WO2019112031A1 - Systemic scleroderma therapeutic agent - Google Patents

Systemic scleroderma therapeutic agent Download PDF

Info

Publication number
WO2019112031A1
WO2019112031A1 PCT/JP2018/045053 JP2018045053W WO2019112031A1 WO 2019112031 A1 WO2019112031 A1 WO 2019112031A1 JP 2018045053 W JP2018045053 W JP 2018045053W WO 2019112031 A1 WO2019112031 A1 WO 2019112031A1
Authority
WO
WIPO (PCT)
Prior art keywords
expression
scleroderma
compound
evaluation
collagen
Prior art date
Application number
PCT/JP2018/045053
Other languages
French (fr)
Japanese (ja)
Inventor
雅巳 大塚
眞一郎 丹羽
大 小倉
Original Assignee
サイエンスファーム株式会社
リンク・ジェノミクス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by サイエンスファーム株式会社, リンク・ジェノミクス株式会社 filed Critical サイエンスファーム株式会社
Publication of WO2019112031A1 publication Critical patent/WO2019112031A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to a preventive and therapeutic agent for systemic scleroderma (SSc). More specifically, the present invention relates to an agent for suppressing and / or treating fibrosis, which is a pathological condition of systemic scleroderma.
  • SSc systemic scleroderma
  • Systemic scleroderma causes fibrosis in the skin dermis, movement disorders due to restricted movement of the limbs / contracture, necrosis in the peripheral area due to ulceration, etc., and the QOL of the patient is significantly reduced, and the lung fibers are advanced by progress.
  • SSc therapeutic agents drugs that suppress inflammation, such as immunosuppressants, steroids, and traditional Chinese medicines, are mainly used, but all of them are symptomatic therapeutic drugs and there is no cure that can be cured.
  • pulmonary antihypertensive agent bosentan Actelion Pharmaceuticals
  • its efficacy is limited, for example, to suppress the onset of finger ulcers, and no therapeutic effect on SSc has been observed.
  • a pharmaceutical composition for treating or preventing systemic scleroderma which comprises a compound that inhibits fibroblast activation in a tissue derived from dermis.
  • a pharmaceutical composition for treating or preventing systemic scleroderma which comprises a compound that inhibits fibroblast activation in a tissue derived from dermis.
  • the above compounds are benzbromarone, albendazole, mebendazole, risedronate sodium hydrate, epalrestat, aluminum flufenamate, zaltoprofen, anlexanox, mefenamic acid, oxaprozin, and 3-[(1E) -2- (1H] -Indol-6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridylmethoxy) phenyl] ethenyl] -1 H-pyrazole, selected from the above The pharmaceutical composition as described in 1] or [2].
  • inflammation is enhanced in dermal tissue of a patient with systemic scleroderma, and cells (fibroblasts) present in the tissue are activated and collagen is Secretion of the extracellular matrix, etc., and this revealed that the focal fibrosis was occurring.
  • a medicament for treating systemic scleroderma a compound that can be used for such a medicament, a method for treating systemic scleroderma using such a compound, a method for producing a medicament and A method of use is provided.
  • a compound having an inhibitory action on fibrosis which is a pathological condition of systemic scleroderma.
  • FIG. 1 shows hematoxylin and eosin (H & E) stained images of skin tissues from scleroderma (SSc) patients and healthy controls (Control).
  • FIG. 2A shows the results of immunohistochemistry (IHC) analysis of skin tissues from scleroderma patients and healthy controls (Control) using an anti- ⁇ -SMA antibody.
  • FIG. 2B shows the results of IHC analysis using anti-Fibronectin antibodies of skin tissues from scleroderma patients and healthy controls (Control).
  • FIG. 2C shows the results of IHC analysis of skin tissue from scleroderma patients and healthy controls (Control) using an anti-ZEB1 antibody.
  • FIG. 1 shows hematoxylin and eosin (H & E) stained images of skin tissues from scleroderma (SSc) patients and healthy controls (Control).
  • FIG. 2A shows the results of immunohistochemistry (IHC) analysis of skin tissues from scleroderma patients and healthy controls (Control) using an anti
  • FIG. 3 is a diagram collectively showing the results of pathological analysis and molecular dynamics analysis in the SSc sample shown in FIGS. 1 and 2A-C.
  • FIG. 4 is a graph showing the results of expression kinetic analysis of various gene groups (differentiation related markers) with respect to primary cultured fibroblasts from skin tissues derived from scleroderma patients and healthy controls (Control).
  • FIG. 5 is a graph showing the results of analysis of expression kinetics of collagen (collagen fibers) of primary culture fibroblasts from skin tissues from scleroderma patients and healthy individuals (Control).
  • FIG. 6 is a graph showing the results of expression kinetic analysis of various differentiation-related markers using healthy fibroblasts and healthy fibroblasts after inflammatory stimulation.
  • FIG. 4 is a graph showing the results of expression kinetic analysis of various gene groups (differentiation related markers) with respect to primary cultured fibroblasts from skin tissues derived from scleroderma patients and healthy controls (Control).
  • FIG. 5 is a graph
  • FIG. 7 is a graph showing the results of expression kinetic analysis of collagen (collagen fiber) using healthy fibroblasts and healthy fibroblasts after inflammatory stimulation.
  • FIG. 8A is a graph showing the evaluation results of evaluation compounds (epalrestat and oxaprozin) on the expression of various differentiation-related marker molecules by scleroderma patient-derived cells at 48 hours after the addition of the evaluation compound.
  • FIG. 8B is a graph showing the results of evaluating the suppressive effects of the evaluation compounds (anlexanox and zaltoprofen) on the expression of various differentiation-related marker molecules using scleroderma patient-derived cells 48 hours after the addition of the evaluation compounds.
  • FIG. 8A is a graph showing the evaluation results of evaluation compounds (epalrestat and oxaprozin) on the expression of various differentiation-related marker molecules by scleroderma patient-derived cells at 48 hours after the addition of the evaluation compound.
  • FIG. 8B is a graph showing the results of evaluating the suppressive effects of
  • FIG. 8C is a graph showing the results of evaluating the suppressive effects of the evaluation compounds (mefenamic acid and aluminum flufenamate) on the expression of various differentiation-related marker molecules 48 hours after the addition of the evaluation compounds using scleroderma patient-derived cells. It is.
  • FIG. 9A is a graph showing the results of evaluating the suppressive activity of epalrestat on various differentiation-related marker molecule expression 120 hours after the addition of the evaluation compound using scleroderma patient-derived cells.
  • FIG. 9B is a graph showing the results of evaluating the suppressive activity of mebendazole on the expression of various differentiation-related marker molecules 120 hours after the addition of the evaluation compound using scleroderma patient-derived cells.
  • FIG. 9A is a graph showing the results of evaluating the suppressive activity of epalrestat on various differentiation-related marker molecule expression 120 hours after the addition of the evaluation compound using scleroderma patient-derived cells.
  • FIG. 9B is a graph showing the results of
  • FIG. 9C is a graph showing the results of evaluation of suppression activity of various types of differentiation-related marker molecules of anbendazole 120 hours after addition of an evaluation compound using scleroderma patient-derived cells.
  • FIG. 10 shows the results of evaluating the inhibitory effect of an evaluation compound (Epalrestat) on collagen secretion increase (factor of fibrosis) specifically recognized in scleroderma patient skin using scleroderma patient-derived cells
  • FIG. 11A shows the results of evaluating the suppressive effects of evaluation compounds (epalrestat and oxaprodin) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects 48 hours after addition of the evaluation compounds. It is a graph.
  • FIG. 10 shows the results of evaluating the inhibitory effect of an evaluation compound (Epalrestat) on collagen secretion increase (factor of fibrosis) specifically recognized in scleroderma patient skin using scleroderma patient-derived cells
  • FIG. 11A shows the results of
  • FIG. 11B shows the results of evaluating the suppressive effect of evaluation compounds (Anlexanox and zaltoprofen) on the expression of various differentiation-related marker molecules by the inflammation stimulation system using fibroblasts from healthy subjects 48 hours after the addition of the evaluation compounds. It is a graph.
  • FIG. 11C shows that the inhibitory effect of the evaluation compounds (aluminum flufenamate and mefenamic acid) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects was evaluated 48 hours after the addition of the evaluation compounds. It is a graph which shows a result.
  • FIG. 12A is a graph showing the results of evaluating the suppressive effect of an evaluation compound (Epalrestat) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects at 120 hours after the addition of the evaluation compound. is there.
  • FIG. 12B is a graph showing the results of evaluating the suppressive effect of an evaluation compound (mebendazole) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects 120 hours after the addition of the evaluation compound. is there.
  • FIG. 12C is a graph showing the results of evaluating the suppressive effect of an evaluation compound (albendazole) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects at 120 hours after addition of the evaluation compound.
  • FIG. 13A is a diagram showing the results of evaluating the suppressive effect of an evaluation compound (epalrestat) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts.
  • FIG. 13B is a diagram showing the results of evaluation of the suppressive effect of an evaluation compound (mebendazole) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts.
  • FIG. 13C is a diagram showing the results of evaluation of the suppressive effect of an evaluation compound (albendazole) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts.
  • FIG. 13A is a diagram showing the results of evaluating the suppressive effect of an evaluation compound (epalrestat) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts.
  • FIG. 13B is a diagram showing the results of evaluation of the suppressive effect of an
  • FIG. 14 is a view schematically showing a protocol for evaluating the efficacy of suppression of fibrosis carried out using a scleroderma disease mouse model.
  • FIG. 15A is a diagram showing an H & E stained image showing the skin thickening inhibitory effect of epalrestat on skin tissue derived from a scleroderma disease model mouse established by administration of bleomycin.
  • FIG. 15B is a graph quantitatively showing the results of FIG. 15A.
  • 16A-D show 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5 against skin tissue of scleroderma disease model mouse induced by administration of bleomycin (Bleo) in Example 7.
  • FIG. 17 is a graph quantitatively showing the results shown in FIG. 16A-D.
  • FIGS. 18A-D show photographs of specimens stained with Masson's trichrome (Masson trichrome) staining in Example 7.
  • FIG. The area stained blue corresponds to the area of fibrosis.
  • the conditions of FIGS. 18A-D correspond to the conditions of FIGS. 16A-D.
  • FIG. 19 is a diagram quantitatively showing the results shown in FIG. 18A-D.
  • FIG. 20 is a diagram showing the results of an evaluation experiment (experiment A) of the inhibitory effect of compound A on the expression (mRNA) of collagens Collagen 1A2 and Fibronectin 1.
  • FIG. 21 is a diagram showing the results of an evaluation experiment (experiment B) of the inhibitory effect of compound A on the expression of collagen Collagen 1A2 protein.
  • FIG. 22 shows the results of an evaluation experiment (experiment C) of the suppressive effect of compound A on the expression of Fibronectin protein.
  • fibroblast activation refers to enhancement of ⁇ -smooth muscle actin (SMA) expression in fibroblasts (myofibroblast formation), enhancement of various collagen (collagen fibers) expression, Alteration of traits in fibroblasts characterized by enhanced expression of fibronectin, other zinc finger E box-bound homeobox 1 (ZEB1), N-cadherin (N-cadherin), enhanced expression of differentiation-related markers such as vimentin (Vimentin) It means what is characteristically found in the dermis of the skin of patients with generalized scleroderma.
  • SMA smooth muscle actin
  • “suppressing fibroblast activation” means that the expression level of differentiation-related markers such as ⁇ SMA, various collagens, fibronectin enhanced by fibroblast activation is decreased by the action of the inhibitor.
  • the expression level of the marker after reduction may not necessarily be low compared to normal cells.
  • Fibroblast activation or suppression thereof is quantified by analyzing the expression dynamics of a group of genes whose expression changes when cells are differentiated ("differentiation related markers"), as described in detail in the examples below. Can be evaluated.
  • the present invention provides a compound that inhibits fibroblast activation in a tissue derived from dermis and a composition containing the compound.
  • the present invention provides a pharmaceutical composition for treating and / or preventing systemic scleroderma, which comprises a therapeutically effective amount of a compound that inhibits fibroblast activation in dermis-derived tissues. Do.
  • the present invention provides a compound that inhibits fibroblast activation in dermis-derived tissues for use in the treatment and / or prevention of systemic scleroderma.
  • the present invention is the use (or method of use of a compound that inhibits fibroblast activation in dermis-derived tissues in the manufacture of a medicament for the treatment and / or prevention of systemic scleroderma. )I will provide a.
  • the present invention relates to a method of treating and / or preventing systemic scleroderma, which comprises administering to a patient in need thereof a compound that inhibits fibroblast activation in a dermis-derived tissue.
  • a method is provided, comprising the step of administering.
  • Specific examples of the “compound that inhibits fibroblast activation in the tissue derived from dermis” used in each of the above embodiments include benzbromarone, albendazole, mebendazole, risedronate sodium hydrate, epalrestat, flufenamic acid Aluminum, zaltoprofen, annexanox, mefenamic acid, oxaprozin, and 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5- (1E) -2- [2-methoxy-4- (2) 2-Pyridylmethoxy) phenyl] ethenyl] -1H-pyrazole, as well as their pharmaceutically acceptable salts and their derivative compounds.
  • the method for confirming the therapeutic effect of the compound of the present invention on systemic scleroderma is not particularly limited.
  • bleomycin-induced pulmonary fibrosis model mice (generally recognized as animal models for systemic scleroderma and pulmonary fibrosis) Using a mouse, for example, Curr Opin Pulm Med. 2011 Sep; 17 (5): 355-61 (G), etc.) to confirm the inhibitory effect of lesion formation.
  • the specific test method using the above-mentioned model mouse is shown in the examples described later.
  • the "pharmaceutically acceptable salts” include those which form pharmaceutically acceptable salts with the specific compounds described above, and are not particularly limited. Specifically, for example, hydrohalide (eg, hydrogen fluoride, hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salt (eg, sulfate, nitrate, persulfate, etc.) Chlorates, phosphates, carbonates, bicarbonates, etc., organic carboxylates (eg, acetates, oxalates, maleates, tartrates, fumarates, citrates, etc.), organic sulfones Acid salts (eg, methane sulfonate, trifluoromethane sulfonate, ethane sulfonate, benzene sulfonate, toluene sulfonate, camphor sulfonate, etc.), amino acid salts (eg, aspartate
  • the "derivative compound” refers to a compound which may be an equivalent of the above specific compound, and includes glycosides or esters (eg, alkyl esters such as methyl ester, ethyl ester etc.), etc. Be done.
  • confirmation about whether a derivative compound is equivalent can be performed by confirming whether it has a function which suppresses the promotion of fibroblast activation in skin dermis tissue by an above-described test.
  • the compounds of the present invention can be formulated, but not limited to, by conventional methods to provide the prophylactic and / or therapeutic agents of the present invention.
  • Preferred dosage forms of the prophylactic and / or therapeutic agent of the present invention are, for example, tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules, syrups Agents, oral solutions, troches, jellies, inhalants, suppositories, injections, ointments, eye drops, eye ointments, eye drops, nasal drops, ear drops, poultices, lotions, external solutions, sprays, External aerosols, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, rectal soft capsules and the like can be mentioned.
  • excipients for example, excipients, binders, disintegrants, coatings, lubricants, coloring agents, flavoring agents and, if necessary, stabilizers, emulsifiers, absorption accelerators, surfactants, etc.
  • PH adjusting agents, preservatives, antioxidants and the like can be used, and the components generally used as raw materials of pharmaceutical preparations can be blended and formulated by a conventional method.
  • the invention also includes a method of preparing a pharmaceutical composition comprising mixing at least one carrier.
  • the components (carriers) used in the formulation are not particularly limited, and examples thereof include animal oils such as soybean oil, beef tallow and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane and solid paraffin; and octyldodecyl myristate, for example.
  • Ester oil such as isopropyl myristate; higher alcohol such as cetostearyl alcohol, behenyl alcohol; silicone resin; silicone oil; polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxy acid Surfactants such as ethylene-cured castor oil, polyoxyethylene polyoxypropylene block copolymer; eg, hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer Water-soluble polymers such as polyethylene glycol, polyvinyl pyrrolidone and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol and sorbitol; sugars such as glucose and sucrose; For example, inorganic powder such as anhydrous silicic acid, aluminum magnesium silicate, aluminum silicate, purified water and the like can be
  • lactose for example, lactose, corn starch, sucrose, glucose, mannitol, sorbite, crystalline cellulose, silicon dioxide etc.
  • a binder for example, polyvinyl alcohol, gelatin, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin , Shellac, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyvinyl acetal diethylamino acetate, corn starch etc., and as a disintegrant, for example, corn starch, low substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate , Croscarmellose sodium, calcium citrate, dextrin, pectin, carboxymethylcellulose calcium
  • lubricants for example, magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, sucrose fatty acid ester
  • an oral preparation may be a compound which is an active ingredient, or a salt or ester thereof, or a hydrate thereof and an excipient, and further, if necessary, for example, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent After adding etc., it is set as a powder, a fine granule, a granule, a tablet, a coated tablet, a capsule etc. by a conventional method. In the case of tablets and granules, for example, sugar coating, etc. may of course be appropriately coated if necessary.
  • a syrup, a preparation for injection, etc. for example, a pH adjusting agent, a solubilizing agent, an isotonizing agent and the like, and a solubilizing agent, a stabilizing agent and the like as necessary, are formulated by a conventional method.
  • the manufacturing method is not particularly limited, and the external preparation can be manufactured by an ordinary method.
  • a base material to be used various raw materials usually used for medicines, quasi-drugs, cosmetics etc. can be used.
  • animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids Raw materials such as silicone oil, silicone oil, surfactant, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, acrylic adhesive clay minerals, purified water etc., and if necessary, pH adjustment Preparations, antioxidants, chelating agents, preservatives and fungicides, coloring agents, flavors and the like can be added.
  • components having differentiation-inducing action for example, components such as blood flow enhancers, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents can also be blended. .
  • the administration mode of the prophylactic and / or therapeutic agent of the present invention is not particularly limited, and may be oral administration or parenteral administration.
  • Parenteral administration includes, for example, rectal administration, nasal administration, pulmonary administration, injection administration (eg, intravenous administration, intraspinal administration, epidural administration, intramuscular administration, subcutaneous administration, intraperitoneal administration
  • the content of the compound of the present invention contained in the preventive and / or therapeutic agent of the present invention is not particularly limited, in the subject to be administered, it exerts the effect of suppressing the enhancement of fibroblast activation in skin dermal tissue. It is preferable to use a dose sufficient for
  • the dosage of the prophylactic and / or therapeutic agent of the present invention varies depending on, for example, the degree of symptoms, age, sex, body weight, administration form, type of salt, specific type of disease, etc.
  • the compound of the present invention which is the active ingredient per day, is orally administered at about 25 mg to about 4.5 g, preferably about 75 mg to about 1.5 g, more preferably about 150 mg to about 600 mg About 25 mg to about 4.5 g, preferably about 75 mg to about 1.5 g, more preferably about 150 mg to about 600 mg, each in one or several divided doses.
  • the administration target of the prophylactic and / or therapeutic agent of the present invention is not particularly limited, but is preferably a mammal including human.
  • Mammals including humans are not particularly limited, and include, for example, humans, monkeys, baboons, chimpanzees, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, goats, pigs, cattle and horses, etc. .
  • compositions of the present invention may, if desired, be provided in the form of a kit comprising a container, such as a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
  • the present invention can also combine separate drug compositions in kit form.
  • the kit may comprise two or more separate pharmaceutical compositions.
  • the compounds of the present invention and anti-inflammatory and / or immunosuppressive agents may be included.
  • the kit will usually include a container for containing the separate compositions, such as, for example, a split bottle or a split foil packet, but the separate compositions may also be included in a single non-split container. it can.
  • the kit form is preferably to administer the separate components in different dosage forms (e.g. orally and parenterally), when the separate components are administered at different dosing intervals, or individual components combined by the prescribing physician It is particularly useful when it is necessary to titrate.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • Blister packs are well known in the packaging industry and are widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs are generally preferred to consist of a sheet of relatively rigid material covered by a foil of transparent plastic material. During the packaging process, recesses are formed in the plastic foil. These indentations are adapted to the size and shape of the individual tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively hard material is sealed against the plastic foil in the foil plane opposite to the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet.
  • the strength of the sheet is preferably such that the tablet or capsule can be removed from the blister pack by manually applying pressure to the recess such that an opening is formed in the sheet at the location of the recess. Tablets or capsules can be removed by the opening
  • the pack or dispenser device may be accompanied by package inserts, product inserts and the like for administration.
  • Containers such as packs or dispensers can be adapted to the notices of government agencies, authorities which regulate the manufacture, use or sale of medicines.
  • Another aspect of the present invention relates to a food or drink, a feed, a food additive, a feed additive or the like, which contains a compound having a function of suppressing an increase in fibroblast activation in a dermis-derived tissue.
  • the food and drink of the present invention are food additives generally used for food and drink, such as sweeteners, colorants, preservatives, thickeners, thickeners, antioxidants, color formers, bleaches, fungicides, One or more of a gum base, a bitter taste agent, an enzyme, a brightener, an acidulant, a seasoning, an emulsifier, a toughening agent, a preparation agent, a flavor, a spice extract and the like may be added.
  • the food and drink of the present invention include health food, functional food, food for specified health, food for infants, food for infants, food for pregnant women, food for elderly people, food for sick people.
  • the form of the food and drink of the present invention is not particularly limited. Specifically, for example, tablets, granules, powders, drinks and the like as so-called nutraceuticals (supplements) can be mentioned.
  • beverages such as tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, etc., buckwheat noodles such as udon, Chinese noodles, instant noodles, etc., candy, candy, gum, chocolate, snacks , Biscuit, jelly, jam, cream, baked goods, confectionery such as bread, breads, fishery products such as kamaboko, ham, sausage, processed foods, dairy products such as processed milk, fermented milk, salad oil, tempura oil, margarine, Oils and fats processed foods such as mayonnaise, shortening, whipped cream and dressings, seasonings such as sauces and sauces, retort pouched foods such as curry, stew, chopsticks, chopsticks and risotto, frozen desserts such as ice cream, she
  • the intake amount of the food and drink of the present invention is not particularly limited, and the form of the food and drink; age, sex, condition and the like of the subject who ingests the food and drink; or the carrier carried or mixed with the carrier of the present invention You may set according to the kind of functional substance, and other conditions.
  • Biopsy sample In order to understand the fibrotic condition occurring in the patient tissue, we used the Biopsy sample to analyze the pathological condition and molecular dynamics in the lesion.
  • Skin tissues from scleroderma patients and healthy (Control) were collected. A formalin fixed paraffin block was made. These tissue samples were deparaffinized by treating with xylene 5 minutes ⁇ 3 times, 100% ethanol 1 minute ⁇ 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes . Staining was performed with Meyer's hematoxylin solution for 4 minutes and eosin solution for 1 minute. Treated in running water for 5 minutes, treated with 70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1 minute, 100% ethanol 1 minute ⁇ 2 times, xylene 5 minutes ⁇ 3 times, using marinol Sealed. After drying at room temperature for 15 minutes or more, observation was performed using an optical microscope.
  • Tissue samples were deparaffinized by treating with xylene 5 minutes ⁇ 3 times, 100% ethanol 1 minute ⁇ 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes.
  • the antigen was activated by microwave treatment for 10 minutes using citrate buffer pH 6.0. After standing at room temperature for 30 minutes, treatment with PBS for 5 minutes for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes was performed to inactivate endogenous peroxidase.
  • the tissue was blocked by standing in a 3% BSA / PBS solution for 30 minutes at room temperature.
  • anti- ⁇ -SMA antibody DAKO # M0851
  • anti-ZEB1 antibody SantaCruz # Sc-10572
  • 4800 times dilution 200 microliter per tissue of the anti-Fibronectin antibody abcam # 45688
  • the tissue reacted with the primary antibody was washed with PBS for 5 minutes x 3 times, and then a biotin-labeled antibody of animal species suitable for the optimally diluted primary antibody was poured onto the slide and allowed to react for 30 minutes at room temperature.
  • FIG. 3 summarizes the above results. Based on these results, systemic sclera of "Scleroderma patient skin has an enhanced activation of fibroblasts in the dermis, which causes the extracellular matrix such as collagen to grow and form a fibrotic lesion".
  • RNA extract lysate was produced.
  • Total RNA was extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and fluorescently labeled using Low Input QuickAmp Labeling kit (Agilent Technologies # 5190-2305). Labeled cRNA was hybridized to Whole Human Genome Oligo-DNA Microarray Kit (Agilent Technologies # G4112F) according to the protocol defined by the manufacturer.
  • the hybridized slides were washed using Gene Expression Wash Pack (Agilent Technologies # 5188-5327) and scanned with an Agilent Microarray Scanner (Agilent Technologies # G2505B). Scanned images were quantified using Feature Extraction software version 9.5.1 (Agilent Technologies). Numerical data were normalized by the Grobal Normalization method.
  • Labeled cRNA was hybridized to Whole Human Genome Oligo-DNA Microarray Kit (Agilent Technologies # G4112F) according to the protocol defined by the manufacturer.
  • the hybridized slides were washed using Gene Expression Wash Pack (Agilent Technologies # 5188-5327) and scanned with an Agilent Microarray Scanner (Agilent Technologies # G2505B). Scanned images were quantified using Feature Extraction software version 9.5.1 (Agilent Technologies). Numerical data were normalized by the Grobal Normalization method.
  • fibroblasts from healthy subjects are activated by inflammatory stimulation, become myofibroblasts ( ⁇ SMA expression), and expression of various differentiation-related markers is enhanced, and fibers from scleroderma patients are observed. It showed the same expression kinetics as blast cells.
  • inflammatory stimulation of normal human fibroblasts leads to increased expression of collagen, and activation of fibroblasts confirmed in scleroderma skin tissue (myofibroblast formation) collagen. Fiber growth was reproduced.
  • a compound having an inhibitory activity was obtained using an inflammatory stimulation activation model system using activated fibroblasts from scleroderma patients and healthy fibroblasts.
  • Plates were seeded with primary culture fibroblasts from scleroderma patient skin tissue and incubated at 37 ° C. The following day, an evaluation compound (test drug) was added to the culture medium to a maximum drug plasma concentration (Cmax), and cells were collected 48 hours later. As a control, a sample to which an evaluation compound was not added and a cultured fibroblast sample derived from a healthy skin tissue were used. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection.
  • RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 ⁇ g of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did.
  • real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed.
  • Specific primers for each gene were purchased from Invitrogen.
  • Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the cultured fibroblast sample derived from the healthy skin tissue was calculated.
  • RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 ⁇ g of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did.
  • real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed.
  • Specific primers for each gene were purchased from Invitrogen.
  • Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the cultured fibroblast sample derived from the healthy skin tissue was calculated.
  • the suppression of (the growth of collagen fibers) was observed, and the remarkable suppression effect on fibroblast activation by albendazole was observed in a concentration-dependent manner.
  • TGF- ⁇ 2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, the evaluation compound (test agent) was brought to the maximum drug plasma concentration (Cmax).
  • Cmax maximum drug plasma concentration
  • the cells were collected after 48 hours.
  • a non-inflammatory stimulus sample untreated
  • a sample to which an evaluation compound was not added were used.
  • the collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection.
  • RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 ⁇ g of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did.
  • real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed.
  • Specific primers for each gene were purchased from Invitrogen.
  • Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
  • the addition of the evaluation compound suppresses the induction of expression of ⁇ SMA (myofibroblast formation) induced by inflammation stimulation, the expression induction of various collagens, and the expression induction of various differentiation-related markers.
  • ⁇ SMA myofibroblast formation
  • the inhibition was observed, and the inhibitory effect on the fibroblast activation by the evaluation compound was observed.
  • TGF- ⁇ 2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, evaluation compounds were added under each of 4 conditions based on Cmax, and cells were recovered after 120 hours did.
  • a non-inflammatory stimulus sample untreated
  • a sample to which an evaluation compound was not added were used.
  • the collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection.
  • RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 ⁇ g of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did.
  • real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed.
  • Specific primers for each gene were purchased from Invitrogen.
  • Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
  • TGF- ⁇ 2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, evaluation compounds were added under each of four conditions based on Cmax, and cell culture was performed after 120 hours. The supernatant was collected.
  • absorbance at 555 nm was measured using Infinite M200 (TECAN).
  • a standard curve was prepared using a standard solution, the content of collagen secreted in the culture solution was calculated, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
  • inflammation stimulation of normal skin tissue-derived fibroblasts confirmed that the secretion of collagen was promoted, and showed properties similar to those of scleroderma patient-derived fibroblasts. Therefore, the inflammatory stimulus on healthy skin tissue-derived fibroblasts reproduces “collagen fiber hyperplasia by fibroblast activation” identified in scleroderma patient tissues.
  • mebendazole significantly suppresses the amount of collagen secreted in the culture supernatant (concentration dependent), and mebendazole appears specifically in scleroderma patient skin. It is suggested that it has an inhibitory effect on collagen fiber proliferation.
  • albendazole As shown in FIG. 13C, the addition of albendazole (ALZ) significantly suppresses the amount of collagen secreted in the culture supernatant, and albendazole is a collagen fiber specifically seen in scleroderma patient skin. It has been suggested that it has an inhibitory effect on hyperplasia of AZA
  • the most frequently used model animal is "a mouse with fibrotic foci formation caused by inflammation by intradermal administration of bleomycin (BLM)", but explored conditions that allow thickening and fibrosis equivalent to SSc patients Optimization was carried out to construct SSc disease model mice.
  • bleomycin was continuously administered subcutaneously daily at a dose of 1.5 mg / day (28 days).
  • the evaluation compound was administered to the mice receiving bleomycin under the following conditions.
  • As a control the same test was conducted on mice that received 0.5% (w / w) aqueous solution of CMC (carboxymethylcellulose) alone.
  • Skin tissues were collected 28 days after the initiation of bleomycin administration, and formalin fixed paraffin blocks were prepared. These tissue samples were deparaffinized by treating with xylene 5 minutes ⁇ 3 times, 100% ethanol 1 minute ⁇ 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes . Staining was performed with Meyer's hematoxylin solution for 4 minutes and eosin solution for 1 minute. Treated in running water for 5 minutes, treated with 70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1 minute, 100% ethanol 1 minute ⁇ 2 times, xylene 5 minutes ⁇ 3 times, using marinol Sealed. After drying at room temperature for 15 minutes or more, observation was performed using an optical microscope.
  • epalrestat significantly suppressed BLM-induced skin thickening (concentration dependent), confirming the therapeutic efficacy of epalrestat against SSc using SSc model mice. .
  • Bleomycin (BLM) -induced scleroderma model construction 150 ml of Bleomycin (Bleo) dissolved in saline (NaCl) at 1 mg / ml on the back of 8- to 10-week-old female C57BL / 6J wild-type mice Every day for 4 weeks, it was injected subcutaneously to induce scleroderma foci of skin hardening due to fibrosis.
  • Oral administration of Compound A At 40 mg / kg or 80 mg / kg of Compound A (: LG283) dissolved in polyethylene glycol was orally administered daily at the same time as Bleo administration (evaluation test group), and compared with placebo group to which only polyethylene glycol was administered.
  • inflammation causes the transformation of fibroblasts in the dermis, and the secretion of matrix molecules such as (1) collagen and (2) Fibronectin is enhanced, resulting in the formation of fibrotic foci in the cell dermis.
  • matrix molecules such as (1) collagen and (2) Fibronectin
  • TGF- ⁇ 1 The action of TGF- ⁇ 1 on human normal cultured dermal fibroblast HFFF induced transformation, and the expression (mRNA) of collagen Collagen 1A2 and Fibronectin 1 was enhanced (quantified by qRT-PCR). By acting on this compound A (4.5 ⁇ M), the expression (mRNA) of collagens Collagen 1A2 and Fibronectin 1 was significantly suppressed (FIG. 20).
  • TGF- ⁇ 1 acts on human normal cultured skin fibroblasts HFFF, transformation is induced and the expression level of collagen Collagen 1A2 protein is enhanced (evaluated by Western Blotting). By acting on this compound A (4.5 ⁇ M), the expression of collagen collagen 1A2 protein was significantly suppressed (FIG. 21).
  • Compound A suppresses the expression of matrix molecules of (1) collagen and (2) fibronectin involved in fibrotic foci formation of scleroderma at gene expression level (experiment A) and protein secretion level (experiments B and C) It was confirmed that Compound A was effective in treating scleroderma.
  • the present invention is useful as a therapeutic and / or prophylactic agent for systemic scleroderma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides a pharmaceutical composition for treating or preventing systemic scleroderma, the composition containing a compound that suppresses the activation of fibroblast cells in tissues derived from dermis.

