WO2022162992A1 - Pharmaceutical composition for treating allergy - Google Patents

Pharmaceutical composition for treating allergy Download PDF

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WO2022162992A1
WO2022162992A1 PCT/JP2021/032406 JP2021032406W WO2022162992A1 WO 2022162992 A1 WO2022162992 A1 WO 2022162992A1 JP 2021032406 W JP2021032406 W JP 2021032406W WO 2022162992 A1 WO2022162992 A1 WO 2022162992A1
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pharmaceutical composition
cell
present
tacrolimus
treating
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PCT/JP2021/032406
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French (fr)
Japanese (ja)
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洋平 向山
宏 森本
允人 久保
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マルホ株式会社
国立研究開発法人理化学研究所
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Priority to JP2022578032A priority Critical patent/JPWO2022162992A1/ja
Publication of WO2022162992A1 publication Critical patent/WO2022162992A1/en

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    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C07D451/04—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof with hetero atoms directly attached in position 3 of the 8-azabicyclo [3.2.1] octane or in position 7 of the 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring system
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Definitions

  • the present invention belongs to the technical field of therapeutic agents for allergic diseases and their symptoms.
  • TECHNICAL FIELD The present invention relates to pharmaceutical compositions and the like containing tacrolimus.
  • Tacrolimus also known as an immunosuppressant used in organ transplantation, binds to calcineurin (calcium-dependent serine-threonine dephosphorylation enzyme) and inhibits dephosphorylation of T-cell nuclear transcription factor (NFAT). Its mechanism of action has been elucidated to suppress nuclear translocation via , and it is currently used for the treatment of late reactions in allergic diseases such as asthma, atopic dermatitis, and rheumatoid arthritis.
  • Patent Document 1 discloses a drug containing tacrolimus as an anti-asthmatic drug that suppresses late-onset asthmatic reactions and airway hyperreactivity.
  • Non-Patent Document 1 discloses the efficacy of tacrolimus for steroid-resistant asthma, and considers that the suppression of T cell activation by tacrolimus is a factor.
  • allergens activate type 2 innate immune cells (ILC2). It is known to cause T cell-independent airway inflammation induced by derived type 2 cytokines.
  • the calcium signal pathway which is the target of tacrolimus, is not involved in this type 2 cytokine production from ILC2 (Non-Patent Document 2).
  • Tacrolimus is not believed to be effective against the early phase of allergic disease or T cell-independent reactions, such as the immediate reaction of protease allergen-induced airway inflammation.
  • the main object of the present invention is to provide a new pharmaceutical composition capable of treating T cell-independent reactions or immediate reactions (early phase symptoms) of allergic diseases.
  • tacrolimus is also effective in treating T cell-independent reactions or immediate reactions in allergic diseases and/or symptoms. completed.
  • Examples of the present invention include the following.
  • [1] A pharmaceutical composition for treating T cell-independent allergic diseases and/or symptoms, containing an effective amount of tacrolimus.
  • [2] The pharmaceutical composition according to [1] above, which is for treating T cell-independent allergic diseases and/or symptoms caused by protease allergens.
  • [3] The pharmaceutical composition according to [1] or [2] above, for treating T cell-independent allergic diseases and/or symptoms in which ILC2 is activated.
  • the present invention can be used to treat immediate reactions (early phase reactions) of allergic diseases and/or symptoms, or non-T cell dependent allergic diseases and/or symptoms. Moreover, according to the present invention, the activation of ILC2 can be suppressed. And according to the present invention, it is possible to inhibit the calcium signal of the pathway in the release and/or production of interleukin 33 (IL-33) from cells, and to suppress the release and/or production of IL-33. .
  • IL-33 interleukin 33
  • Panel A shows eosinophils (upper panel) and activation in the lungs of wild-type (WT) mice, and WT, tacrolimus, and dexamethasone-treated mice 24 hours after nasal administration of papain for 3 consecutive days. These are the results of flow cytometry analysis of eosinophils (lower diagram).
  • FIG. B is a graph quantifying the numbers of eosinophils (left panel) and activated eosinophils (right panel) for each mouse.
  • FIG. C is a photograph showing the results of periodic acid-Schiff (PAS) staining (upper panel) and hematoxylin-eosin (HE) staining (lower panel) of lung tissue in each mouse analyzed in FIG.
  • PAS periodic acid-Schiff
  • HE hematoxylin-eosin
  • FIG. 2 shows the results of RNA-Seq analysis of ILC2 in the lungs of papain-administered mice.
  • FIG. A is a scatter diagram showing the expression level of each gene in ILC2. The horizontal axis represents the expression level (RPM) in tacrolimus-unadministered mice, and the vertical axis represents the expression level (RPM) in tacrolimus-administered mice.
  • Panel B is a heatmap of the ILC2 signature (gene).
  • Panel C is a heatmap of genes associated with ILC2 activation.
  • Panel A shows eosinophils (upper panel) and activated eosinophils in the lungs of WT mice and WT, tacrolimus-treated, and dexamethasone-treated mice 24 hours after 3 consecutive days of intranasal administration of IL-33. These are the results of flow cytometry analysis of spheres (bottom).
  • FIG. B is a graph quantifying the numbers of eosinophils (left panel) and activated eosinophils (right panel) for each mouse.
  • FIG. C is a photograph showing the results of periodic acid-Schiff (PAS) staining (upper panel) and hematoxylin-eosin (HE) staining (lower panel) of lung tissue in each mouse analyzed in FIG.
  • PAS periodic acid-Schiff
  • Figure A is the results of flow cytometry analysis of IL-33-expressing cells in mouse lung epithelial cells (type 1 and type 2 alveolar epithelial cells). It shows that IL-33 localizes to type 2 alveolar epithelial cells.
  • FIG. B shows fluorescence intensity measurement results (photographs) of intracellular calcium concentration over time after papain stimulation (upper diagram) and ionomycin stimulation (lower diagram) in type 2 alveolar epithelial cells. In both the upper and lower figures, the upper row shows microscopic images of cells, and the lower row shows fluorescence intensity imaging images.
  • composition according to the present invention is a pharmaceutical composition containing an effective amount of tacrolimus, which is used to treat non-T cell-dependent allergic diseases. and/or used to treat symptoms.
  • the compositions of the invention can be used to treat allergic diseases and/or symptoms caused by protease allergens.
  • the composition of the present invention can suppress the activation of ILC2 and can be used to treat allergic diseases and/or symptoms in which ILC2 is activated.
  • the composition of the present invention can suppress the release and/or production of IL-33 in cells, and is used to treat allergic diseases and/or symptoms in which the release and/or production of IL-33 is enhanced. can be used for
  • treatment is a concept that includes prevention and improvement.
  • Prevention includes primary prevention as prevention of sensitization, secondary prevention as prevention of disease onset, and tertiary prevention as prevention of exacerbation.
  • the composition of the present invention can be more effective in secondary prevention and tertiary prevention.
  • Prophylaxis also includes prophylaxis in long-term or chronic phase therapy.
  • Treatment in the present invention is not particularly limited as long as it is treatment of T cell-independent allergic diseases and/or symptoms, but usually means acute treatment as treatment for stroke.
  • Acute treatment as an attack treatment includes treatment for allergic reaction or allergic inflammation, which is a common pathology of allergic diseases, and organ-specific treatment as a site of allergic reaction manifestation.
  • T cell-independent allergic diseases and/or symptoms are, in principle, allergic diseases caused by innate immune cells (e.g., type 2 innate immune cells, basophils, mast cells) without the intervention of T cells. and/or symptoms, which correspond to immediate or early phase reactions in the acute phase.
  • innate immune cells e.g., type 2 innate immune cells, basophils, mast cells
  • allergic diseases e.g., airway inflammation
  • T H 2 helper T cells T H 2 helper T cells
  • T H 2 helper T cells T H 2 helper T cells
  • Tacrolimus also known as FK506
  • FK506 has the chemical name (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-5,19-Dihydroxy-3- ⁇ (1E)-2 -[(1R,3R,4R)-4-hydroxy3-methoxycyclohexyl]-1-methylethenyl ⁇ -14,16-dimethoxy-4,10,12,18-tetramethyl-8-(prop-2-en-1-yl )-15,19-epoxy5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-3H-pyrido[2,1-c] It is a 23-membered ring macrolide lactone represented by [1,4]oxaazacyclotricosine-1,7,20,
  • tacrolimus may refer not only to tacrolimus in free form but also to tacrolimus in the form of salts, solvates, and the like.
  • Tacrolimus according to the present invention also includes crystalline phases, amorphous phases, and semi-crystalline phases of tacrolimus.
  • composition of the present invention can contain an appropriate amount of carrier in addition to the active ingredient.
  • carriers can include, for example, solid, semi-solid or liquid diluents, fillers, and other formulation aids. One or more of these carriers can be contained.
  • compositions of the present invention are used to treat allergic diseases and/or conditions. Allergens that cause allergic diseases and/or symptoms are not limited in principle, but interleukin 33 (IL-33) is not included in the allergens referred to herein. Examples of allergens include inhalant antigens, food antigens, contact antigens (dyes, cosmetics, external agents, metals, etc.), drug antigens (antibacterial agents, antiserum, etc.), insect antigens (bee venom, mosquitoes, cockroaches, etc.), vaccines. can be mentioned.
  • IL-33 interleukin 33
  • allergens include inhalant antigens, food antigens, contact antigens (dyes, cosmetics, external agents, metals, etc.), drug antigens (antibacterial agents, antiserum, etc.), insect antigens (bee venom, mosquitoes, cockroaches, etc.), vaccines. can be mentioned.
  • inhaled antigens include, for example, Alternaria, Rhizopus, Malaria, Aspergillus, and mites.
  • food antigens include, for example, papaya, pineapple, ginger, fig, kiwifruit, natto, and mushrooms (maitake, oyster mushroom, etc.).
  • allergen substances contained in at least one of the above allergens that are suitable for treatment with the composition of the present invention include proteases.
  • proteases examples include cysteine proteases, serine proteases, and aspartic proteases (pepsin, etc.).
  • cysteine proteases include, for example, papain, ficin, bromelain, gingipain, actinidin, caspase, and cathepsin.
  • serine proteases examples include trypsin, chymotrypsin, subtilisin, and nattokinase.
  • composition of the present invention can suppress the activation of ILC2 (Group 2 innate lymphoid cells: type 2 innate lymphocytes or type 2 innate lymphocytes).
  • ILC2 Group 2 innate lymphoid cells: type 2 innate lymphocytes or type 2 innate lymphocytes.
  • the composition of the present invention can be used to treat allergic diseases and/or symptoms in which ILC2 is activated.
  • ILC2 is a kind of innate immune cells belonging to the lymphoid system, and when activated, it produces type 2 cytokines (IL-5, IL-13, etc.), hyperplasia of bronchial epithelial cells, enhancement of mucin production, It induces the accumulation and activation of eosinophils.
  • ILC2 can be activated by cytokines from damaged cells, such as epithelial cells. Such cytokines include lipid mediators (eg, LTB4, LTE4, PGD2), TSLP, IL-33, IL-25, and the like. ILC2 activation can also be regulated by neuropeptides (NMU, etc.) and sex hormones.
  • NMU neuropeptides
  • the composition of the present invention can suppress the activation of ILC2, and can be used to treat allergic diseases and/or symptoms associated with the activation.
  • the composition of the present invention can selectively suppress ILC2 activation by lipid mediators or neuropeptides, and more selectively suppress ILC2 activation by LTC4 or NMU.
  • the composition of the present invention can inhibit the release and/or production of IL-33 in cells.
  • the composition of the present invention can be used to treat allergic diseases and/or symptoms in which IL-33 release and/or production are enhanced.
  • IL-33 is a protein mainly present in the nuclei of epithelial cells, and is released outside the cells upon exposure to antigens such as allergens. When released, it activates innate immune cells (eg, ILC2, basophils, mast cells) as an alarmin, causing cytokine-induced T cell-independent inflammation.
  • innate immune cells eg, ILC2, basophils, mast cells
  • the calcium signaling circuit targeted by tacrolimus is not involved in the IL-33-induced cytokine production pathway of innate immune cells.
  • the upstream IL-33 production/release from cells is thought to be mediated by the calcium pathway, and tacrolimus can inhibit the calcium signal in this pathway.
  • composition of the present invention can suppress the release and/or production of IL-33 in cells (e.g., epithelial cells, especially airway epithelial cells, particularly type 2 alveolar epithelial cells), and , can be used to treat allergic diseases and/or conditions in which IL-33 release and/or production is increased.
  • cells e.g., epithelial cells, especially airway epithelial cells, particularly type 2 alveolar epithelial cells
  • IL-33 induces type 2 cytokine production not only in ILC2 but also in type 2 helper T cells (T H 2) (for example, Guo, L., et al., Nature Immunology 2015 No.16: 1051-1059.).
  • T H2 may also be involved in early-phase allergic symptoms (eg, airway inflammation).
  • the composition of the present invention can also be effectively used in this case.
  • the allergic symptoms to be treated by the composition of the present invention are not particularly limited as long as they can occur as acute reactions of allergic diseases. Examples include respiratory allergy symptoms, allergic rhinitis, eosinophilic sinusitis, allergic conjunctivitis, and atopic dermatitis.
  • the above respiratory allergy symptoms include airway inflammation, asthma, goblet cell hyperplasia of the airway epithelium, and increased airway hyperresponsiveness.
  • Airway inflammation suitable for treatment with the composition of the present invention includes, for example, eosinophilic airway inflammation.
  • asthma suitable for treatment with the composition of the present invention include severe asthma that exhibits resistance to high-dose inhaled corticosteroid therapy, and exacerbation of asthma associated with virus (influenza virus, rhinovirus, coronavirus, etc.) infection. be able to.
  • the composition of the present invention can contain an effective amount of any other therapeutic drug for allergic diseases depending on the need and purpose.
  • the therapeutic drug for allergic diseases includes, for example, asthma long-term control drug, therapeutic drug for allergic rhinitis, therapeutic drug for eosinophilic sinusitis, therapeutic drug for allergic conjunctivitis, and therapeutic drug for atopic dermatitis.
  • Asthma long-term control drugs include, for example, corticosteroids, bronchodilators, and molecular-targeted drugs.
  • corticosteroid drugs examples include inhaled steroid drugs (ICS) and oral steroid drugs.
  • ICS include beclomethasone, ciclesonide, fluticasone, mometasone, budesonide and the like.
  • Bronchodilators include, for example, ⁇ 2 agonists, anticholinergics, theophylline sustained - release preparations, and leukotriene receptor antagonists.
  • ⁇ 2 agonists include long - acting ⁇ 2 - agonists (LABA: e.g., salmeterol, formoterol), ultra-long-acting ⁇ 2 - agonists (ultra-LABA: e.g., indacaterol, vilanterol, , olodaterol, carmoterol.) and the like.
  • Anticholinergics include tiotropium, glycopyrronium, acridinium, umeclidinium, trospium, and the like.
  • molecular targeted therapeutic agents include humanized anti-human IgE antibodies, humanized anti-IL-5 antibodies, and anti-IL-13 antibodies.
  • Each of the above therapeutic drugs for allergic diseases may be used alone or in combination of two or more.
  • the route of administration of the composition of the present invention is not particularly limited. Local administration (e.g., inhalation administration, transmucosal administration, nasal administration, ocular administration, transdermal administration), oral administration, other parenteral administration (e.g., intravenous administration, intraportal administration, subcutaneous administration, drip infusion injection administration, etc.), but the composition of the present invention is preferably a topical preparation or an internal preparation (orally administered preparation).
  • Local administration e.g., inhalation administration, transmucosal administration, nasal administration, ocular administration, transdermal administration
  • oral administration other parenteral administration (e.g., intravenous administration, intraportal administration, subcutaneous administration, drip infusion injection administration, etc.)
  • other parenteral administration e.g., intravenous administration, intraportal administration, subcutaneous administration, drip infusion injection administration, etc.
  • the composition of the present invention is preferably a topical preparation or an internal preparation (orally administered preparation).
  • the administration route of the composition of the present invention may be one route or two or more routes among the above.
