CN111803495B - Application of benzydamine hydrochloride in preparation of antitumor drugs - Google Patents

Abstract

The invention discloses application of benzydamine hydrochloride in preparation of antitumor drugs. In particular to the application of benzydamine hydrochloride in preparing the esophagusThe application of the medicine for treating cancer, namely benzydamine hydrochloride, wherein the name of the medicine is as follows: 1-benzyl-3- [3- (dimethylamino) propoxy group]-1H-indazole, english name: benzindamine Hydrochorride molecular formula: c₁₉H₂₄ClN₃Molecular weight of O345.866 CAS number: 132-69-4, the application proves through experiments that when the benzydamine hydrochloride is used for esophageal squamous cell carcinoma cells (KYSE150 cells and KYSE450 cells), the benzydamine hydrochloride can play a role in inhibiting growth and transformation of the esophageal squamous cell carcinoma cells, and the suitable concentration of the benzydamine hydrochloride for inhibiting proliferation and transformation of the esophageal squamous cell carcinoma cells is 2.5-20 mu M.

Description

Application of benzydamine hydrochloride in preparation of antitumor drugs

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of benzydamine hydrochloride in preparation of antitumor medicines.

Background

Worldwide, there are about 140 million new cases of digestive tract malignancy (esophageal cancer) each year, dying about 112 thousands of people, second only to lung cancer. In new cases of upper gastrointestinal malignancies, 74.36% occur in less developed areas and 44.60% occur in china. Esophageal cancer is one of eight common malignant tumors worldwide. Data investigated by the world health organization and the international cancer research institution show that about 45.6 ten thousand new esophageal cancer cases account for 3.2% of all cancers in 2012 all over the world; the number of deaths is about 40 million, accounting for 4.9% of all cancer deaths. China is a high-incidence country of esophageal cancer and a country with the highest esophageal cancer fatality rate, and the incidence rate of the national esophageal cancer is 5 th and 4 th of various tumors in continental areas of China. The incidence of esophageal cancer is obviously different in China, and the absolute high incidence of certain areas is clearly contrasted with the relative low incidence of peripheral areas, so that the most typical epidemiological characteristics of esophageal cancer in China are formed. The histological types of esophageal cancer are classified into esophageal squamous carcinoma and esophageal adenocarcinoma. There are large differences in the level, geographical distribution, time-varying trends and risk factors for the onset of esophageal cancer of different histological types. In China, esophageal squamous cell carcinoma accounts for more than 90% of esophageal carcinoma, and in low-incidence areas such as Europe and America, the histopathological type of the esophageal carcinoma is mainly adenocarcinoma. The time change trends of the incidence rates of the two histological types of esophageal cancers are greatly different, the global esophageal squamous cell carcinoma is in a descending trend, the esophageal adenocarcinoma is in an ascending trend, but the incidence rates of the cancers of the same histological type are different in different regions.

Esophageal cancer is a malignant tumor originated from esophageal epithelial tissue, and the occurrence and development of the esophageal cancer are the result of multi-factor, multi-gene and multi-stage combined action, and are closely related to age, sex, smoking, dietary habits, living environment and genetic factors. Although treatment methods such as surgery, chemotherapy and radiotherapy are widely applied to the treatment of esophageal cancer, the incidence rate and the death rate of esophageal cancer are not improved obviously. This suggests that further study of the mechanisms of esophageal carcinogenesis and progression and intervention in this process are one of the important strategies for reducing the incidence of esophageal epithelial cancers.

The process of human esophageal squamous cell canceration is a dynamic pathological change process from normal, mild atypical hyperplasia, moderate atypical hyperplasia, severe atypical hyperplasia, carcinoma in situ to invasive carcinoma. The process of transforming normal cells into cancer cells takes about 5-10 years, during which epithelial cells can continue to develop into cancer cells, can stop differentiating to be in a stable state, and can even be transformed into normal cells in a reversible way, and the pathological changes before the carcinoma in situ are collectively called as precancerous lesions, so that the theoretical basis is provided for the process of intervening the occurrence and development of the esophageal cancer.

Chemotherapy is one of the traditional methods for treating tumors and plays an important role in treating tumors. At present, the commonly used clinical antitumor drugs have the defects of poor curative effect, large toxic and side effects and the like, and the research and development of new antitumor drugs have the problems of long time consumption, large investment and the like. In recent years, researches show that a large number of non-antitumor drugs which have definite clinical safety and are widely applied to clinical treatment also have antitumor effects, such as astragalus membranaceus, tripterygium wilfordii, metformin, aspirin and the like.

