Polymeric excipients for biopharmaceutical formulations

Abstract

A polyacrylamide-based copolymer reduces or prevents aggregation of biomolecules including proteins, peptides and nucleic acids in aqueous formulations and lipid-based vehicles such as liposomes, lipid nanoparticles, polymerosomes and micelles at hydrophobic interfaces, thereby increasing the thermal stability of the molecules in the formulations. Methods and compositions can be used to treat conditions, including diabetes, the compositions comprising the copolymer and a protein or the copolymer and insulin.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application Serial No. 63 / 011,928 filed on April 17, 2020 and U.S. Provisional Application Serial No. 63 / 159,306 filed on March 10, 2021, the disclosures of which are incorporated herein by reference in their entirety.

[0003] Government Rights

[0004] This invention was made with government support under Grant DK119254 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. Technical Field

[0005] The present disclosure relates to amphiphilic polyacrylamide-based copolymer excipients that can be used to reduce or prevent biomolecules such as proteins and peptides in aqueous formulations and lipid-based mediators from aggregating at hydrophobic interfaces, thereby increasing the thermal stability of the molecules in the formulations. The present disclosure also relates to formulations of monomeric insulin, and co-formulations of insulin and other proteins. Compared to current fast-acting mealtime insulins, formulations containing copolymer excipients exhibit increased stability. Background Art

[0006] There are more than 40 million diabetics in the world, and they rely on insulin replacement therapy through daily subcutaneous insulin injection or insulin infusion pump. These patients cannot produce enough insulin required for promoting cellular glucose uptake for basic metabolic functions, and therefore must deliver calculated insulin doses to manage blood sugar fluctuations. Unfortunately, the pharmacokinetics of current insulin formulations do not simulate endogenous insulin secretion, which can reach peak concentrations within 30 minutes in non-diabetic individuals. Even the "fast-acting" insulin analogs currently designed for mealtime large doses also show a delayed onset of 20 to 30 minutes, a peak effect of 60 to 90 minutes, and a total duration of action of 3 to 4 hours. These dynamics are the result of the mixed association state of the insulin molecules in the formulation. Commercial insulin formulations usually contain a mixture of insulin hexamer, dimer, and monomer. Although monomers are rapidly absorbed into the bloodstream after injection, dimers and hexamers are absorbed more slowly due to their size, and must be dissociated into monomers to become active (Fig. 1). Furthermore, a prolonged duration of insulin action can make it difficult to control postprandial blood glucose excursions and increase the risk of hypoglycemia because insulin may remain in the body even after the mealtime glucose load has passed.

[0007] In order to better control blood glucose at mealtimes, an insulin formulation is needed that can be rapidly absorbed from the subcutaneous space to more closely mimic endogenous postprandial insulin secretion. Monomeric insulin formulations would enable both a faster onset and a shorter duration of action, thereby reducing the risk of postprandial hypoglycemia by eliminating the subcutaneous depot of insulin hexamers ( Figure 1A and Figure 1B ). However, monomeric insulin is unstable in formulations and rapidly aggregates into inactive and immunogenic amyloid fibrils. Currently, zinc and phenolic preservatives are commonly used as excipients in insulin formulations because their tendency to promote the formation of insulin hexamer enables them to act as stabilizers. It is crucial to develop a new class of excipients that can improve insulin stability in the monomeric state so as to achieve feasible ultra-fast-acting insulin formulations.

[0008] Insulin aggregation typically begins at hydrophobic interfaces, such as air-liquid interfaces, where monomers undergo partial unfolding upon adsorption and can nucleate amyloid fibril formation ( Figure 1C ). The monomeric state is most susceptible to the influence of aggregation, because the hydrophobic part that is usually shielded under the dimerization and hexamerization association state is responsible for aggregation. The zinc-free method that is currently used for the stability of monomeric insulin relies on shielding hydrophobic interactions by directly connecting hydrophilic polymers such as poly (ethylene glycol) (PEG) or trehalose glycol polymers to insulin covalently or non-covalently. Although these methods have been proven to be effective in stabilizing the insulin in the formulation, the method causes an increase in the in vivo circulation time, which is undesirable for the insulin formulation of ultra-fast action. Further, although poly (ethylene glycol) polymers are traditionally used for drug delivery due to their water solubility and biocompatibility, recent concerns about immunogenicity have begun to limit their use.

[0009] An alternative approach for insulin stabilization exploits the propensity of amphiphilic polymers to occupy interfaces, preventing insulin-interface interactions ( Figure 1C Poloxamers are used to enhance the performance of commercial insulin formulations ( U400, Sanofi-Aventis). However, these poloxamer excipients include limited chemical space, show a tendency to form microstructures such as micelles in solution, and are prone to transform into gels at high concentrations, and therefore, stable ultrafast monomeric insulin formulations remain uncertain.

[0010] Insulin has been used to treat diabetes for nearly 100 years, but existing fast-acting insulin formulations do not have fast enough pharmacokinetics to maintain strict glycemic control during rapid blood sugar fluctuations, such as meals. The dissociation of the insulin hexamer (the main association state of insulin in fast-acting formulations) is the rate-limiting step that leads to delayed onset and prolonged duration of action. Formulations of insulin monomers will more closely mimic endogenous postprandial insulin secretion, but using known formulation strategies, monomeric insulin is unstable in solution and rapidly aggregates into amyloid fibrils.

[0011] Patients with certain types of diabetes lack sufficient pancreatic beta cell mass and / or function to produce both endogenous insulin and amylin. In non-diabetic individuals, insulin and amylin work synergistically to control postprandial blood glucose; amylin delays gastric emptying and inhibits glucagon action, while insulin promotes cellular glucose uptake. Studies have shown that dual hormone replacement therapy with insulin and amylin results in improved glycemic outcomes in patients with diabetes, including a 0.3% reduction in HbA1c compared to treatment with insulin alone. However, over the past 100 years, treatment of type 1 diabetes has focused primarily on insulin replacement. While there is a commercially available amylin analog (pramlintide), as of 2012, only 1.5% of patients who would benefit from amylin replacement therapy have taken it. This is primarily due to formulation challenges, resulting in the need for cumbersome separate injections of amylin in addition to insulin at mealtimes.

[0012] Amylin is highly unstable and rapidly aggregates to form inactive and immunogenic amyloid fibrils. Pramlintide is an amylin analog with three amino acid modifications designed to reduce its tendency to aggregate into amyloid fibrils, thereby increasing its shelf life, but is formulated at pH = 4, making it incompatible with mixing with insulin formulations (usually pH about 7). Further, in typical clinical administration, insulin and pramlintide have different pharmacokinetics, in contrast to the endogenous co-secretion of the two hormones from beta cells in the same diurnal pattern. When delivered exogenously, the difference in absorption kinetics arises from the different association states of insulin and pramlintide in the formulation ( Fig.15A ). Pramlintide exists only as a monomer, whereas insulin formulations contain a mixture of hexamers, dimers, and monomers. The mixture of insulin association states results in delayed absorption and a prolonged duration of insulin action. Summary of the invention

[0013] The present disclosure provides a copolymer excipient based on polyacrylamide, and the copolymer excipient based on polyacrylamide contains a water-soluble carrier monomer with an acrylamide reactive portion and a functional dopant monomer with an acrylamide reactive portion. It has been found that the copolymer reduces or prevents the biomolecules in the aqueous formulation and the media based on lipids from aggregating at the hydrophobic interface. Therefore, compared with the same formulation without the copolymer based on polyacrylamide, the biomolecules in the formulation containing the copolymer based on polyacrylamide and the media based on lipids show increased stability, such as increased thermal stability. The copolymer based on polyacrylamide can be used with any biomolecule or media based on lipids that are easy to aggregate in aqueous media, and the biomolecule includes but is not limited to proteins, such as antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof. In certain embodiments, the protein is a monoclonal antibody. In certain embodiments, the copolymer based on polyacrylamide reduces or prevents the aggregation of proteins that are easy to aggregate in aqueous media. The polyacrylamide-based copolymers may also be used with lipid-based vehicles (including but not limited to liposomes, lipid nanoparticles, polymersomes, and micelles) that tend to aggregate in aqueous media to prevent or reduce aggregation.

[0014] In some embodiments, the protein is insulin. In some embodiments, polyacrylamide-based copolymers are used to develop ultra-rapidly absorbed insulin lispro (UFAL) formulations that are stable under aging conditions for 25±1 hours, compared to commercial fast-acting insulin lispro formulations ( ) is stable for 5±2 hours. In a pig model of insulin-deficient diabetes, UFAL showed a peak effect at 9±4 minutes, while commercial Peak effect is exhibited at 25 ± 10 minutes. These ultrafast kinetics make UFAL a promising candidate for improving glycemic control and reducing the burden on patients suffering from diabetes.

[0015] According to exemplary embodiments, copolymer excipients based on polyacrylamide enable to realize the stable formulation of monomeric insulin of ultra-fast action. In certain embodiments, these excipients are synthetic copolymers consisting of water-soluble carrier monomers and functional dopant monomers, and the water-soluble carrier monomers are selected to help polymer solubility, and the functional dopant monomers provide the ability to screen the extensive chemical space that is undeveloped in the current surfactant excipients. It is assumed that the dopant monomer promotes polymer-interface interaction, reduces the insulin-insulin interaction at the interface, and therefore improves insulin stability. In certain embodiments, the precise high-throughput synthesis using reverse addition fragmentation transfer (RAFT) polymerization is used to produce a library with more than 100 kinds of copolymers based on polyacrylamide. In the present disclosure, it is demonstrated that copolymers based on polyacrylamide can realize the stable formulation of monomeric insulin lispro, and in the pig model of insulin deficiency diabetes, this ultra-fast absorbed insulin lispro (UFAL) formulation shows 2 times faster pharmacokinetics than commercial fast-acting insulin formulations.

[0016] In addition, in some embodiments, the copolymer based on polyacrylamide enables the stable co-formulation of monomeric insulin lispro and pramlintide to have synchronized ultra-fast insulin-pramlintide pharmacokinetics, which produces better glycemic control in mealtime simulation. This co-formulation has the potential to improve glucose management and reduce the burden on patients in clinical applications whenever fast-acting insulin is used in other ways. The copolymer excipients described herein can also be more widely used to improve the thermal stability of protein formulations (including insulin formulations) as simple "insertion (drop-in)" excipients without changing its biological activity, pharmacokinetics or pharmacodynamics. For example, the copolymer excipients described herein can be used as insert excipients combined with other formulation methods intended to change or regulate the pharmacokinetics of protein formulations.

[0017] The present disclosure also provides a polyacrylamide-based copolymer, wherein the polyacrylamide-based copolymer comprises a water-soluble carrier monomer and a functional dopant monomer, wherein the water-soluble carrier monomer comprises an acrylamide reactive portion, and the functional dopant monomer comprises an acrylamide reactive portion; wherein the weight percentage (wt%) of the water-soluble carrier monomer is about 70% to about 98%; the weight percentage (wt%) of the functional dopant monomer is about 2% to about 30%; and the average molecular weight (M) of the polyacrylamide-based copolymer is about 10%. n ) is from about 1,000 g / mol to about 30,000 g / mol; and the degree of polymerization is from about 10 to about 250.

[0018] As disclosed herein, high throughput controlled radical polymerization technology is implemented to generate a large library of copolymer excipients based on polyacrylamide. Non-limiting examples of copolymers based on polyacrylamide as provided herein include copolymers based on polyacrylamide including a water-soluble carrier monomer and a functional dopant monomer, the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl) acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM), the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl) trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE).

[0019] The present disclosure provides a composition comprising about 0.005 wt % to about 0.2 wt % of a polyacrylamide-based copolymer and about 100 U / mL of insulin or an analog thereof, wherein the polyacrylamide-based copolymer comprises about 70 wt % to about 95 wt % of a MORPH carrier monomer and about 5 wt % to about 30 wt % of a NIP dopant monomer.

[0020] The present disclosure also provides a composition comprising about 0.005 wt % to about 0.2 wt % of a polyacrylamide-based copolymer, about 100 U / mL insulin or an analog thereof, and about 0.01 mg / mL to about 0.1 mg / mL pramlintide, wherein the polyacrylamide-based copolymer comprises about 70 wt % to about 95 wt % of a MORPH carrier monomer and about 5 wt % to about 30 wt % of a NIP dopant monomer.

[0021] Also provided is a method of treating diabetes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the polyacrylamide-based copolymer of the present disclosure.

[0022] Also provided is a method of managing blood glucose levels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the polyacrylamide-based copolymer of the present disclosure.

[0023] Also provided is a method for increasing the stability of a formulation containing a biomolecule, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation.

[0024] Also provided is a method for increasing the stability of a protein formulation, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the protein formulation.

[0025] Also provided is a method for increasing the stability of a formulation containing a lipid-based vehicle, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation.

[0026] Also provided is a method for reducing the aggregation rate of biomolecules in an aqueous composition, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation.

[0027] Also provided is a method for reducing the aggregation rate of a protein in an aqueous composition, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the protein formulation.

[0028] Also provided is a method for reducing the aggregation rate of a lipid-based vehicle in an aqueous composition, the method comprising adding about 0.005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 shows a schematic diagram of the absorption kinetics of insulin in various association states. Figure 1A : Commercial "fast-acting" insulin formulations contain a mixture of insulin hexamers, dimers, and monomers. Only the monomeric form of insulin is active, so the dissociation from the hexamer to the monomer is rate limiting for therapeutic action. For rapid insulin absorption after subcutaneous administration, ultrafast insulin formulations will contain primarily insulin monomers without insulin hexamers. Figure 1B It is shown that a mixture of insulin hexamers, dimers and monomers in commercial fast-acting insulin formulations results in a prolonged duration of insulin action when delivered subcutaneously. Insulin monomers are absorbed in approximately 5 to 10 minutes, dimers are absorbed in 20 to 30 minutes, and hexamers may take 1 to 2 hours to be absorbed and thus result in a prolonged insulin action. A predominantly monomeric insulin formulation will reduce the onset time and allow for a shortened duration of insulin action, thereby better managing blood sugar during meals. Figure 1CIt is shown that ultrafast insulin formulations without hexamers will face stability challenges due to the tendency of insulin monomers to aggregate into amyloid fibrils. At the air-water interface (shown on the left), the unfolding of insulin molecules and the exposure of hydrophobic domains during insulin-insulin interactions promote amyloid fibril formation. Stable polymer excipients are absorbed to the air-liquid interface (shown on the right), hindering the unfolding of insulin molecules and the interfacial nucleation of insulin amyloidosis.

[0030] Figure 2 A protocol for the design of a polymer excipient library is presented. A statistical acrylamide copolymer library with a target degree of polymerization (DP) of 50 was synthesized by using RAFT-controlled copolymerization. The copolymer combinations consisted of one carrier monomer: morpholine acryloyl (MORPH), methoxypropyl acrylamide (MPAM), dimethyl acrylamide (DMA), hydroxyethyl acrylamide (HEAM), or acrylamide (AM). Each copolymer also contained one dopant monomer: tris(hydroxymethyl)-methacrylamide (TRI), acrylamidomethylpropane sulfonic acid (AMP), acrylamidopropyltrimethylammonium chloride (TMA), n-isopropylacrylamide (NIP), tert-butylacrylamide (TBA), or phenylacrylamide (PHE). Each carrier-dopant combination was repeated at low, medium and high dopant loadings: NIP was repeated at 6.7 wt.%, 13.3 wt.% and 20 wt.%, TRI was repeated at 5 wt.%, 10 wt.% and 15 wt.% and AMP, TMA, TBA, PHE were repeated at 3.3 wt.%, 6.7 wt.% and 10 wt.%.

[0031] Figure 3 Demonstrated validation of SEC wt.% measurements 1 H NMR spectra and SEC traces show the properties of the synthesized copolymers with MORPH as carrier and PHE as dopant. 1 H NMR spectrum. Due to the high viscosity of the monomer, MORPH-PHE 6.7％ Insufficient MORPH addition resulted in higher than expected PHE loading in the final copolymer. The SEC trace provided in the inset confirms the experimental loading, where MORPH-PHE 6.7％ MORPH-PHE 3.3％ and MORPH-PHE 10％ The molecular weight of the copolymer was determined by the molecular weight reduction method. The molecular weight reduction method was used to determine the experimental loading of MORPH and PHE in the final copolymer. DRI refers to the differential refractive index measured using SEC.

[0032] Figure 4SEC traces of polymers synthesized from the initial copolymer library are shown. The carrier monomers are, from left to right: acrylamide (AM), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), 4-acryloylmorpholine (MORPH), N-(3-methoxypropyl)acrylamide (MPAM). The copolymers based on AM are measured on aqueous SEC, while the others are measured on DMF SEC. The top row depicts the superposition of all synthetic copolymers. Each subsequent row shows the functional dopant loading of the copolymer. The three copolymers of each carrier-dopant pair are low loading, medium loading and high loading. Functional dopants (abbreviations; low loading, medium loading, and high loading) are as follows: N-[tris(hydroxymethyl)methyl]acrylamide (TRI; 5 wt.%, 10 wt.%, 15 wt.%), (3-acrylamidopropyl)trimethylammonium chloride solution (TMA; 3.3 wt.%, 6.7 wt.%, 10 wt.%), 2-acrylamido-2-methylpropanesulfonic acid (AMP; 3.3 wt.%, 6.7 wt.%, 10 wt.%), N-isopropylacrylamide (NIP; 6.7 wt.%, 13.3 wt.%, 20 wt.%), N-tert-butylacrylamide (TBA; 3.3 wt.%, 6.7 wt.%, 10 wt.%), and N-phenylacrylamide (PHE; 3.3 wt.%, 6.7 wt.%, 10 wt.%).

[0033] Figures 5A to 5G Stability screening of recombinant insulin using a polymer excipient library is demonstrated. Figure 5A Aggregation times of recombinant insulin (100 U / mL) formulated with carrier homopolymer (0.1 wt.%) or Pluronic L-61 control (0.1 wt.%), which has a similar composition to Poloxamer 171 used in commercial insulin formulations, are shown. Aggregation times of recombinant insulin (100 U / mL) formulated with carrier homopolymer (0.1 wt.%) or Pluronic L-61 control (0.1 wt.%), which has a similar composition to Poloxamer 171 used in commercial insulin formulations, are also shown. Figure 5B )、AM( Figure 5C )、HEAM( Figure 5D )、MPAM( Figure 5E ) and MORPH( Fig. 5F ) Aggregation time of recombinant insulin (100 U / mL) formulated with carriers. Dopants and target weight percentages are listed on the x-axis. Figure 5Gis a heat map of the best performing excipients (longest aggregation time) for each carrier-dopant combination. The black dashed squares indicate the top dopant-carrier combinations selected for further screening. These assays evaluate the aggregation of proteins in the formulations over time during stress aging (continuous stirring at 37°C) by monitoring the change in absorbance at 540nm. The data shown are the average aggregation times (n=3; mean ± sd), where aggregation is defined as a 10% increase in absorbance.

[0034] Figure 6 Demonstrated commercial use Aqueous SEC elution profiles of ANTI-IN and UFAL formulations. These traces demonstrate the predominantly monomeric insulin-associated state of UFAL through longer elution times associated with lower effective molecular weights.

[0035] Figures 7A to 7D Shown are SEC traces of copolymers from the second screen targeting DP 50. Fig. 7A Copolymerization of MPAM with PHE is shown. Figure 7B Copolymerization of MPAM with NIP is shown. Figure 7C Copolymerization of MORPH with PHE is shown. Fig.7D Copolymerization of MORPH with NIP is shown.

[0036] Figures 8A to 8D A stable ultra-rapidly absorbed insulin lispro (UFAL) formulation using a polyacrylamide-based copolymer excipient is demonstrated. Fig. 8A shows the MALS determined (top) and insulin-associated states of UFAL (bottom). Figure 8B Is a UFAL stability screen performed with a polymer excipient library. Aggregation time of UFAL (100 U / mL) formulated with polymer excipients (0.01 wt.%) from the second screen is shown. Polyacrylamide-based copolymer excipients include MPAM and MORPH carrier polymers with different weight percentages of dopant PHE (top) or NIP (bottom). Figure 8C Depicted are representative absorbance traces showing the difference in absorbance between the 20 μM MgCl2O3 and the 20 μM MgCl2O3 when compared with UFAL without polymer excipients and Compared with the control, MORPH-NIP 23％ UFAL stability upon formulation. These assays assess aggregation of proteins in formulations over time during stress aging (i.e., continuous stirring at 37°C) by monitoring changes in transmittance at 540 nm. Data shown are mean aggregation times (n=3; mean ± sd), where aggregation is defined as a 10% increase in absorbance. Fig.8D The polymer excipient MORPH-NIP is shown 23％Diffusion-ordered NMR spectroscopy (DOSY) of UFAL of 100 μg / mL was performed. DOSY provides insight into the insulin association state in the formulation and the insulin and polymer diffusion rates. Diffusion properties demonstrate that insulin lispro and MORPH-NIP 23％ diffuse at different rates and are uncorrelated, indicating that disruption of interfacial interactions is the main contributor to the observed stabilization effect.

[0037] Figures 9A to 9B Shown UFAL, aging The in vitro and in vivo bioactivity of the formulations of (shaking at 37°C for 12 hours) and aged UFAL (shaking at 37°C for 12 hours). Fig.9A ) In vitro activity was tested by measuring phosphorylation of Ser473 on AKT. Data shown are mean ± sem of n = 3 experimental replicates. Results were plotted as the ratio of [pAKT] / [AKT] for each sample (n = 3 cell replicates) and EC was plotted using GraphPad Prism8 50 Regression [log(agonist) vs. response (three parameters)]. Fig. 9B ) were used to evaluate the in vivo bioactivity of diabetic male Sprague Dawley rats. Rats fasted for 4 to 6 hours received subcutaneous injections of insulin (1.5 U / kg) and glucose measurements were performed every 30 minutes over 4 hours using a handheld glucose monitor. Sixteen rats were randomly assigned to two groups: (i) (n=8) and (ii) UFAL (n=8). In each group, each rat received both fresh formulation and aged formulation on separate days. The order of administration of formulations was randomized. It was hypothesized that the observed changes in blood glucose but not AKT in aged rats would be due to the presence of fresh formulation and aged formulation. The loss of activity is the result of reversible insulin aggregation. When this aged formulation undergoes significant dilution in the in vitro AKT assay, these aggregates dissociate into active insulin, while the minimum dilution required for accurate dosing in rats is insufficient for this dissociation to occur, and insulin aggregates lead to the observed loss of activity. The data shown are mean ± sem.

[0038] Figures 10A to 10M The pharmacokinetics and pharmacodynamics of monomeric insulin in diabetic pigs are presented. Female diabetic pigs received a subcutaneous regimen consisting of (i) a commercially available or (ii) UFAL formulated with a polymer. Insulin was administered to the pigs based on their individual insulin sensitivity to lower their blood glucose levels by approximately 200 mg / dL. Fig. 10A : Blood glucose measurements in pigs after subcutaneous administration of insulin. Fig. 10B: Pharmacokinetics of insulin lispro in mU / L after subcutaneous (sc) injection. Fig. 10C : Total exposure over 210 minutes represented by the area under the curve. Figures 10D to 10I : Exposure percentage at each time point (AUC t / AUC 210 ). Fig.10J : The pharmacokinetics of each pig were normalized individually to the peak concentration and the normalized values ​​of insulin lispro concentrations were averaged for each treatment group. Figure 10K : Time to reach 50% of peak insulin lispro concentration (onset of action). Fig.10L : Time to reach peak insulin lispro concentration. Figure 10M : The time it takes for insulin lispro to decrease to 50% of its peak concentration. Fig. 10A , 10B and 10J: Error bars indicate mean ± sd for n = 5 for all groups. Figures 10D to 10I : Error bars indicate mean ± sem for n = 5 for all groups. Bonferroni post-hoc test was performed to account for comparison and significance of multiple individual exposure time points and adjusted for α (α = 0.008). Fig. 10C , 10K ( to 10 M) Error bars indicate mean ± sem (α = 0.05) for n = 5 for all groups. Statistical significance was determined by restricted maximum likelihood (REML) repeated measures mixed model.

[0039] Figures 11A to 11B Diabetic female pigs received subcutaneous administration of a therapy comprising (i) a commercially available insulin or (ii) UFAL formulated with polymers. Fig.11A : Scheme of the subcutaneous injection site behind the pig foreleg. Pigs have compact skin and subcutaneous tissue very similar to humans, making them the most relevant preclinical model for studying the pharmacokinetics of biopharmaceuticals following subcutaneous administration. Pigs are large enough to accurately administer insulin using a standard concentration (100 U / mL), ensuring that the observed pharmacokinetics are not distorted by dilution effects. Fig. 11B : Insulin was administered to pigs according to their individual insulin sensitivity to reduce their blood glucose concentration by approximately 200 mg / dL. Blood glucose measurements in pigs after subcutaneous administration of insulin. Error bars indicate mean ± sd for n = 5 for all groups.

[0040] Figures 12A to 12E The pharmacokinetic model of UFAL in humans was demonstrated. Fig. 12A: The model of insulin plasma concentration after injection in human patients was adapted from Wong et al. (J. Diabetes Sci. Technol. (2008) 2: 658-671). Rapid-acting insulin analogs were injected into the subcutaneous space (I inj ), then dissociate and diffuse into the interstitium (k1), before being absorbed into the plasma (k2) and finally cleared (k3). Fig. 12B :The least squares method was used to determine the k1, k2 and k3 of the pigs and the diabetic pigs The normalized pharmacokinetic data of ANTIBASE 1 and UFAL were used for modeling ( FIG14 and Table 6 ). Fig. 12C :Human clinical The pharmacokinetic data were compared with modeled rapid-acting insulin (RAI) analog kinetics (using known human parameters, Table 6) and the predicted kinetics of UFAL in humans. The porcine pharmacokinetic data of UFAL were used to predict the human pharmacokinetics of UFAL. The human pharmacokinetics of UFAL were then plotted by using the estimated k1 and the known k2 and k3 parameters. Fig.12D : Compared with published clinical studies Kinetic comparison. Model predictions for the kinetics of RAI and UFAL, time to 50% of peak rise (left), time to peak (center), and duration of action to 50% of peak fall (right). Fig.12E : The model-predicted time to peak UFAL in humans was compared with human clinical data for commercial rapid-acting insulin formulations (see, e.g., Heise et al., Diabetes Obes. Metab. (2017) 19:208-215; Andersen et al., Diabetes Obes. Metab. (2018) 20:2627-2632; Rave et al., Diabetes Care (2006) 29:1812-1817).

[0041] Fig.13 UFAL and Diabetic female pigs received subcutaneous administration of a therapy comprising (i) a commercial Or (ii) UFAL formulated with a polymer. Insulin was administered to pigs according to their individual insulin sensitivity to reduce their blood glucose concentration by approximately 200 mg / dL. For subcutaneous administration, the area under the moment curve (AUMC) divided by the area under the curve (AUC) equals the mean residence time (MRT) + mean absorption time (MAT). AUMC / AUC = MRT + MAT. Error bars indicate mean ± sem (α = 0.05) for n = 5 for all groups. Statistical significance was determined by a restricted maximum likelihood (REML) repeated measures mixed model.

[0042] Figures 14A to 14C demonstrated with diabetic pigs Pharmacokinetic output of the model fit compared to experimental pharmacokinetic data from UFAL. Fig.14A : Time to reach 50% of the peak rise. Fig. 14B : Time to reach the peak. Fig. 14C : Time to 50% of the peak decrease. Asterisks indicate the pharmacokinetic time points predicted by the model. Data points are Figure 10K to 10M The same as used in .

[0043] Figures 15A to 15F Formulation kinetics and stability were demonstrated. Fig.15A : Current dual hormone replacement of insulin and pramlintide requires two separate injections at mealtimes. This additional injection is not only onerous compared to endogenous co-secretion from β cells, but there is a kinetic mismatch between insulin and pramlintide when exogenously delivering insulin and pramlintide. This is due to the presence of mixed insulin association states in fast-acting insulin formulations, where monomers and dimers are rapidly absorbed, but the slow dissociation of the insulin hexamer results in a prolonged duration of action. Fig. 15B : A single injection co-formulation of monomeric insulin and pramlintide would reduce patient burden and have better pharmacokinetic overlap that more closely mimics endogenous secretion from a healthy pancreas. Fig. 15C : Amphiphilic acrylamide copolymer excipients can be used to stabilize insulin-pramlintide co-formulations. These excipients preferentially adsorb at the air-water interface, displacing insulin and / or pramlintide and preventing nucleation of aggregation events that initiate amyloid fibril formation. Fig.15D : Co-formulation components. Fig.15E : The insulin association state of (top); adapted from 57, compared with insulin lispro containing phenoxyethanol (0.85 wt.%) and glycerol (2.6 wt.%) without zinc (bottom). Fig.15F :(i) (ii) + Pramlintide (1:6 Pramlintide: Insulin Lispro), (iii) Insulin Lispro without zinc (100 U / mL Insulin Lispro, 0.85 wt.% phenoxyethanol, 2.6 wt.% glycerol, 0.1 mg / mL MoNi 23％ ), (iv) co-formulation (100 U / mL insulin lispro, 1:6 pramlintide: insulin lispro, 0.85 wt.% phenoxyethanol, 2.6 wt.% glycerol, 0.1 mg / mL MoNi 23％ ) in a stress aging assay (continuous stirring, 37°C). The change in transmittance is shown from the baseline transmittance. Aggregation is defined as a change in transmittance of >10%.

[0044] Fig.16 The SEC-MALS elution profile of the aggregation state distribution of insulin lispro is shown, where the number average molecular weight and weight average molecular weight are used to calculate the percentage of insulin associated state.

[0045] Fig.17 Shown + Pramlintide and co-formulation (100 U / mL insulin lispro, 1:6 Pramlintide: insulin lispro, 0.85 wt. % phenoxyethanol, 2.6 wt. % glycerol, 0.1 mg / mL MoNi 23％ ) initial transmittance. Stability measurement (i) + initial transmittance values ​​of the pramlintide control group and (ii) the co-formulations ( Fig.15F ) is shown as (average initial Transmittance) - Transmittance change from the initial transmittance of the formulation. The decrease in transmittance of the + Pramlintide sample indicates poor solubility when the two formulations are mixed. In contrast, the initial transmittance of the co-formulation is optically clear to the eye and shows an initial Less difference in transmittance.

