Phenylboronic acid-based insulin derivative as well as preparation method and application thereof

Abstract

The invention provides an insulin derivative based on phenylboronic acid and a preparation method and application thereof, and belongs to the technical field of insulin derivatives. The insulin derivative based on boric acid provided by the invention has a good glucose response release function: a phenylboronic acid group is combined with glycosyl protein to generate a phenylboronic acid ester bond, so that an insulin-glycosyl protein complex is generated in situ in blood. When the concentration of glucose in blood is high, the phenylboronic acid ester bond is specifically cut off by glucose, so that insulin is released. The phenylboronic acid-based insulin derivative provided by the invention realizes stable and rapid glucose-responsive insulin release, can stably and durably maintain the blood glucose of a diabetic subject in a normal range, and hardly causes hypoglycemia.

Description

[0001] The present invention claims the priority of the international patent application filed with the International Patent Office on August 30, 2024, with application number PCT / CN2024 / 115879 and invention name “A phenylboronic acid-based insulin derivative and its preparation method and application”, the entire contents of which are incorporated by reference in the application. Technical Field

[0002] The invention belongs to the technical field of insulin derivatives and relates to an insulin derivative based on phenylboronic acid and a preparation method and application thereof. Background Art

[0003] Diabetes is a chronic disease characterized by high blood sugar, caused by insufficient secretion or utilization of insulin. At present, the main treatment for diabetes is to control blood sugar levels within a reasonable range through scientific and reasonable methods, prevent the occurrence of acute metabolic disorders, and prevent or delay the occurrence and development of complications. Controlling blood sugar levels is the core of diabetes treatment. Currently, diabetes treatment drugs include two categories: oral drugs and injectable drugs.

[0004] Insulin replacement therapy is currently the main treatment for late-stage type I and type II diabetes, but exogenous ordinary insulin replacement is difficult to match the different insulin demands caused by changes in human blood sugar due to various factors (including dietary intake, physical exercise, stress, etc.). This will result in diabetic patients inevitably experiencing symptoms of hyperglycemia or hypoglycemia even under precise dosage design plans.

[0005] Chinese patent application 201280011736.8 discloses a human insulin analog and its acylated product, which successfully improves the hypoglycemic effect of the insulin analog by modifying human insulin with groups such as PEG.

[0006] Chinese patent application 202080026404.1 discloses a glucose-sensitive insulin derivative, which is obtained by modifying the side chain with a phenylboronic acid lactone or phenylboronic acid group.

[0007] Gu Zhen, Wang Jinqiang and others published a research paper titled "Week-longnorm glycaemia in diabetic mice and minipigs via a subcutaneous dose of aglucose-responsive insulin complex" in Nature Biomedical Engineering. By wrapping the insulin-glucose complex with a layer of polymer material modified with phenylboronic acid groups, a smart insulin controlled-release material responsive to blood sugar levels was formed. However, this method relies on a micro-nanostructure complex formed by polymer materials, which has certain biosafety risks.

[0008] In summary, none of the above-mentioned prior arts can provide a responsive insulin derivative based on phenylboronic acid to achieve intelligent release of insulin in vivo in response to glucose concentration. Summary of the invention

[0009] In view of this, the prior art has not yet provided a phenylboronic acid-based insulin derivative to achieve glucose concentration-responsive in vivo insulin intelligent release. The purpose of the present invention is to provide a phenylboronic acid-based insulin derivative and a preparation method and application thereof. Figure 1 As shown, after the phenylboronic acid-based insulin derivative enters the blood, the phenylboronic acid group can form a boronate ester with the glycated protein in situ and bind to each other; at the same time, when the glucose concentration is too high under physiological conditions, the bond between phenylboronic acid and glycated protein is replaced by glucose and cut off, releasing insulin, thereby playing a role in lowering blood sugar. This effect can significantly prolong the time of blood sugar stabilization and almost no hypoglycemia occurs.

[0010] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides an insulin derivative based on phenylboronic acid, wherein the sequence structure of the insulin derivative is:

[0011]

[0012] Among them, the R 1 , R 2 , R 3 are independently selected from H or the structure represented by formula (I) and R 1 , R 2 , R 3 At least one group is a structure represented by formula (I);

[0013]

[0014] The R 4 Selected from halogen, hydrocarbon, nitro, amide, ester, hydrocarbonoxy or nitrile.

