WO2022099991A1

WO2022099991A1 - Polypeptide, derivative thereof, hydrogel, and application thereof in preparing drug for preventing and/or treating type i diabetes

Info

Publication number: WO2022099991A1
Application number: PCT/CN2021/086779
Authority: WO
Inventors: 杨志谋; 王玲; 王忠彦; 李宸; 刘默涵
Original assignee: 南开大学; 中国医学科学院生物医学工程研究所
Priority date: 2020-11-12
Filing date: 2021-04-13
Publication date: 2022-05-19
Also published as: CN112358529A; CN112358529B

Abstract

The present invention belongs to the field of medicine and pharmacy, and provides a polypeptide, a derivative thereof, a hydrogel, and an application in preparing a drug for preventing and/or treating type I diabetes. The present invention provides a polypeptide whose sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and a derivative of the polypeptide and a hydrogel thereof. The polypeptide, the derivative thereof and the hydrogel preparation process of the present invention are simple, the chemical structure of the product is clear, the raw materials used are amino acids daily required for the human body, the costs are low, and biocompatibility is good. The polypeptide, the derivative thereof and the hydrogel are capable of mimicking insulin epitopes, can restore immune tolerance to insulin antigens, have high ability to clear blood sugar and keep blood sugar stable within a normal range, and can effectively reduce the incidence of type 1 diabetes.

Description

Polypeptide and its derivative and hydrogel, and its application in the preparation of medicament for preventing and/or treating type I diabetes

technical field

The present invention relates to the field of medicine and pharmacy, in particular to a polypeptide and its derivatives and hydrogel, and its application in the preparation of medicines for preventing and/or treating type I diabetes.

Background technique

Diabetes is one of the biggest global public health events of the 21st century. The latest statistics in 2015 show that there are 415 million adults with diabetes in the world, of which more than 100 million people have diabetes in my country, accounting for about 10% of the country's total population. Globally, the incidence of type I diabetes is increasing year by year, with an annual increase of about 2 to 3%. There are data reports that in the past 20 years in China, the incidence of diabetes has shown a trend of younger people, and the incidence of diabetes in children and adolescents under the age of 15 has increased by nearly four times. Due to the serious complications and economic burden brought by type 1 diabetes, there are only 105 cases in China with an age of more than 30 years. Because diabetes is often accompanied by serious complications, the average life expectancy of type 1 diabetes patients will be reduced by 10 to 15 years.

The immunopathogenesis of type I diabetes is T cell-mediated attack on beta cells. Abnormally activated T cells selectively attack their own β cells with insulin secretion function. With the progress of this autoimmune process, the number of pancreatic β cells is greatly reduced, which eventually leads to a severe lack of insulin secretion, resulting in glucose metabolism dysfunction. Glucose, which is difficult to use in the patient's body, circulates in the blood for a long time. Over time, excessive levels of glucose in the blood cause damage to various tissues in the body, causing involvement of the heart, kidneys, liver, nerves, eyes and other vital organs. complication.

At present, the mainstream of the treatment of type 1 diabetes is still supplementing exogenous insulin. Over the past 30 years, researchers have introduced a variety of insulin formulations, the development of insulin analogs, self-monitoring of glucose levels, insulin pumps and, more recently, insulin sensor technology for glucose monitoring, enabling both the timing and effectiveness of insulin to act. There has been a great improvement, further strengthening the blood sugar control of diabetic patients. Even though the introduction of these novel treatment concepts significantly reduces the risk of secondary and chronic complications, less than one-third of patients ultimately achieve prevention of secondary end-organ complications such as retinal, renal, and neurological complications. systemic disease) required clinical care goals. Current intensive insulin therapy regimens inject or use insulin pumps according to established algorithms, and can establish a simulated physiological secretion pattern of continuous subcutaneous insulin infusion (CSII), but it is still difficult to achieve ideal results in clinical applications.

