CN116606367A - Long-acting Exendin-9-39 and application thereof in treatment of hypoglycemia and medicament for treating hypoglycemia - Google Patents
Long-acting Exendin-9-39 and application thereof in treatment of hypoglycemia and medicament for treating hypoglycemia Download PDFInfo
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- CN116606367A CN116606367A CN202210611291.2A CN202210611291A CN116606367A CN 116606367 A CN116606367 A CN 116606367A CN 202210611291 A CN202210611291 A CN 202210611291A CN 116606367 A CN116606367 A CN 116606367A
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/575—Hormones
- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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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
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to 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.
Background
Congenital hyperinsulinemia (congenital hyperinsulinism, CHI) is a persistent hyperglycemia that results from excessive or inappropriate secretion of insulin, increased blood insulin concentration, mainly caused by mutation of the gene encoding the function of the islet beta cells, and is also called neonatal persistent hyperinsulinemia (neonatal hypoglycemia). Congenital hyperinsulinemia is difficult to cure and is easy to cause hypoglycemia in young infants, and is characterized by intractable hypoglycemia and relative hyperinsulinemia which is not matched with blood glucose level, accompanied by hypoketosis and hypolipidemia, and the damage of hypoglycemia is greater when the age is smaller. Hypoglycemia is a dangerous condition that, without timely detection and treatment, can cause seizures and permanent brain damage, even death.
One of the most common and severe subtypes of CHI is congenital hyperinsulinemia (K) caused by mutations of the islet beta-cell ATP dependent potassium ion channel (including both SUR1 and kir6.2 subunits) with loss of function ATP -CHI), insulin secretion by the insulin-beta cells of the infant islets of langerhans continues to be large and unregulated, causing severe hypoglycemia, K ATP The infant CHI is insensitive to diazoxide and surgical removal of the pancreas is unique to many patientsAnd (3) selecting one.
Glucagon-like peptide-1 (GLP-1) is a hormone secreted from the intestinal tract, which aids and improves glucose-stimulated insulin secretion, and inhibitors of GLP-1 analogues and GLP-1 degrading enzymes (DDP 4) have been widely used as novel drugs in the treatment of type 2 diabetes. Because congenital hyperinsulinemia is the opposite of diabetes onset, antagonists of the GLP-1 receptor can be used in the treatment of hypoglycemia. Early studies found that GLP-1 receptor antagonism (Exendin-9-39) can significantly raise blood glucose of a potassium channel (SUR 1) knockout mouse disease model, and further clinical trials on patients prove that the drug is effective in hypoglycemic patients, and related articles are published in JBC (2008) and Diabetes (2012).
Exendin-9-39 is a small peptide containing multiple amino acid residues and has the molecular formula C 149 H 234 N 40 O 47 S, molecular weight 3369.8, which is obtained by deleting the first 8 amino acid residues at the N-terminal of the 39 amino acid residues of Exendin-4. Exendin-4 is an activator of GLP-1 receptor, and has the main functions of promoting the combination of GLP-1 and the receptor and inducing the generation of cAMP, thereby playing the role similar to GLP-1: increasing glucose-dependent insulin secretion, inhibiting glucagon secretion, accelerating glucose clearance, delaying gastric emptying, inducing satiety, and suppressing appetite; also can improve peripheral insulin resistance, etc. The Exendin-9-39 which lacks 8 amino acids is an inhibitor of GLP-1 receptor, and has the effect completely opposite to that of Exendin-4, and the effect is mainly that the antagonist of GLP-1 receptor in animals, through binding with GLP-1 receptor, the production of islet cAMP is inhibited, thereby playing the roles of blocking GLP-1, reducing insulin secretion, increasing glucagon secretion, increasing appetite and the like.
However, since Exendin-9-39 has the disadvantages of short half-life, large daily dosage and continuous administration, two derivatives of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02, are designed to increase half-life, extend the metabolism time of the drug itself in vivo and reduce the dosage of administration.
Disclosure of Invention
In order to solve the problems, the invention provides a long-acting Exendin-9-39 and application thereof as a GLP-1 receptor antagonist in the treatment of hypoglycemia and a medicament for treating the hypoglycemia. 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).
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
One aspect of the present invention provides a long-acting Exendin-9-39, wherein the long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, the AR-GLP1R-01 has a structure as shown in FIG. 2, and the AR-GLP1R-02 has a structure as shown in FIG. 3.
