Phenylboronic-acid-based insulin derivative, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of insulin derivatives. Disclosed are a phenylboronic-acid-based insulin derivative, and a preparation method therefor and the use thereof. The provided phenylboronic-acid-based insulin derivative has a good glucose-responsive release function, wherein the phenylboronic acid group binds to a glycosyl protein to generate a phenylboronic acid ester bond, thereby generating an insulin and glycosyl protein complex in situ in blood. When the concentration of glucose in the blood is high, the glucose specifically cleaves the phenylboronic acid ester bond, thereby releasing the insulin. The provided phenylboronic-acid-based insulin derivative achieves stable and rapid glucose-responsive insulin release, which can steadily and persistently maintain the blood glucose of a diabetic subject within the normal range with almost no occurrence of hypoglycemia.

Description

A phenylboronic acid-based insulin derivative, its preparation method and application Technical Field

[0001] This invention belongs to the field of insulin derivative technology, and relates to an insulin derivative based on phenylboronic acid, its preparation method and application. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by insufficient insulin secretion or impaired insulin utilization. Currently, the main treatment for diabetes is to control blood sugar levels within a reasonable range using scientific and rational methods, preventing acute metabolic disorders and preventing or delaying the onset and progression of complications. Controlling blood sugar levels is the core of diabetes treatment. Currently, medications for diabetes treatment include two main categories: oral medications and injectable medications.

[0003] Insulin replacement therapy is currently the main treatment for type I and type II advanced diabetes. However, exogenous regular insulin replacement is difficult to match the different insulin requirements caused by changes in blood glucose levels due to various factors (including dietary intake, physical exercise, stress, etc.). This means that even with a precise dosage design, diabetic patients will still inevitably experience symptoms of hyperglycemia or hypoglycemia.

[0004] Chinese patent application 201280011736.8 discloses a human insulin analog and its acylated product, which modifies human insulin by using groups such as PEG, successfully improving the hypoglycemic effect of the insulin analog.

[0005] Chinese patent application 202080026404.1 discloses a glucose-sensitive insulin derivative, which is obtained by modifying a side chain containing phenylboronic acid lactone or phenylboronic acid groups.

[0006] Gu Zhen, Wang Jinqiang, and others published a research paper titled "Week-long norm glycaemia in diabetic mice and minipigs via a subcutaneous dose of a glucose-responsive insulin complex" in *Nature Biomedical Engineering*. This research describes a smart insulin-controlled release material that responds to blood glucose levels by coating an insulin-glucose complex with a polymer material modified with phenylboronic acid groups. However, this method relies on the micro / nano-structured complex formed by the polymer material, which poses certain biosafety risks.

[0007] In summary, none of the existing technologies described above can provide a responsive insulin derivative based on phenylboronic acid to achieve intelligent in vivo insulin release in response to glucose concentration.

[0008] Summary of the Invention

[0009] In view of this, and considering the current lack of a phenylboronic acid-based insulin derivative to achieve glucose concentration-responsive intelligent insulin release in vivo, the present invention aims to provide a phenylboronic acid-based insulin derivative, its preparation method, and its applications. As shown in Figure 1, after this phenylboronic acid-based insulin derivative enters the bloodstream, the phenylboronic acid groups can bind to glycated proteins in situ to form borate esters. Simultaneously, when glucose concentration is too high under physiological conditions, the bond between phenylboronic acid and glycated proteins is cleaved by glucose, releasing insulin and thus lowering blood glucose levels. This effect can significantly prolong the period of stable blood glucose levels and almost never cause hypoglycemia.

[0010] To achieve the above-mentioned objectives, in one aspect, the present invention provides an insulin derivative based on phenylboronic acid, wherein the sequence structure of the insulin derivative is as follows:

[0011] Wherein, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I) and R 1 R 2 R 3 At least one group in it has the structure shown in formula (I);

[0012] The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

[0013] In the sequence structure, R 1 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin A chain. 1 Group substitution; the R 2 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin B chain. 2 Group substitution; the NHR 3 To perform R-methods on the ε-amino hydrogen of lysine (abbreviated as K) at position 29 of the insulin B chain 3 Group substitution.

