WO2021084791A1 - Composition targeted to adenosine a2a receptor - Google Patents

Composition targeted to adenosine a2a receptor Download PDF

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WO2021084791A1
WO2021084791A1 PCT/JP2020/024876 JP2020024876W WO2021084791A1 WO 2021084791 A1 WO2021084791 A1 WO 2021084791A1 JP 2020024876 W JP2020024876 W JP 2020024876W WO 2021084791 A1 WO2021084791 A1 WO 2021084791A1
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Prior art keywords
adenosine
cells
receptor
activity
test compound
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PCT/JP2020/024876
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French (fr)
Japanese (ja)
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祥 松下
雅章 川野
理英 高木
美枝子 戸叶
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有限会社イムノ
学校法人埼玉医科大学
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Priority to JP2021554066A priority Critical patent/JPWO2021084791A1/ja
Publication of WO2021084791A1 publication Critical patent/WO2021084791A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the present invention is a composition for suppressing the differentiation or activation of Th17 cells by targeting the adenosine A2A receptor, and treating, ameliorating or preventing a disease caused by an overreaction of Th17 cells by targeting the adenosine A2A receptor. And a method for evaluating a compound which is an active ingredient of these compositions.
  • Helper T cells which play a central role in acquired immunity, are classified into Th1 (type 1 helper T cells), Th2 (type 2 helper T cells), Th17 (type 17 helper T cells), etc., based on differences in cytokines produced. Will be done.
  • Th1 / Th2 balance has been considered to be involved in the development of various immune-related diseases.
  • balance bias to Th1 cells can be associated with rheumatoid arthritis, a chronic inflammatory disease, or organ-specific autoimmune diseases (eg, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, glomerulonephritis, hepatitis, liver). It is thought to be involved in disorders, autoimmune hemolytic anemia, leukopenia, thrombocytopenia, demyelinating disease, Hashimoto's thyroiditis, pernicious anemia, psoriasis), and the balance bias to Th2 cells is allergic. It is thought to be involved in sexual disorders and many systemic autoimmune diseases.
  • Th17 cells are involved in exacerbation of autoimmune inflammation by producing IL-17A exclusively, especially the aforementioned type 1 diabetes, inflammatory bowel disease, psoriasis, multiple sclerosis, and joints. It is believed that rheumatism is highly suspected to be due to this bias towards Th17 (Non-Patent Document 1).
  • Th1 bias Th2 bias
  • Th17 bias Various factors are involved in the acquired immune system in a complex manner, and it is still unclear which of the immune-related diseases, Th1 bias, Th2 bias, or Th17 bias, is involved.
  • Et al. Have already found that the action of dopamine is greatly involved in the Th1 / Th2 / Th17 differentiation induction mechanism mediated by dendritic cells (Patent Document 1).
  • Patent Document 1 the action of an antagonist on a dopamine D1-like receptor on a dendritic cell suppresses the synthesis and storage of dopamine in the dendritic cell, resulting in Th17 or Th2 cells of naive T cells. It has been shown that the induction of differentiation into Th1 cells is suppressed and the induction of differentiation into Th1 cells is promoted.
  • the antagonist when allowed to act on dopamine D2-like receptors on dendritic cells, the synthesis and storage of dopamine in dendritic cells is promoted, and as a result, the induction of differentiation of naive T cells into Th1 cells is suppressed. It has also been shown that the induction of differentiation into Th2 cells is promoted.
  • the present inventors not only act on various dopamine D2-like receptor agonists in inducing T cell differentiation, but also act on activated T cells to suppress their IL-17A production. It has also been found that these agonists are effective as therapeutic agents for neutrophil inflammation (Patent Document 2).
  • Interleukin 27 limits autoimmune encephhalomyelitis by suprepressing the development of interleukin 17-producing T cells. Nat Immunol. 2006.7: 929-936.
  • the present invention has been made in view of the above-mentioned prior art, and an object of the present invention is to identify a new receptor involved in the differentiation of helper T cells and to develop a drug targeting the new receptor. ..
  • Th17 cells were generated in a bidirectional mixed lymphocyte culture reaction system (2-way MLR) or a culture system of CD4 + T cells activated by stimulation with CD3 / CD28 by an adenosine or adenosine A2A receptor agonist. It was found that it induces highly selectively and increases the secretion of IL-17A. This suggested a relationship between adenosine A2A receptor and Th17 cell differentiation / activation. To support this, next, adenosine A2A receptor antagonist was added to 2-way MLR or CD3 in the presence of adenosine.
  • the present inventors next examined whether or not the adenosine A2A receptor antagonist can improve the pathological condition of the disease caused by the overreaction of Th17 cells. Specifically, psoriasis, neutrophil airway inflammation, multiple sclerosis, and atopic dermatitis were selected as examples of the disease, and the effect of the adenosine A2A receptor antagonist in each model mouse of these diseases was evaluated. .. As a result, it was found that the antagonist significantly suppressed the activity of these diseases.
  • the present inventors can control the differentiation and activation of Th17 cells by targeting the adenosine A2A receptor, and the treatment of diseases caused by the overreaction of Th17 cells by the antagonist of the receptor, etc. We have found that it is effective as a drug for this purpose, and have completed the present invention.
  • the present invention provides the following in more detail.
  • a composition for treating, ameliorating, or preventing a disease caused by suppression of Th17 cell differentiation or activation or overreaction of Th17 cells which contains an adenosine A2A receptor antagonist as an active ingredient. Stuff.
  • [2] A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
  • a step of contacting the test compound with the adenosine A2A receptor and (b) a step of detecting the binding between the test compound and the adenosine A2A receptor.
  • the test compound binds to the adenosine A2A receptor, it is evaluated that it has an activity of suppressing the differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells. How to be done.
  • a method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells (A) The step of contacting the adenosine A2A receptor ligand with the adenosine A2A receptor in the presence of the test compound, and (b) the step of detecting the binding between the adenosine A2A receptor ligand and the adenosine A2A receptor.
  • test compound inhibits the binding of the adenosine A2A receptor ligand to the adenosine A2A receptor
  • the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated, ameliorated, or prevented.
  • a method that is evaluated as having a probable activity is evaluated.
  • a method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells (A) A step of contacting the test compound with a cell expressing the adenosine A2A receptor, and (b) a step of detecting the activity of the adenosine A2A receptor. Including When the test compound reduces the activity of the adenosine A2A receptor as compared to the case where the test compound is not contacted, the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated. , A method that is assessed as probable to have an ameliorating or prophylactic activity.
  • a method for producing Th17 cells which comprises a step of culturing T cells in the presence of an adenosine A2A receptor agonist.
  • Th17 cells in which T cells are induced or activated by an adenosine A2A receptor agonist are induced or activated by an adenosine A2A receptor agonist.
  • a composition for inducing differentiation or activating Th17 cells which comprises an adenosine A2A receptor agonist as an active ingredient.
  • Neutrophil airway inflammation including neutrophil bronchial asthma, periodontal disease, psoriasis, severe atopic dermatitis, acne vulgaris, endometriosis, chronic sinusitis, and various other autoimmune diseases, etc.
  • neutrophil bronchial asthma periodontal disease
  • psoriasis severe atopic dermatitis
  • acne vulgaris endometriosis
  • chronic sinusitis chronic sinusitis
  • various other autoimmune diseases etc.
  • pathological conditions mainly due to neutrophil inflammation.
  • no silver bullet has been known so far.
  • composition of the present invention targets the adenosine A2A receptor.
  • Th17 cell exerts an inhibitory effect on differentiation and activation. Therefore, the compositions of the present invention are particularly useful as pharmaceuticals based on a novel mechanism of action for the treatment, amelioration, or prevention of diseases caused by Th17 cell overreaction, including neutrophil inflammation. According to the present invention, it is also possible to efficiently evaluate and screen such drug candidate compounds by targeting the adenosine A2A receptor.
  • One-way ANOVA determined that there was a significant difference when the p-value was 0.05 or less, and a Turkey test was performed to analyze the significant difference between the two groups.
  • "*" indicates that the group to which only adenosine (100 ⁇ M) was added and the group to which istradeferin was further added had “P ⁇ 0.05”.
  • One-way ANOVA determined that there was a significant difference when the p-value was 0.05 or less, and a Turkey test was performed to analyze the significant difference between the two groups.
  • the graph shows the degree of auricle thickening, and is represented by a value obtained by subtracting the thickening amount generated in the negative control from the thickness of the auricle of each individual.
  • the unit of the horizontal axis of the graph is mm.
  • the present invention is a composition for treating, ameliorating, or preventing a disease caused by suppression of Th17 cell differentiation or activation or Th17 cell overreaction, and contains an adenosine A2A receptor antagonist as an active ingredient.
  • the composition is provided.
  • Adenosine receptor is a G protein-coupled receptor molecule for adenosine, and in humans, there are four subtypes, A1, A2A, A2B, and A3.
  • A1A Adenosine A2A receptor
  • A2B Adenylate cyclase
  • cAMP adenylate cyclase
  • a signal is transmitted (that is, the adenosine A2A receptor is activated). To do).
  • the "adenosine A2A receptor antagonist" which is the active ingredient of the composition of the present invention is not particularly limited as long as it inhibits the activation of the adenosine A2A receptor. It may be a known drug, or may be obtained by using, for example, the evaluation method of a test compound described later. Known agents include, for example, istradefylline, preladenant, vipadenant, caffeine, SCH-58261, SCH-421348, SCH-442416, VER-6623, VER-6947, VER-7835, ST-1535, ZM-241385.
  • adenosine A2A receptor antagonist for example, a xanthin derivative exhibiting an antagonistic effect on the adenosine A2A receptor disclosed in JP-A-6-211856 or JP-A-2016-0031186;
  • Japanese Patent Publication No. 2013-523711 Japanese Patent Publication No. 2012-505264, Japanese Patent Publication No. 2011-513417, Japanese Patent Publication No. 2011-500833, Japanese Patent Publication No. 2011-500919, Japanese Patent Publication No. 2010-523497, Adenosine A2A receptor disclosed in JP-A-2008-297312, Japanese Patent Application Laid-Open No. 2009-508871, Japanese Patent Application Laid-Open No.
  • Pyrimidine derivative exhibiting antagonism against adenosine A2A receptor for example, amino-quinoxalin derivative exhibiting antagonism against adenosine A2A receptor disclosed in JP-A-2011-514350; eg, JP-A-2011-07869 Examples thereof include anti-adenosine A2A receptor antibodies disclosed in.
  • the "adenosine A2A receptor antagonist” also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has an inhibitory activity thereof.
  • a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on each structure of the adenosine A2A receptor antagonist, for example, an acid addition salt (for example, hydrochloride, bromide).
  • Inorganic acid salts such as hydrogen salts, sulfates, hydrogen sulfates, nitrates, carbonates, hydrogen carbonates, phosphates, monohydrogen phosphates, dihydrogen phosphates, acetates, propionates, lactates, Organic acid salts such as citrate and tartrate), base addition salts (eg alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, zinc salt, aluminum salt, ammonium Salts, tetramethylammonium salts), organic amine addition salts (eg, morpholin, piperidine and other addition salts), amino acid addition salts (eg, lysine, glycine, phenylalanine and other addition salts).
  • the hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 10 molecules of water or a solvent to one molecule of an adenosine A2A receptor antagonist or a salt thereof.
  • the "adenosine A2A receptor antagonist” includes all isomers and isomers such as tautomers, geometric isomers, asymmetric carbon-based optical isomers, and stereoisomers as long as they have inhibitory activity. Contains a mixture. Furthermore, adenosine A2A receptor antagonists are still undergoing metabolism such as oxidation, reduction, hydrolysis, amination, deaminolation, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, and conjugation in vivo. The present invention also includes compounds exhibiting the inhibitory activity, and the present invention also includes compounds that undergo metabolism such as oxidation, reduction, and hydrolysis in vivo to produce adenosine A2A receptor antagonists.
  • adenosine A2A receptor antagonist As mentioned above, many compounds have been developed and are commercially available, so they can be obtained by purchasing. In addition, many reports have been made on methods for producing these compounds, even if they are not commercially available. Therefore, those skilled in the art can appropriately prepare according to the production method.
  • composition of the present invention is used for pharmaceutical compositions (pharmaceutical products, quasi-drugs, veterinary drugs, etc.), foods and drinks (including animal feed), or for research purposes (for example, in vitro or in vivo experiments). It can be in the form of a reagent to be used.
  • composition of the present invention can be used for suppressing the differentiation or activation of Th17 cells, or for treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
  • Vasodilation, sleep, neural activity control, etc. are known as physiological roles of adenosine A2A receptor, but in the present invention, a relationship with Th17 cell differentiation and activation was found. Since the relationship between Th17 cell differentiation / activation and neutrophil inflammation is known (Patent Document 2), the composition of the present invention can be applied to diseases caused by Th17 cell overreaction. It is possible. As the "disease caused by the overreaction of Th17 cells", a disease whose main pathological condition is neutrophil inflammation is suitable.
  • Specific diseases include, for example, neutrophil airway inflammation including neutrophil bronchial asthma, periodontal disease, psoriasis, ulcerative colitis, severe atopic dermatitis, acne vulgaris, and endometriosis. , Chronic sinusitis, and various other autoimmune diseases (eg, rheumatoid arthritis, polysclerosis, type 1 diabetes, nephritis), but are not limited thereto.
  • composition of the present invention When the composition of the present invention is used as a pharmaceutical composition, its dosage form is not particularly limited as long as it contains an adenosine A2A receptor antagonist as an active ingredient.
  • the pharmaceutical composition of the present invention can be formulated by a known pharmaceutical method in various dosage forms including a pharmacologically acceptable carrier.
  • the dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected depending on the desired administration method, for example, an oral solid preparation (tablet, coated tablet, granule, powder, capsule, etc.). , Oral solutions (oral solutions, syrups, elixirs, etc.), external preparations (suppositories, ointments, patches, gels, creams, external powders, sprays, inhalants, etc.), injections (solutions, suspensions, etc.) Turbid liquids, solids for errands, etc.), other oral preparations (gum, troche, sublingual tablets, buccal tablets, adhesives, etc.), oral sprays, oral semi-solids, dentifrices, gargles Examples include agents.
  • an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / odorant, etc. are added to an adenosine A2A receptor antagonist.
  • an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / odorant, etc.
  • excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
  • binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like.
  • disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like.
  • Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like.
  • Examples of the colorant include titanium oxide and iron oxide.
  • Examples of the flavoring / flavoring agent include white sugar, orange peel, citric acid, tartaric acid and the like.
  • the oral solution for example, it can be produced by a conventional method by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to an adenosine A2A receptor antagonist.
  • additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to an adenosine A2A receptor antagonist.
  • Examples of the flavoring / flavoring agent include white sugar, orange peel, citric acid, tartaric acid and the like.
  • Examples of the buffer include sodium citrate and the like.
  • Examples of the stabilizer include tragant, gum arabic, gelatin and the like.
  • the suppository examples include an adenosine A2A receptor antagonist, a known carrier for suppository preparations such as polyethylene glycol, lanolin, cacao butter, and fatty acid triglyceride, and, if necessary, a surfactant such as Tween (registered trademark). After adding an agent or the like, it can be produced by a conventional method.
  • an adenosine A2A receptor antagonist such as polyethylene glycol, lanolin, cacao butter, and fatty acid triglyceride
  • a surfactant such as Tween (registered trademark).
  • ointment for example, a known base, stabilizer, wetting agent, preservative and the like can be blended with an adenosine A2A receptor antagonist and mixed by a conventional method to produce an ointment.
  • Examples of the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like.
  • Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.
  • a cream agent, a gel agent, a paste agent or the like as an ointment can be applied to a known support by a conventional method to produce the patch.
  • the support include woven fabrics made of cotton, rayon, and chemical fibers, non-woven fabrics, films such as soft vinyl chloride, polyethylene, and polyurethane, and foam sheets.
  • a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to an adenosine A2A receptor antagonist, and subcutaneously, intramuscularly, or intravenously by a conventional method. It is possible to manufacture injections for use.
  • Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, sodium phosphate and the like.
  • examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like.
  • examples of the tonicity agent include sodium chloride, glucose and the like.
  • Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.
  • a preparation to be applied to the oral cavity can also be formulated by a known pharmaceutical method according to its properties.
  • composition of the present invention When the composition of the present invention is used as a pharmaceutical composition, one or more other components effective for treating a disease (preferably neutrophil inflammation) caused by an overreaction of Th17 cells. Can be blended. It may also be used in combination with other pharmaceutical compositions effective for this purpose.
  • a disease preferably neutrophil inflammation
  • the administration target of the pharmaceutical composition is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include humans and non-human animals.
  • Non-human animals include, but are not limited to, for example, mice, rats, cows, pigs, monkeys, dogs, cats and the like.
  • the administration method can be appropriately selected according to the dosage form and the like, and examples thereof include oral, external use, and injection.
  • the dose can be appropriately selected according to the type, age, body weight, sex, symptoms, etc. of the administration subject, and for example, as the amount of the adenosine A2A receptor antagonist which is the active ingredient per day for human adults, for example. , The amount selected in the range of 0.04 to 500 mg is considered preferred.
  • the frequency of administration is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the daily dose may be administered once a day or divided into a plurality of doses. You may. It may also be administered, for example, 1 to 4 times a week instead of daily.
  • the food or drink may be, for example, a health food, a functional food, a food for specified health use, a nutritional supplement, a food for the sick, or a food additive.
  • Specific examples of foods and drinks include liquid foods such as drinks, soups, dairy drinks, soft drinks, tea drinks, alcoholic drinks, jelly-like drinks, and functional drinks; oils such as cooking oil, dressings, mayonnaise, and margarine.
  • the food and drink according to the present invention can be produced by a production technique known in the art.
  • one or more components effective for diseases caused by Th17 cell overreaction preferably neutrophil inflammation
  • it may be a multifunctional food or drink by combining it with other ingredients or other functional foods that exhibit this desired function.
  • the food and drink intake targets can be appropriately selected according to the purpose, and examples thereof include humans and non-human animals.
  • Non-human animals include, but are not limited to, for example, mice, rats, cows, pigs, monkeys, dogs, cats and the like.
  • the amount of intake can be appropriately selected according to the type, age, body weight, sex, symptoms, etc. of the subject to be ingested, and for example, as the amount of adenosine A2A receptor antagonist which is an active ingredient per day for human adults, for example. , The amount selected in the range of 0.04 to 500 mg is considered preferred.
  • the intake frequency can be appropriately selected according to the purpose. For example, the daily intake may be taken once a day or may be divided into a plurality of times. It may also be taken, for example, 1 to 4 times a week instead of daily.
  • compositions of the present invention pharmaceutical compositions, foods and drinks, reagents, instruments containing them, etc.
  • instructions thereof are used, for example, to suppress the differentiation and activation of Th17 cells, and / or Th17 cells. It may be labeled as being used for the treatment, amelioration, or prevention of diseases caused by the overreaction of.
  • "marked on a product or instruction manual” means that a label is attached to the main body, container, packaging, etc. of the product, or a manual, package insert, promotional material, or other printed matter that discloses product information. It means that the display is attached to.
  • the present invention provides a method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
  • the first aspect of the evaluation method of the present invention is a method using the binding of the test compound to the adenosine A2A receptor as an index, (a) a step of contacting the test compound with the adenosine A2A receptor, and (b). ) Includes a step of detecting the binding of the test compound to the adenosine A2A receptor.
  • This aspect can be mainly used as a primary evaluation method for the evaluation of the aspects described later.
  • test compound to be the subject of the evaluation method of the present invention is not particularly limited, and for example, a synthetic small molecule compound library, an expression product of a gene library, a peptide library, a polynucleotide library, an antibody, and bacterial release.
  • synthetic small molecule compound library examples include extracts and culture supernatants of substances, cells (microorganisms, plant cells, animal cells), purified or partially purified polypeptides, extracts from marine organisms, plants, or animals.
  • the test compound may be synthesized based on an in silico design based on the three-dimensional structure of the adenosine A2A receptor.
  • the "adenosine A2A receptor" used in the evaluation method of the present invention includes, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Evaluation: NP_000666.2) or a partial peptide thereof. Can be mentioned. However, it should be understood that individual differences may occur in the amino acid sequence of the adenosine A2A receptor.
  • adenosine A2A receptor in addition to the natural protein and its partial peptide, a modified product or a modified product thereof can be used as needed.
  • proteins eg, enzymes such as alkaline phosphatase (SEAP), ⁇ -galactosidase, fluorescent proteins such as green fluorescent protein (GFP), glutathione-S-transferase (GST)).
  • SEAP alkaline phosphatase
  • GFP green fluorescent protein
  • GST glutathione-S-transferase
  • a fusion protein with a tag such as can be used.
  • the adenosine A2A receptor can also be used in the form expressed on the cell surface.
  • Contact between the test compound and the adenosine A2A receptor can be performed by adding the test compound to the evaluation system or the like.
