WO2015037764A1 - In vivo and ex vivo amplification of myeloid-derived immunoregulatory cell and immunoregulatory b lymphocyte by activation of gpcr19 pathway - Google Patents

In vivo and ex vivo amplification of myeloid-derived immunoregulatory cell and immunoregulatory b lymphocyte by activation of gpcr19 pathway Download PDF

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WO2015037764A1
WO2015037764A1 PCT/KR2013/008365 KR2013008365W WO2015037764A1 WO 2015037764 A1 WO2015037764 A1 WO 2015037764A1 KR 2013008365 W KR2013008365 W KR 2013008365W WO 2015037764 A1 WO2015037764 A1 WO 2015037764A1
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cells
mdsc
gpcr19
hydrogen
cell
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Korean (ko)
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성승용
장숙희
김연희
김영주
정희은
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서울대학교 산학협력단
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention treats cells in vivo and externally with GPCR19 agonists to increase bone marrow-derived immunomodulatory cells (3 ⁇ 4 610 0- (1 ⁇ 6 (1 suppressor cel l, MDSC) or immunoregulatory B cell counts).
  • GPCR19-associated disease diseases in which activation of the signaling system via the GPCR19 receptor has a significant effect on the regulation of disease progression and development.
  • the present invention also relates to a method for analyzing the efficacy of a GPCR19 agonist as a therapeutic agent for GPCR19-associated diseases.
  • Sepsis begins with systemic inf lammatory response syndrome (SIRS) due to simultaneous activation of the complement system, coagulation system, and innate immune cell system after microbial infection. Is a disease in which patients die. Therefore, it is easy to assume that sepsis will not be inhibited through the blocking of one or two TTL4 pathways (complementary and coagulat ion pathways), and in most patients the timing of treatment is already active. There are many cases of establ ished sepsi s. It is presumed that the therapeutic agents developed so far aimed at blocking one inflammation-inducing pathway are ineffective in clinical trials.
  • SIRS systemic inf lammatory response syndrome
  • Ligly's Xygris the world's only FDA-licensed product, also has a 28-day survival rate of 6% in patients with poor efficacy, mainly due to APC (act ivated protein C), which regulates blood lactation. Because. After ligation of the colon and connecting sites of the mouse dendritic spine, perforation of the dendritic spine (CLP, cecal ligation and puncture) induces sepsis, which causes CDllb + Gr-l + bone marrow-derived immune suppressor cells (Myeloid-) in the spleen and bone marrow. The number of derived suppressor cells (MDSCs) is significantly increased.
  • MDSC has been reported to have increased numbers in several autoimmune mouse models, as well as sepsis, but there is no clear conclusion as to whether to worsen or inhibit disease progression.
  • MDSC is increased in kidney and blood of active MRL-fas lp ⁇ mice, and ex vivo experiments confirmed that it inhibits the proliferation of CD4 + T cells [Cripps, JG and JD Gorham, (2011) Int Immunopharmacol; Iwata Y., et al. , (2010) Clin Exp Nephrol 14: 411-417.
  • B cells have traditionally been known to produce antibodies and participate in humoral immune response. Recently, B10 immunomodulatory B cells, CD5 + Bla cells, CD + marginal zone B cells and T2-MZP (Tr ans iti ona 1-2-mar gi Regulatory B cells (Bregs), such as na 1 zone precursor B cells, have been reported. Immune Regulatory B Cells Specific surface molecules and target molecules in the cytoplasm have not been identified, so no specific characteristics of these cells have been identified. However, immunoregulatory B cells are mostly derived from B2 B cells (except for CD5 + Bla cells) and have a common secretion of IL-10.
  • immunoregulatory B cells whose immunosuppressive function was confirmed through ex r / ra and in vivo 5 were B10 immunoregulatory B cells and T2-MZP immunoregulatory B cells [Mauri, C. and PA Blair, (2010) Nat Rev Rheumatol, 6 (11): 636-43].
  • T2-MZP immunoregulatory B cells (CD19 +, CD21high, CD23 + , CD24 high, CD93 +, 0 CDld + , IL-10 + ) are the mesenteric lymph nodes (MLNs) of a mouse model of chronic colitis (TCRa-/ —). , Mesenteric lymph node), and it was reported that colitis is inhibited by intravenous injection of these cells isolated from the spleen [Mizoguchi, A., et al. , Immunity 16,219-230, (2002)].
  • T2-MZP immunoregulatory B cells were found to be increased in the spleen of mouse 5 model (CIA, collagen induced arthritis) of rheumatoid arthritis, especially in the spleen of symptomatically improved mice. .
  • IL-10 and CDld knockout mice have been demonstrated to play an important role in inhibiting inflammation by inhibiting IFN-Y secretion T H 1 cell proliferation [Evans, JGet al. , J Immunol 0 178,7868-7878, (2007).
  • B10 immunoregulatory B cells (CD5 +, CDld + , IL-10 +, B220 +) have been reported to be produced in the spleen and lymph nodes of mouse models of several inflammatory and autoimmune diseases [DiLillo, DJ, T. Matsushita, and TF Tedder]. , (2010) Ann NY Acad Sci, 1183: 38-57.]. Inhibitory effects of B10 immunoregulatory B cells were demonstrated by adoptive transfer of ex10 in vivo B10 immunoregulatory B cells into chronic col lagen-induced arthritis (CCIA), a mouse model of rheumatoid arthritis. Adoption of B10 immunoregulatory B cells reduced arthritis index by more than 50% and disease severity by more than 90% [Mauri, C:., Et al. , (2003) J Exp Med, 0 197 (4): 489-501].
  • CCIA chronic col lagen-induced arthritis
  • the present invention relates to a method of using a GPCR19 agonist to artificially amplify these MDSCs and immunoregulatory B cells in vivo and externally, and also to a method of screening a GPCR19 agonist and measuring the efficacy of the drug.
  • the present invention relates to a numerical amplification technique of MDSC and immunoregulatory B lymphocytes having an enhanced anti-inflammatory phenotype different from the one previously known, using deoxycholate (HY2191, Sodiumtaurodeoxycholate) and its derivatives in vivo.
  • the present inventors have tried to develop a technique for artificially amplifying MDSC and immune regulatory B cells with enhanced anti-inflammatory effects in vivo and externally.
  • the present inventors have made diligent research efforts to develop a GPCR19-associated disease treatment agent, a screening method thereof, and a method for analyzing the efficacy of the GPCR19 agonist as a GPCR19-associated disease treatment agent.
  • the present inventors developed a technique for amplifying MDSCXMyeloid-derived suppressor cel l) and immunoregulatory B cells by administering or treating a GPCR19 agonist to a mammalian or bone marrow-derived cell population, and including the GPCR19 agonist as an active ingredient.
  • the present invention was completed by identifying that the composition has anti-inflammatory activity in vivo by mediating MDSCX Myeloid-derived suppressor cel l) and immunoregulatory B cells.
  • an object of the present invention relates to a method of increasing the number of new phenotypes of MDSCs or immunoregulatory B cells with improved anti-inflammatory effects by administering GPCR19 agonists and their derivatives in vivo.
  • the present invention relates to a method of increasing the number of new phenotypes of MDSCs or immunoregulatory B cells, which have been treated with peripheral blood, thereby improving the effect of inhibiting inflammation.
  • Another object of the present invention is to provide a method for analyzing the efficacy of GPCR19 agonists.
  • Another object of the present invention relates to MDSCs having anti-inflammatory activity. Another object of the present invention relates to immunomodulatory B cells having anti-inflammatory activity.
  • Another object of the present invention relates to an anti-inflammatory pharmaceutical composition
  • an anti-inflammatory pharmaceutical composition comprising MDSC or immunoregulatory B cells as an active ingredient.
  • Another object of the present invention is to administer a new phenotype of MDSC or immunoregulatory B cells having improved inflammation inhibitory effect by administering sodium taurodeoxycholate (HY2191, Sod iumtaurodeoxycho late) and its derivatives in vivo or treating bone marrow cells. It is about increasing. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings. [Measures of problem]
  • a method for in vivo screening of a GPCR19-associated disease therapeutic agent comprising the following steps:
  • the present inventors made an intensive study to develop a therapeutic agent for GPCR19-related diseases, a screening method thereof, and a method for analyzing the efficacy of GPCR19 agonists for the treatment of GPCR19-associated diseases.
  • the present inventors developed MDSCCMyeloid-derived suppressor cel l) and immunomodulatory B cells formed by administering or treating GPCR19 agonists to a mammalian or bone marrow-derived cell population, and the composition comprising the same as an active ingredient has anti-inflammatory activity. It was found to have.
  • the present invention provides a method for screening a therapeutic agent for a GPCR19-associated disease.
  • GPC19-associated diseases preferably sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease, ulcerative colitis, type 1 diabetes, Crohn's disease.
  • the in vivo screening method of the GPCR19-associated disease therapeutic agent of the present invention will be described in detail for each step as follows: Step (a): Administration of a physiologically active agent in vivo
  • a physiological reaction modulator in vivo is first administered to a mammal except a human to cause a physiological reaction in vivo.
  • physiological activity modulator used in the present invention means a substance causing a physiological reaction in vivo.
  • the physiologically active regulatory substance in vivo binds to a cell receptor (eg, toll ll receptor) to regulate a signaling response or to various components of the general immune system (eg, vascular endothelial cells, dendritic cells). Cells, T cells, and B cells) to interact with receptors to regulate signaling response.
  • a cell receptor eg, toll ll receptor
  • various components of the general immune system eg, vascular endothelial cells, dendritic cells.
  • Cells, T cells, and B cells to interact with receptors to regulate signaling response.
  • the physiologically active regulatory substance in vivo interacts with various humoral factors or cells of the immune system to secrete specific cytokines or chemokines, or to express the surface molecule expression of living cells.
  • Molecules that increase, form antibodies, or produce or increase specific lymphocyte sites and more preferably, each composed of synthetic organic chemicals, proteins, lipoproteins, glycoproteins, peptides, RNA, DNA, or polysaccharides Molecules, or cellular constructs (bacteria or fungi) or part of a virus, but are not limited to.
  • In vivo physiological activity modulators used in the present invention are substances derived from various kinds, preferably bacteria-derived, fungal-derived, virus-derived, autologous or allergen, and more preferably. Is bacterial or fungal, and more
  • LPS lipopolysaccharide
  • peptidoglycan a molecule derived from bacteria, most preferably lipopolysaccharide (LPS) or peptidoglycan.
  • Bacterial-derived materials for in vivo physiologically active substances used in the method of the present invention can be selected from a group of bacteria that infect mammals and cause various diseases, preferably Salmonella
  • Meningitidis OVe / sser / a meningitidis Neseria Gonorhe 0Ve / sse a gonorrheae, Haemophilus influenzae G3 ⁇ 4i TC3 ⁇ 4! 3 ⁇ 43 ⁇ 4 // i / s influenzae), E. coli (s? Er /? / A coli), Klebsiella pneumoniae, Listeria monocytogenes (/ s er / a monocytogenes), Vibrio cholera cholerae, Clostridium perfrangens (C / ostr / d / n
  • Clostridium botulinum (CVosT / i // ifl7 botulinimi), Domonas Erasions 03 ⁇ 4erac3 ⁇ 4z3 ⁇ 43 ⁇ 4 s aerations, Botelia Burgdorferi 0 / ⁇ 6 // burgdorferi), Shigella flexneri (5 ? / // 3 flexneri), shigella boydi ( ⁇ / ⁇ // a boydii), shigella decentrige (5 ⁇ / ⁇ / / a dysentriae), alloyococcus ortidis ( ⁇ // o / ococ / s otitidis) and groups
  • a fungal-derived material is derived from a fungus that infects mammals and causes various diseases, such as superficial mycosis in the skin and oral cavity of a mammal. It is a substance derived from fungi that cause systemic fungal diseases, such as intestines and brain.
  • Fungal derived materials can be selected from a variety of fungal groups, preferably Candida
  • In vivo biological physiologically active substances used in the method of the present invention can be selected from the group of viruses that infect a mammal and cause a variety of diseases, preferably, adenovirus, alpha virus, kaleici virus, corona Virus, CMV, distemper virus, Ebola virus, enterovirus, EBV, flavivirus (eg Hep C) hapadnavirus (eg Hep B, hepatitis delta, Hep E or F virus), hepatitis A virus (eg , VPl), GBV-C, Herpesviruses (eg Herpes Simplex Virus Protein [eg, Type I Glycoprotein G, gpD, CP27, Varicella Zoster Virus Glycoprotein (eg IE62, gpl or envelope protein)], Immunodeficiency Viruses such as HIV (eg envelope or protease), infective peritonitis virus, influenza Purple virus (eg, influenza A Hemagglut
  • self-derived materials may be derived from the normal constituents of a living body [eg, proteins (cytokines, hormones, interleukins or combinations thereof), nucleic acids, combinations thereof or These fragments are substances that cause autoimmune diseases by causing various signaling reactions to the same individual, causing autoimmune reactions, and stimulating them to form autoantibodies. It is arranged in a different composition from the composition of the substance, which is a self-derived substance that causes autoimmune reactions, preferably insulin ⁇ myelin basic protein, rh factor, acetylcholine receptor, thyroid cell receptor (base membrane protein, thyroid protein).
  • autoimmune diseases caused by the autologous material are preferably rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), lupus nephritis, skin hemorrhage lupus (CLE).
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • CLE skin hemorrhage lupus
  • allergens are allergens known in the art, i.e. all naturally occurring substances known to cause an IgE mediated response upon repeated exposure to an individual or their And mixtures of materials.
  • Naturally occurring allergens preferably pollen allergens (e.g., pollen allergens in trees, weeds, herbs or grass), mite allergens (e.g., house dust mites or storage mites derived), nasal allergies Groups of allergens (eg, allergens derived from inhalation, saliva and venom), animal allergens, fungal allergens and food allergens derived from saliva, hair and dandruff in dogs, cats, rats and mice Selected from, but not limited to, combinations thereof.
  • pollen allergens e.g., pollen allergens in trees, weeds, herbs or grass
  • mite allergens e.g., house dust mites or storage mites derived
  • nasal allergies Groups of allergens eg, allergens derived from inhalation, saliva and venom
  • animal allergens e.g., fungal allergens and food allergens derived from saliva, hair and dandruff in dogs, cats,
  • allergens are selected from the group consisting of allergen extracts, purified allergens, modified allergens, recombinant allergens, recombinant variant allergens, allergen fragments of 30 amino acids or more, or combinations thereof, but are not limited thereto.
  • the in vivo physiologically active substance can be administered to mammals other than humans, preferably mammals, more preferably companion animals (eg dogs, cats), farm animals (eg , Cattle, sheep, pigs or horses or laboratory animals (eg rats, mice or Guinea pigs), and more preferably mouse, rat, dog, animal, goat, sheep, rabbit and most preferably mouse.
  • mammals preferably mammals, more preferably companion animals (eg dogs, cats), farm animals (eg , Cattle, sheep, pigs or horses or laboratory animals (eg rats, mice or Guinea pigs), and more preferably mouse, rat, dog, animal, goat, sheep, rabbit and most preferably mouse.
  • Administration of the physiologically active substance in vivo to the mammal may be carried out through various methods known in the art, preferably orally or parenterally, and in the case of parenteral administration, intravenous infusion, subcutaneous Injection, intramuscular injection, intraperitoneal injection, transdermal administration, and more preferably, orally, intranasally, gastrointestinal tract, external ear canal, vaginal, rectal, intravenous or intraperitoneal injection, skin or eye. Can be administered, most preferably intraperitoneally.
  • the dose of the physiologically active substance in vivo can be determined in the laboratory (rout inely), for example, if lipopolysaccharide (LPS) is used in the mouse as the physiologically active substance in vivo, the dose Is preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, most preferably 20 mg / kg.
  • the GPCR19 agonist test substance used in the method of the present invention interacts with the GPCR19 receptor to produce efficacy, preferably to act on a substance or drug that acts (or similarly) as the substance, or a drug, or a receptor site.
  • Height means molecule.
  • the GPCR19 agonist of the present invention acts as an agonist or partial agonist of the GPCR19 receptor.
  • agonist as used herein generally means a compound that enhances the activity of another molecule or receptor site. In the classical definition, an agonist is a property that causes a biological action by binding to a receptor and altering its receptor state, whether or not it is an orthosteric, allosteric, inverse or co-agonist.
  • agonism is defined as the nature of an agonist or ligand that produces a biological action. More specifically, the GPCR19 agonist is a receptor ligand or compound that binds to GPCR19 and increases the concentration of cyclic adenosine monophosphate (cAMP) by at least 3% in cells expressing the receptor.
  • the GPCR19 agonist used in the present invention comprises a compound of formula (1), a salt or a hydrate thereof, more preferably sodium taurodeoxycholate (HY2191).
  • the dose of sodium taurodeoxycholate is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 10 mg / kg, most preferably 0.5-8 mg /. Use kg.
  • R 2 is hydrogen or ⁇ -hydroxy
  • 3 ⁇ 4 is hydroxy, NH (C3 ⁇ 4) m S0 3 H (m is an integer 0, 1, 2, 3, 4 or 5) or NH (C3 ⁇ 4) n C0 2 H (n is an integer 1, 2, 3, 4 Or 5);
  • R 5 is hydrogen, substituted or unsubstituted alkyl or aryl
  • 3 ⁇ 4 is a ring formed of 3, 4, 5 or 6 atom sizes with hydrogen or carbon with 3 ⁇ 4 and 3 ⁇ 4 attached
  • R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl
  • 3 ⁇ 4 is hydrogen or substituted or unsubstituted alkyl
  • 3 ⁇ 4 is hydrogen or substituted or unsubstituted alkyl
  • R 10 is hydrogen or substituted or unsubstituted alkyl
  • Ru is hydrogen or substituted or unsubstituted alkyl.
  • step (a) Since the method of administering with a mammal other than a human subject to the administration of the GPCR19 agonist test substance used in the method of the present invention is the same as described above in step (a), the contents in common between the two are repeated according to the repetition description. In order to avoid excessive complexity of the specification, the description is omitted.
  • alkyl includes saturated aliphatic groups such as straight chain alkyl groups (eg, methyl, ethyl, propyl, butyl, pentyl, nucleus, heptyl, octyl, nonyl, decyl), branched chain alkyl groups (eg, isopropyl).
  • Cycloalkyl such as cycloaliphatic groups (such as cyclopropyl, cyclopentyl, cyclonuclear, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups.
  • straight or branched chain alkyl has up to 6 carbon atoms in the backbone [eg, dC 6 (straight chain), C 3 -C 6 (branched chain)]. In some instances, straight or branched chain alkyl has up to 4 carbon atoms in the backbone.
