A polymer, an insulin sustained-release preparation containing the polymer, and use thereof

Abstract

The present application discloses a polymer, which comprises a biphenyl boronic acid structure unit capable of binding with glucose and a hydrophilic polymerizable structure unit, wherein the biphenyl boronic acid structure unit can improve the binding capacity and selectivity to glucose. The present application also discloses an insulin sustained-release preparation comprising the above polymer, which has different insulin release rates under different glucose concentrations, can accelerate the release of insulin to reduce blood glucose in a hyperglycemic environment, and can reduce the incorrect release of insulin caused by fructose and other diol substances. The present application further discloses a preparation method of the above insulin sustained-release preparation, which self-assembles the negatively charged insulin and the positively charged biphenyl boronic acid modified polymer into the insulin sustained-release preparation through electrostatic interaction, and has the characteristics of high loading rate. The prepared insulin sustained-release preparation can release insulin, and the released insulin can bind with the insulin receptor in the body to reduce the blood glucose level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a polymer, an insulin sustained-release preparation containing the polymer and application. BACKGROUND

[0002] Diabetes, as a chronic disease, affects more than 537 million people worldwide. At present, the main method for insulin delivery is still subcutaneous injection of insulin solution, which has problems in insulin storage, drug administration comfort and hypoglycemic side effects, and there is an urgent need for a stable, safe and long-acting closed-loop insulin delivery system to improve patient compliance. However, commercially available insulin pumps and in-development insulin microneedle patches, insulin complexes, etc. still have defects and challenges in insulin stability, drug loading rate, release controllability and response selectivity.

[0003] Phenylboronic acid (PBA) responsive to glucose is widely used to construct insulin complexes. However, the binding ability of the commonly used phenylboronic acid structure to glucose is lower than that of fructose, salicylic acid and tetracycline and other common diol interferents in the body. Taking fructose as an example, the concentration of fructose in the human body is usually less than 1 mg / dL, but after a large amount of fructose is ingested in a short time (such as drinking high-fructose beverages, ingesting a large amount of fruit), the level of fructose in the body can rise to 18 mg / dL, which may cause competition between fructose and PBA-based insulin complexes, leading to incorrect release of insulin and causing hypoglycemia.

[0004] Publication No. CN118255997A discloses a polymer, an insulin composition containing the same and application thereof, and the polymer is composed of three different structural units, wherein structural unit III contains a phenylboronic acid structure. However, the polymer constructed in the present application contains only one phenylboronic acid in one monomer, which may cause incorrect release of insulin after binding of the insulin compound to fructose and the like.

[0005] Studies have shown that a double phenylboronic acid (DBA) with a suitable structure can improve the binding capacity and selectivity to glucose, so a strategy based on a DBA glucose-responsive unit can improve the specific release of insulin while reducing the incorrect release of insulin.

[0006] Reference 1 (Zuo Minzan, Wang Leyong. Multiresponsive Supramolecular Theranostic Nanoplatform Based on Pillar[5]arene and Diphenylboronic Acid Derivatives for Integrated Glucose Sensing and Insulin Delivery. Small, 14, e1801942, 2018, doi: 10.1002 / smll.201801942) discloses a specific structure of a diphenylboronic acid derivative, which optimizes its selective recognition of glucose, and then uses a water-soluble pillararene and a diphenylboronic acid derivative to form a water-soluble supramolecular vesicle through host-guest interaction, and encapsulates insulin and glucose oxidase, to construct a multi-responsive supramolecular diagnosis and treatment integrated nanoplatform for glucose recognition and insulin administration. The vesicle co-encapsulating insulin and glucose oxidase can selectively recognize glucose in a hyperglycemic environment, thereby causing the vesicle structure to be destroyed and releasing insulin to regulate blood glucose levels. SUMMARY

[0007] To solve the above technical problems, the present application provides a polymer comprising a diphenylboronic acid structural unit capable of binding to glucose, which can improve the binding capacity and selectivity to glucose.

[0008] A polymer having a structure as shown in formula (1),

[0009]

[0010] A is one of is a connection position

[0011] R1, R2 are each independently selected from at least one of a hydrogen atom, a C1-C6 alkyl group, and a halogen; R3, R4 are each independently selected from a C1-C6 alkyl group, n = 5-800, m = 1-100.