[0046] Figures 18A to 18D The pharmacokinetics and pharmacodynamics in diabetic rats are presented. Fasted male diabetic rats (n=11) were subcutaneously administered (i) (ii) Single injection and pramlintide or (iii) insulin-pramlintide co-formulation. Insulin administration was immediately followed by oral gavage with glucose solution (1 g / kg). Each rat received all treatment groups. The change in blood glucose level from baseline after treatment was Fig.18A Shown in. Figures 18B to 18C Insulin lispro ( Fig.18B ) or pramlintide ( Fig.18C )'s pharmacokinetics.

[0047] Figures 19A to 19M The onset and duration of action in diabetic rats were demonstrated. Fasted male diabetic rats (n=11) were subcutaneously administered (i) (ii) Single injection and pramlintide or (iii) insulin-pramlintide co-formulation. Insulin administration was immediately followed by oral gavage with glucose solution (1 g / kg). Each rat received all treatment groups. The pharmacokinetics of each rat were individually normalized to peak serum levels and to insulin lispro ( Fig.19A ) or pramlintide ( Fig.19J The start of exposure was defined as insulin lispro ( Fig.19B ) or pramlintide ( Figure 19K ) reaches 50% of the peak rise time. Fig.19C ) or pramlintide ( Figure 19L ) peak exposure. The onset of exposure was defined as the peak exposure of insulin lispro ( Fig.19D ) or pramlintide ( Fig.19M ) reaches 50% of the peak rise time. t / AUC 120 The fractional exposure to insulin lispro is expressed as a ratio, Fig.19E , t = 6; at Fig.19F , t = 15; at Figure 19G , t = 30; Fig.19H , t = 45; Fig.19I , t = 60. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post hoc test was applied to account for multiple comparisons (Figures 19b to 19I, 19K, 19M). Bonferroni post hoc test was performed to account for comparison and significance of multiple individual exposure time points and adjusted for α (α = 0.01) ( Figures 19E to 19I ).

[0048] Figures 20A to 20C The area under the curve and exposure ratios for insulin lispro are shown. Total insulin lispro exposure as area under the pharmacokinetic curve ( Fig. 20A ). t / AUC 120 The fractional exposure to insulin lispro is expressed as a ratio, Fig. 20B , t = 3; at Fig. 20C , t = 9. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post hoc test was used to account for multiple comparisons ( Figures 20B to 20C ).

[0049] Figures 21A to 21GThe area under the pramlintide curve and exposure ratios are shown. Total pramlintide exposure as area under the pharmacokinetic curve ( Fig.21A ). t / AUC 120 The fractional exposure of pramlintide is expressed as a ratio, Fig. 20B , t = 6; at Fig. 20C , at t = 9; in FIG. 20D , at t = 15; in FIG. 20E , at t = 30; in FIG. 20F , at t = 45; and in FIG. 20G , at t = 60. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model.

[0050] Figures 22A to 22C The pharmacokinetic overlap of the formulations was demonstrated. The mean normalized serum concentrations of insulin and pramlintide when delivered as two separate injections and when delivered together as a co-formulation (for each rat, n=11 / group) were Fig.22A and 22B The overlap between the two curves is defined as the total time that both the insulin curve and the pramlintide curve (width at half peak height) spend more than 0.5, shown as the ratio of the overlap time to the total width of the two peaks (Overlap ÷ (Insulin lispro + Pramlintide overlap) ( Fig. 22C ). Statistical significance was determined by restricted maximum likelihood repeated measures mixed models.

[0051] Figures 23A to 23C Gastric emptying in diabetic rats is shown. Fasting male diabetic rats were subcutaneously administered (i) (ii) Single injection and pramlintide or (iii) insulin-pramlintide co-formulation. Fig.23A Gastric emptying experiments are depicted in which insulin administration (2 U / kg) was immediately followed by oral gavage with acetaminophen slurry (100 mg / kg). Each rat (n=11) received all treatment groups. Serum acetaminophen concentrations were Fig. 23B The peak exposure time of acetaminophen serum concentration is shown in Fig.23C All data are shown as mean ± SE. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post hoc test was applied to account for multiple comparisons.

[0052] Figures 24A to 24D Fasting male diabetic rats were administered subcutaneously (i) (ii) Single injection and pramlintide or (iii) insulin-pramlintide co-formulation. Fig.24AOral glucose challenge is depicted, in which insulin administration (0.75 U / kg) was immediately followed by oral gavage with glucose solution (2 g / kg). Each rat (n=10) received all treatment groups. The changes in blood glucose levels after administration were Fig. 24B The maximum change in glucose above baseline was Fig.24C The maximum change in glucose below baseline was Fig.24D All data are shown as mean ± SE. Statistical significance was determined by restricted maximum likelihood repeated measures mixed model. Tukey HSD post hoc test was applied to account for multiple comparisons.

[0053] Figures 25A to 25C Shown and Interspecies pharmacokinetics of commercially available and regular human insulin (e.g., R) normalized pharmacokinetics in Fig.25A The peak exposure time for each formulation was Fig.25B The duration of action of each formulation is shown in Fig.25C In rats, the differences in onset and time to peak between the fast-acting insulin formulations and the regular insulin formulations were minimal and difficult to detect. However, in humans, these small differences translate into different differences in onset and time to peak. This suggests that the differences observed in the co-formulations of the present application are consistent with those observed in the co-formulations of the present application. In comparison, the tendency of insulin to act more rapidly may translate into substantial differences in humans.Duration of action is defined here as the peak width at 25% of the peak height (25% drop time - 25% rise time). The rat data for were taken from this study, and the rat data for regular human insulin were adapted from previous work. Data are from three external studies and adapted from presentation in previous work. Human regular human insulin data are from three external studies (Pettis et al., Diabetes Technol. Ther. (2011) 13: 443-450; Andersen et al., Diabetes Obesity & Metabolism (2018) 20: 2627-2632; Plank et al., Diabetes Care (2002) 25: 2053-2057; Linnebjerg et al., Clin. Pharmacokinet. (2020) 59: 1589-1599).

[0054] Figures 26A to 26C Shown and interspecies pharmacokinetics of monomeric insulin lispro formulations. Fig.26A Commercial delivery in rats, pigs, and humans has been demonstrated Normalized pharmacokinetics (left), time to peak (middle), and duration of action (right) of . Fig.26B Shown are the normalized pharmacokinetics (left), time to peak (middle), and duration of action (right) of monomeric insulin delivered in rats, pigs, and humans. It is shown that the onset of action increases and the duration of action is longer as one moves toward species with more complex subcutaneous architecture (rat < pig < human). In contrast, monomeric insulin lispro has very similar onset and duration of action between rat and pig. Since this formulation differs very little between rat and pig, the differences between pig and human are also likely to be small. Further, pramlintide, present only as a monomer, was observed to have very similar kinetics between pig and human. Fig.26C Normalized pharmacokinetics in humans of commercial formulations (left), time to peak exposure for each formulation (middle), and duration of action for each formulation (right) are shown. Even with the shift to faster times to peak for next-generation rapid-acting insulins such as Fiasp and Lyumjev, there has not been a similar increase in reduced duration of action. Duration of action is defined here as peak width at 25% of peak height (time to 25% down - time to 25% up). Rat data are taken from the study described in Example 3 (monomeric insulin lispro in rats was delivered as part of a co-formulation). Pig data are adapted from previous work. Human Data are from four external studies (Pettis et al., Diabetes Diagnostics Technol Ther (2011) 13:443-450; Andersen et al., Diabetes Obesity Metabolism (2018) 20:2627-2632; Plank et al., Diabetes Care (2002) 25:2053-2057; Linnebjerg et al., Clinical Pharmacokinetics (2020) 59:1589-1599). The predicted monomeric insulin lispro for humans has been adapted from pharmacokinetic modeling of previous work in pigs. Fig.26C , data adapted from human clinical studies on: (i) regular human insulin (Rave et al., Diabetes Care (2006) 29:1812-1817; Lindholm et al., Diabetes Care (1999) 22:801-805; Heinemann et al., Diabetic Medicine (1996) 13:625-629); (ii) (Novo Nordisk) (Fath et al., Pediatr. Diabetes (2017) 18:903-910; Heise et al., Clinical Pharmacokinetics (2017) 56:551-559); (iii) Fiasp (Novo Nordisk) (Fath et al., Pediatr. Diabetes (2017) 18:903-910; Heise et al., Clinical Pharmacokinetics (2017) 56:551-559); (iv) (Eli Lily) (Pettis et al., Diabetes Diagnostics Therapeutics (2011) 13:443-450; Andersen et al., Diabetes Obesity Metabolism (2018) 20:2627-2632; Plank et al., Diabetes Care (2002) 25:2053-2057; Linnebjerg et al., Clinical Pharmacokinetics (2020) 59:1589-1599); and (v) Lyumjev (Eli Lilly) (Linnebjerg et al., Clinical Pharmacokinetics (2020) 59:1589-1599; Shiramoto et al., J. Diabetes Invest. (2020) 11:672-680). The predicted monomeric insulin lispro for humans has been adapted from pharmacokinetic modeling of previous work in pigs (Mann et al., Sci. Transl. Med. (2020) 12:eaba6676).

[0055] Figures 27A to 27C The human pharmacokinetics of various insulin formulations are presented, and the normalized pharmacokinetics of pramlintide delivered in rats, pigs, and humans are shown ( Fig.27A ), time to peak value ( Fig.27B ) and duration of action ( Fig.27C). The conservation of ultrafast absorption kinetics from rats to pigs and the similar pramlintide kinetics between pigs and humans confirm the model-predicted kinetics of monomeric insulin lispro in humans. The duration of action is defined here as the peak width at 25% peak height (time to drop 25% - time to rise 25%). The rat data are taken from the study described in Example 3. The pig data are adapted from previous work (Maikawa et al., Nat. Biomed. Eng. (2020) 4: 507-517). The human pramlintide data are adapted from two external studies (Kolterman et al., Diabetologia (1996) 39: 492-499); Riddle et al., Diabetes Obesity Metabolism (2015) 17: 904-907).

[0056] Figures 28A to 28D A scheme showing the cold chain and insulin aggregation mechanisms. To maintain integrity, commercial insulin formulations must currently be shipped and stored in refrigerated containers for long durations of several weeks for global distribution ( Fig.28A ). Fig.28B Aggregation mechanism of commercial insulin formulations is shown. Insulin hexamers are in equilibrium with monomers in the formulation. These monomers interact at the interface, where exposure of hydrophobic domains during insulin-insulin interactions nucleates amyloid fibril formation. Fig.28C Depicted are exemplary polyacrylamide-based copolymer excipients, poly(acryloylmorpholine 77％ -co-N-isopropylacrylamide 23％ )(MoNi 23％ ) chemical structure. Polyacrylamide-based copolymer excipients are amphiphilic copolymers that adsorb to the interface, reduce insulin-insulin interactions and delay the nucleation of insulin amyloidosis ( Fig.28D ).

[0057] Figures 29A to 29C Experimental observations on the stabilization mechanism of polyacrylamide-based copolymer excipients are presented. Fig.29A is a diagram of the proposed stabilization mechanism. In the figure, the monomers at the interface have associative interactions (top). 23％ Occupies the interface where insulin is not present (middle). Compound composition, MoNi 23％ Disruption of insulin-insulin surface interactions provides a mechanism for inhibiting aggregation (bottom). Fig.29B Shows MoNi formulated with formulation excipients 23％(0.01wt.%) and MoNi23% (0.01wt.%) (n=2) surface tension measurements. Fig.29C Shows Interfacial rheology measurements using MoNi 23％ (0.01wt.%) formulated The measurements were below the resolution of the instrument, indicating that there was no protein aggregation at the interface (n=3).

[0058] Fig.30 The surface tension of a 1 mg / mL (0.1 wt.%) polymer formulation is shown. (95U), MoNi23% (0.1wt.%) prepared with glycerol (1.6wt.%) and m-cresol (0.25wt.%), and MoNi23% (0.1wt.%) prepared with (95U) surface tension measurement.

[0059] Figures 31A to 31H Formulations containing polyacrylamide-based copolymers were shown to stabilize insulin. Fig.31A :1mL of commercial or with the addition of a polyacrylamide-based copolymer excipient (i) MoPhe 6％ (ii) MpPhe 8％ (iii) MoNi 23％ of Aliquoted into 2 mL glass vials and aged at 37°C with constant stirring (150 rpm) for 0, 2, 4, and 6 months. Additional 2 week and 1 month time points were added for All formulations were prepared at a concentration of 95 U / mL (diluted so that the copolymer could be added to the commercial middle). Fig.31B Figure 31C shows a transmittance assay used to assess protein aggregation in a formulation over time by monitoring changes in transmittance at 540 nm (n=1 per formulation time point). Figure 31C shows the transmittance at 0 and 6 month time points using or MoNi 23％ After stimulation, in vitro activity was measured by phosphorylation of Ser473 on AKT. Insulin concentrations are shown as Log (ng / mL). Fig.31D The Log (EC 50 ) values. Statistical significance was assessed using an additional sum-of-squares F test to determine the Log(EC 50 The data sets were compared pairwise, and the Bonferroni post hoc test was used to adjust for multiple comparisons (α = 0.008). Figures 31E to 31HCircular dichroism spectra from 200 to 260 nm are depicted for each formulation (diluted to 0.2 mg / mL in PBS) at each time point. For Figure 31C, results are shown as the mean ± s.e. of the ratio of [pAKT] / [AKT] for each sample (n = 3 cell replicates) and EC plotted using GraphPad Prism 8 50 Regression (log(agonist) vs. response (three parameters)). Fig.31D Statistical significance was assessed using an additional sum-of-squares F test to determine the Log(EC 50 The data sets were compared pairwise, and the Bonferroni post hoc test was used to adjust for multiple comparisons (α = 0.008).

[0060] Figures 32A to 32B Transmittance measurements of 5 mg / mL and 1 mg / mL formulations are shown. 1 mL of polyacrylamide-based copolymer excipients (i) MoPhe 6%, (ii) MpPhe 8%, (iii) MoNi 23% were added. Aliquoted into 2 mL glass vials and aged at 37°C with constant stirring (150 rpm) for 0, 2, 4 and 6 months. Fig.32A 5 mg / mL polymer excipient (0.5 wt.%) in the formulation is shown and Fig.32B Shown is 1 mg / mL polymer excipient (0.1 wt / %) in the formulation.

[0061] Figures 33A to 33F Insulin activity after aging of diabetic rats is shown. Fasted diabetic male rats received subcutaneous administration (1.5 U / kg) of each insulin formulation at each aging time point (0, 2, 4, 6 months). ( Fig.33A ), containing MoPhe 6％ of ( Fig.33B ), containing MpPhe 8％ of ( Fig.33C ) or containing MoNi 23％ of ( Fig.33D ). Fig.33E is a comparison of each formulation at t=0 months. In these assays, 32 rats were randomly assigned to one of four formulation groups (n=8), and each rat received a dose of formulation in random order at each aging time point. Blood glucose levels were measured every 30 minutes using a handheld glucose monitor, and changes in blood glucose relative to baseline glucose measurements were plotted. The maximum difference in glucose relative to baseline (Δ glucose) was also plotted for each formulation as a measure of formulation efficacy. Fig.33F It is shown that at t = 0 and t = 6 months, and MoNi 23％ of All data are shown as mean ± s.e. The statistical significance between maximum Δglucose (with rat as a random effect) and formulation age (as a within-subject fixed effect) was assessed using a REML repeated measures mixed model. Formulations were compared using post hoc Tukey HSD tests to determine statistical significance between aging time points.

[0062] Figures 34A to 34B Shows the time points including t=0.5 and t=1 month Fasting diabetic male rats (n=8) received subcutaneous administration (1.5 U / kg) Eight rats were randomly assigned to Groups. Each rat received a dose of formulation in random order at each aging time point. Blood glucose levels were measured every 30 minutes using a handheld glucose monitor, and changes in blood glucose relative to baseline glucose measurements were plotted. The statistical significance between maximum Δ glucose (where rat was a random effect) and formulation age (as a fixed effect within subjects) was assessed using a REML repeated measures mixed model. Post hoc Tukey HSD tests were used for formulations to determine statistical significance between aging time points. Groups that are not connected by the same letter are significantly different.

[0063] Fig.35 The area under the pharmacokinetic curve is shown. Aging (t=6) +MoNi 23％ and aged MoNi 23％ The area under the pharmacokinetic curve of (t = 6) is No differences were observed between the stabilization formulations or the polymer stabilized formulations. In comparison, aging The area under the curve decreases are shown. All data are shown as mean ± SE. A REML repeated measures mixed model was used to assess the statistical significance between AUC (where rat was a random effect) and formulation age (as a fixed effect within subjects). Post hoc Tukey HSD tests were used for formulations to determine statistical significance between aging time points.

[0064] Figures 36A to 36E Demonstrates stress aging in commercial packaging. Fig.36A : The polyacrylamide-based copolymers were tested for their ability to stabilize under stress conditions in commercial packaging. 23％ Stock solution or water (control), add 10mL of U100 R vials were diluted to 95 U / mL (final concentration of 0.01 wt.% copolymer). Addition of copolymer to formulations required dilution. The vials were returned to their original box and taped to a shake plate (150 rpm) in an incubator at 37°C (n=1 per formulation) or 50°C (n=1 per formulation). Samples were observed and imaged daily. Figures 36B to 36C At 37°C ( Fig.36B ) and 50℃( Fig.36C ) after aging or including MoNi 23％ of A single sample (n=1) was tested for each transmittance curve. The blood glucose curves of samples aged at 50°C in fasting diabetic rats are shown ( Fig.36D ) and maximum blood glucose change (Δglucose) ( Fig.36E ). Data are shown as mean ± SE. Statistical significance between maximum Δglucose (with rat as a random effect) and formulation age (as a within-subject fixed effect) was assessed using a REML repeated measures mixed model. Post hoc Tukey HSD tests were used to determine statistical significance between aging time points and groups. DETAILED DESCRIPTION

[0065] The present disclosure describes the synthesis and characterization of a copolymer library including different water-soluble acrylamide monomers and functional dopant monomers of different weight ratios, molecular weights and degrees of polymerization. The copolymers disclosed herein effectively reduce or prevent biomolecules (e.g., proteins and peptides) or lipid-based media in aqueous formulations from aggregating at hydrophobic interfaces, specifically molecules that are prone to aggregation in aqueous media. Proteins and other biomolecules are often used to treat a variety of diseases and conditions; however, maintaining the stability of such formulations and preventing molecular aggregation are key challenges facing the biopharmaceutical industry. Proteins, other biomolecules, and lipid-based media (including but not limited to liposomes, micelles, polymersomes, and lipid nanoparticles) can aggregate due to a variety of factors, including thermal stress, chemical degradation, or exposure to surfaces and interfaces. Non-covalent physical aggregation is mediated by forces such as hydrophobic interactions, van der Waals interactions, hydrogen bonds, and electrostatic forces. Such molecules adsorb to various interfaces, specifically air-water interfaces, and play a key role in inducing aggregation. These aggregates can trigger an immunogenic response in vivo and lead to the production of anti-therapeutic antibodies.

[0066] It has been found that the polyacrylamide-based copolymers of the present disclosure reduce or prevent biomolecules and lipid-based vehicles in aqueous formulations from aggregating at hydrophobic interfaces, thereby producing increased stability. Polyacrylamide-based copolymers can be used with any biomolecule that is prone to aggregation in aqueous media, including but not limited to proteins, such as antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. Monoclonal antibodies (mAbs) are therapeutic proteins used to treat many diseases, but tend to aggregate, making their use a major challenge in formulation development. mAbs spontaneously adsorb to the air-solution interface and experience interfacial stress, which is one of the main causes of aggregation. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine. In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the biomolecule is a nucleic acid, such as mRNA, DNA, siRNA, and miRNA.

[0067] The polyacrylamide-based copolymers may also be used with lipid-based vehicles (including but not limited to liposomes, lipid nanoparticles, polymersomes, and micelles) that tend to aggregate in aqueous media to prevent or reduce aggregation.

[0068] The polyacrylamide-based copolymers of the present disclosure prevent aggregation of proteins, other biomolecules, and lipid-based vehicles such as liposomes, lipid nanoparticles, polymersomes, and micelles by providing an inert barrier at the hydrophobic interface of an aqueous formulation to prevent interactions between molecules such as protein-protein interactions. In some embodiments, the hydrophobic interface is an air-water interface. In some embodiments, the hydrophobic interface is a shell-water interface, including but not limited to a glass-water interface, a rubber-water interface, a plastic-water interface, or a metal-water interface. In some embodiments, the hydrophobic interface is an oil-water interface.

[0069] As described in the present disclosure, copolymers are identified as stabilizers that can serve as formulations containing biomolecules. In some embodiments, the formulation is a protein formulation. The copolymers based on polyacrylamide can also serve as stabilizers for formulations containing lipid-based media (including but not limited to liposomes, lipid nanoparticles, polymersomes and micelles) that are easy to aggregate in aqueous media. In some embodiments, high-throughput screening of a large library of copolymer excipients based on acrylamide is used to identify copolymers. In some embodiments, these copolymers enhance the stability of the formulation without any modification of the molecules in the formulation. For example, copolymers can enhance the stability of protein formulations without any protein modification. In some embodiments, copolymers including water-soluble "carrier" monomers and functional "dopant" monomers serve as stabilizing excipients to reduce the interaction of biomolecules with interfaces such as air-liquid interfaces. In some embodiments, the biomolecule is a protein. In some embodiments, copolymers including water-soluble "carrier" monomers and functional "dopant" monomers serve as stabilizing excipients to reduce the interaction of lipid-based media with interfaces such as air-liquid interfaces.

[0070] Therefore, the present disclosure provides a method for reducing the aggregation of biomolecules or lipid-based media, the lipid-based media including a polyacrylamide-based copolymer excipient as disclosed herein. In some embodiments, the biomolecule is a protein. In some embodiments, the protein is selected from antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants and combinations thereof. In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the lipid-based media is a liposome, a micelle, a polymersome or a lipid nanoparticle. The polyacrylamide-based copolymer of the present disclosure includes a water-soluble carrier monomer comprising an acrylamide-reactive portion and a functional dopant monomer comprising an acrylamide-reactive portion. In some embodiments, the copolymer includes MPAM or MORPH with a NIP or PHE dopant.

[0071] Also provided are methods for increasing the stability of formulations containing biomolecules or lipid-based vehicles. In some embodiments, methods for increasing the thermal stability of formulations containing biomolecules or lipid-based vehicles are provided. In other embodiments, methods for reducing the aggregation rate of biomolecules or lipid-based vehicles in aqueous compositions are provided. The methods comprise adding a copolymer of the present disclosure to the formulation.

[0072] In some embodiments, the biomolecule is a protein. In some embodiments, the protein is insulin. In some embodiments, the concerns about decreased insulin activity or prolonged circulation time typically associated with covalent insulin modifications (e.g., PEGylation) are reduced or eliminated.

[0073] The present disclosure also provides a method for treating an elevated blood sugar level in a subject in need thereof, the method comprising administering to the subject a composition containing a polyacrylamide-based copolymer of the present disclosure and insulin. A method for managing blood sugar levels in a subject in need thereof is also provided, the method comprising administering to the subject a composition containing a polyacrylamide-based copolymer of the present disclosure and insulin.

[0074] definition

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Methods and materials for the present application are described herein; in some aspects of the present disclosure, other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. When a trade name is used herein, unless the context otherwise indicates, the trade name includes product formulations, generic drugs, and active pharmaceutical ingredients of the trade name product.

[0076] The term "polymer" refers to a substance or material composed of repeating monomer subunits.

[0077] As used herein, "acrylamide monomer" refers to a monomeric species having an acrylamide functional group. The term "acrylamide monomer" includes not only monomer acrylamide but also derivatives of monomer acrylamide. Examples of acrylamide monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE).

[0078] The term "polyacrylamide-based copolymer" refers to a polymer formed by the polymerization of two or more monomer species, wherein at least one of the monomer species has an acrylamide functional group (acrylamide monomer), and the monomers are structurally different. In some embodiments, the polyacrylamide-based copolymer is formed by the polymerization of two structurally different acrylamide monomers (two structurally different monomers each having an acrylamide functional group). The resulting copolymer can be an alternating copolymer, in which the monomer species are connected in an alternating manner; a random copolymer, in which the monomer species are connected to each other within the polymer chain without a defined pattern; a block copolymer, in which a polymer block of one monomer species is connected to a polymer block made of another monomer species; and a graft copolymer, in which the main polymer chain consists of one monomer species, and a polymer block of another monomer species is connected to the main polymer chain as a side branch. In some embodiments, the polyacrylamide-based copolymer of the present disclosure is formed by the polymerization of a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer of the present disclosure is a random copolymer.

[0079] As defined herein, the term "water-soluble carrier monomer" refers to an acrylamide monomer species that is a water-soluble species in a polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer is the predominant species in the polyacrylamide-based copolymer. In some embodiments, the water-soluble carrier monomer imparts water solubility to the copolymer. In some embodiments, the water-soluble carrier monomer in the polyacrylamide-based copolymer provides an inert barrier at the interface of the aqueous formulation to prevent protein-protein interactions. In some embodiments, the interface is an air-water interface. In some embodiments, the interface is a shell-water interface, including but not limited to a glass-water interface, a rubber-water interface, a plastic-water interface, or a metal-water interface. In some embodiments, the interface is an oil-water interface. In some embodiments, the interface is an interface between a liquid and a pipe. In some embodiments, the interface is an interface between a liquid and a conduit. In some embodiments, the shell-water interface is located in a pump system. In some embodiments, the shell-water interface is located in a closed loop system. In some embodiments, the water-soluble carrier monomer is non-ionic. Examples of water-soluble carrier monomers include, but are not limited to, acrylamide (AM), N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), and N-hydroxyethylacrylamide (HEAM).

[0080] As used herein, the term "functional dopant monomer" refers to acrylamide monomer species having physicochemical properties (e.g., hydrophobicity, charge) different from those of water-soluble carrier monomers. In some embodiments, the functional dopant monomer in the copolymer based on polyacrylamide promotes the association of the polymer with the interface; such interfaces may include but are not limited to polymer-air-water interface interactions, polymer-protein interactions, polymer-peptide interactions, polymer-micelle interactions, polymer-liposome interactions, and polymer-lipid nanoparticle interactions. The functional dopant monomer can act as a stabilizing portion to promote interaction with biomolecules, such as proteins, peptides, antibodies, antibody-drug conjugates, nucleic acids, lipid particles, and combinations thereof (e.g., to prevent aggregation of biomolecules). Based on its chemical composition, the functional dopant monomer can be further classified into hydrogen bonding monomers, ionic monomers, hydrophobic monomers, and aromatic monomers. Typically, functional dopant monomers are copolymerized at a lower weight percentage than water-soluble carrier monomers.

[0081] The term "polymerization" refers to the process in which monomer molecules undergo chemical reactions to form polymer chains or three-dimensional networks. Different types of polymerization reactions are known in the art, such as addition (chain reaction) polymerization, condensation polymerization, ring-opening polymerization, free radical polymerization, controlled radical polymerization, atom transfer radical polymerization (ATRP), single electron transfer living radical polymerization (SET-LRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, nitroxide-mediated polymerization (NMP) and emulsion polymerization. In some embodiments, RAFT polymerization is used to prepare the copolymers of the present disclosure.

[0082] The term "degree of polymerization" (DP) refers to the number of monomer units in a polymer. The degree of polymerization is calculated by dividing the average molecular weight of a polymer sample by the molecular weight of the monomers. As defined herein, the average molecular weight of a polymer can be expressed by the number average molecular weight (M n ), weight average molecular weight (M w ), Z average molecular weight (M z ) or molecular weight distribution curve (M p ) is represented by the molecular weight at the maximum peak. The average molecular weight of a polymer can be determined by various analytical characterization techniques known to those skilled in the art, such as gel permeation chromatography (GPC), static light scattering (SLS) analysis, multi-angle laser scattering (MALLS) analysis, nuclear magnetic resonance spectroscopy (NMR), intrinsic viscosity method (IV), melt flow index (MFI) and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) and combinations thereof. The degree of polymerization can also be experimentally determined using suitable analytical methods known in the art such as nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy.

[0083] The term "amphiphilic" refers to a chemical substance that has both hydrophilic (water-loving, polar) and lipophilic (lipophilic, non-polar) properties. Examples of common amphiphilic compounds include detergents, soaps, surfactants, lipoproteins, and phospholipids. In some embodiments, the amphiphilic substance is a charged species. In some embodiments, the amphiphilic substance is a neutral species.

[0084] As used herein, "lipid-based vehicles" refer to structures having a protective outer layer of lipids that can be used as drug delivery vehicles. For example, lipid-based vehicles can be used to encapsulate and transport cargo (e.g., therapeutic agents) to biological targets. Examples of lipid-based vehicles include, but are not limited to, liposomes, micelles, polymersomes, and lipid nanoparticles.

[0085] As used herein, "biomolecule" refers to molecules such as proteins, nucleic acids, polysaccharides, and lipids.

[0086] The term "protein" is defined as a class of macromolecules comprising one or more long chains of amino acids. Various proteins can be considered to belong to a protein family based on having similar structural features, having specific biological functions and / or being associated with specific microorganisms, particularly pathogenic microorganisms. Such proteins include, for example, antibodies (immunoglobulins), cytokines, chemokines, enzymes, hormones, vaccine antigens, cancer antigens, adjuvants, nutritional markers, and tissue-specific antigens.

[0087] As used herein, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA) and micro RNA (miRNA).

