KR20170006289A - Composition for preventing or treating immune diseases comprising receptor interacting serine threonine protein kinase inhibitor - Google Patents

Abstract

The present invention relates to a composition comprising an RIPK inhibitor as an active component for treating an immunological disease. In a disease tissue having an immunological disease, RIPK is confirmed to be overly expressed. In the case of injecting an RIPK inhibitor to an animal model having an immunological disease, symptoms of the immunological disease are confirmed to be alleviated and improved, and generation of inflammatory cytokine and morbific cytokine is confirmed to be suppressed. Thus, the RIPK inhibitor can be used to effectively prevent and treat an immunological disease.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating an immunological disease comprising an RIPK inhibitor as an active ingredient,

And a composition for preventing or treating an immunological disease, which comprises a RIPK inhibitor as an active ingredient.

Although diseases caused by immune hyperresponsiveness are increasing worldwide, fundamental causes for the occurrence of these diseases have not been sufficiently elucidated. Currently, as a method of treating diseases due to an excessive immune response, immunosuppressive agents are administered alone or in combination to alleviate or reduce various symptoms caused by the diseases.

Immunosuppressants refer to various substances that are used to reduce or block the ability of a host to make antibodies (humoral immune response) or the ability to cause a cellular immune response to the action of an antigen. Such immunosuppressants can be used not only in organ transplantation fields but also in autoimmune diseases such as rheumatoid arthritis and rheumatoid arthritis, and skin hypersensitivity reactions such as atopy and allergy. Superior immunosuppressants should be able to control the imbalance of the immune response, ensure human safety, and have a low incidence of disease recurrence during long-term treatment.

There are currently used immunosuppressants such as cyclosporin A and FK506. These compounds are derived from natural materials having complex chemical structures, which are uneconomical due to high costs in terms of raw material supply and demand, and various side effects may be caused due to long-term administration Which is a risk. Therefore, there is a desperate need to develop a new immunosuppressive agent which can induce low toxicity and induce immunity and economical production.

On the other hand, there are T cells as one of the cell groups which plays a central role in the immune system as a bio-defense system for 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 these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation with each other.

Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, autoimmune diseases may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

On the other hand, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Tregs) capable of regulating the activity of Th1 cells, , Treg cells have the property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction. Therefore, many studies are being conducted to treat immune diseases through the action of increasing Treg cell activity.

In addition, T17 cells are known to be formed through a process similar to the differentiation of Treg cells in the differentiation process of undifferentiated T cells. In other words, the differentiation of Treg cells and Th17 cells is commonly performed in the presence of TGF-β, whereas IL-6 is not required for Treg cells, while IL-6 is present along with TGF- To differentiate. In addition, differentiated Th17 cells are characterized by secretion of IL-17.

Th17 cells, unlike Treg cells, are involved in the forefront of the inflammatory response seen in immune diseases, thereby maximizing the signal of the inflammatory response and accelerating the progression of the disease. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of therapeutic agents for autoimmune diseases targeting the inhibition of Th17 cell activation has been greatly emphasized.

Immunosuppressants that block the signal transduction pathway in T cells are the most commonly used therapeutic agents for immune diseases. Such immunosuppressants are toxic, infectious, lymphoid, diabetes, tremor, headache, diarrhea, hypertension, nausea , And a disorder of renal function.

In addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, a method of regulating the amount of cytokine secreted from the immune cells and a method of using the antibody targeting the cytokine secreted from the immune cells have been developed . However, this method has a problem in that it takes much time to actually apply to patients through clinical experiments, and that the method using antibody is too expensive in the process of antibody production.

Therefore, it is urgent to develop a therapeutic agent and a treatment method for a new immune disease which has no side effects and is inexpensive and excellent in therapeutic effect.

1. International Patent Publication WO01053793A2

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of an immunological disease comprising RIPK (receptor-interacting serine / threonine protein kinase) inhibitor as an active ingredient.

It is still another object of the present invention to provide a composition for inhibiting bone loss comprising RIPK1 (receptor-interacting serine / threonine protein kinase 1) inhibitor as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating immunodeficiency diseases comprising RIPK3 (receptor-interacting serine / threonine protein kinase 3) inhibitor as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating immune diseases comprising RIPK (receptor-interacting serine / threonine protein kinase) inhibitor as an active ingredient.

In one embodiment of the invention, the RIPK inhibitor may be a RIPK1 inhibitor or a RIPK3 inhibitor.

In one embodiment of the present invention, the RIPK1 receptor-binding serine / threonine protein kinase 1 (RIPK1) inhibitor is a substance that specifically binds to the RIPK1 gene and inhibits the expression of RIPK1 or may specifically bind to the RIPK1 protein, Or the like.

In one embodiment of the present invention, the RIPK1 may be a nucleic acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the protein of RIPK1 may comprise the amino acid sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the substance that inhibits the expression of RIPK1 may be an antisense oligonucleotide, siRNA or shRNA for the gene of RIPK1.

In one embodiment of the present invention, the substance that specifically binds to the protein of RIPK1 and inhibits the activity of RIPK1 may be an antibody or a compound against RIPK1.

In one embodiment of the present invention, the compound may be Necrostatin 1 or Necrostatin 1s.

In one embodiment of the present invention, the receptor-interacting serine / threonine protein kinase 3 (RIPK3) inhibitor is a substance that specifically binds to the RIPK3 gene and inhibits the expression of RIPK3 or may specifically bind to the protein of RIPK3, Or the like.

In one embodiment of the present invention, the RIPK3 comprises a nucleic acid sequence of SEQ ID NO: 3.

In one embodiment of the present invention, the protein of RIPK3 may comprise the amino acid sequence of SEQ ID NO: 4.

In one embodiment of the present invention, the substance that inhibits the expression of RIPK3 may be an antisense oligonucleotide, siRNA or shRNA for the gene of RIPK3.

In one embodiment of the present invention, the substance that specifically binds to the protein of RIPK3 and inhibits the activity of RIPK3 may be an antibody or a compound against RIPK3.

In one embodiment of the present invention, the compound may be GSK'872.

In one embodiment of the present invention, the immunological disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, asthma, dermatitis, psoriasis, cystic fibrosis, It is known that post transplantation late and chronic solid organ rejection, Mltiple Sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma inflammatory bowel disease, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pμlmonary fibrosis, autoimmune diabetes mellitus, autoimmune diabetes mellitus, Diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, restenosis after angioplasty (Post-angioplasty resteno systolic heart disease, chronic obstructive pulmonary diseases (COPD), Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, Angina, cancer metastasis, small artery disease, graft-versus-host disease, or mitochondrial related syndrome.

The present invention also provides a pharmaceutical composition for inhibiting bone loss comprising RIPK1 (receptor-interacting serine / threonine protein kinase 1) inhibitor as an active ingredient.

In one embodiment of the present invention, the RIPK1 inhibitor may be Necrostatin 1 or Necrostatin 1s.

In one embodiment of the present invention, the RIPK1 inhibitor may have an effect of inhibiting osteoclast formation and differentiation.

In one embodiment of the present invention, the disease that can be treated through bone loss suppression may be osteoporosis, Paget's bone disease, rickets, osteomalacia or degenerative bone disease, but is not limited thereto.

The present invention also provides a pharmaceutical composition for the prevention or treatment of immunodeficiency diseases comprising RIPK3 (receptor-interacting serine / threonine protein kinase 3) inhibitor as an active ingredient.

In one embodiment of the present invention, the RIPK3 inhibitor may be GSK'872.

In one embodiment of the present invention, the immunodeficiency disease is a graft rejection reaction of an organ or tissue; Xenotransplant rejection of an organ or tissue; Or allograft rejection.

The present invention also relates to a method for screening a candidate substance for a cell or tissue comprising the step of treating a candidate substance to a cell or tissue comprising the RIPK1 gene or the RIPK1 protein; And measuring the expression level of the RIPK1 gene or the amount of the RIPK1 protein or the activity of the RIPK1 protein.

The present invention demonstrates that the RIPK inhibitor can be used as a new therapeutic agent for an immunological disease. When the RIPK inhibitor is treated in an animal model in which an immune disease has been induced, symptoms of the disease are alleviated and improved, and inflammatory cytokines and diseases The production of human cytokines was inhibited while the production of anti-inflammatory cytokines was increased. Therefore, the present invention is effective in discovering RIPK as a new target for the treatment of immune diseases and utilizing RIPK inhibitor as a new therapeutic agent.

