CN111494371A

CN111494371A - Application of Pyr3

Info

Publication number: CN111494371A
Application number: CN202010240024.XA
Authority: CN
Inventors: 徐迅迪
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-08-07
Anticipated expiration: 2040-03-30
Also published as: WO2021196335A1; CN111494371B

Abstract

The invention relates to the technical field of medicines, in particular to application of Pyr 3. Experiments prove that Pyr3 can significantly reduce the tumor volume of tumor-bearing mice and inhibit the proliferation and migration of tumor cells, and has positive significance for treating cancers. Therefore, the invention provides the application of Pyr3 in preparing anti-tumor medicaments and provides an anti-tumor medicament containing Pyr 3. Studies showed that subcutaneous tumor volumes in Pyr3 group were significantly smaller than NC group from day 24 until day 32 after drug dry prognosis for tumor-bearing animals, with statistical differences (p < 0.05).

Description

Application of Pyr3

Technical Field

The invention relates to the technical field of medicines, in particular to application of Pyr 3.

Background

Cancer is a general term for malignant tumors. Characterized by uncontrolled division of abnormal cells, unlimited proliferation, invasion and destruction of surrounding normal tissues. They can be classified into carcinoma, sarcoma, blastoma, and malignant tumor according to their tissue origin. Cancer from epithelial tissue; sarcomas, which originate in adipose tissue, fibrous tissue, muscle tissue (collectively mesenchymal tissue) and lymphoid reticulum; a so-called blastoma derived from embryonic tissue; malignant tumor such as malignant schwannoma should not be used; there are also cancers specifically referred to as hematologic malignancies and are also known as leukemias. Cancer cells are also metastasized by blood and lymph fluid and can spread directly. The etiology of cancer is complex, and the main inducing factors are chemical carcinogens, special viruses, rays, hormones, free radicals and the like; genetics, diet, and mental state are important contributing factors.

Hepatocellular carcinoma (HCC) is the fifth most common cancer type worldwide and the third leading cause of cancer-related death worldwide. The diagnosis and treatment of HCC has progressed significantly over the past decades, but its long-term prognosis is still less than satisfactory. The poor long-term prognosis of HCC is often due to late diagnosis, high recurrence rate after treatment, and chemotherapy resistance. Therefore, there is an urgent need to further explore more effective and safer anticancer strategies that inhibit the progression of HCC.

1- (4- (2, 3, 3-trichloroacrylamido) phenyl) -5- (trifluoromethyl) -1H-pyrazole-4-carboxylic acid ethyl ester, Pyr3 for short, CAS No. 1160514-60-2; the structural formula is shown as formula I

The TRPC3 channel is one of the members of the TRP (transient receptor potential) channel family, and the TRPC3 channel is a non-selective cation channel, mainly distributing cell membranes, which when open allows cations such as calcium and sodium ions to pass through, thereby participating in intracellular calcium homeostasis. Pyr3 is a TRPC3 channel specific inhibitor and can effectively block the TRPC3 channel, thereby affecting the biological function of the TRPC3 channel. In the prior art, Pyr3 is reported to have an effect on the left ventricular hypertrophy of rats induced by pressure load, and Pyr3 is also reported to improve the prognosis of mice after cerebral hemorrhage and the like. However, the therapeutic effect of Pyr3 or the analogues thereof on cancer has not been reported previously.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide the use of Pyr3, and the research of the present invention finds that Pyr3 can reduce the tumor volume of tumor-bearing mice, inhibit the proliferation and migration of tumor cells, and has a positive significance for the treatment of cancer.

The invention provides application of Pyr3 or an analogue thereof in preparing a medicament for inhibiting tumor cell proliferation.

In embodiments of the invention, said inhibiting tumor cell proliferation comprises: inhibiting the increase in the number of cells and/or inhibiting the formation of clones.

The invention also provides application of Pyr3 or the analogue thereof in preparing a medicament for inhibiting tumor cell migration.

The invention also provides application of Pyr3 or the analogue thereof in preparing a medicament for inhibiting tumor volume increase.

The invention also provides application of Pyr3 or the analogue thereof in preparing a medicament for treating tumors.

In the present invention, the tumor is a malignant tumor selected from the group consisting of carcinoma, sarcoma and/or blastoma.