"treatment" refers to a therapeutic intervention in which a disease or condition has begun to develop with delayed relief from its signs or symptoms. Such as book

As used herein, the term "alleviating" with respect to a disease or condition refers to any observable beneficial effect of the treatment. Advantageous effects

Can be, for example, delayed onset of clinical symptoms of the disease in a susceptible subject, severity of some or all of the clinical symptoms of the disease

A reduction in severity, slower progression of disease, improvement in overall health or well-being of the subject, or by being specific for a particular disease

Other parameters well known in the art. As used herein, the term "prevention" with respect to a disease or condition refers to being at risk of developing the disease. Chemoprevention of cancer refers to the use of natural or synthetic chemicals to arrest, slow or reverse the progression of cancer, thereby reducing the incidence of cancer and mortality.

In recent years, research shows that aspirin can prevent various cancers, reduce the incidence rate of the cancers, remarkably inhibit the growth of various cancer cells and promote the apoptosis of the cancer cells. Although the indazole analog benzydamine hydrochloride is a non-steroidal anti-inflammatory drug, it has various physicochemical properties and pharmacological activities different from those of conventional aspirin-like NSAIDs, but NSAIDs promote the mechanism of action of benzydamine hydrochloride as an effective topical action. The medicine has no report on the aspect of inhibiting the proliferation and the growth of digestive tract tumors such as esophageal cancer, gastric cancer and the like, and has no related patent application.

Disclosure of Invention

The invention aims to provide application of benzydamine hydrochloride in preparation of antitumor drugs.

Based on the purpose, the invention adopts the following technical scheme:

application of benzydamine hydrochloride in preparing an anti-tumor medicament, wherein the anti-tumor medicament is a medicament for treating esophageal cancer. Benzydamine hydrochloride, the Chinese name is: 1-benzyl-3- [3- (dimethylamino) propoxy group]-1H-indazole, english name: benzindamine Hydrochloride formula: c₁₉H₂₄ClN₃Molecular weight of O345.866 CAS number: 132-69-4.

Furthermore, the benzydamine hydrochloride can inhibit the proliferation of esophageal squamous carcinoma cells (KYSE150 and KYSE450 cells) and the quantity and size of formed clones when the concentration is 2.5-20 mu M.

The invention discovers that the benzydamine hydrochloride has obvious anti-tumor growth effect on an esophageal cancer PDX model established by inoculating fresh tumor tissues under the skin of an immunodeficiency SCID mouse, and can play a role in preventing esophageal cancer recurrence, and in the invention, the tumor treatment concentration in the mouse body is 5 mg/kg/day to 50 mg/kg/day.

The invention obviously improves the prevention and treatment effects of the esophageal cancer by utilizing the function of the non-steroidal anti-inflammatory drug benzydamine hydrochloride in the prevention and treatment of the esophageal cancer. The experiment shows that: the benzydamine hydrochloride has obvious effect on treating the esophageal cancer.

The present invention also provides a pharmaceutical composition comprising a compound as described above and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount, e.g., a therapeutically effective amount (including a prophylactically effective amount), of one or more of the foregoing compounds of the invention or salts thereof.

Pharmaceutically acceptable carriers can be routinely used and are limited only by chemical-physical considerations such as solubility and lack of reactivity with the compound and by the route of administration.

Pharmaceutically acceptable carriers, e.g., vehicles, adjuvants, excipients, or diluents, described herein are well known to those skilled in the art and readily available to the public. Preferably, the pharmaceutically acceptable carrier is one which is inert to the active compound and is present in

A carrier that does not have deleterious side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particular active agent and by the particular method used to administer the composition. Thus, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention. Such as formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal and vaginal administration.

According to the invention, the inhibition effect of 1-benzyl-3- [3- (dimethylamino) propoxy ] -1H-indazole in the growth of esophageal cancer cell lines (KYSE150, KYSE450) is verified for the first time through a cell proliferation experiment, and a new thought and basis can be provided for clinical research on drugs for preventing and treating tumors.

Drawings

FIG. 1 shows the structural formula of benzydamine hydrochloride.