[0014] Unless otherwise stated, the term "alkyl" as used herein includes branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms, including all isomers. Common abbreviations for alkyl groups include methyl (e.g., "Me" or CH3), ethyl (e.g., "Et" or CH2CH3), propyl (e.g., "Pr" or CH2CH2CH3), butyl (e.g., "Bu" or CH2CH2CH2CH3," etc.). For example, "C..." 1-4 Alkyl (or "C1-C4 alkyl") refers to a straight-chain or branched alkyl group having a specific number of carbon atoms, including all isomers. 1-4 Alkyl groups include n-, iso-, secondary and tert-butyl, n- and isopropyl, ethyl and methyl. The term "C" is used in this context. 1-20 Alkyl groups have similar meanings. Other common abbreviations for alkyl groups include: isopropyl (i-Pr), n-propyl (n-Pr), n-butyl (n-Bu), and tert-butyl (t-Bu), etc.

[0015] The term "alkoxy" refers to straight-chain and branched alkyl groups with a specified number of carbon atoms connected by oxygen bridges.

[0016] The term "halogen" (or "halogenated") refers to fluorine, chlorine, bromine, and iodine (or fluorinated (F), chlorinated (Cl), brominated (Br), and iodinated (I)).

[0017] Unless otherwise stated, all ranges listed in this article are inclusive. For example, "n is an integer between 0 and 2" means that n can be 0, 1, or 2.

[0018] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0019] Preferably, the R 4 It is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, or nitrile.

[0020] More preferably, the R 4 Selected from halogens, C1-C 20 Alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 alkynyl group, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 Alkyl or nitrile groups.

[0021] More preferably, the structure shown in formula (I) is selected from one of the following structures:

[0022] More preferably, and as an example of the present invention, R 1 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 For H, R 3 It is a 3-fluoro-1-phenylboronic acid-4-acyl group.

[0023] On the other hand, the present invention provides a method for preparing the above-mentioned insulin derivative, comprising the following steps: mixing a phenylboronic acid compound with N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, N,N-diisopropylethylamine and a solvent, reacting, adding an insulin solution, and post-processing to obtain the insulin derivative; the structure of the phenylboronic acid compound is as follows:

[0024] The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

[0025] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0026] Preferably, the R 4 It is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, or nitrile.

[0027] Preferably, the R 4 Selected from halogens, C1-C 20 Alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 alkynyl group, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 Alkyl or nitrile groups.

[0028] Preferably, the structure represented by formula (II) is selected from one of the following structures:

[0029] Preferably, and as an example of the present invention, the phenylboronic acid compound is:

[0030] Preferably, the solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and acetone; the molar ratio of the phenylboronic acid compound, the N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; the reaction temperature is 20-30°C; the insulin concentration in the insulin solution is 0.2-100 mg / mL; the solvent of the insulin solution is selected from phosphate buffer or dimethyl sulfoxide; and the post-treatment is selected from dialysis or precipitation.

[0031] More preferably, the post-treatment is precipitation, specifically precipitation using a precipitant, wherein the precipitant is a mixture of precipitant A and precipitant B, wherein precipitant A is selected from methyl tert-butyl ether and / or diethyl ether; and precipitant B is selected from at least one of isopropyl acetate, acetone and tetrahydrofuran.

[0032] More preferably, the post-treatment is dialysis, which is performed in deionized water; the molecular weight cutoff of the dialysis bag is 1000-3500, and the dialysis time is 12-24 hours; after dialysis, the white flocculent precipitate formed is the insulin derivative.

[0033] In another aspect, the present invention provides the application of the above-mentioned insulin derivative or the insulin derivative prepared by the above-mentioned preparation method in the preparation of drugs for treating diabetes.

[0034] Preferably, the diabetes is type I diabetes and / or type II diabetes.

[0035] In another aspect, the present invention provides a drug in which the active ingredient includes the above-mentioned insulin derivative or the insulin derivative prepared by the above-mentioned preparation method.

