CN112358529A

CN112358529A - Polypeptide, derivative and hydrogel thereof, and application of polypeptide, derivative and hydrogel in preparation of medicine for preventing and/or treating type I diabetes

Info

Publication number: CN112358529A
Application number: CN202011264167.0A
Authority: CN
Inventors: 杨志谋; 王玲; 王忠彦; 李宸; 刘默涵
Original assignee: Nankai University; Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Nankai University; Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-02-12
Anticipated expiration: 2040-11-12
Also published as: CN112358529B; WO2022099991A1

Abstract

The invention belongs to the field of medicines and pharmacy, and provides a polypeptide, a derivative and hydrogel thereof, and application of the polypeptide in preparation of a medicine for preventing and/or treating type I diabetes. The invention provides a polypeptide, wherein the polypeptide sequence is shown as SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, and provides derivatives of the polypeptide and hydrogel thereof. The polypeptide, the derivative and the hydrogel provided by the invention have the advantages that the preparation process is simple, the chemical structure of the product is clear, the used raw materials are all amino acids necessary for human bodies every day, the cost is low, and the biocompatibility is good; the polypeptide, the derivative and the hydrogel thereof can simulate insulin epitope, recover the immune tolerance to insulin antigen, have higher capacity of removing blood sugar, keep the blood sugar stable in a normal range and effectively reduce the incidence rate of type I diabetes.

Description

Polypeptide, derivative and hydrogel thereof, and application of polypeptide, derivative and hydrogel in preparation of medicine for preventing and/or treating type I diabetes

Technical Field

The invention relates to the fields of medicine and pharmacy, in particular to a polypeptide, a derivative and hydrogel thereof, and application of the polypeptide, the derivative and the hydrogel in preparation of a medicament for preventing and/or treating type I diabetes.

Background

Diabetes is one of the largest public health events worldwide in the 21 st century. Recent statistical data in 2015 shows that 4.15 hundred million adults suffer from diabetes globally, wherein the number of diabetic patients in China exceeds 1 hundred million people and accounts for about 10 percent of the total population in China. The incidence of type I diabetes mellitus rises year by year in a global range, and is increased by about 2-3% every year. It is reported that the incidence of diabetes is younger in China over the past 20 years, and the incidence of diabetes is nearly 4 times higher in children and adolescents under 15 years of age. Due to the serious complications and economic burden of type I diabetes, there are only 105 cases of patients with age over 30 years in China. Since diabetes is often associated with severe complications, the average life expectancy of type I diabetic patients will be reduced by 10-15 years.

The immune pathogenesis of type I diabetes is T cell mediated attack on beta cells. The abnormally activated T cells selectively attack beta cells with insulin secretion function, and with the progress of the autoimmune process, the number of the beta cells of the pancreatic islets is greatly reduced, so that the insulin secretion is seriously deficient, and the glucose metabolism dysfunction is caused. Glucose, which is difficult to utilize in a patient, circulates in the blood for a long time, and excessively high levels of glucose in the blood cause damage to various tissues in the body over time, causing complications that involve the heart, kidneys, liver, nerves, eyes, and other vital organs.

The mainstream of the current treatment of type I diabetes is to supplement exogenous insulin. During the last 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, that have greatly improved the duration and effectiveness of insulin action, further enhancing glycemic control in diabetic patients. Even though the risk of secondary and chronic complications is significantly reduced by introducing these new treatment concepts, less than one-third of patients will ultimately achieve the clinical care goals required to prevent secondary end organ complications, such as retinal, renal and neurological diseases. Current insulin potentiation regimens inject or use insulin pumps according to established algorithms to establish a simulated physiologic secretion pattern for Continuous Subcutaneous Insulin Infusion (CSII), but it is still difficult to achieve the desired effect in clinical applications.

Type I diabetes is an autoimmune disease, and immunotherapy has been the focus of basic and clinical research for the past few decades. The destruction mechanism of beta cells, which is the basis of immunotherapy, has also been studied more deeply. With the development of understanding of the pathogenesis of diabetes, the risk of diabetes can be predicted by detecting autoantibodies as biomarkers of autoimmune development before diagnosis of type I diabetes in humans, and insulin epitopes required for preventing or reversing type I diabetes autoimmunity are immunized.

