Application of thonzonium bromide in preparation of medicine for treating microenvironment immune function deficiency type diseases

Abstract

The invention provides application of thonzonium bromide in preparation of a medicine for treating microenvironment immune function deficiency type diseases. Specifically, the invention provides application of thonzonium bromide or a pharmaceutically acceptable salt thereof in preparation of a medicine, and the medicine is used for treating microenvironment immune function deficiency type diseases. It is found that thonzonium bromide can significantly inhibit the number of tumor infiltration Treg cells, promote expression of granzyme B in tumor infiltration CD8 + T cells and remodel a tumor immune microenvironment, and thonzonium bromide can be used independently or combined with other gastrointestinal tumor immunotherapy drugs for immunotherapy of gastrointestinal tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of tongzol bromide in preparing medicines for treating microenvironment immune function deficiency diseases. Background Art

[0002] Gastrointestinal tumors are common tumors of the digestive system. They are malignant tumors with a high mortality rate worldwide, especially in China. The course of the disease is insidious and early diagnosis is extremely difficult. Gastrointestinal tumors have large regional differences. More than 70% of new gastric cancer cases in the world are in developing countries. Currently, there is a lack of effective diagnostic and treatment methods for gastric cancer in the world, and the situation is extremely serious.

[0003] In recent years, immunotherapy represented by immune checkpoint inhibitors (ICI) has brought new hope for the treatment of advanced gastrointestinal tumors. However, the overall response rate of ICI in the treatment of advanced gastric cancer is low, only 10%-26%. The immunosuppressive tumor microenvironment and the exhaustion of effector cells are the main reasons that hinder the effectiveness of immunotherapy. Tumor-infiltrating regulatory T (Treg) cells are the main cell type that shapes the immunosuppressive microenvironment. Their immunosuppressive function hinders the wider efficacy of immunotherapy by inhibiting the activity of effector T cells. Therefore, for the immunotherapy of gastrointestinal tumors, in-depth research on how to reduce the immunosuppression of the tumor microenvironment and enhance the adaptive tumor immune response is the key to improving the response rate of immunotherapy. Elucidating the regulatory patterns and molecular mechanisms of tumor immunosuppressive function and effector cell exhaustion will help develop new strategies for gastrointestinal tumor immunotherapy.

[0004] Thonzonium bromide (TB), CAS No. 553-08-2, molecular formula C32H55N4O+·Br-, is a monocationic detergent, and its structural formula is as follows:

[0005]

[0006] The biological functions of tongzol bromide known so far include coating non-viral vectors as a potential, safe gene delivery method for human gene therapy; acidifying yeast cytoplasm, inhibiting ATP-dependent proton pumps on the vacuole membrane, destroying the function of V-ATPase, and causing pH-sensitive yeast cell growth defects. In pharmaceuticals, tongzol bromide is currently only added to drug solvents as a buffer component. There is no prior art that tongzol bromide is used for gastrointestinal tumor immunotherapy.

[0007] Therefore, there is an urgent need in the art to develop the use of tongzol bromide in the preparation of drugs for treating microenvironmental immune deficiency diseases. Summary of the invention

[0008] The object of the present invention is to provide the use of tongzol bromide in preparing a medicine for treating microenvironment immune function deficiency diseases.

[0009] The first aspect of the present invention provides a use of thonzol bromide or a pharmaceutically acceptable salt thereof for preparing a medicament for treating microenvironmental immune deficiency diseases.

[0010] In another preferred embodiment, the drug is administered to a subject having a phenotypic characteristic selected from the following group:

[0011] (1) The proportion of Treg cells in the blood increases;

[0012] (2) The proportion of Th17 cells in the blood decreases;

[0013] (3) the ratio of the number of Treg cells Sr to the number of Th17 cells Sh in the blood, Ri (Sr / Sh) ≥ 2, preferably ≥ 3, and more preferably ≥ 5;

[0014] (4) or a combination thereof.

[0015] In another preferred embodiment, the proportion is calculated based on the total number of T cells.

[0016] In another preferred embodiment, the drug is also administered to a subject having a phenotypic characteristic selected from the following group:

[0017] Tumor infiltrating CD8 + Granzyme B is lowly expressed in T cells.

[0018] In another preferred embodiment, the low expression of granzyme B refers to the activation of CD8 + Compared with the expression level of granzyme B in T cells F0, the tumor infiltrating CD8 + The expression level of granzyme B in T cells F1 was significantly reduced.

[0019] In another preferred embodiment, the significant reduction refers to that the ratio of Fi to F0 Rf (Fi / F0) is ≤3 / 4, preferably ≤3 / 5, and more preferably ≤1 / 2.

[0020] In another preferred embodiment, the tumor infiltrates CD8 + The positive expression rate of granzyme B in T cells is ≤40%, preferably ≤35%, and more preferably ≤30%.

[0021] In another preferred embodiment, the drug is also administered to a subject having a phenotypic characteristic selected from the following group:

[0022] (1) The proportion of tumor-infiltrating Treg cells increases;

[0023] (2) the proportion of tumor-infiltrating Th17 cells decreased;

[0024] (3) the ratio of the number of tumor-infiltrating Treg cells Sr to the number of tumor-infiltrating Th17 cells Sh Ri (Sr / Sh) ≥ 2, preferably ≥ 3, and more preferably ≥ 5;

[0025] (4) or a combination thereof.

[0026] In another preferred embodiment, the Treg cells are of low Stat3 expression type.

[0027] In another preferred embodiment, the microenvironment immune dysfunction disease includes a tumor disease.

