Method for suppressing immunoreaction

Abstract

The present disclosure provides a method for suppressing an excessively enhanced immunoreaction of a subject. In one aspect, the present disclosure provides a method for suppressing an excessively enhanced immunoreaction of a subject, the method comprising administering to the subject a therapeutically effective amount of a drug that reduces the intracellular concentration of calcium. In one embodiment, the drug may be a drug capable of reducing the intracellular concentration of free calcium ions, a drug capable of suppressing or blocking the calcium ion influx into cells, or a drug capable of discharging calcium ions from cells to reduce the intracellular concentration of calcium ions.

Description

Methods for suppressing immune responses

[0001] The present invention relates to a method and a pharmaceutical composition for suppressing an excessive immune response.

[0002] The immune system protects the body by recognizing and eliminating non-self substances, such as pathogens that invade from the outside and abnormal cells and molecules produced within the body. The immune system is precisely regulated to attack non-self substances, such as pathogens and abnormal cells, while not attacking normal self substances. However, a breakdown in this control mechanism can lead to various intractable diseases caused by an excessive immune response, such as autoimmune diseases such as psoriasis vulgaris and rheumatoid arthritis, and allergic diseases such as atopic dermatitis.

[0003] In the case of atopic dermatitis, steroids are frequently prescribed as a treatment to reduce the immune response, but long-term use is not recommended due to safety concerns. Furthermore, it has become clear that many molecules, such as IL-4, IL-13, IL-31, Janus kinase, and phosphodiesterase, are widely and intricately involved in the immune response in atopic dermatitis, and therapeutic drugs that suppress the function of these molecules have been developed. However, these therapeutic drugs do not provide a high level of satisfaction, perhaps because they only suppress part of the immune response.

[0004] An objective of the present disclosure is to provide a method for suppressing immune responses in subjects with excessively enhanced immune responses, such as patients with psoriasis vulgaris or atopic dermatitis.