[0036] The pharmaceutical products suitable for injection in this invention include sterile aqueous solutions or dispersions. Furthermore, the pharmaceutical product can be in the form of a sterile powder for the ad hoc preparation of such sterile injection solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective liquid so that the injectable pharmaceutical ingredient must remain stable under the production and storage conditions; therefore, it is best to preserve it to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, such as containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0037] The medicaments of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, or the like. Furthermore, the compositions can be in suitable forms for transdermal drug delivery devices. These formulations can be prepared using the medicaments of the present invention through conventional processing methods. For example, a cream or ointment with a desired consistency can be prepared by mixing a hydrophilic material and water, and about 5 wt% to about 10 wt% of the compound.

[0038] The medicament of the present invention can be in a form suitable for rectal administration, wherein the carrier is solid. It is preferable to formulate the mixture into a single-dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be made by first forming a mixture containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0039] Preferably, the drug is a subcutaneous injection drug.

[0040] Preferably, the drug is prepared by resuspending the above-mentioned insulin derivative in sterile phosphate buffer or physiological saline.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The phenylboronic acid-based insulin derivative provided by this invention exhibits excellent glucose-responsive insulin release performance, enabling rapid release at high blood glucose concentrations and sustained, slow release at normal blood glucose concentrations. This invention utilizes the in-situ binding of phenylboronic acid to glycated proteins in vivo, introducing phenylboronic ester bonds that can be cleaved by glucose, thus achieving stable and rapid glucose-responsive insulin release. Under physiological conditions, glucose competitively and reversibly binds to 1,3-diol and phenylboronic acid to achieve a glucose response, prolonging the treatment duration. In diabetic mice, it can rapidly regulate blood glucose, with a stable and sustained effect exceeding 200 hours, and almost no hypoglycemia occurs. Attached Figure Description

[0043] Figure 1 is a schematic diagram of the insulin derivative based on phenylboronic acid and its mechanism of action according to the present invention.

[0044] Figure 2 is a high-performance liquid chromatogram of the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0045] Figure 3 is a Q-TOF mass spectrum of the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0046] Figure 4 is a circular dichroism chromatogram of the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0047] Figure 5 shows scanning electron microscope images of the phenylboronic acid-based insulin derivative synthesized in Example 1 at different magnifications, where the scale bar in (a) is 25 μm and the scale bar in (b) is 100 μm.

[0048] Figure 6 is a MALDI-TOF mass spectrum of the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0049] Figure 7 is the solid-state NMR boron spectrum of the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0050] Figure 8 is a line graph showing the blood glucose levels in type 1 diabetic mice treated with different doses of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0051] Figure 9 is a line graph showing the blood glucose levels in type 1 diabetic mice treated with single-needle and multiple-needle continuous injections of different doses of a commercial long-acting insulin analog (insulin glargine). In this graph, (a) represents a single-needle injection and (b) represents multiple-needle injections.

[0052] Figure 10 shows the hypoglycemia and mortality in type 1 diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1 and the commercial long-acting insulin analog (insulin glargine).

[0053] Wherein, (a) represents the proportion of mice that did not develop hypoglycemia at 4 mg / kg, 8 mg / kg, and 12 mg / kg with the insulin derivative provided in Example 1;

[0054] (b) indicates the survival rate of mice with the insulin derivative provided in Example 1 at 4 mg / kg, 8 mg / kg, and 12 mg / kg;

[0055] (c) represents the proportion of mice that did not experience hypoglycemia after single-injection treatment with insulin glargine at doses of 40 U / kg and 400 U / kg;

[0056] (d) represents the survival rate of mice treated with a single injection of insulin glargine at doses of 40 U / kg and 400 U / kg;

[0057] (e) represents the proportion of mice that did not experience hypoglycemia after multiple injections of insulin glargine;

[0058] (f) represents the survival rate of mice treated with glargine insulin via multiple injections.

[0059] Figure 11 shows the intraperitoneal glucose tolerance test of type 1 diabetic mice treated with the insulin derivative based on phenylboronic acid synthesized in Example 1, where (a) is the test result 1 day after treatment and (b) is the test result 5 days after treatment.