FIG. 1 shows the results of immunohistochemical staining for the amount of RIPK1 expression in the joint tissues of a normal mouse and collagen-induced arthritis animal model (CIA, collagen-induced arthritis).
FIG. 2 shows the results of microarray analysis of the amount of RIPK1 expression in synovial tissues obtained from patients with normal donors, osteoarthritis, and rheumatoid arthritis.
FIG. 3 shows the results of ELISA analysis of the expression levels of IL-17 (a), IFN-y (b) and IL-10 (c) cytokines by RIPK1 inhibitor treatment in spleen cells of anti- .
FIG. 4 shows the results of ELISA analysis of the expression levels of TNF-a (a) and IL-10 (b) cytokines by RIPK1 inhibitor treatment in splenocytes of LPS (lipopolysaccharide) stimulated mice.
FIG. 5 is an analysis of the effect of arthritis treatment by RIPK1 inhibitor treatment on an animal model of collagen-induced rheumatoid arthritis. (A) is an analysis of arthritis score, (b) is an analysis of incidence of arthritis It is.
Figure 6 shows the expression levels of CII specific IgG (a), CII specific IgG1 (b) and CII specific IgG2a (c) in RANK1 inhibitor treated collagen-induced rheumatoid arthritis animal model, As shown in FIG.
FIG. 7 shows the results of MTT assay for cytotoxicity of RIPK1 inhibitor.
FIG. 8 is a graph showing the results of analysis of the number of Th17 cells by FACS (Fluorescence-activated cell sorting), which is an analysis of the inhibitory effect of RIPK1 inhibitor treatment on the differentiation of Th17 cells.
FIG. 9 shows the results of counting the number of stained TRAP + cells and stained TRAP + cells by analyzing the inhibitory effect of osteoclast differentiation according to RIPK1 inhibitor treatment.
FIG. 10 shows the results of ELISA analysis of the expression levels of IL-17 (a) and TNF-α (b) cytokines according to treatment with RIPK1 inhibitor in splenocytes of mice stimulated with anti-CD3 or LPS (lipopolysaccharide) will be.
FIG. 11 shows the expression of TNF-α (a) and IL-17 (b) cytokines in human peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 by RIPK1 inhibitor treatment by ELISA .
FIG. 12 shows the results of ELISA analysis of the expression level of IL-17 cytokine by RIPK3 inhibitor treatment in anti-CD3 stimulated mouse spleen cells.
FIG. 13 shows the result of measuring the body weight of a mouse to examine the inhibitory effect of RIPK3 inhibitor on inflammatory bowel disease in a mouse (B6) model of inflammatory bowel disease. WT: control group; IBD: group not treated with RIPK3 inhibitor as an inflammatory bowel disease animal model; And RIPK3 inhibitor: a group treated with RIPK3 inhibitor.
FIG. 14 shows the results of measuring the degree of proliferation of T cells in order to examine the inhibitory effect of RIPK3 inhibitor on alloresponse.

In the present invention, RIPK1 (receptor-interacting serine / threonine protein kinase 1) is involved in the delivery of cell-death signal (programmed necrosis) by pathogen recognition receptor (PRRS) and DNA damage and death receptor ligation.

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and is capable of binding to a complementary sequence in mRNA, It acts to inhibit. The antisense sequence of the present invention refers to a DNA or RNA sequence complementary to the mRNA of the gene and capable of binding to the mRNA, and the translation, mRNA translocation, maturation, or all other overall biological It is possible to inhibit the essential activity for the function.

In addition, the antisense nucleic acid can be modified at one or more bases, sugars, or backbone locations to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol ., 5, 3, 343-55, 1995). The nucleic acid backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic biantennary bond, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. The antisense nucleic acid may comprise a modified base. Modified bases include hypoxanthane, 6-methyladenine, 5-methylpyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, genentioyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine . In addition, the antisense nucleic acid of the present invention may be chemically combined with one or more moieties or conjugates that enhance the activity and cytotoxicity of the antisense nucleic acid. A cholesterol moiety, a cholesteryl moiety, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, hexylamino- And liposoluble moieties such as Cole sterol moieties. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nuclease and increase the binding affinity of the antisense nucleic acid with the target mRNA.

The antisense oligonucleotides may be synthesized in vitro in a conventional manner and administered in vivo or may allow the synthesis of antisense oligonucleotides in vivo. An example of synthesizing an antisense oligonucleotide in a test tube is to use RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

In the present invention, "RNAi" refers to RNA interference, and in Korean, it means RNA interference. RNA interference is a specific gene suppression phenomenon that is well conserved among most organisms. It is thought to be a type of gene monitoring mechanism used by cells to suppress defenses against transfection, to inhibit transposon, or to remove abnormal mRNA. In particular, gene suppression by small RNA is referred to as RNA interference in a broad sense, and RNA interference in the narrow sense implies mRNA degradation by siRNA. RNA interference also implies a technique for gene silencing using siRNA.

The term "siRNA" in the present invention means a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Publication Nos. 00/44895, 01/36646, 99/32619, 01/29058, 99/07409 and 00/44914). siRNA is provided as an efficient gene knock-down method or gene therapy method since it can inhibit the expression of a target gene.

The siRNA molecule of the present invention may have a structure in which a sense strand (a sequence corresponding to the mRNA sequence of the marker gene) and an antisense strand (a sequence complementary to the mRNA sequence) are positioned on opposite sides to form a double strand, , The siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands. Furthermore, siRNAs are not limited to the complete pairing of double-stranded RNA moieties in RNA pairs, but may be mated by mismatch (corresponding bases are not complementary), bulge (no bases corresponding to one strand) And a portion that is not achieved may be included. In addition, the siRNA terminal structure can be blunt or cohesive at both ends, if the expression of the marker gene can be inhibited by the RNAi effect, and the adhesive terminal structure has a 3'-terminal protruding structure and a 5'- Both end protruding structures are possible.

In addition, the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence is Thereby forming a hairpin structure, which in turn forms a stem-and-loop structure. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

Methods for preparing siRNA include a method of directly synthesizing siRNA in a test tube, introducing the siRNA into a cell through transformation, and a method of expressing siRNA expression vector or PCR-derived siRNA expression cassette into a cell There is a way to convert or infect.

In addition, the composition of the present invention comprising a gene-specific siRNA may include an agent that promotes intracellular infiltration of siRNA. Agents that promote intracellular inflow of siRNA can generally include agents that promote nucleic acid inflow. Examples of such agents include liposomes or one lipophilic one of a number of sterols, including cholesterol, cholate, and deoxycholic acid Can be combined with the carrier. It is also possible to use poly-L-lysine, spermine, polysilazane, polyethylenimine (PEI), polydihydroimidazolenium, a cationic polymer such as polyallylamine and chitosan may be used or a succinylated PLL, a succinylated PEI, polyglutamic acid, Anionic polymers such as polyaspartic acid, polyacrylic acid, polymethacylic acid, dextran sulphate, heparin, hyaluronic acid, and the like, Can be used.

In the present invention, the antibody can be indirectly coupled (for example, covalently bonded) to an existing therapeutic agent directly or through a linker or the like. Therapeutic agents capable of binding to the antibody include, but are not limited to, radionuclides such as 131I, 90Y, 105Rh, 47Sc, 67Cu, 212Bi, 211At, 67Ga, 125I, 186Re, 188Re, 177Lu, 153Sm, 123I, ); A biologically reactive variant or drug, such as methotrexate, adriamycin, and lympokine, such as interferon; Toxins such as lysine, avrine, diphtheria and the like; Heterofunctional antibodies, i.e., antibodies that bind to other antibodies to bind the complex to both cancer cells and effector cells (e. G., K cells such as T cells) Linked or non-conjugated antibody.

The bone supports the body's soft tissues and body weight and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the human body that not only supports the muscles and organs but also stores calcium and other essential minerals, such as phosphorus and magnesium, in the body. Thus, adult bones do not stop growing until the day they die, and the old bones are removed and replaced with new bones. This is called bone remodeling. Such bone reshaping is essential to restore fine bone damage caused by growth and stress and to maintain bone function properly.