  • cycloalkyl has 3 to 8 carbon atoms in the ring structure.
  • substituted alkyl denotes the moiety replaced with a hydrogen atom of one or more substituents on one or more carbons of the hydrocarbon backbone.
  • substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl alkoxycarbons Carbonyl, aminocarbonyl, alkylaminocarbonyl dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate phosphonato, phosphinato, cyano, amino (alkylamino, dialkylamino arylamino, diaryl Acylamino (including amino and alkylarylamino) (including alkylcarbonylamino, arylcarbonylamino, carbamoy
  • aryl includes aromatic groups such as 5- and 6-membered “unconjugated” or single-ring aromatic groups, which may include 0-4 heteroatoms, and one or more aromatic rings. "Junction" or multicyclic systems are included. Examples of aryl groups include benzene, phenyl, pi, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine and pyri Midines and the like.
  • aryl in this specification includes a multicyclic aryl group such as tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedi Oxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benchofuran, purine, benzofuran, deazapurine or indolizine.
  • Aryl groups having heteroatoms in the structure may also be referred to as "aryl heterocycle", “heterocycle”, “heteroaryl” or “heteroaromatic".
  • the aromatic ring may be substituted as described above at one or more ring positions, such as halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl Alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, Phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (alkylcarbonylamino, arylcarbonylamino, carbamoyl And ureido), amidino, im
  • aryl groups may be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic and thus form a multicyclic system (eg, tetralin or methylenedioxyphenyl).
  • lower alkyl includes alkyl groups as defined above having 1 to 10, such as 1 to 6, carbon atoms in the backbone structure.
  • hydroxytoxy or "hydroxyl” herein includes groups having -0H or -0-.
  • the GPCR19 agonist test substance is a GPCR19-associated disease if the degree of production of the cells (0 to (iv) is increased in the mammal after administration of the GPCR19 agonist test substance to a mammal other than a human. Determined as a therapeutic agent.
  • Gr-1 + CDllb + MDSCCMyeloid-derived suppressor cel l Transduction of MDSC (Gr-1 + CDllb + MDSC) cells with increased expression of surface markers Gr-1 + and CDllb + in cell sepsis-induced mice Results have been reported to reduce mortality in sepsis-induced mice.
  • the present inventors confirmed the increase of Gr-1 + CDllb + MDSC cells in the spleen by administering a GPCR19 agonist test substance to sepsis-induced mice, and these Gr-1 + CDllb + MDSC cells were previously known as Gr-1 + CDllb + MDSC cells.
  • Cells with phenotypes with different gene expression patterns were found to be capable of treating sepsis by protonation.
  • the present inventors have found that GPCR19 is involved in higher signaling pathways that regulate the increase of MDSC cells, which in turn treats GPCR19 agonists to induce GPCR19 activation and increases Gr-1 + CDllb + MDSC cells, thereby lowering various signals. It has been suggested that inflammation can be controlled through the delivery pathway.
  • the test substance when administration of the test substance resulted in an increase of Gr-1 + CDllb + MDSC cells compared to the control group not injected with the test substance, it is determined as a treatment for GPCR19-associated disease. More specifically, when the number of Gr-1 + CDllb + MDSC cells increases by 2% or more, preferably more than 0.5%, as compared with the control group which is not injected with the test material, the test substance is GPCR19. -To be determined as a treatment for an associated disease. For example, the number of Gr-1 + CDllb + MDSC cells can be analyzed by FACS. — ⁇ _ ⁇
  • Analysis of the efficacy of the GPCR19 agonist test substance can be performed by measuring the number of Gr-1 + CDllb + MDSC cells that express high or low the specific gene described in (ii) above.
  • the high or low expression of the specific genes described in (ii) in the MDSC cells are expressed more than 1.5 times and less than 1.5 times, respectively, compared to the control (group not treated with the test substance), respectively. Means that.
  • the expression level of the gene is determined by gene expression analysis methods commonly used in the art, such as real-time RT-PCR method or microarray method (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).
  • the sepsis treatment effect of H-MDSC cells used for determination as a GPCR19-associated disease therapeutic agent in sepsis mice showed a significantly higher survival rate of 803 ⁇ 4 compared to other controls.
  • H-MDSC cells produced as a result of the administration of the GPCR19 agonist test substance can be used as a treatment for sepsis or as a treatment for GPCR19-associated diseases.
  • the test substance is determined to be a treatment for GPCR19-associated disease.
  • the number of MDSC cells can be analyzed by FACS. Since the list of high or low expression genes in (i) is the same as described above, the common content between the two is omitted to avoid excessive complexity of the present specification according to the repetitive description.
  • B cells are traditionally known as cells that produce antibodies in the immune response and participate in humoral immune responses. Some of these immunoregulatory B cells are known to regulate inflammatory reactions.
  • the present inventors confirmed the increase of CD23 hi immunoregulatory B cells in the spleen by administering a GPCR19 agonist test substance to sepsis-induced mice. Sepsis treatment effect of the CD23 hi immunoregulatory B cells was also confirmed.
  • the present inventors have found that GPCR19 is involved in a higher signal transduction pathway that regulates the increase of CD23 hi immunoregulatory B cells, thereby treating GPCR19 agonists to induce GPCR19 activation and increasing CD23 hi immunoregulatory B cells. It has been shown that inflammation can be controlled through various signaling pathways.
  • the high or low expression of the specific genes described in (iv) above in CD23 hi immunomodulatory B cells is 1.5-fold and 1.5-fold, respectively, compared to the control (group not treated with test substance), respectively. It means that the expression is less than twice.
  • the expression level of the gene is determined by gene expression analysis methods commonly used in the art, such as real-time RT-PCR method or microarray method (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spr ing Harbor Press (2001)).
  • CD23 hi immunoregulatory B cells with high or low expression of the above-described genes are transformed and amplified in vivo due to the administration of GPCR19 agonists, and the transfected CD23 hi immunoregulatory B cells are administered to sepsis mice.
  • sepsis treatment effect was confirmed, which is shown in more detail in Example 15.
  • the H- produced by GPCR19 agonist test substance preferably in (i) or (ii) Sodium taurodeoxycholate (HY2191), a MDSC cell or GPCR19 agonist test material, was used.
  • the cells produced as a result of administration of H-MDSC cells were named "3 ⁇ 4M” cells, and 48 hours after the administration of sodium taurodeoxycholate, the cells produced in vivo were named "48h B H " cells.
  • the sepsis treatment effect of 3 ⁇ 4M cells used for determination as a GPCR19-associated disease treatment in sepsis mice showed a significantly higher survival rate of 100% compared to other controls (Example 13), and the treatment effect of 48h B H cells was also 80%. The survival rate was (Example 14).
  • These results were obtained by the GPCR19 agonist test substance, preferably 48 hours after administration of 3 ⁇ 4M cells or sodium taurodeoxycholate produced by administration of H-MDSC cells produced in (i) or ( ⁇ ). It is suggested that any one selected from the group consisting of B cells generated later (48h 3 ⁇ 4) can be used as a therapeutic agent for sepsis or a therapeutic agent for GPCR19-associated diseases.
  • an increase in immunoregulatory B cells showing high or low expression of the genes described above in (iv) compared to the control group not injected with the test substance was determined as a treatment for GPCR19-associated disease.
  • the number of immunoregulatory B cells exhibiting high or low expression of the genes described above in (iv) as compared with the control group which is not injected with the test substance as a result of the administration of the test substance is 2% or more, preferably 1% More preferably, by increasing 0.5% or more, the test substance is determined to be a treatment for GPCR19-associated disease.
  • the number of immunoregulatory ⁇ cells can be analyzed by FACS.
  • the test substance is determined to be a treatment for GPCR19-associated disease.
  • the invention comprises the following steps
  • step (a) the cell physiological activity modulator and the GPCR19 agonist test substance are treated to the myeloid cell population.
  • the cell bioactivity modulator used in the method of the present invention is the same as the biologically active crude substance in vivo, and the GPCR19 agonist test substance is also the same as described above, the common contents between the two are described according to the repetition description. In order to avoid excessive complexity, the description thereof is omitted.
  • Bone marrow cell population treated with the cell biological activity regulator and GPCR19 agonist test material is a cell population derived from the bone marrow of the femur and tibia, preferably hematopoietic or myeloid cells (myeloid).
  • hematopoietic lineages including B cells, T cells, NK cells, lymphocyte dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes or red blood cells
  • CMP common myeloid precursor cells
  • CLP common lymphoid progenitor cells
  • MDSC myeloid derived suppressor cells
  • the treatment of the physiologically active substance in vivo to the bone marrow cell population can be treated to the culture of bone marrow cells to be cultured, lipopolysaccharide (LPS) used as one of the physiologically active substance in vivo It is preferably in a concentration of 0.0001 mg / l to 10 mg / m £, more preferably 0.0005 mg / mi to 1 mg / m £, even more preferably 0.001 mg / mi to 0.1 mg / Most preferably 0.001 mg / i.
  • LPS lipopolysaccharide
  • the treatment of the bone marrow cell population with the GPCR19 agonist test substance may be treated with the culture medium of the cultured bone marrow cells, and preferably at a concentration of sodium taurodeoxycollide (HY2191) used as one of the GPCR19 agonist test substances.
  • HY2191 sodium taurodeoxycollide
  • the production level of the cells of (i) to (iv) in the bone marrow cell population treated with the physiological activity modulator and GPCR19 agonist test material in vivo compared to the control group not treated with the test material. Is increased by at least 2%, preferably at least 1%, more preferably at least 0.5%, and the test substance is determined to be a treatment for GPCR19-associated disease.
  • the present invention provides a method for analyzing the efficacy of a GPCR19 agonist in vivo, comprising the following steps:
  • a physiological reaction modulator in vivo is first administered to a mammal except a human to cause physiological reaction in vivo. Since the in vivo physiologically active substance used in step), the method of administration thereof, and the mammal are the same as described above, the common content between the two is to avoid excessive complexity of the present specification according to the repetitive description. Omit the description. Step (b): Administration of GPCR19 Agonist
  • GPCR19 agonist is administered to the mammal to which the physiologically active substance is administered in vivo by the method of step (b).
  • step (b) the GPCR19 agonist, the method of administration thereof and the mammal are the same as described above, so the common content between the two is omitted in order to avoid excessive complexity of the present specification according to the repeated description. do.
  • the production level of the cells of (i) to (iv) was 2% or more in the mammal to which the physiologically active substance and GPCR19 agonist were administered in comparison with the control group to which the GPCR19 agonist was not administered. , Preferably at least 1%, more preferably at least 0.5%, determines that the GPCR19 agonist is efficacious.
  • the present invention provides a method for analyzing in vitro potency of a GPCR19 agonist comprising the following steps:
  • a physiological change of bone marrow cells is first induced by treating the bone marrow cell population with a physiological activity regulator in vivo.
  • physiologically active modulators and GPCR19 agonists methods of treatment and bone marrow cell populations used in step (a) are identical to those described above, and therefore common content between the two is described herein according to the repetition. In order to avoid excessive complexity, the description is omitted.
  • the production of the cells of (i) to (iv) in the bone marrow cell population treated with the cell bioactivity modulator and GPCR19 agonist test material was compared with the control group without the test material.
  • the GPCR19 agonist is determined to be efficacious.
  • the test substance is determined to be a treatment for GPCR19-associated disease.
  • the mammal is a method characterized in that the mouse, rat, dog, monkey, goat, sheep or rabbit.
  • Mammals that can be used in the methods of the present invention can be administered to mammals other than humans and are preferably mammals, more preferably companion animals (eg dogs, cats or birds), farm animals (eg For example, cattle, sheep, pigs, horses or chickens or laboratory animals (eg, rats, mice, guinea pigs or birds), and more preferably mice, rats, dogs, monkeys, goats, sheep, rabbits. And most preferably a mouse.
  • the in vivo physiologically active regulatory substance or cell physiologically active substance used in the method of the present invention is a bacterial-derived fungal-derived, viral-derived, self-derived, or allergen It is a method characterized by the above.
  • the physiological activity modulator or cell physiological activity modulator in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance.
  • the in vivo physiologically active substance or cell physiological activity modulator used in the method of the present invention is a substance that promotes the inflammatory promoting action of target cells by acting on receptors of cells except GPCR19 . Since these are the same as the foregoing, the contents common between the two are omitted in order to avoid excessive complexity of the present specification according to the repetitive description.
  • the GPCR19 agonist used in the process of the invention is a compound of formula (1), a salt or a hydrate thereof:
  • ⁇ In the formula is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen;
  • R 2 is hydrogen or ⁇ -hydroxy;
  • R 3 is hydroxy, NH (C3 ⁇ 4) m S0 3 H (m is integer 0, 1, 2, 3, 4 or 5) or NH (CH 2 ) n C02H (n is integer 0, 1, 2, 3, 4 or 5);
  • R 5 is hydrogen, substituted or unsubstituted alkyl or aryl;
  • R 6 is hydrogen or a ring formed with 3, 4, 5 or 6 atom size with Rs and 3 ⁇ 4 attached carbon;
  • R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl;
  • R 8 is hydrogen or substituted or unsubstituted alkyl;
  • 3 ⁇ 4 is hydrogen or substituted or unsubstituted alkyl;
  • R 10 is hydrogen or substituted or unsubstituted alkyl;
  • R u
  • the common content between the two is to avoid the excessive complexity of the present specification according to the repetitive description, according to a preferred embodiment of the present invention, the method of the present invention Genes that are highly expressed or underexpressed in the MDCR produced by the administration or treatment of the GPCR19 agonist test substance, in vivo physiological activity modulator, cellular bioactivity modulator, and GPCR19 agonist are used in the same manner as described above. Therefore, in order to avoid excessive complexity of this specification according to repetitive description, the content common to both is abbreviate
  • an immunomodulatory B produced by the administration or treatment of a GPC 19 agonist test substance, an in vivo physiological activity modulator, a cell bioactivity modulator and a GPCR19 agonist for use in the method of the invention Since genes that are high or low in the cell are the same as those described above, the common content between the two is omitted to avoid excessive complexity of the present specification according to the repetitive description,
  • the present invention relates to an MDSC which is a cell formed by in vivo physiological activity modulator or cell physiological activity modulator and GPCR19 agonist and has Gr-l + CDllb + surface morphology and has anti-inflammatory activity. Provides (Mye 1 oi d-der i ved suppressor cel l).
  • the treatment of the physiologically active regulatory substance or cell physiologically active substance in vivo used in the present invention is the same as the method described above, and in vivo, for example lipopolysaccharide (LPS)
  • LPS lipopolysaccharide
  • the dosage is preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, and most preferably 20 mg. / kg is administered.
  • the degree is preferably 0.0001 mg / mi to 10 mg / m, more preferably 0.0005 to 1 mg /, even more preferably 0.001 mg / mt to 0.01 mg / and most preferably 0.001 mg / to the culture.
  • Soult taurodeoxycholate is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.1. mg / kg to 10 mg / kg, most preferably 0.5 to 8 mg / kg.
  • the sodium taurodeoxycholate is treated to the bone marrow cell population, it is preferably 0.0001 mg / mt to 100 g / mi, more preferably 0.001 mg / ml to 10 mg / mi, even more preferably 0.01 mg / mi to 1 mg / ml, most preferably 0.05 mg / mC is treated in the culture.
  • the treatment time of, for example, lipopolysaccharide (LPS) with in vivo physiological activity modulators is preferably from 6 hours to 48 hours, most preferably 18 hours.
  • the time for treating sodium taurodeoxycholate as GPCR19 agonist is preferably 6 hours to 96 hours, more preferably 12 hours or 72 hours, and most preferably 48 hours.
  • MDSCs Agonist Myeloid-derived suppressor cells
  • Gr-l + CDllb + surface phenotype Differentiated MDSC cells express the myeloid cell marker CDllb of the macrophage lineage and the marker Grl for granulocytes.
  • Grl + CDllb + can be induced using an antibody against Grl + or CDllb + and selected using flow cytometry.
  • MDSCs with Grl + CDllb + surface traits are Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Highly expresses the Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all Gbp5,
  • in vivo physiological activity control material used in the present invention is characterized in that the bacteria-derived material, fungal-derived material, virus-derived material, autologous material, allergen or cancer cell-derived material It provides MDSC generated by
  • the physiologically active regulatory substance in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance.
  • the physiologically active modulator in vivo is an antigen.
  • the GPCR19 agonist used to make the MDSC in the process of the invention is a compound of formula 1, a salt or a hydrate thereof.
  • the invention is formed by treatment with a physiologically active regulatory substance in vivo and GPCR19 agonist or in vivo physiological
  • a physiologically active regulatory substance in vivo and GPCR19 agonist or in vivo physiological Provided are immunoregulatory B cells (Regulatory B cel l) formed by MDSC treatment formed by treatment with an active modulator and GPCR19 agonist and having a CD23 hi surface trait and anti-inflammatory activity.
  • the number of cells to be administered is preferably 1 X 10 3 to 1 X 10 8 cells, more preferably. Preferably from 1 X 10 5 to 1 X 10 7 cells, most preferably from 1 X 10 6 cells.
  • the H-MDSC administration resulted in immunoregulatory B cells expressing CD23 hi surface morphology markers (eg B HM cells), and administration of these cells to sepsis mice resulted in significantly higher survival compared to controls. It was shown that this immunoregulatory B cells (eg B HM cells) exhibit anti-inflammatory activity.
  • Treatment of a bone marrow cell population with a GPCR19 agonist or a test substance thereof, such as sodium taurodeoxycholate may be treated with a culture medium of the cultured bone marrow cells, preferably 0.0001 mgM to 100 g / t, more preferably Is 0.001 mg / ⁇ to 10 g / ml, even more preferably 0.01 g / l to 1 mg /, most preferably 0.05 mg / is treated to the culture.
  • a culture medium of the cultured bone marrow cells preferably 0.0001 mgM to 100 g / t, more preferably Is 0.001 mg / ⁇ to 10 g / ml, even more preferably 0.01 g / l to 1 mg /, most preferably 0.05 mg / is treated to the culture.
  • CD23 hi a phenotypic marker that is expressed in B cells
  • An early marker is a marker that appears during maturation on the surface of the precursor B cell and remains while the immunoregulatory B cells are alive.
  • CD23 hi immunoregulatory B cells can be stained using CD23 + antibodies and then selected using a flow cytometer.
  • immunoregulatory B cells expressing CD23 hi surface morphological markers are Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc , Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hspala, Srp, ksp , Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhg
  • immunoregulatory B cells expressing CD23 hi surface morphological markers express CD21 and CD23.
  • CD23 (FCeRII) expressed in the immunoregulatory B cells is a type II molecule of the C-lectin group including lymphocyte tracking receptor (MEL-14) and endothelial leukocyte adhesion molecule -KELAM-1).