[0088] The term "antibody" refers to a large Y-shaped protein produced by the immune system for identifying and neutralizing foreign objects such as pathogenic bacteria and viruses. The term "antibody" includes monoclonal antibodies (e.g., full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies, as long as they show desired biological activity), and may also include certain antibody fragments. Antibodies may be human, humanized and / or affinity matured. "Antibody fragments" include only a portion of a complete antibody, wherein in certain embodiments, the portion retains at least one function, typically most or all of the functions, associated with the portion when present in a complete antibody. In one embodiment, the antibody fragment includes the antigen binding site of the complete antibody, and thus retains the ability to bind antigens. In another embodiment, an antibody fragment, such as an antibody fragment including an Fc region, when present in a complete antibody, retains at least one biological function associated with the Fc region, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody, and the in vivo half-life of the monovalent antibody is substantially similar to that of a complete antibody. For example, such antibody fragments may include an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

[0089] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. In addition, compared to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0090] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0091] The term "insulin" refers to the hormone produced by the beta cells in the pancreatic islets that regulate the amount of glucose in the blood. Many eukaryotes, including humans, primates, pigs, cattle, cats, dogs and rodents, all produce insulin. Therefore, as used herein, "insulin" includes insulin produced by humans and its analogs, and insulin produced by other eukaryotes and its analogs, and the eukaryotes include but are not limited to primates, pigs, cattle, cats, dogs and rodents, and also includes purified or synthetic insulin or insulin analogs with similar function and restructuring of structure, unless otherwise indicated. Human insulin protein is composed of 51 amino acids, and the molecular weight is about 5.8 kilodaltons (kDa). Human insulin is a heterodimer of A chain and B chain connected by disulfide bonds.

[0092] Insulin also comprises monomeric form and oligomeric form, such as dimer form and hexameric form. Insulin can exist as a monomer when circulating in blood plasma, and also binds to its receptor when existing in monomeric form. Insulin formulations (or insulin analog formulations) containing protein molecules mainly in the form of monomers and dimers usually have a strong tendency to aggregate and form inactive fibrils. Insulin hexamer is too large to be absorbed, and therefore before insulin is absorbed and plays a role in vivo, hexameric insulin formulations must be decomposed into dimers or monomers. The active form of insulin in the bloodstream is the monomeric form.

[0093] Insulin can be isolated from pancreatic islet extracts of insulin-producing animals or recombinantly expressed in suitable expression systems such as Escherichia coli, yeast, insect cells, and mammalian cells (e.g., Chinese hamster ovary (CHO) cells). Insulin is a highly potent and effective drug for the treatment of acute respiratory syndrome (SARS) and acute respiratory syndrome (SARS). max ), onset time, area under the curve (AUC), insulin can be further characterized as rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin and premixed insulin.

[0094] The term "aggregation" refers to the formation of higher molecular weight amorphous species due to non-covalent adhesion ("aggregation") of smaller species. The aggregation process can be irreversible or reversible. Many biological and synthetic molecules can undergo aggregation, including proteins, peptides, lipid particles, nucleic acids, inorganic nanoparticles and organic nanoparticles (e.g., micelles, lipid nanoparticles, liposomes, polymersomes) that can further include encapsulated species.

[0095] In the case of protein aggregation, the formation of protein aggregates may be due to the inherent disordered nature of proteins or the misfolding of protein molecules, which results in the exposure of hydrophobic residues and surfaces that are usually buried within the interior of the protein's three-dimensional structure. Due to the hydrophobic effect, the exposed hydrophobic parts of misfolded proteins have a tendency to interact with other misfolded protein molecules to protect the exposed hydrophobic surfaces, which may lead to protein aggregation.

[0096] Some biomolecules are more "aggregation-prone" than others. For example, the amino acid sequence and overall three-dimensional structure of a protein are related to its susceptibility to aggregation. For example, transmembrane proteins are more prone to (or prone to) aggregation than non-membrane proteins, specifically when recombinantly expressed without the use of stabilizers. Proteins subjected to conditions other than physiological conditions (37°C, approximately neutral pH, isotonic) may also be more prone to aggregation than in their natural environment. Stress conditions such as temperature fluctuations, light, mechanical disturbances (e.g., shaking), surfaces, ultrasonic vibrations, pH changes, and ionic strength changes can affect protein stability and induce aggregation. Protein aggregation can lead to the formation of subvisible or visible particles (i.e., precipitation). The extent of subvisible protein aggregation can be measured by a variety of analytical methods known in the art, such as size exclusion chromatography (SEC), gel electrophoresis, asymmetric field flow fractionation (AF4), analytical ultracentrifugation (AUC), mass spectrometry (MS), optical microscopy, fluorescence microscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MALLS), flow imaging, turbidity / nephelometer, and transmittance measurement.

[0097] As used herein, the term "reduced aggregation" of a biomolecule or lipid-based medium includes all forms of reduced aggregation. Compared to the same biomolecule or lipid-based medium composition in the absence of a polyacrylamide-based copolymer of the present disclosure, the observed degree of aggregation or amount (e.g., in the composition) can be reduced. Therefore, "reduced aggregation" includes no observable aggregation or reduced aggregation amount (e.g., reduced aggregation protein level). Therefore, compared to the amount of aggregates of the same biomolecule or lipid-based medium in the absence of a polyacrylamide-based copolymer, the amount of aggregates present in the composition can be reduced by at least about 10mol%, about 20mol%, about 30mol%, about 40mol%, about 50mol%, about 60mol%, about 70mol%, about 80mol%, about 90mol% or about 100mol%. Aggregation can be measured by any method known in the art, including but not limited to size exclusion chromatography (SEC), gel electrophoresis, asymmetric field flow fractionation (AF4), analytical ultracentrifugation (AUC), mass spectrometry (MS), optical microscopy, fluorescence microscopy, dynamic light scattering (DLS), multi-angle laser light scattering (MALLS), flow imaging, turbidity / nephelometer, and transmittance measurement.

[0098] As used herein, the term "increased stability," when referring to a formulation containing a biomolecule or a lipid-based vehicle, means that the amount of aggregation is measurably reduced over a fixed period of time under test or fixed storage conditions compared to the amount of aggregates of the same biomolecule or lipid-based vehicle in the absence of the polyacrylamide-based copolymer.

[0099] As used herein, the term "aggregated protein" or "protein aggregate" refers to a collection of disordered or misfolded proteins that are grouped together. Aggregates can be soluble or insoluble. Protein aggregates include but are not limited to inclusion bodies, soluble and insoluble precipitates, soluble non-native oligomers, gels, fibrils, membranes, filaments, protofibrils, amyloid deposits, amyloid fibrils, plaques, and dispersed non-native intracellular oligomers. In some embodiments, the protein in the protein aggregate is a soluble precursor before it aggregates. In a composition containing a polyacrylamide-based copolymer of the present invention, protein aggregation can be prevented. Protein aggregation in a composition containing a polyacrylamide-based copolymer of the present invention can also be reduced compared to a composition containing the same protein but without a polyacrylamide-based copolymer of the present invention. Therefore, a polyacrylamide-based copolymer can reduce or prevent the aggregation of proteins.

[0100] In certain embodiments, the protein is insulin or its analog. As used herein, the term "aggregated insulin" or "insulin aggregates" refers to insulin that has aggregated to form high molecular weight polymers or aggregate particles or amyloid fibrils. The formation of insulin aggregates may be due to, for example, heating or shaking and partial unfolding of insulin. In a composition containing a copolymer based on polyacrylamide of the present disclosure, insulin aggregation can be prevented, i.e., insulin molecules are prevented from aggregating and forming high molecular weight polymers or amyloid fibrils. Aggregation in a composition containing insulin or its analog but not containing a copolymer based on polyacrylamide of the present disclosure can also be reduced. Therefore, copolymers based on polyacrylamide can reduce or prevent the aggregation of insulin or its analog.

[0101] As defined herein, the term "duration of action" is defined as the length of time that the injected insulin exerts its effect of lowering blood glucose levels, i.e., the time frame over which the injected insulin continues to be effective. The term "maximum observed concentration" (C max ) refers to the peak serum concentration reached by an active agent in a specific chamber or test area of ​​the body after the first dose of the active agent is applied and before the second dose is applied. The term "onset time" refers to the amount of time required for the active agent to reach the minimum effective concentration after application. The term "area under the curve" (AUC) refers to the definite integral of the curve describing the concentration of the active agent in the plasma over time, which can be measured by analytical methods such as liquid chromatography-mass spectrometry (LC-MS). AUC reflects the exposure of the active agent after application and is expressed in mg*h / L. The AUC of an active agent depends on the rate at which the active agent is eliminated from the body and the dose applied.

[0102] Polyacrylamide-based copolymers

[0103] The present disclosure provides a polyacrylamide-based copolymer. In some embodiments, the polyacrylamide-based copolymer contains a water-soluble carrier monomer and a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer is amphiphilic.

[0104] In some embodiments, the polyacrylamide-based copolymer includes a nonionic water-soluble acrylamide monomer and a functional acrylamide dopant monomer, and the functional acrylamide dopant monomer is selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromatic functional acrylamide dopant monomer, a hydrogen bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes a nonionic water-soluble acrylamide monomer, and the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the copolymer includes a nonionic water-soluble acrylamide monomer, and the functional acrylamide dopant monomer is an aromatic functional acrylamide dopant monomer. In some embodiments, the copolymer includes a nonionic water-soluble acrylamide monomer, and the functional acrylamide dopant monomer is a hydrogen bonding functional acrylamide dopant monomer. In some embodiments, the copolymer includes a nonionic water-soluble acrylamide monomer, and the functional acrylamide dopant monomer is an ionic functional acrylamide dopant monomer.

[0105] The polyacrylamide-based copolymers disclosed herein contain water-soluble carrier monomers. In some embodiments, the water-soluble carrier monomers are nonionic. In some embodiments, the water-soluble carrier monomers are selected from the group consisting of: N-(3-methoxypropionyl) acrylamide (MPAM), 4-acryloyl morpholine (MORPH), N, N-dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM) and acrylamide (AM) or a combination thereof. In some embodiments, the water-soluble carrier monomers are selected from the group consisting of MPAM and MORPH. In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropionyl) acrylamide (MPAM). In some embodiments, the water-soluble carrier monomer is 4-acryloyl morpholine (MORPH). In some embodiments, the water-soluble carrier monomer is N, N-dimethylacrylamide (DMA). In some embodiments, the water-soluble carrier monomer is N-hydroxyethyl acrylamide (HEAM). In some embodiments, the water-soluble carrier monomer is acrylamide (AM). In some embodiments, the copolymer includes a water-soluble carrier monomer selected from the group consisting of N-(3-methoxypropionyl)acrylamide (MPAM) and 4-acryloylmorpholine (MORPH).

[0106] The polyacrylamide-based copolymer of the present disclosure further contains a functional dopant monomer. In some embodiments, the functional dopant monomer is selected from the group consisting of a hydrophobic functional acrylamide dopant monomer, an aromatic functional acrylamide dopant monomer, a hydrogen bonding functional acrylamide dopant monomer, and an ionic functional acrylamide dopant monomer or a mixture thereof.

[0107] In some embodiments, the functional acrylamide dopant monomer is a hydrophobic functional acrylamide dopant monomer. In some embodiments, the hydrophobic functional acrylamide dopant monomer is N-isopropyl acrylamide (NIP) or N-tert-butyl acrylamide (TBA). In some embodiments, the hydrophobic functional acrylamide dopant monomer is N-isopropyl acrylamide (NIP). In some embodiments, the hydrophobic functional acrylamide dopant monomer is N-tert-butyl acrylamide (TBA). In some embodiments, the functional acrylamide dopant monomer is an aromatic functional acrylamide dopant monomer. In some embodiments, the aromatic functional acrylamide dopant monomer is N-phenyl acrylamide (PHE).

[0108] In some embodiments, the functional acrylamide dopant monomer is a hydrogen bonding functional acrylamide dopant monomer. In some embodiments, the hydrogen bonding functional acrylamide dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylamide (TRI). In some embodiments, the functional acrylamide dopant monomer is an ionic functional acrylamide dopant monomer. In some embodiments, the ionic functional acrylamide dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP) or (3-acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the ionic functional acrylamide dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP). In some embodiments, the ionic functional acrylamide dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA).

[0109] In some embodiments, the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE) or a combination thereof. In some embodiments, the functional dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylamide (TRI). In some embodiments, the functional dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP). In some embodiments, the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the functional dopant monomer is N-isopropylacrylamide (NIP). In some embodiments, the functional dopant monomer is N-tert-butylacrylamide (TBA). In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE).

[0110] In some embodiments, the polyacrylamide-based copolymer includes a water-soluble carrier monomer and a functional dopant monomer, wherein the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE).

[0111] In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropionyl) acrylamide (MPAM). In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropionyl) acrylamide (MPAM), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl] acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl) trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE). In some embodiments, the water-soluble carrier monomer is MPAM, and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is MPAM, and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is MPAM, and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is MPAM, and the functional dopant monomer is NIP. In some embodiments, the water soluble carrier monomer is MPAM and the functional dopant monomer is TBA. In some embodiments, the water soluble carrier monomer is MPAM and the functional dopant monomer is PHE.

[0112] In some embodiments, the water-soluble carrier monomer is 4-acryloylmorpholine (MORPH). In some embodiments, the water-soluble carrier monomer is 4-acryloylmorpholine (MORPH), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE). In some embodiments, the water-soluble carrier monomer is MORPH, and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is MORPH, and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is MORPH, and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is MORPH, and the functional dopant monomer is NIP. In some embodiments, the water soluble carrier monomer is MORPH and the functional dopant monomer is TBA. In some embodiments, the water soluble carrier monomer is MORPH and the functional dopant monomer is PHE.

[0113] In some embodiments, the water-soluble carrier monomer is N,N-dimethylacrylamide (DMA). In some embodiments, the water-soluble carrier monomer is N,N-dimethylacrylamide (DMA), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE). In some embodiments, the water-soluble carrier monomer is DMA, and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is DMA, and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is DMA, and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is DMA, and the functional dopant monomer is NIP. In some embodiments, the water-soluble carrier monomer is DMA, and the functional dopant monomer is TBA. In some embodiments, the water soluble carrier monomer is DMA and the functional dopant monomer is PHE.

[0114] In some embodiments, the water-soluble carrier monomer is N-hydroxyethyl acrylamide (HEAM). In some embodiments, the water-soluble carrier monomer is N-hydroxyethyl acrylamide (HEAM), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl] acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl) trimethylammonium chloride (TMA), N-isopropyl acrylamide (NIP), N-tert-butyl acrylamide (TBA) and N-phenyl acrylamide (PHE). In some embodiments, the water-soluble carrier monomer is HEAM, and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is HEAM, and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is HEAM, and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is HEAM, and the functional dopant monomer is NIP. In some embodiments, the water soluble carrier monomer is HEAM and the functional dopant monomer is TBA. In some embodiments, the water soluble carrier monomer is HEAM and the functional dopant monomer is PHE.

[0115] In some embodiments, the water-soluble carrier monomer is acrylamide (AM). In some embodiments, the water-soluble carrier monomer is acrylamide (AM), and the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA), and N-phenylacrylamide (PHE). In some embodiments, the water-soluble carrier monomer is AM, and the functional dopant monomer is TRI. In some embodiments, the water-soluble carrier monomer is AM, and the functional dopant monomer is AMP. In some embodiments, the water-soluble carrier monomer is AM, and the functional dopant monomer is TMA. In some embodiments, the water-soluble carrier monomer is AM, and the functional dopant monomer is NIP. In some embodiments, the water-soluble carrier monomer is AM, and the functional dopant monomer is TBA. In some embodiments, the water soluble carrier monomer is AM and the functional dopant monomer is PHE.

[0116] In some embodiments, the functional dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylamide (TRI). In some embodiments, the functional dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), and acrylamide (AM). In some embodiments, the functional dopant monomer is TRI, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is TRI, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is TRI, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is TRI, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is TRI, and the water-soluble carrier monomer is AM.

[0117] In some embodiments, the functional dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP). In some embodiments, the functional dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl) acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM). In some embodiments, the functional dopant monomer is AMP, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is AMP, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is AMP, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is AMP, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is AMP, and the water-soluble carrier monomer is AM.

[0118] In some embodiments, the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM). In some embodiments, the functional dopant monomer is TMA, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is TMA, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is TMA, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is TMA, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is TMA, and the water-soluble carrier monomer is AM.

[0119] In some embodiments, the functional dopant monomer is N-isopropylacrylamide (NIP). In some embodiments, the functional dopant monomer is N-isopropylacrylamide (NIP), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM). In some embodiments, the functional dopant monomer is NIP, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is NIP, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is NIP, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is NIP, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is NIP, and the water-soluble carrier monomer is AM.

[0120] In some embodiments, the functional dopant monomer is N-tert-butylacrylamide (TBA). In some embodiments, the functional dopant monomer is N-tert-butylacrylamide (TBA), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl) acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM). In some embodiments, the functional dopant monomer is TBA, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is TBA, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is TBA, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is TBA, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is TBA, and the water-soluble carrier monomer is AM.

[0121] In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE). In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE), and the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl)acrylamide (MPAM), 4-acryloylmorpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) and acrylamide (AM). In some embodiments, the functional dopant monomer is PHE, and the water-soluble carrier monomer is MPAM. In some embodiments, the functional dopant monomer is PHE, and the water-soluble carrier monomer is MORPH. In some embodiments, the functional dopant monomer is PHE, and the water-soluble carrier monomer is DMA. In some embodiments, the functional dopant monomer is PHE, and the water-soluble carrier monomer is HEAM. In some embodiments, the functional dopant monomer is PHE, and the water-soluble carrier monomer is AM.

[0122] In some embodiments, the polyacrylamide-based copolymer includes N-(3-methoxypropionyl) acrylamide (MPAM) or 4-acryloylmorpholine (MORPH) as a water-soluble carrier monomer and N-isopropylacrylamide (NIP) or N-phenylacrylamide (PHE) as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) or acrylamide (AM) as a water-soluble carrier monomer and N-isopropylacrylamide (NIP) or N-phenylacrylamide (PHE) as a functional dopant monomer.

[0123] In some embodiments, the polyacrylamide-based copolymer includes N-(3-methoxypropionyl)acrylamide (MPAM) or 4-acryloylmorpholine (MORPH) as a water-soluble carrier monomer and N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA) or N-tert-butylacrylamide (TBA) as a functional dopant monomer.

[0124] In some embodiments, the polyacrylamide-based copolymer includes N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) or acrylamide (AM) as a water-soluble carrier monomer, and the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride (TMA). In some embodiments, the polyacrylamide-based copolymer includes N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) or acrylamide (AM) as a water-soluble carrier monomer, and the functional dopant monomer is N-tert-butylacrylamide (TBA). In some embodiments, the polyacrylamide-based copolymer includes N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM) or acrylamide (AM) as a water-soluble carrier monomer, and the functional dopant monomer is N-[tris(hydroxymethyl)-methyl]acrylamide (TRI). In some embodiments, the polyacrylamide-based copolymer includes N,N-dimethylacrylamide (DMA), N-hydroxyethylacrylamide (HEAM), or acrylamide (AM) as a water-soluble carrier monomer, and the functional dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid (AMP).

[0125] In some embodiments, the amount of functional dopant monomer used in the copolymerization reaction is designed to maximize dopant loading while producing a functional copolymer having a lower critical solution temperature (LCST) value above 37° C. In some embodiments, this results in a copolymer that remains soluble at all relevant temperatures. In some embodiments, the polyacrylamide-based copolymer includes about 2 wt % to about 30 wt % of a functional dopant monomer, for example, about 5 wt % to about 30 wt %, about 10 wt % to about 30 wt %, about 15 wt % to about 30 wt %, about 20 wt % to about 30 wt %, about 25 wt % to about 30 wt %, about 2 wt % to about 25 wt %, about 5 wt % to about 25 wt %, about 10 wt % to about 25 wt %, about 15 wt % to about 25 wt %, about 20 wt % to about 25 wt %, about 2 wt % to about 20 wt %, about 5 wt % to about 20 wt %, about 10 wt % to about 20 wt %, about 15 wt % to about 20 wt %, about 2 wt % to about 15 wt %, about 5 wt % to about 15 wt %, about 10 wt % to about 15 wt %, about 2 wt % to about 10 wt %, about 5 wt % to about 10 wt %, or about 2 wt % to about 5 wt % of a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes about 2 wt%, about 5 wt%, about 8 wt%, about 10 wt%, about 12 wt%, about 15 wt%, about 18 wt%, about 20 wt%, about 22 wt%, about 25 wt%, about 28 wt%, or about 30 wt% of the functional dopant monomer.

[0126] In some embodiments, the polyacrylamide-based copolymer includes about 70% to about 98% by weight of a water-soluble carrier monomer, such as about 75% to about 98% by weight, about 80% to about 98% by weight, about 85% to about 98% by weight, about 90% to about 98% by weight, about 95% to about 98% by weight, about 70% to about 95% by weight, about 75% to about 95% by weight, about 80% to about 95 ... In some embodiments, the polyacrylamide-based copolymer comprises about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 90%, about 85%, about 90%, about 70%, about 85%, about 75%, about 85%, about 80%, about 85%, about 70%, about 80%, about 75%, about 80%, about 85%, about 70%, about 80%, about 75%, about 80%, about 70%, about 80%, about 75%, about 80%, or about 70% to 75% by weight of a water-soluble carrier monomer. In some embodiments, the polyacrylamide-based copolymer comprises about 70%, about 72%, about 75%, about 78%, about 80%, about 82%, about 85%, about 88%, about 90%, about 92%, about 95%, or about 98% by weight of a water-soluble carrier monomer.

[0127] In some embodiments, the polyacrylamide-based copolymer includes about 70% to about 98% by weight of a water-soluble carrier monomer and about 2% to about 30% by weight of a functional dopant monomer. For example, the polyacrylamide-based copolymer may contain about 70% to about 98% by weight, about 70% to about 95% by weight, about 70% to about 80% by weight, about 80% to about 90% by weight, about 90% to about 98% by weight, about 70% by weight, about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, about 95% by weight, or about 98% by weight of a water-soluble carrier monomer and about 2% to about 30% by weight, about 25% to about 30% by weight, about 25% to about 30% by weight, about 20% to about 25% by weight, about 25% to about 20% by weight, about 20% to about 25% by weight, about 25% to about 20% by weight, about 20% to about 25% by weight, about 20% to about 25% by weight, about 25% to about 25% by weight, about 20 ... to about 20 wt%, about 2 wt% to about 5 wt%, about 5 wt% to about 10 wt%, about 10 wt% to about 15 wt%, about 15 wt% to about 20 wt%, about 20 wt% to about 25 wt%, or about 25 wt% to about 30 wt%, about 2 wt%, about 5 wt%, about 8 wt%, about 10 wt%, about 12 wt%, about 15 wt%, about 18 wt%, about 20 wt%, about 22 wt%, about 25 wt%, about 28 wt%, or about 30 wt% of a functional dopant monomer.

[0128] In some embodiments, the polyacrylamide-based copolymer includes about 2 wt % to about 30 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 5 wt % to about 30 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 10 wt % to about 28 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 5 wt % to about 26 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 5 wt % to about 10 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 10 wt % to about 15 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 15 wt % to about 20 wt % of the functional dopant monomer NIP. In some embodiments, the polyacrylamide-based copolymer includes about 20 wt % to about 26 wt % of the functional dopant monomer NIP.

[0129] In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 2% to about 30% by weight of NIP as a functional dopant monomer. In some embodiments, the copolymer includes MORPH and about 5% to about 30% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 10% to about 28% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 5% to about 26% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 5% to about 10% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 10% to about 15% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 15% to about 20% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 20% to about 25% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 25% to about 30% by weight of NIP. In some embodiments, the copolymer includes MORPH and about 20% to about 28% by weight of NIP. In some embodiments, the copolymer comprises MORPH and about 21 wt % NIP. In some embodiments, the copolymer comprises MORPH and about 22 wt % NIP. In some embodiments, the copolymer comprises MORPH and about 23 wt % NIP. In some embodiments, the copolymer comprises MORPH and about 24 wt % NIP. In some embodiments, the copolymer comprises MORPH and about 25 wt % NIP.

[0130] In some embodiments, the polyacrylamide-based copolymer includes AMP, TMA, TBA or PHE as a functional dopant monomer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 2% to about 16% by weight of the copolymer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 5% to about 15% by weight of the copolymer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 6% to about 14% by weight of the copolymer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 12% to about 15% by weight of the copolymer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 2% to about 5% by weight of the copolymer. In some embodiments, AMP, TMA, TBA or PHE functional dopant monomers are present in about 5% to about 10% by weight of the copolymer.

[0131] In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 2% to about 16% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 4% to about 16% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 6% to about 14% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 8% to about 14% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 10% to about 14% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MORPH as a water-soluble carrier monomer and about 10% to about 12% by weight of PHE as a functional dopant monomer.

[0132] In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 2% to about 16% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 5% to about 15% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 6% to about 10% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 7% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 8% by weight of PHE as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes MPAM as a water-soluble carrier monomer and about 9% by weight of PHE as a functional dopant monomer.

[0133] In some embodiments, the polyacrylamide-based copolymer includes about 3 wt % to about 17 wt % TRI as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes about 7 wt % to about 12 wt % TRI as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes about 4 wt % to about 6 wt % TRI as a functional dopant monomer. In some embodiments, the polyacrylamide-based copolymer includes about 13 wt % to about 17 wt % TRI as a functional dopant monomer.

[0134] In some embodiments, the degree of polymerization (DP) of the polyacrylamide-based copolymer is about 10 to about 500, about 20 to about 200, about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, or about 400 to about 500, or about 10, about 50, about 70, about 100, about 120, about 150, about 170, about 200, about 220, about 250, about 270, about 300, about 320, about 350, about 370, about 400, about 420, about 450, about 470, or about 500. In some embodiments, the DP of the copolymer is about 40. In some embodiments, the DP of the copolymer is about 50. In some embodiments, the DP of the copolymer is about 60. In some embodiments, the DP of the copolymer is about 70. In some embodiments, the DP of the copolymer is about 80. In some embodiments, the DP of the copolymer is about 90. In some embodiments, the copolymer has a DP of about 100.

[0135] In some embodiments, the molecular weight of the polyacrylamide-based copolymer is from about 1,000 g / mol to about 40,000 g / mol, such as from about 1,000 g / mol to about 35,000 g / mol, from about 1,000 g / mol to about 30,000 g / mol, from about 1,000 g / mol to about 25,000 g / mol, from about 1,000 g / mol to about 20,000 g / mol, from about 1,000 g / mol to about 15,000 g / mol, from about 1,000 g / mol to about 10,000 g / mol, from about 1,000 g / mol to about 7,000 g / mol, from about 1,000 g / mol to about 6,000 g / mol. 1, about 1,000 g / mol to about 5,000 g / mol, about 1,000 g / mol to about 4,000 g / mol, about 1,000 g / mol to about 3,000 g / mol, about 3,000 g / mol to about 40,000 g / mol, about 3,000 g / mol to about 35,000 g / mol, about 3,000 g / mol to about 30,000 g / mol, about 3,000 g / mol to about 25,000 g / mol, about 3,000 g / mol to about 20,000 g / mol, about 3,000 g / mol to about 15,000 g / mol, about 3,000 g / mol to about 10,000 g / mol , about 3,000 g / mol to about 7,000 g / mol, about 3,000 g / mol to about 6,000 g / mol, about 3,000 g / mol to about 5,000 g / mol, about 3,000 g / mol to about 4,000 g / mol, about 4,000 g / mol to about 40,000 g / mol, about 4,000 g / mol to about 35,000 g / mol, about 4,000 g / mol to about 30,000 g / mol, about 4,000 g / mol to about 25,000 g / mol, about 4,000 g / mol to about 20,000 g / mol, about 4,000 g / mol to about 15,000 g / mol, about g / mol, about 4,000 g / mol to about 10,000 g / mol, about 4,000 g / mol to about 7,000 g / mol, about 4,000 g / mol to about 6,000 g / mol, about 4,000 g / mol to about 5,000 g / mol, about 5,000 g / mol to about 40,000 g / mol, about 5,000 g / mol to about 35,000 g / mol, about 5,000 g / mol to about 30,000 g / mol, about 5,000 g / mol to about 25,000 g / mol, about 5,000 g / mol to about 20,000 g / mol, about 5,000 g / mol to about 15,000 g / mol, about 5,g / mol, about 5,000 g / mol to about 7,000 g / mol, about 5,000 g / mol to about 6,000 g / mol, about 6,000 g / mol to about 40,000 g / mol, about 6,000 g / mol to about 35,000 g / mol, about 6,000 g / mol to about 30,000 g / mol, about 6,000 g / mol to about 25,000 g / mol, about 6,000 g / mol to about 20,000 g / mol, about 6,000 g / mol to about 15,000 g / mol, about 6,000 g / mol to about 10,000 g / mol, about 6,000 g / mol to about 7,000 g / mol, about 7,000 g / mol to about 40,000 g / mol, about 7,000 g / mol to about 35,000 g / mol, about 7000 g / mol to about 30,000 g / mol, about 7,000 g / mol to about 25,000 g / mol, about 7,000 g / mol to about 20,000 g / mol, about 7,000 g / mol to about 15,000 g / mol, about 7,000 g / mol to about 10,000 g / mol, about 10,000 g / mol to about 40,000 g / mol, about 10,000 g / mol to about 35,000 g / mol, about 7000 g / mol to about 30,000 g / mol, about 7,000 g / mol to about 25,000 g / mol, about 7,000 g / mol to about 20,000 g / mol, about 7,000 g / mol to about 15,000 g / mol, about 7,000 g / mol to about 10,000 g / mol, about 10,000 g / mol to about 40,000 g / mol, about 10,000 g / mol to about 35,000 g / mol g / mol, about 10,000 g / mol to about 30,000 g / mol, about 10,000 g / mol to about 25,000 g / mol, about 10,000 g / mol to about 20,000 g / mol, about 10,000 g / mol to about 15,000 g / mol, about 15,000 g / mol to about 40,000 g / mol, about 15,000 g / mol to about 35,000 g / mol, about 15,000 g / mol to about 30,000 g / mol, about 15,000 g / mol to about 25,000 g / mol, about 15,000 g / mol to about 20,000 g / mol, about 20,000 g / mol g / mol to about 40,000 g / mol, about 20,000 g / mol to about 35,000 g / mol, about 20,000 g / mol to about 30,000 g / mol, about 20,000 g / mol to about 25,000 g / mol, about 25,000 g / mol to about 40,000 g / mol, about 25,000 g / mol to about 35,000 g / mol, about 25,000 g / mol to about 30,000 g / mol, about 30,000 g / mol to about 40,000 g / mol, about 30,000 g / mol to about 35,000 g / mol, or about 35,000 g / mol to about 40,000 g / mol.In some embodiments, the molecular weight of the copolymer is about 1,000 to about 30,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 10,000 to about 20,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 15,000 to about 20,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 20,000 to about 25,000 g / mol. The molecular weight of the copolymer is about 25,000 to about 30,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 30,000 to about 40,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 2,000 to about 10,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 3,000 to about 7,000 g / mol. In some embodiments, the molecular weight of the copolymer is about 4,000 to about 6,000 g / mol. ,

[0136] The present disclosure also provides a polyacrylamide-based copolymer, the polyacrylamide-based copolymer containing a water-soluble carrier monomer including an acrylamide-reactive portion and a functional dopant monomer including an acrylamide-reactive portion. In some embodiments, the polyacrylamide-based copolymer includes about 70% to about 98% of a water-soluble carrier monomer having an acrylamide-reactive portion and about 2% to about 30% of a functional dopant monomer having an acrylamide-reactive portion. In some embodiments, the number average molecular weight (M) of the copolymer is about 100%. n ) is about 1,000 g / mol to about 30,000 g / mol. In some embodiments, the degree of polymerization is about 10 to about 250. In some embodiments, the water-soluble carrier monomer is nonionic. In some embodiments, the functional dopant monomer is hydrophobic. In some embodiments, the copolymer is amphiphilic.

