Use of XMD17109 as an ARIH1 agonist

Abstract

The present application discloses a use of XMD17109 as an ARIH1 agonist. In the present application, XMD17109 as an ARIH1 agonist allows it to be used to prepare drugs for treating diseases associated with low ARIH1 levels, such as non-immunogenic tumors; the present application also provides a method for treating diseases associated with ARIH1 activity, by administering XMD17109 to a subject to activate ARIH1 protein expression levels and reduce PD-L1 levels, thereby enhancing immunotherapy; the present application also provides a method for treating non-immunogenic tumors, by effectively inhibiting the growth of non-immunogenic tumors by combining XMD17109 and PD-L1 antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of XMD17109 as an ARIH1 agonist. Background Art

[0002] Malignant tumors are one of the most serious diseases that endanger human health. With the aging population and the increasing incidence and death toll of tumors induced by environmental factors, safer and more effective treatment measures are urgently needed to prolong the life of patients and improve their quality of life. In 2020, the number of patients with malignant tumors worldwide reached 19.29 million, and it is estimated that the number of patients will reach 21.62 million in 2025. In recent years, with the development of immunotherapies such as CAR-T and monoclonal antibodies, the field of tumor treatment has ushered in a new dawn. Immunotherapy has a good therapeutic effect in hematological tumors, but unfortunately, it is not effective for most solid tumors, especially for "cold" tumors that progress slowly. It is generally believed that "cold tumors" resist immunotherapy by constructing an inhibitory tumor immune microenvironment. Therefore, finding small molecule drugs that can change the immune microenvironment has become the most promising treatment for cold tumors.

[0003] More and more E3 ligases have been identified as key regulators of tumor immune responses and are involved in the process of tumor immunity. There are about 600 E3 ubiquitin ligases in the human body, which regulate the ubiquitination modification and degradation of thousands of substrate proteins by binding to specific substrate proteins. In previous studies, the inventors found that ARIH1 protein reduces the PD-L1 level of tumors by ubiquitinating PD-L1 and degrading it through the lysosomal pathway. At the same time, ARIH1 overexpression can activate the STING-CGAS natural immune pathway. Therefore, increasing the level of ARIH1 can increase innate immunity and adaptive immunity to change the immune microenvironment of the tumor, thereby treating those non-immunogenic tumors. However, there are no drugs targeting the ARIH1 target in the prior art. Therefore, there is an urgent need to develop drugs targeting the ARIH1 target in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of XMD17109.

[0005] Another object of the present invention is to provide a method for activating ARIH1 in vitro.

[0006] Another object of the present invention is to provide a method for preventing and / or treating diseases associated with ARIH1 activity.

[0007] Another object of the present invention is to provide a method for preventing and / or treating non-immunogenic tumors.

[0008] In order to solve the above technical problems, the first aspect of the present invention provides the use of XMD17109 for preparing a medicine or a pharmaceutical composition, wherein the medicine or the pharmaceutical composition is used for one or more uses selected from the following group:

[0009] (i) for increasing the expression level of ARIH1 protein;

[0010] (ii) used to reduce PD-L1 expression levels;

[0011] (iii) for preventing and / or treating diseases associated with ARIH1 activity;

[0012] (iv) for regulating STING levels;

[0013] (v) for increasing the percentage of CD8+ T cells in diseased tissues; and

[0014] (vi) for preventing and / or treating diseases associated with STING activity.

[0015] In some preferred embodiments, the XMD17109 can increase the expression level of ARIH1 protein in a dose-dependent manner.

[0016] In some preferred embodiments, the XMD17109 reduces the expression level of PD-L1 by increasing the expression level of ARIH1 protein.

[0017] In some preferred embodiments, the disease associated with ARIH1 activity is a disease associated with low ARIH1 levels.

[0018] In some preferred embodiments, the diseases associated with low ARIH1 levels include tumors, viral infections, bacterial infections, diseases caused by bacterial or viral infections, nervous system diseases and metabolic diseases.

[0019] In some preferred embodiments, the tumor is a non-immunogenic tumor (eg, mouse 4T1 breast cancer).

[0020] In some preferred embodiments, the neurological disease includes late-onset Parkinson's disease.

[0021] In some preferred embodiments, the metabolic disease includes glycogen storage disease.

[0022] In some preferred embodiments, the XMD17109 increases the percentage of CD8+T cells in diseased tissues by increasing the level of STING.

