Glucose-sensitive insulin with attenuation of hypoglycaemia

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Glucose-sensitive insulin with attenuation of hypoglycaemia

https://doi.org/10.21203/rs.3.rs-2882397/v1

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The risk of inducing hypoglycaemia (low blood glucose) constitutes the main challenge associated with insulin therapy for diabetes. Insulin doses must be adjusted to ensure blood glucose values within normal range, but matching insulin doses to fluctuating glucose levels is difficult since 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 thus 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 hypoglycaemia. Here we report the design and properties of NNC2215, an insulin conjugate with bioactivity that is reversibly responsive to glucose over the physiological range as demonstrated in vitro and in vivo. NNC2215 was engineered by conjugating a glucose-binding macrocycle 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 of NNC2215 increased 12-fold when glucose concentration was increased from 0 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.

Health sciences/Endocrinology/Endocrine system and metabolic diseases/Diabetes/Diabetes complications

Biological sciences/Chemical biology/Protein design

Using insulin to control diabetes comes with the risk of introducing hypoglycaemia, namely blood glucose values below 3.9 mM¹^,².This is due to the fact that blood glucose fluctuations are difficult to predict, because of 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 sugar, 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 sub-optimal glucose control, thus 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 diabetes⁴^–¹⁰. Most papers in the field describe polymer systems that can release insulin from subcutaneous depots in response to glucose fluctuations, but such systems suffer from delayed glucose diffusion to the subcutis, as well as a delay in the released insulin entering the blood circulation. Also, such systems release insulin irreversibly, meaning 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, physiological glucose values vary over a narrow range (from approximately 2 up to 20‑30 mM in people with diabetes), so a rather steep change in insulin bioactivity must be attained in order for the glucose-sensitive insulin to have impact. To achieve such sensitivity to glucose, a chemical group able to bind glucose with maximal sensitivity in the physiological 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 receptor¹¹, but this did not merit pursuing beyond phase 1 clinical trials¹². 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^,¹³^–¹⁵.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 physiological glucose range. 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 use¹⁴. A recently identified macrocycle offers another option for a glucose binding element¹⁶. The macrocycle was designed to provide a glucose-binding cavity that secures a physiologically 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 via a short linker at B1Phe. This combination of glucose binder, glucoside, linker and conjugation sites was found to impart glucose-sensitive bioactivity to NNC2215, which demonstrated a 12-fold increase in insulin receptor binding affinity when glucose was raised from 0 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 in diabetic rats.

Chemistry

The macrocycle¹⁶ (Fig. 1a) was conjugated to desB30 human insulin at the B29Lys N-epsilon amino group via triazole formation¹⁷ between a macrocycle derivative carrying an azido propyl linker from the macrocycle roof, and an alkyne linker attached to B29Lys (by conjugation at pH >10 to obtain the B29 product). Conjugation via the macrocycle roof circumvents the need to orthogonally address one of three carboxylic acids, as would be needed for conjugation to either of the COOH groups on the pillars of the macrocycle. The dendrimers that were used for securing good aqueous solubility of the originally reported macrocycle¹⁶ was found unnecessary when working with insulin-macrocycle conjugates. Besides the macrocycle at B29, an O1-glucoside with a short linker was attached to the B‑chain N-terminal amino group (PheB1) by using the corresponding bromo trifluoromethyl sulfate phenyl ester at pH 7.5¹⁸. The glucoside was used as its O-peracetyl protected building block, and the acetyl groups were removed from the insulin conjugate by gentle saponification. A control compound, NNC2215a, with only the macrocycle in B29 was prepared similarly to NNC2215 by omitting the glucoside step. The conjugation sites on insulin were documented by LCMS analysis of a sample that was treated with trypsin followed by tris(2-carboxyethyl)phosphine (TCEP). The trypsin treatment cleaved NNC2215 after B22Arg to release the B23-B29 fragment, and TCEP cleaved the disulfides of NNC2215 to give separate A and B chains. LCMS analysis of the resulting analytical mixture showed the macrocycle attached to the B23-B29 fragment, and the glucoside attached to the B1-B22 fragment, along with free A-chain (see chemistry details in the Supplementary Methods).

Glucose binding data for free macrocycle and NNC2215

Using an isothermal titration calorimeter (ITC), the free macrocycle of NNC2215 was shown to bind glucose with a K_d of 98 µM (Fig. 1b). Native mass spectrometry (MS) was used to study binding of NNC2215 towards glucose. As expected, the presence of the glucoside in the dual conjugate changed the glucose affinity of NNC2215 relative to the free macrocycle, such that NNC2215 was found to bind glucose with a K_dof 2.1 mM, which matches the hypoglycaemic range for blood glucose. The control compound, NNC2215a with macrocycle only, was found by native MS to bind glucose with a K_d of 0.5 mM. Accordingly, the glucose affinity of NNC2215 and the control compound NNC2215a as measured by native MS was approximately 5-fold and 20-fold weaker when compared to the free macrocycle, thereby demonstrating that suitable switch dynamics had been achieved. The glucose binding and concurrent opening of the switch in NNC2215 and NNC2215a in response to 0 to 20 mM glucose can be followed by the native MS binding plot in Fig. 1c. The steepest part of the binding curve is consistent with the hypoglycaemic range of 0 to 5 mM glucose. Although the native MS data are obtained in the gas phase (mass spectrometry vacuum), such data often reflect the interactions of molecules corresponding to the aqueous solutions from which the complexes are sampled¹⁹.

