阅读说明：本技术 沃替西汀及其盐在制备抗肿瘤药物中的应用 (Application of vortioxetine and salts thereof in preparation of antitumor drugs ) 是由 刘康栋 董子钢 王东浩 赵继敏 赵四敏 张静 江亚南 董子明 于 2020-09-04 设计创作，主要内容包括：本发明公开沃替西汀及其盐在制备抗肿瘤药物中的应用。尤其涉及沃替西汀及其氢溴酸盐在制备治疗食管癌药物中的应用,沃替西汀氢溴酸盐,化学名称为：1-[2-(2,4-二甲基苯硫基)苯基]哌嗪氢溴酸盐,CAS号：960203-27-4,分子式：C<Sub>18</Sub>H<Sub>23</Sub>BrN<Sub>2</Sub>S,分子量：379.358。本申请通过实验证实沃替西汀氢溴酸盐用于食管鳞状细胞癌细胞(KYSE150细胞、KYSE450细胞)时,能够起到抑制食管鳞状细胞癌细胞生长和转化的作用,且沃替西汀氢溴酸盐能够抑制食管鳞状细胞癌细胞增殖和转化的适宜浓度为0.5μM-4μM。(The invention discloses application of vortioxetine and salts thereof in preparation of antitumor drugs. In particular to application of vortioxetine and hydrobromide thereof in preparing a medicament for treating esophageal cancer, wherein the chemical name of vortioxetine hydrobromide is as follows: 1- [2- (2, 4-Dimethylphenylthio) phenyl]Piperazine hydrobromide, CAS number: 960203-27-4, formula: c 18 H 23 BrN 2 S, molecular weight: 379.358. experiments prove that the vortioxetine hydrobromide can play a role in inhibiting the growth and transformation of esophageal squamous cell carcinoma cells when being used for the esophageal squamous cell carcinoma cells (KYSE 150 cells and KYSE450 cells), and the appropriate concentration of the vortioxetine hydrobromide capable of inhibiting the proliferation and transformation of the esophageal squamous cell carcinoma cells is 0.5-4 mu M.)

1. The application of the vortioxetine and the salts thereof in preparing the antitumor drug is characterized in that the antitumor drug is a drug for treating esophageal cancer.

2. The use of claim 1, wherein vortioxetine and its salts are used in the preparation of a medicament for inhibiting esophageal squamous cell proliferation.

3. The use of claim 2, wherein vortioxetine and its salts are capable of inhibiting the proliferation of esophageal squamous cell carcinoma cells and the number and size of colony formations at a concentration of 0.5 μ Μ to 4 μ Μ.

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

5. The use of claim 1, wherein vortioxetine and its salts are used for the preparation of a medicament for inhibiting tumor growth in a humanized transplanted tumor model of esophageal cancer.

6. The use of claim 5, wherein vortioxetine and its salts inhibit the growth of esophageal cancer humanized graft tumor model tumors at a concentration of 1.5 mg/kg/day to 12 mg/kg/day.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of vortioxetine hydrogen and salts thereof in preparation of antitumor medicines.

Background

The treatment of esophageal cancer comprises operations, radiotherapy and chemotherapy, and the operation treatment is the first choice. The preoperative whole-body chemotherapy or concurrent chemoradiotherapy can obviously reduce the tumor stage and the tumor activity, reduce the tumor volume and promote surgical excision. The method can improve the excision rate, eliminate potential micrometastasis of other parts and effectively improve the local control rate. The combined treatment of radiotherapy and chemotherapy can not only improve the sensitivity of tumor tissues to radiotherapy, but also improve the curative effect of chemotherapeutic drugs. Chemotherapy alone can be used to help alleviate signs and symptoms caused by tumors in patients with advanced tumors that have spread beyond the esophagus.

The reuse of medicine, also known as new use of old medicine, means that the existing medicine is used for treating some new diseases, new indications are searched for the existing medicine, the medicine has the characteristics of high efficiency and low cost, and the treatment cost can be greatly reduced. Typical older drugs of traditional medicine are aspirin and metformin. Aspirin was originally used for antipyretic, analgesic and antirheumatic purposes. However, as the research progresses, it is also found that it can reduce blood viscosity, be used for preventing and treating myocardial infarction, and have effects in many aspects such as colorectal cancer, liver cancer, tumor, infectious disease, nervous system disease, etc. Metformin is the most commonly used drug for the treatment of type 2 diabetes. In recent years, metformin has potential effects of resisting tumors, aging, protecting cardiovascular and cerebrovascular systems, protecting nerves or selectively treating polycystic ovarian syndrome and the like. The research and development cost of the novel anti-cancer drugs is high, the time for developing a new drug is usually 10-15 years, but the global tumor burden is more and more serious, and the search for new and affordable anti-cancer drugs through the new use of old drugs is more and more important.

