CN116606367A - Long-acting Exendin-9-39 and its application in the treatment of hypoglycemia and as a drug for treating hypoglycemia - Google Patents

Abstract

The invention discloses a long-acting Exendin-9-39, and application of the long-acting Exendin-9-39 serving as a GLP-1 receptor antagonist in treatment of hypoglycemia and a medicament for treating the hypoglycemia. The long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, wherein the AR-GLP1R-01 has a structure shown in a figure 2, and the AR-GLP1R-02 has a structure shown in a figure 3. The long-acting Exendin-9-39 can inhibit insulin secretion by inhibiting GLP-1 receptor, can raise blood sugar of a mouse model (SUR 1 knockout mouse) with hypoglycemia, and further achieves the effect of treating Congenital Hyperinsulinemia (CHI). Compared with Exendin-9-39, the AR-GLP1R-01 and the AR-GLP1R-02 have long half-lives, lasting action time and obviously improved effect.

Description

Long-acting Exendin-9-39 and its application in the treatment of hypoglycemia and as a treatment for hypoglycemia blood sugar medication

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a long-acting Exendin-9-39 and its application as a GLP-1 receptor antagonist in the treatment of hypoglycemia and as a drug for treating hypoglycemia.

Background technique

Congenital hyperinsulinism hypoglycemia (congenital hyperinsulinism, CHI) is mainly caused by the mutation of the gene encoding the function of pancreatic β-cells, which causes excessive or inappropriate secretion of insulin, and increases the blood insulin concentration, resulting in intractable and persistent hypoglycemia. syndrome, also known as neonatal persistent hyperinsulinemic hypoglycemia (neonatal hypoglycemia). Congenital hyperinsulinemia is difficult to cure, and it is easy to cause hypoglycemia in young infants, which is characterized by persistent hypoglycemia and relative hyperinsulinemia disproportionate to blood sugar levels, accompanied by low ketone body hypolipidemia, age The smaller the risk of hypoglycemia. Hypoglycemia is a dangerous condition that can cause seizures, lasting brain damage, and even death if not detected and treated promptly.

The most common and severe subtype of CHI is congenital hyperinsulinemia (K _ATP -CHI) caused by loss-of-function mutations of ATP-dependent potassium channels (including SUR1 and Kir6.2 subunits) in islet β-cells. ), the pancreatic islet β cells in children continue to secrete a large amount of unregulated insulin, resulting in severe hypoglycemia, and children with K _ATP -CHI are not sensitive to diazoxide. For many patients, surgical resection of the pancreas is the only option.

Glucagon-like peptide-1 (GLP-1) is a hormone secreted by the gut, which can assist and improve glucose-stimulated insulin secretion. GLP-1 analogs interact with GLP-1 degrading enzymes ( DDP4) inhibitors have been widely used as new drugs in the treatment of type 2 diabetes. Because congenital hyperinsulinemia is just opposite to the onset of diabetes, GLP-1 receptor antagonists can be used for the treatment of hypoglycemia. Previous studies have found that GLP-1 receptor antagonism (Exendin-9-39) can significantly increase the blood sugar in the potassium ion channel (SUR1) knockout mouse model, and further clinical trials on patients have proved that the drug is effective in hypoglycemia patients The relevant articles have been published in JBC (2008) and Diabetes (2012) magazines.

Exendin-9-39 is a small peptide containing multiple amino acid residues, the molecular formula is C ₁₄₉ H ₂₃₄ N ₄₀ O ₄₇ S, and the molecular weight is 3369.8. It is derived from the 39 amino acid residues of Exendin-4 and the first 8 amino acids at the N-terminus are deleted. obtained after the residue. Exendin-4 is an activator of the GLP-1 receptor. Its main function is to promote the binding of GLP-1 to the receptor and induce the production of cAMP, thereby exerting a similar effect to GLP-1: increasing glucose-dependent insulin secretion and inhibiting pancreatic secretion. The secretion of glucagon accelerates glucose clearance, delays gastric emptying, induces satiety, and suppresses appetite; it can also improve peripheral insulin resistance, etc. Exendin-9-39, which lacks 8 amino acids, is an inhibitor of GLP-1 receptors, and its effect is completely opposite to that of Exendin-4. Its role is mainly the antagonist of GLP-1 receptors in animals. -1 receptor binding, inhibiting the production of cAMP in islets of pancreas, thereby exerting the effect of blocking GLP-1, reducing the secretion of insulin, increasing the secretion of glucagon, increasing appetite and so on.