  • a known method can be appropriately adopted for "detection of binding between the test compound and the adenosine A2A receptor". For example, a method of contacting a fixed adenosine A2A receptor with a test compound to identify a compound that binds to the adenosine A2A receptor can be mentioned.
  • a means for detecting the binding between the test compound and the adenosine A2A receptor various known means can be used, and one of the preferable means is a biosensor using the surface plasmon resonance phenomenon. ..
  • test compound is a synthetic low molecular weight compound library
  • a high throughput method by combinatorial chemistry technology (Writton NC et al., Science. 26; 273 (5274) 458-464 (1996), Verdine, GL Nature 384 11-13 (1996), Hogan JC, Jr Directed Combinatorial Chemistry. Nature. 384: 17-19 (1996)) can be used.
  • test compound is a polynucleotide library
  • an in vitro selection method can be used (SELEX method) (Tuerk C. & Gold L., Science. 3; 249 (4966): 505). -510 (1990), Green R. et al., Methods Compan Methods Enzymol. 2: 75-86 (1991), Gold L. et al., Annu Rev Biochem 64: 763-97 (1995), Uphoff. Et al., Curr. Opin. Struct. Biol. 6: 281-288 (1996)).
  • test compound a method using FRET (fluorescence resonance energy transfer), an immunoprecipitation method, a yeast two-hybrid system, etc. can also be used.
  • FRET fluorescence resonance energy transfer
  • immunoprecipitation method a method using immunoprecipitation method, a yeast two-hybrid system, etc.
  • test compound when the test compound binds to the adenosine A2A receptor, it is probable that it has an activity of suppressing the differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells. It is evaluated that there is.
  • the second aspect of the evaluation method of the present invention is a method using the inhibition of binding between the adenosine A2A receptor ligand and the adenosine A2A receptor by the test compound as an index, and (a) in the presence of the test compound. It comprises contacting the adenosine A2A receptor ligand with the adenosine A2A receptor and (b) detecting the binding of the adenosine A2A receptor ligand to the adenosine A2A receptor.
  • adenosine A2A receptor ligand for example, adenosine, which is a natural ligand, can be used.
  • the test compound inhibits the binding between the fixed adenosine A2A receptor or the adenosine A2A receptor expressed on the cell surface and the adenosine labeled with a radioactive substance or a fluorescent substance. Whether or not it may be detected by using the sign as an index.
  • the term "inhibition" as used herein includes both complete inhibition and partial inhibition.
  • the test compound inhibits the binding between the adenosine A2A receptor ligand and the adenosine A2A receptor, the activity of suppressing the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated. It is evaluated as having a probability of having an activity of improving or preventing.
  • the third aspect of the evaluation method of the present invention is a method using the suppression of the activity of the adenosine A2A receptor by the test compound as an index, and (a) contacting the test compound with cells expressing the adenosine A2A receptor. Steps, and (b) detecting the activity of the adenosine A2A receptor, including.
  • Examples of the activity of the adenosine A2A receptor include various events that occur in signal transduction from the adenosine A2A receptor in response to a stimulus. Examples of such signal transduction include, but are not limited to, elevation of cAMP and activation of PKA.
  • the test compound is applied to a reaction system in which the adenosine A2A receptor on the cell surface is activated by stimulation of adenosine, which is an adenosine A2A receptor ligand, to obtain the activity. You can evaluate whether it is inhibited. Methods for measuring these activities are known, and measurement may be performed using a usual method or a measurement kit.
  • the test compound when the test compound reduces the activity of the adenosine A2A receptor as compared with the case where the test compound is not contacted, the activity suppresses the differentiation or activation of Th17 cells, or the Th17 cell overreacts. It is evaluated as likely to have the activity of treating, ameliorating, or preventing the resulting disease.
  • the evaluation method of the present invention has been described above, but the evaluation method of the present invention is carried out for a plurality of test compounds to bind to the above-mentioned adenosine A2A receptor, adenosine A2A receptor ligand and adenosine A2A receptor. If a compound is selected based on the inhibition of binding to the body or the suppression of the activity of the adenosine A2A receptor, the activity of suppressing the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells can be treated and improved. , Or compounds with prophylactic activity can be screened. Therefore, the present invention also provides such a screening method.
  • the compound identified by the evaluation method and the screening method of the present invention whether or not to actually suppress the differentiation or activation of Th17 cells, and to treat, ameliorate, or prevent the disease caused by the overreaction of Th17 cells. It is preferable to verify whether or not it has activity. Suppression of Th17 cell differentiation and activation is evaluated by, for example, as described in this example, whether or not the production of Th17 cytokine IL-17A or the like is suppressed by using 2-way MLR or the like. be able to.
  • the activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells can be evaluated in experiments using various model animals as shown in this example, such as psoriasis. Furthermore, in clinical trials, the effect in humans can be actually evaluated.
  • Th17 cytokine IL-17A is produced by adding adenosine or adenosine A2A receptor agonist to a culture system of spleen-derived monocytes or activated CD4 + T cells. It was revealed that it would be induced.
  • the present invention provides a method for producing Th17 cells, which comprises the step of culturing T cells in the presence of an adenosine A2A receptor agonist.
  • the "Th17 cell” produced in the present invention means a helper T cell that produces at least IL-17A.
  • examples of "T cells” that are induced or activated by Th17 cells include naive T cells (naive CD4 + T cells, etc.), effector T cells, and memory T cells.
  • the animal species from which the T cells are derived is not particularly limited, but vertebrates are preferable, and mammals are more preferable. Mammals include, for example, human and non-human mammals. Non-human mammals include, for example, primates such as monkeys and marmosets, rodents such as mice and rats, lagomorphs such as rabbits, ungulates such as pigs, sheep, cows, goats and horses, dogs and cats.
  • the tissue from which lymphocytes are derived is not particularly limited as long as the cells are present, and examples thereof include spleen, peripheral blood, lymph nodes, bone marrow, thymus, umbilical cord blood, and liver.
  • Those skilled in the art can isolate T cells from these tissues using known techniques.
  • Known techniques include, for example, separation with magnetic beads using an antibody against a cell surface marker such as CD4, or flow cytometry. It is also possible to isolate desired T cells using the secretion of cytokines and the expression of functional molecules as indicators.
  • the "adenosine A2A receptor agonist" used to induce or activate Th17 cells is not particularly limited as long as it activates the adenosine A2A receptor (has operative activity). Instead, it may be adenosine, which is a natural ligand for the adenosine A2A receptor. It may also be a known agent that activates the adenosine A2A receptor. Known agents include, for example, PSB0777, ATL-146e, YT-146 (2- (1-octynyl) adenosine), CGS-21680, DPMA (N6- (2- (3,5-dimethoxyphenyl) -2-).
  • examples of the "adenosine A2A receptor agonist” include, for example, Japanese Patent Application Laid-Open No. 2008-285478, Japanese Patent Application Laid-Open No. 2010-503369, Japanese Patent Application Laid-Open No. 2009-534341, Japanese Patent Application Laid-Open No. 2008-534447, Japanese Patent Publication No. 2004- Adenosine derivatives exhibiting operative activity against adenosine A2A receptor disclosed in JP-A-534767 or JP-A-2003-506459; for example, JP-A-2009-534338, JP-A-2009-534340, Disclosure in Japanese Patent Publication No. 2009-534339, Japanese Patent Publication No.
  • Pyrazole derivatives exhibiting operative activity on the body for example, thiophene compounds exhibiting operative activity on adenosine A2A receptor disclosed in JP-A-2003-502434; eg, JP2013-183660.
  • the disclosed anti-adenosine A2A receptor antibody and the like can be mentioned.
  • the "adenosine A2A receptor agonist” also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has its operative activity.
  • a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on each structure of the adenosine A2A receptor agonist and the like.
  • an acid addition salt for example, hydrochloride, bromide
  • hydrochloride, bromide can be selected.
  • Inorganic acid salts such as hydrogen salts, sulfates, hydrogen sulfates, nitrates, carbonates, hydrogen carbonates, phosphates, monohydrogen phosphates, dihydrogen phosphates, acetates, propionates, lactates, Organic acid salts such as citrate and tartrate), base addition salts (eg alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, zinc salt, aluminum salt, ammonium Salts, tetramethylammonium salts), organic amine addition salts (eg, morpholin, piperidine and other addition salts), amino acid addition salts (eg, lysine, glycine, phenylalanine and other addition salts).
  • the hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 10 molecules of water or a solvent to one molecule of an adenosine A2A receptor agonist or a salt thereof
  • the "adenosine A2A receptor agonist” includes all isomers and isomers such as tautomers, geometric isomers, asymmetric carbon-based optical isomers, and stereoisomers as long as they have their operative activity. Contains a mixture. Furthermore, the adenosine A2A receptor agonist undergoes metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, and conjugation, and its operative activity is still active. The present invention also includes compounds that produce adenosine A2A receptor agonists by undergoing metabolism such as oxidation, reduction, and hydrolysis.
  • adenosine A2A receptor agonist As mentioned above, many compounds have been developed and are commercially available, so they can be obtained by purchasing. In addition, many reports have been made on methods for producing these compounds, even if they are not commercially available. Therefore, those skilled in the art can appropriately prepare according to the production method.
  • culturing in the presence of an adenosine A2A receptor agonist is usually carried out by adding an adenosine A2A receptor agonist to a medium for culturing T cells.
  • concentration of the adenosine A2A receptor agonist added to the medium is not particularly limited and can be appropriately adjusted by those skilled in the art based on the type of agonist used, the degree of activity, etc., but for example, 0.01 to 1000 ⁇ M. Is.
  • the "medium” for culturing T cells is not particularly limited as long as it has a composition that maintains the cells and does not suppress the induction of differentiation into Th17 cells and the activation of Th17 cells.
  • basal medium examples include DMEM, ⁇ MEM, ham F12 medium, DMEM / F12 medium, RPMI1640 medium, IMEM, McCoy 5A medium, and EMEM.
  • “Medium additives” include, for example, serum (such as bovine fetal serum), organic compounds (pyruvate, lactic acid, triiodotyronine, paraaminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putresin, and Their salts (eg, sodium salts, etc.), antibiotics (penicillin, streptomycin, gentamycin, amphotericin, etc.), reducing agents (2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), functional proteins (insulin).
  • serum such as bovine fetal serum
  • organic compounds pyruvate, lactic acid, triiodotyronine, paraaminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putresin, and Their salts (eg, sodium salts, etc.)
  • antibiotics penicillin,
  • Transtransferase, lactoferrin, etc. lipids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides (nucleoside, citidine, adenosine 5'-monophosphate, etc.) , Hipoxanthin, thymidin, etc.), metal salts (iron nitrate (III), iron sulfate (II), copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (Glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenic acid, etc.), pH indicators (phenol red, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), but are limited to these. It
  • the target T cell when it is a naive T cell, it may be stimulated and activated as a pretreatment for culturing in the presence of an adenosine A2A receptor agonist, or in the culture. ..
  • stimulation includes stimulation with at least one substance selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, allogeneic antigen-expressing cells, mature dendritic cells, various mitogens, and IL-2. ..
  • the "culture period" of T cells in the presence of an adenosine A2A receptor agonist may be the period until the above-mentioned Th17 cytokine is produced, but is usually 1 day to 1 month, preferably 3 to 14 days. More preferably, it is 5 to 10 days.
  • the "culture environment” is not particularly limited, but the culture temperature is usually 35 to 38 ° C, preferably 37 ° C.
  • the concentration of carbon dioxide is usually 1-10%, preferably 5-10%.
  • the pH of the medium is usually pH 6-8.
  • Th17 cells are contained in the cells induced or activated in this way means that Th17 cytokines are detected by an immunological method such as ELISA, as shown in Examples described later. Can be judged by.
  • the present invention also provides cells produced by the above method, that is, Th17 cells in which T cells are induced or activated by an adenosine A2A receptor agonist.
  • compositions for inducing differentiation or activating Th17 cells which comprises an adenosine A2A receptor agonist as an active ingredient
  • a composition for inducing differentiation or activating Th17 cells which comprises an adenosine A2A receptor agonist as an active ingredient
  • a composition is used as a reagent, it is a pharmacologically acceptable carrier, specifically, a medium, PBS, physiological saline, sterile water, vegetable oil, animal oil, solvent, base, emulsifier, suspension.
  • Surfactants, stabilizers, vehicles, preservatives, binders, diluents, tonicity agents, buffers, solubilizers or other additives can be prepared in appropriate combinations.
  • the difference between the two groups was analyzed using the Student's t-test, but the Wilcoxon rank sum test was used to analyze the comparison of the scores of the degree of motor symptoms of EAE mice, which will be described later. did.
  • the difference between the three groups or more is one-way ANOVA, one-way analysis of variance, where the p-value (probability value, p-value) is 0.05 or less, and there is a significant difference.
  • a Turkey test (Turkey honestly significant difference) was further performed to analyze the significant difference between the two groups.
  • Example 1 Effect of adenosine on 2-way MLR Mononuclear cells were isolated from the splenocytes of BALB / c and SJL / J mice having different H-2. Specifically, spleens are taken from 8-10 week-old female H-2 different BALB / c and SJL / J mice, and the spleens are added to homogenizers (GPE Scientific, Tenbroeck Style Homogenizer, Pestle diameter 15 mm). After adding 5 mL of DMEM, the cells were homogenized by rotating 5 times, and 5 mL of DMEM was further added to transfer a total volume of 10 mL of spleen cells to a 50 mL tube.
  • homogenizers GPE Scientific, Tenbroeck Style Homogenizer, Pestle diameter 15 mm
  • the recovered fraction was transferred to a new 50 mL tube.
  • DMEM was added to this recovered fraction to make a total of 40 mL, suspended, and again at room temperature for 10 minutes at 1,500 r. p. m. Centrifugal at (430 xg). After centrifugation, the supernatant was aspirated with an ejector leaving 2 cm from the lower end, suspended and transferred to a 15 mL tube.
  • DMEM was added to make a total of 12 mL, and 1,500 r. p. m. Centrifugal at (430 xg).
  • the cell pellet is suspended in 2 mL of D10 medium (DMEM supplemented with 10% FCS, 0.1 mg / mL streptomycin, 100 U / mL penicillin, 1 mM sodium pyruvate, and 50 ⁇ M 2-mercaptoethanol).
  • D10 medium DMEM supplemented with 10% FCS, 0.1 mg / mL streptomycin, 100 U / mL penicillin, 1 mM sodium pyruvate, and 50 ⁇ M 2-mercaptoethanol.
  • a part of the prepared cells was added to the turbid and Turku solution, and the number of mononuclear cells was calculated using the Bürkerturk hemocytometer. After calculating, by adding D10 medium mononuclear cells prepared and adjusted to the 3 ⁇ 10 6 cells / ml.
  • adenosine adjusted to 0-1000 ⁇ M was co-cultured, and the concentrations of IFN ⁇ (Th1 cytokine), IL-5 (Th2 cytokine), and IL-17A (Th17 cytokine) in the supernatant 7 days after the start of the culture were subjected to the ELISA method. Quantified.
  • the ELISA method was performed using the Duo Set kit (R & D Systems) according to the attached manual.
  • Example 2 Effect of isstradefylline on 2-way MLR Mononuclear cells were isolated from the splenocytes of BALB / c and SJL / J mice having different H-2 in the same manner as in Example 1. It was adjusted to 3 ⁇ 10 6 cells / ml each. Each cell was mixed with 12-well plate at 1 ml (total: 6 ⁇ 10 6 cells / 2 ml).
  • adenosine A2A receptor antagonist (trade name: Nouriast, generic name: Istradefylline) adjusted to 0.01-1 ⁇ M was co-cultured, and the supernatant 7 days after the start of the culture was used.
  • the IL-17A concentration was quantified by the ELISA method (using R & D Systems Duo Set kit).
  • Fig. 2 concentration-dependent significant suppression of IL-17A production by istradefylline was observed (Fig. 2). That is, it was shown that the induction of IL-17A production by adenosine in 2-way MLR was suppressed by the adenosine A2A receptor antagonist istradefylline in a concentration-dependent manner.
  • Example 3 Effect of adenosine and isstradefylline on CD4 + T cells
  • the spleen was removed from 8-10 week-old BALB / c mice and placed in a 6 cm dish containing 5 mL of DMEM. placed. The spleen was thoroughly kneaded in the medium using tweezers, and the solution containing the lymphocytes eluted in the medium was transferred to a 15 mL tube. The 6 cm dish was washed again with 5 mL DMEM. The supernatant was added to the 15 mL tube to give a total volume of 10 mL.
  • CD4 + T cells were prepared by positive selection using magnetic beads (mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec, 130-049-201)).
  • Adenosine was added to the prepared mouse CD4 + T cells so as to have a final concentration of 100 to 1000 ⁇ M, and an anti-mouse CD3 ⁇ agonist antibody (BioLegend, Clone: 145-2C11) having a final concentration of 1 ⁇ g / mL, and 0. 5 ⁇ g / mL of anti-mouse CD28 agonist antibody (BioLegend, Clone: 37.51) was added, and the cells were seeded and cultured on a 24-well plate at 1 ⁇ 10 6 cells / 500 ⁇ L, and the cell supernatant 7 days after the start of culture was obtained. Recovered. The concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
  • CD4 + T cells activated by receiving CD3 / CD28 stimulation induce IL-17A production in adenosine concentration-dependent manner (graph on the left side of FIG. 3).
  • PSB0777 ammonium salt which is an adenosine A2A receptor agonist
  • adenosine A2A receptor antagonist istradefylin was added to the prepared mouse CD4 + T cells together with adenosine (final concentration, 600 ⁇ M) so that the final concentration was 0.01 to 1 nM.
  • An anti-mouse CD3 ⁇ agonist antibody (BioLegend, Clone: 145-2C11) having a final concentration of 1 ⁇ g / mL and an anti-mouse CD28 agonist antibody (BioLegend, Clone: 37.51) having a final concentration of 0.5 ⁇ g / mL were added.
  • the cells were seeded and cultured on a 24-well plate at 1 ⁇ 10 6 cells / 500 ⁇ L, and the cell supernatant was collected 7 days after the start of the culture.
  • the concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
  • Example 4 Effect of istradefylline on psoriasis model mice Two types of C57BL / 6 mice, "imiquimod (IMQ) cream alone (12.5 mg)” and “IMQ mixed with istradefylline (20 mg)” was applied to the auricle and its thickening (mm) was observed. As a result, a significant suppression of auricular thickening was observed in the group containing isstradefylline as compared with the group in which IMQ cream alone was applied on the 8th day from the start (Fig. 4).
  • Example 5 Effect of Istradefylin on neutrophil airway inflammation model mouse A neutrophil airway inflammation model was prepared according to the protocol shown in FIG. 5A, and the neutrophil airway inflammation model was prepared for IL-17A production of pulmonary lymphocytes. The inhibitory effect of istradefylline was evaluated as follows.
  • DO11.10 mice which are transgenic mice expressing chicken ovoalbumin (OVA) -specific TCR, were subjected to 10 days before pulmonary lymphocyte collection described later (Day).
  • OVA chicken ovoalbumin
  • 6 ⁇ g of istradefylline was dissolved in 100 ⁇ L of ultrapure water (Milli-Q water) or 100 ⁇ L of ultrapure water. The one was orally administered with a gastric sonde.
  • Emulsified 100 ⁇ L (100 ⁇ g as OVA) of OVA-dissolved CFA was subcutaneously injected into the inguinal region of DO11.10 mice.
  • 10 mL PBS (-) in which 0.5 g OVA was dissolved was atomized with a nebulizer and inhaled by DO11.10 mice for 20 minutes. It was.
  • the neutrophil airway inflammation model mouse thus prepared was dislocated from the cervical spine, and then pulmonary lymphocytes were collected. Specifically, the lungs of the mouse were transferred to a 6 cm dish, finely divided using scissors, and D10 medium (DMEM, the final concentration was 10% FCS, 0.1 mg / mL streptamicin, 100 U / mL penicillin, 1 mM, respectively.
  • DMEM D10 medium
  • the pellets are loosened, suspended in 10 mL DMEM, transferred to a new 15 mL tube, and again at room temperature, 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. After removing the supernatant, the cells were finally suspended in D10 medium at a final concentration of 50 ⁇ g / mL gentamicin and 1 ⁇ g / mL amphotericin (D10 + gentamicin + amphotericin medium) at 500 ⁇ L, and prepared in a Turku solution. With some additions, the number of mononuclear cells was calculated using the Bürkertk blood cell count.
  • D10 + gentamicin + amphotericin medium was added to the prepared mononuclear cells, and 1 ⁇ 10 6 cells / 200 ⁇ L were seeded on a round-bottomed 96-well plate. Cell supernatants were collected on Days 1, 2, 3, 4, 5, 6, and 7 (1-7 days after the start of culture). Then, the concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
  • IL-17A production was suppressed in the isstradefylline-administered group as compared with the control group in which ultrapure water was administered. This suggests that administration of istradefylline suppresses neutrophil airway inflammation (Fig. 5B).
