JP2008118870A - Method for screening agent for treating autoimmune disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for screening an anti-autoimmune disease agent. <P>SOLUTION: The method for screening the agent for preventing and/or treating autoimmune disease includes a step for selecting a test agent selectively modifying the expression of a UPR-related factor in a cell in the presence of ER stress. The anti-autoimmune disease agent contains the material selected by the screening method as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an anti-autoimmune disease agent. Furthermore, the present invention relates to a method for searching for and obtaining a novel active ingredient of an anti-autoimmune disease agent.

  A living body usually does not show an immune response to its own constituent components (self-antigen) and maintains immunological homeostasis. Such homeostasis is called innate tolerance. This tolerance state may be broken, resulting in antibodies and lymphocytes that react with self-antigens. This phenomenon is called autoimmunity, and the disease that triggers it is called autoimmune disease. Autoimmune diseases include systemic autoimmune diseases and organ-specific autoimmune diseases. Representative examples include systemic lupus erythematosus and rheumatoid arthritis, and the latter include multiple sclerosis, myasthenia gravis, autoimmune hemostatic anemia, and Mr. Hashimoto's thyroiditis. Systemic autoimmune diseases have long been in the category of collagen disease characterized by an increase in connective tissue growth. In recent years, however, their origin has been studied and at least partly considered to be involved in autoimmunity, but there are still many unclear points. On the other hand, in organ-specific autoimmune diseases, the etiological role of autoantibodies is often clear. Previous studies have suggested that organ-specific autoimmune diseases are caused by antigen-stimulated T lymphocytes. Various thoughts have been made about the autoantibody production mechanism. The first is the theory of involvement of genes involved in the production of specific autoantibodies. The second is that the antigen that is usually present in a very small amount in the blood is released in a large amount for some reason, or the antigen is modified for some reason. Third, there is an abnormality on the lymphocyte side involved in antibody production, which is caused by deviating from the tolerance state. Presumably, these are duplicated and autoantibodies are produced.

  Rheumatoid arthritis is one of the autoimmune diseases. It is a systemic inflammatory disease with unknown causes of multiple organitis, which simultaneously affects multiple organs. It progresses chronically with repeated remissions and exacerbations. If left untreated, the joints are destroyed and deformed, eventually exhibiting motor dysfunction, and sometimes life threatening. However, current medicine states that rheumatoid arthritis cannot be completely cured or prevented. Therefore, the current treatment goal is to diagnose early, start aggressive treatment early, and suppress rheumatic inflammation as quickly and as maximally as possible to prevent the appearance of irreversible changes or By preventing progress, the patient's physical, mental and social quality of life (QOL) is improved.

  The main drugs used to treat rheumatoid arthritis are non-steroidal anti-inflammatory drugs (hereinafter referred to as “NSAIDs”), disease-modifying anti-rheumatic drugs (hereinafter referred to as “DMARDs”), and adrenal cortex Steroid drug.

  NSAIDs are the first drugs used in patients with rheumatoid arthritis, and the analgesic effect is quickly obtained, but the anti-inflammatory effect appears after 1-2 weeks. Although NSAIDs have the effect of reducing the degree of inflammation, NSAIDs do not have the effect of preventing the progression of rheumatic inflammation or the prevention of joint destruction. In addition, many side effects of NSAIDs cannot be separated from their pharmacological action (cyclooxygenase-1 inhibitory activity), and digestive tract disorders often interfere with treatment.

  DMARDs is a general term for drugs aimed at inducing remission by reducing rheumatoid inflammation by correcting immune abnormalities in rheumatoid arthritis. DMARDs are generally slow-acting, but when the effects are manifested, the effects persist for a long period of time. DMARDs have no analgesic effect and are used in combination with fast-acting NSAIDs and corticosteroids at the start of administration. On the other hand, as problems, it is known that rare side effects such as bone marrow suppression, nephritis, interstitial pneumonia appear rarely, and there is an individual difference in the optimum dose. Furthermore, conventional DMARDs often show resistance by continuing administration (escape phenomenon), and the effect is attenuated in 2 to 3 years, and there are many cases in which administration is discontinued.

  Corticosteroids quickly and reliably suppress rheumatic inflammation and, although in a short period of time, significantly improve the quality of life of patients with rheumatoid arthritis, but the effect of long-term continuous use is diminished and withdrawal is difficult (recoil phenomenon, withdrawal syndrome) ), The progression of joint destruction cannot be prevented, and there are many adverse effects such as the induction of infections, adrenal dysfunction, gastrointestinal disorders, and the appearance of serious side effects such as osteoporosis.

  It is known that various cells are involved in the pathogenesis of autoimmune diseases, particularly rheumatoid arthritis. Typical examples include T lymphocytes, macrophage cells, osteoclasts (involved in joint destruction), fibroblasts (involved in joint stiffness), and the like.

Endoplasmic Reticulum (endoplasmic reticulum; hereinafter abbreviated as “ER” in the present specification) is an intracellular membrane system having a filamentous or network-like structure in the vicinity of the nucleus of a cell. Is recognized. The form varies depending on the cell type, such as tubular or flat bag shape. The endoplasmic reticulum is divided into a rough endoplasmic reticulum having a large number of ribosomes attached to the surface and a smooth endoplasmic reticulum having no ribosome. The degree of development of the rough endoplasmic reticulum and the smooth endoplasmic reticulum varies depending on the cell type and the difference in activity. It is clear that pancreatic and salivary gland cells producing a large amount of secreted protein have well-developed rough endoplasmic reticulum, and that steroid hormone-producing cells such as adrenal cortex cells have smooth endoplasmic reticulum. It has become. As recent research progresses, there are various molecular chaperones such as Bip and folding enzymes (catalyzing SS bonds and glycosylation) in ER, and these actions normally fold nascent proteins very efficiently. It has become clear that quality is managed. However, it has been proved probabilistically that the formation of higher-order structures sometimes fails and is processed by the ER-related proteolytic mechanism (ERAD), which is a ubiquitin-proteasome system. It is considered that the quality control mechanism in the ER is established by the folding action and decomposition action of the nascent protein. The ER stress refers to a situation in which such a balance changes, and as a result, abnormal proteins having higher-order structures accumulate in the ER. The cellular response to these is the ER stress response, that is, UPR (Unfolded Protein Response; (Folded protein response). ER stress occurs when (1) the protein folding device or degradation device fails due to environmental factors, etc. (2) Even if the device is normal, the amount of protein exceeding their processing capacity is ER And (3) the case where a protein that cannot originally be normally folded due to genetic variation is synthesized. UPR is a cellular response to the situation where abnormal proteins with higher-order structure accumulate in the ER, and is a concept that includes the following three responses: (1) No further nascent protein is sent into the ER (2) Enhanced ER folding capacity by induction of ER chaperone transcription; (3) Enhanced capacity of degradation mechanism by transcription induction of factors involved in ER-related proteolysis mechanism.
In vivo, ER stress response does not operate under extreme stress conditions, and is considered to occur when a large amount of protein is produced at once, such as antibody production of plasma cells. So far, it has been reported that Cisplatin and Doxorubicin, which are anticancer agents, enhance ER stress, which is considered as one of the mechanisms of cancer cell damage. Patent Document 1 and Patent Document 2 describe the relationship between the regulation of UPR or XBP-1 and the treatment of autoimmune diseases. However, there has been no report that the prevention and / or treatment of autoimmune diseases can be achieved by selectively regulating the activity of UPR in cells in the presence of ER stress.

  As ER stress inducers, there are known Thapsigargin, Tunicamysin, A23187, Cisplatin, Doxorubicin, Bortezomib and the like, but no effect on autoimmune diseases has been reported.

Meanwhile, 3-[(1S) -1- (2-fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole represented by the following formula 1a ( Hereinafter, it is also referred to as “compound (I)” (see, for example, Patent Document 3), and is reported to have an anti-arthritic action on adjuvant arthritic rats and collagen arthritic mice, which are representative rheumatoid arthritis model animals. (For example, see Patent Document 4).
Formula 1a:

However, the action of compound (I) on UPR has never been known so far.

JP-T-2006-515163 US Patent Application Publication No. 2005/0250182 Japanese Patent No. 3237608 International Publication No. 2006/049215 Pamphlet

  The object of the present invention is to provide an excellent anti-autoimmune disease agent based on a novel mechanism of action. It is another object of the present invention to provide a screening method useful for searching for and obtaining candidate substances that are active ingredients of the anti-autoimmune disease agent.

