Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30

Abstract

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

The potential immunogenicity of human insulin and insulin analogues evaluated in a transgenic mouse model

“…The results presented here, with the three insulin analogues A8A, A10V and ASH, provide the first in vivo indication that only one single amino acid substitution in the A-chain loop at position A8 or A10 is sufficient to elicit an immune reaction in responder mice, Analogues A8H, A8A and A10V were prepared with the aim of studying the effect of single substitutions in the A-chain loop. The other insulin analogues used in this study were made by amino acid substitutions or deletions either designed to prevent hexamer formation [25,26] or to improve chemical stability [23,24], without destabilizing their own three-dimensional structure or interfering with their biological activity [23,25,33,43].…”

“…The sites A8 and A10 represent the differences between bovine insulin and porcine insulin. Substitution of A21 and B3 was performed to obtain chemical stability [23,24]. Substitution of B9, B27 or B28 and deletion of B27 were engineered to obtain monomeric analogues [25,26].…”

Application of SIMSTEX to oligomerization of insulin analogs and mutants

“…Furthermore, the mutant B27, an analog in which Thr at B27 is replaced by Arg, does not undergo any significant dimerization but, instead, forms a tetramer and a hexamer to a much higher extent than does human insulin. This insulin mutant has the property of prolonged action in vivo possibly because it has lower solubility (often precipitates at the site of injection into a human) and absorbs slowly [2,3,32]. The mutant A4B27 is more soluble, but its dimerization constant is five times less than that of r-human insulin.…”

Relationship between insulin A chain regions and insulin biological activities

“…It could well be replaced by other amino acid residu es with good retention of biological activities. Better results were observed wh en the side chain of the A21 amino acid is smaller or not present (Gly) [9] . However, deletion of the A21 residue is fatal as the activity of desA21 insu lin showed only less than 1% of the activity.…”