CN112336725A

CN112336725A - Novel medical application of trimethoprim

Info

Publication number: CN112336725A
Application number: CN202011463916.2A
Authority: CN
Inventors: 吴照球; 傅蓉; 任博雪; 李阳
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-02-09

Abstract

The invention relates to a new medical application of trimethoprim, in particular to an application of trimethoprim in preparing a medicament for treating tumors, especially in preparing medicaments for treating tumors such as lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer and the like with high expression of Snail protein.

Description

Novel medical application of trimethoprim

Technical Field

The invention relates to an application of Trimethoprim (Trimethoprim) in inhibiting tumor proliferation and metastasis, belonging to the technical field of medicines.

Background

Malignant tumor is one of the major diseases seriously threatening human life, and according to the latest statistics, the number of newly added tumor patients in 2018 is 2000 ten thousand, and the number of patients dying from cancer is as high as 1000 ten thousand. About 90% of the deaths from malignant tumors are caused by the metastasis of the primary tumor, and the mortality of tumors can be fundamentally reduced only by effectively controlling the occurrence and development of early metastasis of tumors. At present, most of clinical treatment on tumors focuses on surgical treatment and later-stage radiotherapy and chemotherapy, but the effect is not ideal, the toxic and side effects are large, and great pain is brought to patients with advanced cancers.

The zinc finger transcription factor Snail as the core member of Snail family plays an important role not only in physiological process such as embryonic development, but also in tumor proliferation and metastasis process. The Snail is highly expressed in various malignant tumor tissues, such as lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer and pancreatic cancer, and is closely related to poor prognosis, local invasion, relapse, metastasis and the like of malignant tumors. The Snail protein can promote the close connection between tumor cells to be reduced and the motility to be enhanced, thereby leading the tumor cells to have the ability of invading and transferring to the far distance. Meanwhile, the Snail protein can promote the proliferation of tumor cells and inhibit the apoptosis of the tumor cells by high expression, thereby further causing the development of tumors. The research finds that the high expression of the Snail protein is related to various oncogenic signals, such as transforming growth factor beta (TGF beta), Epidermal Growth Factor (EGF), fibroblast growth factor (FGF2), Notch and the like, and is a common downstream node of a plurality of oncogenic related signal pathways, and the high expression of the Snail protein affects various pathways including angiogenesis, cell proliferation, invasion and metastasis, tumor metabolism and the like. Therefore, the targeted Snail protein is developed into an anti-tumor drug to provide a new drug for clinical tumor treatment.

Trimethoprim is a bacterial dihydrofolate reductase inhibitor clinically, and is used for clinically treating infection caused by gram-positive bacteria, such as acute simple urinary tract infection, bacterial prostatitis and pneumonia caused by streptococcus pneumoniae. And can be used for treating infection caused by gram-negative bacteria, such as enteritis, pneumonia, dysentery, typhoid fever, and pertussis. At present, trimethoprim has small adverse reaction in the clinical treatment process, and no report of applying trimethoprim to tumor resistance is found so far.

Disclosure of Invention

According to the invention, a new mechanism of trimethoprim for inhibiting the expression of Snail protein in tumor cells is found through a tumor cell model and a mouse tumor model, so that the proliferation and metastasis capacities of the tumor cells are inhibited.

The invention provides application of trimethoprim in preparing a medicine for treating tumor diseases with high expression of Snail protein.

The chemical structural formula of the trimethoprim is as follows:

furthermore, the trimethoprim can inhibit proliferation and invasion of Snail protein high-expression tumors, and the tumor diseases comprise lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer and pancreatic cancer.

On the other hand, the pharmaceutical composition contains trimethoprim with effective treatment dose, and the pharmaceutical composition is applied to the preparation of the drugs for treating tumor diseases.

On the other hand, the medicinal preparation contains trimethoprim with effective anti-tumor treatment dosage and a pharmaceutically acceptable carrier, adjuvant or vehicle, and is applied to the preparation of the medicament for treating the diseases of the Snail protein high-expression tumor.

In the present invention, when the above-mentioned compound and a pharmaceutically acceptable salt thereof, tablet, capsule, pill, granule, suspension, dripping pill or oral liquid preparation, and a solvate of these compounds (herein, collectively referred to as "therapeutic agent") are administered to a mammal, they may be used alone or, preferably, they may be used in combination with a pharmaceutically acceptable carrier or diluent according to a standard pharmaceutical method. The mode of administration can be by various routes, including oral, parenteral, or topical administration. Parenteral administration as used herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

The invention is proved by experiments that: trimethoprim can obviously reduce the expression of Snail protein in tumor cells.

In the experimental process, the dosage of the trimethoprim is as follows: cells 0.4-1mM, animal 100-200 mg/kg.

The results of the cell experiments show that: trimethoprim is capable of significantly reducing the level of Snail protein expression in a variety of tumor cells. Also, trimethoprim dose-dependently inhibits the proliferation and metastatic ability of a variety of tumor cells.