As a result of intensive studies to solve the above problems, the present inventors have obtained the following surprising findings.
(1) The compound (I) selectively promotes the expression of Bip mRNA in the presence of ER stress, that is, activates UPR. (Example 1).
(2) Compound (I) selectively promotes phosphorylation of eIF2α in the presence of ER stress (Example 2).
(3) Compound (I) selectively suppresses protein expression without suppressing XIAP and ATF4 mRNA expression levels in the presence of ER stress (Example 3).
(4) Compound (I) selectively suppresses Akt1 protein expression in the presence of ER stress. Do not affect the expression level of Akt1 mRNA. Furthermore, the proteasome inhibitor and the caspase 3 inhibitor should not affect the suppression of Akt1 protein expression of Compound (I) (Example 4).
(5) Compound (I) is selectively apoptotic in the presence of ER stress against various cells (T lymphocytes, cells derived from human synovial tissue, etc.) involved in the pathogenesis of autoimmune diseases, particularly rheumatoid arthritis. Show inductive action (Examples 5 and 6).
(6) In animal models of autoimmune diseases and synovial tissues of rheumatoid arthritis patients, UPR is activated in affected areas, that is, ER stress is involved in the pathogenesis of autoimmune diseases (Example 7) .
(7) Compound (I) suppresses limb edema and joint destruction in adjuvant arthritic rats (Example 8).
(8) Compound (I) dose-dependently suppresses the disease state of experimental allergic encephalomyelitis (EAE) mice, which is an animal model of multiple sclerosis (Example 9)

  Factors that vary in relation to ER stress include Bip (Binding Immunoglobulin Protein), XBP-1 (X Box-binding Protein 1), eIF2A (Eukaryotic translation Initiation Factor 2 A), and XIAP (X-chromosome-linked Inhibitor of Apoptosis). ), ATF4 (Activating Transcription Factor 4), IRE1 (Inositol-Requiring Enzyme 1), PERK (PKR-Like ER Kinase) and ATF6 (Activating Transcription Factor 6). For example, in the presence of ER stress, UPR is activated, Bip and spliced XBP-1 expression is induced, and eIF2α is phosphorylated to start translation of ATF4.

The UPR-related factor is a general term for factors that change with the activation of UPR, and many of them are the same as the factors that vary in relation to ER stress. Specific examples of UPR-related factors include Akt, Bip, XBP-1, eIF2α, XIAP, ATF4, and the like.
As a result of intensive studies focusing on Bip and eIF2α, the present inventor has obtained the findings of (1) and (2) above. From these findings, it is considered that a UPR modifier such as compound (I) modifies the expression level of Bip and the phosphorylation level of eIF2α and induces apoptosis of T lymphocytes. It has also been reported that UPR is activated in the absence of fetal calf serum, and UPR modifiers such as compound (I) induce apoptosis in human synovial tissue-derived fibroblasts in the absence of fetal calf serum. It is thought to induce.

  Based on the above findings, the present inventors have found that these compounds (compound (I) and the like) exert an effect of improving the pathological condition of autoimmune diseases through a UPR modifying action, and selectively in the presence of ER stress. It has been convinced that substances that modify UPR can be effectively used as preventive and / or therapeutic agents for autoimmune diseases. The present invention has been completed based on such findings.

That is, the present invention is as follows.
[1] Screening method for anti-autoimmune disease agent comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the expression level of the UPR-related factor in the cell contacted with the test substance and comparing it with the expression level of the UPR-related factor in the cell in which ER stress is induced without contacting the test substance;
(c) In a control cell that does not induce ER stress, the expression level of the UPR-related factor of the cell contacted with the test substance and the UPR relation of the cell not contacted with the test substance are obtained by the same steps as in (a) and (b) above. Comparing the expression level of the factor,
(d) comparing the UPR-related factor expression modification rate as a comparison result of (b) with the UPR-related factor expression modification rate of a control cell as a comparison result of (c); and
(e) A step of selecting a test substance that selectively modifies UPR-related factor expression in cells in the presence of ER stress based on the comparison result of (d) above.
[2] The screening method according to item [1], wherein the UPR-related factor is a factor selected from the group consisting of Akt, Bip, XBP-1, eIF2α, XIAP and ATF4.
[3] The screening method according to item [1], wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses the expression of Akt protein.
[4] The screening method according to item [1], wherein the test substance that modifies the expression of the UPR-related factor is a test substance that enhances the expression of the Bip gene or Bip protein.
[5] The screening method according to item [1], wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses expression of splicing form XBP-1 mRNA or splicing form XBP-1 protein.
[6] The screening method according to item [1], wherein the test substance that modifies UPR-related factor expression is a test substance that promotes phosphorylation of eIF2α.
[7] The screening method according to item [1], wherein the test substance that modifies UPR-related factor expression is a test substance that suppresses expression of XIAP protein.
[8] The screening method according to item [1], wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses the expression of ATF4 protein.

[9] Screening method for anti-autoimmune disease agent comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the apoptosis level of the cell contacted with the test substance and comparing it with the apoptosis level of the cell induced ER stress without contacting the test substance;
(c) In a control cell that does not induce ER stress, the apoptosis level of the cell contacted with the test substance is compared with the apoptosis level of the cell not contacted with the test substance by the same steps as in (a) and (b) above. The process of
(d) comparing the apoptosis induction rate as a comparison result of (b) above with the apoptosis induction rate of a control cell as a comparison result of (c) above, and
(e) A step of selecting a test substance that selectively induces apoptosis in cells in the presence of ER stress based on the comparison result of (d) above.
[10] The test item of [9], wherein at a test substance concentration of 10 μM, a test substance that does not substantially induce apoptosis in the absence of ER stress and exhibits an apoptosis induction rate of 40% or more in the presence of ER stress is selected. Screening method.

[11] The screening method according to any one of items [1] to [10], wherein the cells are mammalian cells.
[12] The screening method according to any one of items [1] to [11], wherein the cell is a disease state-related cell in an autoimmune disease.
[13] The screening method according to any one of items [1] to [11], wherein the cell is a disease state-related cell in rheumatoid arthritis.
[14] The screening method according to any one of items [1] to [11], wherein the cells are cells selected from the group consisting of T cells, synovial cells, B cells, macrophages, fibroblasts, and osteoclasts.
[15] The screening method according to any one of items [1] to [11], wherein the cell is a cell selected from the group consisting of EL-4, SW982 and BCL-1.
[16] The screening method according to any one of items [1] to [15], wherein the anti-autoimmune disease agent is an autoimmune disease preventive agent and / or an autoimmune disease therapeutic agent.
[17] The screening method according to any one of items [1] to [15], wherein the anti-autoimmune disease agent is an agent for improving immunity abnormality.
[18] Autoimmune diseases are rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue disease, Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, Good -pasture syndrome, acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature onset Menopause, Lensogenic uveitis, Exchange vasculitis, Multiple sclerosis, Ulcerative colitis, Primary biliary cirrhosis, Chronic active hepatitis, Autoimmune hemolytic anemia, Paroxysmal hemoglobinuria, Idiopathic The screening method according to any one of Items [1] to [17], which is a disease selected from the group consisting of thrombocytopenic purpura and Sjogren's syndrome.
[19] The screening method according to any one of items [1] to [17], wherein the autoimmune disease is rheumatoid arthritis or multiple sclerosis.
[20] Items [1] to [19], wherein the anti-autoimmune disease agent is an inhibitor of chronic inflammation, an inhibitor of joint destruction, an inhibitor of progression to joint deformation, or an agent of improving physical function in rheumatoid arthritis patients. ] The screening method in any one of.

[21] An anti-autoimmune disease agent comprising, as an active ingredient, a substance selected using the screening method according to any one of items [1] to [15].

[22] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective UPR modifying action in the presence of ER stress.
[23] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective Akt1 protein expression inhibitory action in the presence of ER stress.
[24] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective Bip mRNA or Bip protein expression enhancing action in the presence of ER stress.
[25] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having an action of suppressing the expression of alternative splicing form XBP-1 mRNA or splicing form XBP-1 protein in the presence of ER stress.
[26] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective phosphorylation promoting action of eIF2α in the presence of ER stress.
[27] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective XIAP protein expression inhibitory action in the presence of ER stress.
[28] An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective ATF4 protein expression inhibitory action in the presence of ER stress.
[29] An anti-autoimmune disease agent comprising as an active ingredient a substance having a selective apoptosis-inducing action in the presence of ER stress.

[30] Prevention and / or prevention of autoimmune disease in the mammal, comprising modifying UPR in the mammal to a degree effective for prevention and / or treatment of autoimmune disease on cells in the presence of ER stress. Method of treatment.
[31] Prevention of an autoimmune disease in a mammal, comprising administering a substance having a selective UPR modifying action in the presence of ER stress to the mammal in an amount effective for the prevention and / or treatment of the autoimmune disease. And / or treatment methods.
[32] Use of a substance having a selective UPR modifying action in the presence of ER stress for the manufacture of an anti-autoimmune disease agent.
[33] 3-[(1S) -1- (2-Fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole (compound (I)) or A UPR modifier comprising a pharmaceutically acceptable salt as an active ingredient.