  • CD23 (FCeRII) is a low affinity receptor for IgE.
  • Dendritic cells, B cells, T cells and macrophages in mammals Various blood constituent cell types, including CD23, express CD23 on the cell surface.
  • CD23 molecules are also found in dissolved form in biological fluids. Soluble CD23 (sCD23) molecules are produced by protein cleavage of cell membrane permeable receptors.
  • CD21 is a marker expressed in B cells, complexes with CD19 and TAPA-1 and binds to the complement component 3Cd and is a negative component of activation and proliferation of B cells.
  • the physiologically active substance or cell activity modulator in vivo used in the present invention is a bacteria-derived, fungal-derived, virus-derived, autologous or allergen (al lergen) Characterized by the immunoregulatory B cells produced.
  • the physiologically active regulatory substance or cell activity modulator in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance.
  • LPS lipopolysaccharide
  • the GPCR19 agonist used to make the MDSC or immunoregulatory B cells (48H 3 ⁇ 4 cells) or more specifically CD21 + CD23 + B cells in the method of the present invention is a compound of formula 1 Or salts or hydrates thereof.
  • the present invention is treated with in vivo physiological activity modulators and GPCR19 agonist treated Gr-1 + CDllb + MDSC (H-MDSC), in vivo physiological activity modulators and GPCR19 agonist treated immunomodulatory B cells (B HM cell) formed by the formed Gr- 1 + CDllb + MDSC treatment, more specifically, formed by the in vivo physiological activity regulatory substance and GPCR19 agonist is treated Gr-1 + CDllb + MDSC treatment formed CD21 + CD23 + B cells, or immunoregulatory B cells (48H 3 ⁇ 4 cells) formed by treatment with physiologically active modulators and GPCR19 agonists in vivo (48H 3 ⁇ 4 cells) or more specifically CD21 + formed by treatment with physiologically active modulators and GPCR19 agonists in vivo
  • an anti-inflammatory pharmaceutical composition comprising CD23 + B cells as an active ingredient.
  • an anti-inflammatory pharmaceutical composition containing preferably Gr-1 + CDllb DSC or CD23 hi immunomodulatory B cells (48H 3 ⁇ 4 cells or B HM cells) or B220 + CD21 + CD23 + B cells as an active ingredient of the composition of the present invention to be.
  • the disease that can be prevented or treated with the anti-inflammatory pharmaceutical composition produced by the method of the present invention is preferably sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, Contains gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil
  • a pharmaceutically acceptable carrier included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, Contains gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenz
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives in addition to the above components.
  • lubricants wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives in addition to the above components.
  • suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciencesi 19th ed. 1995).
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraarticular injection, or the like.
  • Appropriate dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction sensitivity. Can be. Exemplary dosages of the pharmaceutical compositions of the invention are 0.001-1000 nig / kg.
  • the pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Can be prepared or incorporated into a multi-dose container. have.
  • the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of axes, powders, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.
  • the present invention is Gr-l + CDllb ⁇ DSCOl-MDSC formed by in vivo physiological activity modulators and GPCR19 agonist treatment, Gr formed by treatment with physiologically active modulators and GPCR19 agonist in vivo - 1 + CDllb + immunoregulatory formed by MDSC treatment B cells (3 ⁇ 4 «cells), and more particularly to CD21 + formed by the controlled in vivo physiological active substance and GPCR19 agonist is treated formed Gr-l + CDlll ⁇ iDSC treatment CD23 + B cells, or immunoregulatory B cells (48H B H cells) formed by treatment with physiologically active regulatory substances and GPCR19 agonists in vivo (48H B H cells) or more specifically CD21 + CD23 formed by treatment with physiologically active regulators and GPCR19 agonists in vivo + B cells comprising the step of administering a pharmaceutically effective amount of the composition to the object (Subject) comprising containing as an active ingredient, the prevention of inflammation, yet Provide treatment.
  • a pharmaceutically effective amount of the composition
  • the method of the present invention utilizes the composition described above, the common content between the two is omitted in order to avoid excessive complexity of the present specification.
  • the number of H-MDSC cells was increased by the administration of sodium taurodeoxycholate (HY2191) in the inflammation-induced mice due to the administration of lipopolysaccharide (LPS) and increased H-MDSC.
  • LPS lipopolysaccharide
  • the number of immunoregulatory B cells (48h 3 ⁇ 4 cells) is increased by administration of sodium taurodeoxycholate (HY2191) in mice induced with inflammation due to administration of lipopolysaccharide (LPS).
  • HY2191 sodium taurodeoxycholate
  • LPS lipopolysaccharide
  • the number of BHM cells is increased by administration of H-MDSC in inflammation-induced mice by administration of lipopolysaccharide (LPS), and the increased 3 ⁇ 4M cells show a significant sepsis treatment effect.
  • LPS lipopolysaccharide
  • the number of B220 + CD21 + CD23 + B cells increased and increased B220 + CD21 + CD23 by administration of H-MDSC in mice induced with inflammation by administration of lipopolysaccharide (LPS). + B cells show significant sepsis treatment effect.
  • LPS lipopolysaccharide
  • the method of the present invention provides a method for screening a therapeutic agent for a GPCR19-associated disease or a method for analyzing the efficacy of a GPCR19 agonast to provide an anti-inflammatory pharmaceutical composition.
  • 1A-1C are graphs showing the survival rate after injection of sodium taurodeoxycholate (HY2191) into various types of sepsis-induced mice to see the treatment of sepsis with sodium taurodeoxycholate (HY2191). An image showing the experimental method was attached at the top of the graph.
  • Figure 1A is a graph showing the survival rate ⁇ when implanted "hayeoteul the HY2191 in LPS induced septic mice after LPS treatment for 30 minutes and 24 hours
  • Figure 1B showing the survival rate hayeoteul injection after CLP induction two hours
  • HY2191 all CLP induced sepsis mouse 1C is a graph showing that HY2191 does not show sepsis treatment effect in mice lacking B cells and T cells (Rag2 K0).
  • 2A-2B are graphs showing inhibition of inflammatory cytokine production in vivo by HY2191.
  • 2A is a graph showing that HY2191 reduces inflammatory cytokine concentrations in blood induced by LPS.
  • 2B is a graph showing that HY2191 decreases the inflammatory cytokine concentration in blood induced by CLP.
  • FIGS. 3A-3B are graphs showing relief of hypotension by sepsis after HY2191 treatment.
  • Figure 3A shows that blood pressure drop induced by LPS was induced by HY2191. Normalized is a graph showing rise in mean blood pressure ( ⁇ Hg).
  • FIG. 3B is a graph showing blood BUN and AST reduction, respectively, that renal failure and liver dysfunction induced by LPS are alleviated by HY2191.
  • FIG. 4 is a graph depicting the absolute numbers of splenocytes, in particular CDllb + cells and B220 + B cells, in LPS induced sepsis mice.
  • FIG. 5 is a graph showing the absolute number of splenic CD4 + Foxp3 + T cel l (regulatory T cel l, Treg) of LPS-induced sepsis mice.
  • FIG. 6 are graphs showing that activation of spleen cells by LPS is inhibited by HY2191.
  • activation of spleen CD11C + cells was expressed by the mean fluorescence intensity (Mean f luoresense intensi ty, MFI) of the CD86 activation marker.
  • activation of splenic B220 + B cells was expressed by the mean fluorescence intensity (Mean f luoresense intensi ty, MFI) of the CD86 activation marker.
  • FIG. 6C is a FACS plot showing the relative percentage of B220 + CD86 + B cells overexpressing CD86, a cell activation marker in spleen B220 + B cells.
  • FIG. 7 is graphs showing that Gr-1 + CDllb + cells (H-MDSC) increase in LPS induced sepsis mice treated with HY2191 and that they show a sepsis treatment effect.
  • FIG. 7A is a graph showing significant increase in Gr-1 + CDllb + MDSC cell numbers in the spleen of LPS induced sepsis mice after 24 and 48 hours in the HY2191 treated group. It is also a graph showing that HY2191 does not change the Gr-1 + CDllb + MDSC cell numbers in mice lacking B cells and T cells (Rag2 K0).
  • 7 is a FACS plot depicting the relative percentage (%) of splenic cells of Bfe H-MDSC.
  • 7C is a FACS histogram showing the immaturity of H-MDSC as the percentage of CD31 + cells that are myeloid cell immature markers.
  • 7C below is a FACS plot showing that H-MDSC is Ly6G + Ly6C inter F4 / 80 low .
  • 7D is a heat-map comparing the expression genes of H-MDSC and L-MDSC. Red indicates genes overexpressing in H-MDSCs and green underexpressed genes compared to L-MDSCs (Gr-1 + CDllb + MDSCs harvested from LPS-induced sepsis mice not treated with HY2191).
  • FIG. 8 is a graph illustrating the survival rate after adopting ive transfer of various types of cells in sepsis-induced mice to see the treatment effect of sepsis. After H-MDSC Harvest and Administration in LPS-Induced Sepsis Mice An image showing the overall experimental method for analyzing the therapeutic effect is attached to the upper part of the graph.
  • FIG. 9 is a graph depicting the absolute number and relative percentage (%) of in vivo immune cells induced by H-MDSC transduced in LPS sepsis induced mice.
  • 9A is a graph of FACS plot images (top) and replicates (bottom) depicting an increase in the relative and absolute numbers of splenic B cells 24 h after H-MDSC transfection.
  • 9B is a graph of FACS plot images (top) and replicates (bottom) depicting an increase in the relative and absolute numbers of spleen and bone marrow CD11B + Gr-1 + F4 / 80 cells after 24 hours of H-MDSC transfusion.
  • FIG. 9A is a graph of FACS plot images (top) and replicates (bottom) depicting an increase in the relative and absolute numbers of spleen and bone marrow CD11B + Gr-1 + F4 / 80 cells after 24 hours of H-MDSC transfusion.
  • FIG. 9C is a FACS plot image (top) and graph (bottom) showing that the relative and absolute numbers of splenic T cells (CD4 + and CD8 + T cells) do not change 24 hours after H-MDSC transfusion.
  • FIG. 9D is a graph showing no significant change in splenic CD11C + dendritic cell absolute number after 24 hours of H-MDSC transfusion.
  • B HM cells splenic B cells proliferated by H-MDSCs.
  • the graph shows the survival rate after ive transfer.
  • FIG. 11 is a graph showing the effect of sepsis treatment in the presence of i-MDSC in B- and T-cell deficient mice (Rag2 K0).
  • 12A-C are graphs illustrating changes in in vivo splenic immune cells induced by 48h 3/4 cells transduced into sepsis-induced mice.
  • 12A is a FACS plot image depicting the activation of splenic B cells by LPS at 24 h after 48 h 3 ⁇ 4 cells transduction.
  • 12B is a FACS plot image showing the activation of splenic T cells (CD4 + and CD8 + T cells) by LPS after 24 h of 48h 3 cells.
  • FIG. 12C is a FACS plot image depicting the relative percentage of CDllb + Ly6G + Ly6C inter cells increased in the spleen 24 hours after transfection of 48h 3/4 cells.
  • FIG. 13 is a FACS plot showing that the phenotype of 48h B H cells differs from previously known immunoregulatory B cells.
  • FIG. 13A is a FACS plot in which 48h 3 cells depict the expression of CDld and CD5, Bla B cell characteristic markers.
  • 13B 48 h B H cells are FACS plots depicting CD93 expression, an immune regulatory T2-MZP B cell characteristic marker.
  • FIG. 13C is a FACS plot in which 48h B H cells depict IgM expression, a characteristic marker of mature B cells, compared to B cells observed in mice induced with sepsis with LPS.
  • L-MDSC harvested in the LPS induced septic mice not treated with HY2191 Gr-l + CDllb + MDSC
  • H-MDSC a Gr-l harvested in the LPS induced septic mice treated with HY2191 + CDllb + MDSC
  • 14B is a graph plotting the absolute number of B220 + CD23 + cell count increase in the spleen after 24 hours of L-MDSC or H-MDSC transduction in mice induced with sepsis with LPS.
  • 15A is a FACS plot showing CD23 expression 48 hours after HY2191 injection in mice in which B220 + CD23 hi CD21 hi cells (CD23 hi 3 ⁇ 4 Cel l) induced sepsis with LPS.
  • 15B is a graph illustrating the survival rate of mice after adoptive transfer into sepsis-induced mice to see sepsis treatment effect of B220 + CD23 hi CD21 int cells (CD23 hi 3 ⁇ 4 Cel l). The timing of harvesting CD23 hi B H cells from LPS-induced sepsis and the overall experimental method to analyze the therapeutic effect after administration are shown in the upper graph.
  • FIG. 16 is a graph depicting the induction of B cell and CDllb + cell proliferation in vivo by mouse adoptive transfer of CD23 hi 3 ⁇ 4 cells in mice induced with sepsis with LPS.
  • CD23 hi B H cells were transfected into mice 24 and 48 hours after the spleen B220 + B cells and CDllb + Gr-1 + absolute numbers were compared with the control (PBS) and CD23 10 B H cells.
  • 17A shows LPS (lmg / ml) and HY2191 (50 mg / ml) in mouse bone marrow cells.
  • mice All experiments used 8-12 week old C57BL / 6 mice (SLC, Japan) to set up LPS (sigma ( US) -induced sepsis model.) Salmonella LPS (sigma, USA) 20 mg / kg (12,000 EU / g) 30 minutes and 24 hours after intraperitoneal administration of mice into the control vein (PBS, WelGENE, Korea) or sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was injected dropwise for 15 minutes using Medfusion2001 (medex, USA) (Medfusion2001, medex, USA) The survival rates of the experimental and control groups were examined over time and the results are shown in Figure 1A.
  • mice in the experimental group administered sodium taurodeoxycholate were 4 times higher in survival after 30 minutes and 5 times higher in 24 hours after the administration of sodium taurodeoxycholate.
  • each of the 10 C57BL / 6 mice contained sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand) was instilled in the tail vein for 20 minutes (experimental group) and the remaining 10 C57BL / 6 mice were administered only PBS in the same way (control).
  • HY2191 sodium taurodeoxycholate
  • HY2191 New Zealand Pharmaceuticals Ltd, New Zealand
  • Example 3 Analysis of the Importance of B Cells and T Cells in LPS-Induced Sepsis Models
  • the same LPS as in Example 1 was used to induce sepsis, and mice using B cells and T cells deficient (Rag2 K0) were used.
  • Sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was tested in Example 1 "light method. As shown in Figure 1C, all subjects died within 48 hours, confirming no change in survival in the experimental and control groups. This suggests that HY2191 requires T cells or B cells to treat sepsis Example 4.
  • Example 4 Sodium taurodeoxycholate (HY2191, sodium taurodeoxycholate HY2191) Inhibitory Effect of Inflammatory Cytokine Production
  • Example 5 Effect of Sodium Taurodeoxycholate (HY2191, Sodium Taurodeoxycholate; HY2191) on Inflammatory Cytokine Changes in a CLP Sepsis Model
  • Example 4 shows changes in inflammatory cytokines that occur upon CLP induction as in Example 2. It confirmed by the same method as the method of. Blood was collected at 4, 24, and 48 hours after CLP to separate serum, and then cytokine expression was confirmed using a cytometric bead array (CBA) kit.
  • CBA cytometric bead array
  • TNF-a, IL-lb, MCP-1 showed a significant decrease by HY2191 administration at 24 h after CLP induction (FIG. 2B).
  • Example 6 Normalization of Blood Pressure Lowering after Sepsis Treatment with Sodium Taurodeoxycholate (HY2191, HY2191)
  • HY2191 organ urea nitrogen (BUN) and aspartate aminotransferase (AST) levels in the blood according to the change in organ damage following HY2191 administration. It was confirmed by (Fig. 3B). LPS induced After 30 minutes, HY2191 or PBS was instilled into the tail and blood was collected at 24 and 48 hours to separate serum. BUN was measured in the isolated serum to observe renal impairment, and AST was measured to evaluate liver impairment. HY2191 was found to significantly reduce the elevation of BUN, indicating kidney damage at 48 hours of LPS induction (FIG. 3B left).
  • Example 9 Identification of T Regulatory Cells Increased by Sodium Taurodeoxycholate (HY2191, HY2191) in LPS-Induced Sepsis Model
  • Example 10 Inhibitory Effect of Sodium Taurodeoxycholate (HY2191, HY2191) on Dendritic Cell Activation in LPS-induced Sepsis Model
  • CDllc (+) / 7AAD ( ⁇ ) cells were selected and analyzed for CD86 expression in dendritic cell costimulatory molecules.
  • the mean fluorescence intensity (Mean f luoresence intensi ty, MFI) expressed CD86 expression ability, and it was confirmed that expression was significantly inhibited by HY2191 after 24 hours after LPS induction.
  • MFI mean fluorescence intensity
  • L-MDSC myeloid derived suppressor cells
  • H-MDSC splenocytes obtained by regeneration of each mouse, and at the same time, the number of cells with high Gr-1 expression (hereinafter referred to as H-MDSC) was significant. Increased (FIG. 7A, FIG. 7B). Immaturity of L-MDSC and H-MDSC cells was confirmed using CD31, a myeloid cell immature marker (upper FIG. 7C). CD31 + Expression in Two Cells No significant difference in cell proportions was observed. L-MDSC and H-MDSC were F4 / 80 lo Gr-1 + with Ly6g + Ly6c + phenotype (below Figure 7C). MDSC with this phenotype showed a significant increase in sepsis mice administered sodium taurodeoxycholate.
  • H-MDSC H-MDSC
  • Grl + CDllb + MDSC L-MDSC
  • genes with increased expression and genes with reduced expression were observed (FIG. 8B, Table 1, and Table 2).
  • the expression differences of genes were compared with IUumina Sentrix MouseRef-8 (vl.1) BeadChip arrays (Ilhimina, USA). Genes with less than 0.05 and a fold change of 2 or more or -2 or less were selected and the list is shown in Tables 1 to 2 below:
  • mice were divided into three groups according to the expression level of CDllb and Gr-1, which were separated and selected by FACS sorter (FIG. 8). The number of cells of the isolated cells was measured by a hemocytometer and mixed in the same amount in PBS so that the number of living cells was 1 X 10 5 and injected into the sepsis-induced tail vein (Fig. 8). At this time, mice were intraperitoneally administered LPS at a lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before cell injection. Five mice were used in the PBS group, the L-MDSC, and the H-MDSC group, respectively, three in the group injected with Gr-l int cells and four in the group injected with Gr- ⁇ cells.
  • mice receiving H-MDSC showed significantly increased survival (80% survival) compared to L-MDSC (40% survival) and other surface phenotype cells.
