WO2015016178A1 - Function inhibitor of apoptosis-associated speck-like protein containing card comprising 1,5-d-anhydrofructose - Google Patents

Function inhibitor of apoptosis-associated speck-like protein containing card comprising 1,5-d-anhydrofructose Download PDF

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WO2015016178A1
WO2015016178A1 PCT/JP2014/069824 JP2014069824W WO2015016178A1 WO 2015016178 A1 WO2015016178 A1 WO 2015016178A1 JP 2014069824 W JP2014069824 W JP 2014069824W WO 2015016178 A1 WO2015016178 A1 WO 2015016178A1
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asc
disease
therapeutic agent
agent according
symptom associated
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French (fr)
Japanese (ja)
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丸山　征郎
聖 野間
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国立大学法人鹿児島大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a function inhibitor of apoptosis-related spec-like card protein (ASC) containing 1,5-D-anhydrofructose as an active ingredient, and a therapeutic agent for diseases or symptoms involving ASC.
  • ASC apoptosis-related spec-like card protein
  • the present invention also relates to an inflammasome pathway inhibitor and a therapeutic agent for a disease or condition involving the inflammasome pathway, which contains 1,5-D-anhydrofructose as an active ingredient.
  • 1,5-D-anhydrofructose acts on starch or starch degradation products of ⁇ -1,4-glucan lyase, an enzyme derived from certain ascomycetes and red algae. Can be produced. 1,5-AF has a double bond in the molecule and is a highly reactive sugar compared to other monosaccharides.
  • 5-AF is a carbohydrate that is expected to develop using its multifunctionality in various fields such as health foods and pharmaceuticals.
  • “Inflammation” is an important field not only in clinical medicine but also in basic medicine and biology because it is a pathological condition underlying human infections and trauma. This inflammation has been identified as “erythema fever” from the symptom-pathomorphological features. In other words, the inflammatory lesion is characterized by a fever, red swelling, and soreness, and this pathological image is a core concept for a long time, which is a concept that should be called a “classical inflammation image”.
  • intercellular mediators called cytokines and their receptors have been discovered. Research on inflammation is a study of elucidation of intercellular networks, intracellular signals, and gene expression. It shifted to. As this brilliant achievement makes it possible to learn from the fact that Dr.
  • caspase 1 leads to maturation of pro-IL-18, pro-IL-1 ⁇ and release of IL-18, IL-1 ⁇ to the outside, thereby immune cells (T cells, B cells, dendritic cells, “Chemotaxis” of neutrophils etc. is initiated, and an inflammatory lesion is formed at the site.
  • T cells, B cells, dendritic cells, “Chemotaxis” of neutrophils etc. is initiated, and an inflammatory lesion is formed at the site.
  • the important point in this regard is firstly the activation of NF- ⁇ B, where pro-IL-1 ⁇ and pro-IL-18 (both precursors) are produced and accumulated in the cell, and then the activated inflammasome Only when procaspase 1 is activated to caspase 1, active caspases activate pro-IL-18 and pro-IL- ⁇ into mature activated IL-18 and IL-1 ⁇ , respectively. It is to be released extracellularly (FIG. 3-1).
  • CARD has a property of polymerizing, so that caspase 1 (CARD ⁇ p20 / p16) and ASC (PYD ⁇ CARD) are bound via CARD, and NLR (PYD / NACHT) is bound to PYD.
  • CARD ⁇ p20 / p16 caspase 1
  • ASC PYD ⁇ CARD
  • NLR PYD / NACHT
  • This ASC is originally a protein that exists in the cytosol and is soluble in a buffer solution containing Triton X-100 (a non-ionic surfactant of the phenyl polyethylene glycol type), but it is activated by apoptosis-inducing agents such as retinoic acid and anticancer agents. Along with the induced apoptosis, it becomes insoluble in a buffer containing Triton X-100 and causes aggregation. The cause of ASC changing from soluble to insoluble in a buffer containing Triton X-100 with apoptosis is considered to be due to a change in conformation. ASC has no effect of inducing apoptosis by itself, but has an effect of promoting apoptosis induced by other factors (see Patent Document 12).
  • NF- ⁇ B and inflammasome are also expressed in cells other than immune cells such as epithelial cells and neurons, and cells of tissues and organs where lymphocyte cells (lymphoid cells) are not resident
  • lymphocyte cells lymphocyte cells
  • a biodefense response is required.
  • the non-myloid tissue catches information on PAMPs and DAMPs, produces and releases IL-18 and IL-1 ⁇ , which are migratory factors of immune cells, and collects myloid cells.
  • a so-called pseudo-lymph node consisting of lymphocytes for biological defense capable of carrying out an immune reaction at that site is constructed.
  • Non-Patent Documents 1 and 2 describe the following matters.
  • Cell groups related to innate immunity and acquired immunity (macrophages, dendritic cells, etc.) express Toll-like receptors (TLRs) on their cell surfaces.
  • TLRs Toll-like receptors
  • DAMPs or PAMPs bind to these receptors, these cells Are activated and transmit various signals, but they merge and one converges to a protein complex called the inflammasome.
  • This complex is composed of Nod-like-receptor (NLR), Apoptosis-associated-spec-like-protein-containing-a-case-recovery-domain (ASC), and procaspase-1.
  • NLR Nod-like-receptor
  • ASC Apoptosis-associated-spec-like-protein-containing-a-case-recovery-domain
  • procaspase-1 Like TRLs, inflammasome is activated by binding of DAMPs and PAMPs to NLRs.
  • Securing IL-1 ⁇ an inflammatory cytokine, requires at least two steps.
  • One is stimulation from TLRs (signal 1), which creates pro-IL-1 ⁇ (a precursor of IL-1 ⁇ ) in the cytoplasm.
  • procaspase 1 is activated to caspase 1 by a signal from inflammasome activation (signal 2), whereby caspase 1 cleaves pro-IL-1 ⁇ , and activated IL-1 ⁇ becomes extracellular.
  • signal 2 signal from inflammasome activation
  • caspase 1 cleaves pro-IL-1 ⁇ , and activated IL-1 ⁇ becomes extracellular.
  • IL-18 is also secreted by a similar mechanism (see Non-Patent Documents 1 and 2).
  • caspase activation not only induces the production and extracellular release of IL-1 ⁇ and IL-18, but also fragmentation of cellular DNA, cell swelling called pyroptosis and cell It causes death (inflammatory programmed cell) death (FIG. 4).
  • cryopyrin-associated periodic fever syndrome the NLRP3 gene, a major component of the inflammasome, is mutated, the inflammasome is activated, and overproduction of IL-1 ⁇ continues. Occur.
  • familial Mediterranean fever, Pyogenicic arthritis with pyderma gregrenosum and acne (PAPA) syndrome, Majeed's syndrome, high IgD syndrome, recurrent hydatidiform mole, DIRA continues to be inflammasome-activated, and IL-1 ⁇ excess Production is known to occur (see Non-Patent Documents 1 and 2).
  • Anakinra, rilonacept, and canakinumab which are inhibitors of IL-1 ⁇ , are used clinically for these diseases, but they are not effective for all patients (see Non-Patent Document 3). Moreover, these IL-1 ⁇ inhibitors, anakinra, rilonacept, and canakinumab, are of course unable to inhibit IL-18.
  • Non-patent Document 2 Non-patent Document 2
  • ASC is a protein that contributes to activation of the innate immune system, suppression of cancer, suppression of natural inflammatory diseases, and the like.
  • JP-T 9-505988 Special Table 2001-89377 French Patent Application Publication No. 2617502 JP 2004-123604 A WO2010 / 082661 WO2004 / 045628 JP 2012-1515 A JP 2007-30146 A JP 2007-91644 A JP 2006-306814 A JP 2005-154425 A JP 2001-275681 A
  • an object of the present invention is to provide a safe and excellent therapeutic agent for a disease or symptom associated with apoptosis-related spec-like card protein (ASC), and a safe and excellent ASC function inhibitor.
  • ASC apoptosis-related spec-like card protein
  • 1,5-AF which is an in vivo sugar metabolite
  • ASC ASC
  • 1,5-AF inhibits the inflammasome pathway.
  • an inhibitory system for the inflammasome pathway has not been found. In other words, 1,5-AF was first discovered as a compound that inhibits the inflammasome pathway.
  • the present invention includes the following inventions.
  • a function inhibitor of apoptosis-related spec-like card protein comprising 1,5-D-anhydrofructose as an active ingredient.
  • ASC apoptosis-related spec-like card protein
  • the present inventors have found that the target protein of 1,5-AF is ASC. Furthermore, the present inventors have found that there is a device for controlling the intracellular inflammatory cascade, particularly in living cells, which is the 1,5-AF pathway. Since 1,5-AF is an in vivo molecule and has no side effects, it is expected to become a great weapon for controlling inflammation. In addition, IL-1 ⁇ inhibitors anakinra, rilonacept, and canakinumab are all proteins and are expensive to produce, but 1,5-AF can be produced at a much lower cost than these protein preparations. These drugs are economically advantageous as compared with these drugs.
  • the agent of the present invention is suitable for a mammal individual having a disease or symptom involving ASC, particularly a disease or symptom involving an inflammasome pathway, specifically, an autoinflammatory disease involving activated procaspase 1
  • a disease or symptom involving ASC particularly a disease or symptom involving an inflammasome pathway, specifically, an autoinflammatory disease involving activated procaspase 1
  • the drug of the present invention it becomes possible to alleviate or suppress diseases or symptoms associated with ASC. Further, according to the agent of the present invention, a disease or symptom involving the inflammasome pathway, particularly a disease or symptom involving active caspase 1 is prevented and / or treated, and the activated innate immunity is significantly alleviated, It becomes possible to suppress.
  • FIG. 1 is a diagram illustrating the relationship between natural inflammation and disease.
  • FIG. 2 is a diagram for explaining the activation of NF- ⁇ B and inflammasome by PAMPs and DAMPs.
  • FIG. 3-1 is a diagram illustrating a pathway in which procaspase 1 is activated through inflammasome pathway activation.
  • FIG. 3-2 is a diagram for explaining the morphology of the inflammasome multiprotein complex.
  • FIG. 4 shows that procaspase 1 causes not only the production and release of IL-1 ⁇ and IL-18, but also DNA fragmentation and programmed cell death called pyroptosis.
  • FIG. 5 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-1 ⁇ production in mouse-derived bone marrow macrophages.
  • FIG. 6 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-1 ⁇ production in mouse-derived bone marrow macrophages.
  • the right side of the graph shows the added substance, and the parenthesis shows the activated NLR.
  • FIG. 7 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-18 production in mouse-derived bone marrow macrophages.
  • the right side of the graph shows the added substance, and the parenthesis shows the activated NLR.
  • FIG. 8 shows the test results of the inhibitory effect of 1,5-AF on IL-18 production in mouse-derived bone marrow macrophages.
  • FIG. 9 is a graph showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by alum or urate.
  • FIG. 10 is a graph showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by nigericin and ATP.
  • FIG. 11 is a diagram showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by ATP.
  • FIG. 12 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by anthrax toxin.
  • FIG. 13 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by flagellin.
  • FIG. 14 is a diagram showing test results of the inhibitory effect of 1,5-AF on inflammasome activation by poly (dA: dT).
  • FIG. 15 is a diagram showing that 1,5-AF suppresses the activation of procaspase 1 to caspase 1.
  • FIG. 16 is a diagram showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by rotenone.
  • FIG. 17 is a diagram showing lung tissue images and cell numbers of the mouse ALI model.
  • FIG. 18 is a diagram showing the relationship between the time after administration of 1,5-AF to the SIRS model and the survival rate.
  • FIG. 19 is a diagram showing that the target protein of 1,5-AF is ASC.
  • FIG. 20a and 20b are diagrams showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation in an asbestosis model experiment.
  • FIG. 21 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by anthrax toxin.
  • FIG. 22 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by ATP, performed using human peripheral blood.
  • 1,5-AF used for the drug of the present invention can be prepared by a known method, for example, the method described in Patent Document 1 above.
  • the agent of the present invention inhibits the function of apoptosis-related spec-like card protein (ASC), particularly the ability to form a complex with caspase 1 and NOD-like receptor.
  • ASC apoptosis-related spec-like card protein
  • ASC having “complex forming ability” indicates that ASC functions as an adapter protein.
  • the agent of the present invention can inhibit the inflammasome pathway, and in particular, can suppress the activation of procaspase 1 to caspase-1.
  • the inflammasome pathway refers to a series of signal transduction pathways performed via the inflammasome (FIG. 3-1). The inflammasome is thought to activate caspase 1 by ASC forming a complex of caspase 1 and NOD-like receptors with the heptamer (FIG. 3-2).
  • the drug of the present invention is a therapeutic drug for a disease or symptom related to ASC, particularly a disease or symptom related to complex formation of ASC, caspase 1 and NOD-like receptor.
  • the agent of the present invention can be used for the purpose of preventing or treating autoinflammatory diseases in which inflammasome is activated.
  • the symptoms are suppressed by suppressing innate immunity of autoinflammatory diseases associated with inflammasome activation, in particular, caspase 1 activation, and therefore used for the purpose of preventing or treating these diseases. Is possible.
  • diseases or symptoms associated with ASC include cancer, immunosuppressive diseases, autoimmune diseases, viral infections, neurodegenerative diseases, endocrine diseases, inflammatory diseases, organ transplantation disorders and radiation disorders.
  • examples of the disease or symptom include a disease or symptom involving the inflammasome pathway, particularly a disease or symptom caused by a series of signal transduction performed through the inflammasome.
  • the disease or symptom involving the inflammasome pathway includes a disease or symptom involving activated procaspase-1.
  • diseases or conditions involving the inflammasome pathway include autoinflammatory diseases, particularly diseases caused by the production of IL-1 ⁇ and / or IL-18.
  • diseases involving the inflammasome pathway include, for example, cryopyrin-related periodic fever syndrome, familial Mediterranean fever, PAPA syndrome (Pyogenic artritis with pyderma grennosum and acne) syndrome, Majeed syndrome, high IgD syndrome, repetitive Hydatidiform mole, DIRA (defectiency of the IL-1 receptor antagonist), gout, Alzheimer's disease, type 2 diabetes, inflammatory bowel disease, rheumatoid arthritis, anthrax infection, acute lung injury (Acute Lung Injury), acute Respiratory Stimulation Syndrome (Acute Repitory Distress syndrome) and Systemic Inflammatory Response Syndrome SIRS (Systemic Infl mmatory Response syndrome), asbestosis and juvenile idiopathic arthritis, and the like.
  • cryopyrin-related periodic fever syndrome familial Mediterranean fever
  • PAPA syndrome Panogenic artritis with pyderma grennosum and acne
  • Majeed syndrome high IgD syndrome
  • repetitive Hydatidiform mole repetitive Hydatidiform
  • Systemic inflammatory response syndrome SIRS includes trauma, burns, pancreatitis, severely invasive postoperative infections and other causes, and bacteria, fungi, parasites, viruses and other infections.
  • SIRS resulting from infection sepsis can be mentioned.
  • Sepsis is a condition in which systemic organ disorders such as intravascular coagulation disorders (bleeding, thrombosis, etc.), decreased urine output, decreased blood pressure, etc. have appeared remarkably in addition to the pathological conditions of systemic reactive inflammation SIRS caused by inflammatory cytokines. .