[0012] Glucose in the form of α-furanose is a special sugar compound that can bind two boronic acid moieties through two pairs of binding sites at its 1,2- and 3,5,6- positions. In the present application, the polymer comprises a structural unit having two phenylboronic acid structures, which can simultaneously bind to two pairs of cis diols (1,2- and 5,6-cis diols of glucose) in a synergistic manner, achieving selective recognition of glucose.

[0013] In the present application, when an electron-withdrawing group (halogen) is introduced on the benzene ring of the phenylboronic acid in formula (1), the electron cloud density is reduced due to the induction effect, the Lewis acidity of the boronic acid group is stronger, the pKa is reduced, and the boronic acid group is more inclined to convert into a tetrahedral form, which can generate stable and hydrophilic phenylboronic acid ester with glucose, and the synthesis of such monomers only needs to replace the phenylboronic acid raw material with an alkyl or halogen-substituted phenylboronic acid raw material.

[0014] Preferably, the structure of the polymer is as shown below,

[0015]

[0016] wherein n = 5-800, m = 1-100, is a connecting position.

[0017] The present application also provides a preparation method of the above-mentioned polymer, which adds and polymerizes the carbon-carbon double bonds on different monomers through a free radical copolymerization reaction, and the method is simple and convenient.

[0018] A preparation method of a polymer, comprising the following steps: performing a free radical copolymerization reaction on a monomer containing A, a monomer containing double phenylboronic acid and an initiator in a solvent, and obtaining the polymer after dialysis and freeze-drying, wherein the structures of the monomer containing A and the monomer containing double phenylboronic acid are respectively as shown in formula (2) and formula (3),

[0019]

[0020] A is one of,

[0021] R1, R2 are each independently selected from at least one of a hydrogen atom, a C1-C6 alkyl group and a halogen; R3, R4 are each independently selected from a C1-C6 alkyl group.

[0022] Preferably, the molar ratio of the monomer containing double phenylboronic acid and the monomer containing A is 1-2:3-19.

[0023] In the present application, by adjusting the molar ratio of the monomer containing A and the monomer containing double phenylboronic acid, a polymer with a double phenylboronic acid content of 5%-40% can be obtained.

[0024] Preferably, the initiator is an azo initiator or an inorganic peroxide initiator.

[0025] In the present application, the azo initiator can be azobisisobutyronitrile (AIBN), azobisisoheptyl nitrile (ABVN) and the like, and the inorganic peroxide initiator can be hydrogen peroxide, ammonium persulfate, potassium persulfate and the like.

[0026] Preferably, the solvent is one or more of alcohol or water.

[0027] Preferably, the total mass of the monomer containing A and the monomer containing diphenylboronic acid to the volume of the solvent is 100-400 mg / mL.

[0028] Preferably, the reaction temperature of the radical copolymerization reaction is 35-75°C, and the reaction time is 3-10 h.

[0029] Preferably, the dialysis step is at least 5 times of dialysis of the solution after the reaction is completed using a dialysis membrane with a molecular weight of 3500-15000 kDa, and each dialysis is performed for 6-8 h.

[0030] The present application also provides an insulin sustained-release preparation containing the above polymer, which has different insulin release rates at different glucose concentrations, and can accelerate the release of insulin to reduce blood glucose in a hyperglycemic environment, and can reduce the incorrect release of insulin caused by fructose and other diol substances.

[0031] An insulin sustained-release preparation, comprising the above polymer and insulin.

[0032] In the present application, the polymer with a quaternary ammonium salt structure with positive electricity can better adsorb negatively charged insulin, and improve the loading rate of insulin in the insulin sustained-release preparation. In a normal blood glucose environment, the combination of glucose and phenylboronic acid groups will reduce the positive charge density on the polymer chain to release a small amount of insulin, thereby providing slow and sustained insulin and allowing high-dose injection. In a hyperglycemic environment, the combination of a large number of phenylboronic acid groups with glucose will accelerate the release of insulin to reduce blood glucose.

[0033] Preferably, the insulin is porcine insulin, human insulin, recombinant human insulin, or FITC-porcine insulin.

[0034] In the present application, the FITC-insulin is obtained by labeling porcine insulin with fluorescein isothiocyanate.

[0035] Preferably, the mass ratio of the polymer to insulin is 1-4:1-4.