However, because Exendin-9-39 has the disadvantages of short half-life, large daily dosage and continuous administration, in order to increase the half-life, prolong the metabolism time of the drug itself in the body, and reduce the dosage, two kinds of Exendin-9-39 were designed. Derivatives of 9-39, AR-GLP1R-01 and AR-GLP1R-02.

Contents of the invention

In view of the above problems, the present invention provides a long-acting Exendin-9-39 and its application as a GLP-1 receptor antagonist in the treatment of hypoglycemia and as a drug for treating hypoglycemia. The long-acting Exendin-9-39 of the present invention can inhibit the secretion of insulin by inhibiting the GLP-1 receptor, and can increase the blood sugar of the hypoglycemic disease mouse model (SUR1 knockout mice), thereby achieving the treatment of congenital hyperinsulinemia and hypoglycemia. The role of glycemia (CHI).

The purpose of the present invention and the solution to its technical problems are achieved by adopting the following technical solutions.

One aspect of the present invention provides a long-acting Exendin-9-39, said long-acting Exendin-9-39 includes AR-GLP1R-01 and AR-GLP1R-02, said AR-GLP1R-01 has As shown in the structure, the AR-GLP1R-02 has the structure shown in Figure 3.

The object of the present invention and the solution to its technical problems are also achieved by adopting the following technical solutions.

Another aspect of the present invention provides the application of a long-acting Exendin-9-39 as a GLP-1 receptor antagonist in the treatment of hypoglycemia.

Preferably, the GLP-1 receptor antagonist is an inhibitor of GLP-1 receptor function.

Preferably, the GLP-1 receptor antagonist contains long-acting Exendin-9-39, and the concentration of the long-acting Exendin-9-39 is between 0.1 μM and 13 μM.

Preferably, the long-acting Exendin-9-39 includes AR-GLP1R-01 and AR-GLP1R-02, the AR-GLP1R-01 has the structure shown in Figure 2, and the AR-GLP1R-02 It has the structure shown in Figure 3.

The object of the present invention and the solution to its technical problems are also achieved by adopting the following technical solutions.

Another aspect of the present invention provides a drug for treating hypoglycemia, which includes a GLP-1 receptor antagonist.

Preferably, the GLP-1 receptor antagonist is an inhibitor of GLP-1 receptor function.

Preferably, the GLP-1 receptor antagonist includes long-acting Exendin-9-39.

Preferably, the long-acting Exendin-9-39 includes AR-GLP1R-01 and AR-GLP1R-02, the AR-GLP1R-01 has the structure shown in Figure 2, and the AR-GLP1R-02 It has the structure shown in Figure 3.

Preferably, the concentration of the long-acting Exendin-9-39 in the GLP-1 receptor antagonist is between 0.1 μM and 13 μM.

By virtue of the above technical solutions, the present invention has at least the following advantages:

(1) AR-GLP1R-01 and AR-GLP1R-02 of the present invention are obtained by connecting fatty acid groups on the side chain of Exendin-9-39, and these two compounds are more stable than Exendin-9-39 , The metabolism time in the body is significantly prolonged.

(2) The present invention provides two kinds of Exendin-9-39 derivatives AR-GLP1R-01 and AR-GLP1R based on the effects of Exendin-9-39 on reducing insulin secretion, increasing glucagon secretion and increasing appetite -02 is used as a GLP-1 receptor antagonist in the field of hypoglycemia treatment. The results show that AR-GLP1R-01 and AR-GLP1R-02 have similar effects to Exendin-9-39, and also inhibit ATP-dependent potassium The problem of excessive secretion of insulin by pancreatic islets caused by ion channel loss of function mutations can achieve the therapeutic purpose of raising CHI blood sugar, and AR-GLP1R-01 and AR-GLP1R-02 have significantly prolonged action time and improved efficacy.

(3) The present invention also provides a drug for treating hypoglycemia. The drug uses AR-GLP1R-01 or AR-GLP1R-02 as the main active substance. The drug can effectively treat hypoglycemia and last for a long time.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention will be described in detail below.