3D structural model of NNC2215 interaction with the insulin receptor

Structural models were built of the insulin receptor binding to NNC2215 with the switch in either the open or closed state by superimposing models of NNC2215 on the insulin/insulin receptor complex PDB 6PXV²⁰^–²⁴. As illustrated in Fig. 2, when the switch is closed, a clash occurs between the C-terminal part of the insulin B-chain and the C-terminal domain of the insulin receptor, termed α-CT. The α-CT domain is known to be a crucial part for insulin binding²⁵. We believe this steric hindrance to be the driving force for the observed lower receptor affinity of NNC2215 at low glucose concentrations, i.e. with the switch populating mainly the closed conformation.

In vitro biology

To study the glucose-sensitive interaction of NNC2215 with the insulin receptor in vitro, an insulin receptor binding assay was used in the absence or presence of physiologically relevant glucose concentrations as well as 1.5% albumin to simulate physiological conditions. The ¹²⁵I-insulin displacement curves of human insulin receptor A (hIR-A)²⁶^,²⁷ binding to NNC2215, as well as to human insulin and to insulin degludec (a long-acting standard insulin)²⁸, are shown at glucose concentrations of 0, 3, 5, 10 and 20 mM (Fig. 3a–c). The displacement curves for NNC2215 shift left as the glucose concentration increases (Fig. 3a), thus showing an increasing affinity of NNC2215 for the hIR-A, whereas the binding affinities of human insulin and insulin degludec are not affected by the glucose concentration (Fig. 3b,c). Relative to the hIR-A affinity of human insulin, the affinity of NNC2215 increases from 0.75% to 2.9%, 4.3%, 6.5% and 9.2% over the glucose concentration range of 0, 3, 5, 10 and 20 mM, whereas insulin degludec has a constant affinity relative to human insulin (Table 1 and Fig. 3d). The increase in hIR-A affinity of NNC2215 from 0 to 20 mM glucose is 12.5‑fold (Table 1), whereas the increase is 3.2-fold from 3 to 20 mM glucose.

The ability of NNC2215 to activate the insulin signalling pathway was studied in Chinese Hamster Ovary cells expressing the cloned human insulin receptor cells (CHO-hIR). Full dose-response curves were obtained for NNC2215 stimulating tyrosine phosphorylation of the human insulin receptor with a potency of 57.1% (95% CI: 41.7-78.1) compared to human insulin. Downstream signalling through Akt and ERK activation had the same balance relative to human insulin with potencies of 68.4% (95% CI: 55.7-84.0) and 48.0% (95% CI: 34.4-66.9), respectively²⁹.

The glucose sensitivity of NNC2215 was reflected in a metabolic endpoint measured ex vivo in rat adipocytes³⁰. The induction of glucose uptake and incorporation into lipids in rat adipocytes, i.e. lipogenesis, was measured in an assay where we took advantage of the enantiomer of the physiologically active D-glucose, namely L-glucose. The rationale for using L-glucose is that cells do not catabolize or take up L-glucose in appreciable amounts, at least not through saturable transport, except in the case of some Gram-negative bacteria and plants under certain conditions³¹. In control experiments, L-glucose did not compete with the uptake of D-glucose into adipocytes, but L-glucose binds the achiral macrocycle of NNC2215 with the same affinity as D-glucose³². There was a 2-fold difference (mean±SD 2.2±0.5) in the ED₅₀ determined for NNC2215-induced ³H-D-glucose conversion into lipid in rat adipocytes at low (3 mM) versus high (20 mM) L-glucose (Fig. 3e). This demonstrates the glucose sensitivity of NNC2215 ex vivo with respect to insulin-induced metabolic response. With insulin degludec, lipogenesis showed no response to L-glucose.

In vivo pharmacology

To investigate the glucose concentration-sensitive activation and deactivation of NNC2215 in vivo, we developed three different protocols. In the simplest protocol, rats were dosed intravenously with NNC2215 followed by L-glucose, thereby triggering NNC2215, resulting in lowering of D-glucose in an L-glucose dose-dependent fashion. Further, the deactivation of NNC2215 at low glucose was investigated in pigs by an acute drop in plasma glucose in comparison to the glucose drop induced by a non-glucose-sensitive insulin (insulin degludec). Finally, the activation of NNC2215 was studied during a glucose challenge in diabetic rats. Insulin degludec was used as comparator in the pig study due to similar pharmacokinetic properties following intravenous administration (Extended Data Table 3), whereas human insulin was used as comparator in the rat study. The pharmacokinetic and pharmacodynamic data from pigs were analysed by applying the minimal model, which describes glucose kinetics and insulin activity in a quantitative manner³³. The model was used to quantify the change in insulin activity of NNC2215 with changing plasma glucose. The in vivo half-life of NNC2215 was determined to be 1.4 h by intravenous dosing to rats and 1.9 h by intravenous dosing to pigs (Extended Data Table 3).

L-glucose study in rats

L-glucose was used to trigger NNC2215 in rats, without triggering endogenous insulin release as dosing of D-glucose would do. Fig. 4a shows how intravenous dosing of NNC2215 followed by L-glucose (3 doses) dose-dependently triggers insulin action of NNC2215, namely by lowering D-glucose. As expected, NNC2215 is simultaneously and dose‑dependently cleared upon the L-glucose triggering (Fig. 4b). The initial peak in D‑glucose upon L-glucose injection (Fig. 4a) is a stress response from the animal, which is also observed with control compounds (data not shown). Notably, the glucometer used to measure D-glucose does not respond to L-glucose, as it is based on the glucose oxidase enzyme.