[0036] The present application also provides a preparation method of the above insulin sustained-release preparation, which self-assembles the negatively charged insulin and the positively charged diphenylboronic acid modified polymer into an insulin sustained-release preparation through electrostatic interaction, and has the characteristic of high loading rate.

[0037] A preparation method of an insulin sustained-release preparation, comprising the following steps: dissolving the above polymer and insulin in acidified water, adjusting the pH to neutral with a lye to form an insulin complex, adding a PBS solution to adjust the insulin concentration, and resuspending after centrifugation to obtain an insulin sustained-release preparation.

[0038] Preferably, the acidified water is hydrochloric acid aqueous solution, and the concentration of the acidified water is 0.01-1M.

[0039] Preferably, the total mass of the polymer and insulin and the mass / volume ratio of the acidified water is 5-20mg / mL.

[0040] Preferably, the alkali solution is sodium hydroxide aqueous solution, and the concentration of the alkali solution is 0.01-1M.

[0041] Preferably, the centrifugal speed is 10000-20000rpm, and the centrifugal time is 5-10min.

[0042] The present application also provides the use of the above-mentioned insulin sustained-release preparation in the preparation of a medicament for treating diabetes, which can release insulin, and the released insulin can bind to insulin receptors in the body to reduce blood glucose level.

[0043] In the present application, the diabetes can refer to a disease with higher blood glucose level, specifically, fasting blood glucose≥7.0mmol / L or 2-hour blood glucose in glucose loading test≥11.1mmol / L or glycosylated hemoglobin≥6.5%.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] (1) The polymer in the present application contains a biphenyl boronic acid structural unit capable of binding with glucose, which can improve the binding capacity and selectivity to glucose.

[0046] (2) The insulin sustained-release preparation in the present application has different insulin release rates under different glucose concentrations, which can accelerate the release of insulin under high blood glucose environment to reduce blood glucose, and the preparation can reduce the erroneous release of insulin caused by fructose and other diol substances.

[0047] (3) The preparation method of the insulin sustained-release preparation in the present application self-assembles the negatively charged insulin and the positively charged biphenyl boronic acid modified polymer into the insulin sustained-release preparation through electrostatic interaction, which has the characteristic of high loading rate. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a specific synthesis route of the monomer containing biphenyl boronic acid in Example 1.

[0049] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum spectrum of compound 8 (AADBA) in Example 1.

[0050] Figure 3 It is the nuclear magnetic resonance carbon spectrum spectrum of compound 8 (AADBA) in Example 1.

[0051] Figure 4 The image shows the proton NMR spectrum of the polymer prepared in Example 1.

[0052] Figure 5 This is a gel permeation chromatography (GPC) spectrum of the polymer prepared in Example 1.

[0053] Figure 6 The image shows the proton NMR spectrum of the polymer prepared in Example 2.

[0054] Figure 7 The image shows the proton NMR spectrum of the polymer prepared in Example 3.

[0055] Figure 8 The image shows the proton NMR spectrum of the polymer prepared in Example 4.

[0056] Figure 9 This is a schematic diagram illustrating the principle of the insulin sustained-release formulation prepared in Example 5.

[0057] Figure 10 The image shows the 1H NMR spectrum of the polymer prepared in Comparative Example 1.

[0058] Figure 11 The graph shows the response of the 1 mg / mL insulin sustained-release formulation obtained in Example 5 to glucose.

[0059] Figure 12 The figures are single-release curves of the insulin sustained-release formulations obtained in Examples 5, 6, 7, 9, 10 and 13 in glucose and fructose solutions, where A to F are single-release curves of Examples 5, 6, 7, 9, 10 and 13 respectively.

[0060] Figure 13 The figures are single-release curves of the insulin sustained-release formulation obtained in Example 5 and the insulin sustained-release formulation containing monophenylboronic acid polymer obtained in Comparative Example 1 in glucose and fructose solutions, where A is the single-release curve of Example 5 and B is the single-release curve of Comparative Example 1.

[0061] Figure 14 The figures show the cumulative release curves of the insulin sustained-release formulation obtained in Example 5 and the insulin sustained-release formulation containing monophenylboronic acid polymer obtained in Comparative Example 1 in vitro in glucose and fructose solutions, respectively. A is the cumulative release curve of the insulin sustained-release formulation obtained in Example 5 in vitro in vitro in multiple cycles, and B is the cumulative release curve of the insulin sustained-release formulation obtained in Comparative Example 1 in vitro in multiple cycles. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0063] All starting materials used in this application were commercially available.