The aim and the technical problems of the invention are also realized by adopting the following technical proposal.
In another aspect, the invention provides the use of long acting Exendin-9-39 as a GLP-1 receptor antagonist in the treatment of hypoglycaemia.
Preferably, the GLP-1 receptor antagonist is an inhibitor that inhibits GLP-1 receptor function.
Preferably, 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 mu M and 13 mu M.
Preferably, the long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, wherein the AR-GLP1R-01 has a structure shown in figure 2, and the AR-GLP1R-02 has a structure shown in figure 3.
The aim and the technical problems of the invention are also realized by adopting the following technical proposal.
In another aspect, the invention provides a medicament for treating hypoglycemia comprising a GLP-1 receptor antagonist.
Preferably, the GLP-1 receptor antagonist is an inhibitor that inhibits GLP-1 receptor function.
Preferably, the GLP-1 receptor antagonist comprises long-acting Exendin-9-39.
Preferably, the long-acting Exendin-9-39 comprises AR-GLP1R-01 and AR-GLP1R-02, wherein the AR-GLP1R-01 has a structure shown in figure 2, and the AR-GLP1R-02 has a structure shown in figure 3.
Preferably, the concentration of said long acting Exendin-9-39 in said GLP-1 receptor antagonist is between 0.1. Mu.M and 13. Mu.M.
By means of the technical scheme, the invention has at least the following advantages:
(1) The AR-GLP1R-01 and the AR-GLP1R-02 are obtained by connecting fatty acid groups on the side chains of Exendin-9-39, and compared with the Exendin-9-39, the two compounds have good stability and obviously prolonged metabolism time in vivo.
(2) Based on the effects of reducing insulin secretion, increasing glucagon secretion and increasing appetite of Exendin-9-39, the invention provides the application of two Exendin-9-39 derivatives AR-GLP1R-01 and AR-GLP1R-02 as GLP-1 receptor antagonists in the field of hypoglycemia treatment, and results show that the AR-GLP1R-01 and AR-GLP1R-02 have similar effects with Exendin-9-39, and also have the problem of inhibiting excessive insulin secretion of islets caused by loss of function mutation of an ATP-dependent potassium ion channel, thereby achieving the aim of treating CHI blood glucose increase, and the acting time of AR-GLP1R-01 and AR-GLP1R-02 is obviously prolonged and the effect is obviously improved.
(3) The invention also provides a medicament for treating hypoglycemia, wherein the medicament takes AR-GLP1R-01 or AR-GLP1R-02 as a main active substance, and the medicament can effectively treat the hypoglycemia and has long maintenance time.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 is a structural formula of Exendin-9-39;
FIG. 2 is the structural formula AR-GLP1R-01;
FIG. 3 is the structural formula AR-GLP1R-02;
FIG. 4 is a schematic of CRISPR/Cas9 technology;
FIG. 5 is a perfusion test of isolated cultured islets;
FIG. 6 is a SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 for pre-drug administration and 1 hour post-drug administration;
FIG. 7 is SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 for the next day, blood glucose values before administration;
FIG. 8 is SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 on day 4, blood glucose levels before and 1 hour after administration;
FIG. 9 is a SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 on day 4, blood glucose levels before administration;
FIG. 10 is a SUR1-KO mouse in vivo test: after 4 days of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, blood glucose levels begin to be fasting from 5 pm to 5 am, after 17 hours of fasting;
FIG. 11 shows blood glucose values for SUR1-KO mice injected subcutaneously with Exendin-9-39 and AR-GLP1R-01 for 16 hours and 24 hours;
FIG. 12 is a perfusion test of isolated cultured islets;
FIG. 13 is a SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-02, and blood glucose changes before and after administration for 1 hr on the first day;
FIG. 14 is a SUR1-KO mouse in vivo test: subcutaneous injection of Exendin-9-39 or AR-GLP1R-01 on day 4, blood glucose levels before and 1 hour after administration;
FIG. 15 is SUR1-KO mouse in vivo test: after 4 days of subcutaneous injection of Exendin-9-39 or AR-GLP1R-01, blood glucose levels begin to be fasting from 5 pm to 5 am, after 17 hours of fasting;
FIG. 16 is an HPLC chromatogram of Exendin-9-39 at various concentrations;
FIG. 17 is a graph showing the blood concentration detection chromatogram of Exendin-9-39 concentration standard curve after SUR1-KO mice were injected;
FIG. 18 is an HPLC chromatogram of different concentrations of AR-GLP1R-01;
FIG. 19 is a graph showing the blood concentration detection chromatogram of AR-GLP1R-01 concentration standard curve after SUR1-KO mice are injected;
FIG. 20 is a chromatogram of different concentrations of AR-GLP1R-02 HPLC;
FIG. 21 is a blood concentration detection chromatogram of AR-GLP1R-02 concentration standard curve after SUR1-KO mice were injected.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The long-acting Exendin-9-39 is a derivative of Exendin-9-39, comprising 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 is used as a GLP-1 receptor antagonist in the treatment of hypoglycemia, and the GLP-1 receptor antagonist is an inhibitor for inhibiting the function of a GLP-1 receptor. 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 mu M and 13 mu M. 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 medicament for treating hypoglycemia according to the present invention comprises a GLP-1 receptor antagonist. GLP-1 receptor antagonists are inhibitors that inhibit GLP-1 receptor function. GLP-1 receptor antagonists comprise long-acting Exendin-9-39. 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 concentration of long acting Exendin-9-39 in the GLP-1 receptor antagonist is between 0.1. Mu.M and 13. Mu.M.