  • splenic cell changes were observed in mice injected with cells (FIG. 9).
  • L-MDSC were protonated.
  • Splenocytes of mice were harvested 24 hours after cell proliferation.
  • FIG. 9A top: representative FACS plot, bottom: replicate graph
  • B220 + cells were significantly increased when H-MDSC was protonated.
  • FIG. 9A top: representative FACS plot, bottom: replicate graph
  • mice The same C57BL / 6 mice as in Example 1 were used. Five mice were used in the PBS group, the normal mouse B cell group, the 48h B L cell group, the 24h 3 ⁇ 4 cell group, the 48h 3 ⁇ 4 cell group, and the B HM cell group. 24 hours after LPS and sodium taurodeoxycholate were administered to secure B HM cells, the mice were sacrificed and spleens were extracted, and MDSCs expressing high CDllb and Gr-1 molecules in these splenocytes were FACS. Sorting and sorting by sorter (upper left in Figure 10).
  • mice 1 ⁇ 10 5 live (7 ⁇ ) CDllb + Gr-1 + cells were suspended in PBS and injected into the sepsis-induced sepsis-induced B6 mice 24 hours ago by CDVbb + Gr to secure B HM cells from these mice.
  • mice were sacrificed to harvest the spleen, and B cells expressing high B220 molecules were separated and selected by FACS sorter (B HM cells, left side of FIG. 10).
  • FACS sorter B HM cells, left side of FIG. 10
  • mice were regenerated 24 hours or 48 hours after administration of LPS and sodium taurodeoxycholate, and the spleen cells expressed high B220 molecules in the splenocytes.
  • mice were isolated and sorted by FACS sorter (24h 3 ⁇ 4 cells or 48h 3 ⁇ 4 cells, middle in FIG. 10).
  • FACS sorter 48h B L Cells, right above FIG. 10
  • 1 x 10 6 (7 ⁇ ) living cells were suspended in PBS to get sepsis These cells were injected into the tail vein of induced B6 mice.
  • the mice adopting the cells were induced sepsis by intraperitoneally administering LPS (20 mg / kg, 12,000 EU / g) 24 hours before B cell injection.
  • H-MDSC and BHM cells produced in C57BL / 6 mice were transfused into Rag2K0 mice without B cells and T cells 24 hours after LPS induction to confirm the effect in the sepsis model.
  • PBS group, B HM cells (1 ⁇ 10 6 , 5 ⁇ 10 6 ), B HM cells (1 ⁇ 10 6 , 5 ⁇ 10 6 ) and 48h H-MDSC (1 ⁇ 10 5 ) groups were used two by one. .
  • 48h 3 ⁇ 4 cells were prepared in the same manner as in Example 13, after LPS induction.
  • mice were left 24 h after protonation of 1 ⁇ 10 6 48h B H cells at 24 h. Regeneration was performed to confirm the activity (CD86 expression) of B cells that are B220 + in splenocytes.
  • CD86 expression was analyzed in B cell co-stimulatory molecules. The expression of CD86 was decreased when 3 ⁇ 4 cells were protonated compared to the control group treated with PBS at 24 hours (FIG. 12A).
  • FIG. 12B it was confirmed that CD69 hi CD62L 10 T cells in LPS administration were reduced in CD4 + T cells or CD8 + T cells among splenocytes.
  • FIG. 12A it was confirmed that CD69 hi CD62L 10 T cells in LPS administration were reduced in CD4 + T cells or CD8 + T cells among splenocytes.
  • Spleen cells are anti-mouse CD11MM1 / 70), anti-mouse B220 (RA3- 6B2), anti-mouse Gr-1 (RB6-8C5), anti-mouse F4 / 80 CI: A3-1), anti-mouse CD23CB3B4 ), Anti-mouse CD21 (7G6) and anti-mouse CD93 (M4.1), and all other antibodies were stained for 15 minutes. After washing, stained cells were stained with 7-amino actinomycin D (7 D) for 10 minutes to identify only living cells and measured using FACS Canto II (BD, USA). It was. Data were analyzed using Flowjo software (Tree Star, Inc., USA).
  • mice 48 hours after administration of LPS and sodium taurodeoxycholate, the mice were sacrificed and the spleens were extracted.
  • CD23 hi B H cells expressing the phenotype of B220 + CD23 hi CD21 hi Gr- ⁇ CDllb— in splenocytes were isolated and selected by FACS sorter.
  • CD23 10 B H cells exhibiting B220 + CD23 lo CD21 hi Gr-rCDllb phenotype were also selected and used as controls.
  • 1 ⁇ 10 5 and 0.5 ⁇ 10 5 live (7 ⁇ ) cells were suspended in PBS and injected into the tail vein of the mouse. At this time, mice were intraperitoneally administered LPS at a lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before cell injection.
  • B220 + B cells 48 h B L cel l isolated after 48 hours without treatment with HY2191 in sepsis-induced mice or CD23 lo CD21 + B220 + B cells (CD23 isolated after 48 hours with HY2191 treatment in sepsis mice) 10 3 ⁇ 4 cel l) specifically in B220 + B cells (48h 3 ⁇ 4 cel l) or CD23 hi CD21 + B220 + B cells (CD23 hi B H cel l) isolated after 48 hours of HY2191 treatment in sepsis mice Differences in expression of genes with increased or decreased expression were compared to I lumina Sentrix MouseRef-8 (vl .1) BeadChi arraysd 1 lumina, USA. Genes with less than 0.05 and a fold change of 2 or less or -2 or less were selected, and Moktok are shown in Tables 3 to 8 below.
  • Example 20 Preparation of H-MDSC in Vitro Using LPS and Sodium Taurodeoxycholate and Analysis of Sepsis Treatment Effect of These Cells
  • the degree of CDllb + Gr-l + MDSC differentiation was compared between MDSC (LexMDSC) harvested by treating only bone marrow cells in vitro with LPS and MDSC (HexMDSC) harvested after treating LPS and HY2191 with bone marrow cells simultaneously.
  • LexMDSC MDSC
  • HexMDSC MDSC
  • the absolute number of CDllb + Gr-1 + cells increased 36% and 34% in the HY2191 treated group compared to the control group, respectively, by flow cytometry (FASC) (FIG. 17A).
  • LexMDSC and HexMDSC which had completed 24-hour incubation, were intravenously injected into C57BL / 6 mice injected with LPS 10 mg / kg 24 hours ago.
  • mice injected with HexMDSC survived, and all of the groups treated with PBS, LexMDSC-treated group and Media-treated group died (FIG. 17B).

Abstract

The present invention provides a screening method for a therapeutic agent of GPCR19-related condition. In addition, the present invention provides an efficacy analysis method for a GPCR19 agonist. Also, the present invention provides a myeloid-derived suppressor cell (MDSC) which is formed by being treated with an in vivo physiological activity regulator and a GPCR19 agonist, and has Gr-1+CD11b+ surface phenotype and anti-inflammatory activity. Also, the present invention provides an immunoregulatory B cell which is formed by being treated with an in vivo physiological activity regulator and a GPCR19 agonist, or is formed by the H-MDSC treatment, and has B220+ surface phenotype and anti-inflammatory activity. Also, the present invention provides an anti-inflammatory pharmaceutical composition comprising an H-MDSC or BHM cell as an active ingredient. Also, the present invention provides an anticancer pharmaceutical composition comprising an H-MDSC, 48H BH or BHM cell as an active ingredient. Also, the present invention provides an anti-inflammatory pharmaceutical composition comprising a GPCR19 agonist which is composed for the purpose of in vivo amplifying an H-MDSC or 48H BH cell. According to the present invention, the number of H-MDSCs is increased by administration of sodium taurodeoxycholate (HY-2191) in a mouse in which inflammation is induced by administration of lipopolysaccharide (LPS), and the increased H-MDSCs reveal a marked therapeutic effect on sepsis. According to the present invention, the number of immunoregulatory B cells (48h BH cells) is increased by administration of sodium taurodeoxycholate (HY-2191) in a mouse in which inflammation is induced by administration of lipopolysaccharide (LPS), and the increased 48h BH cells reveal a marked therapeutic effect on sepsis. According to the present invention, the number of BHM cells is increased by administration of H-MDSC in a mouse in which inflammation is induced by administration of lipopolysaccharide (LPS), and the increased BHM cells reveal a marked therapeutic effect on sepsis.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
GPCR19 경로의 활성화에 의한 골수 유래 면역 조절 세포 및 면역 조절 B 림프구의 체내외 증폭  In Vitro Amplification of Bone Marrow-derived Immune Regulatory Cells and Immune Regulatory B Lymphocytes by Activation of GPCR19 Pathway
【기술분야】 Technical Field
본 특허출원은 2011년 11월 17일에 대한민국 특허청에 제출된 대한민국 특허출원 제 10-2011-0120247 호에 대하여 우선권을 주장하며， 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.  This patent application claims priority to Korean Patent Application No. 10-2011-0120247 filed with the Korean Intellectual Property Office on November 17, 2011, the disclosure of which is incorporated herein by reference.
본 발명은 GPCR19 아고니스트를 생체 내외에서 세포에 처리하여 골수유래 면역조절 세포(¾ 610 0-(1^ ^6(1 suppressor cel l , MDSC) 또는 면역조절 B 세포수를 증가시키고 이 세포들의 항염증 표현형을 증대시키는 방법에 관한 것이다. 본 발명은 GPCR19 수용체를 경유하는 신호전달체계의 활성화가 질환의 진행 및 발생의 조절에 중대한 영향을 미치는 질환 (이하 GPCR19-연관 질환)들의 치료를 목적으로 GPCR19 아고니스트를 스크리닝하는 방법에 관한 것이다. 또한 본 발명은 GPCR19-연관 질환 치료제로 GPCR19 아고니스트의 효능 분석 방법에 관한 것이다.  The present invention treats cells in vivo and externally with GPCR19 agonists to increase bone marrow-derived immunomodulatory cells (¾ 610 0- (1 ^^ 6 (1 suppressor cel l, MDSC) or immunoregulatory B cell counts). FIELD OF THE INVENTION The present invention relates to GPCR19 for the treatment of diseases in which activation of the signaling system via the GPCR19 receptor has a significant effect on the regulation of disease progression and development (hereinafter GPCR19-associated disease). The present invention also relates to a method for analyzing the efficacy of a GPCR19 agonist as a therapeutic agent for GPCR19-associated diseases.
【배경기술】 Background Art
패혈증은 미생물 감염 후 보체계， 혈액응고계, 선천 면역 세포계 등의 동시 다발적 활성화에 기인한 전신성 염증 반웅 증후군 (systemic inf lammatory response syndrome,' SIRS)을 시작으로 점차 환자가 쇼크에 이르러 30~50%의 환자가 사망하는 질환이다. 따라서 이러한 패혈증은 한 두 가지 염증 유발 경로 (TLR4 pathway, complement and coagulat ion pathway) 차단을 통하여 억제되지 않을 것이라는 것은 쉽게 추정 가능하며， 대부분의 환자에서 치료가 시작되는 시기는 이미 염증 유발 경로가 모두 활성화 된 후일 경우 (establ ished sepsi s)가 많다. 지금까지 염증 유발 경로 1개의 차단을 목표로 개발된 치료제가 임상시험에서 효과가 없는 것은 상기의 이유 때문으로 추정된다. 전세계적으로 유일하게 FDA허가를 받아 시판되고 있는 Li l ly사의 Xygris 또한 28일 생존율이 6% 환자에서만 증가되는 저조한 유효성은 혈액웅고기전을 조절하는 APC(act ivated protein C)가 주성분이기 때문이다. 마우스 층수돌기의 대장과 연결 부위를 결찰 한 후 층수 돌기 부분을 천공 하면 (CLP, cecal ligation and puncture) 패혈증이 유도되며， 이로 인하여 비장과 골수에서 CDllb+Gr-l+ 골수유래 면역 억제세포 (Myeloid- derived suppressor cell.MDSC)의 수가 현저히 증가된다. 이 세포들은 CD4+ T 세포의 활성화를 억제하고, CD8+ T세포의 IFNY 분비를 억제한다 [Delano MJ,et al. ,(2007)J Exp Med 204(6)： 1463-1474] . 이러한 보고는 패혈증 환자에서도 MDSC가 면역 억제 상태 유도에 중요한 역할을 함으로써 2차 감염을 용이하게 하여 환자의 사망률을 높일것이라는 예측을 뒷바침하고 있다 (Delano MJ'et al.,(2007)J Exp Med 204(6)： 1463-1474] . 그러나 이러한 예상과는 반대로 5 X 106개의 MDSC를 CLP유도 1시간 전과 유도 24시간후에 정맥주사로 양자이입 (adoptive transfer)하면 패혈증에 의한 전신 염증을 억제함으로써 오히려 사망률을 감소시킨다는 보고도 있다 [Sander, L.E., et al.,(2010) J Exp Med 207(7)： 1453-64] . 따라서 MDSC가 패혈증환자에서 면역억제에 의한 2차감염 빈도를 높여 사망를을 높일지， 아니면 전신염증 반응을 억제하여 오히려 환자의 생존율을 높일지에 대하여는 논란의 여지가 있는 상황이다. Sepsis begins with systemic inf lammatory response syndrome (SIRS) due to simultaneous activation of the complement system, coagulation system, and innate immune cell system after microbial infection. Is a disease in which patients die. Therefore, it is easy to assume that sepsis will not be inhibited through the blocking of one or two TTL4 pathways (complementary and coagulat ion pathways), and in most patients the timing of treatment is already active. There are many cases of establ ished sepsi s. It is presumed that the therapeutic agents developed so far aimed at blocking one inflammation-inducing pathway are ineffective in clinical trials. Ligly's Xygris, the world's only FDA-licensed product, also has a 28-day survival rate of 6% in patients with poor efficacy, mainly due to APC (act ivated protein C), which regulates blood lactation. Because. After ligation of the colon and connecting sites of the mouse dendritic spine, perforation of the dendritic spine (CLP, cecal ligation and puncture) induces sepsis, which causes CDllb + Gr-l + bone marrow-derived immune suppressor cells (Myeloid-) in the spleen and bone marrow. The number of derived suppressor cells (MDSCs) is significantly increased. These cells inhibit the activation of CD4 + T cells and inhibit the IFNY secretion of CD8 + T cells [Delano MJ, et al. , (2007) J Exp Med 204 (6): 1463-1474. These reports support the prediction that MDSC plays an important role in inducing immune suppression in sepsis patients, thereby facilitating secondary infections and increasing patient mortality (Delano MJ 'et al., (2007) J Exp Med 204 (6): 1463-1474.] Contrary to these expectations, however, the 5 x 10 6 MDSCs injected intravenously (1 h before CLP induction and 24 h after induction) suppress the systemic inflammation caused by sepsis. It has also been reported to reduce mortality [Sander, LE, et al., (2010) J Exp Med 207 (7): 1453-64] .Therefore, MDSC increases mortality by increasing the frequency of secondary infection by immunosuppression in sepsis patients. Whether to suppress the systemic inflammatory response or to increase the patient's survival rate is controversial.
MDSC는 패혈증뿐만 아니라 여러 자가 면역 질환 마우스 모델에서도 수적 증가가 보고 되고 있지만, 질환의 진행을 악화시키는지 아니면 억제하는 지에 대한 확실한 결론이 나지 않고 있다. 전신성 흥반성 낭창증의 경우, 증상이 활발한 상태의 MRL-faslp^}우스의 신장과 혈액에서 MDSC가 증가되어 있으며 ex vivo 실험을 통하여 CD4+ T 세포의 증식을 억제함이 확인되었다 [Cripps, J.G. and J.D. Gorham, (2011) Int Immunopharmacol； Iwata Y. , et al . ,(2010) Clin Exp Nephrol 14:411-417] . MDSC has been reported to have increased numbers in several autoimmune mouse models, as well as sepsis, but there is no clear conclusion as to whether to worsen or inhibit disease progression. In case of systemic lupus erythematosus, MDSC is increased in kidney and blood of active MRL-fas lp ^} mice, and ex vivo experiments confirmed that it inhibits the proliferation of CD4 + T cells [Cripps, JG and JD Gorham, (2011) Int Immunopharmacol; Iwata Y., et al. , (2010) Clin Exp Nephrol 14: 411-417.
B 세포 (B cell)는 전통적으로 항체를 생성하여 체액성 면역반웅 (humoral i醒 une response)에 관여한다고 알려져 왔다. 최근에 세포성 면역 반웅과 염증 반웅을 조절하는 기능을 가진 B10 면역조절 B 세포， CD5+Bla 세포， CD + 변연부 (marginal zone) B 세포 및 T2- MZP (Tr ans i t i ona 1 -2-mar g i na 1 zone precursor) B 세포 등의 면역조절 B 세포 (Regulatory B cell, Breg)들이 보고되고 았다. 아직 면역조절 B 세포 특이적인 표면 분자 및 세포질 내 표적 분자가 확인 되지 않아서 상기 세포들의 명확한 특성이 규명 되어 있지 않다. 그러나 면역조절 B 세포는 대부분 B2 B 세포에서 유래되며 (CD5+Bla cell은 예외) IL— 10을 분비하는 공통점을 가지고 있다. 면역조절 B 세포 중 ex r/ra와 in vivo 실험을 5 통하여 면역 억제 기능이 확인된 면역조절 B 세포는 B10 면역조절 B 세포와 T2-MZP 면역조절 B 세포이다 [Mauri, C. and P. A. Blair, (2010) Nat Rev Rheumatol, 6(11) :636-43]. B cells have traditionally been known to produce antibodies and participate in humoral immune response. Recently, B10 immunomodulatory B cells, CD5 + Bla cells, CD + marginal zone B cells and T2-MZP (Tr ans iti ona 1-2-mar gi Regulatory B cells (Bregs), such as na 1 zone precursor B cells, have been reported. Immune Regulatory B Cells Specific surface molecules and target molecules in the cytoplasm have not been identified, so no specific characteristics of these cells have been identified. However, immunoregulatory B cells are mostly derived from B2 B cells (except for CD5 + Bla cells) and have a common secretion of IL-10. Among the immunoregulatory B cells, immunoregulatory B cells whose immunosuppressive function was confirmed through ex r / ra and in vivo 5 were B10 immunoregulatory B cells and T2-MZP immunoregulatory B cells [Mauri, C. and PA Blair, (2010) Nat Rev Rheumatol, 6 (11): 636-43].