Disclosure of Invention

The invention aims to provide a polypeptide capable of simulating an insulin epitope and recovering the immunological tolerance to an insulin antigen, a derivative and a hydrogel thereof, and application of the polypeptide in preparing a medicament for preventing and/or treating type I diabetes.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a polypeptide, wherein the polypeptide sequence is shown as SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence shown as SEQ ID NO: 1 has the structural formula as follows:

the nucleotide sequence shown as SEQ ID NO: 2 is represented by the structural formula:

the nucleotide sequence shown as SEQ ID NO: 3 is represented by the structural formula:

the invention also provides a derivative of the polypeptide, wherein the derivative is formed by connecting a blocking group at the N end of the polypeptide.

Preferably, the end-capping group is acetyl.

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

The invention also provides a hydrogel of the derivative, and the preparation method of the hydrogel comprises the following steps: and (3) placing the derivative in a buffer solution, adjusting the pH to 6.0-8.0, heating to dissolve, and cooling to obtain the hydrogel containing the derivative.

Preferably, the mass-volume ratio of the derivative to the buffer solution is 1 mug: 0.8-1.2 muL.

Preferably, the buffer solution is PBS buffer solution, and the pH value is 5.0-9.0.

Preferably, the hydrogel is a supramolecular hydrogel.

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

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

The technical scheme provided by the invention at least has the following beneficial effects:

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

2. the polypeptide and the derivatives thereof can simulate insulin epitope and restore the immune tolerance to insulin antigen, and intraperitoneal glucose tolerance experiments show that the polypeptide has good sensitivity to glucose, high capacity of clearing blood sugar and no damage to the function of pancreatic islets, can better respond to blood sugar load and keep the blood sugar stable in a normal range.

3. The hydrogel formed by the polypeptide and the derivatives thereof effectively reduces the incidence rate of type I diabetes and maintains the blood glucose value of blood plasma to be stable.

Drawings

FIG. 1 is a NOD mouse incidence curve;

FIG. 2 is a blood glucose level variation curve of NOD mice;

FIG. 3 shows the results of intraperitoneal glucose tolerance measurements in NOD mice;

FIG. 4 shows Treg cells in spleen of NOD mice (CD 4)⁺CD25⁺Foxp3⁺) The level of (c).

Detailed Description

in the present invention, the amino acid sequence shown in SEQ ID NO: 1 is polypeptide (GFFY) consisting of four amino acids of Gly, Phe and Tyr; as shown in SEQ ID NO: 2 is dipeptide (FF) consisting of two amino acids of Phe and Phe; as shown in SEQ ID NO: 3 is polypeptide (GFAY) consisting of four amino acids of Gly, Phe, Ala and Tyr. Wherein GFFY and FF are taken as insulin B:9-23, can simulate insulin epitope and restore the immunological tolerance to insulin antigen.

The invention also provides derivatives of the polypeptides, preferably with a blocking group attached to the N-terminus of the polypeptide.

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

In the present invention, the end-capping group is preferably formed by linking an aromatic ring-containing compound to the N-terminus of the polypeptide via an amide bond.

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

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

The invention also provides a hydrogel containing the derivative, and the preparation method of the hydrogel comprises the following steps: and (3) placing the derivative in a buffer solution, adjusting the pH to 6.0-8.0, heating to dissolve, and cooling to obtain the hydrogel containing the derivative.

In the present invention, the mass-to-volume ratio of the derivative to the buffer is preferably 1. mu.g: 0.8 to 1.2. mu.L, and more preferably 1. mu.g: 1. mu.L.

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 solution, and the pH is preferably adjusted to 6.0 to 8.0, and more preferably 7.4.

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

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

In the present invention, the hydrogel is preferably a supramolecular hydrogel. Supramolecular hydrogels can be formed when the concentration of the derivative of the polypeptide in solution reaches the millimolar range. The supermolecule hydrogel is formed by the mutual aggregation of small molecular compounds with the molecular weight of less than 2000 through non-covalent bond interaction, self-assembly to obtain a network structure and wrapping water molecules. The hydrogel prepared by the method has good solubility, and can form colorless and transparent hydrogel.