The present invention can provide an excellent anti-autoimmune disease agent based on a novel mechanism of action. Furthermore, the present invention can provide a screening method useful for searching for and obtaining candidate substances that are active ingredients of the anti-autoimmune disease agent.

  The present invention relates to 3-[(1S) -1- (2-fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole (compound (I)) Alternatively, hydrates, solvates such as alcohol solvates, and crystal forms of all embodiments relating to pharmaceutically acceptable salts thereof are also included.

  Pharmaceutically acceptable salts include acid addition salts and base addition salts. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, citrate, oxalate, acetate, formate, Organic acids such as propionate, benzoate, trifluoroacetate, fumarate, maleate, tartrate, aspartate, glutamate, methanesulfonate, benzenesulfonate, camphor-sulfonate Salt. Examples of the base addition salt include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt and ammonium salt, and organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt and diisopropylammonium salt. .

  Compound (I) can be produced, for example, by the method described in Patent Document 3.

  Hereinafter, the anti-autoimmune disease agent of the present invention and the screening method thereof will be described in detail.

I. Anti-autoimmune disease drugs In general, “autoimmune diseases” are diseases in which antibodies or lymphocytes that react with the components that make up the tissue of the body are continuously produced in the body, causing tissue damage. It can be classified into the following two types: organ-specific autoimmune diseases and organ-nonspecific autoimmune diseases (systemic autoimmune diseases).

  (1) Organ-specific autoimmune diseases: Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, Good-pasture syndrome, acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous Pemphigoid, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, phakogenic uveitis, exchange vasculitis, multiple sclerosis, ulcerative colitis , Primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, Sjogren's syndrome.

  (2) Non-organ-specific autoimmune disease (systemic autoimmune disease): rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue disease.

  The present invention arbitrarily targets these “autoimmune diseases”. Preferably, rheumatoid arthritis and multiple sclerosis can be exemplified.

  The “anti-autoimmune disease agent” of the present invention is an action to inhibit or suppress the onset of the various autoimmune diseases, an action to delay the onset, an action to reduce or improve the symptoms after the onset. Or having at least one action of healing a disease. In that sense, the “anti-autoimmune disease agent” of the present invention can be rephrased as an autoimmune disease preventive agent, autoimmune disease symptom relieving agent, autoimmune disease ameliorating agent, or autoimmune disease therapeutic agent.

  In the present invention, the “UPR-modifying action” (selective in cells in the presence of ER stress) broadly means the action of modifying the above-mentioned UPR regardless of the mechanism of action. Here, the “action of modifying UPR” includes an action of inhibiting UPR activity and an action of enhancing UPR activity.

  The “substance having the action of modifying (selective) UPR” of the present invention may be any substance having at least one of the above-described actions, and asks what the form or property is. is not. In addition, the “substance having a (selective) UPR modifying action” of the present invention may be any substance that has a modifying action on at least human UPR, and is suitable for UPR derived from mammals other than humans and other biological species. The presence or absence of the modifying action is not particularly limited. Furthermore, the “substance having the action of modifying (selective) UPR” includes embodiments specifically described in the section (II) described later.

  Substances having a (selective) UPR modifying action include proteins, peptides, and low molecular weight compounds. For example, a low molecular weight compound having a (selective) UPR modifying action is performed on the low molecular weight compound library (for example, a low molecular weight compound library such as CamBridge Research Laboratories), etc. Can be obtained.

  The substance having the (selective) UPR modifying action described above is useful as an active ingredient of an anti-autoimmune disease agent. Further, the active ingredient of the anti-autoimmune disease agent is not particularly limited to the substances specifically listed above, and can also be obtained by the screening method of the present invention described below.

Therefore, the anti-autoimmune disease agent targeted by the present invention includes
(1) a preparation containing an effective amount of a substance having a UPR modifying action (selective in cells in the presence of ER stress); and (2) obtained by the screening method described below (in the presence of ER stress) A preparation containing an effective amount of a substance having a UPR-modifying action (selective in the cells);
Are included. Here, the effective amount of the (selective) UPR modifying agent is the blending amount of the UPR modifying agent such that the anti-autoimmune disease agent containing it exhibits the (selective) UPR modifying effect as a result. There is no particular limitation as long as it is. The anti-autoimmune disease agent may contain a pharmaceutically acceptable carrier and various additives in addition to the UPR modifying agent.

  The types of carriers and additives, forms of anti-autoimmune disease agents, and usage (administration form, administration method, dose) of anti-autoimmune disease agents will be described in detail in the section (III) described later.

II. Screening method of active ingredient of anti-autoimmune disease agent As shown in Examples described later, the present inventors suppress the onset of autoimmune disease by selectively modifying UPR activity in cells in the presence of ER stress. And / or a new finding that the disease state can be improved. From this, it is considered that a substance having a UPR modifying action selectively in cells in the presence of ER stress is useful as an active ingredient of an anti-autoimmune disease agent. The screening method of the present invention has been developed on the basis of such findings, and by searching for a substance having a UPR modifying action selectively in cells in the presence of ER stress among test substances, an anti-autoimmune disease The active ingredient of the agent is to be obtained.

  The “substance selectively having a UPR modifying action in cells in the presence of ER stress” targeted by the screening method of the present invention may be any substance that can modify the activity of UPR as a result. It is not limited. Specific examples of the UPR modifying action include 1) an action for inhibiting UPR activity, and 2) an action for enhancing UPR activity. The substance having the UPR modifying action may be any substance having at least one of such UPR modifying actions.

  As a specific example of the UPR modifying action, there can be mentioned an action of modifying the expression of a UPR-related factor. A UPR-related factor is a generic term for factors that change with the activation (or inhibition of activity) of UPR, and many of them are the same as factors that vary in relation to ER stress. Specific examples of UPR-related factors include, for example, Akt, Bip, XBP-1, eIF2α, XIAP, ATF4, etc. More specifically, Akt protein, Bip gene, Bip protein, XBP-1 gene (preferably Include splicing form XBP-1 mRNA), XBP-1 protein (preferably splicing form XBP-1 protein), eIF2α, XIAP protein, ATF4 protein and the like.

As an example of “a substance having an Akt1 protein expression inhibitory action (selective in the presence of ER stress)” in the present invention, a UPR modifying action (selective in a cell in the presence of ER stress) exemplified in the present specification ( Hereinafter, a substance having a “selective UPR modifying action” may be mentioned.
Examples of “substances having an Akt1 protein expression inhibitory action (selective in the presence of ER stress)” in the present invention include substances having a selective UPR modifying action exemplified in the present specification.
Examples of “substances having an action of enhancing expression of Bip mRNA or Bip protein (selective in the presence of ER stress)” in the present invention include substances having a selective UPR modifying action exemplified in the present specification. Can do.
Examples of “substances having an inhibitory action on the expression of XBP-1 mRNA of splicing form (selective in the presence of ER stress) or XBP-1 protein of splicing form” in the present invention are the selections exemplified in the present specification. And substances having a typical UPR modifying action.
Examples of “substances having a phosphorylation promoting action of eIF2α (selective in the presence of ER stress)” in the present invention include substances having a selective UPR modifying action exemplified in the present specification.
Examples of the “substance having an action of suppressing the expression of XIAP protein (selective in the presence of ER stress)” in the present invention include substances having a selective UPR modifying action exemplified in the present specification.
Examples of the “substance having an effect of suppressing the expression of ATF4 protein (selective in the presence of ER stress)” in the present invention include substances having the selective UPR modifying action exemplified in the present specification.
Examples of “substances having an apoptosis-inducing action (selective in the presence of ER stress)” in the present invention include substances having a selective UPR modifying action exemplified in the present specification.

  Candidate substances that can be active ingredients of anti-autoimmune disease agents include nucleic acids, peptides, proteins, organic compounds (including low molecular weight compounds), inorganic compounds, and the like. The screening method of the present invention can be carried out on a substance that can be a candidate substance or a sample containing the substance (collectively referred to as “test substance”). Furthermore, as a sample (test substance) containing a candidate substance, a cell extract, a gene library expression product, a microorganism culture supernatant, a bacterial cell component, and the like can be used.

The “substance having the UPR modifying action” of the present invention may have any structure as long as it is pharmaceutically acceptable, and is not limited to the following, but specific examples thereof include the following.
・ 3-[(1S) -1- (2-Fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole (compound (I)) or its pharmacy Top acceptable salt

  The screening method of the present invention searches for a substance having at least one of the above-mentioned UPR modifying actions from among the test substances as an index, particularly in cells in the presence of ER stress. This is performed by searching for a substance having a UPR modifying action. The test substance thus selected and obtained is useful as an active ingredient of an anti-autoimmune disease agent.

