Sugar-responsive insulin analogue as well as preparation method and application thereof

Abstract

The invention discloses a sugar-responsive insulin analogue as well as a preparation method and application thereof. Specifically, the invention discloses a sugar responsive insulin analogue, which is insulin modified by a structure as shown in a formula I. The modification means that one or more amino groups in the insulin are connected with the structure as shown in the formula I. In the formula I, A is an active structure fragment of a glucose transporter inhibitor, and m is 1-20. The sugar-responsive insulin analogue disclosed by the invention can realize intelligent sugar response adjustment, has a longer half-life period and a wider therapeutic window compared with the existing insulin, and can reduce the introduction of materials and reduce the administration frequency. # imgabs0 #

Description

[0001] This application claims the priority of Chinese patent application No. 2023110201326 filed on August 14, 2023. This application cites the entire text of the above Chinese patent application. Technical Field

[0002] The invention relates to a sugar-responsive insulin analogue and a preparation method and application thereof. Background Art

[0003] About 10% of diabetic patients worldwide need insulin treatment to maintain blood sugar within the normal range. There are many insulin drugs on the market now. Under the existing treatment methods, diabetics need to inject insulin subcutaneously multiple times a day, and then combine it with a regular daily diet to maintain stable fasting blood sugar and postprandial blood sugar. This dosing regimen brings a heavy physical and mental burden to diabetic patients, especially adolescents. In addition, the therapeutic window of insulin is narrow, so a slight discomfort in the dosage of insulin may lead to hypoglycemia, especially nocturnal hypoglycemia; nocturnal hypoglycemia poses a great safety risk. Therefore, we need to design a new type of insulin with a wider therapeutic index, a single injection that can control sugar for a longer time, and better blood sugar control ability.

[0004] Glucose-responsive insulin can simulate the function of human β-cells, releasing insulin quickly when blood sugar is high and slowly when blood sugar is low. Therefore, glucose-responsive insulin can actively track blood sugar fluctuations, dynamically adjust the insulin release rate, meet the dynamic demand for insulin in real time, improve the therapeutic index of insulin, and improve the blood sugar control effect of insulin.

[0005] There are currently three main sugar-responsive mechanisms, including phenylboronic acid derivatives, glucose-binding molecules, and glucose oxidase. However, it is still difficult for sugar-responsive insulin to control blood sugar within a normal range for a long period of time after a single injection. How to achieve both rapid insulin release after a meal and continuous and slow basal insulin release during fasting over a long period of time after a single injection is still a considerable challenge for sugar-responsive insulin. Summary of the invention

[0006] In order to solve the unavoidable hypoglycemia phenomenon of insulin delivery systems in the prior art, the present invention provides a sugar-responsive insulin analog and a preparation method and application thereof. The sugar-responsive insulin of the present invention realizes intelligent sugar response regulation, has excellent hypoglycemic effect under hyperglycemic conditions, can avoid the occurrence of hypoglycemia when overdosed, has a long half-life, a wide therapeutic window, can reduce the introduction of materials, and reduces the frequency of administration.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions.

[0008] The present invention provides a sugar-responsive insulin analog, which is an insulin modified by a structure as shown in Formula I, wherein the modification means that one or more amino groups in the insulin are connected to the structure as shown in Formula I.

[0009]

[0010] In formula I, A is an active structural fragment of a glucose transporter inhibitor, and m is 1-20.

[0011] The plurality is preferably 2 or 3.

[0012] Wherein, m is preferably 1-6, for example 3.

[0013] Wherein, the A can be an active structural fragment among various existing glucose transporter inhibitors that can bind to glucose transporter and inhibit its function. The A is connected to oxygen at a replaceable position, specifically, at position 2, 3 or 4 on the benzene ring structure in the A.

[0014] The A is preferably

[0015]

[0016] The insulin may be any type of insulin currently available in the art, preferably recombinant human insulin, aspart insulin, lispro insulin, detemir insulin, glargine insulin or degludec insulin.

[0017] In a preferred embodiment of the present invention, the structure shown in Formula I is:

[0018]

[0019] The present invention also provides a method for preparing the sugar-responsive insulin analog as described above, which comprises the following steps: (1) reacting the intermediate shown in formula II with a condensing agent in a solvent miscible with water in the presence of a base,

[0020]

[0021] Wherein, A and m are defined as above;

[0022] (2) The product obtained in step (1) is mixed with insulin and reacted.

[0023] The operations and conditions of step (1) and step (2) may be conventional operations and conditions for condensation reactions in the art.

[0024] In step (1), the condensing agent can be any condensing agent suitable for the condensation reaction, preferably N-hydroxysuccinimide or 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). The amount of the condensing agent can be selected according to the amount required for the condensation reaction, and the molar ratio of the amount of the condensing agent to the intermediate shown in Formula II is preferably (0.5-5.0):1, more preferably 0.9:1.

[0025] In step (1), the base may be an organic base, preferably N,N-diisopropylethylamine (DIPEA), triethylamine or 4-dimethylaminopyridine (DMAP). The molar ratio of the base to the insulin may be (1.0-20.0):1, preferably (1.0-3.0):1, and more preferably 2.2:1.

[0026] In step (1), it should be understood by those skilled in the art that the water-miscible solvent can dissolve the intermediate as shown in Formula II. The water-miscible solvent is preferably dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF). The amount of the water-miscible solvent is conventionally selected, preferably so that the concentration of the intermediate as shown in Formula II is 1-200 mg / mL, for example 50 mg / mL.

[0027] In step (1), the reaction temperature may be 4-30°C, preferably 20-25°C, more preferably room temperature. The reaction time may be 1-24h, preferably 2-5h, more preferably 2.5h.

[0028] In step (2), the insulin is the same as described above. The molar ratio of the intermediate shown in formula II to the insulin is preferably (1-20):1, more preferably (1-3):1, for example 2:1.

[0029] In step (2), the mixing method can be to add (such as dropwise add) the reaction solution obtained in step (1) into the aqueous solution of insulin.

[0030] The concentration of the aqueous solution of insulin is preferably 0.2-100 mg / mL, such as 10-15 mg / mL. The pH of the aqueous solution of insulin is preferably 7.4-13.0, more preferably 7.4. The aqueous solution of insulin is preferably a buffer solution containing insulin (such as phosphate buffer).