It is known that two kinds of cells are involved in bone remodeling. One of the cells is osteoblast that produces bone, and the other is osteoclast that destroys bone. Osteoblasts produce RANKL (receptor activator of NF-κB ligand) and OPG (osteoprotegerin), its decoy receptor. When RANKL binds to RANK (receptor activator of NF-κB), which is a receptor on the surface of osteoclast progenitor cells, osteoclast precursor cells are differentiated into osteoclasts, . In detail, the expression of nuclear factor-activated T cells (NFAT), which is known as a key transcription factor that regulates osteoclast differentiation in osteoclast precursor cells by intracellular calcium signaling by RANKL stimulation, Activation of extracellular signal-regulated kinase (ERK), NF-κB, p38 MAP kinase, etc., induces autoamplification of NFAT, This is what will last.

Two types of cytokines such as NF-κB ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF) are essential for osteoclast generation. These cytokines (KOREAN J. FOODSCI. TECHNOL. 40 (6), 674-679 (2008)). In addition, TRAP, which is used as an osteoclast cell marker, is selectively expressed only in activated osteoclasts such as Kappuchin K, which is selectively distributed in osteoclasts and causes bone resorption, and MMP-9, which plays an important role in osteoclast migration do.

The composition according to the present invention can be used as a pharmaceutical composition capable of preventing and treating an immune disease, a bone disease that may be caused by bone loss, and an immunodeficiency disease. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier As shown in FIG. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention, together with a pharmaceutically acceptable carrier as described above, may be formulated into a suitable form according to a method known in the art. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods, and isotonic aqueous solutions or suspensions are preferred for injection formulations typical of parenteral administration formulations. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. Formulations for oral administration also include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the route of administration, subject to be administered, age, gender, individual difference, and disease state. Preferably, the pharmaceutical composition of the present invention may contain the active ingredient in an amount of 1 to 10000 μg / kg body weight / day, more preferably 10 to 1000 mg / kg body weight, / day, which may be repeated several times a day. The composition of the present invention may also be administered in combination with a known compound having an effect of preventing, ameliorating or treating an immunological disease.

The present invention also relates to a method for screening a candidate substance for a cell or tissue comprising the step of treating a candidate substance to a cell or tissue comprising the RIPK1 gene or the RIPK1 protein; And measuring the expression level of the RIPK1 gene or the amount of the RIPK1 protein or the activity of the RIPK1 protein.

The method according to the present invention is a method for discovering a therapeutic agent for a new immune disease disease using RIP-1, wherein the "candidate substance" refers to the amount of the RIPK1 gene, the amount of the RIPK1 protein or the activity of the RIPK1 protein Or < RTI ID = 0.0 > a < / RTI > The sample may include, but is not limited to, compounds, proteins, peptides, oligonucleotides or natural extracts.

The treatment of the candidate substance may be preferably performed by treating the candidate substance with cells or tissue containing the RIPK1 gene or RIPK1 protein and then culturing the candidate substance.

In addition, the intracellular level increase of RIPK1 protein means that the expression of RIPK1 gene is increased or the degradation of RIPK1 protein is inhibited and the amount of RIPK1 protein is increased. The RIPK1 gene expression includes transcription of the RIPK1 gene and translation into a protein. Therefore, when the candidate substance promotes the expression of the RIPK1 gene and the protein level in the cell, the candidate substance can be regarded as a new therapeutic agent for an immune disorder.

The level of the expression level of the gene is preferably a level of mRNA. The level of the mRNA may be measured by RT-PCR, RT-PCR, RNase protection assay, Blots, and DNA chips, but are not limited thereto.

In this case, the marker protein in the biological sample and the antibody specific thereto form a conjugate, that is, an antigen-antibody complex, and the antigen-antibody complex Can be quantitatively measured through the magnitude of the signal of the detection label. Such detection labels can be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules and radioisotopes, but is not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoprecipitation, Oucheroni immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, Complement fixation assay, FACS, and protein chip.

As described above, various methods known in the art may be used to measure the expression level of the RIPK1 gene, the amount of the RIPK1 protein, or the activity of the RIPK1 protein. Preferably, the RT- -PCR), real-time RT-PCR, coimmunoprecipitation, enzyme-linked immunosorbent assays, radioimmunoassay (RIA), immunohistochemistry, Western blotting and flow cytometry FACS) can be used.

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.

Example  1. In normal and arthritic tissues RIPK1 (Receptor-interacting serine / threonine-protein kinase 1)

The present inventors analyzed the expression level of RIPK1 in mouse and arthritis-induced joint tissues by immunohistochemical staining in order to analyze changes in expression of RIPK1 in normal and disease-induced conditions.

First, arthritis-induced mice were injected with 100 μg of CII per mouse in a volume of 50 μl on a tail base by mixing 1: 1 type II collagen (CII) and complete Freund's adjuvant (CFA) in DBA / 1J mice. Two weeks later, mice were induced to arthritis by injecting a mixture of CII and CFA at a ratio of 1: 1 in a volume of 100 μg / 50 μl in a secondary injection, and the normal mice used DBA / 1J mice not treated with CII .

The joint tissue obtained from normal mice and arthritis-induced mice was fixed with 10% formalin, the calcareous material was removed from the bones, embedded in paraffin, and the tissue was adhered to the slides with a thickness of 7 μm. Prior to immunohistochemical staining, xanthrene was passed through a deparaffinization process, and ethanol was allowed to function from a high concentration to a low concentration. At this time, to detect the expression level of RIPK1, immunostaining was performed using an antibody against RIPK1 (Santa Cruz, SC-7881), and then the expression amount of protein was analyzed by optical microscope.

 As a result, it was confirmed that the expression of RIPK1 was remarkably increased in the joint tissues in which rheumatoid arthritis was induced compared to the joint tissues of normal mice (Fig. 1).

Example  2. Osteoarthritis and Rheumatism  Of synovial tissue in patients with arthritis RIPK1  Expression level analysis

Based on the fact that the expression of RIPK1 is increased in comparison with that of normal during the induction of the immune disease, the results of Example 1 above show that, in order to confirm the same clinical results, The expression level of RIPK1 was analyzed by microarray analysis on synovial tissues obtained from patients with rheumatoid arthritis.

As a result, it was confirmed that the expression level of RIPK1 was actually increased when osteoarthritis and rheumatoid arthritis were developed compared to normal subjects (Fig. 2).

Example  3. RIPK1  Modulatory effects of inflammatory cytokines and anti-inflammatory cytokines by inhibitors

As a result of confirming that RIPK1 is associated with immunological disease induction, the inventors of the present invention have found that the inhibitory effect of RIPK1 on the immune diseases can be improved by controlling the inflammatory cytokine and anti-inflammatory cytokine Respectively.

The splenocytes isolated from C57BL / 6 normal mice stimulated with anti-CD3 T-cell activation conditions were treated with 1 μM and 10 μM of RIPK1 inhibitor Necrostatin-1 (Selleckchem, S8037) , And then the culture solution was collected to determine the expression level of IL-17, a representative pathological cytokine, and IFN-γ, an inflammatory cytokine, and IL-10, an anti-inflammatory cytokine, through ELISA analysis. Specifically, for the ELISA analysis, 96-well plates were coated with 2 μg / ml of anti-IL-17, anti-IFN-γ and anti-IL- ), And then IL-17, IFN-γ and IL-10 recombinants were serially diluted by ½ and used as a standard reagent. The cell culture supernatant was added thereto, and the mixture was incubated at room temperature And reacted for 2 hours. Subsequently, biotinylated anti-IL-17, anti-IFN-γ and anti-IL-10 were reacted at room temperature for 2 hours, washed three times and then diluted with ExtraAvidin-Alkaline Phosphatase conjugate. For 2 hours. The control group was stimulated with anti-CD3 only and treated without any treatment.

As a result, the expression of the pathogenic cytokines (IL-17) and inflammatory cytokines (IFN-γ) in the RIPK1 inhibitor-treated group in the T cell-activated condition was lower than that in the RIPK1 inhibitor- (IL-10) with anti-inflammatory activity was increased (FIG. 3).

Therefore, it has been confirmed that a compound inhibiting RIPK1 can be used as a therapeutic agent for treating immune diseases.

