Biosynthetic engineered B28(K)-B29(P) human insulin monomer structure in water and in water/acetonitrile solutions.

Insulin engineering is a process intended to obtain desired therapeutic properties by rationally designed sequence mutations of a wild type insulin (HI) which originally consists of two chains, having 21 and 30 amino acids (AA) in chain A and B, respectively, held together by two disulfide bridges, namely A7–B7 and A20–B19 and intra strand bridge in chain A; A6–A11. It is known that human insulin (HI) circulates in the bloodstream as a monomer and in such a form passes effectively through the capillary membrane and binds to the insulin receptor (DeFelippis et al. 2001). An increase in insulin concentration causes formation of noncovalent dimers and higher oligomers. These entities are present in pharmaceutical preparations. Natural propensity of insulin to self-association has meaningful implication on pharmacokinetic behaviour upon hormone administration. Insulin diffusion into tissues entails progressive dissociation of hexamers into dimers and monomers. This is revealed as an increase in a serum insulin concentration reaching a maximum at approximately 2–3 h after subcutaneous injection and followed by slow regression within 9 h. Such pharmacokinetic behaviour causes inconvenience to patients forcing them to adapt their schedules to a medicine, what adversely affects their quality of life. Therefore, two therapeutic strategies have emerged with the potential to mimic physiological insulin secretion. First, rapid-acting insulin analogues were designed to have a more rapid onset of action than HI preparations. These analogues are suitable for mealtime blood glucose control as they simulate pulsatile insulin secretion during meals. Secondly, long acting insulin analogues characterized by delayed absorption and peakless activity profile were developed to mimic basal insulin secretion between meals and through the night. An example is provided by Lantus, a medicinal product containing ArgB31–ArgB32, A21 ? Gly or recently published recombinant insulin LysB31–ArgB32. The pharmaceutical motivation for the addition of a dibasic tag at the end of the B-chain is to shift the isoelectric point from 5 to 7, thus leading an acidic formulation (at pH 4.0) to undergo isoelectric precipitation at physiological pH in the subcutaneous tissue. Such precipitation is accompanied by zinc insulin hexamer and provides a ‘long-acting depot’, which is redissolved slowly and absorbed into the bloodstream lasting for about 24 h (Lepore et al. 2000). Another way of protraction hypoglycaemic activity of insulin is to attach fatty acids to insulin, which forms a reversible complex with serum albumin. The binding occurs through the fatty acid moiety. As an example, insulin detemir has been developed by the removal of threonine from the position B30 and acylation of the C14 fatty acid chain (myristic acid) at position 29 on the B Electronic supplementary material The online version of this article (doi:10.1007/s10858-013-9713-2) contains supplementary material, which is available to authorized users.