[0137] In some embodiments, the water-soluble carrier monomer is selected from the group consisting of: N-(3-methoxypropionyl) acrylamide (MPAM), 4-acryloyl morpholine (MORPH), N,N-dimethylacrylamide (DMA), N-hydroxyethyl acrylamide (HEAM) and acrylamide (AM). In some embodiments, the water-soluble carrier monomer is selected from the group consisting of N-(3-methoxypropionyl) acrylamide (MPAM) and 4-acryloyl morpholine (MORPH). In some embodiments, the water-soluble carrier monomer is N-(3-methoxypropionyl) acrylamide (MPAM). In some embodiments, the water-soluble carrier monomer is 4-acryloyl morpholine (MORPH).

[0138] In some embodiments, the functional dopant monomer is selected from the group consisting of: N-[tris(hydroxymethyl)-methyl]acrylamide (TRI), 2-acrylamido-2-methylpropanesulfonic acid (AMP), (3-acrylamidopropyl)trimethylammonium chloride (TMA), N-isopropylacrylamide (NIP), N-tert-butylacrylamide (TBA) and N-phenylacrylamide (PHE). In some embodiments, the functional dopant monomer is selected from the group consisting of N-isopropylacrylamide (NIP) and N-phenylacrylamide (PHE). In some embodiments, the functional dopant monomer is N-isopropylacrylamide (NIP). In some embodiments, the functional dopant monomer is N-phenylacrylamide (PHE).

[0139] In some embodiments, the polyacrylamide-based copolymer comprises about 70 wt % to about 95 wt % of the water-soluble carrier monomer MORPH and about 5 wt % to about 30 wt % of the functional dopant monomer NIP, wherein the number average molecular weight (M n ) is about 1,000 g / mol to about 10,000 g / mol, and the degree of polymerization is about 10 to about 100. In some embodiments, the polyacrylamide-based copolymer includes about 74 wt % to about 80 wt % of the water-soluble carrier monomer MORPH and about 20 wt % to about 26 wt % of the functional dopant monomer NIP, wherein the number average molecular weight (M) of the copolymer is about 1,000 g / mol to about 10,000 g / mol, and the degree of polymerization is about 10 to about 100. n ) is about 1,000 g / mol to about 5,000 g / mol, and the degree of polymerization is about 10 to about 50. In some embodiments, the polyacrylamide-based copolymer includes about 77 wt % of the water-soluble carrier monomer MORPH and about 23 wt % of the functional dopant monomer NIP, wherein the number average molecular weight (M) of the copolymer is about 1,000 g / mol to about 5,000 g / mol, and the degree of polymerization is about 10 to about 50. n ) is about 3,200 g / mol, and the degree of polymerization is about 26.

[0140] Compositions containing polyacrylamide-based copolymers

[0141] Compositions containing the polyacrylamide-based copolymers described in the present disclosure are also provided. In some embodiments, the composition includes the polyacrylamide-based copolymers of the present disclosure and a pharmaceutically acceptable excipient.

[0142] In some embodiments, the composition includes a polyacrylamide-based copolymer of the present disclosure and a biomolecule. In some embodiments, the biomolecule is a protein. In some embodiments, the protein is a protein that is prone to aggregation in an aqueous medium. In some embodiments, the copolymer is uncharged, cationic or anionic. In some embodiments, the copolymer is amphiphilic. In some embodiments, the carrier monomer is a water-soluble species and is responsible for both maintaining solubility and providing an inert barrier to prevent biological species from aggregating, such as preventing protein-protein interactions. In some embodiments, the water-soluble carrier monomer is the main species in the polyacrylamide-based copolymer. In some embodiments, the functional dopant monomer is copolymerized at a lower weight percentage and statistically incorporated into the entire resulting copolymer. In some embodiments, these dopants are selected to promote polymer-interface interactions or polymer-protein interactions.

[0143] In some embodiments, the concentration of the copolymer in a composition containing the polyacrylamide-based copolymer of the present disclosure and a biomolecule or lipid-based vehicle is from about 0.0001% to about 5% by weight of the composition, such as from about 0.0001% to about 4% by weight, from about 0.0001% to about 3% by weight, from about 0.0001% to about 2% by weight, from about 0.0001% to about 1% by weight, from about 0.0001% to about 0.5% by weight, from about 0.0001% to about 0.4% by weight, from about 0.0001% to about 0.3% by weight, from about 0.0001% to about 0.2% by weight, from about 0.0001% to about 0.1% by weight, from about 0.0001% to about 0.2% by weight of the composition. % by weight, about 0.005 wt % to about 0.5 wt %, about 0.005 wt % to about 0.6 wt %, about 0.005 wt % to about 0.7 wt %, about 0.005 wt % to about 0.8 wt %, about 0.005 wt % to about 1.0 wt %, about 0.005 wt % to about 0.1 wt %, about 0.005 wt % to about 0.2 wt %, about 0.005 wt % to about 0.1 wt %, about 0.005 wt % to about 0.5 wt %, about 0.005 wt % to about 0.4 wt %, about 0.005 wt % to about 0.3 wt %, about 0.005 wt % to about 0.2 wt %, about 0.005 wt % to about 0.1 wt %, From about 0.005 wt % to about 0.05 wt %, from about 0.005 wt % to about 0.02 wt %, from about 0.005 wt % to about 0.01 wt %, from about 0.01 wt % to about 5 wt %, from about 0.01 wt % to about 4 wt %, from about 0.01 wt % to about 3 wt %, from about 0.01 wt % to about 2 wt %, from about 0.01 wt % to about 1 wt %, from about 0.01 wt % to about 0.5 wt %, from about 0.01 wt % to about 0.4 wt %, from about 0.01 wt % to about 0.3 wt %, from about 0.01 wt % to about 0.2 wt %, from about 0.01 wt % to about 0.1 wt %, from about 0.01 wt % to about 0.05 wt %, from about 0.01 wt % to about 0. 0.02 wt %, about 0.02 wt % to about 5 wt %, about 0.02 wt % to about 4 wt %, about 0.02 wt % to about 3 wt %, about 0.02 wt % to about 2 wt %, about 0.02 wt % to about 1 wt %, about 0.02 wt % to about 0.5 wt %, about 0.02 wt % to about 0.4 wt %, about 0.02 wt % to about 0.3 wt %, about 0.02 wt % to about 0.2 wt %, about 0.02 wt % to about 0.1 wt %, about 0.02 wt % to about 0.05 wt %, about 0.05 wt % to about 5 wt %, about 0.05 wt % to about 4 wt %, about 0.05 wt % to about 3 wt %, about 0.05 wt % to about 2 wt %, about 0.% to about 0.5 wt %, about 0.05 wt % to about 0.4 wt %, about 0.05 wt % to about 0.3 wt %, about 0.05 wt % to about 0.2 wt %, about 0.05 wt % to about 0.1 wt %, about 0.1 wt % to about 5 wt %, about 0.1 wt % to about 4 wt %, about 0.1 wt % to about 3 wt %, about 0.1 wt % to about 2 wt %, about 0.1 wt % to about 1 wt %, about 0.1 wt % to about 0.5 wt %. %, about 0.1 wt % to about 0.4 wt %, about 0.1 wt % to about 0.3 wt %, about 0.1 wt % to about 0.2 wt %, about 0.2 wt % to about 5 wt %, about 0.2 wt % to about 4 wt %, about 0.2 wt % to about 3 wt %, about 0.2 wt % to about 2 wt %, about 0.12 wt % to about 1 wt %, about 0.2 wt % to about 0.5 wt %, about 0.2 wt % to about 0.4 wt %, about 0.2 wt % to about 0.3 wt %, about 0.3 wt % to about 5 wt % , about 0.3 wt % to about 4 wt %, about 0.3 wt % to about 3 wt %, about 0.3 wt % to about 2 wt %, about 0.3 wt % to about 1 wt %, about 0.3 wt % to about 0.5 wt %, about 0.3 wt % to about 0.4 wt %, about 0.4 wt % to about 5 wt %, about 0.4 wt % to about 4 wt %, about 0.4 wt % to about 3 wt %, about 0.4 wt % to about 2 wt %, about 0.4 wt % to about 1 wt %, about 0.4 wt % to about 0.5 wt %, about 0.5 wt % % to about 5 wt %, about 0.5 wt % to about 4 wt %, about 0.5 wt % to about 3 wt %, about 0.5 wt % to about 2 wt %, about 0.5 wt % to about 1 wt %, about 1 wt % to about 5 wt %, about 1 wt % to about 4 wt %, about 1 wt % to about 3 wt %, about 1 wt % to about 2 wt %, about 2 wt % to about 5 wt %, about 2 wt % to about 4 wt %, about 2 wt % to about 3 wt %, about 3 wt % to about 5 wt %, about 3 wt % to about 4 wt %, or about 4 wt % to about 5 wt %. In some embodiments, the copolymer concentration is from about 0.0001 wt % to about 5 wt % of the composition. In some embodiments, the copolymer concentration is 0.001% to about 1% by weight, about 0.005% to about 0.5% by weight, about 0.005% to about 0.02% by weight, about 0.01% to about 0.2% by weight, about 0.1% to about 0.4% by weight, or about 0.2% to about 0.3% by weight of the composition. In some embodiments, the copolymer concentration is about 0.005% by weight of the composition. In some embodiments, the copolymer concentration is about 0.01% by weight of the composition. In some embodiments, the copolymer concentration is about 0.05% by weight of the composition.05 wt %. In some embodiments, the copolymer concentration is about 0.1 wt % of the composition. In some embodiments, the copolymer concentration is about 1 wt % of the composition.

[0144] In some embodiments, the protein is selected from the group consisting of antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof. In some embodiments, the protein is an antibody. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a vaccine. In some embodiments, the protein is a hormone. In some embodiments, the composition exhibits reduced protein aggregation compared to a composition of the same protein without copolymers. In some embodiments, the composition exhibits reduced protein precipitation compared to a composition of the same protein without copolymers. In some embodiments, the composition includes a concentration of the protein that is at least twice, at least three times, at least four times, or at least five times the concentration of the same protein in the composition without the copolymer.

[0145] In some embodiments, the protein is insulin or an analog thereof.Therefore, compositions containing the polyacrylamide-based copolymers of the present disclosure and insulin or an analog thereof are also provided.

[0146] Non-limiting examples of insulin or insulin analogs include insulin lispro, (rapid-acting insulin lispro), insulin glargine, (insulin glargine), insulin detemir, (Insulin Detemir), (rapid-acting human insulin), modern insulin, (Insulin Aspart), (human insulin), M3 (a mixture of soluble insulin and isoproterenol insulin called biphasic isoproterenol insulin), (Neutral bovine insulin), (recombinant human insulin), (Long-acting low-protamine human insulin), 30 (a mixture of 30% soluble insulin and 70% oligoprotamine insulin), 40 (a mixture of 40% soluble insulin and 60% oligoprotamine insulin), 50 (a mixture of 50% soluble insulin and 50% oligoprotamine insulin), insulin aspart, insulin glulisine, oligoprotamine insulin, insulin degludec, insulin icodec, insulin zinc-extended, R (human insulin), R (human insulin), R regular U-500 (concentrated regular insulin), N (intermediate-acting human insulin), N (intermediate-acting human insulin), ( R. N and 70 / 30 (over-the-counter brands) (rapid-acting inhaled insulin), 70 / 30 (a mixture of 70% human oligosaccharide insulin suspension and 30% human insulin injection), 70 / 30 (a mixture of 70% NPH, human oligosperm protein islet suspension and 30% regular human insulin injection), 70 / 30 (a mixture of 70% insulin aspart protamine suspension and 30% insulin aspart injection), 50 / 50 (a mixture of 50% human oligosaccharide insulin suspension and 50% human insulin injection), Mix 75 / 25 (a mixture of 75% insulin lispro protamine suspension and 25% insulin lispro injection), insulin aspart protamine-insulin aspart, insulin lispro protamine-insulin lispro, insulin lispro protamine-insulin lispro, human insulin NPH-regular human insulin, insulin degludec-insulin aspart, and combinations thereof. In some embodiments, the insulin or its analog is human insulin or recombinant human insulin. In some embodiments, the insulin or its analog is non-human insulin (e.g., primate, porcine, cattle, cat, dog, or rodent) or recombinant non-human insulin. In some embodiments, the insulin or its analog is purified or synthetic insulin. In some embodiments, the insulin or its analog is selected from the group consisting of rapid-acting insulin, short-acting insulin, intermediate-acting insulin, long-acting insulin, and premixed insulin. In some embodiments, the insulin or its analog is insulin lispro. In some embodiments, the insulin or its analog is Commercially available fast-acting human insulin analogs, insulin lispro. In certain embodiments, the insulin or its analog is insulin aspart. In certain embodiments, the insulin or its analog is insulin glulisine. In certain embodiments, the insulin or its analog is recombinant human insulin.

[0147] In some embodiments, the insulin or its analog is present in the composition in monomeric form, dimer form, hexameric form, and combinations thereof. In some embodiments, about 1 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 30 mol%, about 1 mol% to about 40 mol%, about 1 mol% to about 50 mol%, about 1 mol% to about 60 mol%, about 1 mol% to about 70 mol%, about 1 mol% to about 80 mol%, about 1 mol% to about 90 mol%, or about 10 mol% to about 100 mol% of the insulin or its analog is present in the composition in monomeric form. For example, about 1mol%, about 2mol%, about 3mol%, about 4mol%, about 5mol%, about 6mol%, about 7mol%, about 8mol%, about 9mol%, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 30mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol%, about 70mol%, about 75mol%, about 80mol%, about 85mol%, about 90mol%, about 95mol%, about 99mol% or about 100mol% of the insulin or its analogue are present in the composition in monomeric form. In some embodiments, about 3mol% of the insulin or its analogue are present in the composition in monomeric form. In some embodiments, insulin or its analogue "substantially" is present in the composition in monomeric form, which means that greater than about 50mol% of the insulin or insulin analogue are present in monomeric form. In some embodiments, the insulin or its analogs include about 50mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 60mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 70mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 80mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 90mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 99mol% or more of insulin in monomeric form. In some embodiments, the insulin or its analogs include about 100mol% of insulin in monomeric form. In some embodiments, insulin or its analogs are "substantially" not present in the composition in monomeric form, which means that less than about 10mol% of insulin or its insulin analogs exist in monomeric form.

[0148] In some embodiments, about 10mol% to about 100mol% of the insulin or its analogue is present in the composition as a dimer. In some embodiments, about 10mol% to about 100mol%, about 20mol% to about 90mol%, about 30mol% to about 80mol%, about 40mol% to about 70mol%, about 50mol% to about 60mol%, about 60mol% to about 100mol%, about 80mol% to about 100mol%, about 90mol% to about 100mol% or about 95mol% to about 100mol% of the insulin or its analogue is present in the composition as a dimer. For example, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 30mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol%, about 70mol%, about 75mol%, about 80mol%, about 85mol%, about 90mol%, about 95mol%, about 97mol%, about 99mol% or about 100mol% of the insulin or its analogue are present in the composition in dimer form. In certain embodiments, insulin or its analogue "substantially" is present in the composition in dimer form, which means that the insulin or insulin analogue greater than about 50mol% exists in dimer form. In certain embodiments, the insulin or its analogue includes about 50mol% or more of the insulin in dimer form. In certain embodiments, the insulin or its analogue includes about 60mol% or more of the insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 70mol% or more of insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 80mol% or more of insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 90mol% or more of insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 97mol% or more of insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 99mol% or more of insulin in dimer form. In some embodiments, the insulin or its analogue comprises about 100mol% of insulin in dimer form.

[0149] In some embodiments, the insulin is not conjugated to poly(ethylene glycol) (PEG) or a trehalose polymer.

[0150] The concentration range of insulin or its analogue can be about 0.34mg / mL (10U / mL) to about 34mg / mL (1000U / mL). In some embodiments, the insulin concentration is about 1.7mg / mL (50U / mL) to about 17mg / mL (500U / mL). In some embodiments, the insulin concentration is about 17mg / mL (500U / mL) to about 34mg / mL (1000U / mL). In some embodiments, the insulin concentration is about 3.4mg / mL (100U / mL). In some embodiments, the insulin concentration is about 6.8mg / mL (200U / mL). In some embodiments, the insulin concentration is about 10.2mg / mL (300U / mL). In some embodiments, the insulin concentration is about 13.6mg / mL (400U / mL). In some embodiments, the insulin concentration is about 17mg / mL (500U / mL). In some embodiments, the insulin concentration is about 20.4mg / mL (600U / mL). In some embodiments, the insulin concentration is about 23.8 mg / mL (700 U / mL). In some embodiments, the insulin concentration is about 27.2 mg / mL (800 U / mL). In some embodiments, the insulin concentration is about 30.6 mg / mL (900 U / mL). In some embodiments, the insulin concentration is about 34 mg / mL (1000 U / mL).

[0151] Therefore, a composition comprising a polyacrylamide-based copolymer of the present disclosure and insulin is also provided. In some embodiments, the polyacrylamide-based copolymer contains a MORPH carrier monomer and a NIP dopant monomer. In some embodiments, the composition contains about 0.005wt% to about 0.2wt% of a polyacrylamide-based copolymer and about 100U / mL insulin or its analog, the polyacrylamide-based copolymer comprising about 70wt% to about 95wt% of a MORPH carrier monomer and about 5wt% to about 30wt% of a NIP dopant monomer. In some embodiments, the composition contains about 0.01wt% of a polyacrylamide-based copolymer and about 100U / mL insulin or its analog, the polyacrylamide-based copolymer comprising about 74wt% to about 80wt% of a MORPH carrier monomer and about 20wt% to about 26wt% of a NIP dopant monomer. In some embodiments, the composition contains about 0.01 wt % of a polyacrylamide-based copolymer including about 77 wt % of a MORPH carrier monomer and about 23 wt % of a NIP dopant monomer and about 100 U / mL of insulin or an analog thereof.

[0152] In some embodiments, the copolymer described in the present disclosure reduces or prevents insulin aggregation. In some embodiments, the copolymer enables the stable formulation of monomeric insulin or its analog in its monomeric form. In some embodiments, about 10mol% to about 100mol% of the insulin molecules or its analogs in the formulation exist as monomeric insulin or its analogs. For example, about 10mol%, about 15mol%, about 20mol%, about 25mol%, about 30mol%, about 35mol%, about 40mol%, about 45mol%, about 50mol%, about 55mol%, about 60mol%, about 65mol%, about 70mol%, about 75mol%, about 80mol%, about 85mol%, about 90mol%, about 95mol%, about 99mol% or about 100mol% of the insulin molecules or its analogs in the formulation exist in the form of monomeric insulin. In some embodiments, more than about 50mol% of the insulin molecules or its analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, more than about 60 mol% of the insulin molecules or their analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, more than about 70 mol% of the insulin molecules or their analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, more than about 80 mol% of the insulin molecules or their analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, more than about 90 mol% of the insulin molecules or their analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, about 100 mol% of the insulin molecules or their analogs in the formulation exist as monomeric insulin or its analogs. In some embodiments, the formulation contains up to about 100 mol% insulin or its analogs present as dimer insulin or its analogs. In some embodiments, less than about 90 mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or its analogs. In some embodiments, less than about 80 mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or its analogs. In some embodiments, less than about 70mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, less than about 60mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, less than about 50mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs.In some embodiments, less than about 40mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, less than about 30mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, less than about 20mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, less than about 10mol% of the insulin molecules or their analogs in the formulation exist as dimer insulin or their analogs. In some embodiments, the formulation is substantially free of dimer insulin or their analogs. In some embodiments, about 0mol% to about 100mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin. In some embodiments, more than about 95mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or their analogs. In some embodiments, less than about 99mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or their analogs. In some embodiments, less than about 90 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 80 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 70 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 60 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 50 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 30 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 20 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 10 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, less than about 5 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, the formulation is substantially free of hexameric insulin or its analogs. In some embodiments, about 0 mol% of the insulin molecules or their analogs in the formulation exist as hexameric insulin or its analogs. In some embodiments, the formulation does not include Zn(II).

[0153] In some embodiments, the stable formulation of insulin in its monomeric form produces ultrafast kinetics in vivo. In some embodiments, the copolymer enables the preparation of ultrafast absorbed insulin lispro (UFAL). In some embodiments, more than 50 mol% of the insulin molecules in the UFAL formulation exist as monomeric insulin. In some embodiments, more than 60 mol% of the insulin molecules in the UFAL formulation exist as monomeric insulin. In some embodiments, more than 70 mol% of the insulin molecules in the UFAL formulation exist as monomeric insulin. In some embodiments, more than 80 mol% of the insulin molecules in the UFAL formulation exist as monomeric insulin. In some embodiments, more than 90 mol% of the insulin molecules in the UFAL formulation exist as monomeric insulin. In some embodiments, less than 30 mol% of the insulin molecules in the UFAL formulation exist as dimer insulin. In some embodiments, less than 20 mol% of the insulin molecules in the UFAL formulation exist as dimer insulin. In some embodiments, less than 10 mol% of the insulin molecules in the UFAL formulation exist as dimer insulin. In some embodiments, the UFAL formulation is substantially free of dimeric insulin. In some embodiments, less than 30 mol% of the insulin molecules in the UFAL formulation exist as hexameric insulin. In some embodiments, less than 20 mol% of the insulin molecules in the UFAL formulation exist as hexameric insulin. In some embodiments, less than 10 mol% of the insulin molecules in the UFAL formulation exist as hexameric insulin. In some embodiments, less than 5 mol% of the insulin molecules in the UFAL formulation exist as hexameric insulin. In some embodiments, the UFAL formulation is substantially free of hexameric insulin.

[0154] In some embodiments, the composition comprises insulin or an analog thereof substantially in monomeric form, wherein administration of the composition to a subject results in a shorter duration of action compared to administration of a composition comprising the same amount of insulin or an analog thereof substantially in dimer form, hexamer form, or a combination thereof, wherein the duration of action is the maximum observed concentration (C max ) of 50% of the time consumed.

[0155] In some embodiments, the composition comprises insulin or an analog thereof substantially in monomeric form, wherein administration of the composition to a subject results in a shorter insulin onset time compared to administration of a composition comprising the same amount of insulin or an analog thereof substantially in dimer form, hexamer form, or a combination thereof, wherein the onset time is the maximum observed concentration (C max ) 50% of the time.

[0156] In some embodiments, the composition comprises insulin or an analog thereof substantially in monomeric form, wherein administration of the composition to a subject results in a greater fraction of total insulin exposure compared to administration of a composition comprising the same amount of insulin or an analog thereof substantially in dimer form, hexamer form, or a combination thereof, wherein the exposure is the fraction of the area under the curve (AUC) at a given time point to the total AUC (AUC 时间 / AUC 总 ).

[0157] In some embodiments, the composition comprises insulin or an analog thereof substantially in monomeric form, wherein administration of the composition to a subject results in a shorter time to maximum observed insulin concentration (Tmax) compared to administration of a composition comprising the same amount of insulin or an analog thereof substantially in dimer form, hexamer form, or a combination thereof. max ).

[0158] In some embodiments, the composition comprising the copolymer of the present disclosure and biomolecule further comprises one or more of a pharmaceutically acceptable carrier, an aqueous buffer, a tension regulator and a preservative. As used herein, pharmaceutically acceptable carriers, tension regulators and preservatives are nontoxic to the recipient at the dosage and concentration employed. In some embodiments, the composition contains a buffer such as phosphate, citrate, succinate, other organic acids and histidine, wherein the term "buffer" refers to a mixture of a weak acid and its conjugate base or vice versa for maintaining the pH of a solution at a near constant value. In some embodiments, the buffer comprises one or more phosphates. In some embodiments, the buffer is sodium phosphate.

[0159] In some embodiments, the composition contains a tonicity adjuster, such as sodium chloride, potassium chloride, mannitol, glucose, glycerol, or magnesium chloride. In some embodiments, the tonicity adjuster is sodium chloride (NaCl) or glycerol. In some embodiments, the tonicity adjuster is glycerol.

[0160] In some embodiments, the composition contains one or more preservatives, such as phenoxyethanol, phenol, meta-cresol, methylparaben, propylparaben and benzyl alcohol. In some embodiments, the one or more preservatives include phenoxyethanol and phenol. In some embodiments, the one or more preservatives include phenoxyethanol and meta-cresol. In some embodiments, the preservative is phenoxyethanol. In some embodiments, the preservative is phenol or meta-cresol.

[0161] In some embodiments, the composition comprising the copolymer of the present disclosure is an aqueous composition.In some embodiments, the composition comprising the copolymer of the present disclosure comprises essentially water.

[0162] In some embodiments, the pH of the composition is from about 4 to about 9, such as from about 4 to about 8, from about 4 to about 7, from about 4 to about 6, from about 4 to about 5, from about 5 to about 9, from about 5 to about 8, from about 5 to about 7, from about 5 to about 6, from about 6 to about 9, from about 6 to about 8, from about 6 to about 7, from about 7 to about 9, from about 7 to about 8, from about 8 to about 9, or about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.4, about 8, about 8.5, or about 9. In some embodiments, the pH of the composition is from about 4 to about 9. In some embodiments, the pH of the composition is from about 6 to about 8. In some embodiments, the pH of the composition is from about 7 to about 8. In some embodiments, the pH of the composition is about 7.4.

[0163] In some embodiments, the copolymer MORPH-NIP 23％ The UFAL is stable to stress aging for more than 24 hours. In some embodiments, the copolymer MORPH-NIP 23％ The stability of UFAL is almost commercially available Four times.

[0164] In some embodiments, the UFAL formulations comprising the copolymers of the present disclosure further comprise one or more of a pharmaceutically acceptable carrier, a tension modifier, and a preservative. In some embodiments, the UFAL formulations comprise a buffer, a preservative, a tension agent, and combinations thereof. In some embodiments, the buffer comprises one or more phosphates. In some embodiments, the buffer is sodium phosphate. In some embodiments, the preservative is phenoxyethanol. In some embodiments, the preservative is phenol or m-cresol. In some embodiments, the tension agent is sodium chloride (NaCl) or glycerol. In some embodiments, the tension agent is glycerol.

[0165] Compositions containing the polyacrylamide-based copolymers of the present disclosure and a lipid-based vehicle are also provided. In some embodiments, the lipid-based vehicle is selected from the group consisting of liposomes, polymersomes, micelles, and lipid nanoparticles.

[0166] In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including monoclonal antibodies. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including antibody drug conjugates. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including protein-based vaccine antigens. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including protein-polysaccharide conjugate vaccine antigens. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including nucleic acids. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including messenger RNA (mRNA). In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including deoxyribonucleic acid (DNA). In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including small interfering RNA (siRNA). In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations including short hairpin RNA (shRNA). In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising microRNA (miRNA). In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising one or more nucleic acids and one or more lipids. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising one or more neutral lipids. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising one or more cationic lipids. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising one or more anionic lipids. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising liposomes. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising lipid nanoparticles. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising micelles. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising polymersomes. In some embodiments, the copolymers of the present disclosure are used as formulation additives in formulations comprising exosomes.

[0167] Insulin-containing co-formulations

[0168] The present disclosure also provides a composition comprising the polyacrylamide-based copolymer of the present disclosure, insulin or an analog thereof, and one or more of a peptide, a protein or a hormone.

[0169] In some embodiments, the co-formulation contains insulin or its analogs, and glucagon, a glucagon-like peptide-1 (GLP-1) receptor agonist, a glucose-dependent insulinotropic polypeptide (GIP) receptor agonist, or a dual GIP and GLP-1 receptor agonist. In some embodiments, the co-formulation comprising the copolymer of the present disclosure and insulin comprises a GLP-1 receptor agonist. In some embodiments, the GLP-1 receptor agonist is selected from the group consisting of lixisenatide, liraglutide, albiglutide, dulaglutide, exenatide, extended release exenatide, and semaglutide. In some embodiments, the co-formulation comprising the copolymer of the present disclosure and insulin comprises a GLP-1 receptor agonist and a GIP receptor agonist. In some embodiments, the co-formulation comprising the copolymer of the present disclosure and insulin comprises a dual GIP and GLP-1 receptor agonist. In some embodiments, the dual GIP and GLP-1 receptor agonist is tirzepatide. In some embodiments, the co-formulations comprising the copolymers of the present disclosure and insulin comprise a GIP receptor agonist. In some embodiments, the co-formulations comprising the copolymers of the present disclosure and insulin comprise glucagon.