  • the dosage forms of the composition of the present invention are inhalants, nasal drops, ophthalmic preparations, external preparations, internal preparations (orally administered preparations), and parenteral preparations, depending on the route of administration. can take any form.
  • the composition of the present invention may be a sustained release formulation.
  • the inhalant is not particularly limited as long as it can be administered using a nebulizer (nebulizer, inhaler, etc.) described below. , for example, liquid sprays, aerosols, and dry powders.
  • Liquid sprays and liquid-based aerosols are prepared by dissolving the active ingredient in distilled water or a sterile aqueous solution such as saline or buffer solution, or by dissolving the active ingredient with an additional excipient. be able to.
  • Such additives include, for example, buffering agents (boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives or preservatives (benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), tonicity Regulators (sodium chloride, etc.), surfactants, solubilizers, stabilizers, analgesic agents, thickeners (e.g., polysaccharides such as lactose, mannitol, maltose; hyaluronic acid, such as sodium hyaluronate and potassium hyaluronate) or salts thereof; mucopolysaccharides such as chondroitin sulfate; polymers such as sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, methylcellulose, and glycerin), emollients, pH adjusters, and lubricants. can.
  • Dry powders and solid-based aerosols are prepared by micronizing the active ingredient to a suitable average particle size, as described below, followed by a similarly finely divided pharmaceutical carrier, for example, an edible carbohydrate such as crystalline lactose. It can be manufactured by mixing. Flavoring agents, preservatives, dispersing agents, coloring agents, perfumes and the like can also be optionally added.
  • Liquid sprays, aerosols, and dry powders are usually administered in the form of fine particles. There is no particular limitation on the average particle size of the fine particles.
  • nasal drops When the composition of the present invention is in the form of nasal drops, examples of such nasal drops include liquid sprays, mists, and aerosols.
  • Nasal drops can be prepared by dissolving the active ingredient in distilled water or sterile aqueous solutions such as saline and buffer solutions, or by adding and dissolving the powder composition prior to use. Additives similar to those used in liquid sprays and the like can also be used in nasal drops.
  • composition of the present invention is in the form of an ophthalmic formulation
  • examples of such ophthalmic formulation include eye drops and ophthalmic ointments.
  • Eye drops can be prepared by dissolving the active ingredient in distilled water or a sterile aqueous solution such as saline or buffer solution, or by adding and dissolving the powder composition prior to use.
  • a sterile aqueous solution such as saline or buffer solution
  • the same additives as those used in the liquid spray and the like can also be used in the eye drops.
  • An ophthalmic ointment can be prepared by mixing an active ingredient and a base by a conventional method.
  • the base include, but are not limited to, vaseline, plastibase, macrogol, and selenium 50.
  • surfactants such as emulsifiers for increasing hydrophilicity and other additives similar to those used in liquid sprays can also be used in ophthalmic ointments.
  • the external preparation is not particularly limited as long as it is a dosage form that allows direct administration of the active ingredient to the topical surface of the skin. formulations, solutions (suspensions, emulsions, lotions, etc.), poultices, tapes, aerosols, and powders for external use.
  • compositions in addition to the active ingredient, various compounding ingredients that are used to prepare ordinary external preparations can be appropriately selected and used.
  • such ingredients include white petrolatum, yellow petrolatum, lanolin, white beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oils, gelling hydrocarbons, bases such as polyethylene glycol, liquid paraffin, and squalane; Solvents and solubilizers; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; preservatives such as parahydroxybenzoic acid esters; moisturizing agents such as glycerin, propylene glycol, and sodium hyaluronate; Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, prop
  • tackifiers such as polyacrylic acid and polyacrylic acid copolymers; cross-linking agents such as aluminum sulfate, aluminum potassium sulfate, aluminum chloride, magnesium aluminometasilicate and dihydroxyaluminum acetate; polyacrylic acid; Thickeners such as sodium, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethylcellulose, carboxymethylcellulose sodium salts, hydroxypropylcellulose, hydroxypropylmethylcellulose; glycerin, polyethylene glycol (macrogol), propylene glycol, 1,3- polyhydric alcohols such as butanediol; surfactants such as polyoxyethylene derivatives; fragrances such as L-menthol; preservatives such as parahydroxybenzoate; Furthermore, if desired, stabilizers, preservatives, absorption enhancers, pH adjusters, and other suitable additives can be added.
  • cross-linking agents such as aluminum sulfate
  • styrene-ethylene ⁇ Adhesives such as acrylic resins such as butylene-styrene block copolymers, (meth)acrylic acid alkyl ester (co)polymers, polyacrylic acid esters, methacrylic acid esters; alicyclic saturated hydrocarbon resins, rosin tackifying resins such as base resins and terpene resins; softeners such as liquid rubber and liquid paraffin; antioxidants such as dibutylhydroxytoluene; polyhydric alcohols such as propylene glycol; absorption enhancers such as oleic acid; Surfactants such as derivatives and other suitable additives
  • a water-containing tape can also be prepared by adding a water-absorbable polymer such as sodium polyacrylate or polyvinyl alcohol and a small amount of purified water. Even in this case, if desired, stabilizers, preservatives, absorption enhancers, pH adjusters and other suitable additives can be added.
  • Bases such as oils, gelling hydrocarbons, polyethylene glycol, liquid paraffin, squalane; oleic acid, isopropyl myristate, diisopropyl adipate, isopropyl sebacate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids , fatty acid esters, fatty alcohols, solvents and solubilizers such as vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylhydroxyanisole; preservatives such as parahydroxybenzoic acid esters; glycerin, Moisturizers such as propylene glycol and sodium hyaluronate; surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glyco
  • excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, etc., or other suitable additives can be added. Even in this case, if desired, various stabilizers, preservatives, absorption enhancers and other suitable additives can be added.
  • the method for preparing the external preparation is not particularly limited, and it can be produced by a conventional method such as thoroughly kneading each component and, if necessary, a base component according to the desired dosage form. Also, poultices and tapes can be prepared by spreading the kneaded mixture on a release paper, drying it, attaching it to a flexible support, and cutting it into a desired size.
  • the external preparations are, for example, ointments, liquids (suspensions, emulsions, lotions, etc.), aerosols and powders for external use, which can be applied directly to the affected skin by coating or the like, or can be supported by a cloth or the like. It can be used by a conventional method such as applying by coating or impregnating the body. In the case of poultices and tapes, these formulations can be used by directly applying them to the affected skin area.
  • composition of the present invention is in the form of an internal preparation
  • examples of such internal preparation include powders, capsules, tablets, dragees, granules, powders, suspensions, liquids, syrups, elixirs, and lozenges. agents can be mentioned.
  • a powder can be produced by appropriately pulverizing the active ingredient. Powders can be prepared by comminuting the active ingredient into a suitable fine powder and then mixing with a similarly finely divided pharmaceutical carrier such as an edible carbohydrate such as starch or mannitol. Optionally, flavoring agents, preservatives, dispersing agents, coloring agents, perfumes and the like can be added.
  • Capsules are manufactured by first filling powders and powders that have been powdered as described above, or granulated as described in the section on tablets, into capsule shells such as gelatin capsules, for example. can do. It can also be produced by mixing a lubricant or glidant such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol with the powder, followed by a filling operation. If disintegrants or solubilizers such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, croscarmellose sodium, carboxymethylstarch sodium, calcium carbonate, sodium carbonate are added, the capsules will be of the drug can be improved.
  • a soft capsule can also be prepared by suspending and dispersing fine powder of the active ingredient in vegetable oil, polyethylene glycol, glycerin, or a surfactant, and wrapping it in a gelatin sheet.
  • Tablets can be made by adding excipients to make a powder mixture, granulating or slugging, and then adding a disintegrant or lubricant before tabletting.
  • a powder mixture can be prepared by mixing the suitably powdered material with the diluents and bases mentioned above.
  • binders e.g. sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, polyvinyl alcohol
  • dissolution retarding agents e.g. paraffin
  • resorbing agents e.g. quaternary salts
  • Adsorbents eg bentonite, kaolin
  • the powder mixture can be first moistened with a binder such as syrup, starch paste, gum arabic, cellulose solution or polymeric substance solution, stirred and mixed, dried and ground into granules.
  • a binder such as syrup, starch paste, gum arabic, cellulose solution or polymeric substance solution
  • stearic acid, stearate, talc, mineral oil, etc. as a lubricant to the granules thus produced, it is possible to prevent them from adhering to each other.
  • Tablets can also be produced by directly compressing after mixing the active ingredient with a flowable inert carrier without going through the granulation or slugging steps as described above.
  • Tablets manufactured in this way can be coated with film or sugar.
  • a clear or translucent protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating consisting of wax can also be used.
  • oral formulations such as solutions, syrups, lozenges, and elixirs, can also be in dosage unit form so that a given amount of the formulation contains a given amount of the active ingredient.
  • Syrups can be prepared by dissolving the active ingredient in a suitable flavored aqueous solution.
  • Elixirs can be prepared using a non-toxic alcoholic carrier.
  • Suspensions can be prepared by dispersing the active ingredient and the like in a non-toxic carrier.
  • solubilizers, emulsifiers e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters
  • preservatives e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters
  • flavoring agents e.g. peppermint oil, saccharin
  • dosage unit formulations for oral administration can be microencapsulated.
  • the formulations can also be coated or embedded in polymers, waxes, etc. to provide extended duration of action or sustained release.
  • composition of the present invention is in the form of preparations for parenteral administration
  • preparations for parenteral administration include injections, infusions, and suppositories.
  • the injection may be an injection kit or drip infusion kit prepared just before use.
  • Parenteral formulations can take the form of liquid dosage unit forms, such as solutions and suspensions, for subcutaneous, intramuscular or intravenous injection.
  • the preparation for parenteral administration is prepared by suspending or dissolving a certain amount of the active ingredient in a non-toxic liquid carrier suitable for the purpose of injection, such as an aqueous or oily medium, and then sterilizing the suspension or solution. It can be manufactured by A non-toxic salt or salt solution can be added to make the injection isotonic. Stabilizers, preservatives, emulsifiers and the like can also be added.
  • an infusion formulation can be prepared.
  • Suppositories can be prepared by dissolving or suspending active ingredients in water-soluble or insoluble solids with a low melting point, such as polyethylene glycol, cocoa butter, semi-synthetic fats and oils, higher esters, or mixtures thereof. can.
  • a low melting point such as polyethylene glycol, cocoa butter, semi-synthetic fats and oils, higher esters, or mixtures thereof.
  • the content of tacrolimus is not particularly limited as long as it is within a range that does not impair the effects of the present invention, that is, within the range of the so-called effective amount. It is suitably in the range of 0.01% to 1% by weight, preferably in the range of 0.01% to 1% by weight.
  • composition of the present invention is an external preparation
  • it is suitably within the range of 0.001% by weight to 10% by weight with respect to the total weight of the composition of the present invention, and 0.01% by weight. It is preferably in the range of ⁇ 0.3% by weight.
  • kit according to the present invention is characterized by combining the composition of the present invention, which is an embodiment of the topical preparation, and a nebulizer. do.
  • nebulizer means both a nebulizer (nebulizer in a narrow sense) for nebulizing a liquid formulation and an inhaler for inhaling an aerosol or dry powder formulation.
  • the nebulizer constituting the kit of the present invention is preferably an atomizer.
  • nebulizers examples include jet nebulizers, ultrasonic nebulizers, and mesh nebulizers.
  • inhalers examples include pressurized metered dose inhalers (pMDI) and dry powder inhalers (DPI).
  • pMDI pressurized metered dose inhalers
  • DPI dry powder inhalers
  • topical formulation that constitutes the kit of the present invention is preferably a liquid, aerosol, or dry powder inhalant.
  • the mouse (C57BL/6J) used in the following Examples was purchased from CLEA Japan Inc. at 8 weeks of age.
  • PE-labeled anti-CD11c antibody, APC-labeled anti-Siglec-F antibody, and BV421-labeled anti-Sca-1 antibody are from BD Bioscience
  • CD326 (EpCAM) antibody was purchased from Biolegend
  • FITC-labeled AQO5 antibody was purchased from Bioss
  • FITC-labeled ST-2 antibody was purchased from Mdbioproducts.
  • Eosinophils were identified by staining with FITC-labeled anti-CD64 antibody, PE-labeled CD11c antibody, PE-Cy7-labeled anti-Gr-1 antibody, and APC-labeled anti-Siglec-F antibody, using FACS calibur (manufactured by BD Biosciences). was used for flow cytometry analysis. Flowjo v10.2 (manufactured by BD Biosciences) was used for data analysis. 7-Amino-Actinomycin D (7AAD) was used to remove dead cells.
  • Results The analysis results are shown in FIG. Each data in FIG. 1B represents the standard deviation, and statistical analysis was performed by two-sided Student's t-test using GraphPad Prism 8 (manufactured by GraphPad Software). Differences at p-values less than 0.05 were considered significant.
  • RNA- Seq transcriptome analysis
  • tacrolimus 200 ⁇ g/day was further intraperitoneally administered for 3 consecutive days, and papain (50 ⁇ l in 40 ⁇ l of PBS/day) was nasally administered at the same time.
  • RNA-Seq Lungs of each mouse were collected, ILC2 was sorted by the same method as in Test Example 1, and total RNA was isolated from the obtained ILC2 using TRIzol (registered trademark) reagent. Subsequently, by using Lexogen's 3'mRNA-Seq Library Prep Kit, mRNA is isolated, fragmented, reverse transcribed, etc., and a cDNA library (adapter-attached library) of 3'specific sequences related to expressed genes is prepared. It was created. 10 ng of Total RNA was used for the library construction. Sequencing was then performed using an Illumina HiSeq1500, sequenced reads were trimmed for adapter sequences, and the entire mouse genome ( mm10).
  • TRIzol registered trademark
  • tacrolimus susceptibility genes include ILC2 signature (genes) and genes associated with ILC2 activation, specifically arginase, GATA3, Tox, IL-13, Klrg1, IL1R1 (IL-33 receptor gene ) etc. are included.
  • tacrolimus can act on ILC2 and suppress the activation of the innate lymphocytes.
  • mice Isolation of Alveolar Epithelial Cells
  • mice IL-33GFP
  • HBSS HBSS containing DNase (0.001%)/collagenase/dispase (2 ⁇ g/ml, manufactured by Roche) at 37° C.
  • the cells were filtered through 100 ⁇ m and 35 ⁇ m cell strainers (manufactured by BD biosciences) and centrifuged at 4° C. for 5 minutes (800 rpm). Cells were resuspended in erythrocyte lysis buffer and washed with HBSS after 3 minutes.
  • FIG. 4A (1-2) Examination and Results of IL-33 Distribution The results of flow cytometry analysis are shown in FIG. 4A. Looking at the distribution of IL-33 in various epithelial tissue cells, IL-33 was localized in type 2 alveolar epithelial cells (CD45-Podoplanin-SP-C+) (Fig. 4A).
  • FIG. 4B An increase in calcium concentration in type 2 alveolar epithelial cells was observed by papain stimulation (Fig. 4B). That is, it is highly likely that papain-stimulated IL-33 production or release in airway epithelial tissue is via the calcium pathway. It is believed that this pathway is targeted by tacrolimus.
  • the composition, etc. of the present invention can exert an effect on immediate reactions (early phase reactions) of allergic diseases and/or symptoms, or T cell-independent allergic diseases and/or symptoms. It is useful as an allergy remedy.
  • the composition of the present invention, etc. can suppress the activation of ILC2, and is therefore also useful as an ILC2 inactivating agent.
  • the compositions of the present invention can inhibit the calcium signal of the pathway in the release and/or production of IL-33 from cells, and can suppress the release and/or production of IL-33. It is also useful as a 33 inhibitor.