Description

全身性強皮症治療剤Therapeutic agent for systemic scleroderma
　本発明は、全身性強皮症（ｓｙｓｔｅｍｉｃ　ｓｃｌｅｒｏｄｅｒｍａ：ＳＳｃ）の予防及び治療薬に関する。より詳しくは、全身性強皮症の病態である線維化の抑制及び／又は治療薬に関する。 The present invention relates to a preventive and therapeutic agent for systemic scleroderma (SSc). More specifically, the present invention relates to an agent for suppressing and / or treating fibrosis, which is a pathological condition of systemic scleroderma.
　全身性強皮症は皮膚真皮において線維化が生じ四肢の可動域制限・拘縮による運動障害、潰瘍化による末梢部の壊死等が生じ、患者のQOLが大幅に低下すると共に、進行により肺線維症、肺動脈性肺高血圧症、腎クリーゼ等の生命予後に係る種々の重篤な疾患を併発する難治性疾患であり、現在2万人規模の患者が国内に存在している。 Systemic scleroderma causes fibrosis in the skin dermis, movement disorders due to restricted movement of the limbs / contracture, necrosis in the peripheral area due to ulceration, etc., and the QOL of the patient is significantly reduced, and the lung fibers are advanced by progress. Disease, pulmonary arterial hypertension, renal crisis, and other intractable diseases accompanied by various serious life-threatening diseases. Currently, there are about 20,000 patients in Japan.
　本疾患の最大の課題は、未だ発症機序が明らかになっておらず、有効な治療薬が存在しない点にある。このため国においては難病に指定し患者への手厚い対応や新たな治療技術の開発が進められている。 The main problem of this disease is that the pathogenesis has not been clarified yet and there is no effective therapeutic agent. For this reason, in the country, it has been designated as intractable disease, and careful response to patients and development of new treatment technology are being promoted.
　現状のＳＳｃ治療薬としては、主に免疫抑制剤、ステロイド、漢方薬等の炎症を抑制する薬剤が用いられているが、いずれも対症療法薬であり根治可能な治療薬は存在しない。近年、肺高血圧症治療薬ボセンタン(Actelion Pharmaceuticals)がしばしば用いられているが、その効能は手指潰瘍の発症抑制に留まるなど有効性が限定的でありＳＳｃに対する治療効果は認められていない。 As the current SSc therapeutic agents, drugs that suppress inflammation, such as immunosuppressants, steroids, and traditional Chinese medicines, are mainly used, but all of them are symptomatic therapeutic drugs and there is no cure that can be cured. In recent years, pulmonary antihypertensive agent bosentan (Actelion Pharmaceuticals) has often been used, but its efficacy is limited, for example, to suppress the onset of finger ulcers, and no therapeutic effect on SSc has been observed.
　また、種々の薬剤の開発が進められているが、多くが他の疾患で線維化の抑制効果を示したとの情報を基にした薬剤の転用であり、ＳＳｃの発症機序の抑制を作用メカニズムとした薬剤はなく、現在までに患者において治療効果の確認に至った薬剤はない。 In addition, although various drugs are being developed, they are diverted from the information based on the information that many showed the suppression effect of fibrosis in other diseases, and the action mechanism suppresses action mechanism of SSc. There is no drug that has been identified, and to date no drug has reached confirmation of its therapeutic effect in patients.
　例えば、最近、特発性肺線維症治療薬として認可された薬ニンテダニブ(Boehringer Ingelheim)は、間質性肺疾患を伴う全身性強皮症患者に対して治験が進められているが、随伴疾患である肺機能の改善を目指した開発であり、ＳＳｃの全身病態に対する有効性は限定的であるといわれている。また、肺高血圧症薬ボセンタン(Actelion Pharmaceuticals)、抗炎症薬トシリズマブ(抗IL-6受容体抗体、中外製薬)などの開発が進められているが、現在のところ最終的に全身性強皮症に対する有効性が確認できた薬剤は存在しない。 For example, the recently approved drug for treating idiopathic pulmonary fibrosis, Boehringer Ingelheim, is being studied in patients with systemic scleroderma with interstitial lung disease, but with concomitant disease This is a development aimed at improving lung function, and the effectiveness of SSc on systemic pathologies is said to be limited. In addition, development of the pulmonary hypertension drug Bosentan (Actelion Pharmaceuticals), the anti-inflammatory drug Tocilizumab (anti-IL-6 receptor antibody, Chugai Pharmaceutical), etc. is underway, but at present it is finally for systemic scleroderma There is no drug whose efficacy has been confirmed.
　したがって、全身性強皮症に対する有効な薬剤が求められている。 Therefore, there is a need for an effective drug for systemic scleroderma.
　上記課題に鑑み、本発明は、以下を提供する。
［１］真皮由来の組織における線維芽細胞活性化を抑制する化合物を含有する、全身性強皮症を治療または予防するための医薬組成物。
［２］上記全身性強皮症が、真皮由来の組織における線維芽細胞活性化によって特徴付けられる、上記［１］に記載の医薬組成物。
［３］上記化合物が、ベンズブロマロン、アルベンダゾール、メベンダゾール、リセドロン酸ナトリウム水和物、エパルレスタット、フルフェナム酸アルミニウム、ザルトプロフェン、アンレキサノクス、メフェナム酸、オキサプロジン、および3 - [（1E）-2-（1H-インドール-6-イル）エテニル] -5 - [（1E）-2- [2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル] -1H-ピラゾールからなる群から選択される、上記［１］または［２］に記載の医薬組成物。 In view of the above problems, the present invention provides the following.
[1] A pharmaceutical composition for treating or preventing systemic scleroderma, which comprises a compound that inhibits fibroblast activation in a tissue derived from dermis.
[2] The pharmaceutical composition according to [1] above, wherein the systemic scleroderma is characterized by fibroblast activation in a tissue derived from dermis.
[3] The above compounds are benzbromarone, albendazole, mebendazole, risedronate sodium hydrate, epalrestat, aluminum flufenamate, zaltoprofen, anlexanox, mefenamic acid, oxaprozin, and 3-[(1E) -2- (1H] -Indol-6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridylmethoxy) phenyl] ethenyl] -1 H-pyrazole, selected from the above The pharmaceutical composition as described in 1] or [2].
　本発明者らによる全身性強皮症患者由来の検体の解析から、全身性強皮症患者の真皮組織において炎症が亢進しており、組織に存在する細胞（線維芽細胞）が活性化されコラーゲン等の細胞外マトリックスの分泌亢進しており、これにより病巣である線維化が生じていることが明らかになった。 According to the present inventors' analysis of a sample derived from a patient with systemic scleroderma, inflammation is enhanced in dermal tissue of a patient with systemic scleroderma, and cells (fibroblasts) present in the tissue are activated and collagen is Secretion of the extracellular matrix, etc., and this revealed that the focal fibrosis was occurring.
　このため炎症によって組織の線維芽細胞の活性化の抑制が有力な治療機序であると考え、線維芽細胞の活性化を誘起し、化合物の抑制活性を評価可能な系を構築し、この系により化合物の活性化抑制能を測定し、これを基にＳＳｃ治療薬を得ることができた。 For this reason, it is thought that suppression of fibroblast activation in tissue is a potential therapeutic mechanism by inflammation, and activation of fibroblasts is induced to construct a system capable of evaluating the inhibitory activity of compounds. Thus, the ability to suppress the activation of the compound was measured, and based on this the SSc therapeutic agent could be obtained.
　本発明によれば、全身性強皮症を治療するための医薬、そのような医薬に使用され得る化合物、ならびにそのような化合物を用いた全身性強皮症の治療方法、医薬の製造方法および使用方法が提供される。 According to the present invention, a medicament for treating systemic scleroderma, a compound that can be used for such a medicament, a method for treating systemic scleroderma using such a compound, a method for producing a medicament and A method of use is provided.
　本発明によれば、全身性強皮症の病態である線維化の抑制作用を有する化合物が提供される。 According to the present invention, there is provided a compound having an inhibitory action on fibrosis which is a pathological condition of systemic scleroderma.
図１は、強皮症（ＳＳｃ）患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織のヘマトキシリン・エオジン（Ｈ＆Ｅ）染色像を示す図である。FIG. 1 shows hematoxylin and eosin (H & E) stained images of skin tissues from scleroderma (SSc) patients and healthy controls (Control). 図２Ａは、強皮症患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織の抗α－ＳＭＡ抗体を用いた免疫組織化学（Ｉｍｍｕｎｏｈｉｓｔｏｃｈｅｍｉｓｔｒｙ：ＩＨＣ）解析の結果を示す図である。FIG. 2A shows the results of immunohistochemistry (IHC) analysis of skin tissues from scleroderma patients and healthy controls (Control) using an anti-α-SMA antibody. 図２Ｂは、強皮症患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織の抗Ｆｉｂｒｏｎｅｃｔｉｎ抗体を用いたＩＨＣ解析の結果を示す図である。FIG. 2B shows the results of IHC analysis using anti-Fibronectin antibodies of skin tissues from scleroderma patients and healthy controls (Control). 図２Ｃは、強皮症患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織の抗ＺＥＢ１抗体を用いたＩＨＣ解析の結果を示す図である。FIG. 2C shows the results of IHC analysis of skin tissue from scleroderma patients and healthy controls (Control) using an anti-ZEB1 antibody. 図３は、図１および図２Ａ－Ｃに示したＳＳｃ検体における病態解析および分子動態解析の結果をまとめて示した図である。FIG. 3 is a diagram collectively showing the results of pathological analysis and molecular dynamics analysis in the SSc sample shown in FIGS. 1 and 2A-C. 図４は、強皮症患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織からの初代培養線維芽細胞について、各種遺伝子群（分化関連マーカー）の発現動態解析を行った結果を示すグラフである。FIG. 4 is a graph showing the results of expression kinetic analysis of various gene groups (differentiation related markers) with respect to primary cultured fibroblasts from skin tissues derived from scleroderma patients and healthy controls (Control). 図５は、強皮症患者および健常者（Ｃｏｎｔｒｏｌ）由来の皮膚組織からの初代培養線維芽細胞について、コラーゲン（膠原線維）の発現動態解析を行った結果を示すグラフである。FIG. 5 is a graph showing the results of analysis of expression kinetics of collagen (collagen fibers) of primary culture fibroblasts from skin tissues from scleroderma patients and healthy individuals (Control). 図６は、健常線維芽細胞および炎症刺激後の健常線維芽細胞を用いた各種分化関連マーカーの発現動態解析の結果を示すグラフである。FIG. 6 is a graph showing the results of expression kinetic analysis of various differentiation-related markers using healthy fibroblasts and healthy fibroblasts after inflammatory stimulation. 図７は、健常線維芽細胞および炎症刺激後の健常線維芽細胞を用いたコラーゲン（膠原線維）の発現動態解析の結果を示すグラフである。FIG. 7 is a graph showing the results of expression kinetic analysis of collagen (collagen fiber) using healthy fibroblasts and healthy fibroblasts after inflammatory stimulation. 図８Ａは、強皮症患者由来細胞を用いて、評価化合物（エパルレスタットおよびオキサプロジン）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 8A is a graph showing the evaluation results of evaluation compounds (epalrestat and oxaprozin) on the expression of various differentiation-related marker molecules by scleroderma patient-derived cells at 48 hours after the addition of the evaluation compound. 図８Ｂは、強皮症患者由来細胞を用いて、評価化合物（アンレキサノクスおよびザルトプロフェン）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 8B is a graph showing the results of evaluating the suppressive effects of the evaluation compounds (anlexanox and zaltoprofen) on the expression of various differentiation-related marker molecules using scleroderma patient-derived cells 48 hours after the addition of the evaluation compounds. 図８Ｃは、強皮症患者由来細胞を用いて、評価化合物（メフェナム酸およびフルフェナム酸アルミニウム）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 8C is a graph showing the results of evaluating the suppressive effects of the evaluation compounds (mefenamic acid and aluminum flufenamate) on the expression of various differentiation-related marker molecules 48 hours after the addition of the evaluation compounds using scleroderma patient-derived cells. It is. 図９Ａは、強皮症患者由来細胞を用いて、エパルレスタットの各種分化関連マーカー分子発現に対する抑制活性を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 9A is a graph showing the results of evaluating the suppressive activity of epalrestat on various differentiation-related marker molecule expression 120 hours after the addition of the evaluation compound using scleroderma patient-derived cells. 図９Ｂは、強皮症患者由来細胞を用いて、メベンダゾールの各種分化関連マーカー分子発現に対する抑制活性を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 9B is a graph showing the results of evaluating the suppressive activity of mebendazole on the expression of various differentiation-related marker molecules 120 hours after the addition of the evaluation compound using scleroderma patient-derived cells. 図９Ｃは、強皮症患者由来細胞を用いて、アンベンダゾールの各種分化関連マーカー分子発現に対する抑制活性を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 9C is a graph showing the results of evaluation of suppression activity of various types of differentiation-related marker molecules of anbendazole 120 hours after addition of an evaluation compound using scleroderma patient-derived cells. 図１０は、強皮症患者由来細胞を用いて、強皮症患者皮膚に特異的に認められたコラーゲン分泌亢進（線維化の要因）に対する評価化合物（エパルレスタット）による抑制効果を評価した結果を示す図である。FIG. 10 shows the results of evaluating the inhibitory effect of an evaluation compound (Epalrestat) on collagen secretion increase (factor of fibrosis) specifically recognized in scleroderma patient skin using scleroderma patient-derived cells FIG. 図１１Ａは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（エパルレスタットおよびオキサプロジン）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 11A shows the results of evaluating the suppressive effects of evaluation compounds (epalrestat and oxaprodin) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects 48 hours after addition of the evaluation compounds. It is a graph. 図１１Ｂは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（アンレキサノクスおよびザルトプロフェン）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 11B shows the results of evaluating the suppressive effect of evaluation compounds (Anlexanox and zaltoprofen) on the expression of various differentiation-related marker molecules by the inflammation stimulation system using fibroblasts from healthy subjects 48 hours after the addition of the evaluation compounds. It is a graph. 図１１Ｃは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（フルフェナム酸アルミニウムおよびメフェナム酸）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後４８時間において評価した結果を示すグラフである。FIG. 11C shows that the inhibitory effect of the evaluation compounds (aluminum flufenamate and mefenamic acid) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects was evaluated 48 hours after the addition of the evaluation compounds. It is a graph which shows a result. 図１２Ａは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（エパルレスタット）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 12A is a graph showing the results of evaluating the suppressive effect of an evaluation compound (Epalrestat) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects at 120 hours after the addition of the evaluation compound. is there. 図１２Ｂは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（メベンダゾール）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 12B is a graph showing the results of evaluating the suppressive effect of an evaluation compound (mebendazole) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects 120 hours after the addition of the evaluation compound. is there. 図１２Ｃは、健常者由来線維芽細胞を用いた炎症刺激系による、評価化合物（アルベンダゾール）による各種分化関連マーカー分子発現に対する抑制効果を、評価化合物の添加後１２０時間において評価した結果を示すグラフである。FIG. 12C is a graph showing the results of evaluating the suppressive effect of an evaluation compound (albendazole) on the expression of various differentiation-related marker molecules by an inflammation stimulation system using fibroblasts from healthy subjects at 120 hours after addition of the evaluation compound. It is. 図１３Ａは、健常由来線維芽細胞を用いた炎症刺激系を用いて、コラーゲン分泌亢進（線維化の要因）に対する評価化合物（エパルレスタット）による抑制効果を評価した結果を示す図である。FIG. 13A is a diagram showing the results of evaluating the suppressive effect of an evaluation compound (epalrestat) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts. 図１３Ｂは、健常由来線維芽細胞を用いた炎症刺激系を用いて、コラーゲン分泌亢進（線維化の要因）に対する評価化合物（メベンダゾール）による抑制効果を評価した結果を示す図である。FIG. 13B is a diagram showing the results of evaluation of the suppressive effect of an evaluation compound (mebendazole) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts. 図１３Ｃは、健常由来線維芽細胞を用いた炎症刺激系を用いて、コラーゲン分泌亢進（線維化の要因）に対する評価化合物（アルベンダゾール）による抑制効果を評価した結果を示す図である。FIG. 13C is a diagram showing the results of evaluation of the suppressive effect of an evaluation compound (albendazole) on collagen secretion enhancement (factor of fibrosis) using an inflammation stimulation system using healthy derived fibroblasts. 図１４は、強皮症疾患モデルマウスを用いて実施した線維化抑制の薬効評価プロトコルを模式的に示す図である。FIG. 14 is a view schematically showing a protocol for evaluating the efficacy of suppression of fibrosis carried out using a scleroderma disease mouse model. 図１５Ａは、ブレオマイシン投与により確立した強皮症疾患モデルマウス由来の皮膚組織に対するエパルレスタットの皮膚肥厚抑制効果を示すＨ＆Ｅ染色像を表す図である。FIG. 15A is a diagram showing an H & E stained image showing the skin thickening inhibitory effect of epalrestat on skin tissue derived from a scleroderma disease model mouse established by administration of bleomycin. 図１５Ｂは、図１５Ａの結果を定量的にグラフに示した図である。FIG. 15B is a graph quantitatively showing the results of FIG. 15A. 図１６Ａ－Ｄは、実施例７においてブレオマイシン（Bleo）投与により誘導した強皮症疾患モデルマウスの皮膚組織に対する3-[（1E）-2-（1H-インドール-6-イル）エテニル]-5-[（1E）-2-[2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル]-1H-ピラゾール（化合物Ａ：ＬＧ２８３）の皮膚肥厚抑制効果を示すＨ＆Ｅ染色像を表す図である。（Ａ）生後8～10週の雌のC57BL/6J野生型マウスの背部に、生理食塩水（NaCl）のみ4週間連日皮下注射したもの。（Ｂ－Ｄ）生後8～10週の雌のC57BL/6J野生型マウスの背部に、生理食塩水 (NaCl) で1 mg/mlに溶解したブレオマイシン (Bleo) 150 mlを、4週間連日皮下注射して線維化による皮膚硬化の強皮症病巣を誘導した後、ポリエチレングリコールのみを投与したplacebo群（Ｂ）、ポリエチレングリコールに溶解した40 mg/kgまたは 80 mg/kgの化合物ＡをBleo投与と同時に連日経口投与した評価試験群（それぞれ（Ｃ）および（Ｄ））を示す。16A-D show 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5 against skin tissue of scleroderma disease model mouse induced by administration of bleomycin (Bleo) in Example 7. It is a figure showing the H & E stained image which shows the skin thickening inhibitory effect of-[(1E) -2- [2-methoxy-4- (2-pyridyl methoxy) phenyl] ethenyl] -1H- pyrazole (compound A: LG283). . (A) Physiological saline (NaCl) alone and subcutaneously injected for 4 weeks every day on the back of 8- to 10-week-old female C57BL / 6J wild-type mice. (B-D) On the back of 8- to 10-week-old female C57BL / 6J wild-type mice, 150 ml of bleomycin (Bleo) dissolved in saline (NaCl) at 1 mg / ml was injected subcutaneously for 4 consecutive weeks After inducing scleroderma foci of skin hardening by fibrosis, placebo group (B) administered only polyethylene glycol, 40 mg / kg or 80 mg / kg of compound A dissolved in polyethylene glycol and Bleo administration The evaluation test group ((C) and (D) respectively) which orally administered simultaneously on the same day is shown. 図１７は、図１６Ａ－Ｄに示した結果を、定量的にグラフに示した図である。FIG. 17 is a graph quantitatively showing the results shown in FIG. 16A-D. 図１８Ａ－Ｄは、実施例７においてMasson’s trichrome（マッソントリクローム）染色により染色した標本の写真を示す図である。青色に染色された領域は線維化の領域に対応する。なお、図１８Ａ－Ｄの条件は、図１６Ａ－Ｄの条件に対応する。18A-D show photographs of specimens stained with Masson's trichrome (Masson trichrome) staining in Example 7. FIG. The area stained blue corresponds to the area of fibrosis. The conditions of FIGS. 18A-D correspond to the conditions of FIGS. 16A-D. 図１９は、図１８Ａ－Ｄに示した結果を、定量的にグラフに示した図である。FIG. 19 is a diagram quantitatively showing the results shown in FIG. 18A-D. 図２０は、コラーゲンCollagen 1A2およびFibronectin1 の発現（mRNA）に対する化合物Ａの抑制効果の評価実験（実験Ａ）の結果を示す図である。FIG. 20 is a diagram showing the results of an evaluation experiment (experiment A) of the inhibitory effect of compound A on the expression (mRNA) of collagens Collagen 1A2 and Fibronectin 1. 図２１は、コラーゲンCollagen 1A2タンパク質の発現に対する化合物Ａの抑制効果の評価実験（実験Ｂ）の結果を示す図である。FIG. 21 is a diagram showing the results of an evaluation experiment (experiment B) of the inhibitory effect of compound A on the expression of collagen Collagen 1A2 protein. 図２２は、Fibronectinタンパク質の発現に対する化合物Ａの抑制効果の評価実験（実験Ｃ）の結果を示す図である。FIG. 22 shows the results of an evaluation experiment (experiment C) of the suppressive effect of compound A on the expression of Fibronectin protein.