Hypoglycaemic study in LYD pigs

To evaluate the in vivo activity of NNC2215 in healthy Landrace-Yorkshire-Duroc (LYD) pigs, and to essentially eliminate the effects from D-glucose fluctuations on endogenous hormone release, somatostatin infusion was used to suppress secretion of both glucagon and insulin. Furthermore, the study was conducted under a primed constant infusion of NNC2215 versus insulin degludec at different rates accompanied by glucagon replacement and constant glucose infusion. After 5 hours of infusion, when approximate steady state plasma concentrations of insulin (degludec or NNC2215) and glucose were achieved, the D-glucose infusion was stopped (Fig. 4c). This procedure resulted in a drop in plasma D-glucose, promoted by the continued insulin infusion, but the drop was observed to be less pronounced with NNC2215 compared to insulin degludec, thereby showing glucose‑sensitive switching of the insulin bioactivity. Evaluation of the full data set utilizing different insulin infusion rates of NNC2215 and insulin degludec shows that after the glucose infusion is turned off, at any given plasma glucose concentration, the drop in plasma glucose is smaller for NNC2215 than for insulin degludec (Extended Data Fig. 6). Data from two representative groups are shown in Fig. 4c, where glucose dropped to about 4.5 mM with NNC2215 versus a drop to about 3 mM glucose for insulin degludec, thus demonstrating protection against hypoglycaemia with NNC2215.

Pharmacokinetic/pharmacodynamic modelling

The best fit to the glucose data for NNC2215 from the hypoglycaemic study in LYD pigs was obtained by implementing a saturable effect of glucose on the insulin sensitivity parameter, SI, and the corresponding SI index, which is defined as 100% at 5.5 mM glucose. A representative fit to the glucose data from a single pig dosed with 1.72 pmol/kg/min NNC2215 is shown in Fig. 4d. For this pig, the plasma glucose dropped from 8.5 mM to 3.8 mM when the glucose was turned off, and the SI index decreased from 140% to 70%, indicating a significant dynamic range for activation of NNC2215 within a physiologically relevant glucose range. For insulin degludec, the SI index was always 100% as indicated by the horizontal dashed line in Fig. 4d. The modelling indicates that within the physiological range of 3 to 15 mM glucose, the relationship between the SI index and plasma glucose is linear with a slope of 14.6% per mM glucose.

Glucose tolerance test in STZ-diabetic rats

Insulinopenic streptozotocin (STZ)-diabetic rats were used to investigate the glucose-induced activation of NNC2215 without interference from endogenous insulin release during an intravenous glucose tolerance test (GTT). Overnight fasted rats were infused intravenously with one of three different constant molar rates of either NNC2215 or human insulin to lower plasma glucose from ≥15 mM to a pre-defined target of 5.7 mM. This glucose target was then maintained by adjusting an intravenous glucose infusion rate. When the glucose infusion rate was at steady state, a GTT was employed by infusing an additional 25 mg/kg/min of glucose for 60 min on top of the individual steady-state glucose infusion rate (Fig. 5a) and the resulting changes in plasma glucose concentrations were then monitored.

A subset of data from STZ-diabetic rats with similar steady-state glucose infusion rate, i.e. with similar insulin effect, prior to the GTT are shown in Fig. 5. As shown in Fig. 5b, during the GTT the increase in plasma glucose was approximately 20% smaller in the NNC2215 group than in the human insulin group (mean±SEM 18.3±1.1 versus 22.9±1.3 mM, p<0.02), indicating a larger insulin effect of NNC2215 at the high glucose concentrations without change in insulin exposure. To quantify how much 50% additional human insulin would reduce the maximal plasma glucose concentrations during the GTT, another group of rats were given 50% additional human insulin on top of its constant infusion rate, i.e. 20 + 10 pmol/kg/min during the GTT. The resulting maximal plasma glucose concentration was significantly reduced (by approximately 34%) by the 50% additional human insulin compared to human insulin at the equimolar infusion rate (mean±SEM 15.1±0.7 versus 22.9±1.3 mM, p<0.0002), and maximal plasma glucose also tended to be further reduced compared to NNC2215 (mean±SEM 15.1±0.7 versus 18.3±1.1 mM, p=0.08) (Fig. 5b).

Analysis of the full dataset showed that during the GTT the reductions in the maximal plasma glucose concentration caused by NNC2215 and by the 50% additional human insulin compared to human insulin at the equimolar infusion rate were comparable across the different insulin doses tested. The glucose-induced increase in insulin activity of NNC2215 during the GTT was estimated to correspond to approximately 30% increase in the human insulin dose (Extended Data Fig. 7).

Insulin was equipped with a glucose-sensitive switch composed of a macrocycle conjugated to LysB29 and an O1-glucoside conjugated to B1Phe, both via short linkers. The binding of NNC2215 to glucose was evaluated by native mass spectrometry analysis, showing the steepest part of the binding curve to match the hypoglycaemic range, i.e. 0 to 5 mM glucose. Glucose-sensitive binding of NNC2215 to the insulin receptor over the physiological glucose concentration range of 0 to 20 mM was measured, and the glucose responsiveness of NNC2215 was found to be much stronger than what has been reported for prior attempts at equipping insulin with glucose-sensitive switches¹. The glucose-promoted modulation of the interaction of NNC2215 with the insulin receptor could be rationalised by 3D molecular modelling studies, visualizing how NNC2215 with the switch in closed state can interfere with binding towards the α-CT domain of the insulin receptor. NNC2215 was also shown to provide glucose-sensitive cellular glucose uptake in the form of lipogenesis, using L-glucose to circumvent the inherent D-glucose sensitivity of lipogenesis.