  • SIRS is a concept mainly focused on inflammatory pathologies and inflammasome activation throughout the body, while sepsis is the activity of inflammasome activity. It can be said that this is a pathological condition that is complicated by multiple organ disorders due to disorders of the blood coagulation system and circulatory disorders.
  • symptoms associated with the inflammasome pathway include inflammatory cell death (pyroptosis) and symptoms associated with DNA fragmentation.
  • the drug of the present invention can be administered by various methods known per se, depending on the dosage form, and the dose, administration site, administration interval, period, etc. are determined depending on the patient's age, weight, medical condition, etc. Or it can determine suitably considering the case where it uses together with another chemical
  • the administration method is not particularly limited as long as 1,5-AF can be quickly delivered to the body or local lesion, but for example, oral administration, injection or infusion, transbronchial method, or application or application Can be mentioned.
  • the dose of the drug of the present invention varies depending on the dosage form, administration method, or symptoms to be prevented or treated.
  • the active ingredient (1, 1, 5-AF) in terms of 1 mg to 500 mg, preferably 10 mg to 100 mg, depending on an appropriate administration frequency such as once or several times a day, continuous infusion, etc., or once every several days. It is possible to administer.
  • a method such as transrespiratory administration may be considered.
  • the dose of the drug of the present invention varies depending on the dosage form, administration method, or symptoms to be prevented or treated.
  • the active ingredient (1, 1, 5-AF) in terms of 0.001 ⁇ g to 10000 mg, preferably 0.01 mg to 5,000 mg, depending on the appropriate administration frequency, such as once or several times a day or once every several days. It is possible to administer.
  • the present invention also relates to a preparation containing the drug of the present invention.
  • a preparation containing the drug of the present invention examples include, but are not particularly limited to, infusion, tablet, capsule, powder, granule, suppository, injection, transdermal absorption agent, cream, paste, gel, spray and the like.
  • the preparation containing the drug of the present invention may further contain necessary components such as a preparation carrier, excipient, stabilizer and the like.
  • the preparation of the present invention can also contain other drugs, other pharmacological components, or nutritional components such as glucose.
  • 1,5-AF has an anti-inflammatory action and also strongly suppresses inflammatory cell death. Therefore, particularly in the treatment of sepsis, combined use of 1,5-AF with an anticoagulant that suppresses coagulation (eg, recombinant thrombomodulin, trade name lycomodulin, etc.) can reduce the combined pathological condition. it can.
  • medical agent of this invention is not restricted to a pharmaceutical use, It is also possible to mix
  • 1,5-AF can be added to foods to take the form of functional foods intended to prevent or treat symptoms in various diseases.
  • the preparation containing the drug of the present invention can be in the form of a quasi-drug or cosmetic for the purpose of treating skin symptoms.
  • the drug of the present invention can be administered to mammals other than humans. In that case, treatment can be performed by administering an appropriate amount of 1,5-AF to the mammal.
  • NLR which is a component of inflammasome, is a pattern recognition receptor that recognizes intracellular pathogens and the like, and the inflammasome is activated by binding DAMPs and PAMPs thereto. Although many NLRs are known, in this example, experiments were conducted using agents that activate NLRP1, NLRP3, NLRC4, and AIM2 as representatives of NLRs.
  • NLR NLRP1 Anthrax PA and LF (ANT) (List Biologist) NLRP3 ATP, nigericin (NIG), urate (MSU), Image Alum Ajuvant (ALU) NLRC4 Flagellin FLA AIM2 poly (dA: dT) (POL) (Sigma), Lipofectamine 2000 (LIP) (Invitrogen).
  • mouse-derived bone marrow macrophages were prepared according to the method of Nakahira, K et al. (Nat. Immunol. 12 (2011) 222-30). Bone marrow stem cells were extracted from the femur and tibia of C57 / BL6 male mice, and DMEM liquid medium (10% FBS and 2% penicillin) containing 25% of the culture supernatant (including M-CSF) extracted from L929 cells. The cells were cultured in streptomycin) for 6 days and differentiated into macrophages. After 6 days, mouse-derived bone marrow macrophages were collected, the number of cells was adjusted, each dish was re-wound and allowed to adhere overnight, and then LPS and an inflammasome-inducing agent were added.
  • pro-IL-1 ⁇ a precursor of IL-1 ⁇
  • pro-IL-18 a precursor of IL-18
  • TLRs TLRs
  • 1,5-AF was incubated for 30 minutes, and a drug that induces inflammasome was further added.
  • NLRP1 When activating NLRP1, Anthrax® PA and LF (ANT) were added at 5 ⁇ g / ml and incubated for 6 hours for induction.
  • induction is carried out by incubating with ATPA5 mM for 1 hour, Nigericin (NIG) 5 ⁇ M for 1 hour, Image Alum Ajuvant (ALU) 200 ⁇ g / ml for 6 hours, and Urate (MSU) 150 ⁇ g / ml for 6 hours did.
  • NOG Nigericin
  • ALU Image Alum Ajuvant
  • MSU Urate
  • FLA flagellin
  • AIM2 In the case of AIM2, it was induced by transfecting poly (dA: dT) (POL) using Lipofectamine 2000 (LIP).
  • IL-1 ⁇ and IL-18 were quantified using an ELISA kit manufactured by R & D.
  • Example 1 The results of Example 1 are shown in Fig. 5-8. From Fig. 5-8, 1,5-AF suppresses the production of IL-1 ⁇ and IL-18 by activation of procaspase 1 through all inflammasome pathways involving NLRP1, NLRP3, NLRC4 and AIM2. You can see that The results shown in FIG. 5-8 will be described below.
  • the NLRP3 inflammasome pathway was activated by adding ATP, nigericin, urate or alum to the medium of bone marrow macrophages stimulated with LPS.
  • the NLRC4 inflammasome pathway was activated by adding flagellin (FLA) obtained by extracting Salmonella cell components to bone marrow macrophages incubated with LPS.
  • FLA flagellin
  • the AIM-2 inflammasome pathway was activated by transfecting poly (dA: dT) (POL) from which DNA was extracted to bone marrow macrophages incubated with LPS using Lipofectamine 2000.
  • Alum (ALU) or urate (MSU) is a representative molecule that activates the NLRP3 pathway. Addition of alum or urate to bone marrow macrophages stimulated with LPS activated NLRP3 inflammasome (Nature Immunology, 2013 May; 14 (5): 454-60). In contrast, in the presence of 1,5-AF, activation of NLRP3 inflammasome by alum or urate was suppressed (see also FIG. 9).
  • Nigericin a kind of bacterial toxin, activates NLRPs and regenerates and releases IL-1 ⁇ and IL-18 via caspase 1 to cause inflammation.
  • NOG Nigericin
  • nigericin was added to bone marrow macrophages stimulated with LPS, the inflammasome was activated.
  • inflammasome activation by nigericin that is, production and release of IL-1 ⁇ and IL-18 were strongly suppressed (see also FIG. 10).
  • ATP activates the inflammasome through the NLRP3 pathway. Inflammasome was activated when ATP was added to bone marrow macrophages stimulated with LPS. In contrast, in the presence of 1,5-AF, inflammasome activation by ATP, ie, production and release of IL-1 ⁇ and IL-18 was suppressed (see also FIG. 11). In addition, compared with anhydroglucose (AG) and fructose, 1,5-AF inhibited inflammasome activation by ATP, that is, production and release of IL-1 ⁇ and IL-18 in a concentration-dependent manner (FIG. 11).
  • AG anhydroglucose
  • fructose 1,5-AF inhibited inflammasome activation by ATP, that is, production and release of IL-1 ⁇ and IL-18 in a concentration-dependent manner
  • Anthrax toxin activates the inflammasome through the NLRP1 pathway.
  • Anthrax toxin causes strong lung injury, and countermeasures are urgently needed due to the danger of being used for bioterrorism.
  • Addition of anthrax toxin to bone marrow macrophages stimulated with LPS activated NLRP1 inflammasome.
  • inflammasome activation by anthrax toxin that is, production and release of IL-1 ⁇ and IL-18 were strongly suppressed (see also FIG. 12).
  • Flagellin (FLA) activates NLRP4 to regenerate and release IL-1 ⁇ and IL-18 via caspase 1 and cause inflammation.
  • Flagellin (FLA) is a type of flagellar protein of flagellar bacteria (H. pylori, etc.) and induces inflammation via Toll-like receptor 5 (Toll like receptor 5) expressed in dendritic cells. To do. When flagellin was added to bone marrow macrophages stimulated with LPS, the inflammasome was activated. In contrast, in the presence of 1,5-AF, inflammasome activation by flagellin, that is, production and release of IL-1 ⁇ and IL-18 were strongly suppressed (see also FIG. 13).
  • dsDNA the host double-stranded DNA activates caspases via AIM2 / ASC, produces and releases IL-1 ⁇ and IL-18, and causes inflammation.
  • Bone marrow macrophages were stimulated with poly (dA: dT) (POL) as dsDNA.
  • dA: dT poly
  • POL poly
  • the production of IL-1 ⁇ in the culture supernatant of the group to which 1,5-AF has been added is remarkably suppressed as compared to the control. It was demonstrated to suppress inflammasome activation from the AIM2 / ASC pathway (see also FIG. 14).
  • Bone marrow stem cells were extracted from the femur and tibia of C57 / BL6 male mice, and DMEM liquid medium (10% FBS and 2% penicillin) containing 25% of the culture supernatant (including M-CSF) extracted from L929 cells. The cells were cultured in streptomycin) for 6 days and differentiated into macrophages. After 6 days, mouse-derived bone marrow macrophages were collected, the number of cells was adjusted, each dish was re-wound and allowed to adhere overnight, and then LPS and an inflammasome-inducing agent were added.
  • LPS was added for the purpose of producing pro-IL-1 ⁇ (IL-1 ⁇ precursor) in the cytoplasm by stimulation from TLRs (signal 1).
  • TLRs signal 1
  • LPS in vivoGen Ultrapure® LPS was used, and stimulation was performed at a concentration of 100 ng / ml for 4 hours.
  • Example 2 The result of Example 2 is shown in FIG. FIG. 15 shows that only caspase-1 was activated by activation of procaspase-1 and maturation of IL-1 ⁇ precursor (proIL-1 ⁇ ) into IL-1 ⁇ , extracellularly, in the presence of both LPS and ATP stimulation. It can be seen that secretion occurred. It was revealed that 1,5-AF suppresses the activation of procaspase 1 to caspase 1 through inflammasome activation.
  • Example 3 Peritoneal macrophages were extracted from wild-type mice, LPS was added to the priming, and then rotenon was added as an agent for inflammasome induction (1 hour) to quantify IL-1 ⁇ in the supernatant. was performed using an ELISA kit.
  • Rotenone is a mitochondrial disorder factor and a neuropathy factor used in experimental Alzheimer's disease and Parkinson's disease. It is known that when the mitochondria are damaged, a large amount of ATP is released from the damaged mitochondria, the inflammasome is activated, and inflammation is caused. In the nervous system, this is known to cause Alzheimer's disease and Parkinson's disease.
  • ALI Intraperitoneal administration of 1,5-AF to a representative model of ALI significantly suppressed lung histology and inflammatory cell infiltration in bronchoalveolar lavage fluid
  • An ALI model was prepared by transtracheally administering LPS to C57 / BL6 wild mice. Intratracheal administration of LPS (endotoxin) causes severe lung injury, which is considered to be a representative model of ALI (Gustavo Matte-Bello, et al. Mol Physiol 295: L379-L399, 2008).
  • Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are typical diseases associated with inflammasome activation.
  • SIRS model was prepared by intraperitoneally administering LPS to C57 / BL6 wild type mice.
  • inflammasome When inflammasome is activated systemically, inflammatory cytokines such as IL-1 ⁇ circulate throughout the body and cause SIRS, which is an important clinical condition.
  • mice SIRS model
  • 1,5-AF a treatment group at 100 mg / kg, 2, 6, 12, 24 hours after LPS administration, and observed for 5 days.
  • the survival rate was significantly improved ( FIG. 18).
  • Mouse bone marrow cells were primed by incubation with LPS for 4 hours, and then incubated with 1,5-AF for 30 minutes, followed by thorough washing of 1,5-AF and then incubation with nigericin (5 ⁇ M) for 1 hour. Inflammatory cell death was examined.
  • 1,5-AF targets apoptosis-related spec-like card protein (ASC)
  • ASC spec-like card protein
  • 1,5-AF has an effect of suppressing the release of IL-1 ⁇ also on human peripheral blood mononuclear cells stimulated with LPS + silica or ATP
  • Human peripheral blood was collected and human peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (Ficoll). PBMCs were dispersed in DMEM + 10% FBS medium at 0.1 ⁇ 10 6 cells / well and used for the experiment. Primed with LPS 500 ng / ml for 3 hours. 1,5-AF (2-10 mg / ml) was added, incubated for 30 minutes and then stimulated with silica (250 ⁇ g / ml) for 2 hours, and then the supernatant was collected.
  • PBMC peripheral blood mononuclear cells
  • 1,5-AF was found to have an effect of suppressing the release of IL-1 ⁇ even on human peripheral blood mononuclear cells stimulated with LPS + ATP (FIG. 22).
  • the drug of the present application can be used as an apoptosis-related spec-like card protein (ASC) regulator in place of the current biological preparation (antibody).
  • ASC apoptosis-related spec-like card protein

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Abstract

Provided are: a safe and excellent therapeutic agent for a disease or symptom in which an apoptosis-associated speck-like protein containing a CARD (ASC) participates; and a safe and excellent function inhibitor of an ASC. A function inhibitor of an ASC that comprises 1,5-D-anhydrofructose as an active ingredient.

Description

１，５－Ｄ－アンヒドロフルクトースを含むアポトーシス関連スペック様カード蛋白質の機能阻害薬Function inhibitor of apoptosis-related spec-like card protein containing 1,5-D-anhydrofructose
　本発明は、１，５－Ｄ－アンヒドロフルクトースを有効成分として含有する、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）の機能阻害薬及びＡＳＣが関与する疾患又は症状の治療薬に関する。本発明はまた、１，５－Ｄ－アンヒドロフルクトースを有効成分として含有する、インフラマソーム経路阻害薬及びインフラマソーム経路が関与する疾患又は症状の治療薬に関する。 The present invention relates to a function inhibitor of apoptosis-related spec-like card protein (ASC) containing 1,5-D-anhydrofructose as an active ingredient, and a therapeutic agent for diseases or symptoms involving ASC. The present invention also relates to an inflammasome pathway inhibitor and a therapeutic agent for a disease or condition involving the inflammasome pathway, which contains 1,5-D-anhydrofructose as an active ingredient.
　１，５－Ｄ－アンヒドロフルクトース（以下、１，５－ＡＦという）は、ある種の子嚢菌や紅藻由来の酵素であるα－１，４－グルカンリアーゼを澱粉あるいは澱粉分解物に作用させることで生産することができる。１，５－ＡＦは、その分子間内に二重結合を有しており、他の単糖類と比較して反応性に富む糖である。 1,5-D-anhydrofructose (hereinafter referred to as 1,5-AF) acts on starch or starch degradation products of α-1,4-glucan lyase, an enzyme derived from certain ascomycetes and red algae. Can be produced. 1,5-AF has a double bond in the molecule and is a highly reactive sugar compared to other monosaccharides.
