Use of tegaserod for the preparation of antitumor medicaments

Abstract

The application discloses tegaserod or a pharmaceutically acceptable salt thereof as a JAK-STAT3 signal pathway inhibitor, an immunomodulator and application thereof in preparation of an antitumor drug. Tegaserod and the pharmaceutically acceptable salt thereof have very good inhibiting effect on the growth of various tumor cells in vivo and in vitro, and are expected to be used in the treatment of various cancers.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications and relates to a new pharmaceutical use of tegaserod, specifically, its use as an inhibitor of the JAK-STAT3 signaling pathway and an immunomodulator in the preparation of antitumor drugs. Background Technology

[0002] Cancer is currently the leading cause of death and harm to human health. Current clinical medications are far from meeting the needs of patients, making the development of anti-cancer drugs an extremely important research direction in the field of drug development.

[0003] JAK-STATs signaling is crucial for cellular signal transduction and various physiological activities. Abnormalities in this family of signals can lead to numerous diseases, including cancer and immune-related disorders. The JAKs family includes four members: JAK1, JAK2, JAK3, and Tyk2. Downstream from JAKs, the STATs family comprises seven members, among which STAT3 is a significant one. It is constitutively activated by aberrant upstream tyrosine kinases in numerous tumor cell lines and human tumors. Abnormal STAT3 signaling participates in the occurrence and development of human tumors by stimulating cell proliferation, promoting angiogenesis, and inhibiting apoptosis. Therefore, inhibiting the JAK-STAT3 signaling pathway is a potentially feasible clinical therapeutic strategy for tumors.

[0004] Currently, clinical treatment strategies for inhibiting the JAK-STAT3 signaling pathway mainly fall into the following categories: First, tyrosine kinase inhibitors targeting upstream signaling molecules of STAT3, including JAK kinase family inhibitors; second, blocking STAT3 gene expression or protein function, such as through dominant-negative STAT proteins or RNAi interference targeting STAT3; and third, using small molecules to inhibit STAT3 activation and dimerization.

[0005] In recent years, with the rapid rise in the cost of new drug development and the gradual decline in the success rate of drug development, coupled with the expiration of patents for most drugs and the difficulty in developing new drugs in the pipeline, pharmaceutical companies have been developing new patent-protected drugs based on existing drugs. "Drug repurposing" has become a hot topic in international drug research and development.

[0006] Tegaserod (Formula I), chemically named 2-[(5-methoxy-1H-indol-3-yl)methylene]-N-pentylcarbazine, is a selective serotonin 4 (5-HT4) receptor agonist. Its maleate salt was approved by the FDA in 2002 for the treatment of irritable bowel syndrome (IBS). In addition, tegaserod has therapeutic effects on various gastrointestinal disorders, including heartburn, flatulence, postoperative intestinal obstruction, abdominal pain and discomfort, epigastric pain, nausea, vomiting, regurgitation, pseudo-intestinal obstruction, and gastroesophageal reflux.

[0007] Summary of the Invention

[0008] This invention unexpectedly discovered that tegaserod has inhibitory activity on the JAK-STAT3 signaling pathway and can inhibit tumor growth, while activating peripheral immune responses and immune responses in the tumor microenvironment, exhibiting in vivo and in vitro antitumor effects against a variety of tumors.

[0009] Based on the above findings, the present invention provides the use of tegaserod or a pharmaceutically acceptable salt thereof in the preparation of antitumor drugs.

[0010] In a preferred embodiment of the present invention, the tumor is a tumor in which the JAK-STAT3 signaling pathway is abnormally activated.

[0011] As a more preferred embodiment of the present invention, the abnormal activation of the JAK-STAT3 signaling pathway is manifested as an increase in the phosphorylation level of JAK1, JAK2, JAK3, Tyk2, or STAT3, preferably an increase in the phosphorylation level of JAK1 Tyr1022 / 1023 sites, JAK2 Tyr1007 / 1008 sites, Tyk2 Tyr1054 / 1055 sites, or STAT3 Tyr705 sites.

[0012] In a preferred embodiment of the invention, the antitumor drug also activates and / or enhances the immune response.

[0013] In a preferred embodiment of the present invention, the immune response is a mammalian immune response.

[0014] In a preferred embodiment of the present invention, the immune response includes a peripheral immune response and / or an immune response in the tumor microenvironment; more preferably, activation and / or enhancement of the peripheral immune response includes an increase in the number of one or more of leukocytes, neutrophils, lymphocytes, and platelets in the peripheral blood of the body; activation and / or enhancement of the anti-tumor immune response in the tumor immune microenvironment includes increasing the infiltration of tumor-infiltrating immune cells (CD45+), or partially or completely increasing the proportion of one or more of cytotoxic T cells (CD8+), helper T cells (CD4+), activated T cells (CD4+CD69+ and CD8+CD69+), tumor-infiltrating inflammatory neutrophils (CD11b+Ly6G+), monocytes / macrophages (CD11b+Ly6C+), and natural killer cells (CD335+).