  • EAE autoimmune encephalomyelitis
  • Example 7 Effect of Istradefylin on atopic dermatitis model mouse
  • the back of NC / Nga mouse was shaved with a hair clipper, and 2% 2,4,6-trinitrochlorobenzene (TNCB) (ethanol and acetone were added 4).
  • Atopic dermatitis model mice were prepared by dropping 150 ⁇ L of TNCB dissolved in a liquid mixed at 1: 1.
  • Istradefylline was mixed with petrolatum to a concentration of 5% to prepare creams, and each cream was applied to the back and its pathological change was observed. Then, the observed pathological conditions were scored (the pathological conditions and scores are shown in the margin of Table 2), and the effect of istradefylline was evaluated.
  • the present invention is useful for the treatment of diseases caused by Th17 cell overreaction, and mainly contributes to the medical field.
  • the present invention also contributes to basic research for regulating Th17 cell differentiation and activation.

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Abstract

The purpose of the invention is to identify a new receptor participating in the differentiation of helper T cells and to develop a drug targeted thereto. It has been discovered that an adenosine A2A receptor participates in control of the differentiation and activation of Th17 cells. It has also been discovered that antagonists for the receptor are effective as drugs for diseases caused by Th17 cell overreaction.

Description

アデノシンＡ２Ａ受容体を標的とする組成物Compositions that target the adenosine A2A receptor
　本発明は、アデノシンＡ２Ａ受容体を標的としてＴｈ１７細胞の分化または活性化を抑制するための組成物、アデノシンＡ２Ａ受容体を標的としてＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、または予防するための組成物、および、これら組成物の有効成分となる化合物の評価方法に関する。 The present invention is a composition for suppressing the differentiation or activation of Th17 cells by targeting the adenosine A2A receptor, and treating, ameliorating or preventing a disease caused by an overreaction of Th17 cells by targeting the adenosine A2A receptor. And a method for evaluating a compound which is an active ingredient of these compositions.
　獲得免疫の中心的役割を担うヘルパーＴ細胞は、産生するサイトカインの違いなどから、Ｔｈ１（タイプ１ヘルパーＴ細胞）、Ｔｈ２（タイプ２ヘルパーＴ細胞）、Ｔｈ１７（タイプ１７ヘルパーＴ細胞）などに分類される。 Helper T cells, which play a central role in acquired immunity, are classified into Th1 (type 1 helper T cells), Th2 (type 2 helper T cells), Th17 (type 17 helper T cells), etc., based on differences in cytokines produced. Will be done.
　従来から、Ｔｈ１／Ｔｈ２バランスの異常は様々な免疫関連疾患の発症に関与すると考えられている。例えば、Ｔｈ１細胞へのバランス偏向は、慢性炎症性疾患である関節リウマチや、臓器特異的自己免疫疾患（例えば、多発性硬化症、１型糖尿病、炎症性腸疾患、糸球体腎炎、肝炎、肝障害、自己免疫性溶血性貧血、白血球減少症、血小板減少症、脱髄疾患、橋本甲状腺炎、悪性貧血、乾癬）などに関与すると考えられており、また、Ｔｈ２細胞へのバランス偏向は、アレルギー性疾患や、多くの全身性自己免疫疾患に関与すると考えられている。一方、Ｔｈ１７細胞は、もっぱらＩＬ－１７Ａを産生することにより、自己免疫性炎症の増悪に関与しており、特に前述の、１型糖尿病、炎症性腸疾患、乾癬、多発性硬化症、および関節リウマチは、このＴｈ１７への偏向に起因する疑いが強いと考えられている（非特許文献１）。 Traditionally, abnormal Th1 / Th2 balance has been considered to be involved in the development of various immune-related diseases. For example, balance bias to Th1 cells can be associated with rheumatoid arthritis, a chronic inflammatory disease, or organ-specific autoimmune diseases (eg, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, glomerulonephritis, hepatitis, liver). It is thought to be involved in disorders, autoimmune hemolytic anemia, leukopenia, thrombocytopenia, demyelinating disease, Hashimoto's thyroiditis, pernicious anemia, psoriasis), and the balance bias to Th2 cells is allergic. It is thought to be involved in sexual disorders and many systemic autoimmune diseases. On the other hand, Th17 cells are involved in exacerbation of autoimmune inflammation by producing IL-17A exclusively, especially the aforementioned type 1 diabetes, inflammatory bowel disease, psoriasis, multiple sclerosis, and joints. It is believed that rheumatism is highly suspected to be due to this bias towards Th17 (Non-Patent Document 1).
　獲得免疫システムには様々な要因が複雑に関与しており、どの免疫関連疾患に、Ｔｈ１偏向、Ｔｈ２偏向、Ｔｈ１７偏向のいずれが関与しているかは、いまだ不明な点も多いが、本発明者らは、既に、樹状細胞を介したＴｈ１／Ｔｈ２／Ｔｈ１７分化誘導機構に、ドーパミンの働きが大きく関与していることを見出している（特許文献１）。この報告においては、樹状細胞上のドーパミンＤ１様受容体に、そのアンタゴニストを作用させると、樹状細胞におけるドーパミンの合成と貯蔵が抑制され、その結果、ナイーブＴ細胞のＴｈ１７細胞またはＴｈ２細胞への分化の誘導が抑制され、Ｔｈ１細胞への分化の誘導が促進されることが示されている。さらに、樹状細胞上のドーパミンＤ２様受容体に、そのアンタゴニストを作用させると、樹状細胞におけるドーパミンの合成・貯蔵が促進され、その結果、ナイーブＴ細胞のＴｈ１細胞への分化の誘導が抑制され、Ｔｈ２細胞への分化の誘導が促進されることも示されている。 Various factors are involved in the acquired immune system in a complex manner, and it is still unclear which of the immune-related diseases, Th1 bias, Th2 bias, or Th17 bias, is involved. Et al. Have already found that the action of dopamine is greatly involved in the Th1 / Th2 / Th17 differentiation induction mechanism mediated by dendritic cells (Patent Document 1). In this report, the action of an antagonist on a dopamine D1-like receptor on a dendritic cell suppresses the synthesis and storage of dopamine in the dendritic cell, resulting in Th17 or Th2 cells of naive T cells. It has been shown that the induction of differentiation into Th1 cells is suppressed and the induction of differentiation into Th1 cells is promoted. Furthermore, when the antagonist is allowed to act on dopamine D2-like receptors on dendritic cells, the synthesis and storage of dopamine in dendritic cells is promoted, and as a result, the induction of differentiation of naive T cells into Th1 cells is suppressed. It has also been shown that the induction of differentiation into Th2 cells is promoted.
　また、本発明者らは、種々のドーパミンＤ２様受容体アゴニストが、Ｔ細胞の分化誘導において作用するのみならず、活性化後のＴ細胞にも作用して、そのＩＬ－１７Ａ産生を抑制することや、これらアゴニストが好中球性炎症の治療薬として有効であることも見出している（特許文献２）。 In addition, the present inventors not only act on various dopamine D2-like receptor agonists in inducing T cell differentiation, but also act on activated T cells to suppress their IL-17A production. It has also been found that these agonists are effective as therapeutic agents for neutrophil inflammation (Patent Document 2).
　しかしながら、ヘルパーＴ細胞の分化や活性化には、ドーパミン受容体以外の受容体からのシグナル伝達も関与している可能性が考えられることから、そのような受容体を同定し、それを標的とした新たな医薬の開発が望まれている。 However, since it is possible that signal transduction from receptors other than dopamine receptors is also involved in the differentiation and activation of helper T cells, we identified such receptors and targeted them. The development of new drugs has been desired.
　なお、アデノシンＡ２Ａ受容体のアゴニストが、喘息、慢性副鼻腔炎、アレルギー性皮膚炎などの治療に有効であるとの報告（特許文献３、段落０００４）や、自己免疫疾患、喘息、アトピー性皮膚炎、乾癬などの治療に有効であるとの報告（特許文献４、請求項１０、１２）がある。 It should be noted that an agonist of adenosine A2A receptor has been reported to be effective in treating asthma, chronic sinusitis, allergic dermatitis, etc. (Patent Document 3, paragraph 0004), autoimmune diseases, asthma, and atopic skin. There are reports that it is effective in treating inflammation, psoriasis, etc. (Patent Document 4, claims 10 and 12).
国際公開２００８－０１６１１８号公報International Publication No. 2008-016118 国際公開２０１８－２２１５６２号公報International Publication No. 2018-221562 特表２００５－５１３０４３号公報Special Table 2005-513043 特開２００６－１６０６２８号公報Japanese Unexamined Patent Publication No. 2006-160628
　本発明は、上記従来技術の状況に鑑みてなされたものであり、その目的は、ヘルパーＴ細胞の分化に関与する新たな受容体を同定し、それを標的とした医薬を開発することにある。 The present invention has been made in view of the above-mentioned prior art, and an object of the present invention is to identify a new receptor involved in the differentiation of helper T cells and to develop a drug targeting the new receptor. ..
　本発明者らは、上記課題を解決すべく鋭意研究を行った。その結果、アデノシンまたはアデノシンＡ２Ａ受容体アゴニストが、双方向混合リンパ球培養反応系（２ウェイＭＬＲ）、または、ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞の培養系において、Ｔｈ１７細胞を高選択的に誘導し、ＩＬ－１７Ａの分泌を増大させることを見出した。これによりアデノシンＡ２Ａ受容体とＴｈ１７細胞の分化・活性化との関係が示唆されたことから、これを裏付けるため、次いで、アデノシンＡ２Ａ受容体アンタゴニストを、アデノシン存在下での２ウェイＭＬＲ、または、ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞の培養系に適用し、その後のＴｈ１７細胞の挙動の検証を行った。その結果、アデノシンＡ２Ａ受容体アンタゴニストの作用により、アデノシンによるＴｈ１７細胞からのＩＬ－１７Ａの分泌が濃度依存的に抑制されることが判明した。これら事実から、アデノシンＡ２Ａ受容体がＴｈ１７細胞の分化・活性化の制御に関与していることが裏付けられた。 The present inventors have conducted diligent research to solve the above problems. As a result, Th17 cells were generated in a bidirectional mixed lymphocyte culture reaction system (2-way MLR) or a culture system of CD4 + T cells activated by stimulation with CD3 / CD28 by an adenosine or adenosine A2A receptor agonist. It was found that it induces highly selectively and increases the secretion of IL-17A. This suggested a relationship between adenosine A2A receptor and Th17 cell differentiation / activation. To support this, next, adenosine A2A receptor antagonist was added to 2-way MLR or CD3 in the presence of adenosine. It was applied to a culture system of CD4 + T cells activated by receiving / CD28 stimulation, and the behavior of Th17 cells thereafter was verified. As a result, it was found that the action of the adenosine A2A receptor antagonist suppresses the secretion of IL-17A from Th17 cells by adenosine in a concentration-dependent manner. These facts support that the adenosine A2A receptor is involved in the regulation of Th17 cell differentiation and activation.
　そこで、本発明者らは、次に、アデノシンＡ２Ａ受容体アンタゴニストが、Ｔｈ１７細胞の過剰反応に起因する疾患の病態を改善できるか否かの検証を行った。具体的には、疾患の例として、乾癬、好中球性気道炎症、多発性硬化症、およびアトピー性皮膚炎を選択し、これら疾患の各モデルマウスにおけるアデノシンＡ２Ａ受容体アンタゴニストの効果を評価した。その結果、当該アンタゴニストが、有意にこれら疾患の活動性を抑制することが判明した。 Therefore, the present inventors next examined whether or not the adenosine A2A receptor antagonist can improve the pathological condition of the disease caused by the overreaction of Th17 cells. Specifically, psoriasis, neutrophil airway inflammation, multiple sclerosis, and atopic dermatitis were selected as examples of the disease, and the effect of the adenosine A2A receptor antagonist in each model mouse of these diseases was evaluated. .. As a result, it was found that the antagonist significantly suppressed the activity of these diseases.
　以上から、本発明者らは、アデノシンＡ２Ａ受容体を標的としてＴｈ１７細胞の分化・活性化を制御することが可能であり、当該受容体のアンタゴニストがＴｈ１７細胞の過剰反応に起因する疾患の治療などのための薬剤として有効であることを見出し、本発明を完成するに至った。 From the above, the present inventors can control the differentiation and activation of Th17 cells by targeting the adenosine A2A receptor, and the treatment of diseases caused by the overreaction of Th17 cells by the antagonist of the receptor, etc. We have found that it is effective as a drug for this purpose, and have completed the present invention.
　本発明は、より詳しくは、以下を提供するものである。 The present invention provides the following in more detail.
　［１］Ｔｈ１７細胞の分化もしくは活性化の抑制またはＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、もしくは予防のための組成物であって、アデノシンＡ２Ａ受容体アンタゴニストを有効成分として含有する組成物。 [1] A composition for treating, ameliorating, or preventing a disease caused by suppression of Th17 cell differentiation or activation or overreaction of Th17 cells, which contains an adenosine A2A receptor antagonist as an active ingredient. Stuff.
　［２］被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性、またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
　（ａ）被検化合物をアデノシンＡ２Ａ受容体に接触させる工程、および
　（ｂ）被検化合物とアデノシンＡ２Ａ受容体との結合を検出する工程、
を含み、
　被検化合物がアデノシンＡ２Ａ受容体と結合する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。 [2] A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
(A) A step of contacting the test compound with the adenosine A2A receptor, and (b) a step of detecting the binding between the test compound and the adenosine A2A receptor.
Including
When the test compound binds to the adenosine A2A receptor, it is evaluated that it has an activity of suppressing the differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells. How to be done.
　［３］被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
　（ａ）被検化合物の存在下で、アデノシンＡ２Ａ受容体リガンドをアデノシンＡ２Ａ受容体に接触させる工程、および
　（ｂ）アデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を検出する工程、
を含み、
　被検化合物がアデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を阻害する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。 [3] A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
(A) The step of contacting the adenosine A2A receptor ligand with the adenosine A2A receptor in the presence of the test compound, and (b) the step of detecting the binding between the adenosine A2A receptor ligand and the adenosine A2A receptor.
Including
When the test compound inhibits the binding of the adenosine A2A receptor ligand to the adenosine A2A receptor, the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated, ameliorated, or prevented. A method that is evaluated as having a probable activity.
　［４］被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
　（ａ）被検化合物をアデノシンＡ２Ａ受容体を発現する細胞に接触させる工程、および
　（ｂ）アデノシンＡ２Ａ受容体の活性を検出する工程、
を含み、
　被検化合物を接触させない場合と比較して、被検化合物がアデノシンＡ２Ａ受容体の活性を低下させる場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。 [4] A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
(A) A step of contacting the test compound with a cell expressing the adenosine A2A receptor, and (b) a step of detecting the activity of the adenosine A2A receptor.
Including
When the test compound reduces the activity of the adenosine A2A receptor as compared to the case where the test compound is not contacted, the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated. , A method that is assessed as probable to have an ameliorating or prophylactic activity.
　［５］Ｔ細胞を、アデノシンＡ２Ａ受容体アゴニストの存在下で培養する工程を含む、Ｔｈ１７細胞の製造方法。 [5] A method for producing Th17 cells, which comprises a step of culturing T cells in the presence of an adenosine A2A receptor agonist.
　［６］アデノシンＡ２Ａ受容体アゴニストによってＴ細胞が分化誘導または活性化されてなる、Ｔｈ１７細胞。 [6] Th17 cells in which T cells are induced or activated by an adenosine A2A receptor agonist.
　［７］アデノシンＡ２Ａ受容体アゴニストを有効成分として含む、Ｔｈ１７細胞に分化誘導または該細胞を活性化させるための組成物。 [7] A composition for inducing differentiation or activating Th17 cells, which comprises an adenosine A2A receptor agonist as an active ingredient.
　好中球性気管支喘息を含む好中球性気道炎症、歯周病、乾癬、重症アトピー性皮膚炎、尋常性ざ瘡、子宮内膜症、慢性副鼻腔炎、その他の各種自己免疫病など、好中球性炎症を主体とする病態は多い。しかしながら、特効薬は、これまで知られていない。 Neutrophil airway inflammation including neutrophil bronchial asthma, periodontal disease, psoriasis, severe atopic dermatitis, acne vulgaris, endometriosis, chronic sinusitis, and various other autoimmune diseases, etc. There are many pathological conditions mainly due to neutrophil inflammation. However, no silver bullet has been known so far.
　ドーパミンＤ２様受容体を介したＴｈ１７細胞の活性化と好中球性炎症との関係については既に報告されているが（特許文献１）、本発明の組成物は、アデノシンＡ２Ａ受容体を標的として、Ｔｈ１７細胞の分化や活性化の抑制効果を発揮するものである。従って、本発明の組成物は、特に、好中球性炎症を含むＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、または予防のための新規作用機序に基づく医薬として有用である。本発明によれば、アデノシンＡ２Ａ受容体を標的として、このような医薬の候補化合物を効率的に評価し、スクリーニングすることも可能となる。 Although the relationship between Th17 cell activation via dopamine D2-like receptors and neutrophil inflammation has already been reported (Patent Document 1), the composition of the present invention targets the adenosine A2A receptor. , Th17 cell exerts an inhibitory effect on differentiation and activation. Therefore, the compositions of the present invention are particularly useful as pharmaceuticals based on a novel mechanism of action for the treatment, amelioration, or prevention of diseases caused by Th17 cell overreaction, including neutrophil inflammation. According to the present invention, it is also possible to efficiently evaluate and screen such drug candidate compounds by targeting the adenosine A2A receptor.
２ウェイＭＬＲにおけるアデノシンの効果を示すグラフである。グラフの縦軸の単位は、ｐｇ／ｍｌである。一元配置分散分析で、ｐ値が０．０５以下で有意差ありと判断し、さらにＴｕｒｋｅｙ検定を行って２群間の有意差を分析した。図中「＊＊」および「＊」は、培地のみに対し、他の群が、「Ｐ＜０．０１」および「Ｐ＜０．０５」であることを各々示す。It is a graph which shows the effect of adenosine in a 2-way MLR. The unit of the vertical axis of the graph is pg / ml. One-way ANOVA determined that there was a significant difference when the p-value was 0.05 or less, and a Turkey test was performed to analyze the significant difference between the two groups. In the figure, "**" and "*" indicate that the other groups have "P <0.01" and "P <0.05" with respect to the medium only, respectively. ２ウェイＭＬＲにおけるノウリアスト（イストラデフィリン）の効果を示すグラフである。グラフの縦軸の単位は、ｐｇ／ｍｌである。一元配置分散分析で、ｐ値が０．０５以下で有意差ありと判断し、さらにＴｕｒｋｅｙ検定を行って２群間の有意差を分析した。図中「＊」は、アデノシン（１００μＭ）のみを加えた群に対し、更にイストラデフェリンを加えた群が「Ｐ＜０．０５」であることを示す。It is a graph which shows the effect of nouriast (istradefylline) in 2-way MLR. The unit of the vertical axis of the graph is pg / ml. One-way ANOVA determined that there was a significant difference when the p-value was 0.05 or less, and a Turkey test was performed to analyze the significant difference between the two groups. In the figure, "*" indicates that the group to which only adenosine (100 μM) was added and the group to which istradeferin was further added had “P <0.05”. ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞の培養系における、アデノシン、ＰＳＢ０７７７、またはイストラデフィリンの効果を示すグラフである。一元配置分散分析で、ｐ値が０．０５以下で有意差ありと判断し、さらにＴｕｒｋｅｙ検定を行って２群間の有意差を分析した。左側および中央のグラフにおいて「＊＊」および「＊」は、培地のみの群に対し、アデノシン（左側のグラフ）またはＰＳＢ０７７７（中央のグラフ）を加えた群が、「Ｐ＜０．０１」および「Ｐ＜０．０５」であることを示す、右側のグラフにおいて「＊＊」は、アデノシン（６００μＭ）を加えた群に対し、更にイストラデフェリンを加えた群が「Ｐ＜０．０１」であることを示す。また、図中「Ｎ．Ｄ．」は未検出であることを示す。It is a graph which shows the effect of adenosine, PSB0777, or isstradefylline in the culture system of CD4 + T cells activated by the stimulation of CD3 / CD28. One-way ANOVA determined that there was a significant difference when the p-value was 0.05 or less, and a Turkey test was performed to analyze the significant difference between the two groups. In the left and center graphs, "**" and "*" indicate "P <0.01" in the group in which adenosine (left graph) or PSB0777 (center graph) is added to the medium-only group. In the graph on the right, which indicates that "P <0.05", "**" is "P <0.01" in the group to which adenosine (600 μM) was added and to the group to which Istradeferin was further added. Indicates that. In addition, "ND" in the figure indicates that it has not been detected. 乾癬モデルマウスにおけるイストラデフィリンの効果を示すグラフである。なお、グラフは、耳介の肥厚度を示し、各個体の耳介の厚さから陰性対照で生じた肥厚分を差し引いた値で表している。グラフの横軸の単位は、ｍｍである。It is a graph which shows the effect of Istradefylline in a psoriasis model mouse. The graph shows the degree of auricle thickening, and is represented by a value obtained by subtracting the thickening amount generated in the negative control from the thickness of the auricle of each individual. The unit of the horizontal axis of the graph is mm. 好中球性気道炎症モデルマウスを作製および解析するためのプロトコルを示す、概略図である。It is the schematic which shows the protocol for making and analyzing the neutrophil airway inflammation model mouse. 好中球性気道炎症モデルマウスにおけるイストラデフィリンの効果を示すグラフである。＊＊Ｐ＜０．０１，＊Ｐ＜０．０５。It is a graph which shows the effect of isstradefylline in the neutrophil airway inflammation model mouse. ** P <0.01, * P <0.05. 多発性硬化症モデルマウスにおけるイストラデフィリンの効果を示すグラフである。It is a graph which shows the effect of isstradefylline in the multiple sclerosis model mouse. 多発性硬化症モデルマウスにおけるイストラデフィリンの効果を示す写真である。It is a photograph showing the effect of Istradefylline in a mouse model of multiple sclerosis. アトピー性皮膚炎モデルマウスにおけるイストラデフィリンの効果を示す写真である。It is a photograph which shows the effect of isstradefylline in atopic dermatitis model mouse. アトピー性皮膚炎モデルマウスにおけるイストラデフィリンの効果を示すグラフである。It is a graph which shows the effect of isstradefylline in atopic dermatitis model mouse.