　１，５－ＡＦは食品に安全に添加される抗酸化剤等としての用途が開示されている（特許文献１及び２参照）。また、この単糖は抗生物質ピロンミクロテシンの前駆体でもある（特許文献３参照）。 1,5-AF has been disclosed for use as an antioxidant that can be safely added to foods (see Patent Documents 1 and 2). This monosaccharide is also a precursor of the antibiotic pyrone microthecin (see Patent Document 3).
　また１，５－ＡＦは、最近では、抗う蝕作用、血小板凝集抑制作用、グリコーゲン分解酵素阻害、アディポネクチンの産生増強、抗腫瘍作用等についても報告されており（特許文献４－１１参照）、１，５－ＡＦは健康食品あるいは医薬品等の様々な分野でもその多機能性を利用した展開が期待される糖質である。 In addition, 1,5-AF has recently been reported for an anti-cariogenic effect, a platelet aggregation inhibitory effect, glycogenolytic enzyme inhibition, adiponectin production enhancement, an antitumor effect, and the like (see Patent Document 4-11). , 5-AF is a carbohydrate that is expected to develop using its multifunctionality in various fields such as health foods and pharmaceuticals.
　「炎症（inflammation）」はヒトの感染症や外傷の基盤をなす病態であるため、臨床医学のみならず、基礎医学、生物学の分野においても重要な分野である。この炎症は症候－病理形態学的特徴から、「紅腫熱疼」として把握されてきた。すなわち炎症巣は、熱を帯びて紅く腫れ、かつ痛むという特徴を有しており、この病態像は長らく中核をなすコンセプトであり、いわば「古典的炎症像」とでも言うべき概念である。しかしこの十数年の研究でサイトカインと称される細胞間のメディエーター類、そしてそれらの受容体が発見され、一挙に炎症の研究は細胞間のネットワークと細胞内のシグナル、遺伝子発現の解明の研究へとシフトしていった。その輝かしい成果で、ブルース・ボイトラー、ジュール・ホフマン、ラルフ・スタインマン各博士らが２０１１年にノーベル賞に輝いたことからも窺い知ることができるように、まさにこの分野は現在オンゴーイングの領域であるといえる。この３氏の研究により、炎症が免疫と密接にリンクしていること、そして細胞－受容体とその下流のシグナルにより、免疫は自然免疫と獲得免疫に２大分類されること等の概念が確立された。これは古典的炎症像を塗り替える、いわば「近代的炎症像」とも言うべき炎症像である。これらの進歩は治療医学に関してもサイトカイン介入療法（抗サイトカイン、又はその受容体のブロッカー、又は抗体）へと発展していった。このうち抗体を用いる療法は「生物学的製剤」と言われ、慢性関節リウマチ等の疾患の治療においては進歩を遂げている。しかしこの生物学的製剤は全ての炎症に対して奏功するわけではなく一部の疾患に限られている。さらに、生物学的製剤が奏功する場合であっても、副作用及びコスト等の多くの問題があることが判明しつつある。 “Inflammation” is an important field not only in clinical medicine but also in basic medicine and biology because it is a pathological condition underlying human infections and trauma. This inflammation has been identified as “erythema fever” from the symptom-pathomorphological features. In other words, the inflammatory lesion is characterized by a fever, red swelling, and soreness, and this pathological image is a core concept for a long time, which is a concept that should be called a “classical inflammation image”. However, in recent decades, intercellular mediators called cytokines and their receptors have been discovered. Research on inflammation is a study of elucidation of intercellular networks, intracellular signals, and gene expression. It shifted to. As this brilliant achievement makes it possible to learn from the fact that Dr. Bruce Voithler, Jules Hoffman, Ralph Steinmann and others won the Nobel Prize in 2011, this field is exactly in the ongoing territory. It can be said that there is. These three studies established the concept that inflammation is closely linked to immunity, and that immunity is broadly classified into innate immunity and acquired immunity based on cell-receptors and downstream signals. It was done. This is an inflammatory image that can be called a “modern inflammatory image”. These advances have also evolved into therapeutic interventions (anti-cytokines, or blockers of their receptors, or antibodies) in therapeutic medicine. Of these, therapies using antibodies are referred to as “biological preparations”, and progress has been made in the treatment of diseases such as rheumatoid arthritis. However, this biologic is not effective for all inflammations and is limited to some diseases. Furthermore, it is becoming clear that there are many problems, such as side effects and costs, even when the biologic is successful.
　一方、上述のサイトカイン類とその受容体からのシグナルは肥満や糖尿病、動脈硬化、高血圧、さらには加齢等によっても微量ではあるが慢性持続的に作動していることが判明し、「自然炎症」なる概念が登場してきつつある（図１）。このような概念の進化の背景には、ＰＡＭＰｓ（Ｐａｔｈｏｇｅｎ Ａｓｓｏｃｉａｔｅｄ Ｍｏｌｅｃｕｌａｒ Ｐａｔｔｅｒｎｓ：病原微生物由来の分子、あるいはその断片)、ＤＡＭＰｓ(Ｄａｍａｇｅ Ａｓｓｏｃｉａｔｅｄ Ｍｏｌｅｃｕｌａｒ Ｐａｔｔｅｒｎｓ：障害を受けた、あるいは病態下で生成された自己の細胞や蛋白、あるいは組織由来の分子等)を認識するＰＲＲｓ（Ｐａｔｔｅｒｎ Ｒｅｃｏｇｎｉｔｉｏｎ Ｒｅｃｅｐｔｏｒｓ）が発見・同定されたこと、そしてそれからのシグナル伝達とそれによって活性化される細胞内マシナリー；ＮＦ-κＢとインフラマソーム（ｉｎｆｌａｍｍａｓｏｍｅ）が明らかになってきたことが挙げられる（図２）。 On the other hand, the signals from the above-mentioned cytokines and their receptors were found to be chronically sustained even though they were traced by obesity, diabetes, arteriosclerosis, hypertension, and aging. The concept of “is emerging” (FIG. 1). The evolution of this concept is based on PAMPs (Pathogen Associated Molecular Patterns: molecules derived from pathogenic microorganisms or fragments thereof), DAMPs (Damage Associated Molecular Patterns: self-affected or generated under pathological conditions) The discovery and identification of PRRs (PatternsｏｒRecognition Receptors) that recognize cells, proteins, or tissue-derived molecules), signal transduction from them, and intracellular machinery activated by them; NF-κB and inflamma It is mentioned that the inflamasome has been revealed (FIG. 2).
　ここに至り、炎症の研究は一挙に細胞内シグナル伝達と細胞内マシナリー、特にインフラマソームの活性化と、その下流に移ってきつつある。従って今後、炎症とその治療の研究は、一斉に細胞内にシフトすることが予想され、事実その兆しは見えつつある。 So, research on inflammation has moved to the downstream of intracellular signal transduction and intracellular machinery, especially inflammasome activation. Therefore, it is expected that research on inflammation and its treatment will shift into cells all at once in the future, and in fact, the signs are being seen.
　上記細胞内炎症発現のプロセスは図２、３－１に示すように、数千個にも上ると考えられ始めたＰＡＭＰｓ、ＤＡＭＰｓが生体内の細胞にあまねく発現している細胞表面、あるいは細胞内のＰＲＲｓによって認識されてシグナルは細胞内に伝達され、一つはＮＦ-κＢへ、あと一つはインフラマソームに収束することが判明してきた。すなわち「情報の収束」である。そして最終的にはＮＦ-κＢとインフラマソームからの情報はさらにプロカスパーゼ１（ｐｒｏｃａｓｐａｓｅ １）のカスパーゼ１への活性化という一点に集中される。すなわち細胞内で炎症発現のためのシグナルのカスケード（瀑状型）反応を構築していると言い得る。 As shown in FIGS. 2 and 3-1, the above-described process of intracellular inflammation is expressed on the cell surface where PAMPs and DAMPs, which have started to be considered to be several thousand, are expressed in cells in the living body, It has been found that a signal is transmitted into the cell by being recognized by the PRRs, and one converges to NF-κB and the other to the inflammasome. That is, “information convergence”. Finally, the information from NF-κB and inflammasome is further concentrated on one point of activation of procaspase 1 (procaspase 1) to caspase-1. In other words, it can be said that a cascade of signals for the expression of inflammation in the cell is constructed.
　カスパーゼ１の生成により、ｐｒｏ－ＩＬ－１８、ｐｒｏ－ＩＬ－１βの成熟化とＩＬ－１８、ＩＬ－１βの細胞外への放出、それによる免疫細胞（Ｔ細胞、Ｂ細胞、樹状細胞、好中球等）の「呼び寄せ（ｃｈｅｍｏｔａｘｉｓ)」が開始され、当該部位に炎症巣が形成されることになる。この点で重要な点は先ず、ＮＦ－κＢの活性化で、ｐｒｏ－ＩＬ－１βとｐｒｏ－ＩＬ－１８（いずれも前駆体）が細胞内に産生・蓄積され、次に活性化インフラマソームがプロカスパーゼ１をカスパーゼ１に活性化してはじめて、活性を持ったカスパーゼがそれぞれ、ｐｒｏ－ＩＬ－１８とｐｒｏ－ＩＬ－βを、成熟型の活性化ＩＬ－１８，ＩＬ－１βに活性化して、細胞外に放出するということである（図３－１）。 Production of caspase 1 leads to maturation of pro-IL-18, pro-IL-1β and release of IL-18, IL-1β to the outside, thereby immune cells (T cells, B cells, dendritic cells, “Chemotaxis” of neutrophils etc. is initiated, and an inflammatory lesion is formed at the site. The important point in this regard is firstly the activation of NF-κB, where pro-IL-1β and pro-IL-18 (both precursors) are produced and accumulated in the cell, and then the activated inflammasome Only when procaspase 1 is activated to caspase 1, active caspases activate pro-IL-18 and pro-IL-β into mature activated IL-18 and IL-1β, respectively. It is to be released extracellularly (FIG. 3-1).
　インフラマソームは、図３－２に示すような７量体を形成することによりカスパーゼ１を活性化すると考えられている。具体的には、ＣＡＲＤには重合する性質があり、そのためカスパーゼ１（ＣＡＲＤ・ｐ２０／ｐ１６）とＡＳＣ（ＰＹＤ・ＣＡＲＤ）とがＣＡＲＤを介して結合し、これにＮＬＲ（ＰＹＤ／ＮＡＣＨＴ）がＰＹＤを介して結合して７量体を形成する。 The inflammasome is thought to activate caspase 1 by forming a heptamer as shown in Fig. 3-2. Specifically, CARD has a property of polymerizing, so that caspase 1 (CARD · p20 / p16) and ASC (PYD · CARD) are bound via CARD, and NLR (PYD / NACHT) is bound to PYD. To form a heptamer.
　このＡＳＣは、本来、サイトゾルに存在し、トリトンＸ－１００（フェニルポリエチレングリコール型のノンイオン界面活性剤）を含む緩衝液に可溶な蛋白質であるが、レチノイン酸や抗癌剤等のアポトーシス誘発剤によって誘発されるアポトーシスに伴い、トリトンＸ－１００を含む緩衝液に不溶化し、凝集を起こす。ＡＳＣがアポトーシスに伴ってトリトンＸ－１００を含む緩衝液に対して溶性から不溶性に変化する原因は、コンフォメーションの変化によるものと考えられる。また、ＡＳＣは、自らアポトーシスを誘発する作用はないが、他の因子によって誘発されるアポトーシスを促進する作用を有する（特許文献１２参照）。 This ASC is originally a protein that exists in the cytosol and is soluble in a buffer solution containing Triton X-100 (a non-ionic surfactant of the phenyl polyethylene glycol type), but it is activated by apoptosis-inducing agents such as retinoic acid and anticancer agents. Along with the induced apoptosis, it becomes insoluble in a buffer containing Triton X-100 and causes aggregation. The cause of ASC changing from soluble to insoluble in a buffer containing Triton X-100 with apoptosis is considered to be due to a change in conformation. ASC has no effect of inducing apoptosis by itself, but has an effect of promoting apoptosis induced by other factors (see Patent Document 12).
　ＮＦ-κＢとインフラマソームは上皮系細胞、神経細胞等、免疫細胞以外の細胞や、免疫担当細胞、すなわちリンパ球細胞（ｌｙｍｐｈｏｉｄ ｃｅｌｌｓ）がレジデントしていない組織や臓器の細胞群にもあまねく発現しているが、これらの非骨髄（ｎｏｎ-ｍｙｅｌｏｉｄ）系細胞も当然、生体内外からの侵襲に会うので、生体防御的応答が要求される。この際に、ｎｏｎ-ｍｙｅｌｏｉｄ組織はＰＡＭＰｓやＤＡＭＰｓの情報をキャッチして、免疫細胞の遊走因子であるＩＬ－１８、ＩＬ－１βを産生・放出して、ｍｙｅｌｏｉｄ系細胞を集簇させることで、その部位に免疫反応を遂行しうる生体防御のためのリンパ球等から成るいわば擬似リンパ節を構築するものと考えられる。 NF-κB and inflammasome are also expressed in cells other than immune cells such as epithelial cells and neurons, and cells of tissues and organs where lymphocyte cells (lymphoid cells) are not resident However, since these non-myeloid cells naturally meet invasion from inside and outside the living body, a biodefense response is required. At this time, the non-myloid tissue catches information on PAMPs and DAMPs, produces and releases IL-18 and IL-1β, which are migratory factors of immune cells, and collects myloid cells. It is considered that a so-called pseudo-lymph node consisting of lymphocytes for biological defense capable of carrying out an immune reaction at that site is constructed.
　非特許文献１及び２には以下の事項が記載されている。自然免疫や獲得免疫に関る細胞群（マクロファージや樹状細胞等）は細胞表面にＴｏｌｌ－ｌｉｋｅレセプター（ＴＬＲｓ）を発現しており、これらの受容体にＤＡＭＰｓやＰＡＭＰｓが結合すると、これらの細胞は活性化され、種々のシグナルが伝達されるが、これらは合流し、１つはインフラマソームと称される蛋白複合体に収束する。この複合体はＮｏｄ－ｌｉｋｅ ｒｅｃｅｐｔｏｒ（ＮＬＲ）、Ａｐｏｐｔｏｓｉｓ－ａｓｓｏｃｉａｔｅｄ ｓｐｅｃｋ－ｌｉｋｅ ｐｒｏｔｅｉｎ ｃｏｎｔａｉｎｉｎｇ ａ ｃａｓｐａｓｅ ｒｅｃｒｕｉｔｍｅｎｔ ｄｏｍａｉｎ（ＡＳＣ）、プロカスパーゼ１から構成される。ＮＬＲにＴＲＬｓ同様、ＤＡＭＰｓやＰＡＭＰｓが結合することにより、インフラマソームが活性化される。 Non-Patent Documents 1 and 2 describe the following matters. Cell groups related to innate immunity and acquired immunity (macrophages, dendritic cells, etc.) express Toll-like receptors (TLRs) on their cell surfaces. When DAMPs or PAMPs bind to these receptors, these cells Are activated and transmit various signals, but they merge and one converges to a protein complex called the inflammasome. This complex is composed of Nod-like-receptor (NLR), Apoptosis-associated-spec-like-protein-containing-a-case-recovery-domain (ASC), and procaspase-1. Like TRLs, inflammasome is activated by binding of DAMPs and PAMPs to NLRs.