[0031] Preferably, the preparation of the aqueous solution of insulin comprises the following steps: adding the insulin to a phosphate buffer (pH may be 5.0-13.0, preferably 6.0-8.0, more preferably 7.4, and the concentration may be 0.01-0.10M), deionized water or pure water, adding an acidic agent to adjust the pH to 3.0-4.0 to dissolve the insulin, and then adding an alkaline agent to adjust the pH to 7.4-13.0. The acidic agent is preferably a hydrochloric acid solution, a phosphoric acid solution or an acetic acid solution, more preferably a hydrochloric acid solution. The alkaline agent is preferably a sodium hydroxide solution, a sodium bicarbonate solution or a sodium carbonate solution, more preferably a sodium hydroxide solution.

[0032] In step (2), the reaction temperature may be 0-30°C, preferably 2-6°C, more preferably 4°C, for example, in an ice bath. The reaction time may be 2-96h, preferably 5min-24h, more preferably 10-18h, for example 16h. The reaction is preferably carried out under stirring.

[0033] After the reaction in step (2) is completed, post-treatment may be performed, preferably dialysis or ion exchange column purification.

[0034] The dialysis can be performed in deionized water using a conventional dialysis bag in the art. The molecular weight cutoff of the dialysis bag is preferably 1k-3.5k. The purpose of the dialysis is to remove the free intermediate shown in Formula II and the organic solvent in the system.

[0035] Wherein, the ion exchange column is preferably an anion exchange column or a dextran gel chromatography column.

[0036] Furthermore, the post-treatment may also include high performance liquid chromatography purification, and the chromatographic conditions are preferably:

[0037] The chromatographic column is an octadecylsilane bonded column, preferably an XBridge-C 18 Preparative column, length 250 mm, inner diameter 19 mm, filler particle size 5 μm;

[0038] Mobile phase A was acetonitrile solution containing 0.1 v% trifluoroacetic acid, and mobile phase B was ultrapure water containing 0.1 v% trifluoroacetic acid;

[0039] Gradient elution is as follows: based on the total volume of the mobile phase as 100%, during 0-60 min, the mobile phase A increases from 30 v% to 40 v%; during 60-65 min, the mobile phase A is maintained at 40 v%; during 65-80 min, the mobile phase A increases from 40 v% to 45 v%; during 80-90 min, the mobile phase A increases from 45 v% to 50 v%;

[0040] The temperature of the chromatographic column is 25° C., the detection wavelengths are 214 nm and 280 nm, the flow rate of the mobile phase is 8 mL / min, and the injection volume can be 1 mL.

[0041] Furthermore, the method for preparing the sugar-responsive insulin analog may further include a freeze-drying step.

[0042] The present invention also provides an intermediate as shown in formula II,

[0043]

[0044] Wherein, m and A are defined as above.

[0045] Wherein, the intermediate as shown in formula II is preferably

[0046]

[0047] The present invention provides a method for preparing an intermediate as shown in formula II, which comprises the following steps: in a solvent, in the presence of a base, subjecting a compound as shown in formula III to a hydrolysis reaction as shown below, thereby preparing an intermediate as shown in formula II;

[0048]

[0049] Among them, R 1 C 1-6 Alkyl, m and A are as defined above.

[0050] The operation and conditions of the hydrolysis reaction can be conventional operations and conditions of hydrolysis reactions in the art, and the present invention is preferably as follows:

[0051] In the hydrolysis reaction, the solvent is preferably an ether solvent, such as THF.

[0052] In the hydrolysis reaction, the base is preferably an alkali metal hydroxide, such as potassium hydroxide.

[0053] In the hydrolysis reaction, the molar ratio of the base to the compound of Formula III is preferably 5:1.

[0054] The reaction temperature of the hydrolysis reaction is preferably room temperature.

[0055] The progress of the hydrolysis reaction can be monitored by conventional detection methods in the art (such as TLC, HPLC, HNMR), and preferably the disappearance or no-reaction of the compound as shown in Formula IV is taken as the end point of the reaction. Preferably, the reaction time of the hydrolysis reaction is 4-5 hours.

[0056] The preparation method of the intermediate shown in formula II may further include a preparation method of a compound shown in formula III. The preparation method of the compound shown in formula III may include the following steps: in a solvent, in the presence of a base, subjecting a compound shown in formula IV and a compound shown in formula V to a substitution reaction as shown below to obtain a compound shown in formula III;

[0057]

[0058] Among them, R 2 for R 1 , m and A are defined as above.

[0059] The operation and conditions of the substitution reaction can be conventional operations and conditions of substitution reactions in the art, and the present invention is preferably as follows:

[0060] In the substitution reaction, the solvent is preferably an amide solvent, such as DMF.

[0061] In the substitution reaction, the base is preferably an alkali metal carbonate, such as potassium carbonate.

[0062] In the substitution reaction, the molar ratio of the base to the compound of formula IV is preferably 1.5:1.

[0063] In the substitution reaction, the molar ratio of the compound shown in Formula IV to the compound shown in Formula IV is preferably (1.2-2.0):1.

[0064] The reaction temperature of the substitution reaction is preferably 60°C.

[0065] The progress of the substitution reaction can be monitored by conventional detection methods in the art (such as TLC, HPLC, HNMR), and preferably the disappearance or no-reaction of the compound shown in Formula IV is taken as the end point of the reaction. Preferably, the reaction time of the substitution reaction is 2-3 hours.

[0066] The present invention also provides a compound as shown below,

[0067]

[0068] The present invention also provides a pharmaceutical composition, which comprises the sugar-responsive insulin analog as described above and pharmaceutical excipients.

[0069] The present invention also provides a use of the sugar-responsive insulin analogue described above in the preparation of a drug for treating diabetes.

[0070] The diabetes may be type 1 diabetes or type 2 diabetes, preferably type 1 diabetes or late stage type 2 diabetes.

[0071] The diabetes treatment drug can be any dosage form suitable for insulin drugs, such as a subcutaneous injection. The subcutaneous injection can be resuspended in phosphate buffer or saline and administered by subcutaneous injection for treatment.

[0072] In the present invention, the room temperature refers to 20-40°C.

[0073] In the present invention, overnight refers to 10-18 hours.