[0015] As a preferred embodiment of the invention, the pharmaceutically acceptable salt is tegaserodara salt.

[0016] In a preferred embodiment of the present invention, the tumor is any of the following: brain tumor, genitourinary system tumor, lymphatic system tumor, gastric cancer, laryngeal cancer, nasopharyngeal carcinoma, skin cancer, bone cancer, blood cancer, leukemia, breast cancer, histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, prostate cancer, cervical cancer, liver cancer, skin cancer, epithelial cell carcinoma, etc., preferably prostate cancer, lung cancer, or colon cancer.

[0017] Furthermore, the tegaserod or its pharmaceutically acceptable salts can enhance clinical efficacy when used in combination with currently used or developing anti-tumor drugs.

[0018] Tegaserod and its pharmaceutically acceptable salts have shown excellent inhibitory effects on the growth of various tumor cells both in vitro and in vivo, and hold promise for the treatment of various cancers. This invention provides cancer patients with a new therapeutic candidate, which may further improve patient efficacy and prognosis. Attached Figure Description

[0019] Figure 1 To investigate the inhibitory effect of tegaserodextrin on reporter gene expression in the STAT3 luciferase drug screening system.

[0020] Figure 2 This is a schematic diagram illustrating how tegaserod can inhibit constitutive activation and IL-6-induced STAT3 activation through immunoblotting experiments.

[0021] Figure 3 This is a schematic diagram illustrating how tegaserod selectively inhibits the phosphorylation of JAK kinase using an immunoblotting assay.

[0022] Figure 4 This is a schematic diagram illustrating the in vitro inhibition of tumor cell growth by tegaserod.

[0023] Figure 5 This is a schematic diagram illustrating how intraperitoneal administration of tegaserod can inhibit tumor growth using an A549 nude mouse xenograft model.

[0024] Figure 6 Oral administration of tegaserod can effectively inhibit the growth of non-small cell lung cancer A549 xenografts in nude mice.

[0025] Figure 7 Oral administration of tegaserod inhibited the growth of DU145 prostate cancer xenografts in nude mice.

[0026] Figure 8 Oral administration of low-dose, low-frequency tegaserod can effectively inhibit the growth of DU145 xenografts in nude mice.

[0027] Figure 9Oral administration of tegaserod activates the immune response in the peripheral and tumor microenvironment.

[0028] Figure 10 Tegaserod activates the immune system to inhibit the growth of colorectal cancer MC38 allograft tumors. Detailed Implementation

[0029] This invention provides the use of tegaserod, its free form, pharmaceutically acceptable salts, prodrugs, and active metabolites in the preparation of antitumor drugs.

[0030] The free form of a specific salt of tegaserod can be separated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous solution of an alkali, such as a dilute aqueous solution of NaOH, potassium carbonate, ammonia, or sodium bicarbonate. The free form differs somewhat from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purposes of the invention, the acid salt and alkali salt are otherwise pharmaceutically equivalent to their respective free forms.

[0031] Pharmaceutically acceptable salts of the present invention can be synthesized from tegaserod using conventional chemical methods. Typically, they are prepared by ion-exchange chromatography or by reacting a free base with a stoichiometric or excess amount of the desired salt in a suitable solvent or combination of solvents. Therefore, pharmaceutically acceptable salts of tegaserod of the present invention comprise conventional, non-toxic salts of the compounds of the present invention formed by the reaction of tegaserod with inorganic or organic acids. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc., as well as salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pyric acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethylsulfonic acid, trifluoroacetic acid, etc., preferably maleate salts.

[0032] This invention relates to tegaserod or a pharmaceutically acceptable salt thereof as a small molecule inhibitor of the JAK-STAT3 signaling pathway, and its use in the preparation of antitumor drugs.

[0033] In one embodiment, this application provides a method for treating hyperproliferative diseases or symptoms such as tumors in humans or other mammals using tegaserod or a pharmaceutically acceptable salt thereof.

[0034] In one embodiment, the compounds and pharmaceutically acceptable salts of the present application may be used to treat or control hyperproliferative diseases such as histiocytic lymphoma, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell carcinoma, prostate cancer, nasopharyngeal carcinoma, epidermal cell carcinoma, cervical cancer, oral cancer, human fibrosarcoma, and leukemia.

