Sugar-responsive protamine insulin, preparation method therefor and use thereof

Abstract

A preparation method for a sugar-responsive protamine insulin and a use thereof. The sugar-responsive protamine insulin is prepared by means of modifying a chemical group on cysteine in protamine, connecting with a phenylboronic acid compound to form a complex, and then mixing with insulin. At normal blood sugar levels, the insulin is released slowly, while under high blood sugar conditions, release is accelerated, thereby achieving effective regulation of blood sugar and avoiding the occurrence of hypoglycemia.

Description

A sugar-responsive protamine insulin and its preparation method and application

[0001] This application claims priority to the prior Chinese application, application number: 202310987884.3, filing date: August 7, 2023; the specification, claims, abstract and set of drawings of that application are all incorporated herein by reference as part of this application. Technical Field

[0002] The present invention belongs to the technical field of drug delivery, and in particular relates to a sugar-responsive protamine insulin and a preparation method and application thereof. Background Art

[0003] Currently, diabetes affects more than 537 million people worldwide, and the number of patients is expected to exceed 783 million by 2045. Diabetes is a common chronic disease characterized by abnormal glucose metabolism due to absolute or relative insufficiency of insulin secretion, with hyperglycemia as the main clinical manifestation. Diabetes is mainly divided into two categories: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Among them, T1DM is mainly caused by damage to the pancreatic beta cells that secrete insulin in the patient's body, and the age of onset is young, manifesting as absolute insulin deficiency. T2DM is mainly caused by insulin resistance, which is more common in the elderly and manifests as relative insulin deficiency. T1DM and late-stage T2DM require daily injection or continuous infusion of exogenous insulin to maintain blood sugar levels within the normal range.

[0004] To reduce the frequency of daily insulin injections, the long-acting insulin preparation protamine zinc insulin (PZI) was developed in 1936. The insulin in PZI can form a complex with excess protamine and zinc, increasing the stability of the preparation, delaying the release of insulin in the body, and prolonging the duration of insulin action. In 1946, the more stable neutral protamine zinc insulin (NPH) was successfully developed. NPH is mainly used to provide basal insulin, which can keep blood sugar stable before meals and at night. Because exogenous insulin is not regulated by endogenous feedback mechanisms, even with a carefully designed dosage regimen and even when patients strictly follow the doctor's diet and lifestyle, diabetic patients will inevitably experience symptoms of hyperglycemia or hypoglycemia.

[0005] Therefore, a glucose-responsive insulin system that can mimic the mechanism of action of endogenous insulin is crucial. This system can provide a treatment method that can adjust the insulin release rate in real time to the patient's blood glucose level, thereby improving the therapeutic effect and reducing adverse reactions. Currently, the glucose-responsive elements used to modify insulin mainly include: glucose oxidase (Glucose oxidase, X), phenylboronic acid (PBA) and glucose-binding molecules.

[0006] In the prior art, phenylboronic acid or phenylboronic acid derivatives are usually compounded with polymers to prepare insulin delivery carriers. For example, patent CN115671050A uses polyacryloyl carbonate-polycaprolactone as raw materials, phenylboronic acid derivatives and dihydroxy to form phenylboronic acid ester complexes, and liver-targeted glucose-responsive nanoparticles are prepared through W / O / W emulsification and UV crosslinking technology. Other researchers have also prepared PBA-modified poly (N-(2-aminoethyl)acrylamide) based on the above strategy, using this material to load insulin, and verified its glucose-responsiveness and blood sugar control effects in animals. However, the above research preparations all use polymer materials, the preparation process is cumbersome, and the biocompatibility and safety of the polymers lack clinical verification.

[0007] Compared with the above polymers, protamine has better biocompatibility and safety, and there are currently no reports on protamine insulin with glucose responsiveness.

[0008] Background Art

[0009] In order to solve the unavoidable hypoglycemia phenomenon of the protamine insulin system in the prior art, the present invention provides a sugar-responsive protamine insulin and its preparation method and application. The sugar-responsive protamine insulin is prepared by modifying chemical groups on the cysteine ​​of protamine, connecting it with a phenylboronic acid compound to form a complex, and mixing it with insulin. The protamine insulin has a better sugar response speed and can achieve intelligent sugar response regulation. It slowly releases insulin when blood sugar is normal and accelerates the release of insulin when blood sugar is high, which can achieve effective regulation of blood sugar and avoid the occurrence of hypoglycemia.

[0010] In one aspect, the present invention provides a sugar-responsive insulin carrier, which comprises protamine and a phenylboronic acid compound, wherein the cysteine ​​in the protamine is directly or indirectly linked to the phenylboronic acid compound, or the phenylboronic acid compound directly or indirectly modifies the cysteine.

[0011] Protamine is a mixture of strongly basic peptides that was originally isolated from the sperm of salmon and other fish species, but is now primarily produced recombinantly through biotechnology. It contains more than two-thirds of the amino acid L-arginine. Because protamine contains amino acids with free basic side chains, it has a certain buffering capacity and is therefore considered a buffering agent.

[0012] In some embodiments, the protamine comprises natural protamine from salmon, herring, trout, or solid-phase synthesized protamine.

[0013] Research has shown that by modifying the cysteine ​​in protamine, the modified protamine can be connected with phenylboronic acid compounds, thereby being used to prepare a sugar-responsive insulin carrier. The carrier can be combined with negatively charged insulin to prepare sugar-responsive protamine insulin. When the glucose content is high, the carrier can combine with insulin, slowly releasing insulin under normal blood sugar conditions and accelerating the release of insulin under high blood sugar conditions.