[0074] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0075] The reagents and raw materials used in the present invention are commercially available.

[0076] The positive and progressive effects of the present invention are:

[0077] The sugar-responsive insulin analog of the present invention can form an insulin reservoir after administration (such as subcutaneous injection) and has good glucose-responsive release performance.

[0078] Under hyperglycemia conditions, glucose-responsive insulin analogs bind weakly to glucose transporters and will dissociate from glucose transporters under the competition of glucose, allowing glucose transporters to function and thus restore blood sugar to normal levels. At the same time, it does not affect the hypoglycemic effect of glucose-responsive insulin analogs.

[0079] When administered in overdose, excess glucose-responsive insulin analogs bind to glucose transporters, reducing the ability of glucose transporters to transport glucose, preventing cells from further uptake of blood glucose, thereby preventing the occurrence of hypoglycemia (see Figure 1 ).

[0080] The sugar-responsive insulin analog of the present invention can realize intelligent sugar-responsive regulation, and has a longer half-life and a wider therapeutic window compared to existing insulin, and can reduce the introduction of materials and reduce the frequency of administration. When the dosage of the preferred embodiment of the present invention is increased to 15 mg / kg, it will not cause hypoglycemia and can maintain the blood sugar of mice at a normal level for more than 40 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1. Schematic diagram of the insulin release mechanism of the sugar-responsive insulin analog of the present invention. Wherein, "1" represents that the binding between the glucose transporter and the sugar-responsive insulin analog is reduced, resulting in more binding of the sugar-responsive insulin analog to the insulin receptor; "2" represents that the binding between the glucose transporter and the sugar-responsive insulin analog is increased, inhibiting the transport function of the glucose transporter; "3" represents that the unbound glucose transporter is reduced, promoting the glucose transporter to transport glucose into the cell.

[0082] Figure 2 .Example 1 Synthesis of modified inhibitor i-562 step 1 of the product (compound 2) of the hydrogen nuclear magnetic resonance spectrum.

[0083] Figure 3 .H NMR spectrum of the product (Compound 3) in Step 2 of the synthesis of the modified inhibitor i-562 in Example 1.

[0084] Figure 4 .H NMR spectrum of the product (Compound 6) in step 3 of the synthesis of the modified inhibitor i-562 in Example 1.

[0085] Figure 5 .H NMR spectrum of the product (Compound 7) in Step 4 of the synthesis of the modified inhibitor i-562 in Example 1.

[0086] Figure 6 .H NMR spectrum of the modified inhibitor i-562 synthesized in Example 1.

[0087] Figure 7 .Mass spectrum of the product (Compound 9) in Step 1 of Example 1 Synthesis of the modified inhibitor i-756.

[0088] Figure 8 .Mass spectrum of the product (Compound 10) in Step 2 of the synthesis of the modified inhibitor i-756 in Example 1.

[0089] Fig. 9 .Mass spectrum of the product (Compound 11) in Step 3 of the synthesis of the modified inhibitor i-756 in Example 1.

[0090] Fig.10 .Mass spectrum of the product (Compound 12) in Step 4 of the synthesis of the modified inhibitor i-756 in Example 1.

[0091] Fig.11 .Mass spectrum of the product (Compound 13) in Step 5 of the synthesis of the modified inhibitor i-756 in Example 1.

[0092] Fig.12 .Mass spectrum of the product (Compound 14) in Step 6 of the synthesis of the modified inhibitor i-756 in Example 1.

[0093] Fig.13 .Mass spectrum of the product (Compound 15) in Step 7 of the synthesis of the modified inhibitor i-756 in Example 1.

[0094] Fig.14 .Mass spectrum of the product (Compound 16) in Step 8 of the synthesis of the modified inhibitor i-756 in Example 1.

[0095] Fig.15 Example 1: Nuclear magnetic resonance hydrogen spectrum of the synthetic modified inhibitor i-756 (the deuterated reagent is CDCl3).

[0096] Fig.16 .Mass spectrum of the product (compound 15c) in step 1 in the synthesis of compound 15d in the synthesis of the modified inhibitor i-756 in Example 1.

[0097] Fig.17 .Mass spectrum of the product (Compound 15d) in Step 2 of the synthesis of Compound 15d in the synthesis of the modified inhibitor i-756 in Example 1.

[0098] Fig.18 .Example 1 Preparative liquid chromatography spectra of the synthetic sugar-responsive insulin analogs i-ins-562, 2i-ins-562 and 3i-ins-562.

[0099] Among them, "1" represents i-ins-562; "2" represents 2i-ins-562; and "3" represents 3i-ins-562.

[0100] Fig.19 .Preparative liquid chromatography spectra of the sugar-responsive insulin analogs i-ins-756, 2i-ins-756 and 3i-ins-756 synthesized in Example 1. Wherein, "1" represents i-ins-756; "2" represents 2i-ins-756; and "3" represents 3i-ins-756.

[0101] Fig. 20 .MALDI-TOF mass spectra of the sugar-responsive insulin analogs i-ins-562, 2i-ins-562 and 3i-ins-562 of the modified inhibitor i-562 synthesized in Example 1; wherein a is the MALDI-TOF mass spectrum of i-ins-562; b is the MALDI-TOF mass spectrum of 2i-ins-562; and c is the MALDI-TOF mass spectrum of 3i-ins-562.

[0102] Fig.21.MALDI-TOF mass spectra of the sugar-responsive insulin analogs i-ins-756, 2i-ins-756 and 3i-ins-756 of the modified inhibitor i-756 synthesized in Example 1; wherein a is the MALDI-TOF mass spectrum of i-ins-756; b is the MALDI-TOF mass spectrum of 2i-ins-756; c is the MALDI-TOF mass spectrum of 3i-ins-756.

[0103] Fig. 22 . Effect Example 1 shows the blood glucose level of type 1 diabetic mice treated with glargine insulin and recombinant human insulin.

[0104] Fig.23 . Effects The blood glucose levels of type 1 diabetic mice treated with subcutaneous injection of the glucose-responsive insulin analog i-ins-562 (15.0 mg / kg) in Example 1.

[0105] Fig.24 . Effects The blood glucose levels of type 1 diabetic mice treated with subcutaneous injection of the glucose-responsive insulin analog i-ins-756 (15.0 mg / kg) in Example 1.