１．用語の定義
　本明細書中、「線維芽細胞活性化」とは、線維芽細胞におけるα平滑筋アクチン（ＳＭＡ）発現の亢進（筋線維芽細胞化）、各種コラーゲン（膠原線維）発現の亢進、フィブロネクチン発現の亢進、その他ジンクフィンガーＥボックス結合ホメオボックス１（ＺＥＢ１）、Ｎ－カドヘリン（N-cadherin）、ビメンチン（Vimentin）等の分化関連マーカーの発現亢進により特徴づけられる線維芽細胞における形質の変化であって、全身性強皮症患者の皮膚の真皮内に特徴的に見られるものを意味する。 1. Definition of Terms In the present specification, “fibroblast activation” refers to enhancement of α-smooth muscle actin (SMA) expression in fibroblasts (myofibroblast formation), enhancement of various collagen (collagen fibers) expression, Alteration of traits in fibroblasts characterized by enhanced expression of fibronectin, other zinc finger E box-bound homeobox 1 (ZEB1), N-cadherin (N-cadherin), enhanced expression of differentiation-related markers such as vimentin (Vimentin) It means what is characteristically found in the dermis of the skin of patients with generalized scleroderma.
　本明細書中、「線維芽細胞活性化を抑制する」とは、線維芽細胞活性化により亢進したαＳＭＡ、各種コラーゲン、フィブロネクチンなどの分化関連マーカーの発現量が、抑制剤の作用により減少することを意味し、減少後の当該マーカーの発現量は正常細胞と比較して必ずしも低くなくてもよい。 In the present specification, “suppressing fibroblast activation” means that the expression level of differentiation-related markers such as αSMA, various collagens, fibronectin enhanced by fibroblast activation is decreased by the action of the inhibitor. The expression level of the marker after reduction may not necessarily be low compared to normal cells.
　線維芽細胞の活性化またはその抑制は、後述の実施例において詳述するように、細胞が分化したときに発現が変化する遺伝子群（「分化関連マーカー」）の発現動態を解析することにより定量的に評価することができる。 Fibroblast activation or suppression thereof is quantified by analyzing the expression dynamics of a group of genes whose expression changes when cells are differentiated ("differentiation related markers"), as described in detail in the examples below. Can be evaluated.
２．本発明の線維芽細胞活性化を抑制する化合物およびその使用 2. Compounds for suppressing fibroblast activation of the present invention and uses thereof
　一実施形態において、本発明は、真皮由来の組織における線維芽細胞活性化を抑制する化合物および当該化合物を含有する組成物を提供する。 In one embodiment, the present invention provides a compound that inhibits fibroblast activation in a tissue derived from dermis and a composition containing the compound.
　別の実施形態において、本発明は、真皮由来の組織における線維芽細胞活性化を抑制する化合物を治療有効量含有する、全身性強皮症の治療及び／又は予防のための医薬組成物を提供する。 In another embodiment, the present invention provides a pharmaceutical composition for treating and / or preventing systemic scleroderma, which comprises a therapeutically effective amount of a compound that inhibits fibroblast activation in dermis-derived tissues. Do.
　さらに別の実施形態において、本発明は、全身性強皮症の治療及び／又は予防に使用されるための、真皮由来の組織における線維芽細胞活性化を抑制する化合物を提供する。 In yet another embodiment, the present invention provides a compound that inhibits fibroblast activation in dermis-derived tissues for use in the treatment and / or prevention of systemic scleroderma.
　さらに別の実施形態において、本発明は、全身性強皮症の治療及び／又は予防のための医薬の製造における、真皮由来の組織における線維芽細胞活性化を抑制する化合物の使用（または使用方法）を提供する。 In yet another embodiment, the present invention is the use (or method of use of a compound that inhibits fibroblast activation in dermis-derived tissues in the manufacture of a medicament for the treatment and / or prevention of systemic scleroderma. )I will provide a.
　さらに別の実施形態において、本発明は、全身性強皮症の治療及び／又は予防方法であって、真皮由来の組織における線維芽細胞活性化を抑制する化合物を、当該治療が必要な患者に投与する工程を包含する、方法を提供する。 In yet another embodiment, the present invention relates to a method of treating and / or preventing systemic scleroderma, which comprises administering to a patient in need thereof a compound that inhibits fibroblast activation in a dermis-derived tissue. A method is provided, comprising the step of administering.
　上記各実施形態において使用される「真皮由来の組織における線維芽細胞活性化を抑制する化合物」の具体例には、ベンズブロマロン、アルベンダゾール、メベンダゾール、リセドロン酸ナトリウム水和物、エパルレスタット、フルフェナム酸アルミニウム、ザルトプロフェン、アンレキサノクス、メフェナム酸、オキサプロジン、および3 - [（1E）-2-（1H-インドール-6-イル）エテニル] -5 - [（1E）-2- [2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル] -1H-ピラゾール、ならびにこれらの薬学的に許容される塩およびこれらの誘導体化合物が含まれる。 Specific examples of the “compound that inhibits fibroblast activation in the tissue derived from dermis” used in each of the above embodiments include benzbromarone, albendazole, mebendazole, risedronate sodium hydrate, epalrestat, flufenamic acid Aluminum, zaltoprofen, annexanox, mefenamic acid, oxaprozin, and 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5- (1E) -2- [2-methoxy-4- (2) 2-Pyridylmethoxy) phenyl] ethenyl] -1H-pyrazole, as well as their pharmaceutically acceptable salts and their derivative compounds.
　本発明の化合物の全身性強皮症の治療効果を確認する方法は、特に限定されないが、例えば、ブレオマイシン誘導肺線維症モデルマウス（全身性強皮症や肺線維症の疾患モデル動物として認知されているマウス：例えば、Curr Opin Pulm Med. 2011 Sep;17(5):355-61（G）等）を用いて、病巣形成の阻害効果を確認することにより行うことができる。上記モデルマウスを用いた具体的な試験方法については、後述の実施例に示されている。 The method for confirming the therapeutic effect of the compound of the present invention on systemic scleroderma is not particularly limited. For example, bleomycin-induced pulmonary fibrosis model mice (generally recognized as animal models for systemic scleroderma and pulmonary fibrosis) Using a mouse, for example, Curr Opin Pulm Med. 2011 Sep; 17 (5): 355-61 (G), etc.) to confirm the inhibitory effect of lesion formation. The specific test method using the above-mentioned model mouse is shown in the examples described later.
　本発明において、「薬学的に許容される塩」とは、上記の具体的な化合物と薬学的に許容される塩を形成するものを含み、特に限定されない。具体的には、例えば、ハロゲン化水素酸塩（例えば、フッ化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩等）、無機酸塩（例えば、硫酸塩、硝酸塩、過塩素酸塩、リン酸塩、炭酸塩、重炭酸塩等）、有機カルボン酸塩（例えば、酢酸塩、シュウ酸塩、マレイン酸塩、酒石酸塩、フマル酸塩、クエン酸塩等）、有機スルホン酸塩（例えば、メタンスルホン酸塩、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩、ベンゼンスルホン酸塩、トルエンスルホン酸塩、カンファースルホン酸塩等）、アミノ酸塩（例えば、アスパラギン酸塩、グルタミン酸塩等）、有機アミン塩（例えば、トリメチルアミン、トリエチルアミン、ピリジン、ピコリン、ジシクロヘキシルアミン、Ｎ，Ｎ’－ジベンジルエチレンジアミン、アルギニン及びリジンなどの有機塩基との塩）、四級アミン塩、アルカリ金属塩（例えば、ナトリウム塩、カリウム塩等）、アルカリ土類金属塩（例えば、マグネシウム塩、カルシウム塩等）、又はアルミニウム塩等が挙げられる。 In the present invention, the "pharmaceutically acceptable salts" include those which form pharmaceutically acceptable salts with the specific compounds described above, and are not particularly limited. Specifically, for example, hydrohalide (eg, hydrogen fluoride, hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salt (eg, sulfate, nitrate, persulfate, etc.) Chlorates, phosphates, carbonates, bicarbonates, etc., organic carboxylates (eg, acetates, oxalates, maleates, tartrates, fumarates, citrates, etc.), organic sulfones Acid salts (eg, methane sulfonate, trifluoromethane sulfonate, ethane sulfonate, benzene sulfonate, toluene sulfonate, camphor sulfonate, etc.), amino acid salts (eg, aspartate, glutamate, etc.) Organic amine salts (eg, trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N, N'-dibenzylethylenediamine, arginine and Salts with organic bases such as sodium), quaternary amine salts, alkali metal salts (eg, sodium salts, potassium salts etc.), alkaline earth metal salts (eg. Magnesium salts, calcium salts etc.), aluminum salts etc. It can be mentioned.
　本発明において、「誘導体化合物」とは、上記の具体的な化合物の等価物であり得る化合物をいい、配糖体又はエステル体（例えば、メチルエステル、エチルエステル等のアルキルエステル等）等も包含される。 In the present invention, the "derivative compound" refers to a compound which may be an equivalent of the above specific compound, and includes glycosides or esters (eg, alkyl esters such as methyl ester, ethyl ester etc.), etc. Be done.
　なお、誘導体化合物が等価物であるかについての確認は、上記した試験により、皮膚真皮組織内の線維芽細胞活性化の亢進を抑制する機能を有するかを確認することにより行うことができる。 In addition, confirmation about whether a derivative compound is equivalent can be performed by confirming whether it has a function which suppresses the promotion of fibroblast activation in skin dermis tissue by an above-described test.
３．本発明の化合物の医薬への適用 3. Application of the compound of the present invention to medicine
　本発明の化合物は、限定されるものではないが、慣用される方法により製剤化し、本発明の予防及び／又は治療剤とすることが可能である。 The compounds of the present invention can be formulated, but not limited to, by conventional methods to provide the prophylactic and / or therapeutic agents of the present invention.
　本発明の予防及び／又は治療剤の好ましい剤形としては、例えば、錠剤、散剤、細粒剤、顆粒剤、ドライシロップ剤、被覆錠剤、口腔内崩壊錠、チュアブル錠、カプセル剤、ソフトカプセル剤、シロップ剤、経口液剤、トローチ剤、ゼリー剤、吸入剤、坐剤、注射剤、軟膏剤、点眼剤、眼軟膏剤、点鼻剤、点耳剤、パップ剤、ローション剤、外用液剤、スプレー剤、外用エアゾール剤、クリーム剤、ゲル剤、テープ剤、バッカル錠、舌下錠、膣坐剤、膣錠、直腸ソフトカプセル剤等が挙げられる。製剤化には、通常用いられる例えば、賦形剤、結合剤、崩壊剤、コーティング剤、滑沢剤、着色剤、矯味矯臭剤や、必要により安定化剤、乳化剤、吸収促進剤、界面活性剤、ｐＨ調製剤、防腐剤、抗酸化剤等を使用することができ、一般に医薬品製剤の原料として用いられる成分を配合して常法により製剤化可能である。本発明は、少なくともひとつの担体を混合させることを特徴とする医薬組成物の調製法も含有する。 Preferred dosage forms of the prophylactic and / or therapeutic agent of the present invention are, for example, tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules, syrups Agents, oral solutions, troches, jellies, inhalants, suppositories, injections, ointments, eye drops, eye ointments, eye drops, nasal drops, ear drops, poultices, lotions, external solutions, sprays, External aerosols, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, rectal soft capsules and the like can be mentioned. For formulation, for example, excipients, binders, disintegrants, coatings, lubricants, coloring agents, flavoring agents and, if necessary, stabilizers, emulsifiers, absorption accelerators, surfactants, etc. PH adjusting agents, preservatives, antioxidants and the like can be used, and the components generally used as raw materials of pharmaceutical preparations can be blended and formulated by a conventional method. The invention also includes a method of preparing a pharmaceutical composition comprising mixing at least one carrier.
　製剤化に用いられる成分（担体）としては、特に限定されないが、例えば大豆油、牛脂、合成グリセライド等の動植物油；例えば、流動パラフィン、スクワラン、固形パラフィン等の炭化水素；例えば、ミリスチン酸オクチルドデシル、ミリスチン酸イソプロピル等のエステル油；例えば、セトステアリルアルコール、ベヘニルアルコール等の高級アルコール；シリコン樹脂；シリコーンオイル；ポリオキシエチレン脂肪酸エステル、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレン硬化ひまし油、ポリオキシエチレンポリオキシプロピレンブロックコポリマー等の界面活性剤；例えば、ヒドロキシエチルセルロース、ポリアクリル酸、カルボキシビニルポリマー、ポリエチレングリコール、ポリビニルピロリドン、メチルセルロース等の水溶性高分子；例えば、エタノール、イソプロパノール等の低級アルコール；例えばグリセリン、プロピレングリコール、ジプロピレングリコール、ソルビトール等の多価アルコール；グルコース、ショ糖等の糖；例えば、無水ケイ酸、ケイ酸アルミニウムマグネシウム、ケイ酸アルミニウム等の無機粉体、精製水等が挙げられる。賦形剤としては、例えば、乳糖、コーンスターチ、白糖、ブドウ糖、マンニトール、ソルビット、結晶セルロース、二酸化ケイ素等が、結合剤としては、例えば、ポリビニルアルコール、ゼラチン、メチルセルロース、エチルセルロース、アラビアゴム、トラガント、ゼラチン、シェラック、ヒドロキシプロピルメチルセルロース、ヒドロキシプロピルセルロース、ポリビニルピロリドン、ポリビニルアセタールジエチルアミノアセテート、トウモロコシデンプン等が、崩壊剤としては、例えば、トウモロコシデンプン、低置換度ヒドロキシプロピルセルロース、クロスポビドン、結晶セルロース、沈降炭酸カルシウム、クロスカルメロースナトリウム、クエン酸カルシウム、デキストリン、ペクチン、カルボキシメチルセルロースカルシウム等が、滑沢剤としては、例えば、ステアリン酸マグネシウム、タルク、ポリエチレングリコール、軽質無水ケイ酸、ショ糖脂肪酸エステル等が、着色剤としては医薬品に添加することが許可されているものが、矯味矯臭剤としては、ココア末、メントール、芳香散、ハッカ油、竜脳、桂皮末等が用いられる。 The components (carriers) used in the formulation are not particularly limited, and examples thereof include animal oils such as soybean oil, beef tallow and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane and solid paraffin; and octyldodecyl myristate, for example. Ester oil such as isopropyl myristate; higher alcohol such as cetostearyl alcohol, behenyl alcohol; silicone resin; silicone oil; polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxy acid Surfactants such as ethylene-cured castor oil, polyoxyethylene polyoxypropylene block copolymer; eg, hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer Water-soluble polymers such as polyethylene glycol, polyvinyl pyrrolidone and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol and sorbitol; sugars such as glucose and sucrose; For example, inorganic powder such as anhydrous silicic acid, aluminum magnesium silicate, aluminum silicate, purified water and the like can be mentioned. As an excipient, for example, lactose, corn starch, sucrose, glucose, mannitol, sorbite, crystalline cellulose, silicon dioxide etc., and as a binder, for example, polyvinyl alcohol, gelatin, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin , Shellac, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyvinyl acetal diethylamino acetate, corn starch etc., and as a disintegrant, for example, corn starch, low substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate , Croscarmellose sodium, calcium citrate, dextrin, pectin, carboxymethylcellulose calcium As lubricants, for example, magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, sucrose fatty acid ester, etc. are permitted to be added to medicines as coloring agents. As a flavoring agent, cocoa powder, menthol, aroma powder, peppermint oil, spinach, cinnamon powder and the like are used.
　例えば、経口製剤は、有効成分である化合物又はその塩もしくはエステルあるいはこれらの水和物と賦形剤、さらに必要に応じて例えば、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤等を加えた後、常法により例えば、散剤、細粒剤、顆粒剤、錠剤、被覆錠剤、カプセル剤等とする。錠剤、顆粒剤の場合には、例えば、糖衣、その他必要により適宜コーティングすることはもちろん差支えない。シロップ剤や注射用製剤等の場合は、例えば、ｐＨ調製剤、溶解剤、等張化剤等と、必要に応じて溶解補助剤、安定化剤等を加えて、常法により製剤化する。また、外用剤の場合は、特に製法が限定されず、常法により製造することができる。使用する基剤原料としては、医薬品、医薬部外品、化粧品等に通常使用される各種原料を用いることが可能で、例えば、動植物油、鉱物油、エステル油、ワックス類、高級アルコール類、脂肪酸類、シリコン油、界面活性剤、リン脂質類、アルコール類、多価アルコール類、水溶性高分子類、アクリル系粘着剤粘土鉱物類、精製水等の原料が挙げられ、必要に応じ、ｐＨ調製剤、抗酸化剤、キレート剤、防腐防黴剤、着色料、香料等を添加することができる。さらに、必要に応じて分化誘導作用を有する成分、例えば、血流促進剤、殺菌剤、消炎剤、細胞賦活剤、ビタミン類、アミノ酸、保湿剤、角質溶解剤等の成分を配合することもできる。 For example, an oral preparation may be a compound which is an active ingredient, or a salt or ester thereof, or a hydrate thereof and an excipient, and further, if necessary, for example, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent After adding etc., it is set as a powder, a fine granule, a granule, a tablet, a coated tablet, a capsule etc. by a conventional method. In the case of tablets and granules, for example, sugar coating, etc. may of course be appropriately coated if necessary. In the case of a syrup, a preparation for injection, etc., for example, a pH adjusting agent, a solubilizing agent, an isotonizing agent and the like, and a solubilizing agent, a stabilizing agent and the like as necessary, are formulated by a conventional method. Moreover, in the case of the external preparation, the manufacturing method is not particularly limited, and the external preparation can be manufactured by an ordinary method. As a base material to be used, various raw materials usually used for medicines, quasi-drugs, cosmetics etc. can be used. For example, animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids Raw materials such as silicone oil, silicone oil, surfactant, phospholipids, alcohols, polyhydric alcohols, water-soluble polymers, acrylic adhesive clay minerals, purified water etc., and if necessary, pH adjustment Preparations, antioxidants, chelating agents, preservatives and fungicides, coloring agents, flavors and the like can be added. Furthermore, if necessary, components having differentiation-inducing action, for example, components such as blood flow enhancers, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents can also be blended. .
　本発明の予防及び／又は治療剤の投与形態は、特に限定されず、経口投与であっても、非経口投与であってもよい。非経口投与としては、例えば、直腸投与、経鼻投与、経肺投与、注射投与（例えば、静脈内投与、脊椎腔内投与、硬膜外腔内投与、筋肉内投与、皮下投与、腹腔内投与、動脈内投与、関節内投与、心臓内投与、嚢内投与、皮内投与、病巣内投与、眼内投与、胸腔内投与、くも膜下投与、子宮内投与、脳室内投与）等が挙げられる。 The administration mode of the prophylactic and / or therapeutic agent of the present invention is not particularly limited, and may be oral administration or parenteral administration. Parenteral administration includes, for example, rectal administration, nasal administration, pulmonary administration, injection administration (eg, intravenous administration, intraspinal administration, epidural administration, intramuscular administration, subcutaneous administration, intraperitoneal administration Intraarterial administration, intraarticular administration, intracardiac administration, intracapsular administration, intradermal administration, intralesional administration, intraocular administration, intrathecal administration, intrathecal administration, intrauterine administration, intraventricular administration, and the like.
　本発明の予防及び／又は治療剤に含まれる、本発明の化合物の含有量は特に限定されないが、投与する対象において、皮膚真皮組織内の線維芽細胞活性化の亢進を抑制する効果を発揮するために十分な用量とすることが好ましい。 Although the content of the compound of the present invention contained in the preventive and / or therapeutic agent of the present invention is not particularly limited, in the subject to be administered, it exerts the effect of suppressing the enhancement of fibroblast activation in skin dermal tissue. It is preferable to use a dose sufficient for