Example  4. In macrophage activation conditions RIPK1  Regulatory Analysis of Inflammatory Cytokines and Anti-Inflammatory Cytokines by Inhibitors

We treated the RIPK1 inhibitor Necrostatin-1 (Selleckchem, S8037) at a concentration of 1 μM and 10 μM, respectively, on splenocytes isolated from C57BL / 6 normal mice stimulated with LPS (lipopolysaccharide) After culturing for 48 hours, the culture solution was collected and the expression level of IL-10, which is a typical inflammatory cytokine, TNF-α and anti-inflammatory cytokine, was confirmed by ELISA analysis. Specifically, for the ELISA analysis, anti-TNF-α and anti-IL-10 were coated at 2 μg / ml in a 96-well plate and then non-specific binding was blocked with a blocking solution (1% BSA / PBST) after the reaction. Then, the TNF-α and IL-10 recombinants were serially diluted by ½ and used as a standard. Cell culture supernatants were added and reacted at room temperature for 2 hours. Then, biotinylated anti-TNF-α and anti-IL-10 were reacted at room temperature for 2 hours and washed three times. ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. Control groups were stimulated with LPS alone and treated without any treatment.

As a result, the group treated with RIPK1 inhibitor significantly inhibited the production of TNF-α, an LPS-induced inflammatory cytokine, compared with the untreated group, while the macrophage- IL-10 < / RTI > (FIG. 4). In other words, RIPK1 inhibitor inhibited the production of inflammatory cytokines but did not inhibit the production of anti-inflammatory cytokines.

Example  5. Rheumatism  Targeted Arthritis Animal Model RIPK1  Analysis of treatment effect by inhibitor

The following experiment was carried out to confirm that the RIPK1 inhibitor is effectively administered to mouse animals induced with an autoimmune disease. The following experiment was carried out: IL-10 knockout to produce arthritis- Mice were injected with CIA (collagen-induced arthritis) as a Type II collagen to produce an arthritis-induced mouse model. Three times a week intraperitoneal RIPK1 inhibitor Necrostatin-1 (Selleckchem, S8037) was administered 10 days after the first immunization 0.02 mg / mouse, and arthritis index was analyzed.

The evaluation criteria for arthritis are as follows.

 -Evaluation standard-

   0 point: No edema or swelling.

   1 point: mild swelling and redness limited to the foot or ankle joint

   2 points: Mild swelling and redness from ankle joint to metatarsal

   3 points: Moderate edema and redness from ankle joint to foot

   4 points: with edema and redness across the entire ankle

   5 points or more: severe symptoms of edema and redness continue

As a result, when arthritis-induced mice were administered RIPK1 inhibitor, the arthritis score was remarkably inhibited compared to the group not administered RIPK1 inhibitor, and the incidence of arthritis was 2.5 times or more (Fig. 5).

Thus, through these results, it has been found that the present inventors can actually treat immune diseases by inhibiting the activity of RIPK1.

Example  6. RIPK1  Analysis of Antibody Modulating Effect by Inhibitor

Blood was collected from the mice administered with the RIPK1 inhibitor for 4 weeks and the serum was separated again. The amount of expression of CII specific IgG, CII specific IgG1 and CII specific IgG2a was measured by ELISA Respectively.

As a result, the expression of CII specific IgG was decreased in the sera of mice treated with RIPK1 inhibitor, and the expression of CII specific IgG1 related to Th2 response was rather increased. In addition, the expression of CII specific IgG2a associated with the Th1 response was also reduced (Figure 6).

Therefore, RIPK1 inhibitors can increase the anti-inflammatory antibodies such as Th2 and inhibit the production of inflammatory antibodies such as Th1.

Example  7. RIPK1  Determination of cytotoxicity of inhibitor

The present inventors have conducted experiments to determine whether RIPK1 inhibitors cause cytotoxicity. Specifically, Necrostatin 1, a RIPK1 inhibitor, was treated with 0.1 μM, 1 μM and 10 μM, respectively, in normal mouse splenocytes, and cultured for 3 days. MTT powder (Thiazolyl Blue Tetrazolium Bromide) mg / ml and treated with 50 μl of each solution for 4 hours. After centrifugation at 1300 rpm, 5 minutes and 4 ° C, 120 μl of the supernatant was removed, 50 μl of DMSO was added, and the absorbance was measured at 540 nm.

As a result, it was confirmed that cytotoxicity was not induced in all the groups treated with RIPK1 inhibitor, and cytotoxicity was not induced in all the groups treated with 0.1 μM, 1 μM, and 10 μM concentrations (FIG. 7).

Example  8. RIPK1  By inhibitor Th17  Inhibition effect of cell differentiation

Experiments were conducted to confirm whether the inhibitory effect of RIPK1 inhibitor on the differentiation of Th17 cells was observed. Specifically, anti-CD3, anti-CD28, anti-IFN-γ, anti-IL-4, IL-6 and TGF-β were treated in splenocytes of normal mice to induce differentiation of Th17 cells , RIPK1 inhibitor Necrostatin 1 was treated with 10 μM, and cells were cultured for 3 days. Cells were collected and stained using anti-mouse CD4 PerCP-Cyanine 5.5 and anti-mouse IL-17 FITC antibody. FACS (Fluorescence-activated cell sorting, BD FACS calibur ^™ ) Respectively.

As a result, it was confirmed that the treatment with RIPK1 inhibitor inhibited the differentiation into Th17 cells as compared with the control group (Fig. 8).

Example  9. RIPK1  Inhibitory effect of inhibitor on osteoclast differentiation

The present inventors conducted an experiment to confirm whether osteoclast differentiation is inhibited by RIPK1 inhibitor. Specifically, IL-10 knockout mice were induced to induce arthritis-induced mouse CIA by Type II collagen. After induction of RIPK-1 inhibitor (Necrostatin 1 ) At 20 mg / kg and 10 mg / kg. At the end of the experiment, 50 ng / ml RANKL (Receptor activator of nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor ), And then treated with 1 μM, 10 μM of RIPK1 inhibitor Necrostatin 1 and then examined for TRAP (Tartrate-resistant acid phosphatase) -positive cells.

As a result, in the osteoclastogenesis differentiation experiment for observing osteoclast differentiation control ability of RIPK-1 inhibitor, TRAP-positive cells associated with the bone destruction mechanism were found to be dose-dependently reduced when RIPK-1 inhibitor was treated ).

Therefore, it was found that RIPK1 inhibitor has an effect of inhibiting osteoclast differentiation and is effective in the treatment of bone diseases.

Example  9. RIPK1  Regulatory Analysis of Pathogenic Cytokines and Inflammatory Cytokines by Inhibitors

The inventors of the present invention have recently found that splenocytes isolated from C57BL / 6 normal mice stimulated with LPS (lipopolysaccharide) stimulating or macrophage activation conditions with T-cell activation condition, anti-CD3, induce RIPK1 inhibitor Necrostatin-1s (Calbiochem, 504297 ) Were cultured at a given concentration for 48 hours. Cultures were collected and the expression level of IL-17, an etiologic cytokine, and TNF-α, an inflammatory cytokine, was confirmed by ELISA analysis. Specifically, for ELISA analysis, anti-IL-17 and anti-TNF-α were coated at 2 μg / ml in a 96-well plate and non-specific binding was blocked with a blocking solution (1% BSA / PBST) . Then, IL-17 and TNF-α recombinants were serially diluted by ½ and used as a standard. Cell culture supernatant was added and reacted at room temperature for 2 hours. Then, biotinylated anti-IL-17 and anti-TNF-α were reacted at room temperature for 2 hours and washed three times. ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. Control groups were stimulated only with anti-CD3 or LPS and treated without any treatment.

As a result, it was confirmed that the group treated with the RIPK1 inhibitor (Necrostain 1s) had an inhibitory effect on the expression level of IL-17, which is a pathological cytokine, and TNF-a, an inflammatory cytokine, .

