Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin

Abstract

Purpose: Basal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements.

Methods: Self-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection.

Results: With depletion of phenol, insulin degludec and another fatty diacid-insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T(3)R(3) conformation in presence of phenol and zinc, changing to T(6) with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly.

Conclusions: Insulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

Fig. 1

Schematic representation of insulin degludec. DesB30 human insulin was acylated at the ε-amino group of Lys^B29 with hexadecandioic acid via a γ-L-glutamic acid linker. The structural elements of the ligand were examined for their impact on the ability of the resulting insulin analogue to form multihexamers (Table I).

Fig. 2

Schematic representation of insulin-zinc hexamer conformation. In a typical pharmaceutical formulation insulin adopts the relaxed (R) conformation. Upon depletion of phenol after injection the poles will subsequently (one pole at a time) adopt the tense (T) conformation, exposing the core and zinc ions. Ultimately zinc dissociates and the hexamer disassembles into dimers and insulin monomers.

Fig. 3

Size exclusion chromatography (SEC) evidence of various insulin degludec self-association states. Insulin degludec was analysed by SEC in the absence of phenol employing a Sepharose 6HR column after formulation with an increasing number of zinc ions per six insulin degludec molecules (panel A). The proportion eluting as multihexamers was closely related to the zinc content. Panel B shows the reciprocal change in the monomer and multihexamer content going from phenol-containing formulations ranging from zero to six zinc ions per six insulin degludec molecules. The traces show the mean and minimum and maximum values (as bars) from duplicate analyses. Near identical values were obtained in each analysis. The lowermost trace in panel A shows SEC of an insulin degludec formulation in the presence of phenol employing a Sepharose 12GL column. Insulin degludec elutes with a peak corresponding to an insulin dihexamer. The small peak that elutes at approximately 44 min corresponds to a molecular weight of 118 kDa. The peak at approximately 78 min does not contain protein. Panel C shows the molar mass distributions of insulin degludec and octadecandioyl-γ-L-Glu desB30 human insulin at two different zinc concentrations in the presence of phenol determined by SEC-MALS. Solid lines in the chromatograms represent traces from the RI detector, dotted lines show the molar mass distribution. The buffer used is without phenol and contains 10 mM Tris–HCl, pH 7.4, 140 mM NaCl.

Fig. 4

Circular dichroism spectroscopy (CDS) assessment at 251 nm of the allosteric interconversion of insulin degludec and human insulin with increasing phenol concentration. The insulin concentration was 0.6 mM containing six Zn²⁺ per six insulin degludec and two Zn²⁺ per six insulin in Tris-perchlorate 10 mM, pH 8.0. Degludec obtains T₃R₃ conformation at 10 mM phenol but remains unchanged thereafter. Human insulin obtains R₆ conformation at 30 mM phenol.

Fig. 5

Schematic representation of the hypothesis for the mode of retarded absorption of insulin degludec: Insulin degludec is injected subcutaneously as a zinc phenol formulation containing insulin degludec dihexamer in the T₃R₃ conformation. Rapid loss of phenol changes the degludec hexamers to T₆ configuration and multi-hexamer chains form. With slow diffusion of zinc, these chains break down into dimers, which quickly dissociate into readily-absorbed monomers.