[0014] Furthermore, the cysteine ​​in the protamine is modified by a chemical group, and the cysteine ​​in the protamine is modified by a chemical group, and the chemical group is connected to the NHS-esterified phenylboronic acid compound through a chemical bond.

[0015] Furthermore, the connection mode between the chemical group and protamine is a thioether bond; and the chemical bond between the chemical group and the NHS-esterified phenylboronic acid compound is an amide bond.

[0016] In some embodiments, the thioether bond is formed by a covalent addition reaction between a cysteine ​​residue of protamine and a maleimide residue of the chemical group.

[0017] In some embodiments, the chemical group is linked to the NHS-esterified phenylboronic acid compound via an amide bond.

[0018] Furthermore, the chemical group includes any one or more of a straight-chain or branched alkyl group, a hydroxyl group, a carboxyl group, an amino group or a thiol group, or any one or more of an alkyl group, a hydroxyl group, a carboxyl group, an amino group or a thiol group substituted at any position of these groups.

[0019] Furthermore, the structural formula of the chemical group includes any one or more of Formula I to Formula IV:

[0020] In some embodiments, the chemical group comprises a compound represented by Formula I, Formula II, Formula III, or Formula IV to modify cysteine ​​in protamine.

[0021] Furthermore, the phenylboronic acid compound includes phenylboronic acid modified with different groups; the modified groups include one or more of alkyl, carboxyl, halogen, nitro and amide.

[0022] Furthermore, the phenylboronic acid compound is modified with a carboxyl group.

[0023] Furthermore, the phenylboronic acid compound is p-carboxylphenylboronic acid or o-carboxylphenylboronic acid which may be substituted or unsubstituted.

[0024] Furthermore, the substituent is one or more of halogen and nitro.

[0025] Furthermore, the phenylboronic acid compound is selected from one or more of the following compounds:

[0026] Furthermore, the phenylboronic acid compound is 4-carboxy-3-fluorophenylboric acid.

[0027] Furthermore, the phenylboronic acid compound reacts with N-hydroxysuccinimide to generate an NHS-esterified phenylboronic acid compound.

[0028] Furthermore, N-hydroxysuccinimide reacts with 4-carboxy-3-fluorophenylboronic acid to activate the carboxyl group of 4-carboxy-3-fluorophenylboronic acid, so that the carboxyl group can further react with the amino group on the chemical group to form an amide bond.

[0029] In some embodiments, the connection between protamine and the phenylboronic acid compound esterified with N-hydroxysuccinimide (NHS) can be that one cysteine ​​is directly or indirectly connected to the phenylboronic acid compound, each cysteine ​​on multiple cysteines is directly or indirectly connected to the phenylboronic acid compound, or some cysteines on multiple cysteines are directly or indirectly connected to the phenylboronic acid compound.

[0030] In some embodiments, there are nine cysteines in the protamine, and 1 to 9 cysteines can be directly or indirectly linked to the phenylboronic acid compound.

[0031] In some embodiments, the reaction formula for modifying the sulfhydryl group of cysteine ​​in protamine by the chemical group is as follows:

[0032] In some embodiments, the reaction formula for the combination of the chemical group-modified protamine and NHS-esterified 4-carboxyl-3-fluorophenylboronic acid is as follows (the chemical group is Formula II):

[0033] Furthermore, the insulin includes any one or more of the ultra-long-acting, long-acting, short-acting, and rapid-acting insulins of recombinant human insulin, lispro insulin, aspart insulin, detemir insulin, glargine insulin, degludec insulin, and icodec.

[0034] In another aspect, the present invention provides a method for preparing a sugar-responsive insulin carrier, the method comprising the following steps:

[0035] (1) reacting the compound solution with the protamine solution to obtain solution A;

[0036] (2) Solution A reacts with a solution of a phenylboronic acid compound esterified with NHS to prepare a sugar-responsive insulin carrier.

[0037] The structural formula of the compound includes any one or more of Formula I to Formula IV:

[0038] Furthermore, the preparation method comprises the following steps:

[0039] (1) reacting a compound represented by Formula I, Formula II, Formula III, or Formula IV or other chemical groups that modify cysteine ​​in protamine with protamine to allow the cysteine ​​in the protein to be linked to the chemical group;

[0040] (2) The modified protamine is chemically linked to an NHS-esterified phenylboronic acid compound, and the phenylboronic acid compound is directly or indirectly linked to the cysteine ​​in the protamine through the modified group to prepare a sugar-responsive insulin carrier.

[0041] In some embodiments, step (1) is to react the solution of the compound represented by Formula I, Formula II, Formula III or Formula IV with a protamine solution, and the solvent of the compound solution or the protamine solution is PBS (0.01M, pH=7.4), PBS (0.1M, pH=7.4), ultrapure water, deionized water, double distilled water, etc., which can dissolve protamine and the compound solution. One or more of them.

[0042] In some embodiments, the preferred solvent is PBS (0.01 M, pH = 7.4) solution.

[0043] Furthermore, the molar ratio of the compound represented by Formula I, Formula II, Formula III or Formula IV to protamine in step (1) is (1-50):1.

[0044] Preferably, the molar ratio is 18:1.

[0045] In some embodiments, the molar ratio of the compound to protamine is 18:1.

[0046] In some embodiments, the reaction temperature in step (1) is 4-40°C.

[0047] In some embodiments, the reaction in step (1) is preferably carried out at room temperature.