　炎症性サイトカインであるＩＬ－１βの分泌には少なくとも２つのステップが必要である。１つはＴＬＲｓからの刺激（シグナル１）で、これによりｐｒｏ－ＩＬ－１β（ＩＬ－１βの前駆体）が細胞質内で作られる。次に、インフラマソームの活性化（シグナル２）からのシグナルでプロカスパーゼ１がカスパーゼ１に活性化され、これによりカスパーゼ１がｐｒｏ－ＩＬ－１βを切断し、活性化ＩＬ－１βが細胞外に分泌され、感染局所への免疫担当症細胞の遊走等、さまざまな免疫応答に関与する。また、ＩＬ－１８も同様のメカニズムで分泌される（非特許文献１及び２参照）。ここで最近の知見で重要な点はカスパーゼの活性化は、ＩＬ－１βとＩＬ－１８の生成と細胞外放出を惹起するのみでなく、細胞ＤＮＡの断片化、ｐｙｒｏｐｔｏｓｉｓと呼ばれる細胞の膨化と細胞死（炎症性ｐｒｏｇｒａｍｍｅｄ ｃｅｌｌ ｄｅａｔｈ）を引き起こすということである（図４）。 Securing IL-1β, an inflammatory cytokine, requires at least two steps. One is stimulation from TLRs (signal 1), which creates pro-IL-1β (a precursor of IL-1β) in the cytoplasm. Next, procaspase 1 is activated to caspase 1 by a signal from inflammasome activation (signal 2), whereby caspase 1 cleaves pro-IL-1β, and activated IL-1β becomes extracellular. And is involved in various immune responses such as migration of immunocompetent cells to the infected area. IL-18 is also secreted by a similar mechanism (see Non-Patent Documents 1 and 2). The important point in recent knowledge is that caspase activation not only induces the production and extracellular release of IL-1β and IL-18, but also fragmentation of cellular DNA, cell swelling called pyroptosis and cell It causes death (inflammatory programmed cell) death (FIG. 4).
　クライオピリン関連周期熱症候群（ｃｒｙｏｐｙｒｉｎ－ａｓｓｏｃｉａｔｅｄ ｐｅｒｉｏｄｉｃ ｓｙｎｄｒｏｍｅ： ＣＡＰＳ）は、インフラマソームの主要成分であるＮＬＲＰ３遺伝子に変異が生じ、インフラマソームが活性化した状態が続き、ＩＬ－１βの過剰産生が起こる。同様に家族性地中海熱、Ｐｙｏｇｅｎｉｃ ａｒｔｈｒｉｔｉｓｗｉｔｈ ｐｙｏｄｅｒｍａ ｇａｎｇｒｅｎｏｓｕｍ ａｎｄ ａｃｎｅ（ＰＡＰＡ）症候群、Ｍａｊｅｅｄ症候群、高ＩｇＤ症候群、反復性胞状奇胎、ＤＩＲＡにおいてもインフラマソームが活性化した状態が続き、ＩＬ－１βの過剰産生が起こることが知られている（非特許文献１及び２参照）。これらの疾患に対してＩＬ－１βの阻害薬であるアナキンラ、リロナセプト、カナキヌマブが臨床で使用されているが、すべての患者に対して効果があるわけではない（非特許文献３参照）。その上、これらのＩＬ－１βの阻害薬アナキンラ、リロナセプト、カナキヌマブは、当然のことながらＩＬ－１８を阻害し得ない。 In cryopyrin-associated periodic fever syndrome (CAPS), the NLRP3 gene, a major component of the inflammasome, is mutated, the inflammasome is activated, and overproduction of IL-1β continues. Occur. Similarly, familial Mediterranean fever, Pyogenicic arthritis with pyderma gregrenosum and acne (PAPA) syndrome, Majeed's syndrome, high IgD syndrome, recurrent hydatidiform mole, DIRA continues to be inflammasome-activated, and IL-1β excess Production is known to occur (see Non-Patent Documents 1 and 2). Anakinra, rilonacept, and canakinumab, which are inhibitors of IL-1β, are used clinically for these diseases, but they are not effective for all patients (see Non-Patent Document 3). Moreover, these IL-1β inhibitors, anakinra, rilonacept, and canakinumab, are of course unable to inhibit IL-18.
　上記した疾患は、遺伝子性のものであるが、内因性の因子であるＤＡＭＰｓや外来性ＰＡＭＰｓによるインフラマソームの活性化を経てプロカスパーゼ１活性化が関係する病気として痛風、アルツハイマー病、２型糖尿病、そしてこれらの病態における急性増悪や急性転化が知られており（非特許文献２）、これらの病気も現状の治療法ではコントロールが効かないことが知られている。 The above-mentioned diseases are genetic, but gout, Alzheimer's disease, type 2 are diseases in which procaspase 1 activation is involved through activation of inflammasome by endogenous factors DAMPs and exogenous PAMPs. It is known that diabetes, and acute exacerbations and transformations in these pathological conditions (Non-patent Document 2), and these diseases are also known to be uncontrollable by current treatment methods.
　このように、インフラマソームからカスパーゼ１の生成が炎症の発現に決定的に重要であることが判明してきたので、現在、インフラマソーム阻害剤やカスパーゼ阻害剤は世界中でいっせいに研究開始されている。そして、確実に症状を抑制でき、かつ、安全で長期に使用し得る薬剤の開発が目指されている。一方ＡＳＣが自然免疫系の活性化、癌の抑制、自然炎症疾患の抑制等に寄与する蛋白質であることも明らかになってきている。 Thus, since it has been found that the production of caspase 1 from inflammasome is critical for the development of inflammation, research on inflammasome inhibitors and caspase inhibitors has been started all over the world. Yes. And development of the medicine which can suppress a symptom surely and is safe and can be used for a long term is aimed at. On the other hand, it has also become clear that ASC is a protein that contributes to activation of the innate immune system, suppression of cancer, suppression of natural inflammatory diseases, and the like.
特表平９－５０５９８８号公報JP-T 9-505988 特表２００１－８９３７７号公報Special Table 2001-89377 仏国特許出願公開第２６１７５０２号French Patent Application Publication No. 2617502 特開２００４－１２３６０４号公報JP 2004-123604 A ＷＯ２０１０／０８２６６１WO2010 / 082661 ＷＯ２００４／０４５６２８WO2004 / 045628 特開２０１２－１５１５号公報JP 2012-1515 A 特開２００７－３０１４６号公報JP 2007-30146 A 特開２００７－９１６４４号公報JP 2007-91644 A 特開２００６－３０６８１４号公報JP 2006-306814 A 特開２００５－１５４４２５号公報JP 2005-154425 A 特開２００１－２７５６８１号公報JP 2001-275681 A
　したがって本発明は、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）が関与する疾患又は症状についての安全でかつ優れた治療薬、及び安全でかつ優れたＡＳＣの機能阻害薬を提供することを課題とする。 Therefore, an object of the present invention is to provide a safe and excellent therapeutic agent for a disease or symptom associated with apoptosis-related spec-like card protein (ASC), and a safe and excellent ASC function inhibitor.
　本発明者らは、生体内糖代謝産物である１，５－ＡＦがＡＳＣを標的蛋白とすることを見出し、本発明に到達した。さらに本発明者らは、１，５－ＡＦがインフラマソーム経路を阻害することを見出した。これまでインフラマソーム経路の阻害系は見出されていなかった。つまり、インフラマソーム経路を阻害する化合物として初めて見出されたのが、１，５－ＡＦである。 The present inventors have found that 1,5-AF, which is an in vivo sugar metabolite, uses ASC as a target protein, and have reached the present invention. Furthermore, the inventors have found that 1,5-AF inhibits the inflammasome pathway. Until now, an inhibitory system for the inflammasome pathway has not been found. In other words, 1,5-AF was first discovered as a compound that inhibits the inflammasome pathway.
　本発明は以下の発明を包含する。 The present invention includes the following inventions.
（１）１，５－Ｄ－アンヒドロフルクトースを有効成分とする、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）の機能阻害薬。 (1) A function inhibitor of apoptosis-related spec-like card protein (ASC) comprising 1,5-D-anhydrofructose as an active ingredient.
（２）ＡＳＣの機能が、カスパーゼ１及びＮＯＤ様受容体との複合体形成能である、上記（１）に記載の阻害薬。 (2) The inhibitor according to (1) above, wherein the function of ASC is the ability to form a complex with caspase 1 and a NOD-like receptor.
（３）複合体がインフラマソームである、上記（２）に記載の阻害薬。 (3) The inhibitor according to (2) above, wherein the complex is an inflammasome.
（４）インフラマソームが関与する経路における、プロカスパーゼ１のカスパーゼ１への活性化を阻害する、上記（３）に記載の阻害薬。 (4) The inhibitor according to (3) above, which inhibits activation of procaspase 1 to caspase 1 in a pathway involving inflammasome.
（５）１，５－Ｄ－アンヒドロフルクトースを有効成分とする、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）が関与する疾患又は症状の治療薬。 (5) A therapeutic agent for a disease or symptom associated with an apoptosis-related spec-like card protein (ASC) comprising 1,5-D-anhydrofructose as an active ingredient.
（６）ＡＳＣが関与する疾患又は症状が、ＡＳＣとカスパーゼ１及びＮＯＤ様受容体との複合体形成が関与する疾患又は症状である、上記（５）に記載の治療薬。 (6) The therapeutic agent according to (5) above, wherein the disease or symptom involving ASC is a disease or symptom involving complex formation between ASC, caspase 1 and NOD-like receptor.
（７）複合体がインフラマソームである、上記（６）に記載の治療薬。 (7) The therapeutic agent according to (6) above, wherein the complex is inflammasome.
（８）ＡＳＣが関与する疾患又は症状が、カスパーゼ１が関与する疾患又は症状である、上記（５）～（７）のいずれかに記載の治療薬。 (8) The therapeutic agent according to any one of (5) to (7) above, wherein the disease or symptom associated with ASC is a disease or symptom associated with caspase-1.
（９）ＡＳＣが関与する疾患又は症状が、ＩＬ－１β及び／又はＩＬ－１８が関与する疾患又は症状である、上記（５）～（８）のいずれかに記載の治療薬。 (9) The therapeutic agent according to any of (5) to (8) above, wherein the disease or symptom associated with ASC is a disease or symptom associated with IL-1β and / or IL-18.
（１０）ＡＳＣが関与する疾患又は症状が、全身性炎症性反応症候群ＳＩＲＳである、上記（５）～（９）のいずれかに記載の治療薬。 (10) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is systemic inflammatory response syndrome SIRS.
（１１）ＡＳＣが関与する疾患又は症状が、慢性関節リウマチである、上記（５）～（９）のいずれかに記載の治療薬。 (11) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is rheumatoid arthritis.
（１２）ＡＳＣが関与する疾患又は症状が、アルツハイマー病である、上記（５）～（９）のいずれかに記載の治療薬。 (12) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is Alzheimer's disease.
（１３）ＡＳＣが関与する疾患又は症状が、クライオピリン関連周期熱症候群である、上記（５）～（９）のいずれかに記載の治療薬。 (13) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is cryopyrine-related periodic fever syndrome.
（１４）ＡＳＣが関与する疾患又は症状が、家族性地中海熱である、上記（５）～（９）のいずれかに記載の治療薬。 (14) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is familial Mediterranean fever.
（１５）ＡＳＣが関与する疾患又は症状が、ＰＡＰＡ症候群である、上記（５）～（９）のいずれかに記載の治療薬。 (15) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is PAPA syndrome.
（１６）ＡＳＣが関与する疾患又は症状が、Ｍａｊｅｅｄ症候群である、上記（５）～（９）のいずれかに記載の治療薬。 (16) The therapeutic agent according to any of (5) to (9) above, wherein the disease or symptom associated with ASC is Majeed's syndrome.
（１７）ＡＳＣが関与する疾患又は症状が、高ＩｇＤ症候群である、上記（５）～（９）のいずれかに記載の治療薬。 (17) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is high IgD syndrome.
（１８）ＡＳＣが関与する疾患又は症状が、反復性胞状奇胎である、上記（５）～（９）のいずれかに記載の治療薬。 (18) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is recurrent hydatidiform mole.
（１９）ＡＳＣが関与する疾患又は症状が、ＤＩＲＡである、上記（５）～（９）のいずれかに記載の治療薬。 (19) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is DIRA.
（２０）ＡＳＣが関与する疾患又は症状が、炭疽菌感染症である、上記（５）～（９）のいずれかに記載の治療薬。 (20) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is anthrax infection.
（２１）ＡＳＣが関与する疾患又は症状が、急性呼吸促拍症候群である、上記（５）～（９）のいずれかに記載の治療薬。 (21) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is acute respiratory stimulation syndrome.
（２２）ＡＳＣが関与する疾患又は症状が、炎症性細胞死である、上記（５）～（９）のいずれかに記載の治療薬。 (22) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is inflammatory cell death.
（２３）ＡＳＣが関与する疾患又は症状が、石綿肺である、上記（５）～（９）のいずれかに記載の治療薬。 (23) The therapeutic agent according to any one of (5) to (9) above, wherein the disease or symptom associated with ASC is asbestosis.
　本発明者らは、１，５-ＡＦの標的蛋白質がＡＳＣであることを見出した。さらに本発明者らは、特に生体細胞には細胞内炎症カスケードを制御するための装置があり、それが１，５-ＡＦ経路であることを見出した。１，５-ＡＦは生体内分子であり、副作用も無いことから、炎症制御の大きな武器になるもと期待される。また、ＩＬ－１βの阻害薬アナキンラ、リロナセプト、カナキヌマブはいずれも蛋白質であり、製造コストが高いが、１，５－ＡＦはこれらの蛋白質製剤に比べると格段に安いコストで製造できるため、本発明の薬剤はこれらと比較して経済的にも有利である。 The present inventors have found that the target protein of 1,5-AF is ASC. Furthermore, the present inventors have found that there is a device for controlling the intracellular inflammatory cascade, particularly in living cells, which is the 1,5-AF pathway. Since 1,5-AF is an in vivo molecule and has no side effects, it is expected to become a great weapon for controlling inflammation. In addition, IL-1β inhibitors anakinra, rilonacept, and canakinumab are all proteins and are expensive to produce, but 1,5-AF can be produced at a much lower cost than these protein preparations. These drugs are economically advantageous as compared with these drugs.
　ＡＳＣが関与する疾患又は症状、特にインフラマソーム経路が関与する疾患又は症状、具体的には活性化プロカスパーゼ１が関与した自己炎症性疾患が起こっている哺乳動物個体に本発明の薬剤を適当量しかるべき方法で投与することにより、自然免疫の中心的役割を果たすＩＬ－１β及び／又はＩＬ－１８生成を有意に抑え、自然免疫の過剰により発症する疾患群の発生を抑制することが可能となる。 The agent of the present invention is suitable for a mammal individual having a disease or symptom involving ASC, particularly a disease or symptom involving an inflammasome pathway, specifically, an autoinflammatory disease involving activated procaspase 1 By administering in an appropriate amount, it is possible to significantly suppress IL-1β and / or IL-18 production, which plays a central role in innate immunity, and to suppress the occurrence of diseases caused by excessive innate immunity It becomes.