Description of drawings

Fig. 1 is Exendin-9-39 structural formula;

Figure 2 is the structural formula of AR-GLP1R-01;

Figure 3 is the structural formula of AR-GLP1R-02;

Figure 4 is a schematic diagram of CRISPR/Cas9 technology;

Figure 5 is a perfusion test for islet isolation and culture;

Figure 6 shows the in vivo test of SUR1-KO mice: the changes in blood sugar before and 1 hour after subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 on the first day of administration;

Figure 7 shows the in vivo test of SUR1-KO mice: the blood glucose level before administration on the second day after subcutaneous injection of Exendin-9-39 or AR-GLP1R-01;

Figure 8 is the in vivo test of SUR1-KO mice: on the 4th day of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, the blood glucose values before and 1 hour after administration;

Figure 9 is the in vivo test of SUR1-KO mice: the blood glucose level before administration on the 4th day of subcutaneous injection of drug Exendin-9-39 or AR-GLP1R-01;

Figure 10 is the in vivo test of SUR1-KO mice: after the 4th day of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, the blood glucose level after fasting for 17 hours from 5:00 pm to the morning of the 5th day;

Figure 11 shows the blood glucose values at 16 hours and 24 hours after subcutaneous injection of Exendin-9-39 and AR-GLP1R-01 in SUR1-KO mice;

Figure 12 is a perfusion test for islet isolation and culture;

Figure 13 is the in vivo test of SUR1-KO mice: subcutaneous injection of Exendin-9-39 or AR-GLP1R-02, blood glucose changes before and 1 hour after the first day of administration;

Figure 14 is the in vivo test of SUR1-KO mice: on the 4th day of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, blood glucose levels before and 1 hour after administration;

Figure 15 is the in vivo test of SUR1-KO mice: after the 4th day of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, the blood glucose level after fasting for 17 hours from 5:00 pm to the morning of the 5th day;

Fig. 16 is the HPLC chromatogram of the Exendin-9-39 of different concentrations;

Figure 17 is Exendin-9-39 concentration standard curve and blood concentration detection chromatogram after injection of SUR1-KO mice;

Figure 18 is the HPLC chromatograms of different concentrations of AR-GLP1R-01;

Figure 19 is the concentration standard curve of AR-GLP1R-01 and the blood concentration detection chromatogram after injection of SUR1-KO mice;

Figure 20 is the chromatogram of different concentrations of AR-GLP1R-02HPLC;

Fig. 21 is the concentration standard curve of AR-GLP1R-02 and the detection chromatogram of blood concentration in SUR1-KO mice after injection.

Detailed ways

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments It is only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

According to the long-acting Exendin-9-39 of the present invention, it is a derivative of Exendin-9-39, including AR-GLP1R-01 and AR-GLP1R-02, and the AR-GLP1R-01 has The structure shown, the AR-GLP1R-02 has the structure shown in Figure 3.

According to the application of the long-acting Exendin-9-39 in the present invention as a GLP-1 receptor antagonist in the treatment of hypoglycemia, the GLP-1 receptor antagonist is an inhibitor that inhibits the function of the GLP-1 receptor. The GLP-1 receptor antagonist contains long-acting Exendin-9-39, and the concentration of the long-acting Exendin-9-39 is between 0.1 μM and 13 μM. Long-acting Exendin-9-39 includes AR-GLP1R-01 and AR-GLP1R-02, described AR-GLP1R-01 has the structure shown in Figure 2, and described AR-GLP1R-02 has the structure shown in Figure 3 structure shown.

According to the medicament for treating hypoglycemia of the present invention, the medicament contains a GLP-1 receptor antagonist. GLP-1 receptor antagonists are inhibitors that inhibit the function of the GLP-1 receptor. GLP-1 receptor antagonists include long-acting Exendin-9-39. Long-acting Exendin-9-39 includes AR-GLP1R-01 and AR-GLP1R-02, described AR-GLP1R-01 has the structure shown in Figure 2, and described AR-GLP1R-02 has the structure shown in Figure 3 structure shown. The concentration of long-acting Exendin-9-39 in the GLP-1 receptor antagonist is between 0.1 μM and 13 μM.