T2-MZP 면역조절 B 세포 (CD19+, CD21high ,CD23+, CD24 high, CD93+,0 CDld+, IL-10+)는 만성 대장염 (chronic colitis)의 마우스 모델 (TCRa-/— )의 장간막림프절 (MLN, Mesenteric lymph node)에서 발현됨이 보고되었고， 비장에서 분리한 이 세포를 정맥주사로 양자이입 (adoptive transfer)하였을 때 대장염이 억제되는 것이 보고되었다 [Mizoguchi， A. ,et al. , Immunity 16,219-230,(2002)]. T2-MZP 면역조절 B 세포는 류마티스 관절염의 마우스5 모델 (CIA, collagen induced arthritis)의 비장， 특히 증상이 호전된 마우스의 비장에서 증가되어 있음이 확인되었고， 양자이입 방법으로 관절염 억제 기능이 증명되었다. IFN-Y 분비 TH 1 세포 증식을 억제하여 염증을 억제하는데 IL-10 및 CDld 발현 증가가 중요한 역할을 함이 IL-10 및 CDld knockout 마우스 실험을 통하여 증명되었다 [Evans, J. G.et al . , J Immunol0 178,7868-7878,(2007)]. T2-MZP immunoregulatory B cells (CD19 +, CD21high, CD23 + , CD24 high, CD93 +, 0 CDld + , IL-10 + ) are the mesenteric lymph nodes (MLNs) of a mouse model of chronic colitis (TCRa-/ —). , Mesenteric lymph node), and it was reported that colitis is inhibited by intravenous injection of these cells isolated from the spleen [Mizoguchi, A., et al. , Immunity 16,219-230, (2002)]. T2-MZP immunoregulatory B cells were found to be increased in the spleen of mouse 5 model (CIA, collagen induced arthritis) of rheumatoid arthritis, especially in the spleen of symptomatically improved mice. . IL-10 and CDld knockout mice have been demonstrated to play an important role in inhibiting inflammation by inhibiting IFN-Y secretion T H 1 cell proliferation [Evans, JGet al. , J Immunol 0 178,7868-7878, (2007).
B10 면역조절 B 세포 (CD5+,CDld+,IL-10+, B220+)는 여러 염증 질환과 자가 면역 질환의 마우스 모델의 비장과 림프절에 생성됨이 보고되었다 [DiLillo, D.J., T. Matsushita, and T.F. Tedder , (2010) Ann N Y Acad Sci,1183: 38-57.]. B10 면역조절 B 세포의 염증 억제 효과는 ex5 vivo에서 생성된 B10 면역조절 B 세포를 류마티스 관절염 마우스 모델인 chronic col lagen- induced arthritis (CCIA)에 양자이입 (adoptive transfer)하여 증명 되었다. B10 면역조절 B 세포의 양자이입으로 관절염 지수 (arthritis index)가 50%이상 감소하였고 질병 중증도 (disease severity)는 90%이상 감소되었다 [Mauri, C：.， et al . , (2003) J Exp Med,0 197 (4): 489-501]. B10 immunoregulatory B cells (CD5 +, CDld + , IL-10 +, B220 +) have been reported to be produced in the spleen and lymph nodes of mouse models of several inflammatory and autoimmune diseases [DiLillo, DJ, T. Matsushita, and TF Tedder]. , (2010) Ann NY Acad Sci, 1183: 38-57.]. Inhibitory effects of B10 immunoregulatory B cells were demonstrated by adoptive transfer of ex10 in vivo B10 immunoregulatory B cells into chronic col lagen-induced arthritis (CCIA), a mouse model of rheumatoid arthritis. Adoption of B10 immunoregulatory B cells reduced arthritis index by more than 50% and disease severity by more than 90% [Mauri, C:., Et al. , (2003) J Exp Med, 0 197 (4): 489-501].
—ᅩ ᅩ ᅩ— -ᅩ그러ᅳ나 아상과—같이—
Figure imgf000005_0001
지속적인 보고에도 불구하고 이들 세포군을 생체 내외에서 약물을 사용하여 인위적으로 증폭하는 기술은 보고되어 있지 못하다. 본 발명은 이들 MDSC와 면역조절 B 세포를 생체 내외에서 인위적으로 증폭시키기 위하여 GPCR19 아고니스트를 사용하는 방법에 관한 것이며, 또한 GPCR19 아고니스트의 스크리닝 및 그 약물의 효능 측정 방법에 관한 것이며, 소듐 타우로데옥시콜레이트 (HY2191 , Sodiumtaurodeoxycholate) 및 그 유도체를 이용하여 생체 내외에서 유전자 발현 및 단백 발현의 표현형이 기존에 알려진 것과는 다른 항염증 표현형이 증대된 MDSC 및 면역조절 B 림프구의 숫적 증폭 기술에 관한 것이다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. — ᅩ ᅩ ᅩ -—- ᅩ But with the sub-—
Figure imgf000005_0001
Despite ongoing reports, no technique has been reported to artificially amplify these cell populations using drugs in vivo or in vivo. The present invention relates to a method of using a GPCR19 agonist to artificially amplify these MDSCs and immunoregulatory B cells in vivo and externally, and also to a method of screening a GPCR19 agonist and measuring the efficacy of the drug. The present invention relates to a numerical amplification technique of MDSC and immunoregulatory B lymphocytes having an enhanced anti-inflammatory phenotype different from the one previously known, using deoxycholate (HY2191, Sodiumtaurodeoxycholate) and its derivatives in vivo. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
【발명의 내용】 [Content of invention]
【해결하려는 과제】  [Problem to solve]
본 발명자들은 생체내외에서 항염증 효과가 증대된 MDSC 및 면역 조절 B세포를 인위적으로 증폭하는 기술을 개발 하고자 노력하였다. 또한 본 발명자들은 GPCR19-연관 질환 치료제와 그의 스크리닝 방법과 GPCR19- 연관 질환 치료제로써 GPCR19 아고니스트의 효능 분석 방법을 개발하고자 예의 연구 노력하였다. 그 결과 본 발명자들은 포유동물 또는 골수 유래 세포집단에 GPCR19 아고니스트를 투여 또는 처리하여 MDSCXMyeloid-derived suppressor cel l ) 및 면역조절 B 세포를 증폭시키는 기술을 개발하였고, GPCR19 아고니스트를 유효성분으로 포함하는 조성물이 MDSCXMyeloid- derived suppressor cel l ) 및 면역조절 B 세포를 매개하여 생체 내에서 항염증 활성을 갖는 것을 규명함으로써， 본 발명을 완성하게 되었다.  The present inventors have tried to develop a technique for artificially amplifying MDSC and immune regulatory B cells with enhanced anti-inflammatory effects in vivo and externally. In addition, the present inventors have made diligent research efforts to develop a GPCR19-associated disease treatment agent, a screening method thereof, and a method for analyzing the efficacy of the GPCR19 agonist as a GPCR19-associated disease treatment agent. As a result, the present inventors developed a technique for amplifying MDSCXMyeloid-derived suppressor cel l) and immunoregulatory B cells by administering or treating a GPCR19 agonist to a mammalian or bone marrow-derived cell population, and including the GPCR19 agonist as an active ingredient. The present invention was completed by identifying that the composition has anti-inflammatory activity in vivo by mediating MDSCX Myeloid-derived suppressor cel l) and immunoregulatory B cells.
따라서 본 발명의 목적은 GPCR19 아고니스트 및 그 유도체를 생체 내에 투여하여 염증 억제 효과가 향상된 새로운 표현형의 MDSC 또는 면역조절 B 세포를 숫적으로 증가시키는 방법에 관한 것이다.  Accordingly, an object of the present invention relates to a method of increasing the number of new phenotypes of MDSCs or immunoregulatory B cells with improved anti-inflammatory effects by administering GPCR19 agonists and their derivatives in vivo.
본ᅩ발명의—목적은 GPGR19-아— Jt나소트를——시험관내쎄 -서一골수세포ᅩ또는— 말초혈액에 처리하여 염증 억제 효과가 향상된 새로운 표현형의 MDSC 또는 면역조절 B세포 수를 증가시키는 방법에 관한 것이다. Of the present invention, o-purpose GPGR19- ah-me sort of Jt-vitro Ste-standing一bone marrow cells or o - The present invention relates to a method of increasing the number of new phenotypes of MDSCs or immunoregulatory B cells, which have been treated with peripheral blood, thereby improving the effect of inhibiting inflammation.
본 발명의 목적은 GPCR19-연관 질환 치료제의 스크리닝 방법을 제공하는 데 있다.  It is an object of the present invention to provide a method for screening a GPCR19-associated disease therapeutic agent.
본 발명의 다른 목적은 GPCR19 아고니스트의 효능 분석 방법을 제공하는 데 있다.  Another object of the present invention is to provide a method for analyzing the efficacy of GPCR19 agonists.
본 발명의 또 다른 목적은 항염증 활성을 갖는 MDSC에 관한 것이다. 본 발명의 또 다른 목적은 항염증 활성을 갖는 면역조절 B 세포에 관한 것이다.  Another object of the present invention relates to MDSCs having anti-inflammatory activity. Another object of the present invention relates to immunomodulatory B cells having anti-inflammatory activity.
본 발명의 또 다른 목적은 MDSC 또는 면역조절 B 세포를 유효성분으로포함하는 항염증 약제학적 조성물에 관한 것이다.  Another object of the present invention relates to an anti-inflammatory pharmaceutical composition comprising MDSC or immunoregulatory B cells as an active ingredient.
본 발명의 또 다른 목적은 소듐 타우로데옥시콜레이트 (HY2191, Sod iumtaurodeoxycho late) 및 그 유도체를 생체 내 투여하거나 골수세포에 처리하여 염증 억제 효과가 향상된 새로운 표현형의 MDSC 또는 면역조절 B 세포의 수를 증가시키는 방법에 관한 것이다. 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명， 청구범위 및 도면에 의해 보다 명확하게 된다. 【과제의 해결 수단】  Another object of the present invention is to administer a new phenotype of MDSC or immunoregulatory B cells having improved inflammation inhibitory effect by administering sodium taurodeoxycholate (HY2191, Sod iumtaurodeoxycho late) and its derivatives in vivo or treating bone marrow cells. It is about increasing. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings. [Measures of problem]
본 발명의 일 양태에 따르면， 본 발명 다음 단계를 포함하는 GPCR19- 연관 질환 치료제의 생체내 스크리닝 방법을 제공한다:  According to one aspect of the present invention, there is provided a method for in vivo screening of a GPCR19-associated disease therapeutic agent comprising the following steps:
(a) 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하는 단계 ;  (a) administering an in vivo physiologically active modulator to a mammal other than a human;
(b) 상기 포유동물에 GPCR19(G-protein coupled receptor 19) 아고니스트 시험물질을 투여하는 단계; 및  (b) administering a G-protein coupled receptor 19 (GPCR19) agonist test substance to the mammal; And
(c) 상기 포유동물에서 (i) Gr-l+CDllb+ MDSCCMyeloid-derived suppressor cell), (ii) Prok2 , Osm, Ltf , lral7, Slc40al, Fcer2a,(c) in said mammal (i) Gr-1 + CDllb + MDSCCMyeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf, lral7, Slc40al, Fcer2a,
Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk,Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk,
Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 (CD23hi Regulatory B cell 혹은 CD23hi BH cell) 또는 (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg3U, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83 Mybl2, IftUO, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592 B3gnt8, 1300014106Rik, Fdps, Ctsa, D隱 d, Sfrs7, Hbb-bhl , Hspala, Srpk3 Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3 Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, LOC100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, LOC100038894 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki , Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl , Itk, Ser inb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 시험물질을 주입하지 않은 대조군과 비교하여 상기 시험물질을 주입한 포유동물에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 증가하면 상기 시험물질은 GPCR19-연관 질환 치료제로 판정한다. 본 발명자들은 GPCR19-연관 질환의 치료제와 그의 스크리닝 방법과 GPCR19-연관 질환의 치료제로 GPCR19 아고니스트의 효능 분석 방법을 개발하고자 예의 연구 노력하였다. 그 결과 본 발명자들은 포유동물 또는 골수 유래 세포집단에 GPCR19 아고니스트를 투여 또는 처리하여 형성된 MDSCCMyeloid-der ived suppressor cel l ) 및 면역조절 B 세포를 개발하였고， 이를 유효성분으로 포함하는 조성물이 항염증 활성을 갖는 것을 규명하였다. 본 발명은 GPCR19-연관 질환의 치료제의 스크리닝 방법을 제공한다. 본 발명의 조성물로 예방 또는 치료가 가능한 질환은 GPC19-연관 질환으로 바람직하게는 패혈증, 자가면역질환， 염증성 질환, 류마티스 관절염， 아토피성 피부염, 알쓰하이머 병, 궤양성 대장염, 제 1형 당뇨병, 크론병， 건선， 건선성 관절염， 강직성 척수염， 다발성 경화증， 캐슬만씨 병， 자가면역성 간염， 자가 면역성 포도막염， 중증 근무력증， 원형 탈모증 및 전신성 흥반성 낭창으로 구성된 군으로부터 선택되나 이에 한정지 않고, 가장 바람직하게는 패혈증이다. 본 발명의 GPCR19-연관 질환 치료제의 생체내 스크리닝 방법을 각각의 단계 별로 상세하게 설명하면 다음과 같다: 단계 (a) : 생체 내 생리적 활성 조절 물질의 투여 High expression of the Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtpfla Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Mc Dll, Ksell3 (iii) CD23 hi immunoregulatory B cells (CD23 hi Regulatory B cells or CD23 hi B H cells) or (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg3U, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83 Mybl2, IftUO, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592 B3gnt8, 1300014106Rik, Fdps, Ctsa, D 隱 dsp Sbbs7 Srpk3 Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3 Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm41000 C55, Cdl80 Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr 9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb or Actb genes or express 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2kg Anxa2, Txndc5, Tmem66, Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selllcs, Dusp4, Selllcs, Dusp4 Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl0, Mxlki, Mxl, Tyx Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2l, Cd6, Analyzing the production of immunoregulatory B cells that underexpress Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Ser inb6b or Cd69 genes; When the production level of the cells of (i) to (iv) is increased in the mammal injected with the test substance compared to the control group not injected with the test substance, the test substance is determined to be a GPCR19-associated disease treatment agent. The present inventors made an intensive study to develop a therapeutic agent for GPCR19-related diseases, a screening method thereof, and a method for analyzing the efficacy of GPCR19 agonists for the treatment of GPCR19-associated diseases. As a result, the present inventors developed MDSCCMyeloid-derived suppressor cel l) and immunomodulatory B cells formed by administering or treating GPCR19 agonists to a mammalian or bone marrow-derived cell population, and the composition comprising the same as an active ingredient has anti-inflammatory activity. It was found to have. The present invention provides a method for screening a therapeutic agent for a GPCR19-associated disease. Diseases that can be prevented or treated with the composition of the present invention are GPC19-associated diseases, preferably sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease, ulcerative colitis, type 1 diabetes, Crohn's disease. Disease, psoriasis, psoriatic arthritis, ankylosing myelitis, multiple sclerosis, castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia areata, and systemic pharyngeal lupus, most preferred It is sepsis. The in vivo screening method of the GPCR19-associated disease therapeutic agent of the present invention will be described in detail for each step as follows: Step (a): Administration of a physiologically active agent in vivo
본 발명의 방법에 있어서， GPCR19-연관 질환의 치료제를 스크리닝하기 위하여 먼저 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하여 생체 내에서 생리적 반웅을 유발한다.  In the method of the present invention, in order to screen a therapeutic agent for a GPCR19-associated disease, a physiological reaction modulator in vivo is first administered to a mammal except a human to cause a physiological reaction in vivo.
본 발명에 사용되는 생체 내 생리적 활성 조절 물질은 생체 내에서 생리적 반웅을 유발하는 물질을 의미한다. 예컨대， 상기 생체 내 생리적 활성 조절 물질은 세포의 리솁터 (Receptor) [예컨대, TLR(tol l-l ike receptor)]에 결합하여 신호전달 반응을 조절하거나 일반적인 면역계의 다양한 성분 (예컨대， 혈관내피세포, 수지상 세포, T세포 및 B세포)의 리셉터와상호작용하여 신호전달 반웅을 조절하는 물질을 포함한다.  In vivo physiological activity modulator used in the present invention means a substance causing a physiological reaction in vivo. For example, the physiologically active regulatory substance in vivo binds to a cell receptor (eg, toll ll receptor) to regulate a signaling response or to various components of the general immune system (eg, vascular endothelial cells, dendritic cells). Cells, T cells, and B cells) to interact with receptors to regulate signaling response.
바람직하게는， 생체 내 생리적 활성 조절 물질은 면역계의 다양한 체액성 인자 또는 세포와 상호작용하여 특이적인 사이토카인 또는 쩨모카인을 을 분비하게 하거나, 생체 세포의 표면 분자 발현을 증가시키거나， 항체를 형성하게 하거나 특이적인 림포사이트 집단을 생성 또는 증가하게 하는 분자이며, 보다 바람직하게는 합성 유기 화학물질, 단백질， 지질단백질, 당단백질， 펩타이드, RNA, DNA 또는 다당류로 구성된 개개의 분자, 또는 세포 구조체 (세균 또는 진균류) 또는 바이러스의 일부로 5 이루어진 군에서 선택되나 이에 한정되지 않는다. Preferably, the physiologically active regulatory substance in vivo interacts with various humoral factors or cells of the immune system to secrete specific cytokines or chemokines, or to express the surface molecule expression of living cells. Molecules that increase, form antibodies, or produce or increase specific lymphocyte sites, and more preferably, each composed of synthetic organic chemicals, proteins, lipoproteins, glycoproteins, peptides, RNA, DNA, or polysaccharides Molecules, or cellular constructs (bacteria or fungi) or part of a virus, but are not limited to.
본 발명에 사용되는 생체 내 생리적 활성 조절 물질은 다양한 종류에서 유래된 물질로 바람직하게는， 박테리아 유래 물질， 진균 유래 물질, 바이러스 유래 물질， 자가 유래 물질, 또는 알러젠 (allergen)이고， 보다 바람직하게는 박테리아 유래 물질 또는 진균 유래 물질이고， 보다 더 In vivo physiological activity modulators used in the present invention are substances derived from various kinds, preferably bacteria-derived, fungal-derived, virus-derived, autologous or allergen, and more preferably. Is bacterial or fungal, and more
10 바람직하게는 박테리아 유래 분자이며， 가장 바람직하게는 리포폴리사카라이드 (LPS) 또는 펩티도글리칸이다. 10 Preferably it is a molecule derived from bacteria, most preferably lipopolysaccharide (LPS) or peptidoglycan.