A relatively simple acute in vivo model was developed using L-glucose dosed to rats that were pre-dosed with NNC2215, thus triggering the insulin effect of NNC2215 without triggering endogenous insulin release. The triggering of NNC2215 by L-glucose resulted in dose-dependent lowering of D-glucose and concurrent L-glucose dose-dependent clearance of NNC2215. This protocol was inspired by the use of alpha-methyl-D-glucopyranose to trigger glucose-sensitive insulin derivatives that rely on the previously described mannose receptor principle¹¹.

Remarkably, the glucose-sensitive insulin receptor binding and cellular effects of NNC2215 translated to an in vivo hypoglycaemia protective effect observed in a novel model with acutely diabetic pigs. When the D-glucose infusion was shut off, we observed a drop in plasma glucose, but the drop was smaller for NNC2215 than for insulin degludec, with the lowest glucose value averaging approximately 4.5 mM for NNC2215 versus below 3 mM for insulin degludec. The difference in glucose drop between NNC2215 and insulin degludec was observed across all dose levels tested and was estimated to be approximately 1.7 mM in the overlapping starting glucose range (Extended Data Fig. 6). This degree of attenuation in the undesirable plasma glucose lowering effect when there is too much insulin at low glucose levels is expected to confer a significant advantage to insulin conjugates like NNC2215 in their ability to reduce the risk of hypoglycaemia inherent to insulin treatment whenever there is a mismatch between insulin dose and actual need.

We also observed an activation of NNC2215 during a glucose challenge in STZ-diabetic rats corresponding to the effect of 30% additional human insulin. Such glucose-induced activation of NNC2215 could likely contribute to reducing some of the glucose excursion after a meal. Therefore, less than full doses of fast-acting insulin may be required with larger meals. Smaller doses of mealtime insulin in combination with NNC2215 could potentially reduce the risk of hypoglycaemia induced by the fast-acting insulin, and could overall allow for tighter glucose control without the fear of hypoglycaemia.

The modelling of pharmacokinetic/pharmacodynamic data in pigs quantified the dynamic range for activation of NNC2215 in vivo. Based on the pharmacokinetic/pharmacodynamic modelling, within the physiological glucose range the insulin activity index varied from around 60% at 3 mM glucose to 290% at 20 mM glucose, i.e. a 5-fold range which corresponds to the 3.2-fold difference in insulin receptor affinity between 3 and 20 mM glucose. Such consistency between in vitro and in vivo glucose sensitivity of NNC2215 lends support to the possible translation to human use for compounds with properties like NNC2215.

In conclusion, insulin conjugates with properties such as NNC2215 hold promise for improved treatment of diabetes by potentially lowering the risk of hypoglycaemia and partly covering the need for fast-acting insulin at mealtime. The combination of these two features should allow for more aggressive insulin titration compared to current insulin therapies to achieve normal glucose levels without increasing the risk of hypoglycaemia. This could improve both the short-term and long-term risks and complications associated with diabetes. On a general note, NNC2215 illustrates how molecular switches can be designed to enable autonomous control of molecular bioactivity in response to changing concentrations of another molecule, even within a narrow range such as is the case for blood glucose levels.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data files (chemical analysis files, Prism files) can be shared upon reasonable request.

Acknowledgements

We thank Kirsten Holmberg, Anja Hansen, Rokhsana J. Andersen, Heidi Jensen, Hildur Gudmundsdottir, Carsten Stokkebye Stenvang, Christine Degnsøe, Mette Okkels, Bent Kolling, Gitte-Mai Nelander, Pia Jensen, Jesper Damgaard, Lisa Hansen, Brian Hansen, Bettina Lyngsø Bengtsen, Sara Louise Riisberg, Merete Hvidt, Wei Liu, Tine Østergaard, Kirsten Borres Jensen, Louise Christiansen, Susanne Jørgensen and animal caretakers for expert technical assistance and Carsten Roepstorff (CR Pharma Consult, Copenhagen, Denmark) for providing medical writing support funded by Novo Nordisk A/S.

Author contributions

T.H.J., T.K., A.R.M., L.L., K.S.H. and P.S. designed and prepared the insulin conjugates. T.H.J., T.K., P.S., A.P.D., R.T., M.T., G.P.H., D.L., M.O. and A.M.C. designed and prepared the macrocycle building blocks. T.H.J., L.L., P.S., S.K. and V.K. designed and prepared the glucoside building block. P.K.N. and G.I. did the biophysical characterizations. D.G. and E.J. performed structural modelling. R.S., B.F.H., T.A.P. and E.N. designed and performed the in vitro biology studies. C.L.B., J.S., C.F., K.M.P., H.H.F.R., L.A., J.K., J.J.F. and A.V.N.W. designed and performed the in vivo studies and analysis.

Funding

This study was funded by Novo Nordisk A/S.