Abstract

The present invention addresses the problem of providing a new pharmaceutical composition that can treat a T cell-independent reaction or an early phase reaction (a symptom in an early phase) of an allergic disease.　Examples of the present invention include a pharmaceutical composition that contains tacrolimus in an effective amount and that is for treating a T cell-independent allergic disease and/or symptom, a pharmaceutical composition for treating a T cell-independent allergic disease and/or symptom in which ILC2s are activated, and a pharmaceutical composition for treating a T cell-independent allergic disease and/or symptom in which the release and/or production of IL-33 is increased.　The present invention can be used for treating an early phase reaction (reaction in an early phase) of an allergic disease and/or symptom or a T cell-independent allergic disease and/or symptom. The present invention is also useful as an ILC2 inactivating agent and an IL-33 inhibitor.

Description

アレルギー治療のための医薬組成物Pharmaceutical composition for treating allergy
（関連出願の相互参照）
　本出願は、２０２１年２月１日に日本国特許庁に出願された日本国出願番号第２０２１－１４０９９号の利益を主張するものである。当該日本国出願は、その出願書類（明細書、特許請求の範囲、図面、要約書）の全体が本明細書に明示されているかのように全ての目的で参照により本明細書に援用される。
　本発明は、アレルギー疾患やその症状の治療薬の技術分野に属する。本発明は、タクロリムスを含有する医薬組成物等に関するものである。 (Cross reference to related applications)
This application claims the benefit of Japanese Application No. 2021-14099 filed with the Japan Patent Office on February 1, 2021. The Japanese application is hereby incorporated by reference for all purposes as if the entire application documents (description, claims, drawings, abstract) were set forth herein. .
The present invention belongs to the technical field of therapeutic agents for allergic diseases and their symptoms. TECHNICAL FIELD The present invention relates to pharmaceutical compositions and the like containing tacrolimus.
　アレルギー疾患においては、一般に、まずＴ細胞非依存的な即時型反応が起こり、続いてＴ細胞依存的な遅発型反応が起こるとされている。臓器移植の際に用いられる免疫抑制剤としても知られるタクロリムス（ＦＫ５０６）は、カルシニューリン（カルシウム依存性セリン・スレオニン脱リン酸化酵素）に結合し、Ｔ細胞核内転写因子（ＮＦＡＴ）の脱リン酸化阻害を介して核内移行を抑制するという作用機序が明らかにされており、現在、喘息、アトピー性皮膚炎、関節リウマチ等のアレルギー疾患における遅発型反応の治療にも用いられている。例えば、特許文献１には、遅発型の喘息反応および気道反応亢進状態を抑制する抗喘息薬として、タクロリムスを含有する薬物が開示されている。また、非特許文献１では、ステロイド抵抗性喘息に対するタクロリムスの有効性が開示されており、タクロリムスによるＴ細胞活性化の抑制がその要因である旨考察されている。 In allergic diseases, it is generally believed that T cell-independent immediate reactions occur first, followed by T cell-dependent delayed reactions. Tacrolimus (FK506), also known as an immunosuppressant used in organ transplantation, binds to calcineurin (calcium-dependent serine-threonine dephosphorylation enzyme) and inhibits dephosphorylation of T-cell nuclear transcription factor (NFAT). Its mechanism of action has been elucidated to suppress nuclear translocation via , and it is currently used for the treatment of late reactions in allergic diseases such as asthma, atopic dermatitis, and rheumatoid arthritis. For example, Patent Document 1 discloses a drug containing tacrolimus as an anti-asthmatic drug that suppresses late-onset asthmatic reactions and airway hyperreactivity. In addition, Non-Patent Document 1 discloses the efficacy of tacrolimus for steroid-resistant asthma, and considers that the suppression of T cell activation by tacrolimus is a factor.
　一方、アレルギー疾患または症状のうちのＴ細胞非依存的な即時型反応、例えば、プロテアーゼアレルゲン誘導気道炎症における即時型反応系においては、アレルゲンは２型自然免疫細胞（ＩＬＣ２）を活性化させ、ＩＬＣ２由来２型サイトカイン誘導によるＴ細胞非依存的な気道炎症を引き起こすことが知られている。このＩＬＣ２からの２型サイトカイン産生には、タクロリムスの標的であるカルシウムシグナル経路は関与していない（非特許文献２）。
  On the other hand, in allergic diseases or symptoms that are T cell-independent immediate responses, for example, in the immediate response system in protease allergen-induced airway inflammation, allergens activate type 2 innate immune cells (ILC2). It is known to cause T cell-independent airway inflammation induced by derived type 2 cytokines. The calcium signal pathway, which is the target of tacrolimus, is not involved in this type 2 cytokine production from ILC2 (Non-Patent Document 2).
特開平０３－２９１２２５号公報JP-A-03-291225
　プロテアーゼアレルゲン誘導気道炎症の即時型反応に代表されるような、アレルギー疾患の早期相、あるいはＴ細胞非依存的な反応については、タクロリムスが有効であるとは考えられていない。
　本発明は、アレルギー疾患のＴ細胞非依存的な反応、あるいは即時型反応（早期相の症状）を治療し得る新たな医薬組成物を提供することを主な課題とする。
  Tacrolimus is not believed to be effective against the early phase of allergic disease or T cell-independent reactions, such as the immediate reaction of protease allergen-induced airway inflammation.
The main object of the present invention is to provide a new pharmaceutical composition capable of treating T cell-independent reactions or immediate reactions (early phase symptoms) of allergic diseases.
　本発明者らは、鋭意検討を重ねた結果、タクロリムスが意外にもアレルギー疾患および／または症状のＴ細胞非依存的な反応、あるいは即時型反応の治療にも有効であることを見出し、本発明を完成するに到った。 As a result of extensive studies, the present inventors have unexpectedly found that tacrolimus is also effective in treating T cell-independent reactions or immediate reactions in allergic diseases and/or symptoms. completed.
　本発明として、例えば、以下のものを挙げることができる。
［１］タクロリムスを有効量含有する、Ｔ細胞非依存型のアレルギー疾患および／または症状を治療するための医薬組成物。
［２］プロテアーゼアレルゲンにより引き起こされたＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、上記［１］に記載の医薬組成物。
［３］ＩＬＣ２が活性化しているＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、上記［１］または［２］に記載の医薬組成物。
［４］ＩＬＣ２の活性化を抑制することでＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、上記［１］～［３］のいずれか一項に記載の医薬組成物。
［５］ＩＬ－３３の放出および/または産生が亢進しているＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、上記［１］～［４］のいずれか一項に記載の医薬組成物。
［６］ＩＬ－３３の放出および／または産生を抑制することでＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、上記［１］～［５］のいずれか一項に記載の医薬組成物。
［７］前記アレルギー症状が呼吸器アレルギー症状である、上記［１］～［６］のいずれか一項に記載の医薬組成物。
［８］前記呼吸器アレルギー症状が気道炎症である、上記［７］に記載の医薬組成物。
［９］前記呼吸器アレルギー症状が喘息である、上記［７］に記載の医薬組成物。
［１０］さらに他のアレルギー治療剤を組み合わせてなる、上記［１］～［９］のいずれか一項に記載の医薬組成物。
［１１］医薬組成物が局所適用型製剤または内服製剤である、上記［１］～［１０］のいずれか一項に記載の医薬組成物。 Examples of the present invention include the following.
[1] A pharmaceutical composition for treating T cell-independent allergic diseases and/or symptoms, containing an effective amount of tacrolimus.
[2] The pharmaceutical composition according to [1] above, which is for treating T cell-independent allergic diseases and/or symptoms caused by protease allergens.
[3] The pharmaceutical composition according to [1] or [2] above, for treating T cell-independent allergic diseases and/or symptoms in which ILC2 is activated.
[4] The pharmaceutical composition according to any one of [1] to [3] above, which is for treating T cell-independent allergic diseases and/or symptoms by suppressing activation of ILC2.
[5] Any one of [1] to [4] above for treating T-cell independent allergic diseases and/or symptoms in which IL-33 release and/or production are enhanced pharmaceutical composition of
[6] Any one of [1] to [5] above for treating T cell-independent allergic diseases and/or symptoms by suppressing the release and/or production of IL-33 pharmaceutical composition of
[7] The pharmaceutical composition according to any one of [1] to [6] above, wherein the allergic symptoms are respiratory allergic symptoms.
[8] The pharmaceutical composition of [7] above, wherein the respiratory allergy symptom is airway inflammation.
[9] The pharmaceutical composition of [7] above, wherein the respiratory allergy symptom is asthma.
[10] The pharmaceutical composition according to any one of [1] to [9] above, which is further combined with another anti-allergic agent.
[11] The pharmaceutical composition according to any one of [1] to [10] above, which is a topical preparation or an internal preparation.
［１２］前記局所適用型製剤とネブライザーとを組み合わせた、キット。
  [12] A kit in which the topical preparation and a nebulizer are combined.
　本発明によれば、アレルギー疾患および／または症状の即時型反応（早期相反応）、あるいはＴ細胞非依存型のアレルギー疾患および／または症状を治療するために用いることができる。また、本発明によれば、ＩＬＣ２の活性化を抑制することができる。そして、本発明によれば、細胞からのインターロイキン３３（ＩＬ－３３）の放出および／または産生における経路のカルシウムシグナルを阻害することができ、ＩＬ－３３の放出および／または産生を抑制し得る。
  The present invention can be used to treat immediate reactions (early phase reactions) of allergic diseases and/or symptoms, or non-T cell dependent allergic diseases and/or symptoms. Moreover, according to the present invention, the activation of ILC2 can be suppressed. And according to the present invention, it is possible to inhibit the calcium signal of the pathway in the release and/or production of interleukin 33 (IL-33) from cells, and to suppress the release and/or production of IL-33. .
図Ａは、野生型（ＷＴ）マウスと、パパインを３日間連続点鼻投与し２４時間後のＷＴ、タクロリムス投与、そしてデキサメタゾン投与の各マウスの肺での好酸球（上図）および活性化好酸球（下図）のフローサイトメトリー解析結果である。図Ｂは、好酸球（左図）および活性化好酸球（右図）の各細胞数を、当該各マウスについて定量したグラフである。図Ｃは、図Ａで解析した各マウスにおける、肺組織の過ヨウ素酸シッフ（ＰＡＳ）染色（上図）およびヘマトキシリンエオジン（ＨＥ）染色（下図）の結果を示した写真である。Panel A shows eosinophils (upper panel) and activation in the lungs of wild-type (WT) mice, and WT, tacrolimus, and dexamethasone-treated mice 24 hours after nasal administration of papain for 3 consecutive days. These are the results of flow cytometry analysis of eosinophils (lower diagram). FIG. B is a graph quantifying the numbers of eosinophils (left panel) and activated eosinophils (right panel) for each mouse. FIG. C is a photograph showing the results of periodic acid-Schiff (PAS) staining (upper panel) and hematoxylin-eosin (HE) staining (lower panel) of lung tissue in each mouse analyzed in FIG. パパイン投与マウスの肺におけるＩＬＣ２のＲＮＡ－Ｓｅｑ解析結果を示した図である。図Ａは、ＩＬＣ２中の各遺伝子の発現量を示す散布図である。横軸は、タクロリムス非投与マウスにおける発現量（ＲＰＭ）を表し、縦軸は、タクロリムス投与マウスにおける発現量（ＲＰＭ）を表す。図Ｂは、ＩＬＣ２シグネチャー（遺伝子）のヒートマップである。図Ｃは、ＩＬＣ２活性化に関連する遺伝子のヒートマップである。FIG. 2 shows the results of RNA-Seq analysis of ILC2 in the lungs of papain-administered mice. FIG. A is a scatter diagram showing the expression level of each gene in ILC2. The horizontal axis represents the expression level (RPM) in tacrolimus-unadministered mice, and the vertical axis represents the expression level (RPM) in tacrolimus-administered mice. Panel B is a heatmap of the ILC2 signature (gene). Panel C is a heatmap of genes associated with ILC2 activation. 図Ａは、ＷＴマウスと、ＩＬ－３３を３日間連続点鼻投与し２４時間後のＷＴ、タクロリムス投与、そしてデキサメタゾン投与の各マウスの肺での好酸球（上図）および活性化好酸球（下図）のフローサイトメトリー解析結果である。図Ｂは、好酸球（左図）および活性化好酸球（右図）の各細胞数を、当該各マウスについて定量したグラフである。図Ｃは、図Ａで解析した各マウスにおける、肺組織の過ヨウ素酸シッフ（ＰＡＳ）染色（上図）およびヘマトキシリンエオジン（ＨＥ）染色（下図）の結果を示した写真である。Panel A shows eosinophils (upper panel) and activated eosinophils in the lungs of WT mice and WT, tacrolimus-treated, and dexamethasone-treated mice 24 hours after 3 consecutive days of intranasal administration of IL-33. These are the results of flow cytometry analysis of spheres (bottom). FIG. B is a graph quantifying the numbers of eosinophils (left panel) and activated eosinophils (right panel) for each mouse. FIG. C is a photograph showing the results of periodic acid-Schiff (PAS) staining (upper panel) and hematoxylin-eosin (HE) staining (lower panel) of lung tissue in each mouse analyzed in FIG. 図Ａは、マウス肺上皮細胞（１型および２型肺胞上皮細胞）における、ＩＬ－３３発現細胞のフローサイトメトリー解析結果である。ＩＬ－３３が２型肺胞上皮細胞に局在していることを示している。図Ｂは、２型肺胞上皮細胞における、パパイン刺激後（上図）およびイオノマイシン刺激後（下図）の時間経過に伴う細胞内カルシウム濃度の蛍光強度測定結果（写真）である。上下図ともに、上段が細胞の顕微鏡画像、下段が蛍光強度イメージング画像を表す。Figure A is the results of flow cytometry analysis of IL-33-expressing cells in mouse lung epithelial cells (type 1 and type 2 alveolar epithelial cells). It shows that IL-33 localizes to type 2 alveolar epithelial cells. FIG. B shows fluorescence intensity measurement results (photographs) of intracellular calcium concentration over time after papain stimulation (upper diagram) and ionomycin stimulation (lower diagram) in type 2 alveolar epithelial cells. In both the upper and lower figures, the upper row shows microscopic images of cells, and the lower row shows fluorescence intensity imaging images.
　以下、本発明について詳述する。
１　本発明に係る医薬組成物
　本発明に係る医薬組成物（以下、「本発明組成物」という。）は、タクロリムスを有効量含有する医薬組成物であって、Ｔ細胞非依存型のアレルギー疾患および／または症状を治療するために用いられることを特徴とする。本発明組成物は、プロテアーゼアレルゲンによって引き起こされたアレルギー疾患および／または症状を治療するために用いることができる。本発明組成物は、ＩＬＣ２の活性化を抑制することができ、ＩＬＣ２が活性化しているアレルギー疾患および／または症状を治療するために用いることができる。また、本発明組成物は、細胞におけるＩＬ－３３の放出および／または産生を抑制することができ、ＩＬ－３３の放出および／または産生が亢進しているアレルギー疾患および／または症状を治療するために用いることができる。 The present invention will be described in detail below.
1. Pharmaceutical composition according to the present invention A pharmaceutical composition according to the present invention (hereinafter referred to as "the composition of the present invention") is a pharmaceutical composition containing an effective amount of tacrolimus, which is used to treat non-T cell-dependent allergic diseases. and/or used to treat symptoms. The compositions of the invention can be used to treat allergic diseases and/or symptoms caused by protease allergens. The composition of the present invention can suppress the activation of ILC2 and can be used to treat allergic diseases and/or symptoms in which ILC2 is activated. In addition, the composition of the present invention can suppress the release and/or production of IL-33 in cells, and is used to treat allergic diseases and/or symptoms in which the release and/or production of IL-33 is enhanced. can be used for