Specific embodiments of the screening method of the present invention are not limited, and examples thereof include the following methods.
Method 1
A method for screening a substance having a selective UPR modifying action in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the expression level of the UPR-related factor in the cell contacted with the test substance and comparing it with the expression level of the UPR-related factor in the cell in which ER stress is induced without contacting the test substance;
(c) In a control cell that does not induce ER stress, the expression level of the UPR-related factor of the cell contacted with the test substance and the UPR relation of the cell not contacted with the test substance are obtained by the same steps as in (a) and (b) above. Comparing the expression level of the factor,
(d) comparing the UPR-related factor expression modification rate as a comparison result of (b) with the UPR-related factor expression modification rate of a control cell as a comparison result of (c); and
(e) A step of selecting a test substance that selectively modifies UPR-related factor expression in cells in the presence of ER stress based on the comparison result of (d) above.

Method 2
A screening method for a substance that selectively suppresses the expression of Akt protein in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the expression level of Akt protein in a cell contacted with a test substance (such as Western blotting) and comparing it with the expression level of Akt protein in a cell in which ER stress was induced without contacting the test substance;
(c) In a control cell that does not induce ER stress, the expression level of Akt protein in the cell contacted with the test substance and the expression of Akt protein in the cell not contacted with the test substance by the same steps as in (a) and (b) above Comparing the amount,
(d) comparing the Akt protein expression modification rate as a comparison result of (b) above with the Akt protein expression modification rate of a control cell as a comparison result of (c), and
(e) A step of selecting a test substance that selectively suppresses the expression of Akt protein in cells in the presence of ER stress based on the comparison result of (d) above.
Method 3
A screening method for a substance that selectively enhances the expression of a Bip gene (or Bip protein) in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) Measures the expression level of Bip gene (or Bip protein) in cells contacted with the test substance (quantitative RT-PCR, Western blotting, etc.), and induces ER stress without contacting the test substance A step of comparing the expression level of Bip gene (or Bip protein) of
(c) In a control cell that does not induce ER stress, the test substance is not contacted with the expression level of the Bip gene (or Bip protein) in the cell contacted with the test substance by the same process as in (a) and (b) above. A step of comparing the expression level of a cell Bip gene (or Bip protein),
(d) A step of comparing the Bip gene (or Bip protein) expression modification rate as a comparison result of (b) with the Bip gene (or Bip protein) expression modification rate of a control cell as a comparison result of (c) ,and
(e) A step of selecting a test substance that selectively enhances the expression of a Bip gene (or Bip protein) in cells in the presence of ER stress based on the comparison result of (d) above.
Method 4
A screening method for a substance that selectively suppresses expression of spliced form XBP-1 mRNA (or spliced form XBP-1 protein) in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) Measure the expression level of splicing form XBP-1 mRNA (or splicing form XBP-1 protein) in the cells contacted with the test substance (quantitative RT-PCR, Western blotting, etc.) Comparing the expression level of the spliced form XBP-1 mRNA (or the spliced form XBP-1 protein) of the cells in which ER stress was induced without contact with
(c) In a control cell that does not induce ER stress, the spliced form XBP-1 mRNA (or the spliced form XBP-1 protein) of the cell contacted with the test substance by the same steps as in (a) and (b) above ) And the expression level of the spliced form XBP-1 mRNA (or the spliced form XBP-1 protein) of the cells not contacted with the test substance,
(d) The expression modification rate of the spliced form XBP-1 mRNA (or the spliced form XBP-1 protein) as a comparison result of the above (b), and the control cell splicing form as a comparison result of the above (c) Comparing the expression modification rate of XBP-1 mRNA (or the spliced form of XBP-1 protein), and
(e) Based on the comparison result of (d) above, select a test substance that selectively suppresses the expression of splicing form XBP-1 mRNA (or splicing form XBP-1 protein) in cells in the presence of ER stress. Process.
Method 5
A screening method for a substance that selectively promotes phosphorylation of eIF2α in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the phosphorylation level of eIF2α in a cell contacted with a test substance and comparing it with the phosphorylation level of eIF2α in a cell in which ER stress was induced without contacting the test substance;
(c) In a control cell that does not induce ER stress, the phosphorylation level of eIF2α of the cell contacted with the test substance and the eIF2α of the cell not contacted with the test substance are obtained by the same steps as in (a) and (b) above. Comparing the phosphorylation level;
(d) comparing the phosphorylation level modification rate of eIF2α as a comparison result of (b) above with the phosphorylation level modification rate of eIF2α of a control cell as a comparison result of (c), and
(e) A step of selecting a test substance that selectively promotes phosphorylation of eIF2α in cells in the presence of ER stress based on the comparison result of (d) above.

Method 6
A screening method for a substance that selectively suppresses expression of XIAP protein in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the expression level of XIAP protein in cells contacted with the test substance (such as Western blotting), and comparing the expression level of XIAP protein in cells induced with ER stress without contacting with the test substance;
(c) In a control cell that does not induce ER stress, the XIAP protein expression level of the cell contacted with the test substance and the XIAP protein expression of the cell not contacted with the test substance by the same steps as in the above (a) and (b) Comparing the amount,
(d) comparing the XIAP protein expression modification rate as a comparison result of (b) with the XIAP protein expression modification rate of a control cell as a comparison result of (c); and
(e) A step of selecting a test substance that selectively suppresses the expression of XIAP protein in cells in the presence of ER stress based on the comparison result of (d) above.
Method 7
A screening method for a substance that selectively suppresses the expression of ATF4 protein in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the ATF4 protein expression level of the cell contacted with the test substance (such as Western blotting), and comparing it with the ATF4 protein expression level of the cell induced ER stress without contacting the test substance;
(c) In a control cell that does not induce ER stress, the expression level of ATF4 protein in the cell contacted with the test substance and the expression of ATF4 protein in the cell not contacted with the test substance by the same steps as in (a) and (b) above Comparing the amount,
(d) comparing the ATF4 protein expression modification rate as a comparison result of (b) above with the ATF4 protein expression modification rate of a control cell as a comparison result of (c), and
(e) A step of selecting a test substance that selectively suppresses the expression of ATF4 protein in cells in the presence of ER stress based on the comparison result of (d) above.

Method 8
A screening method for a substance having a selective apoptosis-inducing action in cells in the presence of ER stress, comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the apoptosis level of the cell contacted with the test substance and comparing it with the apoptosis level of the cell induced ER stress without contacting the test substance;
(c) In a control cell that does not induce ER stress, the apoptosis level of the cell contacted with the test substance is compared with the apoptosis level of the cell not contacted with the test substance by the same steps as in (a) and (b) above. The process of
(d) comparing the apoptosis induction rate as a comparison result of (b) above with the apoptosis induction rate of a control cell as a comparison result of (c) above, and
(e) A step of selecting a test substance that selectively induces apoptosis in cells in the presence of ER stress based on the comparison result of (d) above.

  The “selective action in the presence of ER stress” in the present invention means that the action in a cell in which ER stress is induced is compared with the action in the same type of control cell that does not induce ER stress. Represents being strong. For example, (1) the control cell has almost no such effect and the cell in the presence of ER stress has the effect, (2) the control cell has the effect, but the cell in the presence of ER stress has a higher effect than the control cell. In the case where the action is strong, there may be mentioned.

Various cells can be used as the cells used in the above methods 1 to 8, and examples thereof include mammalian cells. Preferably, the disease state related cell in an autoimmune disease is mentioned, For example, the disease state related cell in rheumatoid arthritis is mentioned.
Specific examples of the disease state-related cells in autoimmune diseases include, for example, T cells, synoviocytes, B cells, macrophages, fibroblasts, osteoclasts and the like. Among these, examples of cells that can be experimentally used in the screening method of the present invention include EL-4 cells, SW982 cells, BCL-1 cells, and the like.

In the above methods 1 to 8, the test substance concentration at the time of screening and the criterion for determining that “it has a selective UPR modifying action in cells in the presence of ER stress” can be appropriately set. . For example, test substance concentrations include, but are not limited to, 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and intermediate concentrations thereof (5 μM, 3 μM, 2 μM, 0.5 μM, 0.3 μM, 0.2 μM) Etc.). The criteria for determining “selectively has a UPR-modifying action in cells in the presence of ER stress” are not limited thereto, but the apoptosis induction rate compared to the control includes, for example, the following: be able to.
(1) At a test substance concentration of 10 μM, apoptosis is not substantially induced in the absence of ER stress, and an apoptosis induction rate of 40% or more is exhibited in the presence of ER stress.

  The screening method for a substance having a selective UPR modifying action in cells in the presence of ER stress in the present invention is not limited to the above embodiment, and can be appropriately designed based on the common general knowledge of those skilled in the art.