Competing interests

T.H.J., T.K., C.L.B., J.S., C.F., P.K.N., E.N., A.R.M., L.L., K.S.H., G.I., E.J., D.G., B.F.H., T.A.P., J.K., K.M.P., H.H.F.R., L.A., J.J.F., A.V.N.W., P.S. and R.S. are employees and shareholders of Novo Nordisk A/S. The other authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material.

Correspondence and requests for materials should be addressed to Thomas Hoeg-Jensen, Novo Nordisk A/S, Global Research Technologies, e-mail: [email protected].

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Hoeg-Jensen, T. Review: Glucose-sensitive insulin. Mol. Metab. 46, 101107 (2021).
ElSayed, N. A. et al. 6. Glycemic targets: Standards of care in diabetes-2023. Diabetes Care 46 (Suppl. 1), S97–S110 (2023).
Brownlee, M. & Cerami, A. A glucose-controlled insulin-delivery system: semisynthetic insulin bound to lectin. Science 206, 1190–1191 (1979).
Veiseh, O., Tang, B. C., Whitehead, K. A., Anderson, D. G., Langer, R. Managing diabetes with nanomedicine: challenges and opportunities. Nat. Rev. Drug Discov. 14, 45–57 (2015).
Zaykov, A. N., Mayer, J. P., DiMarchi, R. D. Pursuit of a perfect insulin. Nat. Rev. Drug Discov. 15, 425–439 (2016).
Bakh, N. A. et al. Glucose-responsive insulin by molecular and physical design. Nat. Chem. 9, 937–944 (2017).
Yu, J., Zhang, Y., Yan, J., Kahkoska, A. R., Gu, Z. Advances in bioresponsive closed-loop drug delivery systems. Int. J. Pharm. 544, 350–357 (2018).
VandenBerg, M. A. & Webber, M. J. Biologically inspired and chemically derived methods for glucose-responsive insulin therapy. Adv. Healthc. Mater. 8, e1801466 (2019).
Disotuar, M. M., Chen, D., Lin, N. P., Chou, D. H. Glucose-responsive insulin through bioconjugation approaches. J. Diabetes Sci. Technol. 14, 198–203 (2020).
Jarosinski, M. A., Dhayalan, B., Rege, N., Chatterjee, D., Weiss, M. A. 'Smart' insulin-delivery technologies and intrinsic glucose-responsive insulin analogues. Diabetologia 64, 1016–1029 (2021).
Kaarsholm, N. C. et al. Engineering glucose responsiveness into insulin. Diabetes 67, 299–308 (2018).
Visser, S. A. G., Kandala, B., Fancourt, C., Krug, A. W., Cho, C. R. A model-informed drug discovery and development strategy for the novel glucose-responsive insulin MK-2640 enabled rapid decision making. Clin. Pharmacol. Ther. 107, 1296–1311 (2020).
Zion, T. C. & Lancaster, T. M. Soluble non-depot insulin conjugates and uses thereof. WO/2010/107520, 23 September 2010 (2010).
Chen, Y. S. et al. Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation. Proc. Natl. Acad. Sci. U S A 118, e2103518118 (2021).
Feringa, B. L., The art of building small: from molecular switches to molecular motors. J. Org. Chem. 72, 6635–6652 (2007).
Tromans, R. A. et al. A biomimetic receptor for glucose. Nat. Chem. 11, 52–56 (2019).
Meldal, M. & Tornøe, C. W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008).
Jensen, K. B. et al. New phenol esters for efficient pH-controlled amine acylation of peptides, proteins, and sepharose beads in aqueous media. Bioconjug. Chem. 33, 172–179 (2022).
Tamara, S., den Boer, M. A., Heck, A. J. R. High-resolution native mass spectrometry. Chem. Rev. 122, 7269–7326 (2022).
Uchikawa, E., Choi, E., Shang, G., Yu, H., Bai, X. C. Activation mechanism of the insulin receptor revealed by cryo-EM structure of the fully liganded receptor–ligand complex. eLife 8, e48630 (2019).
Wagner, A., Diez, J., Schulze-Briese, C., Schluckebier, G. Crystal structure of ultralente – a microcrystalline insulin suspension. Proteins 74, 1018–1027 (2009).
Croll, T. I. et al. Higher-resolution structure of the human insulin receptor ectodomain: Multi-modal inclusion of the insert domain. Structure 24, 469–476 (2016).
Lawrence, M. C. Understanding insulin and its receptor from their three-dimensional structures. Mol. Metab. 52, 101255 (2021).
Gutmann, T. et al. Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain. J. Cell Biol. 219, e201907210 (2020).
Kristensen, C., Andersen, A. S., Ostergaard, S., Hansen, P. H., Brandt, J. Functional reconstitution of insulin receptor binding site from non-binding receptor fragments. J. Biol. Chem. 277, 18340–18345 (2002).
Soos, M. A. et al. Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor. Biochem. J. 235, 199–208 (1986).
Andersen, M., Nørgaard-Pedersen, D., Brandt, J., Pettersson, I., Slaaby, R. IGF1 and IGF2 specificities to the two insulin receptor isoforms are determined by insulin receptor amino acid 718. PLoS One 12, e0178885 (2017).
Jonassen, I. et al. Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin. Pharm. Res. 29, 2104–2114 (2012).
Hansen, B. F. et al. Sustained signalling from the insulin receptor after stimulation with insulin analogues exhibiting increased mitogenic potency. Biochem. J. 315, 271–279 (1996).
Moody, A. J., Stan, M. A., Stan, M., Gliemann, J. A simple free fat cell bioassay for insulin. Horm. Metab. Res. 6, 12–16 (1974).
Ono, K., Takigawa, S., Yamada, K. L-glucose: another path to cancer cells. Cancers 12, 850 (2020).
Tromans, R. A., Samanta, S. K., Chapman, A. M., Davis, A. P. Selective glucose sensing in complex media using a biomimetic receptor. Chem. Sci. 11, 3223–3227 (2020).
Bergman, R. N. Toward physiological understanding of glucose tolerance. Minimal-model approach. Diabetes38, 1512–1527 (1989).