　ここで「治療」とは、予防や改善なども含む概念である。予防には、感作予防としての一次予防、疾患発症予防としての二次予防、そして増悪予防としての三次予防が含まれる。中でも、本発明組成物は、二次予防および三次予防において、より効果を奏し得る。また、予防には、長期治療または慢性期治療における予防も含まれる。本発明における治療は、Ｔ細胞非依存型のアレルギー疾患および／または症状の治療であれば特に制限されないが、通常、発作治療としての急性期治療を意味する。発作治療としての急性期治療には、アレルギー疾患共通の病態であるアレルギー反応あるいはアレルギー性炎症に対する治療と、アレルギー反応発現の場としての臓器特異的な治療とが含まれる。 Here, "treatment" is a concept that includes prevention and improvement. Prevention includes primary prevention as prevention of sensitization, secondary prevention as prevention of disease onset, and tertiary prevention as prevention of exacerbation. Among others, the composition of the present invention can be more effective in secondary prevention and tertiary prevention. Prophylaxis also includes prophylaxis in long-term or chronic phase therapy. Treatment in the present invention is not particularly limited as long as it is treatment of T cell-independent allergic diseases and/or symptoms, but usually means acute treatment as treatment for stroke. Acute treatment as an attack treatment includes treatment for allergic reaction or allergic inflammation, which is a common pathology of allergic diseases, and organ-specific treatment as a site of allergic reaction manifestation.
　「Ｔ細胞非依存型」のアレルギー疾患および／または症状とは、原則として、Ｔ細胞を介さず自然免疫細胞（例えば、２型自然免疫細胞、好塩基球、マスト細胞。）により引き起こされるアレルギー疾患および／または症状を意味し、急性期における即時型反応あるいは早期相反応に該当する。ただし、１．４項にて後述するように、アレルギー疾患（例えば、気道炎症。）では早期相においても２型ヘルパーＴ細胞（ＴＨ２）が関与することがあり得、本明細書および特許請求の範囲における「Ｔ細胞非依存型」は、特記の無い限りこの場合をも含む。 "T cell-independent" allergic diseases and/or symptoms are, in principle, allergic diseases caused by innate immune cells (e.g., type 2 innate immune cells, basophils, mast cells) without the intervention of T cells. and/or symptoms, which correspond to immediate or early phase reactions in the acute phase. However, as described later in Section 1.4, allergic diseases (e.g., airway inflammation) may involve type 2 helper T cells (T H 2) even in the early phase. Unless otherwise specified, the term "T cell-independent" in the claims also includes this case.
１．１　タクロリムス
　本発明組成物は、タクロリムスを有効成分として含有する。タクロリムスは、ＦＫ５０６とも称され、化学名(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-5,19-Dihydroxy-3-{(1E)-2-[(1R,3R,4R)-4-hydroxy3-methoxycyclohexyl]-1-methylethenyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(prop-2-en-1-yl)-15,19-epoxy5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetroneで表される２３員環マクロライド系ラクトンである。タクロリムスは、遊離型または医学的に許容される塩、あるいはそれらの水和物等の溶媒和物またはアナログであり得る。医薬としては通常、構造式 1.1 Tacrolimus The composition of the present invention contains tacrolimus as an active ingredient. Tacrolimus, also known as FK506, has the chemical name (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-5,19-Dihydroxy-3-{(1E)-2 -[(1R,3R,4R)-4-hydroxy3-methoxycyclohexyl]-1-methylethenyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(prop-2-en-1-yl )-15,19-epoxy5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-3H-pyrido[2,1-c] It is a 23-membered ring macrolide lactone represented by [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone. Tacrolimus can be in free form or a pharmaceutically acceptable salt, or a solvate or analogue thereof, such as a hydrate. As a drug, the structural formula is usually
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
で示される１水和物として使用される。本明細書および特許請求の範囲において単にタクロリムスという場合、遊離型のタクロリムスのみでなく、塩、溶媒和物などのタクロリムスであってもよい。また、タクロリムスの結晶相、非晶質相、半結晶相も本発明に係るタクロリムスに含まれる。 used as a monohydrate represented by In the present specification and the scope of claims, simply referring to tacrolimus may refer not only to tacrolimus in free form but also to tacrolimus in the form of salts, solvates, and the like. Tacrolimus according to the present invention also includes crystalline phases, amorphous phases, and semi-crystalline phases of tacrolimus.
　本発明組成物は、有効成分以外に担体を適量含有することができる。当該担体として、例えば、固形、半固形または液状の希釈剤、充填剤、その他の処方用の助剤を挙げることができる。これらの担体を一種または二種以上含有することができる。 The composition of the present invention can contain an appropriate amount of carrier in addition to the active ingredient. Such carriers can include, for example, solid, semi-solid or liquid diluents, fillers, and other formulation aids. One or more of these carriers can be contained.
１．２　アレルゲン
　本発明組成物は、アレルギー疾患および／または症状を治療するために用いられる。当該アレルギー疾患および／または症状を引き起こすアレルゲンに原則として制限はないが、インターロイキン３３（ＩＬ－３３）はここでいうアレルゲンには含まれない。アレルゲンとしては、例えば、吸入抗原、食物抗原、接触抗原（染料、化粧品、外用剤、金属等）、薬物抗原（抗菌薬、抗血清等）、昆虫抗原（ハチ毒、カ、ゴキブリ等）、ワクチンを挙げることができる。 1.2 Allergens The compositions of the present invention are used to treat allergic diseases and/or conditions. Allergens that cause allergic diseases and/or symptoms are not limited in principle, but interleukin 33 (IL-33) is not included in the allergens referred to herein. Examples of allergens include inhalant antigens, food antigens, contact antigens (dyes, cosmetics, external agents, metals, etc.), drug antigens (antibacterial agents, antiserum, etc.), insect antigens (bee venom, mosquitoes, cockroaches, etc.), vaccines. can be mentioned.
　この中、吸入抗原としては、例えば、アルテルナリア、クモノスカビ、ケカビ、コウジ、ダニを挙げることができる。 Among these, inhaled antigens include, for example, Alternaria, Rhizopus, Malaria, Aspergillus, and mites.
　また、食物抗原としては、例えば、パパイヤ、パイナップル、ショウガ、イチジク、キウイフルーツ、納豆、キノコ類（マイタケ、ヒラタケ等）を挙げることができる。 In addition, food antigens include, for example, papaya, pineapple, ginger, fig, kiwifruit, natto, and mushrooms (maitake, oyster mushroom, etc.).
　上記アレルゲンの中の少なくともいずれかに含まれるアレルゲン物質であって、本発明組成物による治療対象として適当なものとしては、例えば、プロテアーゼを挙げることができる。 Examples of allergen substances contained in at least one of the above allergens that are suitable for treatment with the composition of the present invention include proteases.
　当該プロテアーゼとしては、例えば、システインプロテアーゼ、セリンプロテアーゼ、アスパラギン酸プロテアーゼ（ペプシン等）を挙げることができる。 Examples of such proteases include cysteine proteases, serine proteases, and aspartic proteases (pepsin, etc.).
　この中、システインプロテアーゼとしては、例えば、パパイン、フィシン、ブロメライン、ジンギパイン、アクチニジン、カスパーゼ、カテプシンを挙げることができる。 Among them, cysteine proteases include, for example, papain, ficin, bromelain, gingipain, actinidin, caspase, and cathepsin.
　また、セリンプロテアーゼとしては、例えば、トリプシン、キモトリプシン、スブチリシン、ナットウキナーゼを挙げることができる。 Examples of serine proteases include trypsin, chymotrypsin, subtilisin, and nattokinase.
１．３　自然免疫細胞の活性化抑制
　本発明組成物は、ＩＬＣ２（Ｇｒｏｕｐ ２ ｉｎｎａｔｅ ｌｙｍｐｈｏｉｄ ｃｅｌｌ：２型自然免疫細胞あるいは２型自然リンパ球）の活性化を抑制することができる。また、本発明組成物は、ＩＬＣ２が活性化しているアレルギー疾患および／または症状を治療するために用いることができる。 1.3 Suppression of Activation of Innate Immune Cells The composition of the present invention can suppress the activation of ILC2 (Group 2 innate lymphoid cells: type 2 innate lymphocytes or type 2 innate lymphocytes). In addition, the composition of the present invention can be used to treat allergic diseases and/or symptoms in which ILC2 is activated.
　ＩＬＣ２は、リンパ球系に属する自然免疫細胞の一種であり、活性化すると２型サイトカイン（ＩＬ－５、ＩＬ－１３等）を産生し、気管支上皮細胞の過形成、ムチン産生の亢進、肺における好酸球の蓄積や活性化等を誘導する。ＩＬＣ２は、損傷した細胞（例えば、上皮細胞）由来のサイトカインによって活性化され得る。そのようなサイトカインとして、脂質メディエータ（例えば、ＬＴＢ４、ＬＴＥ４、ＰＧＤ２。）、ＴＳＬＰ、ＩＬ－３３、ＩＬ－２５等が挙げられる。また、神経ペプチド（ＮＭＵ等）や性ホルモンによってもＩＬＣ２の活性化は調節され得る。 ILC2 is a kind of innate immune cells belonging to the lymphoid system, and when activated, it produces type 2 cytokines (IL-5, IL-13, etc.), hyperplasia of bronchial epithelial cells, enhancement of mucin production, It induces the accumulation and activation of eosinophils. ILC2 can be activated by cytokines from damaged cells, such as epithelial cells. Such cytokines include lipid mediators (eg, LTB4, LTE4, PGD2), TSLP, IL-33, IL-25, and the like. ILC2 activation can also be regulated by neuropeptides (NMU, etc.) and sex hormones.
　実施例にて後述する通り、タクロリムスは、ＩＬＣ２の活性化に関連する遺伝子の発現を阻害し得る。したがって、本発明組成物は、ＩＬＣ２の活性化を抑制し得、また、当該活性化に伴うアレルギー疾患および／または症状を治療するために用い得る。本発明組成物は、脂質メディエータまたは神経ペプチドによるＩＬＣ２の活性化を選択的に抑制することができ、中でも、ＬＴＣ４またはＮＭＵによるＩＬＣ２活性化をより選択的に抑制することができる。 As described later in the Examples, tacrolimus can inhibit the expression of genes associated with ILC2 activation. Therefore, the composition of the present invention can suppress the activation of ILC2, and can be used to treat allergic diseases and/or symptoms associated with the activation. The composition of the present invention can selectively suppress ILC2 activation by lipid mediators or neuropeptides, and more selectively suppress ILC2 activation by LTC4 or NMU.
１．４　インターロイキン３３（ＩＬ－３３）
　本発明組成物は、細胞におけるＩＬ－３３の放出および／または産生を抑制することができる。また、本発明組成物は、ＩＬ－３３の放出および／または産生が亢進しているアレルギー疾患および／または症状を治療するために用いることができる。 1.4 Interleukin 33 (IL-33)
The composition of the present invention can inhibit the release and/or production of IL-33 in cells. In addition, the composition of the present invention can be used to treat allergic diseases and/or symptoms in which IL-33 release and/or production are enhanced.
　ＩＬ－３３は、主に上皮細胞の核内に存在するタンパク質であり、アレルゲン等の抗原の曝露によって細胞外へ放出される。放出されるとアラーミンとして自然免疫細胞（例えば、ＩＬＣ２、好塩基球、マスト細胞。）を活性化させ、サイトカイン誘導によるＴ細胞非依存的な炎症を引き起こす。
　既述の通り、タクロリムスが標的とするカルシウムシグナル回路は、ＩＬ－３３誘導による自然免疫細胞のサイトカイン産生経路に関与しない。しかし、実施例にて後述する通り、その上流の、細胞からのＩＬ－３３産生・放出においてカルシウム経路を介していると考えられ、タクロリムスは、当該経路におけるカルシウムシグナルを阻害し得る。 IL-33 is a protein mainly present in the nuclei of epithelial cells, and is released outside the cells upon exposure to antigens such as allergens. When released, it activates innate immune cells (eg, ILC2, basophils, mast cells) as an alarmin, causing cytokine-induced T cell-independent inflammation.
As already mentioned, the calcium signaling circuit targeted by tacrolimus is not involved in the IL-33-induced cytokine production pathway of innate immune cells. However, as will be described later in Examples, the upstream IL-33 production/release from cells is thought to be mediated by the calcium pathway, and tacrolimus can inhibit the calcium signal in this pathway.
　これにより、本発明組成物は、細胞（例えば、上皮細胞。中でも、気道上皮細胞。特に、２型肺胞上皮細胞。）におけるＩＬ－３３の放出および／または産生を抑制することができ、また、ＩＬ－３３の放出および／または産生が亢進しているアレルギー疾患および／または症状を治療するために用いることができる。 Thereby, the composition of the present invention can suppress the release and/or production of IL-33 in cells (e.g., epithelial cells, especially airway epithelial cells, particularly type 2 alveolar epithelial cells), and , can be used to treat allergic diseases and/or conditions in which IL-33 release and/or production is increased.
　なお、ＩＬＣ２だけでなく、２型ヘルパーＴ細胞（ＴＨ２）においてもＩＬ－３３により２型サイトカイン産生が誘導される（例えば、Guo, L., et al., Nature Immunology 2015 No.16:1051-1059。）。すなわち、ＴＨ２も早期相のアレルギー症状（例えば、気道炎症。）に関与し得る。本発明組成物は、この場合においても有効に用い得る。 In addition, IL-33 induces type 2 cytokine production not only in ILC2 but also in type 2 helper T cells (T H 2) (for example, Guo, L., et al., Nature Immunology 2015 No.16: 1051-1059.). Thus, T H2 may also be involved in early-phase allergic symptoms (eg, airway inflammation). The composition of the present invention can also be effectively used in this case.
１．５　アレルギー症状
　本発明組成物の治療対象となるアレルギー症状は、アレルギー疾患の急性期の反応として起こり得るものであれば特に制限はない。例えば、呼吸器アレルギー症状、アレルギー性鼻炎、好酸球性副鼻腔炎、アレルギー性結膜炎、アトピー性皮膚炎を挙げることができる。 1.5 Allergic Symptoms The allergic symptoms to be treated by the composition of the present invention are not particularly limited as long as they can occur as acute reactions of allergic diseases. Examples include respiratory allergy symptoms, allergic rhinitis, eosinophilic sinusitis, allergic conjunctivitis, and atopic dermatitis.
　上記呼吸器アレルギー症状としては、気道炎症、喘息、気道上皮の杯細胞過形成、気道過敏性の亢進等が挙げられる。 The above respiratory allergy symptoms include airway inflammation, asthma, goblet cell hyperplasia of the airway epithelium, and increased airway hyperresponsiveness.
　本発明組成物による治療に好適な気道炎症としては、例えば、好酸球性気道炎症を挙げることができる。 Airway inflammation suitable for treatment with the composition of the present invention includes, for example, eosinophilic airway inflammation.
　本発明組成物による治療に好適な喘息としては、例えば、高用量吸入ステロイド薬治療に対して抵抗性を呈する重症喘息、ウイルス（インフルエンザウイルス、ライノウイルス、コロナウイルス等）感染に伴う喘息増悪を挙げることができる。 Examples of asthma suitable for treatment with the composition of the present invention include severe asthma that exhibits resistance to high-dose inhaled corticosteroid therapy, and exacerbation of asthma associated with virus (influenza virus, rhinovirus, coronavirus, etc.) infection. be able to.
１．６　他のアレルギー疾患治療薬との併用
　本発明組成物は、必要や目的に応じて任意の他のアレルギー疾患治療薬を有効量含むことができる。当該アレルギー疾患治療薬としては、例えば、喘息長期管理薬、アレルギー性鼻炎治療薬、好酸球性副鼻腔炎治療薬、アレルギー性結膜炎治療薬、アトピー性皮膚炎治療薬が挙げられる。 1.6 Combined use with other therapeutic agents for allergic diseases The composition of the present invention can contain an effective amount of any other therapeutic drug for allergic diseases depending on the need and purpose. The therapeutic drug for allergic diseases includes, for example, asthma long-term control drug, therapeutic drug for allergic rhinitis, therapeutic drug for eosinophilic sinusitis, therapeutic drug for allergic conjunctivitis, and therapeutic drug for atopic dermatitis.
　喘息長期管理薬としては、例えば、副腎皮質ステロイド薬、気管支拡張薬、分子標的治療薬を挙げることができる。 Asthma long-term control drugs include, for example, corticosteroids, bronchodilators, and molecular-targeted drugs.
　副腎皮質ステロイド薬としては、例えば、吸入ステロイド薬（ＩＣＳ）、経口ステロイド薬を挙げることができる。
　この中、ＩＣＳとしては、具体的には、ベクロメタゾン、シクレソニド、フルチカゾン、モメタゾン、ブデソニド等が挙げられる。 Examples of corticosteroid drugs include inhaled steroid drugs (ICS) and oral steroid drugs.
Among them, specific examples of ICS include beclomethasone, ciclesonide, fluticasone, mometasone, budesonide and the like.
　気管支拡張薬としては、例えば、β２刺激薬、抗コリン薬、テオフィリン徐放製剤、ロイコトリエン受容体拮抗薬を挙げることができる。 Bronchodilators include, for example, β2 agonists, anticholinergics, theophylline sustained - release preparations, and leukotriene receptor antagonists.
　この中、β２刺激薬としては、長時間作用性β２刺激薬（ＬＡＢＡ：例えば、サルメテロール、ホルモテロール。）、超長時間作用性β２刺激薬（ｕｌｔｒａ－ＬＡＢＡ：例えば、インダカテロール、ビランテロール、オロダテロール、カルモテロール。）等が挙げられる。 Among them, β2 agonists include long - acting β2 - agonists (LABA: e.g., salmeterol, formoterol), ultra-long-acting β2 - agonists (ultra-LABA: e.g., indacaterol, vilanterol, , olodaterol, carmoterol.) and the like.
　また、抗コリン薬としては、チオトロピウム、グリコピロニウム、アクリジニウム、ウメクリジニウム、トロスピウム等が挙げられる。 Anticholinergics include tiotropium, glycopyrronium, acridinium, umeclidinium, trospium, and the like.
　分子標的治療薬としては、例えば、ヒト化抗ヒトＩｇＥ抗体、ヒト化抗ＩＬ－５抗体、抗ＩＬ－１３抗体を挙げることができる。 Examples of molecular targeted therapeutic agents include humanized anti-human IgE antibodies, humanized anti-IL-5 antibodies, and anti-IL-13 antibodies.