[0060] Figure 12 is a validation diagram of insulin release in vivo in response to glucose in type 1 diabetic mice treated with the insulin derivative based on phenylboronic acid synthesized in Example 1. In the figure, (a) is the result of the test 1 day after treatment and (b) is the result of the test 5 days after treatment; BGL represents blood glucose level and PIL represents plasma insulin level.

[0061] Figure 13 is a pharmacokinetic curve of type 1 diabetic mice treated with the insulin derivative based on phenylboronic acid synthesized in Example 1; where (a) is intravenous injection and (b) is subcutaneous injection.

[0062] Figure 14 is a biosafety evaluation diagram of type 1 diabetic mice treated with the insulin derivative based on phenylboronic acid synthesized in Example 1.

[0063] Wherein, (a) represents the content of each component in serum, ALT is alanine aminotransferase, AST is aspartate aminotransferase, BUN is blood urea nitrogen, CR is creatinine, ALP is alkaline phosphatase, and ALB is serum albumin.

[0064] (b) indicates the number of cells in the blood: RBC for red blood cells, PLT for platelets, WBC for white blood cells, NEUT for neutrophils, LYMPH for lymphocytes, MONO for monocytes, and EO for eosinophils.

[0065] Figure 15 shows the blood glucose levels of type I diabetic pigs treated with the insulin derivative based on phenylboronic acid synthesized in Example 1; where (a) is the blood glucose level of pig I before administration, (b) is the blood glucose level of pig I after administration, (c) is the blood glucose level of pig II before administration, (d) is the blood glucose level of pig II after administration, (e) is the blood glucose level of pig III before administration, and (f) is the blood glucose level of pig III after administration.

[0066] Figure 16 is a validation diagram of in vivo glucose response and insulin release in type 1 diabetic pigs treated with the phenylboronic acid-based insulin derivative synthesized in Example 1; where (a) shows the blood glucose and plasma insulin levels of pig I 24 hours after treatment, (b) shows the blood glucose and plasma insulin levels of pig II 24 hours after treatment, and (c) shows the blood glucose and plasma insulin levels of pig III 24 hours after treatment. BGL represents blood glucose level, and PIL represents plasma insulin level. Detailed Implementation

[0067] Terminology and Declarations of this Invention:

[0068] 1. As used in this text, the articles “a,” “a kind,” and “the” include plural objects unless otherwise explicitly specified as a single object.

[0069] 2. As used herein, numerical range: Unless otherwise expressly indicated, all ranges or ratios disclosed herein shall be understood to include any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between a minimum value of 1 and a maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0070] 3. As used herein, the terms “comprising,” “including,” “having,” “possessing,” “may,” “containing,” and their variations are open-ended conjunctions or terms that do not exclude the possibility of other components or structures.

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0072] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0073] The manufacturers or models of the reagents or instruments used in the following examples are shown in Table 1.

[0074] Table 1

[0075] Example 1

[0076] Preparation of insulin derivatives based on phenylboronic acid.

[0077] Take 70 mg of 4-carboxy-3-fluorophenylboronic acid and 90 mg of 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroboronic acid ester, place them in a 5 mL sample vial, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours to obtain the first solution. Take 2 g of recombinant human insulin and place it in a 60 mL sample vial, add 20 mL of anhydrous dimethyl sulfoxide, stir until the solution is clear to obtain the recombinant human insulin solution. Add the first solution dropwise to the recombinant human insulin solution, react for 1 hour, and after the reaction is complete, add the solution dropwise to a precipitant of isopropyl acetate:methyl tert-butyl ether = 4:1, stir for one hour, filter to remove the precipitate, and dry to obtain the crude product.

[0078] The crude product was purified by high performance liquid chromatography (HPLC) to obtain an insulin derivative based on phenylboronic acid. The HPLC results are shown in Figure 2.