[0064] Example 1: Preparation of the polymer

[0065] The specific route for the monomer containing bisphenylboronic acid is shown in Figure 1 Compound 2 was first protected with di-tert-butyl dicarbonate in dichloromethane to give compound 2, which was further protected with di-tert-butyl dicarbonate to give compound 3. Compound 3 was brominated with N-bromosuccinimide to give compound 4. Compound 4 was then reacted with dimethylamine in tetrahydrofuran to give compound 5, which was further reacted with 2-bromomethylphenylboronic acid to give compound 6. Compound 6 was deprotected with 3M aqueous HBr to give compound 7, which was then reacted with acryloyl chloride in a basic environment to give compound 8. The specific synthesis steps are shown below.

[0066] (1) Synthesis of compound 2

[0067] Compound 1 (9.55 g, 71 mmol) and triethylamine (TEA, 11.3 mL, 82 mmol) were added to 100 mL of dichloromethane, and di-tert-butyl dicarbonate (25.3 g, 128 mmol) was dissolved in 50 mL of dichloromethane and slowly added to the above mixture. The reaction was stirred at room temperature and monitored by TLC until completion. After 4 hours, the solvent was removed under vacuum. The crude product was recrystallized in n-hexane, washed, and dried to give white solid compound (13.5 g, 80%).

[0068] (2) Synthesis of compound 3

[0069] Compound 2 (13 g, 55 mmol), di-tert-butyl dicarbonate (14.4 g, 66 mmol), and 4-dimethylaminopyridine (DMAP, 0.67 g, 5.5 mmol) were dissolved in 100 mL of anhydrous acetonitrile under nitrogen protection, heated to 60 °C, stirred, and monitored by TLC until completion. After 24 h, the solvent was removed under vacuum. Purification was performed by silica gel column chromatography using petroleum ether / ethyl acetate = 95:5 as the eluent to give colorless oil (14.1 g, 76.0%).

[0070] (3) Synthesis of compound 4

[0071] Compound 3 (5 g, 15 mmol), N-bromosuccinimide (NBS, 5.8 g, 33 mmol), and trace amounts of azobisisobutyronitrile (AIBN) were dissolved in 100 mL of carbon tetrachloride. Nitrogen was bubbled for 30 minutes to remove oxygen. The mixture was heated to 65 °C. The reaction could be initiated by 365 nm UV light, and the reaction was monitored by TLC until completion. After 4 hours of reaction, the solvent was removed under vacuum. The solution was purified by silica gel column chromatography using petroleum ether / ethyl acetate (100:2) as the eluent, yielding a white solid (4.3 g, 60%).

[0072] (4) Synthesis of compound 5: 6 mL of a 2M dimethylamine solution in tetrahydrofuran was cooled to -78°C using an acetone bath on dry ice and allowed to cool for 10 minutes. Simultaneously, compound 4 (1.08 g, 2.75 mmol) was dissolved in 10 mL of tetrahydrofuran and added dropwise to the cooled dimethylamine solution while stirring vigorously. The reaction mixture was then brought to room temperature and stirred for 2 hours. After the reaction was complete, the reaction mixture was poured into 200 mL of ethyl acetate and washed three times with a 1 mol / L potassium carbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate for 30 minutes, and the solvent was removed under vacuum to obtain intermediate 5 (0.84 g, 2.6 mmol, 95.1%).

[0073] (5) Synthesis of compound 6

[0074] The intermediates 5- and 2-bromomethylphenylboronic acid (2.45 g, 8.25 mmol) and potassium carbonate (1.22 g, 8.8 mmol) were dissolved in 10 mL of DMF. Nitrogen was bubbled for 30 minutes to remove oxygen. The mixture was heated to 60 °C and reacted for 12 hours. The reaction solution was then added dropwise to a 1:2 (200 mL) solution of petroleum ether / ethyl acetate to precipitate the product. The filtered residue was dissolved in methanol, and the sample was purified by C18 reversed-phase silica gel column chromatography. Elution with 30% methanol and removal of methanol under reduced pressure were followed by lyophilization to obtain a white solid (1.08 g, 47%).