[0005] As a result of extensive research, the present inventors have found that an immune response in a subject can be suppressed by administering a therapeutically effective amount of a drug that reduces intracellular calcium ion concentration. Based on this finding, the present inventors conducted further research and completed the invention of the present disclosure. Specifically, the present invention encompasses the following aspects: (Item 1) A composition for suppressing an immune response in a subject, the composition comprising an agent that reduces intracellular free calcium ion concentration. (Item 2) The composition described above, wherein the agent is an agent capable of forming a complex with calcium ions or an agent capable of inhibiting or blocking calcium ion influx into the cells. (Item 3) The composition described above, wherein the agent is capable of expelling calcium ions from the cells to reduce the intracellular calcium ion concentration. (Item 4) The composition described above, wherein the cells are immune cells. (Item 5) The composition described above, wherein the immune cells are at least one type selected from the group consisting of T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells. (Item 6) The composition according to any one of the above items, wherein the immune cells are T lymphocytes. (Item 7) The composition according to any one of the above items, wherein the agent is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (In the formula, R 1 ~R 6each independently represent hydrogen or an alkyl group having 3 or less carbon atoms. (Item 8) The composition according to any of the above items, wherein the drug is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof. (Item 9) The composition according to any of the above items, wherein the drug is selected from pyoktanin blue or a pharmaceutically acceptable salt thereof. (Item 10) A composition for treating or preventing an allergic disease in a subject, the composition comprising an agent that reduces intracellular calcium ion concentration. (Item 11) The composition according to any of the above items, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, asthma, seasonal allergic rhinitis, and food allergy. (Item 12) The composition according to any of the above items, wherein the allergic disease is atopic dermatitis. (Item 13) A composition for treating or preventing an autoimmune disease in a subject, the composition comprising an agent that reduces intracellular calcium ion concentration. (Item 14) The composition according to any of the above items, wherein the autoimmune disease is at least one selected from the group consisting of psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis. (Item 15) The composition according to any of the above items, wherein the autoimmune disease is psoriasis vulgaris. (Item 16) The composition according to any of the above items, wherein the composition is administered by topical administration, oral administration, intravenous administration, inhalation, nasal administration, subcutaneous administration, intramuscular administration, sublingual administration, or ophthalmic administration. (Item 17) A calcium ion releasing agent comprising an agent capable of releasing intracellular calcium ions to reduce the intracellular calcium ion concentration, wherein the agent is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (Item 18) A calcium ion releasing agent comprising an agent capable of releasing intracellular calcium ions to reduce the intracellular calcium ion concentration, wherein the agent is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof.(Item 19) The calcium ion releasing agent according to any one of the above items, wherein the calcium ion releasing agent is for the treatment or prevention of ischemic heart disease, high blood pressure, epilepsy, headache, or pruritus. (Item 1A) A method for suppressing an immune response in a subject, comprising administering to the subject a drug that reduces the intracellular free calcium ion concentration. (Item 2A) The method according to any one of the above items, wherein the drug is a drug capable of forming a complex with calcium ions or a drug capable of inhibiting or blocking the influx of calcium ions into the cell. (Item 3A) The method according to any one of the above items, wherein the drug is capable of releasing calcium ions from the cell to reduce the intracellular calcium ion concentration. (Item 4A) The method according to any one of the above items, wherein the cell is an immune cell. (Item 5A) The method according to any one of the above items, wherein the immune cell is at least one type selected from the group consisting of T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells. (Item 6A) The method according to any one of the above items, wherein the immune cells are T lymphocytes. (Item 7A) The method according to any one of the above items, wherein the agent is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (In the formula, R 1 ~R 6each independently represent hydrogen or an alkyl group having 3 or less carbon atoms. (Item 8A) The method according to any one of the above items, wherein the drug is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof. (Item 9A) The method according to any one of the above items, wherein the drug is selected from pyoktanin blue or a pharmaceutically acceptable salt thereof. (Item 10A) A method for treating or preventing an allergic disease in a subject, comprising administering to the subject a drug that decreases intracellular calcium ion concentration. (Item 11A) The method according to any one of the above items, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, asthma, seasonal allergic rhinitis, and food allergy. (Item 12A) The method according to any one of the above items, wherein the allergic disease is atopic dermatitis. (Item 13A) A method for treating or preventing an autoimmune disease in a subject, comprising administering to the subject a drug that decreases intracellular calcium ion concentration. (Item 14A) The method according to any one of the above items, wherein the autoimmune disease is at least one selected from the group consisting of psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis. (Item 15A) The method according to any one of the above items, wherein the autoimmune disease is psoriasis vulgaris. (Item 16A) The method according to any one of the above items, wherein the agent is administered by topical administration, oral administration, intravenous administration, inhalation, nasal administration, subcutaneous administration, intramuscular administration, sublingual administration, or ophthalmic administration. (Item 17A) A method for decreasing intracellular calcium ion concentration by excreting calcium ions from cells, the method comprising contacting the cells with an agent capable of excreting calcium ions from the cells and decreasing the intracellular calcium ion concentration, the agent being a compound represented by formula (1) or a pharmaceutically acceptable salt thereof.(Item 18A) A method for reducing intracellular calcium ion concentration by excreting intracellular calcium ions, the method comprising contacting the cells with an agent capable of excreting intracellular calcium ions and reducing the intracellular calcium ion concentration, wherein the agent is selected from pyoktanin blue, methylene blue, an analog thereof, or a pharmaceutically acceptable salt thereof. (Item 19A) The method according to any of the above items, wherein the method is for the treatment or prevention of ischemic heart disease, high blood pressure, epilepsy, headache, or pruritus. (Item 1B) Use of an agent that reduces intracellular free calcium ion concentration in the manufacture of a medicament for suppressing an immune response in a subject. (Item 2B) The use according to any of the above items, wherein the agent is an agent capable of forming a complex with calcium ions or an agent capable of inhibiting or blocking the influx of calcium ions into the cells. (Item 3B) The use according to any of the above items, wherein the agent is capable of excreting intracellular calcium ions and reducing the intracellular calcium ion concentration. (Item 4B) The use according to any of the above items, wherein the cell is an immune cell. (Item 5B) The use according to any of the above items, wherein the immune cells are at least one type selected from the group consisting of T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells. (Item 6B) The use according to any of the above items, wherein the immune cells are T lymphocytes. (Item 7B) The use according to any of the above items, wherein the agent is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (In the formula, R 1 ~R 6each independently represent hydrogen or an alkyl group having 3 or less carbon atoms. (Item 8B) The use according to any of the above items, wherein the drug is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof. (Item 9B) The use according to any of the above items, wherein the drug is selected from pyoktanin blue or a pharmaceutically acceptable salt thereof. (Item 10B) Use of a drug that decreases intracellular calcium ion concentration in the manufacture of a medicament for treating or preventing an allergic disease in a subject. (Item 11B) Use according to any of the above items, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, asthma, seasonal allergic rhinitis, and food allergy. (Item 12B) Use according to any of the above items, wherein the allergic disease is atopic dermatitis. (Item 13B) Use of a drug that decreases intracellular calcium ion concentration in the manufacture of a medicament for treating or preventing an autoimmune disease in a subject. (Item 14B) The use according to any of the above items, wherein the autoimmune disease is at least one selected from the group consisting of psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis. (Item 15B) The use according to any of the above items, wherein the autoimmune disease is psoriasis vulgaris. (Item 16B) The use according to any of the above items, wherein the medicament is administered by topical administration, oral administration, intravenous administration, inhalation, nasal administration, subcutaneous administration, intramuscular administration, sublingual administration, or ophthalmic administration. (Item 17B) Use of a drug capable of reducing intracellular calcium ion concentration by excreting intracellular calcium ions in the manufacture of a calcium ion exporter, wherein the drug is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (Item 18B) Use of a drug capable of releasing intracellular calcium ions to reduce intracellular calcium ion concentration in the manufacture of a calcium ion releasing agent, wherein the drug is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof.(Item 19B) The use according to any one of the above items, wherein the calcium ion releaser is for the treatment or prevention of ischemic heart disease, high blood pressure, epilepsy, headache, or pruritus. (Item 1C) A drug that reduces intracellular free calcium ion concentration to suppress a subject's immune response. (Item 2C) The drug according to any one of the above items, wherein the drug is capable of forming a complex with calcium ions or inhibiting or blocking the influx of calcium ions into the cell. (Item 3C) The drug according to any one of the above items, wherein the drug is capable of releasing calcium ions from the cell to reduce the intracellular calcium ion concentration. (Item 4C) The drug according to any one of the above items, wherein the cell is an immune cell. (Item 5C) The drug according to any one of the above items, wherein the immune cell is at least one type selected from the group consisting of T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells. (Item 6C) The drug according to any one of the above items, wherein the immune cell is a T lymphocyte. (Item 7C) The agent according to any one of the preceding items, wherein the agent is a compound represented by formula (1) or a pharmaceutically acceptable salt thereof. (In the formula, R 1 ~R 6each independently represent hydrogen or an alkyl group having 3 or less carbon atoms. (Item 8C) The agent according to any of the above items, wherein the agent is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof. (Item 9C) The agent according to any of the above items, wherein the agent is selected from pyoktanin blue or a pharmaceutically acceptable salt thereof. (Item 10C) A agent that reduces intracellular calcium ion concentration for treating or preventing an allergic disease in a subject. (Item 11C) The agent according to any of the above items, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, asthma, seasonal allergic rhinitis, and food allergy. (Item 12C) The agent according to any of the above items, wherein the allergic disease is atopic dermatitis. (Item 13C) A agent that reduces intracellular calcium ion concentration for treating or preventing an autoimmune disease in a subject. (Item 14C) The drug according to any of the above items, wherein the autoimmune disease is at least one selected from the group consisting of psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis. (Item 15C) The drug according to any of the above items, wherein the autoimmune disease is psoriasis vulgaris. (Item 16C) The drug according to any of the above items, wherein the drug is administered by topical administration, oral administration, intravenous administration, inhalation, nasal administration, subcutaneous administration, intramuscular administration, sublingual administration, or ophthalmic administration. (Item 17C) A compound represented by formula (1) or a pharmaceutically acceptable salt thereof for reducing intracellular calcium ion concentration by excreting intracellular calcium ions. (Item 18C) Pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof, for reducing intracellular calcium ion concentration by excreting intracellular calcium ions. (Item 19C) The drug according to any one of the preceding items, wherein the drug is for the treatment or prevention of ischemic heart disease, high blood pressure, epilepsy, headache, or pruritus.

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] According to the present disclosure, symptoms of a subject with an excessively enhanced immune response can be improved by effectively suppressing the immune response.

[0008]

[0033] Figure 1 shows an example of the measurement results of intracellular calcium ion concentration measured in Examples 1 and 2. This figure shows the relative fluorescence intensity over time, with the fluorescence intensity immediately before the addition of A23187 set to 0% and the fluorescence intensity 15 minutes after the addition of A23187 set to 100%. The inhibition rate (%) of drug (1) was calculated from the relative fluorescence intensity of cells added with DMSO and cells added with drug (1). The vertical axis shows the relative fluorescence intensity (%), and the horizontal axis shows time (minutes). The chemical structures of the drugs used in the examples are shown. This figure shows an example of the measurement results of intracellular calcium ion concentration measured in Example 3. This figure shows the relative fluorescence intensity over time, with the fluorescence intensity immediately before the addition of anti-CD3 antibody set to 0% and the fluorescence intensity 3 minutes after the addition of anti-CD3 antibody set to 100%. The inhibition rate (%) of drug (1) was calculated from the relative fluorescence intensity of cells added with DMSO and cells added with drug (1). The vertical axis shows the relative fluorescence intensity (%), and the horizontal axis shows time (minutes). This shows the number of viable mouse spleen cells measured in Example 5 using the OD450 value as an index. Groups with different letters indicate a statistically significant difference (5% or less). The vertical axis shows the OD450 value.