　本発明の予防及び／又は治療剤の投与量は、例えば、症状の程度、年齢、性別、体重、投与形態、塩の種類、疾患の具体的な種類等に応じて異なるが、通常、成人の場合は１日あたり有効成分である本発明の化合物を、経口投与で約２５ｍｇ～約４．５ｇ、好ましくは約７５ｍｇ～約１．５ｇ、さらに好ましくは約１５０ｍｇ～約６００ｍｇを、あるいは、注射投与で約２５ｍｇ～約４．５ｇ、好ましくは約７５ｍｇ～約１．５ｇ、さらに好ましくは約１５０ｍｇ～約６００ｍｇを、それぞれ１回又は数回に分けて投与する。 The dosage of the prophylactic and / or therapeutic agent of the present invention varies depending on, for example, the degree of symptoms, age, sex, body weight, administration form, type of salt, specific type of disease, etc. In the case of oral administration, the compound of the present invention, which is the active ingredient per day, is orally administered at about 25 mg to about 4.5 g, preferably about 75 mg to about 1.5 g, more preferably about 150 mg to about 600 mg About 25 mg to about 4.5 g, preferably about 75 mg to about 1.5 g, more preferably about 150 mg to about 600 mg, each in one or several divided doses.
　本発明の予防及び／又は治療剤の投与対象は、特に限定されないが、ヒトを含む哺乳動物であることが好ましい。ヒトを含む哺乳動物としては、特に限定されないが、例えば、ヒト、サル、マントヒヒ、チンパンジー、マウス、ラット、モルモット、ハムスター、ウサギ、ネコ、イヌ、ヒツジ、ヤギ、ブタ、ウシ及びウマ等が挙げられる。 The administration target of the prophylactic and / or therapeutic agent of the present invention is not particularly limited, but is preferably a mammal including human. Mammals including humans are not particularly limited, and include, for example, humans, monkeys, baboons, chimpanzees, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, goats, pigs, cattle and horses, etc. .
　本発明の組成物は、所望により、有効成分を含有する１つ以上の単位剤形を含有することができるパック又はディスペンサー装置等の容器を備えたキットの形で提供されうる。 The compositions of the present invention may, if desired, be provided in the form of a kit comprising a container, such as a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
　本発明はまた、別々の薬剤組成物をキット形に組み合わせることもできる。このキットは２種類又はそれ以上の別々の薬剤組成物を含んでもよい。例えば、本発明の化合物と、抗炎症剤及び／又は免疫抑制剤を含んでもよい。このキットは、通常、例えば、分割式ボトル又は分割式ホイルパケットのような、別々の組成物を含有するための容器を含むが、別々の組成物を単一の非分割式容器に含めることもできる。キット形は、別々の成分を異なる投与形（例えば、経口的と非経口的）で投与することが好ましい場合、別々の成分を異なる投与間隔で投与する場合、又は処方医師によって組み合わせた個々の成分を滴定する必要がある場合に、特に有用である。 The present invention can also combine separate drug compositions in kit form. The kit may comprise two or more separate pharmaceutical compositions. For example, the compounds of the present invention and anti-inflammatory and / or immunosuppressive agents may be included. The kit will usually include a container for containing the separate compositions, such as, for example, a split bottle or a split foil packet, but the separate compositions may also be included in a single non-split container. it can. The kit form is preferably to administer the separate components in different dosage forms (e.g. orally and parenterally), when the separate components are administered at different dosing intervals, or individual components combined by the prescribing physician It is particularly useful when it is necessary to titrate.
　パックは、例えば、金属もしくはプラスチックホイル、例えば、ブリスターパックを含むことができる。ブリスターパックはパッケージング業界において周知であり、製薬的単位投与形（錠剤、カプセル等）のパッケージングに広く用いられている。ブリスターパックは一般に、透明なプラスチック材料のホイルによって覆われた比較的硬質材料のシートから成るのが好ましい。パッケージング・プロセス中に、該プラスチックホイル中に凹みが形成される。これらの凹みは、パックされる個々の錠剤又はカプセルのサイズ及び形状に合わせてある。次に、錠剤又はカプセルを凹み中に入れて、凹みが形成された方向とは逆のホイル面において、比較的硬質材料のシートをプラスチックホイルに対してシールする。その結果、錠剤又はカプセルが該プラスチックホイルと該シートとの間の凹み中にシールされる。シートの強度は、凹みの場所においてシートに開口が形成されるように手で圧力を凹みに加えることによって、錠剤又はカプセルをブリスターパックから取り出すことができるような強度が好ましい。錠剤又はカプセルを前記開口によって取り出すことができる。 The pack may, for example, comprise metal or plastic foil, such as a blister pack. Blister packs are well known in the packaging industry and are widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs are generally preferred to consist of a sheet of relatively rigid material covered by a foil of transparent plastic material. During the packaging process, recesses are formed in the plastic foil. These indentations are adapted to the size and shape of the individual tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively hard material is sealed against the plastic foil in the foil plane opposite to the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. The strength of the sheet is preferably such that the tablet or capsule can be removed from the blister pack by manually applying pressure to the recess such that an opening is formed in the sheet at the location of the recess. Tablets or capsules can be removed by the opening.
　パック又はディスペンサー装置には、投与のための添付文書、プロダクトインサート等を添付することができる。パック又はディスペンサー等の容器は、医薬の製造、使用又は販売を規制する政府機関、当局の通達に適応させることができる。 The pack or dispenser device may be accompanied by package inserts, product inserts and the like for administration. Containers such as packs or dispensers can be adapted to the notices of government agencies, authorities which regulate the manufacture, use or sale of medicines.
　本発明の別のひとつの態様は、真皮由来の組織における線維芽細胞活性化の亢進を抑制する機能を有する化合物を含有する、飲食品、飼料、食品添加剤、又は飼料添加剤等に関する。 Another aspect of the present invention relates to a food or drink, a feed, a food additive, a feed additive or the like, which contains a compound having a function of suppressing an increase in fibroblast activation in a dermis-derived tissue.
　本発明の前記飲食品は、一般的に飲食品に用いられる食品添加剤、例えば、甘味料、着色料、保存料、増粘安定剤、酸化防止剤、発色料、漂白料、防かび剤、ガムベース、苦味料、酵素、光沢剤、酸味料、調味料、乳化剤、強化剤、製造用剤、香料、香辛料抽出物等の一種以上が添加されてもよい。なお、本発明の前記飲食品には、健康食品、機能性食品、特定保健用食品、乳児用食品、幼児用食品、妊産婦用食品、高齢者用食品、病者用食品が含まれる。 The food and drink of the present invention are food additives generally used for food and drink, such as sweeteners, colorants, preservatives, thickeners, thickeners, antioxidants, color formers, bleaches, fungicides, One or more of a gum base, a bitter taste agent, an enzyme, a brightener, an acidulant, a seasoning, an emulsifier, a toughening agent, a preparation agent, a flavor, a spice extract and the like may be added. The food and drink of the present invention include health food, functional food, food for specified health, food for infants, food for infants, food for pregnant women, food for elderly people, food for sick people.
　本発明の前記飲食品の形態は、特に限定されない。具体的には、例えば、いわゆる栄養補助食品（サプリメント）としての錠剤、顆粒剤、散剤、ドリンク剤等を挙げることができる。これ以外には、例えば、茶飲料、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料等の飲料、そば、うどん、中華麺、即席麺等の麺類、飴、キャンディー、ガム、チョコレート、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、パン等の菓子およびパン類、かまぼこ、ハム、ソーセージ等の水産又は畜産加工食品、加工乳、発酵乳等の乳製品、サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂および油脂加工食品、ソース、たれ等の調味料、カレー、シチュー、丼、お粥、雑炊等のレトルトパウチ食品、アイスクリーム、シャーベット、かき氷等の冷菓などを挙げることができる。 The form of the food and drink of the present invention is not particularly limited. Specifically, for example, tablets, granules, powders, drinks and the like as so-called nutraceuticals (supplements) can be mentioned. Other than this, for example, beverages such as tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, etc., buckwheat noodles such as udon, Chinese noodles, instant noodles, etc., candy, candy, gum, chocolate, snacks , Biscuit, jelly, jam, cream, baked goods, confectionery such as bread, breads, fishery products such as kamaboko, ham, sausage, processed foods, dairy products such as processed milk, fermented milk, salad oil, tempura oil, margarine, Oils and fats processed foods such as mayonnaise, shortening, whipped cream and dressings, seasonings such as sauces and sauces, retort pouched foods such as curry, stew, chopsticks, chopsticks and risotto, frozen desserts such as ice cream, sherbet and shaved ice Can be mentioned.
　本発明の前記飲食品の摂取量は、特に限定されず、飲食品の形態；飲食品を摂取する対象の年齢、性別、状態等；あるいは本発明の前記担体に担持又は前記担体と混合させる前記機能性物質の種類、その他の条件に応じて設定してよい。 The intake amount of the food and drink of the present invention is not particularly limited, and the form of the food and drink; age, sex, condition and the like of the subject who ingests the food and drink; or the carrier carried or mixed with the carrier of the present invention You may set according to the kind of functional substance, and other conditions.
　以下の実施例では、本発明をより具体的に説明するが、本発明の範囲がこれらの実施例に限定されることは意図されない。 The present invention will be more specifically described in the following examples, but the scope of the present invention is not intended to be limited to these examples.
　１) 全身性強皮症の発症機序の解析 1) Analysis of the pathogenesis of generalized scleroderma
　患者組織に起こっている線維化病態の把握のためＢｉｏｐｓｙ検体を用いて病巣における病態解析及び分子動態の解析をおこなった。 In order to understand the fibrotic condition occurring in the patient tissue, we used the Biopsy sample to analyze the pathological condition and molecular dynamics in the lesion.
強皮症(ＳＳｃ)患者検体の形態的観察
　・プロトコル Morphological observation of scleroderma (SSc) patient sample ・ Protocol
　強皮症患者及び健常（Ｃｏｎｔｒｏｌ）由来の皮膚組織を採取した。ホルマリン固定パラフィンブロックを作成した。これらの組織サンプルは、キシレン５分×３回、１００％エタノール１分×２回、９０％エタノール１分、８０％エタノール１分、７０％エタノール１分、水５分間で処理し、脱パラフィンした。マイヤーヘマトキシリン溶液で４分間、エオジン液で１分間染色を行った。流水中で５分処理した後、７０％エタノール１分、８０％エタノール１分、９０％エタノール１分、１００％エタノール１分×２回、キシレン５分×３回で処理を行い、マリノールを用いて封入した。室温で１５分以上乾燥させた後、光学顕微鏡を用いて観察を行なった。 Skin tissues from scleroderma patients and healthy (Control) were collected. A formalin fixed paraffin block was made. These tissue samples were deparaffinized by treating with xylene 5 minutes × 3 times, 100% ethanol 1 minute × 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes . Staining was performed with Meyer's hematoxylin solution for 4 minutes and eosin solution for 1 minute. Treated in running water for 5 minutes, treated with 70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1 minute, 100% ethanol 1 minute × 2 times, xylene 5 minutes × 3 times, using marinol Sealed. After drying at room temperature for 15 minutes or more, observation was performed using an optical microscope.
　・結果 ・ Result
　図１に示すように、ＳＳｃ患者皮膚では、健常者に比べ膠原線維が有意に増生していることが確認された。また、ＳＳｃ患者皮膚では、膠原線維が太く膨化、癒合・均質化し、ＳＳｃ検体の病巣部においてコラーゲン等の細胞外マトリックス増生による高度な線維化が生じていることが確認された。 As shown in FIG. 1, in the skin of SSc patients, it was confirmed that collagen fibers were significantly increased as compared to normal subjects. In addition, in SSc patient skin, it was confirmed that collagen fibers are thickened and coalesced and homogenized, and that high degree of fibrosis is caused by extracellular matrix augmentation such as collagen in the lesion area of the SSc sample.
強皮症(ＳＳｃ)患者検体のＩＨＣ解析
　・プロトコル IHC analysis and protocol of scleroderma (SSc) patient sample
　組織サンプルをキシレン５分×３回、１００％エタノール１分×２回、９０％エタノール１分、８０％エタノール１分、７０％エタノール１分、水５分間で処理し、脱パラフィンした。クエン酸バッファー　ｐＨ６．０を用いて、マイクロウェーブ処理１０分間により抗原を賦活化した。室温で３０分静置した後、ＰＢＳで５分、３％過酸化水素／ＰＢＳ溶液で５分、ＰＢＳで５分処理を行い、内在性ペルオキシダーゼを不活性化した。３％ＢＳＡ／ＰＢＳ溶液中で室温にて３０分静置し、組織をブロッキングした。１次抗体として、１．５％ＢＳＡ／ＰＢＳ溶液にて５００倍に希釈した抗α－ＳＭＡ抗体（ＤＡＫＯ　＃Ｍ０８５１）、４００倍に希釈した抗ＺＥＢ１抗体（ＳａｎｔａＣｒｕｚ　＃Ｓｃ－１０５７２）および４８００倍希釈した抗Ｆｉｂｒｏｎｅｃｔｉｎ抗体（ａｂｃａｍ　＃４５６８８）を１組織当たり２００マイクロリットル滴下し、４℃１７時間で組織と反応させた。１次抗体と反応させた組織は、ＰＢＳで５分×３回洗浄した後、最適に希釈した一次抗体に適した動物種のビオチン標識抗体をスライドに注ぎ、室温にて３０分間反応させた。ＰＢＳで５分×３回洗浄した後、製造業者のプロトコルに従って室温３０分にてＡＢＣ反応を行った。組織をＰＢＳで５分×３回洗浄した後、ＩｍｍＰＡＣＴ　ＤＡＢ　Ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）Ｓｕｂｓｔｒａｔｅ（Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社　＃ＳＫ－４１０５）を用いて、室温３分間で発色を行った。発色させた組織は、Ｎｅｗ　ヘマトキシリン　ＴｙｐｅＭ（武藤化学株式会社　＃３０１４１）を用いて染色した後、流水中で３分静置し、７０％エタノール１分、８０％エタノール１分、９０％エタノール１分、１００％エタノール１分×２回、キシレン５分×３回で処理を行い、マリノールを用いて封入した。室温で１５分以上乾燥させた後、光学顕微鏡を用いて観察を行なった。 Tissue samples were deparaffinized by treating with xylene 5 minutes × 3 times, 100% ethanol 1 minute × 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes. The antigen was activated by microwave treatment for 10 minutes using citrate buffer pH 6.0. After standing at room temperature for 30 minutes, treatment with PBS for 5 minutes for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes was performed to inactivate endogenous peroxidase. The tissue was blocked by standing in a 3% BSA / PBS solution for 30 minutes at room temperature. As primary antibodies, anti-α-SMA antibody (DAKO # M0851) diluted 500 times in 1.5% BSA / PBS solution, anti-ZEB1 antibody (SantaCruz # Sc-10572) diluted 400 times, and 4800 times dilution 200 microliter per tissue of the anti-Fibronectin antibody (abcam # 45688) was dropped and allowed to react with the tissue at 4 ° C. for 17 hours. The tissue reacted with the primary antibody was washed with PBS for 5 minutes x 3 times, and then a biotin-labeled antibody of animal species suitable for the optimally diluted primary antibody was poured onto the slide and allowed to react for 30 minutes at room temperature. After 5 minutes x 3 washes with PBS, the ABC reactions were performed at room temperature for 30 minutes according to the manufacturer's protocol. After washing the tissue with PBS for 5 minutes × 3 times, color development was performed using ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories # SK-4105) at room temperature for 3 minutes. The stained tissue is stained with New Hematoxylin Type M (Muto Fuji Kagaku Co., Ltd. # 30141), and then allowed to stand in flowing water for 3 minutes. 70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1 minute The mixture was treated with 100% ethanol 1 minute x 2 times, xylene 5 minutes x 3 times, and sealed with marinol. After drying at room temperature for 15 minutes or more, observation was performed using an optical microscope.
　・結果 ・ Result
　図２Ａに示すように、強皮症患者皮膚の膠原繊維増生領域では、α－ＳＭＡ陽性細胞が多数存在し、線維芽細胞の活性化（筋線維芽細胞化）が認められた。 As shown in FIG. 2A, in the collagenous fiber-enriched region of the skin of scleroderma patients, many α-SMA positive cells were present, and fibroblast activation (myofibroblast formation) was observed.
　また、図２Ｂに示すように、健常者に比べて強皮症患者皮膚では、細胞外領域に顕著なＦｉｂｒｏｎｅｃｔｉｎの蓄積が見られ、線維化亢進が認められた。 In addition, as shown in FIG. 2B, in the skin of scleroderma patients, significant accumulation of Fibronectin was observed in the extracellular region, and hyperfibrosis was observed in scleroderma patient skin as compared to normal persons.
　さらに、図２Ｃに示すように、強皮症患者皮膚の膠原繊維増生領域では、ＺＥＢ1陽性細胞が多数存在し、線維芽細胞の活性化亢進が認められた。 Furthermore, as shown in FIG. 2C, in the collagenous fiber-growing area of the skin of scleroderma patients, a large number of ZEB1 positive cells were present, and an increase in fibroblast activation was observed.
　図３は、以上の結果をまとめたものである。これらの結果より、『強皮症患者皮膚では真皮内における線維芽細胞の活性化亢進が生じ、これによりコラーゲン等の細胞外マトリックスが増生し線維化病変が形成される』との全身性強皮症の発症機序に関わる有力な知見を得た。 FIG. 3 summarizes the above results. Based on these results, systemic sclera of "Scleroderma patient skin has an enhanced activation of fibroblasts in the dermis, which causes the extracellular matrix such as collagen to grow and form a fibrotic lesion". We obtained strong findings on the pathogenesis of
　この結果から、『真皮内の線維芽細胞活性化の抑制』が線維化病巣を生じる本疾患の有力な治療開発戦略であることが明らかになった。 From this result, it has become clear that "suppression of fibroblast activation in the dermis" is a powerful therapeutic development strategy for this disease causing fibrotic foci.
　２) 全身性強皮症患者由来の線維芽細胞の発現解析 2) Expression analysis of fibroblasts from systemic scleroderma patients
　強皮症患者および健常（Ｃｏｎｔｒｏｌ）の皮膚組織から初代培養線維芽細胞を採取し、分化した時に発現が変化する遺伝子群（以下、「分化関連マーカー」と称する）の発現動態解析を実施した。 Primary cultured fibroblasts were collected from skin tissues of scleroderma patients and healthy (Control), and expression kinetic analysis was performed on a group of genes whose expression changes when differentiated (hereinafter referred to as "differentiation related markers").
　・プロトコル Protocol
　取得した初代培養線維芽細胞をプレートに播種し、７０％程度になった時点でＦＢＳ　Ｆｒｅｅ培地に培地交換し、２４時間後に細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作製した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃７４００４）を用いてトータルＲＮＡを抽出し、Ｌｏｗ　Ｉｎｐｕｔ　ＱｕｉｃｋＡｍｐ　Ｌａｂｅｌｉｎｇ　ｋｉｔ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃５１９０－２３０５）を用いて蛍光ラベルした。ラベル化したｃＲＮＡはＷｈｏｌｅ　Ｈｕｍａｎ　Ｇｅｎｏｍｅ　Ｏｌｉｇｏ－ＤＮＡ　Ｍｉｃｒｏａｒｒａｙ　Ｋｉｔ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃Ｇ４１１２Ｆ）に製造業者の定めるプロトコルに従ってハイブリダイズした。ハイブリダイズしたスライドは、Ｇｅｎｅ　Ｅｘｐｒｅｓｓｉｏｎ　Ｗａｓｈ　Ｐａｃｋ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃５１８８－５３２７）を用いて洗浄し、Ａｇｉｌｅｎｔ　Ｍｉｃｒｏａｒｒａｙ　Ｓｃａｎｎｅｒ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃Ｇ２５０５Ｂ）でスキャニングを行った。スキャン画像は、Ｆｅａｔｕｒｅ　Ｅｘｔｒａｃｔｉｏｎ　ｓｏｆｔｗａｒｅ　ｖｅｒｓｉｏｎ　９．５．１（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用いて数値化した。数値データは、Ｇｒｏｂａｌ　Ｎｏｒｍａｌｉｚａｔｉｏｎ法によって標準化した。 The obtained primary cultured fibroblasts are seeded on a plate, and when it reaches about 70%, the medium is changed to FBS Free medium, and after 24 hours, cells are washed with PBS, RLT buffer 350 uL is added, and RNA extract lysate Was produced. Total RNA was extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and fluorescently labeled using Low Input QuickAmp Labeling kit (Agilent Technologies # 5190-2305). Labeled cRNA was hybridized to Whole Human Genome Oligo-DNA Microarray Kit (Agilent Technologies # G4112F) according to the protocol defined by the manufacturer. The hybridized slides were washed using Gene Expression Wash Pack (Agilent Technologies # 5188-5327) and scanned with an Agilent Microarray Scanner (Agilent Technologies # G2505B). Scanned images were quantified using Feature Extraction software version 9.5.1 (Agilent Technologies). Numerical data were normalized by the Grobal Normalization method.
　・結果 ・ Result
　図４に示すように、強皮症患者由来の線維芽細胞では、αＳＭＡ発現亢進が見られ、線維芽細胞の活性化（＝筋線維芽細胞化）が生じていることが示された。さらに、強皮症患者由来の線維芽細胞では、分化した時に発現が変化する遺伝子群（分化関連マーカー）の発現亢進が認められた。 As shown in FIG. 4, in fibroblasts derived from scleroderma patients, αSMA expression enhancement was observed, and it was shown that fibroblast activation (= myofibroblast formation) occurred. Furthermore, in the fibroblasts derived from scleroderma patients, enhanced expression of genes (differentiation related markers) whose expression changes upon differentiation was observed.
