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

It was previously demonstrated that insulins to which positivecharge has been added by substituting B13 glutamic acid witha glutamine residue, B27 threonine with an arginine or lysineresidue, and by blocking the C-terminal carboxyl group of theB-chain by amidation, featured a prolonged absorption from thesubcutis of rabbits and pigs after injection in solution atacidic pH. The phenomenon is ascribed to a low solubility combinedwith the readiness by which these analogs crystallize as theinjectant is being neutralized in the tissue. However, acidsolutions of insulin are chemically unstable as A21 asparagineboth deamidates to aspartic acid and takes part in formationof covalent dimers via -amino groups of other molecules. Inorder to circumvent the instability, substitutions were introducedin position A21, in addition to those in B13, B27 and B30, challengingthe fact that A21 asparagine has been conserved in this positionthroughout the evolution. Biological potency was retained whenglycine, serine, threonine, aspartic acid, histidine and argininewere introduced in this position, although to a varying degree.In the crystal structure of insulin a hydrogen bond bridgesthe -nitrogen of A21 with the backbone carbonyl of B23 glycine.In order to investigate the importance of this hydrogen bondfor biological activity a gene for the single-chain precursorB-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 prolinewas synthesized. However, this single-chain precursor failedto be properly produced by yeast, pointing to the formationof 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 ofdimers was {small tilde}5–10 times higher than that ofhuman insulin in neutral solution. The rate of absorption ofmost insulins is decreased by increasing the Zn² concentrationof the preparation. However, one analog with A21 glycine showedfirst-order absorption kinetics in pigs with a half-life of{small tilde}25 h, independent of the Zn² concentration. Theday-to-day variation of the absorption of this analog was significantlylower than that of the conventional insulin suspensions, a propertythat might render such an insulin useful in the attempts toimprove glucose control in diabetics by a more predictable deliveryof basal insulin.