[0048] In some embodiments, step (1) further comprises subjecting the chemical group-modified protamine solution to a purification treatment, wherein the purification treatment preferably comprises dialysis and drying.

[0049] The dialysis can be performed in deionized water using a conventional dialysis bag in the art, and the molecular weight cut-off of the dialysis bag is preferably 1k to 3.5k.

[0050] Among them, the drying is preferably freeze-drying.

[0051] In some embodiments, the concentration of the protamine solution is preferably 0.2-100 mg / mL, for example 10 mg / mL.

[0052] In some embodiments, the protamine coupled with the phenylboronic acid compound in step (2) is obtained by grafting protamine with an NHS-esterified phenylboronic acid compound. Specifically, in the protamine coupled with the phenylboronic acid compound, the phenylboronic acid compound is linked to the chemical group via an amide bond, and then the chemical group modified with the phenylboronic acid compound is grafted onto the cysteine ​​side chain of the protamine via a thioether bond.

[0053] The molar ratio of the protamine to the NHS-esterified phenylboronic acid compound is preferably 1:(1-54).

[0054] In some embodiments, the solvent in the protamine solution can be a conventional aqueous solvent in the art that can dissolve protamine, including PBS (0.01M, pH=7.4), PBS (0.1M, pH=7.4), ultrapure water, deionized water, double distilled water, etc., preferably PBS (0.01M, pH=7.4).

[0055] In some embodiments, the concentration of the protamine solution is preferably 1-200 mg / mL, such as 10 mg / mL.

[0056] In some embodiments, the solvent in the solution of the NHS-esterified phenylboronic acid compound can be a conventional water-miscible organic solvent in the art that can dissolve the phenylboronic acid compound, preferably dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), such as DMSO.

[0057] In some embodiments, the carboxyl-modified phenylboronic acid compound is selected from one or more of the following compounds:

[0058] In some embodiments, the phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.

[0059] In some embodiments, the carboxyl-modified phenylboronic acid compound reacts with N-hydroxysuccinimide (NHS) to generate an NHS-esterified phenylboronic acid compound.

[0060] In some embodiments, the mixing method preferably comprises dropping the solution of the NHS-esterified phenylboronic acid compound into the solution of the chemical group-modified protamine.

[0061] In some embodiments, after the mixing, the method further comprises one or more of adjusting the pH to alkaline, stirring, adjusting the pH to acidic, dialysis or freeze-drying.

[0062] In some embodiments, the alkalinity preferably has a pH range of 7.5 to 13.5, such as a pH of 13.

[0063] In some embodiments, the solution for adjusting the pH to alkaline is preferably a sodium hydroxide solution, a sodium carbonate solution or a sodium bicarbonate solution, such as a sodium hydroxide solution.

[0064] In some embodiments, the stirring time is preferably 2 min to 24 h, for example 30 min.

[0065] In some embodiments, the acidic pH range is preferably 2.0 to 6.5, for example, pH 2.

[0066] In some embodiments, the solution for adjusting the pH to acidic is preferably a hydrochloric acid solution, an acetic acid solution or trifluoroacetic acid, such as a hydrochloric acid solution.

[0067] In some embodiments, the dialysis can be performed in deionized water using a conventional dialysis bag in the art. The molecular weight cut-off of the dialysis bag is preferably 1k to 10k. The purpose of the dialysis is to remove free NHS-esterified phenylboronic acid compounds.

[0068] In some embodiments, when the grafting reaction further includes a dialysis step, the freeze-drying step is performed after the dialysis. A white solid is obtained after the freeze-drying step, and the solid can be in powder or flocculent form.

[0069] In another aspect, the present invention provides a sugar-responsive protamine-insulin complex, which is prepared by mixing the sugar-responsive insulin carrier described above with insulin.

[0070] The sugar-responsive protamine-insulin complex provided by the present invention is an amorphous flocculent precipitate that can form an insulin reservoir upon subcutaneous injection. Under hyperglycemic conditions, the binding of high concentrations of glucose to phenylboronic acid groups immediately reduces the positive charge density of the phenylboronic acid-modified protamine, deconstructing the phenylboronic acid ester bond, thereby reducing the electrostatic attraction between insulin and the polymer and promoting insulin release. Under normoglycemic conditions, strong electrostatic forces cause the protamine-insulin complex to release ultra-low amounts of insulin, providing slow and sustained insulin release and reducing the risk of hypoglycemia.

[0071] On the other hand, the present invention provides a method for preparing a sugar-responsive protamine-insulin complex, which comprises the following steps: mixing the sugar-responsive insulin carrier solution and the insulin solution in water, adjusting the pH to 6.5-8.0, and obtaining a sugar-responsive protamine-insulin complex.

[0072] In some embodiments, the pH is preferably 7.4.

[0073] In some embodiments, the mass ratio of the sugar-responsive insulin carrier solution to the insulin solution is (0.5-10):1, preferably (1-2):1, such as 1:1, 2:1 or 4:1.

[0074] In some embodiments, the concentration of the sugar-responsive insulin carrier solution is 1 to 200 mg / mL, for example, 10 mg / mL.

[0075] In some embodiments, the method for preparing the sugar-responsive insulin carrier solution includes the following steps: mixing the sugar-responsive insulin carrier with weakly acidic water to obtain the sugar-responsive insulin carrier solution; wherein the pH range of the weakly acidic water can be 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be hydrochloric acid solution, phosphate buffer, deionized water or pure water, such as hydrochloric acid solution.