　＜アデノシンＡ２Ａ受容体アンタゴニストを有効成分とする組成物＞
　本発明は、Ｔｈ１７細胞の分化もしくは活性化の抑制またはＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、もしくは予防のための組成物であって、アデノシンＡ２Ａ受容体アンタゴニストを有効成分として含有する組成物を提供する。 <Composition containing adenosine A2A receptor antagonist as an active ingredient>
The present invention is a composition for treating, ameliorating, or preventing a disease caused by suppression of Th17 cell differentiation or activation or Th17 cell overreaction, and contains an adenosine A2A receptor antagonist as an active ingredient. The composition is provided.
　「アデノシン受容体」は、アデノシンに対するＧタンパク質共役型の受容体分子であり、ヒトでは、Ａ１、Ａ２Ａ、Ａ２Ｂ、Ａ３の４種類のサブタイプが存在する。「アデノシンＡ２Ａ受容体」は、細胞表面上で刺激を受けると、細胞内でアデニル酸シクラーゼが活性化され、ｃＡＭＰの産生が亢進し、シグナルが伝達される（すなわち、アデノシンＡ２Ａ受容体が活性化する）。 "Adenosine receptor" is a G protein-coupled receptor molecule for adenosine, and in humans, there are four subtypes, A1, A2A, A2B, and A3. When the "adenosine A2A receptor" is stimulated on the cell surface, adenylate cyclase is activated inside the cell, cAMP production is enhanced, and a signal is transmitted (that is, the adenosine A2A receptor is activated). To do).
　本発明の組成物の有効成分となる「アデノシンＡ２Ａ受容体アンタゴニスト」は、アデノシンＡ２Ａ受容体の活性化を阻害するものであれば特に制限はない。公知の薬剤であってもよく、また、例えば、後述の被検化合物の評価法を利用して得られるものであってもよい。公知の薬剤としては、例えば、イストラデフィリン、プレラデナント、ヴィパデナント、カフェイン、ＳＣＨ－５８２６１、ＳＣＨ－４１２３４８、ＳＣＨ－４４２４１６、ＶＥＲ－６６２３、ＶＥＲ－６９４７、ＶＥＲ－７８３５、ＳＴ－１５３５、ＺＭ－２４１３８５、ＡＴＬ－４４４、ＭＳＸ－３、ＰＢＦ－５０９、ＡＺＤ４６３５（ＨＴＬ１０７１）、ＣＰＩ－４４４、ＡＢ９２８、ＢＡＹ－５４５、Ａ２ＡＲアンタゴニスト１、リバーシン、プロキシフィリンなどが挙げられるが、これらに制限されない。これら公知の薬剤の構造式を順に以下に示す。 The "adenosine A2A receptor antagonist" which is the active ingredient of the composition of the present invention is not particularly limited as long as it inhibits the activation of the adenosine A2A receptor. It may be a known drug, or may be obtained by using, for example, the evaluation method of a test compound described later. Known agents include, for example, istradefylline, preladenant, vipadenant, caffeine, SCH-58261, SCH-421348, SCH-442416, VER-6623, VER-6947, VER-7835, ST-1535, ZM-241385. , ATL-444, MSX-3, PBF-509, AZD4635 (HTL1071), CPI-444, AB928, BAY-545, A2AR antagonist 1, reversine, proxyphyllin and the like. The structural formulas of these known drugs are shown below in order.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　さらに、「アデノシンＡ２Ａ受容体アンタゴニスト」としては、例えば、特開平６－２１１８５６号公報、または特開２０１６－００３１８６号公報に開示されている、アデノシンＡ２Ａ受容体に対して拮抗作用を示すキサンチン誘導体；例えば、特表２０１３－５２３７１１号公報、特表２０１２－５０５２６４号公報、特表２０１１－５１３４１７号公報、特表２０１１－５００８３３号公報、特表２０１１－５００８１９号公報、特表２０１０－５２３４９７号公報、特開２００８－２９７３１２号公報、特表２００９－５０８８７１号公報、特表２００８－５２４３３０号公報、特表２００７－５２１２４３号公報、または特表２００５－５０６３５２号公報に開示されている、アデノシンＡ２Ａ受容体に対して拮抗作用を示すピリミジン誘導体；例えば、特表２０１１－５１４３５０号公報に開示されている、アデノシンＡ２Ａ受容体に対して拮抗作用を示すアミノ－キノキサリン誘導体；例えば、特開２０１１－０９７８６９号公報に開示されている、抗アデノシンＡ２Ａ受容体抗体などが挙げられる。 Further, as the "adenosine A2A receptor antagonist", for example, a xanthin derivative exhibiting an antagonistic effect on the adenosine A2A receptor disclosed in JP-A-6-211856 or JP-A-2016-0031186; For example, Japanese Patent Publication No. 2013-523711, Japanese Patent Publication No. 2012-505264, Japanese Patent Publication No. 2011-513417, Japanese Patent Publication No. 2011-500833, Japanese Patent Publication No. 2011-500919, Japanese Patent Publication No. 2010-523497, Adenosine A2A receptor disclosed in JP-A-2008-297312, Japanese Patent Application Laid-Open No. 2009-508871, Japanese Patent Application Laid-Open No. 2008-524330, Japanese Patent Application Laid-Open No. 2007-521243, or Japanese Patent Application Laid-Open No. 2005-506352. Pyrimidine derivative exhibiting antagonism against adenosine A2A receptor; for example, amino-quinoxalin derivative exhibiting antagonism against adenosine A2A receptor disclosed in JP-A-2011-514350; eg, JP-A-2011-07869 Examples thereof include anti-adenosine A2A receptor antibodies disclosed in.
　また、「アデノシンＡ２Ａ受容体アンタゴニスト」には、その阻害活性を有する限り、薬理学上許容される塩、水和物または溶媒和物も含まれる。このような薬理学上許容される塩としては、特に制限はなく、アデノシンＡ２Ａ受容体アンタゴニストの各構造などに応じて適宜選択することができ、例えば、酸付加塩（例えば、塩酸塩、臭化水素塩、硫酸塩、硫酸水素塩、硝酸塩、炭酸塩、炭酸水素塩、リン酸塩、リン酸一水素塩、リン酸二水素塩などの無機酸塩、酢酸塩、プロピオン酸塩、乳酸塩、クエン酸塩、酒石酸塩などの有機酸塩）、塩基付加塩（例えば、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、亜鉛塩、アルミニウム塩、アンモニウム塩、テトラメチルアンモニウム塩）、有機アミン付加塩（例えば、モルホリン、ピペリジンなどの付加塩）、アミノ酸付加塩（例えば、リジン、グリシン、フェニルアラニンなどの付加塩）が挙げられる。また、水和物または溶媒和物としては、特に制限はなく、例えば、アデノシンＡ２Ａ受容体アンタゴニストまたはその塩１分子に対し、０．１～１０分子の水または溶媒が付加したものが挙げられる。 The "adenosine A2A receptor antagonist" also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has an inhibitory activity thereof. Such a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on each structure of the adenosine A2A receptor antagonist, for example, an acid addition salt (for example, hydrochloride, bromide). Inorganic acid salts such as hydrogen salts, sulfates, hydrogen sulfates, nitrates, carbonates, hydrogen carbonates, phosphates, monohydrogen phosphates, dihydrogen phosphates, acetates, propionates, lactates, Organic acid salts such as citrate and tartrate), base addition salts (eg alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, zinc salt, aluminum salt, ammonium Salts, tetramethylammonium salts), organic amine addition salts (eg, morpholin, piperidine and other addition salts), amino acid addition salts (eg, lysine, glycine, phenylalanine and other addition salts). The hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 10 molecules of water or a solvent to one molecule of an adenosine A2A receptor antagonist or a salt thereof.
　さらに、「アデノシンＡ２Ａ受容体アンタゴニスト」には、その阻害活性を有する限り、互変異性体、幾何異性体、不斉炭素に基づく光学異性体、立体異性体などの総ての異性体および異性体混合物が含まれる。さらにまた、アデノシンＡ２Ａ受容体アンタゴニストが生体内で酸化、還元、加水分解、アミノ化、脱アミノ化、水酸化、リン酸化、脱水酸化、アルキル化、脱アルキル化、抱合などの代謝を受けてなおその阻害活性を示す化合物をも包含し、また本発明は生体内で酸化、還元、加水分解などの代謝を受けてアデノシンＡ２Ａ受容体アンタゴニストを生成する化合物をも包含する。 Furthermore, the "adenosine A2A receptor antagonist" includes all isomers and isomers such as tautomers, geometric isomers, asymmetric carbon-based optical isomers, and stereoisomers as long as they have inhibitory activity. Contains a mixture. Furthermore, adenosine A2A receptor antagonists are still undergoing metabolism such as oxidation, reduction, hydrolysis, amination, deaminolation, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, and conjugation in vivo. The present invention also includes compounds exhibiting the inhibitory activity, and the present invention also includes compounds that undergo metabolism such as oxidation, reduction, and hydrolysis in vivo to produce adenosine A2A receptor antagonists.
　なお、アデノシンＡ２Ａ受容体アンタゴニストに関しては、上述のとおり、多くの化合物が開発され市販もされているため、購入することによって入手することができる。また市販されていなくとも、それら化合物の製造方法についても多々報告がなされている。そのため、当業者であれば、当該製造方法に沿って適宜調製することもできる。 As for the adenosine A2A receptor antagonist, as mentioned above, many compounds have been developed and are commercially available, so they can be obtained by purchasing. In addition, many reports have been made on methods for producing these compounds, even if they are not commercially available. Therefore, those skilled in the art can appropriately prepare according to the production method.
　本発明の組成物は、医薬組成物（医薬品、医薬部外品、動物用医薬品など）、飲食品（動物用飼料を含む）、あるいは研究目的（例えば、ｉｎ　ｖｉｔｒｏまたはｉｎ　ｖｉｖｏの実験）に用いられる試薬の形態でありうる。 The composition of the present invention is used for pharmaceutical compositions (pharmaceutical products, quasi-drugs, veterinary drugs, etc.), foods and drinks (including animal feed), or for research purposes (for example, in vitro or in vivo experiments). It can be in the form of a reagent to be used.
　本発明の組成物は、Ｔｈ１７細胞の分化もしくは活性化の抑制、またはＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、もしくは予防に利用することができる。 The composition of the present invention can be used for suppressing the differentiation or activation of Th17 cells, or for treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
　アデノシンＡ２Ａ受容体の生理的役割としては、血管拡張、睡眠、神経活動制御などが知られているが、本発明において、Ｔｈ１７細胞の分化および活性化との関係が見出された。Ｔｈ１７細胞の分化・活性化と好中球性炎症との関係は知られていることから（特許文献２）、本発明の組成物は、Ｔｈ１７細胞の過剰反応に起因する疾患に適用することが可能である。「Ｔｈ１７細胞の過剰反応に起因する疾患」としては、好中球性炎症を主な病態とする疾患が好適である。具体的な疾患としては、例えば、好中球性気管支喘息を含む好中球性気道炎症、歯周病、乾癬、潰瘍性大腸炎、重症アトピー性皮膚炎、尋常性ざ瘡、子宮内膜症、慢性副鼻腔炎、その他の種々の自己免疫病（例えば、関節リウマチや、多発性硬化症、１型糖尿病、腎炎）などが挙げられるが、これらに制限されない。 Vasodilation, sleep, neural activity control, etc. are known as physiological roles of adenosine A2A receptor, but in the present invention, a relationship with Th17 cell differentiation and activation was found. Since the relationship between Th17 cell differentiation / activation and neutrophil inflammation is known (Patent Document 2), the composition of the present invention can be applied to diseases caused by Th17 cell overreaction. It is possible. As the "disease caused by the overreaction of Th17 cells", a disease whose main pathological condition is neutrophil inflammation is suitable. Specific diseases include, for example, neutrophil airway inflammation including neutrophil bronchial asthma, periodontal disease, psoriasis, ulcerative colitis, severe atopic dermatitis, acne vulgaris, and endometriosis. , Chronic sinusitis, and various other autoimmune diseases (eg, rheumatoid arthritis, polysclerosis, type 1 diabetes, nephritis), but are not limited thereto.
　本発明の組成物を医薬組成物として用いる場合、有効成分としてアデノシンＡ２Ａ受容体アンタゴニストを含有するものであれば、その剤形は特に限定されない。本発明の医薬組成物は、公知の製剤学的方法により、薬理学上許容される担体を含む、種々の剤形として製剤化することができる。 When the composition of the present invention is used as a pharmaceutical composition, its dosage form is not particularly limited as long as it contains an adenosine A2A receptor antagonist as an active ingredient. The pharmaceutical composition of the present invention can be formulated by a known pharmaceutical method in various dosage forms including a pharmacologically acceptable carrier.
　医薬組成物の剤型としては、特に制限はなく、例えば、所望の投与方法に応じて適宜選択することができ、例えば、経口固形剤（錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤など）、経口液剤（内服液剤、シロップ剤、エリキシル剤など）、外用剤（坐剤、軟膏剤、貼付剤、ゲル剤、クリーム剤、外用散剤、スプレー剤、吸入剤など）、注射剤（溶液、懸濁液、用事溶解用固形剤など）、その他、口腔用剤（ガム剤、トローチ剤、舌下錠、バッカル錠、付着剤など）、口腔用スプレー剤、口腔用半固形剤、歯磨剤、含嗽剤などが挙げられる。 The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected depending on the desired administration method, for example, an oral solid preparation (tablet, coated tablet, granule, powder, capsule, etc.). , Oral solutions (oral solutions, syrups, elixirs, etc.), external preparations (suppositories, ointments, patches, gels, creams, external powders, sprays, inhalants, etc.), injections (solutions, suspensions, etc.) Turbid liquids, solids for errands, etc.), other oral preparations (gum, troche, sublingual tablets, buccal tablets, adhesives, etc.), oral sprays, oral semi-solids, dentifrices, gargles Examples include agents.
　経口固形剤としては、例えば、アデノシンＡ２Ａ受容体アンタゴニストに、賦形剤、さらには必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤などの添加剤を加え、常法により製造することができる。 As an oral solid preparation, for example, an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / odorant, etc. are added to an adenosine A2A receptor antagonist. Can be manufactured by law.
　賦形剤としては、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸などが挙げられる。結合剤としては、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどが挙げられる。崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖などが挙げられる。滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコールなどが挙げられる。着色剤としては、例えば、酸化チタン、酸化鉄などが挙げられる。矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。 Examples of excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. .. Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like. Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include white sugar, orange peel, citric acid, tartaric acid and the like.
　経口液剤としては、例えば、アデノシンＡ２Ａ受容体アンタゴニストに、矯味・矯臭剤、緩衝剤、安定化剤などの添加剤を加え、常法により製造することができる。 As the oral solution, for example, it can be produced by a conventional method by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to an adenosine A2A receptor antagonist.
　矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。緩衝剤としては、例えば、クエン酸ナトリウムなどが挙げられる。安定化剤としては、例えば、トラガント、アラビアゴム、ゼラチンなどが挙げられる。 Examples of the flavoring / flavoring agent include white sugar, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include tragant, gum arabic, gelatin and the like.
　坐剤としては、例えば、アデノシンＡ２Ａ受容体アンタゴニストに、ポリエチレングリコール、ラノリン、カカオ脂、脂肪酸トリグリセリドなどの公知の坐剤製剤用担体と、必要に応じてツイーン（ＴＷＥＥＮ：登録商標）などの界面活性剤などを加えた後、常法により製造することができる。 Examples of the suppository include an adenosine A2A receptor antagonist, a known carrier for suppository preparations such as polyethylene glycol, lanolin, cacao butter, and fatty acid triglyceride, and, if necessary, a surfactant such as Tween (registered trademark). After adding an agent or the like, it can be produced by a conventional method.
　軟膏剤としては、例えば、アデノシンＡ２Ａ受容体アンタゴニストに、公知の基剤、安定剤、湿潤剤、保存剤などを配合し、常法により混合し、製造することができる。 As the ointment, for example, a known base, stabilizer, wetting agent, preservative and the like can be blended with an adenosine A2A receptor antagonist and mixed by a conventional method to produce an ointment.
　基剤としては、例えば、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィンなどが挙げられる。保存剤としては、例えば、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピルなどが挙げられる。 Examples of the base include liquid paraffin, white petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.
　貼付剤としては、例えば、公知の支持体に軟膏剤としてのクリーム剤、ゲル剤、ペースト剤などを、常法により塗布し、製造することができる。支持体としては、例えば、綿、スフ、化学繊維からなる織布、不織布、軟質塩化ビニル、ポリエチレン、ポリウレタンなどのフィルム、発泡体シートなどが挙げられる。 As the patch, for example, a cream agent, a gel agent, a paste agent or the like as an ointment can be applied to a known support by a conventional method to produce the patch. Examples of the support include woven fabrics made of cotton, rayon, and chemical fibers, non-woven fabrics, films such as soft vinyl chloride, polyethylene, and polyurethane, and foam sheets.
　注射剤としては、例えば、アデノシンＡ２Ａ受容体アンタゴニストに、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤などを添加し、常法により皮下用、筋肉内用、静脈内用などの注射剤を製造することができる。 As an injection, for example, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to an adenosine A2A receptor antagonist, and subcutaneously, intramuscularly, or intravenously by a conventional method. It is possible to manufacture injections for use.
　ｐＨ調節剤および緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。安定化剤としては、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸などが挙げられる。等張化剤としては、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカインなどが挙げられる。 Examples of the pH adjuster and buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.
　口腔内に適用する製剤も、その性状に応じて、公知の製剤学的方法により製剤化することができる。 A preparation to be applied to the oral cavity can also be formulated by a known pharmaceutical method according to its properties.
　本発明の組成物を医薬組成物として用いる場合には、Ｔｈ１７細胞の過剰反応に起因する疾患（好ましくは、好中球性炎症）の治療などに有効な１種もしくは２種以上の他の成分を配合することができる。また、この目的に有効な他の医薬組成物と併用してもよい。 When the composition of the present invention is used as a pharmaceutical composition, one or more other components effective for treating a disease (preferably neutrophil inflammation) caused by an overreaction of Th17 cells. Can be blended. It may also be used in combination with other pharmaceutical compositions effective for this purpose.
　医薬組成物の投与対象としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒトおよび非ヒト動物が挙げられる。非ヒト動物としては、例えば、マウス、ラット、ウシ、ブタ、サル、イヌ、ネコなどが挙げられるが、これらに制限されない。投与方法は、その剤型などに応じて適宜選択することができ、例えば、経口、外用、注射による投与などが挙げられる。投与量は、投与対象の種類、年齢、体重、性別、症状などに応じて適宜選択することができるが、例えば、ヒト成人１日あたり、有効成分であるアデノシンＡ２Ａ受容体アンタゴニストの量として、例えば、０．０４～５００ｍｇの範囲内で選択される量が好ましいと考えられる。投与頻度としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、１日あたりの投与量を、１日に１回で投与してもよいし、複数回に分けて投与してもよい。また、毎日ではなく、例えば、週に１～４回で投与してもよい。 The administration target of the pharmaceutical composition is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include humans and non-human animals. Non-human animals include, but are not limited to, for example, mice, rats, cows, pigs, monkeys, dogs, cats and the like. The administration method can be appropriately selected according to the dosage form and the like, and examples thereof include oral, external use, and injection. The dose can be appropriately selected according to the type, age, body weight, sex, symptoms, etc. of the administration subject, and for example, as the amount of the adenosine A2A receptor antagonist which is the active ingredient per day for human adults, for example. , The amount selected in the range of 0.04 to 500 mg is considered preferred. The frequency of administration is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the daily dose may be administered once a day or divided into a plurality of doses. You may. It may also be administered, for example, 1 to 4 times a week instead of daily.
