Pharmaceutical composition for treating tumors and application thereof

Abstract

The present disclosure relates to the combination treatment of tumors with IN10018 and an epidermal growth factor receptor tyrosine kinase inhibitor.

Description

Drug combination for treating tumors and its use Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and specifically relates to the treatment of tumors using a focal adhesion kinase (FAK) inhibitor IN10018 in combination with other anti-tumor drugs. Background Art

[0002] EGFR, also known as Epidermal Growth Factor Receptor (EGFR) in Chinese. The EGFR gene is responsible for encoding and producing a receptor protein called the epidermal growth factor receptor. The EGFR receptor protein is a transmembrane protein consisting of three parts: one end of the protein is located outside the cell, one part is located in the cell membrane, and the other end is located inside the cell. This allows the EGFR receptor to bind to other proteins outside the cell (called ligands), helping the cell receive signals and respond to their stimulation. The binding of the receptor and ligand is like a key to a lock, so they both have specific binding "partners." When EGFR binds to a ligand, it attaches to another nearby EGFR receptor and forms a complex (dimer), thereby entering an activated state and activating the signaling pathway within the cell. EGFR mutations primarily occur in exons 18 to 21, with deletion mutations in exon 19 and the L858R point mutation in exon 21 being the most common mutation types, accounting for 90% of all mutation types. When a pathogenic gene mutation occurs in EGFR, the EGFR receptor protein will be in a continuously activated state, which causes cells to continuously receive proliferation and survival signals, resulting in excessive cell growth and survival (unable to undergo normal apoptosis), leading to tumor formation.

[0003] Currently available targeted drugs for tumor EGFR mutations include: first-generation targeted drugs icotinib, gefitinib, and erlotinib for exon 19 and 21 mutations; second-generation targeted drugs afatinib for exon 8 and 20 mutations; and third-generation targeted drugs osimertinib (also referred to herein as AZD9291), almonertinib, and alflutinib for T790M mutations. Targeted drugs for ALK mutations include: first-generation targeted drug crizotinib; second-generation targeted drugs ceritinib, alectinib, brigatinib; and third-generation targeted drug lorlatinib. However, resistance to these targeted drugs often develops approximately one year after treatment. Overcoming the resistance to targeted drugs or delaying the onset of resistance is the main goal of anti-tumor drug research and development.

[0004] Summary of the Invention

[0005] In one aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating a tumor in a subject. The structure of IN10018 is as follows:

[0006] Yet another aspect of the present disclosure provides a pharmaceutical combination product of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for use in treating a tumor in a subject.

[0007] In another aspect, the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor to a subject in need thereof.

[0008] In another aspect, the present disclosure provides a kit or a pharmaceutically acceptable composition comprising: (a) IN0018 or a pharmaceutically acceptable salt thereof; and (b) an epidermal growth factor receptor tyrosine kinase inhibitor.

[0009] In another aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a combination drug for treating tumors.

[0010] In another aspect, the present disclosure provides use of IN0018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for use in combination with an epidermal growth factor receptor tyrosine kinase inhibitor in treating tumors.

[0011] In another aspect, the present disclosure provides use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for use in combination with IN0018 or a pharmaceutically acceptable salt thereof in treating tumors.

[0012] In another aspect, the present disclosure provides a kit comprising: IN0018 or a pharmaceutically acceptable salt thereof; and instructions, wherein the instructions indicate that IN0018 or a pharmaceutically acceptable salt thereof can be used for treating tumors in combination with an epidermal growth factor receptor tyrosine kinase inhibitor.

[0013] In another aspect, the present disclosure provides a kit comprising: an epidermal growth factor receptor tyrosine kinase inhibitor; and instructions, wherein the instructions indicate that the epidermal growth factor receptor tyrosine kinase inhibitor can be used in combination with IN0018 or a pharmaceutically acceptable salt thereof to treat tumors.

[0014] Another aspect of the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor to a subject in need thereof.

[0015] Yet another aspect of the present disclosure provides a pharmaceutical combination product of IN0018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for use in treating a tumor in a subject in need thereof.

[0016] Optionally, the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0017] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0018] The CAS number of Gefitinib is 184475-35-2; the CAS number of Erlotinib is 183321-74-6; the CAS number of Icotinib is 610798-31-7; the CAS number of Afatinib is 850140-72-6; the CAS number of Crizotinib is 877399-52-5; the CAS number of Osimertinib (AZD9291) is 1421373-65-0; the CAS number of Almonertinib is 1899921-05-1; and the CAS number of Alflutinib is 1899921-05-1. (Afatinib, also known as Furmonertinib), CAS number 1869057-83-9; EAI045, CAS number 1942114-09-1; JBJ-04-125-02, CAS number 2060610-53-7; BLU945, CAS number 2660250-10-0; BLU701 was jointly developed by Blueprint Medicines Corp and Zai Lab; TQB3804, CAS number 2267329-76-8; BBT-176 was developed by Biliji Biotech Co., Ltd.; ES-072 was developed by Bosheng Pharmaceutical; BPI-361175 was developed by Beida Pharmaceutical; CH7233163 was developed by Chugai Pharmaceutical Co., Ltd.

