Ib Jonassen

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

AbstractPurpose. 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.

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

ABSTRACTPurposeBasal 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.MethodsSelf-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.ResultsWith 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 T3R3 conformation in presence of phenol and zinc, changing to T6 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.ConclusionsInsulins 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.

Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs.

SummaryWe have synthesized insulins acylated by fatty acids in the ε-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r=0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3±2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5±4.3 h (p<0.001), and with less inter-pig variation (p<0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p=0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9±1.0 h and 6.2±1.3 h, respectively (p<0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

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.

Fatty acid acylated insulins display protracted action due to binding to serum albumin

The insulin example demonstrates that it is possible to alter pharmacokinetics and dynamics. By careful choice of ligand and site of substitution it is possible to provide the molecule with the desired characteristics without loosing potency. In this example the following characteristic of insulin has been obtained: 1. A prolonged acting insulin analogue which can be formulated as a neutral soluble preparation 2. Insulin has been provided with albumin binding properties 3. Improved duration of action compared to NPH insulin 4. Improved dynamic profile without sharp peeks of activity, i.e. smoother action