CN115300507B

CN115300507B - Use of I-BRD9 as an ARIH1 agonist

Info

Publication number: CN115300507B
Application number: CN202211011622.5A
Authority: CN
Inventors: 吴荣海; 刘栋
Original assignee: Hangzhou Tianjijishi Biotechnology Co ltd
Current assignee: Hangzhou Tianjijishi Biotechnology Co ltd
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2024-03-01
Anticipated expiration: 2042-08-23
Also published as: CN115300507A

Abstract

The application discloses the use of I-BRD9 as an ARIH1 agonist. In the application, the I-BRD9 is used as an ARIH1 agonist, so that the ARIH1 agonist can be used for preparing medicaments for treating diseases related to the ARIH1 activity, such as non-immune tumors, immunodeficiency diseases and the like; also provided herein are methods of treating diseases associated with ARIH1 activity by administering I-BRD9 to a subject to activate ARIH1 protein expression levels and reduce PD-L1 levels, thereby enhancing immunotherapy; in the method for activating an anti-tumor immune response in a non-immune tumor patient, the percentage of CD8+ T cells in tumor tissue is promoted by administering I-BRD9 to the subject, thereby improving the immune microenvironment of the body.

Description

Use of I-BRD9 as an ARIH1 agonist

Technical Field

The invention relates to the field of biological medicine, in particular to application of I-BRD9 as an ARIH1 agonist.

Background

ARIH1 (Ariadne RBR E3 Ubiquitin Protein Ligase 1) is ubiquitin conjugated enzyme E3 conjugated protein, and can degrade and reduce the PD-L1 level of tumor through the lysosome pathway by ubiquitination of PD-L1, and simultaneously ARIH1 over-expression can activate STING-CGAS natural immune pathway. Thus, increasing ARIH1 levels can increase the innate and adaptive immunity and alter the immune microenvironment of the body, thereby treating diseases associated with immunodeficiency in the body. However, there are no drugs against the ARIH1 target in the prior art. Thus, there is an urgent need in the art to develop drugs directed against the ARIH1 target.

Disclosure of Invention

The invention aims to provide a new application of I-BRD 9.

It is another object of the invention to provide a method of activating ARIH1 in vitro.

It is another object of the present invention to provide a method for preventing and/or treating diseases associated with the activity of ARIH 1.

It is another object of the present invention to provide a method of activating an anti-tumor immune response in a patient with non-immune-derived tumors.

To solve the above technical problem, the present invention provides, in a first aspect, the use of I-BRD9 for the preparation of a medicament or pharmaceutical composition for one or more uses selected from the group consisting of:

(i) For increasing the level of ARIH1 protein expression;

(ii) For reducing PD-L1 expression levels;

(iii) For preventing and/or treating diseases associated with ARIH1 activity;

(iv) For modulating STING levels;

(v) For increasing the percentage of cd8+ T cells in diseased tissue; and

(vi) For preventing and/or treating diseases associated with STING activity.

In some preferred embodiments, the I-BRD9 increases ARIH1 protein expression level dose-dependently.

In some preferred embodiments, the disease associated with ARIH1 activity comprises an non-immunogenic tumor (e.g., 4T1 breast cancer in mice), a viral infection, a bacterial infection, a disease caused by a viral infection or a bacterial infection, a neurological disease (late onset parkinson's disease), and a metabolic disease (glycogenoma), most preferably an non-immunogenic tumor.

In some preferred embodiments, the I-BRD9 reduces PD-L1 expression levels by increasing ARIH1 protein expression levels.

In some preferred embodiments, the I-BRD9 activates an anti-tumor immune response in vivo in a patient with non-immune-derived tumors by increasing the level of ARIH1 protein expression.

In some preferred embodiments, the I-BRD9 increases the percentage of CD8+ T cells in the diseased tissue by increasing STING levels.

In some preferred embodiments, the I-BRD9 activates an anti-tumor immune response in vivo in a patient with non-immune-derived tumor by increasing the percentage of CD8+ T cells in the diseased tissue.