[0106] Fig.25 . Effect Example 2 shows the blood glucose level of type 1 diabetic mice in the intraperitoneal glucose tolerance test.

[0107] Fig.26 .AUC of type 1 diabetic mice in the intraperitoneal glucose tolerance test in Effect Example 2.

[0108] Fig. 27 . Effects The in vivo imaging results of type 1 diabetic mice after subcutaneous injection of the sugar-responsive insulin analog i-ins-562 (1.5 mg / kg) and recombinant human insulin (1.5 mg / kg) in Example 3.

[0109] Fig.28 . Effect Example 4: Subcutaneous injection of the sugar-responsive insulin analog i-ins-562 (0.04 mg / kg) to treat blood glucose levels in miniature pig No. 1 with type 1 diabetes.

[0110] Fig.29 . Effect Example 4: Subcutaneous injection of the sugar-responsive insulin analog i-ins-562 (0.04 mg / kg) to treat blood glucose levels in miniature pig No. 2 with type 1 diabetes.

[0111] Fig.30 . Effect Example 4: Subcutaneous injection of the sugar-responsive insulin analog i-ins-562 (0.04 mg / kg + 0.02 mg / kg) to treat blood glucose levels in miniature pig No. 3 with type 1 diabetes.

[0112] Fig.31 . Effect Example 5: ATP inhibitory effect of the sugar-responsive insulin analog i-ins-562.

[0113] Fig.32 . Effect Example 5 NBDG uptake experimental results of the sugar-responsive insulin analog i-ins-756.

[0114] Fig.33 . Effect Example 6: Plasma insulin concentration in mice after subcutaneous injection of the glucose-responsive insulin analog i-ins-562.

[0115] Fig.34 . Effect Example 6: Plasma insulin concentration in mice after subcutaneous injection of the glucose-responsive insulin analog i-ins-756. DETAILED DESCRIPTION

[0116] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0117] The reagents used in the following examples are shown in Table 1.

[0118] Table 1

[0119]

[0120] The manufacturers or models of the instruments used in the following examples are as follows:

[0121] Vacuum freeze dryer, Labconco;

[0122] Preparative HPLC, Waters 2545,

[0123] Detector: Waters 2489;

[0124] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, ultraflexTOF / TOF, Bruker Daltonics;

[0125] Small animal gas anesthesia machine, R530IE, RWD;

[0126] Small animal in vivo imaging system, Lumina LT Series III, IVIS;

[0127] Blood glucose monitor, Aviva, ACCU-CHEK (Roche Pharmaceuticals, USA);

[0128] Continuous glucose monitoring system, FreeStyle Libre H, Abbott.

[0129] In the following examples, RT means room temperature; DCM means dichloromethane; TsCl means p-toluenesulfonyl chloride; EA means ethyl acetate; THF means tetrahydrofuran; DMF means N,N-dimethylformamide; NBS means N-bromosuccinimide; DIBAL-H means diisobutylaluminum hydride; HATU means N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate; DIPEA means N,N-diisopropylethylamine; MTBE means methyl tert-butyl ether.

[0130] Example 1

[0131] 1. Synthesis and characterization of modified inhibitor i-562

[0132] Step 1:

[0133]

[0134] Triethylene glycol (compound 1, 20 g, 133 mmol) and triethylamine (20.187 g, 199.5 mmol) were added to a two-necked flask, anhydrous DCM was added, the temperature was lowered to 0°C, and then a DCM solution of TsCl (27.93 g, 146.5 mmol) was added, N2 was protected, the mixture was transferred to RT, and the reaction was stirred overnight. After the reaction was completed by TLC and LCMS monitoring, the solvent was removed by distillation under reduced pressure, and compound 2 was purified by column chromatography (the hydrogen spectrum of compound 2 is shown in FIG. Figure 2 ).

[0135] Step 2:

[0136]

[0137] Compound 2 (1 g, 3.28 mmol) was added to a two-necked flask, and anhydrous 10 mL DCM was added. The mixture was protected by N2 and transferred to 0°C. Then, a DCM solution of BF3 (0.0093 g, 0.065 mmol) was added. After 10 min, a DCM solution of methyl diazoacetate (0.328 g, 3.28 mmol) was slowly added dropwise. The mixture was stirred at room temperature and reacted overnight. After the reaction was completed by TLC and LCMS monitoring, 10 mL of water was added, and EA (10 mL x 3) was added for extraction. The mixture was washed three times with saturated brine, dried over anhydrous Na2SO4, and purified by TLC to obtain compound 3 (the hydrogen spectrum of compound 3 is shown in Figure 3 ).

[0138] Step 3:

[0139]

[0140] 4-Chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (compound 5, 100 mg, 0.44 mmol) and triethylamine (132 mg, 1.3 mmol) were added to a two-necked flask, anhydrous THF was added, and then N-(2-hydroxyphenyl)piperazine (compound 4, 85 mg, 0.48 mmol) was added, and N2 protection was applied. The mixture was transferred to 70°C and stirred for 1 h. The solvent was dried by spinning, and 5 mL of DCM and water were added, and the mixture was extracted 3 times. The organic phases were combined, dried over anhydrous Na2SO4, and column chromatography was performed to obtain compound 6 (the hydrogen spectrum of compound 6 is shown in FIG. Figure 4 ).

[0141] Step 4:

[0142]

[0143] Compound 6 (200 mg, 0.537 mmol) and Cs2CO3 (262 mg, 0.805 mmol) were added to a two-necked flask, and DMF solvent was added. N2 was protected, and then a DMF solution of compound 3 (242 mg, 0.644 mmol) was added. The temperature was raised to 60°C for reaction for 3 h. After the reaction was completed by TLC and LCMS monitoring, 10 mL of water was added to the reaction solution after cooling to RT, and the organic phase was extracted with EA (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography was performed to obtain compound 7 (the hydrogen spectrum of compound 7 is shown in Figure 5 ).