Type 1 diabetes is an autoimmune disease, and its immunotherapy has been the focus of basic and clinical research in the past few decades. The mechanism of β-cell destruction as the basis of immunotherapy has also been further explored. With an improved understanding of the pathogenesis of diabetes, it is possible to predict the risk of developing diabetes by detecting autoantibodies as biomarkers for the development of autoimmunity before the diagnosis of type 1 diabetes in humans, and the insulin required to prevent or reverse the autoimmunity of type 1 diabetes Epitopes are immunized.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a polypeptide and its derivatives and hydrogels capable of simulating insulin antigen epitopes and restoring immune tolerance to insulin antigen, and the application in the preparation of medicines for preventing and/or treating type I diabetes.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a polypeptide, and the polypeptide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

The structural formula of the sequence shown in SEQ ID NO: 1 is:

The structural formula of the sequence shown in SEQ ID NO: 2 is:

The structural formula of the sequence shown in SEQ ID NO: 3 is:

The present invention also provides derivatives of the polypeptides, wherein the derivatives are capping groups attached to the N-terminus of the polypeptides.

Preferably, the end capping group is an acetyl group.

Preferably, the end-capping group is formed by connecting the compound containing an aromatic ring to the N-terminus of the polypeptide through an amide bond to form an end-capping group.

The present invention also provides a hydrogel of the derivative, and the preparation method of the hydrogel is as follows: placing the derivative in a buffer, adjusting the pH to 6.0-8.0, heating to dissolve, cooling, and preparing A hydrogel containing the derivative was obtained.

Preferably, the mass-volume ratio of the derivative to the buffer is 1 μg: 0.8-1.2 μL.

Preferably, the buffer is a PBS buffer with a pH of 5.0-9.0.

Preferably, the hydrogel is a supramolecular hydrogel.

The present invention also provides the application of the polypeptide, the derivative or the hydrogel in the preparation of a medicament for preventing and/or treating type I diabetes.

Preferably, the medicine is an injection or an oral preparation.

The beneficial effects of the technical solutions provided by the present invention at least include:

1. The preparation process of the present invention is simple, the chemical structure of the product is clear, the raw materials used are amino acids necessary for the human body every day, and the polypeptide derivatives can be prepared by the method of solid-phase synthesis;

2. The polypeptide and its derivatives can simulate the epitope of insulin and restore the immune tolerance to insulin antigen. Through the intraperitoneal glucose tolerance test, it is found that its sensitivity to glucose is better, and the ability to clear blood sugar is higher, The islet function is not damaged, it can better cope with the glycemic load and keep the blood sugar stable within the normal range.

3. The hydrogel formed by the polypeptide and its derivatives can effectively reduce the incidence of type I diabetes and maintain the stable blood glucose level in plasma.

Description of drawings

Figure 1 shows the incidence curve of NOD mice;

Figure 2 is the change curve of blood glucose level in NOD mice;

Fig. 3 is the intraperitoneal glucose tolerance test result of NOD mice;

Figure 4 shows the level of Treg cells (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ ) in the spleen of NOD mice.

Detailed ways

The structural formula of the sequence shown in SEQ ID NO: 1 is:

The structural formula of the sequence shown in SEQ ID NO: 2 is:

The structural formula of the sequence shown in SEQ ID NO: 3 is:

In the present invention, the amino acid sequence described in SEQ ID NO: 1 is a polypeptide (GFFY) composed of four amino acids Gly, Phe, Phe, and Tyr; the amino acid sequence described in SEQ ID NO: 2 is composed of Phe, A dipeptide (FF) composed of two amino acids of Phe; the amino acid sequence described in SEQ ID NO: 3 is a polypeptide (GFAY) composed of four amino acids of Gly, Phe, Ala, and Tyr. Among them, GFFY and FF are the characteristic peptides of insulin B:9-23, which can mimic insulin antigen epitopes and restore immune tolerance to insulin antigens.

The present invention also provides derivatives of said polypeptides, said derivatives preferably having a capping group attached to the N-terminus of said polypeptide.

In the present invention, the end-capping group is preferably an acetyl group.

In the present invention, the end-capping group preferably connects the compound containing an aromatic ring to the N-terminus of the polypeptide through an amide bond to form an end-capping group.

In the present invention, the compound containing an aromatic ring is preferably 2-naphthaleneacetic acid or 2-(6-methoxy-2-naphthalene)propionic acid, more preferably 2-naphthaleneacetic acid.

In the present invention, the polypeptide or the derivative is synthesized by Fmoc-short peptide solid-phase synthesis method.

The present invention also provides a hydrogel containing the derivative. The preparation method of the hydrogel is as follows: placing the derivative in a buffer, adjusting the pH to 6.0-8.0, heating to dissolve, and cooling, that is, A hydrogel containing the derivative was prepared.