　上記各アレルギー疾患治療薬は、それぞれ一種であっても二種以上の併用であってもよい。 Each of the above therapeutic drugs for allergic diseases may be used alone or in combination of two or more.
１．７　投与経路
　本発明組成物の投与経路に特に制限はない。局所投与（例えば、吸入投与、経粘膜投与、経鼻投与、経眼投与、経皮投与。）、経口投与、その他の非経口投与（例えば、静脈内投与、門脈内投与、皮下投与、点滴投与等の注射投与。）のいずれであってもよいが、本発明組成物は、局所適用型製剤または内服製剤（経口投与製剤）であることが好ましい。 1.7 Route of Administration The route of administration of the composition of the present invention is not particularly limited. Local administration (e.g., inhalation administration, transmucosal administration, nasal administration, ocular administration, transdermal administration), oral administration, other parenteral administration (e.g., intravenous administration, intraportal administration, subcutaneous administration, drip infusion injection administration, etc.), but the composition of the present invention is preferably a topical preparation or an internal preparation (orally administered preparation).
　本発明組成物の投与経路は、上記の中の一経路であっても二経路以上であってもよい。 The administration route of the composition of the present invention may be one route or two or more routes among the above.
１．８　剤型
　本発明組成物の剤型は、上記の通り、その投与経路に応じて吸入剤、点鼻剤、眼科用製剤、外用剤、内服製剤（経口投与製剤）、非経口投与製剤のいずれの形態をもとることができる。本発明組成物は徐放性製剤であってもよい。 1.8 Dosage Forms As described above, the dosage forms of the composition of the present invention are inhalants, nasal drops, ophthalmic preparations, external preparations, internal preparations (orally administered preparations), and parenteral preparations, depending on the route of administration. can take any form. The composition of the present invention may be a sustained release formulation.
１．８．１　吸入剤
　本発明組成物が吸入剤の形態である場合、かかる吸入剤としては、後述するネブライザー（噴霧器、吸入器等）を用いて投与できる剤型であれば特に限定されず、例えば、液体噴霧剤、エアロゾル剤、ドライパウダー剤を挙げることができる。 1.8.1 Inhalant When the composition of the present invention is in the form of an inhalant, the inhalant is not particularly limited as long as it can be administered using a nebulizer (nebulizer, inhaler, etc.) described below. , for example, liquid sprays, aerosols, and dry powders.
　液体噴霧剤および液体基剤のエアロゾル剤は、蒸留水、もしくは生理食塩水、緩衝溶液等の無菌の水溶液に有効成分を溶解することによって、または、さらに添加剤を加えて溶解することによって調製することができる。 Liquid sprays and liquid-based aerosols are prepared by dissolving the active ingredient in distilled water or a sterile aqueous solution such as saline or buffer solution, or by dissolving the active ingredient with an additional excipient. be able to.
　当該添加剤としては、例えば、緩衝剤（ホウ酸、リン酸一水素ナトリウム、リン酸二水素ナトリウム等）、保存剤または保存補助剤（塩化ベンザルコニウム、塩化ベンゼトニウム、クロロブタノール等）、張度調整剤（塩化ナトリウム等）、界面活性剤、可溶化剤、安定化剤、鎮痛増進剤、増粘剤（例えば、ラクトース、マンニトール、マルトース等の多糖；ヒアルロン酸ナトリウム、ヒアルロン酸カリウム等のヒアルロン酸またはその塩；コンドロイチン硫酸等のムコ多糖；ポリアクリル酸ナトリウム、カルボキシビニルポリマー、架橋ポリアクリレート、ポリビニルアルコール、メチルセルロース、グリセリン等のポリマー）、皮膚軟化剤、ｐＨ調整剤、滑沢剤を挙げることができる。
　また、投与直前に、有効成分等を含有した粉末組成物を溶解させることにより調製してもよい。 Such additives include, for example, buffering agents (boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives or preservatives (benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), tonicity Regulators (sodium chloride, etc.), surfactants, solubilizers, stabilizers, analgesic agents, thickeners (e.g., polysaccharides such as lactose, mannitol, maltose; hyaluronic acid, such as sodium hyaluronate and potassium hyaluronate) or salts thereof; mucopolysaccharides such as chondroitin sulfate; polymers such as sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, methylcellulose, and glycerin), emollients, pH adjusters, and lubricants. can.
Alternatively, it may be prepared by dissolving a powder composition containing an active ingredient and the like immediately before administration.
　ドライパウダーおよび固体基剤のエアロゾル剤は、有効成分を下記のような適当な平均粒子径となるように微細化し、次いで同様に細かくした医薬用担体、例えば、結晶性ラクトース等の可食性炭水化物と混合することにより製造することができる。また、任意に風味剤、保存剤、分散剤、着色剤、香料等を添加することができる。 Dry powders and solid-based aerosols are prepared by micronizing the active ingredient to a suitable average particle size, as described below, followed by a similarly finely divided pharmaceutical carrier, for example, an edible carbohydrate such as crystalline lactose. It can be manufactured by mixing. Flavoring agents, preservatives, dispersing agents, coloring agents, perfumes and the like can also be optionally added.
　液体噴霧剤、エアロゾル剤、およびドライパウダー剤は、通常、微細粒子の状態で投与される。当該微細粒子の平均粒子径に特に制限はない。 Liquid sprays, aerosols, and dry powders are usually administered in the form of fine particles. There is no particular limitation on the average particle size of the fine particles.
１．８．２　点鼻剤
　本発明組成物が点鼻剤の形態である場合、かかる点鼻剤としては、例えば、液体スプレー剤、ミスト剤、エアロゾル剤を挙げることができる。 1.8.2 Nasal drops When the composition of the present invention is in the form of nasal drops, examples of such nasal drops include liquid sprays, mists, and aerosols.
　点鼻剤は、蒸留水、もしくは生理食塩水、緩衝溶液等の無菌の水溶液に有効成分を溶解することによって、または粉末組成物を使用前に加えて溶解することによって調製することができる。また、液体噴霧剤等で用いたものと同様な添加剤を点鼻剤においても用いることができる。 Nasal drops can be prepared by dissolving the active ingredient in distilled water or sterile aqueous solutions such as saline and buffer solutions, or by adding and dissolving the powder composition prior to use. Additives similar to those used in liquid sprays and the like can also be used in nasal drops.
１．８．３　眼科用製剤
　本発明組成物が眼科用製剤の形態である場合、かかる眼科用製剤としては、例えば、点眼剤、眼科用軟膏剤を挙げることができる。 1.8.3 Ophthalmic formulation When the composition of the present invention is in the form of an ophthalmic formulation, examples of such ophthalmic formulation include eye drops and ophthalmic ointments.
　点眼剤は、蒸留水、もしくは生理食塩水、緩衝溶液等の無菌の水溶液に有効成分を溶解することによって、または粉末組成物を使用前に加えて溶解することによって調製することができる。また、液体噴霧剤等で用いたものと同様の添加剤を点眼剤においても用いることができる。 Eye drops can be prepared by dissolving the active ingredient in distilled water or a sterile aqueous solution such as saline or buffer solution, or by adding and dissolving the powder composition prior to use. In addition, the same additives as those used in the liquid spray and the like can also be used in the eye drops.
　眼科用軟膏剤は、慣用の方法によって有効成分と基剤とを混合することによって調製することができる。当該基剤として、例えば、ワセリン、プラスチベース、マクロゴール、セレン５０が挙げられるが、それらに限定されない。また、親水性を高める為の乳化剤等の界面活性剤をはじめ、その他液体噴霧剤等で用いたものと同様の添加剤を眼科用軟膏剤においても用いることができる。 An ophthalmic ointment can be prepared by mixing an active ingredient and a base by a conventional method. Examples of the base include, but are not limited to, vaseline, plastibase, macrogol, and selenium 50. In addition, surfactants such as emulsifiers for increasing hydrophilicity and other additives similar to those used in liquid sprays can also be used in ophthalmic ointments.
１．８．４　外用剤
　本発明組成物が外用剤の形態である場合、かかる外用剤としては、皮膚の局所表面に有効成分を直接投与できる剤型であれば特に限定されず、例えば、軟膏剤、液剤（懸濁剤、乳剤、ローション剤等）、パップ剤、テープ剤、エアゾール剤、および外用散剤を挙げることができる。 1.8.4 External preparation When the composition of the present invention is in the form of an external preparation, the external preparation is not particularly limited as long as it is a dosage form that allows direct administration of the active ingredient to the topical surface of the skin. formulations, solutions (suspensions, emulsions, lotions, etc.), poultices, tapes, aerosols, and powders for external use.
　これらの製剤を調製するに際して、有効成分以外に、通常の外用剤を調製するのに使用される各種配合成分を適宜選択して使用することができる。そのような成分として、軟膏剤、クリーム剤、ゲル剤、ないしローション剤の場合には、白色ワセリン、黄色ワセリン、ラノリン、サラシミツロウ、セタノール、ステアリルアルコール、ステアリン酸、硬化油、ゲル化炭化水素、ポリエチレングリコール、流動パラフィン、スクワラン等の基剤；オレイン酸、ミリスチン酸イソプロピル、トリイソオクタン酸グリセリン、クロタミトン、セバシン酸ジエチル、アジピン酸ジイソプロピル、ラウリン酸ヘキシル、脂肪酸、脂肪酸エステル、脂肪族アルコール、植物油等の溶剤および溶解補助剤；トコフェロール誘導体、Ｌ－アスコルビン酸、ジブチルヒドロキシトルエン、ブチルヒドロキシアニソール等の酸化防止剤；パラヒドロキシ安息香酸エステル等の防腐剤；グリセリン、プロピレングリコール、ヒアルロン酸ナトリウム等の保湿剤；ポリオキ シエチレン誘導体、グリセリン脂肪酸エステル、ショ糖脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステル、レシチン等の界面活性剤；カルボキシビニルポリマー、キサンタンガム、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム塩類、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース等の増粘剤等を挙げることができる。さらに、所望により安定剤、保存剤、吸収促進剤、ｐＨ調整剤、その他の適当な添加剤を配合することができる。 When preparing these formulations, in addition to the active ingredient, various compounding ingredients that are used to prepare ordinary external preparations can be appropriately selected and used. In the case of ointments, creams, gels or lotions, such ingredients include white petrolatum, yellow petrolatum, lanolin, white beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oils, gelling hydrocarbons, bases such as polyethylene glycol, liquid paraffin, and squalane; Solvents and solubilizers; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; preservatives such as parahydroxybenzoic acid esters; moisturizing agents such as glycerin, propylene glycol, and sodium hyaluronate; Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, lecithin; carboxyvinyl polymer, xanthan gum, carboxymethylcellulose, carboxymethylcellulose sodium salts, hydroxypropylcellulose, hydroxypropylmethylcellulose and the like. Furthermore, if desired, stabilizers, preservatives, absorption enhancers, pH adjusters, and other suitable additives can be added.
　また、パップ剤については、ポリアクリル酸、ポリアクリル酸共重合体等の粘着付与剤；硫酸アルミニウム、硫酸カリウムアルミニウム、塩化アルミニウム、メタケイ酸アルミン酸マグネシウム、ジヒドロキシアルミニウムアセテート等の架橋剤；ポリアクリル酸ナトリウム、ポリビニルアルコール、ポリビニルピロリドン、ゼラチン、アルギン酸ナトリウム、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム塩類、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース等の増粘剤；グリセリン、ポリエチレングリコール（マクロゴール）、プロピレングリコール、１，３－ブタンジオール等の多価アルコール類；ポリオキシエチレン誘導体等の界面活性剤；Ｌ－メントール等の香料；パラヒドロキシ安息香酸エステル等の防腐剤；精製水等を挙げることができる。さらに、所望により安定剤、保存剤、吸収促進剤、ｐＨ調整剤、その他の適当な添加剤を配合することができる。 As for poultices, tackifiers such as polyacrylic acid and polyacrylic acid copolymers; cross-linking agents such as aluminum sulfate, aluminum potassium sulfate, aluminum chloride, magnesium aluminometasilicate and dihydroxyaluminum acetate; polyacrylic acid; Thickeners such as sodium, polyvinyl alcohol, polyvinylpyrrolidone, gelatin, sodium alginate, carboxymethylcellulose, carboxymethylcellulose sodium salts, hydroxypropylcellulose, hydroxypropylmethylcellulose; glycerin, polyethylene glycol (macrogol), propylene glycol, 1,3- polyhydric alcohols such as butanediol; surfactants such as polyoxyethylene derivatives; fragrances such as L-menthol; preservatives such as parahydroxybenzoate; Furthermore, if desired, stabilizers, preservatives, absorption enhancers, pH adjusters, and other suitable additives can be added.
　テープ剤については、天然ゴム、イソプレンゴム、ポリイソブチレン、ポリブテン、液状ポリイソプレン、スチレン・イソプレン・スチレンブロック共重合体（ＳＩＳブロック共重合体）、スチレン－ブタジエン－スチレンブロック共重合体、スチレン－エチレン・ブチレン－スチレンブロック共重合体や、（メタ）アクリル酸アルキルエステル（共）重合体、ポリアクリル酸エステル、メタクリル酸エステル等のアクリル樹脂等の粘着剤；脂環族飽和炭化水素系樹脂、ロジン系樹脂、テルペン系樹脂等の粘着付与樹脂；液状ゴム、流動パラフィン等の軟化剤；ジブチルヒドロキシトルエン等の酸化防止剤；プロピレングリコール等の多価アルコール；オレイン酸等の吸収促進剤；ポリオキシエチレン誘導体等の界面活性剤、その他の適当な添加剤を配合することができる。また、ポリアクリル酸ナトリウムやポリビニルアルコールのような含水可能な高分子と少量の精製水を加えて含水テープ剤とすることもできる。この場合にあっても、さらに、所望により安定剤、保存剤、吸収促進剤、ｐＨ調整剤、その他の適当な添加剤を配合することができる。 For tape agents, natural rubber, isoprene rubber, polyisobutylene, polybutene, liquid polyisoprene, styrene/isoprene/styrene block copolymer (SIS block copolymer), styrene-butadiene-styrene block copolymer, styrene-ethylene・Adhesives such as acrylic resins such as butylene-styrene block copolymers, (meth)acrylic acid alkyl ester (co)polymers, polyacrylic acid esters, methacrylic acid esters; alicyclic saturated hydrocarbon resins, rosin tackifying resins such as base resins and terpene resins; softeners such as liquid rubber and liquid paraffin; antioxidants such as dibutylhydroxytoluene; polyhydric alcohols such as propylene glycol; absorption enhancers such as oleic acid; Surfactants such as derivatives and other suitable additives can be blended. A water-containing tape can also be prepared by adding a water-absorbable polymer such as sodium polyacrylate or polyvinyl alcohol and a small amount of purified water. Even in this case, if desired, stabilizers, preservatives, absorption enhancers, pH adjusters and other suitable additives can be added.
　エアゾール剤については、軟膏剤、クリーム剤、ゲル剤、懸濁剤、乳剤、液剤およびローション剤等の調製に用いられる白色ワセリン、黄色ワセリン、ラノリン、サラシミツロウ、セタノール、ステアリルアルコール、ステアリン酸、硬化油、ゲル化炭化水素、ポリエチレングリコール、流動パラフィン、スクワラン等の基剤；オレイン酸、ミリスチン酸イソプロピル、アジピン酸ジイソプロピル、セバシン酸イソプロピル、トリイソオクタン酸グリセリン、クロタミトン、セバシン酸ジエチル、ラウリン酸ヘキシル、脂肪酸、脂肪酸エステル、脂肪族アルコール、植物油等の溶剤および溶解補助剤；トコフェロール誘導体、Ｌ－アスコルビン酸、ジブチルヒドロキシトルエン、ブチルヒドロキシアニソール等の酸化防止剤；パラヒドロキシ安息香酸エステル等の防腐剤；グリセリン、プロピレングリコール、ヒアルロン酸ナトリウム等の保湿剤；ポリオキシエチレン誘導体、グリセリン脂肪酸エステル、ショ糖脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステル、レシチン等の界面活性剤；カルボキシビニルポリマー、キサンタンガム、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム塩類、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース等の増粘剤；さらに、各種安定剤、緩衝剤、矯味剤、 懸濁化剤、乳化剤、芳香剤、保存剤、溶解補助剤、その他の適当な添加剤を配合することができる。 For aerosol formulations, white petrolatum, yellow petrolatum, lanolin, white beeswax, cetanol, stearyl alcohol, stearic acid, hardening used in the preparation of ointments, creams, gels, suspensions, emulsions, solutions, lotions, etc. Bases such as oils, gelling hydrocarbons, polyethylene glycol, liquid paraffin, squalane; oleic acid, isopropyl myristate, diisopropyl adipate, isopropyl sebacate, glyceryl triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids , fatty acid esters, fatty alcohols, solvents and solubilizers such as vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylhydroxyanisole; preservatives such as parahydroxybenzoic acid esters; glycerin, Moisturizers such as propylene glycol and sodium hyaluronate; surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, Thickeners such as carboxymethyl cellulose sodium salts, hydroxypropyl cellulose, hydroxypropyl methyl cellulose; furthermore, various stabilizers, buffers, flavoring agents, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, other suitable Additives can be added.