  The candidate substance selected from the test substances by the method for screening a substance having a UPR modifying action selectively in cells in the presence of ER stress as described above further includes, for example, pathological conditions imitating various human autoimmune diseases It can be obtained as an active ingredient of a more practical anti-autoimmune disease agent by subjecting it to a drug efficacy test and safety test using a model animal.

  More specifically, whether a candidate substance selected by the screening method of the present invention is useful as a therapeutic agent for autoimmune diseases and inflammatory diseases, particularly whether it is effective for chronic inflammation such as rheumatoid arthritis, is a pathological condition of rheumatoid arthritis. Model animals (eg, collagen-induced arthritis [Trentham DE, Townes AS, Kang AH: Autoimunity to type II collagens: An experimental model of arthritis J Exp Med 146: 857-868,1977], antigen-induced arthritis, or adjuvant-induced arthritis It can be evaluated by conducting a medicinal effect test using arthritis (any mouse or rat such as Pearson CM: Development of arthritis, periarthritis and periotitis in rats given adjuvant. Proc Soc Expe Biolo Med 91: 95-101, 1956). .

  In particular, by using a rat adjuvant arthritis model, the inhibitory effect (preventive effect, therapeutic effect) on rheumatoid arthritis can be evaluated in detail for the candidate substance. Rat adjuvant arthritis is an experimental arthritis in which T lymphocytes are involved in the onset mechanism, mainly progressing in cartilage / bone changes and fibrosis of joints. Basic research on chronic inflammation and pharmacological research on therapeutic drugs It is widely used as a disease model in Japan. The pathological conditions of adjuvant arthritis are usually (1) a non-specific primary inflammatory phase in which only the injected foot swells (peak: 4 to 5 days later), and (2) an induction phase in which activated lymphocytes increase in the regional lymph nodes ( Period: 5 to 9 days), (3) Exacerbation period of adjuvant arthritis in which polyarthritis develops (peak: 15 to 17 days), and (4) Natural healing period in which inflammation is subdued while leaving joint deformation and destruction Proceed like (period after 25th). Since non-specific primary inflammation due to adjuvant sensitization is also involved in the progression of the pathological condition on the injected foot side, the pathological condition progresses due to the involvement of T lymphocytes on the non-injected foot side, A phenomenon closer to rheumatoid arthritis is considered. Moreover, as for the drug action, the preventive effect administered from the day of inoculation and the therapeutic effect of starting administration after the onset of arthritis are being studied. As an index of drug efficacy evaluation, (1) the volume of each left and right hind limbs, (2) the change in the amount of edema obtained by measuring the volume of each hind limb before and after drug administration, and calculating the volume difference before and after administration, (3) Morphological evaluation of bone by soft X-ray and (4) pathological evaluation by tissue section are generally used.

  In addition, the selected candidate substance is subjected to a medicinal effect test using a disease model animal of multiple sclerosis (for example, an EAE-induced mouse), so that the candidate substance has an inhibitory effect (prophylactic effect, treatment) on multiple sclerosis. Effect).

  The substance thus selected and obtained can be industrially produced by chemical synthesis, biological synthesis (fermentation) or genetic manipulation based on the structural analysis result.

  A substance having a selective UPR modifying action in the presence of ER stress selected using the screening method of the present invention as described above is useful as an active ingredient of the anti-autoimmune disease agent as described above. Here, as the autoimmune disease, specifically, various diseases described in Chapter I of the present specification can be arbitrarily mentioned. In particular, the UPR modifying agent targeted by the present invention is preferably a joint. It can be effectively used for rheumatism and multiple sclerosis.

  The “anti-autoimmune disease agent” of the present invention is an action that inhibits or suppresses the onset of various autoimmune diseases, an action that delays the onset, an action that reduces or improves the symptoms after the onset, or a disease It has at least one action of healing. In that sense, the above-mentioned “anti-autoimmune disease agent” can also be referred to as an autoimmune disease preventive agent, autoimmune disease symptom alleviating agent, autoimmune disease ameliorating agent, or autoimmune disease therapeutic agent. The screening method can be a method for searching for these active ingredients.

  The present invention provides an anti-autoimmune disease agent comprising as an active ingredient a substance having a selective UPR modifying action in the presence of ER stress obtained by the above screening method.

III. About the usage of an anti-autoimmune disease agent, the dosage form, administration method, dosage, etc. of the anti-autoimmune disease agent containing the substance having the UPR modifying action of the present invention as an active ingredient will be described below.

  When the substance having a UPR modifying action is a protein such as a low molecular weight compound, a peptide or an antibody, it can be prepared in the form of a general pharmaceutical composition (pharmaceutical preparation) usually used for these substances. Depending on the dosage, it can be administered orally or parenterally. Generally, the following administration forms and administration methods are mentioned.

  Typical administration forms include solid preparations such as tablets, pills, powders, powders, granules and capsules, and liquids such as solutions, suspensions, emulsions, syrups and elixirs. Can be mentioned. These various forms can be administered orally, depending on the route of administration, as well as nasal, transdermal, rectal (suppository), sublingual, vaginal, injection (transvenous, transarterial, transdermal) It can be classified into parenteral administration agents such as muscle, subcutaneous, intradermal) and infusion. For example, as oral administration agents, for example, tablets, pills, powders, powders, granules, capsules, solutions, suspensions, emulsions, syrups, etc., or as rectal administration agents and vaginal agents, for example It can take the form of tablets, pills, capsules and the like. As a transdermal agent, for example, in addition to a liquid such as lotion, a semi-solid agent such as cream or ointment can be used.

  The injection may take the form of, for example, a solution, a suspension or an emulsion, and specifically, aseptically treated water, water-propylene glycol solution, buffered solution, 0.4% by weight physiological saline. Water etc. can be illustrated. Furthermore, the injection may be frozen or lyophilized after being prepared into a solution, and can be stored thereby. The lyophilized preparation prepared by the latter lyophilization treatment is used by re-dissolving by adding distilled water for injection at the time of use.

  The above-mentioned pharmaceutical composition (pharmaceutical preparation) is prepared by blending a substance having a UPR modifying action and a pharmaceutically acceptable carrier by a conventional method performed in the art. Examples of pharmaceutically acceptable carriers include excipients, diluents, fillers, extenders, binders, disintegrants, wetting agents, lubricants, and dispersants. Further, additives usually used in the field can be blended. Such additives include, for example, stabilizers, bactericides, buffers, thickeners, pH adjusters, emulsifiers, suspending agents, preservatives, fragrances, coloring agents, depending on the form of the pharmaceutical composition to be prepared. It can be appropriately selected from a tonicity adjusting agent, a chelating agent, a surfactant and the like.

  The pharmaceutical composition having such a form can be administered by an appropriate administration route according to the target disease, target organ and the like. For example, it can be administered orally, or it can be administered intravenously, artery, subcutaneously, intradermally or intramuscularly, or directly locally to the affected tissue itself, or transdermally or rectally. .

The dosage and frequency of administration of these pharmaceutical compositions vary depending on the dosage form, the patient's disease and symptoms, the patient's age and weight, etc., and cannot be generally specified, but are usually effective for adults per day The amount of the component is in the range of about 0.0001 to about 1000 mg, preferably in the range of about 0.001 to about 300 mg, more preferably in the range of about 0.1 to about 300 mg, particularly preferably in the range of about 1 to about 240 mg. It can be administered once or several times a day.

Examples Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.
In the examples, the following compounds were used.

Compound (I) : 3-[(1S) -1- (2-fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole (for example, Patent No. 3237608 can be synthesized according to the method described in the specification)
Formula 1a:

Example 1 Examination of UPR modifying activity (1)
The UPR activity-modifying action of compound (I) was evaluated when T lymphocytes were stimulated with ER stress inducer Calcium Ionophore A23187.
EL-4 cells (mouse T lymphocyte cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM: SIGMA) containing 10% fetal bovine serum and used for experiments. Cells were seeded in a 6-well plate at a rate of 1 × 10 ⁶ cells / well, a DMEM medium containing Compound (I) was added as a test compound, and cultured for 2 hours. Subsequently, DMEM medium containing A23187 as a stimulant was added and cultured for 4 or 24 hours. The final concentrations of the test compound and stimulant were adjusted so that Compound (I) was 30 μM and A23187 was 250 ng / mL. After removing the supernatant, total RNA was purified using RNeasy kit (R) (QIAGEN). CDNA was synthesized using TaqMan RT assay reagent (R) (Applied Biosystems). Multiplex Real Time PCR was performed using TaqMan Universal Master mix® (Applied Biosystems) and TaqMan® Gene Expression Assays (Applied Biosystems). The CT method was used for quantitative analysis as a relative value to the expression level of β-actin mRNA. As shown in FIG. 1, Bip mRNA expression was induced over time by A23187 (250 ng / mL) single stimulation. On the other hand, treatment with Compound (I) (30 μM) alone did not induce Bip mRNA expression until at least 24 hours later. In the group to which A23187 was added after 2 hours of pretreatment with Compound (I) (30 μM), Bip mRNA expression induction was higher than that in the group treated with A23187 alone. These results indicate that compound (I) selectively enhances Bip mRNA expression in the presence of ER stress, that is, compound (I) selectively modifies UPR activity in the presence of ER stress. ing.