[0023] In some preferred embodiments, the XMD17109 enhances immunotherapy by increasing the percentage of CD8+T cells in diseased tissues.

[0024] In some preferred embodiments, the XMD17109 can activate the anti-tumor immune response in patients with non-immunogenic tumors, thereby preventing and / or treating non-immunogenic tumors;

[0025] Wherein, the activation of anti-tumor immune response in patients with non-immunogenic tumors is achieved by any one or more of the following mechanisms:

[0026] Increase the percentage of CD8+T cells in tumor tissue; and / or,

[0027] Increase STING levels; and / or,

[0028] Increasing the expression level of ARIH1 protein; and / or,

[0029] Inhibition reduces PD-L1 expression levels.

[0030] In some preferred embodiments, the non-immunogenic tumor is selected from at least one of glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer and stage VI breast cancer, such as stage VI breast cancer (mouse 4T1 breast cancer).

[0031] The second aspect of the present invention provides a method for activating ARIH1 in vitro, the method comprising the steps of: adding XMD17109 or a pharmaceutical composition containing the same to a target cell culture medium, thereby activating the ARIH1 level of the target cell.

[0032] In some preferred embodiments, the molar concentration of XMD17109 in the culture medium is 0.01 to 5 μM; for example, 0.05 μM, 0.2 μM, 1 μM, 2 μM or 5 μM.

[0033] In some preferred embodiments, the method is for non-therapeutic purposes.

[0034] The third aspect of the present invention provides a method for activating STING in vitro, the method comprising the steps of: adding XMD17109 or a pharmaceutical composition containing the same to target cell culture, thereby activating the STING level of the target cell.

[0035] In some preferred embodiments, the molar concentration of XMD17109 in the culture medium is 0.01 to 5 μM; for example, 0.05 μM, 0.2 μM, 1 μM, 2 μM or 5 μM.

[0036] In some preferred embodiments, the method is for non-therapeutic purposes.

[0037] A fourth aspect of the present invention provides a method for activating an anti-tumor immune response in a patient with a non-immunogenic tumor, the method comprising the steps of:

[0038] administering to the subject a therapeutically effective amount of a first drug and a second drug in combination;

[0039] The first drug includes: XMD17109 or a pharmaceutical composition containing the same;

[0040] The second drug includes: immune checkpoint inhibitors.

[0041] In some preferred embodiments, the immune checkpoint inhibitor is selected from at least one of PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

[0042] In some preferred embodiments, the activating anti-tumor immune response in the body of a patient with a non-immunogenic tumor includes increasing the percentage of CD8+T cells in the patient's tumor tissue.

[0043] In some preferred embodiments, the method activates the anti-tumor immune response in patients with non-immunogenic tumors by increasing the percentage of CD8+T cells in the patient's tumor tissue.

[0044] In some preferred embodiments, the anti-tumor immune response includes an innate immune response and an adaptive immune response.

[0045] A fifth aspect of the present invention provides a method for preventing and / or treating non-immunogenic tumors, the method comprising the steps of: administering a therapeutically effective amount of a first drug and a second drug to a subject;

[0046] The first drug includes: XMD17109 or a pharmaceutical composition containing the same;

[0047] The second drug includes: immune checkpoint inhibitors.

[0048] In some preferred embodiments, the immune checkpoint inhibitor is selected from at least one of PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

[0049] In a sixth aspect of the present invention, a method for preventing and / or treating a disease associated with STING activity is provided, the method comprising the steps of:

[0050] A therapeutically effective amount of XMD17109 or a pharmaceutical composition containing the same is co-administered to the subject.

[0051] The seventh aspect of the present invention provides a method for preventing and / or treating a disease associated with ARIH1 activity, the method comprising the steps of:

[0052] A therapeutically effective amount of XMD17109 or a pharmaceutical composition containing the same is co-administered to the subject.

[0053] In some preferred embodiments, in the method, the administration is via the gastrointestinal tract or parenteral administration.

[0054] In some preferred embodiments, the administration method is injection, such as, but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection or intracavity injection, preferably intraperitoneal injection.

[0055] In some preferred embodiments, the administration method is oral administration.

[0056] In some preferred embodiments, the dosage form is a powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder mist, eye drops, nasal drops, eye ointment, gargle, sublingual tablet, adhesive sheet, patch, lotion, liniment, ointment, plaster, paste or patch, such as an injection.

[0057] In some preferred embodiments, the XMD17109 or a pharmaceutical composition containing the same is in unit dosage form.