[0019] Optionally, the IN0018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0020] Optionally, the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, spinal cord cancer, Metastatic tumors caused by malignant cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colorectal cancer; preferably, the tumor is lung cancer or colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, and are not intended to limit the present invention.

[0022] FIG1 shows a white light microscope photograph of lung cancer KPL cells taken after incubation with drugs for 48 hours in Example 1.

[0023] FIG2 shows the percentage of annexin V-positive KPL cells after the lung cancer KPL cells of Example 1 were incubated with drugs for 48 hours.

[0024] FIG3 shows a white light microscope photograph of the lung cancer KPL cells taken after incubation with the drug for 48 hours in Example 2.

[0025] FIG4 shows the percentage of annexin V-positive KPL cells after the lung cancer KPL cells of Example 2 were incubated with drugs for 48 hours.

[0026] FIG5 shows white light microscope pictures of lung cancer KPL cells taken after incubation with drugs for 48 hours in Example 3.

[0027] FIG6 shows the percentage of annexin V-positive KPL cells after the lung cancer KPL cells of Example 3 were incubated with drugs for 48 hours.

[0028] FIG. 7 shows the changes in tumor growth in the BALB / c mouse homograft tumor model of mouse colon cancer CT-26 in Example 4 after administration of different test substances.

[0029] FIG8 shows the changes in body weight of CT-26 tumor-bearing mice after administration of different test substances in Example 4.

[0030] FIG9 shows the tumor growth curves of mice after administration of different test substances in Example 5. The data points represent the average tumor volume within the group, and the error bars represent the standard error (SEM).

[0031] FIG10 shows the tumor data of each treatment group at the end point of the experiment in Example 5. The data points represent the tumor volume of each animal in the group, and the error bars represent the standard error (SEM).

[0032] FIG11 shows the changes in body weight of mice after administration of different test substances in Example 5. The data points represent the mean body weight within the group, and the error bars represent the standard error (SEM).

[0033] FIG12 shows the synergistic killing effect of AZD9291 at different doses in Example 6, combined with 3 μM IN10018 and 5 μM IN10018 for 48 hours on non-small cell lung cancer cells HCC827.

[0034] FIG13 shows the detection of cell apoptosis in HCC827 cells treated with 0.3 nM AZD9291 and 3 μM IN10018 for 48 hours in Example 6. DETAILED DESCRIPTION

[0035] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The present invention may be implemented in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0037] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0040] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0041] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0042] definition

[0043] The following terms and symbols used in this application have the meanings described below, unless the context indicates otherwise.

[0044] As used herein, the term "epidermal growth factor receptor tyrosine kinase inhibitor" refers to an agent that selectively and effectively inhibits epidermal growth factor receptor tyrosine kinase. Examples of epidermal growth factor receptor tyrosine kinase inhibitors include, but are not limited to, Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or pharmaceutically acceptable salts thereof. In some embodiments, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0045] The CAS number of Gefitinib is 184475-35-2; the CAS number of Erlotinib is 183321-74-6; the CAS number of Icotinib is 610798-31-7; the CAS number of Afatinib is 850140-72-6; the CAS number of Crizotinib is 877399-52-5; the CAS number of Osimertinib (AZD9291) is 1421373- 65-0; Almonertinib (Ametinib), CAS No. 1899921-05-1; Alflutinib (Aifetinib, also known as Furmonertinib), CAS No. 1869057-83-9; EAI045, CAS No. 1942114-09-1; JBJ-04-125-02, CAS No. 2060610-53-7; BLU945, CAS No. 2660250-10-0; BLU701 is a product of Blueprint Pharmaceuticals TQB3804, CAS number 2267329-76-8; BBT-176 was developed by Biliji Biotech Co., Ltd.; ES-072 was developed by Bosheng Pharmaceutical; BPI-361175 was developed by Betta Pharmaceuticals; and CH7233163 was developed by Chugai Pharmaceutical Co., Ltd.

[0046] As used herein, "drug combination" or "drug combination product" may refer to a fixed combination in the form of one dosage unit (for example, all active pharmaceutical ingredients are present in one dosage form) or a kit of parts for combined administration, or it may refer to a combination of one drug and instructions indicating that the drug can be used in combination with one or more other drugs.

[0047] As used herein, "combination therapy" or "combination drug" refers to the use of a drug in combination with one or more other drugs to treat a disease, including both the combination of a drug with one or more other drugs and the combination of a drug with instructions indicating that the drug can be used in combination with one or more other drugs.

[0048] "Simultaneous or sequential administration" in this application refers to the simultaneous or sequential administration of two or more drugs within a dosing cycle (e.g., within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours) or at certain time intervals. The modes of drug administration (e.g., oral, intravenous, intramuscular, or subcutaneous administration, etc.) may be the same or different, and the dosing frequency / cycle of the two or more drugs may be the same or different. When the treatment method, product, or use of the present disclosure involves two drugs, the two drugs may be administered simultaneously or separately at certain time intervals.