In some preferred embodiments, the I-BRD9 activates an anti-tumor immune response in a non-immune tumor patient by any one or more of the following:

increasing the percentage of cd8+ T cells in tumor tissue; and/or the number of the groups of groups,

increasing STING levels; and/or the number of the groups of groups,

increasing the ARIH1 protein expression level.

In some preferred embodiments, the I-BRD9 activates an anti-tumor immune response in vivo in a patient with non-immune-derived tumors, thereby effecting prophylaxis and/or treatment of non-immune-derived tumors.

In a second aspect of the invention, there is provided a method of activating ARIH1 in vitro, the method comprising the steps of: adding I-BRD9 or a pharmaceutical composition containing it to a target cell culture medium, thereby activating ARIH1 levels of the target cells.

In some preferred embodiments, the molar concentration of I-BRD9 in the medium is from 0.01 to 5. Mu.M; for example 0.05. Mu.M, 0.2. Mu.M, 1. Mu.M, 2. Mu.M or 5. Mu.M.

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

In a third aspect of the invention, there is provided a method of activating an anti-tumour immune response in a body of a patient having a non-immune tumour, the method comprising the steps of:

administering to the subject a therapeutically effective amount of I-BRD9 or a pharmaceutical composition comprising the same.

In some preferred embodiments, the activating an anti-tumor immune response in a tumor patient without an immune source comprises elevating the percentage of cd8+ T cells in the body.

In some preferred embodiments, the method activates an anti-tumor immune response in vivo in a patient with non-immune-derived tumor by elevating the percentage of cd8+ T cells in vivo.

In some preferred embodiments, the anti-tumor immune response comprises a innate immune response and an adaptive immune response.

In a fourth aspect of the present invention, there is provided a method for preventing and/or treating a disease associated with ARIH1 activity, the method comprising the steps of:

In some preferred embodiments, the disease associated with ARIH1 activity is an immunocompromised tumor.

In some preferred embodiments, the method is administered parenterally or parenterally.

In some preferred embodiments, the mode of administration is administration by injection, such as, without limitation, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, or intracavity injection, preferably intraperitoneal injection.

In some preferred embodiments, the mode of administration is oral.

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

In some preferred embodiments, the I-BRD9, or a pharmaceutical composition containing the same, is in unit dosage form.

The frequency of administration of the compounds or pharmaceutical compositions of the invention is 2 times daily, 1 time every 2 days, 2 times every 3 days, or 1 time every 4 days. The administration is continued for not less than 7 days, preferably not less than 9 days, preferably not less than 11 days, preferably not less than 13 days, most preferably not less than 15 days.

In some preferred embodiments, the method is administered at a daily dose of 0.1 to 1000mg/kg, preferably 0.5 to 50mg/kg, more preferably 1 to 20mg/kg, more preferably 1 to 15mg/kg; more preferably 1 to 10mg/kg; for example: 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg or 6mg/kg.

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

Compared with the prior art, the invention has at least the following advantages:

(1) The invention provides a new application of I-BRD9 as a targeted ARIH1 drug, which can be used as an ARIH1 agonist to prepare drugs for treating diseases related to ARIH1 activity, such as non-immune tumors, immunodeficiency diseases and the like;

(2) The present invention provides methods of treating diseases associated with ARIH1 activity by administering I-BRD9 to a subject to activate ARIH1 protein expression levels and reduce PD-L1 levels, thereby enhancing immunotherapy;

(3) The present invention provides methods for activating an anti-tumor immune response in a subject with a non-immune-derived tumor by administering I-BRD9 to the subject to promote the percentage of CD8+ T cells in the tumor tissue, thereby improving the immune microenvironment of the body.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIG. 1 is a statistical graph of the effect of I-BRD9 on ARIH1 activation in accordance with an embodiment of the present invention;

FIG. 2 is a graph showing in vitro killing statistics of I-BRD9 on 4T1 in an embodiment of the present invention;