[0009] (Definitions) It should be understood that the terms used herein are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of any conflict, the present specification (including definitions) shall prevail.

[0010] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0011] In this specification, the expression "and / or" includes both "and" and "or." That is, the expression "A and / or B" includes both "A or B" and "A and B."

[0012] As used herein, the term "immune response" refers to a response that recognizes and eliminates non-self substances, such as pathogens that invade from outside the body or abnormal cells or molecules produced in the body, and is carried out by immune cells such as T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells. As used herein, the term "suppression of the immune response" means that the immune response is reduced by the composition, agent, or method of the present disclosure compared to before use of the composition, agent, or method of the present disclosure. The reduced immune response can be measured as a decrease in the number of immune cells, a decrease in cytokine and / or chemokine concentrations (e.g., a decrease in IL-2 concentration, a decrease in IL-17 concentration), or a decrease in IgE concentration.

[0013] "Stimulants" is a general term for substances that induce biological responses, such as immune responses. In this specification, "stimulants" refers to substances that increase intracellular calcium ion concentrations. Calcium ions are the primary second messengers in intracellular signaling and play an important role in the physiological functions of various immune cells. Cytoplasmic calcium ion concentrations are tightly regulated and typically maintained at low concentrations. Known stimulants include calcium ionophores such as A23187, which nonspecifically increase intracellular calcium concentrations, and agonists such as antibodies (e.g., anti-CD3 antibodies) and small molecules (e.g., acetylcholine), which specifically bind to cell surface molecules such as CD3 and CD28 and increase intracellular calcium concentrations.

[0014] As used herein, the term "allergic disease" refers to a disease in which the immune response, which is normally intended to eliminate foreign substances from the outside, occurs excessively in response to a specific foreign substance, and examples of such diseases include atopic dermatitis, asthma, seasonal allergic rhinitis, and food allergies.

[0015] As used herein, the term "autoimmune disease" refers to a disease in which immune cells, which normally do not react to one's own tissues, recognize one's own tissues as foreign bodies and target and attack specific tissues or cells, causing inflammation or tissue damage. Examples of such diseases include psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, and ulcerative colitis.

[0016] As used herein, the term "a drug that reduces the intracellular free calcium ion concentration" refers to a drug that reduces the intracellular free calcium ion concentration compared to before use. A reduction in the intracellular free calcium ion concentration can be achieved by inhibiting or blocking the influx of calcium ions into cells through calcium ion channels (e.g., calcium ion channel blockers, calcium ion channel antagonists), forming a complex with calcium ions (e.g., chelating agents), or expelling intracellular calcium ions to the outside of the cells.

[0017] Chelating agents can be hydrophilic or hydrophobic. Hydrophilic chelating agents include EDTA and EGTA, while hydrophobic chelating agents include BAPTA-AM. Hydrophilic chelating agents cannot penetrate into cells, so they form complexes with calcium ions outside the cells, thereby reducing the extracellular free calcium ion concentration and thereby reducing the amount of free calcium ions that flow into the cells. Hydrophobic chelating agents penetrate into cells, form complexes with calcium ions inside the cells, and reduce the intracellular free calcium ion concentration.

[0018] As used herein, "reducing the intracellular free calcium ion concentration" refers to a chelating agent forming a complex with calcium ions within the cells, thereby reducing the intracellular free calcium ions. An example of such a chelating agent is BAPTA-AM (O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester). BAPTA-AM is hydrophobic, so it can penetrate into cells, and when hydrolyzed by esterase within the cells, it can form a complex with calcium ions.

[0019] As used herein, "intracellular calcium ion efflux" refers to the expulsion of intracellular calcium ions to the outside of the cell. Intracellular calcium ion efflux can be measured by the whole-cell clamp method or the patch clamp method. Drugs that can expel intracellular calcium ions and reduce the intracellular calcium ion concentration include, for example, pyoktanin blue (gentian violet, crystal violet, methylrosalinin chloride), methylene blue (3,7-bis(dimethylamino)phenothiazinium chloride trihydrate), or analogs thereof, or pharmaceutically acceptable salts thereof. Such drugs are also referred to as "calcium ion efflux agents."

[0020] High concentrations of intracellular calcium ions cause cytotoxicity, so molecules that release calcium ions into the extracellular space or the endoplasmic reticulum reduce the intracellular calcium ion concentration, maintaining cellular homeostasis. + / Ca 2+ Exchanger, Ca 2+ Pump (Ca 2+ -ATPase), voltage-dependent Ca 2+ Drugs that act directly or indirectly on these calcium ion export molecules can reduce elevated intracellular calcium ion concentrations.

[0021] As used herein, the term "alkyl group" refers to a straight-chain or branched-chain hydrocarbon group formed by removing one hydrogen atom from a saturated aliphatic hydrocarbon.

[0022] As used herein, the term "analog" refers to a compound whose core structure is the same as or very similar to that of the parent compound but has chemical or physical modifications, such as different or additional functional groups. For example, if the parent compound is pyoktanin, the core structure of pyoktanin is the portion excluding the amino and ammonium groups (i.e., the triphenylmethane portion), and a pyoktanin analog is a compound that has a functional group different from the amino or ammonium group, or a functional group modified from the amino or ammonium group. If the parent compound is methylene blue, the core structure of methylene blue is the portion excluding the amino group (i.e., the phenothiazine portion), and a methylene blue analog is a compound that has a functional group different from the amino group, or a functional group modified from the amino group. Analogs have the same or similar biological activity as the parent compound.

[0023] (Preferred Embodiments) A description of preferred embodiments will be given below, but it should be understood that these embodiments are merely examples of the present invention and that the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present invention by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0024] In one aspect, the present disclosure relates to a method for suppressing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that reduces intracellular free calcium ion concentration, thereby suppressing the immune response of the subject. In another aspect, the disclosure relates to a composition for suppressing an immune response in a subject, the composition comprising an agent that reduces intracellular free calcium ion concentration. These methods, agents, and compositions are described below.