Example  10. RIPK1  Regulatory Analysis of Inflammatory Cytokines and Anti-Inflammatory Cytokines by Inhibitors

The present inventors treated RIPK1 inhibitor Necrostatin-1s (Calbiochem, 504297) with anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC) at a given concentration and then cultured for 48 hours , And the amount of IL-10 expressed as an inflammatory cytokine, TNF-α and anti-inflammatory cytokine, was determined by ELISA analysis. Specifically, for ELISA analysis, anti-TNF-α and anti-IL-10 were coated at 2 μg / ml on a 96-well plate and non-specific binding was blocked with a blocking solution (1% BSA / PBST) . Then, the TNF-α and IL-10 recombinants were serially diluted by ½ and used as a standard. Cell culture supernatants were added and reacted at room temperature for 2 hours. Then, biotinylated anti-TNF-α and anti-IL-10 were reacted at room temperature for 2 hours and washed three times. ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. The control group was stimulated with anti-CD3 only and treated without any treatment.

As a result, the group treated with RIPK1 inhibitor (Necrostain 1s) showed an inhibitory effect of TNF-α, which is an inflammatory cytokine, and IL-10, which is an anti-inflammatory cytokine, (Fig. 11).

Example  11. RIPK3  Regulatory Analysis of Pathogenic Cytokines by Inhibitors

The present inventors treated GSK'872 (Calbiochem , 530389), a RIPK3 inhibitor, with splenocytes isolated from C57BL / 6 normal mouse stimulated with anti-CD3, which is a T cell activation condition, at a given concentration, And the amount of IL-17, which is a pathological cytokine, was determined by ELISA analysis. Specifically, for ELISA analysis, 96-well plates were coated with 2 μg / ml of anti-IL-17 first, and non-specific binding was blocked with the blocking solution (1% BSA / PBST) after the reaction. Then, IL-17 recombinant was serially diluted by ½ and used as a standard reagent. Cell culture supernatant was added and reacted at room temperature for 2 hours. Biotinylated anti-IL-17 was reacted at room temperature for 2 hours and washed three times. ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. The control group was stimulated with anti-CD3 only and treated without any treatment.

As a result, it was confirmed that the group treated with RIPK3 inhibitor significantly inhibited the expression level of IL-17, a pathogenic cytokine, compared with the group treated with RIPK3 inhibitor (FIG. 12).

Example  12. RIPK3  Inhibitory effect of inhibitor on inflammatory bowel disease

The present inventors tried to confirm the therapeutic effect by administering RIPK3 inhibitor to an animal model of mice induced inflammatory bowel disease. Specifically, an animal model of inflammatory bowel disease was prepared by adding 3% DSS (dextran sulfate sodium) to negative water of C57BL6 mice. Body weights were measured at the beginning of DSS drug administration, and the RIPK3 inhibitor, GSK'872 (Calbiochem , 530389), was administered orally.

As a result, it was confirmed that RIPK3 inhibitor did not regain body weight in the control (WT, wild type) but slightly increased in weight in inflammatory bowel disease mice (FIG. 13).

Example  13. RIPK3  Inhibition by Inhibitor ( Alloresponse ) Inhibitory effect

We sought to determine whether RIPK3 inhibitors inhibit rejection of tissue grafts. Specifically, the number of 1 × 10 ⁵ of the top in each well of the 96 well in the in vitro woman CD4 + T cell with 1 × 10 ⁵ of the radiation can was irradiated with a woman (homologous homozygous), or donors of (Balb / c, responder) (C57BL / 6, stimulator, allogeneic) derived T cells were removed and mixed culture was performed to induce homologous reaction. In addition, the RIPK3 inhibitor GSK'872 (Calbiochem , 530389) was treated with 1 μM and 10 μM together and cultured for 72 hours. The degree of T cell proliferation was examined by measuring the amount of 3H thymidine in the b-counter by treating 3H thymidine in the cultured cells 16 hours before culturing.

As a result, it was confirmed that RIPK3 inhibitor has an effect of inhibiting alloresponse as compared with the control group (Fig. 14).