[0049] As used herein, the term "treat" refers to administering one or more pharmaceutical substances to a subject suffering from a disease or symptoms of a disease in order to cure, alleviate, relieve, alter, cure, ameliorate, improve, or affect the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.

[0050] The term "tumor" as used herein refers to an abnormal lesion formed when the cells of local tissues lose normal regulation of their growth at the genetic level under the influence of various tumorigenic factors, thereby causing abnormal proliferation of their clonal types. Examples include, but are not limited to: bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer , metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0051] As used herein, the term "subject" or "subject" refers to both mammals and non-mammals. Mammals refer to any member of the class mammalia, including but not limited to humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds. The term "subject" does not limit the subject to a particular age or sex. In some embodiments, the subject is a human.

[0052] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.

[0053] As used herein, the term "pharmaceutically acceptable salt" refers to non-toxic, biologically tolerable acid addition salts suitable for administration to a subject, including, but not limited to, acid addition salts formed with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, and the like; and acid addition salts formed with organic acids, such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts with the formula HOOC-(CH2) n-COOH (wherein n is 0-4) and the like.

[0054] In addition, pharmaceutically acceptable acid addition salts can be prepared by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can readily identify various synthetic methods for preparing non-toxic pharmaceutically acceptable acid addition salts without undue experimentation. In some embodiments, the pharmaceutically acceptable salt of IN10018 is a tartrate salt.

[0055] The term "pharmaceutically acceptable composition" as used herein means that it must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation, and / or compatible with the subject being treated therewith. The term "therapeutically effective amount" as used herein means an amount that is generally sufficient to produce a beneficial therapeutic effect on the subject. The therapeutically effective amount of the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., route of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the subject, health status of the subject, degree of response of the subject to the drug, etc.).

[0056] As used herein, the term "inhibit" refers to a decrease in the baseline activity of a biological activity or process.

[0057] As used herein, the term "kit" refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus species, etc. The kit of the present invention may include (i) one or both of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) an epidermal growth factor receptor tyrosine kinase inhibitor; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat a tumor in a subject.

[0058] The compound of the test kit can be contained in separate containers. Alternatively, two or more compounds are contained in the same container. For example, the test kit can include a first container, a second container, and a package insert, wherein the first container includes at least one dose of a drug including IN10018 or a pharmaceutically acceptable salt thereof, the second container includes at least one dose of an epidermal growth factor receptor tyrosine kinase inhibitor, and the package insert includes instructions for using the drug to treat the tumor of the subject. The first container and the second container can include the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The test kit can also include other materials that can help administer the drug, such as diluents, filters, IV bags and lines, needles, and syringes.

[0059] The precise amount of IN10018, or a pharmaceutically acceptable salt thereof, and the epidermal growth factor receptor tyrosine kinase inhibitor administered to a subject will depend on various factors, such as the specific drug or compound, the pharmaceutical formulation, the route of administration, the type of disease, the condition, the identity of the subject or host being treated, etc., but can nevertheless be routinely determined by one skilled in the art. For example, determining an effective amount will also depend on the extent, severity, and type of cell proliferation. A skilled artisan will be able to determine an appropriate dosage based on these and other factors.

[0060] IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor can be administered by a suitable route such as oral, intravenous, intramuscular or subcutaneous administration.

[0061] For example, when administered orally, the drug can be administered orally with a pharmaceutically acceptable carrier, such as an inert diluent or an assimilable edible carrier. They can be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or mixed directly with the patient's food. For example, the drug can be combined with one or more excipients and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, or wafer. Tablets, lozenges, pills, capsules, etc. may further include: a binder, such as gum tragacanth, gum arabic, corn starch, or gelatin; an excipient, such as dicalcium phosphate; a disintegrant, such as corn starch, potato starch, alginic acid, etc.; a lubricant, such as magnesium stearate; or a sweetener, such as sucrose, fructose, lactose, or aspartame; or a flavoring agent.

[0062] For example, when administered intravenously or intraperitoneally by infusion or injection, solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.

[0063] Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage.

[0064] Sterile injectable solutions can be prepared by incorporating the required amount of the drug into an appropriate solvent with the various other ingredients listed above as required, followed by filtered sterilization. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods may be vacuum drying and freeze drying techniques, which can produce a powder of the active ingredient plus any other desired ingredients that have been previously sterile filtered.

[0065] The amount of IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor required for treatment may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. However, in general, the dosage may be in the range of about 0.1 to about 50 mg / kg body weight per day.

[0066] In some embodiments, IN10018 or a pharmaceutically acceptable salt thereof is administered at a dose of 5 mg / day to 100 mg / day, eg, 20 mg / day, in adults, calculated as the free base.