[0058] In some preferred embodiments, the daily dosage of the first drug is 0.1 to 50 mg / kg.

[0059] In some preferred embodiments, the daily dosage of the second drug is 0.1 to 15 mg / kg.

[0060] The frequency of administration of the compound or pharmaceutical composition of the present invention is 2 times a day, 1 time a day, 1 time every 2 days, 2 times every 3 days or 1 time every 4 days. The continuous administration is not less than 7 days, preferably not less than 9 days, preferably not less than 11 days, preferably not less than 13 days, and most preferably not less than 15 days.

[0061] Compared with the prior art, the present invention has at least the following advantages:

[0062] (1) The present invention provides a new use of XMD17109 as an ARIH1 agonist, so that it can be used to prepare drugs for treating diseases associated with low ARIH1 levels, such as non-immunogenic tumors;

[0063] (2) The present invention provides a method for treating diseases associated with STING activity, wherein XMD17109 is administered to a subject to activate the STING-CGAS pathway, thereby activating the patient's own immune response, increasing the percentage of CD8+ T cells in diseased tissues, and thereby enhancing immunotherapy;

[0064] (3) The present invention provides a method for treating diseases associated with ARIH1 activity, by administering XMD17109 to a subject to activate ARIH1 protein expression levels and reduce PD-L1 levels, thereby enhancing immunotherapy;

[0065] (4) The present invention provides a method for treating non-immunogenic tumors, which effectively inhibits the growth of non-immunogenic tumors by combining XMD17109 and PD-L1 antibodies.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0068] Figure 1 This is a statistical diagram of the effect of XMD17109 on ARIH1 activation according to an embodiment of the present invention;

[0069] Figure 2 This is a statistical diagram of the in vitro killing of 4T1 by XMD17109 in an embodiment of the present invention;

[0070] Figure 3 This is a diagram showing the effect of XMD17109 on the expression level of ARIH1 protein detected by Western Blot in an embodiment of the present invention;

[0071] Figure 4 This is a graph showing the effect of XMD17109 on tumor PD-L1 levels and STING-CGAS pathway-related protein expression levels detected by Western Blot in an embodiment of the present invention;

[0072] Figure 5 It is a tumor growth curve diagram of the application of XMD17109 of the present invention in a mouse 4T1 transplanted tumor model;

[0073] Figure 6 This is a photo of the tumor size in the mouse 4T1 transplanted tumor model in the embodiment of the present invention;

[0074] Figure 7 Schematic diagram of the effect of XMD17109 on tumor weight in a mouse 4T1 transplanted tumor model in an embodiment of the present invention

[0075] Figure 8 is a graph showing the effect of XMD17109 on ARIH1 levels in tumors in an embodiment of the present invention;

[0076] Fig. 9 This is a graph showing the effect of XMD17109 on the number of CD8+T cells in tumor tissues in an embodiment of the present invention;

[0077] Fig.10 This is a statistical diagram of the effect of XMD17109 on the number of CD8+T cells in tumor tissue in an embodiment of the present invention. DETAILED DESCRIPTION

[0078] When the patient's body's immune microenvironment has low or no expression of PD-L1, or interferon (IFN) pathway inactivation and antigen presentation defects, it is usually unresponsive or weakly responsive to tumor immunotherapy. Conventional immunotherapy usually does not work for the treatment of this type of "cold tumor". Therefore, activating the immune response pathway in the patient's body and turning the "cold tumor" into "hot" is a technical problem that urgently needs to be solved in the field. In the inventor's previous research, it has been confirmed that the E3 ubiquitin ligase ARIH1 responsible for the target degradation of PD-L1 can regulate the mechanism of tumor "cold" and "hot" switching, and the activation of ARIH1 can effectively promote PD-L1 degradation (reference ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation. Nat Commun. 2021Apr 20; 12 (1)). Based on this, the inventors conducted a large number of screenings and found that compound XMD17109 has a good activation effect on ARIH1, and can therefore be used as an effective drug for the treatment or adjuvant treatment of "cold tumors". The inventors further established a disease model and confirmed that the combination therapy of compound XMD17109 and PD-L1 antibody can significantly inhibit the growth of non-immunogenic tumor volume and enhance immunotherapy.

[0079] Compound

[0080] In the present invention, the specific structure of the compound "XMD17109" is shown in Formula I below, the CAS number is 1714146-59-4, and XMD17109 can be obtained through commercial channels.