[0144] Step 5:

[0145]

[0146] Compound 7 (760 mg, 1.319 mmol) was dissolved in anhydrous THF, and then an aqueous solution of LiOH (158 mg, 6.59 mmol) was added and stirred at room temperature for 4 h. After the reaction was completed by TLC monitoring, the solvent was dried by spin drying, and the organic phase was extracted with EA three times. The pH of the aqueous phase was adjusted to neutral or weakly acidic with 1M HCl. After stirring for a while, a solid was precipitated. Water and DCM (2% methanol) were added to extract the organic phase, and i-562 was obtained by spin drying (the hydrogen spectrum of i-562 is shown in Figure 6 ).

[0147] 2. Synthesis and characterization of modified inhibitor i-756

[0148] Step 1:

[0149]

[0150] NaH (220 mg) was added to a two-necked flask, and anhydrous toluene and dimethyl carbonate (360 mg) were added at room temperature under argon protection. The temperature was raised to 90°C, and a toluene solution of 4-chloroacetophenone (compound 8, 310 mg) was added at this temperature, and the reaction was continued for 3 hours. The reaction was cooled to room temperature, quenched by adding 1M hydrochloric acid aqueous solution in an ice bath, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain compound 9 (compound 9 mass spectrum see Figure 7 ).

[0151] Step 2:

[0152]

[0153] Compound 9 (200 mg) was added to a single-mouth bottle, DCM was added, NBS (167 mg) was added at room temperature, and the reaction was continued at room temperature for 3 h. After 3 h, the mixture was filtered, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain compound 10 (see the mass spectrum of compound 10 for details). Figure 8 ).

[0154] Step 3:

[0155]

[0156] Compound 10 (270 mg) and 2-amino-5-iodopyridine (244.5 mg) were added to a two-necked flask, and anhydrous acetonitrile was added under nitrogen protection. The reaction was carried out at 80°C overnight. During the reaction, a large amount of white solid was precipitated, which was filtered to obtain compound 11 (see the mass spectrum of compound 11 for details). Fig. 9 ).

[0157] Step 4:

[0158]

[0159] Compound 11 was dissolved in anhydrous tetrahydrofuran, and protected by nitrogen. Four times the equivalent of DIBAL-H was added at -78°C. The reaction was monitored for 10 minutes to complete. Water was added under ice bath to quench the reaction. Due to the poor solubility of the product, EA was added after quenching to extract. Some yellow solids were precipitated and filtered to obtain compound 12 (see the mass spectrum of compound 12 for details). Fig.10 ).

[0160] Step 5:

[0161]

[0162] Compound 12 (1.0 eq), 2-cyanoboric acid (1.5 eq), potassium carbonate (2.0 eq) and triphenylphosphine palladium (0.1 eq) were added to a two-necked flask, and anhydrous DMF was added under argon protection. The mixture was reacted at 100°C overnight. After overnight reaction, the mixture was cooled to room temperature and extracted with ethyl acetate. Some solids were precipitated during the process, and the product was obtained by filtration and pulping. The filtrate was purified by column chromatography to obtain compound 13 (see the mass spectrum of compound 13). Fig.11 ).

[0163] Step 6:

[0164]

[0165] Compound 13 (1.0 eq) was added to a two-necked flask, and anhydrous THF was added under nitrogen protection in an ice bath. Thionyl chloride (3.5 eq) was slowly added dropwise, and then reacted at room temperature for 3 h. The crude product solid (compound 14, mass spectrum see Fig.12 ).

[0166] Step 7:

[0167]

[0168] Compound 15d (1.1 eq) was added to a two-necked flask, and anhydrous DMF and triethylamine (2.0 eq) were added. Under nitrogen protection, the mixture was moved to an ice bath. Compound 14 (1.0 eq) was dissolved in anhydrous DMF and slowly added to the reaction system. The mixture was then moved to room temperature for 6 h. EA extraction was performed, the mixture was dried over anhydrous sodium sulfate, and the mixture was separated by column chromatography to obtain compound 15 (see the mass spectrum of compound 15 for details). Fig.13 ).

[0169] Step 8:

[0170]

[0171] Compound 15 (1.0 eq) and cesium carbonate (1.5 eq) were added to a two-necked flask, and anhydrous DMF and compound 3 (2.0 eq) were added under nitrogen protection, and then the mixture was moved to 60°C for reaction for 2 h. After 2 h, DMF was directly removed under reduced pressure, and the target product (compound 16, mass spectrum see Fig.14 ).

[0172] Step 9:

[0173]

[0174] Compound 16 (1.0 eq) was dissolved in THF, and LiOH (5.0 eq) aqueous solution was added at room temperature, and the reaction was continued at room temperature for 5 h. After the reaction was completed, the mixture was acidified to neutral, the solvent was removed under reduced pressure, and the water was lyophilized. Due to the high polarity of the product, TLC plates were selected for multiple development and purification to obtain i-756 (i-756 hydrogen spectrum is shown in Fig.15 ).

[0175] Synthesis of compound 15d:

[0176] Step 1:

[0177]

[0178] 3-Fluoro-4-hydroxybenzoic acid (compound 15a, 3.6 g, 23 mmol), 1-BOC-piperazine (compound 15b, 10.74 g, 57.6 mmol), and HATU (26.31 g, 69 mmol) were added to a two-necked flask, anhydrous DMF was added, N2 was protected, the temperature was lowered to 0°C, and then DIPEA (14.83 g, 115 mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed by TLC and LCMS monitoring, water was added, citric acid was added, and then EA was added for extraction, washed three times with saturated brine, dried over anhydrous Na2SO4, and dried to obtain an oily state. A large amount of white solid was precipitated after adding a small amount of DCM to obtain compound 15c (see the mass spectrum of compound 15c). Fig.16 ).

[0179] Step 2:

[0180]

[0181] Compound 15c (3 g, 9.24 mmol) was dissolved in anhydrous DCM, and then EA / HCl (92.4 mL) was added and stirred at room temperature overnight. After the reaction was completed by TLC and LCMS monitoring, 30 mL of MTBE was added, stirred and filtered to obtain compound 15d (see the mass spectrum of compound 15d for details). Fig.17 ).

[0182] 3. Synthesis and characterization of glucose-responsive insulin analogs (i-ins-756 and i-ins-562)

[0183] The modified inhibitor (i-562 or i-756) (2 eq) was dissolved in 200 μL of anhydrous N,N-dimethylformamide (DMF), and 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) (1.8 eq) was dissolved in 400 μL of anhydrous DMF, mixed in a 1.5 mL EP tube, 2 μL of DIPEA (2.2 eq) was added, and the mixture was reacted at room temperature for 2.5 h to obtain solution A.

