Stat3 phosphorylation inhibitor, and prophylactic or therapeutic agent for autoimmune disease and coronavirus infection

Abstract

To provide a novel prophylactic or therapeutic agent for coronavirus infection, the agent containing a substance that has not been known for its therapeutic effect on coronavirus infection as an active ingredient.SOLUTION: The present invention provides a prophylactic or therapeutic agent for coronavirus infection, the agent containing stanniocalcin 1 (STC1) as an active ingredient.SELECTED DRAWING: None

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims priority based on Japanese Patent Application No. 2020 - 112680, filed on June 30, 2020, the entire disclosure of which is incorporated herein by reference. The present invention relates to a prophylactic or therapeutic agent for autoimmune diseases and coronavirus infections.

Background Art

[0002] In the respiratory region, the lung injury and fibrosis inhibitory effects and anti - inflammatory effects of mesenchymal stem cells (MSCs) have attracted attention (Non - Patent Document 3). In addition, inflammatory cytokines such as TNF - α, IL - 1β, CCL2, and IL - 6 are important factors in the cytokine storm. In particular, the mechanism by which IL - 6 is induced via the IL - 6 / JAK / STAT signaling pathway etc. is called IL - 6 amplifier (IL - 6 AMP), and is considered to be the main mechanism of cytokine storm that causes the exacerbation of autoimmune diseases typified by rheumatoid arthritis and COVID - 19 etc. (Non - Patent Document 4).

[0003] Autoimmune diseases are a general term for diseases caused by the breakdown of immune tolerance, in which the immune system over - reacts and attacks its own normal cells and / or tissues, resulting in symptoms. There are many diseases classified as autoimmune diseases, and some of them are designated as intractable diseases, so the development of treatment methods is desired.

[0004] In addition, coronaviruses are known as viruses that cause so - called cold symptoms, but there is a problem that strains with high infectivity and toxicity may occur due to mutations. In particular, currently, Coronavirus disease 2019 (Covid - 19), an infectious disease caused by such a mutant strain, severe acute respiratory syndrome coronavirus 2 (SARS - CoV2), so - called novel coronavirus infection, has spread all over the world and there have been many deaths, so the development of its treatment method is urgent.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0007] The problems to be solved by the present invention are to provide a novel inhibitor of STAT3 phosphorylation and IL6AMP. Further, it is also an object of the present invention to provide a new prophylactic or therapeutic agent for diseases that can be treated by suppressing the phosphorylation of STAT3. For example, a further problem to be solved by the present invention is to provide a novel prophylactic or therapeutic agent for autoimmune diseases containing, as an active ingredient, a substance whose therapeutic effect on autoimmune diseases has not been known heretofore. Furthermore, it is to provide a novel prophylactic or therapeutic agent for coronavirus infections containing, as an active ingredient, a substance whose therapeutic effect on coronavirus infections has not been known heretofore.

Means for Solving the Problems

[0008] Under such circumstances, as a result of examining thousands of compounds, the present inventors have found that stanniocalcin 1 (STC1) suppresses the phosphorylation of STAT3, which is downstream of the IL-6 / JAK / STAT signaling pathway. Furthermore, the present inventors have obtained the following findings by using STC1 in relation to the suppression of STAT3 phosphorylation. Specifically, the present inventors have also found that STC1 epigenetically induces the expression of the anti-inflammatory factor SOCS1. Moreover, the present inventors have also found that STC1 suppresses the infection of coronavirus into cells by suppressing the expression of the receptor for the spike protein of coronavirus on the cell surface and a factor important for the fusion of the virus outer membrane and the cell membrane. The present invention is based on these novel findings.

[0009] Therefore, the present invention provides the following items: Item 1. An inhibitor of STAT3 phosphorylation containing stanniocalcin 1 (STC1) as an active ingredient.

[0010] Item 2. An inhibitor of JAK expression or phosphorylation containing stanniocalcin 1 as an active ingredient.

[0011] Item 3. A prophylactic or therapeutic agent for autoimmune diseases containing stanniocalcin 1 as an active ingredient.

[0012] Item 4. A prophylactic or therapeutic agent for influenza virus infection, containing stanniocalcin 1 as an active ingredient.

[0013] Item 5. A prophylactic or therapeutic agent for coronavirus infection, containing stanniocalcin 1 as an active ingredient.

[0014] Item 6. The prophylactic or therapeutic agent for coronavirus infection according to Item 5, wherein the coronavirus has an S protein capable of binding to ACE2.

[0015] Item 7. The prophylactic or therapeutic agent for coronavirus infection according to Item 5 or 6, wherein the coronavirus is a SARS-related coronavirus.

[0016] Item 8. An inhibitor for the expression of ACE2 and TMPRSS2, containing stanniocalcin 1 as an active ingredient.

