Use of baricitinib in the preparation of antitumor drugs

Abstract

The application belongs to the technical field of tumor treatment and prevention, and particularly relates to application of Baloxavir (molecular formula: C 24 H 19 F2N3O4S, molecular weight: 483.487, CAS number: 1985605-59-1) in preparation of esophageal cancer, gastric cancer and other tumor drugs. The application first discovers that Baloxavir has toxic effects on esophageal cancer and gastric cancer cells and can inhibit the proliferation ability of the esophageal cancer and gastric cancer cells. The general design idea of the application is that esophageal cancer and gastric cancer cell drug treatment is carried out in vivo and in vitro, and the application of Baloxavir in esophageal cancer, gastric cancer and other tumors is determined by affecting the growth of esophageal cancer and gastric cancer cells. The results show that appropriate concentration of Baloxavir has toxic effects on esophageal cancer and gastric cancer cells and can inhibit the proliferation ability of the esophageal cancer and gastric cancer cells, thereby providing a new therapeutic drug for treatment and prevention of esophageal cancer, gastric cancer and other tumors.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of Baloxavir in the preparation of antitumor drugs. Background Technology

[0002] According to statistics from the International Agency for Research on Cancer (IARC) of the World Health Organization in 2020, there are more than 19.29 million new cancer cases and nearly 10 million cancer deaths each year. The top 10 cancers by incidence rate globally are: breast cancer (2.26 million, 11.7%), lung cancer (2.2 million, 11.4%), colorectal cancer (1.88 million, 9.8%), prostate cancer (1.41 million, 7.3%), stomach cancer (1.09 million, 5.6%), liver cancer (910,000, 4.7%), cervical cancer (600,000, 3.1%), esophageal cancer (600,000, 3.1%), thyroid cancer (590,000, 3%), and bladder cancer (570,000, 3%). These 10 cancers account for 63% of all new cancer cases. The top ten cancers causing cancer deaths are: lung cancer (1.8 million), colorectal cancer (940,000), liver cancer (830,000), stomach cancer (770,000), breast cancer (680,000), esophageal cancer (540,000), pancreatic cancer (470,000), prostate cancer (380,000), cervical cancer (340,000), and leukemia (310,000). These ten cancers account for 71% of all cancer deaths. Globally, approximately 1.7 million new cases of upper gastrointestinal malignancies (esophageal and stomach cancer) are diagnosed annually, resulting in about 1.3 million deaths. Of these new cases, 80% occur in underdeveloped regions, with more than half occurring in China. The incidence and mortality rates of upper gastrointestinal malignancies increase with age and show significant gender differences, with men being twice as likely as women to develop the disease, and higher rates in underdeveloped regions than in developed regions. China is a high-incidence area for esophageal and stomach cancer. Within China, the incidence and mortality rates of esophageal and stomach cancer vary regionally, with higher rates in rural areas than in urban areas.

[0003] Esophageal cancer ranks sixth among the leading causes of cancer death worldwide. In 2020, there were 600,000 new cases of esophageal cancer globally, resulting in 540,000 deaths. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are the two main histological types of esophageal cancer. ESCC is the most common subtype in developing countries, but EAC is the predominant type in the United States and other Western countries. Despite significant advancements in early diagnosis and detection techniques for esophageal cancer in recent years, and the decades-long clinical application of standard chemotherapy and radiotherapy, the overall survival rate for most patients with advanced-stage cancer has not improved significantly. This suggests that emphasizing only early detection and treatment while neglecting cancer prevention yields minimal results.

[0004] Gastric cancer is one of the most common malignant tumors in China. Although its incidence has decreased slightly recently, the most common site of onset has gradually shifted from the antrum to the fundus and cardia, and its biological behavior has become more aggressive. Overall, treatment efficacy has not improved significantly. Further progress is needed and possible in basic and clinical research aimed at effectively improving the treatment of gastric cancer, including clarifying the mechanisms of its development and progression, increasing early diagnosis rates, advocating standardized comprehensive treatment, and developing and applying new drugs and technologies.

[0005] Chemotherapy is one of the traditional methods of cancer treatment and plays an important role in cancer therapy. Currently, commonly used anti-tumor drugs have drawbacks such as poor efficacy and significant side effects, while developing new anti-tumor drugs is time-consuming and requires substantial investment. Recent studies have found that many non-anti-tumor drugs with well-established clinical safety profiles and widespread clinical application may also possess anti-tumor effects, such as Astragalus membranaceus, Tripterygium wilfordii, metformin, and aspirin.