[0015] In the sequence structure, the R 1 HN is the R-terminal amino hydrogen of the N-terminal of insulin A chain. 1 Group substitution; said R 2 HN is the R-terminal amino hydrogen of the N-terminal of insulin B chain. 2 Group substitution; the NHR 3 To perform R- 3 Group substitution.

[0016] Unless otherwise specified, the term "alkyl" as used herein includes branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms, including all isomers. Common abbreviations for alkyl groups, such as methyl, can be represented by "Me" or CH3, ethyl, can be represented by "Et" or CH2CH3, propyl, can be represented by "Pr" or CH2CH2CH3, butyl, can be represented by "Bu" or CH2CH2CH2CH3, etc. For example, "C 1-4 "C1-C4 alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms, including all isomers. 1-4 Alkyl includes n-, iso-, sec- and tert-butyl, n- and iso-propyl, ethyl and methyl. 1-20 "Alkyl" and the like have similar meanings. In addition, common abbreviations of alkyl include: isopropyl can be represented by "i-Pr", n-propyl can be represented by "n-Pr", n-butyl can be represented by "n-Bu", tert-butyl can be represented by "t-Bu", etc.

[0017] The term "alkoxy" refers to straight and branched chain alkyl groups having the indicated number of carbon atoms attached through an oxygen bridge.

[0018] The term "halogen" (or "halo") refers to fluorine, chlorine, bromine, and iodine (alternatively referred to as fluoro (F), chloro (Cl), bromo (Br), and iodo (I)).

[0019] Unless explicitly stated, all ranges listed herein are inclusive. For example, "the value of n is an integer between 0 and 2" means that n can be 0, 1 or 2.

[0020] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0021] Preferably, the R 4 is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy or nitrile.

[0022] More preferably, the R 4 Selected from halogen, C1-C 20Alkyl, C1-C 20 Cycloalkyl, C1-C 20 Alkenyl, C1-C 20 Alkynyl, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 an alkoxy group or a nitrile group.

[0023] More preferably, the structure represented by formula (I) is selected from one of the following structures:

[0024]

[0025]

[0026] Further preferably, and as an example of the present invention, R 1 is 3-fluoro-1-phenylboronic acid-4-yl, R 2 H, R 3 It is 3-fluoro-1-phenylboronic acid-4-acyl.

[0027] 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-succinimidyl) tetrafluoroborate, N,N-diisopropylethylamine and a solvent, reacting, adding an insulin solution, and post-treating to obtain an insulin derivative; the structure of the phenylboronic acid compound is

[0028]

[0029] The R 4 Selected from halogen, hydrocarbon, nitro, amide, ester, hydrocarbonoxy or nitrile.

[0030] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0031] Preferably, the R 4 is selected from halogen, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, nitro, amide, ester, alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy or nitrile.

[0032] Preferably, the R 4 Selected from halogen, C1-C 20 Alkyl, C1-C 20 Cycloalkyl, C1-C 20 Alkenyl, C1-C 20 Alkynyl, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 an alkoxy group or a nitrile group.

[0033] Preferably, the structure represented by formula (II) is selected from one of the following structures:

[0034]

[0035]

[0036] Preferably, and as an example of the present invention, the phenylboronic acid compound is:

[0037]

[0038] 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-succinimidyl)tetrafluoroborate and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; the reaction temperature is 20-30°C; the concentration of insulin 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.

[0039] More preferably, the post-treatment is precipitation, and the precipitation is specifically performed using a precipitant, the precipitant is a mixture of precipitant A and precipitant B, the precipitant A is selected from methyl tert-butyl ether and / or diethyl ether; the precipitant B is selected from at least one of isopropyl acetate, acetone and tetrahydrofuran.

[0040] More preferably, the post-treatment is dialysis, and the dialysis is performed in deionized water; the molecular weight cutoff of the dialysis bag is 1000-3500, and the dialysis time is 12-24h; after the dialysis, a white flocculent precipitate is formed, which is the insulin derivative.

[0041] In another aspect, the present invention provides the use of the above insulin derivative or the insulin derivative prepared by the above preparation method in the preparation of a drug for treating diabetes.

[0042] Preferably, the diabetes is type I diabetes and / or type II diabetes.

[0043] In yet another aspect, the present invention provides a medicine, the active ingredient of which includes the above-mentioned insulin derivative or the insulin derivative prepared by the above-mentioned preparation method.

[0044] The drugs suitable for injection use in the present invention include sterile aqueous solutions or dispersions. In addition, the drug can be in the form of sterile powders for the extemporaneous preparation of such sterile injections or dispersions. In all cases, the final injection form must be sterile and must be an effective liquid so that the injectable drug component must remain stable under production and storage conditions; therefore, it is best to preserve it to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof.