[0081]

[0082] Pharmaceutical composition

[0083] In the present invention, the compound can be administered alone as a medicine, but is preferably administered in the form of a pharmaceutical composition. Therefore, the present invention further provides a pharmaceutical composition comprising XMD17109 as an active ingredient and a pharmaceutically acceptable carrier or excipient.

[0084] In the present invention, "active ingredient" refers to a compound that is administered to a subject alone or in combination with one or more pharmaceutically acceptable excipients to treat, prevent or alleviate one or more symptoms of a condition, disorder or disease. "Active ingredient" and "active substance" as used herein may be a pharmaceutically acceptable salt, solvate, prodrug, stereoisomer, isotopic variant or tautomer of the compound described herein.

[0085] In the present invention, "pharmaceutically acceptable carriers and excipients" refer to pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, solvents or encapsulation materials. In one embodiment, each ingredient is "pharmaceutically acceptable", which means that it is compatible with other ingredients in the pharmaceutical preparation and is suitable for contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reaction, immunogenicity or other problems or complications, with a reasonable benefit / risk ratio.

[0086] The pharmaceutical composition of the present invention can be formulated with a pharmaceutically acceptable carrier and / or medium as described above, ultimately providing several forms of unit dosage forms and multiple dose forms. Non-limiting examples of preparations include, but are not limited to, oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, topical preparations such as ointments and creams, suppositories and sterile injections, preferably oral preparations or sterile injections.

[0087] use

[0088] After extensive screening, the inventors found that compound XMD17109 has a good activation effect on ARIH1. Therefore, in an embodiment of the present invention, the use of XMD17109 is provided for preparing a drug or a pharmaceutical composition, wherein the drug or pharmaceutical composition is used for one or more uses selected from the following group:

[0089] (i) for increasing the expression level of ARIH1 protein;

[0090] (ii) for reducing PD-L1 expression level; and

[0091] (iii) for preventing and / or treating diseases associated with ARIH1 activity.

[0092] In one embodiment, the diseases associated with ARIH1 activity include tumors (preferably non-immunogenic tumors), viral infections, bacterial infections, diseases caused by bacterial or viral infections, nervous system diseases, and metabolic diseases. Most preferably, they are non-immunogenic tumors.

[0093] In addition, the inventors unexpectedly discovered that compound XMD17109 can also increase STING levels, activate STING-CGAS signaling pathway proteins in the body, and thus promote the initiation and recruitment of CD8+T cells. Therefore, in some other embodiments of the present invention, the use of XMD17109 is provided for preparing a drug or a pharmaceutical composition, wherein the drug or pharmaceutical composition is used for one or more uses selected from the following group:

[0094] (iv) for regulating the levels of stimulator of interferon genes (STING);

[0095] (v) for increasing the percentage of CD8+ T cells in diseased tissues; and

[0096] (vi) for preventing and / or treating diseases associated with STING activity.

[0097] In the present invention, the term "ARIH1 (Ariadne RBR E3 Ubiquitin Protein Ligase 1)" refers to an E3 ubiquitin ligase that targets and degrades PD-L1. Tumor cells evade the attack of the immune system through EGFR-GSK3α-ARIH1 signaling.

[0098] In the present invention, the term "cold tumor" (also known as "non-immunogenic tumor") refers to an individual tumor whose immune microenvironment is usually characterized by low or no expression of PD-L1, inactivated interferon (IFN) pathway, and antigen presentation defects, and which is usually unresponsive or weakly responsive to tumor immunotherapy. Generally, tumors can be roughly divided into cold tumors, hot tumors, and tumor types in between, based on the response of tumor patients to immunotherapy. The immune microenvironment of "hot tumors" has characteristics opposite to those of "cold tumors", and patients with "hot tumors" usually have a better response rate and therapeutic effect to tumor immunotherapy.

[0099] In the present invention, the term "dose dependence" means that the dosage of the drug is correlated with the therapeutic effect, and the therapeutic effect can be improved by adjusting the dosage (within a certain range).

[0100] In the present invention, the term "stimulator of interferon genes (STING)" is a key signal transduction molecule involved in the innate immune response, which is triggered by cytoplasmic DNA from pathogens and hosts, and plays an important role in inducing the secretion of type I interferon and proinflammatory cytokines, defending against viral and intracellular bacterial infections, and regulating the production of spontaneous anti-tumor immune responses in the body. Many diseases are associated with the activity of STING, for example, STING-mediated DNA sensing promotes anti-tumor and autoimmune responses to dying cells.