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

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

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

The sources of the formulations referred to in the following examples are as follows:

the 2-Cl-Trt resin is purchased from Tianjin Nankai and science and technology Limited and has the activity of 1.2 mmol/mL;

n, N-diisopropylethylamine (DIEPA below) was purchased from Adamas, Inc. (Adamas) with a purity of 99%;

benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (hereinafter HBTU) was purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 98%;

trifluoroacetic acid (hereinafter indicated as TFA), purchased from Sigma Aldrich (Sigma-Aldrich), 99% pure;

triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;

anhydrous dichloromethane (hereinafter represented by DCM), tianjin chemical company;

n, N-dimethylformamide (hereinafter referred to as DMF), tianjin chemical company;

methanol, Tianjin Cordcord science and technology;

piperidine, Tianjin chemical Agents;

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

all amino acids were purchased from gill biochemical (shanghai) ltd with a purity of 98%;

naphthylacetic acid was purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;

9-23 Insulin B, purchased from national peptide biology, 99% purity;

NOD/ShiLtJNju mice, 3 weeks old, female, purchased from Nanjing university, Nanjing biomedical research institute.

Example 1

(1) Polypeptide derivative Nap-G^DF^DF^DSynthesis of Y

This example provides a polypeptide having a sequence set forth in SEQ ID NO: 1 (Gly-D-Phe-D-Phe-D-Tyr, G)^DF^DF^DY) of the formula:

the derivative is formed by connecting 2-naphthylacetic acid (Nap) as a blocking group at the N end of the polypeptide to generate a derivative with D configuration. For convenience of description, the D configuration of the derivative is described as Nap-G in the invention^DF^DF^DY。

The polypeptide derivative of this example, Nap-G^DF^DF^DY is synthesized by adopting an Fmoc-short peptide solid phase synthesis method. The method comprises the following specific steps:

1) weighing 0.5mmol of 2-Cl-Trt resin in a solid phase synthesizer, adding 10mL of anhydrous dichloromethane (hereinafter represented by DCM), placing on a shaking table, shaking for 5min, and fully swelling the 2-Cl-Trt resin;

2) removing DCM from the solid phase synthesizer containing 2-Cl-Trt resin by washing the ear with an ear bulb;

3) dissolving 0.75mmol of Fmoc-protected amino acid (Fmoc-D-Tyr (OtBu) -OH) in 10mL of anhydrous DCM, adding 0.75mmol of DIEPA, transferring to the solid phase synthesizer, supplementing 0.75mmol of DIEPA, and reacting at room temperature for 1 h;

4) and (3) sealing: removing reaction liquid in a solid phase synthesizer by using an aurilave, washing with 10mL of anhydrous DCM for 1min each time for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIEPA to methanol being 17: 1: 2, and reacting at room temperature for 10 min;

5) removing reaction liquid in the solid phase synthesizer by using an aurilave, washing by using anhydrous DCM for 5 times, washing by using N, N-dimethylformamide (hereinafter referred to as DMF) for 10mL each time for 1min, washing for 5 times, adding 10mL of DMF containing 20% by volume of piperidine for reaction for 25min, reacting by using 10mL of DMF containing 20% by volume of piperidine for 5min, washing by using DMF for 1min, washing for 5 times, and carrying out next reaction by using 10mL of DMF for 10mL each time for 1 min;

6) adding 1mmol of second Fmoc-protected amino acid (Fmoc-D-Phe-OH), 1.5mmol of HBTU, 2mmol of DIEPA and 10mL of DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;

7) repeating the steps 5) and 6) and adding Fmoc-D-Phe-OH, Fmoc-Gly-OH and a blocking group (2-naphthylacetic acid) in sequence; then washing the mixture for 5 times by using DMF (dimethyl formamide), washing the mixture for 5 times by using dichloromethane, and carrying out the next reaction;

8) the mixture was diluted with 95% TFA, 2.5% TIS, 2.5% H₂Adding 10mL of solution consisting of O in percentage by volume into the solid phase synthesizer, reacting for half an hour, cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by HPLC to obtain the Nap-G^DF^DF^DY。

It should be noted that, in step 8), "10 mL of solution consisting of 95% TFA, 2.5% TIS and 2.5% H2O by volume percentage is added to the above solid phase synthesizer, and the reaction is carried out for half an hour" also can be carried out by preparing a TFA solution with TFA and DCM in a volume ratio of 1: 99 and having a TFA concentration of 1% by volume percentage, and adding the TFA solution into the above solid phase synthesizer 3mL at a time, and adding ten times, each time the reaction time is 1 min.