[0025] The subject is an organism to which the method, agent, or composition of the present disclosure is applied, and the species is not particularly limited. Examples of the species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being preferred. In the case of humans, the subject is not particularly limited as long as the subject has an enhanced immune response. Examples of the subject include psoriasis vulgaris, psoriatic arthritis, atopic dermatitis, asthma, seasonal allergic rhinitis, allergic conjunctivitis, food allergies, systemic lupus erythematosus, contact dermatitis, urticaria, drug eruption, prurigo, thrombocytopenia, granulocytopenia, neonatal hemolytic jaundice, hypersensitivity pneumonitis, lupus nephritis, Hashimoto's disease, Behçet's disease, graft-versus-host disease, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, Graves' disease, type I diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis, and glomerulonephritis.

[0026] The type of cell is not particularly limited as long as it allows measurement of intracellular calcium ion concentration, and examples include immune cells such as T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils, and mast cells (including immortalized cell lines of each immune cell), primary cells such as skin cells, muscle cells, nerve cells, adipocytes, and lung cells, and their immortalized cell lines, as well as various cancer cell lines, with immune cells being preferred. Cell populations such as spleen cells, lymph node cells, and peripheral blood mononuclear cells, including different types of immune cells such as T lymphocytes and B lymphocytes, can also be used.

[0027] Methods for measuring intracellular calcium ion concentrations include the fluorescent dye method using calcium indicators and the patch clamp method. The former is particularly popular due to its simplicity and high sensitivity. Examples of calcium indicators include Fluo4, Fura2, Rhod2, Indo1, and Quin2. The acetoxymethyl (AM) derivatives of these indicators are preferred for their enhanced intracellular permeability. Furthermore, considering the intracellular calcium ion concentration of normal cells before the addition of a stimulant, Fluo4-AM and Fura2-AM are particularly preferred.

[0028] When measuring intracellular calcium ion concentrations using the fluorescent dye method, fluorescent measurement devices include fluorescent plate readers and fluorescent microscopes. Fluorescent plate readers are preferred because they are easy to operate and allow the evaluation of multiple drugs at once. Furthermore, because changes in intracellular calcium ion concentrations often occur immediately after the addition of stimuli, fluorescent plate readers equipped with an injector function are even more preferred.

[0029] Agents that decrease intracellular free calcium ion concentrations can be selected, for example, by the following method: Mouse spleen cells fluorescently labeled with Fluo4-AM are seeded onto a 96-well plate, the plate is placed in a fluorescent plate reader, and fluorescence intensity is measured for 5 to 30 minutes until a plateau is reached. A stimulating substance is then added, and the fluorescence intensity is measured for 5 seconds to 30 minutes. If the fluorescence intensity decreases after the addition of an agent, the agent can be selected as an agent that decreases intracellular calcium ion concentration after its elevation by the stimulating substance. The intracellular free calcium ion concentration can be reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, or at least 50% after application of the disclosed method, agent, or composition compared to before application. In a preferred embodiment, the intracellular free calcium ion concentration can be reduced by at least 30% after application of the disclosed method, agent, or composition compared to before application.

[0030] The agent that reduces the intracellular free calcium ion concentration may be an agent that can form a complex with calcium ions, an agent that can inhibit or block the influx of calcium ions into cells, or an agent that can expel calcium ions from cells to reduce the intracellular calcium ion concentration. Unexpectedly, an agent that can expel calcium ions from cells to reduce the intracellular calcium ion concentration may be advantageously used in the present disclosure.

[0031] Agents capable of forming complexes with calcium ions include, but are not limited to, chelating agents. Chelating agents may be hydrophilic or hydrophobic. The chelating agent may be any chelating agent capable of forming complexes with calcium ions. Examples of hydrophilic chelating agents include, but are not limited to, EDTA and EGTA. Examples of hydrophobic chelating agents include, but are not limited to, BAPTA-AM.

[0032] Agents that can inhibit or block calcium ion influx into cells include, but are not limited to, calcium ion channel blockers, calcium ion channel antagonists, and the like.

[0033] In some embodiments, calcium ion channel blockers or antagonists to calcium ion channels include, but are not limited to, isosorbide dinitrate, verapamil hydrochloride, diltiazem hydrochloride, nifedipine, amlodipine besylate, ethosuximide, lomerizine, crotamiton, and diphenhydramine hydrochloride.

[0034] In another embodiment, the agent is a compound of formula (1)

[0035]

[0036] (In the formula, R 1 ~R 6 each independently represents hydrogen or an alkyl group having 3 or less carbon atoms.) or a pharmaceutically acceptable salt thereof. The compound of formula (1) has the basic skeleton of pyoktanin blue and can exhibit activity equivalent to that of pyoktanin blue (e.g., a decrease in intracellular calcium ion concentration).

[0037] In further embodiments, the agent is selected from pyoktanin blue, methylene blue, or an analog thereof, or a pharmaceutically acceptable salt thereof. The analog of pyoktanin blue can be the compound shown in Figure 2 or a pharmaceutically acceptable salt thereof. Examples of analogs of methylene blue include, but are not limited to, cresyl violet acetate, neutral red, Nile red, toluidine blue O, and safranin O.

[0038] The method for calculating the therapeutically effective amount of a drug that suppresses the immune response of a subject is not particularly limited. For example, the therapeutically effective amount can be easily evaluated by administering various concentrations of the drug to a human or non-human animal with an enhanced immune response and measuring quantitative changes in molecules produced by immune cells or the number of immune cells. When the subject with an enhanced immune response is a human, examples of the disease include the aforementioned diseases. When the subject with an enhanced immune response is a non-human animal, examples of the disease include model animals for human diseases with an enhanced immune response, such as mice treated with imiquimod transdermally, which are model animals for psoriasis vulgaris, and mice treated with ovalbumin transdermally, which are model animals for atopic dermatitis. Molecules produced by immune cells include cytokines such as chemokines, hematopoietic colony-stimulating factors, interleukins (e.g., IL-2, IL-17), interferons, and transforming growth factors, as well as their receptors; chemical mediators such as histamine and serotonin, as well as their receptors; lipid mediators such as prostaglandins and leukotrienes, as well as their receptors; immunoglobulins such as IgG and IgE; and because immune cell-produced molecules are found in body fluids such as blood, cerebrospinal fluid, urine, body secretions, saliva, tears, and sputum, tissues such as the spleen, lymph nodes, and skin, bronchoalveolar lavage fluid, sputum, and the like, the concentrations of immune cell-produced molecules can be quantified by collecting these biological samples.