[0006] Baloxavir (molecular formula: C) 24 H 19 F2N3O4S (molecular weight: 483.487, CAS number: 1985605-59-1) is the active metabolite of Baloxavir marboxil. In February 2018, Baloxavir marboxil (trade name: Xofluza), a novel anti-influenza drug developed by Shionogi Pharmaceutical Co., Ltd. of Japan and co-researched with Roche of Switzerland, received accelerated approval and was approved for marketing in Japan. Baloxavir marboxil is an innovative Cap-dependent endonuclease inhibitor and one of the few new drugs in the world that can inhibit the proliferation of influenza virus. In healthy subjects, a single oral dose of Baloxavir marboxil on an empty stomach rapidly hydrolyzes into the active metabolite Baloxavir. Currently, there are no reports or related patent applications regarding the inhibitory effect of this active metabolite, Baloxavir, on the proliferation and growth of digestive tract tumors such as esophageal and gastric cancer.

[0007]

[0008] Baloxavir Summary of the Invention

[0009] This invention discovers that Baloxavir has an inhibitory effect on the proliferation of esophageal squamous cell carcinoma cells and gastric cancer cells, and can be applied to the preparation of anti-tumor drugs. This invention relates to Baloxavir, a derivative of the FDA-approved drug Baloxavir marboxil, and also the active ingredient of Baloxavir marboxil. Baloxavir (molecular formula: C 24 H 19The application of F2N3O4S (molecular weight: 483.487, CAS No.: 1985605-59-1) in the preparation of antitumor drugs, wherein the antitumor drugs are drugs for treating esophageal squamous cell carcinoma or gastric cancer.

[0010] Furthermore, one of the drugs used to treat esophageal cancer is a drug that inhibits the proliferation of esophageal squamous cell carcinoma cells.

[0011] Furthermore, baloxavir can inhibit the proliferation of esophageal squamous cell carcinoma cells at concentrations of 0.5 μM to 5 μM.

[0012] Furthermore, the esophageal squamous cell carcinoma cells are KYSE150 cells and / or KYSE450 cells.

[0013] Furthermore, the drug used to treat gastric cancer is applied in the preparation of drugs that inhibit the proliferation of gastric cancer cells.

[0014] Furthermore, Baloxavir can inhibit the proliferation of gastric cancer cells at concentrations of 0.5 μM to 5 μM.

[0015] Furthermore, the gastric cancer cells are HGC27 cells and / or AGS cells.

[0016] Application of Baloxavir in the preparation of drugs that inhibit tumor growth in humanized xenograft models of esophageal or gastric cancer.

[0017] Furthermore, Baloxavir can inhibit the growth of tumors in humanized xenograft models of esophageal or gastric cancer at concentrations of 15 mg / kg–30 mg / kg.

[0018] The tumor is a tumor of all mammals. The antitumor drug includes baloxavir, pharmaceutically acceptable salts, esters, or combinations thereof, or combinations with other compounds or drugs. "Antitumor drugs" can be used to prevent tumor development, treat tumors, and prevent tumor recurrence.

[0019] Baloxavir, as an active ingredient in anti-influenza drugs, has been used clinically without requiring clinical safety assessments and has a promising future. Attached Figure Description

[0020] Figure 1 The graph shows the toxicity of Baloxavir to KYSE150 esophageal squamous cell carcinoma cells. Baloxavir was toxic to KYSE150 esophageal squamous cell carcinoma cells at concentrations ranging from 0 to 50 μM. The graph also shows the tumor cell growth curves at different time points with different drug concentrations.

[0021] Figure 2 The figure shows the toxicity of Baloxavir to esophageal squamous cell carcinoma cells KYSE450. Baloxavir showed toxicity to esophageal squamous cell carcinoma cells KYSE450 at concentrations ranging from 0 to 50 μM. The figure also shows the tumor cell growth curves at different time points with different drug concentrations.

[0022] Figure 3 The graph shows the toxicity of Baloxavir to AGS gastric cancer cells. Baloxavir is toxic to AGS gastric cancer cells at concentrations ranging from 0 to 50 μM. The graph also shows the tumor cell growth curves at different time points with different drug concentrations.

[0023] Figure 4 The graph shows the toxicity of Baloxavir to gastric cancer cells HGC27. Baloxavir was toxic to HGC27 gastric cancer cells at concentrations ranging from 0 to 50 μM. The graph also shows the tumor cell growth curves at different time points with different drug concentrations.