(2) Preparation of hydrogel of the polypeptide derivative of this example

Taking 0.5mg of D-configuration short peptide Nap-G^DF^DF^DPlacing Y in a 1.5 ml glass bottle, adding 500 μ l PBS solution (pH 7.4), adjusting pH to 7.4 with sodium carbonate solution, heating to boil to dissolve the compound completely, and cooling to room temperature to obtain Nap-G^DF^DF^DY short peptide hydrogel pharmaceutical formulation.

Example 2

(1) Synthesis of polypeptide derivative Nap-GFFY

This example provides a polypeptide having a sequence set forth in SEQ ID NO: 1 (Gly-Phe-Phe-Tyr, GFFY), the structural formula of which is as follows:

the derivative is formed by connecting 2-naphthylacetic acid (Nap) as a blocking group at the N end of the polypeptide to generate a derivative with an L configuration. For convenience of description, the derivative is described as Nap-GFFY or Nap-G in the present invention^LF^LF^LY。

The polypeptide derivative Nap-GFFY of the present example was synthesized by Fmoc-short peptide solid phase synthesis. The method comprises the following specific steps:

3) dissolving 0.75mmol of Fmoc-protected amino acid (Fmoc-Tyr (OtBu) -OH) in 10mL of anhydrous DCM, adding 0.75mmol of DIEPA, transferring to the solid phase synthesizer, supplementing 0.75mmol of DIEPA, and reacting at room temperature for 1 h;

6) adding 1mmol of second Fmoc-protected amino acid (Fmoc-Phe-OH), 1.5mmol of HBTU, 2mmol of DIEPA and 10mL of DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;

7) repeating the steps 5) and 6) and adding Fmoc-Phe-OH, Fmoc-Gly-OH and a blocking group (2-naphthylacetic acid) in sequence; then washing the mixture for 5 times by using DMF (dimethyl formamide), washing the mixture for 5 times by using dichloromethane, and carrying out the next reaction;

8) the mixture was diluted with 95% TFA, 2.5% TIS, 2.5% H₂Adding 10mL of solution consisting of O in percentage by volume into the solid phase synthesizer, reacting for half an hour, cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by HPLC to obtain the Nap-GFFY.

(2) Preparation of hydrogel of the polypeptide derivative of this example

Putting 0.5mg of L-configuration short peptide Nap-GFFY into a 1.5 ml glass bottle, adding 500 microliters of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain the Nap-GFFY short peptide hydrogel pharmaceutical preparation.

Example 3

(1) Polypeptide derivative Nap-^DF^DSynthesis of F

This example provides a polypeptide having a sequence set forth in SEQ ID NO: 2 (D-Phe-D-Phe,^DF^DF) the structural formula of the derivative is:

and the short peptide Nap-^DF^DF。

(2) 0.5mg of Nap-^DF^DF is put into a 1.5 ml glass bottle, 500 microliter PBS solution (pH 7.4) is added, the pH value is adjusted to 7.4 by sodium carbonate solution, the mixture is heated to boiling to completely dissolve the compound, and the Nap-^DF^DF, preparing the short peptide hydrogel drug preparation.

Example 4

(1) Polypeptide derivative Ac-G^DF^DF^DSynthesis of Y

This example provides the sequences shown in SEQ ID NO: 1 (Gly-D-Phe-D-Phe-D-Tyr, G)^DF^DF^DY) of the formula:

the derivative is formed by connecting acetic acid (Ac) as a blocking group at the N terminal of the polypeptide to generate a derivative with D configuration. For convenience of description, the D configuration of the derivative is adopted in the inventionDescribed as Ac-G^DF^DF^DAnd Y. And Ac-G was synthesized according to Fmoc-solid phase Synthesis method of example 1^DF^DF^DY。

(2) 0.5mg of Ac-G was taken^DF^DF^DAnd placing the Y into a 1.5 ml glass bottle, adding 500 microliters of PBS (pH 7.4), adjusting the pH value to 7.4 by using a sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain the polypeptide solution pharmaceutical preparation.