[0170] In some embodiments, the co-formulations comprising the copolymers disclosed herein and insulin further comprise amylin or an analog thereof. In some embodiments, the amylin analog is pramlintide. According to embodiments of the present disclosure, in the presence of the copolymers described herein, the co-formulations of monomeric insulin or its analogs and pramlintide have ultrafast kinetics, with a high degree of overlap, resulting in improved glucose management after a glucose challenge. In some embodiments, in the presence of the copolymers described herein, the co-formulations of monomeric insulin lispro and pramlintide are shown to have ultrafast kinetics, with a high degree of overlap, resulting in improved glucose management after a glucose challenge. As described in Example 3, the amphiphilic copolymer MoNi 23％ The physical stability of the formulation as a stabilizer in stress aging tests is commercially available twice as much.

[0171] In some embodiments, the composition containing the polyacrylamide-based copolymer of the present disclosure, insulin or an analog thereof, and amylin or an analog thereof is present in a ratio of amylin or an analog thereof to insulin or an analog thereof of about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:6, or about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, or about 1:1.

[0172] The copolymers of the present disclosure also enable formulations of pramlintide to achieve near neutral pH values ​​(about pH 7), whereas existing pramlintide formulations require the pH of the formulation to be at about pH 4, which is incompatible with insulin. In some embodiments, the pH of the co-formulations comprising the copolymers of the present disclosure, insulin or its analogs, and amylin or its analogs is near neutral pH (about pH 7). In some embodiments, the pH of the co-formulations comprising the copolymers of the present disclosure, insulin or its analogs, and amylin or its analogs is about 6 to about 8. In some embodiments, the pH of the co-formulations comprising the copolymers of the present disclosure, insulin or its analogs, and amylin or its analogs is about 6.5. In some embodiments, the pH of the co-formulations comprising the copolymers of the present disclosure, insulin or its analogs, and amylin or its analogs is about 7. In some embodiments, the pH of the co-formulations comprising the copolymers of the present disclosure, insulin or its analogs, and amylin or its analogs is about 7.4. In some embodiments, the amylin analog is pramlintide.

[0173] Therefore, a co-formulation comprising a polyacrylamide-based copolymer of the present disclosure, insulin or its analogue and pramlintide is also provided. In some embodiments, the composition comprises about 0.005 wt % to about 0.2 wt % of a polyacrylamide-based copolymer, about 100 U / mL insulin or its analogue, and about 0.01 mg / mL to about 0.1 mg / mL pramlintide, the polyacrylamide-based copolymer comprising about 70 wt % to about 95 wt % of MORPH carrier monomers and about 5 wt % to about 30 wt % of NIP dopant monomers. In some embodiments, the composition comprises about 0.01 wt % of a polyacrylamide-based copolymer, about 100 U / mL insulin or its analogue, and about 0.5 mg / mL to about 0.6 mg / mL pramlintide, the polyacrylamide-based copolymer comprising about 74 wt % to about 80 wt % of MORPH carrier monomers and about 20 wt % to about 26 wt % of NIP dopant monomers. In some embodiments, the composition comprises about 0.01 wt % of a polyacrylamide-based copolymer, about 100 U / mL insulin or an analog thereof, and about 0.6 mg / mL pramlintide, wherein the polyacrylamide-based copolymer comprises about 77 wt % of a MORPH carrier monomer and about 23 wt % of a NIP dopant monomer. In some embodiments, the insulin or an analog thereof is substantially present in monomeric form. In some embodiments, the composition does not include zinc (II).

[0174] In some embodiments, administration of the composition to a subject exhibits an insulin AUC6 / AUC 120 Only apply or applied alone and insulin AUC6 / AUC after pramlintide 120 At least about 25%, such as about 30%, 40%, 50% or more.

[0175] The embodiments of the present disclosure demonstrate that the copolymers present in the co-formulations do not alter the pharmacokinetic (PK) or pharmacodynamic (PD) properties of the active ingredients within the formulations. In some embodiments, similar to the administration of pramlintide alone, pramlintide in the co-formulations results in delayed gastric emptying. In other embodiments, the combined effect of synchronized ultrafast insulin and pramlintide delivery in the co-formulations of the present disclosure results in a reduction in glucose depletion below baseline measurements while maintaining control of the initial glucose peak in a simulated "mealtime" glucose attack. Therefore, in some embodiments, the co-formulations have the potential to improve glucose management by reducing the risk of postprandial hypoglycemia while reducing patient burden.

[0176] In some embodiments, the use of co-compounds improves bolus insulin delivery. In some embodiments, insulin with ultrafast kinetics is delivered synchronously with pramlintide in an insulin infusion pump and an "artificial pancreas" closed-loop system. In some embodiments, stable insulin-pramlintide co-compounds enable dual hormone therapy to be implemented in a closed-loop system outside of a clinical trial, where it is impractical to use two separate infusion pumps. In some embodiments, the synchronized insulin-pramlintide kinetics and shorter duration of insulin action in the co-compounds of the present disclosure lead to improved autonomous insulin delivery. Typically, closed-loop systems require patients to enter carbohydrate counts at mealtimes, and are not completely autonomous, in part because insulin absorption kinetics are not fast enough to reduce mealtime glucose excursions, and extended duration of insulin action can lead to postprandial hypoglycemia. Therefore, ultrafast insulin-pramlintide co-compounds can react quickly to mealtime spikes, because insulin will take effect immediately, and pramlintide will slow down the appearance of glucose (by delaying gastric emptying). Further, in the case where the duration of insulin action is shorter, the risk of hypoglycemia caused by insulin accumulation will be reduced.

[0177] The present disclosure demonstrates that a stable single-administration insulin-pramlintide co-formulation utilizing monomeric insulin can have synchronized ultrafast insulin-pramlintide pharmacokinetics, which results in better glycemic control in mealtime simulations. Such a co-formulation has the potential to improve glucose management and reduce patient burden in clinical applications, both for direct bolus administration and for use in insulin infusion pumps or artificial pancreas closed-loop systems.

[0178] Properties of copolymer compositions

[0179] The polyacrylamide-based copolymers of the present disclosure have unique properties and, when used in aqueous formulations with biomolecules, can impart beneficial properties to the formulations.

[0180] In some embodiments, the copolymers described in the present disclosure reduce or prevent the aggregation of biomolecules or lipid-based vehicles, such as when formulated in aqueous formulations containing biomolecules or lipid-based vehicles. In some embodiments, the biomolecule is a protein. Therefore, in some embodiments, the copolymers described in the present disclosure reduce or prevent protein aggregation, such as when formulated in aqueous formulations containing proteins. The copolymers can be used with any protein that is prone to aggregation in aqueous media. Non-limiting examples include antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the polyacrylamide-based copolymers reduce or prevent the aggregation of proteins that are prone to aggregation in aqueous media. In some embodiments, the protein is insulin or an analog thereof. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

[0181] The copolymers of the present disclosure can also increase the stability of the formulation. According to embodiments of the present disclosure, the copolymers described herein can be added to improve the stability of the formulation, which contains, for example, biomolecules or lipid-based mediators, such as peptides, proteins and conjugates thereof, nucleic acids and oligonucleotides, liposomes, polymersomes, micelles and lipid nanoparticles. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of protein formulations. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of liposome formulations. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of micelle formulations. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of lipid nanoparticle formulations. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of formulations including one or more nucleic acids. In some embodiments, the addition of the copolymers of the present disclosure increases the stability of formulations including one or more messenger RNAs (mRNAs). In some embodiments, the addition of the copolymers of the present disclosure increases the stability of formulations including one or more small interfering RNAs (siRNAs). In some embodiments, the addition of the copolymers of the present disclosure increases the stability of formulations including one or more deoxyribonucleic acids (DNAs).

[0182] In some embodiments, copolymers of the present disclosure increase the thermal stability of formulations containing biomolecules, in some embodiments, the biomolecules are proteins. Protein formulations generally require expensive refrigerated transportation and storage to prevent the loss of protein integrity. Maintaining the integrity of proteins is a challenge for the pharmaceutical industry, health care providers, and patients in developing countries and low-income areas, in particular, where the cold chain required to maintain protein efficacy and effectiveness is imperfect, overburdened, or non-existent. This causes a large amount of therapeutic protein formulations to be wasted and potentially endangers the life of the patient. Therefore, interruptions in the cold chain may be expensive. Copolymers of the present disclosure can impart long-term stability and / or cold chain flexibility to protein formulations. For example, although commercial protein formulations have a good shelf life when properly stored (i.e., refrigerated), cold chain interruptions can reduce protein biological activity and formulation integrity.

[0183] In some embodiments, the protein formulation containing copolymers of the present invention is stable at a temperature higher than the temperature usually required for cold chain storage over an extended period of time. For example, the protein formulation containing copolymers of the present invention can be stored at room temperature (between about 20°C to about 22°C) or at elevated temperatures such as about 25°C, about 30°C, about 35°C, about 40°C, about 45°C or at higher temperatures, while maintaining protein integrity. In some embodiments, the protein formulation containing copolymers of the present invention can be stored at a temperature higher than the usual required temperature such as at about -20°C or about 0°C, or about 2°C, or about 4°C, or about 8°C, or about 20°C rather than at a lower temperature such as at about -80°C, or about -60°C or about -40°C. In some embodiments, copolymers enable protein formulations to be stored at room temperature or higher temperatures without the need for cold chain storage. In some embodiments, cold chain storage is not required to maintain protein integrity.

[0184] In some embodiments, the use of the copolymers of the present disclosure in protein formulations maintains formulation integrity, bioactivity, pharmacokinetics, and pharmacodynamics for longer periods of time when exposed to conditions such as elevated temperature and agitation compared to the same formulation without the copolymer. In some embodiments, the formulation maintains protein integrity at elevated temperatures for about 1 day, about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, or longer compared to the same formulation without the copolymer of the present disclosure.

[0185] In some embodiments, the copolymers of the present disclosure reduce the aggregation rate of biomolecules in aqueous formulations. In some embodiments, the copolymers of the present disclosure reduce the aggregation rate of proteins in aqueous protein formulations. In some embodiments, the protein is a protein that tends to aggregate in an aqueous medium. In some embodiments, the protein is an antibody or fragment thereof, a cytokine, a chemokine, a hormone, a vaccine antigen, a cancer antigen, an adjuvant, and a combination thereof. In some embodiments, this allows the formulation of a more concentrated protein solution, thereby reducing the volume of the protein solution that needs to be administered to, for example, a patient. In some embodiments, the composition includes a concentration of the protein that is at least twice, at least three times, at least four times, or at least five times the concentration of the same protein in the composition without the copolymer.

[0186] Therefore, the use of copolymers of the present disclosure can impart long-term stability and / or cold chain flexibility to protein formulations. In some embodiments, the protein in the composition comprising the copolymer exhibits increased stability when stored at room temperature compared to a protein composition without the copolymer of the present disclosure. In some embodiments, the increased stability is at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more of the protein composition without the copolymer. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

[0187] Thus, in a particular embodiment, the insulin in the composition comprising the copolymer exhibits increased stability when stored at room temperature compared to an insulin composition not containing the copolymer of the present disclosure. In some embodiments, the increased stability is at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more of the insulin composition not containing the copolymer.

[0188] In some embodiments, the copolymers of the present disclosure maintain protein activity in 6 months of stress aging without changing one or more of formulation pharmacokinetics, protein secondary structure, formulation transparency, and in vivo biological activity. In some embodiments, the copolymer reduces protein aggregation in the formulation when stored at ambient temperature (about 23°C to about 27°C) compared to a protein formulation without the copolymer. In some embodiments, the copolymer reduces protein aggregation in the formulation when stored at 37°C compared to a protein formulation without the copolymer. In some embodiments, the copolymer reduces protein aggregation in the formulation when stored at 50°C compared to a protein formulation without the copolymer. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

[0189] In a specific embodiment, the copolymer of the present disclosure maintains insulin activity during 6 months of stress aging without changing one or more of the formulation pharmacokinetics, protein secondary structure, formulation clarity, and in vivo biological activity. In some embodiments, the copolymer reduces insulin aggregation in the formulation when stored at ambient temperature (about 23°C to about 27°C) compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer reduces insulin aggregation in the formulation when stored at 37°C compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer reduces insulin aggregation in the formulation when stored at 50°C compared to a human insulin formulation without the copolymer. As described in Example 4, when subjected to a harsh stress aging test in standard packaging, a formulation formulated with MoNi23% copolymer R did not aggregate for more than 56 days when subjected to continuous stirring at 37°C and four days of continuous stirring at 50°C, while R aggregated within two days at 37°C and within one day at 50°C. The stress aging conditions used in the studies described herein are intended to simulate typical storage and transportation conditions. Even in hot climates with limited cold chain infrastructure, it is unlikely that a container would be maintained at 50°C for a period of more than 24 hours without any delay of continuous agitation.

[0190] In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37°C by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times compared to a protein formulation without the copolymer. Aggregation time can be assessed by any known method, including, for example, by transmittance measurement. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37°C by at least 10 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37°C by at least 20 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37°C by at least 30 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37°C by at least 40 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 37° C. by at least 50 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50° C. by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50° C. by at least 10 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50° C. by at least 20 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50° C. by at least 30 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50°C by at least 40 times compared to a protein formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the protein formulation when stored at 50°C by at least 50 times compared to a protein formulation without the copolymer. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

[0191] In a specific embodiment, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 10 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 20 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 30 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 40 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 37°C by at least 50 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 50°C by at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 50°C by at least 10 times, as assessed by a transmittance assay, compared to a human insulin formulation without the copolymer. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 50°C by at least 20 times compared to a human insulin formulation without the copolymer, as assessed by a transmittance assay. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 50°C by at least 30 times compared to a human insulin formulation without the copolymer, as assessed by a transmittance assay. In some embodiments, the copolymer increases the aggregation time of the human insulin formulation when stored at 50°C by at least 40 times compared to a human insulin formulation without the copolymer, as assessed by a transmittance assay.In some embodiments, the copolymer increases the aggregation time of a human insulin formulation when stored at 50°C by at least 50-fold compared to a human insulin formulation without the copolymer as assessed by transmittance measurement.

[0192] With separate Compared with R, it was observed that the MoNi23% copolymer The increased stability of R indicates that the MoNi23% copolymer has utility in stabilizing insulin under various stirring conditions (i.e., horizontal stirring) where interfacial conversion may be higher. The conditions evaluated in Example 4 of the present disclosure represent extreme exposure conditions during transportation in uninsulated containers or trucks in the world's hottest climates, where transportation may take weeks to reach the patient.

[0193] In addition to refrigerated transportation in the early stages of the cold chain, maintaining appropriate transportation and storage conditions during local distribution and once it reaches the patient is a challenge in many parts of the world. As described herein, the addition of a polyacrylamide-based copolymer can maintain the integrity of the protein formulation even during severe cold chain interruptions. In some embodiments, this enables the cold chain requirements for protein transportation and storage to be reduced, which is difficult to maintain in resource-poor environments. As disclosed herein, a polyacrylamide-based copolymer as a formulation additive can improve cold chain flexibility, thereby expanding global access to key drugs and vaccines. In some embodiments, the addition of the copolymer maintains the in vitro biological activity of the protein formulation for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, the addition of the copolymer maintains the in vitro biological activity of the protein formulation for at least 1 month. In some embodiments, the addition of the copolymer maintains the in vitro biological activity of the protein formulation for at least 2 months. In some embodiments, the addition of the copolymer maintains the in vitro biological activity of the protein formulation for at least 3 months. In some embodiments, the protein is insulin. In some embodiments, the protein is a monoclonal antibody. In some embodiments, the protein is a hormone. In some embodiments, the protein is a vaccine.

[0194] In some embodiments, the copolymer present in a formulation with a protein does not alter the pharmacokinetic (PK) or pharmacodynamic (PD) properties of the active ingredient in the formulation.

[0195] Methods of using the copolymer compositions

[0196] The present disclosure provides methods of using the compositions and co-formulations described in the present disclosure, which contain polyacrylamide-based copolymers containing water-soluble carrier monomers and functional dopant monomers.

[0197] The present disclosure provides methods for managing the blood glucose level of a subject in need. In some embodiments, a method for treating an elevated blood glucose level of a subject in need is provided. In some embodiments, the elevated blood glucose level is associated with insulin deficiency. In some embodiments, the elevated blood glucose level is associated with food intake (e.g., during mealtime). For example, a method for counteracting elevated glucose levels is provided, which may be accompanied by the intake of macronutrients that increase glucose, such as carbohydrates (i.e., mealtime insulin) . In some embodiments, managing the blood glucose level includes reducing the blood glucose level of a subject in need. In some embodiments, managing the blood glucose level includes regulating the blood glucose level of a subject diagnosed with type 1 or type 2 diabetes. In some embodiments, managing the blood glucose level includes reducing the blood glucose level during mealtime. As used herein, the term "insulin deficiency" refers to reduced insulin levels and / or reduced insulin sensitivity relative to metabolic demand. Subjects with insulin deficiency include, but are not limited to, subjects with diabetes (including, but not limited to, type 1 diabetes, type 1.5 diabetes, type 2 diabetes, gestational diabetes, and post-pancreatectomy diabetes), subjects with hyperglycemia, and subjects with transient hyperglycemia (e.g., transient hyperglycemia caused by stress in otherwise non-diabetic subjects during hospitalization). In humans, blood glucose levels below 100 mg / dL after fasting for at least eight hours, and below 140 mg / dL after two hours of eating are considered normal. Human subjects with normal blood glucose levels described above are considered non-diabetic. The method of the present disclosure comprises administering a therapeutically effective amount of a composition of the present disclosure to a subject in need thereof, the composition comprising a copolymer based on polyacrylamide and insulin or its analog. In some embodiments, the composition is administered by a pump. In some embodiments, the pump is an infusion pump. In some embodiments, the composition is administered by an artificial pancreas closed loop system. In some embodiments, the composition is administered by an automatic insulin delivery system. In some embodiments, the elevated glucose level is associated with insulin deficiency in the subject. In some embodiments, the subject has been diagnosed with type 1 diabetes. In some embodiments, the subject has been diagnosed with type 2 diabetes. In some embodiments, the subject is non-diabetic.In some embodiments, the subject has undergone trauma, surgery, or both.

[0198] A method for increasing the stability of formulations containing biomolecules or lipid-based media is also provided. According to embodiments of the present disclosure, copolymers described herein may be added to improve the stability of formulations, including peptides, proteins and conjugates thereof, nucleic acids and oligonucleotides, liposomes, polymersomes, micelles and lipid nanoparticles. In some embodiments, a method for increasing the stability of protein formulations is provided. In some embodiments, a method for increasing the thermal stability of formulations containing biomolecules or lipid-based media is provided. In some embodiments, a method for increasing the thermal stability of protein formulations is provided. A method for reducing the aggregation rate of biomolecules or lipid-based media in aqueous formulations is also provided. In some embodiments, the molecule is a molecule that tends to aggregate in an aqueous medium. In some embodiments, the protein is an antibody or fragment thereof, a cytokine, a chemokine, a hormone, a vaccine antigen, a cancer antigen, an adjuvant and a combination thereof.

[0199] In some embodiments, the method comprises adding about 0.001 wt% to about 5 wt% of the polyacrylamide-based copolymer of the present disclosure to the formulation containing a biomolecule or a lipid-based vehicle, such as about 0.001 wt% to about 4 wt%, about 0.001 wt% to about 3 wt%, about 0.001 wt% to about 2 wt%, about 0.001 wt% to about 1 wt%, about 0.001 wt% to about 0.5 wt%, about 0.001 wt% to about 0.4 wt%, about 0.001 wt% to about 0.3 wt%, about 0.001 wt% to about 0.2 wt%, about 0.001 wt% to about 0.1 wt%, about 0.001 wt% to about 0.05 wt%, or about 0.001 wt% to about 0.05 wt%. From about 0.001 wt% to about 0.02 wt%, from about 0.001 wt% to about 0.01 wt%, from about 0.001 wt% to about 0.005 wt%, from about 0.005 wt% to about 5 wt%, from about 0.005 wt% to about 4 wt%, from about 0.005 wt% to about 3 wt%, from about 0.005 wt% to about 2 wt%, from about 0.005 wt% to about 1 wt%, from about 0.005 wt% to about 0.5 wt%, from about 0.005 wt% to about 0.4 wt%, from about 0.005 wt% to about 0.3 wt%, from about 0.005 wt% to about 0.2 wt%, from about 0.005 wt% to about 0.1 wt%, from about 0.005 wt% to about 0.05 wt%, from about 0.005 wt% to about 0.05 wt%, .005wt% to about 0.02wt%, about 0.005wt% to about 0.01wt%, about 0.01wt% to about 5wt%, about 0.01wt% to about 4wt%, about 0.01wt% to about 3wt%, about 0.01wt% to about 2wt%, about 0.01wt% to about 1wt%, about 0.01wt% to about 0.5wt%, about 0.01wt% to about 0.4wt%, about 0.01wt% to about 0.3wt%, about 0.01wt% to about 0.2wt%, about 0.01wt% to about 0.1wt%, about 0.01wt% to about 0.05wt%, about 0.01wt% to about 0.02wt%, about 0.02wt% to about 5wt%, about 0.02 wt% to about 4 wt%, about 0.02 wt% to about 3 wt%, about 0.02 wt% to about 2 wt%, about 0.02 wt% to about 1 wt%, about 0.02 wt% to about 0.5 wt%, about 0.02 wt% to about 0.4 wt%, about 0.02 wt% to about 0.3 wt%, about 0.02 wt% to about 0.2 wt%, about 0.02 wt% to about 0.1 wt%, about 0.02 wt% to about 0.05 wt%, about 0.05 wt% to about 5 wt%, about 0.05 wt% to about 4 wt%, about 0.05 wt% to about 3 wt%, about 0.05 wt% to about 2 wt%, about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 0.5wt%, about 0.05wt% to about 0.4wt%, about 0.05wt% to about 0.3wt%, about 0.05wt% to about 0.2wt%, about 0.05wt% to about 0.1wt%, about 0.1wt% to about 5wt%, about 0.1wt% to about 4wt%, about 0.1wt% to about 3wt%, about 0.1wt% to about 2wt%, about 0.1wt% to about 1wt%, about 0.1wt% to about 0.5wt%, about 0.1wt% to about 0.4wt%, about 0.1wt% to about 0.3wt %, about 0.1 wt % to about 0.2 wt %, about 0.2 wt % to about 5 wt %, about 0.2 wt % to about 4 wt %, about 0.2 wt % to about 3 wt %, about 0.2 wt % to about 2 wt %, about 0.2 wt % to about 1 wt %, about 0.2 wt % to about 0.5 wt %, about 0.2 wt % to about 0.4 wt %, about 0.2 wt % to about 0.3 wt %, about 0.3 wt % to about 5 wt %, about 0.3 wt % to about 4 wt %, about 0.3 wt % to about 3 wt %, about 0.3 wt % to about 2 wt %. t%, about 0.3 wt% to about 1 wt%, about 0.3 wt% to about 0.5 wt%, about 0.3 wt% to about 0.4 wt%, about 0.4 wt% to about 5 wt%, about 0.4 wt% to about 4 wt%, about 0.4 wt% to about 3 wt%, about 0.4 wt% to about 2 wt%, about 0.4 wt% to about 1 wt%, about 0.4 wt% to about 0.5 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 4 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%. %, about 0.5 wt % to about 1 wt %, about 1 wt % to about 5 wt %, about 1 wt % to about 4 wt %, about 1 wt % to about 3 wt %, about 1 wt % to about 2 wt %, about 2 wt % to about 5 wt %, about 2 wt % to about 4 wt %, about 2 wt % to about 3 wt %, about 3 wt % to about 5 wt %, about 3 wt % to about 4 wt %, about 4 wt % to about 5 wt %, or about 0.005 wt %, about 0.01 wt % or about 0.1 wt % of the polyacrylamide-based copolymer of the present disclosure is added to the formulation. In some embodiments, the method comprises adding about 0.0005 wt % to about 5 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation. In some embodiments, the method comprises adding about 0.001 wt % to about 1 wt % of the polyacrylamide-based copolymer of the present disclosure to the formulation. In some embodiments, the formulation comprises a biomolecule. In some embodiments, the formulation comprises a protein. In some embodiments, the formulation comprises a lipid-based vehicle. In some embodiments, the lipid-based vehicle comprises a liposome, a lipid nanoparticle, a polymersome, or a micelle.

[0200] Other uses of polyacrylamide-based copolymers

[0201] The polyacrylamide-based copolymers of the present disclosure can be used in any composition or formulation using a surfactant. For example, the copolymers of the present disclosure can be used in applications including but not limited to cosmetic products, hair products, lotions, food products, nutritional products, pigments, and inks. In some embodiments, the copolymers are used to improve food texture or improve mouthfeel. In some embodiments, the copolymers are used in biopharmaceutical compositions for animals or veterinary use. In some embodiments, the presence of the copolymers improves the stability of the formulation.

[0202] Examples

[0203] Example 1 - Synthesis and Characterization of AD / DC Copolymer Library

[0204] Study Design: The pharmacokinetics of an insulin lispro formulation, ultra-rapidly absorbed insulin lispro (UFAL), were compared with a commercial insulin lispro formulation ( ) were compared. Subcutaneous injection (i) or (ii) after UFAL, blood glucose and plasma insulin lispro concentrations were measured on blood samples collected using a handheld blood glucose monitor or ELISA. Randomization: Five pigs were used in this study, and each pig received each formulation once. The order of administration of the formulations was randomized. Blinding: For analysis of pharmacokinetic parameters (t50% rise, time to peak, t50% decline), pharmacokinetic curves were coded and analyzed by blinded investigators. Replication: Five pigs were used in this study, and each pig served as its own control receiving each formulation ( and UFAL) once.

[0205] Material:Solvents N,N-dimethylformamide (DMF; HPLC grade, Alfa Aesar, >99.7%), ethanol (EtOH; certified ACS, Acros, >99.5%), acetone (Sigma, HPLC grade, >99.9%), hexane (Fisher, certified ACS, >99.9%), diethyl ether (Sigma, certified ACS, anhydrous, >99%) and CDCl3 (Acros, >99.8%) were used as received. The monomers N,N-dimethylacrylamide (DMA; Sigma, 99%), N-(3-methoxypropyl)acrylamide (MPAM; Sigma, 95%), 4-acryloylmorpholine (MORPH; Sigma, >97%), acrylamide (AM; Sigma, >99%), and N-hydroxyethylacrylamide (HEAM; Sigma, >97%) were filtered over basic alumina before use. The monomers N-phenylacrylamide (PHE; Sigma, 99%), N-tert-butylacrylamide (TBA; Sigma, 97%), N-isopropylacrylamide (NIPAM; Sigma, >99%), and N-[tris(hydroxymethyl)methyl]acrylamide (TRI; Sigma, 93%) were used as received. (3-Acrylamidopropyl)trimethylammonium (TMA; Sigma, 75%) was washed with ethyl acetate. 2-Acrylamido-2-methylpropanesulfonic acid (AMP; Sigma, 99%) was converted to the sodium salt by equimolar mixing with sodium acetate in methanol and precipitated into acetone. RAFT chain transfer agents 2-cyano-2-propyldodecyl trithiocarbonate (2-CPDT; Strem Chemicals, >97%) and 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanovaleric acid (BM1433; Boron Molecular, >95%) were used as received. Initiator 2,2′-azobis(2-methylpropionitrile) (AIBN; Sigma, >98%) was recrystallized from methanol (MeOH; Fisher, HPLC grade, >99.9%) and dried under vacuum before use. Initiator 4,4-azobis(4-cyanovaleric acid) (ACVA, Sigma, >98%) was used as received. The Z-group removers lauroyl peroxide (LPO; Sigma, 97%) and hydrogen peroxide (H2O2; Sigma, 30%) were used as received.

[0206] Synthesis of the first copolymer library by automated parallel synthesis:Copolymerization of carrier and dopant was carried out using RAFT polymerization ([total monomers] / [CTA]=50, [CTA] / [AIBN]=0.2). MPAM, MORPH and DMA carrier monomers copolymerized with AMP, TMA, NIP, TBA or PHE dopant monomers were polymerized in DMF using 2-CPDT as CTA and AIBN as initiator. MPAM, MORPH and DMA carrier monomers copolymerized with TRI dopant monomer were polymerized in DMF / water mixture using BM1433 as CTA and ACVA as initiator. The total vinyl monomer molar concentration was maintained at 2.72M (MPAM copolymerization), 2.86M (MORPH copolymerization) and 3.84M (DMA copolymerization) so that homopolymerization of carrier monomer in DMF would proceed at a constant 40wt.%. HEAM carrier monomer copolymerized with AMP, TMA, NIP, TBA or PHE dopant monomers was polymerized in DMF / EtOH mixture using 2-CPDT as CTA and AIBN as initiator. HEAM carrier monomer copolymerized with AMP, TMA, NIP, TBA or PHE dopant monomers was polymerized in DMF / EtOH / water mixture using BM1433 as CTA and ACVA as initiator. The total vinyl monomer molar concentration was maintained at 2.58M (HEAM copolymerization) so that the homopolymerization of HEAM in DMF will proceed at a constant 30 wt.%. AM carrier monomer copolymerized with AMP, TMA, NIP, TBA or PHE dopant monomers was polymerized in DMF / water mixture using BM1433 as CTA and ACVA as initiator. AM carrier monomer copolymerized with TRI dopant monomer was polymerized in water using BM1433 as CTA and ACVA as initiator. The total vinyl monomer molar concentration was kept at 4.05 M (AM copolymerization) so that homopolymerization of AM in DMF would proceed at a constant 30 wt.%.

[0207] The reaction mixture was prepared by combining stock solutions: (i) carrier, (ii) dopant, and (iii) CTA and initiator. Stock solutions of carrier monomers were HEAM (555 mg / mL in EtOH), AM (462 mg / mL in water), MPAM (818 mg / mL in DMF), DMA (solvent-free dilution), and MORPH (solvent-free dilution). Stock solutions of dopant monomers were TRI (181 mg / mL in water), PHE (120 mg / mL in DMF), NIP (245 mg / mL in DMF), TBA (122 mg / mL in DMF), AMP (120 mg / mL in DMF), and TMA (124 mg / mL in DMF). Stock solutions of CTA and initiator were prepared such that [CTA] / [initiator] = 5 for AM (310 mg / mL BM1433 in water), HEAM and MPAM (198 mg / mL BM1433 in water and 221 mg / mL 2-CPDT in DMF), MORPH (220 mg / mL BM1433 in water and 247 mg / mL 2-CPDT in DMF), and DMA (220 mg / mL BM1433 in water and 332 mg / mL 2-CPDT in DMF). The reaction mixtures of HEAM, DMA, MPAM, and MORPH were diluted with DMF, while the reaction mixture of AM was diluted with water to achieve the desired vinyl monomer concentration.