[0024] Figure 5 Baloxavir has an inhibitory effect on esophageal squamous cell carcinoma cells. Specifically, baloxavir can inhibit the proliferation of KYSE150 esophageal squamous cell carcinoma cells at concentrations ranging from 0.5 to 5 μM. The figure shows the tumor cell proliferation curves at different time points with different drug concentrations.

[0025] Figure 6 Baloxavir has an inhibitory effect on esophageal squamous cell carcinoma cells. Specifically, baloxavir can inhibit the proliferation of KYSE450 esophageal squamous cell carcinoma cells at concentrations ranging from 0.5 to 5 μM. The figure shows the tumor cell proliferation curves at different time points with different drug concentrations.

[0026] Figure 7 Baloxavir has an inhibitory effect on gastric cancer cells. Specifically, baloxavir can inhibit the proliferation of AGS gastric cancer cells at concentrations ranging from 0.5 to 5 μM. The figure shows the tumor cell proliferation curves at different time points with different drug concentrations.

[0027] Figure 8 Baloxavir has an inhibitory effect on gastric cancer cells. Specifically, baloxavir can inhibit the proliferation of HGC27 gastric cancer cells at concentrations ranging from 0.5 to 5 μM. The figure shows the tumor cell proliferation curves at different time points with different drug concentrations.

[0028] Figure 9 Images of mouse tumors in a human esophageal cancer PDX model induced by Baloxavir;

[0029] Figure 10 This is a graph showing the changes in tumor volume in mice using a human esophageal cancer PDX model induced by Baloxavir.

[0030] Figure 11 This graph shows the changes in tumor weight in mice using a human esophageal cancer PDX model induced by Baloxavir.

[0031] Figures 1 to 11 middle,* p <0.05, ** p <0.01, *** p <0.001. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0033] Application Trial

[0034] Materials and Methods

[0035] 1. Materials

[0036] 1.1 Cells and Reagents

[0037] Esophageal squamous cell carcinoma cells KYSE150 and KYSE450, and gastric cancer cells HGC27 and AGS were obtained from the Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University. KYSE150, KYSE450, and HGC27 cells were cultured in RPMI-1640 medium, while AGS cells were cultured in F12K medium.

[0038] Baloxavir Shanghai Loulan Biotechnology Co., Ltd.

[0039] North China Pharmaceutical Co., Ltd.

[0040] Streptomycin Shandong Lukang Pharmaceutical Co., Ltd.

[0041] RPMI-1640 culture medium, from Israel's Biological Industries.

[0042] F12K culture medium, Dalian Meilun Biotechnology Co., Ltd.

[0043] 0.25% trypsin (Shanghai Beyotime Biotechnology Co., Ltd.)

[0044] Serum-free cell cryopreservation solution Suzhou Xinsaimei Biotechnology Co., Ltd.

[0045] PBS powder, Beijing Solarbio Science & Technology Co., Ltd.

[0046] Fetal bovine serum (BioBio Corporation, USA)

[0047] DMSO Beijing Solarbio Technology Co., Ltd.

[0048] 1.2 Instruments and Equipment:

[0049] 1.5ml centrifuge tubes, Axygen Corporation (USA)

[0050] 15ml centrifuge tubes, Corning Corporation, USA

[0051] 96-well cell culture plate, Wuxi Naisi Biotechnology Co., Ltd.

[0052] 10 cm cell culture dish, Wuxi NiceBiotechnology Co., Ltd.

[0053] Disposable pipettes Guangzhou Jet Biofiltration Co., Ltd.

[0054] In Cell Analyzer 6000, GE (USA)

[0055] Pipettes from Gilson Corporation, USA

[0056] Dry CO2 Incubator Shanghai Yiheng Scientific Instruments Co., Ltd.

[0057] High-speed low-temperature centrifuges from Eppendorf, Germany

[0058] Vacuum suction pumps, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.

[0059] Inverted microscope by Carl Zeiss Jena, Germany

[0060] Snowflake ice maker from SANYO Corporation, Japan

[0061] Mili-Q water purifier from Millipore, USA

[0062] Full-wavelength microplate reader by Thermo Fisher Scientific, USA

[0063] This invention used one human esophageal cancer tissue specimen, numbered EG20 (from Henan Cancer Hospital, male, 46 years old, hospital number 2042083, T2N0M0Ⅱ, moderately differentiated).