The anti-depression drug has long clinical application time and good safety, and can be used for drug reuse. It has been reported that anti-depression drugs have an effective anticancer effect against various malignant tumors in addition to an anti-depression effect. Such as fluoxetine, can significantly reduce rat pituitary adenoma andthe incidence of fibroadenoma of female rats, and has antioxidant effect, and can inhibit skin canceration of mice; citalopram has anti-tumor effect on colon tumor of mice and rats; the clomipramine can enhance the cytotoxicity of the antitumor drug to the drug-resistant tumor; the lorgeilan can inhibit the growth of prostate cancer of mice through an antiproliferative effect. Vortioxetine hydrobromide is a novel clinical antidepressant drug, and the main pharmacological action is antagonism of 5-HT₃、5-HT₇、5-HT_1DReceptor, partial agonism 5-HT_1BReceptor, agonism 5-HT_1AReceptor agent and 5-HT transporter inhibition, the vortioxetine hydrobromide has a positive effect in the acute treatment of depression, and can effectively prevent the recurrence of depression in the maintenance treatment. But currently there is no study between vortioxetine hydrobromide and the tumor. The incidence of depression of tumor patients is high, and if vortioxetine hydrobromide has both antidepressant and anticancer effects, the vortioxetine hydrobromide has good clinical application value.

Disclosure of Invention

The invention aims to provide application of vortioxetine and salts thereof in preparation of antitumor drugs, and particularly relates to application of vortioxetine hydrobromide in preparation of drugs for treating esophageal cancer.

Based on the purpose, the invention adopts the following technical scheme: the chemical name of the FDA approved drug is 1- [2- (2, 4-dimethyl-phenylthio) phenyl]Piperazine hydrobromide (Chinese name: vortioxetine hydrobromide, English name: vortioxetine hydrobromide, CAS number: 960203-27-4, molecular formula: C₁₈H₂₃BrN₂S, molecular weight: 379.358, trade name: xindayue, hydrobromic acid vosulistin)

The tumors are tumors of all mammals.

The anti-tumor medicine comprises vortioxetine, pharmaceutically acceptable salts and esters of vortioxetine, or combinations of vortioxetine and esters of vortioxetine or other compounds and medicines.

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

Furthermore, the vortioxetine hydrobromide can inhibit the proliferation of esophageal squamous carcinoma cells KYSE150 and KYSE450 when the concentration is 0.5-4 mu M.

When the vortioxetine hydrobromide is used for the esophageal cancer human tumor xenograft model, the vortioxetine hydrobromide has small toxic and side effects on animals under 12 mg/kg/day, and can obviously inhibit tumor growth under 1.5-12 mg/kg/day.

Wherein the pharmaceutically acceptable salts, esters or combination thereof of vortioxetine, vortioxetine is suitable for human daily dosage in the range of 0.1-2 mg/kg/day, and the animal is the corresponding equivalent dosage.

In general, vortioxetine hydrobromide or a pharmaceutically acceptable salt thereof is present at a concentration of about 0.5% to about 90% by weight of the total composition, i.e. in an amount sufficient to provide the desired unit dose, when used to treat a tumor patient.

The vortioxetine hydrobromide serving as an antidepressant drug is applied to clinic, does not need clinical safety assessment, and has good application prospect.

The invention discovers the inhibition effect of vortioxetine hydrobromide on tumor cells for the first time, in particular the inhibition effect on esophageal squamous cell carcinoma cells. The research result shows that: the vortioxetine hydrobromide can inhibit the proliferation of esophageal squamous carcinoma cells KYSE150 and KYSE 450. Thereby showing that the vortioxetine hydrobromide has obvious inhibition effect on esophageal squamous cell carcinoma cells. Therefore, vortioxetine hydrobromide provides a reference for preparing antitumor drugs, and preventing, treating and preventing recurrence of tumors.