[0101] In one embodiment, the disease associated with STING activity is an inflammatory disease, such as psoriasis, Crohn's disease, and inflammatory bowel disease.

[0102] In one embodiment, the disease associated with STING activity is an autoimmune disease, for example, selected from systemic lupus erythematosus (SLE), tachycardia-Guterres syndrome, Sjögren's syndrome, and infantile-onset vascular disease associated with STING.

[0103] In one embodiment of the invention, the disease associated with STING activity is cancer. In particular, the cancer may be a cancer caused by chronic inflammatory signals. However, cancer types not associated with chronic inflammatory signals are also relevant targets for treatment. For example, the cancer may be a skin tumor, such as basal cell carcinoma (BCC) or squamous cell carcinoma (SCC).

[0104] Treatment approaches and combination therapies

[0105] In the present invention, the method for treating the above-mentioned indications with compound XMD17109 or a pharmaceutical composition containing the same comprises the steps of:

[0106] A therapeutically effective amount of compound XMD17109 or a pharmaceutical composition containing the same is administered to the subject.

[0107] In the present invention, the term "subject" is defined herein to include animals, such as mammals, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

[0108] In order to adapt to the characteristics of the subjects and the needs of treatment, the method of administering XMD17109 or the pharmaceutical composition to the subjects in the present invention is not limited. The optional administration methods include: enteral administration (oral, sublingual, rectal), parenteral injection (intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection), absorption through the lungs, or absorption through the conjunctiva, nasopharynx, mouth, rectum, urethra or bladder. In one embodiment, the subject is administered by intraperitoneal injection.

[0109] As used herein, a "therapeutically effective amount" of a compound refers to an amount of the compound sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or sufficient to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent that provides a therapeutic effect in the treatment or management of a disease or disorder when used alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

[0110] In the present invention, a therapeutically effective amount of "XMD17109" or "pharmaceutical composition" is administered to the subject, and the therapeutically effective amount here refers to 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, preferably 0.5 to 30 mg / kg, more preferably 0.5 to 20 mg / kg, more preferably 0.5 to 10 mg / kg; more preferably 0.5 to 5 mg / kg; for example: 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg or 1 mg / kg.

[0111] The compound or pharmaceutical composition of the present invention may be optionally used together with one or more other active drugs. When used for specific purposes, such as treating non-immunogenic tumors, activating tumor immune responses in patients with non-immunogenic tumors, etc., the compound or pharmaceutical composition of the present invention is used in combination with at least one anticancer agent. In a preferred embodiment of the present invention, a method for activating anti-tumor immune responses in patients with non-immunogenic tumors is provided, the method comprising the steps of:

[0112] administering to the subject a therapeutically effective amount of a first drug and a second drug in combination;

[0113] The first drug includes: XMD17109 or a pharmaceutical composition containing the same;

[0114] The second drug includes: an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is selected from at least one of PD-1 antibody, PD-L1 antibody and CTLA-4 antibody.

[0115] Immune checkpoints are regulatory molecules that play an inhibitory role in the immune system. They maintain self-tolerance, prevent autoimmune reactions, and minimize tissue damage by controlling the duration and intensity of immune responses. The main tumor-related immune checkpoint molecules are: PD1, CTLA4, Tim3 and LAG3.

[0116] In the present invention, the term "immune checkpoint inhibitor (ICIs)" refers to some monoclonal antibody drugs developed for corresponding immune checkpoints. Preferably, the immune checkpoint inhibitor is a tumor-related immune checkpoint inhibitor, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibitor, programmed cell death protein (PD-1) inhibitor and programmed cell death ligand-1 (PD-L1) inhibitor, etc., which can be used clinically for the treatment of various malignant tumors, such as lung cancer, colorectal cancer, gastric cancer, melanoma, renal cell carcinoma, Hodgkin's lymphoma, etc. In a preferred embodiment of the present invention, the immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody and / or a CTLA-4 antibody.

[0117] The inventors found that when the body has no response or only a weak response to tumor immunotherapy, the use of XMD17109 or a pharmaceutical composition containing it in combination with a PD-L1 antibody can reduce the level of PD-L1 and promote the occurrence of anti-tumor immune responses in the body, which may include natural immune responses and adaptive immune responses. In a preferred embodiment of the present invention, the combined therapy increases the percentage of CD8+T cells in the patient's tumor tissue, and activates the anti-tumor immune response in the body of patients with non-immunogenic tumors by increasing the percentage of CD8+T cells in the patient's tumor tissue, significantly slowing down tumor growth.