[0039] On the other hand, in addition to humans and non-human animals, the therapeutically effective dose of a drug that suppresses immune responses can be calculated using cultured immune cells. For example, the drug is added to mouse spleen cells or the human T cell line Jurkat cells that have been treated with A23187 (and, if necessary, 12-O-tetradecanoylphorbol 13-acetate (TPA)), anti-CD3 antibody, or anti-CD28 antibody, and the cells are cultured for 30 minutes to 3 days. The therapeutically effective dose of the drug can be roughly calculated by comparing the concentration of molecules produced by immune cells, such as interleukin-2, in the culture supernatant and the number of immune cells with those without the drug.

[0040] In one aspect, the present disclosure also includes a pharmaceutical composition containing, as an active ingredient, an agent that reduces intracellular calcium ion concentration.

[0041] The pharmaceutical composition is a composition comprising a drug that reduces intracellular calcium ion concentration mixed with a pharmaceutically acceptable carrier, excipient, and / or pharmaceutical additive, food additive, or cosmetic additive, and has the ability to suppress immune responses, which is important for normalizing immune responses by suppressing excessive immune responses.

[0042] The pharmaceutical composition can be used to treat diseases caused by excessively enhanced immune responses, and examples of the diseases to which it can be applied include allergic diseases such as atopic dermatitis, and autoimmune diseases such as psoriasis vulgaris and rheumatoid arthritis.

[0043] Pharmaceutically acceptable carriers, excipients, and / or pharmaceutical additives can be appropriately selected depending on the specific application of the composition. The form of the composition can also be, for example, various solid, liquid, and other forms depending on the specific application. Examples include oral preparations such as powders, fine granules, granules, tablets, syrups, capsules, suspensions, emulsions, extracts, and pills, injections, topical liquids, transdermal preparations such as ointments, suppositories, and topical preparations.

[0044] The dosage varies depending on the age, sex, weight, severity of the disease, administration method, etc. of the subject, but typically, for an adult human, the daily dose is about 1 mg to about 2 g of the active ingredient, preferably about 5 mg to about 1 g of the active ingredient. The daily dose can be administered once or in divided doses.

[0045] In a further aspect, the present disclosure provides a calcium ion-releasing agent comprising an agent capable of releasing intracellular calcium ions to reduce the intracellular calcium ion concentration. The agent contained in the calcium ion-releasing agent may be a compound represented by formula (1) above or a pharmaceutically acceptable salt thereof, or may be pyoktanin blue, methylene blue, an analog thereof, or a pharmaceutically acceptable salt thereof.

[0046] Such calcium ion exporters can be used for the treatment or prevention of diseases or conditions that benefit from the release of intracellular calcium ions, such as ischemic heart disease, high blood pressure, epilepsy, headache, or pruritus. Since calcium ion channel blockers and calcium ion channel antagonists are known to be effective in treating ischemic heart disease, high blood pressure, epilepsy, headache, and pruritus, the agents of the present disclosure that decrease the intracellular free calcium ion concentration are also expected to be effective in the prevention or treatment of these diseases or conditions.

[0047] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1. Analysis of the correlation between the effect of decreasing the intracellular calcium ion concentration of a human T cell line after the increase in the calcium ion concentration and the effect of suppressing immune responses (hereinafter referred to as "both effects"). (a) Measurement of intracellular calcium ion concentration. Jurkat E6.1 cells (purchased from KAC Corporation, catalog number EC88042803-F0) were cultured at 5 x 10 5The cells were suspended in 100 μL of Loading Buffer (Fluo 4-AM concentration: 5 μg / mL) included in the Calcium Kit-Fluo 4 (DOJINDO, catalog number: CS22) for intracellular calcium ion measurement per well and incubated at 37°C for 1 hour under 5% CO2. The fluorescently labeled cells were then centrifuged (11,000 x g, 30 seconds) to remove the supernatant, washed twice with PBS(-), and suspended in 100 μL of Calcium Kit-Fluo 4 Recording Medium. The same number of cells were seeded into each well of a 96-well plate and incubated at room temperature for 30 minutes. The plate was then centrifuged (1,000 rpm, 5 minutes) to allow the cells to contact the plate bottom.

[0048] The plate was placed in a BioTek Synergy H1 Multimode Reader (Agilent) and fluorescence intensity (excitation wavelength 488 nm, detection wavelength 528 nm, measured every 15 seconds) was measured until stable. Next, calcium ionophore A23187 (Sigma, catalog number: C7522) was added to a final concentration of 1 μM and fluorescence intensity was measured for 15 minutes. Then, 10 μM drug was added and fluorescence intensity was measured for another 15 minutes. The drug and A23187 were dissolved in DMSO and added, and the DMSO concentration in the recording medium was kept constant between wells.

[0049] As shown in Figure 1, the relative fluorescence intensity was calculated by setting the fluorescence intensity immediately before the addition of A23187 to 0% and the fluorescence intensity 15 minutes after the addition of A23187 to 100%. The drug inhibition rate of the A23187-induced increase in intracellular calcium concentration was calculated using the following formula, and the highest value 15 minutes after the addition of the drug was taken as the drug inhibition rate.

[0050] Inhibition rate (%) = (1 - (relative fluorescence intensity) 薬剤 / relative fluorescence intensity DMSO )) x100 relative fluorescence intensity DMSO : Relative fluorescence intensity of wells to which DMSO was added Relative fluorescence intensity 薬剤The inhibitory rates of the drugs on the increase in intracellular calcium concentration caused by A23187 stimulation are shown in Table 1. The chemical structures of the drugs are shown in Figure 2.

[0051] (b) Measurement of immune response: 1 x 10 per well 5 Jurkat E6.1 cells were suspended in RPMI-1640 medium (Fujifilm Wako, catalog number: 189-02025) containing 10% fetal bovine serum and seeded into each well of a 96-well plate. Then, 0.25 μM A23187, 12.5 ng / mL TPA (AdipoGen Life Science, catalog number: AG-CN2-0010-M001), and 0.125 μM TPA were added to the medium and cultured at 37°C under 5% CO2 for 2 days. After incubation, the 96-well plate was centrifuged at 1000 rpm for 5 minutes to collect the culture supernatant. IL-2 concentrations in the culture supernatant were quantified using a human IL-2 ELISA kit (Diaclone, catalog number: 950.010.096). The inhibitory rate of the drug on the enhancement of immune response (IL-2 concentration as an index) induced by A23187 and TPA stimulation was calculated according to the following formula.

[0052] Suppression rate (%) = ((IL-2 concentration DMSO + A23187 + TPA - IL-2 concentration drug + A23187 + TPA) / (IL-2 concentration DMSO + A23187 + TPA - IL-2 concentration DMSO )) x 100 IL-2 concentration DMSO + A23187 + TPA: IL-2 concentration in the culture supernatant of cells to which DMSO, A23187 and TPA were added IL-2 concentration Drug + A23187 + TPA: IL-2 concentration in the culture supernatant of cells to which drug, A23187 and TPA were added IL-2 concentration DMSO : IL-2 concentration in the culture supernatant of cells to which only DMSO was added. The calculated inhibition rates are shown in Table 1.