Thus, it has been confirmed that RIPK3 inhibitors can effectively inhibit post-graft rejection and allogeneic transplantation of T cells, which may occur during transplantation, and successfully induce transplant-induced disease treatment.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Composition for preventing or treating immune diseases          comprising receptor interacting serine theronin protein kinase          inhibitor <130> PN1606-205 <150> KR 10-2015-0096322 <151> 2015-07-07 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 4096 <212> DNA <213> Homo sapiens <400> 1 agactgctcg tcaagtgtgg gaaaagctcc gtggcgtcac aagctactat ataaaaggcg 60 gtgcccgccg gggccgagtg ggagtccgcg gcgagcgcag cagcagggcc cggtcctgcg 120 cctcgggagt cggcgtccag gctcggagcg cgacacggag actaggtggc agggtacagc 180 tctgccgggg ggggaaaaag tggtaccatt ttgggcgttc ttgagcttca gaatgcaacc 240 agacatgtcc ttgaatgtca ttaagatgaa atccagtgac ttcctggaga gtgcagaact 300 ggacagcgga ggctttggga aggtgtctct gtgtttccac agaacccagg gactcatgat 360 catgaaaaca gtgtacaagg ggcccaactg cattgagcac aacgaggccc tcttggagga 420 ggcgaagatg atgaacagac tgagacacag ccgggtggtg aagctcctgg gcgtcatcat 480 agaggaaggg aagtactccc tggtgatgga gtacatggag aagggcaacc tgatgcacgt 540 gctgaaagcc gagatgagta ctccgctttc tgtaaaagga aggataattt tggaaatcat 600 tgaaggaatg tgctacttac atggaaaagg cgtgatacac aaggacctga agcctgaaaa 660 tatccttgtt gataatgact tccacattaa gatcgcagac ctcggccttg cctcctttaa 720 gatgtggagc aaactgaata atgaagagca caatgagctg agggaagtgg acggcaccgc 780 taagaagaat ggcggcaccc tctactacat ggcgcccgag cacctgaatg acgtcaacgc 840 aaagcccaca gagaagtcgg atgtgtacag ctttgctgta gtactctggg cgatatttgc 900 aaataaggag ccatatgaaa atgctatctg tgagcagcag ttgataatgt gcataaaatc 960 tgggaacagg ccagatgtgg atgacatcac tgagtactgc ccaagagaaa ttatcagtct 1020 catgaagctc tgctgggaag cgaatccgga agctcggccg acatttcctg gcattgaaga 1080 aaaatttagg cctttttatt taagtcaatt agaagaaagt gtagaagagg acgtgaagag 1140 tttaaagaaa gagtattcaa acgaaaatgc agttgtgaag agaatgcagt ctcttcaact 1200 tgattgtgtg gcagtacctt caagccggtc aaattcagcc acagaacagc ctggttcact 1260 gcacagttcc cagggacttg ggatgggtcc tgtggaggag tcctggtttg ctccttccct 1320 ggagcaccca caagaagaga atgagcccag cctgcagagt aaactccaag acgaagccaa 1380 ctaccatctt tatggcagcc gcatggacag gcagacgaaa cagcagccca gacagaatgt 1440 ggcttacaac agagaggagg aaaggagacg cagggtctcc catgaccctt ttgcacagca 1500 aagaccttac gagaattttc agaatacaga gggaaaaggc actgcttatt ccagtgcagc 1560 cagtcatggt aatgcagtgc accagccctc agggctcacc agccaacctc aagtactgta 1620 tcagaacaat ggattatata gctcacatgg ctttggaaca agaccactgg atccaggaac 1680 agcaggtccc agagtttggt acaggccaat tccaagtcat atgcctagtc tgcataatat 1740 cccagtgcct gagaccaact atctaggaaa tacacccacc atgccattca gctccttgcc 1800 accaacagat gaatctataa aatataccat atacaatagt actggcattc agattggagc 1860 ctacaattat atggagattg gtgggacgag ttcatcacta ctagacagca caaatacgaa 1920 cttcaaagaa gagccagctg ctaagtacca agctatcttt gataatacca ctagtctgac 1980 ggataaacac ctggacccaa tcagggaaaa tctgggaaag cactggaaaa actgtgcccg 2040 taaactgggc ttcacacagt ctcagattga tgaaattgac catgactatg agcgagatgg 2100 actgaaagaa aaggtttacc agatgctcca aaagtgggtg atgagggaag gcataaaggg 2160 agccacggtg gggaagctgg cccaggcgct ccaccagtgt tccaggatcg accttctgag 2220 cagcttgatt tacgtcagcc agaactaacc ctggatgggc tacggcagct gaagtggacg 2280 cctcacttag tggataaccc cagaaagttg gctgcctcag agcattcaga attctgtcct 2340 cactgatagg ggttctgtgt ctgcagaaat tttgtttcct gtacttcata gctggagaat 2400 ggggaaagaa atctgcagca aaggggtctc actctgttgc caggctggtc tcaaacttct 2460 ggactcaagt gatcctcccg cctcggcctt ccaaagtgct gggatatcag gcactgagcc 2520 actgcgccca gccaacaatc cgctctgagg aaagcgtaag caggaagacc tcttaatggc 2580 atagcaccaa taaaaaaatg actcctagtt gtgtttggaa agggagagaa gagatgtctg 2640 aggaaggtca tgttctttca gcttatggca tttcctagag ttttgttgaa gcaagaagaa 2700 aaactcagag aatataaaat caacttttaa aattgtgtgc tctcttcttc acgtaggctc 2760 ctgttaaaaa caaagtgcag tcagattcta agccctgttc agagacttcg tggatcacag 2820 ctgcagctca ccgccacatc acaggatccg ttaacgttaa tacccaatac tctgtcagcc 2880 actgtaggct ctaagaacca cgtgcagtct tcagcccatt aaattatcga ttatttttta 2940 atgaattgaa tttatattga gtcttcaaat taactgaatg gatttaaagg ggtaccaagg 3000 aggggggaaa catcagaatt tcccaggcag ttgttgcaag gaattggtac taaccgtgac 3060 tacaacaaaa attcttgatt gacttttaaa gttatttcct ggcattctgg taccttcacc 3120 cagcctgagt gccctggaga gggaacagga aatgctgatc tctacccctg ggtgagacca 3180 gaacctcagg gctgatactg ttgagtggct tcctcggttt actctgtgta ctgtgaaagt 3240 attttcatat tttttctgtg tgccagagtg aaaaaggaca gcttctgagt gtggtaattg 3300 tgcctctagc acccagcctt tcaaagccca cctgaaacct gggggtggat gaaagaacta 3360 gaatagaaga ctgaagctgg gtaggccgct cagtgtccac tggcattttg ctaaaccgac 3420 aaggaaggct gtgtgcttag ctctccccag agggagggcg agaagggtgt ggtgatggtc 3480 aatctggctg tcggaacaga ttctggtgtc ttgggctgat aacagtgttg ttgattctga 3540 ttgtgaatcc cctcaactct agcagacaca tacacacccc tgaaatgggg ctgcagagca 3600 ggctgtctca gccttgccac tgtcggcatc tcggcctggg taattctgtt gtggggactg 3660 tcctgttcct tgtaggatgt ttagtagcat ccctgccccc acctactaga tgccaggggc 3720 actgttctcc ccagcccccc gccccagttg tgacaatagt ctctaaacat tgtcaaatgg 3780 tccaaggaaa ggggaaaatt gccccggttg agaagagcac tgctgtaaag taatgagcct 3840 cggctctcct gtctgcacct gtccggttac tacttggcca ccacgcagcc ttggctccta 3900 cagcccaaaa gggagaatgg agggaggctc caggctttgc tggaggggcc tgggtgagtt 3960 ctgtttgctc cttgtaccac catccaaatg gtgttatcaa atctcttaga ttccaaagag 4020 gttgaataat taatgttcaa aggcaagagg gcaaggcatt ttttaacact ttttaaaata 4080 aaaatttata ccacaa 4096 <210> 2 <211> 671 <212> PRT <213> Homo sapiens <400> 2 Met Gln Pro Asp Met Ser Leu Asn Valle Lys Met Lys Ser Ser Asp   1 5 10 15 Phe Leu Glu Ser Ala Glu Leu Asp Ser Gly Gly Phe Gly Lys Val Ser              20 25 30 Leu Cys Phe His Arg Thr Gln Gly Leu Met Ile Met Lys Thr Val Tyr          35 40 45 Lys Gly Pro Asn Cys Ile Glu His Asn Glu Ala Leu Leu Glu Glu Ala      50 55 60 Lys Met Met Asn Arg Leu Arg His Ser Arg Val Val Lys Leu Leu Gly  65 70 75 80 Val Ile Ile Glu Glu Gly Lys Tyr Ser Leu Val Met Glu Tyr Met Glu                  85 90 95 Lys Gly Asn Leu Met His Val Leu Lys Ala Glu Met Ser Thr Pro Leu             100 105 110 Ser Val Lys Gly Arg Ile Ile Leu Glu Ile Ile Glu Gly Met Cys Tyr         115 120 125 Leu His Gly Lys Gly Val Ile His Lys Asp Leu Lys Pro Glu Asn Ile     130 135 140 Leu Val Asp Asn Asp Phe His Ile Lys Ile Ala Asp Leu Gly Leu Ala 145 150 155 160 Ser Phe Lys Met Trp Ser Lys Leu Asn Asn Glu Glu His Asn Glu Leu                 165 170 175 Arg Glu Val Asp Gly Thr Ala Lys Lys Asn Gly Gly Thr Leu Tyr Tyr             180 185 190 Met Ala Pro Glu His Leu Asn Asp Val Asn Ala Lys Pro Thr Glu Lys         195 200 205 Ser Asp Val Tyr Ser Phe Ala Val Val Leu Trp Ala Ile Phe Ala Asn     210 215 220 Lys Glu Pro Tyr Glu Asn Ala Ile Cys Glu Gln Gln Leu Ile Met Cys 225 230 235 240 Ile Lys Ser Gly Asn Arg Pro Asp Val Asp Asp Ile Thr Glu Tyr Cys                 245 250 255 Pro Arg Glu Ile Ile Ser Leu Met Lys Leu Cys Trp Glu Ala Asn Pro             260 265 270 Glu Ala Arg Pro Thr Phe Pro Gly Ile Glu Glu Lys Phe Arg Pro Phe         275 280 285 Tyr Leu Ser Gln Leu Glu Glu Ser Val Glu Glu Asp Val Lys Ser Leu     290 295 300 Lys Lys Glu Tyr Ser Asn Glu Asn Ala Val Val Lys Arg Met Gln Ser 305 310 315 320 Leu Gln Leu Asp Cys Val Ala Val Ser Ser Ser Arg Ser Ser Ser Ser                 325 330 335 Thr Glu Gln Pro Gly Ser Leu His Ser Ser Gln Gly Leu Gly Met Gly             340 345 350 Pro Val Glu Glu Ser Trp Phe Ala Pro Ser Leu Glu His Pro Gln Glu         355 360 365 Glu Asn Glu Pro Ser Leu Gln Ser Lys Leu Gln Asp Glu Ala Asn Tyr     370 375 380 His Leu Tyr Gly Ser Arg Met Asp Arg Gln Thr Lys Gln Gln Pro Arg 385 390 395 400 Gln Asn Val Ala Tyr Asn Arg Glu Glu Glu Arg Arg Arg Arg Val Ser                 405 410 415 His Asp Pro Phe Ala Gln Gln Arg Pro Tyr Glu Asn Phe Gln Asn Thr             420 425 430 Glu Gly Lys Gly Thr Ala Tyr Ser Ser Ala Ser Ser His Gly Asn Ala         435 440 445 Val His Gln Pro Ser Gly Leu Thr Ser Gln Pro Gln Val Leu Tyr Gln     450 455 460 Asn Asn Gly Leu Tyr Ser Ser His Gly Phe Gly Thr Arg Pro Leu Asp 465 470 475 480 Pro Gly Thr Ala Gly Pro Arg Val Trp Tyr Arg Pro Ile Pro Ser His                 485 490 495 Met Pro Ser Leu His Asn Ile Pro Val Pro Glu Thr Asn Tyr Leu Gly             500 505 510 Asn Thr Pro Thr Met Pro Phe Ser Ser Leu Pro Pro Thr Asp Glu Ser         515 520 525 Ile Lys Tyr Thr Ile Tyr Asn Ser Thr