[0076] In some embodiments, the concentration of the insulin solution may be 1-100 mg / mL, for example, 10 mg / mL.

[0077] In some embodiments, the method for preparing the insulin solution includes the following steps: mixing insulin with weakly acidic water to obtain an insulin solution; wherein the pH range of the weakly acidic water can be 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be hydrochloric acid solution, phosphate buffer, deionized water or pure water, such as hydrochloric acid solution.

[0078] In some embodiments, after the sugar-responsive insulin carrier solution is mixed with the insulin solution, the pH is adjusted to 6.5 to 7.4, for example, to 7.4.

[0079] In some embodiments, a white flocculent precipitate is formed after the pH adjustment, which is a sugar-responsive protamine-insulin complex. The sugar-responsive protamine-insulin complex is soluble or readily soluble in weakly acidic water and insoluble or slightly soluble in weakly alkaline water.

[0080] In some embodiments, the step of centrifugation may be further included after adjusting the pH. The purpose of the centrifugation is to collect the white flocculent precipitate and remove the uncomplexed insulin in the supernatant.

[0081] In some embodiments, the diabetes may be type 1 diabetes or type 2 diabetes; preferably type 1 diabetes or late-stage type 2 diabetes.

[0082] In some embodiments, the sugar-responsive protamine-insulin complex can be resuspended in phosphate buffer or physiological saline and administered by subcutaneous injection for treatment.

[0083] Without violating the common sense in the art, the conditions of the above-mentioned embodiments can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0084] The "room temperature" in the present invention refers to 18°C ​​to 25°C.

[0085] The reagents and raw materials used in the present invention are commercially available.

[0086] The beneficial effects of the present invention are:

[0087] 1. The protamine analog modified with a phenylboronic acid compound designed in the present invention is a biodegradable positively charged polymer capable of charge reversal, which enables it to bind to insulin and achieve stable and efficient glucose-responsive insulin release;

[0088] 2. The use of protamine, which is already in clinical use, solves the biocompatibility and safety issues of the insulin-carrying system, improves the stability of insulin, and prolongs the duration of blood sugar control;

[0089] 3. By selecting appropriate compounds to modify protamine, the sugar-responsive insulin carrier constructed has a faster and more sensitive sugar response speed after being combined with insulin. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 is a hydrogen nuclear magnetic resonance spectrum of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS);

[0091] Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the reaction product (compound 2) of protamine modified with chemical group formula I and FPBA-NHS;

[0092] FIG3 is a hydrogen nuclear magnetic resonance spectrum of compound 4 in Example 2;

[0093] FIG4 is a hydrogen nuclear magnetic resonance spectrum of compound 6 in Example 3;

[0094] FIG5 is a hydrogen nuclear magnetic resonance spectrum of compound 8 in Example 4;

[0095] FIG6 is a transmission electron microscopy image of the sugar-responsive protamine-insulin complex I in Example 5;

[0096] FIG7 is a transmission electron microscopy image of the sugar-responsive protamine-insulin complex III in Example 5;

[0097] FIG8 is a glucose-responsive insulin release curve of the glucose-responsive protamine-insulin complex I in Example 5;

[0098] FIG9 is a glucose-responsive insulin release curve of the glucose-responsive protamine-insulin complex III in Example 5;

[0099] FIG10 shows the blood glucose results of diabetic mice after administration of commercially available protamine insulin (NPH) in Example 6.

[0100] FIG11 shows the blood glucose results of diabetic mice after administration of the sugar-responsive protamine-insulin complex I in Example 6.

[0101] FIG12 shows the blood glucose results of diabetic mice after administration of the glucose-responsive protamine-insulin complex III in Example 6. DETAILED DESCRIPTION

[0102] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0103] Example 1: Modification of protamine with chemical group I and 4-carboxyl-3-fluorophenylboronic acid to prepare a sugar-responsive insulin carrier, thereby preparing a sugar-responsive protamine-insulin complex

[0104] The preparation process of the sugar-responsive protamine-insulin complex provided in this example is as follows:

[0105] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS)

[0106] Chemical reaction formula for synthesizing FPBA-NHS:

[0107] 10 g of 4-carboxy-3-fluorophenylboronic acid (FPBA) and 10 g of N-hydroxysuccinimide (NHS) were weighed into a 200 mL round-bottom flask. 150 mL of N,N-dimethylformamide (DMF) was added. Then, 15 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), 2.2 g of 4-dimethylaminopyridine (DMAP), and 12.5 mL of triethylamine were added to the mixture. The mixture was allowed to react in an ice-water bath for two days. After two days, the pale pink mixture was transferred to a 250 mL separatory funnel, 150 mL of dichloromethane was added, and the mixture was extracted three times with 50 mL of 1 mol / L HCl. The lower dichloromethane layer was collected and an appropriate amount of anhydrous MgSO₄ was added to remove excess water. After removing anhydrous MgSO4 by filtration, dichloromethane was removed by rotary evaporation to obtain a white powder product with the structural formula: Using deuterated dimethyl sulfoxide as solvent, 1 The results were characterized by H-NMR (see Figure 1).

[0108] 2. Preparation of protamine modified with chemical group formula I

[0109] The chemical reaction formula of protamine modified with chemical group formula I is:

[0110] 50 mg of protamine (1.0 eq, 5 mL of PBS) and 120 mg of MAL-KK-NH2 (18.0 eq, 5 mL of PBS) were placed in a 25 mL round-bottom flask and reacted overnight at room temperature. The reaction mixture was dialyzed four times against 4 L of deionized water. The dialyzed product was freeze-dried to obtain Compound 1 as a white powder.