[0053] (c) Correlation analysis of both effects As shown in Table 1, it was shown that there is a correlation between the inhibitory rate of the drug on the increase in intracellular calcium ion concentration due to A23187 stimulation and the inhibitory rate of the drug on the enhanced immune response due to A23187 and TPA stimulation.

[0054]

[0055] Example 2. Correlation Analysis of Both Effects in Mouse Spleen Cells (a) Preparation of Mouse Spleen Cells. Spleens were collected from 7-week-old male BALB / c mice (CLEA Japan) after cervical dislocation and minced in cold Hank's Balanced Salt Solution (Fujifilm Wako, catalog number: 084-08965). The spleen pieces were transferred to a 70 μm cell strainer (pluriSelect Life Science, catalog number: 43-50070-03) and crushed with the plunger of a 2.5 mL disposable syringe. After washing with cold PBS(-), the spleen cell suspension was centrifuged at 1000 rpm at 4°C for 5 minutes to recover the pellet. Next, cold red blood cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) was added to the pellet, which was then left on ice for 5 minutes and centrifuged. The pellet was washed with PBS(-) and then suspended in RPMI-1640 containing 10% fetal bovine serum to prepare mouse spleen cells.

[0056] (b) Measurement of Intracellular Calcium Ion Concentration. Mouse spleen cells were fluorescently labeled according to the method described in Example 1 (a), suspended in 100 μL of Recording Medium, and seeded in equal numbers into each well of a 96-well plate. The plate was then centrifuged (1,000 rpm, 5 minutes) to allow the cells to contact the bottom of the plate. The plate was placed in a BioTek Synergy H1 Multimode Reader, and after the fluorescence intensity stabilized, A23187 was added at a final concentration of 1 μM and the fluorescence intensity was measured for 5 minutes. Next, 10 μM of drug or DMSO was added, and the fluorescence intensity was measured for another 5 minutes.

[0057] The relative fluorescence intensity was calculated by setting the fluorescence intensity immediately before the addition of A23187 as 0% and the fluorescence intensity 5 minutes after the addition of A23187 as 100%. The drug inhibition rate was calculated according to the formula described in Example 1 (a), and the highest value 5 minutes after the addition of the drug was taken as the drug inhibition rate. The drug inhibition rates for the increase in intracellular calcium concentration induced by A23187 stimulation are shown in Table 2.

[0058] (c) Measurement of immune response: 5x10 per well 5Mouse spleen cells were suspended in RPMI-1640 medium (Fujifilm Wako, catalog number: 189-02025) containing 10% fetal bovine serum and seeded into each well of a 96-well plate. Next, 0.2 μM A23187, 10 ng / mL TPA, and 1 μM of each drug were added to the medium and cultured at 37°C under 5% CO2 for 2 days (200 μL of medium was used). After incubation, the 96-well plate was centrifuged at 1000 rpm for 5 minutes to recover 100 μL of culture supernatant. The number of mouse spleen cells (containing 100 μL of culture medium) was measured. (1) Viable Cell Counting: Viable cell counts of mouse spleen cells were measured using the Cell Counting Kit-8 (Dojindo, catalog number: 341-07761). The reagents included in this kit were added to a culture medium containing mouse spleen cells, and after incubation at 37°C under 5% CO for 1 hour, the absorbance at 450 nm (OD450) was measured. The inhibitory effect of the drug on the enhancement of the immune response (as measured by cell count) induced by A23187 and TPA stimulation was calculated using the following formula:

[0059] Inhibition rate (%) = ((OD450DMSO + A23187 + TPA - OD450drug + A23187 + TPA) / (OD450DMSO + A23187 + TPA - OD450 DMSO )) x 100 OD450DMSO + A23187 + TPA: Absorbance at 450 nm of cells to which DMSO, A23187 and TPA were added OD450Drug + A23187 + TPA: Absorbance at 450 nm of cells to which drug, A23187 and TPA were added OD450 DMSO : Absorbance at 450 nm of cells treated with DMSO only. The calculated inhibition rates are shown in Table 2. (2) Measurement of IL-2 concentration in culture supernatants. IL-2 concentrations in culture supernatants were quantified using a mouse IL-2 ELISA kit (Diaclone, catalog number: 860.000.096). The drug inhibition rates for the enhancement of immune responses (IL-2 concentration as an index) induced by A23187 and TPA stimulation were calculated according to the following formula:

[0060] Suppression rate (%) = ((IL-2 concentration DMSO + A23187 + TPA - IL-2 concentration drug + A23187 + TPA) / (IL-2 concentration DMSO + A23187 + TPA - IL-2 concentration DMSO )) x 100 IL-2 concentration DMSO + A23187 + TPA: IL-2 concentration in the culture supernatant of cells to which DMSO, A23187 and TPA were added IL-2 concentration Drug + A23187 + TPA: IL-2 concentration in the culture supernatant of cells to which drug, A23187 and TPA were added IL-2 concentration DMSO : IL-2 concentration in the culture supernatant of cells to which only DMSO was added. The calculated inhibition rates are shown in Table 2.

[0061] (d) Correlation analysis of both effects As shown in Table 2, it was shown that there is a correlation between the inhibitory rate of the drug on the increase in intracellular calcium ion concentration due to A23187 stimulation and the inhibitory rate of the drug on the enhancement of immune responses (cell count and IL-2 concentration) due to A23187 and TPA stimulation.

[0062]

[0063] Example 3. Effect of Agent (1) on Mouse Spleen Cells Stimulated with Anti-CD3 Antibody (a) Measurement of Intracellular Calcium Ion Concentration Mouse spleen cells fluorescently labeled according to the method described in Example 1 (a) were added with 5 μg / mL anti-CD3 antibody (BioLegend, catalog number: 100302) and incubated on ice for 30 minutes. After washing with PBS(-), the cells were suspended in Recording Medium and seeded in equal numbers into each well of a 96-well plate and incubated at room temperature for 30 minutes. The plate was then centrifuged (1,000 rpm, 5 minutes) to allow the cells to contact the bottom of the plate. The plate was placed in a BioTek Synergy H1 Multimode Reader, and after the fluorescence intensity stabilized, a final concentration of 20 μg / mL goat anti-hamster IgG antibody (Jackson ImmunoResearch, catalog number: 564-74561, hereafter referred to as the secondary antibody) was added, and the fluorescence intensity was measured for 3 minutes. Next, 0.5 to 3 μM of the drug (1) or DMSO was added, and the fluorescence intensity was measured for another 5 minutes.