[0028] In another preferred embodiment, the tumor includes a gastrointestinal tumor.

[0029] In another preferred embodiment, the gastrointestinal tumor is selected from the following group:

[0030] Colon cancer, pancreatic cancer, gastric cancer, liver cancer, neuroendocrine cancer, esophageal cancer, or a combination thereof.

[0031] In another preferred embodiment, the gastrointestinal tumor includes cancers in which the number of Treg cells Sr infiltrating the blood or tumor is relatively high and / or the number of Th17 cells Sh infiltrating the blood or tumor is relatively low.

[0032] In another preferred embodiment, the high number of Treg cells Sr means that compared with the number of Treg cells Sr0 in the healthy control, the number of Treg cells Sri in the cancer patient is significantly increased.

[0033] In another preferred embodiment, the significant increase refers to a ratio of Sri to Sr0, Rr (Sri / Sr0) ≥ 2, preferably ≥ 3, and more preferably ≥ 5.

[0034] In another preferred embodiment, the low Th17 cell number Sh means that compared with the Th17 cell number Sh0 in the healthy control, the Th17 cell number Shi in the cancer patient is significantly reduced.

[0035] In another preferred embodiment, the significant reduction refers to that the ratio of Shi to Sh0 Rh (Shi / Sh0) is ≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 4.

[0036] In another preferred embodiment, compared with the normal control value, the ratio of Sr to Sh Ri (Sr / Sh) in the cancer patient is significantly increased.

[0037] In another preferred embodiment, the significant increase refers to that the ratio of Ri to the ratio R0 of Sr to Sh in healthy controls RRH (Ri / R0) is ≥2, preferably ≥3, and more preferably ≥5.

[0038] A second aspect of the present invention provides a kit, comprising:

[0039] (1) a detection reagent, wherein the detection reagent is used to detect whether there is a microenvironmental immune function defect; and

[0040] (2) A pharmaceutical composition comprising thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient.

[0041] In another preferred embodiment, the kit further comprises instructions, which record using the detection reagent to detect whether the subject has a microenvironmental immune function deficiency disease;

[0042] If microenvironmental immune function deficiency is detected, a pharmaceutical composition containing thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient is administered to the subject.

[0043] In another preferred embodiment, the pharmaceutical composition further contains other gastrointestinal tumor immunotherapy drugs.

[0044] In another preferred embodiment, the other gastrointestinal tumor immunotherapy drugs are selected from: tegafur, eufotaxime, flutolan, fluorouracil, mitomycin, cisplatin, doxorubicin, etoposide, calcium leucovorin, paclitaxel, oxaliplatin, topoisomerase inhibitors, xeloda, epidermal growth factor receptor inhibitors, angiogenesis inhibitors, cell cycle inhibitors, apoptosis promoters, matrix metalloproteinase inhibitors, BCG, lentinan, interleukin, interferon and tumor necrosis factor.

[0045] In another preferred embodiment, the other gastrointestinal tumor immunotherapy drugs also include a Stat3 promoter.

[0046] In another preferred embodiment, the pharmaceutical composition is administered to a subject selected from the group consisting of a rodent or a primate.

[0047] In another preferred embodiment, the subject is a patient with microenvironment immune dysfunction.

[0048] In another preferred embodiment, the first active ingredient accounts for 1-99wt% of the total weight of the pharmaceutical composition; preferably, 10-90wt%; more preferably, 30-70wt%.

[0049] In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

[0050] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral or parenteral dosage form.

[0051] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0052] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0053] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection, inhalation, tincture, powder, granule, capsule, oral solution, tablet, pill, suspension, emulsion, lozenge, or pill.

[0054] A third aspect of the present invention provides a medicine kit, comprising:

[0055] (I) a first formulation in a first container, the first formulation comprising (a) tonazocium bromide or a pharmaceutically acceptable salt thereof as a first active ingredient, and (b) a pharmaceutically acceptable carrier;

[0056] (II) optionally a second formulation in a second container, the second formulation comprising (c) a Stat3 promoter as a second active ingredient; and

[0057] (III) instructions, wherein the instructions indicate that the drug kit is used for the treatment of microenvironmental immune deficiency diseases.

[0058] In another preferred embodiment, the Stat3 promoter includes: an expression cassette or vector for expressing Stat3, a small molecule compound that promotes Stat3 expression, or a combination thereof.

[0059] In another preferred embodiment, the first preparation and the second preparation are independent of each other.

[0060] In another preferred embodiment, the first preparation is a lyophilized preparation or a liquid preparation.

[0061] In another preferred embodiment, the medicine kit further comprises (III) a third preparation optionally located in a third container, wherein the third preparation comprises other gastrointestinal tumor immunotherapy drugs.

[0062] The fourth aspect of the present invention provides an in vitro non-therapeutic method for inducing immune cell differentiation, comprising the steps of: culturing T cells in the presence of bromocriptine or a pharmaceutically acceptable salt thereof, thereby reducing the proportion of Treg cells in the T cells and / or increasing the proportion of Th17 cells in the immune cells.

[0063] In another preferred embodiment, the T cells include CD4 + T cells or tumor infiltrating T cells.

[0064] The fifth aspect of the present invention provides an in vitro non-therapeutic method for regulating immune cell function, comprising the steps of:

[0065] In the presence of tongzol bromide or a pharmaceutically acceptable salt thereof, CD8 + T cells, CD8 + The expression percentage of granzyme B in T cells increases, thereby regulating the function of immune cells.