In some embodiments, the site of tumorigenesis comprises one or more of head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gall bladder, colon, rectum, ovary, cervix, uterus, prostate, bladder, testis.

In some embodiments, the tumor is liver cancer.

In some embodiments, the liver cancer is hepatocellular carcinoma or hepatoblastoma.

In a specific embodiment, Huh7 cells were used to verify the therapeutic effect of Pyr3 on hepatocellular carcinoma. The therapeutic effect of Pyr3 on hepatoblastoma was verified with HepG2 cells.

In the present invention, the Pyr3 or its analogues were treated at a concentration of 1. mu. mol/L-4. mu. mol/L for the cell experiments, and the dose of Pyr3 or its analogues was 10 mg/kg. d for the animal experiments^-1. The administration mode is intraperitoneal injection, and the solvent for injection is DMSO.

The invention also provides a medicament for treating tumors, which comprises Pyr3 or an analogue thereof.

The medicine also comprises pharmaceutically acceptable auxiliary materials.

The medicine of the invention can also comprise other medicines with anti-tumor function.

In some embodiments of the present invention, the pharmaceutically acceptable excipient is one or a mixture of two or more of a flavoring agent, an osmotic pressure regulator, a filler, a lubricant, a preservative, a suspending agent, an edible pigment, a diluent, an emulsifier, a disintegrant, or a plasticizer.

Preferably, the medicament is an oral preparation or an injection. The oral preparation can be tablet, pill, oral liquid, capsule, syrup, dripping pill or granule. Preferably, the capsule is a hard capsule or a soft capsule. The tablet is oral tablet or buccal tablet. The oral tablet refers to a tablet for oral administration, and most of the tablets have the drug which is absorbed through the gastrointestinal tract to exert the effect, and some tablets have the drug which is locally exerted in the gastrointestinal tract. In some embodiments provided herein, the oral tablet is a compressed tablet, a dispersible tablet, an effervescent tablet, a chewable tablet, a coated tablet, or a sustained release tablet. The injection comprises powder injection or injection.

The invention also provides a method of treating a tumour comprising administering Pyr3 or an analogue thereof.

In the invention, the Pyr3 analogue is a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer or clathrate of Pyr 3.

The agents of the invention may be used in combination therapy, i.e. in combination with one or more other agents, wherein the combination comprises Pyr3 administered together with the other agent or sequentially. For example, the other agent may be administered before, during, or after Pyr3 or an analog thereof. The mode of administration may be the same or different, for example Pyr3 and the other agents may both be administered in injectable form, may both be administered orally, or may be administered orally, one administered by injectable form and the other.

Experiments prove that Pyr3 can significantly reduce the tumor volume of tumor-bearing mice and inhibit the proliferation and migration of tumor cells, and has positive significance for treating cancers. Therefore, the invention provides the application of Pyr3 in preparing anti-tumor medicaments and provides an anti-tumor medicament containing Pyr 3. Studies showed that subcutaneous tumor volumes in Pyr3 group were significantly smaller than NC group from day 24 until day 32 after drug dry prognosis for tumor-bearing animals, with statistical differences (P < 0.05).

Drawings

FIG. 1 shows the results of subcutaneous tumor experiments in mice, wherein A represents the tumor mass in the control group (NC) and in which Pyr3 was administered; b is a curve of tumor volume change of two groups of mice with time as an abscissa;

FIG. 2 shows the results of MTT experiments, wherein A shows the proliferation of HepG2 cells; b shows proliferation of Huh7 cells;

FIG. 3 shows the results of a plate cloning experiment; wherein A represents the cloning formation of each group of HepG2 cells; b shows the clonogenic profiles of the groups of Huh7 cells;

FIG. 4 shows scratch test results; wherein, A represents the migration condition of HepG2 cells in each group; b shows migration of Huh7 cells in each group.

Detailed Description

The invention provides the application of Pyr3, and a person skilled in the art can use the content for reference and appropriately modify the technological parameters to realize the Pyr 3. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

In the examples of the present invention, 6-week-old male NOD/SCID mice were used, provided by schlaikeda experimental animals ltd, the license number: SCXK (Xiang) 2016-. Mice were housed in SPF-scale laboratories. Pyr3 (code: B7494) was purchased from ApexBio.