[0111] 3. Preparation of Sugar-Responsive Insulin Carriers

[0112] The chemical reaction formula of protamine modified with chemical group formula I and FPBA-NHS is as follows:

[0113] The preparation process is as follows:

[0114] Compound 1 (1.0eq) was dissolved in 5mL of deionized water to obtain an aqueous solution of compound 1. 4-Carboxyl-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 2.0eq) was weighed and dissolved in 5mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 1 was ice-cooled, and FPBA-NHS solution was added dropwise thereto. 1mol / L sodium hydroxide solution was added dropwise to the mixed solution to control the pH of the mixture to about 13.0, and stirred for about 30 minutes. 1mol / L hydrochloric acid solution was then added dropwise to the clear solution to allow the mixture to first become white precipitate and then become clear, with a pH of about 2.0. The reacted mixture was dialyzed 4 times with 4L of deionized water, and the product was freeze-dried after dialysis to obtain a white powder or flocculent compound 2. 550μL of deuterated water containing 1μL of trifluoroacetic acid was used as the solvent, and the mixture was dried by 1 The results were characterized by H-NMR (see Figure 2).

[0115] 4. Preparation of Sugar-responsive Protamine-Insulin Complex I

[0116] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately, weigh 10 mg of compound 2 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution to adjust the pH to approximately 2.0-3.0. Take 100 μL of each solution and mix them in an EP tube. Add 3.0 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 7.4. Afterwards, centrifuge to remove uncomplexed insulin, obtaining a white precipitate. Add 1 mL of PBS (0.01 M, pH 7.4) to the precipitate to obtain 1 mg / mL (wherein the mass is based on the mass of insulin) of insulin-protamine complex I dispersed in PBS.

[0117] Example 2: Preparation of Sugar-Responsive Insulin Carrier by Modifying Protamine with Chemical Group II and 4-Carboxyl-3-Fluorophenylboronic Acid, and Thus Preparation of Sugar-Responsive Protamine-Insulin Complex

[0118] The preparation process of the sugar-responsive protamine-insulin complex provided in this example is as follows:

[0119] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS). The preparation method is as shown in Example 1.

[0120] 2. Preparation of protamine modified with chemical group II

[0121] The chemical reaction formula of protamine modified with chemical group II:

[0122] 50 mg of protamine (1.0 eq, 5 mL of PBS) and MAL-KKK-NH2 (18.0 eq, 5 mL of PBS) were placed in a 25 mL round-bottom flask and reacted overnight at room temperature. The reaction mixture was dialyzed four times against 4 L of deionized water. The dialyzed product was freeze-dried to obtain compound 3 as a white powder.

[0123] 3. Preparation of Sugar-Responsive Insulin Carriers

[0124] The sugar-responsive insulin carrier prepared in this example is KKK-protamine-4-carboxyl-3-fluorophenylboronic acid (KKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0125] The preparation process is as follows:

[0126] Compound 3 (1.0eq) was dissolved in 5mL of deionized water to obtain an aqueous solution of compound 3. 4-Carboxyl-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 3.0eq) was weighed and dissolved in 5mL of dimethyl sulfoxide (DMSO) to obtain an FPBA-NHS solution. The aqueous solution of compound 3 was ice-cooled, and FPBA-NHS solution was added dropwise thereto. 1mol / L sodium hydroxide solution was added dropwise to the mixed solution to control the pH of the mixture to about 13.0, and stirred for about 30 minutes. 1mol / L hydrochloric acid solution was then added dropwise to the clear solution to allow the mixture to first become white precipitate and then become clear, with a pH of about 2.0. The reacted mixture was dialyzed 4 times with 4L of deionized water, and the product was freeze-dried after dialysis to obtain a white powder or flocculent compound 4. 550μL of deuterated water containing 1μL of trifluoroacetic acid was used as the solvent, and the mixture was purified by 1 The results were characterized by H-NMR (see Figure 3).

[0127] 4. Preparation of Sugar-responsive Protamine-Insulin Complex II

[0128] The reaction formula is as follows:

[0129] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately, weigh 10 mg of compound 4 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution and adjust the pH to approximately 2.0-3.0. Take 100 μL of each solution and mix them in an EP tube. Add 3.0 μL of 1 mol / L sodium hydroxide solution and adjust the pH to 7.4. Then, centrifuge to remove uncomplexed insulin, obtaining a white precipitate. Add 1 mL of PBS (0.01 M, pH 7.4) to the precipitate to obtain 1 mg / mL (wherein the mass is based on the mass of insulin) of insulin-protamine complex II dispersed in PBS.

[0130] Example 3: Modification of protamine with chemical group III and 4-carboxyl-3-fluorophenylboronic acid to prepare a sugar-responsive insulin carrier, thereby preparing a sugar-responsive protamine-insulin complex

[0131] The preparation process of the sugar-responsive protamine-insulin complex provided in this example is as follows:

[0132] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS). The preparation method is as shown in Example 1.

[0133] 2. Preparation of protamine modified with chemical group III

[0134] The chemical reaction formula of protamine modified with chemical group III:

[0135] 50 mg of protamine (1.0 eq, 5 ml PBS) and MAL-KKKK-NH2 (18.0 eq, 5 ml PBS) were placed in a 25 mL round-bottom flask and reacted overnight at room temperature. The reaction mixture was dialyzed four times against 4 L of deionized water. The dialyzed product was freeze-dried to obtain compound 5 as a white powder.