Example 1: Preparation of AR-GLP1R-01 and AR-GLP1R-02

The AR-GLP1R-01 and AR-GLP1R-02 described in this example are Exendin-9-39, collectively referred to as long-acting Exendin-9-39.

The structure of Exendin-9-39 is shown in Figure 1, and its amino acid sequence is:

H-Asp-Leu-Ser-Lys-D-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro- Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

The AR-GLP1R-01 and AR-GLP1R-02 of this example are obtained by linking the side chains of the following structure I to the 12th and 27th lysine side chains of Exendin-9-39, the principle It is to prolong the metabolism time of the drug itself in the body.

AR-GLP1R-01 and AR-GLP1R-02 in this example were prepared by entrusting a third-party institution, which is Chengdu Shengnuo Biotechnology Co., Ltd.

Example 2: Basic technical principles and steps of making SUR1 knockout mice

1. Principle

Through CRISPR/Cas9 gene knockout technology, crRNA combines with tracrRNA (trans-activating RNA) through base pairing to form double-stranded RNA. This tracrRNA:crRNA binary complex directs the Cas9 protein to cleave double-stranded DNA at specific sites targeted by the crRNA guide sequence. At the site complementary to the crRNA guide sequence, the HNH nuclease domain of the Cas9 protein cuts the complementary strand while the Cas9 RuvC-like domain cuts the non-complementary strand, realizing the function of knocking out the target gene and preparing a gene knockout mouse model .

2. Vector design, construction and purification

Using the CRISPR Design tool of the Massachusetts Institute of Technology (http://crispr.mit.edu/), design a pair of 20bp oligonucleotide chain sequences for the target DNA according to the level of Score for the preparation of sgRNA, and Primers were designed in this target region for subsequent gene identification in mice. The designed sequences were synthesized into PAGE products.

The synthesized two single-stranded oligonucleotide sgRNA sequences were annealed (95°C for 5 minutes and then naturally cooled to room temperature) to form a double-stranded DNA, which was linked with the pGK1.1 linear vector under the action of T4 DNA ligase to construct the sgRNA expression vector , Transform the recombinant plasmid into DH5a competent cells, screen and identify positive cloned plasmids through the kanamycin resistance of pGK1.1linear vector and the sequencing of target DNA, select the correct colony clone, and extract the plasmid after expansion Used to prepare templates for in vitro transcription.

3. In vitro transcription

The sgRNA expression vector was linearized by Dra I digestion, extracted and purified with phenol chloroform, dissolved in nuclease-free water as a template for in vitro transcription, and synthesized in vitro by T7 RNA polymerase according to the MEGAshortscript Kit (Ambion, AM1354) kit sgRNA.

4. Microinjection of Cas9/sgRNA

Mix the transcribed sgRNA and cas9 and adjust the injection concentration to microinject the mixture into the cytoplasm of C57BL/6 mouse fertilized eggs using a TE2000U microinjector, and then transplant the fertilized eggs into the uterus of a pseudopregnant C57BL/6 female mouse , waiting for the F0 generation mice to be born.

5. Identification of F0 mice

When the F0 generation mice were 5-7 days after birth, the mice were marked by the toe clipping method, and the DNA was extracted from the clipping mouse tail tissue by the phenol-chloroform method, and identified according to the primers designed in the target region in the above experiment (1). Select PCR-positive samples for sequencing.

6. Inheritability detection of F0 generation mice

The F0 generation mice with correct PCR and sequencing were mated with wild-type C57BL/6 mice to generate F1 generation mice, and the F1 generation mice were identified according to the identification method of F0 generation mice, and the positive F1 generation heterozygotes obtained Mice can be genetically stable.

7. Abcc8 gene production scheme

This model uses CRISPR/Cas9 technology to edit the Abcc8 gene. The schematic diagram of the principle is shown in Figure 4.

(1) Primer information:

(2) PCR amplification system:

Reaction composition Volume (μL) gDNA template 2.0 10×Taq Buffer(mg ²⁺ plus) 2.0 dNTP mix (10mM) 0.5 Primer mix (10μM) 0.5 Taq DNA polymerase (5U/μL) 0.5 Milli-Q H ₂ O To 20μL

(3) PCR amplification program

Example 3: Isolation, purification and culture of islets of SUR1-KO mice

1. Isolation and purification of islets from SUR1-KO mice include the following steps:

(1) After the mouse was anesthetized, it was fixed on the operating board, the abdominal cavity was opened, and the entrance of the bile duct into the duodenum was blocked with a hemostat, and then a 2 mg/mL collagenase solution was injected into the bile duct with a 5 mL syringe and a 31.5 needle, until Stop when the pancreas is fully inflated.

