the co-formulation. In a glucose challenge, rats receiving the co-formulation had reduced deviation from baseline glucose compared to treatment with either Humalog alone or separate injections of Humalog and pramlintide. Together these results suggest that a stable co-formulation of monomeric insulin and pramlintide has the potential to improve mealtime glucose management and reduce patient burden in the treatment of diabetes.

Zn(II) ions co-secreted with insulin suppress inherent amyloidogenic properties of monomeric insulin

Insulin, a 51-residue peptide hormone, is an intrinsically amyloidogenic peptide, forming amyloid fibrils in vitro. In the secretory granules, insulin is densely packed together with Zn(II) into crystals of Zn2Insulin6 hexamer, which assures osmotic stability of vesicles and prevents fibrillation of the peptide. However, after release from the pancreatic β-cells, insulin dissociates into active monomers, which tend to fibrillize not only at acidic, but also at physiological, pH values. The effect of co-secreted Zn(II) ions on the fibrillation of monomeric insulin is unknown, however, it might prevent insulin fibrillation. We showed that Zn(II) inhibits fibrillation of monomeric insulin at physiological pH values by forming a soluble Zn(II)–insulin complex. The inhibitory effect of Zn(II) ions is very strong at pH 7.3 (IC50=3.5 μM), whereas at pH 5.5 it progressively weakens, pointing towards participation of the histidine residue(s) in complex formation. The results obtained indicate that Zn(II) ions might suppress fibrillation of insulin at its release sites and in circulation. It is hypothesized that misfolded oligomeric intermediates occurring in the insulin fibrillation pathway, especially in zinc-deficient conditions, might induce autoantibodies against insulin, which leads to β-cell damage and autoimmune Type 1 diabetes.