[0045] The medicine of the present invention can be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, powder or the like. In addition, the composition can be in a suitable form for transdermal administration devices. These prescriptions can be prepared using the medicine of the present invention by 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 5wt% to about 10wt% of the compound.

[0046] The medicine of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture is made into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a composition containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0047] Preferably, the drug is a subcutaneous injection drug.

[0048] Preferably, the preparation method of the drug is to resuspend the above insulin derivative using sterile phosphate buffer or physiological saline.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The phenylboronic acid-based insulin derivative provided by the present invention has good glucose-responsive release performance, and can release insulin quickly at high blood sugar concentrations and continuously and slowly at normal blood sugar concentrations. The present invention utilizes phenylboronic acid to bind to glycosylated proteins in the body in situ, introduces a phenylboronic acid ester bond that can be cut by glucose, and achieves stable and rapid glucose-responsive insulin release. Under physiological conditions, the competitive reversible binding of glucose with 1,3-diol and phenylboronic acid achieves sugar response and prolongs the treatment time. In diabetic mice, blood sugar can be regulated quickly, the effect is stable and lasting for more than 200 hours, and hypoglycemia hardly occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a schematic diagram of the insulin derivative based on phenylboronic acid of the present invention and its mechanism of action.

[0052] Figure 2 It is the HPLC chromatogram of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0053] Figure 3 It is the Q-TOF mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0054] Figure 4 is the circular dichroism spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0055] Figure 5 These are scanning electron micrographs of different magnifications of the phenylboronic acid-based insulin derivative synthesized in Example 1, wherein the scale bar in (a) is 25 μm, and the scale bar in (b) is 100 μm.

[0056] Figure 6 It is the MALDI-TOF mass spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0057] Figure 7 This is the solid-state NMR boron spectrum of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0058] Figure 8 It is a line graph of blood glucose levels in type I diabetic mice treated with different doses of the phenylboronic acid-based insulin derivative synthesized in Example 1.

[0059] Fig. 9 The blood glucose level line graph of type I diabetic mice treated with single injection and multiple continuous injections of different doses of commercial long-acting insulin analogs (insulin glargine), where (a) represents a single injection and (b) represents multiple injections.

[0060] Fig.10 This is a diagram showing hypoglycemia and mortality in type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1 and a commercial long-acting insulin analog (insulin glargine);

[0061] Wherein, (a) represents the proportion of mice that did not experience hypoglycemia at 4 mg / kg, 8 mg / kg, and 12 mg / kg of the insulin derivative provided in Example 1;

[0062] (b) represents the survival rate of mice at 4 mg / kg, 8 mg / kg and 12 mg / kg of the insulin derivative provided in Example 1;

[0063] (c) represents the proportion of mice that did not develop hypoglycemia after single injection treatment with glargine insulin at a dose of 40 U / kg and 400 U / kg;

[0064] (d) represents the survival rate of mice treated with single injection of glargine insulin at a dose of 40 U / kg and 400 U / kg;

[0065] (e) represents the proportion of mice that did not develop hypoglycemia after multiple injections of glargine insulin;

[0066] (f) shows the survival rate of mice treated with multiple injections of insulin glargine.

[0067] Fig.11 The graphs are intraperitoneal glucose tolerance test of type 1 diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1, wherein (a) is the test result 1 day after treatment, and (b) is the test result 5 days after treatment.

[0068] Fig.12 This is a verification graph of in vivo sugar-responsive insulin release in type 1 diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1, wherein (a) is the test result 1 day after treatment, and (b) is the test result 5 days after treatment; BGL represents blood glucose level, and PIL represents plasma insulin level.

[0069] Fig.13 The pharmacokinetic curve of the insulin derivative based on phenylboronic acid synthesized in Example 1 in mice with type I diabetes treated; wherein (a) is intravenous injection, and (b) is subcutaneous injection.

[0070] Fig.14 This is a diagram of the biosafety evaluation of type I diabetic mice treated with the phenylboronic acid-based insulin derivative synthesized in Example 1;

[0071] 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;

[0072] (b) represents the cell content in the blood, RBC is red blood cells, PLT is platelets, WBC is white blood cells, NEUT is neutrophils, LYMPH is lymphocytes, MONO is monocytes, and EO is eosinophils.