　図５に示すように、強皮症患者由来の線維芽細胞では、種々のコラーゲンの発現が顕著に亢進していた。従って、強皮症患者皮膚では線維芽細胞の活性化によって種々のコラーゲン発現亢進が生じ、線維化の進展に寄与していることが示された。 As shown in FIG. 5, in the fibroblasts derived from scleroderma patients, the expression of various collagens was significantly enhanced. Therefore, in scleroderma patient skin, it was shown that activation of fibroblasts causes various types of collagen upregulation, contributing to the progress of fibrosis.
　３) 健常線維芽細胞を用いた炎症刺激による発現動態解析 3) Analysis of expression kinetics by inflammation stimulation using healthy fibroblasts
　炎症刺激後の健常の初代培養線維芽細胞からＲＮＡを抽出し、発現変化を検証した。 RNA was extracted from healthy primary cultured fibroblasts after inflammation stimulation, and the expression change was verified.
　・プロトコル Protocol
　取得した初代培養線維芽細胞をプレートに播種し、７０％程度になった時点で成長因子ＴＧＦ－β２（ｅＢｉｏｓｃｉｅｎｃｅ社製、１４－８３６８）を、培地に対して５ｎｇ／ｍＬになるように添加し、４８時間後に細胞を回収した。回収した細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作製した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃７４００４）を用いてトータルＲＮＡを抽出し、Ｌｏｗ　Ｉｎｐｕｔ　ＱｕｉｃｋＡｍｐ　Ｌａｂｅｌｉｎｇ　ｋｉｔ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃５１９０－２３０５）を用いて蛍光ラベルした。ラベル化したｃＲＮＡはＷｈｏｌｅ　Ｈｕｍａｎ　Ｇｅｎｏｍｅ　Ｏｌｉｇｏ－ＤＮＡ　Ｍｉｃｒｏａｒｒａｙ　Ｋｉｔ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃Ｇ４１１２Ｆ）に製造業者の定めるプロトコルに従ってハイブリダイズした。ハイブリダイズしたスライドは、Ｇｅｎｅ　Ｅｘｐｒｅｓｓｉｏｎ　Ｗａｓｈ　Ｐａｃｋ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃５１８８－５３２７）を用いて洗浄し、Ａｇｉｌｅｎｔ　Ｍｉｃｒｏａｒｒａｙ　Ｓｃａｎｎｅｒ（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社　＃Ｇ２５０５Ｂ）でスキャニングを行った。スキャン画像は、Ｆｅａｔｕｒｅ　Ｅｘｔｒａｃｔｉｏｎ　ｓｏｆｔｗａｒｅ　ｖｅｒｓｉｏｎ　９．５．１（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用いて数値化した。数値データは、Ｇｒｏｂａｌ　Ｎｏｒｍａｌｉｚａｔｉｏｎ法によって標準化した。 The obtained primary cultured fibroblasts were seeded on a plate, and when it reached about 70%, growth factor TGF-β2 (eBioscience, 14-8368) was added to the culture medium to a concentration of 5 ng / mL. After 48 hours, cells were recovered. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection. Total RNA was extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and fluorescently labeled using Low Input QuickAmp Labeling kit (Agilent Technologies # 5190-2305). Labeled cRNA was hybridized to Whole Human Genome Oligo-DNA Microarray Kit (Agilent Technologies # G4112F) according to the protocol defined by the manufacturer. The hybridized slides were washed using Gene Expression Wash Pack (Agilent Technologies # 5188-5327) and scanned with an Agilent Microarray Scanner (Agilent Technologies # G2505B). Scanned images were quantified using Feature Extraction software version 9.5.1 (Agilent Technologies). Numerical data were normalized by the Grobal Normalization method.
　・結果 ・ Result
　図６に示すように、健常者由来線維芽細胞は、炎症刺激により活性化され、筋線維芽細胞化（αＳＭＡ発現）し、各種分化関連マーカーの発現亢進が認められ、強皮症患者由来線維芽細胞と同様の発現動態を示した。 As shown in FIG. 6, fibroblasts from healthy subjects are activated by inflammatory stimulation, become myofibroblasts (αSMA expression), and expression of various differentiation-related markers is enhanced, and fibers from scleroderma patients are observed. It showed the same expression kinetics as blast cells.
　図７に示すように、健常者由来線維芽細胞の炎症刺激により、コラーゲンの発現亢進が見られ、強皮症皮膚組織で確認された線維芽細胞の活性化（筋線維芽細胞化）による膠原繊維増生が再現された。 As shown in FIG. 7, inflammatory stimulation of normal human fibroblasts leads to increased expression of collagen, and activation of fibroblasts confirmed in scleroderma skin tissue (myofibroblast formation) collagen. Fiber growth was reproduced.
　４) 全身性強皮症の治療薬の取得 4) Acquisition of a remedy for systemic scleroderma
　これら結果を基に、強皮症患者由来の活性化した線維芽細胞および健常由来線維芽細胞を用いた炎症刺激活性化モデル系を用いて、その抑制活性を保有する化合物の取得を行なった。 Based on these results, a compound having an inhibitory activity was obtained using an inflammatory stimulation activation model system using activated fibroblasts from scleroderma patients and healthy fibroblasts.
　約１，２００種の既存薬の評価の結果、線維芽細胞活性化に対する抑制活性の高い治療薬候補化合物１１種を取得した。
　１．ベンズブロマロン
　２．アルベンダゾール
　３．メベンダゾール
　４．リセドロン酸ナトリウム水和物
　５．エパルレスタット
　６．フルフェナム酸アルミニウム
　７．ザルトプロフェン
　８．アンレキサノクス
　９．メフェナム酸
　１０．オキサプロジン
　１１．3 - [（1E）-2-（1H-インドール-6-イル）エテニル] -5 - [（1E）-2- [2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル] -1H-ピラゾール(3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl] ethenyl]-1H-pyrazole) As a result of evaluation of about 1,200 existing drugs, 11 kinds of candidate therapeutic drug compounds having high inhibitory activity on fibroblast activation were obtained.
1. Benz bromarone 2. Albendazole 3. Mebendazole 4. Risedronate sodium hydrate 5. Epalrestat 6. Aluminum flufenamate 7. Zaltoprofen 8. Ann Rexanox 9. Mefenamic acid 10. Oxaprozin 11.3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5- (1E) -2- [2-methoxy-4- (2-pyridylmethoxy) phenyl] ethenyl] -1H-Pyrazole (3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridinyloxy) phenyl] ethenyl ] -1H-pyrazole)
　４）-１ ＳＳｃ患者由来細胞を用いた線維芽細胞抑制効果の評価（発現解析）
　強皮症患者由来細胞を用いて、評価化合物による線維化の要因である線維芽細胞活性化に対する抑制効果の評価を実施した。 4) -1 Evaluation of fibroblast suppressive effect using SSc patient-derived cells (expression analysis)
The scleroderma patient-derived cells were used to evaluate the suppressive effect on fibroblast activation that is a factor of fibrosis by the evaluation compound.
　・プロトコル Protocol
　プレートに強皮症患者皮膚組織由来の初代培養線維芽細胞を播種し、 ３７℃にてインキュベートした。翌日、評価化合物（被験薬剤）を最大薬物血漿中濃度（Ｃｍａｘ）になるように培地中に添加し、４８時間後に細胞を回収した。コントロールとして、評価化合物を未添加のサンプルおよび健常皮膚組織由来の培養線維芽細胞サンプルを用いた。回収した細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作成した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃　７４００４）を用いてトータルＲＮＡを抽出し、得られたＲＮＡ２μｇからＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　ＳｕｐｅｒＭｉｘ　ｆｏｒ　ｑＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ社　＃１１７５２－２５０）を用いてｃＤＮＡを作製した。得られたｃＤＮＡ５０ｎｇを鋳型として、リアルタイムＰＣＲ（ＡＢＩ　Ｐｒｉｓｍ　７５００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ）を製造業者のプロトコルに従って行い、各遺伝子のｍＲＮＡの発現を定量分析した。各遺伝子に対する特異的プライマーはＩｎｖｉｔｒｏｇｅｎ社より購入した。各遺伝子Ｃｔ（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）値は内在性コントロールによりノーマライズ後、健常皮膚組織由来の培養線維芽細胞サンプルに対する割合を算出した。 Plates were seeded with primary culture fibroblasts from scleroderma patient skin tissue and incubated at 37 ° C. The following day, an evaluation compound (test drug) was added to the culture medium to a maximum drug plasma concentration (Cmax), and cells were collected 48 hours later. As a control, a sample to which an evaluation compound was not added and a cultured fibroblast sample derived from a healthy skin tissue were used. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection. Total RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 μg of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did. Using 50 ng of the obtained cDNA as a template, real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen. Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the cultured fibroblast sample derived from the healthy skin tissue was calculated.
　・結果 ・ Result
　図８Ａ～図８Ｃに示すように、評価化合物の添加によって、強皮症患者由来線維芽細胞で特異的に認められたαSMA発現亢進（筋線維芽細胞化）の抑制、種々コラーゲン分子および各種分化関連マーカー分子発現の抑制が認められ、評価化合物による線維芽細胞活性化に対する抑制効果が認められた。 As shown in FIG. 8A to FIG. 8C, suppression of αSMA expression enhancement (myofibroblast formation) specifically recognized in fibroblasts derived from scleroderma patients by addition of an evaluation compound, various collagen molecules and various differentiation The suppression of the expression of the related marker molecule was observed, and the inhibitory effect on the fibroblast activation by the evaluation compound was observed.
　・プロトコル Protocol
　プレートに強皮症患者皮膚組織由来の初代培養線維芽細胞を播種し、 ３７℃にてインキュベートした。翌日、評価化合物（被験薬剤）をＣｍａｘを基準に各４条件で添加し、１２０時間後に細胞を回収した。コントロールとして、評価化合物を未添加のサンプルおよび健常皮膚組織由来の培養線維芽細胞サンプルを用いた。回収した細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作成した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃　７４００４）を用いてトータルＲＮＡを抽出し、得られたＲＮＡ２μｇからＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　ＳｕｐｅｒＭｉｘ　ｆｏｒ　ｑＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ社　＃１１７５２－２５０）を用いてｃＤＮＡを作製した。得られたｃＤＮＡ５０ｎｇを鋳型として、リアルタイムＰＣＲ（ＡＢＩ　Ｐｒｉｓｍ　７５００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ）を製造業者のプロトコルに従って行い、各遺伝子のｍＲＮＡの発現を定量分析した。各遺伝子に対する特異的プライマーはＩｎｖｉｔｒｏｇｅｎ社より購入した。各遺伝子Ｃｔ（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）値は内在性コントロールによりノーマライズ後、健常皮膚組織由来の培養線維芽細胞サンプルに対する割合を算出した。 Plates were seeded with primary culture fibroblasts from scleroderma patient skin tissue and incubated at 37 ° C. The next day, an evaluation compound (test drug) was added based on Cmax under each of 4 conditions, and cells were recovered after 120 hours. As a control, a sample to which an evaluation compound was not added and a cultured fibroblast sample derived from a healthy skin tissue were used. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection. Total RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 μg of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did. Using 50 ng of the obtained cDNA as a template, real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen. Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the cultured fibroblast sample derived from the healthy skin tissue was calculated.
　・結果 ・ Result
　図９Ａに示すように、エパルレスタットの添加によって、強皮症患者由来皮膚線維芽細胞で特徴的に見られたαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制、およびＦｉｂｒｏｎｅｃｔｉｎ（分化関連マーカー）発現亢進の抑制が認められ、エパルレスタットによる線維芽細胞活性化に対する顕著な抑制効果が濃度依存的に認められた。 As shown in FIG. 9A, by addition of epalrestat, suppression of αSMA expression enhancement characteristically seen in scleroderma patient-derived skin fibroblasts (= suppression of myofibroblast activation), various collagen expression enhancement (= Suppression of collagen fiber growth) and suppression of Fibronectin (differentiation related marker) expression elevation were observed, and a remarkable suppression effect on fibroblast activation by epalrestat was observed in a concentration-dependent manner.
　図９Ｂに示すように、メベンダゾールの添加によって、強皮症患者由来皮膚線維芽細胞で特徴的に見られたαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制、およびＦｉｂｒｏｎｅｃｔｉｎ（分化関連マーカー）発現亢進の抑制が認められ、メベンダゾールによる線維芽細胞活性化に対する顕著な抑制効果が濃度依存的に認められた。 As shown in FIG. 9B, the addition of mebendazole suppresses the enhancement of αSMA expression characteristically seen in scleroderma patient-derived skin fibroblasts (= suppression of myofibroblast activation), various collagen expression enhancement (= Suppression of collagenous fiber proliferation and suppression of Fibronectin (differentiation related marker) expression elevation were observed, and a remarkable suppression effect on fibroblast activation by mebendazole was observed in a concentration-dependent manner.
　図９Ｃに示すように、アルベンダゾールの添加によって、強皮症患者由来皮膚線維芽細胞で特徴的に見られたαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制が認められ、アルベンダゾールによる線維芽細胞活性化に対する顕著な抑制効果が濃度依存的に認められた。 As shown in FIG. 9C, the addition of albendazole suppresses the enhancement of αSMA expression characteristically seen in scleroderma patient-derived skin fibroblasts (= suppression of myofibroblast activation), various collagen expression enhancements The suppression of (the growth of collagen fibers) was observed, and the remarkable suppression effect on fibroblast activation by albendazole was observed in a concentration-dependent manner.
　４）-２ SSc患者由来細胞を用いた線維芽細胞抑制効果の評価（培養液中コラーゲンの定量）
　強皮症患者由来細胞を用いて、強皮症患者皮膚に特異的に認められたコラーゲン分泌亢進（線維化の要因）に対する評価化合物による抑制効果の評価を実施した。 4) -2 Evaluation of fibroblast suppression effect using SSc patient-derived cells (quantification of collagen in culture fluid)
The scleroderma patient-derived cells were used to evaluate the inhibitory effect of the evaluation compound on the collagen secretion (factor of fibrosis) specifically recognized in the skin of scleroderma patients.
　・プロトコル Protocol
　プレートに強皮症患者皮膚組織由来の初代培養線維芽細胞を播種し、 ３７℃にてインキュベートした。翌日、評価化合物（被験薬剤）をＣｍａｘを基準に各４条件で添加し、１２０時間後に細胞の培養上清を回収した。コントロールとして、評価化合物を未添加のサンプルおよび健常皮膚組織由来の培養線維芽細胞サンプルを用いた。Ｓｉｒｃｏｌ　Ｃｏｌｌａｇｅｎ　Ａｓｓａｙ　Ｋｉｔ（Ｂｉｏｃｏｌｏｒ社　＃Ｓ１０００）を用いて、製造業者のプロトコルに従ってＤｙｅ　ｒｅａｇｅｎｔおよびＡｌｋａｌｉ　ｒｅａｇｅｎｔと反応させた後、Ｉｎｆｉｎｉｔｅ　Ｍ２００（ＴＥＣＡＮ）を用いて５５５ｎｍの吸光度を測定した。標準溶液を用いて検量線を作成し、培養液中に分泌されたコラーゲン含有量を算出後、健常皮膚組織由来の培養線維芽細胞サンプルに対する割合を算出した。 Plates were seeded with primary culture fibroblasts from scleroderma patient skin tissue and incubated at 37 ° C. On the next day, an evaluation compound (test drug) was added based on Cmax under each of 4 conditions, and after 120 hours, the culture supernatant of cells was collected. As a control, a sample to which an evaluation compound was not added and a cultured fibroblast sample derived from a healthy skin tissue were used. After reacting with Dye reagent and Alkali reagent according to the manufacturer's protocol using Sircol Collagen Assay Kit (Biocolor # S1000), absorbance at 555 nm was measured using Infinite M200 (TECAN). A standard curve was prepared using a standard solution, and after calculating the content of collagen secreted in the culture solution, the ratio to the cultured fibroblast sample derived from healthy skin tissue was calculated.
　・結果 ・ Result
　図１０に示すように、健常皮膚組織由来の線維芽細胞に比べて、強皮症患者由来の皮膚線維芽細胞では、コラーゲンの分泌促進が認められ、強皮症患者皮膚で見られる膠原繊維の増生の要因であることが示唆された。 As shown in FIG. 10, compared to fibroblasts derived from healthy skin tissue, skin fibroblasts derived from scleroderma patients show enhanced secretion of collagen, and the collagenous fibers found in scleroderma patient skin It is suggested that it is a factor of growth.
　エパルレスタット（ＥＬＳ）の添加によって、培養上清中に分泌されるコラーゲン量の顕著な抑制が認められ（濃度依存的）、エパルレスタットは強皮症患者皮膚で特異的に見られる膠原繊維の増生の抑制効果を有することが示唆された。 With the addition of epalrestat (ELS), significant suppression of the amount of collagen secreted in the culture supernatant was observed (concentration dependent), and epalrestat suppressed the growth of collagen fibers specifically seen in scleroderma patient skin It was suggested to have an effect.
　５）-１炎症誘発系を用いた線維芽細胞抑制活性の評価（発現解析） 5) -1 Evaluation of fibroblast suppressive activity using a proinflammatory system (expression analysis)
　健常由来線維芽細胞を用いた炎症刺激系による、評価化合物による線維芽細胞活性化に対する抑制効果の評価を実施した。 Evaluation of the suppressive effect on the fibroblast activation by the evaluation compound by the inflammation stimulation system using healthy derived fibroblasts was carried out.
　・プロトコル Protocol
　プレートに健常初代培養線維芽細胞を播種し、３７℃にてインキュベートした。翌日、ＴＧＦ－β２（ｅＢｉｏｓｃｉｅｎｃｅ社製、１４－８３６８）を培地に対して５ｎｇ／ｍＬになるように添加し、同時に評価化合物（被験薬剤）を最大薬物血漿中濃度（Ｃｍａｘ）になるように培地中に添加し、４８時間後に細胞を回収した。コントロールとして、非炎症刺激サンプル（未処理）および評価化合物を未添加のサンプル（炎症刺激のみ）を用いた。回収した細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作成した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃　７４００４）を用いてトータルＲＮＡを抽出し、得られたＲＮＡ２μｇからＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　ＳｕｐｅｒＭｉｘ　ｆｏｒ　ｑＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ社　＃１１７５２－２５０）を用いてｃＤＮＡを作製した。得られたｃＤＮＡ５０ｎｇを鋳型として、リアルタイムＰＣＲ（ＡＢＩ　Ｐｒｉｓｍ　７５００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ）を製造業者のプロトコルに従って行い、各遺伝子のｍＲＮＡの発現を定量分析した。各遺伝子に対する特異的プライマーはＩｎｖｉｔｒｏｇｅｎ社より購入した。各遺伝子Ｃｔ（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）値は内在性コントロールによりノーマライズ後、非炎症刺激サンプル（未処理）に対する割合を算出した。 Plates were seeded with healthy primary fibroblasts and incubated at 37 ° C. The next day, TGF-β2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, the evaluation compound (test agent) was brought to the maximum drug plasma concentration (Cmax). The cells were collected after 48 hours. As a control, a non-inflammatory stimulus sample (untreated) and a sample to which an evaluation compound was not added (inflammation stimulus alone) were used. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection. Total RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 μg of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did. Using 50 ng of the obtained cDNA as a template, real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen. Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
　・結果 ・ Result
　図１１Ａ～図１１Ｃに示すように、評価化合物の添加によって、炎症刺激によって誘起されるαSMAの発現誘導（筋線維芽細胞化）の抑制、種々コラーゲンの発現誘導および各種分化関連マーカーの発現誘導の抑制が認められ、評価化合物による線維芽細胞活性化に対する抑制効果が認められた。 As shown in FIGS. 11A to 11C, the addition of the evaluation compound suppresses the induction of expression of αSMA (myofibroblast formation) induced by inflammation stimulation, the expression induction of various collagens, and the expression induction of various differentiation-related markers. The inhibition was observed, and the inhibitory effect on the fibroblast activation by the evaluation compound was observed.
　・プロトコル Protocol
　プレートに健常初代培養線維芽細胞を播種し、 ３７℃にてインキュベートした。翌日、ＴＧＦ－β２（ｅＢｉｏｓｃｉｅｎｃｅ社製、１４－８３６８）を培地に対して５ｎｇ／ｍＬになるように添加し、同時に評価化合物をＣｍａｘを基準に各４条件で添加し、１２０時間後に細胞を回収した。コントロールとして、非炎症刺激サンプル（未処理）および評価化合物を未添加のサンプル（炎症刺激のみ）を用いた。回収した細胞をＰＢＳで洗浄し、ＲＬＴ　ｂｕｆｆｅｒ　３５０ｕＬを加えて、ＲＮＡ採取用ライセートを作成した。ライセートからＲＮｅａｓｙ　Ｍｉｃｒｏ　ｋｉｔ（ＱＩＡＧＥＮ社　＃　７４００４）を用いてトータルＲＮＡを抽出し、得られたＲＮＡ２μｇからＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ　Ｆｉｒｓｔ－Ｓｔｒａｎｄ　Ｓｙｎｔｈｅｓｉｓ　ＳｕｐｅｒＭｉｘ　ｆｏｒ　ｑＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ社　＃１１７５２－２５０）を用いてｃＤＮＡを作製した。得られたｃＤＮＡ５０ｎｇを鋳型として、リアルタイムＰＣＲ（ＡＢＩ　Ｐｒｉｓｍ　７５００　Ｓｅｑｕｅｎｃｅ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ）を製造業者のプロトコルに従って行い、各遺伝子のｍＲＮＡの発現を定量分析した。各遺伝子に対する特異的プライマーはＩｎｖｉｔｒｏｇｅｎ社より購入した。各遺伝子Ｃｔ（Ｔｈｒｅｓｈｏｌｄ　Ｃｙｃｌｅ）値は内在性コントロールによりノーマライズ後、非炎症刺激サンプル（未処理）に対する割合を算出した。 Plates were seeded with healthy primary fibroblasts and incubated at 37 ° C. The next day, TGF-β2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, evaluation compounds were added under each of 4 conditions based on Cmax, and cells were recovered after 120 hours did. As a control, a non-inflammatory stimulus sample (untreated) and a sample to which an evaluation compound was not added (inflammation stimulus alone) were used. The collected cells were washed with PBS, and 350 uL of RLT buffer was added to prepare a lysate for RNA collection. Total RNA is extracted from the lysate using RNeasy Micro kit (QIAGEN # 74004), and cDNA is prepared from 2 μg of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen # 11752-250) did. Using 50 ng of the obtained cDNA as a template, real-time PCR (ABI Prism 7500 Sequence Detection System) was performed according to the manufacturer's protocol, and the expression of mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen. Each gene Ct (Threshold Cycle) value was normalized by the endogenous control, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