FIG. 2 shows the toxic effect of benzydamine hydrochloride in the esophageal squamous carcinoma cells KYSE150 and KYSE 450;

FIG. 3 is the proliferation curve of esophageal squamous carcinoma cells at different time points with different concentrations of benzydamine hydrochloride;

FIG. 4 is the results of the statistics of the number of clone of KYSE450, KYSE150, drug and control group for inhibiting esophageal squamous carcinoma cell with benzydamine hydrochloride;

FIG. 5 is a photograph comparison result of tumor mass after mouse sacrifice after xenograft esophageal cancer model mouse of esophageal squamous carcinoma patient tumor tissue is treated with benzydamine hydrochloride for 40 days;

FIG. 6 shows the statistical results of tumor mass weighing after mice are sacrificed 40 days after xenografted esophageal cancer model mice of tumor tissues of esophageal squamous carcinoma patients are treated with benzydamine hydrochloride;

FIG. 7 is a statistical result of tumor volume in a mouse after the xenograft esophageal cancer model mouse of the tumor tissue of an esophageal squamous carcinoma patient is treated by benzydamine hydrochloride for 40 days;

FIG. 8 shows the statistical results of the weights of the xenograft esophageal cancer model mice with tumor tissues of esophageal squamous carcinoma patients treated with benzydamine hydrochloride for 40 days;

p <0.05, p <0.01, p <0.001 in fig. 3, 4, 7.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Application test

Materials and methods

1. Material

1.1 esophageal cancer cell lines

The esophageal cancer cell line used in the present invention was from the pathophysiology research laboratory of the basic medical college of Zhengzhou university.

1.2 reagents

Penicillin North China pharmaceutical Co Ltd

Streptomycin Shandong anti-medicine GmbH

RPMI-1640 Medium Israel Biological Industries, Inc

DMEM Medium Israel Biological Industries, Inc

0.25% pancreatin Shanghai Bitian Biotech Co., Ltd

Serum-free cell freezing medium Suzhou Xinsaimei Biotech limited

DAPI Beijing Solaibao Tech Co Ltd

Agar powder, American B & D

PBS powder Beijing Solaibao Tech Co Ltd

BME powder SIGMA-ALDRICH

L-Glutamine Beijing Soilebao Tech Co., Ltd

NaHCO₃Kaiton chemical reagent Co., Ltd, Tianjin City

Paraformaldehyde powder Tianjin City Guangfu Fine chemical research institute

Fetal bovine serum BI Ltd

Bendamine hydrochloride powder carbofuran

Sodium pentobarbital (national pharmaceutical group chemical agents limited) 0.4%;

a 500ml bottle of physiological saline (Chenxin pharmaceutical Co., Ltd.);

1.3 instruments and equipment:

1.5ml centrifuge tubes Axygen Inc. USA

15ml centrifuge tubes, Corning Inc

96-well cell culture plate tin-free Kangsi Biotech Co Ltd

10 cm cell culture dish tin-free Kangsi Biotech Ltd

15 cm cell culture dish Thermo Fisher Scientific Co., USA

Disposable pipette Guangzhou Jiete biofiltration GmbH

In Cell Analyzer 6000 USA GE

Pipettor Gilson Corp

Drying CO₂Shanghai-Hengchang scientific instruments Co Ltd

High speed cryogenic centrifuge Eppendorf Co, Germany

Vacuum suction Pump manufactured by Ribel instruments Ltd, Haiman, Inc

Inverted microscope, Carl Zeiss Jena, Germany

Nippon SANYO company of snow flake Ice maker

Milli-Q pure Water instruments Millipore, USA

A Thermo clean bench;

ophthalmic scissors, surgical forceps, a scalpel, a drug dissolving needle and an injector;

1.4 Experimental animals

SCID mice of 4-5 weeks old were purchased from Beijing Witonglihua laboratory animal technology Co., Ltd and were bred in a constant temperature and pressure environment alternating day and night for 12 hours. The experiment can be carried out when the weight of the mouse is about 18-20 g. The experimental animals are raised in an animal facility of a synergetic innovation center of cancer chemoprevention Henan province of Zhengzhou university under the conditions of constant temperature (25-27), constant humidity (45% -50%), fresh air and special pathogen free (SPF level) feeding room with dust removal and sterilization, the feed subjected to aseptic treatment is freely ingested by the animals, padding subjected to high-temperature sterilization is replaced once every three days, cages and drinking water are subjected to ultraviolet sterilization once every three days, and sterile distilled water is drunk. The operation of changing the feeding supplies strictly follows the aseptic principle.