Example 2 Examination of UPR modifying activity (2)
EL-4 cells (mouse T lymphocyte cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM: SIGMA) containing 10% fetal bovine serum and used for experiments. Cells were seeded in a 6 cm dish at a rate of 2 × 10 ⁶ cells / dish, a DMEM medium containing a test compound was added, and the cells were cultured for 2 hours. Subsequently, DMEM medium containing A23187 was added. The final concentrations of the test compound and stimulant were adjusted so that Compound (I) was 30 μM and A23187 was 250 ng / mL. The culture was further continued for 15 minutes, 30 minutes, 1 hour or 4 hours. A protease inhibitor (SIGMA) and a phosphatase inhibitor (SIGMA) -added RIPA buffer (0.2 mL / dish) were added, stirred with vortex, and allowed to stand on ice for 10 minutes. Centrifugation was performed at 14,000 rpm for 5 minutes (4 ° C.), and the supernatant was used as a cell lysate. Cell lysate was applied to a 10% polyacrylamide gel (50 μg / lane) and electrophoresed at 145 V for about 2 hours. Proteins were adsorbed from the gel to the PVDF membrane using a water tank type blotting device (100 V, 1 hour). The membrane was immersed in 5% skim milk and treated at room temperature for 1 hour. After washing the membrane, an anti-phospho-eIF2α antibody (Cell Signaling) or an anti-eIF2α antibody (Santa Cruz) was added and allowed to react overnight at 4 ° C. After washing the membrane, a secondary antibody (GE Healthcare) was reacted at room temperature for 1 hour. The membrane was washed and chemiluminescent using Super signal west pico (PIERCE) to detect the signal. The membrane was stripped using Re-Blot Plus (Chemicon) and reused. As shown in FIG. 2, it was revealed that the phosphorylation level of eIF2α (Ser51) increased with time in the group to which A23187 was added after 2 hours pretreatment with Compound (I) (30 μM). On the other hand, no clear temporal change was observed in the eIF2α (Ser51) phosphorylation level of the compound (I) alone treatment group and the A23187 alone treatment group. These results indicate that Compound (I) selectively promotes phosphorylation of eIF2α in the presence of ER stress, that is, Compound (I) selectively modifies UPR activity in the presence of ER stress. ing.

Example 3 Examination of UPR modifying activity (3)
EL-4 cells (mouse T lymphocyte cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM: SIGMA) containing 10% fetal bovine serum and used for experiments. Cells were seeded in a 6 cm dish at a rate of 2 × 10 ⁶ cells / dish, a DMEM medium containing a test compound was added, and the cells were cultured for 2 hours. Subsequently, DMEM medium containing A23187 was added. The final concentrations of the test compound and stimulant were adjusted so that Compound (I) was 30 μM and A23187 was 250 ng / mL. The culture was further continued for 24 hours. A protease inhibitor (SIGMA) and a phosphatase inhibitor (SIGMA) -added RIPA buffer (0.2 mL / dish) were added, stirred with vortex, and allowed to stand on ice for 10 minutes. Centrifugation was performed at 14,000 rpm for 5 minutes (4 ° C.), and the supernatant was used as a cell lysate. Cell lysate was applied to a 10% polyacrylamide gel (50 μg / lane) and electrophoresed at 145 V for about 2 hours. Proteins were adsorbed from the gel to the PVDF membrane using a water tank type blotting device (100 V, 1 hour). The membrane was immersed in 5% skim milk and treated at room temperature for 1 hour. After the membrane was washed, anti-XIAP antibody (Cell Signaling), anti-ATF4 antibody (Santa Cruz) or anti-β-actin antibody (SIGMA) was added and reacted at 4 ° C. overnight. After washing the membrane, a secondary antibody (GE Healthcare) was reacted at room temperature for 1 hour. The membrane was washed and chemiluminescent using Super signal west pico (PIERCE) to detect the signal. The membrane was stripped using Re-Blot Plus (Chemicon) and reused. As shown in FIG. 3, in the group to which A23187 was added after 2 hours pretreatment with Compound (I) (30 μM), it was revealed that the protein expression levels of XIAP and ATF4 were decreased. At this time, the expression level of β-actin was not affected. On the other hand, no clear change was observed in the expression level of XIAP and ATF4 protein in the A23187 single treatment group. These results indicate that compound (I) selectively suppresses XIAP and ATF4 protein expression in the presence of ER stress, that is, compound (I) selectively modifies UPR activity in the presence of ER stress. Is shown.

Example 4 Examination of Akt1 protein expression inhibitory action
EL-4 cells (mouse T lymphocyte cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM: SIGMA) containing 10% fetal bovine serum and used for experiments. Cells are seeded in a 6cm dish at a rate of 2 × 10 ⁶ cells / dish, and DMEM medium containing test compound, proteasome inhibitor MG-262 (Calbiochem) and caspase inhibitor ZVAD-fmk (Calbiochem) is added. And cultured for 2 hours. Subsequently, DMEM medium containing A23187 was added. The final concentrations of the test compound and stimulant were adjusted so that Compound (I) was 30 μM and A23187 was 250 ng / mL. The culture was further continued for 24 hours. A protease inhibitor (SIGMA) and a phosphatase inhibitor (SIGMA) -added RIPA buffer (0.2 mL / dish) were added, stirred with vortex, and allowed to stand on ice for 10 minutes. Centrifugation was performed at 14,000 rpm for 5 minutes (4 ° C.), and the supernatant was used as a cell lysate. Cell lysate was applied to a 10% polyacrylamide gel (50 μg / lane) and electrophoresed at 145 V for about 2 hours. Proteins were adsorbed from the gel to the PVDF membrane using a water tank type blotting device (100 V, 1 hour). The membrane was immersed in 5% skim milk and treated at room temperature for 1 hour. After washing the membrane, anti-Akt1 antibody (Cell Signaling), anti-ERK2 antibody (Santa Cruz) or anti-β-actin antibody (SIGMA) was added. Anti-Akt1 antibody was reacted overnight at 4 ° C., and anti-ERK2 antibody and anti-β-actin antibody were reacted at room temperature for 1 hour. After washing the membrane, a secondary antibody (GE Healthcare) was reacted at room temperature for 1 hour. The membrane was washed and chemiluminescent using Super signal west pico (PIERCE) to detect the signal. The membrane was stripped using Re-Blot Plus (Chemicon) and reused. As shown in FIG. 4, in the group to which A23187 was added after 2 hours pretreatment with compound (I) (30 μM), it was revealed that the protein expression level of Akt1 was decreased. At this time, the expression levels of ERK2 and β-actin were not affected. On the other hand, no clear change was observed in the expression level of Akt1 protein in the single treatment groups of Compound (I) and A23187. In addition, no clear change was observed in the expression level of Akt1 mRNA. Furthermore, the Akt1 protein expression-reducing action of compound (I) was not canceled by the proteasome inhibitor or caspase inhibitor addition group. These results suggest that compound (I) selectively suppresses the expression level of Akt1 protein at the translational level, not at the transcriptional or proteolytic level, in the presence of ER stress.

Example 5 Examination of apoptosis-inducing action on T lymphocytes
EL-4 cells (mouse T lymphocyte cell line) were cultured in Dulbecco's Modified Eagle Medium (DMEM: SIGMA) containing 10% fetal bovine serum and used for experiments. Cells were seeded in a 96-well plate at a rate of 1 × 10 ⁴ cells / well, and a DMEM medium containing a test compound and a DMEM medium containing A23187 were added. The final concentrations of the test compound and stimulant were adjusted so that Compound (I) was 5-30 μM and A23187 was 250 ng / ml. After culturing for 66 hours, 20 μl of alamar blue® (BIOSOURCE) was added to each well, further cultured for 6 hours, and fluorescence intensity was measured using a fluorescence plate reader (Ex: 544 nm, Em: 590 nm). The fluorescence intensity was used as an index of the number of cells, and the value obtained by the following formula was used as Apoptotic cells (%) (apoptosis induction rate).
Apoptotic cells (%) = (1- (FI (sample) -FI (blank)) / (FI (DMSO) -FI (blank))) x 100
FI (sample): Fluorescence intensity of each sample addition well
FI (blank): Fluorescence intensity of medium-added well
Fluorescence intensity of FI (DMSO): DMSO (0.1%) added well As shown in FIG. 5, compound (I), which is a UPR modifier, induced EL-4 cell apoptosis in a dose-dependent manner in the presence of A23187. These results indicate that UPR is involved in maintaining the survival of T cells in the presence of ER stress, and that compound (I) selectively induces apoptosis by modifying UPR activity in cells under ER stress. It strongly suggests that it induces.