[0075] (6) Synthesis of compound 7

[0076] Compound 6 was dissolved in 5 mL of pure water, and 1 mL of hydrobromic acid was added to remove the Boc protecting group. The mixture was stirred overnight at room temperature. The pH of the reaction solution was adjusted to 4–5 with 1 mol / L sodium hydroxide solution. The sample was purified by C18 reversed-phase silica gel column chromatography, eluted with pure water, and lyophilized to obtain a white solid (687 mg, 98%).

[0077] (7) Synthesis of compound 8

[0078] Compound 7 (150 mg, 0.23 mmol) was dissolved in a mixture of H2O:THF = 2:1, total solvent volume was 6 mL, 146.28 mg of Na2CO3(1.38 mmol) was added, the reaction bottle was ice-bathed, 62.45 mg of acryloyl chloride (0.69 mmol) was dissolved in a small amount of anhydrous THF and added dropwise to the reaction bottle, after the dropwise addition was completed, the reaction was allowed to proceed for 3 h, the pH of the reaction was detected, if the pH was acidic, sodium carbonate was added to adjust the pH to alkaline. The THF in the reaction solution was removed under reduced pressure, the pH was adjusted to 4-5 with 0.1 M HBr aqueous solution, and the sample was purified by C18 reverse phase silica gel column chromatography, eluted with 30% methanol, and freeze-dried to obtain a white solid, which was compound 8 (hereinafter referred to as AADBA) (159.8 mg, 98.7%), the hydrogen spectrum and carbon spectrum of which are shown in Figure 2 and Figure 3 .

[0079] (9) Preparation of the polymer

[0080] AADBA (100 mg, 0.142 mmol) was added to a reaction bottle, 1200 μL of ethanol and 60 μL of water were added as solvents, N2 was bubbled to remove oxygen, 142.03 mg of N-vinylpyrrolidone (136.6 μL, 1.278 mmol) was added, 2.42 mg of AIBN was dissolved therein (1% of the total mass of AADBA and N-vinylpyrrolidone), and the temperature was raised to 65°C, and the reaction was allowed to proceed for 5 h. The polymer was separated by dialysis with a 7000 kDa dialysis membrane, and the water was changed 5 times. After dialysis was completed, the polymer was freeze-dried to obtain a white solid (192 mg, 79.3%), the hydrogen spectrum of the polymer is shown in Figure 4 , and the gel permeation chromatography (GPC) spectrum of the polymer is shown in Figure 5 , wherein the weight average molecular weight (Mw) of the polymer is 25032, and the polydispersity coefficient (PD) is 1.78.

[0081] Examples 2-4: Preparation of the polymer

[0082] The preparation methods of Examples 2-4 are the same as those of Example 1, and the differences are shown in the following table.

[0083] Table 1: Differences in preparation methods of Examples 1-4

[0084]

[0085] Example 5: Preparation of an insulin sustained-release preparation

[0086] FITC-porcine insulin (1 mg) and the polymer obtained in Example 1 (1 mg) were dissolved in 0.2 mL of HCl aqueous solution (0.1 M), mixed, and NaOH aqueous solution (1 M) was added to adjust the pH to 7.4, to obtain an insulin sustained-release preparation, the schematic diagram of the principle is shown in Figure 9indicated.

[0087] The supernatant was removed after centrifugation, and the pellet was resuspended in 1 mL of PBS. The washing was repeated three times, and the insulin concentration of the supernatant was measured each time. The final insulin sustained-release preparation was prepared to have an insulin concentration of 1 mg / mL, and was used in subsequent experiments.

[0088] Examples 6 to 13: Preparation of insulin sustained-release preparations

[0089] The preparation methods of Examples 6 to 13 were the same as those of Example 5, except for the differences shown in the following table.

[0090] Table 2: Differences in the preparation methods of Examples 5 to 13

[0091]

[0092] The test method for the insulin loading rate was to measure the insulin concentration of the supernatant of the three washes during the preparation of the insulin sustained-release preparation in Examples 5 to 13, and to obtain the total amount of washed-out insulin. The loading rate = (total amount of added insulin - total amount of washed-out insulin) / total amount of added insulin * 100%.

[0093] As can be seen from Table 2, different insulin sustained-release preparations (i.e., Examples 5 to 13) can be prepared by changing the mass ratio of insulin to polymer and changing the content of AADBA in the polymer, and the insulin loading rates of these insulin sustained-release preparations are all higher than 70%.