The invention is further illustrated by the following examples:

example 1: subcutaneous tumor model test

Clean-grade feeding, inoculating HepG2 liver cancer cells 2 x 10^ 6/mouse neck and back subcutaneous after 1 week adaptation period until subcutaneous tumor volume reaches about 100mm³The mice were then divided equally into two groups (NC group: DMSO solvent group, TRPC3 inhibitor group: Pyr3 group), 5 mice each, two groups of mice were each intraperitoneally injected with the solvents DMSO and Pyr3(10mg/kg) daily for 32 days, tumor volumes were measured every 3-4 days, and subcutaneous tumor specimens were collected after 32 days.

As shown in fig. 1A and 1B, from day 24 to day 32 of the drug-dry prognosis, the subcutaneous tumor volume of Pyr3 group was significantly smaller than that of NC group, and the difference was statistically significant (P < 0.05).

Example 2: MTT assay

Hepatoma cells HepG2 and Huh7 at 5 × 10³Cells/well were plated in 96-well plates and intervened with Pyr3 at different concentrations for 24, 48, 72h next, 20 μ L MTT (5mg/ml, st louis, usa) was added to each well and incubation continued for 4h the crystal violet was solvent shaken with 150 μ L DMSO the absorbance measurements were taken at 490nm wavelength.

As shown in FIGS. 2A and 2B, 4uM Pyr3 and 1uM Pyr3 intervene in hepatoma cells HepG2 and Huh7, respectively, the proliferation capacity of hepatoma cells is obviously reduced compared with that of the NC group, and the difference is statistically significant (P < 0.0001).

Example 3: plate cloning experiment

Hepatoma cells HepG2 and Huh7 were seeded in 6-well plates (1.5 × 10) in 2m L complete medium³/well) and then cultured in air containing 5% CO2 at 37C for 2 weeks. During which the medium was changed every two days with a different Pyr3 concentration. After 2 weeks the cells were stained with 0.5% crystal violet and counted at 50 x magnification.

As shown in FIGS. 3A and 3B, 4uM Pyr3 and 1uM Pyr3 intervene in hepatoma cells HepG2 and Huh7, respectively, the proliferation capacity of hepatoma cells is obviously reduced compared with that of the NC group, and the difference is statistically significant (P < 0.01).

Example 4: scratch test

In the scratch experiment, hepatoma cells HepG2 and Huh7 were seeded into 6-well plates and cultured to a confluent monolayer. Monolayers were scored in parallel with 10ul sterile tip heads. The medium was replaced by 1ml of serum-free medium containing Pyr3 at various concentrations. The scratch healing process was photographed at 0, 24 and 48 hours, respectively.

As shown in FIGS. 4A and 4B, 4uM Pyr3 and 1uM Pyr3 intervene in hepatoma cells HepG2 and Huh7, respectively, the migration capacity of hepatoma cells is obviously reduced compared with that of the NC group, and the difference is statistically significant (P < 0.01).

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

Claims

Use of Pyr3 or an analogue thereof in the manufacture of a medicament for inhibiting tumor cell proliferation.
2. The use of claim 1, wherein said inhibiting tumor cell proliferation comprises: inhibiting the increase in the number of cells and/or inhibiting the formation of clones.
Use of Pyr3 or an analogue thereof in the manufacture of a medicament for inhibiting migration of tumour cells.
Use of Pyr3 or an analogue thereof in the manufacture of a medicament for inhibiting tumor volume growth.
Use of Pyr3 or an analogue thereof in the manufacture of a medicament for the treatment of a tumour.
6. The use according to any one of claims 1 to 5, wherein the tumour is a malignant tumour selected from the group consisting of carcinomas, sarcomas and/or blastomas.
7. The use of claim 6, wherein the site of tumorigenesis comprises one or more of head and neck, brain, thyroid, esophagus, pancreas, lung, liver, stomach, breast, kidney, gall bladder, colon, rectum, ovary, cervix, uterus, prostate, bladder, testis.
8. The use of any one of claims 1 to 7, wherein the tumor is liver cancer; the liver cancer is hepatocellular carcinoma or hepatoblastoma.
9. The use according to any one of claims 1 to 8, wherein the dose of Pyr3 or the like is 10mg/kg d^-1。
10. A medicament for the treatment of tumours, comprising Pyr3 or an analogue thereof.