Example 6 Examination of Apoptosis-Inducing Action on Human Synovial Tissue-Derived Cells Human synovial tissue-derived cell line SW982 cells were cultured in L-15 Medium (SIGMA) containing 10% fetal bovine serum and used for experiments. SW982 was seeded at 4 × 10 ⁴ / well in a 96-well flat bottom plate without serum. The test substance (compound (I)) was added at the same time as the seeding of cells and cultured at 200 μl / well. After 18 hours, 20 μl of alamar blue® (BIOSOURCE) was added to each well, further cultured for 6 hours, and fluorescence intensity was measured using a fluorescence plate reader (Ex: 544 nm, Em: 590 nm). The fluorescence intensity was used as an index of the number of cells, and the value obtained by the following formula was used as Apoptotic cells (%) (apoptosis induction rate).
Apoptotic cells (%) = (1- (FI (sample) -FI (blank)) / (FI (DMSO) -FI (blank))) x 100
FI (sample): Fluorescence intensity of each sample addition well
FI (blank): Fluorescence intensity of medium-added well
Fluorescence intensity of FI (DMSO): DMSO (0.1%) added well As shown in FIG. 6, compound (I), which is a UPR modifier, induced SW982 cell apoptosis in a dose-dependent manner in the absence of fetal calf serum. These results indicate that UPR is involved in maintaining the survival of human synovial cells in the presence of ER stress, and that compound (I) selectively modifies UPR activity in cells under ER stress. It strongly suggests that apoptosis is induced.

Example 7 Evaluation of UPR activation in rheumatoid arthritis patients synovium and adjuvant-induced arthritic rat joint affected area
The expression levels of Bip and XBP-1 mRNA in the synovial tissue of rheumatoid arthritis patients were analyzed using Genelogic database. As a control, synovial tissue from osteoarthritis patients was used. In addition, the Bip and XBP-1 mRNA expression levels in the affected joints of adjuvant arthritic rats widely used for pharmacological evaluation of anti-inflammatory and anti-rheumatic agents were compared with those of normal rats. Adjuvant arthritis was induced by injecting a liquid paraffin suspension (1 mg / 200 μl) of Mycobacterium butyricum (adjuvant) into the right hind footpad of Lewis rats (male, 6 weeks old). Total RNA was extracted from the left hind leg 17 days after adjuvant injection, and cDNA was further synthesized from the total RNA. The cDNA was subjected to DNA chip analysis and the expression of Bip and XBP-1 was quantified.
As shown in FIG. 7, the expression levels of Bip and XBP-1 mRNA were increased in the synovial tissue of rheumatoid arthritis patients. In addition, as shown in FIG. 8, an increase in expression was also observed in the affected part of the rat with adjuvant arthritis. These results strongly suggest that UPR is activated in the synovial tissue of patients with rheumatoid arthritis and in the affected area of rats with adjuvant-induced arthritis.

Example 8 Effect on Adjuvant-Induced Arthritis The adjuvant-induced arthritis model is a typical chronic inflammation model induced in rats and is widely used for pharmacological evaluation of anti-inflammatory agents and anti-rheumatic agents. Thus, this model was used to examine the onset-preventing effect of Compound (I). In the onset prevention test, the hindlimb volume was measured over time during the administration period, and the effect of the drug over a long period of time was examined. In addition, hind limb volume or edema amount was used as an index of drug efficacy evaluation.
(experimental method)
A liquid paraffin suspension (1 mg / 200 μl) of Mycobacterium butyricum (adjuvant) was injected subcutaneously into the right hind footpad of Lewis rats (male, 6 weeks old) to induce arthritis. In the onset prevention test, the drug was administered once a day for 21 consecutive days from the day of injection of the adjuvant. The volume of the left and right hind limbs was measured by the daily water replacement method.
As a control, only 0.5% methylcellulose containing no test compound was administered in the same manner as described above.
(result)
When adjuvant is injected, arthritis develops in the left hind limb around 10 days, and the limb volume increases to around 17 days, reaching a maximum. FIG. 9 shows the left and right hind limb volumes when compound (I) (20-80 mg / kg) was orally administered for 21 days from the day of adjuvant injection.
As shown in FIG. 9, in the onset prevention test in which administration was started before the left hind limb (non-injected limb) exhibited edema, Compound (I) showed a remarkable edema-inhibiting effect.

Example 9 Effect on Experimental Allergic Encephalomyelitis The efficacy of Compound (I) was evaluated using experimental allergic encephalomyelitis mice, which are animal models of multiple sclerosis. Induction of experimental allergic encephalomyelitis was according to the method of Bradley et al. (J. Clinical Investigation, 107: 995-1006, 2001). That is, a 7-week-old female SJL / J mouse (purchased from Charles River) was subcutaneously injected with 150 μg of PLP partial peptide (PLP _139-151 ) mixed with Freund's complete adjuvant. The severity of symptoms was expressed as a score (pathological score) according to the following criteria.
0: Asymptomatic 0.5: Mild paralysis of the tail 1: Tail paralysis 2: Mild hind limb paralysis 3: Moderate to strong hind limb paralysis or Mild fore limb paralysis 4: Complete hind limb paralysis or Moderate to strong forelimb paralysis 5: Paralysis of the extremities or in the deadline stage 6: Death Compound (I) is suspended in 0.5% methylcellulose solution, 8mg / ml (80mg / kg administration group), 4mg / ml 40 mg / kg administration group) and 2 mg / ml (20 mg / kg administration group) were prepared, and 0.1 ml was orally administered per 10 g of mouse body weight. In the prednisolone group as a positive control, prednisolone was suspended in a 0.5% methylcellulose solution to prepare a 0.3 mg / ml administration solution, and 0.1 ml was orally administered per 10 g body weight of a mouse. Only 0.5% methylcellulose solution was subcutaneously administered to the control group. Administration was started from the day of sensitization and was performed once a day for 40 days. In the Normal group, neither PLP _139-151 sensitization nor test substance administration was performed.
The results are shown in Table 1, FIG. 10 and FIG. Compound (I) suppressed the disease state score at any dose, and the effect was dose-dependent. In the control group, all cases (9/9 animals) developed and 4/9 animals died of nerve palsy during the study period, whereas in the compound (I) administration group, 80 mg / kg administration group There were no deaths except 1 of 10 animals died. Prednisolone strongly suppressed the onset (only 2 out of 10 animals), but unlike the control group and the compound (I) -administered group, the spontaneous increase in body weight due to growth was strongly suppressed. This result is an effect accompanying continuous administration of prednisolone, and becomes a problem when long-term administration is performed for a chronic disease state. That is, Compound (I) dose-dependently increases the pathological condition of experimental allergic encephalomyelitis without the strong suppression of weight gain during long-term administration seen in the group administered with prednisolone, which is a typical steroid. It was shown to suppress.

Table 1. Effects in experimental allergic encephalomyelitis mice
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Test substance Dose n Incidence rate Mortality rate
(mg / kg)
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Control group 9 9/9 4/9
Normal group 10 0/10 0/10
Prednisolone 3 10 2/10 0/10
Compound (I) 20 10 8/10 0/10
40 10 7/10 0/10
80 10 8/10 1/10
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INDUSTRIAL APPLICABILITY According to the present invention, a screening method useful for searching for and obtaining candidate substances that are active ingredients of anti-autoimmune diseases based on a novel mechanism of action can be provided. Furthermore, the present invention can provide an excellent anti-autoimmune disease agent based on a novel mechanism of action.