　外用散剤については、バレイショデンプン、コメデンプン、トウモロコシデンプン、タルク、酸化亜鉛等の賦形剤またはその他の適当な添加剤を配合することができる。この場合にあっても、さらに、所望により各種安定剤、保存剤、吸収促進剤、その他の適当な添加剤を配合することができる。 For powders for external use, excipients such as potato starch, rice starch, corn starch, talc, zinc oxide, etc., or other suitable additives can be added. Even in this case, if desired, various stabilizers, preservatives, absorption enhancers and other suitable additives can be added.
　上記外用剤を調製する方法としては特に限定されず、所望の剤型に応じて、各成分および必要に応じた基剤成分をよく混練する等の常法により製造することができる。また、パップ剤やテープ剤の調製においては、混練した混合物を剥離紙上に展延、乾燥し、さらに柔軟な支持体と貼り合わせ、所望の大きさに裁断することにより調製することができる。 The method for preparing the external preparation is not particularly limited, and it can be produced by a conventional method such as thoroughly kneading each component and, if necessary, a base component according to the desired dosage form. Also, poultices and tapes can be prepared by spreading the kneaded mixture on a release paper, drying it, attaching it to a flexible support, and cutting it into a desired size.
　上記外用剤は、例えば、軟膏剤、液剤（懸濁剤、乳剤、ローション剤等）、エアゾール剤および外用散剤の場合には、皮膚患部に塗布等により直接適用したり、あるいは、布等の支持体に塗布または含浸させて適用したりするなど、常法により使用することができる。また、パップ剤やテープ剤の場合には、これらの製剤を皮膚患部に直接貼付することにより使用することができる。 The external preparations are, for example, ointments, liquids (suspensions, emulsions, lotions, etc.), aerosols and powders for external use, which can be applied directly to the affected skin by coating or the like, or can be supported by a cloth or the like. It can be used by a conventional method such as applying by coating or impregnating the body. In the case of poultices and tapes, these formulations can be used by directly applying them to the affected skin area.
１．８．５　内服製剤（経口投与製剤）
　本発明組成物が内服製剤の形態である場合、かかる内服製剤としては、例えば、末剤、カプセル剤、錠剤、糖衣剤、顆粒剤、散剤、懸濁剤、液剤、シロップ剤、エリキシル剤、トローチ剤を挙げることができる。 1.8.5 Oral formulation (oral administration formulation)
When the composition of the present invention is in the form of an internal preparation, examples of such internal preparation include powders, capsules, tablets, dragees, granules, powders, suspensions, liquids, syrups, elixirs, and lozenges. agents can be mentioned.
　末剤は、有効成分を適当な細かさにすることにより製造することができる。
　散剤は、有効成分を適当な細かさにし、次いで同様に細かくした医薬用担体、例えば、澱粉、マンニトールのような可食性炭水化物と混合することにより製造することができる。任意に風味剤、保存剤、分散剤、着色剤、香料等を添加することができる。 A powder can be produced by appropriately pulverizing the active ingredient.
Powders can be prepared by comminuting the active ingredient into a suitable fine powder and then mixing with a similarly finely divided pharmaceutical carrier such as an edible carbohydrate such as starch or mannitol. Optionally, flavoring agents, preservatives, dispersing agents, coloring agents, perfumes and the like can be added.
　カプセル剤は、まず上述のようにして粉末状となった末剤や散剤あるいは錠剤の項で述べるように顆粒化したものを、例えば、ゼラチンカプセルのようなカプセル外皮の中へ充填することにより製造することができる。滑沢剤や流動化剤、例えば、コロイド状のシリカ、タルク、ステアリン酸マグネシウム、ステアリン酸カルシウム、固形のポリエチレングリコールを粉末状のものに混合し、その後充填操作を行うことにより製造することもできる。崩壊剤や可溶化剤、例えば、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、低置換度ヒドロキシプロピルセルロース、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、炭酸カルシウム、炭酸ナトリウムを添加すれば、カプセル剤が摂取されたときの医薬の有効性を改善することができる。また、有効成分の微粉末を植物油、ポリエチレングリコール、グリセリン、界面活性剤中に懸濁分散し、これをゼラチンシートで包んで軟カプセル剤とすることもできる。 Capsules are manufactured by first filling powders and powders that have been powdered as described above, or granulated as described in the section on tablets, into capsule shells such as gelatin capsules, for example. can do. It can also be produced by mixing a lubricant or glidant such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol with the powder, followed by a filling operation. If disintegrants or solubilizers such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, croscarmellose sodium, carboxymethylstarch sodium, calcium carbonate, sodium carbonate are added, the capsules will be of the drug can be improved. A soft capsule can also be prepared by suspending and dispersing fine powder of the active ingredient in vegetable oil, polyethylene glycol, glycerin, or a surfactant, and wrapping it in a gelatin sheet.
　錠剤は、賦形剤を加えて粉末混合物を作り、顆粒化もしくはスラグ化し、次いで崩壊剤または滑沢剤を加えた後、打錠することにより製造することができる。
　粉末混合物は、適当に粉末化された物質を上述の希釈剤やベースと混合することにより製造することができる。必要に応じて、結合剤（例えば、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシプロピルメチルセルロース、ゼラチン、ポリビニルピロリドン、ポリビニルアルコール）、溶解遅延化剤（例えば、パラフィン）、再吸収剤（例えば、四級塩）、吸着剤（例えばベントナイト、カオリン）等を添加することができる。 Tablets can be made by adding excipients to make a powder mixture, granulating or slugging, and then adding a disintegrant or lubricant before tabletting.
A powder mixture can be prepared by mixing the suitably powdered material with the diluents and bases mentioned above. Optionally binders (e.g. sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone, polyvinyl alcohol), dissolution retarding agents (e.g. paraffin), resorbing agents (e.g. quaternary salts), Adsorbents (eg bentonite, kaolin) and the like can be added.
　粉末混合物は、まず結合剤、例えば、シロップ、澱粉糊、アラビアゴム、セルロース溶液または高分子物質溶液で湿らせ、攪拌混合し、これを乾燥、粉砕して顆粒とすることができる。このように粉末を顆粒化する代わりに、まず打錠機にかけた後、得られる不完全な形態のスラグを破砕して顆粒にすることも可能である。このようにして作られる顆粒に、滑沢剤としてステアリン酸、ステアリン酸塩、タルク、ミネラルオイル等を添加することにより、互いに付着することを防ぐことができる。
　また、錠剤は、上述のように顆粒化やスラグ化の工程を経ることなく、有効成分を流動性の不活性担体と混合した後に直接打錠することによっても製造することができる。 The powder mixture can be first moistened with a binder such as syrup, starch paste, gum arabic, cellulose solution or polymeric substance solution, stirred and mixed, dried and ground into granules. As an alternative to granulating the powder in this manner, it is also possible to first run it through the tableting machine and then break the resulting imperfectly formed slugs into granules. By adding stearic acid, stearate, talc, mineral oil, etc. as a lubricant to the granules thus produced, it is possible to prevent them from adhering to each other.
Tablets can also be produced by directly compressing after mixing the active ingredient with a flowable inert carrier without going through the granulation or slugging steps as described above.
　こうして製造された錠剤にフィルムコーティングや糖衣を施すことができる。シェラックの密閉被膜からなる透明または半透明の保護被覆、糖や高分子材料の被覆およびワックスよりなる磨上被覆をも用いることができる。 Tablets manufactured in this way can be coated with film or sugar. A clear or translucent protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating consisting of wax can also be used.
　他の経口投与製剤、例えば、液剤、シロップ剤、トローチ剤、エリキシル剤もまたその一定量が有効成分を一定量含有するように用量単位形態にすることができる。 Other oral formulations, such as solutions, syrups, lozenges, and elixirs, can also be in dosage unit form so that a given amount of the formulation contains a given amount of the active ingredient.
　シロップ剤は、有効成分を適当な香味水溶液に溶解して製造することができる。エリキシル剤は、非毒性のアルコール性担体を用いることにより製造することができる。
　懸濁剤は、有効成分等を非毒性担体中に分散させることにより製造することができる。必要に応じて、可溶化剤や乳化剤（例えば、エトキシ化されたイソステアリルアルコール類、ポリオキシエチレンソルビトールエステル類）、保存剤、風味付与剤（例えば、ペパーミント油、サッカリン）等を添加することができる。
　必要であれば、経口投与のための用量単位処方をマイクロカプセル化することができる。当該処方はまた、被覆をしたり、高分子・ワックス等中に埋め込んだりすることにより作用時間の延長や持続放出をもたらすこともできる。 Syrups can be prepared by dissolving the active ingredient in a suitable flavored aqueous solution. Elixirs can be prepared using a non-toxic alcoholic carrier.
Suspensions can be prepared by dispersing the active ingredient and the like in a non-toxic carrier. If necessary, solubilizers, emulsifiers (e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters), preservatives, flavoring agents (e.g. peppermint oil, saccharin) and the like can be added. can.
If desired, dosage unit formulations for oral administration can be microencapsulated. The formulations can also be coated or embedded in polymers, waxes, etc. to provide extended duration of action or sustained release.
１．８．６　非経口投与製剤
　本発明組成物が非経口投与製剤の形態である場合、かかる非経口投与製剤としては、例えば、注射剤、点滴製剤、坐剤を挙げることができる。注射剤は、用時調製の注射用キットないし点滴用キットであってもよい。 1.8.6 Preparations for Parenteral Administration When the composition of the present invention is in the form of preparations for parenteral administration, examples of such preparations for parenteral administration include injections, infusions, and suppositories. The injection may be an injection kit or drip infusion kit prepared just before use.
　非経口投与製剤は、皮下・筋肉または静脈内注射用とした液状用量単位形態、例えば、溶液や懸濁液の形態をとることができる。当該非経口投与製剤は、一定量の有効成分を、注射の目的に適合する非毒性の液状担体、例えば、水性や油性の媒体に懸濁しまたは溶解し、次いで当該懸濁液または溶液を滅菌することにより製造することができる。注射液を等張にするために非毒性の塩や塩溶液を添加することができる。また、安定剤、保存剤、乳化剤等を添加することもできる。同様に点滴製剤とすることもできる。 Parenteral formulations can take the form of liquid dosage unit forms, such as solutions and suspensions, for subcutaneous, intramuscular or intravenous injection. The preparation for parenteral administration is prepared by suspending or dissolving a certain amount of the active ingredient in a non-toxic liquid carrier suitable for the purpose of injection, such as an aqueous or oily medium, and then sterilizing the suspension or solution. It can be manufactured by A non-toxic salt or salt solution can be added to make the injection isotonic. Stabilizers, preservatives, emulsifiers and the like can also be added. Similarly, an infusion formulation can be prepared.
　坐剤は、有効成分を低融点の水に可溶または不溶の固体、例えば、ポリエチレングリコール、カカオ脂、半合成の油脂、高級エステル類またはそれらの混合物に溶解または懸濁させて製造することができる。 Suppositories can be prepared by dissolving or suspending active ingredients in water-soluble or insoluble solids with a low melting point, such as polyethylene glycol, cocoa butter, semi-synthetic fats and oils, higher esters, or mixtures thereof. can.
　本発明組成物において、タクロリムスの含有量は、本発明の効果を損なわない範囲内、いわゆる有効量の範囲内であれば特に制限はないが、本発明組成物の総重量に対して０．００１重量％～１０重量％の範囲内であることが適当であり、０．０１重量％～１重量％の範囲内であることが好ましい。 In the composition of the present invention, the content of tacrolimus is not particularly limited as long as it is within a range that does not impair the effects of the present invention, that is, within the range of the so-called effective amount. It is suitably in the range of 0.01% to 1% by weight, preferably in the range of 0.01% to 1% by weight.
　また、本発明組成物が外用剤である場合には、本発明組成物の総重量に対して０．００１重量％～１０重量％の範囲内であることが適当であり、０．０１重量％～０．３重量％の範囲内であることが好ましい。
  Also, when the composition of the present invention is an external preparation, it is suitably within the range of 0.001% by weight to 10% by weight with respect to the total weight of the composition of the present invention, and 0.01% by weight. It is preferably in the range of ~0.3% by weight.
２　本発明に係るキット
　本発明に係るキット（以下、「本発明キット」という。）は、上記局所適用型製剤の態様である本発明組成物とネブライザーとを組み合わせたものであることを特徴とする。 2 Kit according to the present invention The kit according to the present invention (hereinafter referred to as "the kit of the present invention") is characterized by combining the composition of the present invention, which is an embodiment of the topical preparation, and a nebulizer. do.
　ここで「ネブライザー」とは、液体の製剤を霧状にして噴霧するための噴霧器（狭義のネブライザー）、およびエアロゾル状またはドライパウダー状の製剤を吸入するための吸入器、のいずれをも意味する。この中でも、本発明キットを構成するネブライザーは、噴霧器であることが好ましい。 Here, the term "nebulizer" means both a nebulizer (nebulizer in a narrow sense) for nebulizing a liquid formulation and an inhaler for inhaling an aerosol or dry powder formulation. . Among these, the nebulizer constituting the kit of the present invention is preferably an atomizer.
　噴霧器としては、例えば、ジェット式ネブライザー、超音波式ネブライザー、メッシュ式ネブライザーが挙げられる。 Examples of nebulizers include jet nebulizers, ultrasonic nebulizers, and mesh nebulizers.
　吸入器としては、例えば、加圧噴霧式定量吸入器（ｐＭＤＩ）、ドライパウダー吸入器（ＤＰＩ）が挙げられる。 Examples of inhalers include pressurized metered dose inhalers (pMDI) and dry powder inhalers (DPI).
　また、本発明キットを構成する局所適用型製剤は、液体状、エアロゾル状、またはドライパウダー状の吸入剤であることが好ましい。
  In addition, the topical formulation that constitutes the kit of the present invention is preferably a liquid, aerosol, or dry powder inhalant.
　以下、実施例および試験例（実施例等）を掲げて本発明を詳しく説明するが、本発明は以下の実施例等の態様に限定されるものではない。 The present invention will be described in detail below with reference to examples and test examples (examples, etc.), but the present invention is not limited to the following embodiments.
　なお、以下の実施例等で用いたマウス（Ｃ５７ＢＬ/６Ｊ）は、８週齢のものを日本クレア社より購入した。
　また、ＰＥ標識抗ＣＤ１１ｃ抗体、ＡＰＣ標識抗Ｓｉｇｌｅｃ－Ｆ抗体、ＢＶ４２１標識抗Ｓｃａ－１抗体はＢＤ Ｂｉｏｓｃｉｅｎｃｅ社より、ＦＩＴＣ標識坑ＣＤ６４抗体、ＰＥ標識抗ＣＤ４５，ＣＤ３１抗体、ＡＰＣ標識ＣＤ４，Ｐｏｄｏｐｌａｎｉｎ抗体、ＰＥ－Ｃｙ７標識抗Ｇｒ１（Ｌｙ－６Ｃ/６Ｇ），ＣＤ３２６（ＥｐＣＡＭ）抗体は、Ｂｉｏｌｅｇｅｎｄ社より、ＦＩＴＣ標識ＡＱＯ５抗体はＢｉｏｓｓ社より、ＦＩＴＣ標識ＳＴ－２抗体はＭｄｂｉｏｐｒｏｄｕｃｔｓ社より、それぞれ購入した。 The mouse (C57BL/6J) used in the following Examples was purchased from CLEA Japan Inc. at 8 weeks of age.
In addition, PE-labeled anti-CD11c antibody, APC-labeled anti-Siglec-F antibody, and BV421-labeled anti-Sca-1 antibody are from BD Bioscience, FITC-labeled anti-CD64 antibody, PE-labeled anti-CD45, CD31 antibody, APC-labeled CD4, Podoplanin antibody, PE-Cy7-labeled anti-Gr1 (Ly-6C/6G), CD326 (EpCAM) antibody was purchased from Biolegend, FITC-labeled AQO5 antibody was purchased from Bioss, and FITC-labeled ST-2 antibody was purchased from Mdbioproducts.
［試験例１]　タクロリムスによる気道炎症早期相の抑制
　タクロリムスが、パパイン誘導気道炎症早期相を抑制するかどうかを調べるため、タクロリムス投与マウスにパパインを点鼻投与して試験・検討を行った（Ｐａｐａｉｎ＋ＦＫ５０６、実施例１）。参照として、何も処置を行わない野生型（ＷＴ）マウス（Ｕｎｔｒｅａｔｅｄ）、パパイン点鼻投与のみを行ったＷＴマウス（Ｐａｐａｉｎ）、そしてパパイン点鼻投与前にデキサメタゾンを投与したマウス（Ｐａｐａｉｎ＋Ｄｅｘ）をそれぞれ用意した。 [Test Example 1] Suppression of early phase of airway inflammation by tacrolimus In order to investigate whether tacrolimus suppresses the early phase of papain-induced airway inflammation, papain was nasally administered to tacrolimus-administered mice and tested and examined (Papain + FK506). , Example 1). As references, wild-type (WT) mice (Untreated) without any treatment, WT mice (Papain) only administered papain nasal drops, and mice (Papain+Dex) administered dexamethasone before intranasal papain administration. prepared.
（１）薬剤処理
　タクロリムス２００μｇまたはデキサメタゾン１００μｇを、気道炎症誘導を行う３日前より３日間連続で、マウスに対して腹腔内投与した。 (1) Drug treatment 200 μg of tacrolimus or 100 μg of dexamethasone was intraperitoneally administered to mice for three consecutive days starting three days before induction of airway inflammation.
