KR101273747B1 - Composition for preventing and treating cancer or autoimmune diseases comprising STA-21 - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of cancer or immune diseases comprising STA-21 as an active ingredient, and more specifically, the present invention contains STA-21 as a compound or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to a method for preventing and treating cancer or immune disease, an immunosuppressive composition, a method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells using STA-21, and a method for activating regulatory T cells. The STA-21 compound according to the present invention has an excellent effect of inhibiting the differentiation of cytotoxic Th17 cells that produce and secrete inflammatory cytokines, and has the property of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response. It is useful as a pharmaceutical composition or immunosuppressant that can prevent and treat immune diseases such as autoimmune disease, inflammatory disease and transplant rejection disease caused by abnormal regulation of immune response because it has an excellent effect of activating immunoregulatory T cell (Treg). There is an effect that can be used.

Description

Composition for preventing and treating arthritis containing STA-21 as an active ingredient STA-21}

The present invention relates to a pharmaceutical composition containing STA-21 as an active ingredient, and more particularly to a composition for the prevention and treatment of cancer or immune diseases containing STA-21 as an active ingredient.

T cells are one of a group of cells that play a central role in the immune system as a biological defense system against various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among them, the main function of Th1 cells is involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations are balanced with each other so that they are not activated with each other.

Therefore, most of the immune diseases can be attributed to the imbalance between these two immune cells, for example, when an abnormal increase in the activity of Th1 cells may cause an immune disease, an abnormal increase in the activity of Th2 cells It is known that immune diseases occur due to hypersensitivity reactions.

Meanwhile, according to a recent study on the differentiation of Th1 cells, the existence of a new group of immunoregulatory T cells (Tregs), which can control Th1 cell activity, is known, and studies on the treatment of immune diseases using the same are emerging. In addition, since Treg cells inhibit the function of abnormally activated immune cells to control the inflammatory response, many experiments have been reported to treat immune diseases and inflammatory diseases by increasing the activity of Treg cells.

In addition to the Treg cells, another group made during the differentiation is Th17 cells, which are known to be formed through a process similar to the differentiation of Treg cells during the differentiation of undifferentiated T cells. That is, differentiation of Treg cells and Th17 cells is performed in the presence of TGF-β in common but does not require IL-6 in Treg cells, whereas IL-6 is present in combination with TGF-β in Th17 cells. Differentiate in situations In addition, differentiated Th17 cells are characterized by the secretion of IL-17.

Unlike Treg cells, Th17 cells have been found to be involved in the forefront of inflammatory reactions seen in immune diseases, maximizing the signal of inflammatory responses and accelerating disease progression. Therefore, in the case of immune diseases which are not controlled by Treg cells among immune diseases, the development of therapeutic agents for immune diseases that target the inhibition of Th17 cell activity has been highlighted.

Currently used immunosuppressive drugs are immunosuppressants that block signal transduction pathways in T cells. These immunosuppressive agents are toxic, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. There is a problem that side effects such as renal failure occur.

In addition to the treatment of immune diseases by inhibiting the activation of T cells, a treatment for controlling the amount of cytokines secreted from immune cells and a therapeutic method using antibodies targeting cytokines secreted from immune cells have been developed. There is. However, this method has to take a long time to apply to the patients after the actual clinical trials, the method using the antibody has a problem that too much cost in the antibody manufacturing process.

Therefore, there is a need for the development of a new immune disease treatment agent that is inexpensive and has excellent therapeutic effect.

The present inventors first identified that the STA-21 compound inhibits STAT3, thereby inhibiting the activity of Th17, a pathogen cell, and increasing the activity of Treg, an immunoregulatory cell, thereby making STA-21 a therapeutic agent for immune-related diseases. Confirming the fact that it can be used as, the present invention was completed.

Accordingly, an object of the present invention is to prevent cancer or immune diseases comprising STA-21 compound or a pharmaceutically acceptable salt thereof as an active ingredient to effectively prevent and treat immune diseases and cancers caused by abnormalities of various immune responses. And it provides a therapeutic composition.

Another object of the present invention is to reduce or inhibit the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating undifferentiated T cells with a STA-21 compound or a pharmaceutically acceptable salt thereof. To provide a way.

Another object of the present invention is a method of activating regulatory T cells comprising treating a Regulatory T cell (Treg) with an STA-21 compound or a pharmaceutically acceptable salt thereof in vitro. To provide.

