Use of a biomimetic hydrogel depot technology for sustained delivery of GLP-1 receptor agonists reduces burden of diabetes management.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone and neurotransmitter secreted from intestinal L cells in response to nutrients to stimulate insulin and block glucagon secretion in a glucose-dependent manner. Long-acting GLP-1 receptor agonists (GLP-1 RAs) have become central to treating type 2 diabetes (T2D); however, these therapies are burdensome, as they must be taken daily or weekly. Technological innovations that enable less frequent administrations would reduce patient burden and increase patient compliance. Herein, we leverage an injectable hydrogel depot technology to develop a GLP-1 RA drug product capable of months-long GLP-1 RA delivery. Using a rat model of T2D, we confirm that one injection of hydrogel-based therapy sustains exposure of GLP-1 RA over 42 days, corresponding to a once-every-4-months therapy in humans. Hydrogel therapy maintains management of blood glucose and weight comparable to daily injections of a leading GLP-1 RA drug. This long-acting GLP-1 RA treatment is a promising therapy for more effective T2D management.

Polyacrylamide-based hydrogel coatings improve biocompatibility of implanted pump devices.

The introduction of transcutaneous and subcutaneous implants and devices into the human body instigates fouling and foreign body responses (FBRs) that limit their functional lifetimes. Polymer coatings are a promising solution to improve the biocompatibility of such implants, with potential to enhance in vivo device performance and prolong device lifetime. Here we sought to develop novel materials for use as coatings on subcutaneously implanted devices to reduce the FBR and local tissue inflammation in comparison to gold standard materials such as poly(ethylene glycol) and polyzwitterions. We prepared a library of polyacrylamide-based copolymer hydrogels, which were selected from materials previously shown to exhibit remarkable antifouling properties with blood and plasma, and implanted them into the subcutaneous space of mice to evaluate their biocompatibility over the course of 1 month. The top performing polyacrylamide-based copolymer hydrogel material, comprising a 50:50 mixture of N-(2-hydroxyethyl)acrylamide (HEAm) and N-(3-methoxypropyl)acrylamide (MPAm), exhibited significantly better biocompatibility and lower tissue inflammation than gold standard materials. Moreover, when applied to polydimethylsiloxane disks or silicon catheters as a thin coating (45 ± 1 µm), this leading copolymer hydrogel coating significantly improved implant biocompatibility. Using a rat model of insulin-deficient diabetes, we showed that insulin pumps fitted with HEAm-co-MPAm hydrogel-coated insulin infusion catheters exhibited improved biocompatibility and extended functional lifetime over pumps fitted with industry standard catheters. These polyacrylamide-based copolymer hydrogel coatings have the potential to improve device function and lifetime, thereby reducing the burden of disease management for people regularly using implanted devices.