Gly Ile Gln Ile Gly Ala Tyr     530 535 540 Asn Tyr Met Glu Ile Gly Gly Thr Ser Ser Ser Leu Leu Asp Ser Thr 545 550 555 560 Asn Thr Asn Phe Lys Glu Glu Pro Ala Ala Lys Tyr Gln Ala Ile Phe                 565 570 575 Asp Asn Thr Thr Ser Leu Thr Asp Lys His Leu Asp Pro Ile Arg Glu             580 585 590 Asn Leu Gly Lys His Trp Lys Asn Cys Ala Arg Lys Leu Gly Phe Thr         595 600 605 Gln Ser Gln Ile Asp Glu Ile Asp His Asp Tyr Glu Arg Asp Gly Leu     610 615 620 Lys Glu Lys Val Tyr Gln Met Leu Gln Lys Trp Val Met Arg Glu Gly 625 630 635 640 Ile Lys Gly Ala Thr Val Gly Lys Leu Ala Gln Ala Leu His Gln Cys                 645 650 655 Ser Arg Ile Asp Leu Leu Ser Ser Leu Ile Tyr Val Ser Gln Asn             660 665 670 <210> 3 <211> 4016 <212> DNA <213> Homo sapiens <400> 3 gcgggactgt agaggcgcct ataagggaag ttgttcagtc aactcggaaa aagggtaaca 60 acccggaaag tagactcacc gtcttggtct agagactgac ccctgcacag acagacccct 120 tcccctctct gcgaaaggac caagccccag aagtcactcc atctcctacg gctcgcaatt 180 tccagaggcc ccctggcacc ttccagcctg atgtcgtgcg tcaagttatg gtgagtaggg 240 agtggcatgt cgatcgcagc ctccgacatt cacacccaga gagccccaca gactcgctca 300 gagaacgtct ttgagtccgg ggacggtggg ggccggcgag ggaggctggg gattgcagtg 360 agaacgccga gtcacagggg cgcggggagc agcgctgagg cacggcacgg ccggctagtg 420 ggcgacaccc ctctctcagg cccagcggtg cccccgcccc cttggtgtcc atcgaggaac 480 tggagaacca ggagctcgtc ggcaaaggcg ggttcggcac agtgttccgg gcgcaacata 540 ggaagtgggg ctacgatgtg gcggtcaaga tcgtaaactc gtgagtgacc ccggttgacc 600 ccagccgcaa tctggccaaa aaggtggagc cactgtgctc tggggcctga atggcgaagg 660 gagggatttt cccacgaccc cggtgcgact ccagctctct cctggagtgt aggaaggcga 720 tatccaggga ggtcaaggcc atggcaagtc tggataacga attcgtgctg cgcctagaag 780 gggttatcga gaaggtgaac tgggaccaag atcccaagcc ggctctggtg actaaattca 840 tggagaacgg ctccttgtcg gggctgctgc agtcccagtg ccctcggccc tggccgctcc 900 tttgccgcct gctgaaagaa gtggtgcttg ggatgtttta cctgcacgac cagaacccgg 960 tgctcctgca ccgggacctc aagccatcca acgtcctgct ggacccagag ctgcacgtca 1020 aggtcagctg gtctacaccc ctctcagcct caagacaagg ccccagagct gctctagcca 1080 ggcccgccgg acactagacc tccagagccc tgtctattga ataggcgtag cctctctctc 1140 tggacacagg gctgccaggg gacaaagccc agccaggagt tttagctcct gcctaccagg 1200 tgatagaggc cctgagccac acgagtcatt tccaccagct gccccaccag cccctccaaa 1260 gagtcatggc tgacccagcc ctggagactc agaccttgac ctagccctac cacaaggcac 1320 ctagatctac catccaaggg aacctcttcc cttgacctcc acactccctt tacaaagctg 1380 aacctgcacc ttctgggggc aggggggttc ctggaggctc tctggccccc ttgtcttccc 1440 tacctcccac ttctttcctt cctttgcagc tggcagattt tggcctgtcc acatttcagg 1500 gaggctcaca gtcagggaca gggtccgggg agccaggggg caccctgggc tacttggccc 1560 cagaactgtt tgttaacgta aaccggaagg cctccacagc cagtgacgtc tacaggtaag 1620 gatcccggcc caaacacaca gccaggattc ctacacttct ctgggccctc ccaggggatg 1680 atgtatagga aaggagtctt gccagtgatt ggaccgatcc tcagctttgt gtctcctgca 1740 gcttcgggat cctaatgtgg gcagtgcttg ctggaagaga agttgagtgt aagactctag 1800 gaggattctg ggatccttaa ccccaggtgc ccttctcttg aaagccccaa ggcttcccct 1860 tcccgctcac ctacttagac tgccctcctc cctcctcgtg cccccacatt acactccctg 1920 cctctttcca tgatgaagca gataattccc ttttaccctg aatgccttct cttctcaccc 1980 tcctccccta ctccagtgcc aaccgaacca tcactcgtgt acgaagcagt gtgcaacagg 2040 cagaccggc cttcattggc tgagctgccc caagccgggc ctgagactcc cggcttagaa 2100 ggactgaagg agctaatgca gctctgctgg agcagtgagc ccaaggacag accctccttc 2160 cagggtgagc tggccagtta gctggatcta ggataagctt gggtctgtct gcagggggct 2220 tttctcagga aaagaagatt tgctcacctg cactccactc tgctgtccct ctagaatgcc 2280 taccaaaaac tgatgaagtc ttccagatgg tggagaacaa tatgaatgct gctgtctcca 2340 cggtgagtgc caacatcacc cctccccagg agctggtcag ggatggcaca gcgtggctac 2400 ctggccatcc aatgactcac cactgaccac tgctttttat gcgttgacct ttgtacccac 2460 cgtccccagc ccacccctga gttcccagac aacagattat ggaccgagag actactccct 2520 tgggtttact ctggctcatc caagtcatac ccagcaaact cctcccatcc ctgcaggtaa 2580 aggatttcct gtctcagctc aggagcagca ataggagatt ttctatccca gagtcaggcc 2640 aaggagggac agaaatggat ggctttagga gaaccataga aaaccagcac tctcgtaatg 2700 atgtcatggt ttctgagtgg ctaaacaaac tgaatctaga ggagcctccc agctctgttc 2760 ctaaaaaatg cccgagcctt accaagagga gcagggcaca agaggagcag gttccacaag 2820 cctggacagc aggcacatct tcagattcga tggcccaacc tccccagact ccagagacct 2880 caactttcag aaaccagatg cccagcccta cctcaactgg aacaccaagt cctggacccc 2940 gagggaatca ggtgagacat tgacaggatt aggggatgca ctaagagtcc tataaatgct 3000 gtccccttgc cagtgaaaac ttggggctga gaggagtgca gtgggagtgc caggctccag 3060 ggcagaactg gatgctctgt gctgtttgca gggggctgag agacaaggca tgaactggtc 3120 ctgcaggacc ccggagccaa atccagtaac aggtacccat tctctacctc tttcttttcc 3180 ctgttcaatt cttccaccag actcctcctc cagacctata tctagattca gggtactttg 3240 tggaggggag gggagaagat gatctaactc aaccttctca tttcataaat gggaaaaccg 3300 acaccccctg agaaaggagc cctctgaggt cacacaacaa agcttggatt tgtccctgaa 3360 tcttctaact ttccacaaca ccagctctgt gagcccccta agttccattg tttcagcagt 3420 ccagaggacc tcccagcaaa aaagatcagg gtctcaaatg tcaattgcca ctctctagag 3480 cttctgctaa cacaattctg gagaggagct ctgaggggag gagcacaggg cacaccttga 3540 cacagccact gacaagtaga gacttcatac tccccaccca cccccaactt ggcctttctg 3600 acagctaaac cctcctgcca ccatcactat ttctcttaaa gggcgaccgc tcgttaacat 3660 atacaactgc tctggggtgc aagttggaga caacaactac ttgactatgc aacagacaac 3720 tgccttgccc acatggggct tggcaccttc gggcaagggg aggggcttgc agcacccccc 3780 accagtaggt tcgcaagaag gccctaaaga tcctgaagcc tggagcaggc cacagggttg 3840 gtataatcat agcgggaaat aaagcacctt ccaagcttgc ctccaagagt tacgagttaa 3900 ggaagagtgc caccccttga ggcccctgac ttccttctag ggcagtctgg cctgcccaca 3960 aactgacttt gtgacctgtc ccccaggagt caataaacat gatggaatgc tagtca 4016 <210> 4 <211> 518 <212> PRT <213> Homo sapiens <400> 4 Met Ser Cys Val Lys Leu Trp Pro Ser Gly Ala Pro Ala Pro Leu Val   1 5 10 15 Ser Ile Glu Glu Leu Glu Asn Gln Glu Leu Val Gly Lys Gly Gly Phe              20 25 30 Gly Thr Val Phe Arg Ala Gln His Arg Lys Trp Gly Tyr Asp Val Ala          35 40 45 Val Lys Ile Val Asn Ser Lys Ala Ile Ser Arg Glu Val Lys Ala Met      50 55 60 Ala Ser Leu Asp Asn Glu Phe Val Leu Arg Leu Glu Gly Val Ile Glu  65 70 75 80 Lys Val Asn Trp Asp Gln Asp Pro Lys Pro Ala Leu Val Thr Lys Phe                  85 90 95 Met Glu Asn Gly Ser Leu Ser Gly Leu Leu Gln Ser Gln Cys Pro Arg             100 105 110 Pro Trp Pro Leu Leu Cys Arg Leu Leu Lys Glu Val Val Leu Gly Met         115 120 125 Phe Tyr Leu His Asp Gln Asn Pro Val Leu Leu His Arg Asp Leu Lys     130 135 140 Pro Ser Asn Val Leu Leu Asp Pro Glu Leu His Val Lys Leu Ala Asp 145 150 155 160 Phe Gly Leu Ser Thr Phe Gln Gly Gly Ser Gln Ser Gly Thr Gly Ser                 165 170 175 Gly Glu Pro Gly Gly Thr Leu Gly Tyr Leu Ala Pro Glu Leu Phe Val             180 185 190 Asn Val Asn Arg Lys Ala Ser Thr Ala Ser Asp Val Tyr Ser Phe Gly         195 200 205 Ile Leu Met Trp Ala Val Leu Ala Gly Arg Glu Val Glu Leu Pro Thr     210 215 220 Glu Pro Ser Leu Val Tyr Glu Ala Val Cys Asn Arg Gln Asn Arg Pro 225 230 235 240 Ser Leu Ala Glu Leu Pro Gln Ala Gly Pro Glu Thr Pro Gly Leu Glu                 245 250 255 Gly Leu Lys Glu Leu Met Gln Leu Cys Trp Ser Ser Glu Pro Lys Asp             260 265 270 Arg Pro Ser Phe Gln Glu Cys Leu Pro Lys Thr Asp Glu Val Phe Gln         275 280 285 Met Val Glu Asn Asn Met Asn Ala Ala Val Ser Thr Val Lys Asp Phe     290 295 300 Leu Ser Gln Leu Arg Ser Ser Asn Arg Arg Phe Ser Ile Pro Glu Ser 305 310 315 320 Gly Gln Gly Gly Thr Glu Met Asp Gly Phe Arg Arg Thr Ile Glu Asn                 325 330 335 Gln His Ser Arg Asn Asp Val Met Val Ser Glu Trp Leu Asn Lys Leu             340 345 350 Asn Leu Glu Glu Pro Pro Ser Ser Val Pro Lys Lys Cys Pro Ser Leu         355 360 365 Thr Lys Arg Ser Ser Ala Gln Glu Glu Gln Val Pro Gln Ala Trp Thr     370 375 380 Ala Gly Thr Ser Ser Asp Ser Ala Gln Pro Pro Gln Thr Pro Glu 385 390 395 400 Thr Ser Thr Phe Arg Asn Gln Met Pro Ser Pro Thr Ser Thr Gly Thr                 405 410 415 Pro Ser Pro Gly Pro Arg Gly Asn Gln Gly Ala Glu Arg Gln Gly Met             420 425 430 Asn Trp Ser Cys Arg Thr Pro Glu Pro Asn Pro Val Thr Gly Arg Pro         435 440 445 Leu Val Asn Ile Tyr Asn Cys Ser Gly Val Gln Val Gly Asp Asn Asn     450 455 460 Tyr Leu Thr Met Gln Gln Thr Thr Ala Leu Pro Thr Trp Gly Leu Ala 465 470 475 480 Pro Ser Gly Lys Gly Arg Gly Leu Gln His Pro Pro Pro Val Gly Ser                 485 490 495 Gln Glu Gly Pro Lys Asp Pro Glu Ala Trp Ser Arg Pro Gln Gly Trp             500 505 510 Tyr Asn His Ser Gly Lys         515