[0064] As shown in Figure 3, the relative fluorescence intensity was calculated by setting the fluorescence intensity immediately before the addition of the secondary antibody to 0% and the fluorescence intensity 3 minutes after the addition of the secondary antibody to 100%. The drug inhibition rate was calculated according to the formula described in Example 1(a), and the highest value 5 minutes after the addition of the drug was taken as the drug inhibition rate. The inhibition rate of drug (1) against the increase in intracellular calcium concentration due to stimulation with anti-CD3 antibody is shown in Table 3.

[0065] As a result, it was shown that drug (1) inhibited the increase in intracellular calcium concentration caused by anti-CD3 antibody stimulation in a concentration-dependent manner.

[0066]

[0067] (b) Measurement of immune response 200 μL of 3 μg / mL anti-CD3 antibody (BioLegend, catalog number: 100302) or PBS(-) was added per well to a 96-well plate and allowed to stand at 37°C for 2 hours. After washing each well twice with PBS(-), 5 × 10 mouse spleen cells prepared by the method described in Example 2(a) were added per well. 5 Each well was seeded with 1000 μL of drug (1). Drug (1) was then added at various concentrations and cultured at 37°C under 5% CO2 for 2 days (200 μL of medium was used). After culture, the 96-well plate was centrifuged at 1000 rpm for 5 minutes to recover 100 μL of culture supernatant, and the number of mouse spleen cells (containing 100 μL of culture medium) was measured. (1) Measurement of viable cell count: OD450 was measured according to the method described in Example 2(c)(1). The inhibitory rate of drug (1) at each concentration on the enhancement of immune response (as indicated by cell count) induced by anti-CD3 antibody stimulation was calculated according to the following formula:

[0068] Inhibition rate (%) = ((OD450 anti-CD3 antibody + DMSO - OD450 anti-CD3 antibody + drug (1)) / (OD450 anti-CD3 antibody + DMSO - OD450 PBS(-) + DMSO)) x 100 OD450 anti-CD3 antibody + DMSO: OD450 of cells coated with anti-CD3 antibody and added with DMSO OD450 anti-CD3 antibody + drug (1): OD450 of cells coated with anti-CD3 antibody and added with drug (1) OD450 PBS(-) + DMSO: OD450 of cells coated with PBS(-) and added with DMSO The calculated inhibition rates are shown in Table 4. (2) Measurement of IL-2 concentration in culture supernatant The IL-2 concentration was measured according to the method described in Example 2(c)(2). The inhibition rate of each concentration of drug (1) on the enhancement of immune response (IL-2 concentration as an index) stimulated by anti-CD3 antibody was calculated according to the following formula.

[0069] Inhibition rate (%) = ((IL-2 concentration anti-CD3 antibody + DMSO - IL-2 concentration anti-CD3 antibody + drug (1)) / (IL-2 concentration anti-CD3 antibody + DMSO - IL-2 concentration PBS(-) + DMSO)) x 100 IL-2 concentration anti-CD3 antibody + DMSO: IL-2 concentration in the culture supernatant of cells coated with anti-CD3 antibody and treated with DMSO IL-2 concentration anti-CD3 antibody + drug (1): IL-2 concentration in the culture supernatant of cells coated with anti-CD3 and treated with drug (1) IL-2 concentration PBS(-) + DMSO: IL-2 concentration in the culture supernatant of cells coated with PBS(-) and treated with DMSO The calculated inhibition rates are shown in Table 4. (3) Measurement of IL-17 concentration in culture supernatant The IL-17 concentration in the culture supernatant was quantified using a mouse IL-17 Quantikine ELISA kit (R&D SYSTEMS, catalog number: M1700). The inhibitory rate of each concentration of drug (1) against the enhancement of immune response (IL-17 concentration as an index) stimulated by anti-CD3 antibody was calculated according to the following formula.

[0070] Inhibition rate (%) = ((IL-17 concentration anti-CD3 antibody + DMSO - IL-17 concentration anti-CD3 antibody + drug (1)) / (IL-17 concentration anti-CD3 antibody + DMSO - IL-17 concentration PBS(-) + DMSO)) x 100 IL-17 concentration anti-CD3 antibody + DMSO: IL-17 concentration in the culture supernatant of cells coated with anti-CD3 antibody and added with DMSO IL-17 concentration anti-CD3 antibody + drug (1): IL-17 concentration in the culture supernatant of cells coated with anti-CD3 and added with drug (1) IL-17 concentration PBS(-) + DMSO: IL-17 concentration in the culture supernatant of cells coated with PBS(-) and added with DMSO The calculated inhibition rates are shown in Table 4.

[0071] (d) Inhibitory effect of drug (1) on enhanced immune response The inhibitory rate of drug (1) on enhanced immune response (cell count, IL-2 concentration, IL-17 concentration) induced by anti-CD3 antibody is shown in Table 4. The results showed that drug (1) concentration-dependently inhibited the immune response induced by anti-CD3 antibody.

[0072]

[0073] Example 4. Inhibitory effect of drug (1) on immune response in autoimmune disease model animals (a) Preparation of psoriasis vulgaris model animals and drug application Imiquimod, an agonist of toll-like receptor 7 (TLR7), is known to activate immune responses by acting on immune cells and is used as a therapeutic agent for genital warts and actinic keratosis. Normal mice transdermally administered with imiquimod exhibit clinical symptoms similar to those of human psoriasis vulgaris patients, and are therefore widely used as model animals for psoriasis vulgaris. In these model animals, IL-17 secreted by activated T cells promotes epidermal cell proliferation, resulting in epidermal thickening.

[0074] Male BALB / c mice (8 weeks old) obtained from CLEA Japan were anesthetized with isoflurane and their backs were shaved. 100 μL of 0.02% or 0.1% (solvent: distilled water) drug (1) was applied to the backs once daily for a total of six doses. As a control, 100 mg of 0.1% tacrolimus ointment (Sun Pharma, trade name: Tacrolimus Ointment 0.1% "PP") was applied to the backs once daily for a total of six doses. Next, 3 hours after drug (1) or tacrolimus application, 62.5 mg of 5% Veselna Cream (Mochida Pharmaceutical, identification code: MO651) was applied to the backs once every two days for a total of four doses. Plasma and dorsal skin samples were collected the day after the final application.

[0075] (b) Measurement of Plasma IL-17 Concentration Plasma IL-17 concentrations were measured according to the method described in Example 3(3), and the measured values ​​are shown in Table 5.