Example 5

(1) Polypeptide derivative Nap-G^DF^DA^DSynthesis of Y

This example provides a polypeptide having a sequence set forth in SEQ ID NO: 1 (Gly-D-Phe-D-Ala-D-Tyr, G)^DF^DA^DY) of the formula:

the derivative is formed by connecting 2-naphthylacetic acid (Nap) as a blocking group at the N end of the polypeptide to generate a derivative with D configuration. For convenience of description, the derivative is described herein as Nap-G^DF^DA^DY。

The polypeptide derivative of this example, Nap-G^DF^DA^DY is synthesized by adopting an Fmoc-short peptide solid phase synthesis method. The method comprises the following specific steps:

6) adding 1mmol of second Fmoc-protected amino acid (Fmoc-D-Ala-OH), 1.5mmol of HBTU, 2mmol of DIEPA and 10mL of DMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;

8) the mixture was diluted with 95% TFA, 2.5% TIS, 2.5% H₂Adding 10mL of solution consisting of O in percentage by volume into the solid phase synthesizer, reacting for half an hour, cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by HPLC to obtain the Nap-G^DF^DA^DY。

(2) Preparation of hydrogel of the polypeptide derivative of this example

Taking Nap-G^DF^DA^DPlacing Y in a 1.5 ml glass bottle, adding 500 microliters of PBS (pH 7.4), adjusting pH to 7.4 with sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain Nap-G^DF^DA^DY short peptide solution pharmaceutical preparation.

Comparative example 1

Aluminum adjuvanted diabetes vaccine Alum + InsulinB:9-23 (i.e., Alum + Ins 2)_9-23) Preparation of

(1) 1mg of Insulin B:9-23 polypeptide from domestic peptide organisms with a purity of 99% was placed in a 1.5 mL glass vial, 400. mu.l of PBS solution (pH 7.4) was added, and the pH was adjusted to 7.4 with sodium carbonate solution to dissolve it completely, giving a 2.5mg/mL solution of Insulin B:9-23 polypeptide.

(2) 62.5. mu.l of 200mg/mL aluminum adjuvant was added, and the volume was adjusted to 250. mu.l with a PBS solution (pH 7.4), to obtain an aluminum adjuvant dispersion.

(3) 200 microliters of 2.5mg/mL Insulin B:9-23 polypeptide solution was added to the aluminum adjuvant dispersion prepared in (2), and the mixture was dissolved in 500 microliters with a PBS solution (pH 7.4) and physically mixed to obtain an aluminum-adjuvanted diabetic vaccine Alum + Insulin B: 9-23. (the final concentration of aluminum adjuvant was 25mg/mL, and the concentration of Insulin B:9-23 polypeptide was 1 mg/mL).

Comparative example 2

Sterile 1 XPBS

Weighing 8g NaCl, 0.2g KCl and 1.44g Na₂HPO₄And 0.24g KH₂PO₄Dissolving the mixture in 800mL of distilled water, adjusting the pH value of the solution to 7.4 by using HCl, and finally adding distilled water to a constant volume of 1L. After sterilization in an autoclave, the mixture was stored at room temperature (25 ℃ C.) or 4 ℃ in a refrigerator.

EXAMPLE 1 immunoassay

3 week-old NOD mice were kept stable for one week in a new breeding environment, and then randomly divided into groups at 4 weeks of age, and 20 mice in each group were administered with the drug formulation by injecting 80. mu.L of Nap-G in a total volume of 1mg/mL subcutaneously into axillary regions each time^DF^DF^DY、Nap-GFFY、Nap-^DF^DF、Nap-G^DF^DA^DY、Ac-G^DF^DF^DY, Alum + Insulin B:9-23, weekly, in the Control group, 80. mu.L of sterile 1 XPBS was injected. After 5 weeks of continuous dosing, One was administered weekly starting at week 11

Blood glucose was measured once by a glucometer (Lifescan, usa) and the blood glucose level statistics are shown in fig. 2. If the value is higher than 11.1mmol/L for two consecutive days, the animal is considered to have diabetes. And recording the morbidity of each group of mice, and counting the morbidity, wherein the result is shown in figure 1.