FIG. 3 is a graph showing the effect of Western Blot detection I-BRD9 on the expression level of tumor PD-L1 and STING-CGAS pathway-associated proteins in an embodiment of the present invention;

FIG. 4 is a graph showing the tumor growth of the present invention I-BRD9 applied to a mouse 4T1 engraftment tumor model;

FIG. 5 is a graph showing the change in weight of mice on a model of 4T1 transplantation tumor in mice for I-BRD9 in an embodiment of the present invention;

FIG. 6 is a schematic representation of tumor size of the application of I-BRD9 in a mouse 4T1 engraftment tumor model in accordance with an embodiment of the present invention;

FIG. 7 is a graph showing tumor weight statistics of the application of I-BRD9 in a mouse 4T1 transplanted tumor model in an embodiment of the present invention;

FIG. 8 is a statistical plot of the effect of I-BRD9 on CD8+ T cells of tumor tissue in an embodiment of the invention.

Detailed Description

There is no drug-related record in the prior art that acts directly on the ARIH1 target. The inventor of the present invention has conducted extensive and intensive studies and has found that a known compound I-BRD9 can be used as an ARIH1 agonist to effectively increase the expression level of ARIH1 protein and repair an immune microenvironment, so that the compound I-BRD9 can be used for treating diseases associated with ARIH1 activity, such as non-immune tumors and the like.

Compounds of formula (I)

In the invention, the specific structure of the compound "I-BRD9" is shown in the following formula I, the CAS number is 1714146-59-4, and the I-BRD9 can be obtained through a commercial channel.

Pharmaceutical composition

In the present invention, the compounds may be administered alone as a pharmaceutical product, but are preferably administered in the form of a pharmaceutical composition. Accordingly, the present invention further provides a pharmaceutical composition comprising I-BRD9 as active ingredient and a pharmaceutically acceptable carrier or excipient.

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

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 encapsulating materials. In one embodiment, each component is "pharmaceutically acceptable" in the sense that it is compatible with the other components of the pharmaceutical formulation and is suitable for contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The pharmaceutical compositions of the present invention may be formulated with pharmaceutically acceptable carriers and/or vehicles as described above, ultimately providing several forms, both unit dosage forms and multi-dose forms. Non-limiting examples of formulations include, but are not limited to, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, topical formulations such as ointments and creams, suppositories and sterile injectable solutions, preferably oral formulations or sterile injectable solutions.

Use of the same

The present inventors have found, through extensive screening, that compound I-BRD9 has a good activating effect on ARIH1, and thus in an embodiment of the present invention, there is provided the use of I-BRD9 for the preparation of a medicament or pharmaceutical composition for one or more uses selected from the group consisting of:

(i) For increasing the level of ARIH1 protein expression;

(ii) For reducing PD-L1 expression levels; and

(iii) For the prevention and/or treatment of diseases associated with ARIH1 activity.

In one embodiment, the disease associated with ARIH1 activity is an non-immunogenic tumor (e.g., 4T1 breast cancer in mice), a viral infection, a bacterial infection, a disease caused by a viral infection or a bacterial infection, a neurological disease (late onset parkinson's disease), and a metabolic disease (glycogenoma), most preferably an non-immunogenic tumor.

Furthermore, the inventors have unexpectedly found that compound I-BRD9 also increases STING levels, activates STING-CGAS signaling pathway proteins in the body, and thereby promotes the initiation and recruitment of cd8+ T cells, and thus in other embodiments of the invention, there is provided the use of I-BRD9 for the preparation of a medicament or pharmaceutical composition for one or more uses selected from the group consisting of:

(iv) For modulating interferon gene stimulatory factor (stimulator of interferon genes, STING) levels;

(v) For increasing the percentage of cd8+ T cells in diseased tissue; and

(vi) For preventing and/or treating diseases associated with STING activity.

In the present invention, the term "ARIH1 (ariadone RBR E3 Ubiquitin Protein Ligase 1)" refers to the E3 ubiquitin ligase that target-degrades PD-L1. Tumor cells will evade the mechanism of immune system attack through EGFR-GSK3 alpha-ARIH 1 signaling.