[0066] A sixth aspect of the present invention provides a method for immunotherapy of gastrointestinal tumors, the method comprising the steps of:

[0067] A safe and effective amount of tonazocium bromide or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, is administered to a subject in need thereof.

[0068] In another preferred embodiment, the method further comprises administering a Stat3 promoter to the subject simultaneously or sequentially.

[0069] In another preferred embodiment, the administration comprises oral administration.

[0070] In another preferred embodiment, the subject is a human or non-human mammal.

[0071] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0072] 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

[0073] Figure 1 Shown are the results of the effect of tongzol bromide on the tumor immune microenvironment in the present invention, wherein Figure A represents the tumor weight and photos of the control group (Ctrl) and the tongzol bromide (TB) treatment group; Figure B represents the 10× single-cell sequencing flow chart: flow cytometry sorting of tumor-infiltrating CD45+ immune cells in the control group and the tongzol bromide (TB) treatment group mice, and 10×Genomic single-cell sequencing; Figure C represents the UMAP graph of single-cell sequencing of the control group (Ctrl) and the tongzol bromide (TB) treatment group; Figure D represents the UMAP graph of single-cell sequencing immune cell clustering in the control group (Ctrl) and the tongzol bromide (TB) treatment group; Figure E represents the proportion of each immune cell subpopulation in the tumor infiltration of the control group (Ctrl) and the tongzol bromide (TB) treatment group, wherein NK: natural killer cell, GMP: granulocyte-monocyte group cell, CD4 + T: CD4 positive T cells, CD8 +T: CD8 positive T cell, B: B cell; Mono: monocyte, Mφ: macrophage, Neu: neutrophil, DC: dendritic cell; Figure F shows the percentage of tumor infiltrating effector CD8+T cells in the control group (Ctrl) and the tongzolamide (TB) treatment group, where GzmB: granzyme B, it: intratumoral injection.

[0074] Figure 2 Shown are the results of the effect of tongzol bromide of the present invention on tumor-infiltrating Treg / Th17 cells, wherein Figure A represents the UMAP diagram of single-cell sequencing T cell clustering and the proportion of each subgroup in the control group (Ctrl) and the tongzol bromide (TB) treatment group; Figure B represents the proportion of tumor-infiltrating Treg cells in the control group (Ctrl) and the tongzol bromide (TB) treatment group detected by flow cytometry, and the right figure is its statistical diagram; Figure C represents the proportion of tumor-infiltrating Th17 cells in the control group (Ctrl) and the tongzol bromide (TB) treatment group detected by flow cytometry, and the right figure is its statistical diagram; it: intratumoral injection.

[0075] Figure 3 Shown are the results of the effect of tongzol bromide of the present invention on peripheral immune homeostasis, wherein Figure A represents the ratio of Treg and Th17 cells in the draining lymph nodes (dLN) of mice in the control group (Ctrl) and the tongzol bromide (TB) treatment group detected by flow cytometry; Figure B represents the ratio of Treg and Th17 cells in the colon tissue (Colon) of mice in the control group (Ctrl) and the tongzol bromide (TB) treatment group detected by flow cytometry; Figure C represents the ratio of Treg and Th17 cells in the lung tissue (Lung) of mice in the control group (Ctrl) and the tongzol bromide (TB) treatment group detected by flow cytometry.

[0076] Figure 4 Shown are the results of the effect of tazolamide on Treg and Th17 cell differentiation of the present invention, wherein Figure A represents the active up-regulated transcription factors in tumor-infiltrating Treg cells in the control group (Ctrl) and the tazolamide (TB) treatment group; Figure B represents the in vitro induction efficiency of Treg cells when treated with different concentrations of tazolamide (TB); Figure C represents the in vitro induction efficiency of Th17 cells when treated with different concentrations of tazolamide (TB); Figures D and E respectively represent the in vitro differentiation efficiency of Treg and Th17 cells when treated with different concentrations of Stat3 inhibitor Stattic alone; Figures F and G respectively represent the in vitro differentiation efficiency of Treg and Th17 cells when treated with tazolamide (TB) and Stat3 inhibitor Stattic together. DETAILED DESCRIPTION

[0077] After in-depth research, the inventors unexpectedly discovered for the first time that bromoquine, added as a buffer component to the drug solvent, can significantly inhibit the number of tumor-infiltrating Treg cells and promote the number of tumor-infiltrating CD8 +The expression of T cell granzyme B reshapes the tumor immune microenvironment and can be used alone or in combination with other gastrointestinal tumor immunotherapy drugs to treat gastrointestinal tumors. On this basis, the inventors have completed the present invention.

[0078] the term

[0079] As used herein, the term “including” or “comprising” includes “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0080] As used herein, the term "microenvironmental immune deficiency disease" refers to a disease in which the ratio Ri (Sr / Sh) of the number of Treg cells Sr to the number of Th17 cells Sh in the patient's blood or tissues, especially in tumor tissues, is significantly high. The ratio Ri (Sr / Sh) is ≥2, preferably ≥3, more preferably ≥5; the ratio Ri (Sr / Sh) is ≤30, preferably ≤20, more preferably ≤10.

[0081] As used herein, the term "gastrointestinal tumors" refers to malignant tumors occurring in the digestive tract and digestive system, including: colon cancer, pancreatic cancer, gastric cancer, liver cancer, neuroendocrine cancer and esophageal cancer, etc.