Sustained Delivery of GLP-1 Receptor Agonists from Injectable Biomimetic Hydrogels Improves Treatment of Diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone and neurotransmitter secreted from intestinal L-cells in response to nutrients to stimulate insulin and block glucagon secretion in a glucose-dependent manner. GLP-1 in itself is rapidly degraded, but long-acting GLP-1 receptor agonists (GLP-1 RAs) have become central in the treatment of T2D because of the beneficial effects extending also beyond glucose control. Currently, these therapeutics must be injected either daily or weekly or taken daily orally, leaving room for technological innovations that enable less frequent administrations, which will reduce patient burden and increase patient compliance. An ideal GLP-1 RA drug product would provide continuous therapy for upwards of four months from a single administration to match the cadence with which T2D patients typically visit their physician. In this work, we leveraged an injectable hydrogel depot technology to develop a long-acting GLP-1 RA drug product. By modulating the hydrogel properties to tune GLP-1 RA retention within the hydrogel depot, we engineered formulations capable of months-long GLP-1 RA delivery. Using a rat model of T2D, we confirmed that a single injection of hydrogel-based therapies exhibits sustained exposure of GLP-1 RA over 42 days, corresponding to a once-every four month therapy in humans. Moreover, these hydrogel therapies maintained optimal management of blood glucose and weight comparable to daily injections of a leading GLP-1 RA drug molecule. The pharmacokinetics and pharmacodynamics of these hydrogel-based long-acting GLP-1 RA treatments are promising for development of novel therapies reducing treatment burden for more effective management of T2D. Progress and Potential: While insufficient access to quality healthcare is problematic for consistent management of Type II diabetes (T2D), poor adherence to burdensome treatment regimens is one of the greatest challenges for disease management. Glucagon-like peptide 1 (GLP1) drugs have become central to the treatment of T2D due to their many beneficial effects beyond improving glucose control. Unfortunately, while optimization of GLP1 drugs has reduced treatment frequency from daily to weekly, significant patient burden still leads to poor patience compliance. In this work we developed an injectable hydrogel technology to enable GLP1 drugs only requiring administration once every four months. We showed in a rat model of T2D that one injection of a hydrogel-based therapy improves management of blood glucose and weight when compared with daily injections of the leading drug used clinically. These hydrogel-based GLP1 treatments are promising for reducing treatment burden and more effectively managing T2D. Future Impact: A GLP-1-based drug product providing four months of continuous therapy per administration could be transformational for the management of Type II diabetes (T2D). One of the most challenging aspects of diabetes management with GLP-1 mimics is maintenance of consistent levels of the drugs in the body, which is complicated by poor patient compliance on account of the high frequency of dosing required for current treatments. By leveraging a unique sustained release hydrogel depot technology we develop a months-long GLP-1 drug product candidate that has the potential to reduce patient burden and improving diabetes management. Overall, the hydrogel technology we describe here can dramatically reduce the frequency of therapeutic interventions, significantly increasing patient quality of life and reducing complications of diabetes management.Our next steps will focus on optimization of the drug formulations in a swine model of T2D, which is the most advanced and translationally-relevant animal model for these types of therapeutics. The long-term vision for this work is to translate lead candidate drug products towards clinical evaluation, which will also require comprehensive safety evaluation in multiple species and manufacturing our these materials according to Good Manufacturing Practices. The months-long-acting GLP-1 drug product that will come from this work has the potential to afford thus far unrealized therapeutic impact for the hundreds of millions of people with diabetes worldwide.

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient.

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors.

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Ultra-Fast Insulin-Pramlintide Co-Formulation for Improved Glucose Management in Diabetic Rats.

Dual-hormone replacement therapy with insulin and amylin in patients with type 1 diabetes has the potential to improve glucose management. Unfortunately, currently available formulations require burdensome separate injections at mealtimes and have disparate pharmacokinetics that do not mimic endogenous co-secretion. Here, amphiphilic acrylamide copolymers are used to create a stable co-formulation of monomeric insulin and amylin analogues (lispro and pramlintide) with synchronous pharmacokinetics and ultra-rapid action. The co-formulation is stable for over 16 h under stressed aging conditions, whereas commercial insulin lispro (Humalog) aggregates in 8 h. The faster pharmacokinetics of monomeric insulin in this co-formulation result in increased insulin-pramlintide overlap of 75 ± 6% compared to only 47 ± 7% for separate injections. The co-formulation results in similar delay in gastric emptying compared to pramlintide delivered separately. In a glucose challenge, in rats, the co-formulation reduces deviation from baseline glucose compared to insulin only, or separate insulin and pramlintide administrations. Further, comparison of interspecies pharmacokinetics of monomeric pramlintide suggests that pharmacokinetics observed for the co-formulation will be well preserved in future translation to humans. Together these results suggest that the co-formulation has the potential to improve mealtime glucose management and reduce patient burden in the treatment of diabetes.

Affinity-Directed Dynamics of Host-Guest Motifs for Pharmacokinetic Modulation via Supramolecular PEGylation.

Proteins are an impactful class of therapeutics but can exhibit suboptimal therapeutic performance, arising from poor control over the timescale of clearance. Covalent PEGylation is one established strategy to extend circulation time but often at the cost of reduced activity and increased immunogenicity. Supramolecular PEGylation may afford similar benefits without necessitating that the protein be permanently modified with a polymer. Here, we show that insulin pharmacokinetics can be modulated by tuning the affinity-directed dynamics of a host-guest motif used to non-covalently endow insulin with a poly(ethylene glycol) (PEG) chain. When administered subcutaneously, supramolecular PEGylation with higher binding affinities extends the time of total insulin exposure systemically. Pharmacokinetic modeling reveals that the extension in the duration of exposure arises specifically from decreased absorption from the subcutaneous depot governed directly by the affinity and dynamics of host-guest exchange. The lifetime of the supramolecular interaction thus dictates the rate of absorption, with negligible impact attributed to association of the PEG upon rapid dilution of the supramolecular complex in circulation. This modular approach to supramolecular PEGylation offers a powerful tool to tune protein pharmacokinetics in response to the needs of different disease applications.