[0067] The epidermal growth factor receptor tyrosine kinase inhibitor is administered in a dosage range of 2-500 mg per day in adults. In a specific embodiment, osimertinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-500 mg per day in adults, for example, 80 mg, calculated as osimertinib; almonertinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-250 mg per day in adults, for example, 110 mg, calculated as almonertinib; and alflutinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-250 mg per day in adults, for example, 80 mg, calculated as alflutinib.

[0068] Technical and scientific terms used herein without specific definition have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] In some embodiments, the present disclosure further discloses the following:

[0070] 1. Use of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:

[0071] 2. A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for treating a tumor in a subject, wherein the structure of IN10018 is as follows:

[0072] 3. A method for treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor, wherein the structure of IN10018 is as follows:

[0073] 4. The use, pharmaceutical combination or method according to any one of embodiments 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.

[0074] 5. The use, pharmaceutical combination or method according to any one of embodiments 1 to 4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0075] 6. The use, pharmaceutical combination or method according to any one of embodiments 1 to 5, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0076] 7. The use, pharmaceutical combination product or method according to any one of embodiments 1 to 6, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuro Endocrine cancer, ovarian cancer, salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0077] 8. A kit or pharmaceutically acceptable composition comprising:

[0078] (a) IN10018 or a pharmaceutically acceptable salt thereof; and

[0079] (b) epidermal growth factor receptor tyrosine kinase inhibitors,

[0080] The structure of IN10018 is as follows:

[0081] 9. The kit or composition according to embodiment 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0082] 10. The kit or composition according to any one of embodiments 8-9, for use as a medicament.

[0083] 11. The kit or composition according to any one of embodiments 8-10, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.

[0084] 12. A method of treating a tumor in a subject, wherein the method comprises administering to the subject simultaneously or sequentially a compound of the kit or composition according to any one of embodiments 8-11.

[0085] 13. The method of embodiment 12, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer , salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0086] 14. A method for treating a tumor in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor; wherein the structure of IN10018 is as follows:

[0087] 15. The method according to embodiment 14, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0088] 16. The method according to embodiment 14 or 15, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0089] 17. The method of any one of embodiments 14-16, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0090] 18. A method as described in any one of embodiments 14-17, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, Ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0091] 19. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a tumor in a subject, wherein the IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject, wherein the structure of IN10018 is as follows:

[0092] 20. The use according to embodiment 19, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0093] 21. The use according to embodiment 19 or 20, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0094] 22. The use according to any one of embodiments 19-21, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0095] 23. The use according to any one of embodiments 19 to 22, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovariectomy, The invention relates to a method for treating a thyroid cancer in a manner suitable for the treatment of thyroid cancer. The method comprises the following steps: treating a thyroid cancer in a manner suitable for the treatment of thyroid cancer: first, treating a thyroid cancer in a manner suitable for the treatment of thyroid cancer; ...

[0096] 24. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the epidermal growth factor receptor tyrosine kinase inhibitor and IN10018 or a pharmaceutically acceptable salt thereof are administered to the subject, wherein the structure of IN10018 is as follows:

[0097] 25. The use according to embodiment 24, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib, EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0098] 26. The use according to embodiment 24 or 25, wherein the pharmaceutically acceptable salt of IN10018 is IN10018 tartrate.

[0099] 27. The use according to any one of embodiments 24-26, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0100] 28. The use according to any one of embodiments 24 to 27, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, Ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0101] 29. Use of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for combined treatment of tumors; the structure of IN10018 is as follows:

[0102] 30. Use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors in combination with an epidermal growth factor receptor tyrosine kinase inhibitor; the structure of IN10018 is as follows:

[0103] 31. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating tumors in combination with IN10018 or a pharmaceutically acceptable salt thereof; the structure of IN10018 is as follows:

[0104] 32. The use according to any one of embodiments 29 to 31, wherein the IN10018 or a pharmaceutically acceptable salt thereof is IN10018 tartrate,

[0105] 33. The use according to any one of embodiments 29 to 32, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib, EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0106] 34. The use according to any one of embodiments 29 to 33, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

[0107] 35. The use according to any one of embodiments 29 to 34, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, Ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

[0108] Example

[0109] The following examples are provided to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0110] The experimental methods in the following examples without specifying specific conditions can be carried out according to conventional conditions of such reactions or according to conditions recommended by the manufacturers.

[0111] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources.

[0112] The abbreviations used in the examples have the following meanings:

[0113] Example 1: Study of IN10018 and AZD9291 in lung cancer KPL cells

[0114] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When the cells were in good condition, the culture medium was plated in a 24-well plate. After 24 hours of cell spreading, four groups were set up: the first group was a negative control group, with culture medium added; the second group was treated with IN10018 at a concentration of 10 μM; the third group was treated with AZD9291 at a concentration of 10 μM; and the fourth group was a combination of IN10018 and AZD9291, with IN10018 (10 μM) and AZD9291 (10 μM), respectively. The drugs were mixed and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0115] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0116] Microscopic observation of cells revealed poor cell status in the AZD9291 monotherapy group and the IN10018 combination group, with the combination group showing the worst results, with more cell death. Cells in the negative control group and IN10018 group were in better condition (see Figures 1 and 2 for details).