1.5 Experimental methods

1.5.1 cytotoxicity assays: the esophageal squamous carcinoma cells are planted in a 96-well plate KYSE150 with about 8000 cells per well and KYSE450 with about 12000 cells per well (KYSE150 cells: 10% FBS/RPMI-1640; KYSE450 cells: 10% FBS/DMEM), and 3 cells are put in7℃，5% CO₂The incubator is used for culturing for 12-16 h. Then adding different amounts of benzydamine hydrochloride respectively to make the final concentrations of the medicine in the culture medium be 0, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M and 100. mu.M respectively, and treating for 24h and 48 h. After the cells were removed, 100. mu.l/well of a 4% paraformaldehyde solution was added to fix the cells at room temperature for 30 min, and then 100. mu.l/well of DAPI staining solution (DAPI stock: 1 XPBS = 1: 5000 dilution, Beijing Soilebao Tech. Co., Ltd.) was added thereto at 37 ℃ with 5% CO₂The incubator (2) is cultured for 30 min. The number of cells was counted by a high content cell imaging analysis system and the statistical results were analyzed, and the results are shown in fig. 2.

1.5.2 cell proliferation Experimental study make internal disorder or usurp

The esophageal squamous carcinoma cells are planted in a 96-well plate, KYSE150 has about 3000 cells per well, KYSE450 has about 5000 cells per well (KYSE150 cells: 10% FBS/RPMI-1640; KYSE450 cells: 10% FBS/DMEM), and the plate is placed at 37 ℃ and 5% CO₂The incubator is used for culturing for 12-16 h. Adding different amounts of benzydamine hydrochloride to make final concentration of the medicine in culture medium 0, 2.5, 5, 10, 20 μ M, respectively, adding 100 μ l/well 4% paraformaldehyde solution at 0, 24, 48, 72, 96h to fix cells at room temperature for 30 min, adding 100 μ l/well DAPI staining solution (DAPI stock solution: 1 XPBS = 1: 5000 dilution, Beijing Solebao Tech Co., Ltd.) at 37 deg.C and 5% CO₂The incubator (2) is cultured for 30 min. The number of cells was counted and the statistics were analyzed using a high content cell imaging analysis system, and the results are detailed in fig. 3.

1.5.3 Soft agar colony formation assay

Cells (8000 cells per well) are inoculated on a 6-well plate, the lower layer gel is agar gel added with benzydamine hydrochloride with corresponding concentration, and the upper layer gel is agar gel added with benzydamine hydrochloride with corresponding concentration and esophageal squamous carcinoma cells KYSE150 and KYSE 450. The solidified agar was then placed at 37 ℃ in 5% CO₂Cultured in an incubator for 7-14 days. The number of cell clones was counted using a high content cell imaging analysis system and the results were analyzed and shown in detail in FIG. 4.

1.5.4 establishment of human esophagus cancer immunodeficiency mouse planting tumor model

The esophageal squamous carcinoma tissue selection standard is as follows: fresh tumor tissue from patients who did not receive any radiotherapy or chemotherapy treatment before surgery (patient, male, 46 years old, 2042083, T2NOMOII, squamous cell carcinoma staging, taken from tumor hospital, south of Henan province) was placed in serum-free 1640 medium for storage and transport to the laboratory within 90 min after tumor tissue isolation. Before tissue inoculation, tumor tissues were washed with PBS containing penicillin streptomycin (PBS: 50:1 double antibody) and placed on ice for inoculation. The method comprises the steps of firstly injecting 0.4% sodium pentobarbital into a mouse to enable the mouse to enter an anesthesia state, then cutting tissues into small pieces of 3 with the thickness of 10-15 mm, planting the small pieces under the skin of the back and the neck of the mouse by using forceps, and returning the small pieces to a sterile breeding room after the mouse is anesthetized and revived. After the wound on the neck and the back of the mouse healed after about 3 to 5 days, the tumor volume of the mouse is measured once every fixed time until the tumor volume reaches 1000 mm³At that time, mice were sacrificed and tumor tissue was removed. Subcultured to the subcutaneous stage of new SCID mice in the same manner (passage 2). When the transplanted tumor is stably transferred to 3 generations, the success establishment of the esophageal cancer transplanted tumor model is proved.

1.5.5 Benzamine hydrochloride inhibition of tumor growth in human esophageal cancer xenograft mice

One or two weeks after inoculation, the mice begin to be grouped when the tumor nodules on the backs of the mice grow to about 200 cubic millimeters, namely the mice are uniformly distributed to each group according to the volume of the tumors, and each group contains more than 10 components. The 3 groups of mice were individually gavaged with normal saline, benzydamine hydrochloride 5mg/kg, benzydamine hydrochloride 50mg/kg (benzydamine hydrochloride dissolved in normal saline to the desired concentration). Mouse tumor volumes were recorded every 4 days. When the tumor volume of the control mice had grown to about 1000 mm, the experiment was terminated, tumor tissue was taken out, and the tumor weight was weighed and photographed, and the results are shown in fig. 5 to 8.

