Insulin and its analogues have a propensity to undergo stress-induced conformational changes leading to various aggregated forms including amyloid-type fibrils. Fibrillation is inhibited – but not prevented – by stabilisation of insulin with zinc into a hexameric structure or other excipients. Glargine, the most commonly used longer-acting insulin analogue, has been designed to be insoluble at the body’s neutral pH and therefore precipitates at the site of injection. Like native insulin, however, glargine is also reported to fibrillate and causes injection amyloidosis. Therefore, development of non-fibrillogenic insulin analogues with long half-lives are highly desirable.
An insulin analogue with one free thiol (Cys residue) unit at the N-terminus of the B-chain was synthesised chemically using Cys(tBu) at the N-terminus of the B-chain. After 3 successive regioselective disulfide bond formations (to produce non-glycoinsulin), the tBu on the side chain of the extra Cys was cleaved with trifluoromethanesulfonic acid. Pleasingly, the resulting peptide possessing one free thiol group was very stable in aqueous solution and was purified as a single species. Mono-labelled glycoinsulin was then selectively obtained for the first time by reacting bromo-acetamidyl undecasaccharides with thiol insulin using our optimised chemical method.
Preliminary in vitro study (binding affinity) shows that disialo-glycoinsulin is active at the insulin receptor. Glycoinsulin analogue lowered blood glucose over a 3 hour insulin tolerance test and there was no significant difference between any of the synthetic compounds and controls (actrapid and non-glycoinsulin). More importantly, time-lapse atomic force microscopy clearly demonstrated that insulin forms fibrils at all time points tested (6-24 hr), whereas no fibrils were detected with glycoinsulin over the same period.