As can be seen from FIG. 1, the Control group had total onset by week 36, with onset starting at week 11, and Nap-G^DF^DF^DNo mice developed in group Y all the time, Nap-G^DF^DA^DY、Nap-GFFY、Nap-^DF^DF、Ac-G^DF^DF^DY, Alum + Insulin B:9-23 groups had incidence rates of 60%, 30%, 15%, respectively, and the onset time was earlier at week 14 than that of Control group. The combination of the figure 4 shows that the polypeptide provided by the invention, the derivative thereof and the hydrogel can reduce the incidence rate of the type I diabetes of the mice and delay the incidence time of the type I diabetes of the mice. Nap-G^DF^DF^DThe incidence of disease of the Y group is lower than that of Nap-G^DF^DA^DY and Nap-^DF^DF indicates the importance of the GFFY amino acid sequence (shown in SEQ ID NO: 1). By Nap-G^DF^DF^DY、Nap-^DF^DF、Ac-G^DF^DF^DThe incidence of Y is obviously lower than that of Nap-GFFY, and the effect of inhibiting the incidence of the hydrogel formed by the D-configuration polypeptide is obviously better than that of the hydrogel formed by the L-configuration polypeptide (although only Nap-G is provided in the application)^DF^DF^DExamples and experimental examples of D and L configurations of the Y and Nap-GFFY groups are provided to illustrate the problem, but such a phenomenon has been tested in Nap-^DF^DF, Nap-FF and Ac-G^DF^DF^DY and Ac-GFFY groups are also present, thus giving D configurationThe conclusion that the polypeptide is better in suppressing the incidence of disease than the L-configuration polypeptide). Nap-G^DF^DF^DThe incidence of disease of the Y group is lower than that of Ac-G^DF^DF^DAs seen in panel Y, the hydrogel formed by self-assembly was better than the unassembled polypeptide solution.

As can be seen from FIG. 2, the blood glucose levels in the Control group gradually increased from week 11 to week 20, and then rapidly increased from week 25 to week 36 until the blood glucose level reached 27 mmol/L; and Nap-G^DF^DF^DThe blood sugar value of the group Y is always kept to be 7mmol/L steadily; Nap-G^DF^DA^DThe blood sugar level of group Y starts to rise at week 16, starts to fall from week 20, and keeps stable until week 26, and reaches 12mmol/L at the end of week 36; Ac-G^DF^DF^DThe blood sugar values of the group Y and the group Alum + Insulin B:9-23 start to rise at the 14 th week and decline from the 24 th week until the 25 th week, and the blood sugar values reach 7mmol/L at the 36 th week; the blood glucose values of the Nap-GFFY group increased three times at week 14, week 25 and week 32, with three large fluctuations, and the final blood glucose value was 17mmol/L at week 36; nap-^DF^DF gradually increased in blood glucose at 16 weeks, reached a maximum of 16mmol/L at 23 weeks, then decreased, returned to 7mmol/L at 24 weeks, and remained steady. The polypeptide, the derivative and the hydrogel provided by the invention can better respond to the blood sugar load, have higher cleaning capability on blood sugar, and maintain the blood sugar of blood plasma to be stable at a certain level, especially Nap-G^DF^DF^DThe Y polypeptide hydrogel always keeps the blood sugar level to be 7mmol/L, and has higher capabilities of reducing the blood sugar level and maintaining the blood sugar level of blood plasma.

Experimental example 2 glucose tolerance test

3 week-old NOD mice were kept stable for one week in a new breeding environment, and then randomly divided into groups at 4 weeks of age, and 20 mice in each group were administered with the drug formulation by injecting 80. mu.L of Nap-G in a total volume of 1mg/mL subcutaneously into axillary regions each time^DF^DF^DY、Nap-GFFY、Nap-^DF^DF、Nap-G^DF^DA^DY、Ac-G^DF^DF^DY, Alum + Insulin B:9-23, weekly, and in the Control group, 80. mu.L of sterile 1 XPBS was injected for 5 weeks. The intraperitoneal glucose tolerance of NOD mice was tested at 14 weeks of age of the mice, respectively. Mice were fasted overnight for 8 hours before the experiment. The mice were weighed in advance and were injected intraperitoneally with sterile glucose injection (10%) at a dose of 2 g/kg. Used at 0, 15, 30, 45, 60, 90, 120 minutes after injection respectively

The glucometer (Lifescan, usa) measures the tail vein blood glucose concentration (glucose oxidase method). As can be seen from FIG. 3, blood glucose levels reached the highest in all groups at 15 minutes, and in the Control group, 20mmol/L, Nap-G^DF^DF^DLess than 15mmol/L of group Y, and then gradually decreases. After 120 minutes, the blood glucose level of the Control group was still around 15mmol/L, Nap-GFFY, and Nap-G^DF^DA^DY is recovered to 10mmol/L, Nap-^DF^DF and Ac-G^DF^DF^DY is recovered to about 7mmol/L, Nap-G^DF^DF^DThe Y and Alum + Insulin B:9-23 groups recovered to less than 10mmol/L after 30 minutes and substantially to the level before the injection of glucose after 90 minutes, approximately 5 mmol/L. The polypeptide and the derivative thereof have good sensitivity to glucose and high blood sugar removing capability, and can better respond to blood sugar load and keep blood sugar stable in a normal range.