[0094] Comparative Example 1: Preparation of an insulin sustained-release preparation containing a monophenylboronic acid polymer

[0095] (1) Preparation of a phenylboronic acid compound

[0096] After 4-bromomethylphenylboronic acid (160 mg, 0.745 mmol) was dissolved in DMF (5 mL) and added dropwise to dimethylaminopropyl acrylamide (232.8 mg, 1.49 mmol) dissolved in DMF (5 mL), it was stirred at room temperature for 5 h. After the reaction was completed, the reaction solution was added dropwise into more than 20 times the volume of ethyl acetate to precipitate the precipitate. Stirring was maintained during the dropwise addition. After the precipitation was completed, it was suction-filtered and dried in a vacuum drying oven to obtain a crude product. The crude product was dissolved in a small amount of water and purified by C18 reverse-phase silica gel column chromatography, eluted with 10% methanol, and lyophilized to obtain a white transparent solid, which was a phenylboronic acid compound (hereinafter referred to as AAPBA) (159.8 mg, 58.0%).

[0097] (2) Preparation of a polymer

[0098] In a reaction bottle, AAPBA (50 mg, 0.227 mmol) obtained from step (1) was added, 400 μL of ethanol and 20 μL of water were added as solvents, N2 was bubbled to remove oxygen, 101 mg (97.1 μL, 0.909 mmol) of N-vinylpyrrolidone was added, 1.51 mg of AIBN was dissolved therein (1% of the total mass of AAPBA and N-vinylpyrrolidone), and the temperature was raised to 65°C, and the reaction was carried out for 5 h. Separation was carried out by dialysis using a 7000 kDa dialysis membrane, and water was changed 5 times. After dialysis was completed, the white solid was obtained by lyophilization (132 mg, 87.4%), and the hydrogen spectrum of the polymer is shown in Figure 10 .

[0099] (3) Preparation of an insulin sustained-release preparation containing a monoboronic acid-containing polymer

[0100] FITC-porcine insulin (1 mg) and the polymer obtained from step (2) (1 mg) were dissolved in 0.2 mL of an aqueous HCl solution (0.1 M), mixed, and an aqueous NaOH solution (1 M) was added to adjust the pH to 7.4, to obtain an insulin sustained-release preparation containing a monoboronic acid-containing polymer.

[0101] The volume was made up to 1 mL with PBS (10 mM, pH 7.4), the supernatant was removed after centrifugation, and the washing was repeated three times by resuspending in 1 mL of PBS. The insulin concentration of the supernatant was measured each time, and the final insulin sustained-release preparation was prepared to have an insulin concentration of 1 mg / mL for use in subsequent experiments.

[0102] Sample analysis

[0103] I. Test of the glucose responsiveness of the insulin sustained-release preparation

[0104] Preparation of a standard curve: 1 mg of FITC-porcine insulin was weighed into an EP tube, 0.1 mL of an HCl solution (0.1 M) was added, 0.15 mL of an NaOH solution (0.1 M) was added to completely dissolve the insulin, and the volume was made up to 1 mL with PBS (10 mM, pH 7.4) to obtain a FITC-porcine insulin solution having a concentration of 1 mg / mL. Using the FITC-porcine insulin stock solution having a concentration of 1 mg / mL, insulin solutions having concentrations of 1, 5, 10, 20, 40, 80, and 160 μg / mL were prepared, 100*3 μL of each was taken, and the fluorescence intensity at 525 nm was measured using an enzyme marker after excitation at 495 nm. Finally, a linear standard curve of the fluorescence intensity versus the insulin concentration was prepared.

[0105] The insulin sustained release formulation of 1 mg / mL obtained from Example 5 was subjected to cyclic exposure to glucose solutions at concentrations of 100 and 400 mg / dL (n=3). At the beginning of the experiment, the insulin sustained release formulation (equivalent to 1 mg of insulin) was centrifuged and then mixed with 1 mL of glucose solution (400 mg / dL). After 10 minutes of incubation at 37°C with shaking at 400 rpm, the suspension was centrifuged again and the supernatant was collected to determine the insulin concentration, then 1 mL of glucose solution (100 mg / dL) was added, after 10 minutes, the suspension was centrifuged again and the supernatant was collected to determine the insulin concentration, then 1 mL of glucose solution (400 mg / dL) was added, and this process was repeated three times. The insulin concentration of the supernatant in the EP tube was quantitatively determined using fluorescence method, and the concentration of insulin was calculated using a standard curve.