　本発明の組成物を飲食品として用いる場合、当該飲食品は、例えば、健康食品、機能性食品、特定保健用食品、栄養補助食品、病者用食品、あるいは食品添加物でありうる。飲食品の具体例としては、ドリンク類、スープ類、乳飲料、清涼飲料水、茶飲料、アルコール飲料、ゼリー状飲料、機能性飲料などの液状食品；食用油、ドレッシング、マヨネーズ、マーガリンなどの油分を含む製品；飯類、麺類、パン類などの炭水化物含有食品；ハム、ソーセージなどの畜産加工食品；かまぼこ、干物、塩辛などの水産加工食品；漬物などの野菜加工食品；ゼリー、ヨーグルトなどの半固形状食品；みそ、発酵飲料などの発酵食品；洋菓子類、和菓子類、キャンディー類、ガム類、グミ、冷菓、氷菓などの各種菓子類；カレー、あんかけ、中華スープなどのレトルト製品；インスタントスープ、インスタントみそ汁などのインスタント食品や電子レンジ対応食品などが挙げられる。さらには、粉末、穎粒、錠剤、カプセル剤、液状、ペースト状またはゼリー状に調製された健康飲食品も挙げられる。 When the composition of the present invention is used as a food or drink, the food or drink may be, for example, a health food, a functional food, a food for specified health use, a nutritional supplement, a food for the sick, or a food additive. Specific examples of foods and drinks include liquid foods such as drinks, soups, dairy drinks, soft drinks, tea drinks, alcoholic drinks, jelly-like drinks, and functional drinks; oils such as cooking oil, dressings, mayonnaise, and margarine. Products containing; Carbohydrate-containing foods such as rice, noodles and bread; Processed livestock foods such as ham and sausage; Processed marine foods such as kamaboko, dried food and salt; Processed vegetable food such as pickles; Half of jelly and yogurt Solid foods; Fermented foods such as miso and fermented beverages; Western confectionery, Japanese confectionery, candy, gums, gummy, chilled confectionery, ice confectionery and other confectionery; curry, ankake, Chinese soup and other retort products; instant soup, Examples include instant foods such as instant miso soup and foods compatible with microwave ovens. Further, healthy foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly can be mentioned.
　本発明における飲食品の製造は、当該技術分野に公知の製造技術により実施することができる。飲食品においては、Ｔｈ１７細胞の過剰反応に起因する疾患（好ましくは、好中球性炎症）に有効な１種もしくは２種以上の成分を配合してもよい。また、この目的の機能を発揮する他の成分あるいは他の機能性食品と組み合わせることによって、多機能性の飲食品としてもよい。 The food and drink according to the present invention can be produced by a production technique known in the art. In foods and drinks, one or more components effective for diseases caused by Th17 cell overreaction (preferably neutrophil inflammation) may be blended. In addition, it may be a multifunctional food or drink by combining it with other ingredients or other functional foods that exhibit this desired function.
　飲食品の摂取対象としては、目的に応じて適宜選択することができ、例えば、ヒトおよび非ヒト動物が挙げられる。非ヒト動物としては、例えば、マウス、ラット、ウシ、ブタ、サル、イヌ、ネコなどが挙げられるが、これらに制限されない。摂取量は、摂取対象の種類、年齢、体重、性別、症状などに応じて適宜選択することができるが、例えば、ヒト成人１日あたり、有効成分であるアデノシンＡ２Ａ受容体アンタゴニストの量として、例えば、０．０４～５００ｍｇの範囲内で選択される量が好ましいと考えられる。摂取頻度は、目的に応じて適宜選択することができ、例えば、１日あたりの摂取量を、１日に１回で摂取してもよいし、複数回に分けて摂取してもよい。また、毎日ではなく、例えば、週に１～４回で摂取してもよい。 The food and drink intake targets can be appropriately selected according to the purpose, and examples thereof include humans and non-human animals. Non-human animals include, but are not limited to, for example, mice, rats, cows, pigs, monkeys, dogs, cats and the like. The amount of intake can be appropriately selected according to the type, age, body weight, sex, symptoms, etc. of the subject to be ingested, and for example, as the amount of adenosine A2A receptor antagonist which is an active ingredient per day for human adults, for example. , The amount selected in the range of 0.04 to 500 mg is considered preferred. The intake frequency can be appropriately selected according to the purpose. For example, the daily intake may be taken once a day or may be divided into a plurality of times. It may also be taken, for example, 1 to 4 times a week instead of daily.
　本発明の組成物の製品（医薬組成物、飲食品、試薬、これらを含む器具など）またはその説明書は、例えば、Ｔｈ１７細胞の分化および活性化の抑制に用いられる旨、および／またはＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、または予防に用いられる旨の表示を付したものでありうる。ここで「製品または説明書に表示を付した」とは、製品の本体、容器、包装などに表示を付したこと、あるいは製品の情報を開示する説明書、添付文書、宣伝物、その他の印刷物などに表示を付したことを意味する。 The products of the compositions of the present invention (pharmaceutical compositions, foods and drinks, reagents, instruments containing them, etc.) or instructions thereof are used, for example, to suppress the differentiation and activation of Th17 cells, and / or Th17 cells. It may be labeled as being used for the treatment, amelioration, or prevention of diseases caused by the overreaction of. Here, "marked on a product or instruction manual" means that a label is attached to the main body, container, packaging, etc. of the product, or a manual, package insert, promotional material, or other printed matter that discloses product information. It means that the display is attached to.
　＜被検化合物の活性評価法＞
　本発明は、被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法を提供する。 <Activity evaluation method of test compound>
The present invention provides a method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
　－第一の態様－
　本発明の評価方法の第一の態様は、被検化合物とアデノシンＡ２Ａ受容体との結合を指標とする方法であり、（ａ）被検化合物をアデノシンＡ２Ａ受容体に接触させる工程、および（ｂ）被検化合物とアデノシンＡ２Ａ受容体との結合を検出する工程、を含む。本態様は、主として、後述の態様の評価のための一次的評価法として利用することができる。 -First aspect-
The first aspect of the evaluation method of the present invention is a method using the binding of the test compound to the adenosine A2A receptor as an index, (a) a step of contacting the test compound with the adenosine A2A receptor, and (b). ) Includes a step of detecting the binding of the test compound to the adenosine A2A receptor. This aspect can be mainly used as a primary evaluation method for the evaluation of the aspects described later.
　本発明の評価方法の対象とする「被検化合物」としては特に制限はなく、例えば、合成低分子化合物ライブラリー、遺伝子ライブラリーの発現産物、ペプチドライブラリー、ポリヌクレオチドライブラリー、抗体、細菌放出物質、細胞（微生物、植物細胞、動物細胞）の抽出液および培養上清、精製または部分精製ポリペプチド、海洋生物、植物、または動物から採取した抽出物などが挙げられる。被検化合物は、アデノシンＡ２Ａ受容体の立体構造を基にしたｉｎ　ｓｉｌｉｃｏでのデザインを基に合成したものであってもよい。 The "test compound" to be the subject of the evaluation method of the present invention is not particularly limited, and for example, a synthetic small molecule compound library, an expression product of a gene library, a peptide library, a polynucleotide library, an antibody, and bacterial release. Examples include extracts and culture supernatants of substances, cells (microorganisms, plant cells, animal cells), purified or partially purified polypeptides, extracts from marine organisms, plants, or animals. The test compound may be synthesized based on an in silico design based on the three-dimensional structure of the adenosine A2A receptor.
　本発明の評価方法において使用する「アデノシンＡ２Ａ受容体」は、ヒトタンパク質の場合は、例えば、配列番号：１に記載のアミノ酸配列からなるタンパク質（ＮＣＢＩ　Ｒｅｆｅｒｅｎｃｅ　Ｓｅｑｕｅｎｃｅ：　ＮＰ＿０００６６６．２）やその部分ペプチドが挙げられる。但し、アデノシンＡ２Ａ受容体のアミノ酸配列には、個体差が生じうることを理解されたい。 In the case of a human protein, the "adenosine A2A receptor" used in the evaluation method of the present invention includes, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Evaluation: NP_000666.2) or a partial peptide thereof. Can be mentioned. However, it should be understood that individual differences may occur in the amino acid sequence of the adenosine A2A receptor.
　アデノシンＡ２Ａ受容体は、前記天然型のタンパク質やその部分ペプチドに加え、必要に応じて、その改変体や修飾体などを用いることができる。例えば、検出や精製を容易にするために、他の蛋白質（例えば、アルカリフォスファターゼ（ＳＥＡＰ）、β－ガラクトシダーゼなどの酵素、緑色蛍光蛋白質（ＧＦＰ）などの蛍光蛋白質、グルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）などのタグ）との融合蛋白質を用いることができる。また、アデノシンＡ２Ａ受容体は、細胞表面に発現させた形態で用いることもできる。 As the adenosine A2A receptor, in addition to the natural protein and its partial peptide, a modified product or a modified product thereof can be used as needed. For example, to facilitate detection and purification, other proteins (eg, enzymes such as alkaline phosphatase (SEAP), β-galactosidase, fluorescent proteins such as green fluorescent protein (GFP), glutathione-S-transferase (GST)). A fusion protein with a tag such as) can be used. The adenosine A2A receptor can also be used in the form expressed on the cell surface.
　被検化合物とアデノシンＡ２Ａ受容体との「接触」は、当該評価系への被検化合物の添加などにより行うことができる。 "Contact" between the test compound and the adenosine A2A receptor can be performed by adding the test compound to the evaluation system or the like.
　「被検化合物とアデノシンＡ２Ａ受容体との結合の検出」には、公知の手法を適宜採用することができる。例えば、固定したアデノシンＡ２Ａ受容体に、被検化合物を接触させ、アデノシンＡ２Ａ受容体に結合する化合物を同定する方法が挙げられる。被検化合物とアデノシンＡ２Ａ受容体との結合を検出する手段としては、様々な公知の手段を利用することができるが、好適な手段の一例として、表面プラズモン共鳴現象を利用したバイオセンサーが挙げられる。 A known method can be appropriately adopted for "detection of binding between the test compound and the adenosine A2A receptor". For example, a method of contacting a fixed adenosine A2A receptor with a test compound to identify a compound that binds to the adenosine A2A receptor can be mentioned. As a means for detecting the binding between the test compound and the adenosine A2A receptor, various known means can be used, and one of the preferable means is a biosensor using the surface plasmon resonance phenomenon. ..
　被検化合物が合成低分子化合物ライブラリーである場合には、例えば、コンビナトリアルケミストリー技術によるハイスループット法（Ｗｒｉｇｈｔｏｎ　Ｎ．Ｃ．ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ．２６；２７３（５２７４）４５８－４６４（１９９６）、Ｖｅｒｄｉｎｅ，Ｇ．Ｌ．Ｎａｔｕｒｅ　３８４　１１－１３　（１９９６）、Ｈｏｇａｎ　Ｊ.Ｃ．，Ｊｒ　Ｄｉｒｅｃｔｅｄ　ｃｏｍｂｉｎａｔｏｒｉａｌ　ｃｈｅｍｉｓｔｒｙ．Ｎａｔｕｒｅ．３８４：１７－１９（１９９６））を利用することができる。 When the test compound is a synthetic low molecular weight compound library, for example, a high throughput method by combinatorial chemistry technology (Writton NC et al., Science. 26; 273 (5274) 458-464 (1996), Verdine, GL Nature 384 11-13 (1996), Hogan JC, Jr Directed Combinatorial Chemistry. Nature. 384: 17-19 (1996)) can be used.
　また、被検化合物がポリヌクレオチドライブラリーである場合には、例えば、インビトロセレクション法を利用することができる（ＳＥＬＥＸ法）（Ｔｕｅｒｋ　Ｃ．　＆　Ｇｏｌｄ　Ｌ．，Ｓｃｉｅｎｃｅ．３；２４９（４９６８）：５０５－５１０（１９９０）、Ｇｒｅｅｎ　Ｒ．ｅｔ　ａｌ．，Ｍｅｔｈｏｄｓ　Ｃｏｍｐａｎ　Ｍｅｔｈｏｄｓ　Ｅｎｚｙｍｏｌ．２：７５－８６（１９９１）、Ｇｏｌｄ　Ｌ．ｅｔ　ａｌ．，Ａｎｎｕ　Ｒｅｖ　Ｂｉｏｃｈｅｍ　６４：７６３－９７　（１９９５）、Ｕｐｈｏｆｆ　Ｋ．Ｗ．　ｅｔ　ａｌ．，Ｃｕｒｒ．Ｏｐｉｎ．Ｓｔｒｕｃｔ．Ｂｉｏｌ．６：２８１－２８８（１９９６））。 When the test compound is a polynucleotide library, for example, an in vitro selection method can be used (SELEX method) (Tuerk C. & Gold L., Science. 3; 249 (4966): 505). -510 (1990), Green R. et al., Methods Compan Methods Enzymol. 2: 75-86 (1991), Gold L. et al., Annu Rev Biochem 64: 763-97 (1995), Uphoff. Et al., Curr. Opin. Struct. Biol. 6: 281-288 (1996)).
　その他、被検化合物の種類などに応じて、ＦＲＥＴ（蛍光共鳴エネルギー移動）を利用した方法、免疫沈降法、酵母ツーハイブリッドシステムなどを利用することもできる。 In addition, depending on the type of test compound, a method using FRET (fluorescence resonance energy transfer), an immunoprecipitation method, a yeast two-hybrid system, etc. can also be used.
　以上の結果、被検化合物がアデノシンＡ２Ａ受容体と結合する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される。 As a result of the above, when the test compound binds to the adenosine A2A receptor, it is probable that it has an activity of suppressing the differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells. It is evaluated that there is.
　－第二の態様－
　本発明の評価方法の第二の態様は、被検化合物によるアデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合の阻害を指標とする方法であり、（ａ）被検化合物の存在下で、アデノシンＡ２Ａ受容体リガンドをアデノシンＡ２Ａ受容体に接触させる工程、および（ｂ）アデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を検出する工程、を含む。 -Second aspect-
The second aspect of the evaluation method of the present invention is a method using the inhibition of binding between the adenosine A2A receptor ligand and the adenosine A2A receptor by the test compound as an index, and (a) in the presence of the test compound. It comprises contacting the adenosine A2A receptor ligand with the adenosine A2A receptor and (b) detecting the binding of the adenosine A2A receptor ligand to the adenosine A2A receptor.
　「アデノシンＡ２Ａ受容体リガンド」としては、例えば、天然リガンドであるアデノシンを用いることができる。この評価方法においては、例えば、固定したアデノシンＡ２Ａ受容体や細胞表面に発現させたアデノシンＡ２Ａ受容体と、放射性物質や蛍光物質などの標識を行ったアデノシンとの結合を、被検化合物が阻害するか否かを、当該標識を指標に検出すればよい。ここでいう「阻害」には、完全な阻害および部分的な阻害の双方が含まれる。 As the "adenosine A2A receptor ligand", for example, adenosine, which is a natural ligand, can be used. In this evaluation method, for example, the test compound inhibits the binding between the fixed adenosine A2A receptor or the adenosine A2A receptor expressed on the cell surface and the adenosine labeled with a radioactive substance or a fluorescent substance. Whether or not it may be detected by using the sign as an index. The term "inhibition" as used herein includes both complete inhibition and partial inhibition.
　以上の結果、被検化合物がアデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を阻害する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される。 As a result of the above, when the test compound inhibits the binding between the adenosine A2A receptor ligand and the adenosine A2A receptor, the activity of suppressing the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated. It is evaluated as having a probability of having an activity of improving or preventing.
　－第三の態様－
　本発明の評価方法の第三の態様は、被検化合物によるアデノシンＡ２Ａ受容体の活性の抑制を指標とする方法であり、（ａ）被検化合物をアデノシンＡ２Ａ受容体を発現する細胞に接触させる工程、および（ｂ）アデノシンＡ２Ａ受容体の活性を検出する工程、
を含む。 -Third aspect-
The third aspect of the evaluation method of the present invention is a method using the suppression of the activity of the adenosine A2A receptor by the test compound as an index, and (a) contacting the test compound with cells expressing the adenosine A2A receptor. Steps, and (b) detecting the activity of the adenosine A2A receptor,
including.
　本発明の評価方法の指標となるアデノシンＡ２Ａ受容体の活性としては、刺激に応答したアデノシンＡ２Ａ受容体からのシグナル伝達において生じる様々な事象が挙げられる。このようなシグナル伝達としては、例えば、ｃＡＭＰの上昇、ＰＫＡの活性化などが挙げられるが、これらに制限されない。本発明の評価方法においては、例えば、アデノシンＡ２Ａ受容体リガンドであるアデノシンの刺激により、細胞表面上のアデノシンＡ２Ａ受容体を活性化させてる反応系に、被検化合物を適用して、当該活性が阻害されるかを評価すればよい。これら活性の測定方法は公知であり、通常の方法や測定キットを用いて測定すればよい。 Examples of the activity of the adenosine A2A receptor, which is an index of the evaluation method of the present invention, include various events that occur in signal transduction from the adenosine A2A receptor in response to a stimulus. Examples of such signal transduction include, but are not limited to, elevation of cAMP and activation of PKA. In the evaluation method of the present invention, for example, the test compound is applied to a reaction system in which the adenosine A2A receptor on the cell surface is activated by stimulation of adenosine, which is an adenosine A2A receptor ligand, to obtain the activity. You can evaluate whether it is inhibited. Methods for measuring these activities are known, and measurement may be performed using a usual method or a measurement kit.
　以上の結果、被検化合物を接触させない場合と比較して、被検化合物がアデノシンＡ２Ａ受容体の活性を低下させる場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性、またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される。 As a result of the above, when the test compound reduces the activity of the adenosine A2A receptor as compared with the case where the test compound is not contacted, the activity suppresses the differentiation or activation of Th17 cells, or the Th17 cell overreacts. It is evaluated as likely to have the activity of treating, ameliorating, or preventing the resulting disease.
　以上、本発明の評価方法について説明したが、複数の被検化合物に対して、本発明の評価方法を実施して、上記のアデノシンＡ２Ａ受容体への結合、アデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合の阻害、あるいはアデノシンＡ２Ａ受容体の活性の抑制を指標に化合物を選択すれば、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する化合物をスクリーニングすることができる。従って、本発明は、このようなスクリーニング方法をも提供する。 The evaluation method of the present invention has been described above, but the evaluation method of the present invention is carried out for a plurality of test compounds to bind to the above-mentioned adenosine A2A receptor, adenosine A2A receptor ligand and adenosine A2A receptor. If a compound is selected based on the inhibition of binding to the body or the suppression of the activity of the adenosine A2A receptor, the activity of suppressing the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells can be treated and improved. , Or compounds with prophylactic activity can be screened. Therefore, the present invention also provides such a screening method.
　本発明の評価方法やスクリーニング方法によって同定された化合物については、実際に、Ｔｈ１７細胞の分化もしくは活性化を抑制するか否か、Ｔｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有するか否かを検証することが好ましい。Ｔｈ１７細胞の分化や活性化の抑制は、例えば、本実施例に記載の通り、２ウェイＭＬＲなどを利用して、Ｔｈ１７サイトカインであるＩＬ－１７Ａなどの産生が抑制されるか否かにより評価することができる。また、Ｔｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性は、本実施例において、乾癬などを例に示したように、各種モデル動物による実験において評価することができる。さらに、臨床試験において、実際にヒトにおける効果を評価することができる。 With respect to the compound identified by the evaluation method and the screening method of the present invention, whether or not to actually suppress the differentiation or activation of Th17 cells, and to treat, ameliorate, or prevent the disease caused by the overreaction of Th17 cells. It is preferable to verify whether or not it has activity. Suppression of Th17 cell differentiation and activation is evaluated by, for example, as described in this example, whether or not the production of Th17 cytokine IL-17A or the like is suppressed by using 2-way MLR or the like. be able to. In addition, the activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells can be evaluated in experiments using various model animals as shown in this example, such as psoriasis. Furthermore, in clinical trials, the effect in humans can be actually evaluated.
　＜Ｔｈ１７細胞の製造方法等＞
　後述の実施例に示すとおり、アデノシンまたはアデノシンＡ２Ａ受容体アゴニストを、脾臓由来の単核球または活性化したＣＤ４＋Ｔ細胞の培養系に添加することによって、Ｔｈ１７サイトカインであるＩＬ－１７Ａの産生が誘導されることを明らかにした。 <Method of manufacturing Th17 cells, etc.>
As shown in Examples below, the production of the Th17 cytokine IL-17A is produced by adding adenosine or adenosine A2A receptor agonist to a culture system of spleen-derived monocytes or activated CD4 + T cells. It was revealed that it would be induced.