[0076] (c) Measurement of epidermal thickness. The collected skin was fixed overnight at room temperature in Mildphor 10NM (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number: 130-10527). The fixed skin was dehydrated and embedded in paraffin, and 5-μm-thick skin sections were prepared using a LEICA SM2010R (Leica Biosystems). The prepared skin sections were deparaffinized, stained with hematoxylin and eosin, and mounted in mounting medium (Merck, catalog number: 1.07961.0100). Images of the hematoxylin and eosin-stained sections were then captured using a BZ-X710 All-in-One Fluorescence Microscope (Keyence Corporation), and epidermal thickness was measured using image analysis software BZ-X analyzer (Keyence Corporation). The measurements are shown in Table 5.

[0077] (d) Inhibitory effect on immune response The measured plasma IL-17 concentration and skin thickness are shown in Table 5 (each measured value is the average value). As a result, it was shown that drug (1) concentration-dependently inhibited the immune response in a psoriasis vulgaris model animal with an enhanced immune response. This indicates that the immune response of a subject with an enhanced immune response can be inhibited by administering a drug that reduces intracellular calcium ion concentration after the calcium ion concentration has increased due to a stimulant.

[0078]

[0079] Example 5. Effects of Drug (1) and Calcium Chelators on Imiquimod-Induced Enhanced Immune Response O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), which chelates intracellular calcium ions and reduces the intracellular free calcium ion concentration, is a compound in which all carboxyl groups of BAPTA have been converted to acetoxymethyl ester (AM). After being readily taken up into cells, it is decomposed by intracellular esterases and functions as a calcium chelator, BAPTA, within the cells. Furthermore, BAPTA produced intracellularly is retained intracellularly without being transported outside the cells.

[0080] Mouse spleen cells prepared by the method described in Example 2(a) were added at 5 x 10 per well. 5 The cells were seeded into a 96-well plate. DMSO or final concentrations of 5 μM BAPTA-AM, 1 μM drug (1), or 5 μg / mL imiquimod were then added, and the cells were cultured at 37°C under 5% CO2 for 3 days (200 μL of medium was used). After culture, the 96-well plate was centrifuged at 1000 rpm for 5 minutes, and the mouse spleen cells (containing 100 μL of culture medium) and 100 μL of culture supernatant were collected. The OD450 of the mouse spleen cells (containing 100 μL of culture medium) was measured according to the method described in Example 2(c)(1), and the measured values ​​are shown in Figure 4.

[0081] As shown in Figure 4, in the group without imiquimod ((-) Imiquimod), there was no statistically significant difference between the DMSO group and the other groups, indicating no cytotoxicity due to BAPTA-AM or drug (1). On the other hand, in the group with imiquimod ((+) Imiquimod), BAPTA-AM and drug (1) statistically significantly reduced the cell number increased by imiquimod. This indicates that the immune response of subjects whose immune response has been enhanced by imiquimod can be suppressed by administering a drug that reduces intracellular free calcium ion concentration.

[0082] Calcium ion channel blockers or antagonists can also inhibit the influx of free calcium ions into cells, suggesting that they similarly suppress immune responses. Because elevated calcium ion concentrations play an important role in immune cell activation, a decrease in calcium ion concentration is thought to have an inhibitory effect on immune responses. For example, elevated calcium ion concentrations in T cells and B cells promote cell proliferation and activation, while elevated calcium ion concentrations in mast cells promote degranulation. Furthermore, elevated calcium ion concentrations in dendritic cells promote migration, which is one indicator of activation.

[0083] As described above, the present invention has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications, and literature cited in this specification should be incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims the benefit of priority from Japanese Patent Application No. 2023-160695, filed on September 25, 2023, the contents of which are incorporated herein by reference.

[0084] INDUSTRIAL APPLICABILITY The present disclosure can provide a method for effectively suppressing an immune response in a subject with an excessively enhanced immune response, and can be used in the pharmaceutical and cosmetic industries.

Claims

1. A composition for suppressing an immune response in a subject, the composition comprising an agent that reduces intracellular free calcium ion concentration.

2. The composition of claim 1, wherein the agent is an agent capable of forming a complex with calcium ions or an agent capable of inhibiting or blocking the influx of calcium ions into the cell.

3. The composition according to claim 1, wherein the drug is capable of expelling calcium ions from the cells to reduce the calcium ion concentration in the cells.

4. The composition according to any one of claims 1 to 3, wherein the cell is an immune cell.

5. The composition according to claim 3, wherein the immune cells are at least one selected from the group consisting of T lymphocytes, B lymphocytes, dendritic cells, macrophages, Langerhans cells, eosinophils, basophils, neutrophils and mast cells.

6. The composition of claim 5, wherein the immune cell is a T lymphocyte.

7. The composition of claim 1, wherein the drug is a compound represented by formula (1) or a pharma- ceutically acceptable salt thereof. (In the formula, R 1 ~R 6 each independently represents hydrogen or an alkyl group having 3 or less carbon atoms.

8. The composition of claim 1, wherein the agent is selected from pyoktanin blue, methylene blue, or an analogue thereof, or a pharma- ceutically acceptable salt thereof.

9. The composition of claim 6, wherein the agent is selected from pyoktanin blue or a pharma- ceutically acceptable salt thereof.

10. A composition for treating or preventing an allergic disease in a subject, the composition comprising an agent that reduces intracellular calcium ion concentration.

11. The composition according to claim 10, wherein the allergic disease is at least one selected from the group consisting of atopic dermatitis, asthma, seasonal allergic rhinitis and food allergy.

12. The composition according to claim 10, wherein the allergic disease is atopic dermatitis.

13. A composition for treating or preventing an autoimmune disease in a subject, the composition comprising an agent that reduces intracellular calcium ion concentration.

14. The composition according to claim 13, wherein the autoimmune disease is at least one selected from the group consisting of psoriasis vulgaris, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease and ulcerative colitis.

15. The composition of claim 13, wherein the autoimmune disease is psoriasis vulgaris.

16. The composition of claim 1, wherein the composition is administered by topical administration, oral administration, intravenous administration, inhalation, intranasal administration, subcutaneous administration, intramuscular administration, sublingual administration, or ophthalmic administration.

17. A calcium ion exporter comprising an agent capable of exporting calcium ions from within a cell and reducing the intracellular calcium ion concentration, the agent being a compound represented by formula (1) or a pharma- ceutical acceptable salt thereof.

18. A calcium ion exporter comprising an agent capable of exporting intracellular calcium ions and decreasing the intracellular calcium ion concentration, the agent being selected from pyoktanin blue, methylene blue, or an analogue thereof, or a pharma- ceutical acceptable salt thereof.

19. The calcium ion exporter according to claim 17 or 18, which is for the treatment or prevention of ischemic heart disease, hypertension, epilepsy, headache, or pruritus.

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