阅读说明：本技术 奥替溴铵在制备抗肿瘤药物中的应用 (Application of otilonium bromide in preparing antitumor drugs ) 是由 刘康栋 包卓 董子钢 赵继敏 江亚南 于 2020-09-04 设计创作，主要内容包括：本发明公开奥替溴铵在制备抗肿瘤药物中的应用,奥替溴铵(Otilonium Bromide),化学名称：N,N-二乙基-N-甲基-2-[[4[[2-(辛基)苯甲酰]氨基]苯甲酰]氧基]乙烷铵溴化物,CAS号为26095-59-0,分子式为C<Sub>29</Sub>H<Sub>43</Sub>BrN<Sub>2</Sub>O<Sub>4</Sub>,分子量为563.56。所述抗肿瘤药物为治疗食管癌或胃癌的药物。本申请通过实验证实奥替溴铵用于食管鳞状细胞癌细胞(KYSE150细胞、KYSE450细胞)或胃癌细胞(HGC27细胞、AGS细胞)时,能够起到抑制食管鳞状细胞癌细胞或胃癌细胞生长和转化的作用,且奥替溴铵能够抑制食管鳞状细胞癌细胞或胃癌细胞增殖和转化的适宜浓度为2.5μM~20μM。(The invention discloses an application of Otilonium Bromide in preparing antitumor drugs, wherein the Otilonium Bromide (Otilonium Bromide) has the chemical name: n, N-diethyl-N-methyl-2- [ [4[ [2- (octyl) benzoyl ] methyl ester]Amino group]Benzoyl radical]Oxy radical]Ethylammonium bromide with CAS number 26095-59-0 and molecular formula C 29 H 43 BrN 2 O 4 And the molecular weight is 563.56. The anti-tumor drug is a drug for treating esophageal cancer or gastric cancer. Experiments prove that the otilonium bromide can play a role in inhibiting the growth and transformation of esophageal squamous cell carcinoma cells (KYSE 150 cells and KYSE450 cells) or gastric cancer cells (HGC 27 cells and AGS cells) when used for the esophageal squamous cell carcinoma cells or the gastric cancer cells, and the proper concentration of the otilonium bromide in inhibiting the proliferation and transformation of the esophageal squamous cell carcinoma cells or the gastric cancer cells is 2.5-20 mu M.)

1. The application of the otilonium bromide in preparing the antitumor drug is characterized in that the antitumor drug is a drug for treating esophageal cancer or gastric cancer.

2. The use according to claim 1, wherein the use of otilonium bromide for the preparation of a medicament for inhibiting the proliferation of esophageal squamous cell carcinoma cells.

3. The use according to claim 2, wherein the amount of otilonium bromide that can inhibit the proliferation and colony formation of esophageal squamous carcinoma cells is 2.5-20 μ M.

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

5. The use according to claim 1, wherein the use of otilonium bromide in the preparation of a medicament for inhibiting gastric cancer cell proliferation.

6. The use according to claim 5, wherein the otilonium bromide is capable of inhibiting the proliferation of gastric cancer cells at a concentration of 2.5 μ M to 20 μ M.

7. The use according to claim 6, wherein the gastric cancer cells are HGC27 cells and/or AGS cells.

8. The use according to claim 1, wherein the use of otilonium bromide in the preparation of a medicament for inhibiting the growth of tumors in a humanized transplantation tumor model of esophageal cancer or gastric cancer.

9. The use according to claim 8, wherein the otilonium bromide can inhibit the growth of the tumor of the esophageal cancer or gastric cancer humanized transplantation tumor model at the concentration of 5 mg/kg-20 mg/kg.

Technical Field

The invention belongs to the technical field of tumor treatment and prevention, and particularly relates to application of otilonium bromide in preparation of antitumor drugs.

Background

Cancer is a serious threat to human health. The incidence of cancer has been increasing in recent years, and this disease is second to cardiovascular and cerebrovascular diseases. Cancer treatment is one of the difficult and difficult problems in the world at present, not only is complete cure difficult, but also the improvement of the life cycle of patients has a lot of difficulties.

Cancer has become the leading cause of death in china and is a serious public health problem. Statistics show that Chinese esophageal cancer and gastric cancer are the most common types of cancer and the most common causes of cancer death.

Worldwide, the incidence of esophageal cancer is eighth in malignant tumors, the mortality rate is sixth, about 456000 new cases are generated every year, esophageal cancer has certain ethnic and regional characteristics, the incidence of esophageal cancer is higher in east Asia, east Africa, south Africa and south Europe, and nearly 70% of esophageal cancer occurs in China. Esophageal cancer is the sixth most common tumor causing cancer death in China, the prognosis is very poor, and the five-year survival rate is only 15-20%. Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC) are the two major histological types of esophageal cancer. ESCC is the most common subtype in developing countries, but esophageal cancer is predominantly EAC in the united states and other western countries. Esophageal cancer typically has progressive dysphagia, which is characterized by difficulty swallowing dry food, followed by semifluid food, and finally, water and saliva.