Drawings

FIG. 1 is the chemical structure of vortioxetine hydrobromide;

FIG. 2 shows the cytotoxic effect of vortioxetine hydrobromide on esophageal squamous carcinoma cells KYSE150 and KYSE450 at a concentration range of 1-50 μ M;

FIG. 3 shows the proliferation inhibiting effect of vortioxetine hydrobromide on esophageal squamous carcinoma cells KYSE150 and KYSE 450; *p<0.05,**p<0.01, ***p<0.001；

FIG. 4 shows inhibition of KYSE150 and KYSE450 clone formation of esophageal squamous carcinoma cells by vortioxetine hydrobromide, wherein the control and the dosing are shown in the figureStatistical results of the number of group clones; *p<0.05, **p<0.01, ***p<0.001；

Figure 5 is a graph of the change in tumor tissue volume and weight of mice treated with vortioxetine hydrobromide. Wherein, when the concentration of the vortioxetine hydrobromide is in the normal oral dose of an adult (converted into the oral dose of a mouse of 1.5 mg/kg/d), the vortioxetine hydrobromide can obviously inhibit the growth of the tumor of a SCID esophageal squamous cell carcinoma PDX model mouse, and has no influence on the weight of the mouse; *p<0.05, **p<0.01,***p<0.001。

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.

Materials and methods

1. Material

1.1 cells and reagents

The esophageal squamous carcinoma cells KYSE150 and KYSE450 are from the pathophysiology research laboratory of the basic medical college of Zhengzhou university, and the vortioxetine hydrobromide is purchased from Beijing carbofuran technology Limited and has the purity of 98.5 percent.

1.2 instruments and equipment: high content cell imaging analysis system (GE Healthcare), micropipette (Eppendorf, germany), clean bench (suzhou purification ltd), cell culture box (ThermoScientific, usa), 4 ℃ refrigerator (hel appliances ltd, henna), analytical balance (aohaos instruments ltd).

2 method

2.1 cytotoxicity assay

Planting esophageal squamous carcinoma cells in 96-well plate, KYSE150 about 8000 cells per well, KYSE450 about 12000 cells per well, placing at 37 deg.C and 5% CO₂The incubator is used for culturing for 12-16 h. The drug powder was dissolved in DMSO to prepare vortioxetine hydrobromide at various concentrations (0mM, 3.125 mM, 6.25 mM, 12.5mM, 25 mM, 50 mM, 75 mM, 100 mM), and 0.5. mu.L each was added to 500. mu.L of the cell culture medium (5 wells per concentration, 100. mu.L of the medium per well, KYSE150 cells: 10% FBS/RPMI-1640; KYSE450 cells: 10% FBS/DMEM), vortioxetine hydrobromide at final concentrations in the medium of 0. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, treated cells for 24 h and 48 h. Taking out, adding 100 μ L of 4% paraformaldehyde solution into each well of 96-well plate, fixing cells at room temperature for 30 min, adding DAPI staining solution (prepared by DAPI: PBS at a ratio of 1: 1000) at 37 deg.C 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.

2.2 cell proliferation assay

Planting esophageal squamous carcinoma cells in a 96-well plate, placing about 3000 cells in KYSE150 per well and about 5000 cells in KYSE450 per well at 37 ℃ in 5% CO₂The incubator is used for culturing for 12-16 h. The drug powder was dissolved in DMSO to prepare vortioxetine hydrobromide at various concentrations (0mM, 0.5 mM, 1 mM, 2 mM, 4 mM), 0.5. mu.L each was added to 500. mu.L of the cell culture medium (5 wells per concentration, 100. mu.L of the medium per well, KYSE150 cells: 10% FBS/RPMI-1640; KYSE450 cells: 10% FBS/DMEM), and the final concentration of vortioxetine hydrobromide in the medium was 0. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 4. mu.M, and the cells were treated for 24, 48, 72, 96 h. Taking out, adding 100 μ L of 4% paraformaldehyde solution into each well of 96-well plate, fixing cells at room temperature for 30 min, adding DAPI staining solution (prepared by DAPI: PBS at a ratio of 1: 1000) at 37 deg.C 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 counted results were analyzed, and the results are shown in fig. 3.

2.3 cell anchoring independent growth experiment

Cells (8000 cells per well) are inoculated on a 6-well plate, the lower layer gel is agar gel added with vortioxetine hydrobromide with corresponding concentration, and the upper layer gel is agar gel added with vortioxetine hydrobromide 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. Counting the number of cell clones by using high content cell imaging analysis system, and analyzing the counting result, such asAs shown in fig. 4.