[0208] The parallel synthesis of the copolymer excipient (also referred to as AC / DC excipient) based on polyacrylamide is carried out on a Chemspeed Swing XL automatic synthesizer robot equipped with a 4-needle tool and an iSynth reactor. The reaction is carried out in an 8mL disposable ISynth reactor vial. All aspiration and distribution of reagent solutions are carried out using a 4-needle tool, which is equipped with 2×10mL and 2×1mL syringes equipped with a septum puncture needle, wherein 1mL and 10mL syringes are used in this particular experiment. All solvent lines are perfused with 60mL (6 strokes of syringe volume) degassed DMF. For two 1mL syringes, the typical aspiration and distribution rate of reagents is 10 ml / min. During aspiration using a 4-needle tool, an air gap of 50 μL and an additional volume of 50 μL are used for a 1mL syringe, and an air gap of 50 μL and an additional volume of 100 μL are used for a 10mL syringe. After each reagent dispensing mission, the needle and lines were flushed with 3 mL of internal and external volume of pre-filled solvent for 1 mL syringes and 20 mL of internal and external volume of pre-filled solvent for 10 mL syringes. The DMF reservoir was degassed by continuous nitrogen sparging. All stock solutions were prepared in reagent vials with septum caps and degassed by purging with argon for 15 minutes before transfer to the Chemspeed. The atmosphere inside the Chemspeed was reduced to <1% oxygen by purging with nitrogen while the exhaust port was closed. The reactor vials were exposed to a stream of nitrogen until the reaction started. Calculated aliquots of stock solutions and solvents were transferred to the reactor by an automated liquid handling system. After dispensing, the reactor vials were manually sealed in an inert atmosphere, removed from the Chemspeed, manually shaken to combine the reagents, and heated to 65 °C in an oven for 24 hours, after which the reactor vials were cooled to room temperature and exposed to air.

[0209] The procedure for removing CTA Z groups from AC / DC excipients containing MORPH, DMA, HEAM and MPAM copolymers was adapted from the literature. The reactor vial was diluted to 6 mL with DMF. LPO (2 eq.) and AIBN (20 eq.) were added to the reaction mixture, which was sealed with a PTFE seal cap. While heating at 90°C, the reaction mixture was purged with nitrogen for 10 minutes and then heated at 90°C for 12 hours. The procedure for removing CTA Z groups from AC / DC excipients containing AM copolymers was adapted from the literature. The reactor vial was diluted to 5 mL with miliQ water. H2O2 (20 eq.) was added to the reaction vial, which was sealed and heated to 60°C for 12 hours. The resulting copolymer was isolated by precipitation as outlined below.

[0210] AC / DC excipients synthesized with AM and HEAM carriers were precipitated twice from acetone. AC / DC excipients synthesized with DMA and MORPH were precipitated twice from diethyl ether. AC / DC excipients synthesized with MPAM were precipitated twice from a mixture of diethyl ether and hexane (3:1 ratio). The number average molecular weight (M) of AC / DC excipients containing MORPH, MPAM, DMA, and HEAM was calculated. n ) and weight average molecular weight (M w ) and dispersibility were determined using SEC in DMF with poly(ethylene glycol) standards. The AM of AC / DC excipients containing AM was determined using aqueous SEC-MALLS n 、M w and dispersion.

[0211] Synthesis of the Second Polymer Library: The typical procedure for synthesizing MORPH-NIP AC / DC excipient is as follows, and is almost identical for all other carrier / dopant combinations, wherein only the selection and concentration of carrier / dopant are changed.MORPH (645mg, 4.57mmol, 41.5 equivalents), NIPAM (105mg, 0.93mmol, 8.5 equivalents), 2CPDT (38mg, 0.11mmol, 1 equivalent) and AIBN (3.6mg, 0.02mmol, 0.2 equivalent) are combined and in the 8mL scintillation vial equipped with PTFE spacer, be diluted to cumulative volume 2.25mL (33.3w / v vinyl monomer concentration) with DMF.Purge reaction mixture with nitrogen 10 minutes, and then heat 12 hours at 65 ℃. To remove the Z end of the resulting polymer, AIBN (360 mg, 2.2 mmol, 20 equiv) and LPO (88 mg, 0.22 mmol, 2 equiv) were added to the reaction mixture, which was then bubbled with nitrogen for 10 minutes and heated at 90°C for 12 hours. In SEC analysis, the Z group removal was confirmed by the ratio of the refractive index to the UV (λ = 310 nm) intensity. The resulting polymer was precipitated three times from diethyl ether and dried overnight under vacuum. The resulting composition and molecular weight were determined by 1 H NMR spectroscopy and SEC were determined using poly(ethylene glycol) standards.

[0212] Copolymer Molecular Weight Characterization: M of copolymers with HEAM, DMA, MPAM and MORPH carrier monomers n 、M w and the dispersion is at 35 °C and 1.0 ml min -1The precipitate was purified by flow cytometry on two size exclusion chromatography columns (Analytical Mixed Bed Low DVB, ID 7.8 mm, M 2000) in a mobile phase of 0.1 M LiBr in N,N-dimethylformamide (DMF) at a flow rate of 1.5 % (Dionex Ultimate 3000 pump, degasser and autosampler (Thermo Fisher Scientific)). w Range 200-600,000 gmol - 1 (Jordi Labs)) followed by SEC running poly(ethylene glycol) standards (American Polymer Standards Corporation).

[0213] After passing through a size exclusion chromatography column (Superose 6 Increase 10 / 300 GL, 5,000-5,000,000 gmol -1 (GE Healthcare)) Then, the M of the copolymers with AM were determined by SEC-MALLS in a mobile phase of phosphate buffered saline containing 300 ppm sodium azide. n 、M w and dispersion. Detection consisted of an Optilab T-rEX (Wyatt Technology Corporation) refractive index detector operating at 658 nm and a TREOS II light scattering detector (Wyatt Technology Corporation) operating at 659 nm. In this medium, the dn / dc value of the AM copolymer was assumed to be 0.185.

[0214] Method used to determine experimental VM wt.% values

[0215] During the initial AC / DC copolymer library synthesis, Chemspeed handling of sticky monomers (HEAM, MPAM, MORPH) resulted in monomer loadings that differed from the target monomer loadings selected for copolymerization. The experimental weight percentages were determined by the peak molecular weight (M) of the SEC traces. p ) is approximately obtained. Since only the sticky monomer is affected, M p The variation in M ​​is due to insufficient addition of carrier monomer (in addition to minor variations in the permeation volume for different weight percentages of dopant monomer relative to carrier). Therefore, to calculate the experimental weight percentage, for a given carrier / dopant pair, determine M p，max .

[0216] result

[0217] High-throughput synthesis of polyacrylamide libraries: Combinatorial synthesis of AC / DC excipient libraries ( Figure 2 ). The carrier monomer is the main species and is responsible for both maintaining solubility and providing an inert barrier to prevent insulin-insulin interactions. Functional dopants copolymerized at lower weight percentages are statistically incorporated into the entire resulting copolymer. These dopants are selected by design to promote polymer-interface interactions or polymer-insulin interactions. The library targets copolymers with a degree of polymerization (DP) of 50, resulting in a molecular weight similar to insulin and well below the glomerular filtration threshold of synthetic polymers.

[0218] The experimental degree of polymerization (DP) of the carrier monomer is approximately calculated using equation (S1). This value is used to approximate the experimental weight percentage (wt%). Figure 3 As shown, for the copolymerization of MORPH (support) and PHE (dopant), the results calculated using this method are given by 1 The results of this comparison are shown in Table 1. 1 The experimental weight percent values ​​of the carrier monomer and the functional dopant monomer for each copolymer were determined by H NMR and SEC and are summarized in Table 2.

[0219]

[0220] Table 1: Pass 1 H NMR spectroscopy to verify SEC wt.% measurements

[0221] Carrier wt.% Dopant wt.% <![CDATA[wt.% a ]]> <![CDATA[wt.% b ]]> <![CDATA[M p ]]> (Target) (Target) (Experimental, NMR) <![CDATA[(Experiment, M p )]]> MORPH 96.7 PHE 3.3 4.5 3.6 2900 MORPH 93.3 PHE 6.7 11.7 10.7 1850 MORPH 90 PHE 10 11.3 10 3100

[0222] a After precipitation 1 1H NMR calculated experimental wt. % (δ = 3.3-3.7, 8H).

[0223] b Using M determined by SEC p Values ​​(peak molecular weight) experimental wt.% calculated according to Eq. 1.

[0224] Table 2: SEC and MALS characterization and analysis of polymers synthesized in the initial AC / DC library

[0225]

[0226]

[0227]

[0228] aM determined by DMF size exclusion chromatography n (number average molecular weight), M w (weight average molecular weight) and (Dispersity) Calibrated using polyethylene glycol standards for HEAM, MPAM, MORPH, and DMA. n and M w Determined using aqueous SEC-MALS using a dn / dc value of 0.185.

[0229] b Experimental wt. % values ​​were determined by SEC using Equation S1.

[0230] The library was produced by parallel synthesis using a Chemspeed Swing XL automated synthesizer (liquid handling robot in an inert environment). RAFT polymerization was used because it provides precise copolymerization stoichiometry, low dispersity, and controllable molecular weight for a wide range of monomers. Due to the range and availability of commercially available water-soluble monomers (carriers) and functional monomers (dopants) and polymer stability, polyacrylamide derivatives are used for both carrier monomers and dopant monomers. Although monomeric acrylamide derivatives typically exhibit acute toxicity, when properly purified from their monomer precursors, polyacrylamide derivatives exhibit high biocompatibility. In addition, the reactivity ratio between various acrylamide monomers is close to 1, producing copolymers with little or no dopant gradient composition. Carrier monomers include acrylamide (AM), hydroxyethyl acrylamide (HEAM), dimethyl acrylamide (DMA), acryloyl morpholine (MORPH), and methoxypropyl acrylamide (MPAM) because they are nonionic and water-soluble (arranged in order of increasing hydrophobicity). Dopant monomers include tris(hydroxymethyl)-methyl acrylamide (TRI), acrylamidomethyl propane sulfonic acid (AMP), acrylamidopropyl trimethyl ammonium chloride (TMA), n-isopropyl acrylamide (NIP), tert-butyl acrylamide (TBA), and phenyl acrylamide (PHE). Based on their chemical composition, these functional dopants can be further classified into hydrogen bonding (TRI) monomers, ionic (AMP, TMA) monomers, hydrophobic (NIP, TBA) monomers, and aromatic (PHE) monomers.

[0231] For a given carrier-dopant pair, a library of 90 AC / DC excipients was synthesized by combinatorial copolymerization of carrier monomer and dopant monomer at each of three different compositions. NIP was copolymerized at 6.7 wt.%, 13.3 wt.%, or 20 wt.%. TRI was copolymerized at 5 wt.%, 10 wt.%, or 15 wt.%. AMP, TMA, TBA, and PHE were copolymerized at 3.3 wt.%, 6.7 wt.%, or 10 wt.%. These values ​​were chosen to maximize the dopant loading while producing functional copolymers with lower critical solution temperature (LCST) values ​​above 37°C to ensure that they remain soluble at all relevant temperatures. The polymers were characterized by NMR and SEC (Table 2, Figure 3 and Figure 4 ). While RAFT polymerization offers many synthetic advantages, it produces polymers with a reactive trithiocarbonate chain transfer agent (CTA) attached at the Z terminus. Therefore, the CTA moiety on the synthesized AC / DC excipient was removed prior to using the copolymer in subsequent assays to ensure its inertness.

[0232] Example 2 - Ultra-rapidly absorbed insulin lispro (UFAL) formulation and in vitro and in vivo evaluation

[0233] Methods for determining mammalian cell viability: NIH / 3T3 mouse fibroblasts from ATCC were cultured in DMEM containing 10wt.% FBS and 1wt.% penicillin-streptomycin in a 37°C, 5% CO2 incubator. 3T3s at passage 9 were seeded in 96-well plates at 5000 cells per well and cultured in 100 μL of culture medium for 24 hours. Subsequently, 100 μL of MORPH-NIP containing different concentrations was used. 23％ The medium was replaced with the medium of , and incubated for 24 hours. The medium containing the polymer was then aspirated from each well. Each well was then washed with 100 μL of PBS, and 100 μL of new medium and 10 μL of WST reagent were filled into each well. After incubation in the WST solution for 3 hours, the absorbance was read using a plate reader (λ = 450 nm). All experiments were performed in triplicate. Cell viability was calculated using equation S2, where A 孔 , A 对照 and A WST are absorbance measurements of cells cultured with polymer in culture medium, cells cultured without polymer, and WST.

[0234]

[0235] In vitro insulin cell activity assay:C2C12 mouse muscle myoblasts (ATCC CRL-1772) were cultured to detect phosphorylated AKT 1 / 2 / 3 (pS473) compared to total Akt1 using the AlphaLISA SureFire Ultra (Perkin-Elmer) kit to confirm insulin functional activity through the AKT phosphorylation pathway. Before use, cells were confirmed to be free of mycoplasma contamination. Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L D-glucose, L-glutamine, and 110 mg / L sodium pyruvate (Gibco) was supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin to prepare complete culture medium. Cells were seeded in a volume of 200 μL / well in a 96-well tissue culture plate at a density of 25,000 cells / well and grown for 24 hours. Prior to insulin stimulation, cells were washed twice with 200 μL of unsupplemented DMEM and starved overnight in 100 μL of unsupplemented DMEM. The medium was then removed and cells were stimulated with 100 μL of insulin diluted to the desired concentration in unsupplemented DMEM (i). (ii) UFAL, (iii) aged (shaking at 37°C for 12 hours), (iv) aged UFAL (shaking at 37°C for 12 hours) for 30 minutes while incubating at 37°C. Wash cells twice with 100 μL of cold 1X Tris-buffered saline, then add 100 μL of lysis buffer to each well and shake at room temperature for at least 10 minutes to completely lyse the cells. For each assay, transfer 30 μL of lysate to a 96-well white half-area plate. The assay was completed according to the manufacturer's protocol. The plates were incubated at room temperature and read using a Tecan Infinite M1000 PRO plate reader 18 to 20 hours after the addition of the final assay reagents. The results were plotted as the ratio of [pAKT] / [AKT] for each sample (n=3 cell replicates) and EC was plotted using GraphPad Prism 8 50 Regression [log(agonist) vs. response (three parameters)].

[0236] In vitro insulin stability:The method for the aggregation assay of recombinant human insulin was adapted from Webber et al. (Proc. Natl. Acad. Sci. USA 113, 14189-14194 (2016)). Briefly, the formulation samples (3.4 mg / mL) were plated in a transparent 96-well plate at 150 μL per well (n=3 / group) and sealed with an optically transparent and heat-stable seal (VWR). The plate was immediately placed in the plate reader and cultured with continuous shaking at 37°C. The absorbance readings were read every 10 minutes at 540 nm for 100 hours (BioTek Syner-gyH1 microplate reader). The aggregation of insulin causes light scattering, which results in an increase in the measured absorbance. The aggregation time (A) is defined as the time at which the absorbance is observed to increase by more than 10% compared to the absorbance at zero time. After 100 hours, the plate was removed from the plate reader and transferred to an incubator shaker where the plate was shaken for continued stress aging. Absorbance readings were taken periodically over a period of up to 30 days.

[0237] For the initial high-throughput stability screening, recombinant human insulin (Gibco) was formulated in phosphate buffered saline (0.9 wt.% NaCl) and AC / DC excipients at a concentration of 1 mg / mL or 10 mg / mL added to the recombinant insulin formulations for a final insulin concentration of 3.4 mg / mL. Each plate contained a recombinant insulin control without added polymer.

[0238] For the secondary stability screening of UFAL formulations, the control group included: (i) commercial (Eli Lilly); (ii) zinc-free insulin lispro including phosphate buffer, glycerol (2.6 wt.%) and phenoxyethanol (0.85 wt.%). Zinc (II) was removed from a commercial insulin formulation by competitive binding by the addition of ethylenediaminetetraacetic acid (EDTA), which exhibited a dissociation binding constant (K) close to attomolar concentrations. D ~10 -18 M). EDTA was added to the formulation (1 equivalent relative to zinc) to isolate zinc from the formulation. After zinc isolation, the zinc / EDTA complex and other formulation excipients were removed using a PD MidiTrap G-10 gravity column (GE Healthcare). Insulin lispro was concentrated using an Amino Ultra 3K centrifugal unit (Millipore) and then reconstituted at 100 U / mL with phosphate buffer (10 mM), glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%), and AC / DC excipient (0.01 wt.%).

[0239] NMR DOSY: 1H 2D DOSY spectra were obtained in a solution of 3.4 mg / mL insulin lispro with 40 wt.% D2O in a phosphate buffer solution, glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%), and MORPH-NIP 23％ The data were recorded for UFAL formulations of 1,2-dimethoxy- ... 1 The DOSY time and gradient pulse were set to 132 milliseconds (Δ) and 3 milliseconds (δ), respectively. All NMR data were processed using MestReNova 11.0.4 software.

[0240] Streptozotocin-induced diabetic rat model: Male Sprague Dawley rats (Charles River) were used for the experiments. Animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals; all protocols were approved by the Animal Care and Use Committee of Stanford University. The protocol for STZ induction was adapted from that of Wu and Huan (Curr. Protoc. Pharmacol. (2008) 5: Unit 5.47). Briefly, male Sprague Dawley rats of 160 to 230 g (8 to 10 weeks) were weighed and fasted for 6 to 8 hours before treatment with STZ. STZ was diluted to 10 mg / mL in sodium citrate buffer immediately before injection. STZ solution was injected intraperitoneally into each rat at 65 mg / kg. Within 24 hours after injection with STZ, rats were provided with water containing 10% sucrose. After STZ treatment, hyperglycemia of rat blood glucose levels was tested daily by tail vein blood collection using a handheld Bayer ContourNext glucose monitor (Bayer). Diabetes was defined as 3 consecutive blood glucose measurements >400 mg / dL in nonfasted rats.

[0241] In vivo pharmacodynamics in diabetic rats: Diabetic rats were fasted for 4 to 6 hours. Rats were subcutaneously injected with 1.5 U / kg of (i) (ii) UFAL, (iii) aged (shaking at 37°C for 12 hours), (iv) aged UFAL (shaking at 37°C for 12 hours). To prepare the aged samples, 150uL of each formulation was placed in a 96-well plate under constant stirring. 16 rats were used in this study and they were randomly assigned to two groups (i) (ii) UFAL. In these groups, each rat received a dose of fresh and aged versions of the formulation on a separate experimental day. The order of administering the formulation was randomized. Insulin was diluted 10 times in phosphate buffered saline before injection to allow accurate administration of small volumes. Before injection, baseline blood glucose was measured. After injection, blood was sampled once every 30 minutes for 4 hours. Blood glucose was measured using a handheld blood glucose monitor (Bayer Contour Next).

[0242] Biocompatibility in Diabetic Rats: For diabetic rats (i) (n=5) or (ii) UFAL (n=5) treatment for 7 consecutive days. The formulation was administered subcutaneously at a dose of 1.5 U / kg. Blood was collected on days 0 and 7 for blood chemistry testing. Chemical analyses were performed on a Siemens Dimension Xpand analyzer. A medical technologist performed all tests, including specified dilutions and replicates, and reviewed all data.

[0243] In vivo pharmacokinetics and pharmacodynamics of ultra-rapidly absorbed insulin lispro (UFAL) in a porcine model Research:

[0244] Study Design: The pharmacokinetics of an insulin lispro formulation, ultra-rapidly absorbed insulin lispro (UFAL), were compared with a commercial insulin lispro formulation ( ) were compared. Subcutaneous injection (i) or (ii) after UFAL, blood glucose and plasma insulin lispro concentrations were measured on blood samples collected using a handheld blood glucose monitor or ELISA. Randomization: Five pigs were used in this study, and each pig received each formulation once. The order of administration of the formulations was randomized. Blinding: For analysis of pharmacokinetic parameters (t50% rise, time to peak, t50% decline), pharmacokinetic curves were coded and analyzed by blinded investigators. Replication: Five pigs were used in this study, and each pig served as its own control receiving each formulation ( and UFAL) once.

[0245] Streptozotocin-induced diabetes in pigs: Five female Yorkshire pigs (pork meal) were used for animal studies, which were conducted in accordance with the Guide to the Care and Use of Laboratory Animals and the Animal Welfare Act. All protocols were approved by the Stanford Institutional Animal Care and Use Committee. Type 1 diabetes in pigs (25 to 30 kg) was induced using streptozotocin (STZ) (MedChemExpress). STZ was injected intravenously at a dose of 125 mg / kg, and the animals were monitored for 24 hours. Food and 5% glucose solution were administered as needed to prevent hypoglycemia. Diabetes was defined as fasting blood glucose above 300 mg / dL.

[0246] In vivo pharmacokinetics and pharmacodynamics in diabetic pigs: Five diabetic pigs were fasted for 4 to 6 hours. The pigs were injected subcutaneously with 2 to 4 U of the following formulation: (i) (100 U / mL, Eli Lilly) or (ii) ultra-rapid absorption insulin lispro (UFAL) (100 U / mL Zn-free insulin lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, 0.01 wt.% MORPH-NIP 23％ ). Determine dosage based on insulin sensitivity value of individual pig, wherein target is blood sugar reduction about 200mg / dL.Individual pig of each treatment group receives identical dosage.Pig receives every kind of formulation once every other day, and the order of treatment group is randomized.Before injection, sample baseline blood from venous catheter, and measure using handheld glucose monitor (Bayer Contour Next).After injection, in the first 60 minutes, sample blood sample from venous catheter every 5 minutes, then sample once every 30 minutes, until 4 hours.Blood is collected in K2EDTA plasma tube (Greiner-BioOne), for ELISA analysis.Use insulin lispro ELISA kit (Mercodia company) to quantify plasma insulin lispro concentration.

[0247] Pharmacokinetic Modeling: The pharmacokinetic model used in this analysis was derived from a literature report. The concentration of injected insulin (I inj ), interstitial balance (I eq ) and plasma (I p ) is solved numerically as outlined below.

[0248]

[0249]

[0250]

[0251] Initialize the concentration so that at t = 0, all insulin is present in I inj Normalized insulin plasma concentrations generated by minimizing the model from 0 to 90 min experimental time points were compared with UFAL and The sum of squared errors (SSE) between the normalized porcine plasma insulin concentrations and the kinetic rate constants were fit to the normalized porcine pharmacokinetic curve. It was assumed that k2 and k3 were species-dependent, while k1 was species- and formulation-dependent. While minimizing the SSE, k 1，UFAL，猪There is no room for improvement; so that for a given k2 and k3, k 1，UFAL，猪 Higher values ​​of k result in progressively smaller SSEs at the edges. 1，UFAL，猪 Then it is set to 100,000 minutes -1 The rate constants were optimized by first performing a grid search using SciPy's optimize brute force function and then minimizing the SSE by using the L-BFGS-B method using SciPy's optimize minimize function. 1，UFAL，人 , assuming the following relationship:

[0252]

[0253] k 2，人 and k 3，人 The values ​​of were used as reported in the literature.

[0254] Statistical analysis: Unless otherwise stated, all results are expressed as mean ± SD. Figures 10D to 10F Shown are mean ± standard error of the mean. All statistical analyses were performed as general linear models (GLM) in JMP Professional 14. Comparisons between formulations ( Fig. 10D =Analysis of the responses of the subjects (all P < 0.05) was performed using a restricted maximum likelihood (REML) repeated measures mixed model. Appropriate transformations were applied as needed to meet the assumptions of the methods (i.e., homogeneity of variance, normality of errors, and linearity). Time to 50% of peak, time to peak, and time to 50% of peak decline were log transformed for analysis corrected for heterogeneity of variance. Pigs were included in the model as a random effects blocking (control) factor to account for variation in individual pig responses. Statistical significance was considered p < 0.05 for Fig. 10D To I, post hoc Bonferroni correction was used to account for multiple comparisons, and significance was adjusted to α = 0.008.

[0255] result

[0256] High-throughput screening of insulin stabilizing excipients: The potential of AC / DC excipients prepared as described in Example 1 as stabilizing excipients for insulin was evaluated using an absorbance-based stress aging assay, in which unstable insulin aggregates scatter light and increase the absorbance of the solution. In these assays, the aggregation time was defined as the time it took for the absorbance of the formulation to increase by 10%. Recombinant insulin was formulated in PBS at standard formulation concentrations (100 U / mL; 3.4 mg / mL) and was incubated with (i) no polymer excipient, (ii) Pluronic L-61 (which is chemically and physically similar to The results were as follows: (i) 100 mg / mL AC / DC excipients (the most similar commercially available polymer used in U400, poloxamer 171), (ii) 1 mg / mL AC / DC excipients, or (iii) 10 mg / mL AC / DC excipients. In this assay, the recombinant insulin control without the polymer excipient aggregated within 13 ± 8 hours. The formulation containing Pluronic L-61 (1 mg / mL) extended aggregation to 27 ± 2 hours, demonstrating the efficacy of commercial polymers as excipients to prevent insulin aggregation. The use of a water-soluble carrier homopolymer excipient (1 mg / mL) had no effect on insulin stability ( Figure 5A ), demonstrating that free hydrophilic polymers are not sufficient to prevent insulin aggregation. This finding is supported by previous work showing that other hydrophilic polymers such as poly(ethylene glycol) (PEG) do not improve insulin stability.

[0257] The stability of insulin when formulated with AC / DC excipients is highly chemically dependent. Each AC / DC excipient was formulated with insulin and tested for stability over a period of up to one month ( Figure 5B To G and Table 3). The formulations comprising AC / DC excipients and MPAM and MORPH carrier chemicals showed the overall highest improvement of insulin stabilization, especially when combined with NIP, TBA and PHE dopants. Although many carrier-dopant combinations showed long-term stability at 1wt.% formulation concentration, attempts were made to engineer copolymers that can stabilize insulin at the minimum concentration of formulations. The AC / DC excipients comprising MPAM-PHE, MPAM-TBA, MPAM-TRI and MORPH-TBA (0.1wt.%) stabilized insulin for stress aging over 100 hours. Therefore, the stability of these formulations is 7 times that of the stability of independent recombinant insulin, and is 3 times that of the stability of the formulation containing Pluronic L-61. In addition, the AC / DC excipients comprising MPAM-NIP, MPAM-AMP and MORPH-PHE (0.1wt.%) stabilized insulin for 30 days of stress aging, which terminated at the time point. The stability of these formulations is 50 times that of insulin alone and 24 times that of formulations containing Pluronic L-61. These selected carriers and dopants are the most hydrophobic among the monomers screened, indicating that the amphiphilic water-soluble copolymers are the most effective in preventing insulin aggregation.

[0258] Table 3: Days before aggregation of recombinant insulin formulated with AC / DC excipients at two excipient concentrations (1 mg / mL and 10 mg / mL)

[0259]

[0260]

[0261] nt indicates not tested.

[0262] Stabilizing Monomeric Insulin by Careful Screening: Based on the initial recombinant insulin stability screen, copolymers including MPAM or MORPH carriers with NIP or PHE dopants proved to be the most promising candidates for stabilizing monomeric insulin. Previous work by the group demonstrated that the balance between insulin association states can be altered by changing the formulation excipients, where a formulation that is approximately 70% monomeric can be achieved with a zinc-free insulin lispro formulation containing glycerol and phenoxyethanol. This formulation favors insulin monomers and completely dissociates insulin hexamers. Mainly hexamers Representative SEC traces of predominantly monomeric zinc-free UFAL demonstrate the associated state of insulin in the formulation ( Figure 6 ). However, insulin monomers are unstable in formulations and require additional stabilizing excipients to translate. Further, it may be prudent to use copolymer excipients at as low a concentration as possible to reduce chronic exposure to the excipients and to use typical insulins frequently to control diabetes.

[0263] To address this need, a second AC / DC excipient library was synthesized to evaluate additional carrier-dopant ratios for the best performing candidate monomers: (i) MPAM and MORPH as monomers and (ii) NIP and PHE as dopants. Standard synthetic practice was followed to characterize the chemistry by SEC and 1 H NMR spectroscopy yielded this secondary library consisting of copolymers of DP 50 with MORPH or MPAM as carrier and either (i) NIP at a loading of 14, 17, 20, 23, or 26 wt.% or (ii) PHE at a loading of 6, 8, 10, 12, or 14 wt.% ( FIG. 7 , Table 4 ).

[0264] Table 4: SEC and DP 50 of polymers synthesized during the second screening 1 H NMR analysis

[0265]

[0266]

[0267] a The M was determined using size exclusion chromatography n (number average molecular weight), M w (weight average molecular weight) and (Dispersity) Calibrated using polyethylene glycol standards.

[0268] bWeight percentages calculated from post-precipitation NMR spectra of MORPH (δ = 3.3-3.7, 8H) and PHE (δ = 7.6, 2H).

[0269] c The weight percentage was difficult to determine due to spectral overlap. The weight percentage was estimated from the NMR spectrum after precipitation by measuring the left half of the higher resolution peak of NIP (δ = 4.0, 0.5H), doubling it, and subtracting it from the unresolved peaks of MORPH and NIP (δ = 3.2-4.2, 7H (MORPH) 1H (NIP)).

[0270] d Weight percentages calculated from post-precipitation NMR spectra of MPAM (δ = 3.1-3.5, 7H) and PHE (δ = 7.6, 2H).

[0271] e Weight percentages calculated from post-precipitation NMR spectra of MPAM (δ = 3.2, 3H) and NIP (δ = 3.8, 1H).