　・結果 ・ Result
　図１２Ａに示すように、エパルレスタットの添加によって、炎症刺激によって誘発されるαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制、およびＦｉｂｒｏｎｅｃｔｉｎ（分化関連マーカー）発現亢進の抑制が認められ、エパルレスタットによる線維芽細胞活性化に対する顕著な抑制効果が濃度依存的に認められた。 As shown in FIG. 12A, the addition of epalrestat suppresses the inflammatory stimulus-induced increase in αSMA expression (= suppresses myofibroblast activation), suppresses various collagen expression increases (enrichment of collagen fibers), and Fibronectin. (Differentiation-related markers) Suppression of enhanced expression was observed, and a remarkable suppression effect on fibroblast activation by epalrestat was observed depending on concentration.
　図１２Ｂに示すように、メベンダゾールの添加によって、炎症刺激によって誘発されるαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制、およびＦｉｂｒｏｎｅｃｔｉｎ（分化関連マーカー）発現亢進の抑制が認められ、メベンダゾールによる線維芽細胞活性化に対する顕著な抑制効果が濃度依存的に認められた。 As shown in FIG. 12B, the addition of mebendazole suppresses the increase in αSMA expression induced by inflammatory stimulation (= suppression of myofibroblast activation), suppression of various collagen expression increases (enrichment of collagen fibers), and Fibronectin. (Differentiation-related markers) Suppression of enhanced expression was observed, and a remarkable inhibitory effect on fibroblast activation by mebendazole was observed depending on concentration.
　図１２Ｃに示すように、アルベンダゾールの添加によって、炎症刺激によって誘発されるαＳＭＡ発現亢進の抑制（＝筋線維芽細胞活性化の抑制）、種々コラーゲン発現亢進（膠原繊維の増生）の抑制、およびＦｉｂｒｏｎｅｃｔｉｎ（分化関連マーカー）発現亢進の抑制が認められ、アルベンダゾールによる線維芽細胞活性化に対する顕著な抑制効果が認められた。 As shown in FIG. 12C, by addition of albendazole, suppression of αSMA expression enhancement (= suppression of myofibroblast activation), suppression of various collagen expression enhancement (proliferation of collagen fibers), and the like are promoted. Suppression of the upregulation of Fibronectin (differentiation related marker) was observed, and a remarkable suppression effect on fibroblast activation by albendazole was observed.
　５）-２炎症誘発系を用いた線維芽細胞抑制活性の評価（培養液中コラーゲンの定量） 5) -2 Evaluation of fibroblast inhibitory activity using a proinflammatory system (quantification of collagen in culture fluid)
　健常由来線維芽細胞を用いた炎症刺激系を用いて、コラーゲン分泌亢進（線維化の要因）に対する評価化合物による抑制効果の評価を実施した。 Evaluation of the inhibitory effect of the evaluation compound on collagen secretion enhancement (factor of fibrosis) was carried out using an inflammatory stimulation system using healthy derived fibroblasts.
　・プロトコル Protocol
　プレートに健常初代培養線維芽細胞を播種し、 ３７℃にてインキュベートした。翌日、ＴＧＦ－β２（ｅＢｉｏｓｃｉｅｎｃｅ社製、１４－８３６８）を培地に対して５ｎｇ／ｍＬになるように添加し、同時に評価化合物をＣｍａｘを基準に各４条件で添加し、１２０時間後に細胞の培養上清を回収した。コントロールとして、非炎症刺激サンプル（未処理）および評価化合物を未添加のサンプル（炎症刺激のみ）を用いた。Ｓｉｒｃｏｌ　Ｃｏｌｌａｇｅｎ　Ａｓｓａｙ　Ｋｉｔ（Ｂｉｏｃｏｌｏｒ社　＃Ｓ１０００）を用いて、製造業者のプロトコルに従ってＤｙｅ　ｒｅａｇｅｎｔおよびＡｌｋａｌｉ　ｒｅａｇｅｎｔと反応させた後、Ｉｎｆｉｎｉｔｅ　Ｍ２００（ＴＥＣＡＮ）を用いて５５５ｎｍの吸光度を測定した。標準溶液を用いて検量線を作成し、培養液中に分泌されたコラーゲン含有量を算出し、非炎症刺激サンプル（未処理）に対する割合を算出した。 Plates were seeded with healthy primary fibroblasts and incubated at 37 ° C. The next day, TGF-β2 (eBioscience, 14-8368) was added to the medium to 5 ng / mL, and at the same time, evaluation compounds were added under each of four conditions based on Cmax, and cell culture was performed after 120 hours. The supernatant was collected. As a control, a non-inflammatory stimulus sample (untreated) and a sample to which an evaluation compound was not added (inflammation stimulus alone) were used. After reacting with Dye reagent and Alkali reagent according to the manufacturer's protocol using Sircol Collagen Assay Kit (Biocolor # S1000), absorbance at 555 nm was measured using Infinite M200 (TECAN). A standard curve was prepared using a standard solution, the content of collagen secreted in the culture solution was calculated, and the ratio to the non-inflammatory stimulus sample (untreated) was calculated.
　・結果 ・ Result
　図１３Ａに示すように、常皮膚組織由来線維芽細胞に対する炎症刺激によって、コラーゲンの分泌促進が確認され、強皮症患者由来線維芽細胞と同様の性質を示した。よって、健常皮膚組織由来線維芽細胞に対する炎症刺激によって、強皮症患者組織で確認された「線維芽細胞の活性化による膠原繊維増生」が再現された。 As shown in FIG. 13A, inflammation stimulation of normal skin tissue-derived fibroblasts confirmed that the secretion of collagen was promoted, and showed properties similar to those of scleroderma patient-derived fibroblasts. Therefore, the inflammatory stimulus on healthy skin tissue-derived fibroblasts reproduces “collagen fiber hyperplasia by fibroblast activation” identified in scleroderma patient tissues.
　エパルレスタット（ＥＬＳ）の添加によって、培養上清中に分泌されるコラーゲン量の顕著な抑制が認められ（濃度依存的）、エパルレスタットは強皮症患者皮膚で特異的に見られる膠原繊維の増生の抑制効果を有することが示唆された。 With the addition of epalrestat (ELS), significant suppression of the amount of collagen secreted in the culture supernatant was observed (concentration dependent), and epalrestat suppressed the growth of collagen fibers specifically seen in scleroderma patient skin It was suggested to have an effect.
　図１３Ｂに示すように、メベンダゾール（ＭＢＺ）の添加によって、培養上清中に分泌されるコラーゲン量の顕著な抑制が認められ（濃度依存的）、メベンダゾールは強皮症患者皮膚で特異的に見られる膠原繊維の増生の抑制効果を有することが示唆された。 As shown in FIG. 13B, the addition of mebendazole (MBZ) significantly suppresses the amount of collagen secreted in the culture supernatant (concentration dependent), and mebendazole appears specifically in scleroderma patient skin. It is suggested that it has an inhibitory effect on collagen fiber proliferation.
　図１３Ｃに示すように、アルベンダゾール（ＡＬＺ）の添加によって、培養上清中に分泌されるコラーゲン量の顕著な抑制が認められ、アルベンダゾールは強皮症患者皮膚で特異的に見られる膠原繊維の増生の抑制効果を有することが示唆された。 As shown in FIG. 13C, the addition of albendazole (ALZ) significantly suppresses the amount of collagen secreted in the culture supernatant, and albendazole is a collagen fiber specifically seen in scleroderma patient skin. It has been suggested that it has an inhibitory effect on hyperplasia of
　６) ＳＳｃ疾患モデルマウスを用いたエパルレスタットの線維化抑制の薬効評価 6) Efficacy evaluation of epalrestat's suppression of fibrosis using SSc disease model mice
　評価化合物の有効性の解析を、疾患モデルマウスを用いて実施した（図１４）。 Analysis of the efficacy of the evaluation compound was performed using a disease model mouse (FIG. 14).
　最も頻度高く使用されるモデル動物は「ブレオマイシン（ＢＬＭ）の真皮内投与による炎症に惹起される線維化病巣形成のマウス」であるが、ＳＳｃ患者と同等の肥厚・線維化が形成できる条件を探索し最適化を図り、ＳＳｃ疾患モデルマウスの構築を行なった。 The most frequently used model animal is "a mouse with fibrotic foci formation caused by inflammation by intradermal administration of bleomycin (BLM)", but explored conditions that allow thickening and fibrosis equivalent to SSc patients Optimization was carried out to construct SSc disease model mice.
　確立されたＳＳｃ疾患モデルマウスを用いて、評価化合物の有効性（線維化抑制効果）の評価を行なった。 Using the established SSc disease model mice, the efficacy of the evaluation compounds (fibrosis suppression effect) was evaluated.
　・プロトコル Protocol
　Ｃ５７ＢＬ／６Ｊマウス（雌、１０－１２週齢）に対して、ブレオマイシンを１．５ｍｇ／日となるように毎日皮下に連続投与した（ ２８日間） 。
　ブレオマイシンを投与したマウスに対して、評価化合物を下記条件により投与した。コントロールとして、０．５％（ｗ／ｗ）ＣＭＣ（カルボキシメチルセルロース）水溶液のみを投与したマウスについても同様に試験した。
　・投与経路：経口投与
　・投与開始：ブレオマイシン投与開始３日前から
　・投与期間：３１日間連続投与
　・投与量：エパルレスタットが１００または３００ｍｇ／体重ｋｇ／日となるように投与した。 To C57BL / 6J mice (female, 10 to 12 weeks of age), bleomycin was continuously administered subcutaneously daily at a dose of 1.5 mg / day (28 days).
The evaluation compound was administered to the mice receiving bleomycin under the following conditions. As a control, the same test was conducted on mice that received 0.5% (w / w) aqueous solution of CMC (carboxymethylcellulose) alone.
Administration route: oral administration start of administration: 3 days before start of administration of bleomycin administration period: continuous administration for 31 days administration amount: Epalrestat was administered at 100 or 300 mg / kg body weight / day.
　ブレオマイシン投与開始から２８日後に皮膚組織を採取し、ホルマリン固定パラフィンブロックを作成した。これらの組織サンプルは、キシレン５分×３回、１００％エタノール１分×２回、９０％エタノール１分、８０％エタノール１分、７０％エタノール１分、水５分間で処理し、脱パラフィンした。マイヤーヘマトキシリン溶液で４分間、エオジン液で１分間染色を行った。流水中で５分処理した後、７０％エタノール１分、８０％エタノール１分、９０％エタノール１分、１００％エタノール１分×２回、キシレン５分×３回で処理を行い、マリノールを用いて封入した。室温で１５分以上乾燥させた後、光学顕微鏡を用いて観察を行なった。 Skin tissues were collected 28 days after the initiation of bleomycin administration, and formalin fixed paraffin blocks were prepared. These tissue samples were deparaffinized by treating with xylene 5 minutes × 3 times, 100% ethanol 1 minute × 2 times, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, water 5 minutes . Staining was performed with Meyer's hematoxylin solution for 4 minutes and eosin solution for 1 minute. Treated in running water for 5 minutes, treated with 70% ethanol 1 minute, 80% ethanol 1 minute, 90% ethanol 1 minute, 100% ethanol 1 minute × 2 times, xylene 5 minutes × 3 times, using marinol Sealed. After drying at room temperature for 15 minutes or more, observation was performed using an optical microscope.
　・結果 ・ Result
　図１５Ａ、Ｂに示すように、エパルレスタット（ＥＬＳ）は、ＢＬＭによって誘発される皮膚肥厚を顕著に抑制（濃度依存的）し、ＳＳｃモデルマウスを用いたエパルレスタットのＳＳｃに対する治療有効性が確認された。 As shown in FIGS. 15A and B, epalrestat (ELS) significantly suppressed BLM-induced skin thickening (concentration dependent), confirming the therapeutic efficacy of epalrestat against SSc using SSc model mice. .
　７）ＳＳｃ疾患モデルマウスを用いた3-[（1E）-2-（1H-インドール-6-イル）エテニル]-5-[（1E）-2-[2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル]-1H-ピラゾール（化合物Ａ：ＬＧ２８３）の線維化抑制の薬効評価 7) 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridyl) using the SSc disease model mouse Efficacy evaluation of inhibition of fibrosis of (methoxy) phenyl] ethenyl] -1H-pyrazole (Compound A: LG283)
　・プロトコル Protocol
　Bleomycin (BLM) 誘導性強皮症モデル構築
　生後8～10週の雌のC57BL/6J野生型マウスの背部に、生理食塩水 (NaCl) で1 mg/mlに溶解したブレオマイシン (Bleo) 150 mlを、4週間連日皮下注射し、線維化による皮膚硬化の強皮症病巣を誘導した。 Bleomycin (BLM) -induced scleroderma model construction 150 ml of Bleomycin (Bleo) dissolved in saline (NaCl) at 1 mg / ml on the back of 8- to 10-week-old female C57BL / 6J wild-type mice Every day for 4 weeks, it was injected subcutaneously to induce scleroderma foci of skin hardening due to fibrosis.
　化合物Ａの経口投与 
　ポリエチレングリコールに溶解した40 mg/kgまたは 80 mg/kgの化合物Ａ（：ＬＧ２８３）をBleo投与と同時に連日経口投与し（評価試験群）、ポリエチレングリコールのみを投与したplacebo群と比較した。 Oral administration of Compound A
At 40 mg / kg or 80 mg / kg of Compound A (: LG283) dissolved in polyethylene glycol was orally administered daily at the same time as Bleo administration (evaluation test group), and compared with placebo group to which only polyethylene glycol was administered.
　皮膚の線維化の評価
　注射した局所の皮膚を採取し、Ｈ＆Ｅ染色またはマッソントリクローム（Masson’s trichrome）染色を行い、（1）真皮の厚さ、および（2）線維化の領域を測定した。 Evaluation of Skin Fibrosis The injected local skin was collected, subjected to H & E staining or Masson's trichrome staining, and (1) thickness of dermis, and (2) area of fibrosis was measured.
　・結果
　BLMを連日28日間皮内投与後、背部皮膚の（1）真皮の厚さをＨ＆Ｅ染色像（図１６Ａ～Ｄ）を用いて測定し、結果を図１７のグラフに示した。化合物Ａの投与によって、強皮症病巣である線維化による真皮の肥厚が抑制され、それが投与濃度依存的に抑制される結果が得られ、化合物Ａの治療有効性が確認された。 Results After intradermal administration of BLM for 28 days a day, the thickness of (1) dermis of the back skin was measured using H & E stained images (FIGS. 16A to D), and the results are shown in the graph of FIG. The administration of Compound A suppressed the thickening of the dermis due to fibrosis, which is a scleroderma lesion, and the result was suppressed depending on the dose concentration, and the therapeutic efficacy of Compound A was confirmed.
　また（2）線維化の領域であるMasson’s trichrome染色標本上で青色に染色（図１８Ａ～Ｄ）された面積を測定することにより評価を行い、結果を図１９のグラフに示した。化合物Ａの投与により、強皮症病巣領域が減少すること、それが投与濃度依存的に減少する結果が得られ、化合物Ａの治療有効性が確認された。 (2) Evaluation was carried out by measuring the area stained in blue (FIG. 18A to D) on the Masson's trichrome-stained sample, which is an area of fibrosis, and the results are shown in the graph of FIG. The administration of compound A resulted in a decrease in scleroderma lesion area, and a result that the dose concentration-dependently was reduced, and the therapeutic efficacy of compound A was confirmed.
　なお、図中に示される統計学的な有意差の有無（ｐ値）は、unpaired t-testを用いて解析した（以下の実施例においても同様）。 The presence or absence (p value) of statistical significance shown in the figure was analyzed using unpaired t-test (the same applies to the following examples).
　８）3-[（1E）-2-（1H-インドール-6-イル）エテニル]-5-[（1E）-2-[2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル]-1H-ピラゾール（化合物Ａ：ＬＧ２８３）のマトリックス分子発現抑制効果の評価 8) 3-[(1E) -2- (1H-indol-6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridylmethoxy) phenyl] ethenyl]- Evaluation of matrix molecule expression suppression effect of 1H-pyrazole (compound A: LG283)
　強皮症においては炎症により真皮内の線維芽細胞が形質転換し、（1）コラーゲン、（2）Fibronectin等のマトリックス分子の分泌が亢進し細胞真皮に線維化病巣が形成されることから、化合物Ａ（：ＬＧ２８３）がこれらのマトリックス分子の分泌を抑制する効果があるか否かを下記の実験により評価した。 In scleroderma, inflammation causes the transformation of fibroblasts in the dermis, and the secretion of matrix molecules such as (1) collagen and (2) Fibronectin is enhanced, resulting in the formation of fibrotic foci in the cell dermis. Whether A (: LG 283) had the effect of suppressing the secretion of these matrix molecules was evaluated by the following experiment.
　実験Ａ）コラーゲンCollagen 1A2およびFibronectin1 の発現（mRNA）に対する抑制効果の評価 Experiment A) Evaluation of the suppressive effect on the expression (mRNA) of collagen Collagen 1A2 and Fibronectin 1
ヒト正常培養皮膚線維芽細胞HFFFにTGF-β1を作用すると形質転換が誘導され、コラーゲンCollagen 1A2とFibronectin1 の発現（mRNA）が亢進した（qRT-PCRにて定量）。これに化合物Ａ（4.5μM）を作用することで、コラーゲンCollagen 1A2およびFibronectin1 の発現（mRNA）が有意に抑制された（図２０）。 The action of TGF-β1 on human normal cultured dermal fibroblast HFFF induced transformation, and the expression (mRNA) of collagen Collagen 1A2 and Fibronectin 1 was enhanced (quantified by qRT-PCR). By acting on this compound A (4.5 μM), the expression (mRNA) of collagens Collagen 1A2 and Fibronectin 1 was significantly suppressed (FIG. 20).
　実験Ｂ）コラーゲンCollagen 1A2タンパク質の発現に対する抑制効果の評価 Experiment B) Evaluation of the inhibitory effect on the expression of collagen Collagen 1A2 protein
　ヒト正常培養皮膚線維芽細胞HFFFにTGF-β1を作用すると形質転換が誘導され、コラーゲンCollagen 1A2タンパク質の発現量が亢進した（Western Blottingにて評価）。これに化合物Ａ（4.5μM）を作用することでコラーゲンCollagen 1A2タンパク質の発現が有意に抑制された（図２１）。  When TGF-β1 acts on human normal cultured skin fibroblasts HFFF, transformation is induced and the expression level of collagen Collagen 1A2 protein is enhanced (evaluated by Western Blotting). By acting on this compound A (4.5 μM), the expression of collagen collagen 1A2 protein was significantly suppressed (FIG. 21).
　実験Ｃ）Fibronectinタンパク質の発現に対する抑制効果の評価
　ヒト正常培養皮膚線維芽細胞HFFFにTGF-β1を作用すると形質転換が誘導され、Fibronectinタンパク質の発現量が亢進した（Western Blottingにて評価）。これに化合物Ａ（4.5μM）を作用することでFibronectinタンパク質の発現が有意に抑制された（図２２）。 Experiment C) Evaluation of inhibitory effect on expression of Fibronectin protein Transformation of human normal cultured dermal fibroblasts HFFF was induced by the action of TGF-β1, and the expression level of Fibronectin protein was enhanced (as assessed by Western Blotting). The action of Compound A (4.5 μM) on this significantly suppressed the expression of Fibronectin protein (FIG. 22).
　強皮症の線維化病巣形成に係る（1）コラーゲン、（2）Fibronectinのマトリックス分子の発現を化合物Ａは遺伝子発現レベル（実験Ａ）とタンパク質分泌レベル（実験Ｂ，Ｃ）で抑制することが確認され、化合物Ａが強皮症の治療に効果があることが検証された。 Compound A suppresses the expression of matrix molecules of (1) collagen and (2) fibronectin involved in fibrotic foci formation of scleroderma at gene expression level (experiment A) and protein secretion level (experiments B and C) It was confirmed that Compound A was effective in treating scleroderma.
　なお、時間、労力、コスト等の理由から、実施例４において言及した１１種の線維芽細胞活性化に対する抑制活性の高い治療薬候補化合物の全てについて、実施例６および７に示したようなＳＳｃ疾患モデルマウスを用いた線維化抑制の薬効評価試験結果を本明細書中に示すことはできなかったが、それは必ずしも１１種のうちの薬効評価試験結果が示されなかった化合物がＳＳｃ疾患モデルマウスを用いた線維化抑制の薬効評価試験において効果が見られないことを意味するものではなく、当業者であれば、上記１１種の化合物のうち本明細書中にＳＳｃモデルマウス薬効評価試験の結果が示されなかった化合物についても、本明細書全体の記載および本出願の有効出願日における本発明の属する技術分野の技術常識を考慮して、薬効試験評価結果が記載された化合物と同様に有効な結果を示すであろうことは理解できるであろう。 In addition, for the reasons of time, labor, cost, etc., SSc as shown in Examples 6 and 7 for all the therapeutic drug candidate compounds having high inhibitory activity on the activation of fibroblasts mentioned in Example 4. Although the efficacy evaluation test results of fibrosis suppression using disease model mice could not be shown in this specification, it is not necessarily the compound of 11 kinds of efficacy evaluation test results showing SSc disease model mice It does not mean that no effect is seen in the efficacy evaluation test of inhibition of fibrosis using the above-mentioned, and those skilled in the art can evaluate the results of the SSc model mouse efficacy evaluation test in the present specification among the above 11 compounds. With respect to the compounds for which the compound (1) is not indicated, the pharmacological efficacy test in view of the general description of the present specification and the common general knowledge of the technical field to which the present invention belongs We will show effective results as with compounds described evaluation results will be appreciated.
　本発明は、全身性強皮症の治療および／または予防薬として有用である。 The present invention is useful as a therapeutic and / or prophylactic agent for systemic scleroderma.