2.4 construction of esophageal squamous carcinoma humanized transplantation tumor model (PDX immunodeficiency mouse model)

Female SCID mice of 6-8 weeks old are selected, and esophageal squamous cell carcinoma EG20 tumor tissues (patient, male, 46 years old, 2042083, T2NOMOII, middle differentiation squamous cell carcinoma, obtained from tumor hospital of Henan province) are implanted in the back after anesthesia. When the growth state of the mice is normal, dividing the tumor-bearing mice into 3 groups randomly, and taking the first group of the gavage normal saline as a control group. Vortioxetine hydrobromide, a clinical drug, vortioxetine hydrobromide tablet (trade name: cardiac deamidate) was ground into powder and dissolved in physiological saline to form a desired concentration as a drug group. The second group is a vortioxetine hydrobromide low dose group (1.5 mg/kg/d) and the third group is a vortioxetine hydrobromide high dose group (12 mg/kg/d), according to which vortioxetine hydrobromide is administered orally by gavage once daily, weighed twice a week and measured for tumor volume = (long diameter × short diameter)/2. When the tumor tissue of the control group mouse reaches 1000 mm³When the tumor tissue is taken out, the mice are sacrificed, the tumor weight is weighed and is arranged according to the size sequence of the tumor tissue, and the images are photographed and retained. The data obtained from the experiment were plotted and analyzed by SPSS to determine whether there was a difference and whether there was a statistical significance, the results are shown in fig. 5.

Results of the experiment

1) The vortioxetine hydrobromide has a cytotoxic effect on esophageal squamous cell carcinoma cells, and the result of fig. 2 shows that the vortioxetine hydrobromide has a cytotoxic effect on esophageal squamous cell carcinoma cells KYSE150 and KYSE450 when the concentration range is 1-50 μ M; in order to increase the cell survival rate at different time points by adding different concentrations of vortioxetine hydrobromide at 100% of the control group, as can be seen from fig. 2, it can be found that the influence of vortioxetine hydrobromide of 0-4 μ M on the esophageal cancer cell lines KYSE150 and KYSE450 is very small, so that 4 μ M of cells is selected as the highest dose in the subsequent experiments of the esophageal cancer cell lines, and the inhibition effect of vortioxetine hydrobromide on KYSE150 and KYSE450 cells at 0-4 μ M has time and dose dependence.

2) Vortioxetine hydrobromide dosed tumor cell proliferation curves at different concentrations at different time points on esophageal squamous carcinoma cells (see fig. 3). Drawing (A)The results of 3 show that: the vortioxetine hydrobromide can inhibit the proliferation of esophageal squamous carcinoma cells KYSE150 and KYSE450 cells after being cultured for 72 hours in the concentration range of 0.5-4 mu Mp<0.05, **p<0.01, ***p<0.001。

3) FIG. 4 shows that vortioxetine hydrobromide inhibits the formation of clone KYSE150 and KYSE450 of esophageal squamous carcinoma cells. Wherein, the cloning number is obviously reduced along with the increase of the dosing concentration, and the cloning is obviously reduced. FIG. 4 is a statistical result of the number of clones in the drug-added and control groups and a clone micrograph of the corresponding group, specifically, the clonogenic phase of KYSE450 cells can be significantly inhibited when the concentration of vortioxetine hydrobromide is 0.5-4 μ M, and the clonogenic phase of KYSE150 cells can be significantly inhibited when the concentration is 2-4 μ M. *p<0.05, **p<0.01, ***p<0.001。

4) Figure 5 is a graph of the change in tumor tissue volume and weight of mice treated with vortioxetine hydrobromide. Toxicity experiments show that after vortioxetine hydrobromide is orally taken, the body weight of mice in the high-dose group and the low-dose group is not obviously changed, which indicates that vortioxetine hydrobromide has no obvious toxic effect on the mice. Wherein, when the concentration of the vortioxetine hydrobromide is in the normal oral dose of an adult (converted into the oral dose of 1.5 mg/kg/d and 12 mg/kg/d of a mouse), the vortioxetine hydrobromide can obviously inhibit the growth of the tumor of a SCID esophageal squamous cell carcinoma PDX model mouse, and the tissue volume and the weight of the tumor are obviously reduced, thereby indicating that the vortioxetine hydrobromide can inhibit the proliferation of the esophageal squamous cell carcinoma in vivo. *p<0.05, **p<0.01, ***p<0.001。

In summary, it can be seen that: the research shows that vortioxetine hydrobromide has a cytotoxic effect on esophageal squamous carcinoma cells KYSE150 and KYSE450, can inhibit the proliferation of the esophageal squamous carcinoma cells KYSE150 and KYSE450, can inhibit the formation of clones of the esophageal squamous carcinoma cells KYSE150 and KYSE450, and can inhibit the proliferation of esophageal squamous carcinoma in vivo.

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.