[0272] Using the AC / DC excipient synthesized in the secondary screen, UFAL formulations with 0.01 wt.% (0.1 mg / mL) copolymer excipient were prepared and insulin aggregation was assessed under stress conditions using the same assay as the initial screen (Table 5 in Supplementary Appendix A). Commercial Formulations of Insulin Lispro Under these conditions, the UFAL aggregated within 6 hours. UFAL without AC / DC excipients aggregated within 1.3 ± 0.3 hours, demonstrating the severe instability of the insulin monomer in solution. All UFAL formulations stabilized with MORPH-PHE or MPAM-PHE AC / DC excipients showed stability to stress aging at least equivalent to that of commercial MPAM (MPAM-PHE 14％ ) and MORPH with 12 wt.% PHE (MORPH-PHE 12％ ) were the best candidates, extending the stability of UFAL formulations to 27±2 hours and 25±5 hours, respectively ( Figure 8B to C). MPAM-NIP copolymers showed limited efficacy in stabilizing monomeric insulin; however, In contrast, the MORPH-NIP copolymer prolonged the stability of monomeric insulin. In fact, the MORPH (MORPH-NIP 23％ ) extended the stability of the UFAL formulation to more than 25 ± 1 hours. The best candidate AC / DC excipient after the second screening was MPAM-PHE14％ 、MORPH-PHE 12％ and MORPH-NIP 23％ Although these copolymers demonstrated high efficacy, MPAM-PHE 14％ and MORPH-PHE 12％ It was also demonstrated that the solubility and LCST-like phase separation behavior at physiological temperature decreased when present at higher concentrations. 23％ was selected as the best candidate for stabilizing UFAL formulations. In vitro and in vivo bioactivity assays were used to confirm the transmittance data and confirm the integrity of UFAL before and after aging. 473 UFAL showed no loss of activity after 12 hours of stress aging in a cellular assay of phosphorylation or in diabetic rats with reduced blood glucose levels ( FIG. 9 ).

[0273] To verify the presence of MORPH-NIP 23％ The formulations did not shift the association state equilibrium of insulin lispro away from the monomeric form, as determined by NMR DOSY ( Fig.8D ). NMR DOSY indicated the diffusion rate of insulin lispro under formulation conditions (100 U / mL insulin lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol and 0.1 wt.% MORPH-NIP 23％ ) has 2.0×10 -10 m 2 s -1 The diffusion rate of 1.0 nm corresponds to a hydrodynamic radius of 1.2 nm, which corresponds to the value reported in the literature for insulin monomers (28). NMR DOSY also provides insights into the stabilization mechanism of polymer excipients. MORPH-NIP 23％ Diffusion at a slower rate than insulin suggests that its stabilization mechanism is independent of excipient-insulin complexation and co-diffusion. These data support the hypothesis that copolymer-interface interactions are the primary mechanism driving stabilization of monomeric insulin in formulations.

[0274] Pharmacokinetics and pharmacodynamics of UFAL formulations in diabetic pigs: To evaluate the ultrafast potential of monomeric insulin formulations, pharmacokinetic studies were performed in a porcine model of insulin-deficient diabetes. or (ii) UFAL (100 U / mL insulin lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol and 0.1 wt.% MORPH-NIP 23％) were treated with 2-4 U of lispro insulin, depending on the insulin sensitivity of each pig. The initial blood glucose level of the pigs was between 330 and 430 mg / dL, and the insulin dose was selected to lower blood glucose to approximately 100 mg / dL. The insulin dose given to each pig was consistent between treatment groups and was The reduction in blood glucose was similar in both HF and UFAL treatments ( Fig. 10A and Figure 11). Plasma concentrations of insulin lispro were measured over time by enzyme-linked immunosorbent assay (ELISA) to assess the pharmacokinetics following subcutaneous injection of each treatment group. No difference in total exposure (AUC 210 ) Fig. 10C ). By looking at the AUC t / AUC 210 The percentage of exposure at different time points was analyzed. This analysis showed that In comparison, the exposure of UFAL at the 10-min and 20-min time points increased ( Figures 10D to 10I ). Mean residence time (MRT) is often reported for formulation pharmacokinetics. For intravenous administration, MRT is usually described as the area under the moment curve (AUMC) divided by the area under the curve (AUC); however, when the drug is administered subcutaneously, the mean absorption time (MAT) must also be considered. When an absorption period exists, AUMC / AUC = MAT + MRT. Calculate and plot the ratio of the area under the moment curve (AUMC) to the area under the pharmacokinetic plot curve (AUMC / AUC), showing the relationship between UFAL and There was no difference between treatments ( Fig.13 ). This is not surprising, as the expected The blood clearance of both ALEXANDER® and UFAL (both are insulin lispro) was similar, and the magnitude of MAT was smaller compared to MRT, thereby masking the differences between the formulations.

[0275] Alternatively, exposure metrics are often reported for rapid-acting insulin formulations to describe the pharmacokinetics of the formulation. The "onset of action" rate of rapid-acting insulin is usually determined using two metrics: (i) the time to rise to 50% of the normalized peak height after administration (expressed as "rise 50%") and (ii) the time to peak insulin plasma concentration. Normalized plasma concentration measurements are used to compare commercial insulins with The time to peak insulin lispro concentration in the UFAL-treated group ( Figures 10J to 10M ). Pig The pharmacokinetics of UFAL are almost twice that of humans. Fast, the time for UFAL to reach 50% of the peak value (5±2 minutes) is (12±6 minutes), and the time to peak of UFAL (9±4 minutes) was The exposure duration of UFAL (defined as the time to reach a 50% decrease in the normalized peak height at the peak exposure concentration (time to reach 50% peak decrease)) (28 ± 8 minutes) was 2.8 times that of the baseline exposure duration (defined as the time to reach a 50% decrease in the normalized peak height at the peak exposure concentration) (28 ± 8 minutes). 1 / 1.9 of the exposure duration (54±21 minutes).

[0276] Modeling the pharmacokinetics of UFAL in humans: To better understand how the rapid onset and short duration of action demonstrated by UFAL in pigs translates to humans, the pharmacokinetic model of Wong et al. (Journal of Diabetes Science and Technology (2008) 2: 658-671) was adapted to approximate the pharmacokinetics of UFAL in humans. The model was constructed so that injection into the subcutaneous space (I inj ) in rapid-acting insulin analogs ( ) dissociates and diffuses into the interstitium with a rate constant k1 eq ) is absorbed into plasma (I p ) and is subsequently cleared by several mechanisms that can nevertheless be approximated by a single elimination constant k3 ( Fig. 12A ). It is assumed that k2 and k3 are species-dependent, k1 is formulation- and species-dependent, and the ratio of k1 between formulations is species-independent. Because the UFAL formulation is composed of insulin monomers and dimers, the time required to reach equilibrium in the interstitium is expected to be significantly lower than In fact, when fitting the experimental porcine pharmacokinetics of subcutaneously administered UFAL, k1 tends to infinity, meaning that UFAL effectively bypasses the first model compartment and insulin monomers immediately reach equilibrium in the subcutaneous space (Table 6). The porcine pharmacokinetic data of the two were fitted in Fig. 12B The infinite k1 determined for porcine UFAL was converted to a human pharmacokinetic model and used to estimate UFAL pharmacokinetics while maintaining k2 and k3 values ​​reported in the literature ( Fig. 12C ).

[0277] Table 6: Rate constants used to simulate PK profiles in Example 2.

[0278]

[0279] a Human pharmacokinetic rate constants from Wong et al. Journal of Diabetes Science and Technology (2008) 2: 658-671.

[0280] The model predicts a human UFAL onset time (i.e., 50% rise) of 2.5 minutes, peak exposure at 10 minutes, and exposure duration (i.e., 50% decline) of 28 minutes ( Fig.12D ). In contrast, using parameters reported in the literature, the model predicts that “rapid-acting” insulin analogs (RAI), such as The onset of action was 14 minutes, the peak exposure was 43 minutes, and the exposure duration was 157 minutes ( Fig. 12C Although the RAI model underestimates the time to onset of exposure (t50% rise), the predicted curve robustly captures the published Clinical pharmacokinetic data on the peak and duration of exposure were used. Therefore, pharmacokinetic modeling predicted that UFAL would exhibit more than 4 times the kinetics of current "fast-acting" insulin formulations. Further comparison with clinical data on fast-acting insulin formulations demonstrated that UFAL was predicted to be even faster than second-generation fast-acting insulin formulations such as Fiasp (Novo Nordisk) and BioChaperone Lispro (Adocia) ( Fig.12E ).

[0281] In diabetic pigs, this UFAL formulation exhibited ultrafast pharmacokinetics with an onset of action of (commercial "fast-acting" insulin formulations using the same insulin molecule, insulin lispro), and the duration of exposure is These results suggest that this UFAL formulation more closely mimics endogenous insulin secretion in healthy individuals and highlight the promise of this formulation for enhanced diabetes management. Fiasp, a faster-acting version of the commercial insulin aspart formulation, has also shown a number of clinical benefits with incremental improvements in pharmacokinetics over current “fast-acting” insulin formulations. While Fiasp showed a modest reduction in time to peak of 10 minutes and duration of action of 15 minutes compared to “fast-acting” insulin formulations, postprandial glucose excursions and HbA1c levels in diabetic patients were still reduced with Fiasp. In contrast, in diabetic pigs, insulin pharmacokinetics were observed to be twice that of humans, comparable to In comparison, UFAL reduced peak exposure by 16 minutes and reduced exposure duration by 26 minutes. The results observed in diabetic pigs, combined with model-predicted human UFAL pharmacokinetics, suggest that UFAL may have absorption kinetics unprecedented in an injectable insulin formulation. If achieved in human clinical studies, these kinetics would approach those of commercially available inhaled insulins. However, In contrast, UFAL is an injectable formulation that enables more accurate dosing regimens and compatibility with pumps and closed-loop systems, offering UFAL the potential to improve postprandial glycemic control in diabetic patients.

[0282] Taken together, these studies identify copolymer excipients for protein stabilization and demonstrate their utility in stabilizing ultrafast-absorbing insulin formulations. Initial cytotoxicity experiments demonstrated that the copolymer excipients were not toxic at doses an order of magnitude higher than those used in insulin formulations ( Fig.13 Preliminary biocompatibility studies of UFAL in diabetic rats also confirmed the cytotoxicity results, indicating that UFAL should be well tolerated (Figure 14).

[0283] Example 3: Ultra-rapid insulin-pramlintide co-formulation

[0284] Material: The amphiphilic acrylamide copolymer excipient acryloylmorpholine was prepared according to the procedure described above in Example 1, UFAL 77％ -N-isopropylacrylamide 23％ (MoNi 23％ ). (Eli Lilly) and pramlintide (BioTang) were purchased and used as received. For zinc-free insulin lispro, zinc (II) was removed from insulin lispro by competitive binding by adding ethylenediaminetetraacetic acid (EDTA), and the commercial insulin formulations exhibited a dissociation binding constant (K) close to attomolar concentrations. D ~10 -18 M). EDTA was added to the formulation (4 equivalents relative to zinc) to isolate zinc from the formulation, and then insulin lispro was separated using a PD MidiTrap G-10 gravity column (GE Healthcare) for buffer exchange into water. The solution was then concentrated using an Amino Ultra 3K centrifugal unit (Millipore) and reconstituted in 10 mM phosphate buffer (pH = 7.4) with 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich.

[0285] Online Size Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS):As previously reported, SEC-MALS was used to obtain the insulin association state composition of monomeric insulin formulations (Maikawa et al., Adv. Ther. (2019) 75:1900094). Zinc-free insulin lispro was evaluated in a buffer containing glycerol (2.6%) and phenoxyethanol (0.85%). Briefly, the number average molecular weight (MW) and dispersity of the formulations were Obtained using size exclusion chromatography (SEC), the SEC is performed using a Dionex Ultimate 3000 instrument (including a pump, an autosampler, and a column chamber), the instrument is equipped with a Dawn Heleos II multi-angle light scattering detector and an Optilabrex refractive index detector. The chromatographic column is a Superose 6Increase 10 / 300GL from General Electric Healthcare. Astra 6.0 software is used to analyze the data. The fraction of each insulin association state is obtained by fitting the experimentally obtained number average molecular weight and weight average molecular weight to equations 1 and 2 below. m, d, and h represent the molar fractions of monomer, dimer, and hexameric insulin, respectively, and I represents the molecular weight of monomer lispro insulin. The solver is constrained to make m+d+h=1, while m, d, and h are still between 0 and 1.

[0286] M n =m*I+d*2I+h*6I (1)

[0287]

[0288] In vitro stability assessment of insulin and pramlintide: Aggregation assays for assessing stability were adapted from Webber et al. (Proceedings of the National Academy of Sciences of the United States of America (2016) 113: 14189-14194). Briefly, formulations were aliquoted in clear 96-well plates at 150 μL per well (n=3 / group) and sealed with optically clear and heat-stable seals (VWR). Plates were incubated at 37°C in a microplate reader (BioTek Synergy H1 microplate reader) with continuous stirring (567 cpm). Absorbance readings were taken at 540 nm every 10 minutes for the duration of the experiment. The formation of insulin or pramlintide aggregates results in light scattering and a decrease in sample transmittance (aggregation time = time to reach a 10% change in transmittance). Controls included: (i) (100U / mL); (ii) (100 U / mL) + pramlintide (1:6 insulin lispro: pramlintide); (iii) zinc-free insulin lispro (100 U / mL insulin lispro, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, pH = 7.4). Evaluation of the effect of 0.1 mg / mL MoNi 23％Stability of mixed insulin-pramlintide co-formulation (100 U / mL insulin lispro, 1:6 insulin lispro:pramlintide, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, pH = 7.4).

[0289] Streptozotocin-induced diabetic rat model: Male Sprague Dawley rats (Charles River) were used for the experiments. Animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals; all protocols were approved by the Animal Care and Use Committee of Stanford University. The protocol for streptozotocin (STZ) induction was adapted from that of Wu and Huan. In brief, male Sprague Dawley rats of 160 to 230 g (8 to 10 weeks) were weighed and fasted at 6 to 8 hours in the morning before treatment with STZ. STZ was diluted to 10 mg / mL in sodium citrate buffer immediately before injection. STZ solution was injected intraperitoneally into each rat at 65 mg / kg. Within 24 hours after injection with STZ, rats were provided with water containing 10% sucrose. After STZ treatment, hyperglycemia of rat blood glucose levels was tested daily by tail vein blood collection using a handheld Bayer Contour Next glucose monitor (Bayer). Diabetes was defined as 3 consecutive blood glucose measurements >400 mg / dL in non-fasted rats.

[0290] In vivo pharmacokinetics and pharmacodynamics in diabetic rats: Prior to injection, diabetic rats were fasted for 4 to 6 hours. For pharmacokinetic experiments, rats were injected with 1 U of insulin formulation (approximately 2 U / kg) and then immediately (<30 seconds after injection) given a 1 g / kg glucose solution by oral gavage. The formulations tested were: (i) (ii) and a single injection of pramlintide (1:6 pramlintide: insulin lispro, pH = 4); (iii) insulin-pramlintide co-formulation (100 U / mL insulin lispro, 1:6 insulin lispro: pramlintide, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, 0.1 mg / mL MoNi 23％, pH = 7.4). Each cohort of 11 rats received each formulation once, and the order of administration of the formulations was randomized. To allow accurate dosing and avoid dilution effects (dilution favors insulin monomers), the formulations were diluted twice (10 μL formulation + 10 μL formulation buffer) immediately before administration. After injection, blood glucose measurements were performed using a handheld glucose monitor (Bayer Contour Next), and additional blood (Sarstedt serum tubes) was collected for ELISA analysis. Time points were recorded every 3 minutes for the first 30 minutes, then every 5 minutes for 30 minutes, and then at 75 minutes, 90 minutes, and 120 minutes. Serum pramlintide concentrations were quantified using a human amylin ELISA kit (Millipore Sigma). Serum lispro insulin concentrations were quantified using NorthernLights Mercodia Lispro NL-ELISA. A second pharmacodynamic experiment was performed in an attempt to better match insulin doses and oral glucose doses to better simulate mealtime glucose management. The same formulation was tested, but the dosage was changed to 0.75U / kg insulin, which was delivered subcutaneously immediately before oral gavage of 2g / kg glucose. Undiluted (100U / mL) formulations were accurately administered using 10 μL Hamilton syringes. Ten rats per cohort received each formulation once, and the order in which the formulations were given was randomized. Glucose was measured only, and time points were measured every 5 minutes in the first hour, and then at 75 minutes, 90 minutes, and 120 minutes.

[0291] Gastric emptying in diabetic rats: Acetaminophen was used as a model compound for assessing gastric emptying during a meal. Diabetic rats were fasted for 4 to 6 hours before the start of the experiment. Rats were then injected subcutaneously with one of the following formulations (2 U / kg): (i) (ii) and a single injection of pramlintide (1:6 pramlintide: insulin lispro, pH = 4); (iii) insulin-pramlintide co-formulation (100 U / mL insulin lispro, 1:6 insulin lispro: pramlintide, 2.6 wt.% glycerol, 0.85 wt.% phenoxyethanol, 0.1 mg / mL MoNi 23％, pH = 7.4). To allow accurate dosing and avoid dilution effects (dilution favors insulin monomers), the formulation was diluted twice (10 μL formulation + 10 μL formulation buffer) immediately before administration. Each cohort of 11 rats received each formulation once, and the order of administration of the formulations was randomized. Acetaminophen was administered immediately after insulin administration by oral gavage as a slurry in phosphate buffer (100 mg / kg). (Before oral gavage, the tip of the feeding tube was immersed in a glucose solution to reduce the pressure of administration). Blood samples were collected for ELISA (Neogen) at -30, 0, 15, 30, 60, 90, 120 and 150 minutes after injection.

[0292] statistics: Unless otherwise stated, all results are expressed as mean ± standard error (SE). All statistical analyses were performed as general linear models in JMP Professional Edition 14. Comparisons between formulations were performed using a restricted maximum likelihood repeated measures mixed model. When formulation was a significant fixed effect, multiple comparisons were performed using a post hoc Tukey HSD test and adjusted p values ​​were reported. Rats were included in the model as a variable as a random effect blocking (control) factor to illustrate individual changes in rat responses. (Each rat received each formulation and served as its own control). Statistical significance was considered to be P < 0.05. For Figures 18H to 18L, post hoc Bonferroni corrections were applied to illustrate formulation comparisons at multiple exposure time points (in addition to Tukey HSD corrections), and significance was adjusted to α = 0.01.

[0293] result

[0294] Stability of insulin-pramlintide co-formulations: MoNi 23％ Characterization of molecular weight and monomer composition can be found in Table 7. The results shown in Example 2 demonstrate that MoNi 23％ The propensity of insulin and pramlintide to aggregate to form amyloid fibrils, primarily initiated at hydrophobic interfaces, makes them ideal for use as stabilizing excipients for monomeric insulin. 23％ Therefore, assuming that the same MoNi 23％ It may also be used to physically stabilize ultra-rapid mealtime insulin-pramlintide co-formulations so that a single formulation has increased pharmacokinetic overlap between the two hormones.

[0295] Table 7: MoNi copolymer excipient characterization.

[0296]

[0297] aDetermined by size exclusion chromatography using polyethylene glycol sample calibration.

[0298] b Due to spectral overlap, the weight percentage is difficult to determine. The weight percentage was estimated from the NMR spectrum after precipitation by measuring the left half of the higher resolution peak of N-isopropylacrylamide (δ = 4.0, 0.5H), doubling it, and subtracting it from the unresolved peaks of Mo and Ni (δ = 3.2-4.2, 7H(Mo)1H(Ni)).

[0299] Zinc-free insulin lispro (2.6 wt.%) and phenoxyethanol (0.85 wt.%) in the presence of glycerol as a tonicity agent and antimicrobial agent produced a formulation with a high monomer content. This result was confirmed in the current study where 83% monomer, 17% dimer and 0% hexamer ( Fig.15E and Fig.16 ). In contrast, commercial >99% hexamer. For SEC-MALS measurements, only small molecule excipients were used to test the insulin association state alone, because pramlintide and MoNi 23％ The excipients have similar molecular weights and would prevent calculation of the monomer content in the formulations by SEC-MALS. Diffuse ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) showed that the addition of MoNi 23％ No changes in insulin association status. The presence of pramlintide is not expected to alter insulin association status.

[0300] The insulin-pramlintide co-formulation was composed of zinc-free insulin lispro (100 U / mL), pramlintide (1:6 molar ratio of pramlintide: insulin lispro), glycerol (2.6 wt.%), phenoxyethanol (0.85 wt.%) and MoNi 23％ (0.1 mg / mL) in phosphate buffer at pH 7 ( Fig.15D ). A pramlintide ratio of 1:6 was chosen to compare with previous work using CB[7]-PEG stabilized insulin-pramlintide co-formulations in diabetic pigs. Further, a ratio of 1:6 is similar to the high endogenous insulin-pramlintide ratios reported in the literature and is within the range of ratios indicated to be most effective by computer experiments. Formulation stability was assessed using a stress aging assay. As insulin and / or pramlintide aggregates form, the aggregates scatter light, which can be measured by absorbance. As defined herein, formulation aggregation is defined as a change in transmittance of 10% or greater. The co-formulations tested showed stability for 16.2±0.1 hours, which is a commercially available (aggregated after 8.2±0.5 hours) twice ( Fig.15F). Pramlintide was added directly to After mixing, a translucent formulation is formed immediately. Compared with Fig.17 ). This mixture reaches an aggregation threshold after 8±3 hours, which is highly variable due to variable initial transmittance. Zinc-free insulin lispro alone is mainly monomeric and highly unstable, rapidly aggregating after 5.7±0.1 hours.

[0301] Pharmacokinetics and Pharmacodynamics in Diabetic Rats: After establishing the stability of the insulin-pramlintide coformulation, its in vivo pharmacokinetics were evaluated to determine whether the use of monomeric insulin would result in increased pharmacokinetic overlap between insulin and pramlintide. The co-formulations were tested with controls of single injections of insulin and pramlintide (Figure 18). Each rat was given a high dose of each formulation (2 U / kg) followed by oral gavage with glucose solution (1 g / kg). Similar levels of glucose depletion were observed in all three formulations, however, compared to Glucose depletion with the coformulations tended to be more rapid in onset (faster glucose depletion) and shorter in duration (faster glucose recovery) than with the single injection control ( Fig.18A This trend is also reflected in the pharmacokinetics of insulin lispro, where a more rapid onset of action and a shorter time to peak effect were observed for the co-formulations ( Figures 19A to 19C There was a difference in duration of action between the two formulations, defined as a 50% decrease from the peak (F2,20 = 7.07, P = 0.0048). The duration of action of the co-formulation was shorter (22 ± 2 minutes) compared with the single injection (34 ± 3 minutes, P = 0.0034) and was similar to that of the single injection (22 ± 2 minutes, P = 0.0034). (27±2 minutes, P=0.24), there was a trend of shorter duration of action ( Figures 19A to 19D ). Use the exposure ratio - the fraction of the area under the curve (AUC) at a given time point to the total area (AUC t / AUC 120 ), which also confirmed a faster onset of action. At 6 minutes, 15 minutes, and 30 minutes, the Compared with single injection, the co-formulation showed a greater total exposure fraction ( Figures 19E to 19I There were no differences in the area under the exposure curve for either insulin lispro or pramlintide between the two formulations ( FIG. 20 ). As expected, no differences were observed between the kinetics of pramlintide delivered by injection alone and the co-formulations ( Figures 19J to 19M , Figure 21).

[0302] The leftward shift of the pramlintide insulin pharmacokinetic curve of the coformulation insulin lispro was confirmed by superimposing the curves of pramlintide insulin delivered by either single injection or coformulation and comparing the overlap time (Figure 22). Overlap is defined as the ratio of overlap to total peak width at half peak height (overlap ÷ (insulin lispro + pramlintide overlap). As hypothesized, delivering monomeric insulin with pramlintide in a coformulation resulted in increased overlap (0.75 ± 0.06) (0.47 ± 0.07, F1,10 = 6.96, P = 0.025) compared to single injections. Fig. 10C The faster insulin kinetics and increased overlap between insulin and pramlintide observed in the co-formulations more closely mimic insulin-pramlintide secretion during a meal.

[0303] Gastric emptying of acetaminophen in diabetic rats: Once faster insulin kinetics and increased overlap between insulin and pramlintide were observed for the co-formulations of the present disclosure, the next step was to determine whether the insulin kinetics were comparable to those of the co-formulations alone. Or use alone Whether the co-formulations have mealtime benefits compared to standard administration of pramlintide. Acetaminophen was used as a model cargo to confirm the functionality of pramlintide by testing its ability to delay gastric emptying after administration of the formulation (Figure 23). In contrast, pramlintide in both single administrations and in co-formulation resulted in delayed gastric emptying. The time to peak acetaminophen concentration was delayed to 76 ± 5 minutes for single injection and 68 ± 6 minutes for co-formulation, compared to 35 ± 5 minutes for single injection and co-formulation, demonstrating that there was no difference in the time to peak acetaminophen between single injection and co-formulation ( Fig.23C ).

[0304] Mealtime Glucose Challenge in Diabetic Rats: The co-formulations were further tested in a simulated mealtime challenge with a low dose of subcutaneous insulin (0.75 U / kg) and a high dose of glucose (2 g / kg) administered by oral gavage ( FIG. 24 ). In contrast to glucose measurements in the insulin-dominated pharmacokinetic experiments, this experiment was designed to reduce the insulin dose and increase the glucose load to better simulate mealtime glucose management. All three formulations controlled the glucose peak similarly ( Fig.24C ). However, for insulin and pramlintide with MoNi 23％ In the immediate co-formulation of the copolymers, a rapid insulin onset combined with delayed gastric emptying and a short duration of action was observed, resulting in tighter control of this mealtime glucose spike while also reducing the magnitude of glucose depletion to below baseline levels ( Fig. 24B and Fig.24D). In contrast, while delayed gastric emptying with the formulation alone resulted in rapid glucose depletion and control of mealtime glucose spikes, it also resulted in greater glucose depletion below baseline. Both insulin and pramlintide produced similar glucose profiles as when administered alone, but glucose depletion was delayed because glucose release was not delayed. These results suggest that the co-formulation can achieve good postprandial glycemic control while also reducing postprandial hypoglycemia.

[0305] This study demonstrates that co-formulations of monomeric insulin lispro and pramlintide have ultrafast kinetics and a high degree of overlap, which results in improved glucose management after a glucose challenge. 23％ As a stabilizer and physically stable in stress aging tests, it is commercially available Similar to pramlintide alone, pramlintide in the coformulation resulted in delayed gastric emptying. Further, the combined effect of synchronized ultra-rapid insulin and pramlintide delivery in the coformulation resulted in a reduction in glucose depletion below baseline measurements while maintaining control of the initial glucose peak in a simulated "mealtime" glucose challenge. These results suggest that the coformulation has the potential to improve glucose management by reducing the risk of postprandial hypoglycemia while reducing patient burden.

[0306] and The data for rats showed only a trend towards an increase in the onset of action (50% of the peak rise) and time to peak for insulin lispro in the co-formulation compared to injection alone. However, the AUC ratios representing the fractional exposure at different time points showed a decrease in the onset of action (50% of the peak rise) and time to peak for insulin lispro in the co-formulation compared to injection alone and before 30 minutes post-injection. These observations were surprising because the study was conducted in diabetic rats, which have a faster insulin absorption rate compared to humans because their skin is loose, resulting in a larger surface area for subcutaneous absorption ( Figures 25A to 25C ). In fact, studies comparing rapid-acting insulin analogs with significantly different onset times to recombinant human insulin observed no differences when compared in rats. Previous studies of monomeric insulin lispro in diabetic pigs have shown that the onset and time to peak effect of monomeric insulin lispro are Furthermore, the Comparison of monomeric insulin lispro and pramlintide kinetics confirmed previous modeling suggesting that these ultrafast kinetics would be conserved across species (for humans) Figures 26A to 26C and Figures 27A to 27C ). In the range from rats (13±1 minutes) to pigs (25±4 minutes), While the time to peak was nearly doubled, monomeric insulin lispro (delivered as part of a co-formulation in rats) had similar times to peak in both species (11 ± 1 minutes in rats and 9 ± 2 minutes in pigs) (Figure 26). The preservation of the time to peak exposure from rats to pigs is very promising for translating these ultrafast insulin kinetics to human trials and will result in faster kinetics than current commercial formulations (Figure 26).

[0307] Example 4: Copolymers as "Intercalating" Excipients for Insulin Formulations

[0308] Material: R (Eli Lilly) was purchased and used as received. Solvents N,N-dimethylformamide (DMF; HPLC grade, Alfa Aesar, >99.7%), hexane (Fisher, certified ACS, >99.9%), ether (Sigma, certified ACS, anhydrous, >99%), and CDCl3 (Acrols, >99.8%) were used as received. Monomers N-(3-methoxypropionyl)acrylamide (MPAM; Sigma, 95%), 4-acryloylmorpholine (MORPH; Sigma, >97%) were filtered over basic alumina before use. Monomers N-phenylacrylamide (PHE; Sigma, 99%) and N-isopropylacrylamide (NIPAM; Sigma, >99%) were used as received. RAFT chain transfer agents 2-cyano-2-propyldodecyl trithiocarbonate (2-CPDT; Strem Chemicals, >97%) and 4-((((2-carboxyethyl)thio)thiocarbonyl)thio)-4-cyanopentanoic acid (BM1433; Boron Molecular, >95%) were used as received. Initiator 2,2′-azobis(2-methylpropionitrile) (AIBN; Sigma, >98%) was recrystallized from methanol (MeOH; Fisher, HPLC grade, >99.9%) and dried under vacuum before use. Z group removers lauroyl peroxide (LPO; Sigma, 97%) and hydrogen peroxide (H2O2; Sigma, 30%) were used as received. Streptozotocin (99.58%) was purchased from MedChem Express. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich.

[0309] Surface tension:The time-resolved surface tension of the air-solution interface was measured with a platinum / iridium Wilhelmy plate connected to an electronic balance (KSVNima, Finland). The Wilhelmy plate was partially immersed in an aqueous solution in a Petri dish and the surface tension of the interface was recorded within 50 minutes from the formation of the new interface. The equilibrium surface tension values ​​(t = 50 minutes) were reported because these values ​​more closely describe the environment in the vial stored before stirring. Two replicates were taken and averaged.

