Abstract
The risk of inducing hypoglycaemia (low blood glucose) constitutes the main challenge associated with insulin therapy for diabetes1,2.
Insulin doses must be adjusted to ensure that blood glucose values are within the normal range, but matching insulin doses to fluctuating glucose levels is difficult because even a slightly higher insulin dose than needed can lead to a hypoglycaemic incidence, which can be anything from uncomfortable to life-threatening.
It has therefore been a long-standing goal to engineer a glucose-sensitive insulin that can auto-adjust its bioactivity in a reversible manner according to ambient glucose levels to ultimately achieve better glycaemic control while lowering the risk of hypoglycaemia3. Here we report the design and properties of NNC2215, an insulin conjugate with bioactivity that is reversibly responsive to a glucose range relevant for diabetes, as demonstrated in vitro and in vivo. NNC2215 was engineered by conjugating a glucose-binding macrocycle4 and a glucoside to insulin, thereby introducing a switch that can open and close in response to glucose and thereby equilibrate insulin between active and less-active conformations. The insulin receptor affinity for NNC2215 increased 3.2-fold when the glucose concentration was increased from 3 to 20 mM. In animal studies, the glucose-sensitive bioactivity of NNC2215 was demonstrated to lead to protection against hypoglycaemia while partially covering glucose excursions.
Main
Using insulin to control diabetes comes with the risk of introducing hypoglycaemia, namely blood glucose values below 3.9 mM (refs. 1,2). This is due to the fact that blood glucose fluctuations are difficult to predict owing to many factors, such as the character and timing of meals, exercise, infections and changing individual insulin sensitivity. People with diabetes must therefore adjust their daily doses of insulin (both basal and meal insulin) to account for these factors. However, to avoid events of low blood glucose, which can be dangerous especially during the night, many opt for conservative insulin doses. Compromising insulin doses due to the fear of hypoglycaemia subsequently results in suboptimal glucose control, thereby increasing the risk of complications arising from long-term hyperglycaemia. To facilitate improved glycaemic control without the risk of hypoglycaemia, the idea of engineering an insulin that can modify its bioactivity in response to varying blood glucose levels has been pursued since the 1970s3. Despite many publications and patents, to date, no mechanism has proven to solve the issue to the extent that it can be applied to treat diabetes5,6,7,8,9,10,11. Most papers in the field describe polymer systems that can release insulin from subcutaneous (s.c.) depots in response to glucose fluctuations, but such systems are limited by delayed glucose diffusion to the subcutis, as well as a delay in the released insulin entering the blood circulation. Moreover, such systems release insulin irreversibly, meaning that, once the insulin is released from the depot, it is no longer glucose sensitive. A better approach seems to be equipping insulin itself with glucose-responsive properties, so it can respond to glucose in a reversible manner. Notably, glucose values vary over a narrow range (from approximately 2 to 20–30 mM in people with diabetes), so a rather steep change in insulin bioactivity must be attained for the glucose-sensitive insulin to have an impact. To achieve such sensitivity to glucose, a chemical group able to bind to glucose with maximal sensitivity in this glucose range will be required. One system was based on oligofucose/mannose insulin conjugates that can be cleared from the circulation in an equilibrium between glucose-sensitive binding to the mannose receptor versus insulin binding to the insulin receptor12, but this did not merit pursuing beyond phase I clinical trials13. The glucose response was found to be shallow, and high clearance at the mannose receptor led to a very low in vivo potency, implicating the eventual need for prohibitively high insulin doses.
The concept of introducing a glucose-sensitive switch into the insulin molecule has been pursued over many years1,14,15,16. A switch involves dual conjugation of a glucose-binding motif plus a binding partner onto insulin such that, at low glucose, the switch will induce a closed less-active state, equilibrating towards an open more-active state with higher glucose concentrations. The glucose-binding motif must therefore have an affinity for both glucose and the binding partner within the narrow glucose range that occurs in people with diabetes (approximately 2 to 20–30 mM). Furthermore, the two components of the switch must be attached to insulin in a manner that ensures that, in the closed state, there is a lower insulin bioactivity by altering the insulin conformation and/or blocking the receptor binding surfaces of insulin. This switch idea has been pursued by using boronates as glucose binders, but the glucose sensitivity of such designs has so far been too limited for pharmacological use. The best previous example of a carbohydrate-sensitive switch working with insulin showed sensitivity to fructose at high concentrations (50 mM), but the compound was insensitive to glucose15. A recently identified macrocycle offers another option for a glucose-binding element4. The macrocycle was designed to provide a glucose-binding cavity that secures a relevant affinity for glucose as well as selectivity over other carbohydrates and potentially interfering small molecules. Here we describe the molecular design of NNC2215, an insulin with a glucose switch by incorporating the macrocycle at B29Lys and introducing an O1-glucoside through a short linker at B1Phe (Fig. 1a). This combination of glucose binder, glucoside, linker and conjugation sites was found to impart glucose-sensitive bioactivity to NNC2215, which demonstrated a 12.5-fold increase in insulin receptor binding affinity when glucose was raised from 0 to 20 mM and a 3.2-fold increase when raised from 3 to 20 mM. Furthermore, NNC2215 was shown to be glucose sensitive in vivo, to attenuate hypoglycaemia in pigs and to reduce the glucose excursions during glucose tolerance tests (GTTs) in diabetic rats.
Published: 16 October 2024
Authors: Thomas Hoeg-Jensen, Thomas Kruse, Christian L. Brand, Jeppe Sturis, Christian Fledelius, Peter K. Nielsen, Erica Nishimura, Alice R. Madsen, Lennart Lykke, Kim S. Halskov, Simona Koščová, Vladislav Kotek, Anthony P. Davis, Robert A. Tromans, Michael Tomsett, Guillem Peñuelas-Haro, Daniel J. Leonard, Michael G. Orchard, Andy Chapman, Gaetano Invernizzi, Eva Johansson, Daniele Granata, Bo F. Hansen, Thomas A. Pedersen, …Rita Slaaby
Link: https://www.nature.com/articles/s41586-024-08042-3
Source: Nature
Note: Nutrigenomics Institute is not responsible for the opinions expressed in this article.
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