Example 1: preparation of AR-GLP1R-01 and AR-GLP1R-02
AR-GLP1R-01 and AR-GLP1R-02 described in the embodiment are Exendin-9-39, and are collectively called long-acting Exendin-9-39.
The structure of Exendin-9-39 is shown in figure 1, and the 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 attaching side chains of the following structure I to lysine side chains at positions 12 and 27 of Exendin-9-39, the principle of which is to extend the metabolism time of the drug itself in vivo.
The AR-GLP1R-01 and AR-GLP1R-02 of the present example were prepared by a third party agency, which is the company of Chengdu Santa Biotechnology Co., ltd.
Example 2: basic technical principle and steps for preparing SUR1 gene knockout mice
1. Principle of
By CRISPR/Cas9 gene knockout technology, crRNA binds to tracrRNA (trans-activating RNA) by base pairing, forming double stranded RNA. This tracrRNA, crRNA binary complex, directs Cas9 protein to cleave double-stranded DNA at specific sites of the crRNA guide sequence target. At the site complementary to the crRNA guide sequence, the HNH nuclease domain of Cas9 protein cleaves the complementary strand and the Cas9 RuvC-like domain cleaves the non-complementary strand, thus achieving the function of knocking out the gene of interest, and preparing a gene knockout mouse model.
2. Design, construction and purification of vectors
A pair of oligonucleotide strand sequences of 20bp length for target DNA was designed according to the Score using CRISPR Design tool (http:// CRISPR. Mit. Edu /) from the university institute of Japan for the preparation of sgRNA, and primers were designed in the target region for subsequent gene identification in mice. The designed sequences were synthesized into PAGE products.
Annealing the synthesized 2 single-stranded oligonucleotide sgRNA sequences (naturally cooling to room temperature after 5min at 95 ℃) to form double-stranded DNA, linking with pGK1.1linear vector under the action of T4 DNA ligase to construct sgRNA expression vector, transforming recombinant plasmid into DH5a competent cells, screening and identifying positive cloning plasmid through kanamycin resistance of pGK1.1linear vector and sequencing of target DNA, selecting correct colony clone, and extracting plasmid for preparing in vitro transcription template after expanding culture.
3. In vitro transcription
The expression vector for sgRNA was linearized by DraI cleavage, purified by phenol-chloroform extraction, dissolved in nuclease-free water as template for in vitro transcription, and the sgRNA was synthesized by T7 RNA polymerase in vitro according to the MEGAshortscript Kit (Ambion, AM 1354) kit.
4. Microinjection of Cas9/sgRNA
Mixing the transcribed sgRNA and cas9, adjusting injection concentration, microinjecting the mixture into cytoplasm of fertilized eggs of C57BL/6 mice by using a TE2000U microinjection instrument, transplanting the fertilized eggs into uterus of pseudopregnant C57BL/6 female mice, and waiting for birth of F0-generation mice.
5. Identification of F0 mice
At 5-7 days after birth of F0 generation mice, marking the mice by a toe shearing method, extracting DNA from the sheared rat tail tissues by a phenol chloroform method, identifying the primers designed in the target area according to the experiment (1), and selecting a PCR positive sample for sequencing.