In the present invention, the mass-volume ratio of the derivative to the buffer is preferably 1 μg:0.8-1.2 μL, more preferably 1 μg:1 μL.

In the present invention, the buffer is preferably a PBS buffer, and the pH is preferably 5.0 to 9.0, more preferably 6.0 to 8.0, and still more preferably 7.4.

In the present invention, the derivative is placed in a buffer, and the pH is preferably adjusted to 6.0 to 8.0, more preferably 7.4.

In the present invention, the heating is preferably heated to boiling to completely dissolve the derivative.

In the present invention, the cooling is preferably cooling to room temperature (25°C).

In the present invention, the hydrogel is preferably a supramolecular hydrogel. When the concentration of the derivative of the polypeptide in solution reaches a millimolar level, a supramolecular hydrogel can be formed. The so-called supramolecular hydrogel is a gel formed by the aggregation of small molecular compounds with a molecular weight of less than 2000 through non-covalent bonds, self-assembly to obtain a network structure and encapsulation of water molecules. The hydrogel prepared by the invention has good solubility, and can form colorless and transparent hydrogel.

The present invention also provides the application of the polypeptide, the derivative of the polypeptide or the hydrogel in the preparation of a medicament for preventing and/or treating type I diabetes.

In the present invention, the drug is preferably an injection or an oral preparation.

The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

The formulation sources involved in the following examples are as follows:

2-Cl-Trt resin was purchased from Tianjin Nankai Hecheng Technology Co., Ltd., with an activity of 1.2 mmol/mL;

N,N-diisopropylethylamine (represented by DIEPA below), purchased from Adamas, with a purity of 99%;

Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (represented by HBTU below), purchased from Sigma-Aldrich, with a purity of 98%;

Trifluoroacetic acid (represented by TFA below), purchased from Sigma-Aldrich, with a purity of 99%;

Triisopropylsilane (represented by TIS below), purchased from Sigma-Aldrich, with a purity of 99%;

Anhydrous dichloromethane (represented by DCM below), Tianjin Chemical Reagent Company;

N,N-Dimethylformamide (represented by DMF below), Tianjin Chemical Reagent Company;

Methanol, Tianjin Concord Technology Company;

Piperidine, Tianjin Chemical Reagent Company;

DMF containing 20% piperidine by volume (20% piperidine + 80% DMF);

All amino acids were purchased from Gill Biochemical (Shanghai) Co., Ltd. with a purity of 98%;

Naphthalene acetic acid was purchased from Sigma-Aldrich, with a purity of 99%;

Insulin B: 9-23, purchased from China Peptide Bio, with a purity of 99%;

NOD/ShiLtJNju mice, 3 weeks old, female, were purchased from Nanjing University-Nanjing Institute of Biomedicine.

Example 1

(1) Synthesis of polypeptide derivative Nap-G ^D F ^D F ^D Y

This example provides a derivative of the polypeptide (Gly-D-Phe-D-Phe-D-Tyr, G ^D F ^D F ^D Y) whose sequence is shown in SEQ ID NO: 1, and its structural formula is:

In this derivative, 2-naphthylacetic acid (Nap) is connected to the N-terminus of the polypeptide as a capping group to generate a derivative with a D configuration. For the convenience of description, the D configuration of the derivative is described as Nap-G ^D F ^D F ^D Y in the present invention.

The polypeptide derivative Nap-G ^D F ^D F ^D Y of this example was synthesized by Fmoc-short peptide solid-phase synthesis method. Specific steps are as follows:

1) Weigh 0.5 mmol of 2-Cl-Trt resin in a solid-phase synthesizer, add 10 mL of anhydrous dichloromethane (represented by DCM below), place it on a shaking table and shake for 5 min to fully swell the 2-Cl-Trt resin ;

2) Use ear washing balls to remove DCM from the solid-phase synthesizer equipped with 2-Cl-Trt resin;

3) Dissolve 0.75 mmol of Fmoc-protected amino acid (Fmoc-D-Tyr(OtBu)-OH) in 10 mL of anhydrous DCM, add 0.75 mmol of DIEPA, then transfer to the above solid-phase synthesizer, and add 0.75 mmol of DIEPA, reacted at room temperature for 1h;