[0073] Fig.15 This is a graph of blood glucose levels in piglets with type I diabetes treated with the phenylboronic acid-based insulin derivative synthesized in Example 1; wherein, (a) is the blood glucose level of piglet I before administration, (b) is the blood glucose level of piglet I after administration, (c) is the blood glucose level of piglet II before administration, (d) is the blood glucose level of piglet II after administration, (e) is the blood glucose level of piglet III before administration, and (f) is the blood glucose level of piglet III after administration.

[0074] Fig.16The figure is a verification diagram of the in vivo sugar response insulin release of type I diabetic piglets treated with the phenylboronic acid-based insulin derivative synthesized in Example 1; wherein (a) is the blood glucose level and plasma insulin level of piglet I 24 hours after treatment, (b) is the blood glucose level and plasma insulin level of piglet II 24 hours after treatment, and (c) is the blood glucose level and plasma insulin level of piglet III 24 hours after treatment. Wherein, BGL represents blood glucose level, and PIL represents plasma insulin level. DETAILED DESCRIPTION

[0075] Terms and Claims of the Present Invention:

[0076] 1. As used herein, the articles “a”, “an” and “the” include plural referents unless expressly limited to one or more referents otherwise.

[0077] 2. As used herein, numerical range: Unless otherwise expressly indicated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0078] 3. As used herein, the terms "comprises," "including," "have," "have," "may," "contain" and variations thereof are open-ended conjunctions or terms that do not exclude the possibility of additional compositions or structures.

[0079] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0080] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0081] The manufacturers or models of the reagents or instruments used in the following examples are shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085] Example 1

[0086] Preparation of insulin derivatives based on phenylboronic acid.

[0087] Take 70 mg of 4-carboxyl-3-fluorophenylboronic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, 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 bottle, add 20 mL of anhydrous dimethyl sulfoxide, stir until the solution is clear, and obtain a recombinant human insulin solution. Add the first solution dropwise to the recombinant human insulin solution and react for 1 hour. After the reaction is completed, add the solution dropwise to a precipitant of isopropyl acetate: methyl tert-butyl ether = 4:1 and stir for one hour, filter out the precipitate, and dry to obtain a crude product.

[0088] The crude product was purified by high performance liquid chromatography (HPLC) to obtain an insulin derivative based on phenylboronic acid. The HPLC results are as follows: Figure 2 shown.

[0089] The structures of the obtained insulin derivatives based on phenylboronic acid were characterized by Q-TOF mass spectrometry, circular dichroism, scanning electron microscopy, MALDI-TOF mass spectrometry and solid-state boron nuclear magnetic resonance spectroscopy. The results are as follows: Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 shown.

[0090] From the results of the above experimental characterization, it can be seen that the structure of the insulin derivative based on phenylboronic acid is:

[0091]

[0092] Among them, R 1 and R 3 is 3-fluoro-1-phenylboronic acid-4-yl, R 2 For H.

[0093] The R 1 HN is the R-terminal amino hydrogen of the N-terminal of insulin A chain. 1 Group substitution; said R 2 HN is the R-terminal amino hydrogen of the N-terminal of insulin B chain. 2 Group substitution; the NHR 3 To perform R- 3 Group substitution.

[0094] Effect Example 1

[0095] Rodent efficacy studies

[0096] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected, and after fasting overnight, streptozocin (STZ) solution was intraperitoneally injected under light-proof conditions at a dose of 120 mg / kg. Blood glucose of mice was measured one week and two weeks after injection. If blood glucose>300 mg / dL, the model was successfully established. Otherwise, STZ solution was intraperitoneally injected again after two weeks at a dose of 50 mg / kg, and blood glucose of mice was measured again after one week and two weeks.

[0097] 2. In vivo hypoglycemic study in type 1 diabetic mice

[0098] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose of 400-500 mg / dL were selected for evaluation of the therapeutic effect. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose level of the diabetic mice remained in the normal range of less than 200 mg / dL for more than 214 hours, which was significantly longer than that of glargine insulin. The specific results are shown in Fig. 9 Compared with the commercially available long-acting insulin (insulin glargine), the insulin analog prepared in Example 1 showed a faster blood sugar lowering effect and a more stable blood sugar control ability, and no diabetic mice showed hypoglycemia (less than 50 mg / dL) (see Figure 8 ).