The results of animal experiments show that: the trimethoprim can obviously inhibit the proliferation of subcutaneous xenograft tumor of the mouse, has no obvious toxicity to each main organ of the mouse under the treatment dosage and has no influence on the weight of the mouse. Meanwhile, trimethoprim can obviously inhibit tumor metastasis in mice. The invention has the advantages that:

(1) the invention discovers that trimethoprim has the effects of inhibiting tumor proliferation and invasion for the first time, and has no obvious toxicity under the effective dose.

(2) The invention discovers for the first time that trimethoprim can inhibit the expression of Snail protein and play an anti-tumor role.

Drawings

FIG. 1 is a graph showing the effect of trimethoprim on the expression of Snail protein in human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human hepatoma cells HepG2, and human pancreatic cancer cells Panc-1 in the examples;

FIG. 2 is the effect of trimethoprim on the viability of human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human hepatoma cells HepG2, human pancreatic cancer cells Panc-1 in the examples;

FIG. 3 is a graph showing the effect of trimethoprim on the proliferation capacity of subcutaneous mouse transplants. Wherein^**P<0.01vs vehicle group, (GraphPad prism8.0, two-sided Student's t test);

FIG. 4 shows the effect of trimethoprim on the invasiveness of human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-1 in the examples. Wherein^*P<0.05,^**P<0.01vs vehicle group, (GraphPad prism8.0, two-sided Student's t test);

FIG. 5 is a graph showing the effect of trimethoprim on the ability of mice to metastasize to the liver. Wherein^**P<The 0.01vs vehicle group, (GraphPad prism8.0, two-sided Student's t test).

FIG. 6 is a graph showing the effect of trimethoprim on the body weight and the pathology of heart, liver, spleen, lung and kidney in tumor-bearing mice in the examples.

FIG. 7 shows the pharmacological effects of trimethoprim (4g/kg) on heart, liver, spleen, lung and kidney in mice given orally in one time in the examples.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

Drugs and reagents: the trimethoprim used in the experiment is purchased from Beijing Solebao scientific and technological Limited, and other reagents are commercially available analytical pure reagents.

Assay of Snail protein expression in cells: treating human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-148 h with a solvent or trimethoprim (0.4-1mM), collecting the cells, and detecting the expression of Snail protein in the cells according to a Western Blot technology of a protein extraction kit operation instruction.

Cell viability assay: the cell viability is determined by a method of respectively treating human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2, human pancreatic cancer cells Panc-148 h and CCK-8 by using a solvent or trimethoprim (0.03-1 mM).

Determination of Transwell invasion: after the human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-148 h were treated with a solvent or trimethoprim (0.4mM, 0.8mM), the treated cells were made into a single cell suspension and dispersed in a serum-free culture solution, 200. mu.l of the cell suspension was taken and added to the upper chamber, and 1ml of fresh complete medium was added to the lower chamber. After further culturing for 48h, 0.1% crystal violet is stained for several seconds, washed clean with clear water, inverted, ventilated and dried, observed under an optical microscope and photographed.

Determination of proliferation of subcutaneous xenograft tumors in mice: taking HCT116 cells in logarithmic growth phase, inoculating the cells to the subcutaneous back of a nude mouse, and observing the size of the transplanted tumor in real time until the tumor volume reaches 100mm³Then (TV is 3.14 multiplied by length multiplied by width ^ s²/6), experimental nude mice were randomly divided into 3 groups of 6 mice each. Vehicle or trimethoprim (100mg/kg, 200mg/kg) was continuously administered orally for 14 days, the subcutaneous tumor volume of the mice was measured daily, and the body weight of the mice was measured. At the end of the treatment period, mice were isolated for subcutaneous tumors, and heart, liver, spleen, lung, and kidney were h.e. stained.

Determination of tumor metastasis in vivo:2 x 10 to⁶Several GFP-labeled HCT116 cells in the logarithmic growth phase were inoculated into the spleen of 8-week-old male nude mice. Mice were randomized post-operatively into 2 groups of 6 mice each. Vehicle or trimethoprim (200mg/kg) was administered orally for 21 consecutive days starting on the third day after surgery, and at the end of the treatment period, the nude mice were sacrificed and livers were removed for analysis.

And (3) measuring the maximum drug resistance: 24 mice of 8 weeks old, each half male and female, were divided into 2 groups, and vehicle or trimethoprim (4g/kg) was orally administered at one time, and after 3 weeks of observation, the mice were sacrificed, and heart, liver, spleen, lung, and kidney were stained with h.e. to observe pathological changes.

Example 1

Snail protein expression assay: treating human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-148 h by using a solvent or trimethoprim (0.4-1.0mM), collecting the cells, and detecting the expression of Snail in the cells according to a Western Blot technology of a protein extraction kit operation instruction. The test result is shown in figure 1, and the trimethoprim can obviously inhibit the expression of Snail protein in tumor cells and is dose-dependent.