[0017] Item 9. An inhibitor for IL6AMP, containing stanniocalcin 1 as an active ingredient.

[0018] Item 10. An inhalant, being the STAT3 phosphorylation inhibitor according to Item 1, the JAK expression or phosphorylation inhibitor according to Item 2, the prophylactic or therapeutic agent for autoimmune diseases according to Item 3, the prophylactic or therapeutic agent for influenza virus infection according to Item 4, the prophylactic or therapeutic agent for coronavirus infection according to any one of Items 5 to 7, the inhibitor for the expression of ACE2 and TMPRSS2 according to Item 8, or the inhibitor for IL6AMP according to Item 9.

Advantages of the Invention

[0019] According to the present invention, it is possible to provide a novel inhibitor of STAT3 phosphorylation and IL6AMP. Further, the present invention provides a new prophylactic or therapeutic agent for diseases that can be treated by suppressing the phosphorylation of STAT3. For example, the present invention provides a novel prophylactic or therapeutic agent for autoimmune diseases, which contains a substance that has not been known for its therapeutic effect on autoimmune diseases as an active ingredient. Furthermore, by using STC1, which is a substance that has not been known for its therapeutic effect on coronavirus infections, as an active ingredient, it is possible to provide a new prophylactic or therapeutic agent for coronavirus infections. Specifically, according to the present invention, since the infection of coronavirus can be suppressed through the suppression of the expression of ACE2 and TMPRSS2, it is useful for the prevention and treatment of coronavirus infections. In addition, the present inventors have already found that STC1 significantly suppresses pulmonary fibrosis (Patent Document 1). Therefore, according to the present invention, not only the prevention or treatment of coronavirus infections, but also the prevention of pulmonary fibrosis when coronavirus infections deteriorate, and the treatment and progression suppression effect of pulmonary fibrosis when it has occurred are expected, so it is more useful. In addition, since a drug that can suppress pulmonary fibrosis does not necessarily exhibit the prevention or treatment of coronavirus infections, such an effect of the present invention is unexpected.

Brief Description of Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] STAT3 phosphorylation inhibitor The present invention provides a STAT3 phosphorylation inhibitor containing stanniocalcin 1 as an active ingredient. STAT3 (signal transducer and activator of transcription 3) is a protein belonging to the STAT protein family and is known to be phosphorylated and activated in the IL-6 / JAK / STAT signaling pathway and the like. Typically, examples of STAT3 include those with the amino acid sequence registered under NCBI accession No. NP_001356441.1. STC1, which is the active ingredient of the present invention, is a known ingredient described in Patent Document 1 and the like, and is a dimeric glycoprotein hormone. In the present invention, STC1 may be a natural STC1 molecule obtained from animal cells or tissues, etc., a recombinant STC1 having the same amino acid sequence as natural STC1, a recombinant STC1 produced based on an STC1 gene modified by genetic engineering techniques, or a synthesized STC1. Further, in the present invention, STC1 may be a functional equivalent such as an active fragment of STC1. Therefore, in the present invention, the term "stanniocalcin 1" (STC1) is used in the sense of including these various STC1s unless otherwise specified.

[0022] In addition, as STC1 used in the present invention, those having an amino acid sequence derived from a human or modified based on the amino acid sequence derived from a human without impairing the function (for example, one or several (for example, 2, 2 - 3, 2 - 4, 2 - 5) amino acids are deleted, added and / or substituted) are preferable. The amino acid sequence and base sequence of human STC1 are known and are registered as registration numbers NP#003146.1 and NM#003155.2. For example, the amino acid sequence of human STC1 registered as registration number NP_003146.1 is shown in SEQ ID NO: 1. Also, for many STC1s derived from animals other than humans, the amino acid sequence and base sequence are known. All of these are known to have high homology to human STC1 (Table 1).

[0023]

Table 1

[0024] Therefore, in one embodiment of the present invention, examples of usable STC1 include those having 80% or more, preferably 90% or more, more preferably 94% or more homology to the amino acid sequence of human STC1. Typically, it is more preferable to use a natural or recombinant STC1 (rSTC1) having an amino acid sequence that is 100% identical to the human STC1 sequence (for example, the human STC1 sequence derived from 293 cells). In the present invention, these STC1s can be used alone or in combination of two or more.

[0025] Since STC1 and its salts, which are the active ingredients of the present invention, may exist in the form of hydrates or solvates, these hydrates and solvates are also included in the compounds that are the active ingredients of the present invention.

[0026] Examples of the solvent forming the solvate include alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, and DMSO. These solvents can be used alone or in combination of two or more as a mixed solvent.

[0027] In the present invention, STC1 itself, which is the active ingredient of the present invention, may be used as an inhibitor of STAT3 phosphorylation, or may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizing agents, thickening agents, etc.).