[0082] As used in this article, the term "gastric cancer" can refer to various classifications of gastric cancer known in the prior art, such as gross morphological classification, which can be divided into early gastric cancer and advanced gastric cancer; according to histopathological classification, it can be divided into adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, carcinoid, etc., and the vast majority are gastric adenocarcinoma; according to the site of onset, it can be divided into gastric fundus and cardia cancer, gastric body cancer and gastric antrum cancer, etc.; according to molecular classification, it includes EBV infection type, microsatellite instability type, genome stable type and chromosomal instability type; according to the cause of the disease, it can include gastric cancer caused by Helicobacter pylori infection, etc.

[0083] As used herein, the "safe and effective amount" refers to an amount sufficient to improve or alleviate in some way the symptoms associated with the disease without causing serious side effects. Such an amount can be administered as a single dose, or can be administered according to an effective treatment regimen. The amount administered may cure the disease, but administration is usually to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement. For example, the content of bromoquine in a single dose can be 0.1-100 mg.

[0084] Gastrointestinal cancer immunotherapy

[0085] Gastrointestinal tumors are common tumors of the digestive system. They are malignant tumors with a high mortality rate in the world, especially in China. The course of the disease is hidden, and early diagnosis is extremely difficult. Gastrointestinal tumors have large regional differences. More than 70% of new gastric cancer cases in the world are in developing countries. The main clinical symptoms of gastrointestinal tumors are: abdominal pain of varying severity, intestinal bleeding, intestinal obstruction, abdominal mass and weight loss; malignant tumors have more and more severe symptoms, while benign tumors have milder symptoms or no symptoms at all; CT shows the presence of tumors. At present, there is a lack of effective diagnosis and treatment methods for gastric cancer in the world, and the situation is extremely serious.

[0086] Immunotherapy represented by immune checkpoint inhibitors (ICI) has brought new hope for the treatment of advanced gastrointestinal tumors. However, the overall response rate of ICI in the treatment of advanced gastric cancer is low, only 10%-26%. The immunosuppressive tumor microenvironment and exhaustion of effector cells are the main reasons that hinder the effectiveness of immunotherapy. Tumor-infiltrating regulatory T (Treg) cells are the main cell type that shapes the immunosuppressive microenvironment. Their immunosuppressive function hinders the wider efficacy of immunotherapy by inhibiting the activity of effector T cells. Therefore, for the immunotherapy of gastrointestinal tumors, in-depth research on how to reduce the immunosuppression of the tumor microenvironment and enhance the adaptive tumor immune response is the key to improving the response rate of immunotherapy. Elucidating the regulatory patterns and molecular mechanisms of tumor immunosuppressive function and effector cell exhaustion will help develop new strategies for gastrointestinal tumor immunotherapy.

[0087] Tronazolidinone

[0088] Thonzonium bromide (TB), CAS No. 553-08-2, molecular formula C32H55N4O+·Br-, is a monocationic detergent, and its structural formula is as follows:

[0089]

[0090] Tongzho bromide can be formulated in a pharmaceutical composition as an active ingredient in the pharmaceutical composition that plays a therapeutic role. In addition, the pharmaceutical composition may optionally contain other drugs for treating gastric cancer and optional pharmaceutically acceptable carriers. Oral chemotherapy drugs commonly used in the treatment of gastric cancer include but are not limited to tegafur, eufodine and flutolan, etc. Intravenous chemotherapy drugs commonly used in the treatment of gastric cancer include but are not limited to fluorouracil, mitomycin, cisplatin, doxorubicin, etoposide and calcium leucovorin, etc. Other drugs commonly used in the treatment of gastric cancer also include paclitaxel, oxaliplatin, topoisomerase inhibitors and Xeloda, etc. Targeted therapeutic drugs for gastric cancer may also be used, including but not limited to epidermal growth factor receptor inhibitors, angiogenesis inhibitors, cell cycle inhibitors, apoptosis promoters and matrix metalloproteinase inhibitors, etc. In other embodiments, tongzho bromide or its pharmaceutical composition may be administered together with immunotherapy for gastric cancer. Suitable immunotherapy includes non-specific biological response modifiers such as BCG, Lentinan, etc.; cytokines such as interleukin, interferon, tumor necrosis factor, etc.; and adoptive immunotherapy such as the clinical application of activated lymphocyte killer cells (LAK) and tumor infiltrating lymphocytes (TIL).

[0091] The patient may be given bromozolamide or its pharmaceutical composition by conventional methods in the art. Commonly used routes of gastric cancer treatment drugs include but not preceded by oral administration, intravenous administration, peritoneal administration, arterial cannulation area perfusion administration, etc.

[0092] In certain embodiments, the present invention also provides a pharmaceutical composition, in particular a pharmaceutical composition for treating gastric cancer, which contains tongzol bromide as an active ingredient and a pharmaceutically acceptable excipient. It should be understood that existing pharmaceutical preparations may contain tongzol bromide, but tongzol bromide in these pharmaceutical preparations is only added as an excipient such as a buffer component. In the present invention, the tongzol bromide in the pharmaceutical composition is the active ingredient of the pharmaceutical composition and serves the purpose of treatment. In certain embodiments, the pharmaceutical composition may also contain other gastric cancer therapeutic drugs, such as the gastric cancer therapeutic drugs described above. In certain embodiments, the active ingredient in the pharmaceutical composition of the present invention is only tongzol bromide. In certain embodiments, the active ingredient in the pharmaceutical composition of the present invention also includes other gastric cancer therapeutic drugs, especially Stat3 promoters.