[0136] 3. Preparation of Sugar-Responsive Insulin Carriers

[0137] The sugar-responsive insulin carrier prepared in this example is KKKK-protamine-4-carboxyl-3-fluorophenylboronic acid (KKKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0138] The preparation process is as follows:

[0139] Compound 5 (1.0eq) was dissolved in 5mL of deionized water to obtain an aqueous solution of compound 5. Separately, 4-carboxyl-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 4.0eq) was weighed and dissolved in 5mL of dimethyl sulfoxide (DMSO) to obtain a FPBA-NHS solution. The aqueous solution of compound 5 was ice-cooled, and FPBA-NHS solution was added dropwise thereto. 1mol / L sodium hydroxide solution was added dropwise to the mixed solution to control the pH of the mixture to about 13.0, and stirred for about 30 minutes. 1mol / L hydrochloric acid solution was then added dropwise to the clear solution to allow the mixture to first become white precipitate and then become clear, with a pH of about 2.0. The reacted mixture was dialyzed 4 times with 4L of deionized water, and the product was freeze-dried after dialysis to obtain a white powder or flocculent compound 6. 550μL of deuterated water containing 1μL of trifluoroacetic acid was used as the solvent, and the mixture was dried by 1 The results were characterized by H-NMR (see Figure 4).

[0140] 4. Preparation of Sugar-responsive Protamine-Insulin Complex III

[0141] Weigh 10 mg of insulin and dissolve it in 1 mL of water. Separately, weigh 10 mg of compound 6 and dissolve it in 1 mL of water. Add 15 μL of 1 mol / L hydrochloric acid solution to each solution and adjust the pH to approximately 2.0-3.0. Take 100 μL of each solution and mix them in an EP tube. Add 3.0 μL of 1 mol / L sodium hydroxide solution and adjust the pH to 7.4. Then, centrifuge to remove uncomplexed insulin, obtaining a white precipitate. Add 1 mL of PBS (0.01 M, pH 7.4) to the precipitate to obtain 1 mg / mL (wherein the mass is based on the mass of insulin) of insulin-protamine complex III dispersed in PBS.

[0142] Example 4: Preparation of Sugar-Responsive Insulin Carrier by Modifying Protamine with Chemical Group IV and 4-Carboxyl-3-Fluorophenylboronic Acid, Thus Preparing Sugar-Responsive Protamine-Insulin Complex

[0143] The preparation process of the sugar-responsive protamine-insulin complex provided in this example is as follows:

[0144] 1. Preparation of 4-carboxy-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS). The preparation method is as shown in Example 1.

[0145] 2. Preparation of protamine modified with chemical group IV

[0146] The chemical reaction formula of protamine modified with chemical group IV is:

[0147] 50 mg of protamine (1.0 eq, 5 ml PBS) and MAL-KKKKKK-NH2 (18.0 eq, 5 ml PBS) were placed in a 25 mL round-bottom flask and reacted overnight at room temperature. The reaction mixture was dialyzed four times with 4 L of deionized water. The dialyzed product was freeze-dried to obtain compound 7 as a white powder.

[0148] 3. Preparation of Sugar-Responsive Insulin Carriers

[0149] The sugar-responsive insulin carrier prepared in this example is KKKKKK-protamine-4-carboxyl-3-fluorophenylboronic acid (KKKKKK-protamine-FPBA), and its chemical reaction formula is as follows:

[0150] The preparation process is as follows:

[0151] Compound 7 (1.0eq) was dissolved in 5mL of deionized water to obtain an aqueous solution of compound 7. Separately, 4-carboxyl-3-fluorophenylboronic acid-N-hydroxysuccinimide (FPBA-NHS, 6.0eq) was weighed and dissolved in 5mL of dimethyl sulfoxide (DMSO) to obtain a FPBA-NHS solution. The aqueous solution of compound 7 was ice-cooled, and FPBA-NHS solution was added dropwise thereto. 1mol / L sodium hydroxide solution was added dropwise to the mixed solution to control the pH of the mixture to about 13.0, and stirred for about 30 minutes. 1mol / L hydrochloric acid solution was then added dropwise to the clear solution to allow the mixture to first become white precipitate and then become clear, with a pH of about 2.0. The reacted mixture was dialyzed 4 times with 4L of deionized water, and the product was freeze-dried after dialysis to obtain a white powder or flocculent compound 8. 550μL of deuterated water containing 1μL of trifluoroacetic acid was used as the solvent, and the mixture was dried by 1 Characterization by H-NMR (see Figure 5)

[0152] 4. Preparation of Sugar-responsive Protamine-Insulin Complex IV

[0153] 10 mg of insulin was dissolved in 1 mL of water, and 10 mg of compound 8 was dissolved in 1 mL of water. 15 μL of 1 mol / L hydrochloric acid solution was added to each solution to adjust the pH to approximately 2.0-3.0. 100 μL of each solution was mixed in an EP tube, and 3.0 μL of 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.4. Uncomplexed insulin was then removed by centrifugation to obtain a white precipitate. 1 mL of PBS (0.01 M, pH 7.4) was added to the precipitate to obtain an insulin-protamine complex IV dispersed in PBS at a concentration of 1 mg / mL (wherein the mass is based on the mass of insulin). Among all the complexes, Complex IV had the highest proportion of phenylboronic acid grafting, which resulted in a high level of free insulin in the Complex IV solution and, in turn, a low insulin loading rate. Therefore, Complex IV will not be discussed further, and the complexes in Examples 1 and 3 are preferred for discussion.