6. Heritability detection of F0 mice
And mating the F0 generation mice with correct PCR and sequencing with wild type C57BL/6 mice to generate F1 generation mice, and identifying the F1 generation mice according to an identification method of the F0 generation mice to obtain positive F1 generation heterozygote mice which can be inherited stably.
7. Abcc8 Gene production protocol
The model adopts CRISPR/Cas9 technology to carry out gene editing on the Abcc8 gene, and the principle schematic diagram is shown in figure 4.
(1) Primer information:
(2) PCR amplification system:
| reaction composition | Volume (mu L) |
| gDNA template | 2.0 |
| 10×Taq Buffer(mg 2+ plus) | 2.0 |
| dNTP mixture (10 mM) | 0.5 |
| Primer mix(10μM) | 0.5 |
| Taq DNA polymerase (5U/. Mu.L) | 0.5 |
| Milli-Q H 2 O | To 20μL |
(3) PCR amplification procedure
Example 3: islet isolation and purification and culture of SUR1-KO mice
1. The islet isolation and purification of SUR1-KO mice comprises the following steps:
(1) After the mice are anesthetized, the mice are fixed on an operation plate, the abdominal cavity is opened, a hemostat is used for locking the bile duct into the entrance of the duodenum, then a 5mL syringe and a 31.5-gauge needle are used for injecting 2mg/mL collagenase solution into the bile duct, and the mice are stopped after pancreas is fully filled.
(2) After removing the pancreas, removing fat and non-pancreatic tissues, transferring the pancreas into a 50mL centrifuge tube, pouring 3mL of 2mg/mL collagenase solution, and shaking in a water bath at 37 ℃ for 4-5 minutes.
(3) Immediately after the shaking is finished, hanks buffer is added into a 50mL centrifuge tube to 50mL, then the centrifuge is carried out, the supernatant is removed, 5mL of Histopaque-1119 solution is added into the rest tissue sediment, and the shaking and the mixing are carried out uniformly.
(4) 5mL of Histopaque-1077 solution was slowly added, followed by 5mL of Hanks buffer and centrifugation.
(5) Islet tissue was removed from between the layered fluids, and purified islets were picked under a dissecting microscope, washed with Hanks buffer and cultured.
2. Islet culture procedure for SUR1-KO mice:
1. preparation of RPMI 1640 culture solution: the preparation method comprises the following raw materials in milliliters: 10 parts of glucose, 2 parts of glutamine, 10 parts of fetal bovine serum, 24 parts of sodium bicarbonate, 100units/mL of penicillin and 10 mug/mL of streptomycin, and the pH is adjusted to 7.2.
2. Culturing the isolated and purified islets with RPMI 1640 medium at 37deg.C with 5% CO 2 And an incubator with 95% air humidity overnight.
Example 4: examination of islet perfusion and insulin secretion
(1) The reaction solution: krebs buffer
A) The method comprises the following steps NaCl:107g was dissolved in 1000mL of water.
B)：KCl(5.96g)+NaHCO 3 (32.256g)+MgCl 2 -6H 2 O (3.25 g) was dissolved in 1000mL of water.
C)：CaCl 2 -2H 2 O:5.168g was dissolved in 1000mL of water.
Krebs buffer：
BSA(0.25％)：2.0g
HEPES：1.906g(10mM)
(2) Stimulation fluid configuration
30mM KCl solution; amino Acid Mixture (AAM)
AAM(100mL)：
| Amino acids | Content (mg) | Amino acids | Content (mg) |
| Alanine (Ala) | 155.7 | Isoleucine (Ile) | 49.32 |
| Arginine (Arg) | 157.6 | Leucine (leucine) | 85 |
| Aspartic acid | 21.8 | Lysine | 270.2 |
| Citrulline | 65.16 | Methionine | 28.64 |
| Glutamic acid | 70.128 | Ornithine | 46.52 |
| Glycine (Gly) | 89.48 | Phenylalanine (Phe) | 54.2 |
| Histidine | 48.422 | Proline (proline) | 160.68 |
| Serine (serine) | 238.89 | Threonine (Thr) | 128.64 |
| Valine (valine) | 94.16 | Tryptophan | 60.44 |
(4) Islet count: 120 islets were collected in islet cells and placed in a 37 ℃ thermostatic waterbath.