[0106] Figure 11 The test chart of the glucose responsiveness of the insulin sustained release formulation of 1 mg / mL obtained from Example 5. As shown in the figure, in the three high glucose-normal glucose cycles, the amount of insulin released by the insulin sustained release formulation in the high glucose solution was greater than that in the normal glucose solution, which fully verified the ability of the insulin sustained release formulation to release insulin in response to glucose, thereby providing long-acting ultra-low-dose insulin to maintain blood glucose stable at normal blood glucose, and providing a one-time high-dose of insulin to reduce blood glucose to the normal range when blood glucose is elevated. The amount released each time will decrease slightly, on the one hand because the reserve of insulin will decrease as the insulin is released, and on the other hand because the centrifugation causes the insulin sustained release formulation to aggregate.

[0107] II. Single Kinetic Release Test of Insulin Sustained Release Formulation

[0108] First, the insulin sustained release formulations of Examples 5, 6, 7, 9, 10 and 13 were respectively prepared into 160 μg / mL PBS solutions as mother liquor. The samples were added to the enzyme marker hole board according to the following groups:

[0109] PBS group: 50 μL of mother liquor + 125 μL of PBS;

[0110] 100G group: 50 μL of mother liquor + 17.5 μL of 10 mg / mL glucose + 107.5 μL of PBS;

[0111] 400G group: 50 μL of mother liquor + 70 μL of 100 mg / mL glucose + 55 μL of PBS;

[0112] 20F group: 50 μL of mother liquor + 3.5 μL of 10 mg / mL fructose + 121.5 μL of PBS;

[0113] 100F group: 50 μL stock solution + 17.5 μL 10 mg / mL fructose + 107.5 μL PBS; after the solution was prepared, the 96-well plate was sent to the microplate reader, and the total test duration was set to 12 h, the test was performed every 5 min, the solution was shaken for 10 s during each test, the fluorescence intensity at 525 nm under excitation at 495 nm was measured each time, and finally the change of the fluorescence intensity with time was plotted, and the results are shown in FIG. 10A-F. Figure 12

[0114] The 1 mg / mL insulin sustained-release preparation obtained in Example 5 was respectively mixed with sugar solutions of different concentrations in a 96-well plate to finally prepare a release solution with a total volume of 100 μL per well and an insulin concentration of 500 μg / mL. Six release groups were set, including PBS, 100 mg / dL glucose (100G), 400 mg / dL glucose (400G), 20 mg / dL fructose (20F), 100 mg / dL fructose (100F), and a mixture of 20 mg / dL fructose and 100 mg / dL glucose (100G+20F). The insulin sustained-release preparation (1 mg / mL) containing monobenzoic acid polymer obtained in Comparative Example 1 was mixed with sugar solutions of different concentrations in a 96-well plate to finally prepare a release solution with a total volume of 100 μL per well and an insulin concentration of 500 μg / mL. Two release groups were set, including 100 mg / dL fructose (100F) and 400 mg / dL glucose (400G). After the solution was prepared, the 96-well plate was sent to the microplate reader, and the total test duration was set to 4 h, the test was performed every 2.5 min, the solution was shaken for 10 s during each test, the fluorescence intensity at 525 nm under excitation at 495 nm was measured each time, and finally the change of the fluorescence intensity with time was plotted, and the results are shown in FIG. 11A-F. Figure 13

[0115] Figure 12 FIGS. 10A-F are single release curves of the insulin sustained-release preparations obtained in Examples 5, 6, 7, 9, 10, and 13 in glucose and fructose solutions, respectively. Figure 13 Figure 12 Figure 13 As shown in FIGS. 10A-F and 11A-F, the response of the insulin sustained-release preparation of Comparative Example 1 to glucose was obviously lower than the response to fructose, and the responses of the insulin sustained-release preparations obtained in Examples 5, 6, 7, 9, 10, and 13 to glucose and fructose were close, and even the response to glucose was better than the response to fructose, indicating that the insulin sustained-release preparation containing dibenzoic acid can reduce the false recognition of fructose and the like, and can avoid the false release of insulin as much as possible.