4) Blocking: remove the reaction solution in the solid-phase synthesizer with ear washing balls, then wash with 10 mL of anhydrous DCM for 1 min each, wash 5 times in total, add the prepared volume ratio of anhydrous DCM:DIEPA:methanol= 17:1:2 solution 20mL, react at room temperature for 10min;

5) Remove the reaction solution in the solid-phase synthesizer with ear-washing balls, first wash with anhydrous DCM, use 10 mL of DCM each time, wash for 1 min, wash 5 times in total, and then wash with N,N-dimethylformamide ( The following is represented by DMF) washing, the amount of DMF is 10 mL each time, and the washing time is 1 min, washing 5 times in total, adding 10 mL of DMF containing 20% piperidine by volume, reacting for 25 minutes, and then using 10 mL of piperidine containing 20% by volume. The DMF reaction of pyridine was carried out for 5 min, and then washed with DMF, the amount of DMF was 10 mL each time, and the washing time was 1 min, washed 5 times in total, and the next step was carried out;

6) the added amino acid (Fmoc-D-Phe-OH) 1mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mL DMF of the second Fmoc protection, add the prepared solution into the above-mentioned solid-phase synthesizer, react 2h;

7) Repeat steps 5) and 6) to sequentially add Fmoc-D-Phe-OH, Fmoc-Gly-OH and end-capping group (2-naphthaleneacetic acid); then wash with DMF for 5 times and dichloromethane for 5 times, and proceed to the next step;

8) 10 mL of a solution consisting of 95% TFA, 2.5% TIS, and 2.5% H ₂ O by volume was added to the above solid-phase synthesizer, reacted for half an hour, and the product was cut from the 2-Cl-Trt resin, Concentrate in vacuo and remove the solvent to give the crude product, which is then separated and purified by HPLC to prepare Nap-G ^D F ^D F ^D Y.

It should be noted that, in step 8), the step of "adding 10 mL of a solution consisting of 95% TFA, 2.5% TIS, 2.5% H2O by volume percentage to the above solid-phase synthesizer, and reacting for half an hour" can also be performed by adding TFA According to the volume ratio of 1:99 with DCM, a TFA solution with a TFA volume percentage concentration of 1% was prepared, and each 3 mL of the TFA solution was added to the above-mentioned solid-phase synthesizer for ten times, and each reaction time was 1 min. operation to complete.

(2) Preparation of Polypeptide Derivative Hydrogel of this Example

Take 0.5 mg of D-configuration short peptide Nap-G ^D F ^D F ^D Y and put it in a 1.5 ml glass bottle, then add 500 microliters of PBS solution (pH=7.4), and use sodium carbonate solution to equalize its pH value. Adjusted to 7.4, heated to boiling to completely dissolve the compound, and cooled to room temperature to obtain the Nap-G ^D F ^D F ^D Y short peptide hydrogel pharmaceutical preparation.

Example 2

(1) Synthesis of polypeptide derivative Nap-GFFY

This embodiment provides a derivative of the polypeptide (Gly-Phe-Phe-Tyr, GFFY) whose sequence is shown in SEQ ID NO: 1, and its structural formula is:

In this derivative, 2-naphthylacetic acid (Nap) is connected to the N-terminus of the polypeptide as a capping group to generate a derivative with an L configuration. For the convenience of description, the derivative is described as Nap-GFFY or Nap-GL F ^L F ^L Y in the present invention ^.

The polypeptide derivative Nap-GFFY of this example was synthesized by Fmoc-short peptide solid-phase synthesis method. Specific steps are as follows:

3) Dissolve 0.75 mmol of Fmoc-protected amino acid (Fmoc-Tyr(OtBu)-OH) in 10 mL of anhydrous DCM, add 0.75 mmol of DIEPA, then transfer to the above solid-phase synthesizer, and add 0.75 mmol of DIEPA, reacted at room temperature for 1h;

5) Remove the reaction solution in the solid-phase synthesizer with ear-washing balls, first wash with anhydrous DCM, use 10 mL of DCM each time, wash for 1 min, wash 5 times in total, and then wash with N,N-dimethylformamide ( The following is represented by DMF) washing, the amount of DMF is 10 mL each time, and the washing time is 1 min, washing 5 times in total, adding 10 mL of DMF containing 20% piperidine by volume, reacting for 25 minutes, and then using 10 mL of piperidine containing 20% by volume. The DMF reaction of pyridine was carried out for 5 min, and then washed with DMF. The dosage of DMF was 10 mL each time, and the washing time was 1 min. After washing 5 times, the next step was carried out;