[0064] 1.3 Laboratory Animals

[0065] In this embodiment, Cb-17 SCID immunodeficient mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The license number is: SCXK(Beijing) 2012-0001, SPF level, 5-6 weeks old, weighing 16-18 g, female mice. The mouse feed was purchased from Beijing Huafu Biotechnology Co., Ltd. The experimental animals were housed in the experimental animal room of the School of Basic Medicine, Zhengzhou University, and were raised in a specific pathogen-free (SPF level) breeding room with constant temperature (25-27 °C), constant humidity (45%-50%), fresh air, dust removal and disinfection. Six animals were housed in each breeding box. The sterile-treated feed was provided for the animals to freely ingest. The bedding was changed every three days after high-temperature disinfection. The cages and drinking water were disinfected at high temperature every three days, and sterile distilled water was used for drinking. When changing the breeding supplies, the operation was strictly carried out in accordance with the aseptic principle. The experimental animals were raised under a 12:12 hour light / dark cycle and had free access to food and water.

[0066] 2. Method

[0067] 2.1 Cytotoxicity experiment: The cells were seeded into 96-well plates, with 8000 KYSE150 cells per well, 10000 KYSE450 cells per well, 8000 AGS cells per well, and 6000 HGC27 cells per well. First, Baloxavir was dissolved in DMSO, and then human esophageal squamous carcinoma KYSE150 and KYSE450 cells, human gastric cancer AGS and HGC27 cells were treated with different concentrations of Baloxavir, respectively, so that the final concentration of Baloxavir in RPMI-1640 medium or F12K medium was 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM. Six replicates were set for each concentration. After 24 h and 48 h, MTT was added, and the cells were incubated in a cell culture incubator at 37 °C and 5% CO2 for 2 hours. DMSO was added to measure the absorbance value, and the cell growth curve graph at different time points for each concentration was plotted to evaluate the effect of Baloxavir on the growth of esophageal squamous carcinoma and gastric cancer cells.

[0068] 2.2 Cell proliferation assay: Cells were seeded into 96-well plates, with 3000 cells per well for KYSE150, 5000 cells per well for KYSE450, 3000 cells per well for AGS, and 2000 cells per well for HGC27. Baloxavir was first dissolved in DMSO, and then human esophageal squamous cell carcinoma KYSE150 and KYSE450 cells, and human gastric cancer AGS and HGC27 cells were treated with different concentrations of baloxavir. The final concentrations of baloxavir in RPMI-1640 or F12K medium were 0 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM, respectively. Each concentration was used in 6 replicates. MTT was added after different time points (0, 24, 48, 72, and 96 h), and the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 2 hours. DMSO was added and the absorbance was measured. Cell growth curves of each concentration at different time points were plotted to evaluate the effect of baloxavir on the proliferation of esophageal squamous cell carcinoma and gastric cancer cells.

[0069] 2.3 Establishment of a human esophageal cancer immunodeficient mouse tumor-implanting model

[0070] The selection criteria for esophageal squamous cell carcinoma tissue were as follows: fresh tumor tissue from patients who had not received any radiotherapy or chemotherapy before surgery. Within 90 minutes of tumor tissue removal, it was transported to the laboratory in serum-free 1640 medium under refrigeration. Before tissue inoculation, the tumor tissue was rinsed with PBS containing penicillin and streptomycin (PBS: penicillin-streptomycin 50:1) and placed on ice, awaiting inoculation. Mice were anesthetized by intraperitoneal injection of 0.4% sodium pentobarbital, and the tissue was cut into 10-15 mm pieces. 3 Small pieces of tissue were implanted subcutaneously into the back of the neck of mice using forceps. After the mice recovered from anesthesia, they were returned to a sterile rearing room. Approximately 3-5 days later, after the wounds on the back of the neck had healed, the tumor volume was measured at fixed intervals. The tumor was considered complete when it reached 1000 mm². 3 Mice were then sacrificed and tumor tissue was removed. The tumor was then passaged subcutaneously into new SCID mice in the same manner (generation 2). When the xenograft was stably passaged to generation 3, the esophageal cancer xenograft model was considered successfully established.