Synthesis of buildings blocks, insulin conjugate NNC2215 and control compound NNC2215a

Detailed synthesis of NNC2215 and the control compound NNC2215a is described in the Supplementary Methods. In brief, desB30 human insulin was conjugated at B29 with O‑succinimidyl-pentyn-1-oxycarbonyl at pH >10, and at B1 with O-peracetyl-D-glucosyl-beta-ethylenoxy-acetic acid using Br CF₃ sulfonate phenolic active ester at pH 7.5. The acetyl groups were removed by gentle saponification. The macrocycle reagent was made from 3‑(3,5‑dimethylphenyl)propanoic acid by benzylic bromination, azidation, reduction of COOH to alcohol, and the azides were reduced to the amines, which were Boc-protected. The propyl alcohol was activated as the mesylate and transformed to the propyl azide. The three Boc-amino groups were transformed to the isocyanates using triflic anhydride, and the macrocycle was closed by reaction of the isocyanates with the previously described trisamino half-macrocycle reagent¹⁶. The roof azide macrocycle was triazole-coupled under Cu(I) catalysis with the given B29-alkyne insulin to give NNC2215, which was purified by HLPC. For chemical characterization, see the Supplementary Methods.

Glucose affinity of the free macrocycle propyl azide by calorimetry

The concentration of the free macrocycle (Extended Data Fig. 4c) in the cell was 50 uM. The concentration of glucose added in the syringe was 3 mM. The injection volume was 10 uL. The stirring speed was 310 rpm. All in 10 mM phosphate buffer, pH 7.4 at 25^oC. The obtained K_a was 10,200 M^-1, and K_d was 98 µM. Data were acquired on a MicroCal VP‑ITC microcalorimeter and processed using MicroCal software (MicroCal VP-ITC Analysis Add‑On Software Package 7.20 for ORIGIN 7.0).

Affinity of NNC2215 towards glucose by native mass spectrometry

Glucose solutions (18.75 µM, 37.5 µM, 75 µM, 150 µM, 300 µM, 625 µM, 1.25 mM, 2.5 mM, 5 mM, 10 mM, 20 mM) in presence of NNC2215 (0.5 mg/mL) were prepared in 75 mM NH₄Ac, pH 7.4. The compounds were buffer exchanged into 75 mM NH₄Ac, pH7.4 by Amicon^® Ultra centrifugal devices with 3000 Da molecular weight cut-off filters. Direct infusion using a standard ESI probe was carried out on a UPLC-ESI-MS Synapt G2-S (Waters) system with a UPLC flow-through needle. No column was used, and the system was configured to allow sample flow directly from the sample manager to the electrospray ionization probe. Positive ionization mode was used and the samples were sprayed using a flow of 30 µL/min and an injection volume of 20 µL. The capillary voltage was 1.2 kV, and the source temperature was 85˚C, while the desolvation temperature was 80˚C. A Genedata workflow was used to quantify the ratio bound using the intensity (TIC) of the bound glucose complex versus the unbound form, i.e. M + 1 glucose/(M + (M + 1 glucose)). Ratio bound (%) versus the glucose concentration (mM) was plotted using GraphPad Prism. The apparent glucose equilibrium dissociation constant K_d was determined from the binding curves by fitting the data with a One-site Total binding model.

3D modelling studies

The structural models of NNC2215 were built employing Maestro (Schrödinger release 2021‑2). The macrocycle model was adapted from the models shown by Tromans et al.¹⁶ and connected to insulin at B29²⁸ with the corresponding linker. The glucoside was built modifying B1. For the open configuration, the modifications were applied to the receptor‑bound insulin in PDB entry 6PXV (chain E²⁰), and for the closed conformation insulin from the PDB entry 2VJZ²¹ was modified. Both models underwent structural preparation and energy minimisation using the Maestro suite. In order to relax the system and test the stability of the closed conformation, a molecular dynamics (MD) simulation in explicit solvent for the latter model was run, consisting of 200 ps of equilibration and then 100 ns of production run. The final coordinates of the simulation were used for the corresponding model. Both open and closed models were superimposed on the bound insulin in 6PXV (chain E) in order to show how the closed conformation would hinder the interaction with the insulin receptor. Fig. 2 has been obtained using ChimeraX.

In vitro biology

Insulin receptor affinity measurements

The following materials were used: SPA PVT Antibody-Binding Beads, Anti-Mouse Reagent (GE Healthcare, RPNQ0017), HSA (Sigma-Aldrich A1887 lot# SLCBR2530) and antibody hIR 83.7 (produced at Novo Nordisk A/S, licensed from Prof. K. Siddle, University of Cambridge, UK²⁶).