[0093] In certain embodiments, the present invention also provides a medicine kit containing tongzol bromide as a therapeutically active ingredient. The medicine kit may also contain other pharmaceutically acceptable excipients, and optionally Stat3 promoters or other gastric cancer therapeutic drugs. For example, the medicine kit may contain independently packaged tongzol bromide, pharmaceutically acceptable excipients, and optional Stat3 promoters or other gastric cancer therapeutic drugs. When it is necessary to use the medicine kit for administration, the pharmaceutically acceptable excipients can be used to formulate tongzol bromide into corresponding preparations, such as injections or perfusion solutions, and administered simultaneously or sequentially with the optionally provided Stat3 promoters or other gastric cancer therapeutic drugs. Alternatively, the medicine kit may contain a tongzol bromide preparation that has been formulated into a pharmaceutical preparation (i.e., containing tongzol bromide and pharmaceutically acceptable excipients) and optional other gastric cancer therapeutic drugs.

[0094] In certain embodiments, the present invention also includes the use of tongzol bromide in the preparation of gastric cancer immunotherapy drugs. In certain embodiments, the present invention also includes tongzol bromide or a pharmaceutical composition thereof for treating gastric cancer. The gastric cancer can be various gastric cancers known in the art, including the gastric cancer described above.

[0095] Kit and administration method

[0096] The kit of the present invention comprises:

[0097] (1) a detection reagent, wherein the detection reagent is used to detect microenvironment immune function defects;

[0098] (2) A pharmaceutical composition comprising thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient.

[0099] A preferred kit also includes instructions, which state that the detection reagent is used to detect whether the subject has a microenvironmental immune function deficiency disease;

[0100] If microenvironmental immune function deficiency is detected, a pharmaceutical composition containing thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient is administered to the subject.

[0101] Pill Box

[0102] The kit of the present invention comprises:

[0103] (I) a first preparation in a first container, the first preparation comprising (a) tonazocium bromide or a pharmaceutically acceptable salt thereof as a first active ingredient, and (b) a pharmaceutically acceptable carrier;

[0104] (II) optionally a second formulation in a second container, the second formulation comprising (c) a Stat3 promoter as a second active ingredient; and

[0105] (III) instructions, indicating that the drug kit is used for the treatment of microenvironmental immune deficiency diseases.

[0106] A preferred first formulation and second formulation are independent of each other.

[0107] A preferred first formulation is a lyophilized formulation or a liquid formulation.

[0108] A preferred kit further comprises (III) a third preparation optionally located in a third container, wherein the third preparation comprises other gastrointestinal tumor immunotherapy drugs.

[0109] The main advantages of the present invention include:

[0110] The present invention provides a use of tonazolidinone bromide in the preparation of a drug for treating microenvironmental immune deficiency diseases. The present invention finds that tonazolidinone bromide can significantly reduce the number of tumor-infiltrating Treg cells, inhibit the expression level of Stat3 in Treg cells, increase the number of tumor-infiltrating Th17 cells, and promote the expression of tumor-infiltrating CD8 + The expression of T cell granzyme B reshapes the tumor immune microenvironment, and bromoquine can be effectively used for the immunotherapy of gastrointestinal tumors. On this basis, the present invention was completed.

[0111] The present invention will be 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 carried out according to conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0112] Basic experimental methods:

[0113] 1. Cell culture:

[0114] MC-38 and MFC cells were obtained from commercial sources and cultured in RPMI1640 (Invitrogen) and DMEM (Invitrogen) culture medium, respectively, supplemented with 10% serum, 100 μg / ml penicillin, and 100 μg / ml streptomycin. The cells were cultured at 37°C and a carbon dioxide concentration of 5%.

[0115] 2. Mouse subcutaneous tumor formation experiment:

[0116] Trypsin digestion of mouse gastric cancer MFC (3×10 6 ) cell line, wash the cells once with serum-free DMEM medium, centrifuge at 300g for 3 minutes at room temperature, discard the supernatant; resuspend the cells with pre-cooled serum-free DMEM medium and adjust the cell concentration to 3×10 8 / ml, and each mouse was subcutaneously injected with 100μl of cell suspension; the length and width of the tumor were measured every other day, and the mice were killed at the end of the experiment, and the tumor was weighed.

[0117] 3. Single-cell sequencing experiment:

[0118] The sorted CD45 + The immune cells were collected by centrifugation at 300g at room temperature for 3 minutes, resuspended and counted, and cell viability was confirmed by staining with Benchtop Blue to ensure that the cell viability was greater than 90%; 20,000 cells were immediately used to construct a single-cell cDNA library using the Chromium Next GEM Single Cell3′Reagent Kits v3.1 kit from 10×Genomics; the constructed library was sequenced on the Illumina Nova Seq 6000 platform, and the mouse sequencing data was compared with the mm10 gene library.

[0119] 4. In vivo treatment experiment of TB:

[0120] Dissolve the compound bromoprolin in PBS to a concentration of 10 mg / ml; take subcutaneous tumor-bearing mice and wait until the tumor volume grows to 50 mm 3 At the time of the experiment, TB was injected into the tumor every other day at a dose of 50 mg / kg in the experimental group, and an equal volume of PBS was injected into the control group;

[0121] Mice were housed in a culture room dedicated to infectious disease animal models and were exposed to a 12-h light and 12-h dark circadian rhythm. Animal culture and animal experiments were performed in accordance with the relevant regulations and animal welfare policies of the Animal Management Committee of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Studies, Chinese Academy of Sciences.

[0122] Preparation of required reagents:

[0123] Phosphate buffered saline (PBS): Dissolve 0.2 g KCl, 8 g NaCl, 0.24 g KH2PO4 and 1.44 g Na2HPO4 in 800 ml distilled water. Adjust the pH of the solution to 7.4 with HCl and make up to 1 L. Sterilize by high pressure steam or filter.