[0028] Examples of isotonic agents include saccharides such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonic agents can be used alone or in combination of two or more.

[0029] Examples of chelating agents include edetates such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate; ethylenediaminetetraacetate salts; nitrilotriacetic acid or its salts; sodium hexametaphosphate; and citric acid. These chelating agents can be used alone or in combination of two or more.

[0030] Examples of stabilizers include sodium bisulfite.

[0031] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0032] Examples of preservatives include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride; alkyl polyaminoethyl glycine; chlorobutanol; polyquaternium; polyhexamethylene biguanide; and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0033] Examples of the antioxidant include sodium bisulfite, dried sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol, and the like. These antioxidants can be used alone or in combination of two or more.

[0034] Examples of the solubilizing agent include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, and the like. These solubilizing agents can be used alone or in combination of two or more.

[0035] Examples of the thickening agent include polyethylene glycol, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, and the like. These thickening agents can be used alone or in combination of two or more.

[0036] In an embodiment of the pharmaceutical composition, the content of STC1 in the composition is not particularly limited, and can be appropriately set from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more, etc.

[0037] The dosage form is not particularly limited, and examples include oral administration agents such as tablets, pills, capsules, powders, granules, syrups; parenteral administration agents such as injections (intravenous injection, intramuscular injection, local injection, etc.), gargles, drip infusions, external preparations (ointments, creams, patches, inhalants), suppositories, and other various dosage forms. Among the above dosage forms, preferred ones include external preparations, etc., and among them, inhalants (transairway administration agents) are included.

[0038] In the present invention, the dosage of STC1 varies depending on the administration route, the age, weight, symptoms, etc. of the patient and cannot be uniformly defined. However, the daily dosage for adults is usually about 5000 mg or less, preferably about 1000 mg or less. The lower limit of the dosage of STC1 is not particularly limited either. For example, as the dosage of STC1, the daily dosage for adults can be appropriately set usually in the range of 0.1 mg or more, preferably 0.5 mg or more. When administered once a day, this amount may be included in one preparation. When administered three times a day, one-third of this amount may be included in one preparation.

[0039] The STAT3 phosphorylation inhibitor of the present invention is administered to patients such as mammals. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep, etc.

[0040] Agent for preventing or treating autoimmune diseases The present invention provides a prophylactic or therapeutic agent for autoimmune diseases containing stanniocalcin 1 as an active ingredient. When cytokines such as IL6 bind to receptors, phosphorylation of JAK and STAT is induced, and transcription of acute-phase proteins, etc. is induced (Non-Patent Document 5). SOCS indirectly suppresses the phosphorylation of STAT by suppressing the phosphorylation of JAK. The IL6 / JAK / STAT pathway is considered to be an important pathway involved in the onset of autoimmune diseases such as rheumatoid arthritis and is a target for drug development. Here, as described above, the present inventors have now found that STC1 suppresses the phosphorylation of STAT3 downstream of the IL-6 / JAK / STAT signaling pathway. The present invention is based on these new findings. Regarding the use of STC1, its hydrates and solvates, which are the active ingredients of the present invention, etc., is as described above.

[0041] Regarding the target diseases in the present invention, examples of autoimmune diseases include rheumatoid arthritis, Castleman's disease, Crohn's disease, Wegener's granulomatosis, sarcoidosis, atopic dermatitis, chronic pyoderma, psoriasis, hyper IgE syndrome with pneumonia, etc.

[0042] In the present invention, STC1 itself, which is the active ingredient of the present invention, may be used as a prophylactic or therapeutic agent for autoimmune diseases, or may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers. As the carrier, those described above for the STAT3 phosphorylation inhibitor can be used in the same manner in the amounts described above.

[0043] In addition, the above pharmaceutical composition may further contain, in addition to STC1, a compound that is said to have a prophylactic or therapeutic effect on autoimmune diseases. Examples of compounds known to have a prophylactic or therapeutic effect on autoimmune diseases include, for example, corticosteroids (such as prednisolone), JAK inhibitors (such as tofacitinib), calcineurin inhibitors (such as cyclosporine and tacrolimus), DNA synthesis inhibitors (such as cyclophosphamide), mTOR inhibitors (such as sirolimus), IMDH inhibitors (such as mycophenolate mofetil), antibody preparations (such as tocilizumab and rituximab), and the like. These compounds can be used alone or in combination of two or more. Regarding the content of STC1 in the composition, the dosage form, the dosage, the patients to be administered, etc. in the embodiment of the pharmaceutical composition are the same as those described above for the STAT3 phosphorylation inhibitor.

[0044] Agent for preventing or treating coronavirus infections The present invention provides a prophylactic or therapeutic agent for coronavirus infection containing STC1. Regarding the use of STC1, its hydrates and solvates, which are the active ingredients of the present invention, etc., are as described above.