(5) The prepared reaction solutions are respectively placed in a water bath kettle (37 ℃), and are inserted into corresponding sample injection pipes, and the following instrument procedures are carried out:
(6) Secreted insulin was collected from Waters Fraction Collector, 10 μl of solution after islet stimulation experiments was removed and transferred into 384 well plates; antibodies were added according to the HTRF insulin assay kit instructions, after shaking and mixing, incubated at room temperature for 2 hours, hormone secretion values were calculated according to standard curves using the HTRF program reading of the Clariostar microplate reader of BMG, and the effect of the stimulus or drug on insulin secretion was confirmed by measuring insulin secretion.
Example 5: long-acting Exendin-9-39 animal experiment
(1) Experimental animals: 41 SUR1-KO mice
(2) Experimental compounds: exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02;
(3) Experimental instrument: blood glucose meter
(4) Experimental grouping:
a. experiment group 1: 10 SUR1-KO mice were injected with Exendin-9-39;
b. experiment group 2: 10 SUR1-KO mice were injected with AR-GLP1R-01;
c. experiment group 3: 10 SUR1-KO mice were injected with AR-GLP1R-02;
d. control group: 10 SUR1-KO mice were injected with physiological saline.
(5) The experimental method comprises the following steps:
A. taking out Exendin-9-39 and two derivatives AR-GLP1R-01 and AR-GLP1R-02 at-80deg.C, dissolving in sterile physiological saline to concentration of 108 μmol/L, and packaging at-80deg.C for storage.
B. Subcutaneous injections were made at 2 μl/g body weight, 2 times/day (10 a.m. and 5 a.m.) and blood glucose values were measured 1h before and after the first injection on the first, second and fourth days. The control group was normal saline.
C. After the continuous treatment for 4 days and the secondary injection on the fourth day, the grains of the mice are taken away, the padding is replaced, the water is not stopped, and the fasting is started.
D. Blood glucose values were measured after 16h of fasting.
E. If the test is to be injected only once, the doses and methods of injection are as described in A and B.
Example 6: concentration of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 in serum of SUR1-KO mice
Measurement
The concentrations of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 in the serum of SUR1-KO mice were determined by liquid chromatography.
(1) Experimental conditions:
A. drug concentration: 1080 mu mol/L
B. Injection method and dosage: c57BL/6 mice were intraperitoneally injected at a weight of 2. Mu.L/g
C. Blood taking time: 0.5h,2h,10h
D. The blood taking method comprises the following steps: blood is taken from the inner canthus of the eye of the mouse, 10 mu L of DPP4 inhibitor is added into the blood, the mixture is kept still, and upper serum is taken after centrifugation for 3min at 3000r/min at 4 ℃ and then stored at-20 ℃.
(2) Serum treatment:
the protein in the serum was treated in a ratio of serum to acetonitrile=1:1.5 and the filtration membrane was subjected to a liquid loading.
(3) Experimental instrument:
(4) Setting parameters of an experimental instrument:
mass spectral parameters
Chromatographic parameters
Examples7: experimental results and analysis
Comparison study of AR-GLP1R-01 and Exendin-9-39
1. Study of the influence of Exendin-9-39 and AR-GLP1R-01 on isolated islet function:
FIG. 5 is a perfusion test of isolated cultured islets showing the dynamic secretion of insulin, the stimulus being an amino acid cocktail (AAM), climbing a slope at a concentration of from 0 to 12 mM; islets are SUR1 knockout mouse islets. As shown in FIG. 5, SUR1-KO mice had elevated basal insulin secretion and were sensitive to insulin secretion stimulated by amino acid cocktail (AAM), which was consistent with the results of previous studies. 100nM Exendin-9-39 can reduce basal insulin secretion which is too high and AAM-stimulated insulin secretion, also consistent with earlier studies. The same concentration of AR-GLP1R-01 (100 nM) acts similarly to Exendin-9-39, and can significantly inhibit basal insulin and AAM-stimulated insulin secretion. The results show that the AR-GLP1R-01 has the same action as Exendin-9-39, and can solve the problems of excessive basal insulin secretion and excessive insulin secretion reaction to amino acid stimulation caused by the damage of islet ATP dependent potassium ion channels.
2. Animal experiment results of Exendin-9-39 and AR-GLP1R-01
Living animal experiments were performed using Exendin-9-39 and AR-GLP 1R-01. The experimental design is that Exendin-9-39 and AR-GLP1R-01 are subcutaneously injected twice daily (10 am and 5 pM), and the administration is continued for 4 days, wherein the administration concentration is 216pM/g body weight.