[0117] Example 2: Study of IN10018 and Almonertinib in Lung Cancer KPL Cells

[0118] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell expansion, four groups were set up: the first group served as a negative control group, receiving culture medium; the second group received IN10018 at a concentration of 10 μM; the third group received almonertinib at a concentration of 4.7 μM; and the fourth group received a combination of IN10018 and almonertinib, with IN10018 (10 μM) and almonertinib (4.7 μM), respectively. The drugs were mixed and incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0119] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5ul Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0120] Microscopic observation of cells revealed poor cell status in the almonertinib monotherapy group and the combination group, with the combination group showing the worst results, with more cell death. Cells in the negative control group and IN10018 group were in better condition. See Figures 3 and 4 for details.

[0121] Example 3: Study of IN10018 and Alflutinib in Lung Cancer KPL Cells

[0122] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell expansion, four groups were set up: the first group served as a negative control group, receiving culture medium; the second group received IN10018 at a concentration of 10 μM; the third group received alflutinib at a concentration of 4.7 μM; and the fourth group received a combination of IN10018 and alflutinib, with IN10018 (10 μM) and alflutinib (4.7 μM), respectively. The drugs were mixed and incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0123] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5ul Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0124] Microscopic observation of cells revealed poor cell status in the Alflutinib monotherapy group and the combination group, with the combination group showing the worst results, with more cell death. Cells in the negative control group and IN10018 group were in better condition. See Figures 5 and 6 for details.

[0125] Example 4: In vivo antitumor efficacy study of AZD9291 in a subcutaneous BALB / c mouse colon cancer CT-26 cell transplant model

[0126] Experimental Materials:

[0127] Mice: Female BALB / c mice aged 6-8 weeks were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Upon arrival, the animals were acclimated to the experimental environment before the experiment began. The animals were housed in an SPF-grade animal room using IVC (independent ventilation system) cages (5 per cage). All cages, bedding, and drinking water were sterilized before use. All experimental personnel were required to wear protective clothing and latex gloves when working in the animal room. Cages, feed, and drinking water were changed twice a week. The housing environment and lighting conditions were as follows:

[0128] Temperature: 20-26℃

[0129] Humidity: 40-70%

[0130] Photoperiod: 12 hours of light, 12 hours of no light

[0131] Cages: Made of polycarbonate, 300 mm × 180 mm × 150 mm. Bedding is corn cobs, changed twice a week.

[0132] Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.

[0133] Drinking water: Experimental animals can drink sterile water freely.

[0134] Cage identification: The animal information card for each cage should indicate the number of animals in the cage, sex, strain, receipt date, dosing regimen, experiment number, group and start date of the experiment.

[0135] Animal identification: Experimental animals were identified with ear tags.

[0136] Compound information is shown in Table 1

[0137] Table 1. Compound information

[0138] Colon cancer cells CT-26 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60043) were maintained and passaged by Instar Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA two to three times weekly. When cells were in the exponential growth phase and at 80%-90% saturation, they were harvested, counted, and plated.

[0139] Cell seeding and grouping

[0140] 0.1 mL contains 3×10 5 A cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. 3 At 12 days after cell inoculation, the patients were randomly divided into groups according to the tumor volume and the group information is shown in Table 2.

[0141] Table 2. Dosage regimen of the test substance in the CT-26 mouse transplant tumor model Note: 1. Number of mice in each group; 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight decreases by more than 15%, dosing should be stopped; dosing should be resumed after the body weight recovers to 10% of the decrease.

[0142] Preparation of test substances

[0143] See Table 3 for details

[0144] Table 3. Test substance configuration method

[0145] Daily observation of experimental animals

[0146] Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight changes, physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0147] Experiment terminated

[0148] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0149] Tumor measurements and experimental parameters

[0150] Tumor diameter was measured with a vernier caliper three times a week. Tumor volume was calculated using the formula: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0151] The tumor growth inhibition rate (TGI) (%) was calculated based on the tumor volume on the first day after grouping according to the following formula: TGI (%) = [1 - (average tumor volume of a given dosing group - average tumor volume of that dosing group at the start of treatment) / (average tumor volume of the vehicle control group - average tumor volume of the vehicle control group at the start of treatment)] × 100%.

[0152] Statistical analysis

[0153] Statistical analysis was performed using Prism GraphPad software based on tumor volume and weight at the end of the study. Multiple group comparisons were performed using two-way ANOVA with Fisher's LSD test. P < 0.05 was considered significant.

[0154] Experimental results

[0155] The in vivo efficacy of the test substance AZD9291 combined with IN10018 in a CT-26 mouse colon cancer cell subcutaneous transplant tumor model in BALB / c mice. After cell inoculation, tumor growth was observed daily. Groups were divided according to tumor volume on the 11th day after inoculation. The average tumor volume in the group was approximately 38mm. 3 Due to tumor burden, the control group was euthanized on day 26 after inoculation, which was the 15th day after group administration, and the entire experiment was terminated.