6) amino acid (Fmoc-Phe-OH) 1mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mL DMF of the second Fmoc protection added, add the prepared solution into the above-mentioned solid-phase synthesizer, react 2h;

7) Repeat steps 5) and 6) and add Fmoc-Phe-OH, Fmoc-Gly-OH and end-capping group (2-naphthaleneacetic acid) in turn; then wash with DMF for 5 times and dichloromethane for 5 times, Carry out the next reaction;

8) 10 mL of a solution consisting of 95% TFA, 2.5% TIS, and 2.5% H ₂ O by volume was added to the above solid-phase synthesizer, reacted for half an hour, and the product was cut from the 2-Cl-Trt resin, Concentration in vacuo and removal of solvent gave crude product, which was then separated and purified by HPLC to prepare Nap-GFFY.

(2) Preparation of Polypeptide Derivative Hydrogel of this Example

Take 0.5 mg of L-configuration short peptide Nap-GFFY into a 1.5 ml glass bottle, add 500 microliters of PBS solution (pH=7.4), adjust its pH to 7.4 with sodium carbonate solution, and heat it to boiling The compound was completely dissolved, and the Nap-GFFY short peptide hydrogel pharmaceutical preparation was obtained after cooling to room temperature.

Example 3

(1 ⁾ Synthesis of Polypeptide Derivative Nap- ^DFDF

This example provides a derivative of a polypeptide (D-Phe-D-Phe, ^D F ^D F) whose sequence is shown in SEQ ID NO: 2, and its structural formula is:

And the short peptide Nap- ^DFDF was prepared according to the Fmoc-solid phase synthesis method provided in Example 1 ^.

(2) Take 0.5mg ^Nap - ^DFDF into a 1.5ml glass bottle, add 500 microliters of PBS solution (pH=7.4), adjust its pH to 7.4 with sodium carbonate solution, heat to boiling to make the compound After being completely dissolved and cooled to room temperature, the ^Nap - ^DFDF short peptide hydrogel pharmaceutical preparation is obtained.

Example 4

(1) Synthesis of Polypeptide Derivative Ac-G ^D F ^D F ^D Y

In this derivative, acetic acid (Ac) is connected to the N-terminus of the polypeptide as a capping group to generate a derivative with a D configuration. For the convenience of description, the D configuration of the derivative is described as Ac-G ^D F ^D F ^D Y in the present invention. And according to the Fmoc-solid phase synthesis method of Example 1, Ac-G ^D F ^D F ^D Y was synthesized.

(2) Take 0.5 mg of Ac-G ^D F ^D F ^D Y into a 1.5 ml glass bottle, add 500 microliters of PBS solution (pH=7.4), adjust its pH to 7.4 with sodium carbonate solution, heat The compound is completely dissolved to boiling, and the polypeptide solution pharmaceutical preparation is obtained after cooling to room temperature.

Example 5

(1) Synthesis of polypeptide derivative Nap-G ^D F ^D A ^D Y

This example provides a derivative of the polypeptide (Gly-D-Phe-D-Ala-D-Tyr, G ^D F ^D A ^D Y) whose sequence is shown in SEQ ID NO: 1, and its structural formula is:

In this derivative, 2-naphthylacetic acid (Nap) is connected to the N-terminus of the polypeptide as a capping group to generate a derivative with a D configuration. For the convenience of description, the derivative is described as Nap-G ^D F ^D A ^D Y in the present invention.

The polypeptide derivative Nap-G ^D F ^D A ^D Y of this example was synthesized by Fmoc-short peptide solid-phase synthesis method. Specific steps are as follows:

6) amino acid (Fmoc-D-Ala-OH) 1mmol, HBTU 1.5mmol, DIEPA 2mmol and 10mL DMF of the second Fmoc protection added, add the prepared solution into the above-mentioned solid-phase synthesizer, react 2h;

8) 10 mL of a solution consisting of 95% TFA, 2.5% TIS, and 2.5% H ₂ O by volume was added to the above solid-phase synthesizer, reacted for half an hour, and the product was cut from the 2-Cl-Trt resin, Concentrate in vacuo and remove solvent to give crude product, which is then separated and purified by HPLC to prepare Nap-G ^D F ^D A ^D Y.