[0099] Meanwhile, the dose-effect relationship showed that the insulin derivative prepared in Example 1 did not cause hypoglycemia in the range of 4-12 mg / kg, while 40% of the mice developed hypoglycemia when glargine insulin was at 400 U / kg (about 13.8 mg / kg in mass units). Moreover, when glargine insulin was used to control blood sugar for one week (i.e., injected once a day at 40 U / kg), all mice developed hypoglycemia, and 40% of the mice died due to severe hypoglycemia (see Fig.10 ).

[0100] 3. Intraperitoneal Glucose Tolerance Test in Type I Diabetic Mice

[0101] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose of 400-500 mg / dL (n=5) were selected for evaluation of glucose control effect. On the first and fifth days after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), 1.5 g / kg of glucose was intraperitoneally injected into the mice, and the blood glucose of the mice was detected and recorded every 15 minutes for continuous monitoring for 2 hours.

[0102] At the same time, a group of healthy C57BL / 6J mice were selected and intraperitoneally administered with 1.5 g / kg of glucose. The blood glucose of the mice was tested and recorded every 15 minutes and monitored continuously for 2 hours as a control.

[0103] The results showed that when the mice were stimulated with sugar on the first day after administration, their blood sugar levels could be maintained within the normal blood sugar range (50-200 mg / dL), and the blood sugar control effect (assessed by the area under the blood sugar-time curve) was better than that of normal mice. When the mice were stimulated with sugar on the fifth day after administration, their blood sugar levels showed a significant peak at 15 minutes, but returned to normal (less than 200 mg / dL) after 1 hour, and remained stable for 2 hours (see Fig.11 ), the sugar control effect is still better than that of normal mice.

[0104] 4. Verification of glucose-responsive insulin release in type 1 diabetic mice

[0105] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice (n=5) with blood glucose of 400-500 mg / dL were selected for in vivo sugar response insulin release evaluation. On the first and fifth days after subcutaneous injection of the insulin analog prepared in Example 1 (12 mg / kg), 3.0 g / kg of glucose was intraperitoneally injected into the mice, and the blood glucose of the mice was detected and recorded every 15 minutes for 2 hours, and blood samples were taken at 0, 15, 30, 60, and 120 minutes (orbital blood, 20 μL, and plasma was obtained after centrifugation).

[0106] The insulin content in plasma was detected by recombinant human insulin ELISA kit.

[0107] The results showed that after the sugar stimulation, the blood glucose of mice showed an obvious peak (higher than 300mg / kg), and the insulin concentration in the plasma also changed synchronously with the change of blood glucose, and both peaked at 30 minutes. On the first day after administration, the peak insulin value increased by 4-5 times compared with the initial value; on the fifth day after administration, the peak insulin value increased by 2-3 times. At the same time, when the blood glucose gradually dropped to the initial value, the insulin concentration in the plasma also decreased and returned to the initial level, verifying the sugar-responsive insulin release (see Fig.12 ).

[0108] 5. Pharmacokinetics of the insulin derivatives prepared in Example 1 in mice treated with type I diabetes

[0109] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose 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 (orbital blood, 20 μL, and plasma was collected after centrifugation). The blood samples were continuously monitored for 17 days (see Fig.13 ).

[0110] At the same time, a group of healthy C57BL / 6J mice were selected and intravenously administered with 1 nmol / kg of the insulin derivative prepared in Example 1. Blood samples were collected at predetermined time points (orbital blood, 20 μL, and plasma was collected after centrifugation). The mice were monitored continuously for 21 days (see Fig.13 ).

[0111] The plasma insulin concentration-time curve data were analyzed by non-compartmental model analysis using Winnonlin. The half-life of the insulin derivative prepared in Example 1 reached 79.0±17.7 hours after subcutaneous injection in a diabetic mouse model, and 93.7.0±9.5 hours after intravenous injection in healthy mice, which is 1000 times that of normal human insulin (half-life of about 5 minutes).

[0112] 6. Biosafety evaluation of insulin derivatives prepared in Example 1 in mice treated with type 1 diabetes

[0113] A type I diabetic mouse model was established according to the above method, and type I diabetic mice (n=5) with blood glucose of 400-500 mg / dL were selected for biosafety evaluation.

[0114] Before administration, blood samples were collected from mice. After subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), blood samples were collected on the 7th day. Blood-related biochemical indices and blood routine tests were performed.

[0115] At the same time, a group of healthy C57BL / 6J mice were selected, blood samples were collected, and blood-related biochemical indicators and blood routine tests were also performed as a control.

[0116] The results showed that after subcutaneous injection of the insulin derivative prepared in Example 1, the various indicators of the mice were similar to those of healthy mice (see Fig.14 ).