[0184] 30 mg (1 eq) of recombinant human insulin was dissolved in 2 mL of phosphate buffer (0.01 M, pH = 7.4), 1 mol / L hydrochloric acid solution was added to adjust the pH to 3-4 to dissolve the insulin, and then an equal amount of 1 mol / L sodium hydroxide solution was added to obtain solution B.

[0185] The above solution A was added to solution B and reacted in an ice bath for 16 hours. The solution was dialyzed (MW: 3500) with 4 L of deionized water for 3 times and freeze-dried to obtain a sugar-responsive insulin analog in the form of a white powder.

[0186] The obtained product was further purified by preparative high performance liquid chromatography under the following chromatographic conditions:

[0187] XBridge Preparative Columns (C 18 , 5μm spherical mixed particles, 19mm×250mm);

[0188] Mobile phase A was an acetonitrile solution containing 0.1 v% trifluoroacetic acid, and mobile phase B was an ultrapure aqueous solution containing 0.1 v% trifluoroacetic acid;

[0189] Gradient elution is as follows: based on the total volume of the mobile phase as 100%, during 0-60 min, the mobile phase A increases from 30 v% to 40 v%; during 60-65 min, the mobile phase A is maintained at 40 v%; during 65-80 min, the mobile phase A increases from 40 v% to 45 v%; during 80-90 min, the mobile phase A increases from 45 v% to 50 v%;

[0190] The temperature of the chromatographic column is 25° C., the detection wavelengths are 214 nm and 280 nm, the flow rate of the mobile phase is 8 mL / min, and the injection volume can be 1 mL.

[0191] Collect one, two or three modified inhibitor-modified sugar-responsive insulin analogs. For i-562: 25-45 min to collect one modified inhibitor-modified sugar-responsive insulin analog (i-ins-562), 50-70 min to collect two modified inhibitor-modified sugar-responsive insulin analogs (2i-ins-562), 75-90 min to collect three modified inhibitor-modified sugar-responsive insulin analogs (3i-ins-562) (see Fig.18 ); for i-756: 15-35 min to collect a sugar-responsive insulin analog modified with one modification inhibitor (i-ins-756), 40-50 min to collect a sugar-responsive insulin analog modified with two modification inhibitors (2i-ins-756), 55-70 min to collect a sugar-responsive insulin analog modified with three modification inhibitors (3i-ins-756) (see Fig.19), collect each eluate, remove acetonitrile by rotary evaporation at low temperature, and then freeze-dry. The MALDI-TOF mass spectra (time of flight mass spectrometry) of the prepared sugar-responsive insulin analogs are as follows: Fig. 20 and Fig.21 As shown, the matrix used was sinapinic acid (SA).

[0192] Effect Example: Animal Efficacy Study

[0193] Effect Example 1. Animal efficacy study of glucose-responsive insulin analogs (i-ins-756 and i-ins-562)

[0194] C57BL / 6 mice (purchased from Hangzhou Medical College) were induced with type 1 diabetes at a dose of 120 mg / kg streptozotocin. Diabetic mice were maintained on a standard diet and a circadian cycle of 12 hours light and 12 hours dark.

[0195] According to the above method, a type 1 diabetic mouse model was established, and type 1 diabetic mice with blood glucose higher than 300 mg / dL were selected for evaluation of the therapeutic effect. Three groups of 5 mice were selected in each group, and phosphate buffer, recombinant human insulin (1.5 mg / kg) and glargine insulin (80 U / kg) were injected subcutaneously. Blood glucose was measured using a blood glucose meter. The results are as follows: Fig. 22 As shown. Fig. 22 It can be seen that the normal blood sugar level (less than 200 mg / dL) of mice injected with recombinant human insulin was only maintained for less than 4 hours; the blood sugar of mice injected with glargine insulin was maintained at a normal level for about 10 hours, but hypoglycemia occurred 1.5h, 6h, 9h and 12h after administration. This shows that the current long-acting insulin has a certain risk of hypoglycemia.

[0196] In order to study the hypoglycemic risk reduction function and hypoglycemic effect of i-ins-562 and i-ins-756, an overdose of glucose-responsive insulin analogues was selected for the experiment. Two groups of model mice, 5 mice in each group, were subcutaneously injected with 15 mg / kg of i-ins-562 and i-ins-756, and blood glucose was measured using a blood glucose tester. The results are shown in Figure 2. Fig.23 and 24 shown.

[0197] Depend on Fig.23 and Fig.24 It can be seen that when the dose was increased to 1.5 mg / kg (10 times the dose of recombinant human insulin), no hypoglycemia (blood sugar below 50 mg / dL) was observed in mice in the i-ins-562 group and the i-ins-756 group. Fig.23It can be seen that a dose of 15 mg / kg of i-ins-562 can maintain the blood glucose of mice at a normal level for more than 40 hours, which is far longer than the action time of the currently commercially available long-acting insulin.

[0198] Effect Example 2: Intraperitoneal glucose tolerance test of glucose-responsive insulin analogs (i-ins-756 and i-ins-562)

[0199] In order to further study the blood sugar regulating effect of i-ins-562, two groups of model mice and one group of normal mice, with 5 mice in each group, were subjected to intraperitoneal glucose tolerance test (IPGTT) 4 hours after subcutaneous injection of i-ins-562 (dosage was 1.5 mg / kg) and recombinant human insulin (dosage was 1.5 mg / kg). After intraperitoneal injection of glucose (3 g / kg), the blood sugar of mice in the subcutaneous injection of recombinant human insulin group increased sharply and remained above 300 mg / dL within 2 hours; although the blood sugar of the healthy mouse group did not increase sharply, its recovery was slower. In contrast, the i-ins-562 group did not show a sharp increase in blood sugar and returned to normal blood sugar in 40 minutes (see Fig.25 ). The area under the curve (AUC) was calculated (see Fig.26 ), which confirmed that i-ins-562 has a better blood sugar regulating effect than unmodified recombinant human insulin, and that type 1 diabetic mice injected with i-ins-562 have faster blood sugar regulation ability than healthy mice.