[0156] On the 15th day after group administration, the tumor volume of the control group was 1546.6±1038.8mm 3 The tumor volumes of the IN10018 (25 mg / kg), AZD9291 (20 mg / kg), and AZD9291+IN10018 (20+25 mg / kg) treatment groups were 1766.9±732.5 mm 3 ,1046.3±407.8mm 3 and 540.4±218.0mm 3 , see Table 4 for details. The comprehensive tumor volume was compared with the control group. The tumor inhibition rates TGI of the IN10018 (25 mg / kg), AZD9291 (20 mg / kg) and AZD9291+IN10018 (20+25 mg / kg) groups were -14.5% (p=0.3079), 33.2% (p=0.0216) and 66.6% (p<0.0001), respectively. See Table 4 for details. The comprehensive tumor volume was compared with the AZD9291+IN10018 (20+25 mg / kg) combination group for statistical analysis. The P values ​​of the control group, IN10018 (25 mpk) monotherapy and AZD9291 (20 mpk) monotherapy groups were p<0.0001, p<0.0001 and p=0.0202, respectively. The tumor volume of each dose group at different time periods is shown in Figure 7.

[0157] Table 4 Evaluation of the tumor inhibition effect of the test substance on the BALB / c mouse transplant tumor model of mouse colon cancer CT-26 cells (based on the data on the 15th day after group administration) Notes: 1. Data are calculated based on the number of days after dosing and grouping. Mean ± standard error. 2. *: p < 0.05, ****: p < 0.0001, vs. control group, two-way ANOVA. 3. *: p < 0.05, ****: p < 0.0001, vs. AZD9291 + IN10018 (20 + 25 mg / kg) group, two-way ANOVA.

[0158] The experiment was conducted according to the dosing regimen. During the experiment, the animals' activities such as food intake and drinking were observed daily, and their body weights were recorded three times a week. The animal weight curves are shown in Figure 8. Throughout the dosing period, the animals in each group showed no significant weight loss and were in good condition.

[0159] in conclusion

[0160] Compared with the blank control group, both the AZD9291 (20 mg / kg) and AZD9291+IN10018 (20+25 mg / kg) groups had significant tumor growth inhibition effects, and there were statistical differences compared with the control group. Comprehensively considering the entire dosing cycle, the tumor volume of the AZD9291+IN10018 (20+25 mg / kg) group was always smaller than that of the IN10018 (25 mg / kg) group and the AZD9291 (20 mg / kg) group, and there were statistical differences compared with these two groups. It had a better effect of inhibiting tumor growth compared with the IN10018 (25 mg / kg) and AZD9291 (20 mg / kg) single-drug groups. At the same time, the weight of the animals changed well, and no abnormalities were found in their activities, drinking water, eating, and mental state during the entire dosing cycle, indicating that the animals tolerated the combination of AZD9291+IN10018 (20+25 mg / kg).

[0161] Example 5: In vivo antitumor efficacy study of AZD9291 and IN10018 in a subcutaneous xenograft model of human non-small cell lung cancer HCC827 cells in BALB / c-nude mice

[0162] Experimental Materials

[0163] Experimental methods and steps

[0164] Human non-small cell lung cancer cells HCC827 (from Shanghai Cell Bank, catalog number: TCHu153) were maintained and passaged by Nanjing Yunqiao Pure Biotechnology Co., Ltd. The cells were cultured in a monolayer in vitro using RPMI-1640 medium + 10% FBS at 37°C and 5% CO2. They were routinely digested and passaged using trypsin-EDTA two to three times a week. When the cells were in the exponential growth phase and the saturation was 80%-90%, the cells were harvested, counted, and inoculated. 0.1 mL of 5 × 10 6 A cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. 3Around 16 days after cell inoculation, the mice were randomly divided into groups according to tumor volume and given medication. The grouping information is shown in Table 5.

[0165] Table 5. Animal groups and dosing regimens for in vivo efficacy experiments Note: 1. N is the number of mice in each group. 2. The dosing volume is 10 mL / kg based on the mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to 10% of the decrease.

[0166] Daily observation of experimental animals

[0167] The use and welfare of experimental animals were carried out in accordance with AAALAC guidelines. Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight changes, physical signs, or other abnormalities. Within-group mortality and adverse reactions were recorded based on the number of animals in each group.

[0168] Experiment terminated

[0169] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0170] Tumor measurements and experimental parameters

[0171] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured with a vernier caliper three times a week. The formula for calculating tumor volume is: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0172] The tumor inhibition efficacy of the compound was evaluated using the TGI (%), which reflects the rate of tumor growth inhibition. The tumor growth inhibition rate (TGI) (%) was calculated using the following formula, referring to the tumor volume on the first day after grouping: TGI (%) = [1 - (average tumor volume of a given dosing group - average tumor volume of that dosing group at the start of treatment) / (average tumor volume of the solvent control group - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0173] Statistical analysis

[0174] Statistical analysis was performed using Prism Graphpad software based on tumor volume at the end of the study. Comparisons between multiple groups were performed using two-way ANOVA with Fisher's LSD test, with P < 0.05 considered significant. Comparisons between two groups were performed using t-test with Mann-Whitney test, with P < 0.05 considered significant.