FIG. 6 is a SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), changes in blood glucose before and after 1 hour of administration on the first day. Exendin-9-39 and AR-GLP1R-01 significantly raised blood glucose in SUR1-KO mice. As shown in FIG. 6, blood glucose of the disease mouse model was significantly elevated by AR-GLP1R-01 and Exendin-9-39 effects, both after 1 hour of injection, compared to blood glucose of basal non-administered SUR1-KO mice.
FIG. 7 is SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), the following day of administration, blood glucose values before administration. FIG. 7 shows that basal blood glucose elevation was not sustained in Exendin-9-39 treated groups as in the blank, but that the effect of AR-GLP1R-01 was maintained to 10 AM (about 17 hours) the second day after 2 doses were injected the first day, indicating that AR-GLP1R-01 could improve blood glucose in the disease mouse model for a long period of time and was maintained to the fourth day, before the next morning dosing.
FIG. 8 is SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), day 4 of administration, blood glucose values before administration and 1 hour after administration. As shown in FIG. 8, after the administration of the Exendin-9-39 group twice daily for 3 days, the basal blood sugar of the Exendin-9-39 group is the same as that of the blank control, and the blood sugar can be obviously increased after the injection of the Exendin-9-39 for 1 hour, which indicates that the short-time treatment effect of the Exendin-9-39 can be repeatedly generated. Compared with the Exendin-9-39 group and the blank control group, the AR-GLP1R-01 not only can maintain the elevation of basal blood sugar value observed on the next day, but also has the capability of continuously elevating blood sugar for a short time after 1 hour of injection.
FIG. 9 is a SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), day 4 of administration, blood glucose values before administration. FIG. 9 shows that AR-GLP1R-01 significantly increased basal blood glucose in SUR1-KO mice, but Exendin-9-39 did not improve basal blood glucose, again confirming that the long-term elevation of blood glucose by AR-GLP1R-01 was significantly better than Exendin-9-39.
FIG. 10 is a SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-01, dose: 216pM/g body weight), after 4 th day of administration, 5 pM began to empty stomach to 5 am, blood glucose levels after 17 hours empty stomach. As shown in FIG. 10, AR-GLP1R-01 significantly increased basal blood glucose in SUR1-KO mice after two daily administrations over 4 days, but Exendin-9-39 did not improve basal blood glucose. Meanwhile, AR-GLP1R-01 can obviously improve fasting blood glucose of a mouse model for treating group diseases. The long-term effect of AR-GLP1R-01 is obviously superior to that of Exendin-9-39, and the short-term effect (1 hour after injection) is equivalent to that of Exendin-9-39.
To further verify the conclusion of this test, the effect of one injection was observed for 24 hours, as shown in FIG. 11, for 20 SUR1-KO mice (female-male ratio 1:1), after the blood glucose level of the base (not administered) was measured, exendin-9-39 and AR-GLP1R-01 were subcutaneously injected at a dose of 216pM/g body weight, blood glucose was observed for 16 hours and 24 hours after administration, the short-term effect of Exendin-9-39 on elevation of blood glucose could not be maintained for 16 and 24 hours, but the effect of AR-GLP1R-01 on one administration could be maintained for 24 hours.
Comparison study of (two) AR-GLP1R-02 and Exendin-9-39
1. Study of the influence of Exendin-9-39 and AR-GLP1R-02 on isolated islet function:
FIG. 12 is a perfusion test of isolated cultured islets showing the dynamic secretion of insulin, the stimulus being an amino acid cocktail (AAM), climbing a slope at a concentration of from 0 to 12 mM; islets are SUR1 knockout mouse islets. As shown in FIG. 12, SUR1-KO mice had elevated basal insulin secretion, was sensitive to insulin secretion stimulated by amino acid cocktail (AAM), and was consistent with the results of previous studies. 100nM Exendin-9-39 can reduce basal insulin secretion which is too high and AAM-stimulated insulin secretion, also consistent with earlier studies. The same concentration of AR-GLP1R-02 (100 nM) acts similarly to Exendin-9-39, and both significantly inhibits basal insulin and AAM-stimulated insulin secretion. The AR-GLP1R-02 has the same action as Exendin-9-39, so that the problems of excessive basal insulin secretion and excessive insulin secretion response to amino acid stimulation caused by the damage of an insulin ATP dependent potassium ion channel can be solved.