In the present invention, the term "dose-dependent" means that the dosage of the drug is correlated with the therapeutic effect, and the therapeutic effect can be improved (to a certain extent) according to the dosage adjustment.

In the present invention, the terms "interferon gene stimulatory factor" and "STING" are used interchangeably and are key signal transduction molecules involved in innate immune responses.

Tumor immunogenicity refers to the ability of a tumor antigenic substance to elicit an immune response in a host. In the present invention, the term "non-immunogenic tumor" refers to a type of tumor that has no or few immune cells in the tumor tissue of the patient's body, and the patient suffering from the tumor does not respond or responds poorly to tumor immunotherapy by having the characteristics of abnormal immune microenvironment of the body, inactivation of the Interferon (IFN) pathway, and defective antigen presentation. In a preferred embodiment of the invention, the compound I-BRD9 or a pharmaceutical composition containing the same can activate the immune response of a non-immune tumor body, so that patients have better response rate and treatment effect on tumor immunotherapy.

Therapeutic method

In the present invention, the method for treating the above indications by the compound I-BRD9 or a pharmaceutical composition containing the same comprises the steps of:

administering to the subject a therapeutically effective amount of compound I-BRD9 or a pharmaceutical composition comprising the same.

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, and the like. In a particular embodiment, the subject is a human.

The manner of administering I-BRD9 or pharmaceutical compositions to a subject of the present invention is not limited, and alternative modes of administration include: enteral administration (oral, sublingual, rectal), parenteral injection (intravenous, subcutaneous, intramuscular, intraperitoneal), pulmonary absorption, or absorption via the conjunctiva, nasopharynx, oral, rectal, urinary tract, or bladder, in one embodiment, the subject is administered by intraperitoneal injection.

As used herein, a "therapeutically effective amount" of a compound means an amount of the compound that is sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic effect in the treatment or management of a disease or disorder. 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.

In the present invention, a therapeutically effective amount of I-BRD9 or pharmaceutical composition, herein a therapeutically effective amount of 0.1 to 1000mg/kg, preferably 0.5 to 50mg/kg, more preferably 1 to 20mg/kg, more preferably 1 to 15mg/kg, is administered to a subject human; more preferably 1 to 10mg/kg; for example: 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg or 6mg/kg.

In a preferred embodiment of the invention, the compound or pharmaceutical composition of the invention is in the form of a powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder mist, eye drops, nose drops, eye ointments, gargle, sublingual tablet, adhesive patch, lotion, liniment, ointment, plaster, paste or patch, for example injection.

The compound or pharmaceutical composition of the invention is administered 2 times daily, 1 time daily, 2 times daily, 3 times daily, or 1 time daily 4 days. The administration is continued for not less than 7 days, preferably not less than 9 days, preferably not less than 11 days, preferably not less than 13 days, most preferably not less than 15 days.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.

Example 1 effect of I-BRD9 on ARIH1 activation

In this example, a fluorescent reporter system was used, followed by a luciferase reporter tag to the ARIH1 gene, which allows the intracellular protein signal of ARIH1 to be shown by a luminescent signal. The specific implementation steps are as follows:

293T transfected ARIH1 fluorescent reporter gene was plated into 384 well plates, 5000 cells per well, and then 5. Mu.M final concentration of test drug I-BRD9 was added, and after 24 hours of drug addition treatment, 10. Mu.M final luminescent bottom Furimazine was added, and the luminescence values were read on the cells 5, see FIG. 1.

As shown in FIG. 1, I-BRD9 can significantly increase the luminescence read compared to the DMSO control, and the p-value is about 0.0006 with significant statistical differences when performing a two-tailed T-test. The results show that I-BRD9 has an effect of activating ARIH 1.