[0035] The tegaserod and its pharmaceutically acceptable salts described in this application can be used to treat the following diseases and other diseases not listed below, according to the methods described below:

[0036] 1) A method for treating breast cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

[0037] 2) A method for treating respiratory tract cancers in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, small cell lung cancer, non-small cell lung cancer, bronchial adenoma, and pleural pulmonary blastoma.

[0038] 3) A method of treating brain cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, brainstem and subocular gliomas, cerebellar and cerebral astrocytomas, ependymocytomas, and neuroectodermal and pineal tumors.

[0039] 4) A method for treating tumors of the male or female reproductive organs of humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, and intrauterine tumors.

[0040] 5) A method of treating tumors of the digestive tract of humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, rectal cancer, small bowel cancer, or salivary gland cancer.

[0041] 6) A method of treating tumors of the urethra in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. These include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureteral cancer, invasive papillary urothelial carcinoma of the bladder, or urethral cancer.

[0042] 7) A method of treating eye cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, intraocular melanoma and retinoblastoma.

[0043] 8) A method of treating liver cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, hepatocellular carcinoma (stem cell carcinoma with or without fibrous plate changes), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

[0044] 9) A method of treating skin cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merck cell carcinoma, and non-melanoma cell carcinoma.

[0045] 10) A method of treating head and neck cancer in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, cancers of the larynx, hypopharynx, nasopharynx, oropharynx, and lips and oral cavity.

[0046] 11) A method of treating lymphomas in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, AIDS-related lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Hodgkin's disease, and central nervous system lymphomas.

[0047] 12) A method of treating sarcomas in humans or other mammals using a pharmaceutically acceptable composition of tegaserod or a pharmaceutically acceptable salt thereof. Including, but not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, Linba sarcoma, and rhabdomyosarcoma.

[0048] 13) A method of treating leukemia in humans or other mammals using a pharmaceutical composition of tegaserod or a pharmaceutically acceptable salt thereof. This includes, but is not limited to, acute myeloid leukemia, acute leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0049] According to standard pharmaceutical techniques, tegaserod or a pharmaceutically acceptable salt thereof of the present invention can be administered to mammals, preferably humans, alone or in combination with pharmaceutically acceptable receptors, excipients or diluents in a pharmaceutical composition. It can be administered orally, subcutaneously, intramuscularly, intraperitoneally, intravenously, rectally and topically, through the eyes, lungs, nose, or parenterally.

[0050] The active metabolites of tegaserod or its pharmaceutically acceptable salts involved in this application, as well as the prodrugs that can be converted in vivo into the structures of the compounds involved in this application and their pharmaceutically acceptable salts, are also included in the claims of this application.

[0051] The term "immune response" refers to the body's defensive and recognition response to foreign or mutated self-components. Depending on where it occurs or acts within the body, immune responses typically include systemic and local immune responses.

[0052] The term "peripheral immune response" refers to a systemic immune response, which involves the broad activation of the immune system in places far from the tumor itself. This includes responses of immune cells circulating in the bloodstream to foreign or mutated autologous or autologous DAMP (Danger associated Molecular Pattern) released by the autologous cells, such as activation, phenotypic changes, or proliferation. It also includes immune responses in lymph node nodules, spleen, or intestinal-associated lymphoid tissues.

[0053] The tumor microenvironment is composed of tumor cells and tumor-infiltrating immune cells, neovascularization and its endothelial cells, tumor-associated fibroblasts, and extracellular matrix. It can promote tumor progression, increase tumor invasiveness, evade host immune responses, and counteract treatment responses. The term "immune response in the tumor microenvironment" refers to a local immune response, typically encompassing the composition, activity, and function of immune cells within the tumor microenvironment. In this invention, the immune response in the tumor microenvironment includes changes in the levels of tumor-internal immune cells (CD45+), cytotoxic T cells (CD8+), helper T cells (CD4+), activated T cells (CD4+CD69+ and CD8+CD69+), and tumor-infiltrating inflammatory neutrophils (CD11b+Ly6G+), monocytes / macrophages (CD11b+Ly6C+), and natural killer cells (CD335+). Activation and / or enhancement of the immune response in the tumor microenvironment refers to an increase in the number or proportion of all or some of the aforementioned cells.

[0054] Tegaserod can be used in combination with other known medications for treating or improving similar symptoms. When used in combination, the original medication's administration method and dosage remain unchanged, while tegaserod is taken simultaneously or subsequently. When tegaserod is taken concurrently with one or more other medications, a pharmaceutical composition containing one or more known medications and tegaserod is preferred. Drug combination also includes taking tegaserod with one or more other known medications during overlapping time periods. When tegaserod is used in combination with one or more other medications, the dose of tegaserod or the known medication may be lower than the dose when they are used alone.

[0055] Drugs or active ingredients that can be used in combination with tegaserod to treat tumors include, but are not limited to:

[0056] Estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxins / cell inhibitors, antiproliferative agents, proteotransferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, etc.