[0045] Regarding the target disease in the present invention, the coronavirus is intended to mean a virus belonging to the family Coronaviridae. Examples of coronaviruses include those belonging to the genus Alphacoronavirus, genus Betacoronavirus, etc., preferably those belonging to the genus Betacoronavirus, etc. Examples of the genus Alphacoronavirus include the genus Duvinacovirus (e.g., HCoV-229E, etc.), the genus Setracovirus (e.g., HCoV-NL63, etc.). Examples of the genus Betacoronavirus include the genus Embecovirus (e.g., HCoV-OC43, HCoV-HKU1, etc.), the genus Sarbecovirus, the genus Merbecovirus (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), etc.), etc., preferably the genus Sarbecovirus, etc. Examples of the genus Sarbecovirus include severe acute respiratory syndrome-related coronavirus (SARSr-CoV), etc. Examples of SARSr-CoV include severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), etc.

[0046] In the case of coronaviruses such as SARS-CoV2, they have Spike protein (S protein) on their envelope, and it is known that the S protein binds to the receptor (ACE2) on the cell membrane and then undergoes proteolysis by Transmembrane protease, serine 2 (TMPRSS2), a protease, which is important for the fusion of the viral outer membrane and the cell membrane. It is considered that ACE2 and TMPRSS2 are essential in airway cells for the infection of SARS-CoV-2 (Non-Patent Document 1, Non-Patent Document 2). The inventors of the present invention found that the expression of ACE2 and TMPRSS2 is enhanced by lung injury, and by using STC1, which is an active ingredient of the preventive or therapeutic agent of the present invention, the enhanced expression of ACE2 and TMPRSS2 during the lung injury can be suppressed. Therefore, the preventive or therapeutic agent for coronavirus infection is particularly effective against coronaviruses having S protein, typically coronaviruses having S protein that can bind to ACE2.

[0047] In the present invention, STC1 itself, which is the active ingredient of the present invention, may be used as a prophylactic or therapeutic agent for coronavirus infection, or may be used as a pharmaceutical composition in combination with various pharmaceutically acceptable carriers. As the carrier, those described above regarding the STAT3 phosphorylation inhibitor can be used in the same manner.

[0048] Further, in addition to STC1, the above pharmaceutical composition may further contain a compound having a prophylactic or therapeutic effect on coronavirus infection. Compounds known to have a prophylactic or therapeutic effect on coronavirus infection include, for example, favipiravir, remdesivir, ivermectin, ciclesonide, tocilizumab, camostat, nafamostat. These compounds can be used alone or in combination of two or more. Regarding the content of STC1 in the composition, the dosage form, the dosage, the patient to be administered, etc. in the embodiment of the pharmaceutical composition are the same as those described above regarding the STAT3 phosphorylation inhibitor.

[0049] STC1, which is an active ingredient of a prophylactic or therapeutic agent for coronavirus infectious disease of the present invention, prevents, treats and / or alleviates coronavirus infectious disease by suppressing the expression of at least ACE2 and TMPRSS2. Accordingly, the present invention also provides an agent for suppressing the expression of ACE2 and TMPRSS2, which contains STC1. Further, the present inventors have found that STC1 significantly suppresses pulmonary fibrosis, and a pharmaceutical composition for preventing, treating and / or suppressing the progression of pulmonary fibrosis containing STC1 as an active ingredient has been patented (Patent Document 1). However, although pulmonary fibrosis may occur when coronavirus infectious disease deteriorates, it is known that pulmonary fibrosis can be caused by a variety of factors, and coronavirus infectious disease is only one of them, and it is clear that a compound capable of treating pulmonary fibrosis does not necessarily suppress the infection of coronavirus itself. Further, there has been no report so far on the relationship between the expression of ACE2 and TMPRSS2 and STC1. Therefore, the effect of suppressing the expression of ACE2 and TMPRSS2 by STC1 in the present invention and the prophylactic or therapeutic effect on coronavirus infectious disease are unpredictable from the prior art. Further, it has been reported that TMPRSS2 also plays an important role in the infection of influenza (type A, type B) and other respiratory-related viruses (Non-Patent Document 6). Accordingly, the agent for suppressing the expression of ACE2 and TMPRSS2 of the present invention can also be used as a prophylactic or therapeutic agent for respiratory-related viruses (for example, coronavirus, influenza virus). Incidentally, the agent for suppressing the expression of ACE2 and TMPRSS2, the active ingredient, dosage form, dosage, etc. of the prophylactic or therapeutic agent for respiratory-related viruses are the same as those of the prophylactic or therapeutic agent for coronavirus infectious disease. IL-6 AMP inhibitor When SARS-CoV2 binds to ACE2, angiotensin 2, which should normally bind to and be degraded by ACE2, induces TNFα, IL6, etc. through a receptor called AT1R. As a result, STAT3 is activated, and the synthesis of IL6 is further enhanced. Since IL6 itself amplifies IL6, it is called IL6 amplifier (AMP) and is thought to be involved in the aggravation of COVID19. Also, the pathway in which the virus is recognized by innate immunity and NfkB is activated is thought to act additively to IL6AMP. As described above, STC1, which is the active ingredient of the present invention, suppresses IL-6 AMP by inhibiting the IL-6 / JAK / STAT signaling pathway. Therefore, the present invention also provides an inhibitor of IL-6 AMP containing STC1. Incidentally, the active ingredient, dosage form, dosage, etc. of the inhibitor of IL-6 AMP are the same as those described above.