(2) Peel off the pancreas, clean up fat and non-pancreatic tissue, transfer the pancreas to a 50mL centrifuge tube, pour 3mL of 2mg/mL collagenase solution, and shake in a 37°C water bath for 4-5 minutes.

(3) Immediately after shaking, add Hanks buffer to 50mL in a 50mL centrifuge tube, then centrifuge, remove the supernatant, add 5mL of Histopaque-1119 solution to the remaining tissue pellet, shake and mix.

(4) Slowly add 5mL Histopaque-1077 solution, then slowly add 5mL Hanks buffer, and then centrifuge.

(5) Remove islet tissue from the layered liquid, pick and purify islets under a dissecting microscope, wash with Hanks buffer and culture.

2. Islet culture steps of SUR1-KO mice:

1. Preparation of RPMI 1640 culture medium: in milliliters, the following raw materials are included: 10 parts of glucose, 2 parts of glutamine, 10 parts of fetal bovine serum, 24 parts of sodium bicarbonate, 100 units/mL of penicillin, 10 μg/mL of streptomycin , adjust the pH to 7.2.

2. Use RPMI 1640 culture medium to culture the isolated and purified islets overnight in an incubator at 37°C, 5% CO ₂ and 95% air humidity.

Example 4: Inspection of islet perfusion and insulin secretion

(1) Reaction solution: Krebs buffer

A): NaCl: 107g dissolved in 1000mL water.

B): KCl(5.96g)+NaHCO ₃ (32.256g)+MgCl ₂ -6H ₂ O(3.25g) was dissolved in 1000mL water.

C): CaCl ₂ -2H ₂ O: 5.168g dissolved in 1000mL water.

Krebs buffer:

BSA (0.25%): 2.0g

HEPES: 1.906g (10mM)

(2) Stimulation solution configuration

30mM KCl solution; amino acid mixture (AAM)

AAM (100mL):

amino acid Content (mg) amino acid Content (mg) Alanine 155.7 Isoleucine 49.32 arginine 157.6 Leucine 85 aspartic acid 21.8 Lysine 270.2 Citrulline 65.16 Methionine 28.64 glutamic acid 70.128 Ornithine 46.52 Glycine 89.48 Phenylalanine 54.2 Histidine 48.422 proline 160.68 serine 238.89 threonine 128.64 Valine 94.16 Tryptophan 60.44

(4) Counting of islets: 120 islets were collected in the islet chamber and placed in a constant temperature water bath at 37°C.

(5) Place the prepared reaction solution in a water bath (37°C) and insert the corresponding sampling tube. The following is the instrument procedure:

(6) Collect the secreted insulin by the Waters Fraction Collector, take 10 μL of the solution after the islet stimulation experiment, and transfer it into a 384-well plate; add the antibody according to the instructions of the HTRF insulin assay kit, shake and mix, and incubate at room temperature for 2 hours. The Clariostar microplate reader HTRF program reads, calculates the hormone secretion value according to the standard curve, and confirms the influence of stimulants or drugs on insulin secretion by measuring insulin secretion.

Example 5: Animal experiment of long-acting Exendin-9-39

(1) Experimental animals: 41 SUR1-KO mice

(2) Experimental compounds: Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02;

(3) Experimental equipment: blood glucose meter

(4) Experimental grouping:

a. Experimental group 1: 10 SUR1-KO mice, injected with Exendin-9-39;

b. Experimental group 2: 10 SUR1-KO mice injected with AR-GLP1R-01;

c. Experimental group 3: 10 SUR1-KO mice injected with AR-GLP1R-02;

d. Control group: 10 SUR1-KO mice injected with normal saline.

(5) Experimental method:

A. Take out Exendin-9-39 and two derivatives AR-GLP1R-01 and AR-GLP1R-02 at -80°C, dissolve them in sterile saline to a concentration of 108 μmol/L, and store in -80°C ,spare.