　従って、本発明は、Ｔ細胞を、アデノシンＡ２Ａ受容体アゴニストの存在下で培養する工程を含む、Ｔｈ１７細胞の製造方法を提供する。 Therefore, the present invention provides a method for producing Th17 cells, which comprises the step of culturing T cells in the presence of an adenosine A2A receptor agonist.
　本発明において製造される「Ｔｈ１７細胞」は、少なくともＩＬ－１７Ａを産生するヘルパーＴ細胞を意味する。本発明の方法において、Ｔｈ１７細胞に分化誘導または活性化される「Ｔ細胞」としては、ナイーブＴ細胞（ナイーブＣＤ４＋Ｔ細胞など）、エフェクターＴ細胞、記憶Ｔ細胞が挙げられる。Ｔ細胞が由来とする動物種は特に制限されないが、脊椎動物が好ましく、哺乳動物がより好ましい。哺乳動物としては、例えば、ヒトおよび非ヒト哺乳動物が挙げられる。非ヒト哺乳動物としては、例えば、サル、マーモセットなどの霊長類、マウス、ラットなどのげっ歯類、ウサギなどのウサギ目、ブタ、ヒツジ、ウシ、ヤギ、ウマなどの有蹄目、イヌ、ネコなどのネコ目が挙げられる。また、リンパ球が由来とする組織としては、該細胞が存在する限り特に制限はないが、例えば、脾臓、末梢血、リンパ節、骨髄、胸腺、臍帯血、肝臓が挙げられる。当業者であれば、これらの組織から公知の手法を用い、Ｔ細胞を単離することが出来る。公知の手法としては、例えば、ＣＤ４などの細胞表面マーカーに対する抗体を用いた、磁気ビーズによる分離、またはフローサイトメトリーが挙げられる。また、サイトカインの分泌や機能性分子の発現を指標に、所望のＴ細胞を単離することも出来る。 The "Th17 cell" produced in the present invention means a helper T cell that produces at least IL-17A. In the method of the present invention, examples of "T cells" that are induced or activated by Th17 cells include naive T cells (naive CD4 + T cells, etc.), effector T cells, and memory T cells. The animal species from which the T cells are derived is not particularly limited, but vertebrates are preferable, and mammals are more preferable. Mammals include, for example, human and non-human mammals. Non-human mammals include, for example, primates such as monkeys and marmosets, rodents such as mice and rats, lagomorphs such as rabbits, ungulates such as pigs, sheep, cows, goats and horses, dogs and cats. There are cat eyes such as. The tissue from which lymphocytes are derived is not particularly limited as long as the cells are present, and examples thereof include spleen, peripheral blood, lymph nodes, bone marrow, thymus, umbilical cord blood, and liver. Those skilled in the art can isolate T cells from these tissues using known techniques. Known techniques include, for example, separation with magnetic beads using an antibody against a cell surface marker such as CD4, or flow cytometry. It is also possible to isolate desired T cells using the secretion of cytokines and the expression of functional molecules as indicators.
　本発明の方法において、Ｔｈ１７細胞に分化誘導または活性化されるために用いられる「アデノシンＡ２Ａ受容体アゴニスト」は、アデノシンＡ２Ａ受容体を活性化する（作動活性を有する）ものであれば特に制限はなく、アデノシンＡ２Ａ受容体の天然のリガンドであるアデノシンであってもよい。また、アデノシンＡ２Ａ受容体を活性化する公知の薬剤であってもよい。公知の薬剤としては、例えば、ＰＳＢ０７７７、ＡＴＬ－１４６ｅ、ＹＴ－１４６（２－（１－オクチニル）アデノシン）、ＣＧＳ－２１６８０、ＤＰＭＡ（Ｎ６－（２－（３，５－ジメトキシフェニル）－２－（２－メチルフェニル）エチル）アデノシン）、レガデノソン、ＵＫ－４３２，０９７、リモネン、ゼアチンリボシド、ＮＥＣＡ（５’－（Ｎ－エチルカルボキサミド）アデノシン）、ビノデノソン、カンナビジオールが挙げられるが、これらに制限されない。これら公知の薬剤の構造式を順に以下に示す。 In the method of the present invention, the "adenosine A2A receptor agonist" used to induce or activate Th17 cells is not particularly limited as long as it activates the adenosine A2A receptor (has operative activity). Instead, it may be adenosine, which is a natural ligand for the adenosine A2A receptor. It may also be a known agent that activates the adenosine A2A receptor. Known agents include, for example, PSB0777, ATL-146e, YT-146 (2- (1-octynyl) adenosine), CGS-21680, DPMA (N6- (2- (3,5-dimethoxyphenyl) -2-). (2-Methylphenyl) ethyl) adenosine), regadenoson, UK-432,097, limonene, zeatinriboside, NECA (5'-(N-ethylcarboxamide) adenosine), binodenoson, cannavidiol, but not limited to these. .. The structural formulas of these known drugs are shown below in order.
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000025
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Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000031
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Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034
　さらに、「アデノシンＡ２Ａ受容体アゴニスト」としては、例えば、特開２００８－２８５４７８号公報、特表２０１０－５０３６３９号公報、特表２００９－５３４３４１号公報、特表２００８－５３４４４７号公報、特表２００４－５３４７６７号公報、または特表２００３－５０６４５９号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すアデノシン誘導体；例えば、特表２００９－５３４３３８号公報、特表２００９－５３４３４０号公報、特表２００９－５３４３３９号公報、特表２００９－５３４３３６号公報、特表２００９－５０１７４６号公報、特表２００８－５２６９１１号公報、特表２００５－５１３０４３号公報、または特表２００５－５００３５５号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すプリン誘導体；例えば、特表２００５－５００３５５号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すピリジン誘導体；特表２００５－５０６３５２号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すピリミジン誘導体；例えば、特表２００３－５０６４６０号公報、特表２００３－５０６４６１号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すピラゾール誘導体；例えば、特表２００３－５０２４３４号公報に開示されている、アデノシンＡ２Ａ受容体に対して作動活性を示すチオフェン化合物；例えば、特開２０１３－１８３６６０号公報に開示されている、抗アデノシンＡ２Ａ受容体抗体などが挙げられる。 Further, examples of the "adenosine A2A receptor agonist" include, for example, Japanese Patent Application Laid-Open No. 2008-285478, Japanese Patent Application Laid-Open No. 2010-503369, Japanese Patent Application Laid-Open No. 2009-534341, Japanese Patent Application Laid-Open No. 2008-534447, Japanese Patent Publication No. 2004- Adenosine derivatives exhibiting operative activity against adenosine A2A receptor disclosed in JP-A-534767 or JP-A-2003-506459; for example, JP-A-2009-534338, JP-A-2009-534340, Disclosure in Japanese Patent Publication No. 2009-534339, Japanese Patent Publication No. 2009-534336, Japanese Patent Publication No. 2009-501746, Japanese Patent Publication No. 2008-526911, Japanese Patent Application Laid-Open No. 2005-513403, or Japanese Patent Application Laid-Open No. 2005-550355 Purine derivatives exhibiting operative activity against adenosine A2A receptor; for example, pyridine derivatives exhibiting operative activity against adenosine A2A receptor disclosed in JP-A-2005-500355; A pyrimidine derivative exhibiting operative activity against adenosine A2A receptor disclosed in JP-506352; for example, adenosine A2A receptor disclosed in JP-A-2003-506460 and JP-A-2003-506461. Pyrazole derivatives exhibiting operative activity on the body; for example, thiophene compounds exhibiting operative activity on adenosine A2A receptor disclosed in JP-A-2003-502434; eg, JP2013-183660. The disclosed anti-adenosine A2A receptor antibody and the like can be mentioned.
　また、「アデノシンＡ２Ａ受容体アゴニスト」には、その作動活性を有する限り、薬理学上許容される塩、水和物または溶媒和物も含まれる。このような薬理学上許容される塩としては、特に制限はなく、アデノシンＡ２Ａ受容体アゴニストの各構造などに応じて適宜選択することができ、例えば、酸付加塩（例えば、塩酸塩、臭化水素塩、硫酸塩、硫酸水素塩、硝酸塩、炭酸塩、炭酸水素塩、リン酸塩、リン酸一水素塩、リン酸二水素塩などの無機酸塩、酢酸塩、プロピオン酸塩、乳酸塩、クエン酸塩、酒石酸塩などの有機酸塩）、塩基付加塩（例えば、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、亜鉛塩、アルミニウム塩、アンモニウム塩、テトラメチルアンモニウム塩）、有機アミン付加塩（例えば、モルホリン、ピペリジンなどの付加塩）、アミノ酸付加塩（例えば、リジン、グリシン、フェニルアラニンなどの付加塩）が挙げられる。また、水和物または溶媒和物としては、特に制限はなく、例えば、アデノシンＡ２Ａ受容体アゴニストまたはその塩１分子に対し、０．１～１０分子の水または溶媒が付加したものが挙げられる。 The "adenosine A2A receptor agonist" also includes a pharmacologically acceptable salt, hydrate or solvate as long as it has its operative activity. Such a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on each structure of the adenosine A2A receptor agonist and the like. For example, an acid addition salt (for example, hydrochloride, bromide) can be selected. Inorganic acid salts such as hydrogen salts, sulfates, hydrogen sulfates, nitrates, carbonates, hydrogen carbonates, phosphates, monohydrogen phosphates, dihydrogen phosphates, acetates, propionates, lactates, Organic acid salts such as citrate and tartrate), base addition salts (eg alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, zinc salt, aluminum salt, ammonium Salts, tetramethylammonium salts), organic amine addition salts (eg, morpholin, piperidine and other addition salts), amino acid addition salts (eg, lysine, glycine, phenylalanine and other addition salts). The hydrate or solvate is not particularly limited, and examples thereof include those obtained by adding 0.1 to 10 molecules of water or a solvent to one molecule of an adenosine A2A receptor agonist or a salt thereof.
　さらに、「アデノシンＡ２Ａ受容体アゴニスト」には、その作動活性を有する限り、互変異性体、幾何異性体、不斉炭素に基づく光学異性体、立体異性体などの総ての異性体および異性体混合物が含まれる。さらにまた、アデノシンＡ２Ａ受容体アゴニストが酸化、還元、加水分解、アミノ化、脱アミノ化、水酸化、リン酸化、脱水酸化、アルキル化、脱アルキル化、抱合などの代謝を受けてなおその作動活性を示す化合物をも包含し、また本発明は、酸化、還元、加水分解などの代謝を受けてアデノシンＡ２Ａ受容体アゴニストを生成する化合物をも包含する。 Furthermore, the "adenosine A2A receptor agonist" includes all isomers and isomers such as tautomers, geometric isomers, asymmetric carbon-based optical isomers, and stereoisomers as long as they have their operative activity. Contains a mixture. Furthermore, the adenosine A2A receptor agonist undergoes metabolism such as oxidation, reduction, hydrolysis, amination, deamination, hydroxylation, phosphorylation, dehydration, alkylation, dealkylation, and conjugation, and its operative activity is still active. The present invention also includes compounds that produce adenosine A2A receptor agonists by undergoing metabolism such as oxidation, reduction, and hydrolysis.
　なお、アデノシンＡ２Ａ受容体アゴニストに関しては、上述のとおり、多くの化合物が開発され市販もされているため、購入することによって入手することができる。また市販されていなくとも、それら化合物の製造方法についても多々報告がなされている。そのため、当業者であれば、当該製造方法に沿って適宜調製することもできる。 As for the adenosine A2A receptor agonist, as mentioned above, many compounds have been developed and are commercially available, so they can be obtained by purchasing. In addition, many reports have been made on methods for producing these compounds, even if they are not commercially available. Therefore, those skilled in the art can appropriately prepare according to the production method.
　本発明において、「アデノシンＡ２Ａ受容体アゴニストの存在下で培養」とは、通常、Ｔ細胞を培養する培地に、アデノシンＡ２Ａ受容体アゴニストを添加することによって行なわれる。アデノシンＡ２Ａ受容体アゴニストの培地への添加濃度としては、特に制限はなく、当業者であれば、用いるアゴニストの種類、活性の程度などに基づき、適宜調整しうるが、例えば、０．０１～１０００μＭである。Ｔ細胞を培養する「培地」としては、当該細胞を維持し、またＴｈ１７細胞への分化誘導、Ｔｈ１７細胞の活性化を抑制しない組成であれば、特に制限はないが、当業者であれば、基礎培地に、培地添加物を適宜含有させることにより、調製することができる。「基礎培地」としては、例えば、ＤＭＥＭ、αＭＥＭ、ハムＦ１２培地、ＤＭＥＭ／Ｆ１２培地、ＲＰＭＩ１６４０培地、ＩＭＥＭ、マッコイ５Ａ培地、ＥＭＥＭが挙げられる。「培地添加物」としては、例えば、血清（ウシ胎仔血清など）、有機化合物（ピルビン酸、乳酸、トリヨードチロニン、パラアミノ安息香酸、エタノールアミン、コルチコステロン、プロゲステロン、リポ酸、プトレシン、およびそれらの塩（例えば、ナトリウム塩など）、抗生物質（ペニシリン、ストレプトマイシン、ゲンタマイシン、アンホテリシンなど）、還元剤（２－メルカプトエタノール、カタラーゼ、スーパーオキシドジスムターゼ、Ｎ－アセチルシステインなど）、機能性蛋白質（インスリン、トランスフェリン、ラクトフェリンなど）、脂質（コレステロールなど）、アミノ酸（アラニン、Ｌ－グルタミン、非必須アミノ酸など）、ペプチド（グルタチオン、還元型グルタチオンなど）、ヌクレオチド（ヌクレオシド、シチジン、アデノシン５’－一リン酸、ヒポキサンチン、チミジンなど）、金属塩（硝酸鉄（III）、硫酸鉄（II）、硫酸銅、硫酸亜鉛等）、無機塩類（ナトリウム、カリウム、カルシウム、マグネシウム、リン、塩素など）、炭素源（グルコース、ガラクトース、フルクトース、スクロース等）、ビタミン、無機化合物（亜セレン酸など）、ｐＨ指示薬（フェノールレッドなど）、緩衝化合物（ＨＥＰＥＳ、重炭酸ナトリウムなど）が挙げられるが、これらに限定されるものではない。 In the present invention, "culturing in the presence of an adenosine A2A receptor agonist" is usually carried out by adding an adenosine A2A receptor agonist to a medium for culturing T cells. The concentration of the adenosine A2A receptor agonist added to the medium is not particularly limited and can be appropriately adjusted by those skilled in the art based on the type of agonist used, the degree of activity, etc., but for example, 0.01 to 1000 μM. Is. The "medium" for culturing T cells is not particularly limited as long as it has a composition that maintains the cells and does not suppress the induction of differentiation into Th17 cells and the activation of Th17 cells. It can be prepared by appropriately adding a medium additive to the basal medium. Examples of the "basic medium" include DMEM, αMEM, ham F12 medium, DMEM / F12 medium, RPMI1640 medium, IMEM, McCoy 5A medium, and EMEM. "Medium additives" include, for example, serum (such as bovine fetal serum), organic compounds (pyruvate, lactic acid, triiodotyronine, paraaminobenzoic acid, ethanolamine, corticosterone, progesterone, lipoic acid, putresin, and Their salts (eg, sodium salts, etc.), antibiotics (penicillin, streptomycin, gentamycin, amphotericin, etc.), reducing agents (2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine, etc.), functional proteins (insulin). , Transtransferase, lactoferrin, etc.), lipids (cholesterol, etc.), amino acids (alanine, L-glutamine, non-essential amino acids, etc.), peptides (glutathione, reduced glutathione, etc.), nucleotides (nucleoside, citidine, adenosine 5'-monophosphate, etc.) , Hipoxanthin, thymidin, etc.), metal salts (iron nitrate (III), iron sulfate (II), copper sulfate, zinc sulfate, etc.), inorganic salts (sodium, potassium, calcium, magnesium, phosphorus, chlorine, etc.), carbon sources (Glucose, galactose, fructose, sucrose, etc.), vitamins, inorganic compounds (selenic acid, etc.), pH indicators (phenol red, etc.), buffer compounds (HEPES, sodium bicarbonate, etc.), but are limited to these. It's not a thing.
　本発明の方法において、対象とするＴ細胞がナイーブＴ細胞である場合には、アデノシンＡ２Ａ受容体アゴニストの存在下での培養の前処理として、または当該培養において、刺激を与え活性化してもよい。かかる「刺激」としては、抗ＣＤ３抗体、抗ＣＤ２８抗体、同種異系抗原発現細胞、成熟樹状細胞、各種マイトゲン、およびＩＬ－２からなる群から選択される少なくとも１の物質による刺激が挙げられる。 In the method of the present invention, when the target T cell is a naive T cell, it may be stimulated and activated as a pretreatment for culturing in the presence of an adenosine A2A receptor agonist, or in the culture. .. Such "stimulation" includes stimulation with at least one substance selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, allogeneic antigen-expressing cells, mature dendritic cells, various mitogens, and IL-2. ..
　アデノシンＡ２Ａ受容体アゴニストの存在下でのＴ細胞の「培養期間」としては、上述のＴｈ１７サイトカインが産生するまでの期間であればよいが、通常１日～１月間、好ましくは３～１４日間、より好ましくは５～１０日間である。「培養環境」としては、特に制限はないが、培養の温度は、通常３５～３８℃、好ましくは３７℃の条件である。二酸化炭素の濃度は、通常１～１０％、好ましくは５～１０％である。また、培地のｐＨは通常ｐＨ６～８である。 The "culture period" of T cells in the presence of an adenosine A2A receptor agonist may be the period until the above-mentioned Th17 cytokine is produced, but is usually 1 day to 1 month, preferably 3 to 14 days. More preferably, it is 5 to 10 days. The "culture environment" is not particularly limited, but the culture temperature is usually 35 to 38 ° C, preferably 37 ° C. The concentration of carbon dioxide is usually 1-10%, preferably 5-10%. The pH of the medium is usually pH 6-8.
　そして、このようにして、分化誘導または活性化された細胞に、Ｔｈ１７細胞が含まれていることは、後述の実施例に示すとおり、ＥＬＩＳＡなどの免疫学的手法により、Ｔｈ１７サイトカインを検出することによって判断することができる。 The fact that Th17 cells are contained in the cells induced or activated in this way means that Th17 cytokines are detected by an immunological method such as ELISA, as shown in Examples described later. Can be judged by.
　また、本発明は、前記方法により製造される細胞、すなわち、アデノシンＡ２Ａ受容体アゴニストによってＴ細胞が分化誘導または活性化されてなる、Ｔｈ１７細胞を提供する。 The present invention also provides cells produced by the above method, that is, Th17 cells in which T cells are induced or activated by an adenosine A2A receptor agonist.
　さらにまた、アデノシンＡ２Ａ受容体アゴニストを有効成分として含む、Ｔｈ１７細胞に分化誘導または該細胞を活性化させるための組成物を提供する。かかる組成物を、試薬として用いる場合には、薬理学上許容される担体、具体的には、培地、ＰＢＳ、生理食塩水、滅菌水、植物油、動物油、溶剤、基剤、乳化剤、懸濁剤、界面活性剤、安定剤、ベヒクル、防腐剤、結合剤、希釈剤、等張化剤、緩衝剤、溶解補助剤あるいはその他の添加剤などを、適宜組み合わせて調製することができる。 Furthermore, a composition for inducing differentiation or activating Th17 cells, which comprises an adenosine A2A receptor agonist as an active ingredient, is provided. When such a composition is used as a reagent, it is a pharmacologically acceptable carrier, specifically, a medium, PBS, physiological saline, sterile water, vegetable oil, animal oil, solvent, base, emulsifier, suspension. , Surfactants, stabilizers, vehicles, preservatives, binders, diluents, tonicity agents, buffers, solubilizers or other additives can be prepared in appropriate combinations.
　以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.