[0175] Experimental results

[0176] After cell inoculation, tumor growth was observed every day. Groups were divided according to tumor volume on the 16th day after inoculation. The average tumor volume in the group was about 147 mm 3 Due to tumor burden, the control group was euthanized on day 48 after inoculation, i.e., day 32 after group administration; the high-dose AZD9291 (3 mg / kg) and the combination group related to the IN10018 25 mg / kg group were euthanized on day 53 after inoculation, i.e., day 37 after group administration, and the entire experiment was terminated.

[0177] On day 32 after group administration, the tumor volume of the control group was 1805.6±722.8mm 3 The tumor volumes of the AZD9291 (3 mg / kg), IN10018 (25 mg / kg) and AZD9291+IN10018 (3+25 mg / kg) treatment groups were 20.3±4.7 mm 3 ,79.2±51.8mm 3 and 9.0±7.0mm 3 Comparing the overall tumor volume with the control group, the tumor inhibition rates (TGI) for the AZD9291 (3 mg / kg), IN10018 (25 mg / kg), and AZD9291 + IN10018 (3 + 25 mg / kg) groups were 107.7% (p < 0.0001), 104.1% (p < 0.0001), and 108.4% (p < 0.0001), respectively. See Table 5-1 for details. The tumor volumes of each dose group at different time periods are shown in Figure 9.

[0178] On day 32 after group administration, the control group was euthanized due to excessive tumor volume, and the AZD9291 (3 mg / kg), IN10018 (25 mg / kg) and AZD9291 + IN10018 (3 + 25 mg / kg) groups continued to be administered and observed until day 37. Combined with the tumor volume data on day 37 after group administration, the tumor volume of the AZD9291 (3 mg / kg) and IN10018 (25 mg / kg) single-drug groups was 17.0 ± 6.7 mm, respectively. 3 and 69.6±46.6mm3 The tumor volume of the AZD9291+IN10018 (3+25 mg / kg) combination group was 5.8±6.5 mm 3 The overall tumor volume was compared with the AZD9291 + IN10018 (3 + 25 mg / kg) combination group. The P values ​​for the AZD9291 (3 mg / kg) and IN10018 (25 mg / kg) monotherapy groups were p = 0.0317 and p = 0.0079, respectively, both showing statistically significant differences. See Table 5-2 for details. The tumor volumes of each dose group at different time periods are shown in Figure 9. The tumor volumes of each dose group at the end of the experiment are shown in Figure 10.

[0179] Table 5-1 Evaluation of the tumor inhibition effect of the test substance on the BALB / c-nude mouse transplant tumor model of human non-small cell lung cancer HCC827 cells (based on the data on the 32nd day after group administration) Note: 1. Calculated based on the number of days after administration in each group. Data are mean ± standard deviation (mean ± SD); 2. ****: p < 0.0001, vs. control group, two-way ANOVA;

[0180] Table 5-2 Evaluation of the tumor inhibition effect of the test substance on the BALB / c-nude mouse transplant tumor model of human non-small cell lung cancer HCC827 cells (based on the data on the 37th day after group administration) Note: 1. Calculated based on the number of days after administration, data are mean ± standard deviation (mean ± SD); 2. *: p < 0.05, **: p < 0.01, vs. AZD9291 + IN10018 (3 + 25 mg / kg), t-test, Mann Whitney test

[0181] The experiment was conducted according to the dosing schedule. During the experiment, the animals' activities such as food intake and drinking were observed daily, and their weights were recorded three times a week. After 28 days of group administration, the average body weight of the control group changed from 20.0g on the day of group administration (Day 0) to 19.4g, with a body weight growth rate of -3.3%. The average body weight of the IN10018 (25mg / kg) treatment group changed from 21.3g on Day 0 to 20.7g on Day 28, with a body weight change rate of -2.6%.

[0182] After 37 days of group administration, the average body weight of the AZD9291 (3 mg / kg) and IN10018 (25 mg / kg) single-drug groups changed from 19.9 g and 21.3 g on Day 0 to 19.4 g and 20.3 g on Day 37, respectively, with body weight change rates of -2.2% and -4.5%, respectively; the average body weight of the AZD9291+IN10018 (3+25 mg / kg) group changed from 20.0 g on Day 0 to 19.5 g on Day 37, with a body weight change rate of -2.3%.

[0183] Throughout the dosing cycle, animals in all groups showed no significant weight loss and remained in good condition. See Table 5-3 for details. The weight changes and rates of change for the groups receiving AZD9291 (3 mg / kg) and IN10018 over different time periods are shown in Figure 11.

[0184] Table 5-3. Evaluation of the test substance on body weight changes in the BALB / c-nude mouse transplant model of human non-small cell lung cancer HCC827 cells (based on data on day 28 / 37 after group administration) Note: 1. Calculated based on the number of days after group administration: number of animals surviving on day 0 / number of animals surviving on day 15; 2. Data are mean ± standard deviation (mean ± SD); 3. Body weight change rate = [1-(Wt-W0) / W0]*100%.