B. Subcutaneous injection at 2 μL/g body weight, 2 times/day (10:00 am and 5:00 pm), and measure the blood glucose level before the first injection and 1 hour after the injection on the first day, the second day and the fourth day . The control group was normal saline.

C. Continuous treatment for 4 days, after the second injection on the fourth day, the mouse food was taken away, the bedding was replaced, water was not stopped, and food fasting began.

D. After fasting for 16 hours, measure blood sugar.

E. If it is a test with only one injection, the injection dose and method are the same as those described in A and B.

Example 6: Concentrations of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 in SUR1-KO mouse serum determination

The concentrations of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 in the serum of SUR1-KO mice were detected by liquid chromatography.

(1) Experimental conditions:

A. Drug concentration: 1080μmol/L

B. Injection method and dose: intraperitoneal injection into C57BL/6 mice at 2 μL/g body weight

C. Blood sampling time: 0.5h, 2h, 10h

D. Blood collection method: blood was collected from the inner canthus of the mouse eye, 10 μL of DPP4 inhibitor was added to the blood and allowed to stand still, centrifuged at 3000 r/min for 3 minutes at 4°C, the upper serum was collected and stored at -20°C.

(2) Serum treatment:

Treat the protein in the serum with the ratio of serum: acetonitrile = 1:1.5 and filter the membrane to obtain the sample liquid.

(3) Experimental equipment:

(4) Experimental instrument parameter settings:

Mass Spectrometry Parameters

Chromatographic parameters

Embodiment 7 : experimental result and analysis

(1) Comparative study of AR-GLP1R-01 and Exendin-9-39

1. Study on the effect of Exendin-9-39 and AR-GLP1R-01 on the function of isolated islets:

Figure 5 is a perfusion test of isolated and cultured islets, showing the dynamic secretion of insulin, the stimulus is amino acid mixture (AAM), the concentration is climbing from 0 to 12mM; the islets are islets of SUR1 knockout mice. As shown in Figure 5, the basal insulin secretion of SUR1-KO mice was elevated and sensitive to insulin secretion stimulated by amino acid mixture (AAM), which was consistent with previous research results. 100nM Exendin-9-39 can reduce excessive basal insulin secretion and AAM-stimulated insulin secretion, which is also consistent with previous research results. The same concentration of AR-GLP1R-01 (100nM) has a similar effect to Exendin-9-39, both of which can significantly inhibit the insulin secretion stimulated by basal insulin and AAM. It shows that AR-GLP1R-01 has the same effect as Exendin-9-39, and both can improve the excessive secretion of basal insulin and the excessive response to amino acid-stimulated insulin secretion caused by the damage of islet ATP-dependent potassium channel.

2. Animal experiment results of Exendin-9-39 and AR-GLP1R-01

Live animal experiments were performed using Exendin-9-39 and AR-GLP1R-01. The test design is to inject Exendin-9-39 and AR-GLP1R-01 subcutaneously twice a day (10 am and 5 pm) for 4 consecutive days, and the administration concentration is 216pM/g body weight.

Figure 6 shows the in vivo test of SUR1-KO mice: subcutaneous injection of drugs (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), changes in blood sugar before and 1 hour after drug administration on the first day. Exendin-9-39 and AR-GLP1R-01 significantly increased blood glucose in SUR1-KO mice. As shown in Figure 6, compared with the blood glucose of SUR1-KO mice without basic drug administration, AR-GLP1R-01 and Exendin-9-39 had the same effect 1 hour after injection, and both significantly increased the blood glucose of the disease mouse model.

Fig. 7 shows the in vivo test of SUR1-KO mice: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), the blood glucose level before administration on the second day of administration. Figure 7 shows that before administration on the second morning, the basal blood sugar of the Exendin-9-39 treatment group was the same as that of the blank control, and the effect of raising blood sugar could not be sustained, while the effect of AR-GLP1R-01 was injected 2 doses on the first day After that, it can be maintained until 10 am the next day (about 17 hours), indicating that AR-GLP1R-01 can improve the blood sugar of the disease mouse model for a long time, and the effect is maintained until the fourth day.