[0057] In one implementation scheme, drugs or active ingredients that can be used in combination with tegaserod to treat tumors include, but are not limited to: interleukin, alendronate, interferon, atrazonoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglucopyranoside, amifostine, amrubicin, azithromycin, anatoxazole, dolasetron, aranesp, arglabin, arsenic trioxide, anoxin, 5-azacytidine, azathioprine, BCG or TICE, betamethasone acetate, betamethasone phosphate preparation, bexarotine, bleomycin sulfate, bromouridine, bortezomib, busulfan, calcitonin, alectozumab injection, capecitabine, carboplatin, and casrocin. Cefesone, Simmo-interleukin, Daunorubicin, Chlorbutamol, Cisplatin, Cladribine, Cladribine, Chlordroxylphosphate, Cyclophosphamide, Cytarabine, Dacarbazine, Actinomycin D, Daunorubicin Liposome, Dexamethasone, Dexamethasone Phosphate, Estradiol Valerate, Dernexyl Interleukin-2, Depomere, Delorelin, Delazosone, Diethylstilbestrol, Diflucan, Docetaxel, Deoxyfluorouracil, Doxorubicin, Drolamine Chromium-166-chitosan complex, eligard, raburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetopin, erythropoietin, etoposide, levamisole tablets, estradiol preparations, 17-β-estradiol, estradiol sodium phosphate, ethinylestradiol, amifostine, hydroxyphosphate, vanbex, etoposide, fazodazole, tamoxifen preparations, filgrastim, phenacetin, fenexilate, fluorouracil Fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, flumethasone, flutamide, formestan, 1-β-D-arasulofuranylcytidine-5'-stearoyl phosphate, formustin, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisilone hydrochloride, histamine relin, and acetaminophen, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, tetanisoprostol, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B. Interferon α-n1, Interferon α-n3, Interferon β, Interferon γ-1a, Interleukin-2, Intron A, Iressa, Irinotecan, Keterene, Lentinan sulfate, Letrozole, Levofloxacin, Levofloxacin acetate, Levotetraimidazole, Levolecithin calcium salt, Levothyroxine sodium, Levothyroxine sodium preparations, Lomustine, Clonidamine, Drowanesol, Nitrogen mustard, Mecobalamin, Medroxyprogesterone acetate, Medroxyprogesterone acetate, Melphalan, Esterified estrogen, 6-Mercaptopurine, Mesna, Methotrexate, Methylaminolevulinate, Mitofosine, Minocycline, Mitomycin C, Mitotane, Mitoxantrone, Tralostertan, Doxorubicin citrate liposomes, Nedaplatin, PolyvinylpyrrolidoneDiolated filgrastim, olprednisolone, interleukin, neupogen, nilumethoxazole, tamoxifen, NSC-631570, recombinant human interleukin-1-β, octreotide, odannillone hydrochloride, dehydrocortisone oral solution, oxaliplatin, paclitaxel, prednisolone sodium phosphate preparation, pegaspargase, pegastin, pentostatin, streptomycin preparation, pilucarpine hydrochloride, prarubicin, porphyrin sodium, prednimustine, stiprednisolone, prednisone, premarin. Procarbazine, Recombinant Human Erythropoietin, Raltitrexed, Ribeye, Rhenium-186 Etidronate, Rituximab, Rituximab-A, Romotide, Pilocarpine Hydrochloride Tablets, Octreotide, Samostin, Semustine, Cizonan, Sobuzosen, Methylprednisolone Sodium, Paphosphatase, Stem Cell Therapy, Levothyroxine, Strontium Chloride-89, Levothyroxine Sodium, Tamoxifen, Tansulosin, Tastolactone, Doxorubicin, Tecithiazide, Temozolomide, Teniposide Testosterone propionate, Methyltestosterone, Thioguanine, Thiotepa, Thyroid-stimulating hormone, Tiludronate, Topotecan, Toremifene, Tosimomab, Trastuzumab, Triosmectin, Retinoic acid, Methotrexate tablets, Trimethylmelamine, Trimethoprim, Triptorelin acetate, Triptorelin dinaphthylnaphthyl acid, Ufodin, Urine, Pentorubicin, Visorcinol, Vincristine, Vincristine, Vincrylene, Vinorelin, Verrucidiazin, Dextromethorphan, Nettostatin, Scyproterone acetate, Paclitaxel protein stabilizer, Acolbifene, Interferon R-LB, Affinitak, Aminopterin, Azoxifen Asoprisnil, Atamitan, Atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, Cetuximab, Clinatropin, Cyproterone Acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edotecarin, Eflunomide, Ecinotecan, Fenivel-Amine, Histamine Dihydrochloride, Histamine Relin Hydrogel Implant, Holmium-166DOTMP, Ibandronate, Interferon-γ, Intron-PEG

