Physico-chemical properties of co-formulated fast-acting insulin with pramlintide.

Since the discovery of amylin its use has been discouraged by the inadequacy of the protocol involving multiple injections in addition to insulin. We aimed here to develop a combined fixed-dose formulation of pramlintide with fast-acting insulin. We have investigated the compatibility of regular and fast-acting insulin analogues (Aspart, AspB28, and LisPro, LysB28ProB29) with the amylin analogue pramlintide by using electrospray ionization - ion mobility spectrometry-mass spectrometry (ESI-IMS-MS), kinetic aggregation assays monitored by thioflavin T, and transmission electron microscopy (TEM) in the evaluation of the aggregation product. Insulin interacts with pramlintide, forming heterodimers as probed by ESI-IMS-MS. While their interaction is likely to delay the amyloid aggregation of pramlintide in phosphate-buffered solution pH 7.0, they do not prevent aggregation at this condition. At acidic sodium acetate solution pH 5.0, combination of pramlintide and the fast-acting insulin analogues become stable against amyloid aggregation. The co-formulated product at high concentration of both pramlintide (600 µg/mL,150 µM) and LisPro insulin (50 IU/mL, 300 µM) showed also stability against amyloid aggregation. These data indicate the physico-chemical short-term stability of the co-formulated preparation of LisPro insulin with pramlintide, which could bring benefits for the combined therapy.

Rheological properties of regular insulin and aspart insulin Langmuir monolayers at the air/water interface: condensing effect of Zn2+ in the subphase.

The interfacial behavior of regular insulin (Reg-insulin) and aspart insulin (Asp-insulin) was critically affected by the presence of Zn(2+) in the subphase. This cation induced a condensed-like behavior in the compression isotherms, especially apparent for Reg-insulin films when observed by Brewster angle microscopy. Immediately after spreading, Reg-insulin, but not Asp-insulin, showed bright patches that moved in a gaseous-like state. Moreover, Zn(2+) caused marked variations of the surface electrostatics of both insulin monolayers and considerable hysteresis of their molecular organization. By oscillatory compression-expansion cycles, we observed in all cases the development of a dilatational response to the surface perturbation, and both monolayers exhibited well-defined shear moduli in the presence of Zn(2+), which was higher for Reg-insulin. Development of a shear modulus indicates behavior resembling a nominal solid, more apparent for Reg-insulin than for Asp-insulin, suggesting the presence of viscoelastic networks at the surface.

A T3R3 hexamer of the human insulin variant B28Asp.

Insulin shows a complex equilibrium between monomers and hexamers, involving varying conformers and association states. We sought to perform a structural characterization of the fast-acting human insulin variant B28Asp ("aspart"). Small-angle X-ray scattering measurements reveal similar globular behavior in both the aspart and regular human insulin, with a Rg of 19Å and a Dmax of approximately 50Å, indicating similar mean quaternary assembly distribution. Crystallographic assays revealed a T3R3 assembly of the aspart insulin formed by the TR dimer in the asymmetric unit, with all the first 8 residues of the B chain in the R-state monomer in helical conformation and the participation of its B3Asn in the stabilization of the hexamer. Our data provide access to novel structural information on aspart insulin such as an aspart insulin dimer in solution, the aspart insulin in T conformation and a pure R-state conformer establishing a T3R3 assembly, providing further insight on the stepwise conformational transition and assembly of this fast-insulin.