BHK cells over-expressing human insulin receptor A (hIR-A) were lysed in 50 mM Hepes pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA and 10% glycerol. The cleared cell lysate was batch absorbed with wheat germ agglutinin (WGA)-agarose (Lectin from Triticum vulgaris-Agarose, L1394, Sigma-Aldrich, Steinheim, Germany) for 90 min. The receptors were washed with 20 volumes 50 mM Hepes pH 8.0, 150 mM NaCl and 0.1% Triton X-100, where after the receptors were eluted with 50 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5 M n-Acetyl Glucosamine and 10% glycerol²⁷. All buffers contained Complete protease inhibitor mixture (Roche Diagnostic GmbH, Mannheim, Germany).

Binding studies were performed with dilution series of ligands in 100 mM Hepes, 100 mM NaCl, 10 mM MgSO₄ and 0.025% (v/v) Tween 20, pH 7.4, 1.5% HSA with 0, 3, 5, 10 or 20 mM D-glucose in triplicates for each dilution in 96-well Isoplates (PerkinElmer, 6005049), 5000 CPM Tyr A14-125-I-insulin, 25 µl SPA beads, 25 ng IR 83-7 antibody, 0.006 µl hIR-A per well. After 22 h incubation at 22^oC the bound radioactivity was quantified by counting in a Microbeta2 2450 Microplate counter (Perkin Elmer), essentially as described previously²⁷.

The IC₅₀ and relative affinities to human insulin were calculated. Each point in the competition curve was a measure of triplicates with the mean and SD. The IC₅₀ of the one site binding model was fitted with non-linear regression algorithm using GraphPad Prism 8.0.2 (GraphPad Software Inc., San Diego, CA). The top, bottom and slope were set to be equal for all compounds in each experiment. Since the logarithmic cannot be calculated for zero, the concentration without unlabelled ligand was set to 1x10^-14 M in the calculations. On each plate human insulin was included for calculation of the relative affinities of NNC2215 and insulin degludec for each set of plates. The average and SD for three independent experiments were calculated using Excel. The fold changes in relative affinity to hIR-A were calculated from 0 or 3 mM glucose to 20 mM glucose in each experiment and the average and SD calculated.

hIR phosphorylation and AKT and ERK assays

CHO-hIR cells²⁹ were stimulated with increasing concentrations of human insulin and NNC2215 (0-1 µM) for 10 min. After stimulation, cells were homogenised in lysis buffer. Insulin receptor phosphorylation (pIR) was measured with the InsR(pY1158) ThermoFisher ELISA kit according manufacturer’s instructions. Phosphorylation of AKT (pAKT) and ERK (pERK) were measured using AlphaScreen, SureFire AKT1/2/3 (p-Ser473) and SureFire ERK1/2 p-T202/Y204 Assay Kits from PerkinElmer according to manufacturer’s instructions and Hansen et al.²⁹

Lipogenesis in primary mouse adipocytes

In primary rat mouse adipocytes isolated from epididymal fat pads, the effect of NNC2215 and insulin degludec on lipogenesis was determined by measuring the incorporation of [³H]‑labelled glucose into fat as described previously³⁰ with a slight modification: L-Glucose was added to a final concentration of 3 mM or 20 mM.

In vivo studies

L-glucose rat model

Non-fasted male Sprague-Dawley rats were dosed intravenously with NNC2215 (4.5 nmol/kg) at time point zero. Thirty minutes later, the rats received an additional intravenous dosing of either vehicle or 0.5, 1 or 2 g/kg L-glucose (n=7 per group). Blood was drawn from the tongue vein and plasma collected at timepoints 0, 25, 35, 45, 60, 90 and 120 min for quantification of D-glucose and NNC2215 concentrations. The rats had no access to food during the experiment.

Hypoglycaemia study in pigs

Female LYD pigs were used. Prior to the experiments, all animals were instrumented with two venous catheters, one for infusion and one for sampling. Animals were subjected to constant intravenous infusions of somatostatin (1 µg/kg/min) – to suppress endogenous insulin and glucagon secretion – and glucagon (0.45 pmol/kg/min) to replace the suppressed glucagon secretion. Suppression of insulin secretion was verified by measuring C-peptide levels, which documented that endogenous insulin secretion was below basal level throughout the experiment in spite of plasma glucose being elevated (data not shown). Primed constant infusions of either NNC2215 or insulin degludec and infusion of glucose were given as indicated below. Suitable priming doses of each insulin analogue were estimated based on their pharmacokinetics following intravenous administration.

The duration of the experiment was 540 min. At time 0, infusions of hormones and glucose were started. Glucose was infused at 6 mg/kg/min from 0 to 360 min after which it was turned off for 90 min and restarted at 6 mg/kg/min for the last 90 min of the experiment. To investigate a range of plasma glucose concentrations prior to turning off the glucose infusion, different insulin infusion rates were used (NNC2215: 1.30, 1.44, 1.58, 1.72, 1.86, 2.00 and 2.14 pmol/kg/min; insulin degludec: 0.7, 0.9 and 1.1 pmol/kg/min).

Pharmacokinetic/pharmacodynamic modelling

The pharmacokinetic parameters for the 2-compartment model of NNC2215 in LYD pigs from the hypoglycaemia study are shown in Extended Data Table 1.