[0200] Effect Example 3: In vivo imaging test of sugar-responsive insulin analogs (i-ins-756 and i-ins-562)

[0201] In vivo imaging was performed after subcutaneous injection of i-ins-562 (1.5 mg / kg) and recombinant human insulin (1.5 mg / kg) into type 1 diabetic mice (5 mice in each group) (see Fig. 27 ). The fluorescence intensity of mice in the recombinant human insulin group decreased rapidly within 4 hours after subcutaneous injection of the drug, and began to disappear after 6 hours; while the fluorescence intensity of mice in the i-ins-562 group decreased more slowly and began to disappear 22 hours after administration. During this period, the fluorescence intensity of mice in the i-ins-562 group was significantly higher than that in the recombinant human insulin group, confirming that i-ins-562 has a longer duration of action than recombinant human insulin. In vivo imaging showed that after subcutaneous injection of i-ins-562, the fluorescence basically disappeared 22 hours later, indicating that the metabolism and disappearance of the efficacy of i-ins-562 are synchronized and will not cause drug accumulation at the injection site.

[0202] Effect Example 4: Study on the hypoglycemic effect of glucose-responsive insulin analog i-ins-562 in diabetic type 1 miniature pigs

[0203] Six-month-old Bama miniature pigs were given an intravenous injection of STZ (150 mg / kg) to induce type 1 diabetes. The miniature pigs were fed twice a day and their blood sugar was controlled with insulin glargine (Lantus). They could be used for research after their blood sugar was stable for 1 month. Blood sugar was monitored using a dynamic blood glucose monitoring system (FreeStyle Libre H, Abbott Laboratories, Inc., USA), and the blood sugar of the miniature pigs was above 200 mg / dL. The blood sugar of the miniature pigs is usually controlled with insulin glargine. After 48 hours of discontinuation of insulin glargine, the glucose-responsive insulin analog i-ins-562 was given to test the blood sugar control effect of the glucose-responsive insulin analog i-ins-562.

[0204] Fig.28 and Fig.29 The results showed that subcutaneous injection of the glucose-responsive insulin analog i-ins-562 at a dose of 0.04 mg / kg in miniature pigs 1 and 2 could reduce blood sugar to below 200 mg / mL, and almost no blood sugar was below 40 mg / dL. The glucose-responsive insulin analog i-ins-562 could regulate blood sugar in the two miniature pigs to below 200 mg / dL for more than 80 hours.

[0205] Fig.30 The results showed that miniature pig No. 3 was subcutaneously injected with a glucose-responsive insulin analog i-ins-562 at a dose of 0.04 mg / kg. 24 hours later, i-ins-562 was subcutaneously injected again at a dose of 0.02 mg / kg. Miniature pig No. 3 regulated blood sugar to below 200 mg / dL for more than 140 hours. Since miniature pigs are more clinically relevant than mice, this long-term blood sugar control ability and postprandial transient hyperglycemia regulation ability verified in the miniature pig model indicate that this glucose-responsive insulin analog is very likely to regulate human blood sugar in the same way.

[0206] The sugar-responsive insulin analogue capable of long-acting insulin release prepared by the present invention is verified by in vitro glucose response and by using mouse and minipig animal models, which confirms that the sugar-responsive insulin analogue of the present invention can release insulin in a sugar-responsive manner and avoid the occurrence of hypoglycemia under overdose conditions.

[0207] Effect Example 5: In vitro cell experiment study

[0208] (1) ATP uptake experiment of glucose-responsive insulin analog (i-ins-562)

[0209] A549 cells were plated in a 96-well plate overnight, and the culture medium was replaced with sugar-free DMEM medium and incubated overnight. The culture medium was removed, and DMEM medium containing i-ins-562 (10 μM) and glucose concentrations of 0.1, 1, and 10 mM was added to the 96-well plate and incubated in an incubator for 1 hour. After 1 hour, the culture medium was removed and the cells were incubated with The cell viability detection system measures chemiluminescence. The results of ATP inhibition are as follows Fig.31 When i-ins-562 was added, the ATP uptake of cells decreased, and with the increase of glucose concentration, the ATP inhibition rate of cells gradually weakened.

[0210] (2) NBDG uptake experiment of glucose-responsive insulin analog (i-ins-756)

[0211] A549 cells were plated in 48-well plates overnight. The culture medium was removed and DMEM medium containing i-ins-756 (20 μM), insulin (20 μM) or phloretin (250 μM) was added to the 48-well plates and incubated in the incubator for 1 hour. After 1 hour, NBDG solution (67 μM) was added to the 48-well plates and incubated for 2 hours. The uptake of NBDG by cells was calculated using flow cytometry ( Fig.32 ). When i-ins-756 was added, the NBDG uptake of cells was reduced.

[0212] Effect Example 6: In vivo glucose response experiment of glucose-responsive insulin analogs (i-ins-756 and i-ins-562)

[0213] C57BL / 6J type I diabetic mice were subcutaneously injected with i-ins-562 and i-ins-756 (1.5 mg / kg). After 4 h of treatment, the mice were intraperitoneally injected with glucose (3.0 g / kg). A 40 μL blood sample was taken and transferred into a 1.5 mL heparin anticoagulant tube. Blood was collected before and at predetermined time intervals (0, 15, 30, 60, and 120 min) after glucose injection. The obtained blood was centrifuged and the plasma insulin level was quantitatively determined by human insulin enzyme-linked immunosorbent assay (ELISA) ( Fig.33 and Fig.34 ).

Claims

1. A glucose-responsive insulin analog, characterized in that It is an insulin modified by the structure shown in Formula I, wherein the modification means that one or more amino groups in the insulin are connected to the structure shown in Formula I, In formula I, A is an active structural fragment of a glucose transporter inhibitor, and m is 1-20.

2. The glucose-responsive insulin analog according to claim 1, characterized in that The glucose-responsive insulin analog satisfies one or more of the following conditions: (1) The plurality is 2 or 3; (2) m is 1-6, for example, 3; (3) A is (4) The insulin is recombinant human insulin, insulin aspart, insulin lispro, insulin detemir, insulin glargine or insulin degludec.