　本発明の薬剤によれば、ＡＳＣが関与する疾患又は症状を緩和、抑制することが可能となる。また本発明の薬剤によれば、インフラマソーム経路が関与する疾患又は症状、特には活性型カスパーゼ１が関与する疾患又は症状を予防及び／又は治療し、活性化した自然免疫を有意に緩和、抑制することが可能となる。 According to the drug of the present invention, it becomes possible to alleviate or suppress diseases or symptoms associated with ASC. Further, according to the agent of the present invention, a disease or symptom involving the inflammasome pathway, particularly a disease or symptom involving active caspase 1 is prevented and / or treated, and the activated innate immunity is significantly alleviated, It becomes possible to suppress.
図１は、自然炎症と疾患との関係を説明する図である。FIG. 1 is a diagram illustrating the relationship between natural inflammation and disease. 図２は、ＰＡＭＰｓ及びＤＡＭＰｓによるＮＦ－κＢ及びインフラマソームの活性化を説明する図である。FIG. 2 is a diagram for explaining the activation of NF-κB and inflammasome by PAMPs and DAMPs. 図３－１は、インフラマソーム経路活性化を経てプロカスパーゼ１が活性化される経路を説明する図である。FIG. 3-1 is a diagram illustrating a pathway in which procaspase 1 is activated through inflammasome pathway activation. 図３－２は、インフラマソーム多重蛋白質複合体の形態を説明する図である。FIG. 3-2 is a diagram for explaining the morphology of the inflammasome multiprotein complex. 図４は、プロカスパーゼ１が、ＩＬ－１β、ＩＬ－１８の産生・放出のみならず、ＤＮＡの断片化、ｐｙｒｏｐｔｏｓｉｓと呼ばれるプログラム化された細胞死をも引き起こすことを示した図である。FIG. 4 shows that procaspase 1 causes not only the production and release of IL-1β and IL-18, but also DNA fragmentation and programmed cell death called pyroptosis. 図５はマウス由来骨髄マクロファージにおけるＩＬ－１β生成に対する１，５－ＡＦの抑制効果の試験結果を示す図である。グラフ右は添加した物質で、括弧内は活性化されるＮＬＲを示す。FIG. 5 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-1β production in mouse-derived bone marrow macrophages. The right side of the graph shows the added substance, and the parenthesis shows the activated NLR. 図６は、マウス由来骨髄マクロファージにおけるＩＬ－１β生成に対する１，５－ＡＦの抑制効果の試験結果を示す図である。グラフ右は添加した物質で、括弧内は活性化されるＮＬＲを示す。FIG. 6 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-1β production in mouse-derived bone marrow macrophages. The right side of the graph shows the added substance, and the parenthesis shows the activated NLR. 図７は、マウス由来骨髄マクロファージにおけるＩＬ－１８生成に対する１，５－ＡＦの抑制効果の試験結果を示す図である。グラフ右は添加した物質で、括弧内は活性化されるＮＬＲを示す。FIG. 7 is a graph showing the test results of the inhibitory effect of 1,5-AF on IL-18 production in mouse-derived bone marrow macrophages. The right side of the graph shows the added substance, and the parenthesis shows the activated NLR. 図８は、マウス由来骨髄マクロファージにおけるＩＬ－１８生成に対する１，５－ＡＦの抑制効果の試験結果を示す図である。グラフ右は添加した物質で、括弧内は活性化されるＮＬＲを示す。FIG. 8 shows the test results of the inhibitory effect of 1,5-AF on IL-18 production in mouse-derived bone marrow macrophages. The right side of the graph shows the added substance, and the parenthesis shows the activated NLR. 図９は、アラム又は尿酸塩によるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 9 is a graph showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by alum or urate. 図１０は、ニゲリシン及びＡＴＰによるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 10 is a graph showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by nigericin and ATP. 図１１は、ＡＴＰによるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 11 is a diagram showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by ATP. 図１２は、炭疽菌毒素によるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 12 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by anthrax toxin. 図１３は、フラジェリンによるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 13 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by flagellin. 図１４は、ｐｏｌｙ（ｄＡ：ｄＴ）によるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 14 is a diagram showing test results of the inhibitory effect of 1,5-AF on inflammasome activation by poly (dA: dT). 図１５は、１，５－ＡＦがプロカスパーゼ１のカスパーゼ１への活性化を抑制することを示す図である。FIG. 15 is a diagram showing that 1,5-AF suppresses the activation of procaspase 1 to caspase 1. 図１６は、ロテノンによるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 16 is a diagram showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation by rotenone. 図１７は、マウスＡＬＩモデルの肺組織像及び細胞数を示す図である。FIG. 17 is a diagram showing lung tissue images and cell numbers of the mouse ALI model. 図１８は、ＳＩＲＳモデルに１，５－ＡＦを投与した後の時間と生存率との関係を示す図である。FIG. 18 is a diagram showing the relationship between the time after administration of 1,5-AF to the SIRS model and the survival rate. 図１９は、１，５－ＡＦの標的蛋白質がＡＳＣであることを示す図である。FIG. 19 is a diagram showing that the target protein of 1,5-AF is ASC. 図２０ａ及びｂは、石綿肺のモデル実験におけるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。20a and 20b are diagrams showing the test results of the inhibitory effect of 1,5-AF on inflammasome activation in an asbestosis model experiment. 図２１は、炭疽菌毒素によるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 21 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by anthrax toxin. 図２２は、ヒト末梢血を用いて行った、ＡＴＰによるインフラマソーム活性化に対する１，５－ＡＦの抑制効果の試験結果を示す図である。FIG. 22 shows the test results of the inhibitory effect of 1,5-AF on inflammasome activation by ATP, performed using human peripheral blood.
　本発明の薬剤に使用される１，５－ＡＦは、既に公知の方法、例えば、上記特許文献１に記載の方法によって調製可能である。 1,5-AF used for the drug of the present invention can be prepared by a known method, for example, the method described in Patent Document 1 above.
　本発明の薬剤は、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）の機能、特には、カスパーゼ１及びＮＯＤ様受容体との複合体形成能を阻害する。ここでＡＳＣが「複合体形成能」を有するとは、ＡＳＣがアダプター蛋白質として機能することを示す。特には複合体がインフラマソームである場合、本発明の薬剤は、インフラマソーム経路を阻害し、特にはプロカスパーゼ１のカスパーゼ１への活性化を抑制することができる。ここで、インフラマソーム経路とはインフラマソームを介して行われる一連のシグナル伝達経路を示す（図３－１）。インフラマソームは、ＡＳＣがカスパーゼ１及びＮＯＤ様受容体と７量体の複合体を形成することによりカスパーゼ１を活性化すると考えられている（図３－２）。 The agent of the present invention inhibits the function of apoptosis-related spec-like card protein (ASC), particularly the ability to form a complex with caspase 1 and NOD-like receptor. Here, ASC having “complex forming ability” indicates that ASC functions as an adapter protein. In particular, when the complex is an inflammasome, the agent of the present invention can inhibit the inflammasome pathway, and in particular, can suppress the activation of procaspase 1 to caspase-1. Here, the inflammasome pathway refers to a series of signal transduction pathways performed via the inflammasome (FIG. 3-1). The inflammasome is thought to activate caspase 1 by ASC forming a complex of caspase 1 and NOD-like receptors with the heptamer (FIG. 3-2).
　また、本発明の薬剤は、ＡＳＣが関与する疾患又は症状、特には、ＡＳＣとカスパーゼ１及びＮＯＤ様受容体との複合体形成が関与する疾患又は症状の治療薬である。特には複合体がインフラマソームである場合、本発明の薬剤は、インフラマソームが活性化した自己炎症性疾患の予防若しくは治療を目的として使用することができる。この場合インフラマソーム活性化、特にはカスパーセ１活性化に伴う自己炎症性疾患の自然免疫を抑えることによりその症状を抑えるものであり、よって、これらの疾患の予防若しくは治療を目的として使用することが可能である。 In addition, the drug of the present invention is a therapeutic drug for a disease or symptom related to ASC, particularly a disease or symptom related to complex formation of ASC, caspase 1 and NOD-like receptor. In particular, when the complex is inflammasome, the agent of the present invention can be used for the purpose of preventing or treating autoinflammatory diseases in which inflammasome is activated. In this case, the symptoms are suppressed by suppressing innate immunity of autoinflammatory diseases associated with inflammasome activation, in particular, caspase 1 activation, and therefore used for the purpose of preventing or treating these diseases. Is possible.
　ＡＳＣが関与する疾患又は症状としては、例えば癌、免疫抑制性疾患、自己免疫疾患、ウイルス感染症、神経変性疾患、内分泌疾患、炎症性疾患、臓器移植障害及び放射線障害等が挙げられる。 Examples of diseases or symptoms associated with ASC include cancer, immunosuppressive diseases, autoimmune diseases, viral infections, neurodegenerative diseases, endocrine diseases, inflammatory diseases, organ transplantation disorders and radiation disorders.
　さらに、上記疾患又は症状としては、インフラマソーム経路が関与する疾患又は症状、特には、インフラマソームを介して行われる一連のシグナル伝達により引き起こされる疾患又は症状が挙げられる。インフラマソーム経路が関与する疾患又は症状としては、活性化プロカスパーゼ１が関与した疾患又は症状が挙げられる。また、インフラマソーム経路が関与する疾患又は症状としては、自己炎症性疾患、特にはＩＬ－１β及び／又はＩＬ－１８の生成により引き起こされる疾患が挙げられる。具体的には、インフラマソーム経路が関与する疾患としては、例えば、クライオピリン関連周期熱症候群、家族性地中海熱、ＰＡＰＡ症候群（Ｐｙｏｇｅｎｉｃ ａｒｔｈｒｉｔｉｓｗｉｔｈ ｐｙｏｄｅｒｍａ ｇａｎｇｒｅｎｏｓｕｍ ａｎｄ ａｃｎｅ）症候群、Ｍａｊｅｅｄ症候群、高ＩｇＤ症候群、反復性胞状奇胎、ＤＩＲＡ（ｄｅｆｉｃｉｅｎｃｙ ｏｆ ｔｈｅ ＩＬ－１ ｒｅｃｅｐｔｏｒ ａｎｔａｇｏｎｉｓｔ）、痛風、アルツハイマー病、２型糖尿病、炎症性腸疾患、慢性関節リウマチ、炭疽菌感染症、急性肺障害（Ａｃｕｔｅ Ｌｕｎｇ Ｉｎｊｕｒｙ）、急性呼吸促拍症候群（Ａｃｕｔｅ Ｒｅｐｉｒａｔｏｒｙ Ｄｉｓｔｒｅｓｓ ｓｙｎｄｒｏｍｅ）及び全身性炎症性反応症候群ＳＩＲＳ（Ｓｙｓｔｅｍｉｃ Ｉｎｆｌａｍｍａｔｏｒｙ Ｒｅｓｐｏｎｓｅ ｓｙｎｄｒｏｍｅ）、石綿肺及び若年性特発性関節炎等が挙げられる。 Furthermore, examples of the disease or symptom include a disease or symptom involving the inflammasome pathway, particularly a disease or symptom caused by a series of signal transduction performed through the inflammasome. The disease or symptom involving the inflammasome pathway includes a disease or symptom involving activated procaspase-1. In addition, diseases or conditions involving the inflammasome pathway include autoinflammatory diseases, particularly diseases caused by the production of IL-1β and / or IL-18. Specifically, diseases involving the inflammasome pathway include, for example, cryopyrin-related periodic fever syndrome, familial Mediterranean fever, PAPA syndrome (Pyogenic artritis with pyderma grennosum and acne) syndrome, Majeed syndrome, high IgD syndrome, repetitive Hydatidiform mole, DIRA (defectiency of the IL-1 receptor antagonist), gout, Alzheimer's disease, type 2 diabetes, inflammatory bowel disease, rheumatoid arthritis, anthrax infection, acute lung injury (Acute Lung Injury), acute Respiratory Stimulation Syndrome (Acute Repitory Distress syndrome) and Systemic Inflammatory Response Syndrome SIRS (Systemic Infl mmatory Response syndrome), asbestosis and juvenile idiopathic arthritis, and the like.
　全身性炎症性反応症候群ＳＩＲＳには、外傷、熱傷、膵炎、侵襲の強い術後等感染を伴わない様々な原因によるものと、細菌、真菌、寄生虫、ウイルス等による感染を伴うものがあり、特に感染に起因するＳＩＲＳとして敗血症（sepsis）を挙げることができる。敗血症は、炎症性サイトカインによる全身の反応性炎症ＳＩＲＳの病態に加えて、血管内凝固障害（出血や血栓等）、尿量減少、血圧低下等の全身の臓器障害が顕著に出現した病態である。つまり、いずれもインフラマソームの活性化に起因する病態であるが、ＳＩＲＳは主として全身の炎症性病態、インフラマソームの活性化に主眼を置いた概念であり、一方敗血症はインフラマソームの活性化に引き続き、血液凝固系の障害や循環障害等によって多臓器障害等を合併している病態であるといえる。 Systemic inflammatory response syndrome SIRS includes trauma, burns, pancreatitis, severely invasive postoperative infections and other causes, and bacteria, fungi, parasites, viruses and other infections. In particular, as a SIRS resulting from infection, sepsis can be mentioned. Sepsis is a condition in which systemic organ disorders such as intravascular coagulation disorders (bleeding, thrombosis, etc.), decreased urine output, decreased blood pressure, etc. have appeared remarkably in addition to the pathological conditions of systemic reactive inflammation SIRS caused by inflammatory cytokines. . In other words, all are pathologies caused by inflammasome activation, but SIRS is a concept mainly focused on inflammatory pathologies and inflammasome activation throughout the body, while sepsis is the activity of inflammasome activity. It can be said that this is a pathological condition that is complicated by multiple organ disorders due to disorders of the blood coagulation system and circulatory disorders.
　さらには、インフラマソーム経路が関与する症状としては、炎症性細胞死（ｐｙｒｏｐｔｏｓｉｓ）及びＤＮＡの断片化に伴う症状が挙げられる。 Furthermore, symptoms associated with the inflammasome pathway include inflammatory cell death (pyroptosis) and symptoms associated with DNA fragmentation.
　本発明の薬剤は、その剤形に応じてそれ自体公知の種々の方法で投与することが可能であり、その投与量、投与部位、投与する間隔、期間等は、患者の年齢や体重、病状あるいは他の薬剤や治療法と併用した場合等を考慮して適宜決定することができる。投与方法としては、速やかに体内、あるいは病巣局所に１，５－ＡＦを送達することができる限り特に制限されないが、例えば、経口投与、注射や点滴、経気管支等の方法、あるいは貼付、塗布を挙げることができる。 The drug of the present invention can be administered by various methods known per se, depending on the dosage form, and the dose, administration site, administration interval, period, etc. are determined depending on the patient's age, weight, medical condition, etc. Or it can determine suitably considering the case where it uses together with another chemical | medical agent and treatment method. The administration method is not particularly limited as long as 1,5-AF can be quickly delivered to the body or local lesion, but for example, oral administration, injection or infusion, transbronchial method, or application or application Can be mentioned.