Furthermore, another object of the present invention is to provide an anticancer or immunosuppressive composition comprising the STA-21 compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of cancer or immune diseases comprising the STA-21 compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In one embodiment of the present invention, the STA-21 can reduce or inhibit the differentiation of undifferentiated T cells into Th17 cells.

In one embodiment of the present invention, the STA-21 may promote or increase the activity or amplification of Regulatory T cells (Treg).

In one embodiment of the present invention, the cancer is liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer and pancreatic cancer composition for the prevention and treatment of cancer diseases, characterized in that selected from the group consisting of.

In one embodiment of the present invention, the immune disease may be selected from the group consisting of autoimmune diseases, inflammatory diseases and transplant rejection of cells, tissues or organs.

In one embodiment of the present invention, the immune disease is Behcet's disease, multiple myositis / skin myositis, autoimmune hematopoiesis, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatitis, Goodfiction syndrome, autoimmunity Meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes, dystrophic epidermal detachment, epididymitis, glomerulonephritis, Graves' disease, Guillain Barre syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, It can be chosen from obesity.

In one embodiment of the present invention, the STA-21 compound may comprise a concentration of 1uM ~ 30uM.

The present invention also provides a method of reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating the undifferentiated T cells with a STA-21 compound or a pharmaceutically acceptable salt thereof. To provide.

In one embodiment of the present invention, the reduction or inhibition of differentiation of undifferentiated T cells into Th17 cells may be achieved through inhibition of production of IL-17 cytokines.

In addition, the present invention provides a method of activating regulatory T cells comprising treating a regulatory T cell (Treg) with an STA-21 compound or a pharmaceutically acceptable salt thereof in vitro. to provide.

In one embodiment of the present invention, activated regulatory T cells may have increased expression of Foxp3.

Furthermore, the present invention provides an anticancer or immunosuppressive composition comprising the STA-21 compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The STA-21 compound according to the present invention has an excellent effect of inhibiting the differentiation of cytotoxic Th17 cells that produce and secrete inflammatory cytokines, and has the property of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response. It is useful as a pharmaceutical composition or immunosuppressant that can prevent and treat immune diseases such as autoimmune disease, inflammatory disease and transplant rejection disease caused by abnormal regulation of immune response because it has an excellent effect of activating immunoregulatory T cell (Treg). Not only can it be used, but it has no toxicity and side effects and has a stable effect in the body.

1 is a graph showing the results of FACs stains performed to compare the expression of IL-17 and Tregs in mouse spleen cells 3 days after differentiation into Th17 cells according to an embodiment of the present invention.
Figure 2 shows the results of measuring the secretion amount of IL-17 cytokines generated after treatment of the splenocytes isolated from the mouse under Th17 differentiation conditions and treatment with STA-21 for each concentration. The graph shown.
Figure 3, in one embodiment of the present invention, to examine whether there is an action to activate the Treg cells, immunoregulatory cells, cells subjected to Th17 differentiation environment when STA-21 is treated by concentration using a mouse splenocyte model Gene expression levels of IL-17 and Foxp3 in the graphs showing the results of analysis via RT-PCR.
4 is a graph showing the arthritis treatment effect and its incidence by injecting STA-21 into IL-1RaKO mouse, an autoimmune arthritis animal model, and inflammation and cartilage damage in the joint tissue of each group. It is a graph observed by the chemical staining method.
5 is a confocal result of analysis of the expression level of IL-17 and Foxp3 genes of each group by RT-PCR and Th17 and Treg factors in the animal model spleen ex vivo cells of FIG. 4 according to one embodiment of the present invention. Staining and graphs quantifying cells that are IL-17 + and cells that are Foxp3 + are shown.

STAT3, a member of the Signal Transducers and Activators of Transcription (STAT) family member, known to regulate various intracellular functions, has the leucine zipper of N terminus, DNA-binding domain, and SH2 transactivation domain at the C-terminal end as small domains. STA-21 is a major transcription factor of IL-17-producing T cell (Th17), which is activated by IL-6 and TGF-β, leading to increased expression of another major transcription factor, orphan nuclear receptors RORγ and RORα. Binding to the known SH2 domain of STAT3 can directly inhibit STAT3 DNA binding activity, STAT3 dimerization and STAT3-dependent luciferase activity.

Therefore, the present invention is the first to the effect that the STA-21 compound can inhibit the STAT3 to reduce the production of IL-17 in the pathogen cell Th17, as well as to increase the expression of Foxp3, a key factor of the Treg immunoregulatory cell As it turned out, the present invention is characterized by providing a composition for the prevention and treatment of cancer or immune diseases with STA-21 as an active ingredient.