[0058] ixabepilone, keyhole hemocyanin, L-651582, lanafamib, lasoxifene, libra, lonafamib, miprexifen, minocycline, MS-209, liposome MTP-PE, MX-6, nafarelin, nemorubicin, neovastatin, nortratriptyline, olimolsen, onco-TCS, osidem, paclitaxel polyglutamate, sodium pamoate, PN-

[0059] 401, QS-21, Quasi-Yang, R-1549, Raloxifene, Leopard Frog Enzyme, 13-cis-retinoic acid, Saplatin, Ciocalcitriol, T-138067, Tarceva, docosahexaenoic acid paclitaxel, Thymosin α1, Gazofuran, Tipifanib, Tirazamine, TLK-286, Toremifen, Trans-MID-lo7R, Vasopida, Vatalanib, Vertepofen, Vinpocetine, Z-100, and Zoledronic acid or combinations thereof.

[0060] The present invention will be further illustrated below with reference to specific embodiments. The purpose of the embodiments provided is to help to further understand the present invention. The specific materials, methods and other methods used are for the purpose of describing the present invention and do not constitute a limitation on the scope of application of the present invention.

[0061] The experimental materials and general experimental methods used in the examples are as follows. Unless otherwise specified, they shall be used in accordance with the manufacturer's instructions.

[0062] 1. Antibodies and reagents

[0063] Antibodies such as p-Tyr705-STAT3, p-Tyr1022 / 1023-JAK1, p-Tyr1007 / 1008-JAK2, and p-Tyr1054 / 1055-Tyk2 were purchased from Cell Signaling Technology, and α-Tubulin antibody was purchased from Santa Cruz. Recombinant human IL-6 cytokine was purchased from Peprotech. CD11b-PE-Cyanine 7 and CD8a-PerCP-eFluor TM 710, CD45-APC-eFluor 780, Rat IgG1 kappa Isotype Control (eBRG1), and Anti-Mo CD32 / CD16 antibody were purchased from Invitrogen. CD11b-AF488 and CD4-Brilliant Violet 510 were also purchased. TM CD206-PE-Cyanine 7 and CD69-PE antibodies were purchased from Biolegend. Ly-6G-FITC and Ly-6C-APC antibodies, erythrocyte lysis buffer, mouse tumor dissociation kit, and human tumor dissociation kit were purchased from Medtronic. Fixable Viability Dye (eFluor TM506) and Ki67 antibodies were purchased from BD. IDEXX Procyte Dx* Reagent kit and ProCyte Dx* Stain Pack were purchased from IDEXX Small Animal Company. p-Tyr705-STAT3 antibody was purchased from Cell Signaling Technology. Matrigel basement membrane matrix was purchased from Corning.

[0064] 2. Cell Culture

[0065] HeLa and SKA cells (A549 cells constructed using the STAT3-luciferase reporter gene) were cultured in DMEM medium, while DU145, A549, DLD1, H460, and MC38 cells were cultured in RPMI 1640 medium. During routine culture, the culture medium was supplemented with 10% fetal bovine serum (FBS), 100 IU / ml penicillin, and 100 mg / ml streptomycin. All cells were cultured at 37°C under 5% CO2.

[0066] 3. STAT3-dependent luciferase reporter gene detection

[0067] SKA cells were loaded at 1×10 4 The cells were seeded at a density of / wells into white 96-well plates and incubated overnight at 37°C with 5% CO2. The test drug was added at the desired concentration, and the cells were treated for 24 hours. A luciferase assay kit (Promega) was used. The activity of luciferase was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0068] 4. Western blot for protein immunoblotting

[0069] Cells were harvested and lysed using RIPA buffer. After separation on an SDS-PAGE gel, proteins were transferred to a nitrocellulose membrane (GE Healthcare). Primary antibodies were added to bind the proteins to the membrane, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Abixin). Finally, Immobilon assay was performed. TM The resulting immune complexes were detected using Western chemiluminescence HRP substrate (Millipore) and imaged using a Tanon 5200 imaging system.

[0070] 5. Flow cytometry analysis of tumor cell cycle

[0071] DU145 cells were loaded at 5 × 10 5Seeds were planted at a density of / well into 6-well plates, and different concentrations of the test drug were added as needed, or serum-free medium was used for culture. For cell cycle assays, cells were harvested after 24 hours of treatment and stained with a cell cycle staining kit (Lianke Bio, catalog number: CCS012); for apoptosis assays, cells were treated for 48 hours and then stained with eBioscience. TM Annexin V-FITC apoptosis kit (purchased from Invitrogen) was used for staining. Finally, the stained cells were analyzed by flow cytometry.

