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

PURPOSE: To study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot. METHODS: Formulations 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. RESULTS: Simulating 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. CONCLUSION: 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.

Ligand-controlled assembly of hexamers, dihexamers, and linear multihexamer structures by the engineered acylated insulin degludec.

Insulin degludec, an engineered acylated insulin, was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultralong glucose-lowering effect in excess of 40 h in humans. We describe the structure, ligand binding properties, and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in the R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol, insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state that interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimics that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(6) state and an average molar mass, M(0), of 59.7 × 10(3) kg/mol. This novel concept of self-assemblies of insulin controlled by zinc and phenol provides the basis for the slow action profile of insulin degludec. To the best of our knowledge, this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers, and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer for allosteric half-site reactivity.

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

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.

Biochemical and physiological properties of a novel series of long-acting insulin analogs obtained by acylation with cholic acid derivatives.

PURPOSE: This study was conducted to assess the suitability of insulin analogs acylated by various cholic acid derivatives for use as basal insulin, and to test the most promising of these, LysB29(Nepsilon-lithocholyl-gamma-Glu) des(B30) human insulin (NN344) in pigs. METHODS: Circular dichroism spectroscopy and size-exclusion chromatography were used to explore the physicochemical properties of the analogs, and affinities for albumin and insulin receptors were determined. After subcutaneous injection in pigs, disappearance half-times were measured, and the plasma profile and glucose-lowering effect in a euglycemic clamp were assessed for NN344. RESULTS: NN344 showed glucose-lowering activity lasting more than 24 h. Glucose infusion rate was essentially constant from 5 to 19 h after injection. NN344 seemed to be a dodecamer in the presence of zinc ions and phenol. Without phenol, the apparent molecular mass was >5000 kDa. Formation of such a self-assembly at the site of s.c. injection and its subsequent slow decomposition might explain the long duration of action of NN344. A measurable affinity for albumin of the lithocholic acid ligand may also contribute to the prolonged action. CONCLUSIONS: NN344 is a candidate for a neutral soluble basal insulin that might offer people with diabetes a prolonged duration, smooth, and predictable basal insulin supplement.

Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose-responsive polymer-based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15-31 mM (K(d)), which match physiological glucose fluctuations. The dose-responsive glucose-mediated release of the novel insulins was demonstrated using glucamine-derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%-122%) which is unusual for insulin LysB29 derivatives.

The mechanism of protraction of insulin detemir, a long-acting, acylated analog of human insulin.

PURPOSE: Insulin detemir has been found in clinical trials to be absorbed with very low variability. A series of experiments were performed to elucidate the underlying mechanisms. METHODS: The disappearance from an injected subcutaneous depot and elimination studies in plasma were carried out in pigs. Size-exclusion chromatography was used to assess the self-association and albumin binding states of insulin detemir and analogs. RESULTS: Disappearance T50% from the injection depot was 10.2+/-1.2 h for insulin detemir and 2.0+/-0.1 h for a monomeric acylated insulin analog. Self-association of acylated insulin analogs with same albumin affinity in saline correlated with disappearance rate and addition of albumin to saline showed a combination of insulin detemir self association and albumin binding. Intravenous kinetic studies showed that the clearance and volume of distribution decreased with increasing albumin binding affinity of different acylated insulin analogs. CONCLUSIONS: The protracted action of detemir is primarily achieved through slow absorption into blood. Dihexamerization and albumin binding of hexameric and dimeric detemir prolongs residence time at the injection depot. Some further retention of detemir occurs in the circulation where albumin binding causes buffering of insulin concentration. Insulin detemir provides a novel principle of protraction, enabling increased predictability of basal insulin.

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins.

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.