On the other hand, STA-21, the active ingredient of the composition according to the present invention enters the cell and is directly delivered to cancer cells or solid cancers, while inhibiting the growth of cancer cells, but very low toxicity to normal cells to be used for the treatment of cancer In particular, experimental results in human breast cancer have been reported to have excellent ability to kill breast cancer cells but few side effects in normal fibroblasts (Hui, S. et al., PNAS , vol. 102 no. 13 (2005) 4700-4705).

However, it is not known at all that STA-21 can be used for the treatment of immune diseases.

Therefore, in the present invention, the STA-21 compound can be used for the prevention and treatment of immune diseases, and especially when the STA-21 is injected into an animal model of autoimmune arthritis, it is found that the activity of improving and treating arthritis symptoms. First identified.

On the other hand, the immune system controls specific immune responses to autoantigens under normal conditions, and also suppresses immune responses against external antigens. For example, the pregnant woman's response to fetuses and immunity to chronic infections Reaction. These phenomena are known to be induced by clonal deletion, clone anergy and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen specific immunotolerance can be induced. Investigating some patients who accidentally acquired immunotolerance against transplanted antigens or experimentally induced animal models showed that all three of these mechanisms are involved in immunological tolerance. Is attracting attention as an important cell involved in controlling almost all immune responses of living body such as autoimmune, tumoral immunity, infectious immune response as well as transplantation immune response.

In particular, immunoregulatory T cells, ie immunoregulatory T lymphocytes (Tregs), whose presence has recently been identified, can be largely divided into natural and adaptive Treg cells, and CD4 + CD25 + T cells, which are natural Tregs, are cells. Has been given immunosuppressive function when it is newly created in the thymus, and is present at a frequency of 5 to 10% of peripheral CD4 + T lymphocytes in normal individuals. Although the immunosuppressive mechanism of this cell has not yet been elucidated yet, it has recently been shown that the expression control gene of Foxp3 plays an important role in the differentiation and activity of this cell. In addition, peripheral T cells can be differentiated into cells exhibiting an immunosuppressive effect upon being stimulated by a self or external antigen under a specific environment, which is called an adaptive or inducible Treg and secretes IL-10 Tr1 that secretes TGF-beta, Th3 and CD8 Ts that secrete TGF-beta, and the like.

As described in the prior art, T cells are also differentiated into Th17 cells through differentiation in addition to Treg cells. Th17 cells are formed in the presence of TGF-β in common with Treg cells, but IL-6 is expressed in Treg cells. On the other hand, Th17 cells are characterized by differentiating in the presence of IL-6 with TGF-β and secreting IL-17.

In addition, Th17 cells have cytotoxic properties that maximize signal of inflammatory response and accelerate disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the methods for treating immune diseases.

The present inventors confirmed that the STA-21 compound can treat immune diseases by regulating T cells, specifically, differentiating T cells into Treg cells and Th17 cells, but one embodiment of the present invention. According to the present invention, T cells were isolated from mice to compare the expression of IL-17 and Tregs in cells differentiated into Th17 cells by stimulating conditions that can differentiate into Th17 cells and STA-21 compounds at different concentrations. One spleen cell was collected and treated with each concentration of STA-21 compound to proceed FACs staining.

As a result, when STA-21 was treated, Foxp3 expression was significantly increased in proportion to the concentration of STA-21, and IL-17 expression was proportional to the concentration of STA-21 compared to the control group not treated with STA-21. Gradually decreasing (see FIG. 1).

Therefore, the present inventors have found that the STA-21 compound reduces the production of IL-17 in the pathogen cell Th17 while increasing the expression of Foxp3, the main factor of the immunoregulatory cell Treg.

According to another embodiment of the present invention, at the same time to treat the stimulation conditions for the differentiation of undifferentiated T cells present in the spleen to Th17 cells in the spleen cells isolated from the mouse at each concentration of STA-21 compound After treatment and incubation, the amount of intracellularly expressed IL-17 cytokine was measured. In general, the inflammatory cytokine IL-17 is produced from Th17 cells and is differentiated through the amount of IL-17 produced. The degree of differentiation of cells into Th17 cells can be measured.

As a result, in the group treated with STA-21, the differentiation into Th17 cells was suppressed by Th17 cell differentiation conditions, and the production of IL-17 cytokines was significantly reduced compared to the group not treated with STA-21. . In addition, the amount of IL-17 cytokine produced was found to decrease in proportion to the concentration of treated STA-21 (see FIG. 2).