[0072] 6. Cell viability assay

[0073] Cells were seeded at a density of 3,000 cells / well in 96-well plates. After 18 hours, different concentrations of the test drug were added to react with the cells. After 72 hours, 10 μl of resazurin (1 mg / ml) was added to each well and the cells were incubated for another 3 hours, followed by... The fluorescence value at a wavelength of 595 nm (excitation wavelength of 544 nm) was measured using a multi-functional microplate reader (purchased from Molecular Device).

[0074] 7. Nude mouse tumor-suppressing animal model and in vivo antitumor activity assay of the drug

[0075] Female NRMI nu / nu athymic nude mice (SPF grade, 6 weeks old, weighing 17-20g) were purchased from Jicui Biotechnology and housed in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle, according to standard requirements. Animal experiments were approved by the Laboratory Animal Committee of Ocean University of China and complied with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1996).

[0076] Nude mice were subcutaneously implanted with A549 cell suspension (female mice) or DU145 matrigel cell suspension (male mice). After the cells grew and formed palpable solid tumors, the model mice were randomly divided into groups of 7-10 mice each, and treated with medication according to the experimental group. Animal weight was measured periodically throughout the experiment. Tumor volume was calculated as: Tumor volume = 0.5 × length × width × width.

[0077] 8. Flow cytometry analysis of immune cell surface markers

[0078] After the experiment was terminated, the mice were euthanized with CO2, and an appropriate amount of tumor tissue was taken and placed in a dissociation tube containing mouse tumor dissociation reagent (MC38 xenograft) or human tumor dissociation reagent (DU145 xenograft). The dissociation was performed using a tissue dissociator (Medini). After red blood cell lysis and dead cell staining, the cells were incubated with blocking solution at 4°C, and the corresponding antibodies were added. The results were then analyzed by FACSAriaIII flow cytometer.

[0079] 9. Blood cell analysis

[0080] After tumor-bearing mice were euthanized by CO2, blood was collected from their hearts and placed in EDTA anticoagulant tubes for blood cell component analysis using the IDEXX ProCyte Dx fully automated hematology analyzer.

[0081] 10. Immunohistochemistry

[0082] Paraffin sections were dewaxed with xylene, washed with ethanol at various levels and then with water, antigen retrieval was performed, and the sections were incubated with blocking solution at 37°C for primary antibody treatment, secondary antibody treatment, DAB staining solution, and treated with hematoxylin, differentiation solution and ammonia water. After treatment with ethanol at various levels and xylene, the sections were mounted with neutral resin.

[0083] 11. Transcriptome sequencing

[0084] A suitable amount of tumor tissue was collected, flash-frozen in liquid nitrogen, and then subjected to eukaryotic mRNA sequencing by MegiGene. Using the Illumina Novaseq 6000 sequencing platform, all mRNAs transcribed from specific eukaryotic tissues or cells at a specific time point were sequenced. Library construction was performed using the Illumina Truseq™ RNA sample prep kit. Raw data underwent quality control, sequence alignment, transcript assembly, functional annotation, and expression level analysis. Significantly differentially expressed genes (p-value < 0.05) were analyzed using DESeq2 differential analysis software, and KEGG signaling pathway analysis was performed on the significantly differentially expressed genes.

[0085] Example 1: Tigaserod showed inhibitory activity in a constitutive STAT3-activated luciferase cell model.

[0086] Using a constitutive STAT3-activated luciferase expression cell model (see Chinese Patent 1407357), it was found that tigaserodextrin significantly inhibited STAT3, with an IC50 concentration of 100%. 50 5.06 μM ( Figure 1 ).

[0087] Example 2: Tigaserod inhibits STAT3 activation

[0088] Different concentrations of tegaserod were added to normally cultured cells, and after 2 hours of treatment, cell protein lysates were collected for protein immunoblotting. The activation status of STAT3 was observed by the phosphorylation status of the STAT3 Tyr705 site.

[0089] The results showed that tegaserod was effective in DU145 ( Figure 2 A) and A549 Figure 2 B) Both of these constitutively activated STAT3 cell lines were able to inhibit STAT3 activation in a dose-dependent manner 2 hours after drug treatment.

[0090] IL-6 is a key cytokine activating STAT3 signaling. HeLa cells were treated with different concentrations of tegaserod for 2 hours, followed by stimulation with 5 ng / ml IL-6 for 10 minutes. Cells were then harvested, total protein was extracted, and STAT3 phosphorylation was detected by Western spectroscopy (Y705). Figure 2 As shown in C, in HeLa cells, tegaserod also inhibited IL-6-induced STAT3 activation in a dose-dependent manner.