（２）パパインによる気道炎症誘導
　パパイン（Ｓｉｇｍａ Ａｌｄｒｉｃｈ社製）５０μｇをマウスに対して３日間連続点鼻投与し、気道炎症を誘導した。最終投与から２４時間後に各マウスの肺を採取した。 (2) Induction of Airway Inflammation by Papain 50 μg of papain (manufactured by Sigma Aldrich) was nasally administered to mice for 3 consecutive days to induce airway inflammation. Lungs from each mouse were harvested 24 hours after the final dose.
（３）肺細胞調整とフローサイトメトリー解析
　マウスの肺をｇｅｎｔｌｅＭＡＣＳ Ｄｉｓｓｏｃｉａｔｏｒ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社製）で細かく刻み、コラゲナーゼＤ（１００ｍｇ/ｍｌ、Ｒｏｃｈｅ社製）およびＤＮａｓｅ　I（２ｍｇ/ｍｌ、Ｒｏｃｈｅ社製)含ＨＢＳＳで３７℃にて３０分間インキュベートを行った。その後、パーコールを用いた密度勾配遠心法により、肺組織より白血球を抽出した。
　好酸球の同定は、ＦＩＴＣ標識抗ＣＤ６４抗体、ＰＥ標識ＣＤ１１ｃ抗体、ＰＥ-Ｃｙ７標識抗Ｇｒ-１抗体、ＡＰＣ標識抗Ｓｉｇｌｅｃ-Ｆ抗体を用いて染色を行い、ＦＡＣＳ ｃａｌｉｂｕｒ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用いてフローサイトメトリー解析を行った。データ解析には、Ｆｌｏｗｊｏ ｖ１０．２（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用いた。
　なお、死細胞の除去には、７－Ａｍｉｎｏ－Ａｃｔｉｎｏｍｙｃｉｎ Ｄ（７ＡＡＤ）を用いた。 (3) Lung Cell Preparation and Flow Cytometry Analysis Mouse lungs were minced with a gentleMACS Dissociator (manufactured by Miltenyi Biotec), collagenase D (100 mg/ml, manufactured by Roche) and DNase I (2 mg/ml, manufactured by Roche). Incubation was performed at 37° C. for 30 minutes with HBSS. After that, leukocytes were extracted from the lung tissue by density gradient centrifugation using Percoll.
Eosinophils were identified by staining with FITC-labeled anti-CD64 antibody, PE-labeled CD11c antibody, PE-Cy7-labeled anti-Gr-1 antibody, and APC-labeled anti-Siglec-F antibody, using FACS calibur (manufactured by BD Biosciences). was used for flow cytometry analysis. Flowjo v10.2 (manufactured by BD Biosciences) was used for data analysis.
7-Amino-Actinomycin D (7AAD) was used to remove dead cells.
（４）組織学的解析
　肺組織を４％（ｖ/ｖ）パラホルムアルデヒドで固定して２０％スクロースを浸透させた後、ＯＴＣコンパウンド（Ｓａｋｕｒａ Ｆｉｎｅｔｅｋ社製）で包埋し、凍結ブロックを作成した。凍結ブロックは５μｍの厚さに切断し、ＰＡＳ染色またはＨＥ染色を行った。 (4) Histological Analysis Lung tissue was fixed with 4% (v/v) paraformaldehyde, permeated with 20% sucrose, and then embedded with OTC compound (manufactured by Sakura Finetek) to create a frozen block. . Frozen blocks were cut to a thickness of 5 μm and subjected to PAS staining or HE staining.
（５）結果
　解析結果を図１に示す。なお、図１Ｂの各データはそれぞれ標準偏差を表記し、ＧｒａｐｈＰａｄ Ｐｒｉｓｍ ８（ＧｒａｐｈＰａｄ Ｓｏｆｔｗａｒｅ社製）を用いて両側スチューデントｔ検定で統計解析を行った。ｐ値０．０５未満における差を有意であると見なした。 (5) Results The analysis results are shown in FIG. Each data in FIG. 1B represents the standard deviation, and statistical analysis was performed by two-sided Student's t-test using GraphPad Prism 8 (manufactured by GraphPad Software). Differences at p-values less than 0.05 were considered significant.
　タクロリムス投与マウスでは肺での好酸球炎症が減衰した（図１Ａ、Ｂ）。また、気管支周辺の好酸球の集積や杯細胞の過形成の減少が見られた（図１Ｃ）。この結果は、デキサメタゾン（ＩＬＣ２を減衰させ気道炎症を抑えるコルチコステロイド）の投与を行ったマウスの結果と同等程度であった（図１Ａ～Ｃ）。これらの結果より、タクロリムスは気道炎症早期相を抑制することが明らかとなった。 Eosinophilic inflammation in the lungs was attenuated in tacrolimus-administered mice (Fig. 1A, B). There was also a decrease in peribronchial eosinophil accumulation and goblet cell hyperplasia (Fig. 1C). This result was comparable to that of mice treated with dexamethasone, a corticosteroid that attenuates ILC2 and reduces airway inflammation (FIGS. 1A-C). These results demonstrate that tacrolimus suppresses the early phase of airway inflammation.
［試験例２］　タクロリムスによるＩＬＣ２活性化の抑制
　ＩＬＣ２活性化に対するタクロリムスの効果を調べるため、タクロリムスおよびパパインをマウスに投与し、その後、当該マウスの肺ＩＬＣ２細胞に対してトランスクリプトーム解析（ＲＮＡ－Ｓｅｑ）を行った（Ｐａｐａｉｎ＋ＦＫ５０６、実施例２）。参照として、何も処置を行わないＷＴマウス（Ｃｏｎｔｒｏｌ）およびパパイン点鼻投与のみを行ったＷＴマウス（Ｐａｐａｉｎ）を用意し、それぞれ同様にＲＮＡ－Ｓｅｑを行った。 [Test Example 2] Suppression of ILC2 activation by tacrolimus In order to examine the effect of tacrolimus on ILC2 activation, tacrolimus and papain were administered to mice, and then transcriptome analysis (RNA- Seq) was performed (Papain+FK506, Example 2). As a reference, WT mice (Control) without any treatment and WT mice (Papain) only administered intranasal papain were prepared, and RNA-Seq was performed in the same manner.
（１）薬剤処理
　タクロリムス（２００μｇ／ｄａｙ）を、マウスに対して３日間連続で腹腔内投与した。 (1) Drug treatment Tacrolimus (200 μg/day) was intraperitoneally administered to mice for 3 consecutive days.
（２）パパイン投与
　上記投与の最終から２日後より、３日間連続にて、タクロリムス（２００μｇ／ｄａｙ）をさらに腹腔内投与し、同時にパパイン（５０μｌ ｉｎ ４０μｌ ｏｆ ＰＢＳ／ｄａｙ）を点鼻投与した。 (2) Papain Administration Two days after the last administration, tacrolimus (200 µg/day) was further intraperitoneally administered for 3 consecutive days, and papain (50 µl in 40 µl of PBS/day) was nasally administered at the same time.
（３）ＲＮＡ－Ｓｅｑ
　各マウスの肺を採取し、試験例１と同様の手法によりＩＬＣ２をソーティングし、得られたＩＬＣ２から、ＴＲＩｚｏｌ（登録商標）試薬を用いてＴｏｔａｌ ＲＮＡを単離した。続いて、Ｌｅｘｏｇｅｎ社製３’ｍＲＮＡ－Ｓｅｑ Ｌｉｂｒａｒｙ Ｐｒｅｐ Ｋｉｔを用いることにより、ｍＲＮＡの単離、断片化、逆転写等を経て、発現遺伝子に係る３’特異的配列のｃＤＮＡライブラリ（アダプタ付きライブラリ）を作成した。当該ライブラリ構築のために、１０ｎｇのＴｏｔａｌ ＲＮＡを使用した。そして、Ｉｌｌｕｍｉｎａ社製ＨｉＳｅｑ１５００を用いてシーケンシングを行い、配列決定されたリードをアダプタ配列についてトリミングし、高速マッピングプログラムＳＴＡＲ２.７.０ｃ（Dobin et al, Bioinformatics 2021）を使用して全マウスゲノム（ｍｍ１０）にマッピングした。 (3) RNA-Seq
Lungs of each mouse were collected, ILC2 was sorted by the same method as in Test Example 1, and total RNA was isolated from the obtained ILC2 using TRIzol (registered trademark) reagent. Subsequently, by using Lexogen's 3'mRNA-Seq Library Prep Kit, mRNA is isolated, fragmented, reverse transcribed, etc., and a cDNA library (adapter-attached library) of 3'specific sequences related to expressed genes is prepared. It was created. 10 ng of Total RNA was used for the library construction. Sequencing was then performed using an Illumina HiSeq1500, sequenced reads were trimmed for adapter sequences, and the entire mouse genome ( mm10).
（４）結果
　解析結果を図２に示す。パパイン投与により２倍以上の誘導を示す４７９の遺伝子について、タクロリムスはその誘導を阻害した。これらのタクロリムス感受性遺伝子には、ＩＬＣ２シグネチャー（遺伝子）やＩＬＣ２活性化に関連する遺伝子が含まれ、具体的には、アルギナーゼ、ＧＡＴＡ３、Ｔｏｘ、ＩＬ－１３、Ｋｌｒｇ１、ＩＬ１Ｒ１（ＩＬ－３３受容体遺伝子）等が含まれることが分かる。 (4) Results The analysis results are shown in FIG. Tacrolimus inhibited the induction of 479 genes showing a 2-fold or greater induction by papain administration. These tacrolimus susceptibility genes include ILC2 signature (genes) and genes associated with ILC2 activation, specifically arginase, GATA3, Tox, IL-13, Klrg1, IL1R1 (IL-33 receptor gene ) etc. are included.
　これらの結果より、タクロリムスはＩＬＣ２に作用し、当該自然リンパ球の活性化を抑制し得ることが明らかである。 From these results, it is clear that tacrolimus can act on ILC2 and suppress the activation of the innate lymphocytes.
［試験例３]　タクロリムスによるＩＬ－３３経路への作用の検討１
　タクロリムスが、ＩＬ－３３誘導によるＩＬＣ２の２型サイトカイン産生を抑制しているのかどうかを調べるため、タクロリムス投与マウスにＩＬ－３３を点鼻投与して試験・検討を行った（ＩＬ－３３＋ＦＫ５０６）。参照として、何も処置を行わないＷＴマウス（Ｕｎｔｒｅａｔｅｄ）、ＩＬ－３３点鼻投与のみを行ったＷＴマウス（ＩＬ－３３）、そしてＩＬ－３３点鼻投与前にデキサメタゾンを投与したマウス（ＩＬ－３３＋Ｄｅｘ）をそれぞれ用意した。 [Test Example 3] Investigation 1 of the action of tacrolimus on the IL-33 pathway
In order to investigate whether tacrolimus suppresses IL-33-induced ILC2 type 2 cytokine production, IL-33 was intranasally administered to tacrolimus-administered mice and tested/examined (IL-33+FK506). As references, WT mice that received no treatment (Untreated), WT mice that received IL-33 intranasal administration only (IL-33), and mice that received dexamethasone before IL-33 intranasal administration (IL-33). 33+Dex) were prepared respectively.
（１）薬剤処理
　試験例１と同様に、タクロリムス２００μｇまたはデキサメタゾン１００μｇを、気道炎症誘導を行う３日前より３日間連続で、マウスに対して腹腔内投与した。 (1) Drug treatment As in Test Example 1, 200 μg of tacrolimus or 100 μg of dexamethasone was intraperitoneally administered to mice for 3 consecutive days starting 3 days before airway inflammation induction.
（２）ＩＬ－３３による気道炎症誘導
　ＩＬ-３３（Ｒ＆Ｄ ｓｙｓｔｅｍ社製）１００ｎｇをマウスに対して３日間連続点鼻投与し、気道炎症を誘導した。最終投与から２４時間後に各マウスの肺を採取した。 (2) Induction of Airway Inflammation by IL-33 100 ng of IL-33 (manufactured by R&D system) was nasally administered to mice for 3 consecutive days to induce airway inflammation. Lungs from each mouse were harvested 24 hours after the final dose.
（３）解析
　試験例１と同様にして、肺細胞調整およびフローサイトメトリー解析、ならびに組織学的解析を行った。 (3) Analysis In the same manner as in Test Example 1, lung cell preparation, flow cytometry analysis, and histological analysis were performed.
（４）結果
　解析結果を図３に示す。なお、図３Ｂの各データも図１Ｂと同様に、それぞれ標準偏差を表記し、両側スチューデントｔ検定で統計解析を行った。ｐ値０．０５未満における差を有意であると見なした。 (4) Results The analysis results are shown in FIG. As in FIG. 1B, each data in FIG. 3B was also represented by standard deviation, and statistical analysis was performed by two-sided Student's t-test. Differences at p-values less than 0.05 were considered significant.
　パパイン誘導時と異なり、好酸球炎症の減衰（図３Ａ、Ｂ）、気管支周辺の好酸球の集積や杯細胞の過形成の減少が見られなかった（図３Ｃ）。一方、デキサメタゾン投与マウスでは、パパイン誘導時と同様であった（図３Ａ～Ｃ）。これらの結果より、タクロリムスは、ＩＬ－３３シグナルの下流経路を標的とせずに気道炎症早期相（あるいはＩＬＣ２の活性化）を抑制することが明らかとなった。 Unlike the time of papain induction, attenuation of eosinophilic inflammation (Fig. 3A, B), accumulation of eosinophils around the bronchi and reduction of goblet cell hyperplasia were not observed (Fig. 3C). In contrast, dexamethasone-treated mice were similar to papain induction (FIGS. 3A-C). These results demonstrate that tacrolimus suppresses the early phase of airway inflammation (or ILC2 activation) without targeting the downstream pathway of IL-33 signaling.
［試験例４]　タクロリムスによるＩＬ－３３経路への作用の検討２
　タクロリムスが、細胞からのＩＬ－３３放出または産生を標的としているのかどうかを調べるため、肺胞上皮細胞におけるＩＬ－３３の分布について試験・検討を行った。タクロリムスの標的回路であるカルシウムシグナル経路の駆動には、カルシウム流入が必要であるため、続いて、細胞内カルシウム濃度変化について試験・検討を行った。 [Test Example 4] Investigation 2 of the action of tacrolimus on the IL-33 pathway
To investigate whether tacrolimus targets IL-33 release or production from cells, the distribution of IL-33 in alveolar epithelial cells was examined. Since calcium influx is required to drive the calcium signaling pathway, which is the target circuit of tacrolimus, we next examined changes in intracellular calcium concentration.
（１－１）肺胞上皮細胞の単離
　まず、マウス（ＩＬ－３３ＧＦＰ）をネンブタールで麻酔下状態にし、１０ｍｌのＨＢＳＳで灌流を行った。１ｍｌのＤｉｓｐａｓｅ（Ｇｉｂｃｏ社製）をマウスの気管に滴下注入した後、１％低融点アガロース１ｍｌを注入し、すぐに氷でマウスの肺を冷やした。
　次に、マウスの肺をｇｅｎｔｌｅＭＡＣＳ Ｄｉｓｓｏｃｉａｔｏｒ（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ社製）で細かく刻んだ後、ＤＮａｓｅ（０．００１％）・コラゲナーゼ／ディスパーゼ（２μｇ／ｍｌ、Ｒｏｃｈｅ社製）含ＨＢＳＳで３７℃にて４５分間インキュベート（８０ｒｐｍ）し、１００μｍと３５μｍセルストレーナー（ＢＤ ｂｉｏｓｃｉｅｎｃｅｓ社製）で細胞を濾して４℃にて５分間遠心を行った（８００ｒｐｍ）。細胞を赤血球溶解バッファーで再懸濁し、３分後ＨＢＳＳで洗浄した。
　そして、ＦＩＴＣ標識抗ＡＱＯ５抗体、ＰＥ標識抗ＣＤ４５，ＣＤ３１抗体、ＢＶ４２１標識抗Ｓｃａ－１抗体、ＰＥ－Ｃｙ７標識抗ＣＤ３２６（ＥｐＣＡＭ）抗体、Ｂｉｏｔｉｎ標識抗Ｐｏｄｏｐｌａｎｉｎ抗体を用いて染色し、ＦＡＣＳ Ａｒｉａ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用いてセルソーティングを行い、１型および２型肺胞上皮細胞を単離した。 (1-1) Isolation of Alveolar Epithelial Cells First, mice (IL-33GFP) were anesthetized with Nembutal and perfused with 10 ml of HBSS. After instilling 1 ml of Dispase (manufactured by Gibco) into the trachea of the mouse, 1 ml of 1% low-melting point agarose was injected, and the lungs of the mouse were immediately cooled with ice.
Next, the mouse lungs were minced with a gentleMACS Dissociator (manufactured by Miltenyi Biotec) and then treated with HBSS containing DNase (0.001%)/collagenase/dispase (2 μg/ml, manufactured by Roche) at 37° C. for 45 minutes. After incubation (80 rpm), the cells were filtered through 100 μm and 35 μm cell strainers (manufactured by BD biosciences) and centrifuged at 4° C. for 5 minutes (800 rpm). Cells were resuspended in erythrocyte lysis buffer and washed with HBSS after 3 minutes.
Then, staining with FITC-labeled anti-AQO5 antibody, PE-labeled anti-CD45, CD31 antibody, BV421-labeled anti-Sca-1 antibody, PE-Cy7-labeled anti-CD326 (EpCAM) antibody, Biotin-labeled anti-Podoplanin antibody, FACS Aria (BD Biosciences) was used to isolate type 1 and type 2 alveolar epithelial cells.
（１－２）ＩＬ－３３分布の検討および結果
　フローサイトメトリー解析結果を図４Ａに示す。各種上皮組織細胞におけるＩＬ－３３の分布を見てみると、ＩＬ－３３は２型肺胞上皮細胞（ＣＤ４５－Ｐｏｄｏｐｌａｎｉｎ－ＳＰ－Ｃ＋）に局在していた（図４Ａ）。 (1-2) Examination and Results of IL-33 Distribution The results of flow cytometry analysis are shown in FIG. 4A. Looking at the distribution of IL-33 in various epithelial tissue cells, IL-33 was localized in type 2 alveolar epithelial cells (CD45-Podoplanin-SP-C+) (Fig. 4A).
（２－１）カルシウム流入解析
　単離した２型肺胞上皮細胞を、３７℃、５％ＣＯ２の条件下で、ＨＢＳＳで３０分間インキュベートし、０．１％Ｒｈｏｄ４－ＡＭ ＨＢＳＳで同様にインキュベートを行った。インキュベート後ＨＢＳＳで３回洗浄を行い、その後０．１％ ＤＲＡＱ７ ＨＢＳＳに懸濁し、μ－Ｄｉｓｈ ３５ｍｍ Ｑｕａｄ(Ｉｂｉｄｉ社製)を用いて解析を行った。
　カルシウム流入解析は、肺胞上皮細胞を１００μＭパパイン（Ｓｉｇｍａ　Ａｌｄｒｉｃｈ社製）または５μＭイオノマイシンで刺激し、時間経過に伴う細胞内カルシウム濃度をＴＲＴＩＣの蛍光強度測定により行なった。蛍光の検出はＢｉｏ－ｓｔａｔｉｏｎ ＩＭ－Ｑ（２０×，４１４μｍ×３１１μｍ、Ｎｉｋｏｎ社製）を用いた。 (2-1) Calcium Influx Analysis Isolated type 2 alveolar epithelial cells were incubated with HBSS for 30 minutes at 37° C. and 5% CO 2 , and similarly incubated with 0.1% Rhod4-AM HBSS. did After incubation, the cells were washed three times with HBSS, suspended in 0.1% DRAQ7 HBSS, and analyzed using μ-Dish 35 mm Quad (manufactured by Ibidi).
For calcium influx analysis, alveolar epithelial cells were stimulated with 100 μM papain (manufactured by Sigma Aldrich) or 5 μM ionomycin, and the intracellular calcium concentration over time was measured by TRTIC fluorescence intensity measurement. Bio-station IM-Q (20×, 414 μm×311 μm, manufactured by Nikon) was used for fluorescence detection.
（２－２）解析結果
　解析結果を図４Ｂに示す。パパイン刺激により２型肺胞上皮細胞内のカルシウム濃度上昇が見られた（図４Ｂ）。すなわち、パパイン刺激による気道上皮組織でのＩＬ－３３産生または放出において、カルシウム経路を介している可能性が高い。当該経路が、タクロリムスの標的となっていると考えられる。
  (2-2) Analysis Results The analysis results are shown in FIG. 4B. An increase in calcium concentration in type 2 alveolar epithelial cells was observed by papain stimulation (Fig. 4B). That is, it is highly likely that papain-stimulated IL-33 production or release in airway epithelial tissue is via the calcium pathway. It is believed that this pathway is targeted by tacrolimus.
　本発明組成物等は、上記の通り、アレルギー疾患および／または症状の即時型反応（早期相反応）、あるいはＴ細胞非依存型のアレルギー疾患および／または症状に対して効果を発揮しうるため、アレルギー治療薬として有用である。本発明組成物等は、ＩＬＣ２の活性化を抑制することができるため、ＩＬＣ２不活化剤としても有用である。また、本発明組成物等は、細胞からのＩＬ－３３の放出および／または産生における経路のカルシウムシグナルを阻害することができ、ＩＬ－３３の放出および／または産生を抑制し得るため、ＩＬ－３３抑制剤としても有用である。