[0071] 2.4 Baloxavir inhibits tumor growth in mice xenografted with human esophageal cancer

[0072] One or two weeks after inoculation, when the tumor nodules on the backs of mice grew to approximately 200 cubic millimeters, they were divided into groups, i.e., mice were evenly distributed into each group according to tumor size. Baloxavir was dissolved in physiological saline, and the three groups of mice were administered physiological saline, 15 mg / kg Baloxavir, and 30 mg / kg Baloxavir by gavage, respectively. Tumor volume was recorded every 3 days. When the tumor volume in the control group mice reached approximately 1000 cubic millimeters (approximately 33 days), the experiment was terminated, the tumor tissue was removed, the tumor weight was measured, and photographs were taken.

[0073] Experimental results

[0074] 1. Cytotoxicity assay results indicate that, as Figure 1 , 2 As shown in Figures 3 and 4, Baloxavir exhibits toxicity against esophageal squamous cell carcinoma and gastric cancer cells. At 24 hours, the IC50 of KYSE150, KYSE450, AGS, and HGC27 reached 50% at concentrations of 20.593 μM, 21.150 μM, 27.596 μM, and 12.937 μM; at 48 hours, the IC50 of KYSE150, KYSE450, AGS, and HGC27 reached 50% at concentrations of 12.107 μM, 13.587 μM, 14.702 μM, and 5.845 μM. Safe drug concentrations were then screened, and 0 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM were determined for further proliferation experiments.

[0075] 2. Cell proliferation experiments showed that, for example... Figure 5 , 6 As shown in Figures 7 and 8, the inhibitory effect of baloxavir on the proliferation of esophageal squamous cell carcinoma cells and gastric cancer cells increased with increasing baloxavir concentration. When KYSE150, KYSE450, AGS, and HGC27 cells were treated with 5 μM of the drug for 96 h, the cell proliferation inhibition rates were 64.72%, 81.46%, 58.69%, and 79.23%, respectively.

[0076] 3. Baloxavir inhibits tumor growth in mice xenografted with human esophageal cancer, such as... Figure 9 The mouse tumor images shown indicate that high-dose Baloxavir (30 mg / kg) and low-dose Baloxavir (15 mg / kg) tumors were significantly inhibited compared to the control group. Figure 10 Tumor volume was significantly inhibited in both the high-dose Baloxavir group (30 mg / kg) and the low-dose Baloxavir group (15 mg / kg) compared to the control group. Figure 11As shown, tumor weight decreased in mice in both the high-dose Baloxavir group (30 mg / kg) and the low-dose Baloxavir group (15 mg / kg). Therefore, Baloxavir can inhibit tumor growth in mice in a human esophageal squamous cell carcinoma xenograft model at concentrations of 15 mg / kg / day and 30 mg / kg / day.

[0077] In summary, this study found that Baloxavir has toxic effects on esophageal squamous cell carcinoma KYSE150 and KYSE450 cells, as well as gastric cancer AGS and HGC27 cells, and can inhibit the proliferation of these cells. Baloxavir can also inhibit the growth of human esophageal cancer xenografted mouse tumors (EG20). These findings suggest that Baloxavir may be effective in preventing and treating esophageal cancer, gastric cancer, and other tumors.

Claims

1. The application of baloxavir in the preparation of antitumor drugs, characterized in that, The antitumor drug is a drug for treating esophageal squamous cell carcinoma or gastric cancer.

2. The application according to claim 1, characterized in that, The drugs used to treat esophageal squamous cell carcinoma are those that inhibit the proliferation of esophageal squamous cell carcinoma cells.

3. The application according to claim 2, characterized in that, Baloxavir can inhibit the proliferation and number of colonies of esophageal squamous cell carcinoma cells at concentrations of 0.5 μM to 5 μM.

4. The application according to claim 3, characterized in that, The esophageal squamous cell carcinoma cells were KYSE150 cells and / or KYSE450 cells.

5. The application according to claim 1, characterized in that, Among them, the drugs used to treat gastric cancer are those that inhibit the proliferation of gastric cancer cells.

6. The application according to claim 5, characterized in that, Baloxavir can inhibit the proliferation of gastric cancer cells at concentrations of 0.5 μM to 5 μM.

7. The application according to claim 6, characterized in that, The gastric cancer cells are HGC27 cells and / or AGS cells.

8. The application according to claim 1, characterized in that, Application of baloxavir in the preparation of drugs that inhibit tumor growth in humanized xenograft models of esophageal or gastric cancer.

9. The application according to claim 8, characterized in that, Baloxavir can inhibit the growth of tumors in humanized xenograft models of esophageal or gastric cancer at doses of 15 mg / kg to 30 mg / kg.

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