2. Animal experiment results of Exendin-9-39 and AR-GLP 1R-02:
living animal experiments were performed using Exendin-9-39 and AR-GLP1R-02. The test design is that Exendin-9-39 and AR-GLP1R-01 are injected subcutaneously twice daily (10 am and 5 pM), and the administration is continued for 4 days, wherein the administration concentration is 216pM/g body weight.
FIG. 13 is a SUR1-KO mouse in vivo test: subcutaneous injection of drug (Exendin-9-39 or AR-GLP1R-02, dose: 216pM/g body weight), changes in blood glucose before and after 1 hour of administration on the first day. As shown in FIG. 13, exendin-9-39 can improve blood glucose in disease model for a short period of time, but AR-GLP1R-02 did not increase after the first dose, compared to blood glucose in SUR1-KO mice without basal administration for 1 hour.
After 3 days of administration, the basal blood glucose of the AR-GLP1R-02 group is equivalent to that of the other two groups compared with that of a blank solvent control group and that of an Exendin-9-39 treatment group, and the basal blood glucose of the AR-GLP1R-02 group is consistent with that of the Exendin-9-39 after 1 hour of administration, so that the blood glucose of a model mouse can be raised in a short time. After the 4 th afternoon administration, mice began the fasting test, and the overnight fasting blood glucose condition was observed on the 5 th morning, as shown in fig. 15, and AR-GLP1R-02 could improve the fasting blood glucose in the treatment group disease mice model after the 4 th day twice daily administration. The AR-GLP1R-02 is similar to Exendin-9-39, the short-time effect (1 hour after injection) is equivalent to Exendin-9-39, and the drug effect can be maintained to be fasting, so that the long-time effect is superior to Exendin-9-39.
(III) Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 blood concentration detection
(1) Absorption peaks of Exendin-9-39, AR-GLP1R-01 and AR-GLP1R-02 different concentration standards: for Exendin-9-39, as shown in FIGS. 16 and 17; for AR-GLP1R-01, as shown in FIGS. 18 and 19; for AR-GLP1R-02, as shown in FIGS. 20 and 21; a standard graph is drawn based on the chromatogram and the area under the peak of the standard.
(2) The results of the blood concentration calculation are shown in Table 1.
As a result of detection by liquid chromatography mass spectrometry, it was found that Exendin-9-39 had an absorption peak of about 4.9 minutes and AR-GLP1R-01 and AR-GLP1R-02 had an absorption peak of about 7.7 minutes. The test mice (SUR 1-KO) were subcutaneously injected with the drug at a concentration of 216pM/g body weight, respectively. Blood was then taken at 0.5, 2 and 10 hours post-dose to determine plasma concentration. From the results of the calculation of the concentrations of the three components in serum based on the areas of the absorption peaks, it can be seen that at 0.5h, the concentration of Exendin-9-39 in serum is lower than that of AR-GLP1R-01 and AR-GLP1R-02,2 hours, and that no Exendin-9-39 can be detected in serum, but AR-GLP1R-01 and AR-GLP1R-02 can still be detected at 10 hours, indicating that the half-lives of AR-GLP1R-01 and AR-GLP1R-02 after side chains are superior to those of Exendin-9-39, and also demonstrating that the liquid chromatography mass spectrometry can detect the concentrations of the three samples in serum.
TABLE 1 blood concentration
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the 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.
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| PCT/CN2023/073843 WO2023231433A1 (en) | 2022-05-31 | 2023-01-30 | Long-acting exendin-9-39 and use thereof in hypoglycemia treatment and as drug for treating hypoglycemia |
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| DE59712555D1 (en) * | 1996-06-05 | 2006-04-06 | Roche Diagnostics Gmbh | EXENDIN ANALOGUE, PROCESS FOR THE PRODUCTION THEREOF AND THE MEDICAMENTS CONTAINING THEREOF |
| ES2908838T3 (en) * | 2007-01-08 | 2022-05-04 | Univ Pennsylvania | GLP-1 receptor antagonist for use in the treatment of congenital hyperinsulinism |
| UA116217C2 (en) * | 2012-10-09 | 2018-02-26 | Санофі | Exendin-4 derivatives as dual glp1/glucagon agonists |
| TW201625668A (en) * | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| JP2019535734A (en) * | 2016-11-21 | 2019-12-12 | アイガー・バイオファーマシューティカルズ・インコーポレイテッドＥｉｇｅｒ Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． | Buffered formulation of exendin (9-39) |
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