Experimental example 3

3 week-old NOD mice were kept stable for one week in a new breeding environment, and then randomly divided into groups at 4 weeks of age, and 20 mice in each group were administered with the drug formulation by injecting 80. mu.L of Nap-G in a total volume of 1mg/mL subcutaneously into axillary regions each time^DF^DF^DY、Nap-GFFY、Nap-^DF^DF、Nap-G^DF^DA^DY、Ac-G^DF^DF^DY, Alum + Insulin B:9-23, weekly, in the Control group, 80. mu.L of sterile 1 XPBS was injected. The administration was continued for 5 weeks. At 14 weeks of mouse week age, the mice are collectedSpleens of each group of mice were ground and dispersed into single cells, filtered through a 70 μm filter, and the filtrate was subjected to gradient centrifugation (800g/30 min). Erythrocytes were subsequently removed by lysis with an erythrocyte lysate. The lymphocytes obtained by centrifugation were first incubated overnight with Anti-mouse CD4 antibody and Anti-mouse CD25 antibody, and then incubated with Anti-mouse Foxp3 antibody for 30 minutes, followed by flow analysis. As can be seen from FIG. 4, the polypeptide hydrogel (Nap-G) provided by the present invention^DF^DF^DY、Nap-G^LF^LF^LY、Nap-^DF^DF) Can remarkably improve CD4 in spleen⁺CD25⁺Foxp3⁺The level of Treg cells indicates that the mice can correct the self-immunity of the mice and establish the immune tolerance to self-antigens under the action of the polypeptide hydrogel. In addition, the mice in the Alum + Insulin B:9-23 group failed to establish self-antigen immune tolerance, which also accords with the previous report and clinical test, and the action mechanism of the aluminum adjuvant for treating the type I diabetes is to induce T cells to shift from proinflammatory Th1 to anti-inflammatory Th2 instead of causing immune tolerance.

The examples show that the polypeptide, the derivative and the hydrogel provided by the invention can effectively reduce the incidence rate of type I diabetes, and the onset cycle of the diabetes of NOD mice is delayed from 11 weeks to 14 weeks, and the non-onset proportion is increased to 40%. And can well control the blood sugar level; further, an intraperitoneal glucose tolerance experiment shows that the glucose-sensitive chitosan hydrogel has good glucose sensitivity and high blood sugar clearing capacity, can better cope with blood sugar load and keeps the blood sugar stably in a normal range. By detecting CD4⁺CD25⁺Foxp3⁺The Treg cell level indicates that the function of the pancreatic islet is not further damaged, and further indicates that the polypeptide hydrogel provided by the invention can correct autoimmunity and establish immune tolerance to autoantigens.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of southern kayak

<120> polypeptide, derivative and hydrogel thereof, and application thereof in preparation of drugs for preventing and/or treating type I diabetes

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Phe Phe Tyr

1

<210> 2

<211> 2

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Phe Phe

1

<210> 3

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Phe Ala Tyr

1

Claims

1. A polypeptide having a sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3 is shown in the specification;

2. a derivative of the polypeptide of claim 1, wherein the derivative is a terminal capping group attached to the N-terminus of the polypeptide.

3. The derivative of claim 2, wherein the end-capping group is an acetyl group.

4. The derivative of claim 2, wherein the end-capping group is formed by linking an aromatic ring-containing compound to the N-terminus of the polypeptide via an amide bond.

5. The hydrogel of claim 4, wherein said hydrogel is prepared by a method comprising: and (3) placing the derivative in a buffer solution, adjusting the pH to 6.0-8.0, heating to dissolve, and cooling to obtain the hydrogel containing the derivative.

6. The hydrogel according to claim 5, wherein the mass-to-volume ratio of the derivative to the buffer is 1 μ g:0.8 to 1.2 μ L.

7. The hydrogel according to claim 5 or 6, wherein the buffer is PBS buffer and the pH is 5.0 to 9.0.

8. The hydrogel of claim 5, wherein the hydrogel is a supramolecular hydrogel.

9. Use of the polypeptide of claim 1, the derivative of any one of claims 2 to 4 or the hydrogel of any one of claims 5 to 8 for the preparation of a medicament for the prevention and/or treatment of type I diabetes.

10. The use of claim 9, wherein the medicament is an injection or an oral formulation.