　なお、各実施例において、２群間の差はＳｔｕｄｅｎｔのｔ検定を使用して分析したが、後述のＥＡＥマウスの運動症状の程度のスコアの比較に関してはＷｉｌｃｏｘｏｎの順位和検定を使用して分析した。３群以上の差は、一元配置分散分析（ｏｎｅ－ｗａｙ　ＡＮＯＶＡ，ｏｎｅ－ｗａｙ　ａｎａｌｙｓｉｓ　ｏｆ　ｖａｒｉａｎｃｅ）で、ｐ値（確率値，ｐ　ｖａｌｕｅ）が０．０５以下を有意差ありとし、ｏｎｅ－ｗａｙ　ＡＮＯＶＡで有意差ありと判定された場合は、さらにＴｕｒｋｅｙ検定（Ｔｕｒｋｅｙ　ｈｏｎｅｓｔｌｙ　ｓｉｇｎｉｆｉｃａｎｔ　ｄｉｆｆｅｒｅｎｃｅ）を行って２群間の有意差を分析した。各コントロールと比較して、Ｐ値が０．０５以下の群には＊印、０．０１以下の群には＊＊印を付加した。分析にはＫａｌｅｉｄａＧｒａｐｈソフトウェアを使用した。Ｐ値＜０．０５を統計的に有意であると見做した。 In each example, the difference between the two groups was analyzed using the Student's t-test, but the Wilcoxon rank sum test was used to analyze the comparison of the scores of the degree of motor symptoms of EAE mice, which will be described later. did. The difference between the three groups or more is one-way ANOVA, one-way analysis of variance, where the p-value (probability value, p-value) is 0.05 or less, and there is a significant difference. When it was determined that there was a significant difference in the above, a Turkey test (Turkey honestly significant difference) was further performed to analyze the significant difference between the two groups. Compared with each control, a * mark was added to the group having a P value of 0.05 or less, and a ** mark was added to the group having a P value of 0.01 or less. KaleidaGraph software was used for the analysis. A P-value <0.05 was considered statistically significant.
　［実施例１］２ウェイＭＬＲにおけるアデノシンの効果
　Ｈ－２の異なるＢＡＬＢ／ｃとＳＪＬ／Ｊマウスの脾細胞から単核球を分離した。具体的には、８－１０週齢のメスのＨ－２の異なるＢＡＬＢ／ｃとＳＪＬ／Ｊマウスから脾臓を取り出し、その脾臓をホモジナイザー（ＧＰＥ　Ｓｃｉｅｎｔｉｆｉｃ，Ｔｅｎｂｒｏｅｃｋ　Ｓｔｙｌｅ　Ｈｏｍｏｇｅｎｉｚｅｒ，Ｐｅｓｔｌｅ　ｄｉａｍｅｔｅｒ　１５ｍｍ）に加えて、５ｍＬのＤＭＥＭを加えた後、５回転させてホモジナイズして、さらに５ｍＬのＤＭＥＭを加えて、総量１０ｍＬの脾臓細胞を５０ｍＬチューブに移した。７ｍＬのＦｉｃｏｌｌ（Ｆｉｃｏｌｌ－Ｐａｑｕｅ　ｐｒｅｍｉｕｍ　１．０８４，ＧＥ　ｈｅａｌｔｈｃａｒｅ）を脾臓細胞溶液の入った５０ｍＬチューブの下端にピペットエイドを用いて、脾臓細胞溶液とＦｉｃｏｌｌの界面を乱さない様に静かに加え、室温で３０分１，５００ｒ．ｐ．ｍ．（４３０ｘｇ）で遠心した。遠心後、５０ｍＬチューブの下端から１ｃｍ離れた上部から、５０ｍＬチューブの中央付近のＦｉｃｏｌとＤＭＥＭの境界付近に白いバンドとして出現するＰｅｒｉｐｈｅｒａｌ　ｂｌｏｏｄ　ｍｏｎｏｎｕｃｌｅａｒ　ｃｅｌｌｓ（ＰＢＭＣｓ）の層までをパスツールピペットで回収し、その回収画分を新しい５０ｍＬチューブに移した。この回収画分に、合計４０ｍＬになるようにＤＭＥＭを加えて懸濁し、再び、室温で１０分１，５００ｒ．ｐ．ｍ．（４３０ｘｇ）で遠心した。遠心後、下端から２ｃｍを残して上清をアスピレーターで吸引し、懸濁して１５ｍＬチューブに移した。合計１２ｍＬになるようにＤＭＥＭを加え、室温で１０分１，５００ｒ．ｐ．ｍ．（４３０ｘｇ）で遠心した。遠心後、細胞ペレットを２ｍＬのＤ１０培地（ＤＭＥＭに、最終濃度が１０％　ＦＣＳ、０．１ｍｇ／ｍＬ　ストレプトマイシン、１００Ｕ／ｍＬ　ペニシリン、１ｍＭ　ピルビン酸ナトリウム、５０μＭ　２－メルカプトエタノールを加えた培地）で懸濁し、チュルク溶液に調製した細胞の一部を加えて、ビュルケルチュルク血球計算版を用いて単核球の細胞数を算出した。算出した後、調製した単核球にＤ１０培地を加えて、各３×１０６細胞／ｍｌに調整した。そして、ＭＬＲ反応を誘導するために、調製したＢＡＬＢ／ｃ由来の単核球、および、ＳＪＬ／Ｊ由来の単核球を１ｍｌずつ１２ウェルプレートで混合した（全体：６×１０６細胞／２ｍｌ）。 [Example 1] Effect of adenosine on 2-way MLR Mononuclear cells were isolated from the splenocytes of BALB / c and SJL / J mice having different H-2. Specifically, spleens are taken from 8-10 week-old female H-2 different BALB / c and SJL / J mice, and the spleens are added to homogenizers (GPE Scientific, Tenbroeck Style Homogenizer, Pestle diameter 15 mm). After adding 5 mL of DMEM, the cells were homogenized by rotating 5 times, and 5 mL of DMEM was further added to transfer a total volume of 10 mL of spleen cells to a 50 mL tube. 7 mL of Ficoll (Ficoll-Paque premium 1.084, GE healthcare) was gently added to the lower end of a 50 mL tube containing the spleen cell solution using a pipette aid so as not to disturb the interface between the spleen cell solution and Ficoll, and at room temperature. 30 minutes at 1,500 r. p. m. Centrifugal at (430 xg). After centrifugation, collect from the upper part 1 cm away from the lower end of the 50 mL tube to the layer of Peripheral blood mononuclear cells (PBMCs) appearing as a white band near the boundary between Ficoll and DMEM near the center of the 50 mL tube with a pass tool pipette. The recovered fraction was transferred to a new 50 mL tube. DMEM was added to this recovered fraction to make a total of 40 mL, suspended, and again at room temperature for 10 minutes at 1,500 r. p. m. Centrifugal at (430 xg). After centrifugation, the supernatant was aspirated with an ejector leaving 2 cm from the lower end, suspended and transferred to a 15 mL tube. DMEM was added to make a total of 12 mL, and 1,500 r. p. m. Centrifugal at (430 xg). After centrifugation, the cell pellet is suspended in 2 mL of D10 medium (DMEM supplemented with 10% FCS, 0.1 mg / mL streptomycin, 100 U / mL penicillin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol). A part of the prepared cells was added to the turbid and Turku solution, and the number of mononuclear cells was calculated using the Bürkerturk hemocytometer. After calculating, by adding D10 medium mononuclear cells prepared and adjusted to the 3 × 10 6 cells / ml. Then, in order to induce the MLR reaction, prepared BALB / c-derived mononuclear cells and SJL / J-derived mononuclear cells were mixed in 12-well plates (1 ml each) (total: 6 × 10 6 cells / 2 ml). ).
　次いで、０－１０００μＭに調整したアデノシンを共培養し、培養開始から７日後の上清のＩＦＮγ（Ｔｈ１サイトカイン）、ＩＬ－５（Ｔｈ２サイトカイン）、ＩＬ－１７Ａ（Ｔｈ１７サイトカイン）の濃度をＥＬＩＳＡ法にて定量した。なお、ＥＬＩＳＡ法は、Ｄｕｏ　Ｓｅｔ　ｋｉｔ（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ）を用い、その添付のマニュアルに従って行なった。 Next, adenosine adjusted to 0-1000 μM was co-cultured, and the concentrations of IFNγ (Th1 cytokine), IL-5 (Th2 cytokine), and IL-17A (Th17 cytokine) in the supernatant 7 days after the start of the culture were subjected to the ELISA method. Quantified. The ELISA method was performed using the Duo Set kit (R & D Systems) according to the attached manual.
　その結果、ＩＬ－１７Ａ産生の濃度依存性の有意な上昇、ならびに、ＩＬ－５産生およびＩＦＮγ産生の濃度依存性の有意な低下が認められた（図１）。 As a result, a significant increase in the concentration dependence of IL-17A production and a significant decrease in the concentration dependence of IL-5 production and IFNγ production were observed (Fig. 1).
　［実施例２］２ウェイＭＬＲにおけるイストラデフィリンの効果
　実施例１に記載の方法と同様にして、Ｈ－２の異なるＢＡＬＢ／ｃとＳＪＬ／Ｊマウスの脾細胞から単核球を分離し、各３×１０６細胞／ｍｌに調整した。それぞれの細胞を１ｍｌずつ１２ウェルプレートで混合した（全体：６×１０６細胞／２ｍｌ）。 [Example 2] Effect of isstradefylline on 2-way MLR Mononuclear cells were isolated from the splenocytes of BALB / c and SJL / J mice having different H-2 in the same manner as in Example 1. It was adjusted to 3 × 10 6 cells / ml each. Each cell was mixed with 12-well plate at 1 ml (total: 6 × 10 6 cells / 2 ml).
　そして、アデノシン（１００μＭ）共存下で、０．０１－１μＭに調整したアデノシンＡ２Ａ受容体アンタゴニスト（商品名：ノウリアスト、一般名：イストラデフィリン）を共培養し、培養開始から７日後の上清のＩＬ－１７Ａ濃度をＥＬＩＳＡ法（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ　Ｄｕｏ　Ｓｅｔ　ｋｉｔ使用）にて定量した。 Then, in the coexistence of adenosine (100 μM), an adenosine A2A receptor antagonist (trade name: Nouriast, generic name: Istradefylline) adjusted to 0.01-1 μM was co-cultured, and the supernatant 7 days after the start of the culture was used. The IL-17A concentration was quantified by the ELISA method (using R & D Systems Duo Set kit).
　その結果、イストラデフィリンによる濃度依存性の有意なＩＬ－１７Ａ産生抑制が認められた（図２）。すなわち、２ウェイＭＬＲにおける、アデノシンによるＩＬ－１７Ａ産生の誘導は、濃度依存的にアデノシンＡ２Ａ受容体アンタゴニストであるイストラデフィリンにより、抑制されることが示された。 As a result, concentration-dependent significant suppression of IL-17A production by istradefylline was observed (Fig. 2). That is, it was shown that the induction of IL-17A production by adenosine in 2-way MLR was suppressed by the adenosine A2A receptor antagonist istradefylline in a concentration-dependent manner.
　［実施例３］ＣＤ４＋Ｔ細胞におけるアデノシンおよびイストラデフィリンの効果
　脾臓細胞を調製するために、８－１０週齢のＢＡＬＢ／ｃマウスから脾臓を摘出し、５ｍＬのＤＭＥＭの入った６ｃｍディッシュに置いた。ピンセットを用いて脾臓を培地内で、良く揉みほぐし、培地に溶出されたリンパ球を含む溶液を１５ｍＬチューブに移した。もう一度６ｃｍディッシュを５ｍＬのＤＭＥＭで洗浄した。上清をその１５ｍＬチューブに加え、総量１０ｍＬとした。すぐに１５ｍＬチューブの底にたまる組織断片を残して、上清を再び新しい１５ｍＬチューブに移し、室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離し、リンパ球を含むペレットを得た。上清を除いて、ペレットをほぐした。その後、赤血球を除くために、２５０μｌのＮＨ４Ｃｌ－トリス溶液を加えかき混ぜた。その後、すばやく１０ｍＬのＤＭＥＭを加え、室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離し、リンパ球を含むペレットを得た。上清を除いて、ペレットをほぐした後、再び１０ｍＬのＤＭＥＭを加えた。変性した赤血球を極力吸わないようにしてピペットでリンパ球を含む培地を新しい１５ｍＬチューブに移して、再び室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離した。上清を除いた後、ペレットをほぐした。もう一度１０ｍＬのＤＭＥＭで懸濁し、室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離した。上清を除いて、最後に２ｍＬのＤ１０培地（ＤＭＥＭに、最終濃度が１０％　ＦＣＳ、０．１ｍｇ／ｍＬ　ストレプトマイシン、１００Ｕ／ｍＬ　ペニシリン、１ｍＭ　ピルビン酸ナトリウム、５０μＭ　２－メルカプトエタノールを加えた培地）で懸濁した。その後、マニュアルに従って磁気ビーズ（ｍｏｕｓｅ　ＣＤ４　（Ｌ３Ｔ４）　ＭｉｃｒｏＢｅａｄｓ（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ，１３０－０４９－２０１））を用いたポジティブセレクションで、ＣＤ４＋Ｔ細胞を調製した。 [Example 3] Effect of adenosine and isstradefylline on CD4 + T cells To prepare spleen cells, the spleen was removed from 8-10 week-old BALB / c mice and placed in a 6 cm dish containing 5 mL of DMEM. placed. The spleen was thoroughly kneaded in the medium using tweezers, and the solution containing the lymphocytes eluted in the medium was transferred to a 15 mL tube. The 6 cm dish was washed again with 5 mL DMEM. The supernatant was added to the 15 mL tube to give a total volume of 10 mL. Immediately transfer the supernatant to a new 15 mL tube again, leaving tissue fragments that accumulate at the bottom of the 15 mL tube, at room temperature, 1,200 r. p. m. Centrifugation at (270 xg) for 5 minutes gave pellets containing lymphocytes. The supernatant was removed and the pellet was loosened. Then, 250 μl of NH 4 Cl-Tris solution was added and stirred to remove erythrocytes. Then, 10 mL of DMEM was quickly added to room temperature at 1,200 r. p. m. Centrifugation at (270 xg) for 5 minutes gave pellets containing lymphocytes. After removing the supernatant and loosening the pellets, 10 mL of DMEM was added again. Transfer the medium containing lymphocytes to a new 15 mL tube with a pipette so as not to inhale the denatured red blood cells as much as possible, and again at room temperature, 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. After removing the supernatant, the pellet was loosened. Suspend again in 10 mL DMEM, room temperature, 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. Finally, 2 mL of D10 medium (medium containing 10% FCS, 0.1 mg / mL streptomycin, 100 U / mL penicillin, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol in DMEM) after removing the supernatant. Suspended in. Then, according to the manual, CD4 + T cells were prepared by positive selection using magnetic beads (mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec, 130-049-201)).
　調製したマウスＣＤ４＋Ｔ細胞に、最終濃度が１００～１０００μＭになるようにアデノシンを加え、最終濃度が、１μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ３εアゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：１４５－２Ｃ１１）、および、０．５μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ２８アゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：３７．５１）を加えて、１×１０６細胞／５００μＬで２４穴プレートに播種して培養し、培養開始から７日後の細胞上清を回収した。回収した細胞上清に含まれるＩＬ－１７Ａの濃度をマニュアルに従ってＥＬＩＳＡ法で定量した（Ｄｕｏ　ｓｅｔ　ｋｉｔ，Ｒ＆Ｄ　ｓｙｓｔｅｍｓ）。 Adenosine was added to the prepared mouse CD4 + T cells so as to have a final concentration of 100 to 1000 μM, and an anti-mouse CD3ε agonist antibody (BioLegend, Clone: 145-2C11) having a final concentration of 1 μg / mL, and 0. 5 μg / mL of anti-mouse CD28 agonist antibody (BioLegend, Clone: 37.51) was added, and the cells were seeded and cultured on a 24-well plate at 1 × 10 6 cells / 500 μL, and the cell supernatant 7 days after the start of culture was obtained. Recovered. The concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
　その結果、ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞は、アデノシンの濃度依存的に、ＩＬ－１７Ａ産生が誘導されることが示唆された（図３　左側のグラフ）。 As a result, it was suggested that CD4 + T cells activated by receiving CD3 / CD28 stimulation induce IL-17A production in adenosine concentration-dependent manner (graph on the left side of FIG. 3).
　また、上記のアデノシン単独に代えて、調製したマウスＣＤ４＋Ｔ細胞に、最終濃度が０．０１から１０μＭになるようにアデノシンＡ２Ａ受容体アゴニストであるＰＳＢ０７７７アンモニウム塩（ＴＯＣＲＩＳ）を加え、最終濃度が、１μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ３εアゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：１４５－２Ｃ１１）、および、０．５μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ２８アゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：３７．５１）を加えて、１×１０６細胞／５００μＬで２４穴プレートに播種して培養し、培養開始から７日後の細胞上清を回収した。回収した細胞上清に含まれるＩＬ－１７Ａの濃度をマニュアルに従ってＥＬＩＳＡ法で定量した（Ｄｕｏ　ｓｅｔ　ｋｉｔ，Ｒ＆Ｄ　ｓｙｓｔｅｍｓ）。その結果、ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞は、ＰＳＢ０７７７の濃度依存的に、ＩＬ－１７Ａ産生が誘導されることが示唆された（図３　真ん中のグラフ）。 Further, instead of the above-mentioned adenosine alone, PSB0777 ammonium salt (TOCRIS), which is an adenosine A2A receptor agonist, was added to the prepared mouse CD4 + T cells so that the final concentration was 0.01 to 10 μM, and the final concentration was adjusted. Add 1 μg / mL anti-mouse CD3ε agonist antibody (BioLegend, Clone: 145-2C11) and 0.5 μg / mL anti-mouse CD28 agonist antibody (BioLegend, Clone: 37.51) to 1 × 10 6 The cells were seeded on a 24-well plate at 500 μL and cultured, and the cell supernatant 7 days after the start of the culture was collected. The concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems). As a result, it was suggested that the CD4 + T cells activated by the stimulation of CD3 / CD28 induce IL-17A production in a concentration-dependent manner of PSB0777 (the graph in the middle of FIG. 3).
　さらに、調製したマウスＣＤ４＋Ｔ細胞に、アデノシン単独に代えて、アデノシン（最終濃度、６００μＭ）と共に、最終濃度が０．０１～１ｎＭになるようにアデノシンＡ２Ａ受容体アンタゴニストであるイストラデフィリンを加え、最終濃度が、１μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ３εアゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：１４５－２Ｃ１１）、および、０．５μｇ／ｍＬの抗ｍｏｕｓｅ　ＣＤ２８アゴニスト抗体（ＢｉｏＬｅｇｅｎｄ，Ｃｌｏｎｅ：３７．５１）を加えて、１×１０６細胞／５００μＬで２４穴プレートに播種して培養し、培養開始から７日後の細胞上清を回収した。回収した細胞上清に含まれるＩＬ－１７Ａの濃度をマニュアルに従ってＥＬＩＳＡ法で定量した（Ｄｕｏ　ｓｅｔ　ｋｉｔ，Ｒ＆Ｄ　ｓｙｓｔｅｍｓ）。 Furthermore, instead of adenosine alone, adenosine A2A receptor antagonist istradefylin was added to the prepared mouse CD4 + T cells together with adenosine (final concentration, 600 μM) so that the final concentration was 0.01 to 1 nM. , An anti-mouse CD3ε agonist antibody (BioLegend, Clone: 145-2C11) having a final concentration of 1 μg / mL and an anti-mouse CD28 agonist antibody (BioLegend, Clone: 37.51) having a final concentration of 0.5 μg / mL were added. The cells were seeded and cultured on a 24-well plate at 1 × 10 6 cells / 500 μL, and the cell supernatant was collected 7 days after the start of the culture. The concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
　その結果、ＣＤ３／ＣＤ２８刺激を受けて活性化したＣＤ４＋Ｔ細胞はアデノシンの添加によって、ＩＬ－１７Ａの産生が誘導されるが、その誘導は、アデノシンＡ２Ａ受容体アンタゴニストであるイストラデフィリンによって濃度依存的に抑制されることが示された（図３　右側のグラフ）。 As a result, the production of IL-17A was induced in the CD4 + T cells activated by the stimulation of CD3 / CD28 by the addition of adenosine, and the induction was concentrated by the adenosine A2A receptor antagonist istradefylline. It was shown to be suppressed in a dependent manner (graph on the right side of FIG. 3).
　［実施例４］乾癬モデルマウスにおけるイストラデフィリンの効果
　Ｃ５７ＢＬ／６マウスに「イミキモド（ＩＭＱ）クリーム単独（１２．５ｍｇ）」と「ＩＭＱにイストラデフィリン（２０ｍｇ）を混合したクリーム」の２種類を耳介に塗布してその肥厚（ｍｍ）を観察した。その結果、開始から８日目においてＩＭＱクリーム単独を塗布した群と比較して、イストラデフィリンを含んだ群で有意な耳介肥厚抑制が観察された（図４）。 [Example 4] Effect of istradefylline on psoriasis model mice Two types of C57BL / 6 mice, "imiquimod (IMQ) cream alone (12.5 mg)" and "IMQ mixed with istradefylline (20 mg)" Was applied to the auricle and its thickening (mm) was observed. As a result, a significant suppression of auricular thickening was observed in the group containing isstradefylline as compared with the group in which IMQ cream alone was applied on the 8th day from the start (Fig. 4).
