Svend Havelund

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.

Alanine Scanning Mutagenesis of Insulin

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the α-helix B8–B19 to the β-turn B20–B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.

Chemical Stability of Insulin. 1. Hydrolytic Degradation During Storage of Pharmaceutical Preparations

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (α-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8–A9 was observed. Formation of this split product is species dependent: bovine > porcine > human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations.

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4–45°C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures ≥25°C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.

Quality by design – Spray drying of insulin intended for inhalation

Quality by design (QBD) refers to a holistic approach towards drug development. Important parts of QBD include definition of final product performance and understanding of formulation and process parameters. Inhalation of proteins for systemic distribution requires specific product characteristics and a manufacturing process which produces the desired product. The objective of this study was to understand the spray drying process of insulin intended for pulmonary administration. In particular, the effects of process and formulation parameters on particle characteristics and insulin integrity were investigated. Design of experiments (DOE) and multivariate data analysis were used to identify important process parameters and correlations between particle characteristics. The independent parameters included the process parameters nozzle, feed, and drying air flow rate and drying air temperature along with the insulin concentration as a formulation parameter. The dependent variables included droplet size, geometric particle size, aerodynamic particle size, yield, density, tap density, moisture content, outlet temperature, morphology, and physical and chemical integrity. Principal component analysis was performed to find correlations between dependent and independent variables. Prediction equations were obtained for all dependent variables including both interaction and quadratic terms. Overall, the insulin concentration was found to be the most important parameter, followed by inlet drying air temperature and the nozzle gas flow rate. The insulin concentration mainly affected the particle size, yield and tap density, while the inlet drying air temperature mainly affected the moisture content. No change was observed in physical and chemical integrity of the insulin molecule.

Ionization behavior of native and mutant insulins: pK perturbation of B13-Glu in aggregated species.

Upscale titration from pH 2.5 to 11.2 is used as a means for probing solvent accessibility of ionizing groups in zinc-free preparations of native and mutant insulins. Stoichiometry and pK alpha values of ionizing groups in the titration curves are determined by iterative curve fitting. Under denaturing conditions, the titration curve of human insulin is in good agreement with that predicted from the sum of unperturbed titrations of the constituent ionizing groups and yields an apparent isoionic point of 5.3. Under nondenaturing conditions where aggregation and precipitation occur, titrations show that only five out of six carboxylate residues of human insulin ionize in the expected region. Consequently, one carboxylate ionization is masked and the apparent isoionic point located at pH 6.4. Correlation between ionization behavior and patterns of aggregation and solubility is established by titrations of mutant insulins and of dilute native insulin. Titration of an unusually soluble species, B25-Phe----His, shows that precipitation is not responsible for the masked carboxylate ionization of native insulin. Titrations of mutants B13-Glu----Gln and B9-Ser----Asp show that the masked ionization probably originates from monomer-monomer interactions in the insulin dimer. We conclude that the B13-Glu side chain is responsible for the masked carboxylate ionization in aggregated forms of human insulin.

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.

Adsorption of insulin on solid surfaces in relation to the surface properties of the monomeric and oligomeric forms

Abstract Adsorption of insulin on clean (hydrophilic) silica, methylated (hydrophobic) silica, and silica with a wettability gradient was studied by in situ ellipsometry. Both human and modified (monomeric) insulin were used. The adsorption was related to the surface properties of the different oligomeric forms of insulin. Adsorption of insulin, carrying a negative net charge, on the hydrophilic, negatively charged silica surface is dependent on the concentration, ionic strength, and type of ions added. No adsorption of monomeric insulin on the hydrophilic surface was observed. It is suggested that it is the hexameric form of insulin that adsorbs to the hydrophilic surface and that the adsorption is governed by electrostatic forces. Adsorption of insulin on the hydrophobic surface is independent of the concentration and, to a large extent, independent of the ionic strength and type of ions added. The amounts adsorbed of human insulin and monomeric insulin are the same on the hydrophobic surface. There is strong evidence that it is the monomeric form of insulin that adsorbs to the hydrophobic surface and that the driving force is hydrophobic interaction.