[0050] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example

[0051] In this example, human recombinant (r)STC1 was manufactured by BioVender. Specifically, a human-derived STC1 gene with a TAG structure added was introduced into 293 cells, and rSTC1 was isolated using the TAG sequence. In the following examples, the above human recombinant (r)STC1 may be simply referred to as STC1.

[0052] Example 1 In the bleomycin-induced lung injury model, intratracheal administration of STC1 suppresses the expression of ACE2 and TMPRSS2 In this example, a mouse bleomycin (BLM) intratracheal administration lung injury model was used (Figure 1). Specifically, on Day0, C57BL6 / J mice (female, 8 weeks old), six per group, were anesthetized by intraperitoneal administration of an anesthetic (a mixture of 300 μg of ketamine hydrochloride + 160 ng of xylazine diluted in 100 μL of physiological saline). Then, the neck was incised to expose the trachea, and the outer sheath of a 23G Surflo needle was inserted into the trachea. Further, the following drugs were administered intratracheally from the Surflo sheath to the tracheas of the mice in each group. (i) Saline group (control): 50 μL of saline (ii) BLM group: 50 μL of bleomycin-containing saline (bleomycin concentration 0.4 mg / ml) (iii) BLM+STC1 group: 50 μL of bleomycin-containing saline (bleomycin concentration 0.4 mg / mL) On Day 1, in the same manner as above, the mice were anesthetized, and the following drugs were respectively administered into the trachea of the mice in each group from a surflo catheter. (i) Saline group (control): 50 μL of saline (ii) BLM group: 50 μL of saline (iii) BLM+STC1 group: 50 μL of STC1-containing saline (STC1 concentration 40 μg / mL) On Day 3, three mice in each group were anesthetized in the same manner as above, and then the mice were euthanized by cutting the carotid artery, and then the lungs were excised. Furthermore, on Day 14, three mice in each group were anesthetized in the same manner as above, and then the mice were euthanized by cutting the carotid artery, and then the lungs were excised. Western Blotting was performed on the excised lungs. Antibodies used were anti-ACE2 antibody (E-11, 2E2) and anti-TMPRSS2 antibody (H8) from SantaCruz. The results of western blotting for each group are shown in Figure 2A. As shown in Figure 2A, the increase in ACE2 and TMPRSS2 induced by bleomycin lung injury was strongly inhibited by STC1.

[0053] Quantitative PCR was also performed using the ABI7500 RealTime PCR system from Applied Biosystem. The results are shown in Figure 2B. As shown in Figure 2B, the expression of ACE2 and TMPRSS2 was enhanced by lung injury (comparison between Saline group and Bleo group). And as is clear from the comparison between the Bleo group and the Bleo+STC1 group in Figure 2B, the administration of STC1 via the airway suppressed the increase in the expression of ACE2 and TMPRSS2 during lung injury. As described above, since the presence of ACE2 and TMPRSS2 in airway cells is essential for the infection of the coronavirus, it is considered that STC1 is effective for the prevention and treatment of coronavirus infection based on the above results. Further, as described above, since the expression of ACE2 and TMPRSS2 was enhanced due to lung injury, it is considered that the expression of ACE2 and TMPRSS2 is renewed due to lung injury, whereby the coronavirus easily enters the cell via ACE2 and TMPRSS2, and furthermore, a vicious cycle in which the coronavirus infection worsens occurs. According to the present invention, it is useful because it can suppress the progression of the above vicious cycle by suppressing the expression of ACE2 and TMPRSS2.

[0054] Example 2 Investigation of the metabolic modification effect of STC1 by metabolome analysis Regarding the effect of STC1 on SMAD2, 3, 7 The influence of STC1 on the mitochondrial TCA cycle and its peripheral metabolism is shown in FIG. 3.