　本発明の一実施形態において、本発明の薬剤の投与量は、その剤形、投与方法、又は予防若しくは治療しようとする症状により異なるが、例えば、体重１ｋｇあたりの投与量として有効成分（１，５－ＡＦ）換算で１ｍｇ～５００ｍｇ、好ましくは１０ｍｇ～１００ｍｇとすることができ、１日１回又は数回、あるいは持続点滴等、さらには数日毎に１回というような、適当な投与頻度によって投与することが可能である。また肺障害が強い場合には経気道投与等の方法も考えられる。 In one embodiment of the present invention, the dose of the drug of the present invention varies depending on the dosage form, administration method, or symptoms to be prevented or treated. For example, the active ingredient (1, 1, 5-AF) in terms of 1 mg to 500 mg, preferably 10 mg to 100 mg, depending on an appropriate administration frequency such as once or several times a day, continuous infusion, etc., or once every several days. It is possible to administer. In addition, when the lung injury is strong, a method such as transrespiratory administration may be considered.
　本発明の一実施形態において、本発明の薬剤の投与量は、その剤形、投与方法、又は予防若しくは治療しようとする症状により異なるが、例えば、体重１ｋｇあたりの投与量として有効成分（１，５－ＡＦ）換算で０．００１μｇ～１００００ｍｇ、好ましくは０．０１ｍｇ～５，０００ｍｇとすることができ、１日１回又は数回、あるいは数日毎に１回というような、適当な投与頻度によって投与することが可能である。 In one embodiment of the present invention, the dose of the drug of the present invention varies depending on the dosage form, administration method, or symptoms to be prevented or treated. For example, the active ingredient (1, 1, 5-AF) in terms of 0.001 μg to 10000 mg, preferably 0.01 mg to 5,000 mg, depending on the appropriate administration frequency, such as once or several times a day or once every several days. It is possible to administer.
　本発明は、本発明の薬剤を含む製剤にも関する。本発明の製剤の形態としては、例えば、点滴、錠剤、カプセル剤、散剤、顆粒剤、坐剤、注射剤、経皮吸収剤、クリーム、ペースト、ゲル、スプレー等が挙げられるが、特に制限されない。また本発明の薬剤を含む製剤は、さらに必要な成分、例えば、製剤担体や賦形剤、安定剤等を含有することもできる。 The present invention also relates to a preparation containing the drug of the present invention. Examples of the form of the preparation of the present invention include, but are not particularly limited to, infusion, tablet, capsule, powder, granule, suppository, injection, transdermal absorption agent, cream, paste, gel, spray and the like. . The preparation containing the drug of the present invention may further contain necessary components such as a preparation carrier, excipient, stabilizer and the like.
　さらに、本発明の効果を奏する限り、本発明の製剤は、他の薬剤あるいはその他の薬理成分あるいはブドウ糖等の栄養成分を含むことも可能である。上述したように、例えば１，５－ＡＦは抗炎症作用を有し、さらに炎症性細胞死をも強く抑制する。よって、特に敗血症の治療においては、１，５－ＡＦを、凝固を抑制する抗凝固剤（例えば、遺伝子組換えトロンボモジュリン、商品名リコモジュリン等）と併用することにより、複合した病態を軽減することができる。 Furthermore, as long as the effects of the present invention are exhibited, the preparation of the present invention can also contain other drugs, other pharmacological components, or nutritional components such as glucose. As described above, for example, 1,5-AF has an anti-inflammatory action and also strongly suppresses inflammatory cell death. Therefore, particularly in the treatment of sepsis, combined use of 1,5-AF with an anticoagulant that suppresses coagulation (eg, recombinant thrombomodulin, trade name lycomodulin, etc.) can reduce the combined pathological condition. it can.
　また本発明の薬剤は、医薬品用途に限られるものではなく、医薬部外品、化粧品、食品、飲料、飼料等に配合することも可能である。例えば、１，５－ＡＦを食品に添加して、各種疾患における症状の予防あるいは治療を目的とした機能性食品のような形態をとることもできる。 Moreover, the chemical | medical agent of this invention is not restricted to a pharmaceutical use, It is also possible to mix | blend with a quasi-drug, cosmetics, a foodstuff, a drink, feed, etc. For example, 1,5-AF can be added to foods to take the form of functional foods intended to prevent or treat symptoms in various diseases.
　本発明の薬剤を含む製剤は、皮膚症状の治療を目的とする医薬部外品あるいは化粧品等の形態をとることも可能である。 The preparation containing the drug of the present invention can be in the form of a quasi-drug or cosmetic for the purpose of treating skin symptoms.
　本発明の薬剤は、人間以外の哺乳動物にも投与することができる。その場合、哺乳動物に対し、１，５－ＡＦを適量投与することによって治療を行うことができる。 The drug of the present invention can be administered to mammals other than humans. In that case, treatment can be performed by administering an appropriate amount of 1,5-AF to the mammal.
　以下、実施例により本発明を更に詳細に説明するが、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[１]１，５－ＡＦは、ＮＬＲＰ１、ＮＬＲＰ３、ＮＬＲＣ４及びＡＩＭ２が関連する全てのインフラマソーム経路を介したプロカスパーゼ１の活性化を抑制した
［実施例１］
　インフラマソームの構成成分であるＮＬＲは、細胞内の病原体等を認識するパターン認識受容体であり、これにＤＡＭＰｓやＰＡＭＰｓが結合することによりインフラマソームが活性化される。ＮＬＲは多くのものが知られているが、本実施例においてはＮＬＲの代表としてＮＬＲＰ１、ＮＬＲＰ３、ＮＬＲＣ４、ＡＩＭ２を活性化する薬剤を用いて実験を行った。各ＮＬＲを活性化するものとして以下の薬剤を用いた：
　ＮＬＲＰ１　Ａｎｔｈｒａｘ ＰＡとＬＦ（ＡＮＴ）（Ｌｉｓｔ Ｂｉｏｌｏｇｉｓｔ社）
　ＮＬＲＰ３　ＡＴＰ、ｎｉｇｅｒｉｃｉｎ（ＮＩＧ）、尿酸塩（ＭＳＵ）、Ｉｍｊｅｃｔ Ａｌｕｍ Ａｊｕｖａｎｔ（ＡＬＵ）
　ＮＬＲＣ４　フラジェリンＦＬＡ
　ＡＩＭ２　ｐｏｌｙ（ｄＡ：ｄＴ）（ＰＯＬ）（Ｓｉｇｍａ社）、リポフェクタミン２０００（ＬＩＰ）（Ｉｎｖｉｔｒｏｇｅｎ社）。 [1] 1,5-AF inhibited procaspase 1 activation via all inflammasome pathways involving NLRP1, NLRP3, NLRC4 and AIM2 [Example 1]
NLR, which is a component of inflammasome, is a pattern recognition receptor that recognizes intracellular pathogens and the like, and the inflammasome is activated by binding DAMPs and PAMPs thereto. Although many NLRs are known, in this example, experiments were conducted using agents that activate NLRP1, NLRP3, NLRC4, and AIM2 as representatives of NLRs. The following agents were used to activate each NLR:
NLRP1 Anthrax PA and LF (ANT) (List Biologist)
NLRP3 ATP, nigericin (NIG), urate (MSU), Image Alum Ajuvant (ALU)
NLRC4 Flagellin FLA
AIM2 poly (dA: dT) (POL) (Sigma), Lipofectamine 2000 (LIP) (Invitrogen).
　本実施例において、Ｎａｋａｈｉｒａ，Ｋらの方法（Ｎａｔ．Ｉｍｍｕｎｏｌ．１２ （２０１１）２２２－３０）に従ってマウス由来骨髄マクロファージを作製した。Ｃ５７／ＢＬ６の雄性マウスの大腿骨及び脛骨より、骨髄幹細胞を抽出し、Ｌ９２９細胞より抽出した培養上清液（Ｍ－ＣＳＦを含む）を２５％含むＤＭＥＭ液体培地（１０％ＦＢＳ及び２％ペニシリンストレプトマイシン含有）中で６日間培養し、マクロファージへと分化させた。６日間経過後に、マウス由来骨髄マクロファージを回収し、細胞数を調節し、各デッシュにまきなおし、オーバーナイトで接着させた後に、ＬＰＳ、及びインフラマソームを誘導する薬剤を添加した。 In this example, mouse-derived bone marrow macrophages were prepared according to the method of Nakahira, K et al. (Nat. Immunol. 12 (2011) 222-30). Bone marrow stem cells were extracted from the femur and tibia of C57 / BL6 male mice, and DMEM liquid medium (10% FBS and 2% penicillin) containing 25% of the culture supernatant (including M-CSF) extracted from L929 cells. The cells were cultured in streptomycin) for 6 days and differentiated into macrophages. After 6 days, mouse-derived bone marrow macrophages were collected, the number of cells was adjusted, each dish was re-wound and allowed to adhere overnight, and then LPS and an inflammasome-inducing agent were added.
　最初に、ＴＬＲｓからの刺激（シグナル１）でｐｒｏ－ＩＬ－１β（ＩＬ－１βの前駆体）及びｐｒｏ－ＩＬ－１８（ＩＬ－１８の前駆体）を細胞質内で作らせる目的で、ＬＰＳの添加を行った。ＬＰＳは、Ｉｎｖｉｔｒｏｇｅｎ社のＵｌｔｒａｐｕｒｅ ＬＰＳを使用し、１００ｎｇ／ｍｌ濃度で４時間刺激を行った。 First, in order to make pro-IL-1β (a precursor of IL-1β) and pro-IL-18 (a precursor of IL-18) in the cytoplasm upon stimulation from TLRs (signal 1), The addition was made. LPS was Invitrogen's Ultrapure® LPS, and stimulation was performed at a concentration of 100 ng / ml for 4 hours.
　その後、１，５－ＡＦを３０分間インキュベートし、さらにインフラマソームを誘導する薬剤を添加した。 Thereafter, 1,5-AF was incubated for 30 minutes, and a drug that induces inflammasome was further added.
　ＮＬＲＰ１を活性化する場合は、Ａｎｔｈｒａｘ ＰＡとＬＦ（ＡＮＴ）を５ μｇ／ｍｌずつ入れ６時間インキュベートして誘導した。ＮＬＲＰ３の場合は、ＡＴＰ ５ｍＭで１時間、ニゲリシン（Ｎｉｇｅｒｉｃｉｎ：ＮＩＧ） ５μＭで１時間、Ｉｍｊｅｃｔ Ａｌｕｍ Ａｊｕｖａｎｔ（ＡＬＵ） ２００μｇ／ｍｌで６時間、尿酸塩（ＭＳＵ） １５０μｇ／ｍｌで６時間インキュベートして誘導した。ＮＬＲＣ４の場合は、フラジェリン（ＦＬＡ）２．５μｇ／ｍｌで６時間インキュベートして誘導した。ＡＩＭ２の場合は、リポフェクタミン２０００（ＬＩＰ）を使用し、ｐｏｌｙ（ｄＡ：ｄＴ）（ＰＯＬ）をトランスフェクションさせることで誘導した。ＩＬ－１βやＩＬ－１８の定量はＲ＆Ｄ社のＥＬＩＳＡキットを用いて行った。 When activating NLRP1, Anthrax® PA and LF (ANT) were added at 5 μg / ml and incubated for 6 hours for induction. In the case of NLRP3, induction is carried out by incubating with ATPＡ5 mM for 1 hour, Nigericin (NIG) 5 μM for 1 hour, Image Alum Ajuvant (ALU) 200 μg / ml for 6 hours, and Urate (MSU) 150 μg / ml for 6 hours did. In the case of NLRC4, it was induced by incubation with flagellin (FLA) 2.5 μg / ml for 6 hours. In the case of AIM2, it was induced by transfecting poly (dA: dT) (POL) using Lipofectamine 2000 (LIP). IL-1β and IL-18 were quantified using an ELISA kit manufactured by R & D.
　実施例１の結果を図５－８に示す。図５－８より、１，５－ＡＦは、ＮＬＲＰ１、ＮＬＲＰ３、ＮＬＲＣ４及びＡＩＭ２が関連する全てのインフラマソーム経路を介したプロカスパーゼ１の活性化によるＩＬ－１β、ＩＬ－１８の生成を抑制したことがわかる。図５－８に示す結果について以下に説明する。 The results of Example 1 are shown in Fig. 5-8. From Fig. 5-8, 1,5-AF suppresses the production of IL-1β and IL-18 by activation of procaspase 1 through all inflammasome pathways involving NLRP1, NLRP3, NLRC4 and AIM2. You can see that The results shown in FIG. 5-8 will be described below.
　ＬＰＳで刺激した骨髄マクロファージのメディウム中にＡＴＰ、ニゲリシン、尿酸塩又はアラムを添加することによりＮＬＲＰ３インフラマソーム経路を活性化した。ＬＰＳでインキュベートした骨髄マクロファージに対して、サルモネラ菌の菌体成分を抽出したフラジェリン（ＦＬＡ）を添加することによりＮＬＲＣ４インフラマソーム経路を活性化した。ＬＰＳでインキュベートした骨髄マクロファージに対して、ＤＮＡを抽出したｐｏｌｙ（ｄＡ：ｄＴ）（ＰＯＬ）を、リポフェクタミン２０００を使用してトランスフェクションすることによりＡＩＭ－２インフラマソーム経路を活性化した。 The NLRP3 inflammasome pathway was activated by adding ATP, nigericin, urate or alum to the medium of bone marrow macrophages stimulated with LPS. The NLRC4 inflammasome pathway was activated by adding flagellin (FLA) obtained by extracting Salmonella cell components to bone marrow macrophages incubated with LPS. The AIM-2 inflammasome pathway was activated by transfecting poly (dA: dT) (POL) from which DNA was extracted to bone marrow macrophages incubated with LPS using Lipofectamine 2000.
　アラム（ＡＬＵ）又は尿酸塩(ＭＳＵ)は、ＮＬＲＰ３経路を活性化する代表的な分子である。ＬＰＳで刺激した骨髄マクロファージにアラム又は尿酸塩を加えるとＮＬＲＰ３インフラマソームが活性化された（Ｎａｔｕｒｅ Ｉｍｍｕｎｏｌｏｇｙ, ２０１３ Ｍａｙ；１４（５）：４５４－６０）。これに対し、１，５－ＡＦが存在する場合には、アラム又は尿酸塩によるＮＬＲＰ３インフラマソームの活性化は抑制された（図９も参照）。 Alum (ALU) or urate (MSU) is a representative molecule that activates the NLRP3 pathway. Addition of alum or urate to bone marrow macrophages stimulated with LPS activated NLRP3 inflammasome (Nature Immunology, 2013 May; 14 (5): 454-60). In contrast, in the presence of 1,5-AF, activation of NLRP3 inflammasome by alum or urate was suppressed (see also FIG. 9).
　細菌毒素の一種であるニゲリシン（ＮＩＧ）はＮＬＲＰｓを活性化し、カスパーゼ１を介してＩＬ－１β及びＩＬ－１８を再生、放出させて、炎症を引き起こす。ＬＰＳで刺激した骨髄マクロファージにニゲリシンを加えるとインフラマソームが活性化された。これに対し、１，５－ＡＦが存在する場合には、ニゲリシンによるインフラマソーム活性化、すなわちＩＬ－１β及びＩＬ－１８の産生、放出は強く抑制された（図１０も参照）。 Nigericin (NIG), a kind of bacterial toxin, activates NLRPs and regenerates and releases IL-1β and IL-18 via caspase 1 to cause inflammation. When nigericin was added to bone marrow macrophages stimulated with LPS, the inflammasome was activated. In contrast, in the presence of 1,5-AF, inflammasome activation by nigericin, that is, production and release of IL-1β and IL-18 were strongly suppressed (see also FIG. 10).