　［実施例５］好中球性気道炎症モデルマウスにおけるイストラデフィリンの効果
　図５Ａに示すプロトコールに沿って、好中球性気道炎症モデルを作製し、その肺リンパ球のＩＬ－１７Ａ産生に対する、イストラデフィリンの抑制効果を、以下のようにして評価した。 [Example 5] Effect of Istradefylin on neutrophil airway inflammation model mouse A neutrophil airway inflammation model was prepared according to the protocol shown in FIG. 5A, and the neutrophil airway inflammation model was prepared for IL-17A production of pulmonary lymphocytes. The inhibitory effect of istradefylline was evaluated as follows.
　先ず、好中球性気道炎症モデルを作製するため、ニワトリのオボアルブミン（ＯＶＡ）特異的ＴＣＲを発現するトランスジェニックマウスであるＤＯ１１．１０マウスに、後述の肺リンパ球採取より１０日前から（Ｄａｙ－１０，－８，－６，－４，－２，および，－１）において、１００μＬの超純水（Ｍｉｌｌｉ－Ｑ水）、または、１００μＬの超純水に６μｇのイストラデフィリンを溶解したものを胃ゾンデで経口投与した。Ｄａｙ－８において、ＯＶＡに対する獲得免疫を誘導する目的で、ＯＶＡタンパク質（Ｓｉｇｍａ）をＰＢＳ　ｗｉｔｈｏｕｔ　ｍａｇｎｅｓｉｕｍ　ａｎｄ　ｃａｌｃｉｕｍ（ＰＢＳ（－））に溶解した２ｍｇ／ｍＬ　ＯＶＡ　ｉｎ　ＰＢＳ（－）を完全フロイントアジュバント（ＣＦＡ）（ＤＩＦＣＯ，ａｄｊｕｖａｎｔ　ｃｏｍｐｌｅｔｅ　ｆｒｅｕｎｄ，Ｈ３７　Ｒａ）を体積として等量混和し（例えば、５００μＬのＯＶＡ溶液と５００μＬのＣＦＡを混和させた）、２つの２ｍＬのシリンジ（Ｉｎｊｅｃｔ，Ｂ．Ｂｒａｕｎ　Ｍｅｄｉｃａｌ）をシリンジコネクター（ＧＰ　Ｓｙｒｉｎｇｅ　Ｃｏｎｎｅｃｔａ，Ｎｉｐｒｏ）で繋ぎ、ｔｗｏ－ｓｙｒｉｎｇｅ法で乳化させた。乳化した１００μＬ（ＯＶＡとして１００μｇ）のＯＶＡ溶解ＣＦＡをＤＯ１１．１０マウスの鼠径部に皮下注射した。Ｄａｙ－１（皮下注射７日後）に、ＯＶＡに対する獲得免疫を再活性化させる目的で、０．５ｇ　ＯＶＡを溶解した１０ｍＬ　ＰＢＳ（－）をネブライザーで霧化し、ＤＯ１１．１０マウスに２０分間吸入させた。 First, in order to prepare a model of neutrophilic airway inflammation, DO11.10 mice, which are transgenic mice expressing chicken ovoalbumin (OVA) -specific TCR, were subjected to 10 days before pulmonary lymphocyte collection described later (Day). In -10, -8, -6, -4, -2, and -1), 6 μg of istradefylline was dissolved in 100 μL of ultrapure water (Milli-Q water) or 100 μL of ultrapure water. The one was orally administered with a gastric sonde. In Day-8, for the purpose of inducing acquired immunity to OVA, 2 mg / mL OVA in PBS (-) in which OVA protein (Sigma) was dissolved in PBS with out syringe and syringe (PBS (-)) was completely Freund's adjuvant (CFA). ) (DIFCO, adjuvant complete fluid, H37 Ra) is mixed in equal amounts by volume (for example, 500 μL of OVA solution and 500 μL of CFA are mixed), and two 2 mL syringes (Inject, B. Brown Medical) are syringed. It was connected with a connector (GP Syringe Connecta, Nipro) and emulsified by the two-syringe method. Emulsified 100 μL (100 μg as OVA) of OVA-dissolved CFA was subcutaneously injected into the inguinal region of DO11.10 mice. On Day-1 (7 days after subcutaneous injection), for the purpose of reactivating acquired immunity to OVA, 10 mL PBS (-) in which 0.5 g OVA was dissolved was atomized with a nebulizer and inhaled by DO11.10 mice for 20 minutes. It was.
　そして、Ｄａｙ０において、このようにして調製した好中球性気道炎症モデルマウスを頚椎脱臼した後、肺リンパ球を採取した。具体的には、マウスの肺を、６ｃｍディッシュに移し、はさみを用いて細かくし、Ｄ１０培地（ＤＭＥＭに、最終濃度が各々、１０％　ＦＣＳ、０．１ｍｇ／ｍＬ　ストレプトマイシン、１００Ｕ／ｍＬ　ペニシリン、１ｍＭ　ピルビン酸ナトリウム、５０μＭ　２－メルカプトエタノールを加えた培地）に、最終濃度が各々、５０μｇ／ｍＬ　ゲンタマイシン、１μｇ／ｍＬ　アンホテリシン、０．０５Ｕ／ｍＬ　Ｃｌｏｓｔｒｉｄｉｕｍ　ｈｉｓｔｏｌｙｔｉｃｕｍ由来コラゲナーゼ（ＳＩＧＭＡ）を加えた培地、１０ｍＬ加え、３７℃で１時間インキュベートした。インキュベートした後、肺組織断片を含む培地を、５０ｍＬチューブを接続したセルストレーナー（ＦＡＬＣＯＮ、４０μｍ　ｍｅｓｈ）で濾過し、セルストレーナーに残存している肺組織断片を、セルスクレイパー（ＩＷＡＫＩ）を用いてセルストレーナー上で擦り潰し、肺組織を擦り潰したセルストレーナーに１０ｍＬのＤＭＥＭを通過させて共洗いした。共洗いした後、５０ｍＬチューブに回収した肺細胞を含む総量２０ｍＬの溶液を、室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離した。そして、上清を除いた後、ペレットをほぐし、１０ｍＬのＤＭＥＭで懸濁し、室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離した。上清を除いた後、ペレットをほぐし、１０ｍＬのＤＭＥＭで懸濁して、新しい１５ｍＬチューブに移し、再び室温、１，２００ｒ．ｐ．ｍ．（２７０ｘｇ）で５分間遠心分離した。上清を除いて、最後にＤ１０培地に、最終濃度が各々、５０μｇ／ｍＬゲンタマイシン、１μｇ／ｍＬ　アンホテリシンを加えた培地（Ｄ１０＋ゲンタマイシン＋アンホテリシン培地）、５００μＬで懸濁し、チュルク溶液に調製した細胞の一部を加えて、ビュルケルチュルク血球計算版を用いて単核球の細胞数を算出した。 Then, on Day 0, the neutrophil airway inflammation model mouse thus prepared was dislocated from the cervical spine, and then pulmonary lymphocytes were collected. Specifically, the lungs of the mouse were transferred to a 6 cm dish, finely divided using scissors, and D10 medium (DMEM, the final concentration was 10% FCS, 0.1 mg / mL streptamicin, 100 U / mL penicillin, 1 mM, respectively. Medium with sodium pyruvate and 50 μM 2-mercaptoethanol), and 10 mL of medium with final concentrations of 50 μg / mL gentamicin, 1 μg / mL amphotelicin, and 0.05 U / mL Crystaldium histolyticum-derived collagenase (SIGMA), respectively. , 37 ° C. for 1 hour. After incubation, the medium containing the lung tissue fragment is filtered with a cell strainer (FALCON, 40 μm mesh) connected to a 50 mL tube, and the lung tissue fragment remaining in the cell strainer is celled using a cell scraper (IWAKI). It was mashed on a strainer, and 10 mL of DMEM was passed through a cell strainer with mashed lung tissue for co-washing. After co-washing, a total volume of 20 mL of the solution containing the collected lung cells in a 50 mL tube was added to room temperature at 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. Then, after removing the supernatant, the pellets were loosened and suspended in 10 mL DMEM at room temperature of 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. After removing the supernatant, the pellets are loosened, suspended in 10 mL DMEM, transferred to a new 15 mL tube, and again at room temperature, 1,200 r. p. m. Centrifuge at (270 xg) for 5 minutes. After removing the supernatant, the cells were finally suspended in D10 medium at a final concentration of 50 μg / mL gentamicin and 1 μg / mL amphotericin (D10 + gentamicin + amphotericin medium) at 500 μL, and prepared in a Turku solution. With some additions, the number of mononuclear cells was calculated using the Bürkertürk blood cell count.
　算出した後、調製した単核球にＤ１０＋ゲンタマイシン＋アンホテリシン培地を加えて、１×１０６細胞／２００μＬずつ丸底９６穴プレートに播種した。Ｄａｙ　１，２，３，４，５，６，および，７（培養開始から１～７日後）において細胞上清を回収した。そして、回収した細胞上清に含まれるＩＬ－１７Ａの濃度をマニュアルに従ってＥＬＩＳＡ法で定量した（Ｄｕｏ　ｓｅｔ　ｋｉｔ，Ｒ＆Ｄ　ｓｙｓｔｅｍｓ）。 After calculation, D10 + gentamicin + amphotericin medium was added to the prepared mononuclear cells, and 1 × 10 6 cells / 200 μL were seeded on a round-bottomed 96-well plate. Cell supernatants were collected on Days 1, 2, 3, 4, 5, 6, and 7 (1-7 days after the start of culture). Then, the concentration of IL-17A contained in the collected cell supernatant was quantified by the ELISA method according to the manual (Duo set kit, R & D systems).
　その結果、イストラデフィリン投与群では、超純水を投与したコントロール群と比較して、ＩＬ－１７Ａ産生が抑制された。このことからイストラデフィリンの投与によって、好中球性気道炎症が抑制されることが示唆された（図５Ｂ）。 As a result, IL-17A production was suppressed in the isstradefylline-administered group as compared with the control group in which ultrapure water was administered. This suggests that administration of istradefylline suppresses neutrophil airway inflammation (Fig. 5B).
　［実施例６］多発性硬化症モデルマウスにおけるイストラデフィリンの効果
　マウスの多発性硬化症モデルである、実験的自己免疫性脳脊髄炎（ＥＡＥ）モデルを調製すべく、マウス髄鞘のミエリンタンパク質を標的としたＴヘルパーペプチドである、ＰＬＰ　１３９－１５１ペプチド（アミノ酸配列：ＨＣＬＧＫＷＬＧＨＰＤＫＦ、配列番号：２）（ＴＯＣＲＩＳ）を完全フロイントアジュバント（ＤＩＦＣＯ，ａｄｊｕｖａｎｔ　ｃｏｍｐｌｅｔｅ　ｆｒｅｕｎｄ，Ｈ３７　Ｒａ）と等量混和し、エマルジョンを作製した。具体的には、ＰＬＰ　１３９－１５１　ペプチドをＰＢＳ　ｗｉｔｈｏｕｔ　ｍａｇｎｅｓｉｕｍ　ａｎｄ　ｃａｌｃｉｕｍ（ＰＢＳ（－））に溶解した１ｍｇ／ｍＬ　ＰＬＰ　１３９－１５１　ペプチド　ｉｎ　ＰＢＳ（－）を完全フロイントアジュバント（ＤＩＦＣＯ，ａｄｊｕｖａｎｔ　ｃｏｍｐｌｅｔｅ　ｆｒｅｕｎｄ，Ｈ３７　Ｒａ）を体積として等量混和し（例えば、５００μＬのＰＬＰ　１３９－１５１　ペプチド溶液と５００μＬの完全フロイントアジュバントを混和させた）、２つの２ｍＬのシリンジ（Ｉｎｊｅｃｔ，Ｂ．Ｂｒａｕｎ　Ｍｅｄｉｃａｌ）をシリンジコネクター（ＧＰ　Ｓｙｒｉｎｇｅ　Ｃｏｎｎｅｃｔａ，Ｎｉｐｒｏ）で繋ぎ、ｔｗｏ－ｓｙｒｉｎｇｅ法で乳化させた。乳化した２００μＬ（ＰＬＰ　１３９－１５１　ペプチドとして１００μｇ）のＰＬＰ　１３９－１５１　ペプチド溶解完全フロイントアジュバントをＳＪＬ／Ｊマウス（６週齢、雌）の鼠径部１か所に皮下注射し、ＥＡＥマウスを誘導した。１回あたり、超純水１００μＬ、または、超純水１００μＬに６μｇのイストラデフィリン溶解したものを、週３回胃ゾンデで経口投与し、運動症状の程度をスコアで記録した（症状とスコアの関係については、下記表１に示す）。得られた結果を図６Ａに示す。 [Example 6] Effect of Istradefylin on Multiple Sclerosis Model Mice To prepare an experimental autoimmune encephalomyelitis (EAE) model, which is a mouse multiple sclerosis model, the myelin protein in the mouse myelin sheath. PLP 139-151 peptide (amino acid sequence: HCLGKWLGHPDKF, SEQ ID NO: 2) (TOCRIS), which is a T helper peptide targeted at Was produced. Specifically, 1 mg / mL PLP 139-151 peptide in PBS (-) in which the PLP 139-151 peptide was dissolved in PBS without syringe and syringe (PBS (-)) was added to a complete Freund's adjuvant (DIFCO, adjuvant syringe, H37). Ra) was mixed in equal amounts by volume (eg, 500 μL of PLP 139-151 peptide solution mixed with 500 μL of complete Freund's adjuvant), and two 2 mL syringes (Inject, B. Brown Medical) were combined with a syringe connector (GP). It was connected by Syringe Connecta, Nipro) and emulsified by the two-syringe method. 200 μL of emulsified (100 μg as PLP 139-151 peptide) PLP 139-151 peptide-dissolved complete Freund's adjuvant was subcutaneously injected into one inguinal region of SJL / J mice (6 weeks old, female) to induce EAE mice. .. Each dose was orally administered in 100 μL of ultrapure water or 6 μg of isstradefylline dissolved in 100 μL of ultrapure water with a gastric sonde three times a week, and the degree of motor symptoms was recorded as a score (symptoms and scores). The relationship is shown in Table 1 below). The obtained results are shown in FIG. 6A.
Figure JPOXMLDOC01-appb-T000035
Figure JPOXMLDOC01-appb-T000035
　さらに、皮下注射２０日後にマウスの頸椎を脱臼させた後、脊髄を摘出した。新組織化学研究所に依頼し、ＨＥ染色および抗ｍｏｕｓｅＣＤ３ε抗体（Ｃｅｌｌ　Ｓｉｇｎａｌｉｎｇ　ＴＥＣＨＮＯＬＯＧＹ　＃９９９４０）を用いた免疫染色を行った。得られた結果を図６Ｂに示す。 Furthermore, 20 days after the subcutaneous injection, the cervical spine of the mouse was dislocated, and then the spinal cord was removed. We requested the New Institute of Tissue Chemistry to perform HE staining and immunostaining using anti-mouseCD3ε antibody (Cell Signaling TECHNOLOGY # 99940). The obtained results are shown in FIG. 6B.
　その結果、水のみを投与したコントロール群と比較して、イストラデフィリン投与群では運動症状の増悪が抑制され（図６Ａ）、病理学的にもリンパ球（ＣＤ３陽性Ｔ細胞）の浸潤が抑制された（図６Ｂ）。 As a result, the exacerbation of motor symptoms was suppressed in the isstradefylline-administered group (Fig. 6A), and the infiltration of lymphocytes (CD3-positive T cells) was pathologically suppressed as compared with the control group in which only water was administered. (Fig. 6B).
　［実施例７］アトピー性皮膚炎モデルマウスにおけるイストラデフィリンの効果
　ＮＣ／Ｎｇａマウスの背部をバリカンで剃毛し、２％　２，４，６－トリニトロクロロベンゼン（ＴＮＣＢ）（エタノールとアセトンを４：１に混合した液体にＴＮＣＢを溶解したもの）を１５０μＬ滴下することにより、アトピー性皮膚炎モデルマウスを調製した。イストラデフィリンを５％の濃度になるようにワセリンに混合してクリームを調製し、各クリームを背部に塗布し、その病態変化を観察した。そして、観察された病態をスコア化し（病態の項目およびスコアは、表２の欄外に示す）、イストラデフィリンによる効果を評価した。 [Example 7] Effect of Istradefylin on atopic dermatitis model mouse The back of NC / Nga mouse was shaved with a hair clipper, and 2% 2,4,6-trinitrochlorobenzene (TNCB) (ethanol and acetone were added 4). Atopic dermatitis model mice were prepared by dropping 150 μL of TNCB dissolved in a liquid mixed at 1: 1. Istradefylline was mixed with petrolatum to a concentration of 5% to prepare creams, and each cream was applied to the back and its pathological change was observed. Then, the observed pathological conditions were scored (the pathological conditions and scores are shown in the margin of Table 2), and the effect of istradefylline was evaluated.
Figure JPOXMLDOC01-appb-T000036
Figure JPOXMLDOC01-appb-T000036
　その結果、開始から１０日目において、誘導群と比較してクリーム塗布群で有意な病態抑制が観察された（図７Ａ）。また、表２および図７Ｂに示すとおり、皮膚炎スコアにおいても、クリーム塗布群は、誘導群に対し、統計学上有意な抑制効果を示した。 As a result, on the 10th day from the start, significant suppression of pathological condition was observed in the cream-applied group as compared with the induction group (Fig. 7A). In addition, as shown in Table 2 and FIG. 7B, the cream-applied group also showed a statistically significant inhibitory effect on the induction group in the dermatitis score.
　本発明は、Ｔｈ１７細胞の過剰反応に起因する疾患の治療などに有用であり、主として、医療分野に貢献するものである。また、本発明は、Ｔｈ１７細胞の分化や活性化の調節を行う基礎研究においても貢献するものである。 The present invention is useful for the treatment of diseases caused by Th17 cell overreaction, and mainly contributes to the medical field. The present invention also contributes to basic research for regulating Th17 cell differentiation and activation.

Claims (4)

  1. 　Ｔｈ１７細胞の分化もしくは活性化の抑制またはＴｈ１７細胞の過剰反応に起因する疾患の治療、改善、もしくは予防のための組成物であって、アデノシンＡ２Ａ受容体アンタゴニストを有効成分として含有する組成物。 A composition for treating, ameliorating, or preventing a disease caused by suppression of Th17 cell differentiation or activation or overreaction of Th17 cells, which contains an adenosine A2A receptor antagonist as an active ingredient.
  2. 　被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
    　（ａ）被検化合物をアデノシンＡ２Ａ受容体に接触させる工程、および
    　（ｂ）被検化合物とアデノシンＡ２Ａ受容体との結合を検出する工程、
    を含み、
    　被検化合物がアデノシンＡ２Ａ受容体と結合する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。     A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
    (A) A step of contacting the test compound with the adenosine A2A receptor, and (b) a step of detecting the binding between the test compound and the adenosine A2A receptor.
    Including
    When the test compound binds to the adenosine A2A receptor, it is evaluated that it has an activity of suppressing the differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells. How to be done.
  3. 　被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
    　（ａ）被検化合物の存在下で、アデノシンＡ２Ａ受容体リガンドをアデノシンＡ２Ａ受容体に接触させる工程、および
    　（ｂ）アデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を検出する工程、
    を含み、
    　被検化合物がアデノシンＡ２Ａ受容体リガンドとアデノシンＡ２Ａ受容体との結合を阻害する場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。     A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
    (A) The step of contacting the adenosine A2A receptor ligand with the adenosine A2A receptor in the presence of the test compound, and (b) the step of detecting the binding between the adenosine A2A receptor ligand and the adenosine A2A receptor.
    Including
    When the test compound inhibits the binding of the adenosine A2A receptor ligand to the adenosine A2A receptor, the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated, ameliorated, or prevented. A method that is evaluated as having a probable activity.
  4. 　被検化合物が、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性、またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性を評価する方法であって、
    　（ａ）被検化合物をアデノシンＡ２Ａ受容体を発現する細胞に接触させる工程、および
    　（ｂ）アデノシンＡ２Ａ受容体の活性を検出する工程、
    を含み、
    　被検化合物を接触させない場合と比較して、被検化合物がアデノシンＡ２Ａ受容体の活性を低下させる場合、Ｔｈ１７細胞の分化もしくは活性化を抑制する活性またはＴｈ１７細胞の過剰反応に起因する疾患を治療、改善、もしくは予防する活性を有する蓋然性があると評価される方法。     A method for evaluating the probability that a test compound has an activity of suppressing differentiation or activation of Th17 cells or an activity of treating, ameliorating, or preventing a disease caused by an overreaction of Th17 cells.
    (A) A step of contacting the test compound with a cell expressing the adenosine A2A receptor, and (b) a step of detecting the activity of the adenosine A2A receptor.
    Including
    When the test compound reduces the activity of the adenosine A2A receptor as compared to the case where the test compound is not contacted, the activity that suppresses the differentiation or activation of Th17 cells or the disease caused by the overreaction of Th17 cells is treated. , A method that is assessed as probable to have an ameliorating or prophylactic activity.
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