Accordingly, the present inventors have found that the STA-21 compound reduces or inhibits the differentiation of undifferentiated T cells into Th17 cells, which are pathogenic cells producing IL-17, an inflammatory cytokine.

Furthermore, the present inventors measured the expression levels of IL-17 and Foxp3 genes following the treatment of STA-21 in order to investigate how STA-21 affects the activity or proliferation of immunoregulatory T cells (Treg).

Foxp3 is mainly present in regulatory T cells derived from the thymus and is a transcriptional factor present in cells with CD4 + CD25 + labeling antigen, and its function is to express Foxp3. Recognition of IL-2 against T cells that are potentially responsive to antigens and that may potentially cause autoimmune among CD4 + CD25- T cells that do not express Foxp3 differentiated from the thymus at the time of antigen recognition against T cells It has a role as a suppressor T cell that suppresses production and cell division. In addition, Foxp3 stimulates not only IL-2 but also IL-4, which is influenced by NFAT, which is a transcription factor, in CD25- T cells through regulatory T cells expressing Foxp3 and cell-cell contact with them, Lt; RTI ID = 0.0 > IFN-gamma. ≪ / RTI > Therefore, in the case of T cells expressing Foxp3 having such a function, it is applied to the treatment of immune diseases through the action of inhibiting or regulating the immune response, and furthermore, the CD4 T cells expressing Foxp3 present in humans. To apply self-antigen specific T cell clones to cell therapy by increasing their number through high concentration of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibodies There have been attempts. Therefore, confirming the expression of Foxp3 is an indicator to measure the activity or amplification of Treg cells.

According to another embodiment of the present invention, the splenocytes isolated from the mouse are treated with stimulation conditions capable of differentiating into Th17 cells and simultaneously treated and cultured with STA-21, followed by amplification or activity of differentiated Treg cells. As a result of measuring the amount of IL-17 and Foxp3 expressed, the amount of Foxp3 mRNA expressed in Treg cells was significantly increased in the group treated with STA-21 compared to the group not treated with STA-21. The degree of increase was proportional to the concentration of the treated STA-21. In addition, the production of IL-17 in the pathogen cell Th17 was also shown to be reduced by the treatment of STA-21 (see Figure 3).

Therefore, the present inventors have found that the STA-21 compound can be used to prevent or treat immune diseases by activating or amplifying regulatory T cells (Treg).

Therefore, the STA-21 compound of the present invention is characterized by preventing or treating immune diseases through the action of reducing or inhibiting the differentiation of undifferentiated T cells into pathological Th17 cells. That is, the present invention can prevent and treat immune diseases by suppressing immune or inflammatory responses.

In addition, the STA-21 compounds of the present invention can activate or amplify Regulatory T cells (Tregs) that can normally regulate excessive immune responses, and at the same time inhibit Th17 cell differentiation, which secretes inflammatory cytokines. Since the activity is excellent, there is a characteristic that can prevent and treat diseases related to immunity or inflammation.

In the present invention, the term "activation" means that all the mechanisms of Treg cells including both regulatory T cells (Tregs), that is, natural and adaptive Treg cells in vivo It is meant to be promoted or promoted, which means that an immunomodulatory action such as an immunosuppressive reaction is promoted or promoted so that an immune response in a living body is maintained in a normal state.

In addition, the term "expansion" refers to the differentiation and proliferation of undifferentiated T cells into regulatory T cells, 'differentiation' refers to a phenomenon in which structures or functions are specialized while the cells divide and grow, In other words, the shape or function of a cell or tissue of an organism is changed in order to perform a given task, and 'proliferation' refers to the division of cells and the expansion of homogeneous ones. It means to increase.

Therefore, the present invention can provide a composition for the prevention and treatment of immune diseases comprising the STA-21 compound or a pharmaceutically acceptable salt thereof as an active ingredient, the STA-21 compound is represented by the formula It may be a compound having.

[Formula 1]

In addition, the STA-21 compound according to the present invention may be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The STA-21 compound according to the present invention can be used that is isolated from nature or prepared by chemical synthesis known in the art.

When the STA-21 compound is separated from nature, it can be obtained by using a method of extracting and separating conventional materials, and the STA-21 compound which can be used in the present invention is not limited to that obtained from the method. . In one embodiment of the present invention was used to buy what is sold in the market.