Claims (3)

  1. 　真皮由来の組織における線維芽細胞活性化を抑制する化合物を含有する、全身性強皮症を治療または予防するための医薬組成物。 A pharmaceutical composition for treating or preventing systemic scleroderma, comprising a compound that inhibits fibroblast activation in a dermis-derived tissue.
  2. 　前記全身性強皮症が、真皮由来の組織における線維芽細胞活性化によって特徴付けられる、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the systemic scleroderma is characterized by fibroblast activation in tissues derived from dermis.
  3. 　前記化合物が、ベンズブロマロン、アルベンダゾール、メベンダゾール、リセドロン酸ナトリウム水和物、エパルレスタット、フルフェナム酸アルミニウム、ザルトプロフェン、アンレキサノクス、メフェナム酸、オキサプロジン、および3 - [（1E）-2-（1H-インドール-6-イル）エテニル] -5 - [（1E）-2- [2-メトキシ-4-（2-ピリジルメトキシ）フェニル]エテニル] -1H-ピラゾールからなる群から選択される、請求項１または２に記載の医薬組成物。 The above compounds are benzbromarone, albendazole, mebendazole, risedronate sodium hydrate, epalrestat, aluminum flufenamate, zaltoprofen, anlexanox, mefenamic acid, oxaprozin, and 3- (1E) -2- (1H-indole- The compound according to claim 1 or 2, selected from the group consisting of 6-yl) ethenyl] -5-[(1E) -2- [2-methoxy-4- (2-pyridylmethoxy) phenyl] ethenyl] -1H-pyrazole. Pharmaceutical composition as described in-.
PCT/JP2018/045053 2017-12-08 2018-12-07 Systemic scleroderma therapeutic agent WO2019112031A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017-235864 2017-12-08
JP2017235864 2017-12-08