[0124] 5. Preparation of single cell suspension of mouse immune organs:

[0125] The mice were killed by cervical dislocation, soaked in 75% ethanol for 5 minutes, removed and placed on a sterile operating table, dissected, spleen and lymph removed, and placed in pre-cooled 1640 complete culture medium; the connective tissue and fat on the spleen and lymphatic tissue were removed, the piston of a 2ml sterile syringe was removed, and the spleen was ground 5 times in a culture dish using the flat end to gently draw circles to destroy the cortex and medulla and release immune cells; the cell suspension obtained above was filtered through a 70μm cell sieve, centrifuged at 300g for 5 minutes to collect the cells, and the supernatant was discarded. 1ml of red blood cell lysis solution (erythrocyte lysis) was added to each spleen, lysed at room temperature for 5 minutes, and centrifuged at 300g for 5 minutes to collect the cells. The supernatant was discarded, washed once with 1640 complete culture medium, and then centrifuged at 1×10 7 Resuspend at 100 μl / ml and set aside.

[0126] 6. Preparation of single cell suspension of mouse tumor tissue:

[0127] Mice with subcutaneous tumors were euthanized in a carbon dioxide killing box, soaked in 75% ethanol for 5 minutes, and then taken out and placed on a sterile operating table. The mice were dissected, the tumor tissue was taken out, the fat tissue and skin were removed, and placed in pre-cooled 1640 complete culture medium; 100 μL of pre-cooled flow staining solution was added to a sterile 1.5 ml EP tube, and the tumor tissue was placed in it, and the tumor tissue was cut into pieces with ophthalmic scissors; the tissue fragments were transferred to a 15 ml centrifuge tube, and 1 ml of digestion solution prepared with 1640 complete culture medium (containing 1% FBS, 1% penicillin-streptomycin, 1 mg / ml collagenase II and 200 μg / ml DNase I) was added per 100 mg of tumor, and digested in a 37°C water bath for 30 minutes, inverting and mixing every 10 minutes; after digestion, an equal volume of 1640 complete culture medium was added to each tube to terminate digestion. Filter the tumor tissue digestion suspension with a 70 μm cell strainer, and grind the tissue pieces left on the strainer with the back of a 2 ml syringe. Rinse the strainer with 1640 complete culture medium, centrifuge the obtained tumor suspension at 300 g for 5 min, and collect single cells. Discard the supernatant, wash once with 1640 complete culture medium, and centrifuge at 1×10 7 Resuspend at 100 μl / ml and set aside.

[0128] 7. CD4 + T cell magnetic bead sorting experiment:

[0129] The spleen of the mouse was taken and the red blood cells were lysed and resuspended in PBS containing 2% FBS to prepare a single cell suspension. The cell concentration was adjusted to 1×10 8 / ml. Used Stemcell mice CD4 +T cell sorting kit was used for sorting: 10 μL blocking antibody and 50 μL primary antibody were added to each milliliter of cell suspension, and the mixture was stained at room temperature for 7.5 minutes; then 50 μL secondary antibody was added, and the mixture was stained at room temperature for 2.5 minutes; then 75 μL vortexed magnetic beads were added and the mixture was stained at room temperature for 2.5 minutes; after staining, the cells were placed on a magnetic rack, the supernatant was poured out after 2.5 minutes, and the cells were collected by centrifugation at 300 g for 5 minutes for later use.

[0130] 8. Flow cytometry experiment:

[0131] Take 100 μL of the prepared single cell suspension (1×10 6 cells), centrifuged at room temperature at 300g for 5 minutes, and discarded the supernatant; added 50μL of CD16 / CD32 antibody (5μg / ml) prepared by flow staining solution to each tube, incubated on ice for 30min, blocked Fc receptors and reduced nonspecific binding; added 1mL of flow staining solution for washing, centrifuged at room temperature at 300g for 5 minutes, and discarded the supernatant; added 50μL of surface flow staining antibody (5μg / ml) prepared by flow staining solution to each tube, incubated on ice in the dark for 30min; added 1ml of flow staining solution for washing to each tube, centrifuged at room temperature at 300g Centrifuge for 5 minutes and discard the supernatant; add 100 μL cytoplasmic staining fixation / perforation buffer to each tube of sample and incubate on ice in the dark for 1 hour; centrifuge at 300g at room temperature for 5 minutes, discard the supernatant, add intracellular staining antibody (5 μg / ml) diluted in cytoplasmic staining buffer to each tube of sample, and incubate on ice in the dark for 1 hour; wash the cells with flow staining buffer, centrifuge at 300g at room temperature for 5 minutes, discard the supernatant; finally, resuspend the cells with 300 μL flow cytometry buffer, detect on BD Fortessa, and analyze the data using FlowJo software.

[0132] 9. In vitro induction experiment of Treg and Th17 cells:

[0133] Take the mice sorted by magnetic beads CD4 + T cells, 1×10 5 The cells were plated in a 96-well plate at 100 μg / well. The Treg culture conditions were as follows: RPMI1640 complete culture medium + 50 μM β-mercaptoethanol + 20 ng / mL mouse IL-2 + 5 ng / mL mouse TGF-β; the Th17 culture conditions were as follows: RPMI1640 complete culture medium + 50 μM β-mercaptoethanol + 30 ng / mL mouse IL-6 + 5 ng / mLmouse TGF-β + 10 ng / mL mouse TNF-α. The cells were cultured at 37°C with a carbon dioxide concentration of 5%. The differentiation efficiency was detected by flow cytometry on the fifth day.