In addition, a suitable solvent that can be used to extract the STA-21 compound from the nature may be water or an organic solvent, preferably purified water, methanol (methanol), ethanol (proolol), isopropanol (isopropanol), butanol, acetone, ether, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Various solvents, such as (cyclohexane), can be used individually or in mixture.

In addition, the separation and purification of the STA-21 compound of the present invention from the extract obtained by the above method is performed by methods known in the art, for example, column chromatography packed with various synthetic resins such as silica gel or activated alumina. Column chromatography and high performance liquid chromatography (HPLC) may be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

On the other hand, in general, cancer is a step in which mutations are induced in DNA by carcinogens, a benign tumor appears, a benign tumor is converted into a malignant tumor, and an increase in the degree of malignancy and metastasis It is known to proceed by a multi-step as step, and the treatment of such cancer is used to inhibit the cell proliferation of cancer cells or to induce apoptosis of cancer cells.

In the present invention, the type of cancer is not limited thereto, but may be liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer, and pancreatic cancer.

In addition, the STA-21 compound according to the present invention can reduce or inhibit the production of IL-17 in the pathological cell Th17, while cancer or immune disease through the action of promoting or enhancing the activity or amplification of regulatory T cells. It can be prevented and treated, the composition for the prevention and treatment of cancer or immune diseases according to the invention may comprise a STA-21 compound in a concentration of 1uM ~ 30uM, preferably in a concentration of 1uM ~ 20uM Can be.

In the present invention, the "immune disease" refers to a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to the pathology of a mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplant rejection of cells, tissues, or organs.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when there is a problem in inducing or maintaining such self-tolerance, an immune response occurs to autoantigens, thereby attacking one's own tissues. This disease is referred to as an "autoimmune disease". It is called.

In addition, "inflammatory disease" refers to tumor necrosis factor-α (TNF-α) and IL-1 (excessively stimulated by the immune system such as macrophages due to excessive stimulation of the human immune system due to harmful stimuli such as inflammation-induced factors or irradiation). interleukin-1), IL-6, prostaglandin (prostagladin), luecotriene or nitric oxide (nitric oxide, NO) refers to a disease caused by proinflammatory substances (inflammatory cytokines).

On the other hand, in order to successfully transplant the organ, the recipient's immune rejection response to the transplanted cells and organs must be overcome. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. Immunosuppressive agents have been used to reduce these transplant immune rejection responses. A common goal of these immunosuppressive agents is to inhibit T cell-mediated immune responses to grafts. Recently, regulatory T cells have been used to suppress immune responses, Methods to treat refractory diseases have been attempted.

In addition, the type of immune disease in the present invention is not limited to this, Behcet's disease, multiple myositis / skin myositis, autoimmune hematopoiesis, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatitis, gut pie Syndrome, autoimmune meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, dystrophic bullous epidermal detachment, epididymitis, glomerulonephritis , Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, sclerosis, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia , Ulcerative colitis, obesity, and the like.

Therefore, the composition according to the present invention can be used as a pharmaceutical composition that can prevent and treat cancer or immune diseases.

The term “treatment”, unless stated otherwise, means to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of the disease or condition. The term "treatment" as used herein refers to the act of treating when "treating" is defined as above. Thus, “treatment” or “therapeutic therapy” for cancer or immune disease in a mammal may include one or more of the following:

(1) inhibits the growth of cancer or immune disease, ie inhibits its development,

(2) preventing the spread of cancer or immune disease, ie preventing metastasis,

(3) reduce cancer or immune disease.

(4) prevent the recurrence of cancer or immune disease, and

(5) Palliating the symptoms of cancer or immune disease

The composition for preventing and treating cancer or immune disease according to the present invention may include a pharmaceutically effective amount of a STA-21 compound or a salt thereof alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. . The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of cancer or immune disease.

The pharmaceutically effective amount of the STA-21 compound or salt thereof according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the extent of symptoms of cancer or immune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

Also, as used herein, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing and treating cancer or immune disease according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is determined by the route of administration, the age of the patient, It may be appropriately selected according to several factors such as sex, weight and severity of the patient, and the composition for preventing and treating cancer or immune disease according to the present invention has the effect of preventing, improving or treating the symptoms of cancer or immune disease. It can be administered in combination with a known compound.

Accordingly, the present invention can provide a medicament for the prevention and treatment of cancer or immune diseases, including a composition containing a STA-21 compound or a salt thereof as an active ingredient, and the present invention further provides a STA-21 compound or a salt thereof. It is possible to provide an immunosuppressive composition comprising as a component.