(2) Preparation of Polypeptide Derivative Hydrogel of this Example

Take Nap-G ^D F ^D A ^D Y into a 1.5 ml glass bottle, add 500 microliters of PBS solution (pH=7.4), adjust the pH value to 7.4 with sodium carbonate solution, and heat to boiling to make the compound After completely dissolving and cooling to room temperature, the Nap-G ^D F ^D A ^D Y short peptide solution pharmaceutical preparation is obtained.

Comparative Example 1

Preparation of Alum+InsulinB:9-23 (ie Alum+Ins2 _9-23 ) of Alum-adjuvanted Diabetic Vaccine

(1) Take 1 mg of Insulin B:9-23 polypeptide with a purity of 99% purchased from China Peptide Biology and place it in a 1.5 ml glass bottle, add 400 microliters of PBS solution (pH=7.4), and adjust its pH with sodium carbonate solution The value was adjusted to 7.4 to allow complete dissolution to obtain a solution of Insulin B:9-23 polypeptide with a concentration of 2.5 mg/mL.

(2) Take 62.5 microliters of 200 mg/mL aluminum adjuvant, and dilute to 250 microliters with a PBS solution (pH=7.4) to obtain an aluminum adjuvant dispersion.

(3) 200 microliters of 2.5 mg/mL Insulin B:9-23 polypeptide solution was added to the aluminum adjuvant dispersion prepared in (2), and the volume was adjusted to 500 microliters with PBS solution (pH=7.4), Physical mixing was performed, and the resulting mixture was the aluminum-adjuvanted diabetes vaccine Alum+Insulin B: 9-23. (The final concentration of aluminum adjuvant was 25 mg/mL, and the concentration of Insulin B:9-23 polypeptide was 1 mg/mL).

Comparative Example 2

Sterile 1x PBS

Weigh 8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ and 0.24g KH ₂ PO ₄ , dissolve them in 800 mL of distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to 1L. After sterilizing in an autoclave, store at room temperature (25°C) or in a 4°C refrigerator.

Experimental Example 1 Immunoassay

After 3-week-old NOD mice were stabilized in the new rearing environment for one week, they were randomly divided into groups from 4-week-old mice, and 20 mice in each group were given the drug formulation, with a total volume of 80 μL 1 mg/time subcutaneously injected into the axilla. mL of Nap-G ^D F ^D F ^D Y, Nap-GFFY, Nap- ^D F ^D F, Nap-G ^D F ^D A ^D Y, Ac-G ^D F ^D F ^D Y, Alum+Insulin B:9- 23. Once a week, the control group was injected with 80 μL of sterile 1×PBS. After 5 weeks of continuous dosing, use One weekly starting at Week 11

A blood glucose meter (Lifescan, USA) was used to measure blood glucose once, and the blood glucose level statistics were shown in Figure 2. If the value was above 11.1 mmol/L for two consecutive days, the animal was considered diabetic. The incidence of mice in each group was recorded and the incidence rate was counted. The results are shown in Figure 1.

It can be seen from Figure 1 that by the 36th week, all the mice in the Control group had the disease, and the onset time started from the 11th week, while the Nap-G ^D F ^D F ^D Y group had no disease in the whole process, and the Nap-G ^D F ^D A The incidence rates of ^D Y, Nap-GFFY, Nap- ^D F ^D F, Ac-G ^D F ^D F ^D Y, Alum+Insulin B: 9-23 groups were 60%, 60%, 30%, 30%, 15%, respectively %, and the onset time was later than that of the Control group, the earliest was in the 14th week. Combined with the level of Treg cells in the spleen of mice in Figure 4, it is shown that the polypeptide and its derivatives and hydrogel provided by the present invention can reduce the incidence of type 1 diabetes in mice and delay the onset time of type 1 diabetes in mice. The Nap-G ^D F ^D F ^D Y group had a lower incidence than Nap-G ^D F ^D A ^D Y and Nap- ^D F ^D F indicating the importance of the GFFY amino acid sequence (as shown in SEQ ID NO: 1). The incidence of Nap-G ^D F ^D F ^D Y, Nap- ^D F ^D F, and Ac-G ^D F ^D F ^D Y was significantly lower than that of Nap-GFFY. It can be seen that the water formed by D-configuration polypeptides The effect of the gel in inhibiting the incidence is significantly better than that of the hydrogel formed by the L-configuration polypeptide (although only the implementation of the D configuration and the L configuration of the Nap-G ^D F ^D F ^D Y and Nap-GFFY groups is provided in this application. Examples and experimental examples are used to illustrate this problem, but after experiments, such a phenomenon also exists in the Nap- ^D F ^D F and Nap-FF, Ac-G ^D F ^D F ^D Y and Ac-GFFY groups, so it is concluded that D The conclusion that the conformation polypeptide is better than the L conformation polypeptide in inhibiting the morbidity rate). The incidence of the Nap-G ^D F ^D F ^D Y group was lower than that of the Ac-G ^D F ^D F ^D Y group. It can be seen that the hydrogel formed by self-assembly is better than the unassembled polypeptide solution.