[0091] Example 3: Tigaserod inhibits JAK kinase family activity.

[0092] STAT3 is typically activated by phosphorylation of JAK kinase following autophosphorylation by its upstream JAK kinase. In DU145 cells, different concentrations of tegaserod were added, and after 2 hours of incubation, it was found that tegaserod at a concentration of 7.5 μM inhibited phosphorylation of JAK1 Tyr1022 / 1023 and JAK2 Tyr1007 / 1008 sites in DU145 cells. At a higher concentration (15 μM), it also inhibited phosphorylation of TYK2 Tyr1054 / 1055 sites. Figure 3 This indicates that tegaserod is a JAK kinase family inhibitor, exhibiting higher affinity for JAK1 and JAK2 than for TYK2. Tegaserod downregulates STAT3 phosphorylation in tumor cells by specifically inhibiting JAK kinase activation.

[0093] Example 4: Inhibitory effect of tegaserod on tumor cell growth.

[0094] Tegaserod has an inhibitory effect on the growth of tumor cells from various sources. Figure 4 Cell viability was measured 72 hours after tegaserod treatment to reflect the drug's inhibitory effect on cell growth. The IC50 values ​​for tegaserod's inhibitory effect on cell growth were determined. 50The values ​​were as follows: 2.5 μM for prostate cancer DU145 cells, 3.1 μM for lung cancer H460 cells, 5.7 μM for lung cancer A549 cells, and 8.7 μM for DLD1 colon cancer cells.

[0095] Example 5: Intraperitoneal injection of tegaserod inhibited the growth of xenografts in nude mice.

[0096] In a nude mouse A549 lung cancer xenograft model, intraperitoneal injection of tegaserod inhibited the growth of xenograft cells in the model animals. Figure 5 (A and 5B). Compared with the control group, 5 mg / kg tegaserod (ip) effectively inhibited the growth of A549 tumor cells, and its antitumor effect was comparable to that of the 100 mg / kg gefitinib (po) group. Throughout the experiment, there was no significant change in body weight in any of the mice. Figure 5 C) No obvious organ damage was observed after dissection at the end of the experiment, indicating that tegaserod has no obvious toxicity and good drug safety.

[0097] Example 6: Oral administration of tegaserod can effectively inhibit the growth of non-small cell lung cancer A549 xenografts in nude mice.

[0098] Given that tegaserod is administered orally in the treatment of constipation-predominant irritable bowel syndrome, we tested whether oral administration of tegaserod could effectively inhibit tumor growth in vivo. Figure 6 A and Figure 6 The tumor weight and volume results for B mice both indicate that oral administration of tegaserod can inhibit the growth of A549 nude mouse xenografts, and Figure 6 The C data showed that no weight loss occurred in mice at the oral administration dose, indicating that the dosage was safe and no obvious drug toxicity was observed.

[0099] Example 7: Oral administration of tegaserod inhibited the growth of DU145 prostate cancer xenografts in nude mice.

[0100] In addition, we selected the human prostate cancer cell line DU145 with constitutive STAT3 activation and also examined the effect of tegaserod on the growth of xenografts in nude mice. Figure 7 Results A and 7B indicate that oral administration of tegaserod effectively inhibits the growth of DU145 xenografts in nude mice, with no significant drug toxicity. Figure 7 C). Figure 7 Immunohistochemical results of phosphorylated STAT3 in D and 7E indicate that tegaserod can effectively inhibit the phosphorylation level of STAT3 in DU145 nude mouse xenografts and has the ability to target the JAK / STAT3 signaling pathway in vivo.

[0101] Example 8: Oral administration of low-dose, low-frequency tegaserod effectively inhibited the growth of DU145 xenograft tumors in nude mice.

[0102] In both A549 and DU145 nude mouse xenograft models, we observed no dose-dependent relationship between the tumor inhibition rate and the dose of tegaserod used; high-dose tegaserod did not enhance its tumor-suppressive activity. Next, we tried different dosing frequencies to examine whether further reducing the dosing frequency in the high-dose group still effectively inhibited tumor growth with oral tegaserod. Figure 8 Results A and 8B both indicate that reducing the dosing frequency to once every two days for oral administration resulted in stronger tumor-suppressive activity of tegaserod, and Figure 8 The results of mouse weight analysis showed that tegaserod did not have significant drug toxicity at this concentration and administration frequency. Figure 8 Immunohistochemical results of Ki67, a cell proliferation marker for D and 8E cells, also indicated that tumor proliferation decreased after treatment with tegaserod at an oral dose of 1.25 mg / kg twice daily. These results demonstrate that low-dose, low-frequency oral administration of tegaserod can effectively inhibit tumor growth.

[0103] Example 9: Oral administration of tegaserod activates the immune response in the peripheral and tumor microenvironment.