1.6 results of the experiment

The invention selects the esophageal cancer cell line, so the cytotoxicity test is firstly carried out on the esophageal cancer cell line, and the result is shown in figure 2. As can be seen from FIG. 2, it can be found that the effect of 0-20 μ M benzydamine hydrochloride on the esophageal cancer cell lines KYSE150 and KYSE450 is very small, so that 20 μ M of the cells is selected as the highest dose in the subsequent experiments of the esophageal cancer cell lines, and the inhibition effect of the benzydamine hydrochloride on the KYSE150 and KYSE450 cells has time and dose dependence at 0-25 μ M.

The benzydamine hydrochloride has an inhibiting effect on esophageal squamous carcinoma cells (see figure 3). Among them, the results of fig. 3 show that: the concentration range of the benzydamine hydrochloride is as follows: the growth of the esophageal squamous carcinoma cells KYSE150 and KYSE450 can be inhibited when the concentration is 2.5-20 mu M, the growth inhibition effect on the KYSE150 cells after 96h of culture is obvious when the concentration is 20 mu M, and the growth inhibition effect on the KYSE450 cells after 72h of culture is obvious.

FIG. 4 shows that benzydamine hydrochloride inhibits KYSE150 and KYSE450 clone formation of esophageal squamous carcinoma cells. The clone number is obviously reduced along with the increase of the dosing concentration, the clone number is obviously reduced, the inhibition effect on the formation of KYSE150 clone is obvious when the concentration is 10 mu M and 20 mu M, and the inhibition effect on the formation of KYSE450 clone is obvious when the concentration is 2.5 mu M, 5 mu M, 10 mu M and 20 mu M. FIG. 4 is a photomicrograph of clones, control and counter-drug of the corresponding groupsp<0.05，**p<0.01，***p<0.001。

Fig. 5 to 8 show the therapeutic effect of benzydamine hydrochloride on tumors in the PDX model of human esophageal cancer. As shown in FIG. 5, the tumor growth of the mice in the group was significantly inhibited by the high dose of benzydamine hydrochloride (50mg/kg) compared to the control group. As shown in FIG. 6, the tumor weight decreased in the high dose (50mg/kg) group of mice with benzydamine hydrochloride. FIG. 7 shows that the tumor volumes of the low dose group (5mg/kg) and the high dose group (50mg/kg) of benzydamine hydrochloride are more obviously inhibited by p <0.05 compared with the control group. FIG. 8 shows that the body weight of the mice was not significantly different among the administration groups. Therefore, the tumor growth treatment effect of the high-dose benzydamine hydrochloride on the esophageal cancer PDX mouse model is obvious.

In conclusion, the inhibition effect of the benzydamine hydrochloride in the esophageal cancer cell lines is verified through cell proliferation experiments. The invention firstly determines the safe concentration of the administration through a cytotoxicity experiment; and then, the compound benzydamine hydrochloride is given to the esophageal cancer cell lines for treatment, and the proliferation conditions of the cells are detected at 0h, 24h, 48h, 72h and 96h after the treatment, so that the compound benzydamine hydrochloride is determined to have the inhibition effect on the esophageal cancer cell lines. The soft agar clone formation experiment shows that the benzydamine hydrochloride can inhibit the formation of clone of esophageal squamous carcinoma KYSE150 and KYSE450 cells. In an esophageal cancer PDX model, the benzydamine hydrochloride has obvious tumor treatment effect on an esophageal cancer transplantation tumor mouse, and helps clinical research, prevention and treatment, relapse prevention, esophageal cancer and other tumors and other tumor medicaments.

The above embodiments are only for illustrating the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention within the knowledge of those skilled in the art should be considered as the protection scope of the present application.

Claims (4)

1. The application of the benzydamine hydrochloride in preparing the anti-tumor medicament is characterized in that the anti-tumor medicament is a medicament for treating esophageal cancer.

2. The use of claim 1, wherein the medicament for the treatment of esophageal cancer is for the preparation of a medicament for inhibiting proliferation of esophageal squamous cell carcinoma cells.

3. The use as claimed in claim 2 wherein the esophageal squamous cancer cells are KYSE150 cells and/or KYSE450 cells.

4. The use of claim 1, wherein the medicament for the treatment of esophageal cancer is for the preparation of a medicament for inhibiting tumor growth in a humanized graft tumor model of esophageal cancer.

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