Glucose challenge study in diabetic rats

Healthy Sprague Dawley male rats (350-400 g) were surgically instrumented with permanent arterial (blood sampling) and venous (insulin and glucose infusions) catheters under isoflurane anaesthesia and recovered from surgery with analgesia (carprofen, 5 mg/rat/day, subcutaneous) for 7-8 days before made acutely diabetic with streptozotocin (65 mg/kg, subcutaneous). Three to four days after streptozotocin treatment, rats were fasted overnight and subjected to one of three different constant intravenous infusion rates of either NNC2215 (42, 84, 100 pmol/kg/min; primed, see below) ) or human insulin (20, 25, 30 pmol/kg/min) to lower plasma glucose from ≥15 mM to a pre-defined target of 5.7 mM. Appropriate priming doses of NNC2215 to obtain steady state in plasma exposure within the duration of the study was calculated using its pharmacokinetics after intravenous administration in rats. When the glucose target was reached, it was clamped by adjusting the intravenous glucose infusion rate (GIR). When GIR had been at steady state for at least 30 min, a GTT was employed by infusing an additional 25 mg/kg/min of glucose for 60 min on top of the individual steady‑state GIR without clamping the plasma glucose. Plasma glucose concentrations were measured every 10 min throughout by the glucose oxidase method (YSI 2900). To mimic the effect of a glucose sensitive insulin and to quantify its extra effect during the GTT, another group of streptozotocin diabetic rats was given 50% additional human insulin on top of the constant human insulin infusion (20 + 10 pmol/kg/min; Human insulin +50%) during the GTT. The maximal plasma glucose concentrations during the GTT were compared by one‑way ANOVA and pairwise comparisons by Tukey’s post hoc test.

Pharmacokinetic and pharmacodynamic studies in LYD pig and rat

NNC2215 or insulin degludec were dosed intravenously to LYD pigs, and NNC2215 or human insulin were dosed intravenously to rats. Blood was sampled at selected time points and plasma was prepared and analysed for insulin analogue concentration (see below) and glucose concentration (using the glucose oxidase method).

Quantification of insulin concentrations in plasma samples

The plasma samples were analysed for levels of NNC2215, insulin degludec and human insulin using luminescence oxygen channeling immunoassay (LOCI)/Alpha-LISA, which is a homogeneous immunoassay method without washing steps. In the LOCI/Alpha-LISA, streptavidin coated donor beads are utilised (Alpha-LISA donor beads, Perkin Elmer, Waltham, Massachusetts, USA) in addition to acceptor beads (Alpha-LISA acceptor beads, Perkin Elmer, Waltham, Massachusetts, USA), which were conjugated with a monoclonal antibody (mAb) specific for the insulin analogues of interest (NNC2215, insulin degludec or human insulin). A second mAb recognising another part of the insulin analogues of interest was biotinylated and utilised in the LOCI/Alpha-LISA assay. The antibody pairs used for determination of the three analytes were as follows: NNC2215 (mAb HUI-018 conjugated to acceptor beads and biotinylated pAb 4080E); insulin degludec (mAb NN454-1F31 conjugated to acceptor beads and biotinylated mAb S1); human insulin (mAb HUI-018 conjugated to acceptor beads and biotinylated mAb OXI-005). All the indicated antibodies are Novo Nordisk in-house generated antibodies. A dilution row of either NNC2215, insulin degludec or human insulin were prepared as calibrators in species-specific plasma pools in appropriate concentration ranges. The calibrator curves for each of the insulin analogues are used for the quantification of the specific analytes in unknown samples. Plasma samples containing the analytes of interest were incubated with antibody coated acceptor beads in addition to the described biotinylated mAb (Bio-mAb) in 384-well plates. After 1 hour (insulin degludec and human insulin) or 24 hours (NNC2215) of incubation, the streptavidin coated donor beads were added to the wells. The acceptor beads, the analyte of interest, the Bio-mAb and the donor beads all form a complex within the solution. Illumination of the complex releases singlet oxygen atoms from the donor beads. These are channelled into the acceptor beads and trigger a chemiluminescene response, which is measured in an Envision plate reader (Perkin Elmer, Waltham, Massachusetts, USA). The amount of light is proportional to the concentration of the analyte. The lower limit of quantification was determined to be 42 pM in LYD pig and 27 pM in rat for NNC2215, 15 pM in LYD pig for insulin degludec and 2.6 pM in rat for human insulin.

Methods references

34. Hoeg-Jensen, T. et al. Glucose sensitive insulins and uses thereof. Patent application WO2020058322, 26 March 2020.

Table 1: Glucose-sensitivity of NNC2215 activity with respect to hIR-A binding and downstream metabolic response.

	hIR-A affinity relative to human insulin^a
	D-glucose concentration (mM)
	0	3		5	10		20
NNC2215	0.75±0.1	2.9±0.4		4.3±0.4	6.5±1.0		9.2±0.9
Insulin degludec	0.36±0.01	0.35±0.03		0.36±0.03	0.37±0.03		0.36±0.04
	hIR-A affinity relative to human insulin					Lipogenesis
	Fold change 3 to 20 mM D-glucose		Fold change 0 to 20 mM D-glucose			Fold change 3 to 20 mM L-glucose
NNC2215	3.2±0.3		12.5±1.7			2.2±0.5
Insulin degludec	1.1±0.2		1.0±0.1			1.1±0.2

Data are mean±SD of three independent measurements.

^a Determined from the IC₅₀ in the absence or presence of 0, 3, 5, 10 and 20 mM D-glucose.

Yes there is potential Competing Interest. The Novo Nordisk employees are shareholders of Novo Nordisk A/S. However, the manuscipt does not cover commercial products, but research work.

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Glucose-sensitive insulin with attenuation of hypoglycaemia

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