[0118] In a preferred embodiment of the present invention, the dosage form of the compound or pharmaceutical composition of the present invention is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder spray, eye drops, nasal drops, eye ointment, gargle, sublingual tablet, adhesive sheet, patch, lotion, liniment, ointment, plaster, paste or patch, such as injection.

[0119] The frequency of administration of the compound or pharmaceutical composition of the present invention is 2 times a day, 1 time a day, 1 time every 2 days, 2 times every 3 days or 1 time every 4 days. The continuous administration is not less than 7 days, preferably not less than 9 days, preferably not less than 11 days, preferably not less than 13 days, and most preferably not less than 15 days.

[0120] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. 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. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0121] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0122] Example 1: Effect of XMD17109 on ARIH1 activation

[0123] In this embodiment, a fluorescent reporter system is used, and the ARIH1 gene is followed by a luciferase reporter tag, so that the protein level of ARIH1 in the cell is expressed through a fluorescent signal. The specific implementation steps are as follows:

[0124] 293T (purchased from ECACC) were transfected with ARIH1 fluorescent reporter gene and plated in 384-well plates with 5000 cells per well. Then, the test drug was added at a final concentration of 5 μM. After 24 hours of drug treatment, the luminescent substrate Furimazine was added at a final concentration of 10 μM, and the luminescence value was read on cytation5.

[0125] like Figure 1 As shown, XMD17109 significantly increased the luminescence reading compared with the DMSO control group. A two-tailed T test showed that the p value was 0.000134, which was statistically significant, indicating that XMD17109 had a significant activation effect on ARIH1.

[0126] Example 2: Effect of XMD17109 on cell viability of 4T1 cells in vitro

[0127] XMD17109 was previously reported as a selective inhibitor of ERK5 and a BET family inhibitor. In order to explore its killing effect on cold tumor cells 4T1 in vitro, the cell viability of 4T1 cells after treatment with XMD17109 was determined in this example. The specific implementation steps are as follows:

[0128] Cells were plated in 384-well plates (CORNING 3764) with 5000 cells per well. XMD17109 was added at a final concentration of 5 μM for 24 hours. The cell viability was determined by luminescence cell viability assay kit.

[0129] like Figure 2 As shown in the figure, it was found that it did not cause significant changes in 4T1 cell viability at a concentration of 5 μM, suggesting that it may be safe. XMD17109's anti-tumor effect in vivo is achieved through changes in the immune microenvironment.

[0130] Example 3: XMD17109 activates ARIH1 in 4T1 cells

[0131] In order to further determine the activation effect of XMD17109 on ARIH1 at the cellular level, the present invention measured the changes in ARIH1 protein levels under different treatment concentrations of XMD17109. The specific implementation steps are as follows:

[0132] 4T1 cells were plated in 12-well plates, with 100,000 cells per well. After 24 hours of growth, XMD17109 was added at final concentrations of 0.05, 0.2, 1, and 2 μM for 24 hours, and the samples were collected with 100 μL 2XSDS loading buffer. Heated at 100°C for 10 minutes. Immunohistochemistry: 10 μL of each sample was loaded, electrophoresed at 100V for 2 hours; transferred to PVDF membrane at 300 mA for 1 hour. Blocked with 5% skim milk at room temperature for 1 hour, antibodies β-Actin and ARIH1 (Goat) were diluted to a PBST solution containing 5% BSA at a certain dilution ratio (1:2000 for β-Actin antibody and 1:1000 for ARIH1 antibody) and incubated overnight at 4 degrees, and washed 3 times with PBST (phosphate buffer + 0.1% Tween20) for 10 minutes each time. Secondary antibodies Goat anti-Mouse IgG (H+L); donkey anti-goat were diluted 1:20000 in 5% skim milk and incubated at room temperature for 1 hour, washed 3 times with PBST for 10 minutes each time. Chemiluminescent reagents were used for color development and imaging was performed using the Tanon 4600 gel imaging system.

[0133] like Figure 3 As shown, the upregulation of ARIH1 was concentration-dependent, and with the increase of XMD17109 concentration, the ARIH1 protein level was gradually increased.

