Development of glucose-responsive 'smart' insulin systems

Abstract

Purpose of review: The complexity of modern insulin-based therapy for type I and type II diabetes mellitus and the risks associated with excursions in blood-glucose concentration (hyperglycemia and hypoglycemia) have motivated the development of 'smart insulin' technologies (glucose-responsive insulin, GRI). Such analogs or delivery systems are entities that provide insulin activity proportional to the glycemic state of the patient without external monitoring by the patient or healthcare provider. The present review describes the relevant historical background to modern GRI technologies and highlights three distinct approaches: coupling of continuous glucose monitoring (CGM) to deliver devices (algorithm-based 'closed-loop' systems), glucose-responsive polymer encapsulation of insulin, and molecular modification of insulin itself.

Recent findings: Recent advances in GRI research utilizing each of the three approaches are illustrated; these include newly developed algorithms for CGM-based insulin delivery systems, glucose-sensitive modifications of existing clinical analogs, newly developed hypoxia-sensitive polymer matrices, and polymer-encapsulated, stem-cell-derived pancreatic β cells.

Summary: Although GRI technologies have yet to be perfected, the recent advances across several scientific disciplines that are described in this review have provided a path towards their clinical implementation.

Figure 1. Cartoon of structure of ultra-stable insulin analogs

(A) Single chain insulin (SCI) contains the native A-domain (dark gray) and B-domain (light gray) and the three native disulfide linkages (yellow) of insulin. In addition, SCI analogs contain a foreshortened “C-domain” (6-8 amino acids in length) (orange) that connects the C-terminal strand of the B-domain (green) to the N-terminus of the A-domain. This domain dampens the conformational fluctuations of the molecule thus increasing its thermodynamic stability and rendering it resistant to fibrillation (54). (PDB ID: 2LWZ) (B) 4SS-insulin is a two-chain insulin analog (A-chain, dark gray, B-chain, light gray) that contains an engineered fourth disulfide linkage (red asterisk) between the A- and B-chains in addition to its three native disulfide linkages (yellow). The analog was reported to be resistant to fibrillation and have increased temperature stability (55). (PDB ID: 4EFX)

Figure 2. Schematic of phenylboronic acid-monosaccharide complexation

Phenylboronic acid (PBA) comprises an aryl ring containing a boronic acid substituent group. When the boronic acid moiety is ionized to phenylboronate, the group may form reversible ester bonds with cis-diols, including those found on monosaccharides (59). The pK_a of unmodified PBA is 8.0 and it has an affinity for glucose of approximately 10 mM. Both of these properties may be modulated by introducing electron-withdrawing substituents to the aryl ring (red circle marked with “X”) (60). A variety of groups, including nitro-, fluoro-, carboxyl, and sulfone groups have been shown to lower pK_a of PBA to below physiological pH (7.4), affinities of these derivatives for glucose range from 8-14 mM (17).

Figure 3. Cartoon representation of polymer-based GRIs

(A) Insulin is encapsulated within a polymeric matrix in large implants, transdermal patches, or micro- or nanoparticles. The native or derivatized insulin analog is typically sequestered within a water-containing cavity within the hydrophobic, water impermeable polymer matrix. Such matrices are compact during hypo- or euglycemia, but swell during hypoglycemia to release sequestered insulin (B). Several different methods have been used to create glucose-responsive polymers (C). (Top panels, C) PBA (green hexagon) and glucose have been incorporated into polymeric scaffolds to maintain the compactness of the matrix. This interaction is competitively disfavored as ambient glucose concentrations rise, causing the polymer to swell (top panels, C). (Middle panels, C) Immobilized or co-encapsulated GBPs (red shapes) have been used as agents that stabilize the compactness of polymeric matrices in a glucose-dependent fashion. (Bottom panels, C) Matrices that are sensitive to the byproducts of co-encapsulated or immobilized GoD (red polygon), gluconic acid and H₂O₂, along with the resulting decrease in pH and local hypoxia, have been developed. Oftentimes, polymeric matrices will undergo conformational changes resulting from changes in the protonation state of components (blue circles, red lines) of the scaffold or from reactions that are catalyzed by the byproducts of GoD.

Figure 4. Cartoon representation of derivatization of the insulin molecule in GRI technology

The insulin hormone comprises two polypeptide chains that are designated “A” and “B” and are connected by two disulfide linkages spanning residues A7 and B7 and A20 and B19. Brownlee, et al created a molecular GRI by coupling mono- and disaccharides to the N-termini of one or both polypeptide chains of insulin (red). These analogs were bound to ConA (red star) before administration. ConA was expected to sequester the insulin in the SQ space during euglycemic conditions (left, A) and allow it to liberated by competitive binding of ambient glucose (blue hexagons) during hyperglycemia (right, A) (11) (66). (B) Kashyap and colleagues created a GRI system using the clinical analog insulin glargine. Glargine contains an addition of two arginine residues at the C-terminus of its B-chain and a substitution of Asn^A21 for glycine (77). These modifications shift the isoelectric point of the protein to near physiological conditions causing it to form precipitates after SQ injection. In this GRI system glargine was co-injected with GoD (red polygon), which was expected to lower the local pH by oxidizing glucose to gluconic acid at a rate proportional to the glycemic state of the patient increasing the solubility and, hence, the bioavailability of glargine. (C-E) A number of groups have modified the ɛ-amino group of Lys^B29 with PBA derivatives (green hexagons) to create GRI systems. (C) Hoeg-Jensen and colleagues directly coupled Lys^B29 with PBA derivatives and demonstrated the ability of the analog to couple to diol-containing polymer carriers (orange rectangles with red circles) in a glucose-dependent fashion (17). (D) The same group also derivatized residue B29 with a molecule containing a PBA and a polyol group (black lines with red circles) and demonstrated the ability of the analog to form multi-hexameric complexes in vitro that could dissociate in a glucose-dependent fashion (90). (C) Most recently, Chou and colleagues created a GRI that contained a PBA derivative coupled via a fatty acyl linker (black, jagged line with green hexagon) to Lys^B29, it was hypothesized that this analog would bind to albumin (blue oval) under normoglycemic conditions and become liberated during hyperglycemia as free glucose complexed with the PBA molecule and decreased the affinity of the analog for albumin (86). Although this analog did show glucose-responsiveness in a mouse model, glucose-dependent association or dissociation from albumin was not confirmed.

Figure 5. Structural representation of insulin IR binding

(A) The ectodomain of insulin receptor (IR) (anterior monomer of homodimer shown in color), the molecular target of insulin. The L1 and αCT (purple) domains of IR comprise the insulin binding site. (B) In its “closed” or storage conformation, insulin cannot engage its binding site due to a steric clash between αCT (purple) and the C-terminal B-chain of insulin (brown). (C) To engage its primary binding site, insulin must undergo a conformational change in which the C-terminal B-chain detaches from the hydrophobic core of the globular protein via unfolding of a β-turn spanning residues B20-B23 (red box in B and C). (D) The detachment of the C-terminal B-chain from the hydrophobic core of insulin allows the molecule to intercalate between the L1 and αCT domains and occupy its binding site.