In addition, the present invention can provide a method for reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro, comprising treating the undifferentiated T cells with a STA-21 compound or a salt thereof. The reduction or inhibition of the differentiation of the undifferentiated T cells into Th17 cells may be achieved through the inhibition of production of IL-17 cytokines.

In addition, the present invention can provide a method for activating regulatory T cells comprising the step of treating a STA-21 compound or a salt thereof in a regulatory T cell (Treg) in vitro, The regulatory T cells activated according to the present invention may increase the expression of Foxp3.

The method of reducing or inhibiting the differentiation of undifferentiated T cells into Th17 cells in vitro and the method of activating regulatory T cells in the above-described method according to the present invention may be described above. The medium for culturing cells may be treated with the STA-21 compound directly, or the composition containing the STA-21 compound according to the present invention as an active ingredient to the culture medium. In addition, at this time, the concentration of the STA-21 compound that can be treated in the culture medium may be treated so that the final concentration is 1μM ~ 30μM, in one embodiment of the present invention, the concentration of the STA-21 compound is the final 3.75μM, 7.5 It was added to the culture medium of the cells to be μM and 15 μM.

Furthermore, the composition for preventing and treating cancer or immune disease according to the present invention is excellent in the activity or amplification effect of immune regulatory T cells (Treg) and excellent in inhibiting the differentiation of Th17 cells, which are pathogenic cells producing inflammatory cytokines. In addition, there is no toxicity and side effects to the drug can be used safely even if taken for a long time, and has a stable characteristic for the body.

Therefore, the present invention can provide a food composition capable of improving or preventing the symptoms of cancer or immune diseases containing the STA-21 compound or a salt thereof as an active ingredient, wherein the food composition according to the present invention is cancer or immune. Food, which is effective for improving or preventing disease symptoms, for example, it can be easily utilized as a main ingredient, side ingredients, food additives, functional food or beverage of food.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Th17  In a cell STA By -21 IL -17 lesson Treg Analysis of the effect on the expression of

In order to determine whether the STA-21 compound has an effect of treating cancer or immune disease, the present inventors examined the change in the secretion of IL-17 cytokines and expression of Treg cells by STA-21 in Th17 cells associated with these diseases. Th17 cells were obtained by differentiating from CD4 + T cells as described below.

<1-1> Th17  Induction of cell differentiation and STA -21 processing

The present inventors investigated whether the STA-21 compound has an activity of inhibiting the production of the inflammatory cytokine IL-17 in Th17 cells, which are pathogenic cells. For this, first, spleen cells were obtained from the mouse, that is, the spleen extracted from the mouse was finely chopped spleen tissue using a glass slide, and then red blood cells in the spleen were removed with a erythrocyte hemolysis solution. Splenocytes were obtained by adding PBS buffer solution and washing by centrifugation. Then, a single cell 1 × 10 ⁶ isolated from the spleen was dispensed into a 24-well plate coated with 1 μg / mL of anti-CD3 antibody, and anti-CD28 was a condition capable of stimulating Th17 cells. Antibodies were treated with 1 μg / mL, TGF-β 2ng / ml, IL-6 20ng / ml, anti-IL-4 10ng / ml, and anti-IFNr 10ng / ml simultaneously, and the cells were cultured for 3 days. The cells were cultured by treating STA-21 compounds at concentrations of 3.75 μM, 7.5 μM, and 15 μM, respectively. In this case, a group not treated with the STA-21 compound was used as a control group.

<1-2> FACs  Expression analysis through

Splenocytes of the cultured mice were collected and washed with FACs buffer for flow cytometry, which is used in the art, and then reacted with blocking at 4 ° C. for 15 minutes to inhibit nonspecific binding and then washed with perm wash buffer. Anti Foxp3-FITC and anti IL-17 PE were added thereto, reacted at 4 ° C. for 30 minutes, and washed with perm wash buffer. After staining cells were washed with FACs buffer, flow cytometry (FACs, fluorescent-activated cell sorter) was used to analyze the expression level of IL-17 cytokine and Foxp3 by STA-21 compound treatment.

As a result, as shown in Figure 1, when treated with 3.75μM STA-21 expression level Foxp3 was 6.34, IL-17 expression level was 1.71. In addition, when STA-21 was treated with a concentration of 7.5 μM, Foxp3 expression was 9.2, IL-17 expression was 1.5, and when 15 μM STA-21 was treated, Foxp3 expression was 16.6. The expression of IL-17 was 0.91. On the other hand, the control group not treated with STA-21 showed Foxp3 expression of 3.82 and IL-17 expression of 2.07.