Example 2 in vitro Effect of I-BRD9 on cell viability of 4T1 cells

In this example, cell viability after treatment of 4T1 cells with I-BRD9 was determined. The specific implementation steps are as follows:

cells were plated in 384-well plates (CORNING 3764), 5000 cells per well, and 5 μm final I-BRD9 was added, and incubated in a cell incubator for 24 hours using CellTiter-The luminous cell viability detection kit is used for determining the cell viability, and is shown in figure 2.

As shown in FIG. 2, the I-BRD9 does not kill cells in vitro at an action concentration of 5 mu M, which suggests that the mechanism by which I-BRD9 can act in vivo is not killed, but rather exerts an anti-tumor effect by activating the immune system, with good safety.

Example 3I-BRD 9 activates ARIH1 in 4T1 cells, down-regulates PD-L1 levels, activates STING-CGAS signaling pathway

To further determine the activation of ARIH1 by I-BRD9 at the cellular level, changes in ARIH1 protein levels at different I-BRD9 treatment concentrations were measured in this example. The specific implementation steps are as follows:

4T1 cells were plated in 12-well plates of 100000 cells per well and grown for 12 hours, and then treated with 1. Mu.M final I-BRD9 for 0, 12, 24, and 48 hours, respectively, with 100. Mu.L of 2XSDS loading buffer. Heated at 100℃for 10 minutes. Immune hybridization: 10 mu L of each sample is subjected to electrophoresis for 100V for 2 hours; transfer 300mA to PVDF membrane for 1 hour. 5% skim milk was blocked for 1 hour at room temperature, antibodies β -action, ARIH1 (coat), STING, p-STING (Ser 366), PD-L1 were incubated overnight at 4℃in PBST solution with 5% BSA at the dilution ratio described above, and PBST (phosphate buffer+0.1% Tween 20) was washed 3 times for 10 minutes each. Secondary antibodies (coat anti-Mouse IgG (h+l), coat anti-rabit IgG (h+l) and donkey anti-coat were diluted to 5% skimmed milk at a dilution ratio of 1:20000 and incubated for 1H at room temperature, pbst washed 3 times for 10 minutes each time, developed using chemiluminescent reagents and imaged using a tenability gel imaging system (Tanon 4600), the results of which are shown in fig. 3.

As shown in FIG. 3, ARIH1 was gradually upregulated, PD-L1 was gradually downregulated, and p-STING levels were gradually upregulated as I-BRD9 treatment time was prolonged. The above results demonstrate the role of I-BRD9 in activating ARIH1 and downstream signaling pathways.

The reagents and sources used in this example are shown in Table 1 below:

TABLE 1

Example 4, tumor growth curves of I-BRD9 in the mouse 4T 1-engrafted tumor model

In order to verify the in vivo efficacy of I-BRD9 for enhancing immunotherapy, in this example, a model animal experiment of transplantation tumor of mouse triple negative breast cancer cells 4T1 was performed, and the specific implementation steps are as follows:

inoculation of 6-8 week old BALB/c female mice with 5X10 ⁵ The number of 4T1 cells (cells were mixed in 50% PBS+50% matrigel) to subcutaneous breast pad, tumor size was measured with vernier calipers 5 days after inoculation, tumor volumes were calculated at the square of length multiplied by width and grouped into six mice each with similar average initial tumor size, and then administration was performed. The mice model for the transplanted tumor was divided into 3 groups, which were respectively a solvent control group, a PD-L1 antibody single-dose group (100. Mu.g each injected intraperitoneally every three days), and an I-BRD9 single-dose group (10 mg/kg/day intraperitoneally, I-BRD 9). As shown in fig. 4, the I-BRD9 single administration group significantly slowed down the tumor growth compared to the solvent control group, suggesting a cold tumor treatment effect.

The reagents and sources used in this example are shown in Table 2 below:

TABLE 2

Reagent(s)	Source
		I-BRD9	# T6859, shanghai Tao Shu
PD-L1 antibodies	#BE0101；Bioxcell

Example 5 influence of I-BRD9 on body weight of mice model for 4T 1-grafted tumor

In this example, in the mouse transplantation tumor model experiment, the body weight of the mouse was measured once every two days, and the result is shown in fig. 5.