[0116] ​​​​III. In vitro cumulative release test of insulin sustained-release formulations across multiple cycles

[0117] The insulin sustained-release formulation obtained in Example 5 was resuspended in PBS at pH 7.4 to achieve a final insulin concentration of 1 mg / dL. It was added to EP tubes to prepare final glucose concentrations of 100 mg / dL and 400 mg / dL, fructose concentrations of 20 mg / dL and 100 mg / dL, and a mixture of glucose and fructose concentrations of 100 mg / dL and 20 mg / dL, with a final insulin concentration of 500 μg / mL (n = 3). The above steps were repeated with the insulin sustained-release formulation containing monophenylboronic acid polymer obtained in Comparative Example 1 to obtain a release group with a final glucose concentration of 400 mg / dL and a fructose concentration of 100 mg / dL. Each release experiment lasted 1 hour, with 10 cycles. After each cycle, 90% of the supernatant was collected by centrifugation, and the same volume and concentration of insulin-free glucose solution were added. 100 μL of the release supernatant was added to a 96-well plate, and the fluorescence intensity of FITC-insulin was detected using a microplate reader. The insulin concentration was calculated using a standard curve, plotted as described above. The cumulative release was calculated using the following formula:

[0118] Q n =C1×V1+C2×V2×0.9+C3×V3×0.9+……+C n ×V n ×0.9

[0119] Q n C represents the cumulative release amount in the nth cycle. n V represents the insulin concentration in the supernatant measured during the nth cycle. n This represents the total volume of the liquid released during the nth cycle.

[0120] Figure 14 The figures show the cumulative release curves of the insulin sustained-release formulation obtained in Example 5 and the insulin sustained-release formulation containing monophenylboronic acid polymer obtained in Comparative Example 1 in vitro through multiple cycles in glucose and fructose solutions, respectively. During long-term, multiple cumulative release processes, the insulin sustained-release formulation obtained in Example 5 maintained its responsiveness to different concentrations of glucose solution. Furthermore, compared to Comparative Example 1, the insulin sustained-release formulation of Example 5 showed better selectivity for glucose, reducing erroneous insulin release due to fructose.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polymer, characterized in that, The structure of the polymer is shown in formula (1). ， A is , , , One of them, " is the connection position, R1 and R2 are each independently selected from one of hydrogen atoms, C1~C6 alkyl groups, and halogens; R3 and R4 are each independently selected from C1~C6 alkyl groups, n=5~800, m=1~100.

2. The method for preparing the polymer according to claim 1, characterized in that, Includes the following steps: A monomer containing A, a monomer containing bisphenylboronic acid, and an initiator were subjected to a free radical copolymerization reaction in a solvent. The polymer was obtained by dialyzing and lyophilization. The structures of the monomer containing A and the monomer containing bisphenylboronic acid are shown in formula (2) and formula (3), respectively. ， A is , , In one of the following, R1 and R2 are each independently selected from at least one of hydrogen atoms, C1-C6 alkyl groups, and halogens; R3 and R4 are each independently selected from C1-C6 alkyl groups.

3. The method for preparing the polymer according to claim 2, characterized in that, The molar ratio of the monomer containing bisphenylboronic acid to the monomer containing A is 1~2:3~19.

4. The method for preparing the polymer according to claim 2, characterized in that, The initiator is an azo initiator or an inorganic peroxide initiator.

5. The method for preparing the polymer according to claim 2, characterized in that, The total mass ratio of the monomer containing A and the monomer containing bisphenylboronic acid to the volume of the solvent is 100~400 mg / mL.

6. The method for preparing the polymer according to claim 2, characterized in that, The free radical copolymerization reaction is carried out at a temperature of 35-75 °C for 3-10 h.

7. A sustained-release insulin formulation, characterized in that, Includes insulin and the polymer of claim 1.

8. The insulin sustained-release formulation according to claim 7, characterized in that, The insulin is porcine insulin, human insulin, recombinant human insulin, or FITC-porcine insulin; the mass ratio of the polymer to insulin is 1~4:1~4.

9. The method for preparing the insulin sustained-release formulation according to claim 7 or 8, characterized in that, The process includes the following steps: dissolving the polymer described in claim 1 with insulin in acidified water, adjusting the pH to neutral with an alkaline solution to form an insulin complex, adding PBS solution to adjust the insulin concentration, centrifuging and resuspending to obtain a sustained-release insulin formulation.

10. The use of the insulin sustained-release formulation according to claim 7 or 8 in the preparation of a medicament for treating diabetes.

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