[0134] 10. Data analysis:

[0135] SAS software package (9.1.3) was used to analyze the data, and the statistical data were mean ± standard deviation. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze continuous variables. The confidence interval was P < 0.05.

[0136] Example 1: Tongzho bromide has a remodeling effect on the tumor immune microenvironment of gastric cancer

[0137] 1. Experimental purpose: To determine the effect of tongzol bromide on the immune microenvironment of gastric cancer

[0138] 2. Experimental methods: The experimental methods for cell culture, subcutaneous tumor formation in mice, and intratumoral administration of bromocriptine were as described in Basic Experimental Methods 1, 2, and 4. The single-cell sequencing experiment was as described in Basic Experimental Methods 3.

[0139] 3. Experimental results and analysis:

[0140] First, the therapeutic effect of systemic administration of tongzol bromide on tumors was evaluated by intratumoral injection. Mouse intestinal cancer MC38 cells were inoculated in wild-type mice, and one week later, PBS was injected intratumorally as a control group, or 1mg / kg or 5mg / kg of tongzol bromide was injected as a drug treatment group. The results showed that tongzol bromide significantly inhibited tumor growth in mice in a dose-dependent manner ( Figure 1 A).

[0141] The tumor-infiltrating CD45 + Immune cells, 10×Genomics single-cell sequencing ( Figure 1 B). UMAP grouping of immune cells showed that compared with the control group, the immune cell subpopulations in the tongzol bromide group were shifted, indicating that tongzol bromide has a remodeling effect on the tumor immune microenvironment ( Figure 1 C). Furthermore, UMAP clustering showed that a total of 9 immune cell subsets were identified, including natural killer cells (NK), granulocyte-monocyte histocytes (GMP), CD4 + T cells, CD8 + T cells, B cells, monocytes (Mono), macrophages Neutrophils (Neu) and dendritic cells (DC) ( Figure 1 D).

[0142] The statistical analysis of the proportion of cells in different subpopulations is shown in Table 1 and Figure 1 As shown in E:

[0143] Table 1

[0144]

[0145] The results showed that bromocriptine promoted CD8 + Infiltration of T cells, B cells, monocytes, and macrophages in tumors inhibits CD4 + T cell tumor infiltration ( Figure 1 E).

[0146] Furthermore, flow cytometry showed that intratumoral injection of bromocriptine could significantly promote tumor-infiltrating CD8 + The expression of T cell granzyme B (GzmB) is shown in Table 2 and Figure 1 As shown in F:

[0147] Table 2

[0148]

[0149] The results showed that compared with the GzmB in the peripheral blood of normal people or the control group without drug treatment + T cells account for CD8 + Compared with the percentage of cells in the subcutaneous tumor tissue of mice, TB significantly promoted the tumor infiltrating CD8 + T cell granzyme B (GzmB) expression; promotes tumor infiltration CD8 + Anti-tumor activity of T cells Figure 1 F).

[0150] Example 2: Regulation of the balance of tumor-infiltrating Treg / Th17 cells by bromoproma

[0151] 1. Experimental purpose: To determine the effect of tonazolidinone on tumor-infiltrating T cell subsets

[0152] 2. Experimental methods: Flow cytometry was as described in Basic Experimental Methods 8. Single cell sequencing was the same as in Example 1.

[0153] 3. Experimental results and analysis:

[0154] As the single-cell sequencing results in Example 1 showed, tongzolamide reshaped the tumor immune microenvironment and significantly promoted the tumor infiltrating CD8 + T cell activity, the inventors further analyzed the effect of bromocriptine on T cell subsets that play a core role in tumor immunity. Figure 2 As shown. UMAP subpopulation analysis of T cells identified a total of 6 subpopulations, including regulatory T cells (Treg), Th17 cells, and initial T cells. Effector T cells (Teff), effector memory T cells (Tem) and exhausted T cells (Tex) ( Figure 2 A). The proportion of each subgroup was statistically analyzed, and the results are shown in Table 3:

[0155] Table 3

[0156]

[0157] The results showed that bromocriptine inhibited tumor-infiltrating Treg cells and promoted tumor-infiltrating Th17 cells ( Figure 2 A).

[0158] Furthermore, the effect of tongzolamide on intratumoral Treg / Th17 cells was verified in the mouse subcutaneous tumor formation and intratumoral administration model. The flow cytometry results are shown in Table 4:

[0159] Table 4

[0160]

[0161]

[0162] The results showed that compared with the control group without drug treatment, 25mg / kg and 50mg / kg of bromoamine could inhibit tumor infiltrating Treg cells in a dose-dependent manner. Figure 2 B) and promoted tumor-infiltrating Th17 cells in a dose-dependent manner ( Figure 2 C).

[0163] Example 3: Tongzol bromide has no significant effect on peripheral immune homeostasis

[0164] 1. Experimental purpose: The balance of Treg / Th17 cells is crucial to tissue immune homeostasis.

[0165] 2. Experimental method: Mice were injected with 5X10 5 Mouse intestinal cancer cells (MC38 cells) were injected into the tail vein 1 week later, with an injection of 1mg / kg or 5mg / kg of bromoamine. The control group was injected with PBS for a total of 4 injections. After 2 weeks, the mice were killed, and the proportion and number of Treg / Th17 cells infiltrating in the draining lymph nodes (dLN), colon (Colon) and lung (Lung) tissues were counted.