[0079] The structures of the obtained insulin derivatives based on phenylboronic acid were characterized by Q-TOF mass spectrometry, circular dichroism spectroscopy, scanning electron microscopy, MALDI-TOF mass spectrometry, and solid-state nuclear magnetic resonance boron spectroscopy. The results are shown in Figures 3, 4, 5, 6, and 7, respectively.

[0080] The experimental characterization results above indicate that the structure of the phenylboronic acid-based insulin derivative is as follows:

[0081] Among them, R 1 and R 3 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 For H.

[0082] The R 1 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin A chain. 1 Group substitution; the R 2 HN represents the terminal amino hydrogen atom at the N-terminus of the insulin B chain. 2 Group substitution; the NHR 3 To perform R-methods on the ε-amino hydrogen of lysine (abbreviated as K) at position 29 of the insulin B chain 3 Group substitution.

[0083] Example of effect 1

[0084] Rodent pharmacology studies

[0085] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected and fasted overnight. Then, under light-protected conditions, they were intraperitoneally injected with streptozocin (STZ) solution at a dose of 120 mg / kg. Blood glucose levels were measured one and two weeks after injection. A blood glucose level >300 mg / dL indicated successful model establishment. Otherwise, STZ solution was injected intraperitoneally again at a dose of 50 mg / kg two weeks later, and blood glucose levels were measured again one and two weeks later.

[0086] 2. In vivo glucose-lowering study in type 1 diabetic mice

[0087] A type 1 diabetic mouse model was established using the method described above. Type 1 diabetic mice with blood glucose levels of 400-500 mg / dL were selected for evaluation of the treatment effect. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose levels of the diabetic mice remained below 200 mg / dL for more than 214 hours, significantly longer than with glargine insulin (see Figure 9 for details). Compared with commercially available long-acting insulin (glargine insulin), the insulin analog prepared in Example 1 showed a faster blood glucose reduction effect and more stable blood glucose control, with no diabetic mice exhibiting hypoglycemia (below 50 mg / dL) (see Figure 8).

[0088] Meanwhile, the dose-response relationship showed that the insulin derivative prepared in Example 1 did not cause hypoglycemia in the range of 4-12 mg / kg, while glargine insulin at 400 U / kg (equivalent to approximately 13.8 mg / kg) caused hypoglycemia in 40% of the mice. Furthermore, when glargine insulin was used to control blood glucose for one week (i.e., injected once daily at 40 U / kg), all mice developed hypoglycemia, and 40% of the mice died due to severe hypoglycemia (see Figure 10).

[0089] 3. Intraperitoneal glucose tolerance test in type I diabetic mice

[0090] A type 1 diabetic mouse model was established using the method described above. Type 1 diabetic mice with blood glucose levels of 400-500 mg / dL (n=5) were selected for evaluation of the blood glucose control effect. On days 1 and 5 after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), mice were intraperitoneally injected with 1.5 g / kg glucose. Blood glucose levels were measured and recorded every 15 minutes for 2 consecutive hours.

[0091] Meanwhile, a group of healthy C57BL / 6J mice were selected and administered 1.5 g / kg of glucose intraperitoneally. The blood glucose levels of the mice were measured and recorded every 15 minutes for 2 hours as a control.

[0092] The results showed that glucose stimulation on day 1 after drug administration maintained blood glucose levels in mice within the normal range (50-200 mg / dL), and the glucose control effect (evaluated by the area under the blood glucose-time curve) was superior to that in normal mice. On day 5 after drug administration, glucose stimulation resulted in a significant peak in blood glucose levels at 15 minutes, which returned to normal (below 200 mg / dL) after 1 hour and remained stable for 2 hours (see Figure 11), again demonstrating superior glucose control compared to normal mice.

[0093] 4. Verification of insulin release in response to glucose in type 1 diabetic mice

[0094] A type 1 diabetic mouse model was established using the method described above. Type 1 diabetic mice with blood glucose levels of 400-500 mg / dL (n=5) were selected for in vivo glucose response insulin release evaluation. On days 1 and 5 after subcutaneous injection of the insulin analog prepared in Example 1 (12 mg / kg), mice were intraperitoneally injected with 3.0 g / kg glucose. Blood glucose levels were measured and recorded every 15 minutes for 2 consecutive hours. Blood samples (20 μL from the orbital cavity, plasma collected after centrifugation) were collected at 0, 15, 30, 60, and 120 minutes.