Claims (22)

A pharmaceutical composition for the prevention or treatment of an immunological disease comprising RIPK (receptor-interacting serine / threonine protein kinase) inhibitor as an active ingredient. The method according to claim 1,
Wherein the RIPK inhibitor is a RIPK1 inhibitor or a RIPK3 inhibitor. 3. The method of claim 2,
The receptor-interacting serine / threonine protein kinase 1 (RIPK1) inhibitor is a substance that specifically binds to the RIPK1 gene and inhibits the expression of RIPK1 or specifically binds to the protein of RIPK1 to inhibit the activity of RIPK1 . The method of claim 3,
Wherein said RIPK1 comprises the nucleic acid sequence of SEQ ID NO: 1. The method of claim 3,
Wherein the protein of RIPK1 consists of the amino acid sequence of SEQ ID NO: 2. The method of claim 3,
Wherein the substance that inhibits the expression of RIPK1 is an antisense oligonucleotide, siRNA or shRNA for the gene of RIPK1. The method of claim 3,
Wherein the substance that specifically binds to the protein of RIPK1 and inhibits the activity of RIPK1 is an antibody or compound to RIPK1. 8. The method of claim 7,
Wherein said compound is Necrostatin 1 or Necrostatin 1s. 3. The method of claim 2,
The receptor-interacting serine / threonine protein kinase 3 (RIPK3) inhibitor is a substance that specifically binds to the RIPK3 gene and inhibits the expression of RIPK3, or specifically binds to the protein of RIPK3 to inhibit the activity of RIPK3 . 10. The method of claim 9,
Wherein said RIPK3 consists of the nucleic acid sequence of SEQ ID NO: 3. 10. The method of claim 9,
Wherein the protein of RIPK3 consists of the amino acid sequence of SEQ ID NO: 4. 10. The method of claim 9,
Wherein the substance that inhibits the expression of RIPK3 is an antisense oligonucleotide, siRNA or shRNA for the gene of RIPK3. 10. The method of claim 9,
Wherein the substance that specifically binds to the protein of RIPK3 and inhibits the activity of RIPK3 is an antibody or a compound against RIPK3. 14. The method of claim 13,
Wherein said compound is GSK'872. The method according to claim 1,
The immunological diseases are selected from the group consisting of rheumatoid arthritis, osteoarthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late chronic solid organ rejection, Mltiple Sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease Inflammatory bowel dieses, autoimmune diabetes, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, , Rhinitis, Ischemia-reperfusion injury, Post-angioplasty restenosis, Chronic obstructive heart disease (Ch coronary artery disease, angina, cancer metastasis, chronic obstructive pulmonary disease (COPD), Graves disease, gastrointestinal allergies, conjunctivitis, atherosclerosis, coronary artery disease, Aortic disease, graft-versus-host disease, and mitochondrial related syndrome. A pharmaceutical composition for inhibiting bone loss comprising RIPK1 (receptor interacting serine / threonine protein kinase 1) inhibitor as an active ingredient. 17. The method of claim 16,
Wherein the RIPK1 inhibitor is Necrostatin 1 or Necrostatin 1s. 17. The method of claim 16,
Wherein said RIPK1 inhibitor has the ability to inhibit osteoclast formation and differentiation. 17. The method of claim 16,
Wherein the disease that can be treated through bone loss inhibition is any one selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia and degenerative bone disease. A pharmaceutical composition for the prevention or treatment of immunodeficiency diseases comprising RIPK3 (receptor-interacting serine / threonine protein kinase 3) inhibitor as an active ingredient. 21. The method of claim 20,
Wherein said RIPK3 inhibitor is GSK'872. 21. The method of claim 20,
The immunodeficiency disease may include graft rejection of an organ or tissue; Xenotransplant rejection of an organ or tissue; Or allograft rejection.

Images