It is a graph which shows the Bip mRNA expression level when mouse | mouth T lymphocyte system cell strain EL-4 cell is stimulated with Calcium Ionophore A23187. The results are shown as the mean and standard deviation of 3 wells for each group. The results of the compound (I) added group (30 μM) and the control group are shown. The result of Western blotting is shown about the eIF2 (alpha) phosphorylation level when mouse | mouth T lymphocyte system cell strain EL-4 cell is stimulated with Calcium Ionophore A23187. The results of the compound (I) added group (30 μM) and the control group are shown. The results of Western blotting are shown for XIAP and ATF4 protein expression levels when mouse T lymphocyte cell line EL-4 cells were stimulated with Calcium Ionophore A23187. The results of the compound (I) added group (30 μM) and the control group are shown. A: Western blotting results are shown for the expression level of Akt1 protein when mouse T lymphocyte cell line EL-4 cells were stimulated with Calcium Ionophore A23187. B: Western blotting results are shown for Akt1 protein expression levels in the proteasome inhibitor or caspase inhibitor addition group. The results of the compound (I) added group (30 μM) and the control group are shown. It is a graph which shows the apoptosis induction rate when mouse | mouth T lymphocyte system cell strain EL-4 cell is stimulated with Calcium Ionophore A23187. Results are shown as the mean and standard deviation of 3 wells in each group. The results of the compound (I) addition group (5, 10, 20 and 30 μM) and the control group are shown. In the graph, ● represents the A23187 added group, and ○ represents the A23187 non-added group. It is a graph which shows the apoptosis of SW982 cell which is a human synovial tissue origin cell. Results are shown as the mean and standard deviation of 3 wells in each group. The results of the compound (I) added group (maximum concentration 30 μM) and the control group are shown. In the graph, ● represents a group not added with fetal calf serum, and ○ represents a group added with fetal calf serum. 2 shows Bip and XBP-1 mRNA expression levels in the synovium of rheumatoid arthritis patients. Shown as minimum, 25% quantile, median, 75% quantile, and maximum. The expression level of Bip and XBP-1 mRNA in the joint affected part of an adjuvant-induced arthritis rat is shown. In the graph, ● represents the expression level of Bip mRNA, and ▲ represents the expression level of XBP-1 mRNA. In both cases, the joint affected part shows the measurement results of 4 samples, and the normal site shows the measurement results of 3 samples. It is a graph which shows the change of the amount of edema of a bilateral hind limb in the adjuvant induction arthritis of a rat. The results of the compound (I) administration group (20-80 mg / kg) and the control group are shown. In the graph, ▪ indicates the results of the compound (I) (20 mg / kg) administration group. The symbol ▲ shows the results of the compound (I) (40 mg / kg) administration group. ● shows the results of the compound (I) (80 mg / kg) administration group. ◇ indicates the result of the test compound non-administered group (control group). Each group is shown as the mean and standard deviation of 10 cases. It is a graph which shows a pathological condition score when compound (I) is tested using the experimental allergic encephalomyelitis mouse which is an animal model of multiple sclerosis. □ indicates the results of the compound (I) (20 mg / kg) administration group. The symbol ▲ shows the results of the compound (I) (40 mg / kg) administration group. (1) shows the results of the compound (I) (80 mg / kg) administration group. X shows the result of a prednisolone (3 mg / kg) administration group. ● shows the results of the test compound non-administered group (control group). + Indicates the result of the normal control group. The control group is shown as an average of 9 cases, and the other groups are shown as an average of 10 cases. It is a graph which shows the effect on weight gain when testing compound (I) using experimental allergic encephalomyelitis mice which are animal models of multiple sclerosis. □ indicates the results of the compound (I) (20 mg / kg) administration group. The symbol ▲ shows the results of the compound (I) (40 mg / kg) administration group. (1) shows the results of the compound (I) (80 mg / kg) administration group. X shows the result of a prednisolone (3 mg / kg) administration group. ● shows the results of the test compound non-administered group (control group). + Indicates the result of the normal control group. The control group is shown as an average of 9 cases, and the other groups are shown as an average of 10 cases.

Claims (33)

A screening method for an anti-autoimmune disease agent comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the expression level of the UPR-related factor in the cell contacted with the test substance and comparing it with the expression level of the UPR-related factor in the cell in which ER stress is induced without contacting the test substance;
(c) In a control cell that does not induce ER stress, the expression level of the UPR-related factor of the cell contacted with the test substance and the UPR relation of the cell not contacted with the test substance are obtained by the same steps as in (a) and (b) above. Comparing the expression level of the factor,
(d) comparing the UPR-related factor expression modification rate as a comparison result of (b) with the UPR-related factor expression modification rate of a control cell as a comparison result of (c); and
(e) A step of selecting a test substance that selectively modifies UPR-related factor expression in cells in the presence of ER stress based on the comparison result of (d) above.   The screening method according to claim 1, wherein the UPR-related factor is a factor selected from the group consisting of Akt, Bip, XBP-1, eIF2α, XIAP and ATF4.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses the expression of Akt protein.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that enhances the expression of a Bip gene or a Bip protein.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses expression of XBP-1 mRNA in the splicing form or XBP-1 protein in the splicing form.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that promotes phosphorylation of eIF2α.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses the expression of XIAP protein.   The screening method according to claim 1, wherein the test substance that modifies the expression of the UPR-related factor is a test substance that suppresses the expression of ATF4 protein. A screening method for an anti-autoimmune disease agent comprising the following steps (a) to (e):
(a) contacting the test substance with cells in which ER stress is induced,
(b) measuring the apoptosis level of the cell contacted with the test substance and comparing it with the apoptosis level of the cell induced ER stress without contacting the test substance;
(c) In a control cell that does not induce ER stress, the apoptosis level of the cell contacted with the test substance is compared with the apoptosis level of the cell not contacted with the test substance by the same steps as in (a) and (b) above. The process of
(d) comparing the apoptosis induction rate as a comparison result of (b) above with the apoptosis induction rate of a control cell as a comparison result of (c) above, and
(e) A step of selecting a test substance that selectively induces apoptosis in cells in the presence of ER stress based on the comparison result of (d) above. The screening method according to claim 9, wherein, at a test substance concentration of 10 μM, a test substance that does not substantially induce apoptosis in the absence of ER stress and exhibits an apoptosis induction rate of 40% or more in the presence of ER stress is selected.   The screening method according to claim 1, wherein the cell is a mammalian cell.   The screening method according to claim 1, wherein the cell is a pathological condition-related cell in an autoimmune disease.   The screening method according to any one of claims 1 to 11, wherein the cell is a pathological condition-related cell in rheumatoid arthritis.   The screening method according to any one of claims 1 to 11, wherein the cell is a cell selected from the group consisting of T cells, synoviocytes, B cells, macrophages, fibroblasts and osteoclasts.   The screening method according to any one of claims 1 to 11, wherein the cell is a cell selected from the group consisting of EL-4, SW982 and BCL-1.   The screening method according to any one of claims 1 to 15, wherein the anti-autoimmune disease agent is an autoimmune disease preventive agent and / or an autoimmune disease therapeutic agent.   The screening method according to any one of claims 1 to 15, wherein the anti-autoimmune disease agent is an agent for improving immunity abnormality.   Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue disease, Hashimoto's thyroiditis, primary myxedema, thyroid poisoning, pernicious anemia, Good-pasture syndrome , Acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, lens Primary uveitis, exchange vasculitis, multiple sclerosis, ulcerative colitis, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenia The screening method according to claim 1, wherein the screening method is a disease selected from the group consisting of purpura and Sjogren's syndrome.   The screening method according to any one of claims 1 to 17, wherein the autoimmune disease is rheumatoid arthritis or multiple sclerosis.   The screening according to any one of claims 1 to 19, wherein the anti-autoimmune disease agent is an inhibitor of chronic inflammation, an inhibitor of joint destruction, an inhibitor of progression to joint deformation, or an agent of improving body function in rheumatoid arthritis patients. Method.   The anti-autoimmune disease agent which contains the substance selected using the screening method in any one of Claims 1-15 as an active ingredient.   An anti-autoimmune disease agent comprising as an active ingredient a substance having a selective UPR modifying action in the presence of ER stress.   An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective inhibitory effect on Akt1 protein expression in the presence of ER stress.   An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective Bip mRNA or Bip protein expression enhancing action in the presence of ER stress.   An anti-autoimmune disease agent comprising, as an active ingredient, a substance having an inhibitory action on the expression of alternatively spliced form XBP-1 mRNA or spliced form XBP-1 protein in the presence of ER stress.   An anti-autoimmune disease agent comprising as an active ingredient a substance having a selective phosphorylation promoting action of eIF2α in the presence of ER stress.   An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective XIAP protein expression inhibitory action in the presence of ER stress.   An anti-autoimmune disease agent comprising, as an active ingredient, a substance having a selective ATF4 protein expression inhibitory action in the presence of ER stress.   An anti-autoimmune disease agent comprising as an active ingredient a substance having a selective apoptosis-inducing action in the presence of ER stress.   A method for preventing and / or treating an autoimmune disease in a mammal, comprising modifying UPR in a mammal to a degree effective for preventing and / or treating an autoimmune disease on cells in the presence of ER stress.   Administration of a substance having a selective UPR modifying action in the presence of ER stress to a mammal in an amount effective for the prevention and / or treatment of the autoimmune disease, and / or prevention of the autoimmune disease of the mammal Method of treatment.   Use of a substance having a selective UPR modifying action in the presence of ER stress for the production of an anti-autoimmune disease agent.   Effective with 3-[(1S) -1- (2-fluorobiphenyl-4-yl) ethyl] -5-{[amino (morpholin-4-yl) methylene] amino} isoxazole or a pharmaceutically acceptable salt thereof UPR modifier included as a component.

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