[0055] The influence of STC1 on the TGFβ / SMAD signaling system was analyzed by western blotting and quantitative PCR. Specifically, human alveolar epithelial cell lines A549, H1299, and human fibroblast cell lines MRC5, HFL1 were cultured for 24 hours in a cell culture medium supplemented with recombinant STC1 (50 ng / ml) or without supplementation. As the antibodies, anti-SMAD7 antibody (B8) from Santa Cruz and Phospho-Smad Antibody Sampler Kit from Cell Signaling Technology were used. For quantitative PCR, an ABI7500 Real Time PCR system from Applied Biosystem was used. The results are shown in FIGS. 4A to D.

[0056] As shown in Figure 4, STC1 suppressed the phosphorylation of SMAD2 / 3 necessary for lung injury and pulmonary fibrosis. This effect was due to STC1 inducing SMAD7, an inhibitory SMAD. Excessive and persistent secretion of TGFbeta induces chronic antioxidant stress and inflammatory cytokines via SMAD2 / 3 activation. These are involved in the cytokine storm related to the exacerbation of coronavirus infections such as COVID19 (lung injury, etc.). Therefore, from the above results, it is considered that suppression of SMAD2 / 3 phosphorylation by STC1 can avoid lung injury in coronavirus infections and severe pneumonia by suppressing the persistent secretion of inflammatory cytokines. Figure 5 shows the effect of STC1 on the methylation of the promoter region of the SMAD7 gene. The effect of STC1 on the methylation of the promoter region of the SMAD7 gene was analyzed by quantitative methylation-specific PCR (qMSP). Specifically, genomic DNA extracted from each cell was treated with bisulfite (Fujifilm-Wako, EpiSight TM Bisulfite Conversion Kit Ver.2), and performed using a polymerase for methylation-specific PCR (Takara Episcope MSP kit) and an ABI7500 RealTime PCR system. As shown in Figure 5, STC1 also induced demethylation of SMAD7. Figure 6 shows the effect of STC1 on the acetylation of SMAD7 protein. The effect of STC1 on SMAD7 in the lungs of mice was analyzed by fluorescence immunohistochemistry on excised mouse lungs. Specifically, the lungs used in Example 1 were sectioned thinly, fixed to a preparation, and a combination of an SMAD7 antibody (Santa Cruz B8), an anti-acetylated lysine antibody (Cell Signaling Technology (Ac-K-103), nuclear staining (DACO DAPI), Alexa-488 antibody and Alexa594 antibody from Molecular Devices was used and examined using a phase-contrast microscope. The results are shown in Fig. 6. As shown in Fig. 6, STC1 also induced the acetylation of the lysine group of SMAD7. By being acetylated, SMAD7 can be stably expressed. That is, it became clear that STC1 enhanced the expression of SMAD7 through epigenetic regulation of SMAD7.

[0057] Example 3 In addition, hematoxylin and eosin staining was performed using the lung specimens excised from the mice in each group obtained in Example 1. The results are shown in Fig. 7. As shown in Fig. 7, STC1 inhibited the lung inflammation and fibrosis caused by bleomycin.