Gastric cancer (gastric carcinoma) is a malignant tumor originating from the epithelium of the gastric mucosa, an important cancer in the world, with over 100 million new cases in 2018, and an estimated 78.3 million deaths. In chinese cancer, the incidence of gastric cancer is second and mortality is third. The incidence of gastric cancer is obviously different regionally, and the incidence rate of gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. Gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like.

In recent years, despite the great progress in the early diagnosis and early detection of esophageal and gastric cancers, and the clinical application of standard chemotherapy and radiotherapy for decades, the overall survival rate of most patients in the middle and late stages of the clinic is not improved significantly, suggesting that only early detection and treatment of tumors are emphasized, while tumor prevention is ignored, with little success.

Although treatment regimens for cancer are rapidly evolving, treatment is challenged by drug toxicity and postoperative recurrence due to drug resistance. New safer and more effective treatment options are therefore highly desirable. With the proposal of the concept of 'Old Drugs used newly' (Liu Q. Editorial: Old Drugs Learn New locks: Advances and applications for Drug reproduction. Curr Top Med Chem 2016; 16: 3627. 3628.), more and more doctors and scientists think that clinical non-anti-tumor Drugs which are popularized and applied can be explored and developed into New anti-cancer Drugs if the basic and clinical verification is carried out. The method can save the development cost and time of the new medicine and also can ensure the physicochemical property, the pharmacokinetics and the safety of the medicine to a certain extent.

Otilonium bromide is an antimuscarinic and can be used as an analgesic, anti-inflammatory, and uterine contraction-inhibiting agent. Otilonium bromide has selective and strong spasmolytic effect on the smooth muscle of the digestive tract, so that the otilonium bromide is suitable for all hyperkinesia, different reasons and different parts and spasmodic reaction caused by pathological atrophy of smooth muscle fibers. The composition can be used for treating gastrointestinal spasm and motor dysfunction (irritable bowel syndrome, gastritis, gastroduodenitis, enteritis, and esophageal lesion). However, the effect of the drug on inhibiting the proliferation and growth of digestive tract tumors such as esophageal cancer and gastric cancer is not reported at present, and no related patent application is available.

Disclosure of Invention

The invention aims to provide a new application of otilonium bromide in the technical field of medicines, namely an application of otilonium bromide in preparing antitumor medicines. In particular to the application of the compound in clinical treatment as a medicament for treating and preventing esophageal cancer and gastric cancer.

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

application of otilonium bromide in preparing antitumor drugs. Otilonium Bromide (Otilonium Bromide), chemical name: n, N-diethyl-N-methyl-2- [ [4[ [2- (octyl) benzoyl ] methyl ester]Amino group]Benzoyl radical]Oxy radical]Ethylammonium bromide with CAS number 26095-59-0 and molecular formula C₂₉H₄₃BrN₂O₄Molecular weight of 563.56, structural formula:. The anti-tumor drug is a drug for treating esophageal cancer or gastric cancer.

The anti-tumor medicament is prepared into medicinal formulations, wherein the formulations comprise sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquid, buccal agents, granules, medicinal granules, pills, pellets, suspensions, powder, vinum, preparations, drops, injection, powder injections, creams, sustained-release agents, targeting agents and the like, and the anti-tumor medicament is used as a medicament for preventing, treating and preventing recurrent cancers.

The administration mode of the antitumor drug comprises oral administration, injection, implantation, external application, spraying, inhalation or the combination of the oral administration, the injection, the implantation, the external application, the spraying and the inhalation.

The anti-tumor drug can be applied to preventing tumor occurrence, treating tumor and preventing tumor recurrence.

Specifically, the application of the otilonium bromide in the aspect of medicines for treating and preventing esophageal cancer or gastric cancer is realized by treating cells with a clinical non-anticancer application medicine otilonium bromide through a cytotoxicity experiment, a cell proliferation experiment and a soft agar clone formation experiment by using an esophageal cancer cell line KYSE150, KYSE450 and a gastric cancer cell line HGC27 and AGS, and the finding that the otilonium bromide has a toxic effect on esophageal squamous cell KYSE150 cells and KYSE450 and gastric cancer cell line HGC27 and AGS cells at 25 mu M, has the functions of inhibiting the proliferation and clone formation capacities of esophageal cancer cell lines at the concentration of 2.5-20 mu M, and has the functions of inhibiting the proliferation and clone formation capacities of the gastric cancer cell lines at the concentration of 2.5-20 mu M.

Furthermore, the invention utilizes an esophageal cancer PDX (pdx) (Patientderiversedxenogrft) model, namely a subcutaneous transplanted tumor model which is established on an immunodeficient mouse by utilizing cancer tissues of a cancer patient and grows by depending on a microenvironment provided by the mouse, shows a remarkable anti-tumor growth effect and can play a role in preventing esophageal cancer relapse. In the invention, the tumor treatment concentration of the otilonium bromide in SCID mice is 5g/kg-20mg/kg, the otilonium bromide has no obvious influence on the body weight of the mice, is the acceptable safe drug concentration, and can inhibit the growth of esophageal cancer or gastric cancer humanized transplantation tumor model tumors within the concentration range.