As can be seen from Figure 2, the blood glucose level of the Control group began to gradually increase slowly from the 11th week, and then decreased in the 20th week, and then the blood glucose value increased rapidly from the 25th week to the 36th week. The final blood glucose value reached 27mmol /L; while the blood glucose level in the Nap-G ^D F ^D F ^D Y group remained stable at 7 mmol/L; the blood glucose level in the Nap-G ^D F ^D A ^D Y group began to rise at the 16th week, and from the 20th week decreased until the 26th week and remained stable, and the final blood glucose value reached 12mmol/L by the 36th week; the blood glucose value of the Ac-G ^D F ^D F ^D Y group and Alum+Insulin B:9-23 group began to rise at the 14th week. , decreased from the 24th week, remained stable after the 25th week, and finally reached 7 mmol/L in the 36th week; the blood glucose value of the Nap-GFFY group increased in the 14th, 25th and 32nd weeks. There were three large fluctuations and the final blood glucose value at 36 weeks was 17mmol/L; the blood glucose value of Nap- ^DFDF gradually increased at 16 weeks, reached a maximum of 16mmol/L at 23 weeks, then decreased, and returned to ^7mmol /L at 24 weeks , and keep it steady. It shows that the polypeptides and their derivatives and hydrogels provided by the present invention can better cope with the glycemic load, have a high scavenging ability to blood sugar, and maintain a stable plasma blood sugar at a certain level, especially Nap-G ^D F ^D F ^D Y Polypeptide hydrogel always keeps blood sugar stable at 7mmol/L, and has a high ability to lower blood sugar and maintain stable plasma blood sugar.

Experimental Example 2 Glucose tolerance test

After 3-week-old NOD mice were stabilized in the new rearing environment for one week, they were randomly divided into groups from 4-week-old mice, and 20 mice in each group were given the drug formulation, with a total volume of 80 μL 1 mg/time subcutaneously injected into the axilla. mL of Nap-G ^D F ^D F ^D Y, Nap-GFFY, Nap- ^D F ^D F, Nap-G ^D F ^D A ^D Y, Ac-G ^D F ^D F ^D Y, Alum+Insulin B:9- 23. Once a week, the control group was injected with 80 μL of sterile 1×PBS for 5 weeks. The intraperitoneal glucose tolerance of NOD mice was measured at 14 weeks of age, respectively. Mice were fasted overnight for 8 hours prior to experimentation. The mice were weighed in advance, and were injected intraperitoneally with sterile glucose injection (10%) at a dose of 2 g/kg. Use at 0, 15, 30, 45, 60, 90, 120 minutes after injection

Blood glucose concentration in tail vein (glucose oxidase method) was measured with a blood glucose meter (Lifescan, USA). As can be seen from Figure 3, the blood glucose levels of all groups reached the highest at 15 minutes, the control group was as high as 20 mmol/L, and the Nap-G ^D F ^D F ^D Y group was less than 15 mmol/L, and then gradually decreased. After 120 minutes, the blood glucose level of the Control group was still around 15mmol/L, Nap-GFFY, and Nap-G ^D F ^D A ^D Y recovered to 10 mmol/L, Nap- ^D F ^D F and Ac-G ^D F ^D F ^D Y recovered to about 7 mmol/L, Nap-G ^D F ^D F ^D Y and Alum+Insulin B: 9-23 group recovered to below 10 mmol/L after 30 minutes, and basically recovered to the level before glucose injection after 90 minutes level, roughly 5mmol/L. It shows that the polypeptide and its derivatives provided by the present invention have good sensitivity to glucose, high blood sugar clearance ability, can better cope with blood sugar load, and keep blood sugar stable within the normal range.