  As described above, the composition, etc. of the present invention can exert an effect on immediate reactions (early phase reactions) of allergic diseases and/or symptoms, or T cell-independent allergic diseases and/or symptoms. It is useful as an allergy remedy. The composition of the present invention, etc. can suppress the activation of ILC2, and is therefore also useful as an ILC2 inactivating agent. In addition, the compositions of the present invention can inhibit the calcium signal of the pathway in the release and/or production of IL-33 from cells, and can suppress the release and/or production of IL-33. It is also useful as a 33 inhibitor.

Claims (12)

  1. タクロリムスを有効量含有する、Ｔ細胞非依存型のアレルギー疾患および／または症状を治療するための医薬組成物。 A pharmaceutical composition for treating T cell-independent allergic diseases and/or symptoms, containing an effective amount of tacrolimus.
  2. プロテアーゼアレルゲンにより引き起こされたＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、請求項１に記載の医薬組成物。 2. The pharmaceutical composition according to claim 1, for treating T-cell independent allergic diseases and/or conditions caused by protease allergens.
  3. ＩＬＣ２が活性化しているＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、請求項１または２に記載の医薬組成物。 3. The pharmaceutical composition according to claim 1 or 2, for treating T cell-independent allergic diseases and/or symptoms in which ILC2 is activated.
  4. ＩＬＣ２の活性化を抑制することでＴ細胞非依存型のアレルギー疾患および/または症状を治療するための、請求項１～３のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 3, which is for treating T cell-independent allergic diseases and/or symptoms by suppressing the activation of ILC2.
  5. ＩＬ－３３の放出および／または産生が亢進しているＴ細胞非依存型のアレルギー疾患および／または症状を治療するための、請求項１～４のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 4, for treating T cell-independent allergic diseases and/or conditions in which IL-33 release and/or production are enhanced.
  6. ＩＬ－３３の放出および／または産生を抑制することでＴ細胞非依存型のアレルギー疾患および/または症状を治療するための、請求項１～５のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5, for treating T cell-independent allergic diseases and/or symptoms by suppressing the release and/or production of IL-33.
  7. 前記アレルギー症状が呼吸器アレルギー症状である、請求項１～６のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 6, wherein said allergic symptom is a respiratory allergic symptom.
  8. 前記呼吸器アレルギー症状が気道炎症である、請求項７に記載の医薬組成物。 8. The pharmaceutical composition according to claim 7, wherein said respiratory allergy symptom is airway inflammation.
  9. 前記呼吸器アレルギー症状が喘息である、請求項７に記載の医薬組成物。 8. The pharmaceutical composition of claim 7, wherein said respiratory allergy condition is asthma.
  10. さらに他のアレルギー治療剤を組み合わせてなる、請求項１～９のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 9, which is further combined with another anti-allergic agent.
  11. 医薬組成物が局所適用型製剤または内服製剤である、請求項１～１０のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 10, which is a topical preparation or an internal preparation.
  12. 前記局所適用型製剤とネブライザーとを組み合わせた、キット。

          A kit comprising a combination of the topical formulation and a nebulizer.
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JPH03291225A (en) * 1990-04-09 1991-12-20 Fujisawa Pharmaceut Co Ltd Antiasthmatic

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03291225A (en) * 1990-04-09 1991-12-20 Fujisawa Pharmaceut Co Ltd Antiasthmatic

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* Cited by examiner, † Cited by third party
Title
CAI TING; QIU JINXIN; JI YAN; LI WENJING; DING ZHAOYUN; SUO CAIXIA; CHANG JIALI; WANG JINGJING; HE RUI; QIAN YOUCUN; GUO XIAOHUAN;: "IL-17-producing ST2+group 2 innate lymphoid cells play a pathogenic role in lung inflammation", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 143, no. 1, 1 January 2019 (2019-01-01), AMSTERDAM, NL , pages 229 - 224, XP085572588, ISSN: 0091-6749, DOI: 10.1016/j.jaci.2018.03.007 *
HAMASAKI YUHEI: "Degranulation inhibitory effect of cyclosporin D (CSD) -comparison with other immunosuppressive agents/anti-inflammatory agents", JAPANESE JOURNAL OF PEDIATRIC ALLERGY AND CLINICAL IMMUNOLOGY, vol. 14, no. 2, 1 June 2000 (2000-06-01), Tokyo , pages 182 - 188, XP055954120, ISSN: 0914-2649, DOI: 10.3388/jspaci.14.182 *

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