Example 2

Cell viability assay: treating human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-148 hours by using a solvent or trimethoprim (0.03-1mM), and determining the cell activity by using a CCK-8 method. The test results are shown in fig. 2, where trimethoprim is capable of dose-dependent inhibition of tumor cell proliferation.

Example 3

Determination of proliferation of subcutaneous xenograft tumors: taking HCT116 cells in logarithmic growth phase at 1 × 10⁶Inoculating the cells into the subcutaneous layer of the back of a nude mouse, and observing the size of the transplanted tumor in real time until the volume is about 100mm³Then (TV is 3.14 multiplied by length multiplied by width ^ s²6), dividing the experimental nude mice into 3 groups randomly, and dividing each group into 6 mice. Vehicle or trimethoprim (100mg/kg, 200mg/kg) was continuously orally administered for 14 days, the state of nude mice was observed every day, the volume of subcutaneous transplanted tumor of the mice was measured, and the body weight of the mice was measured. To be treatedAt the end of the treatment cycle, subcutaneous tumors were isolated and h.e staining of heart, liver, spleen, lung, kidney was done. The test results are shown in FIGS. 3 and 6, in which^**P<The 0.01vs vehicle group is used,^N.S _.p > 0.05vs vehicle group, (GraphPad prism8.0, two-sided Student's t test). The results show that the trimethoprim can obviously inhibit the proliferation of subcutaneous tumor cells of the mice, and the therapeutic dose has no obvious influence on the body weight and main organs of the mice.

Example 4

Determination of Transwell invasion: after the human colorectal cancer cells HCT116, human breast cancer cells SUM159, human gastric cancer cells MGC-803, human lung cancer cells A549, human liver cancer cells HepG2 and human pancreatic cancer cells Panc-148 h were treated with a solvent or trimethoprim (0.4mM, 0.8mM), the treated cells were made into a single cell suspension and dispersed in a serum-free culture solution, 200. mu.l of the cell suspension was taken and added to the upper chamber, and 1ml of fresh complete medium was added to the lower chamber. After further culturing for 48h, 0.1% crystal violet is stained for several seconds, washed clean with clear water, inverted, ventilated and dried, observed under an optical microscope and photographed. The test results are shown in FIG. 4, in which^*P<0.05,^**P<The 0.01vs vehicle group, (GraphPad prism8.0, two-sided Student's t test). The results show that the trimethoprim can obviously inhibit the invasive and metastatic capacities of various tumor cells.

Example 5

Determination of tumor metastasis in vivo: 2 x 10 to⁶Several GFP-labeled HCT116 cells in the logarithmic growth phase were inoculated into the spleen of 8-week-old male nude mice. Mice were randomized post-operatively into 2 groups of 6 mice each. Vehicle or trimethoprim (200mg/kg) was administered orally for 21 consecutive days starting on the third day after surgery, and at the end of the treatment period, nude mice were sacrificed and livers were removed for analysis. The results of the test are shown in FIG. 5, in which^**P<The 0.01vs vehicle group, (GraphPad Prism8.0, two-sided Student's t test). From the results, it can be seen that trimethoprim can significantly inhibit tumor metastasis in mice.

Example 6

24 mice of 8 weeks old, each half male and female, were divided into 2 groups, and vehicle or trimethoprim (4g/kg) was orally administered at one time, and after 3 weeks of observation, the mice were sacrificed, and heart, liver, spleen, lung, and kidney were stained with h.e. to observe pathological changes. As shown in FIG. 7, the test results showed that the maximum tolerated dose (4g/kg) had no significant effect on the major organs of the mice.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. Application of trimethoprim in preparing medicine for treating tumor is disclosed.

2. The use according to claim 1, wherein the tumor disease is a Snail protein high expression tumor disease.

3. The use of claim 2, wherein said Snail high expression tumor disease comprises lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer.

4. Use according to claim 1, wherein the trimethoprim has the following chemical formula:

5. a pharmaceutical composition comprising an anti-tumour therapeutically effective amount of trimethoprim or a pharmaceutically acceptable salt or ester thereof.

6. The use of the pharmaceutical composition of claim 5 in the preparation of a medicament for the treatment of a tumor disease in which Snail protein is highly expressed.

7. The use of claim 6, wherein the tumor diseases with high expression of Snail protein comprise lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer and pancreatic cancer.

8. A pharmaceutical formulation comprising a therapeutically effective amount of trimethoprim and a pharmaceutically acceptable carrier, adjuvant or vehicle.

9. Use of a pharmaceutical formulation according to claim 8 in the manufacture of a medicament for the treatment of diseases of Snail high expression tumours.

10. The use of claim 9, wherein the Snail high expression tumor disease comprises lung cancer, liver cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer.