Glucose - Responsive Smart Insulin

Glucose-binding proteins

Concanavalin A (Con A), a lectin was used by Brownlee and Cerami (25) in their initial model of glucose-responsive insulin. Glucose responsiveness in this system can be achieved through two mechanisms. First is through immobilization of a sugar-modified insulin on Con A, which is released from the injected subcutaneous deposit upon displacement by ambient glucose. The second is by incorporating Con A lectin with its tetrameric structure and four sugar-binding sites as a cross-linker in a polymer backbone. Competitive binding of glucose with Con A will disrupt the structural integrity of the polymer and release the insulin contained within it (Fig. 2). The clinical utility of Con A is limited by its non-physiological glucose affinity (competition for glucose binding at ambient glucose levels that are higher than typical diabetic range), immunogenicity and mitogenic potential (26-28).

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Fig. 2:

(A&B) Concanavalin A forms an important structural component of the matrix that entraps insulin molecules. When it interacts with the ambient glucose molecules the interaction is interrupted leading to disintegration of the matrix and release of insulin from the polymer matrix.

Fig. 2 (C&D): Concanavalin A forms an integral part of the polymer matrix along with glucose conjugated insulin molecule. Ambient glucose displaces the insulin glucose conjugate by competitive binding to concanavalin A, thereby effecting the release of insulin from the polymer.

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Glucose oxidase

This enzyme catalyses glucose oxidation. This reaction results in the formation of gluconic acid, H₂O₂ and drop in pH. The resulting change in pH leads to structural changes in polymers that are built to respond to pH by virtue of their acidic/basic functional groups. A polymer with predominantly acidic group would shrink and one with basic groups would swell. Some polymers are built to degrade in an acidic environment thus releasing the contained insulin. (Fig. 1, 3). Glucose oxidase (GoD) based polymersomes incorporated on cross-linked hyaluronic acid microneedle arrays are being developed for painless transcutaneous delivery (29). Experimental data suggests these glucose sensing system can lower blood glucose within one hour followed by maintenance up to five hours without any hypoglycemia in mice pre- treated with insulin.

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Fig. 3:

(A) shows a vesicle (polymer matrix) with the encapsulated insulin.

Fig. 3 (B): shows production of gluconic acid by interaction of glucose oxidase enzyme contained within the vesicle with the ambient glucose molecule (resulting in an acidic environment). The vesicle being acidic shrinks in an acidic environment and the pores increase in size resulting in escape of insulin.

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Fig. 3:

(C) shows the same reaction resulting in production of gluconic acid from glucose oxidase contained within the vesicle and ambient glucose (pink) and the disintegration of the vesicle matrix with release of insulin in an acidic environment.

Fig. 3 (D): shows the same reaction with a basic vesicle matrix demonstrating swelling and release of contained insulin in response to an acidic environment due to production of gluconic acid by glucose oxidase enzyme acting on ambient glucose.

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Phenylboronic acid

Glucose responsiveness property of Phenylboronic acid (PBA) is achieved by its formation of reversible esters with cis- diol molecules. The ambient glucose acts as a competitive inhibitor of diol molecules bound to PBA. In PBA based system, diols are integrated with insulin and immobilized on a PBA based polymer scaffold. With hyperglycemia, diolinsulin ester bond with PBA is dissociated and insulin is released in proportion to glucose levels. PBA has also been used, like Con A lectin as a component maintaining the structural integrity of the polymer backbone, competitive binding to glucose would cause degradation of a polymer leading to insulin secretion (Fig. 4). The advantage of PBA over Con A is due to its affinity for glucose which is in the physiological range (11). However, it has a tendency for spontaneous degradation (11) and the interaction, which is mediated by the diol group, is not specific to glucose, thereby limiting its clinical translational value.

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Fig. 4:

(A&B) Phenylboronic acid (PBA) is an integral part of the polymer structure that houses insulin. The interaction in the polymer is mediated through another monosaccharide unit which gets competitively displaced by ambient glucose causing disruption of the structural attachments of PBA and disintegration of the matrix with release of insulin.

Fig. 4 (C&D): PBA and glucose conjugated insulin form an important component of the polymer structure. The ambient glucose displaces the insulin glucose conjugate from the PBA molecule by competitive inhibition thereby freeing the glucose conjugated insulin from the matrix.

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Currently, polymer based GRI systems have several limitations. These challenges include limited particle stability, non-physiological range of action and too slow or too rapid response. Strategies being considered to overcome these hurdles include: use of multiple glucose sensing mechanisms like GoD and PBA in polymer matrix, variations in particle size, permeability of surface area and use of multilayered polymer matrices. Another interesting innovation is the integration of glucose-responsive system with externally positioned β-cell capsules. Ye et al (30) fashioned beta cells embedded in an alginate based microgel capsule, fitted with a microneedle array made of hyaluronic acid. The principle is that the interstitial glucose permeates through the microneedle patch and reaches the β-cells stimulating insulin release. However, it was found that the diffusion of glucose through the microneedle patch is inadequate, resulting in an insignificant insulin release. To overcome this, the microneedles, are embedded with α-amylose as well as self-assembling polymers containing GoD, α-amylase, and glucoamylase enzymes. The GoD in these microneedles converts interstitial glucose into gluconic acid leading to pH mediated disintegration of the polymers on the microneedle arrays. Release of α-amylase from within the polymers hydrolyses α-amylose embedded on the microneedle into disaccharides and tri-saccharides. Glucoamylase converts these saccharides to glucose. Thus effectively the glucose concentration in the vicinity of the microneedles/beta cell capsule structure increases, providing stimulus to beta cells for insulin secretion. This strategy of amplifying glucose signal overcomes the disadvantage of beta cell capsules, i.e. poor response at physiological glucose levels (Fig. 5). A single patch of this type of GRS was effective in Type 1 diabetic mice for up to 10 hours (30).

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Fig. 5:

β cell capsule with an α-amylase and polymers (encapsulating glucose oxidase, α-amylase, glucoamylase enzyme) embedded micro needle patch. This system helps to amplify the glucose concentration in the vicinity of the β cells so as to elicit a more robust insulin secretory response.

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In brief, all the three technologies discussed above were based on sequestering insulin in a glucose-responsive matrix followed by appropriate release of insulin as per the ambient glucose.

Molecular GRI Analog Systems

Molecular GRI are different class of system wherein the glucose responsiveness is inbuilt in the insulin molecule itself. The strategy involves altering the structure of insulin to modulate its pharmacokinetics.

GoD based approach uses GoD-insulin compound wherein a cysteine based linkage is disrupted by the enzymatic glucose oxidation to affect insulin release. GoD-based acidification at local injection site can also increase the bioavailability of glargine due to its enhanced solubility at acidic pH (31). Both the approaches have not resulted in any clinically significant effects due to adverse effects. The important drawback is the low Km of GoD for glucose leads to excessive insulin release at even low ambient glucose levels and results in hypoglycemia. Besides, tissue damage can occur at the local injection sites due to liberation of H₂O₂ (32). The in-vitro efficacy of GoD-based glargine, in reality, did not translate to improved glycemic control in pilot animal studies (33).

PBA based approaches to generate molecular GRI analog have been attempted. Insulin conjugated to a diol or sugar molecule is anchored to PBA molecule through a reversible ester bond. The interstitial glucose, competitively binds to PBA molecule thus freeing the diol labelled insulin for action. A second approach is through addition of a PBA tag to the insulin analogue detemir. Detemir has long half-life consequent to its myristic acid chain mediated binding to albumin and slow release from albumin. This release remains unrelated to the glucose concentration in blood. Addition of a PBA molecule to detemir in such a way that the interaction of detemir with albumin becomes glucose-responsive has been attempted. However, both the above approaches were unable to achieve the desired result in-vitro (34).

Zion and Lancaster proposed another novel alternative strategy for a GRI, based on endogenous lectin–based clearance (35). In this approach, addition of saccharides to the native insulin molecule results in an analog that can bind to insulin receptor as well as the mannose binding receptor. The mannose receptor (MR) normally binds and transports proteins and pathogens tagged for intracellular destruction and degradation through lysosomes without eliciting any immune response. Glucose is a competitive inhibitor of MR binding. At high ambient glucose level, insulin binding to MR and destruction is decreased. As a result more insulin is available for normal action through insulin receptor. In hypoglycemia there is a reverse sequence of events, i.e. higher fraction of circulating modified insulin is destroyed via uninhibited binding to MR (Fig. 6). Kaarsholm et al (36) were able to demonstrate in vitro glucose responsiveness of this approach, with the molecule MK 2640. However, the insulin analogue though safe and well tolerated, had poor in vitro potency for clinical use. Further modifications in this approach are underway, to address the potency of these formulations.

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Fig. 6:

Insulin carbohydrate conjugates (ICC) compete with ambient glucose for binding to the mannose receptors (MR) on hepatocyte cell surface. At low ambient glucose levels (Fig. 6B), more molecules of ICC bind to the MR and are subjected to lysosomal degradation, thereby clearing excess insulin and preventing hypoglycemia. At high ambient glucose levels (Fig. 6A) there is little attachment to the MR and therefore minimal degradation.

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