[0058] Example 4 Evaluation of the effect of STC1 on STAT3 phosphorylation, IL-6AMP, etc. <Method> ·Cell experiment Human-derived monocyte cells THP1 cells were cultured in RPMI medium (Sigma) containing 10% fetal bovine serum (Nichirei) and 1% penicillin streptomycin (Sigma) for 24 hours, and then differentiated into macrophages by adding 10 μM phorbol 12-myristate 13-acetate (PMA). Twenty-four hours after adding PMA, three groups were prepared: a group without adding human recombinant IL6, a group adding 10 ng / ml of IL6, and a group adding 50 ng / ml of IL6. Further, for each of these groups, a group without adding 50 ng / ml of STC1 and a group adding 50 ng / ml of STC1 were prepared, that is, six groups of 3×2 were prepared. After culturing for 24 hours under these conditions, Western blot, immunoprecipitation, PCR, methylation-specific PCR, and suppression of specific gene transcription using siRNA were performed. ·Animal experiment In this example, a mouse model of bleomycin (BLM)-induced airway lung injury was used. Specifically, according to the method described in Example 1, mouse models of bleomycin (BLM)-induced airway lung injury were prepared, and the lungs were excised from three mice in each group on Day 3 and Day 14. ·Method of Western blot After electrophoresis of the total extract of THP1 cells or mouse excised lung extract with RIPA buffer on an SDS-PAGE gel, the proteins derived from cells or tissues were transferred to a PVDF membrane, incubated with a primary antibody and an HRP-conjugated secondary antibody, developed with a chromogenic substrate, and the protein expression was measured with a chemiluminometer. The primary antibodies used in Western blot were as follows. Anti-STAT3 antibody (Cell Signaling Technology (CST), 12640), anti-pSTAT3 antibody (CST, 9145), anti-SOCS1 antibody (CST, 3950), anti-IL6 antibody (CST, 12153), anti-JAK1 antibody (CST, 3344), anti-pJAK1 antibody (CST, 74129), anti-Ubiquitin antibody (CST, 58395), anti-ACE2 antibody (Santa Cruz (SC), 390851), anti-TMPRSS2 antibody (SC, 515727), anti-βACTIN antibody (R&D, MAB8929). HRP-conjugated antibodies compatible with each animal species were used as secondary antibodies. · Method of immunoprecipitation After binding Protein A / G plus agarose (SC, 2003) and anti-JAK1 antibody (SC, 1677), the total extract of THP1 cells or mouse excised lung extract with RIPA buffer was added to recover JAK1 and the proteins binding to JAK1 in these extracts. By boiling with a reducing solution, the JAK1 protein and the proteins binding to JAK1 were separated. · Semi-quantitative PCR mRNA was separated from THP1 cells and mouse lung tissues using the RNeasy mini kit (QIAGEN), and cDNA reverse-transcribed from mRNA was prepared using the high capacity cdna kit (Applied Biosystems). Primers were designed using primer blast (NCBI), and semi-quantitative PCR was performed using SYBR Green PCR Master Mix and the ABI7500 real-time PCR system (Applied Biosystems). · Methylation-specific PCR Methylation and demethylation primers for the promoter region of the SOCS1 gene were designed using Methyl Primer Express software (Applied Biosystems). Genomic DNA extracted from THP1 cells using the DNeasy Blood & Tissue Kit (QIAGEN) was subjected to bisulfite treatment (Fujifilm-Wako, EpiSight TM Bisulfite Conversion Kit Ver.2), and performed using a polymerase for methylation-specific PCR (Takara Episcope MSP kit) and an ABI7500 RealTime PCR system. ·siRNA Predesigned siRNA (Ambion) was used for SOCS1-specific siRNA and control siRNA. The transfection reagent used was Lipofectamine RNAiMAX (Invitrogen).

[0059] <Results> (THP1 cell data) 1) STC1 strongly suppresses IL6-induced STAT3 phosphorylation in THP1 cells The results of measuring the presence or absence of STAT3 and phosphorylated STAT3 in the total cell extracts of THP1 cells with or without the addition of IL6 (10 ng / ml), IL6 (50 ng / ml), and / or STC1 (50 ng / ml) according to the above method, as well as THP1 cells without these additions (control), are shown in Fig. 9. As shown in Fig. 9, STC1 suppressed STAT3 phosphorylation induced by IL6. That is, STC1 is useful for the treatment of cytokine syndrome and various autoimmune diseases by suppressing IL6-induced STAT3 phosphorylation.

[0060] 2) STC1 suppresses the enhancement of IL6 production (IL6 amp) induced by IL6 in THP1 cells The results of measuring the presence or absence of IL6 in the total cell extracts of THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method and THP1 cells to which these were not added (control) by Western blot are shown in Fig. 10. As shown in Fig. 10, STC1 suppressed the enhancement of IL6 production (IL6 amp) induced by IL6. That is, STC1 is useful for the treatment of cytokine syndrome and various autoimmune diseases by suppressing IL6-induced IL6 amplification (IL6 amp).

[0061] 3) STC1 induces SOCS1 in THP1 cells The Western blot of SOCS1 (Fig. 11A), semi-quantitative PCR of SOCS1 mRNA using total cell extract mRNA (Fig. 11B), and the results of measuring the degree of methylation of the SOCS1 gene promoter region using total cell extract DNA by methylation-specific PCR (Fig. 11C) in the total cell extracts of THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method and THP1 cells to which these were not added (control) are shown. As shown in Fig. 11A, STC1 enhanced the production of SOCS1 protein. Also, as shown in Fig. 11B, STC1 enhanced the mRNA production of SOCS1. As shown in Fig. 11C, STC1 suppressed the methylation of the SOCS1 gene promoter region. The results of Figs. 11A - C all imply the enhanced production of SOCS1 protein by STC1. That is, STC1 is useful for the treatment of cytokine syndrome and various autoimmune diseases by inducing SOCS1 which has the effect of suppressing JAK.

[0062] 4) STC1 suppresses the expression and phosphorylation of JAK1 in THP1 cells The results of measuring the presence or absence of JAK1 and phosphorylated JAK1 in the total cell extracts of THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method, and THP1 cells to which these were not added (control), by Western blot, are shown in Fig. 12. As shown in Fig. 12, STC1 suppressed the expression and phosphorylation of JAK1.