[0095] The insulin content in plasma was detected using a recombinant human insulin ELISA kit.

[0096] The results showed that after glucose stimulation, mice exhibited a significant peak in blood glucose (above 300 mg / kg), and plasma insulin concentration also changed synchronously with blood glucose levels, peaking at 30 minutes. On day 1 after administration, the peak insulin level was 4-5 times higher than the initial value; on day 5, the peak insulin level was 2-3 times higher. Simultaneously, as blood glucose gradually decreased to its initial value, plasma insulin concentration also decreased back to its initial level, verifying glucose-responsive insulin release (see Figure 12).

[0097] 5. Pharmacokinetics of type 1 diabetic mice treated with the insulin derivative prepared in Example 1

[0098] A type 1 diabetic mouse model was established using the method described above. Type 1 diabetic mice with blood glucose levels of 400-500 mg / dL (n=5) were selected for pharmacokinetic evaluation. After subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), blood samples were collected at predetermined time points (20 μL of blood was collected from the orbital cavity; plasma was collected after centrifugation). Monitoring was conducted continuously for 17 days (see Figure 13).

[0099] Meanwhile, a group of healthy C57BL / 6J mice were selected and intravenously administered 1 nmol / kg of the insulin derivative prepared in Example 1. Blood samples were collected at predetermined time points (20 μL of blood was drawn from the orbital cavity, and plasma was collected after centrifugation). The mice were monitored continuously for 21 days (see Figure 13).

[0100] Plasma insulin concentration-time curve data were analyzed using a non-compartmental model via Winnonlin. In a diabetic mouse model, the insulin derivative prepared in Example 1 had a half-life of 79.0 ± 17.7 hours after subcutaneous injection; while in healthy mice, the insulin derivative prepared in Example 1 had a half-life of 93.7 ± 9.5 hours after intravenous injection, which is 1000 times that of normal human insulin (half-life of about 5 minutes).

[0101] 6. Biosafety evaluation of type 1 diabetic mice treated with the insulin derivative prepared in Example 1

[0102] A type 1 diabetic mouse model was established using the method described above, and 5 type 1 diabetic mice with blood glucose levels of 400-500 mg / dL were selected for biosafety evaluation.

[0103] Blood samples were collected from mice prior to drug administration. Blood samples were collected on day 7 after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg). Blood-related biochemical indicators and routine blood tests were performed.

[0104] Meanwhile, a group of healthy C57BL / 6J mice were selected, and blood samples were collected. Blood-related biochemical indicators and routine blood tests were performed as controls.

[0105] The results showed that after subcutaneous injection of the insulin derivative prepared in Example 1, all indicators of the mice were similar to those of healthy mice (see Figure 14).

[0106] Example 2

[0107] Drug efficacy studies in non-rodent animals

[0108] 1. Establishment of a type I diabetic pig model

[0109] Six-month-old Bama pigs were selected, fasted overnight, and then injected intravenously with STZ solution at a dose of 150 mg / kg. Blood glucose levels were measured one and two weeks after the injection. If the blood glucose level was >300 mg / dL, the model was considered to have been successfully established.

[0110] 2. In vivo glucose-lowering study in type I diabetic piglets

[0111] A type 1 diabetic pig model was established using the method described above, and the treatment effect was evaluated in type 1 diabetic pigs. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose levels of the diabetic pigs were measured using a continuous glucose monitoring system (CGMS).

[0112] The results showed that subcutaneous injection of the insulin derivative prepared in Example 1 achieved therapeutic effects for 8, 5, and 6 days in piglets I, II, and III (see Figure 15).