본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질로서 박테리아 유래 물질은 포유동물에 감염되어 여러가지 질병을 일으키는 박테리아 군으로부터 선택이 가능하며， 바람직하게는 살모넬라 Bacterial-derived materials for in vivo physiologically active substances used in the method of the present invention can be selected from a group of bacteria that infect mammals and cause various diseases, preferably Salmonella
15 티피뮤리움 typhi murium) , 살모넬라 엠반다카 (5*a/»o/?e// mbandaka) , 살모넬라 엔테라이티디스 (5a½o/?e// enter it idis) , 스트렙토코커스 뉴모니아 (S re ococ /s pneumoniae) , 스타필로코쿠스 오레우스 {Staphylococcus aureus) , 타플릴로코쿠스 에피더미디스 ( Staphlylococcus epidermidis)， 네이세리아15 typhi murium), Salmonella embandaka (5 * a / »o /? E // mbandaka), Salmonella enteritidis (5a½o /? E // enter it idis), Streptococcus pneumoniae (S re ococ / s pneumoniae), Staphylococcus aureus, Staphlylococcus epidermidis, Neisseria
20 메닌기티디스 OVe/sser/a meningitidis) , 네이세리아 고노르헤에 0Ve/sse a gonorrheae) , 헤모필루스 인플루엔제 G¾i TC¾!¾¾//i/s influenzae) , 대장균 ( s?er/ ?/a coli) , 클레브시엘라 뉴모니아 {Klebsiella pneumoniae) , 리스테리아 모노시토겐스 ( /s er/a monocytogenes) , 비브리오 콜레레 cholerae) , 클로스트리디움 퍼프란젠스 (C/ostr/d/ n20 Meningitidis OVe / sser / a meningitidis), Neseria Gonorhe 0Ve / sse a gonorrheae, Haemophilus influenzae G¾i TC¾! ¾¾ // i / s influenzae), E. coli (s? Er /? / A coli), Klebsiella pneumoniae, Listeria monocytogenes (/ s er / a monocytogenes), Vibrio cholera cholerae, Clostridium perfrangens (C / ostr / d / n
25 perfringens) , 클로스트리디움 보틀리늄 (CVosT /i//ifl7 botulinimi) , 수도모나스 에라션스 0¾erac¾z¾¾ s aerations) , 보텔리아 부르그도르테리 0 /了6// burgdorferi) , 시겔라 플렉스네리 (5?/ //3 flexneri) , 시겔라 보이디 (^/^//a boydii) , 시겔라 디센트리게 (5Λ/^/ /a dysentriae) , 알로이오코쿠스 오티티디스 (^//o/ococ /s otitidis) 및 그룹25 perfringens), Clostridium botulinum (CVosT / i // ifl7 botulinimi), Domonas Erasions 0¾erac¾z¾¾ s aerations, Botelia Burgdorferi 0 / 了 6 // burgdorferi), Shigella flexneri (5 ? / // 3 flexneri), shigella boydi (^ / ^ // a boydii), shigella decentrige (5Λ / ^ / / a dysentriae), alloyococcus ortidis (^ // o / ococ / s otitidis) and groups
30 B 스트렙토코시 (s re ioco :/)이고, 가장 바람직하게는 살모넬라30 B streptococci (s re ioco: /), most preferably Salmonella
——ᅳ 티꾀뮤라움 ( 5a7/TO //a bandaka) 및 살모넬라 엔테라이티디스 (5a/z ;2e//a enteritidis)으로 이루어진 군에서 선택되나 이에 한정되지 않는다. ——Jean Timumurium (5a7 / TO // a bandaka) and Salmonella enteritidis (5a / z; 2e // a enteritidis).
본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질로서 진균 유래 물질은 포유동물에 감염되어 여러 가지 질병을 일으키는 진균에서 유래하는 물질로, 예컨대 포유동물의 피부， 구강에 표재성 진균증 (superficial mycosis)을 일으키고, 내장， 뇌 등에 전신성 진균증을 일으키는 진균에서 유래한 물질이다. 진균 유래 물질은 다양한 진균 군으로부터 선택이 가능하며， 바람직하게는 칸디다  As a biologically physiologically active substance used in the method of the present invention, a fungal-derived material is derived from a fungus that infects mammals and causes various diseases, such as superficial mycosis in the skin and oral cavity of a mammal. It is a substance derived from fungi that cause systemic fungal diseases, such as intestines and brain. Fungal derived materials can be selected from a variety of fungal groups, preferably Candida
아스페르길루스 G4 ?e/ 7/ s) , 크립토코커스 (C^ ococcws) , 무코르 (Mucor) , 리조푸스 Rhizopus) , 압시디아 , 노카르디아 GVoca/ /a)， 히스토플라스마 Oy/s i¾o/aa7?a), 블라스토마이세스 ( / as cmjces) , 콕시디오이 데스 ( Coccidioides) , 토리코피톤 ( Trichophyton) , 미크로스포륨Aspergillus G4? E / 7 / s), Cryptococcus (C ^ ococcws), Mucor, Rhizopus, Apsidia, Nocardia GVoca / / a, Histoplasma Oy / s i¾o / aa7? a), blastomaes (/ as cmjces), Coccidioides, Trichophyton, Microsporium
{Microsporu ) , 에피더모피톤 ^//ᅳ/ ？ ^ ， 스포로트릭스 (5/κτσ Ατ/ ) , 혹入 균 Demat iaceous fungi) , 말라세치아 뉴모사이스 ^쓰 {Pneumocystis) , 페니실륨 , 알타나리아 / er^a /a) , 클라도스포륨 ( Cladosporium)， 보트리티느 Botrytis)， 어에로바시듐 {Aureobasidiu )， 프자晉 0¾sar/uro) , 트리코데르口 K Trichoderma) , 헬민쏘스포륨
Figure imgf000011_0001
, 뉴로스포라 0Ve ro wra) , 월레미아 {Wallemia) 및 로도토를라 0¾θί/σ σπ// )으로 이루어지는 군에서 선택되나 이에 한정되지ᅵ ^는다. ― (Microsporu), Epidermophyton ^ // ᅳ /？ ^, Sporotrix (5 / κτσ Ατ /), Demat iaceous fungi, Malaceccia pneumocystis (Pneumocystis), Penicillium, Altanaria / er ^ a / a), Cladosporium, Botrytis, Aerobasidiu, Pzaret 0¾sar / uro), Trichoder 口 K Trichoderma), Helminso Sporium
Figure imgf000011_0001
, Neunda ^ neuro spokes la 0Ve ro wra), May remiah {Wallemia) and the soil but also i selected from the group consisting of La 0¾θί / σ σπ //) so limited. ―
본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질로서 바이러스 유래 물질은 포유동물에 감염되어 다양한 질병을 일으키는 바이러스 군으로부터 선택이 가능하며， 바람직하게는， 아데노바이러스， 알파바이러스， 칼리시 바이러스， 코로나바이러스， CMV, 디스템퍼 바이러스， 에볼라 바이러스， 엔테로바이러스, EBV, 플라비바이러스 (예컨대， Hep C) 해파드나바이러스 (예컨대, Hep B, 간염 델타 물질， Hep E 또는 F바이러스), A형 간염 바이러스 (예컨대, VPl), GBV-C, 허피스바이러스 (예컨대， 허피스 심플렉스 바이러스 단백질 [예컨대, 타입 I 당단백질 G, gpD, CP27, 수두 대상포진 바이러스 당단백질 (예컨대 IE62, gpl 또는 외피 단백질)]， 면역결핍 바이러스， 예컨대, HIV (예컨대， 외피 또는 단백질분해효소), 감염성 복막염 바이러스， 인플루엔자 바이러스 (예컨대, 인플루엔자 A 헤마글루티닌， 뉴라미니다제 또는 핵단백질) , LCMV, 백혈병 바이러스， 마버그 바이러스， 오르토믹소바이러스， 파필로마 바이러스 [예컨대, HPV (예컨대， HPV 캡시드 단백질) , 파라인플루엔자 바이러스 (예컨대, 헤마글루티닌 /뉴라미니다제) ]， 파라믹소바이러스 [예컨대， RSV (예컨대， F 또는 G 단백질 ) ]， 파보바이러스， 페스티바이러스， 피코나바이러스 [예컨대, 폴리오바이러스 캡시드 폴리펩티드 (예컨대， VP1 , VP2 또는 VP3 , 또는 Hep A 항원) ]， 폭스 바이러스 (예컨대, 백시니아 바이러스 폴리펩티드， 예컨대， 외피 단백질) , 공수병 바이러스 (예컨대， 공수병 바이러스 당단백질 G) , 레오바이러스， 레트로바이러스， 리노바이러스 (예컨대, 인간 리노바이러스 캡시드)， 루벨라 바이러스 (예컨대, 캡시드 단백질) 및 로타바이러스로 이루어지는 군에서 선택되나 이에 한정되지 않는다. In vivo biological physiologically active substances used in the method of the present invention can be selected from the group of viruses that infect a mammal and cause a variety of diseases, preferably, adenovirus, alpha virus, kaleici virus, corona Virus, CMV, distemper virus, Ebola virus, enterovirus, EBV, flavivirus (eg Hep C) hapadnavirus (eg Hep B, hepatitis delta, Hep E or F virus), hepatitis A virus (eg , VPl), GBV-C, Herpesviruses (eg Herpes Simplex Virus Protein [eg, Type I Glycoprotein G, gpD, CP27, Varicella Zoster Virus Glycoprotein (eg IE62, gpl or envelope protein)], Immunodeficiency Viruses such as HIV (eg envelope or protease), infective peritonitis virus, influenza Purple virus (eg, influenza A Hemagglutinin, neuraminidase or nucleoprotein), LCMV, leukemia virus, Marburg virus, orthomyxovirus, papilloma virus [eg HPV (eg HPV capsid protein), parainfluenza virus (eg hemaggle) Rutinin / neuramidase)], paramyxoviruses [eg RSV (eg F or G protein)], parvoviruses, pestiviruses, piconaviruses [eg poliovirus capsid polypeptides (eg VP1, VP2 or VP3, or Hep A antigen)], pox virus (eg vaccinia virus polypeptide, eg envelope protein), rabies virus (eg rabies virus glycoprotein G), leovirus, retrovirus, rhinovirus (eg human reno) Virus capsid), rubella virus (eg, capsid protein) and rotavirus Which is selected from the group, but not limited to this.
본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질로서 자가 유래 물질은 생체의 정상 구성 성분 [예컨대， 단백질 (시 "이토카인， 호르몬， 인터루킨 또는 이들의 흔합물)， 핵산, 이들의 흔합물 또는 이들의 조각]에 있어서 동일 개체에 다양한 신호전달 반웅을 일으키어 자가면역반웅을 야기하여 자가 항체를 형성하도록 자극함으로써 자가면역질환을 일으키는 물질이다. 자가면역반웅을 일으키는 자가 유래 물질은 자연발생적인 자가 유래 물질의 구성과는 상이한 구성으로 배열되어 있다. 자가면역반웅을 일으키는 자가 유래 물질로， 바람직하게는 인슐린ᅳ 미예린 염기성 단백질, rh 인자, 아세틸콜린 수용체, 갑상성 세포 수용체 (기초막 단백질, 갑상선 단백질 PM-1 또는 글루탐산 디카복실라제 (64K) ) 및 카복시펩티다제 H로 구성되는 군으로부터 선택되나 이에 한정되지 않는다. 자가 유래 물질이 야기하는 자가면역질환은 바람직하게는， 류머티스성 관절염 (RA) , 다발성 경화증 (MS) , 전신 홍반 루푸스 (SLE) , 루푸스 신장염， 피부 흥반 루푸스 (CLE) , 자가면역성 간염， 연소성 류머티스성 관절염， 전염성 간염， 원발성 담즙성 간경화， 건선, 피부염， 아토피성 피부염， 전신성 피부경화증， 전신성 경화증， 크론병， 궤양성 대장염， 호흡 곤란 증후군， 성인성 호흡 곤란 증후군， ARDS , 수막염, 뇌염， 포도막염, 사구체신염， 천포창， 대식세포 활성화 증후군, 습진, 천식, 죽상동맥경화증， 백혈구 부착 결핍증, 진성 당뇨병， 제 I형 진성 당뇨병 인슬린 의존성 진성 당뇨병， 알러지성 바염—，ᅭ장거——이식과——관 ¾돤 자자 반웅，—레이 증후군， 갑상선염, 하시모토 갑상선염， 알러지성 뇌척수염， 쇼그렌 증후군， 유년 발병형 당뇨병, 사이토카인 및 T-림프구에 의해 매개되는 급성 및 지연형 과민증과 관련되는 면역 응답， 결핵, 사르코이드증 다발근육염， 육아종증， 혈관염， 악성 빈혈 (애디슨병)， 백혈구 누출을 수반하는 질환， 증추 신경계 (CNS) 염증성 장애， 다발성 장기 손상 증후군， 용혈 빈혈， 한랭글로빈혈증， 큼즈 (Coombs) 양성 빈혈， 중증 근육무력증， 항원 -항체 복합체 매개 질환， 항-사구체 기저막 질환, 항-인지질 증후군, 알러지성 신경염， 그레이브스병， 램버트 -이튼 근무력 증후군， 수포성 유사천포창, 천포창， 자가면역성 다중내분비병증， 라이터 질환， 근육강직 증후군, 베쳇병， 거대세포성 동맥염， 면역 복합체 신장염, IgA 산장병증， IgM 다발신경병증, 면역성 혈소판감소성 자반 ( ITP) 및 자가면역성 혈소판감소증으로 이루어지는 군에서 선택되나 이에 한정되지 않는다. In vivo physiological activity modulators used in the methods of the invention, self-derived materials may be derived from the normal constituents of a living body [eg, proteins (cytokines, hormones, interleukins or combinations thereof), nucleic acids, combinations thereof or These fragments are substances that cause autoimmune diseases by causing various signaling reactions to the same individual, causing autoimmune reactions, and stimulating them to form autoantibodies. It is arranged in a different composition from the composition of the substance, which is a self-derived substance that causes autoimmune reactions, preferably insulin ᅳ myelin basic protein, rh factor, acetylcholine receptor, thyroid cell receptor (base membrane protein, thyroid protein). PM-1 or glutamic acid dicarboxylase (64K)) and carboxypeptidase H The autoimmune diseases caused by the autologous material are preferably rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), lupus nephritis, skin hemorrhage lupus (CLE). ), Autoimmune hepatitis, combustible rheumatoid arthritis, infectious hepatitis, primary biliary cirrhosis, psoriasis, dermatitis, atopic dermatitis, systemic sclerosis, systemic sclerosis, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome ARDS, meningitis, encephalitis, uveitis, glomerulonephritis, pemphigus, macrophage activation syndrome, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, diabetes mellitus, type I diabetes mellitus inslin dependent diabetes mellitus, allergic baitis— ， ᅭ 거 ——transplantation and ― 관 관 돤 자 반 반 ， — Ray syndrome ， Thyroiditis, Hashimoto thyroiditis, allergic encephalomyelitis, Sjogren's syndrome, immune response associated with acute and delayed hypersensitivity mediated by juvenile onset diabetes, cytokines and T-lymphocytes, tuberculosis, sarcoidosis polymyositis, granulomatosis, vasculitis , Pernicious anemia (Addison's disease), disease with leukocyte leakage, central nervous system (CNS) inflammatory disorders, multiple organ damage syndrome, hemolytic anemia, cold globinemia, Coombs-positive anemia, severe myasthenia, antigen-antibody Complex-mediated disease, anti-glomerular basement membrane disease, anti-phospholipid syndrome, allergic neuritis, Graves' disease, Lambert-Eton's work syndrome, bullous pseudocystic swelling, pemphigus, autoimmune multiple endocrine disease, lighter disease, muscle stiffness syndrome, Stomach disease, Cytomegaloid arteritis, Immune complex nephritis, IgA antosis, IgM polyneuropathy, It is selected from the group consisting of immune thrombocytopenic purpura (ITP) and autoimmune thrombocytopenia.
본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질로서 알러젠 (al lergen)은 당업계에 공지된 알레르기， 즉， 개체에 반복적으로 노출시 IgE 매개 반응을 유발하는 것으로 알려져 있는 모든 자연 발생적 물질 또는 이들 물질의 흔합물을 포함한다. 자연 발생적 알레르기 유발 물질로， 바람직하게는 꽃가루 알레르기 유발 물질 (예컨대, 수목， 잡초, 초본 또는 잔디의 꽃가루 알레르기 유발 물질)， 진드기 알레르기 유발 물질 (예컨대， 집 먼지진드기 또는 저장 진드기 유래 물질)， 곤층 알레르기 유발 물질 (예컨대， 흡입성， 타액 및 독액 유래 알레르기 유발 물질)， 개， 고양이， 랫트 및 마우스의 타액， 털 및 비듬에서 유래하는 동물 알레르기 유발 물질， 진균 알레르기 유발 물질 및 음식물 알레르기 유발 물질로 이루어진 군에서 선택되나 이에 한정되지 않고 이들의 흔합물을 포함한다. 이들 알러젠의 형태로는 알러젠 추출물， 정제된 알러젠, 변형된 알러젠, 재조합 알러젠， 재조합 변이체 알러젠， 30 아미노산 이상의 알러젠 단편 또는 이들의 조합 형태물로 이루어지는 군에서 선택되나 이에 한정되지 않는다.  As an in vivo physiologically active substance used in the methods of the present invention, allergens are allergens known in the art, i.e. all naturally occurring substances known to cause an IgE mediated response upon repeated exposure to an individual or their And mixtures of materials. Naturally occurring allergens, preferably pollen allergens (e.g., pollen allergens in trees, weeds, herbs or grass), mite allergens (e.g., house dust mites or storage mites derived), nasal allergies Groups of allergens (eg, allergens derived from inhalation, saliva and venom), animal allergens, fungal allergens and food allergens derived from saliva, hair and dandruff in dogs, cats, rats and mice Selected from, but not limited to, combinations thereof. The forms of these allergens are selected from the group consisting of allergen extracts, purified allergens, modified allergens, recombinant allergens, recombinant variant allergens, allergen fragments of 30 amino acids or more, or combinations thereof, but are not limited thereto.
본 발명의 방법에 있어서， 상기 생체 내 생리적 활성 조절 물질은 인간을 제외한 포유동물에게 투여가 가능하며 바람직하게는 포유동물， 보다 바람직하게는 반려 동물 (예컨대, 개， 고양이)， 농장용 동물 (예컨대， 소， 양， 돼지 또는 말) 또는 실험실용 동물 (예컨대， 랫트， 마우스 또는 기니피그)이고， 보다 더 바람직하게는 마우스， 랫트, 개, 원승이， 염소， 양， 토끼이고 가장 바람직하게는 마우스이다. In the method of the present invention, the in vivo physiologically active substance can be administered to mammals other than humans, preferably mammals, more preferably companion animals (eg dogs, cats), farm animals (eg , Cattle, sheep, pigs or horses or laboratory animals (eg rats, mice or Guinea pigs), and more preferably mouse, rat, dog, animal, goat, sheep, rabbit and most preferably mouse.