[0310] Interfacial Rheology: Interfacial shear rheology was measured using a Discovery HR-3 rheometer (TA Instruments) with an interfacial geometry including a Du Noüy ring made of platinum / iridium wire (CSC Scientific, Fairfax, VA, catalog number 70542000). Prior to each experiment, the Du Noüy ring was rinsed with ethanol and water and flame treated to remove organic contaminants. The solution chamber consisted of a double-walled Couette flow cell with an inner Teflon cylinder and an outer glass beaker. Time sweeps were performed with a strain of 1% (within the linear range) and a frequency of 0.05 Hz (so low that instrument inertia was not significant). The interfacial composite shear viscosity was measured for 30 minutes. The experiments were repeated in triplicate.

[0311] Polymer Synthesis: The polymers were synthesized by reversible addition-fragmentation transfer as described in Example 1 above. 23％The program of AC / DC excipient is as follows, and almost identical for all other carrier / dopant combinations, wherein only the selection and concentration of carrier / dopant are changed.MORPH (566mg, 4.02mmol, 36.5 equivalents), NIPAM (168mg, 1.485mmol, 13.5 equivalents), 2CPDT (38mg, 0.11mmol, 1 equivalent) and AIBN (3.6mg, 0.02mmol, 0.2 equivalent) are combined and in the 8mL scintillation vial equipped with PTFE spacer, be diluted to cumulative volume 2.25mL (33.3w / v vinyl monomer concentration) with DMF.Purge reaction mixture with nitrogen 10 minutes, and then heat 12 hours at 65 ℃. To remove the Z end of the resulting copolymer, AIBN (360 mg, 2.2 mmol, 20 equiv) and LPO (88 mg, 0.22 mmol, 2 equiv) were added to the reaction mixture, which was then sparged with nitrogen for 10 minutes and heated at 90°C for 12 hours. In size exclusion chromatography (SEC) analysis, Z group removal was confirmed by the ratio of refractive index to UV (λ = 310 nm) intensity. The resulting copolymer was precipitated three times from diethyl ether and dried overnight under vacuum.

[0312] In vitro insulin stability assay (accelerated aging): 50 μL of AC / DC excipient (MoNi 23％ 、MpPhe 8％ 、MoPhe 6％ ) or 50 μL of milli-Q water was added to 1 mL of R (Eli Lilly 100U) (JG Finneran, 2.0mL clear RAMTM wide opening vial, 12 x 32mm, 9mm threads) and capped to produce 95U as a control or formulated with 0.01, 0.1 or 0.5wt.% AC / DC excipients The vials were incubated at 37°C and agitated at 150 RPM for 2, 4, and 6 months (in addition, only The control was stirred at 2 weeks and 1 month). The preparation of the formulations was staggered so that all samples reached their endpoint age at the same time. The vials were refrigerated until the selected aging time points were reached and tested. After the initial transmittance experiments, all further experiments were performed with formulations with 0.01 wt.% AC / DC excipient to minimize copolymer concentration. In addition, 500 μL of 2.1 mg / mL MoNi 23％ or milli-Q water to 10 mL of unadulterated (Eli Lilly 100U) to produce 95U Control and formulations with 0.01wt% AC / DC excipients. These vials are placed in original individual packaging boxes together with instructions. These packages are incubated at 37°C or 50°C until the opacity changes significantly. Take out 300 to 400 μL aliquots every 24 hours for the first 7 days and refrigerate. Afterwards, take intermittent aliquots to preserve the volume. Every 24 hours, take a picture of the bottom of the vial to track the change in opacity. The method for aggregation determination of recombinant human insulin is adapted from Webber et al. The formulation samples are plated in a transparent 96-well plate at a concentration of 150 μL per well, and the absorbance reading is read at 540nm (BioTek Synergy H1 microplate reader). The aggregation of insulin causes light scattering, which results in an increase in the measured absorbance. Aggregation time (t A ) is defined as the time point when a 10% increase in transmittance is observed from time zero.

[0313] Circular Dichroism: Circular dichroism was used to verify that aging of the AC / DC excipients did not result in changes in the secondary structure of insulin. Control or unaged with 0.01 wt.% AC / DC excipient Assessment of aging (0.5, 1, 2, 4 and 6 months) or aged with 0.01 wt.% AC / DC excipient (2, 4 and 6 months). Formulation samples were diluted to 0.2 mg / mL in PBS (pH=7.4). Samples were allowed to equilibrate at room temperature for 15 minutes prior to measurement. Near-UV circular dichroism spectroscopy was performed at 20°C using a J-815 CD spectropolarimeter (Jasco Corporation) in the wavelength range of 200 to 260 nm using a 0.1 cm pathlength cell.

[0314] In vitro insulin cell activity assay: The AKT phosphorylation pathway was used to test in vitro insulin activity using the AlphaLISA SureFire Ultra (PerkinElmer) kit to detect phosphorylated AKT 1 / 2 / 3 (pS473) compared to total Akt1. Aging (t = 6 months), +MoNi 23％ and aging +MoNi 23％ (t=6 months) formulation. +MoNi 23％ and aging +MoNi 23％(t = 6 months) formulation. C2C12 mouse muscle myoblasts (ATCC CRL-1772) were cultured and confirmed to be mycoplasma-free before use. Dulbecco's modified Eagle's medium (DMEM) (Gibco; 4.5 g / L D-glucose, L-glutamine, 110 mg / L sodium pyruvate) was supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin. Cells were grown in 96-well tissue culture plates for 24 hours (seeding density = 25,000 cells / well in 200 μL medium). Prior to insulin stimulation, cells were washed twice with 200 μL of unsupplemented DMEM and starved overnight in 100 μL of unsupplemented DMEM. The medium was then removed and the cells were incubated with 100 μL of insulin diluted in unsupplemented DMEM (i) (ii) Aging (t = 6 months), (iii) +MoNi 23％ (iv) Aging +MoNi 23％ (t = 6 months) Stimulate cells for 30 minutes while incubating at 37 ° C. Wash cells twice with 100 μL of cold 1X Tris-buffered saline, then add 100 μL of lysis buffer to each well and shake at room temperature for at least 10 minutes to completely lyse the cells. For each assay, 30 μL of lysate was transferred to a 96-well white half-area plate. The assay was completed according to the manufacturer's protocol. The plate was incubated at room temperature and read using a Tecan Infinite M1000PRO plate reader 18 to 20 hours after adding the final assay reagent. The results were plotted as the ratio of [pAKT] / [AKT] for each sample (n = 3 cell replicates), and EC was plotted using GraphPad Prism 8 50 Regression (log(agonist) vs. response(three parameters)).

[0315] Ethical approval for studies involving animal experiments: All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals; all protocols (protocol number 32873) were approved by the Stanford University Animal Care and Use Committee prior to study performance.

[0316] Streptozotocin (STZ)-induced diabetic rat model:Male Sprague Dawley rats (Charles River) were used for the experiment. Animal studies were performed according to the Laboratory Animal Care and Use Guide; all protocols were approved by the Animal Care and Use Committee of Stanford University. The protocol for STZ induction was adapted from the protocol of Wu and Huan and has been described previously. Male Sprague Dawley rats of 180 to 250 g (8 to 10 weeks) were weighed and fasted in the morning of treatment (6 to 8 hours) before treatment with STZ in the afternoon. Pre-weighed STZ was protected from light and diluted to 10-20 mg / mL in 1 mL sodium citrate buffer (pH=4.5) immediately before injection. Rats were intraperitoneally injected with STZ solution (65 mg / kg). Within 24 hours after applying STZ, rats were given water containing 10% sucrose. After three days of treatment with STZ, hyperglycemia of blood glucose levels in rats was tested by tail vein blood collection using a handheld Bayer ContourNext glucose monitor (Bayer). Blood glucose monitoring was subsequently performed every day. Diabetes was defined as 3 consecutive blood glucose measurements >300 mg / dL in nonfasted rats.

[0317] In vivo pharmacodynamics in diabetic rats: Diabetic rats were fasted for 4 to 6 hours. For the initial blood glucose study, rats were injected subcutaneously (1.5 U / kg) with the following formulations: (i) or (ii) with 0.01 wt.% AC / DC excipient (MoNi 23％ 、MpPhe 8％ 、MoPhe 6％ )of Six aging time points were tested at 0, 0.5, 1, 2, 4, and 6 months. Formulations with AC / DC excipients were tested at 0, 2, 4, and 6 month aging time points. The preparation of the formulations was staggered so that all samples reached their endpoint age at the same time and all aging time points could be compared in the same group of rats. Thirty-two rats with fasting blood glucose levels >300 mg / dL were randomly assigned to the formulation groups (8 rats / group) and each rat received the formulation at all aging levels (the order in which the rats received the aging time points was also randomized). For blood glucose studies after aging of the formulations at 50°C, rats were injected subcutaneously (1.5 U / kg) with the following formulations: (i) (ii) Aging (t = 1 day); (iii) +MoNi 23％ ; or (iv) aged +MoNi 23％ (t = 4 days). 16 rats with fasting blood glucose level > 300 mg / dL were randomly divided into Control group or MoNi 23％ Group. In both groups, the order of administration of the aged formulations was also randomized, and the formulations were applied on separate experimental days. Before injection, baseline blood glucose was measured. After injection, blood was sampled every 30 minutes within 5 hours. Blood glucose was measured using a handheld blood glucose monitor. The maximum change in measured blood glucose relative to baseline was used as a measure of the bioactivity of each formulation to assess the in vivo bioactivity after aging.

[0318] In vivo pharmacokinetics in diabetic rats: Diabetic rats were fasted for 4 to 6 hours. For the initial pharmacokinetic studies, rats were injected subcutaneously (1.5 U / kg) with the following formulations: (i) (ii) Aging (t = 6 months); (iii) +MoNi 23％ ; or (iv) aged +MoNi 23％ (t=6 months). 16 diabetic rats were randomly assigned to formulation groups: or +MoNi 23％ (8 rats / group). In each group, rats received fresh (t=0 months) or aged (t=6 months) formulations in a randomized order. After subcutaneous injection, blood was sampled every 15 minutes for 2 hours and collected in serum tubes (Sarstedt) for ELISA analysis. Serum insulin concentrations were quantified using a human insulin ELISA kit (Mercodia).

[0319] statistics: Unless otherwise stated, all data are presented as mean ± standard error. For in vitro activity assays (AKT), EC was plotted using GraphPad Prism 8 50 Regression (log(agonist) vs. response (three parameters)). GraphPadPrism8 Extra Sum of Squares F-test was used to test Log(EC 50 ) were different. Data sets were compared pairwise, and Bonferroni post hoc tests were used to adjust for multiple comparisons (α = 0.008). For blood glucose measurements, REML repeated measures mixed models were used to test for differences in formulations at different aging time points (JMP Pro 14). Rat was included as a random effect, and formulation age was included as a within-subject fixed effect. Formulations were compared using post hoc Tukey HSD tests to determine statistical significance between aging time points.

[0320] result

[0321] Characterization of the synthesized copolymers: The composition and molecular weight of the synthesized copolymers were determined by 1 H NMR spectroscopy and SEC were confirmed with poly(ethylene glycol) standards (Table 8).

[0322] Table 8: Characterization of copolymers

[0323]

[0324] a Determined by size exclusion chromatography using polyethylene glycol sample calibration.

[0325] b The weight percentage was difficult to determine due to spectral overlap. The weight percentage was estimated from the post-precipitation NMR spectrum by measuring the left half of the higher resolution peak of Nipam (δ = 4.0, 0.5H), doubling it, and subtracting it from the unresolved peaks of MORPH and Nipam (δ = 3.2-4.2, 7H (MORPH) 1H (Nipam)).

[0326] c Weight percentages calculated from post-precipitation NMR spectra of Morph (δ = 3.3-3.7, 8H) and Phe (δ = 7.6, 2H).

[0327] d Weight percentages calculated from post-precipitation NMR spectra of Mp (δ = 3.1-3.5, 7H) and Phe (δ = 7.6, 2H).

[0328] AC / DC excipient insulin stabilization mechanism: Three amphiphilic AC / DC excipients that were shown to stabilize monomeric insulin were tested. These excipients consisted of copolymers of acryloylmorpholine (MORPH or Mo) or methoxypropylacrylamide (MPAM) as hydrophilic carrier monomers and N-isopropylacrylamide (NIP or Ni) or phenylacrylamide (PHE) as hydrophobic dopant monomers. To test whether these excipients preferentially occupied air-water interfaces and thus inhibited insulin-insulin interactions occurring at these interfaces ( FIG. 28 ), time-resolved surface tension and interfacial rheology experiments were performed in conjunction with commercially available R (Eli Lilly) co-formulated model AC / DC excipient, poly(acryloylmorpholine 77％ -co-N-isopropylacrylamide 23％ )(MoNi 23％ ) (Figure 29).

[0329] R, containing MoNi 23％ (0.01wt.%) R and containing the same formulation excipients (i.e., without insulin R)MoNi 23％ The equilibrium surface tension measurement of the solution of MoNi (0.01 wt.%) revealed 23％ The presence of R (about 42 to 47 mN / m, Fig.29B ). In addition, MoNi 23％ A ten-fold increase in the concentration (0.1 wt.%) further reduced the surface tension of the formulation ( Fig.30 ). 23％ Add to The reduction in surface tension during Compared with MoNi 23％ and When mixed together, more species are present at the interface. In the case of MoNi 23％ The decrease in surface tension with increasing concentration shows that the surface is 23％ However, and MoNi 23％ and MoNi 23％ The surface tension of the formulations with the formulation excipients was the same, indicating that Similar numbers of molecular species exist at the interface. In summary, these surface tension experiments help to demonstrate that MoNi 23％ Preferentially adsorbs and controls the air-water interface.

[0330] Interfacial shear rheology measurements show that compared to R, MoNi 23％ (0.01wt.%) added to R reduces the interfacial complex viscosity to below the detection limit of the instrument, which exhibits values ​​between 0.002 and 0.003 Pa·s·m ( Fig.29C ). The complex viscosity of 1.37 indicates that associative insulin-insulin interactions can consume viscous energy at the interface. Although not quantitative, the reduction in interfacial complex viscosity to below the instrument detection limit suggests that the addition of MoNi 23％ The insulin-insulin interaction at the interface is disrupted.

[0331] When this complex interface is subjected to interfacial stress and agitation, it is likely that these insulin-insulin associations can nucleate amyloid fibril formation and lead to aggregation. Surface tension and interfacial rheology experiments together suggest a mechanism for AC / DC-enhanced insulin stabilization, in which preferential adsorption of AC / DC excipients to air-water interfaces disrupts insulin-insulin interactions.

[0332] AC / DC excipients for long-term insulin stability: The AC / DC excipient MoNi was evaluated 23％ , poly(acryloylmorpholine 94％ -Co-phenylacrylamide 6％ )(MoPhe 6％ ) and poly(methoxypropylacrylamide 92％ -Co-phenylacrylamide 8％ )(MpPhe 8％ ) acts as a simple "insertion" excipient to stabilize the stress aging process R ability. Preparation has only or a formulation with an AC / DC excipient added thereto The formulations were prepared and aged at 37°C with constant stirring (150 rpm on an orbital shaking plate) for 0, 2, 4, or 6 months. The preparation of the formulations was staggered so that all samples reached their end-point age at the same time. Both visual inspection and transmittance determination were used to determine whether the insulin had aggregated (Figure 31). Insulin aggregation scatters light, and therefore aggregation can be defined as a change in transmittance greater than 10%. After 2 weeks of stress aging, individual In contrast, all AC / DC excipients containing 0.01, 0.1 or 0.5 wt.% MoPhe 6％ 、MpPhe 8％ and MoNi 23％ The insulin formulations did not show any signs of insulin aggregation during the 6-month study, except for 0.5 wt.% MpPhe 8％ In addition ( Fig.31B and Figure 32). Therefore, in order to minimize the amount of copolymer excipient in the formulation, only 0.01 wt.% formulation was used for the rest of the studies reported herein.

[0333] To further validate the transmittance results of evaluating only insulin aggregation, Or containing MoNi 23％ (0.01wt.%) After stimulation of C2C12 cells at both the 0-month and 6-month time points, the expression of Ser on protein kinase B (AKT) was measured. 473 Phosphorylation of α-glucose was used to assess in vitro activity (Figures 31C to 31D). +MoNi 23％ The formulations showed equivalent biological activity ( t=0Log(EC 50 )=2.252±0.158;MoNi 23％ , t=0Log(EC 50 )=2.448±0.186;MoNi 23％ , t = 6Log(EC 50 )=2.405±0.158), while the aging R exhibited an almost complete loss of biological activity ( t=6Log(EC 50 )=3.606±0.139)(Figures 31C to 31D).

[0334] While these in vitro AKT assay results supported the transmittance data, the integrity of the insulin formulations was further confirmed using circular dichroism to visualize the insulin secondary structure at each formulation time point ( Figures 31E to 31H The formulations stabilized with AC / DC excipients showed no change in secondary structure after stress aging, whereas the All structural features were lost at 1 month. These data confirm both the transmittance and in vitro activity data.

[0335] Bioactivity of aged insulin in diabetic rats: To assess the integrity of aged insulin formulations in an in vivo functional environment, formulation activity was assessed in diabetic rats. Administration of streptozotocin was used to induce insulin-dependent diabetes in a cohort of 32 male rats. The rats were randomly assigned to one of four formulation groups: (i) or including (ii) MoPhe 6％ (iii) MpPhe 8％ or (iv) 0.01wt.% MoNi 23％ of And each rat receives the formulation at each aging time point (0, 2, 4, 6 months). The preparation of the formulation is staggered so that all samples reach their terminal age at the same time, and all aging time points can be compared in the same group of rats. Fasting rats are injected with insulin (1.5U / kg) subcutaneously, and blood glucose levels are measured every 30 minutes. The active formulation causes blood glucose to significantly decline from extreme hyperglycemia, reaching a minimum value within the normal blood glucose range between 60 to 100 minutes after administration (Figure 33 and Figure 34). After this stage, as insulin is removed, blood glucose levels begin to rise. In contrast, the formulation that appears to aggregate in the in vitro transmittance determination after aging does not show this obvious reduction of glucose that reminds people of insulin action, but causes glucose levels to gradually decrease. The gradual reduction of glucose may indicate that some insulin is initially captured in reversible aggregates, and over time, these aggregates dissociate and cause a slow-acting insulin effect. For each formulation, the maximum difference in blood glucose from baseline to the lowest glucose level is plotted as a measure of formulation effectiveness. All copolymer-stabilized formulations showed no differences in activity between aging time points, but individual The aged time points showed significant differences (F3,21 = 23.83, P < 0.0001), with post hoc Tukey HSD test revealing that the (t = 0 months) compared to the 2, 4 and 6 months of aging These observations were confirmed by evaluation of insulin pharmacokinetic data, in which no fresh R(t=0month) +MoNi 23％ There was a difference between the initial (t = 0 months) and after 6 months of aging, but the aging (t = 6 months) exposure reduction ( Fig.33F and Fig.35 These data suggest that AC / DC excipients may be useful as a novel excipient for insulin administration without altering insulin pharmacokinetics or pharmacodynamics. R and other commercial formulations.

[0336] High temperature aging of insulin formulations: To determine the ability of AC / DC excipients to improve the cold chain resilience of insulin, MoNi 23％ The extent of stability under extreme manufacturing and distribution conditions (37°C and 50°C, with constant stirring) (Figure 36). The temperatures chosen represent the temperature of a hot summer day (37°C) and the maximum temperature range that an unrefrigerated or insulated shipping container or truck might reach during peak summer (50°C). R, the glass vials are packed and shipped in cardboard boxes ( Fig.36A and Figure 37). 23％ (0.01wt.%) added to new vials R (diluted from 100 U / mL to 95 U / mL to allow for the addition of copolymer; control vials diluted with water), and the vials were then placed back into the original cardboard package with the package insert ( FIG. 37A ). The cardboard package was secured on a rotary shaker in a temperature-controlled incubator and agitated at 150 RPM ( FIG. 37B ).

[0337] Visual inspection combined with transmissivity measurement was used as the primary measure of insulin integrity ( Fig.36A ). These measurements are consistent with earlier experiments demonstrating that transmittance reads correlate well with both in vitro and in vivo functional activity measurements. It began to show a visual change of opacity on the first day and became completely opaque by the second day. In contrast, when MoNi 23％ When formulated, the insulin formulation showed no visual change in opacity until day 56 and maintained less than 10% change in transmittance over 56 days. In contrast, MoNi 23％ The stability of the formulation under these extreme conditions extended past 4 days, before the formulation became turbid on day 5. These qualitative observations are consistent with the quantitative transmittance readings ( Figures 36B to 36C ).

[0338] To verify functional insulin activity after aging at 50°C in vivo, these formulations were evaluated in diabetic rats. Measurement (i) (t = 0 days), (ii) aged (t = 1 day), (iii) containing MoNi 23％ of (t = 0 days) or (iv) containing MoNi 23％ Aging (t=4 days) Ability to reduce glucose levels in fasting diabetic rats. After subcutaneous administration of the formulation (1.5 U / kg), blood glucose levels were measured every 30 minutes ( Fig.36D ). (t = 0 days), containing MoNi 23％ of (t = 0 days) and MoNi 23％ Aging (t = 4 days) demonstrated an initial drop in blood glucose that reached a minimum between 60 and 100 minutes after injection. These results are consistent with the active formulations in earlier experiments. This characteristic drop in glucose was observed in aging mice. (t = 1 day), which is consistent with the inactive formulation in earlier experiments. The maximum difference in glucose from baseline was also plotted for each formulation as a measure of formulation potency ( Fig.36E ).

[0339] Statistical analysis identified differences in potency between these formulations (F3, 18.18 = 10.71, P = 0.0003), whereby a post hoc Tukey HSD test revealed that aging alone was superior to the other formulations. In contrast, after aging at 50°C for 4 days, the unaged Unaged MoNi 23％ of And aged MoNi 23％ of There was no statistical difference between them.

[0340] Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A random copolymer based on polyacrylamide, comprising the following components: A water-soluble carrier monomer, wherein the water-soluble carrier monomer is selected from N -(3-methoxypropyl)acrylamide and 4-acryloylmorpholine; and A functional dopant monomer which is N -isopropylacrylamide; wherein N - The weight percentage of isopropyl acrylamide is 10% to 28%.

2. The copolymer according to claim 1, wherein the weight percentage of the water-soluble carrier monomer is 70% to 98%, and the water-soluble carrier monomer and N The sum of the weight percentages of -isopropylacrylamide is 100%.

3. The copolymer according to claim 1, wherein N - The weight percentage of isopropyl acrylamide is 20% to 26%.

4. The copolymer according to claim 3, wherein N -The weight percentage of isopropyl acrylamide is 23%.

5. The copolymer according to claim 4, wherein the water-soluble carrier monomer is 4-acryloylmorpholine.

6. The copolymer according to claim 1, wherein the water-soluble carrier monomer is N -(3-methoxypropyl)acrylamide.

7. The copolymer according to claim 1, wherein the copolymer has the following composition: 74 to 80 wt % of 4-acryloylmorpholine, and 20 to 26 wt % N -isopropylacrylamide; The copolymer has a number average molecular weight of 1000 g / mol to 5000 g / mol; and The copolymer has a degree of polymerization of 10 to 50.

8. The copolymer according to claim 7, wherein the copolymer has the following composition: 77% by weight of 4-acryloylmorpholine; and 23% by weight N -Isopropyl acrylamide.

9. The copolymer according to claim 6, wherein N - The weight percentage of isopropyl acrylamide is 20% to 26%.

10. A copolymer based on polyacrylamide, comprising: A water-soluble carrier monomer, wherein the water-soluble carrier monomer is selected from N -(3-methoxypropyl)acrylamide and 4-acryloylmorpholine; as well as A functional dopant monomer, wherein the functional dopant monomer is N -phenylacrylamide.

11. The copolymer according to claim 10, wherein the water-soluble carrier monomer is N -(3-methoxypropyl)acrylamide.

12. The copolymer according to claim 10, wherein the water-soluble carrier monomer is 4-acryloylmorpholine.

13. The copolymer according to any one of claims 10 to 12, wherein the copolymer comprises 10 to 30 wt % of N -phenylacrylamide.

14. The copolymer according to claim 1 or 10, wherein the degree of polymerization is 10 to 500.

15. The copolymer of claim 1 or 10, wherein the copolymer has a molecular weight of 1,000 to 40,000 g / mol.

16. The copolymer according to claim 1 or 10, wherein: A degree of polymerization of 10 to 500; and The molecular weight of the copolymer is 1,000 to 40,000 g / mol.

17. The copolymer according to claim 1 or 10, wherein A degree of polymerization of 20 to 200; and The molecular weight of the copolymer is 2,000 to 10,000 g / mol.

18. A random copolymer based on polyacrylamide, comprising: A water-soluble carrier monomer, wherein the water-soluble carrier monomer is selected from N -(3-methoxypropyl)acrylamide, 4-acryloylmorpholine, N , N -Dimethylacrylamide, N - hydroxyethyl acrylamide and acrylamide; and A functional dopant monomer selected from N -[Tris(hydroxymethyl)-methyl]acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, (3-acrylamidopropyl)trimethylammonium chloride, N -isopropyl acrylamide, N -tert-Butylacrylamide and N -phenylacrylamide; wherein: The weight percentage of the water-soluble carrier monomer is 70% to 98%; The weight percentage of the functional dopant monomer is 2% to 30%; The polyacrylamide-based copolymer has a number average molecular weight of 1,000 g / mol to 10,000 g / mol; and The degree of polymerization is 10 to 250.

19. The copolymer according to claim 18, wherein the water-soluble carrier monomer is selected from N -Hydroxyethyl acrylamide.

20. The copolymer according to claim 18 or 19, wherein the functional dopant monomer is selected from N -[Tris(hydroxymethyl)-methyl]acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, (3-acrylamidopropyl)trimethylammonium chloride and N -tert-Butylacrylamide.

21. A random copolymer based on polyacrylamide, comprising: A water-soluble carrier monomer, wherein the water-soluble carrier monomer is N - hydroxyethyl acrylamide; and A functional dopant monomer selected from N -[Tris(hydroxymethyl)-methyl]acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, (3-acrylamidopropyl)trimethylammonium chloride, N -isopropyl acrylamide, N -tert-Butylacrylamide and N -phenylacrylamide.

22. A random copolymer based on polyacrylamide, comprising: Water-soluble carrier monomer, the water-soluble carrier monomer is: N -(3-methoxypropyl)acrylamide, 4-acryloylmorpholine, N , N -Dimethylacrylamide, N - hydroxyethyl acrylamide and acrylamide; and A functional dopant monomer selected from N -[Tris(hydroxymethyl)-methyl]acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, (3-acrylamidopropyl)trimethylammonium chloride and N -tert-Butylacrylamide.

23. The copolymer according to claim 22, wherein the water-soluble carrier monomer is N , N -Dimethylacrylamide.

24. The copolymer according to claim 22, wherein the water-soluble carrier monomer is N -Hydroxyethyl acrylamide.

25. The copolymer of claim 22, wherein the water-soluble carrier monomer is acrylamide.

26. The copolymer according to claim 21 or 22, wherein the functional dopant monomer is N -[Tris(hydroxymethyl)-methyl]acrylamide.

27. The copolymer of claim 21 or 22, wherein the functional dopant monomer is 2-acrylamido-2-methylpropanesulfonic acid.

28. The copolymer of claim 21 or 22, wherein the functional dopant monomer is (3-acrylamidopropyl)trimethylammonium chloride.

29. The copolymer according to claim 21 or 22, wherein the functional dopant monomer is N -tert-Butylacrylamide.

30. The copolymer of claim 21, wherein the functional dopant monomer is N -Isopropyl acrylamide.

31. The copolymer of claim 21, wherein the functional dopant monomer is N -phenylacrylamide.

32. The copolymer of claim 21, wherein the functional dopant monomer is selected from N -[Tris(hydroxymethyl)-methyl]acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, (3-acrylamidopropyl)trimethylammonium chloride and N -tert-Butylacrylamide.

33. The copolymer according to claim 22, wherein the water-soluble carrier monomer is N -(3-methoxypropyl)acrylamide.

34. The copolymer of claim 22, wherein the water-soluble carrier monomer is 4-acryloylmorpholine.

35. The copolymer according to claim 21 or 22, wherein the degree of polymerization is 10 to 500.

36. The copolymer of claim 21 or 22, wherein the copolymer has a molecular weight of 1,000 to 40,000 g / mol.

37. The copolymer according to claim 21 or 22, wherein the degree of polymerization is from 10 to 500, and The molecular weight of the copolymer is 1,000 to 40,000 g / mol.

38. The copolymer according to claim 21 or 22, wherein the degree of polymerization is from 20 to 200, and The molecular weight of the copolymer is 2,000 to 10,000 g / mol.

39. A composition comprising the copolymer according to any one of claims 1, 10, 18, 21 and 22 and a pharmaceutically acceptable excipient.

40. A composition comprising the copolymer of any one of claims 1, 10, 18, 21 and 22, wherein the composition is a cosmetic product, a hair product, a lotion, a food product, a veterinary product or a nutritional product.

41. A composition comprising the copolymer of any one of claims 1, 10, 18, 21 and 22 and a protein.

42. The composition of claim 41, wherein the protein is a protein that tends to aggregate in aqueous media.

43. The composition of claim 41, wherein the protein is selected from the group consisting of antibodies and fragments thereof, cytokines, chemokines, hormones, vaccine antigens, cancer antigens, adjuvants, and combinations thereof.

44. The composition of claim 43, wherein the protein is a monoclonal antibody.

45. The composition of claim 41, wherein the protein is insulin or an analog thereof.

46. ​​A method for increasing the thermal stability of a protein formulation, the method comprising adding 0.0005 wt% to 5 wt% of the copolymer according to any one of claims 1, 10, 18, 21 and 22 to the protein formulation.

47. A method for increasing the stability of a protein formulation, the method comprising adding 0.005 wt% to 5 wt% of the copolymer according to any one of claims 1, 10, 18, 21 and 22 to the protein formulation.

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