Experimental example 3

After 3-week-old NOD mice were stabilized in the new rearing environment for one week, they were randomly divided into groups from 4-week-old mice, and 20 mice in each group were given the drug formulation, with a total volume of 80 μL 1 mg/time subcutaneously injected into the axilla. mL of Nap-G ^D F ^D F ^D Y, Nap-GFFY, Nap- ^D F ^D F, Nap-G ^D F ^D A ^D Y, Ac-G ^D F ^D F ^D Y, Alum+Insulin B:9- 23. Once a week, the control group was injected with 80 μL of sterile 1×PBS. Dosing for 5 consecutive weeks. When the mice were 14 weeks old, the spleens of the mice in each group were taken, ground and dispersed into single cells, filtered through a 70 μm filter, and the filtrate was subjected to gradient centrifugation (800 g/30 minutes). Red blood cells are subsequently removed by lysis with red blood cell lysate. Lymphocytes obtained by centrifugation were first incubated with anti-mouse CD4 antibody and anti-mouse CD25 antibody overnight, and then incubated with Anti-mouse Foxp3 antibody for 30 minutes before flow cytometry analysis. It can be seen from Figure 4 that the polypeptide hydrogels (Nap-G ^D F ^D F ^D Y, Nap-G ^L F ^L F ^L Y, Nap- ^D F ^D F) provided by the present invention can significantly increase the CD4 ⁺ in the spleen The level of CD25 ⁺ Foxp3 ⁺ Treg cells indicated that under the action of polypeptide hydrogel, mice could correct their own immunity and establish immune tolerance to self-antigens. In addition, the mice in the Alum+Insulin B:9-23 group failed to establish immune tolerance to self-antigens, which is also in line with previous reports and clinical trials. The mechanism of aluminum adjuvant therapy for type 1 diabetes is to induce T cells to promote Inflammatory Th1 turns to anti-inflammatory Th2 instead of causing immune tolerance.

It can be seen from the above examples that the polypeptides and their derivatives and hydrogels provided by the present invention can effectively reduce the incidence of type 1 diabetes, delay the onset period of diabetes in NOD mice from 11 weeks to 14 weeks, and the proportion of non-onset diabetes. raised to 40%. And it can control the blood sugar level well; further through the intraperitoneal glucose tolerance test, it is found that it has good sensitivity to glucose, high blood sugar clearance ability, can better cope with glycemic load, and keep blood sugar stable within the normal range. By detecting the level of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Treg cells, it shows that the islet function is not further damaged, and further shows that the polypeptide hydrogel provided by the present invention can correct autoimmunity and establish immune tolerance to self-antigens.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims

A polypeptide, characterized in that the polypeptide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

The structural formula of the sequence shown in SEQ ID NO: 1 is:

The structural formula of the sequence shown in SEQ ID NO: 2 is:

The structural formula of the sequence shown in SEQ ID NO: 3 is:
The derivative of the polypeptide of claim 1, wherein the derivative is a capping group attached to the N-terminus of the polypeptide.
The derivative of claim 2, wherein the capping group is an acetyl group.
The derivative according to claim 2, wherein the end-capping group is formed by connecting the compound containing an aromatic ring to the N-terminus of the polypeptide through an amide bond to form an end-capping group.
The hydrogel containing the derivative of claim 4, characterized in that, the preparation method of the hydrogel is as follows: placing the derivative in a buffer, adjusting the pH to 6.0-8.0, heating to dissolve, cooling, and then preparing A hydrogel containing the derivative was obtained.
The hydrogel of claim 5, wherein the mass-volume ratio of the derivative to the buffer is 1 μg: 0.8-1.2 μL.
The hydrogel according to claim 5 or 6, wherein the buffer is a PBS buffer with a pH of 5.0-9.0.
The hydrogel of claim 5, wherein the hydrogel is a supramolecular hydrogel.
Application of the polypeptide according to claim 1, the derivative according to any one of claims 2 to 4, or the hydrogel according to any one of claims 5 to 8 in the preparation of a drug for preventing and/or treating type I diabetes .
The application according to claim 9, wherein the medicine is an injection or an oral preparation.