상기 포유동물에 생체 내 생리적 활성 조절 물질의 투여는 당업계에 공지된 다양한 방법을 통해 실시할 수 있으며， 바람직하게는 경구 또는 비경구로 투여할 수 있고， 비경구 투여인 경우에는 정맥내 주입， 피하 주입， 근육 주입， 복강 주입, 경피 투여 등으로 투여할 수 있고, 보다 바람직하게는 구강내, 비강내, 위장관내, 외이도내， 질내， 직장내， 정맥내 또는 복강내 주입， 피부 또는 안구에 투여할 수 있고， 가장 바람직하게는 복강내 투여한다 .  Administration of the physiologically active substance in vivo to the mammal may be carried out through various methods known in the art, preferably orally or parenterally, and in the case of parenteral administration, intravenous infusion, subcutaneous Injection, intramuscular injection, intraperitoneal injection, transdermal administration, and more preferably, orally, intranasally, gastrointestinal tract, external ear canal, vaginal, rectal, intravenous or intraperitoneal injection, skin or eye. Can be administered, most preferably intraperitoneally.
한편， 생체 내 생리적 활성 조절 물질의 투여량은 실험실에서 통상적 (rout inely)으로 정할 수 있으며， 예를 들어 생체 내 생리적 활성 조절 물질로서 마우스에 리포폴리사카라이드 (LPS)가 이용되는 경우, 투여량은 바람직하게는 0.001 mg/kg 내지 100 mg/kg이고, 보다 바람직하게는 0. 1 mg/kg 내지 50 mg/kg이고, 가장 바람직하게는 20 mg/kg을 사용한다. 단계 (b) : GPCR19 아고니스트시험물질의 투여  On the other hand, the dose of the physiologically active substance in vivo can be determined in the laboratory (rout inely), for example, if lipopolysaccharide (LPS) is used in the mouse as the physiologically active substance in vivo, the dose Is preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, most preferably 20 mg / kg. Step (b): Administration of GPCR19 Agonist Test Substance
본 발명의 방법에 사용되는 GPCR19 아고니스트 시험물질은 GPCR19 수용체와 상호작용하여 효능을 일으키는， 바람직하게는 그 물질이 갖는 작용과 같은 (또는 유사한) 작용을 하는 물질 또는 약제， 혹은 수용부위의 활성을 높이는 분자를 의미한다. 본 발명의 GPCR19 아고니스트는 GPCR19 수용체의 효능제 또는 부분적 효능제로서 작용한다. 본 명세서의 용어 "효능제"는 일반적으로 다른 분자 또는 수용체 부위의 활성을 강화하는 화합물을 의미한다. 고전적인 정의에서， 효능제는 오르토스테릭 효능제, 알로스테릭 효능제, 역 효능제 또는 공동-효능제의 여부와 관계없이， 수용체에 결합하여 그것의 수용체 상태를 변경시킴으로써 생물학적 작용을 일으키는 특성을 갖는다. 결론적으로， 효능작용은 생물학적 작용을 생성하는 효능제 또는 리간드의 특성으로 정의된다. 보다 구체적으로, GPCR19 아고니스트는 GPCR19에 결합하여 수용체를 발현하는 세포에서 사이클릭 아데노신 모노포스페이트 (cAMP)의 농도를 3 % 이상 증가시키는 수용체 리간드또는 화합물이다. 본 발명의 바람직한 구현예에 따르면， 본 발명에 사용되는 GPCR19 아고니스트는 하기 화학식 1의 화합물, 그의 염 또는 수화물을 포함하고， 보다 바람직하게는 소듐 타우로데옥시콜레이트 (HY2191)이다. The GPCR19 agonist test substance used in the method of the present invention interacts with the GPCR19 receptor to produce efficacy, preferably to act on a substance or drug that acts (or similarly) as the substance, or a drug, or a receptor site. Height means molecule. The GPCR19 agonist of the present invention acts as an agonist or partial agonist of the GPCR19 receptor. The term "agonist" as used herein generally means a compound that enhances the activity of another molecule or receptor site. In the classical definition, an agonist is a property that causes a biological action by binding to a receptor and altering its receptor state, whether or not it is an orthosteric, allosteric, inverse or co-agonist. Has In conclusion, agonism is defined as the nature of an agonist or ligand that produces a biological action. More specifically, the GPCR19 agonist is a receptor ligand or compound that binds to GPCR19 and increases the concentration of cyclic adenosine monophosphate (cAMP) by at least 3% in cells expressing the receptor. According to a preferred embodiment of the present invention, the GPCR19 agonist used in the present invention comprises a compound of formula (1), a salt or a hydrate thereof, more preferably sodium taurodeoxycholate (HY2191).
소듐 타우로데옥시콜레이트의 투여량으로 바람직하게는 0.01 mg/kg 내지 100 mg/kg이고, 보다 바람직하게는 0. 1 mg/kg 내지 10 mg/kg이고, 가장 바람직하게는 0.5 - 8 mg/kg을 사용한다.  The dose of sodium taurodeoxycholate is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 10 mg / kg, most preferably 0.5-8 mg /. Use kg.
화학식 1  Formula 1
Figure imgf000015_0001
Figure imgf000015_0001
상기 화학식에서 은 수소, 하이드록시, 치환 또는 비치환 알킬 또는 할로겐; R2는 수소 또는 α -하이드록시; ¾은 하이드록시, NH(C¾)mS03H(m은 정수 0， 1 , 2 , 3 , 4 또는 5) 또는 NH(C¾)nC02H(n은 정수 으 1， 2， 3， 4 또는 5) ; 는 수소， 알킬 또는 할로겐; R5는 수소， 치환 또는 비치환 알킬 또는 아릴이고; ¾은 수소 또는 ¾ 및 ¾가 부착된 탄소와 함께 3， 4, 5 또는 6개 원자 크기로 형성된 고리이고; R7은 수소, 하이드록시， 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; R10은 수소 또는 치환 또는 비치환 알킬; Ru은 수소 또는 치환 또는 비치환 알킬이다. In the formula is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; R 2 is hydrogen or α-hydroxy; ¾ is hydroxy, NH (C¾) m S0 3 H (m is an integer 0, 1, 2, 3, 4 or 5) or NH (C¾) n C0 2 H (n is an integer 1, 2, 3, 4 Or 5); Is hydrogen, alkyl or halogen; R 5 is hydrogen, substituted or unsubstituted alkyl or aryl; ¾ is a ring formed of 3, 4, 5 or 6 atom sizes with hydrogen or carbon with ¾ and ¾ attached; R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; R 10 is hydrogen or substituted or unsubstituted alkyl; Ru is hydrogen or substituted or unsubstituted alkyl.
본 발명의 방법에 사용되는 GPCR19 아고니스트 시험물질의 투여 대상인 인간을 제외한 포유동물과 투여하는 방법은 단계 (a)에서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 생략한다.  Since the method of administering with a mammal other than a human subject to the administration of the GPCR19 agonist test substance used in the method of the present invention is the same as described above in step (a), the contents in common between the two are repeated according to the repetition description. In order to avoid excessive complexity of the specification, the description is omitted.
본 명세서의 용어 "알킬''에는 포화 지방족 기， 예컨대 직쇄 알킬 기 (예컨대, 메틸, 에틸， 프로필, 부틸, 펜틸， 핵실, 헵틸, 옥틸， 노닐, 데실)， 분지쇄 알킬 기 (예컨대, 이소프로필， te -부틸， 이소부틸) , 시클로알킬 (예컨대， 지환족) 기 (예컨대， 시클로프로필， 시클로펜틸， 시클로핵실, 시클로헵틸， 시클로옥틸)， 알킬 치환 시클로알킬 기 및 시클로알킬 치환 알킬 기가 포함된다. 특정 실시예에서 , 직쇄 또는 분지쇄 알킬은 골격 내에 6개 이하의 탄소 원자를 갖는다 [예컨대， d-C6 (직쇄) , C3-C6 (분지쇄) ] . 일부 예에서 , 직쇄 또는 분지쇄 알킬은 골격 내에 4개 이하의 탄소 원자를 갖는다. 또한， 시클로알킬은 고리 구조 내에 3 내지 8개의 탄소 원자를 갖는다. As used herein, the term "alkyl" includes saturated aliphatic groups such as straight chain alkyl groups (eg, methyl, ethyl, propyl, butyl, pentyl, nucleus, heptyl, octyl, nonyl, decyl), branched chain alkyl groups (eg, isopropyl). , Te-butyl ， isobutyl) Cycloalkyl (such as cycloaliphatic) groups (such as cyclopropyl, cyclopentyl, cyclonuclear, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups. In certain embodiments, straight or branched chain alkyl has up to 6 carbon atoms in the backbone [eg, dC 6 (straight chain), C 3 -C 6 (branched chain)]. In some instances, straight or branched chain alkyl has up to 4 carbon atoms in the backbone. In addition, cycloalkyl has 3 to 8 carbon atoms in the ring structure.
본 명세서의 용어 "치환 알킬"은， 탄화수소 골격의 1개 이상의 탄소 상의 1개 이상의 수소 원자가 치환기로 대체된 알킬 잔기를 '나타낸다. 이러한 치환기에는， 예컨대 알킬， 알케닐， 알키닐， 할로겐， 히드록실 알킬카르보닐옥시， 아릴카르보닐옥시, 알콕시카르보닐옥시 아릴옥시카르보닐옥시, 카르복실레이트, 알킬카르보닐, 아릴카르보닐 알콕시카르보닐， 아미노카르보닐， 알킬아미노카르보닐 디알킬아미노카르보닐， 알킬티오카르보닐, 알콕실, 포스페이트 포스포네이토, 포스피네이토， 시아노, 아미노 (알킬아미노, 디알킬아미노 아릴아미노, 디아릴아미노 및 알킬아릴아미노 포함) 아실아미노 (알킬카르보닐아미노, 아릴카르보닐아미노, 카르바모일 ^ 우레이도 포함) , 아미디노， 이미노， 술프히드릴， 알킬티오， 아릴티오 티오카르복실레이트， 술페이트， 알킬술피닐， 술포네이토 술파모일 술폰아미도, 니트로， 트리플루오로메틸， 시아노， 아지도, 헤테로시클릴 알킬아릴， 또는 방향족 또는 헤테로방향족 잔기가 포함될 수 있다. The term of the terms "substituted alkyl", denotes the moiety replaced with a hydrogen atom of one or more substituents on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl alkoxycarbons Carbonyl, aminocarbonyl, alkylaminocarbonyl dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate phosphonato, phosphinato, cyano, amino (alkylamino, dialkylamino arylamino, diaryl Acylamino (including amino and alkylarylamino) (including alkylcarbonylamino, arylcarbonylamino, carbamoyl ^ ureido), amidino, imino, sulfhydryl, alkylthio, arylthio thiocarboxylate, sulf Fate, Alkylsulfinyl, Sulfonatosulfamoyl Sulfonamido, Nitro, Trifluoromethyl, Cyano, Azido, Heterocyclyl Alkylaryl or aromatic or heteroaromatic moieties may be included.
본 명세서의 용어 "아릴 "에는 방향족성을 가진 기， 예컨대 0 내지 4개의 헤테로원자를 포함할 수 있는 5 및 6원의 "비접합" 또는 단일 -고리 방향족 기, 및 1개 이상의 방향족 고리를 갖는 "접합" 또는 멀티시클릭 계가 포함된다. 아릴 기의 예로는 벤젠， 페닐, 피를， 푸란, 티오펜， 티아졸， 이소티아졸， 이미다졸, 트리아졸, 테트라졸， 피라졸， 옥사졸, 이속사졸， 피리딘， 피라진， 피리다진 및 피리미딘 등이 포함된다. 또한， 본 명세서의 용어 "아릴''에는 멀티시클릭 아릴기， 예컨대 트리시클릭， 비시클릭, 예컨대 나프탈렌， 벤즈옥사졸， 벤조디옥사졸， 벤조티아졸， 벤조이미다졸 , 벤조티오펜， 메틸렌디옥시페닐, 퀴놀린， 이소퀴놀린, 나프티리딘， 인돌, 벤쵸푸란， 퓨린, 벤조푸란, 데아자퓨린 또는 인돌리진이 포함된다. 고리 구조 내에 헤테로원자를 갖는 아릴 기는 "아릴 헤테로사이클"， "헤테로사이클"， "헤테로아릴" 또는 "헤테로방향족"으로도 지칭될 수 있다. 방향족 고리는 하나 이상의 고리 위치에서 상기 기재된 바와 같은 치환기 , 예컨대 할로겐, 히드록실， 알콕시, 알킬카르보닐옥시， 아릴카르보닐옥시， 알콕시카르보닐옥시， 아릴옥시카르보닐옥시， 카르복실레이트, 알킬카르보닐 알킬아미노카르보닐， 아르알킬아미노카르보닐, 알케닐아미노카르보닐, 알킬카르보닐, 아릴카르보닐， 아르알킬카르보닐， 알케닐카르보닐, 알콕시카르보닐， 아미노카르보닐， 알킬티오카르보닐， 포스페이트 , 포스포네이토, 포스피네이토， 시아노, 아미노 (알킬아미노， 디알킬아미노, 아릴아미노, 디아릴아미노 및 알킬아릴아미노 포함) , 아실아미노 (알킬카르보닐아미노， 아릴카르보닐아미노， 카르바모일 및 우레이도 포함)， 아미디노， 이미노, 술프히드릴, 알킬티오， 아릴티오, 티오카르복실레이트， 술페이트， 알킬술피닐, 술포네이토, 술파모일, 술폰아미도, 니트로, 트리플루오로메틸， 시아노， 아지도， 헤테로시클릴, 알킬아릴， 또는 방향족 또는 헤테로방향족 잔기로 치환될 수 있다. 또한， 아릴 기는 지환족 또는 헤테로시클릭 고리와 융합 또는 가교될 수 있고, 이들은 방향족이 아니므로 멀티시클릭 계 (예컨대， 테트랄린 또는 메틸렌디옥시페닐)를 형성한다. 탄소의 개수가 달리 특정되어 있지 않다면， "저급 알킬' '에는 골격 구조 내에 1 내지 10개， 예컨대 1 내지 6 개의 탄소 원자를 갖는， 상기 정의된 바와 같은 알킬기가 포함된다. As used herein, the term "aryl" includes aromatic groups such as 5- and 6-membered "unconjugated" or single-ring aromatic groups, which may include 0-4 heteroatoms, and one or more aromatic rings. "Junction" or multicyclic systems are included. Examples of aryl groups include benzene, phenyl, pi, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine and pyri Midines and the like. In addition, the term "aryl" in this specification includes a multicyclic aryl group such as tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedi Oxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benchofuran, purine, benzofuran, deazapurine or indolizine. Aryl groups having heteroatoms in the structure may also be referred to as "aryl heterocycle", "heterocycle", "heteroaryl" or "heteroaromatic". The aromatic ring may be substituted as described above at one or more ring positions, such as halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl Alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, Phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (alkylcarbonylamino, arylcarbonylamino, carbamoyl And ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, Sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. In addition, aryl groups may be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic and thus form a multicyclic system (eg, tetralin or methylenedioxyphenyl). Unless the number of carbons is specified otherwise, "lower alkyl" includes alkyl groups as defined above having 1 to 10, such as 1 to 6, carbon atoms in the backbone structure.
본 명세서의 용어 "히드톡시" 또는 "히드록실" 에는 -0H 또는 -0- 를 갖는 기가포함된다.  The term "hydroxytoxy" or "hydroxyl" herein includes groups having -0H or -0-.
본 명세서의 용어 "할로겐"에는 불소, 브롬， 염소 또는 요오드가 포함된다. 단계 (c) : GPCR19-연관 질환 치료제로서의 GPCR19 아고니스트 시험물질의 생체내 유효성 판정  The term "halogen" herein includes fluorine, bromine, chlorine or iodine. Step (c): In vivo determination of GPCR19 agonist test substance as GPCR19-associated disease therapeutic
본 발명의 방법에 있어서， GPCR19 아고니스트 시험물질을 인간을 제외한 포유동물에 투여한 후 포유동물에서 상기 ( 0 내지 ( iv)의 세포의 생성 정도가 증가하면 GPCR19 아고니스트 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. ( i ) Gr-l+CDllb+ MDSCCMyeloid-der ived suppressor cel l ) 세포 패혈증 유도 마우스에 표면형질 마커 Gr-1+ 및 CDllb+ 의 발현이 증가된 MDSC(Gr-l+CDllb+ MDSC) 세포의 양자이입 결과는 패혈증 유도 마우스에서 사망를을 감소시킨 보고가 있다. In the method of the present invention, the GPCR19 agonist test substance is a GPCR19-associated disease if the degree of production of the cells (0 to (iv) is increased in the mammal after administration of the GPCR19 agonist test substance to a mammal other than a human. Determined as a therapeutic agent. (i) Gr-1 + CDllb + MDSCCMyeloid-derived suppressor cel l) Transduction of MDSC (Gr-1 + CDllb + MDSC) cells with increased expression of surface markers Gr-1 + and CDllb + in cell sepsis-induced mice Results have been reported to reduce mortality in sepsis-induced mice.
이에 본 발명자들은 패혈증 유도 마우스에 GPCR19 아고니스트 시험물질 투여하여 비장에서의 Gr-l+CDllb+ MDSC 세포의 증가를 확인하였고, 이들 Gr-l+CDllb+ MDSC 세포는 기존에 알려진 Gr-l+CDllb+ MDSC 세포와는 유전자 발현 양상이 다른 표현형을 가지는 세포들이었으며, 양자이입에 의하여 패혈증 치료능이 있었다. 본 발명자들은 MDSC 세포의 증가를 조절하는 상위 신호 전달 경로에 GPCR19가 관련되어 있음을 규명하였고， 이에 GPCR19 아고니스트를 처리하여 GPCR19 활성화를 유도하고， Gr-l+CDllb+ MDSC 세포를 증가시킴으로 하위 다양한 신호전달 경로를 통하여 염증을 제어할 수 있음을 제시하였다. Therefore, the present inventors confirmed the increase of Gr-1 + CDllb + MDSC cells in the spleen by administering a GPCR19 agonist test substance to sepsis-induced mice, and these Gr-1 + CDllb + MDSC cells were previously known as Gr-1 + CDllb + MDSC cells. Cells with phenotypes with different gene expression patterns were found to be capable of treating sepsis by protonation. The present inventors have found that GPCR19 is involved in higher signaling pathways that regulate the increase of MDSC cells, which in turn treats GPCR19 agonists to induce GPCR19 activation and increases Gr-1 + CDllb + MDSC cells, thereby lowering various signals. It has been suggested that inflammation can be controlled through the delivery pathway.