Figure 8 is the in vivo test of SUR1-KO mice: subcutaneous injection of drugs (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), on the 4th day of drug administration, blood glucose before and 1 hour after drug administration value. As shown in Figure 8, after 3 days of medication twice a day, before the morning of the fourth day, the basal blood glucose of the Exendin-9-39 group was the same as that of the blank control group. One hour after the injection of Exendin-9-39, the blood glucose was still significantly increased, indicating that the short-term therapeutic effect of Exendin-9-39 can occur repeatedly. Compared with the Exendin-9-39 group and the blank control group, AR-GLP1R-01 can not only maintain the elevated basal blood sugar value observed on the second day, but also has the ability to continue to raise blood sugar for a short time after 1 hour of injection.

Fig. 9 is the in vivo test of SUR1-KO mice: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), blood glucose level before administration on day 4 of administration. Figure 9 shows that AR-GLP1R-01 significantly increased the basal blood sugar of SUR1-KO mice, but Exendin-9-39 had no effect on the basal blood sugar, reconfirming the long-term elevating effect of AR-GLP1R-01 on blood sugar Significantly better than Exendin-9-39.

Figure 10 is the in vivo test of SUR1-KO mice: subcutaneous injection of drugs (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), after the 4th day of medication, fasting from 5 pm to the morning of the 5th day , the blood glucose value after fasting for 17 hours. As shown in Figure 10, after 4 days of administration twice a day, AR-GLP1R-01 significantly increased the basal blood glucose of SUR1-KO mice, but Exendin-9-39 did not improve fasting blood glucose. At the same time, AR-GLP1R-01 can significantly improve the fasting blood glucose of the disease mouse model in the treatment group. It shows that the long-term effect of AR-GLP1R-01 is obviously better than that of Exendin-9-39, and the short-term effect (1 hour after injection) is equivalent to that of Exendin-9-39.

In order to further verify the conclusions of this experiment, the effect of one injection for 24 hours was observed, as shown in Figure 11, for 20 SUR1-KO mice (female to male ratio 1:1), after measuring the basal (non-medicated) blood glucose value , subcutaneously inject Exendin-9-39 and AR-GLP1R-01, the dose is 216pM/g body weight, observe the blood glucose 16 hours and 24 hours after the administration, the short-term effect of Exendin-9-39 on raising blood sugar cannot be maintained until 16 and 24 hours, but AR-GLP1R-01 once administered, the effect of raising blood sugar can be maintained to 24 hours.

(2) Comparative study of AR-GLP1R-02 and Exendin-9-39

1. Study on the effect of Exendin-9-39 and AR-GLP1R-02 on the function of isolated islets:

Figure 12 is a perfusion test of isolated and cultured islets, showing the dynamic secretion of insulin, the stimulus is amino acid mixture (AAM), the concentration is climbing from 0 to 12mM; the islets are islets of SUR1 knockout mice. As shown in Figure 12, the basal insulin secretion of SUR1-KO mice was elevated and sensitive to insulin secretion stimulated by amino acid mixture (AAM), which was consistent with previous research results. 100nM Exendin-9-39 can reduce excessive basal insulin secretion and AAM-stimulated insulin secretion, which is also consistent with previous research results. The same concentration of AR-GLP1R-02 (100nM) has a similar effect to Exendin-9-39, both of which can significantly inhibit the insulin secretion stimulated by basal insulin and AAM. It shows that AR-GLP1R-02 has the same effect as Exendin-9-39, and both can improve the excessive secretion of basal insulin and the excessive response to amino acid-stimulated insulin secretion caused by the damage of the islet ATP-dependent potassium channel.

2. Animal experiment results of Exendin-9-39 and AR-GLP1R-02:

Live animal experiments were performed using Exendin-9-39 and AR-GLP1R-02. The experimental design is subcutaneous injection of Exendin-9-39 and AR-GLP1R-01, twice a day (10 am and 5 pm), for 4 consecutive days, and the administration concentration is 216pM/g body weight.

Figure 13 shows the in vivo test of SUR1-KO mice: subcutaneous injection of drugs (Exendin-9-39 or AR-GLP1R-02, dose: 216pM/g body weight), changes in blood sugar before and 1 hour after drug administration on the first day. As shown in Figure 13, compared with the blood glucose of untreated SUR1-KO mice, Exendin-9-39 can temporarily improve the blood glucose of the disease model after injection for 1 hour, but after the first dose of AR-GLP1R-02, Blood sugar is not elevated.