Publications (1)

Publication Number Publication Date
WO2019112031A1 true WO2019112031A1 (en) 2019-06-13

Family

ID=66751504

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/045053 WO2019112031A1 (en) 2017-12-08 2018-12-07 Systemic scleroderma therapeutic agent

Country Status (1)

Country Link
WO (1) WO2019112031A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2021387517B2 (en) * 2020-11-30 2024-02-15 Innovo Therapeutics Inc. Pharmaceutical composition for preventing or treating wound or scar, comprising benzbromarone

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009513520A (en) * 2003-07-08 2009-04-02 イーエムテーエム ゲーエムベーハー Use of inhibitors of enzymes having the activity of aminopeptidase N and / or dipeptidyl peptidase IV for the treatment and prevention of skin diseases including hyperproliferation of fibroblasts and changes in differentiation state, and medicaments containing them Use of preparation
US20100093613A1 (en) * 2007-03-09 2010-04-15 Kunkel Eric J Methods for identifying agents and their use for the prevention or stabilization of fibrosis
WO2017073060A1 (en) * 2015-10-29 2017-05-04 国立大学法人東北大学 Collagen production inhibitor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009513520A (en) * 2003-07-08 2009-04-02 イーエムテーエム ゲーエムベーハー Use of inhibitors of enzymes having the activity of aminopeptidase N and / or dipeptidyl peptidase IV for the treatment and prevention of skin diseases including hyperproliferation of fibroblasts and changes in differentiation state, and medicaments containing them Use of preparation
US20100093613A1 (en) * 2007-03-09 2010-04-15 Kunkel Eric J Methods for identifying agents and their use for the prevention or stabilization of fibrosis
WO2017073060A1 (en) * 2015-10-29 2017-05-04 国立大学法人東北大学 Collagen production inhibitor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOTO, SHIGERU: "MEDICAL DICTIONARY", vol. 2, 2001, ISHIYAKU, article "the dermis" *
IMOKOWA, GENJI: "Onset mechanism of aging and photoaging (wrinkle)", FUNCTIONAL FOOD, vol. 2, no. 4, 2009, pages 358 - 365 *
PREHM,P. ET AL.: "Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporter", BIOCHEMICAL PHARMACOLOGY, vol. 68, 2004, pages 1401 - 1410, XP004549996, doi:10.1016/j.bcp.2004.06.017 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2021387517B2 (en) * 2020-11-30 2024-02-15 Innovo Therapeutics Inc. Pharmaceutical composition for preventing or treating wound or scar, comprising benzbromarone
US11951089B2 (en) 2020-11-30 2024-04-09 Innovo Therapeutics Inc. Pharmaceutical composition for preventing or treating wound or scar, comprising benzbromarone

Similar Documents

Publication Publication Date Title
JP6974669B2 (en) Combinations including B-Raf inhibitors, EGFR inhibitors and possibly PI3K-α inhibitors
JPWO2017047769A1 (en) Toll-like receptor 7 or toll-like receptor 9 activation inhibitor
AU2015384339B2 (en) Composition for preventing or treating valve calcification, containing DPP-4 inhibitor
TW200803900A (en) Compositions and methods for reducing food cravings
JP6860949B2 (en) How to treat cancer
CN108025035A (en) The composition and method of relevant cancer are activated with ETBR for treating
JP2022531939A (en) How to treat cancer
US20190011457A1 (en) Biomarkers for diagnosing keloid skin or keloid scar, and use thereof
JP2018529637A (en) PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF IL-6-MEDIATED DISEASE CONTAINING HAMANUS FLOWER EXTRACT AS ACTIVE INGREDIENT
WO2019112031A1 (en) Systemic scleroderma therapeutic agent
JP2023549678A (en) Combination of AHR inhibitors with PDx inhibitors or doxorubicin
JP2012072136A (en) Composition for promoting intracellular metabolism, and pharmaceutical preparation for preventing and/or treating saccharometabolism or lipid metabolism disease, functional food, and health food containing the composition
KR20170036928A (en) IKK Pharmaceutical Composition for Preventing or Treating Chronic Rhinosinusitis Comprising IKK Inhibitor
KR101777770B1 (en) Pharmaceutical composition comprising ursolic acid acetate for preventing or treating STAT3 mediated disease and use thereof
JP2019511495A (en) Treatment of CDKL5 disorders with the GSK3.BETA.
JP2019077643A (en) Wnt EXPRESSION INHIBITOR
US20110130374A1 (en) Small Pyrimidine Derivatives and Methods of Use Thereof
KR102625224B1 (en) Composition for preventing, improving or treating autoimmune diseases comprising pyrazol-one derivatives
WO2015182565A1 (en) Therapeutic agent for lung disease
EP4285938A1 (en) Pharmaceutical composition for treating or preventing malignant breast cancer
JP6212563B2 (en) Combination medicine comprising phosphatidylinositol 3-kinase inhibitor and aromatase inhibitor
JP7511741B2 (en) Use of 2,3,5-substituted thiophene compounds for the prevention, amelioration or treatment of gastrointestinal stromal tumors
WO2022185742A1 (en) Prophylactic or therapeutic composition for disseminated intravascular coagulation syndrome
KR20230143965A (en) Composition for preventing or treating pulmonary fibrosis comprising taurodeoxycholic acid or pharmaceutically acceptable salts thereof as an active ingredient
US20200163942A1 (en) Composition for preventing or treating cancer comprising a vascular disrupting agent and immune checkpoint inhibitor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18886234

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18886234

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP