Stable Monomeric Insulin Formulations Enabled by Supramolecular PEGylation of Insulin Analogues

Abstract

Current "fast-acting" insulin analogues contain amino acid modifications meant to inhibit dimer formation and shift the equilibrium of association states toward the monomeric state. However, the insulin monomer is highly unstable and current formulation techniques require insulin to primarily exist as hexamers to prevent aggregation into inactive and immunogenic amyloids. Insulin formulation excipients have thus been traditionally selected to promote insulin association into the hexameric form to enhance formulation stability. This study exploits a novel excipient for the supramolecular PEGylation of insulin analogues, including aspart and lispro, to enhance the stability and maximize the prevalence of insulin monomers in formulation. Using multiple techniques, it is demonstrated that judicious choice of formulation excipients (tonicity agents and parenteral preservatives) enables insulin analogue formulations with 70-80% monomer and supramolecular PEGylation imbued stability under stressed aging for over 100 h without altering the insulin association state. Comparatively, commercial "fast-acting" formulations contain less than 1% monomer and remain stable for only 10 h under the same stressed aging conditions. This simple and effective formulation approach shows promise for next-generation ultrafast insulin formulations with a short duration of action that can reduce the risk of post-prandial hypoglycemia in the treatment of diabetes.

Keywords: PEGylation; diabetes; drug delivery; insulin; polymers; supramolecular.

Figure 1.

Schematic of contributions of insulin association state on absorption kinetics. a), Non-covalent PEGylation is enabled by the strong specific binding between CB[7]-PEG and the N-terminal phenylalanine on insulin (K_D =0.5 μM). Under high concentrations in formulation insulin will be greater than 98% bound, but after dilution upon subcutaneous injection less than 1% of CB[7]-PEG will remain associated with insulin. b,c) Schematic illustration of insulin action over time following subcutaneous administration, whereby insulin association states in formulation dictate insulin activity at various time points. Commercial “fast-acting” insulin formulations comprise a mixture of insulin association states (i.e., hexamers, dimers, and monomers) that result in extended duration of insulin action when delivered subcutaneously. Insulin monomers are absorbed in approximately 5–10 min, dimers are absorbed in 20–30 min, and hexamers can take 1–2 h and cause prolonged insulin action (b). By eliminating insulin hexamers from formulations and shifting the equilibrium to a higher ratio of monomers, future formulations could achieve ultrafast onset and shorter duration of action, which would improve meal-time insulin responses and reduce post-prandial glucose excursions (c).

Figure 2.

Association state of lispro with different formulation excipients. Zinc-free insulin lispro association states when formulated in i) phosphate buffer, sodium chloride (0.9%) (LS), ii) phosphate buffer with glycerol (2.6%) (LG), iii) phosphate buffer with glycerol (2.6%) and meta-cresol (0.315%) (LGM), and iv) phosphate buffer with glycerol (2.6%) and phenoxyethanol (0.85%) (LGPhE). Formulations were compared against a formulation of commercial Humalog. a) SEC-MALS elution profiles. b) Number-averaged molecular weight of the distribution of insulin lispro association states. c–g) Ratio of monomers, dimers, and hexamers in each formulation.

Figure 3.

Association state of aspart with different formulation excipients. Zinc-free insulin aspart association states when formulated in i) phosphate buffer, sodium chloride (0.9%) (AS), ii) phosphate buffer with glycerol (2.6%) (AG), iii) phosphate buffer with glycerol (2.6%) and metacresol (0.315%) (AGM), and iv) phosphate buffer with glycerol (2.6%) and phenoxyethanol (0.85%) (AGPhE). Formulations were compared against a formulation of commercial Novolog. a) SEC-MALS elution profiles. b) Number-averaged molecular weight of the distribution of insulin aspart association states. c–g) Ratio of monomers, dimers, and hexamers in each formulation.

Figure 4.

Formulation with CB[7]-PEG stabilizes zinc-free lispro. In vitro stability of insulin lispro under different formulation conditions with a molar ratio of CB[7]-PEG:Lispro of 0:1 (teal), 3:1 (blue), and 5:1 (orange) against a commercial Humalog control (black). a) Lispro in phosphate buffer with saline (0.9%). b) Lispro in phosphate buffer with glycerol (2.6%). c) Lispro in phosphate buffer with glycerol (2.6%) and phenol (0.25%). d) Lispro in phosphate buffer with glycerol (2.6%) and meta-cresol (0.315%). e) Lispro in phosphate buffer with glycerol (2.6%) and phenoxyethanol (0.85%). f) Comparison of stability by aggregation times (t_A), defined as the time to a change in transmittance (λ=540 nm) of 10% or greater following stressed aging (i.e., continuous agitation at 37° C). Data shown are average transmittance traces for n =3 samples per group and error bars ((f) only) are standard deviation.

Figure 5.

Formulation with CB[7]-PEG stabilizes zinc-free aspart. In vitro stability of insulin aspart under different formulation conditions with a molar ratio of CB[7]-PEG:Aspart of 0:1 (teal), 3:1 (blue), and 5:1 (orange) against a commercial Novolog control (black). a) Aspart in phosphate buffer with saline (0.9%). b) Aspart in phosphate buffer with glycerol (2.6%). c) Aspart in phosphate buffer with glycerol (2.6%) and phenol (0.25%). d) Aspart in phosphate buffer with glycerol (2.6%) and meta-cresol (0.315%). e) Aspart in phosphate buffer with glycerol (2.6%) and phenoxyethanol (0.85%). f) Comparison of stability by aggregation times (t_A), defined as the time to a change in transmittance (λ = 540 nm) of 10% or greater following stressed aging (i.e., continuous agitation at 37 °C). Data shown are average transmittance traces for n =3 samples per group and error bars ((f) only) are standard deviation.

Figure 6.

Diffusion of insulin analogues in various formulations with CB[7]-PEG. Diffusion-ordered NMR spectroscopy (DOSY) provides insight into the formation of protein/CB[7]-PEG complexes and their rates of diffusion in formulation. Diffusion characteristics demonstrate that lispro and aspart diffuse at a similar rate under both a) commercial Humalog (navy blue) and Novolog (light blue) in the presence of zinc ion and b) LGPhE (red) and AGPhE (pale red) in the presence of CB[7]-PEG (0.6 equiv.). Increased diffusion was observed for insulin c) LGPhE and d) AGPhE formulated with 0.6 equiv. CB[7]-PEG (red) compared to commercial formulation conditions (blue).