[0104] Given the efficacy of tegaserod under low-dose, low-frequency administration and its concentration-independent antitumor activity, we hypothesize that tegaserod may act as an immunomodulator, modulating the tumor immune microenvironment in vivo, thereby producing a dual-regulatory effect to inhibit tumor growth. To verify this hypothesis, we first examined the changes in the composition of immune cells in peripheral blood after tegaserod administration. Figure 9 Blood analyzer data from A showed that, similar to the positive control drug gefitinib, tegaserod could stimulate an increase in the total number of white blood cells, neutrophils, lymphocytes, and platelets in peripheral blood, effectively stimulating the peripheral immune response.

[0105] Furthermore, we used flow cytometry to detect the composition and proportion of immune cells within DU145 nude mouse xenografts. Figure 9 Results showed that tegaserod administration increased the proportion of intratumoral immune cells (CD45+), tumor-infiltrating inflammatory neutrophils (CD11b+Ly6G+), monocytes / macrophages (CD11b+Ly6C+), and natural killer cells (CD335+), thereby inhibiting tumor cell growth. The positive control drug gefitinib also exhibited similar immunomodulatory effects.

[0106] To further investigate the immunomodulatory role of tegaserod in the antitumor process, we used transcriptome sequencing technology to perform mRNA sequencing on DU145 nude mouse xenografts and obtained the overall mRNA expression of mouse cells in DU145 nude mouse xenografts by comparing with the mouse reference genome. Figure 9 As shown in Figure C, a large number of significantly altered genes were found in the stromal cells of mouse tumor samples from the tegaserod treatment group. KEGG signaling pathway analysis revealed that… Figure 9 D) Significant upregulation was observed in both cytokine-cytokine receptor interaction and antigen processing and presentation. This result is consistent with... Figure 9 The flow cytometry results for B were consistent, demonstrating that tegaserod exerts its anti-tumor effect by modulating the tumor immune microenvironment.

[0107] These results indicate that tegaserod can activate the composition and function of immune cells both systemically and in the tumor microenvironment, thereby exerting a dual anti-tumor effect.

[0108] Example 10: Tegaserod activates the immune system to inhibit the growth of colorectal cancer MC38 allograft tumors.

[0109] In a nude mouse xenograft model, we found that tegaserod possesses immune-activating and anti-tumor effects. Nude mice have some acquired immune cell deficiencies and an underdeveloped immune system; therefore, we further investigated the anti-tumor effects and immunomodulatory effects of tegaserod in immunocompetent mice. The results showed that tegaserod can effectively inhibit the growth of MC38 colorectal cancer xenografts. Figure 10 A and 10B), and there was no obvious drug toxicity ( Figure 10 C). Flow cytometry results (Figure 10D) showed that tegaserod promoted the infiltration of immune cells (CD45+) within the tumor. This result is consistent with the results from the DU145 nude mouse xenograft model, both indicating the immune-activating effect of tegaserod. Simultaneously, the tegaserod group showed increased infiltration of cytotoxic T cells (CD8+) and helper T cells (CD4+) within the tumor, and an increased proportion of activated T cells (CD4+CD69+ and CD8+CD69+). Figure 10 D). These results indicate that tegaserod can activate the tumor-killing activity of acquired immune cells, thereby exerting an anti-tumor effect.

[0110] It should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the relevant conditions of the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of tegaserod or its pharmaceutically acceptable salts in the preparation of antitumor drugs; wherein, The tumor is breast cancer, colon cancer, prostate cancer, cervical cancer, or liver cancer.

2. The application as described in claim 1, characterized in that, The tumor showed abnormal activation of the JAK-STAT3 signaling pathway.

3. The application as described in claim 2, characterized in that, The aberrant activation of the JAK-STAT3 signaling pathway manifests as an increase in the phosphorylation levels of JAK1, JAK2, JAK3, Tyk2, or STAT3.

4. The application as described in claim 3, characterized in that, The increase in phosphorylation level refers to an increase in phosphorylation level at JAK1 Tyr1022 / 1023 sites, JAK2 Tyr1007 / 1008 sites, TYK2 Tyr1054 / 1055 sites, or STAT3 Tyr705 sites.

5. The application as described in claim 1, characterized in that, The antitumor drug activates and / or enhances the immune response.

6. The application as described in claim 5, characterized in that, The immune response is a mammalian immune response.

7. The application as described in claim 6, characterized in that, The immune response includes peripheral immune responses and / or immune responses in the tumor microenvironment.

8. The application as described in claim 1, characterized in that, The pharmaceutically acceptable salt is tegaserodextroseate.

9. The application as described in claim 1, characterized in that, The tumor is either prostate cancer or colon cancer.

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