　ＡＴＰはＮＬＲＰ３経路でインフラマソームを活性化する。ＬＰＳで刺激した骨髄マクロファージにＡＴＰを加えるとインフラマソームが活性化された。これに対し、１，５－ＡＦが存在する場合には、ＡＴＰによるインフラマソーム活性化、すなわちＩＬ－１β及びＩＬ－１８の産生、放出は抑制された（図１１も参照）。また、アンヒドログルコース（ＡＧ）及びフルクトースと比較して、１，５－ＡＦはＡＴＰによるインフラマソーム活性化、すなわちＩＬ－１β及びＩＬ－１８の産生、放出を濃度依存性に抑制した（図１１参照）。 ATP activates the inflammasome through the NLRP3 pathway. Inflammasome was activated when ATP was added to bone marrow macrophages stimulated with LPS. In contrast, in the presence of 1,5-AF, inflammasome activation by ATP, ie, production and release of IL-1β and IL-18 was suppressed (see also FIG. 11). In addition, compared with anhydroglucose (AG) and fructose, 1,5-AF inhibited inflammasome activation by ATP, that is, production and release of IL-1β and IL-18 in a concentration-dependent manner (FIG. 11).
　炭疽菌毒素（ａｎｔｈｏｒａｘ ｔｏｘｉｎ：ＡＮＴ）はＮＬＲＰ１経路でインフラマソームを活性化する。炭疽菌毒素は強い肺障害を起こし、バイオテロに使用される危険性からその対策が急がれている。ＬＰＳで刺激した骨髄マクロファージに炭疽菌毒素を加えるとＮＬＲＰ１インフラマソームが活性化された。これに対し、１，５－ＡＦが存在する場合には、炭疽菌毒素よるインフラマソーム活性化、すなわちＩＬ－１β及びＩＬ－１８の産生、放出は強く抑制された（図１２も参照）。 Anthrax toxin (ANT) activates the inflammasome through the NLRP1 pathway. Anthrax toxin causes strong lung injury, and countermeasures are urgently needed due to the danger of being used for bioterrorism. Addition of anthrax toxin to bone marrow macrophages stimulated with LPS activated NLRP1 inflammasome. In contrast, in the presence of 1,5-AF, inflammasome activation by anthrax toxin, that is, production and release of IL-1β and IL-18 were strongly suppressed (see also FIG. 12).
　フラジェリン（ＦＬＡ）はＮＬＲＰ４を活性化し、カスパーゼ１を介してＩＬ－１β及びＩＬ－１８を再生、放出させて、炎症を引き起こす。フラジェリン（ＦＬＡ）は、鞭毛を有する細菌（ピロリ菌等）の鞭毛の構成タンパク質の一種で、樹状細胞等に発現しているトール様受容体５（Ｔｏｌｌ ｌｉｋｅ ｒｅｃｅｐｔｏｒ ５）を介して炎症を惹起する。ＬＰＳで刺激した骨髄マクロファージにフラジェリンを加えるとインフラマソームが活性化された。これに対し、１，５－ＡＦが存在する場合には、フラジェリンによるインフラマソーム活性化、すなわちＩＬ－１β及びＩＬ－１８の産生、放出は強く抑制された（図１３も参照）。 Flagellin (FLA) activates NLRP4 to regenerate and release IL-1β and IL-18 via caspase 1 and cause inflammation. Flagellin (FLA) is a type of flagellar protein of flagellar bacteria (H. pylori, etc.) and induces inflammation via Toll-like receptor 5 (Toll like receptor 5) expressed in dendritic cells. To do. When flagellin was added to bone marrow macrophages stimulated with LPS, the inflammasome was activated. In contrast, in the presence of 1,5-AF, inflammasome activation by flagellin, that is, production and release of IL-1β and IL-18 were strongly suppressed (see also FIG. 13).
　宿主の２重鎖ＤＮＡ（ｄｓＤＮＡ）はＡＩＭ２／ＡＳＣ経由でカスパーゼを活性化して、ＩＬ－１β及びＩＬ－１８を産生放出し、炎症を引き起こすことが判明している。ｄｓＤＮＡとしてｐｏｌｙ（ｄＡ：ｄＴ）（ＰＯＬ）で骨髄マクロファージを刺激した。１，５－ＡＦが存在する場合には、コントロールに比べ、１，５－ＡＦを添加した群の培養上清中のＩＬ－１βの産生は顕著に抑制されており、１，５－ＡＦがＡＩＭ２／ＡＳＣ経路からのインフラマソーム活性化を抑制していることが実証された（図１４も参照）。 It has been found that the host double-stranded DNA (dsDNA) activates caspases via AIM2 / ASC, produces and releases IL-1β and IL-18, and causes inflammation. Bone marrow macrophages were stimulated with poly (dA: dT) (POL) as dsDNA. When 1,5-AF is present, the production of IL-1β in the culture supernatant of the group to which 1,5-AF has been added is remarkably suppressed as compared to the control. It was demonstrated to suppress inflammasome activation from the AIM2 / ASC pathway (see also FIG. 14).
[２]（ｉ）ＬＰＳとＡＴＰの両方の刺激があったもののみプロカスパーゼ－１のカスパーゼ１への活性化とＩＬ－１β前駆体（ｐｒｏＩＬ－１β）のＩＬ－１βへの成熟化、細胞外への分泌が起きた
（ｉｉ）１，５－ＡＦはインフラマソームの活性化を介したプロカスパーゼ１からカスパーゼ１への活性化を抑制した
［実施例２］
　実施例１同様に、本実験はＮａｋａｈｉｒａ，Ｋらの方法（Ｎａｔ．Ｉｍｍｕｎｏｌ．１２ （２０１１）２２２－３０）に従ってマウス由来骨髄マクロファージを作製し、実験を行った。Ｃ５７／ＢＬ６の雄性マウスの大腿骨及び脛骨より、骨髄幹細胞を抽出し、Ｌ９２９細胞より抽出した培養上清液（Ｍ－ＣＳＦを含む）を２５％含むＤＭＥＭ液体培地（１０％ＦＢＳ及び２％ペニシリンストレプトマイシン含有）中で６日間培養し、マクロファージへと分化させた。６日間経過後に、マウス由来骨髄マクロファージを回収し、細胞数を調節し、各デッシュにまきなおし、オーバーナイトで接着させた後に、ＬＰＳ、及びインフラマソームを誘導する薬剤を添加した。 [2] (i) Activation of procaspase-1 to caspase-1 and maturation of IL-1β precursor (proIL-1β) to IL-1β, cells only stimulated by both LPS and ATP (Ii) 1,5-AF suppressed secretion from procaspase 1 to caspase 1 through inflammasome activation [Example 2]
In the same manner as in Example 1, mouse-derived bone marrow macrophages were prepared according to the method of Nakahira, K et al. (Nat. Immunol. 12 (2011) 222-30). Bone marrow stem cells were extracted from the femur and tibia of C57 / BL6 male mice, and DMEM liquid medium (10% FBS and 2% penicillin) containing 25% of the culture supernatant (including M-CSF) extracted from L929 cells. The cells were cultured in streptomycin) for 6 days and differentiated into macrophages. After 6 days, mouse-derived bone marrow macrophages were collected, the number of cells was adjusted, each dish was re-wound and allowed to adhere overnight, and then LPS and an inflammasome-inducing agent were added.
　最初に、ＴＬＲｓからの刺激（シグナル１）でｐｒｏ－ＩＬ－１β（ＩＬ－１βの前駆体）を細胞質内で作らせる目的で、ＬＰＳの添加を行った。ＬＰＳは、ｉｎｖｉｖｏＧｅｎ社のＵｌｔｒａｐｕｒｅ ＬＰＳを使用し、１００ｎｇ／ｍｌ濃度で４時間刺激を行った。 First, LPS was added for the purpose of producing pro-IL-1β (IL-1β precursor) in the cytoplasm by stimulation from TLRs (signal 1). For LPS, in vivoGen Ultrapure® LPS was used, and stimulation was performed at a concentration of 100 ng / ml for 4 hours.
　その後、１，５－ＡＦを３０分間インキュベートし、さらにインフラマソームを誘導する薬剤を添加した。ＮＬＲＰ３の場合は、ＡＴＰ ５ｍＭで時間インキュベートし誘導した。各サンプルの細胞と上清各分を電気泳動にかけ、カテプシンＢ抗体（Ｓａｎｔａ Ｃｒｕｚ社、ｓｃ－１３９８５）、ＡＳＣ抗体（Ｓａｎｔａ Ｃｒｕｚ社、ｓｃ－２２５１４－Ｒ）、カスパーゼ－１抗体（Ｓａｎｔａ Ｃｒｕｚ社、ｓｃ－５１４）、ＩＬ－１β抗体（Ｂｉｏｖｉｓｉｏｎ社、５１２９－１００）、β－アクチン抗体（Ｓｉｇｍａ社、Ａ１９７８）を用いて、ウエスタンブロッティングを行った。 Thereafter, 1,5-AF was incubated for 30 minutes, and a drug that induces inflammasome was further added. In the case of NLRP3, it was induced by incubation with 5 mM ATP for 5 hours. The cells and supernatant of each sample were subjected to electrophoresis, and cathepsin B antibody (Santa Cruz, sc-13985), ASC antibody (Santa Cruz, sc-22514-R), caspase-1 antibody (Santa Cruz, sc-514), IL-1β antibody (Biovision, 5129-100) and β-actin antibody (Sigma, A1978) were used for Western blotting.
　実施例２の結果を図１５に示す。図１５より、ＬＰＳとＡＴＰの両方の刺激があったもののみプロカスパーゼ－１のカスパーゼ１への活性化とＩＬ－１β前駆体（ｐｒｏＩＬ－１β）のＩＬ－１βへの成熟化、細胞外への分泌が起きたことがわかる。そして、１，５－ＡＦはインフラマソームの活性化を介したプロカスパーゼ１からカスパーゼ１への活性化を抑制することが明らかとなった。 The result of Example 2 is shown in FIG. FIG. 15 shows that only caspase-1 was activated by activation of procaspase-1 and maturation of IL-1β precursor (proIL-1β) into IL-1β, extracellularly, in the presence of both LPS and ATP stimulation. It can be seen that secretion occurred. It was revealed that 1,5-AF suppresses the activation of procaspase 1 to caspase 1 through inflammasome activation.
[３]１，５－ＡＦは実験的アルツハイマー病やパーキンソン病に使われる神経障害因子であるロテノンによるインフラマソーム活性化を抑制した
[実施例３]
　ワイルドタイプマウスより腹腔マクロファージを抽出し、これにＬＰＳを添加してプライミングした後に、インフラマソームを誘導する薬剤としてロテノン（ｒｏｔｅｎｏｎ）を添加（１時間）して上清中のＩＬ－１βの定量をＥＬＩＳＡキットを用いて行った。ロテノンは、ミトコンドリア障害因子であり、実験的アルツハイマー病やパーキンソン病に使われる神経障害因子である。ミトコンドリアが障害されると、障害ミトコンドリアから大量のＡＴＰが放出されて、インフラマソームが活性化されて炎症が惹起されることが知られている。神経系の場合にはこのことがアルツハイマー病やパーキンソン病の原因となることがわかっている。 [3] 1,5-AF inhibited inflammasome activation by rotenone, a neuropathic factor used in experimental Alzheimer's disease and Parkinson's disease
[Example 3]
Peritoneal macrophages were extracted from wild-type mice, LPS was added to the priming, and then rotenon was added as an agent for inflammasome induction (1 hour) to quantify IL-1β in the supernatant. Was performed using an ELISA kit. Rotenone is a mitochondrial disorder factor and a neuropathy factor used in experimental Alzheimer's disease and Parkinson's disease. It is known that when the mitochondria are damaged, a large amount of ATP is released from the damaged mitochondria, the inflammasome is activated, and inflammation is caused. In the nervous system, this is known to cause Alzheimer's disease and Parkinson's disease.
　１，５－ＡＦは、ロテノンによるインフラマソーム活性化、すなわちＩＬ－１βの産生、放出を抑制した（図１６）。 1,5-AF inhibited rotenone-induced inflammasome activation, that is, production and release of IL-1β (FIG. 16).
[４]１，５－ＡＦをＡＬＩの代表的モデルに腹腔内投与すると顕著に肺組織像と気管支肺胞洗浄液中の炎症性細胞浸潤が抑制された
[実施例４]
　Ｃ５７／ＢＬ６ワイルドマウスにＬＰＳを経気管投与してＡＬＩモデルを作製した。ＬＰＳ(エンドトキシン)を経気管投与すると激しい肺障害が惹起され、これはＡＬＩの代表的モデルとされている（Ｇｕｓｔａｖｏ Ｍａｔｕｔｅ－Ｂｅｌｌｏ，ｅｔ ａｌ. “Ａｎｉｍａｌ ｍｏｄｅｌｓ ｏｆ ａｃｕｔｅ ｌｕｎｇ ｉｎｊｕｒｙ”, Ａｍ Ｊ Ｐｈｙｓｉｏｌ Ｌｕｎｇ Ｃｅｌｌ Ｍｏｌ Ｐｈｙｓｉｏｌ ２９５： Ｌ３７９－Ｌ３９９， ２００８）。急性肺障害(ａｃｕｔｅ ｌｕｎｇ ｉｎｊｕｒｙ, ＡＬＩ)や急性呼吸促拍症候群(ａｃｕｔｅ ｒｅｓｐｉｒａｔｏｒｙ ｄｉｓｔｒｅｓｓ ｓｙｎｄｒｏｍｅ, ＡＲＤＳ)はインフラマソーム活性化に伴う代表的な疾患である。 [4] Intraperitoneal administration of 1,5-AF to a representative model of ALI significantly suppressed lung histology and inflammatory cell infiltration in bronchoalveolar lavage fluid
[Example 4]
An ALI model was prepared by transtracheally administering LPS to C57 / BL6 wild mice. Intratracheal administration of LPS (endotoxin) causes severe lung injury, which is considered to be a representative model of ALI (Gustavo Matte-Bello, et al. Mol Physiol 295: L379-L399, 2008). Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are typical diseases associated with inflammasome activation.
　作製したマウス（ＡＬＩモデル）に１，５－ＡＦを腹腔内投与すると顕著に肺組織像と気管支肺胞洗浄液中の炎症性細胞浸潤が抑制された（図１７）。 When 1,5-AF was intraperitoneally administered to the prepared mouse (ALI model), pulmonary histology and inflammatory cell infiltration in bronchoalveolar lavage fluid were markedly suppressed (FIG. 17).
[５]１，５－ＡＦをＳＩＲＳモデルに投与すると顕著に生存率が改善された
[実施例５]
　Ｃ５７／ＢＬ６ワイルドタイプマウスにＬＰＳを腹腔内投与してＳＩＲＳモデルを作製した。全身性にインフラマソームが活性化されるとＩＬ－１β等の炎症性サイトカインが全身を循環しＳＩＲＳを引きおこし、臨床上も重要な病態となっている。 [5] Survival improved significantly when 1,5-AF was administered to SIRS model
[Example 5]
A SIRS model was prepared by intraperitoneally administering LPS to C57 / BL6 wild type mice. When inflammasome is activated systemically, inflammatory cytokines such as IL-1β circulate throughout the body and cause SIRS, which is an important clinical condition.