Therefore, the results of the present inventors have found that when STA-21 is treated, Foxp3 expression is increased, whereas IL-17 expression is decreased, and this expression change is proportional to the treatment concentration of STA-21. Foxp3 expression was further increased and IL-17 expression was further reduced.

ELISA Through IL -17 Cytokine Expression Measurement

Furthermore, the present inventors investigated whether the STA-21 compound has an activity of inhibiting the production of inflammatory cytokine IL-17 in Th17 cells, which are pathogenic cells, by the following method. Culture supernatants of the cultured cells were collected and IL-17 expression levels were examined using IL-17 and sandwich ELISA. To this end, the monoclonal anti-IL-17 was first treated in a 96-well plate at 2 μg / mL and reacted overnight at 4 ° C., after which the non-specific binding was blocked with a blocking solution (1% BSA / PBST). After that, IL-17 recombinant was serially diluted by 1/2, and used as a standard. The cell culture supernatant was added and reacted at room temperature for 2 hours. Thereafter, the biotinylated anti-IL-17 was reacted at room temperature for 2 hours and washed four times. Then, the ExtraAvidin-Alkaline Phosphatase conjugate was diluted and added and reacted at room temperature for 2 hours. Thereafter, PNPP / DEA solution was added, followed by color development, and absorbance at 405 nm was measured.

As a result, as shown in FIG. 2, IL-17, an inflammatory cytokine produced from Th17 cells, which are pathogenic cells, was found to be inhibited by the treatment of the STA-21 compound of the present invention, and the concentration of STA-21 was increased. Depending on the production of IL-17 was shown to be reduced.

RT - PCR  Through the way Treg  In a cell STA -21 impact analysis

Controlled T lymphocytes, or Treg (regulatory T lymphocytes) cells, are known to play a key role in the maintenance of immune tolerance (Nat. Rev. Immunol. 2009, 7: 305) and are produced primarily in the thymus and are transcription factors Expression of Foxp3 is required and Foxp3 is known to play a role in inhibiting the production of inflammatory cytokines.

In order to investigate whether the STA-21 compound according to the present invention has an action of activating the activity of Treg cells, which are immunoregulatory cells, the present inventors stimulated spleen cells of mice with Th17 cell stimulation conditions of Example 1 The expression level of IL-17 and Foxp3 expressed from the cells, ie, mRNA, was measured by reverse transcriptase polymerase reaction. That is, after treatment with STA-21 at a concentration of 0μM, 3.75μM, 7.5μM, 15μM to the cells in Th17 differentiation conditions, respectively, RNA was extracted from the cells according to methods known in the art, and then Foxp3 and Polymerase chain reaction was carried out using primers capable of amplifying IL-17. The reaction compound for RT-PCR was 25 μl in total. The composition of the solution was 2.5 μl of 10 × reaction buffer, 0.5 mM dNTP. And each primer pair having the sequence shown in Table 1 was used, amplification was 94 ℃ 30 seconds in the denaturation step, 60 ℃ 30 seconds in the annealing step, 72 ℃ in the extension step using a Dual-bay Thermal cycler system instrument The reaction for 30 seconds was repeated 25 times.

Primer sequence primer Primer sequence SEQ ID NO:  IL-17 sense  5'-TCT CAT CCA GCA AGA GAT CC-3 ' One  IL-17 antisense  5'-AGT TTG GGA CCC CTT TAC AC-3 ' 2  Foxp3 sense  5'-GGC CCT TCT CCA GGA CAG A-3 ' 3  Foxp3 antisense  5'-GCT GAT CAT GGC TGG GTT GT-3 ' 4

As a result, as shown in FIG. 3, the expression of IL-17 was found to gradually decrease in concentration-dependent manner by the treatment of STA-21, whereas the expression of Foxp3 gradually increased in concentration-dependent manner by the treatment of STA-21. Appeared.

Therefore, through the above results, the present inventors confirmed that the STA-21 compound according to the present invention inhibits cell differentiation into Th17, which is a pathogenic cell, and simultaneously increases the expression of Foxp3. It has been found that the action of activating Treg cells can prevent and treat cancer or immune diseases.

Autoimmune Arthritis Animal Model STA -21 impact analysis

In order to observe the effect of STA-21 on arthritis, sixteen-week-old IL-1RaKO male mice, which are naturally induced arthritis animals, were evaluated by arthritis.