[0134] The reagents used in this example and their sources are shown in Table 1 below:

[0135] Table 1

[0136] Reagents source Antibody β-Actin #M1210-2;HUABIO ARIH1(Goat) #EB05812,1:1000;Everestbiotech Secondary AntibodyGoat anti-Mouse IgG Thermo Fisher Scientific, #31430 Secondary antibodyDonkey anti-goat #A0181,Beyotime Chemiluminescent reagents #4AW001-500,4A649 Biotech,Co.

[0137] Example 4: XMD17109 downregulates PD-L1 levels in 4T1 tumor cells and activates the STING-CGAS pathway

[0138] In order to further detect the effect of XMD17109 on ARIH1 downstream signal detection, in this example, the PD-L1 level of 4T1 cells was detected. In addition, the STING-CGAS signaling pathway level was also detected in this example. The specific implementation steps are as follows:

[0139] 100,000 4T1 cells were plated in a 12-well plate, and XMD17109 was added to each well at a final concentration of 1 μM. After treatment for 0, 6, 12, and 24 hours, 100 μL of 2XSDS loading buffer was added to the wells. The samples were heated at 100°C for 10 minutes. Immunohistochemistry: 10 μL of each sample was loaded, electrophoresis was performed at 100V for 2 hours, and transfer was performed at 300 mA for 1 hour. Blocked with 5% skim milk at room temperature for 1 hour, the antibodies β-Actin, STING, p-STING (Ser366), and PD-L1 were diluted in a PBST solution containing 5% BSA at a certain dilution ratio (1:2000 for β-Actin antibody, 1:1000 for STING antibody, 1:1000 for p-STING (Ser366) antibody, and 1:2000 for PD-L1 antibody) and incubated overnight at 4 degrees, and washed 3 times with PBST (phosphate buffer + 0.1% Tween20) for 10 minutes each time. The secondary antibodies (Goat anti-Mouse IgG (H+L), Goat anti-Rabbit IgG (H+L) were incubated at room temperature for 1 hour at a dilution ratio of 1:20000, and washed 3 times with PBST for 10 minutes each time. Chemiluminescent reagents were used for color development, and imaging was performed using the Tianneng gel imaging system (Tanon 4600).

[0140] like Figure 4 As shown, XMD17109 gradually downregulated the PD-L1 level and activated the STING-CGAS signaling pathway protein as the treatment time increased.

[0141] The reagents used in this example and their sources are shown in Table 2 below:

[0142] Table 2

[0143]

[0144] Example 5. Tumor growth curve of XMD17109 combined with PD-L1 in the mouse 4T1 transplanted tumor model

[0145] In order to verify the in vivo efficacy of XMD17109 in enhancing immunotherapy, in this example, a transplanted tumor model of mouse triple-negative breast cancer cell 4T1 was conducted. The specific implementation steps are as follows:

[0146] BALB / c female mice aged 6-8 weeks were inoculated with 5x10 5The number of 4T1 cells (cells mixed in 50% PBS + 50% matrix gel) was inoculated into the subcutaneous mammary pad. After 7 days of inoculation, the tumor size was measured, and the tumor volume was calculated by multiplying the length by the square of the width and then divided into groups, with six mice in each group, so that the initial tumor size of each group was similar, and then the drug treatment was carried out. The mouse transplant tumor model was divided into four groups, namely the solvent control group, the PD-L1 antibody single drug group (#BE0101; Bioxcell, 100μg per mouse intraperitoneal injection once every 3 days), the XMD17109 single drug group (5mg / kg / day intraperitoneal injection, XMD17109, #T1842, Shanghai Taoshu), and the XMD17109 and PD-L1 combination group (the dosage was consistent with the above single drug groups).

[0147] like Figure 5 As shown, the PD-L1 antibody monotherapy group and the XMD17109 monotherapy group did not have a significant effect on tumor growth, while the combination therapy group significantly slowed tumor growth, indicating that it enhanced the effect of immunotherapy.

[0148] Example 6. Effect of XMD17109 combined with PD-L1 on tumor size and weight in the mouse 4T1 transplant tumor model

[0149] At the end of the mouse transplant tumor model (16 days after inoculation), the transplanted tumor was removed and weighed. The results are shown in Figure 6 and Figure 7 .