[0154] Example 5: Verification of the Effect of Sugar-Responsive Protamine-Insulin Complex

[0155] 1. Study on the morphology of sugar-responsive protamine complexes

[0156] In this example, the morphology of the sugar-responsive protamine-insulin complexes prepared in Examples 1 and 3 was observed by transmission electron microscopy:

[0157] The glucose-responsive protamine-insulin complexes I and III prepared in Examples 1 and 3 were weighed, water was added to an insulin equivalent concentration of 1 mg / mL, and ultrasonic cell disruptors were used at 100 W for 1 minute. The cells were dispersed and suspended in water and then dropped onto a copper mesh. 10 μL of 5% uranyl acetate solution was added to the mesh, allowed to stand for 10 minutes, and the solution was removed with filter paper. Transmission electron microscopy (TEM) of the samples revealed similar morphologies for the glucose-responsive protamine-insulin complexes prepared in Examples 1 and 3. TEM images of the glucose-responsive protamine-insulin complexes in Examples 1 and 3 are shown in Figures 6 and 7.

[0158] As can be seen from Figures 6 and 7, the sugar-responsive protamine-insulin complex has a porous and loose microstructure. The formed protamine-insulin complex has an irregular shape and a size of micromole order, and insulin and protamine are uniformly distributed in the complex.

[0159] 2. In vitro glucose responsiveness verification of glucose-responsive protamine-insulin complex

[0160] Dissolve 0.1 mg of water-soluble Cy5-NHS ester in 0.02 mL of DMSO. Add 10 mg of insulin to 5 mL of 0.1 M sodium bicarbonate solution. Mix the two solutions, stir overnight at room temperature, and dialyze three times against 4 L of deionized water in the dark. Freeze-dry to obtain Cy5-labeled insulin.

[0161] Cy5-labeled insulin was used with the sugar-responsive insulin carriers prepared in Examples 1 and 3 to prepare 1 mg / mL sugar-responsive protamine-insulin complexes (steps as shown in Examples 1 and 3, respectively). Different amounts of 0.4 g / mL glucose solution were added to the solution to obtain different initial glucose concentrations (0, 100, 200, 400 mg / dL). 50 μL of the complex was drawn at 0, 15, 30, 60, 120, and 180 minutes, respectively, and centrifuged. 20 μL of the supernatant was then mixed with 180 μL of PBS (0.01 M, pH = 7.4). The absorbance of the mixture at 640 nm of excitation light and 670 nm of emission light was measured using a microplate reader, and the insulin concentration in the supernatant was calculated using a pre-established standard curve. The results are shown in Figures 8 and 9, respectively. In phosphate buffer at pH 7.4, the insulin concentration in the supernatant was determined using Cy5-labeled insulin, and the glucose-responsive insulin release performance of the complex was evaluated at different glucose concentrations (0, 100, 200, and 400 mg / dL).

[0162] Figures 8 and 9 show that the addition of glucose to the PBS solution significantly increases the insulin release rate and equilibrium insulin concentration. High glucose concentrations may result in more glucose binding to the FPBA residues on the complex, thereby reducing the positive charge density and diminishing the attraction between the negatively charged insulin and the positively charged polymer under physiological conditions, promoting insulin release. Comparison of the glucose-responsive protamine-insulin complexes I and III reveals that increasing the proportion of phenylboronic acid modification on the protamine increases the insulin release rate.

[0163] However, it should be noted that the faster the insulin is released, the better the final blood sugar control effect is not necessarily the case. The insulin release rate is only one factor that affects the blood sugar control effect. Other factors such as the metabolism of the drug in the body and the sensitivity of tissues to insulin also play a vital role. Therefore, rigorous experiments are still needed to further verify the blood sugar control effects of Complex I and Complex III. Based on this, in subsequent experiments, glucose-responsive protamine insulin complex I and complex III were used to conduct in-depth in vivo blood sugar regulation studies to comprehensively evaluate their blood sugar regulation performance in practical applications.

[0164] Example 6: In vivo blood glucose regulation study of sugar-responsive protamine-insulin complexes

[0165] Streptozotocin (STZ) was dissolved in citric acid / sodium citrate buffer to prepare a 10 mg / mL solution. The resulting solution was stored on ice in the dark and used immediately upon preparation. C57BL / 6J mice (male, approximately 25 g, clean grade) were selected and fasted overnight. STZ solution was then injected intraperitoneally in the dark at a dose of 120 mg / kg. Blood glucose was measured two weeks after injection, and the diabetic mice with successful modeling (blood glucose of 300-500 mg / dL) were used for the subsequent study. The insulin equivalent dose of the complex was determined by converting the dose of commercially available drugs. Blood glucose was monitored before and after treatment until blood glucose returned to the initial level. Blood was collected from the tip of the tail, and plasma glucose concentration was measured using a glucometer.

[0166] Three groups (n=5) of C57BL / 6J diabetic mice with blood glucose levels between 300 and 500 mg / dL were selected. The control group received commercially available protamine insulin (NPH), while the experimental groups received glucose-responsive protamine insulin complexes I and III. Both the control and experimental groups were administered a subcutaneous dose of 6.0 mg / kg insulin equivalent. Following administration, blood glucose levels were measured using an ACCU-CHEK glucometer and the results were recorded. Figures 10-12 show the blood glucose levels of diabetic mice after administration of the NPH, Complex I, and Complex III groups. Following subcutaneous administration of the complexes, blood glucose levels in the diabetic mice returned to normal. The duration of normoglycemia in the NPH (control), Complex I, and Complex III groups reached 2.3±0.975, 11.4±4.099, and 8.8±3.174 hours, respectively. Furthermore, no hypoglycemia (<50 mg / dL) was observed in the diabetic mice receiving either complex. Compared with the NPH group (control group), the normal blood sugar maintenance time of diabetic mice in the complex I and complex III groups was prolonged, and the incidence of hypoglycemia was reduced.