As shown in FIG. 5, the weight change of the I-BRD9 treatment group and the control group is not obviously different, which proves that the I-BRD9 has better safety and does not cause the weight of the 4T1 transplanted tumor mice to be obviously reduced.

Example 6, size of tumor and tumor weight of I-BRD9 in mouse 4T 1-transplanted tumor model

In this example, the tumors were peeled off at the end point of the mouse tumor model (21 days after inoculation), and the tumors were weighed and the structure was as shown in FIG. 6.

As shown in FIG. 6, the tumors of the I-BRD 9-dosed group are significantly smaller. Compared with the control group, the tumor weight is obviously reduced, the average value of the solvent group is 1477mg, the average value of the PD-L1 group is 886mg, and the average value of the I-BRD9 group is 749mg. The I-BRD9 group is about 50.7% of the solvent control group, the tumor inhibition rate is 49.3%, and the I-BRD9 can obviously inhibit the tumor growth and has obvious treatment effect.

Example 7, I-BRD9 increases tumor infiltration activating CD8+ cell fraction

Tumors were harvested and processed into single cell suspensions by collagenase type I and Dnase I digestion at 37 ℃ for 45 minutes. After filtration through a 45 μm filter (BD Bioscience), the isolated cells were stained with the specific surface marker antibody anti-CD 45-Percp-Cy5.5, anti-CD 3-PE-Cy7 and anti-CD 8-FITC were placed in PBS at 4℃for 30 min. Intracellular staining of GzmB was as follows: cells were washed, then fixed and permeabilized with a fixation/permeabilization kit, and finally stained with anti-APC GzmB. To properly compensate for the flow cytometry channel, the compensation was adjusted using a single staining sample. Stained cells were analyzed on a flow cytometer (Beckman Coulter Cytoflex) and data was analyzed using cytpert2.3 software. As shown in FIG. 8, the proportion of CD8+ T cells in the I-BRD9 treated group is significantly increased, indicating that I-BRD9 activates an anti-tumor immune response.

The reagents and sources used in this example are shown in Table 3 below:

TABLE 3 Table 3

Reagent(s)	Source
		Collagenase type I	#2350118，Gibco
Dnase I	#143582，Roche
		anti-CD 45-Percp-Cy5.5	#103132；Biolegend
anti-CD 3-PE-Cy7	#100320；Biolegend
		anti-CD 8-FITC	#100706；Biolegend
Immobilization/permeation kit	#421403；Biolegend
		anti-APC GzmB	#372204；Biolegend

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

Use of i-BRD9 for the preparation of a medicament or pharmaceutical composition for the prevention and/or treatment of triple negative breast cancer.
2. The use according to claim 1, wherein the I-BRD9 prevents and/or treats triple negative breast cancer by one or more means selected from the group consisting of:

(i) Increasing the level of ARIH1 protein expression;

(ii) Reducing PD-L1 expression levels;

(iii) Modulating STING levels; and

(iv) Increasing the percentage of cd8+ T cells in diseased tissue.
3. The use according to claim 2, wherein the I-BRD9 increases the level of ARIH1 protein expression dose-dependently.
4. The use according to claim 3, wherein the I-BRD9 activates an anti-tumor immune response in vivo in a patient with triple negative breast cancer by any one or more of the following:

increasing the percentage of cd8+ T cells in tumor tissue; and/or the number of the groups of groups,

increasing STING levels; and/or the number of the groups of groups,

increasing the ARIH1 protein expression level.
Use of 5.I-BRD9 for the preparation of an agent that activates ARIH1 for non-therapeutic purposes in vitro.
6. The use according to claim 5, wherein the in vitro non-therapeutic purpose activating ARIH1 comprises the steps of: adding I-BRD9 or a pharmaceutical composition containing it to a target cell culture medium, thereby activating ARIH1 levels of the target cells.
7. Use according to claim 6, characterized in that the molar concentration of I-BRD9 in the medium is 0.01 to 5 μm.