After 3 days of medication, compared with before medication on the 4th day and 1 hour after medication (as shown in Figure 14), compared with the blank solvent control group and the Exendin-9-39 treatment group, the basal blood glucose of the AR-GLP1R-02 group was comparable to that of the other two groups Correspondingly, the short-term effect of AR-GLP1R-02 administered for 1 hour is consistent with that of Exendin-9-39, which can temporarily increase the blood sugar of model mice. After taking medicine in the afternoon of the fourth day, the mice started the fasting test, and observed the overnight fasting blood glucose in the morning of the fifth day. As shown in Figure 15, after taking medicine twice a day for four days, AR-GLP1R-02 can improve the treatment group. Fasting blood glucose in the mouse model. It shows that AR-GLP1R-02 is similar to Exendin-9-39, and its short-term effect (1 hour after injection) is equivalent to that of Exendin-9-39, and the drug effect can also be maintained after fasting, indicating that the long-term effect is better than Exendin-9- 39.

(3) Blood concentration detection of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02

(1) Absorption peaks of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 standards with different concentrations: for Exendin-9-39, as shown in Figure 16 and Figure 17; for AR-GLP1R-01 , as shown in Figure 18 and Figure 19; for AR-GLP1R-02, as shown in Figure 20 and Figure 21; draw a standard curve based on the chromatogram and the area under the peak of the standard.

(2) See Table 1 for the calculation results of blood drug concentration.

From the detection results of liquid chromatography mass spectrometry, it can be seen that the absorption peak of Exendin-9-39 appeared at about 4.9 minutes, and the absorption peaks of AR-GLP1R-01 and AR-GLP1R-02 appeared at about 7.7 minutes. The test mice (SUR1-KO) were subcutaneously injected with the drug, and the administration concentration was 216pM/g body weight. Afterwards, blood samples were taken at 0.5 hour, 2 hours and 10 hours after administration to determine the blood drug concentration. The concentrations of the three components in serum were calculated according to the areas of the absorption peaks. It can be seen from the summarized results that at 0.5h, the concentration of Exendin-9-39 in serum was lower than that of AR-GLP1R-01 and AR-GLP1R-02 , Exendin-9-39 could not be detected in the serum after 2 hours, but AR-GLP1R-01 and AR-GLP1R-02 could still be detected at 10 hours, indicating that AR-GLP1R-01 and AR after the side chain - The half-life of GLP1R-02 is better than that of Exendin-9-39, which also proves that this liquid chromatography-mass spectrometry method can detect the concentrations of the three samples in serum.

Table 1 blood concentration

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this field Those skilled in the art, without departing from the scope of the technical solution of the present invention, may use the method and technical content disclosed above to make some changes or modifications to equivalent embodiments with equivalent changes, but if they do not depart from the technical solution of the present invention, Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims (10)

1. The long-acting Exendin-9-39 is characterized in that the long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, wherein the AR-GLP1R-01 has a structure shown in a figure 2, and the AR-GLP1R-02 has a structure shown in a figure 3.

2. Use of long acting Exendin-9-39 as a GLP-1 receptor antagonist in the treatment of hypoglycaemia.

3. The use according to claim 2, wherein the GLP-1 receptor antagonist is an inhibitor of GLP-1 receptor function.

4. The use according to claim 2, wherein the GLP-1 receptor antagonist comprises long-acting Exendin-9-39, and the concentration of the long-acting Exendin-9-39 is between 0.1 μm and 13 μm.

5. The use according to claim 2, wherein said long acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, said AR-GLP1R-01 having the structure shown in fig. 2 and said AR-GLP1R-02 having the structure shown in fig. 3.

6. A medicament for treating hypoglycemia, comprising a GLP-1 receptor antagonist.

7. The medicament according to claim 6, characterized in that the GLP-1 receptor antagonist is an inhibitor of GLP-1 receptor function.

8. The medicament according to claim 6, wherein the GLP-1 receptor antagonist comprises long acting Exendin-9-39.

9. The drug of claim 6, wherein the long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, wherein the AR-GLP1R-01 has a structure as shown in FIG. 2, and wherein the AR-GLP1R-02 has a structure as shown in FIG. 3.

10. The medicament according to claim 8, characterized in that the concentration of the long acting Exendin-9-39 in the GLP-1 receptor antagonist is between 0.1 μm and 13 μm.