[0166] 3. Experimental results and analysis: The draining lymph nodes, intestines and lung tissues of the control group and mice treated with different doses of tongzol bromide were subjected to flow cytometry to detect the ratio and number of Treg / Th17 cell infiltration in peripheral tissues. The results showed that different doses of tongzol bromide had no significant effect on the number of Treg and Th17 cell infiltration in the draining lymph nodes (dLN) ( Figure 3 A). Similarly, different doses of bromocriptine had an effect on colon tissue (Colon, Figure 3 B) and lung tissue (Lung, Figure 3C) had no significant effect on the number of Treg and Th17 cell infiltration. In summary, tongzol bromide had no significant effect on peripheral immune homeostasis.

[0167] Example 4: Bromide inhibits Treg cell differentiation

[0168] 1. Experimental purpose: To study the mechanism of bromocriptine in regulating Treg / Th17 balance

[0169] 2. Experimental methods: In an in vitro induction system, the effects of bromocriptine on the differentiation of Treg and Th17 cells were detected. The experimental methods were the same as those described in basic experimental methods 6 and 9.

[0170] 3. Experimental results and analysis: In order to explore the mechanism of bromotidine regulating Treg / Th17 balance, the inventors analyzed the transcription factor activity in Treg cells based on the single-cell sequencing data in Example 1. The results showed that bromotidine significantly promoted the transcriptional activity of Stat3 in Treg cells ( Figure 4 A).

[0171] It is known that Stat3 promotes Th17 cell differentiation and inhibits Treg cell differentiation. Therefore, the inventors further used an in vitro induced differentiation model to detect the effect of bromotidine on Treg and Th17 cell differentiation. The results showed that bromotidine inhibited iTreg cell differentiation in a concentration gradient-dependent manner ( Figure 4 B), and promote Th17 cell differentiation ( Figure 4 C).

[0172] Furthermore, combined with the Stat3 pathway inhibitor Stattic (MCE, Shanghai), we detected whether the regulation of Treg / Th17 differentiation balance by bromocriptine depends on the Stat3 pathway.

[0173] The results showed that Stattic alone could promote Treg cell differentiation in a dose-dependent manner ( Figure 4 D) and inhibit Th17 cell differentiation ( Figure 4 E); After combined with bromocriptine, Stattic was able to reverse the phenotype of bromocriptine in a dose-dependent manner, which inhibited iTreg cell differentiation and promoted Th17 cell differentiation ( Figure 4 F and 4G).

[0174] The above results indicate that the regulation of Treg / Th17 differentiation balance by bromocriptine depends on the Stat3 signaling pathway.

[0175] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. The use of tongzol bromide or a pharmaceutically acceptable salt thereof, characterized in that: Used for preparing medicines for treating microenvironment immune function deficiency diseases.

2. The use according to claim 1, characterized in that The drug is administered to a subject having a phenotypic characteristic selected from the group consisting of: (1) The proportion of Treg cells in the blood increases; (2) The proportion of Th17 cells in the blood decreases; (3) the ratio of the number of Treg cells Sr to the number of Th17 cells Sh in the blood, Ri (Sr / Sh) ≥ 2, preferably ≥ 3, and more preferably ≥ 5; (4) or a combination thereof.

3. The use according to claim 1, characterized in that The medicament is also administered to a subject having a phenotypic characteristic selected from the group consisting of: Tumor infiltrating CD8 + Granzyme B expression is low in T cells.

4. The use according to claim 3, characterized in that The subject has tumor-infiltrating CD8 + The positive expression rate of granzyme B in T cells is ≤40%, preferably ≤35%, and more preferably ≤30%.

5. The use according to claim 1, characterized in that The medicament is also administered to a subject having a phenotypic characteristic selected from the group consisting of: (1) The proportion of tumor-infiltrating Treg cells increases; (2) the proportion of tumor-infiltrating Th17 cells decreased; (3) the ratio of the number of tumor-infiltrating Treg cells Sr to the number of tumor-infiltrating Th17 cells Sh Ri (Sr / Sh) ≥ 2, preferably ≥ 3, and more preferably ≥ 5; (4) or a combination thereof.

6. A kit, characterized in that: The kit comprises: (1) a detection reagent, wherein the detection reagent is used to detect whether there is a microenvironmental immune function defect; and (2) A pharmaceutical composition comprising thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient.

7. The kit according to claim 6, characterized in that The kit also includes instructions, which record the use of the detection reagent to detect whether the subject has a microenvironmental immune function deficiency disease; If microenvironmental immune function deficiency is detected, a pharmaceutical composition containing thonzol bromide or a pharmaceutically acceptable salt thereof as the first active ingredient is administered to the subject.

8. A medicine box, characterized in that: The kit comprises: (I) a first formulation in a first container, the first formulation comprising (a) tonazocium bromide or a pharmaceutically acceptable salt thereof as a first active ingredient, and (b) a pharmaceutically acceptable carrier; (II) optionally a second formulation in a second container, the second formulation comprising (c) a Stat3 promoter as a second active ingredient; and (III) instructions, wherein the instructions indicate that the drug kit is used for the treatment of microenvironmental immune deficiency diseases.

9. A non-therapeutic in vitro method for inducing immune cell differentiation, characterized in that: Includes steps: In the presence of thonzyl bromide or a pharmaceutically acceptable salt thereof, T cells are cultured, thereby reducing the proportion of Treg cells in the T cells and / or increasing the proportion of Th17 cells in the immune cells.

10. An in vitro non-therapeutic method for regulating immune cell function, characterized in that: Includes steps: In the presence of tongzol bromide or a pharmaceutically acceptable salt thereof, CD8 + T cells, CD8 + The expression percentage of granzyme B in T cells increases, thereby regulating the function of immune cells.

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