[0185] Experimental Conclusion

[0186] Compared with the blank control group, the AZD9291 (3 mg / kg), IN10018 (25 mg / kg) and AZD9291+IN10018 (3+25 mg / kg) groups all showed significant tumor growth inhibition, with statistically significant differences compared to the control group. Across the entire dosing cycle, the tumor volume of the AZD9291+IN10018 (3+25 mg / kg) combination group was consistently smaller than that of the AZD9291 (3 mg / kg) and IN10018 (25 mg / kg) monotherapy groups, and there were also statistically significant differences compared to the two monotherapy groups, indicating that the AZD9291+IN10018 (3+25 mg / kg) combination group had a better effect in inhibiting tumor growth.

[0187] Example 6: IN10018 combined with AZD-9291 produces synergistic tumor killing effect

[0188] In vitro killing curves of AZD9291 in lung cancer HCC827 cells were determined, and IC50 values ​​were determined. Cytotoxic effects of different doses of AZD-9291 were examined in combination with 3μM and 5μM IN10018. Flow cytometry and Annexin V staining were also used to assess apoptosis in cells treated with either drug alone or in combination.

[0189] Experimental antibody: recombinant Alexa 647 fluorescent Anti-Calreticulin (anti-calreticulin) antibody (Abcam, ab196159), FAK antibody (CST, 3285S), Phospho-eIF2α (Ser51) (CST, 3398), eif2α (CST, 5324), DDIT3 (HUABIO, ET1703-05), HRP-labeled Alpha tubulin (α-tubulin) antibody (Proteintech, HRP-66031).

[0190] 1. IN10018 combined with AZD9291 produces synergistic tumor killing effect

[0191] 5000 HCC827 cells were plated in a 96-well plate. After 24 hours, a fixed concentration of IN10018 was added in combination with varying concentrations of AZD9291. IN10018 concentrations were 3 μM and 5 μM; AZD9291 was diluted to a maximum concentration of 1 μM using a 3-fold dilution gradient for a total of nine concentrations. After 72 hours of drug-cell incubation, 10 μL of CCK8 solution was added to each well. After incubation for 2 hours at 37°C in a 5% CO2 incubator, the absorbance was read using a microplate reader set at 450 nm. The values ​​were compared with the DMSO control group and plotted using Graphpad 8.0. See Figure 12 for details.

[0192] 2. IN10018 combined with AZD9291 promotes cell apoptosis

[0193] HCC827 cells were treated with 0.3 nM AZD9291 and 3 μM IN10018. After 48 hours, cells were harvested, stained with an Annexin V kit, and analyzed by flow cytometry. Early and late apoptosis were compared with the DMSO control group and plotted using Graphpad 8.0. See Figure 13 for details. The results showed that both early and late apoptosis were significantly enhanced in the combination group compared to the single-drug group.

[0194] All references mentioned in this application are incorporated herein by reference in their entirety, just as if each reference were listed separately. It should be understood that after reading the disclosure of this application, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the claims appended hereto.

[0195] This application claims priority to Chinese patent application No. 202210730136.2 filed on June 24, 2022, and the contents of the above-mentioned Chinese patent application disclosure are hereby incorporated by reference in their entirety as a part of this application.

Claims

1. Use of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the structure of IN10018 is as follows:

2. A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor for treating a tumor in a subject, wherein the structure of IN10018 is as follows:

3. A method for treating tumors, comprising administering to a subject in need thereof simultaneously or sequentially a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and an epidermal growth factor receptor tyrosine kinase inhibitor, wherein the structure of IN10018 is as follows:

4. The use, pharmaceutical combination or method according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt of IN10018 is a tartrate salt.

5. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

6. The use, pharmaceutical combination or method according to any one of claims 1 to 5, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the epidermal growth factor receptor tyrosine kinase inhibitor are administered to the subject simultaneously or sequentially.

7. The use, pharmaceutical combination or method according to any one of claims 1 to 6, wherein the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroblastoma, Metastases caused by endocrine cancer, ovarian cancer, salivary gland cancer, spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, head and neck cancer or colon cancer; preferably, the tumor is lung cancer or colon cancer.

8. A kit or pharmaceutically acceptable composition comprising: (a) IN10018 or a pharmaceutically acceptable salt thereof; and (b) epidermal growth factor receptor tyrosine kinase inhibitors, The structure of IN10018 is as follows:

9. The kit or composition according to claim 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Gefitinib, Erlotinib, Icotinib, Afatinib, Crizotinib, Osimertinib (AZD9291), Almonertinib, Alflutinib (also known as Furmonertinib), EA1045, JBJ-25-02, BLU945, BLU701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof, preferably Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

10. The kit or composition according to any one of claims 8 to 9 for use as a medicament.

11. The kit or composition according to any one of claims 8 to 10, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.

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