3. The glucose-responsive insulin analog according to claim 1 or 2, characterized in that The structure shown in Formula I is:

4. A method for preparing a sugar-responsive insulin analogue according to any one of claims 1 to 3, characterized in that: It includes the following steps: (1) reacting the intermediate represented by formula II with a condensing agent in a solvent miscible with water in the presence of a base, Wherein, the definitions of A and m are as described in any one of claims 1 to 3; (2) The product obtained in step (1) is mixed with insulin and reacted.

5. The method for preparing a sugar-responsive insulin analogue according to claim 4, characterized in that: The method for preparing the sugar-responsive insulin analog satisfies one or more of the following conditions: (1) In step (1), the condensing agent is N-hydroxysuccinimide or TSTU; (2) In step (1), the molar ratio of the condensing agent to the intermediate of formula II is (0.5-5.0):1, preferably 0.9:1; (3) In step (1), the base is an organic base, preferably N,N-diisopropylethylamine, triethylamine or 4-dimethylaminopyridine; (4) In step (1), the molar ratio of the base to the insulin is (1.0-20.0):1, preferably (1.0-3.0):1, and more preferably 2.2:1; (5) In step (1), the solvent miscible with water is dimethyl sulfoxide or N,N-dimethylformamide; (6) In step (1), the amount of the water-miscible solvent is such that the concentration of the intermediate represented by formula II is 1-200 mg / mL, for example 50 mg / mL; (7) In step (1), the reaction temperature is 4-30°C, preferably 20-25°C, and more preferably room temperature; (8) In step (2), the insulin is as described in claim 2; (9) In step (2), the molar ratio of the intermediate represented by formula II to the insulin is (1-20):1, preferably (1-3):1, for example 2:1; (10) In step (2), the mixing method is to add the reaction solution obtained in step (1) to the aqueous solution of insulin; preferably, the concentration of the aqueous solution of insulin is 0.2-100 mg / mL, for example, 10-15 mg / mL; the pH of the aqueous solution of insulin is preferably 7.4-13.0, more preferably 7.4; (11) In step (2), the reaction temperature may be 0-30°C, preferably 2-6°C, and more preferably 4°C; (12) After the reaction in step (2) is completed, post-treatment is further performed; the post-treatment is preferably dialysis or ion exchange column purification; the dialysis can be performed in deionized water using a dialysis bag, and the molecular weight cutoff of the dialysis bag is preferably 1k-3.5k; the ion exchange column is preferably an anion exchange column or a dextran gel chromatography column.

6. The method for preparing a sugar-responsive insulin analogue according to claim 5, characterized in that: The method for preparing the sugar-responsive insulin analog satisfies one or more of the following conditions: (1) In step (2), the aqueous solution of insulin is a buffer solution containing insulin; (2) In step (2), the preparation of the aqueous solution of insulin comprises the following steps: adding the insulin to a phosphate buffer, deionized water or pure water, adding an acidic agent to adjust the pH to 3.0-4.0 to dissolve the insulin, and then adding an alkaline agent to adjust the pH to 7.4-13.0; wherein the acidic agent is preferably a hydrochloric acid solution, a phosphoric acid solution or an acetic acid solution; and the alkaline agent is preferably a sodium hydroxide solution, a sodium bicarbonate solution or a sodium carbonate solution; (3) After the reaction in step (2) is completed, the post-treatment further comprises purification by high performance liquid chromatography, and the chromatographic conditions are preferably: the chromatographic column is an octadecylsilane bonded column, preferably an XBridge-C 18 Preparative column, length 250 mm, inner diameter 19 mm, filler particle size 5 μm; Mobile phase A was acetonitrile solution containing 0.1 v% trifluoroacetic acid, and mobile phase B was ultrapure water containing 0.1 v% trifluoroacetic acid; Gradient elution is as follows: based on the total volume of the mobile phase as 100%, during 0-60 min, the mobile phase A increases from 30 v% to 40 v%; during 60-65 min, the mobile phase A is maintained at 40 v%; during 65-80 min, the mobile phase A increases from 40 v% to 45 v%; during 80-90 min, the mobile phase A increases from 45 v% to 50 v%; The column temperature is 25°C, the detection wavelengths are 214nm and 280nm, the flow rate of the mobile phase is 8mL / min, and the injection volume can be 1mL; (4) After the reaction in step (2) is completed, the method for preparing the sugar-responsive insulin analog further comprises a freeze-drying step.

7. An intermediate as shown in formula II, in, m and A are defined as in any one of claims 1 to 3; The intermediate shown in formula II is preferably 8. A method for preparing an intermediate as shown in formula II, characterized in that: The method comprises the following steps: in a solvent, The presence of alkali Under the condition, the compound shown in formula III is subjected to the hydrolysis reaction shown below to prepare the intermediate shown in formula II; Among them, R 1 C 1-6 Alkyl, m and A are as defined in any one of claims 1 to 3; The solvent is preferably an ether solvent, such as THF; the base is preferably an alkali metal hydroxide, such as potassium hydroxide; the molar ratio of the base to the compound represented by formula III is preferably 5:1; and the reaction temperature of the hydrolysis reaction is preferably room temperature.

9. The method for preparing the intermediate of formula II as claimed in claim 8, characterized in that: The preparation method of the compound shown in formula III comprises the following steps: in a solvent, in the presence of a base, subjecting the compound shown in formula IV and the compound shown in formula V to a substitution reaction as shown below to obtain the compound shown in formula III; Among them, R 2 for R 1 As claimed in claim 8, the definitions of m and A are as described in any one of claims 1 to 3; The solvent is preferably an amide solvent, such as DMF; the base is preferably an alkali metal carbonate, such as potassium carbonate; the molar ratio of the base to the compound shown in formula IV is preferably 1.5:1; the molar ratio of the compound shown in formula IV to the compound shown in formula IV is preferably (1.2-2.0):1; the reaction temperature of the substitution reaction is preferably 60°C.

10. A compound as shown below:

11. A pharmaceutical composition, characterized in that The invention comprises the sugar-responsive insulin analogue according to any one of claims 1 to 3 and a pharmaceutical excipient.

12. Use of the sugar-responsive insulin analogue according to any one of claims 1 to 3 in the preparation of a drug for treating diabetes; the diabetes may be type 1 diabetes or type 2 diabetes, preferably type 1 diabetes or late-stage type 2 diabetes.

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