[0167] Compared to existing controlled-release insulin systems, the glucose-responsive protamine-insulin complexes I and III of the present invention demonstrate significant advantages in terms of accuracy of blood glucose response, controllable insulin release, and biocompatibility. Existing controlled-release insulin systems suffer from issues such as slow response, poor biocompatibility, and unstable insulin release. Complexes I and III provided by the present invention effectively address these challenges, making them viable alternatives.

[0168] In the complexes I and III provided by the present invention, according to the study of the complex morphology in Example 5, they all have a porous and loose microstructure, and insulin and protamine are evenly distributed, which is conducive to the release of insulin. The results of the in vivo blood glucose regulation study of Example 6 showed that compared with commercially available protamine insulin (NPH), both complexes I and III can significantly prolong the normal blood glucose maintenance time of diabetic mice and reduce the incidence of hypoglycemia. And on the whole, complex I can more accurately and quickly regulate the release of insulin according to blood glucose levels, has a longer blood glucose control time, and thus more effectively maintains blood glucose stability, so that its maintenance time of normal blood glucose in diabetic mice reaches 11.4±4.099 hours. Therefore, the present invention prefers the structure of complex I.

[0169] The embodiments described in the present invention are all preferred embodiments and features. Any person skilled in the art can make some changes and variations based on the essence of the description of the present invention. These changes and variations are also considered to fall within the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. A sugar-responsive insulin carrier, characterized in that: The invention comprises protamine and phenylboronic acid compounds, wherein the cysteine ​​in the protamine is directly or indirectly connected with the phenylboronic acid compound, or the phenylboronic acid compound directly or indirectly modifies the cysteine ​​in the protamine.

2. The vector according to claim 1, characterized in that The cysteine ​​in the protamine is modified by a chemical group, and the chemical group is connected to the NHS-esterified phenylboronic acid compound through a chemical bond.

3. The carrier according to claim 2, characterized in that The chemical group is connected to protamine in a thioether bond.

4. The carrier according to claim 2, characterized in that The chemical bond between the chemical group and the NHS-esterified phenylboronic acid compound is an amide bond.

5. The carrier according to claim 2, characterized in that The chemical groups include any one or more of straight-chain or branched alkyl, hydroxy, carboxyl, amino or thiol, or any one or more of alkyl, hydroxy, carboxyl, amino or thiol substituted at any position of these groups.

6. The carrier according to claim 5, characterized in that The structural formula of the chemical group includes any one or more of Formula I to Formula IV:

7. The carrier according to claim 6, characterized in that The phenylboronic acid compounds include phenylboronic acid modified with different groups; the modified groups include one or more of alkyl, carboxyl, halogen, nitro and amide.

8. The carrier according to claim 7, characterized in that The phenylboronic acid compound is modified with a carboxyl group.

9. The carrier according to claim 8, characterized in that The phenylboronic acid compound is p-carboxylphenylboronic acid or o-carboxylphenylboronic acid which is substituted or unsubstituted with a substituent.

10. The carrier according to claim 9, characterized in that The substituent may be one or more of halogen and nitro.

11. The carrier according to claim 10, characterized in that The phenylboronic acid compound is selected from one or more of the following compounds:

12. The carrier according to claim 11, characterized in that The phenylboronic acid compound is 4-carboxy-3-fluorophenylboric acid.

13. The carrier according to claim 12, wherein N-hydroxysuccinimide reacts with 4-carboxy-3-fluorophenylboronic acid to activate the carboxyl group of 4-carboxy-3-fluorophenylboronic acid, so that it can further react with the amino group on the chemical group to form an amide bond.

14. The carrier according to claim 1, characterized in that The insulin includes any one or more of recombinant human insulin, insulin lispro, insulin aspart, insulin detemir, insulin glargine, insulin degludec, and ultra-long-acting, long-acting, short-acting, and rapid-acting insulins of icodec.

15. A method for preparing a sugar-responsive insulin carrier, characterized in that: The following steps are involved: (1) reacting the compound solution with the protamine solution to obtain solution A; (2) reacting solution A with a solution of a phenylboronic acid compound esterified with NHS; The structural formula of the compound includes any one or more of Formula I to Formula IV:

16. The preparation method according to claim 15, characterized in that: The molar ratio of the compound in step (1) to protamine is (1-50):

1.

17. The preparation method according to claim 16, characterized in that: In step (1), the concentration of the protamine solution is 0.2 to 100 mg / mL.

18. A sugar-responsive protamine-insulin complex, characterized in that: The method is prepared by mixing the carrier according to any one of claims 1 to 14 and insulin.

19. A method for preparing a sugar-responsive protamine-insulin complex, characterized in that: The sugar-responsive insulin carrier according to any one of claims 1 to 14 is mixed with insulin, and the pH is adjusted to 6.5 to 8.0 to obtain a sugar-responsive protamine insulin complex.

20. The preparation method according to claim 19, characterized in that: The mass ratio of the sugar-responsive insulin carrier solution to the insulin solution is (0.5-10):1.

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