[0117] Effect Example 2

[0118] Non-rodent efficacy studies

[0119] 1. Construction of Type I Diabetes Piglet Model

[0120] Six-month-old Bama pigs were selected and after overnight fasting, STZ solution was intravenously injected at a dose of 150 mg / kg. Blood sugar levels of the piglets were measured one week and two weeks after the injection. If blood sugar levels were >300 mg / dL, the model was successfully established.

[0121] 2. In vivo hypoglycemic study in type 1 diabetic piglets

[0122] According to the above method, a type I diabetic piglet model was established, and the therapeutic effect was evaluated on the type I diabetic piglets. After subcutaneous injection of the insulin derivative prepared in Example 1, the blood glucose of the diabetic piglets was tested by a continuous blood glucose monitoring system (CGMS).

[0123] The results showed that the insulin derivative prepared in Example 1 was injected subcutaneously to achieve a therapeutic effect of 8, 5, and 6 days in piglets I, II, and III (see Fig.15 ).

[0124] 3. Verification of glucose-responsive insulin release in type 1 diabetic piglets

[0125] According to the above method, a type I diabetic piglet model was established, and type I diabetic piglets were selected for in vivo sugar response insulin release evaluation. On the first day after subcutaneous injection of the insulin derivative prepared in Example 1 (12 mg / kg), 10% glucose (at a rate of 1 L / h) was intravenously injected into the piglets until the blood sugar of the piglets was higher than 300 mg / dL, and then the injection of glucose was stopped. The blood sugar of the piglets was tested and blood samples were taken every 15 minutes, and the monitoring was continued for 2 hours.

[0126] The insulin content in plasma was detected by recombinant human insulin ELISA kit.

[0127] The results showed that after the sugar stimulation, the piglets' blood sugar showed an obvious peak (higher than 300 mg / kg), and the insulin concentration in the plasma also changed synchronously with the change of blood sugar, and peaked when the blood sugar reached the peak. In both piglets, the insulin peak value increased by 2-3 times compared with the initial value. At the same time, when the blood sugar gradually dropped to the initial value, the insulin concentration in the plasma also decreased and returned to the initial level, verifying the sugar-responsive insulin release (see Fig.16 ).

[0128] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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: Among them, the R 1 , R 2 , R 3 are independently selected from H or the structure represented by formula (I), and R 1 , R 2 , R 3 At least one group is a structure represented by formula (I); The R 4 Selected from halogen, hydrocarbon, nitro, amide, ester, hydrocarbonoxy or nitrile.

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 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 halogen, C1-C 20 Alkyl, C1-C 20 Cycloalkyl, C1-C 20 Alkenyl, C1-C 20 Alkynyl, C1-C 20 Cycloalkenyl, C1-C 20 Cycloalkynyl, nitro, amide, ester, C1-C 20 an alkoxy group or a nitrile group.

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

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

7. The method for preparing an insulin derivative according to any one of claims 1 to 6, characterized in that: The following steps are involved: The phenylboronic acid compound is mixed with N,N,N′,N′-tetramethyl-O-(N-succinimidyl) tetrafluoroborate, N,N-diisopropylethylamine and a solvent, reacted, added with an insulin solution, and post-treated to obtain an insulin derivative; the structure of the phenylboronic acid compound is The R 4 Selected from halogen, hydrocarbon, nitro, amide, ester, hydrocarbonoxy or nitrile.

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-succinimidyl) tetrafluoroborate and the N,N-diisopropylethylamine is 1:0.8-1.2:1.1-1.5; The reaction temperature is 20-30°C; The concentration of insulin in the insulin solution is 0.2-100 mg / mL; The solvent of the insulin solution is selected from phosphate buffer or dimethyl sulfoxide; The post-treatment is selected from the use of dialysis or precipitation.

9. The preparation method according to claim 8, characterized in that: The post-treatment is precipitation, and the precipitation is specifically performed using a precipitant, the precipitant is a mixture of precipitant A and precipitant B, the precipitant A is selected from methyl tert-butyl ether and / or diethyl ether; the precipitant 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, and the dialysis is performed in deionized water; the molecular weight cutoff of the dialysis bag is 1000-3500, and the dialysis time is 12-24h; after the dialysis, a white flocculent precipitate is formed, which is the insulin derivative.

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

12. The use according to claim 11, characterized in that: The diabetes mellitus is type I diabetes mellitus and / or type II diabetes mellitus.

13. A drug, characterized in that The active ingredient comprises the insulin derivative 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.

Images