[0063] 5) In the presence of IL6, STC1 promotes the binding of SOCS1 and JAK1 and promotes the ubiquitination of JAK1 in THP1 cells (immunoprecipitation) The results of immunoprecipitating the total cell extracts of THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method, and THP1 cells to which these were not added (control), are shown in Fig. 13. For immunoprecipitation, THP1 whole cell lysates were used, and JAK1 and JAK1-binding proteins were recovered using a JAK antibody and A / G agarose gel, and Western blot was performed after adding a reducing agent and boiling. As shown in Fig. 13, SOCS1 that binds to JAK1 decreases in the presence of IL6, but SOCS1 that binds to JAK1 increases in the presence of STC1. Accordingly, it was revealed that STC1 also promotes the ubiquitination of JAK1. That is, STC1 promotes the binding of JAK1 and SOCS1 and enhances the ubiquitination (degradation of JAK1) of JAK1.

[0064] 6) STC1 suppresses the expression of ACE2 and TMPRSS2 induced by IL6 in THP1 cells The results of Western blotting the total cell extracts of THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method, and THP1 cells to which these were not added (control), are shown in Fig. 14. As shown in Fig. 14, STC1 suppressed the expression of ACE2 and TMPRSS2. Therefore, STC1 may reduce the chance of SARS-CoV2 infection of cells by suppressing the expression of ACE2 and TMPRSS2.

[0065] 7) STC1 suppresses the expression of ACE2 and TMPRSS2 in a SOCS1-dependent manner In THP1 cells to which IL6 (10 ng / ml), IL6 (50 ng / ml) and / or STC1 (50 ng / ml) were added according to the above method, and THP1 cells to which these were not added (control), the results of Western blotting when SOCS1 was suppressed using siRNA are shown in Fig. 15. siCTR is the control group and shows the behavior when SOCS1 is the same as normal. As shown in Fig. 15, the inhibitory effect of STC1 on the induction of ACE2 and TMPRSS2 in THP1 cells disappeared when SOCS1 was knocked down using siRNA. This indicates that the inhibitory effect of STC1 on ACE2 and TMPRSS2 is SOCS1-dependent. (Animal model: Mouse bleomycin intratracheal administration model, a state in which inflammation has occurred in the lungs)

[0066] 8) STC1 suppresses the induction of JAK1 expression, phosphorylation of JAK1, and upregulation of ACE2 and TMPRSS2 expression induced in mouse lungs by intratracheal administration of bleomycin According to the above method, physiological saline or physiological saline in which bleomycin was dissolved was intratracheally administered to animals (C57BL6 mice). In the intratracheal administration group of physiological saline in which bleomycin was dissolved, physiological saline or STC1-dissolved physiological saline solution was intratracheally administered the next day. The results of collecting lung tissue and performing Western blotting 3 days and 14 days later are shown in Fig. 16. As shown in Fig. 16, STC1 suppressed the phosphorylation of JAK1, the phosphorylation of STAT3, the increased expression of ACE2, and the increased expression of TMPRSS2 caused by bleomycin-induced inflammation. Therefore, intratracheal administration of STC1 suppresses the JAK / STAT3 pathway and at the same time suppresses the expression of ACE2 and TMPRSS2. That is, by suppressing the cytokine release syndrome in COVID19 and suppressing the expression of ACE2 and TMPRSS2, it may be possible to suppress the infection and aggravation of COVID19. Fig. 17 summarizes the mechanism of action of STC1 clarified this time.

Claims

1. A STAT3 phosphorylation inhibitor containing stanniocalcin 1 (STC1) as an active ingredient.

2. A JAK expression or phosphorylation inhibitor containing stanniocalcin 1 as an active ingredient.

3. A prophylactic or therapeutic agent for autoimmune diseases containing stanniocalcin 1 as an active ingredient.

4. A prophylactic or therapeutic agent for influenza virus infection containing stanniocalcin 1 as an active ingredient.

5. A prophylactic or therapeutic agent for coronavirus infection containing stanniocalcin 1 as an active ingredient.

6. The prophylactic or therapeutic agent for coronavirus infection according to claim 5, wherein the coronavirus has an S protein capable of binding to ACE2.

7. The prophylactic or therapeutic agent for coronavirus infection according to claim 5 or 6, wherein the coronavirus is a SARS-related coronavirus.

8. An inhibitor of the expression of ACE2 and TMPRSS2 containing stanniocalcin 1 as an active ingredient.

9. An inhibitor of IL6AMP containing stanniocalcin 1 as an active ingredient.

10. The STAT3 phosphorylation inhibitor according to claim 1, the JAK expression or phosphorylation inhibitor according to claim 2, the prophylactic or therapeutic agent for autoimmune diseases according to claim 3, the prophylactic or therapeutic agent for influenza virus infection according to claim 4, the prophylactic or therapeutic agent for coronavirus infection according to any one of claims 5 to 7, the inhibitor of the expression of ACE2 and TMPRSS2 according to claim 8, or the inhibitor of IL6AMP according to claim 9, which is an inhalant.

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