<4-1> Arthritis in Animal Models STA -21 effect

To investigate the effects of STA-21 in animal models of spontaneous arthritis, dissolve STA-21 powder in DMSO, calculate the injection volume using 20g of a mouse, and use the saline as the solvent to inject the total injection volume. Was diluted to 200 μl. The experimental group was injected with STA-21 0.5 mg / kg a total of 8 times at 2 day intervals, and the negative control group was injected with the same dilution with saline in the amount of DMSO used to dilute the STA-21 group.

As a result, as shown in Figures 4a and 4b, a significant arthritis treatment effect was observed after STA-21 injection, this was maintained for a long time even after 8 injections of STA-21, the degree of Incidence was also observed the same as the arthritis evaluation.

<4-2> Tissue staining of animal models of arthritis

To confirm the tissue staining results, the joints of the control and experimental mice of Example 4 were taken, fixed in 10% neutral buffered formalin, and demineralized with EDTA. Subsequently, paraffin was embedded and articular tissue was made into 7 μm thick sections and attached to the slides. Before the basic dyeing, ethanol was dehydrated from high to low concentrations by deparaffinization using xylene, and hematoxylin and eosin staining were performed. Next, Safranin O and Toluidine blue methods, which can detect proteoglycans contained in cartilage, were quantified in terms of inflammation and destruction of cartilage tissue.

As a result, as shown in Figure 4c, it was observed that the result of tissue staining of the mouse joint was reduced the degree of inflammation and cartilage destruction in the STA-21 treatment group compared to the negative control group. Therefore, when the STA-21 injection according to the present invention through the above results, it was confirmed that the excellent effect on the treatment of arthritis in the animal model.

ex - vivo in STA -21 IL -17 lesson Treg Impact on

We performed PCR and confocal staining to determine the effect of STA-21 on IL-17 and Tregs in ex-vivo using splenocytes in animal models.

<5-1> In splenocytes STA -21 processing IL -17 lesson Foxp3 Gene expression

In order to determine the effect of STA-21 in ex-vivo, the spleen cell RNA was directly obtained in the animal model of Example 4, and the expression of IL-17 and Foxp3 genes was confirmed by RT-PCR in the same manner as in Example 3. It was.

As a result, as shown in Fig. 5a, the IL-17 gene expression was lower in the STA-21 injection group than in the negative control group, but Foxp3 expression was more than three times higher.

<5-2> In splenocytes STA -21 is Th17 and Treg Influence on Factors

In order to determine the effect of STA-21 on Th17 and Treg factors in ex-vivo, the confocal staining method was used. The spleens of the experimental and control mice of Example 4 were taken to embed the OCT compound, and the tissue rapidly cooled using liquid nitrogen was attached to the slide with a thickness of 7 μm using a frozen slicer. Sections attached to the slides were fixed with acetone and blocked nonspecific reaction for 30 minutes with 10% normal goat serum. The first antibodies are FITC-labeled anti-Foxp3 Ab, PE-labeled anti-IL-17 Ab APC-labeled anti-CD4 Ab, Allophycocyanin-labeled anti-CD25 Ab (Biolegend), biotinylated anti-CD4 Ab (BD Biosciences, San Jose, CA) was diluted 1: 100 in PBS (pH 7.5) and reacted overnight at 4 ° C. After washing with PBS the next day, streptavidin cy-3 was reacted at room temperature for 2 hours. Stained tissues were analyzed by confocal microscopy (LSM 510 Meta. Zeiss, Go¨ttingen, Germany), and the number of IL-17 + cells and Foxp3 + cells were quantified under the microscope and presented graphically.

As a result, the number of IL-17 + cells was reduced by more than 50% in the STA-21 injection group compared to the negative control group, while the number of Foxp3 + cells was significantly increased in the STA-21 injection group compared to the negative control group.

Finally, through the above results, the present inventors confirmed that the STA-21 compound according to the present invention inhibits cell differentiation into Th17, which is a pathogenic cell, whereas STA-21 has an activity of increasing expression of Foxp3. By activating the Treg cells, which are immunoregulatory cells, it was found that cancer and immune diseases including autoimmune arthritis can be prevented and treated.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

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Claims (12)

delete delete delete delete delete delete delete delete delete Treatment of Foxp3 (forkhead box P3) comprising the step of treating the regulatory T cell (Treg) with a STA-21 compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof in vitro A method of activating regulatory T cells, wherein expression is increased.
[Formula 1]

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