The invention has the beneficial effects that: the invention discloses that the otilonium bromide can be used for treating and preventing esophageal cancer, gastric cancer and other tumors for the first time, inhibiting the growth and proliferation of esophageal cancer and gastric cancer cells, inhibiting the in-vitro clone formation, and having obvious effect on the prevention of esophageal cancer humanized transplantation tumor models (PDX). The implementation of the invention widens the medical application of the otilonium bromide, provides a new idea for treating human esophageal cancer, gastric cancer and other tumors, and has important significance for treating, preventing and recrudescence of the tumors.

Drawings

FIG. 1 is a graph showing the results of the effect of otilonium on esophageal cancer cells; a is an experimental result graph of toxicity of the otilonium bromide to esophageal cancer cells KYSE150 and KYSE 450; b is an analysis chart of the influence of the otilonium bromide on the proliferation activity of esophageal cancer cells KYSE150 and KYSE 450;

FIG. 2 is a graph showing the effect of otilonium bromide on the capability of KYSE150 and KYSE450 in vitro clone formation of esophageal cancer cells;

figure 3 is a graph of the results of the effect of otilonium on gastric cancer cells; a is a graph of the experimental results of toxicity of otilonium bromide to gastric cancer cells HGC27 and AGS; b is an analysis chart of the effect of the otilonium bromide on the proliferation activity of gastric cancer cells HGC27 and AGS;

FIG. 4 shows the tumor growth therapeutic effect of otilonium bromide on a mouse model with esophageal cancer number EG20, A shows the trend of the weight change of mice during administration of otilonium bromide-treated PDX mouse model with tumor tissues of esophageal squamous carcinoma patients; b is a graph of the change trend of the tumor volume of a mouse in the administration period after the PDX mouse model of the tumor tissue of an esophageal squamous cell carcinoma patient is treated by otilonium bromide; c, treating a PDX mouse model of the tumor tissue of the esophageal squamous carcinoma patient by otilonium bromide, and weighing the weight statistical result of the tumor after the mouse is killed;

d is a tumor picture of a control group and an administration group after mice are killed after a PDX mouse model of the tumor tissue of an esophageal squamous cell carcinoma patient is treated by otilonium bromide;

in the context of the figures 1 to 4,*p<0.05, **p<0.01, ***p<0.001。

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention. The experimental procedures in the examples, unless otherwise specified, were carried out by conventional techniques in the art and the experimental reagents were all purchased commercially.

Materials and methods

Material

Esophageal squamous carcinoma cells KYSE150 and KYSE450 and gastric carcinoma cells HGC27 and AGS from Zhengzhou university basic medical college pathophysiology research room

Cell lines: KYSE150 cell line, wherein the cell line adopts 1640 culture medium containing 10% FBS, and is constant in temperature of 37 ℃ and 5% CO₂Culturing under the condition for later use.

KYSE450 cell strain, wherein the cell strain adopts DMEM medium containing 10% FBS, is constant in temperature of 37 ℃ and contains 5% CO₂Culturing under the condition for later use.

HGC27 cell line, the cell line adopts 1640 culture medium containing 10% FBS, the temperature is constant at 37 ℃, and 5% CO₂Culturing under the condition for later use.

AGS cell line, wherein the cell line adopts F12K culture medium containing 10% FBS, and is constant in temperature of 37 ℃ and 5% CO₂Culturing under the condition for later use.

Tumor tissue

The present invention used 1 human esophageal cancer tissue specimen, numbered EG20 (from tumor hospital, patient, male, 46 years old, hospital number 2042083, T2N0M0 ii, department of south fluvial province, mid-differentiation).

Laboratory animal

SCID CB-17 mice in this example were purchased from Experimental animals technology, Inc. of Wei Tongliwa, Beijing. The license number is: SCXK (jing) 2012-0001. SPF grade, SCID mice are 6-8 weeks old, 16-18 g in weight, female. Mouse feeds were purchased from Aojieli feeds, Inc., Beijing, Ke. The license number is SCXK (Jing) 2014-0010.

Reagent

RPMI-1640 medium, DMEM medium 500ml one bottle (Biological Industries); F12K (Gibco); FBS (biological industries); Trypsin-EDTA pancreatin digestive juice (Shanghai Bin Yuntian Biotechnology Co., Ltd.); PBS (beijing solibao technologies ltd); a cell culture dish; pentobarbital sodium (national pharmaceutical group chemical agents limited): weighing sodium pentobarbital, dissolving in sterile distilled water, and filtering; double resistance: penicillin and streptomycin were purchased from north China pharmaceutical Co., Ltd, both at a concentration of 500U/ml; physiological saline 500 ml/bottle (Chenxin pharmaceutical Co., Ltd.); otilonium bromide: the in vitro experimental drug was purchased from MCE, and the in vivo experimental drug was purchased from Berlin chemical, Germany.

Apparatus and equipment

A medical heat preservation refrigerator for Haler; an electronic balance; a Thermo clean bench; an electronic balance; cell culture case, ophthalmology scissors, surgical forceps, aseptic culture dish, scalpel, medicine dissolving needle, syringe.