An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins

Abstract

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side‐chain of HI by pH‐controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

‘Smart’ insulin-delivery technologies and intrinsic glucose-responsive insulin analogues

“…4) [114]. In the same study [114], in glucose clamp models, some insulin analogues demonstrated GRI activity, but their in vitro hydrolysis rates were >6 h, suggesting that lag time might be an issue.…”

“…The strategy biased for glucose reactivity over fructose by virtue of aldehyde capture based on the increased reactivity of acyclic glucosyl aldehyde over fructosyl ketone and the fact that fasting glucose concentrations are about >1000-fold greater than those of fructose (Fig. 4) [114]. In the same study [114], in glucose clamp models, some insulin analogues demonstrated GRI activity, but their in vitro hydrolysis rates were >6 h, suggesting that lag time might be an issue.…”

“…In a similar manner to the above, Jensen and colleagues [114] took advantage of albumin binding as a plasma depot to demonstrate novel GRI activity acceleration, which was mediated by aldehyde-responsive capture of released insulin. The strategy biased for glucose reactivity over fructose by virtue of aldehyde capture based on the increased reactivity of acyclic glucosyl aldehyde over fructosyl ketone and the fact that fasting glucose concentrations are about >1000-fold greater than those of fructose (Fig.…”

“…Neither D-fructose nor other saccharides accommodate these requirements for interaction with the biomimetic receptor complex [125]. (b) Jensen and colleagues [114] demonstrated a novel chemical approach to D-glucose selectivity by exploiting its open chain, acyclic D-glucosyl aldehyde. A series of masked cleavable hydrazones or thiazolidines were tuned for aldehyde reactivity (carbonyl groups shown in blue).…”

“…Increasing concentrations of glucose (from normoglycaemia to hyperglycaemia) are proposed to drive this shift. In contrast, D-fructose's acyclic form (Dfructosyl ketone [shown in blue]) is reactive to the unmasked linkers but not generally found in the body [114]. (c) PBAs bind most strongly to aligned 1,2-diol elements, such as those found in β-D-fructofuranose; respective conformational equilibria, thus, favour selective binding to fructose [84].…”