[0150] like Figure 6 and Figure 7 As shown, the tumors in the XMD17109 (5 mg / kg / day intraperitoneal injection) combined with PD-L1 (100 μg per mouse intraperitoneal injection once every 3 days) group were significantly smaller, and the tumor weight was significantly reduced compared with the control group and XMD17109 alone. The mean values ​​of the solvent group were 455 mg, the mean values ​​of the PD-L1 group were 444 mg, the mean values ​​of XMD17109 were 722 mg, and the combination group was 257 mg. The XMD17109 combined with PD-L1 group was approximately 56% of the solvent control group, and the tumor inhibition rate was 44%, which was statistically significant, indicating a significant therapeutic effect of the combination of XMD17109 and PD-L1.

[0151] Example 7: XMD17109 increases ARIH1 protein levels in tumor tissues

[0152] In the mouse transplant tumor model, this example further verifies the effect of XMD17109 on activating ARIH1 in tumor tissues and performs immunoblotting of tumor tissues. The specific steps are as follows;

[0153] After the mouse tumor was removed, 50 mg of tumor tissue was transferred to 500 μL RIPA lysis buffer (with protease inhibitors), and the tumor tissue was lysed on a Jingxin tissue homogenizer (60 Hz, 90 s), then centrifuged at 12000 rpm for 10 min, the supernatant was taken, and an equal volume of 2XSDS loading buffer was added to collect the sample, and heated at 100°C for 10 minutes. Immunohistochemistry: 10 μL of each sample was loaded, electrophoresed at 100V for 2h; transferred to a PVDF membrane at 300mA for 1h. Blocked with 5% skim milk at room temperature for 1 hour, antibodies (β-Actin, ARIH1 (Goat) and PD-L1) were diluted in PBST solution containing 5% BSA at a certain dilution ratio (1:2000 for beta-actin antibody, 1:1000 for ARIH1 antibody, and 1:2000 for PD-L1 antibody) at 4 degrees overnight, and washed 3 times with PBST (phosphate buffer + 0.1% Tween20) for 10 minutes each time. Secondary antibodies (Goat anti-Mouse IgG (H+L) and donkey anti-goat were diluted in 5% skim milk at a dilution ratio of 1:20000 and incubated at room temperature for 1 hour, and washed 3 times with PBST for 10 minutes each time. Chemiluminescent reagents were used for color development, and imaging was performed using the Tianneng gel imaging system (Tanon 4600).

[0154] like Figure 8 As shown, the ARIH1 protein level in the tumor tissue of mice treated with XMD17109 (lane7-12) was significantly higher than that in the tumor tissue of mice in the solvent control group (lane1-6). Correspondingly, the PD-L1 level in the mice in the XMD17109 administration group was significantly lower than that in the solvent control group. The above results indicate that after XMD17109 is administered by intraperitoneal injection system in mice, the ARIH1 level in the tumor is activated.

[0155] The reagents used in this example and their sources are shown in Table 3 below:

[0156] Table 3

[0157]

[0158]

[0159] Example 8: XMD17109 increases CD8+ positive cell infiltration into tumor tissue

[0160] To further explore the effect of XMD1709 on changing the immune microenvironment, in this example, CD8+ positive immune cell tissue staining was performed on tumor tissue samples in a mouse transplant tumor model. The specific implementation steps are as follows:

[0161] Tumor tissues were immersed in 4% paraformaldehyde solution, then embedded in paraffin and sliced ​​at 4 μm thickness, and stained with CD8α (#98941, 1:200, Cell Signaling Technology) antibody. Immunohistochemical sections were imaged using an Olympus BX61 optical microscope.

[0162] like Fig. 9 and Fig.10 As shown, the XMD17109 monotherapy group and the XMD17109 combined with a-PD-L1 antibody therapy group significantly increased the infiltration of CD8+ positive T cells. Through manual counting statistics, it can be seen that XMD17109 can significantly increase the number of CD8+ positive T cells infiltrating tumor tissues after administration, indicating that XMD17109 can enhance tumor immunotherapy.

[0163] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. Use of drug combination, It is characterized in that The drug combination comprises a first drug and a second drug, wherein the first drug is XMD17109, and the second drug is a PD-L1 antibody, and the drug combination is used to prepare a drug or a drug composition, and the drug or drug composition is used for one or more uses selected from the following group: for preventing and / or treating triple-negative breast cancer.

2. The use according to claim 1, It is characterized in that The XMD17109 can (i) increase the expression level of ARIH1 protein; (ii) reduce the expression level of PD-L1; (iii) regulate the STING level; and (v) increase the percentage of CD8+T cells in diseased tissues.

3. The use according to claim 2, It is characterized in that XMD17109 can increase the expression level of ARIH1 protein in a dose-dependent manner.

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