Investigation of the Physico-Chemical Properties that Enable Co-Formulation of Basal Insulin Degludec with Fast-Acting Insulin Aspart.

Svend Havelund,Thomas Hoeg-Jensen,Ib Jonassen,Ulla Ribel,Per-Olof Wahlund,František Hubálek

doi:10.1007/s11095-014-1614-x

Abstract

PurposeTo study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot.MethodsFormulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography.ResultsSimulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers.ConclusionInsulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.

Highlights

Insulin naturally associates into a hexameric structure in the presence of zinc ions, thought to be an adaptation for efficient storage within pancreatic beta-cell vesicles [1]
Insulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart
This problem has hitherto prevented the co-formulation of two different insulin analogs that have distinct and desired PK/PD profiles because these profiles would have been compromised by such interactions

Summary

Introduction

Insulin naturally associates into a hexameric structure in the presence of zinc ions, thought to be an adaptation for efficient storage within pancreatic beta-cell vesicles [1]. Hexamers are relatively large structures that do not diffuse across capillary membranes as readily as monomers do, and to provide a therapeutic insulin dose in a tolerable injection volume requires insulin to be formulated at concentrations at which it is hexameric [3]. This means that a subcutaneous depot is formed in which dilution occurs slowly, the dissociation of hexamers and absorption of insulin into the circulation is retarded, rising to and falling from a peak over a period of a few hours. An example is insulin aspart (B28 aspartic acid human insulin) in which proline is substituted by aspartic acid at the B28 position

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