[0113] 3. Verification of insulin release in response to glucose in type I diabetic piglets

[0114] A type 1 diabetic pig model was established using the method described above. Type 1 diabetic piglets were selected for in vivo glucose response insulin release evaluation. On day 1 after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), piglets were intravenously injected with 10% glucose (at a rate of 1 L / h) until their blood glucose level exceeded 300 mg / dL, at which point glucose injection was stopped. Blood glucose levels were measured and blood samples were taken from the piglets every 15 minutes, and monitoring was continued for 2 hours.

[0115] The insulin content in plasma was detected using a recombinant human insulin ELISA kit.

[0116] The results showed that after glucose stimulation, the piglets exhibited a significant peak in blood glucose (above 300 mg / kg), and the plasma insulin concentration also changed synchronously with the blood glucose level, peaking at the same point as blood glucose reached its peak. In both piglets, the peak insulin level was 2-3 times higher than the initial value. Simultaneously, as blood glucose gradually decreased to its initial value, the plasma insulin concentration also decreased back to its initial level, verifying the glucose-responsive insulin release (see Figure 16).

[0117] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An insulin derivative based on phenylboronic acid, characterized in that, The sequence structure of the insulin derivative is as follows: Wherein, the R 1 R 2 R 3 Each is independently selected from the structure shown in H or formula (I), and R 1 R 2 R 3 At least one group in it has the structure shown in formula (I); The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

2. The insulin derivative according to claim 1, characterized in that, The halogen is selected from fluorine, chlorine or bromine.

3. The insulin analogue according to claim 1, characterized in that, The R 4 It is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, or nitrile.

4. The insulin derivative according to claim 3, characterized in that, The R 4 Selected from halogens, C1-C 20 Alkyl, C1-C 20 cycloalkyl, C1-C 20 alkenyl, C1-C 20 Alkyne group, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 Alkyl or nitrile groups.

5. The insulin derivative according to claim 4, characterized in that, The structure shown in formula (I) is selected from one of the following structures:

6. The insulin derivative according to claim 5, characterized in that, R 1 It is 3-fluoro-1-phenylboronic acid-4-acyl, R 2 For H, R 3 It is a 3-fluoro-1-phenylboronic acid-4-acyl group.

7. A method for preparing the insulin derivative according to any one of claims 1-6, characterized in that, Includes the following steps: A phenylboronic acid compound was mixed with N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, N,N-diisopropylethylamine, and a solvent. The mixture was reacted, followed by the addition of an insulin solution and post-treatment to obtain an insulin derivative. The structure of the phenylboronic acid compound is as follows: The R 4 It is selected from halogen, hydrocarbon, nitro, amide, ester, hydroxyl, or nitrile groups.

8. The preparation method according to claim 7, characterized in that, The solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, and acetone; The molar ratio of the phenylboronic acid compound, the N,N,N′,N′-tetramethyl-O-(N-succinimide)tetrafluoroborate, and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; The reaction temperature is 20-30℃; The concentration of insulin in the insulin solution is 0.2-100 mg / mL; The solvent for the insulin solution is selected from phosphate buffer or dimethyl sulfoxide; The post-treatment is selected from dialysis or precipitation.

9. The preparation method according to claim 8, characterized in that, The post-treatment is precipitation, specifically precipitation using a precipitating agent. The precipitating agent is a mixture of precipitating agent A and precipitating agent B. Precipitating agent A is selected from methyl tert-butyl ether and / or diethyl ether; precipitating agent B is selected from at least one of isopropyl acetate, acetone, and tetrahydrofuran.

10. The preparation method according to claim 8, characterized in that, The post-treatment is dialysis, which is performed in deionized water; the molecular weight cutoff of the dialysis bag is 1000-3500, and the dialysis time is 12-24 hours; after dialysis, a white flocculent precipitate is formed, which is the insulin derivative.

11. The use of the insulin derivative according to any one of claims 1-6 or the insulin derivative prepared by the preparation method according to any one of claims 7-10 in the preparation of a drug for treating diabetes.

12. The application according to claim 11, characterized in that, The diabetes refers to type 1 diabetes and / or type 2 diabetes.

13. A drug, characterized in that, The active ingredient includes the insulin derivative as described in any one of claims 1-6 or the insulin derivative prepared by the preparation method described in any one of claims 7-10.

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