　作製したマウス（ＳＩＲＳモデル）に治療群として１，５－ＡＦを１００ｍｇ／ｋｇ、ＬＰＳ投与２、６、１２、２４時間後に投与し、５日間観察したところ、顕著に生存率が改善された（図１８）。 The prepared mice (SIRS model) were treated with 1,5-AF as a treatment group at 100 mg / kg, 2, 6, 12, 24 hours after LPS administration, and observed for 5 days. The survival rate was significantly improved ( FIG. 18).
[６]１，５－ＡＦはプロカスパーゼ１のカスパーゼ１への活性化を抑制して炎症性細胞死を抑制した
[実施例６]
　プロカスパーゼ１のカスパーゼ１への活性化は、単にｐｒｏ－ＩＬ－１βとｐｒｏ－ＩＬ－１８のＩＬ－１β、ＩＬ－１８への活性化で免疫担当細胞を病巣部へリクルートするのみでなく、プログラム化された炎症性細胞死（ｐｙｒｏｐｔｏｓｉｓ）をも引き起こす。これらは新しい炎症の概念である。本発明者らは、１，５－ＡＦがプロカスパーゼ１のカスパーゼ１への活性化を抑制して、炎症性細胞死を抑制することを検証した。 [6] 1,5-AF inhibited inflammatory cell death by inhibiting the activation of procaspase 1 to caspase 1
[Example 6]
Activation of procaspase 1 to caspase 1 not only recruits immunocompetent cells to the lesion by activation of pro-IL-1β and pro-IL-18 to IL-1β and IL-18, It also causes programmed inflammatory cell death. These are new inflammation concepts. The present inventors have verified that 1,5-AF suppresses inflammatory cell death by suppressing the activation of procaspase 1 to caspase 1.
　マウス骨髄細胞をＬＰＳで４時間インキュベーションしてプライミングし、次に１，５－ＡＦと３０分間インキュベーションした後に、完全に１，５－ＡＦを洗い去り、次にニゲリシン（５μＭ）で１時間インキュベーションして、炎症性細胞死を検討した。 Mouse bone marrow cells were primed by incubation with LPS for 4 hours, and then incubated with 1,5-AF for 30 minutes, followed by thorough washing of 1,5-AF and then incubation with nigericin (5 μM) for 1 hour. Inflammatory cell death was examined.
　３つのサンプルについての結果を表１に示す。ＬＰＳとニゲリシンを加えたものは、炎症性細胞死が誘導されて、生細胞数はＬＰＳ単独に比べ、約１／６に減少していたが、１，５―ＡＦ（１０ｍｇ／ｍｌ）を添加した場合、炎症性細胞死した細胞数は約１／３程度に収まり、細胞死は抑制され、生細胞数は５０％近くまで増加した。すなわち１，５－ＡＦによりニゲリシンによる炎症性細胞死は抑制された。
Figure JPOXMLDOC01-appb-T000001
 The results for the three samples are shown in Table 1. When LPS and nigericin were added, inflammatory cell death was induced, and the number of living cells was reduced to about 1/6 compared with LPS alone, but 1,5-AF (10 mg / ml) was added. In this case, the number of inflammatory cell deaths was reduced to about 1/3, cell death was suppressed, and the number of living cells increased to nearly 50%. That is, 1,5-AF suppressed inflammatory cell death by nigericin.
Figure JPOXMLDOC01-appb-T000001
[７]１，５－ＡＦはアポトーシス関連スペック様カード蛋白質（ＡＳＣ）を標的とする
［実施例７］
　ＡＳＣを発現させたＨＥＫ細胞をエンドトキシン刺激し（第１刺激）、次にｐｏｌｙ（ｄＡ：ｄＴ）を加えた（第２刺激）場合、図１９の列５のようにＡＳＣモノマーは、ダイマー、トリマー及びテトラマーを形成した。しかしながら、１，５－ＡＦの存在下では、このようなＡＳＣモノマーの重合体は観察されなかった（図１９の６列参照）。つまり、１，５－ＡＦはＡＳＣの重合を抑制していることが示唆された。結果として１，５－ＡＦは図３－２に示されるＡＳＣ重合阻害を介してカスパーゼ１の生成を阻害して、結果的にＩＬ－１ｂ、ＩＬ－１８の産生を抑制しているものといえる。 [7] 1,5-AF targets apoptosis-related spec-like card protein (ASC) [Example 7]
When HEK cells expressing ASC were stimulated with endotoxin (primary stimulation) and then added with poly (dA: dT) (second stimulation), as shown in column 5 of FIG. And tetramers formed. However, in the presence of 1,5-AF, such a polymer of ASC monomer was not observed (see column 6 in FIG. 19). In other words, it was suggested that 1,5-AF suppressed ASC polymerization. As a result, it can be said that 1,5-AF inhibits the production of caspase 1 through inhibition of ASC polymerization shown in FIG. 3-2 and consequently suppresses the production of IL-1b and IL-18. .
[８]１，５－ＡＦは、シリカによるインフラマソーム活性化を抑制した
［実施例８］
　マウス骨髄単核球を培養し、シリカ(珪酸マグネシウム：５０μｇ/ｍｌ)による刺激した後、１，５-ＡＦ（５μｇ/ｍｌ）を添加して１時間後、ＩＬ－１８の濃度を測定した（図２０ａ）。同様に、各濃度のシリカで骨髄単核球を刺激した後、５時間後に１，５－ＡＦ（２～１０ｍｇ/ｍｌ）加え、ＩＬ－１βの濃度を測定した（図２０ｂ）。 [8] 1,5-AF inhibited inflammasome activation by silica [Example 8]
Mouse bone marrow mononuclear cells were cultured, stimulated with silica (magnesium silicate: 50 μg / ml), 1,5-AF (5 μg / ml) was added, and one hour later, the concentration of IL-18 was measured ( FIG. 20a). Similarly, after stimulating bone marrow mononuclear cells with each concentration of silica, 1,5-AF (2 to 10 mg / ml) was added 5 hours later, and the concentration of IL-1β was measured (FIG. 20b).
　１，５－ＡＦは、シリカによるインフラマソーム活性化、すなわちＩＬ－１８、ＩＬ－１βの産生、放出を抑制した（図２０）。 1,5-AF suppressed inflammasome activation by silica, that is, production and release of IL-18 and IL-1β (FIG. 20).
　石綿肺のモデル実験において、炎症を抑制し、石綿肺防止剤として利用できることが分かった。 In an asbestos lung model experiment, it was found that inflammation can be suppressed and used as an asbestosis inhibitor.
[９]１，５－ＡＦは、炭疽菌毒素によるインフラマソーム活性化ＩＬ－１βの産生、放出を抑制した
［実施例９］
　マウス骨髄単核球を炭疽菌毒素（ａｎｔｈｏｒａｘ ｔｏｘｉｎ：１ｍｇ/ｍｌ）又はアラム（５０μｇ/ｍｌ）で４時間培養刺激した後、１，５－ＡＦ（５ｍｇ/ｍｌ）を添加して、２時間後のＩＬ－１βの濃度を測定した（図２１）。アラムはポジティブコントロールとして使用した。 [9] 1,5-AF inhibited production and release of inflammasome-activated IL-1β by anthrax toxin [Example 9]
Mouse bone marrow mononuclear cells were stimulated with anthrax toxin (anthorax toxin: 1 mg / ml) or alum (50 μg / ml) for 4 hours, 1,5-AF (5 mg / ml) was added, and 2 hours later The concentration of IL-1β was measured (FIG. 21). Alum was used as a positive control.
　１，５－ＡＦは、炭疽菌毒素によるインフラマソーム活性化、すなわちＩＬ－１βの産生、放出を抑制した（図２１）。 1,5-AF inhibited inflammasome activation by anthrax toxin, that is, production and release of IL-1β (FIG. 21).
　炭疽菌によるバイオテロへの防御剤として利用できることが分かった。 It was found that it can be used as a protective agent against bioterrorism by anthrax.
[１０]１，５－ＡＦは、ＬＰＳ＋シリカ又はＡＴＰで刺激したヒト末梢血単核細胞に対してもＩＬ－１βの放出を抑制する効果を有する
［実施例１０］
　ヒト末梢血を採取し、Ｌｙｍｐｈｏｐｒｅｐ（Ｆｉｃｏｌｌ）を使用してヒト末梢血単核細胞（ＰＢＭＣ）を分離した。ＰＢＭＣを、ＤＭＥＭ＋１０％ＦＢＳ培地に当たり０．１ｘ１０６個/ウェルずつまいて、実験に用いた。ＬＰＳ ５００ｎｇ／ｍｌで３時間プライミング処理した。１，５－ＡＦ（２～１０ｍｇ/ｍｌ）を加え、３０分インキュベートした後にシリカ（２５０μｇ／ｍｌ）で２時間刺激した後、上清を回収した。 [10] 1,5-AF has an effect of suppressing the release of IL-1β also on human peripheral blood mononuclear cells stimulated with LPS + silica or ATP [Example 10]
Human peripheral blood was collected and human peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (Ficoll). PBMCs were dispersed in DMEM + 10% FBS medium at 0.1 × 10 6 cells / well and used for the experiment. Primed with LPS 500 ng / ml for 3 hours. 1,5-AF (2-10 mg / ml) was added, incubated for 30 minutes and then stimulated with silica (250 μg / ml) for 2 hours, and then the supernatant was collected.
［実施例１１］
　ヒト末梢血を採取し、Ｌｙｍｐｈｏｐｒｅｐ（Ｆｉｃｏｌｌ）を使用してヒト末梢血単核細胞（ＰＢＭＣ）を分離した。ＰＢＭＣを、ＤＭＥＭ＋１０％ＦＢＳ培地に当たり０．１ｘ１０６個/ウェルずつまいて実験に用いた。ＬＰＳ ５００ｎｇ／ｍｌで３時間プライミング処理した。１，５－ＡＦ（２～１０ｍｇ/ｍｌ）を加え、３０分インキュベートした後にＡＴＰ（２～５ｍＭ）で1時間刺激した後、上清を回収した。 [Example 11]
Human peripheral blood was collected and human peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (Ficoll). PBMCs were dispersed in DMEM + 10% FBS medium at 0.1 × 10 6 cells / well and used for the experiment. Primed with LPS 500 ng / ml for 3 hours. 1,5-AF (2-10 mg / ml) was added, incubated for 30 minutes and then stimulated with ATP (2-5 mM) for 1 hour, and then the supernatant was collected.
　１，５－ＡＦは、ＬＰＳ＋ＡＴＰで刺激したヒト末梢血単核細胞に対してもＩＬ－１βの放出を抑制する効果を有することが分かった（図２２）。 1,5-AF was found to have an effect of suppressing the release of IL-1β even on human peripheral blood mononuclear cells stimulated with LPS + ATP (FIG. 22).
　本願の薬剤は、現在の生物学的製剤（抗体）に代わるアポトーシス関連スペック様カード蛋白質（ＡＳＣ）制御剤として使用することができる。 The drug of the present application can be used as an apoptosis-related spec-like card protein (ASC) regulator in place of the current biological preparation (antibody).
　本明細書で引用した全ての刊行物、特許及び特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into this specification as they are.

Claims (23)

  1. 　１，５－Ｄ－アンヒドロフルクトースを有効成分とする、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）の機能阻害薬。 A function inhibitor of apoptosis-related spec-like card protein (ASC) containing 1,5-D-anhydrofructose as an active ingredient.
  2. 　ＡＳＣの機能が、カスパーゼ１及びＮＯＤ様受容体との複合体形成能である、請求項１に記載の阻害薬。 The inhibitor according to claim 1, wherein the function of ASC is the ability to form a complex with caspase 1 and a NOD-like receptor.
  3. 　複合体がインフラマソームである、請求項２に記載の阻害薬。 The inhibitor according to claim 2, wherein the complex is an inflammasome.
  4. 　インフラマソームが関与する経路における、プロカスパーゼ１のカスパーゼ１への活性化を阻害する、請求項３に記載の阻害薬。 The inhibitor according to claim 3, which inhibits activation of procaspase 1 to caspase 1 in a pathway involving inflammasome.
  5. 　１，５－Ｄ－アンヒドロフルクトースを有効成分とする、アポトーシス関連スペック様カード蛋白質（ＡＳＣ）が関与する疾患又は症状の治療薬。 A therapeutic agent for diseases or symptoms involving apoptosis-related spec-like card protein (ASC), which contains 1,5-D-anhydrofructose as an active ingredient.
  6. 　ＡＳＣが関与する疾患又は症状が、ＡＳＣとカスパーゼ１及びＮＯＤ様受容体との複合体形成が関与する疾患又は症状である、請求項５に記載の治療薬。 The therapeutic agent according to claim 5, wherein the disease or symptom involving ASC is a disease or symptom involving complex formation between ASC, caspase 1 and NOD-like receptor.
  7. 　複合体がインフラマソームである、請求項６に記載の治療薬。 The therapeutic agent according to claim 6, wherein the complex is inflammasome.
  8. 　ＡＳＣが関与する疾患又は症状が、カスパーゼ１が関与する疾患又は症状である、請求項５～７のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 7, wherein the disease or symptom associated with ASC is a disease or symptom associated with caspase-1.
  9. 　ＡＳＣが関与する疾患又は症状が、ＩＬ－１β及び／又はＩＬ－１８が関与する疾患又は症状である、請求項５～８のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 8, wherein the disease or symptom associated with ASC is a disease or symptom associated with IL-1β and / or IL-18.
  10. 　ＡＳＣが関与する疾患又は症状が、全身性炎症性反応症候群ＳＩＲＳである、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is systemic inflammatory response syndrome SIRS.
  11. 　ＡＳＣが関与する疾患又は症状が、慢性関節リウマチである、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is rheumatoid arthritis.
  12. 　ＡＳＣが関与する疾患又は症状が、アルツハイマー病である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is Alzheimer's disease.
  13. 　ＡＳＣが関与する疾患又は症状が、クライオピリン関連周期熱症候群である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is cryopyrine-related periodic fever syndrome.
  14. 　ＡＳＣが関与する疾患又は症状が、家族性地中海熱である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is familial Mediterranean fever.
  15. 　ＡＳＣが関与する疾患又は症状が、ＰＡＰＡ症候群である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is PAPA syndrome.
  16. 　ＡＳＣが関与する疾患又は症状が、Ｍａｊｅｅｄ症候群である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is Majeed's syndrome.
  17. 　ＡＳＣが関与する疾患又は症状が、高ＩｇＤ症候群である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is high IgD syndrome.
  18. 　ＡＳＣが関与する疾患又は症状が、反復性胞状奇胎である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is repetitive hydatidiform mole.
  19. 　ＡＳＣが関与する疾患又は症状が、ＤＩＲＡである、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is DIRA.
  20. 　ＡＳＣが関与する疾患又は症状が、炭疽菌感染症である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is anthrax infection.
  21. 　ＡＳＣが関与する疾患又は症状が、急性呼吸促拍症候群である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is acute respiratory stimulation syndrome.
  22. 　ＡＳＣが関与する疾患又は症状が、炎症性細胞死である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is inflammatory cell death.
  23. 　ＡＳＣが関与する疾患又は症状が、石綿肺である、請求項５～９のいずれか１項に記載の治療薬。 The therapeutic agent according to any one of claims 5 to 9, wherein the disease or symptom associated with ASC is asbestosis.
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