Multi-State Dynamic Coordination Complexes Interconverted through Counterion-Controlled Phase Transfer.

We studied a series of dynamic weak-link approach (WLA) complexes that can be shuttled between two immiscible solvents and switched between two structural states via ion exchange. Here, we established that hydrophobic anions transfer cationic, amphiphilic complexes from the aqueous phase to the organic phase, while a chloride source reverses the process. As a result of the dynamic metal coordination properties of WLA complexes, the denticity of these complexes (mono- to bi-) can be modulated as they partition into different phases. In addition, we discovered that heteroligated complexes bearing ligands of different donor strengths preferentially rearrange into two homoligated complexes that are phase-partitioned to maximize the number of stronger coordination bonds. This behavior is not observed in systems with one solvent, highlighting the dynamic and stimuli-responsive nature of hemilabile ligands in a multiphasic solvent environment. Taken together, this work shows that the highly reconfigurable WLA modality can enable the design of biphasic reaction networks or chemical separations driven by straightforward salt metathesis reactions.

Engineering biopharmaceutical formulations to improve diabetes management.

Insulin was first isolated almost a century ago, yet commercial formulations of insulin and its analogs for hormone replacement therapy still fall short of appropriately mimicking endogenous glycemic control. Moreover, the controlled delivery of complementary hormones (such as amylin or glucagon) is complicated by instability of the pharmacologic agents and complexity of maintaining multiple infusions. In this review, we highlight the advantages and limitations of recent advances in drug formulation that improve protein stability and pharmacokinetics, prolong drug delivery, or enable alternative dosage forms for the management of diabetes. With controlled delivery, these formulations could improve closed-loop glycemic control.

A Redox-Switchable, Allosteric Coordination Complex.

A redox-regulated molecular tweezer complex was synthesized via the weak-link approach. The PtII complex features a redox-switchable hemilabile ligand (RHL) functionalized with a ferrocenyl moiety, whose oxidation state modulates the opening of a specific coordination site. Allosteric regulation by redox agents gives reversible access to two distinct structural states-a fully closed state and a semi-open state-whose interconversion was studied via multinuclear NMR spectroscopy, cyclic voltammetry, and UV-vis-NIR spectroscopy. Two structures in this four-state system were further characterized via SCXRD, while the others were modeled through DFT calculations. This fully reversible, RHL-based system defines an unusual level of electrochemical control over the occupancy of a specific coordination site, thereby providing access to four distinct coordination states within a single system, each defined and differentiated by structure and oxidation state.

An Allosterically Regulated, Four-State Macrocycle.

Macrocycles capable of host-guest chemistry are an important class of structures that have attracted considerable attention because of their utility in chemical separations, analyte sensing, signal amplification, and drug delivery. The deliberate design and synthesis of such structures are rate-limiting steps in utilizing them for such applications, and coordination-driven supramolecular chemistry has emerged as a promising tool for rapidly making large classes of such systems with attractive molecular recognition capabilities and, in certain cases, catalytic properties. A particularly promising subset of such systems are stimuli-responsive constructs made from hemilabile ligands via the weak-link approach (WLA) to supramolecular coordination chemistry. Such structures can be reversibly toggled between different shapes, sizes, and charges based upon small-molecule and elemental-anion chemical effectors. In doing so, one can deliberately change their recognition properties and both stoichiometric and catalytic chemistries, thereby providing mimics of allosteric enzymes. The vast majority of structures made to date involve two-state systems, with a select few being able to access three different states. Herein, we describe the synthesis of a new allosterically regulated four-state macrocycle assembled via the WLA. The target structure was made via the stepwise assembly of ditopic bidentate hemilabile N-heterocyclic carbene thioether (NHC,S) and phosphino thioether (P,S) ligands at PtII metal nodes. The relatively simple macrocycle displays complex dynamic behavior when addressed with small-molecule effectors, and structural switching can be achieved with several distinct molecular cues. Importantly, each state was fully characterized by multinuclear NMR spectroscopy and, in some cases, single-crystal X-ray diffraction studies and density functional theory computational models. This new structure opens the door to complex multicue switching reminiscent of multistate chemoswitches that could be important in controlling stoichiometric and catalytic transformations as well as generating molecular logic systems.

Palladium(II) Weak-Link Approach Complexes Bearing Hemilabile N-Heterocyclic Carbene-Thioether Ligands.

A new class of homoligated palladium(II) weak-link approach (WLA) complexes bearing hemilabile N-heterocyclic carbene (NHC)-thioether ligands is reported that, unlike previous tweezer-like WLA complexes, expand and contract in a linear fashion when switching between configurational states. These complexes can be chemically switched between a trans open state and a trans closed state via the addition or subsequent extraction of Cl-. These bis(NHC) complexes also display unusual isomerization behavior. For example, an NMR spectroscopic investigation into the solution-state configuration of the open complex reveals the presence of interconverting syn,trans and anti,trans isomers, and a kinetic study shows that the barrier is large enough to isolate, store, and study the anti,trans isomer at room temperature. Notably, the linker length between the NHC and thioether moieties can be tailored with additional -CH2- groups, which affords considerable control over the geometric changes imposed by switching. Therefore, this class of complexes may be useful in the construction of allosterically regulated supramolecular assemblies and materials.

Reversible and Selective Encapsulation of Dextromethorphan and ß-Estradiol Using an Asymmetric Molecular Capsule Assembled via the Weak-Link Approach.

An allosterically regulated, asymmetric receptor featuring a binding cavity large enough to accommodate three-dimensional pharmaceutical guest molecules as opposed to planar, rigid aromatics, was synthesized via the Weak-Link Approach. This architecture is capable of switching between an expanded, flexible "open" configuration and a collapsed, rigid "closed" one. The structure of the molecular receptor can be completely modulated in situ through the use of simple ionic effectors, which reversibly control the coordination state of the Pt(II) metal hinges to open and close the molecular receptor. The substantial change in binding cavity size and electrostatic charge between the two configurations is used to explore the capture and release of two guest molecules, dextromethorphan and ß-estradiol, which are widely found as pollutants in groundwater.

A concerted two-prong approach to the in situ allosteric regulation of bifunctional catalysis.

Herein, we report the reversible in situ "on-off" allosteric regulation of hydrogen-bond-donating (HBD)-Lewis base co-catalytic activity via a concerted two-prong methodology entailing cooperative acid-base chemistry and a structurally addressable coordination complex. Specifically, a heteroligated Pt(ii) weak-link approach (WLA) tweezer complex containing both a hemilabile squaramide-piperidine-based catalytic ligand and a sodium sulfonate hydrogen-bond-accepting (HBA) ligand was synthesized. Due to the hemilabile nature of the catalyst-containing ligand, the heteroligated complex can be reversibly toggled in situ between a flexible, semi-open state and a rigid, fully closed state upon the addition of elemental ion cues. 1H NMR spectroscopy titration studies show that in the semi-open state interligand hydrogen-bonding prevents substrate recognition by the squaramide unit, while in the fully closed state ligand-ligand interactions are prevented. This results in a catalytically active closed state, whereas in the semi-open state, when the piperidine tertiary amine is deliberately protonated, no catalytic activity is observed. Reversible interconversion between the active fully closed state and the dormant protonated semi-open state is achieved in the presence of substrate upon the concerted addition and abstraction of both a proton and a coordinating elemental anion. In this work, allosteric regulation of catalytic activity is demonstrated for both the Michael addition of nitroethane to ß-nitrostyrene and the ring-opening of l-(-)-lactide. Taken together, this work details a potentially generalizable platform for the "on-off" allosteric regulation of a family of HBD-Lewis base co-catalysts capable of catalyzing a broad scope of reactions, including the living ring-opening polymerization of cyclic esters.