따라서 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 Gr-l+CDllb+ MDSC 세포의 증가가 나타나면， 이는 GPCR19-연관 질환의 치료제로 판정한다. 보다 구체적으로， 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 Gr-l+CDllb+ MDSC 세포의 수가 2% 이상， 바람직하게는 이상， 보다 바람직하게는 0.5% 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 예를 들어， Gr-l+CDllb+ MDSC 세포의 수는 FACS로 분석할—수 있다. — ― _ ―  Therefore, when administration of the test substance resulted in an increase of Gr-1 + CDllb + MDSC cells compared to the control group not injected with the test substance, it is determined as a treatment for GPCR19-associated disease. More specifically, when the number of Gr-1 + CDllb + MDSC cells increases by 2% or more, preferably more than 0.5%, as compared with the control group which is not injected with the test material, the test substance is GPCR19. -To be determined as a treatment for an associated disease. For example, the number of Gr-1 + CDllb + MDSC cells can be analyzed by FACS. — ― _ ―
( i i ) GPCR19 아고니스트 시험물질의 유효성 분석은 상기 ( i i )에 기재된 특정 유전자를 고발현 또는 저발현하는 Gr-l+CDllb+ MDSC 세포수를 측정함으로써 실시할 수 있다. (ii) Analysis of the efficacy of the GPCR19 agonist test substance can be performed by measuring the number of Gr-1 + CDllb + MDSC cells that express high or low the specific gene described in (ii) above.
본 발명의 바람직한 구현예에 따르면， MDSC 세포에서 상기 ( i i )에 기재된 특정 유전자의 고발현 또는 저발현은 대조군 (시험물질을 처리하지 않은 군)과 비교하여 각각 1.5 배 이상 및 1.5 배 이하 발현 되는 것을 의미한다.  According to a preferred embodiment of the present invention, the high or low expression of the specific genes described in (ii) in the MDSC cells are expressed more than 1.5 times and less than 1.5 times, respectively, compared to the control (group not treated with the test substance), respectively. Means that.
유전자의 발현정도는 당업계에서 통상적으로 이용되는 유전자발현 분석 방법， 예컨대 실시간 RT-PCR 방법 또는 마이크로어레이 방법 (참조: Sambrook , J . et al . , Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spr ing Harbor Press(2001) )에 따라 실시할 수 있다. The expression level of the gene is determined by gene expression analysis methods commonly used in the art, such as real-time RT-PCR method or microarray method (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).
상술한 유전자들의 고발현 또는 저발현되는 MDSC 세포는 GPCR19 아고니스트로 투여로 인해 형질전환 및 생체내 증폭되고， 형질전환 및 생체내 증폭된 MDSC 세포를 패혈증 마우스에 투여한 결과 패혈증 치료효과를 확인하였고 이는 실시예 1에서 보다 상세하게 제시되고 있다. GPCR19 아고니스트 시험물질로 바람직하게는, 소듐 타우로데옥시콜레이트 (HY-2191)를 사용하였고， 상기 시험물질의 투여로 인해 생성된 MDSC 세포는 "H-MDSC" 세포로 명명하였다. 패혈증 마우스에 GPCR19-연관 질환 치료제로써 판정을 위해 사용된 H-MDSC 세포의 패혈증 치료효과는 다른 대조군에 비해서 현저하게 높은 80¾의 생존율을 보였다. 이와 같은 결과는, GPCR19 아고니스트 시험물질의 투여 결과로 생성된 H- MDSC 세포를 패혈증의 치료제 또는 GPCR19-연관 질환 치료제로 사용 될 수 있음을 제시한다.  MDSC cells with high or low expression of the above-described genes were transformed and amplified in vivo due to the administration of GPCR19 agonist. This is shown in more detail in Example 1. As the GPCR19 agonist test substance, sodium taurodeoxycholate (HY-2191) was preferably used, and MDSC cells generated by administration of the test substance were named "H-MDSC" cells. The sepsis treatment effect of H-MDSC cells used for determination as a GPCR19-associated disease therapeutic agent in sepsis mice showed a significantly higher survival rate of 80¾ compared to other controls. These results suggest that H-MDSC cells produced as a result of the administration of the GPCR19 agonist test substance can be used as a treatment for sepsis or as a treatment for GPCR19-associated diseases.
따라서 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 상기 ( i i )에서 상술한 유전자들의 고발현 또는 저발현을 보이는 Gr-l+CDllb+ MDSC 세포의 증가가 나타나면, 이는 GPCR19-연관 질환의 치료제로 판정한다. 보다 구체적으로， 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 상기 ( i i )에서 상술한 유전자들의 고발현 또는 저발현을 보이는 MDSC 세포의 수가 2% 이상, 바람직하게는 1% 이상, 보다 바람직하게는 0.5% 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 예를 들어， MDSC 세포의 수는 FACS로 분석할 수 있다. 상기 ( i i )에서 고발현 또는 저발현된 유전자의 리스트는 앞서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.  Therefore, as a result of administration of the test substance, an increase in Gr-l + CDllb + MDSC cells showing high or low expression of the genes described above in (ii) was observed compared to the control group not injected with the test substance. Judging by the treatment. More specifically, the number of MDSC cells showing high or low expression of the genes described in (ii) above 2%, preferably 1% or more, as compared with the control group which did not inject the test substance as a result of the administration of the test substance. More preferably, by 0.5% or more, the test substance is determined to be a treatment for GPCR19-associated disease. For example, the number of MDSC cells can be analyzed by FACS. Since the list of high or low expression genes in (i) is the same as described above, the common content between the two is omitted to avoid excessive complexity of the present specification according to the repetitive description.
( i i i ) CD23hi면역조절 B 세포 (CD23hi Regulatory B cel l ) (iii) CD23 hi regulatory B cell (CD23 hi Regulatory B cel l)
B 세포는 면역 반응에 있어서 전통적으로 항체를 생성하여 체액성 면역반웅 (humoral immune response)에 관여하는 세포로 알려져 있다. 이중 일부 면역조절 B 세포는 염증 반웅을 조절하는 것으로 알려져 있다.  B cells are traditionally known as cells that produce antibodies in the immune response and participate in humoral immune responses. Some of these immunoregulatory B cells are known to regulate inflammatory reactions.
이에 본 발명자들은 패혈증 유도 마우스에 GPCR19 아고니스트 시험물질 투여하여 비장에서의 CD23hi면역조절 B 세포의 증가를 확인하였고, 상기 CD23hi 면역조절 B 세포의 패혈증 치료 효과도 확인 하였다. 본 발명자들은 CD23hi 면역조절 B 세포의 증가를 조절하는 상위 신호 전달 경로에 GPCR19가 관련되어 있음을 규명하였고， 이에 GPCR19 아고니스트를 처리하여 GPCR19 활성화를 유도하고, CD23hi 면역조절 B 세포를 증가시킴으로 하위 다양한 신호전달 경로를 통하여 염증을 제어할 수 있음을 제시하였다. Therefore, the present inventors confirmed the increase of CD23 hi immunoregulatory B cells in the spleen by administering a GPCR19 agonist test substance to sepsis-induced mice. Sepsis treatment effect of the CD23 hi immunoregulatory B cells was also confirmed. The present inventors have found that GPCR19 is involved in a higher signal transduction pathway that regulates the increase of CD23 hi immunoregulatory B cells, thereby treating GPCR19 agonists to induce GPCR19 activation and increasing CD23 hi immunoregulatory B cells. It has been shown that inflammation can be controlled through various signaling pathways.
따라서 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 CD23hi 면역조절 B 세포의 증가가 나타나면， 이는 GPCR19-연관 질환의 치료제로 판정한다. 보다 구체적으로， 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 CD23hi 면역조절 B 세포의 수가 2% 이상， 바람직하게는 1% 이상， 보다 바람직하게는 0.5¾> 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 예를 들어, CD23hi면역조절 B 세포의 수는 FACS로 분석할 수 있다. ( iv) GPCR19 아고니스트 시험물질의 분석은 상기 ( iv)에 기재된 특정 유전자를 고발현 또는 저발현하는 CD23hi 면역조절 B 세포를 측정함으로써 실시할 수 있다. Therefore, when administration of the test substance resulted in an increase of CD23 hi immunomodulatory B cells compared to the control group not injected with the test substance, it is determined as a treatment for GPCR19-associated disease. More specifically, when the number of CD23 hi immunomodulatory B cells increases by 2% or more, preferably 1% or more, and more preferably 0.5¾>, compared to the control group that is not injected with the test material, Is determined as a therapeutic agent for GPCR19-associated diseases. For example, the number of CD23 hi immunoregulatory B cells can be analyzed by FACS. (iv) Analysis of the GPCR19 agonist test substance can be carried out by measuring CD23 hi immunomodulatory B cells expressing or expressing a specific gene described in (iv) above.
본 발명의 바람직한 구현 예에 따르면 , CD23hi 면역조절 B 세포에서 상기 ( iv)에 기재된 특정 유전자의 고발현 또는 저발현은 대조군 (시험물질을 처리하지 않은 군)과 비교하여 각각 1.5 배 이상 및 1.5 배 이하 저발현 되는 것을 의미한다. According to a preferred embodiment of the invention, the high or low expression of the specific genes described in (iv) above in CD23 hi immunomodulatory B cells is 1.5-fold and 1.5-fold, respectively, compared to the control (group not treated with test substance), respectively. It means that the expression is less than twice.
유전자의 발현정도는 당업계에서 통상적으로 이용되는 유전자발현 분석 방법, 예컨대 실시간 RT-PCR 방법 또는 마이크로어레이 방법 (참조: Sambrook, J . et al . , Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spr ing Harbor Press(2001) )에 따라 실시할 수 있다.  The expression level of the gene is determined by gene expression analysis methods commonly used in the art, such as real-time RT-PCR method or microarray method (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spr ing Harbor Press (2001)).
상술한 유전자들의 고발현 또는 저발현되는 CD23hi면역조절 B 세포는 GPCR19 아고니스트의 투여로 인해 형질전환 및 생체내 증폭 되고， 형질전환 및 생체내 증폭된 CD23hi 면역조절 B 세포를 패혈증 마우스에 투여한 결과 패혈증 치료효과를 확인하였고 이는 실시예 15에서 보다 상세하게 제시되고 있다. CD23hi 면역조절 B 세포의 형질 전환 및 생체내 증폭을 위하여 GPCR19 아고니스트 시험물질에 의하여， 바람직하게는 ( i ) 또는 ( i i )에서 생성된 H- MDSC 세포 또는 GPCR19 아고니스트 시험물질인 소듐 타우로데옥시콜레이트 (HY2191)를 사용하였다. H-MDSC 세포를 투여한 결과 생성된 세포는 "¾M" 세포로 명명 하였고， 소듐 타우로데옥시콜레이트를 투여한 후 48시간 뒤에 생체내에서 생성된 세포는 "48h BH" 세포로 명명하였다. 패혈증 마우스에 GPCR19-연관 질환 치료제로써 판정을 위해 사용된 ¾M 세포의 패혈증 치료효과는 다른 대조군에 비해서 현저하게 높은 100%의 생존율을 보였고 (실시예 13)， 48h BH 세포의 치료효과 역시 80%의 생존율을 보였다 (실시예 14 ) . 이와 같은 결과는， GPCR19 아고니스트 시험물질에 의하여， 바람직하게는 ( i ) 또는 ( Π )에서 생성된 H-MDSC 세포를 투여하여 생성된 ¾M 세포 또는 소듐 타우로데옥시콜레이트를 투여한후 48시간 뒤에 생성된 B 세포 (48h ¾)로 이루어진 군에서 선택된 어느 하나를 패혈증의 치료제 또는 GPCR19-연관 질환 치료제로 사용 될 수 있음을 제시한다. CD23 hi immunoregulatory B cells with high or low expression of the above-described genes are transformed and amplified in vivo due to the administration of GPCR19 agonists, and the transfected CD23 hi immunoregulatory B cells are administered to sepsis mice. As a result, sepsis treatment effect was confirmed, which is shown in more detail in Example 15. For transformation and in vivo amplification of CD23 hi immunoregulatory B cells, the H- produced by GPCR19 agonist test substance, preferably in (i) or (ii) Sodium taurodeoxycholate (HY2191), a MDSC cell or GPCR19 agonist test material, was used. The cells produced as a result of administration of H-MDSC cells were named "¾M" cells, and 48 hours after the administration of sodium taurodeoxycholate, the cells produced in vivo were named "48h B H " cells. The sepsis treatment effect of ¾M cells used for determination as a GPCR19-associated disease treatment in sepsis mice showed a significantly higher survival rate of 100% compared to other controls (Example 13), and the treatment effect of 48h B H cells was also 80%. The survival rate was (Example 14). These results were obtained by the GPCR19 agonist test substance, preferably 48 hours after administration of ¾M cells or sodium taurodeoxycholate produced by administration of H-MDSC cells produced in (i) or (Π). It is suggested that any one selected from the group consisting of B cells generated later (48h ¾) can be used as a therapeutic agent for sepsis or a therapeutic agent for GPCR19-associated diseases.
따라서 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 상기 ( iv)에서 상술한 유전자들의 고발현 또는 저발현을 보이는 면역조절 B 세포의 증가가 나타나면, 이는 GPCR19-연관 질환의 치료제로 판정한다. 보다 구체적으로, 시험물질의 투여 결과 시험물질을 주입하지 않은 대조군과 비교하여 상기 ( iv)에서 상술한 유전자들의 고발현 또는 저발현을 보이는 면역조절 B 세포의 수가 2% 이상, 바람직하게는 1% όᅵ상, 보다 바람직하게는 0.5% 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 예를 들어， 면역조절 Β 세포의 수는 FACS로 분석할 수 있다.  Therefore, as a result of administration of the test substance, an increase in immunoregulatory B cells showing high or low expression of the genes described above in (iv) compared to the control group not injected with the test substance, was determined as a treatment for GPCR19-associated disease. do. More specifically, the number of immunoregulatory B cells exhibiting high or low expression of the genes described above in (iv) as compared with the control group which is not injected with the test substance as a result of the administration of the test substance is 2% or more, preferably 1% More preferably, by increasing 0.5% or more, the test substance is determined to be a treatment for GPCR19-associated disease. For example, the number of immunoregulatory Β cells can be analyzed by FACS.
상기 ( iv)의 MDSC또는 면역조절 B 세포에서 고발현 또는 저발현되는 유전자 리스트는 앞서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.  Since the list of genes that are expressed high or low in the MDSC or immunoregulatory B cells of (iv) is the same as described above, the common content between the two is to avoid excessive complexity of the present specification according to the repetitive description, The description is omitted.
결과적으로 본 발명의 방법에 있어서, 단계 (c)에서 상술한 내용과 같이 GPCR19 아고니스트 시험물질을 처리하지 않은 대조군과 비교하여 GPCR19 아고니스트 시험물질을 투여한 군에서 상기 ( i ) 내지 ( iv)의 세포의 생성 정도가 2% 이상， 바람직하게는 1¾ 이상， 보다 바람직하게는 0 , 5¾> 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 본 발명의 다른 양태에 따르면, 본 발명은 다음 단계를 포함하는As a result, in the method of the present invention, (i) to (iv) in the group administered with the GPCR19 agonist test substance as compared to the control group did not treat the GPCR19 agonist test substance as described above in step (c) If the production of cells increases by 2% or more, preferably 1¾ or more, more preferably 0, 5¾> or more, the test substance is determined to be a treatment for GPCR19-associated disease. According to another aspect of the invention, the invention comprises the following steps
GPCR19-연관 질환의 치료제의 시험관내 스크리닝 방법을 제공한다: Provided are in vitro screening methods for the treatment of GPCR19-associated diseases:
(a) 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 시험물질을 골수세포 집단 (bone marrow cell population)에 처리하는 단계;  (a) treating the bone marrow cell population with a physiological activity modulator and a GPCR19 agonist test substance in vivo;
(b) 상기 골수세포 집단에서 (i) Gr-l+CDllb+ MDSC(Myeloid-derived suppressor cell), (i i ) Prok2, Osm, Ltf , Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 또는 (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4mol , Cyp51, Sqle, L0C100040592, B3gnt8, 1300014106Rik, Fdps, Ctsa, D隱 d, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, LOC100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik: Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, lrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4 Rgsl, 0asl2, I17r, Us l8, Mxl, LOC 100048346, Mx2, Tyki , Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat , Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Rlf9, Pexlla, D14Ertd668e, Mlkl , Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 시험물질을 처리하지 않은 대조군과 비교하여 상기 시험물질을 처리한 골수세포 집단에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. 단계 (a): 세포 생리 활성 조절 물질 및 GPCR19 아고니스트 시험물질의 시험관내 골수세포에 처리 (b) (i) Gr-1 + CDllb + Myeloid-derived suppressor cells (MDSC), (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h , Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2 , Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd861FflaRik Smp004, HiflaRik , D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 or MxlSC (iii) CD23 hi immunomodulatory B cells or (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cbl 83, Mybl2 , If t 140, Snxll, Swap70, Ccbp2, Sc4m ol, Cyp51, Sqle, L0C100040592, B3gnt8, 1300014106Rik, Fdps, Ctsa, D 隱 d, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwh3, Cr4, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, LOC100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 Lmnbl Highly expressing the Blvrb or Actb gene or 1190002H23Rik : Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndcra, Tmndc5 Am4 , L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, lrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcolb2, Srpr2, Tsr2in , Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4 Rgsl, 0asl2, I17r, Us l8, Mxl, LOC 100048346, Mx2, Tyki, Hba-al, Lgals3bp, Ifitm3as Cfll3f2 , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Rlf9, Plllae Analyzing the production of immunoregulatory B cells that express low levels of Itk, Serpinb6b or Cd69; When the degree of production of the cells of (i) to (iv) in the bone marrow cell population treated with the test substance was increased compared to the control group compared to the control group not treated with the test substance, the test substance was associated with GPCR19-associated disease. Judging by the treatment. Step (a): Treatment of In Vitro Bone Marrow Cells with Cellular Biological Modulators and GPCR19 Agonist Test Substances
본 발명의 방법에 있어서， 단계 (a)에서 세포 생리 활성 조절 물질 및 GPCR19 아고니스트 시험물질을 골수세포 집단에 처리한다.  In the method of the present invention, in step (a), the cell physiological activity modulator and the GPCR19 agonist test substance are treated to the myeloid cell population.
본 발명의 방법에 사용되는 세포 생리 활성 조절 물질은 상기 생체내 생리 활성 조질물질과 동일하고， GPCR19 아고니스트 시험물질 역시 상술한 내용과 동일하기 때문에 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.  Since the cell bioactivity modulator used in the method of the present invention is the same as the biologically active crude substance in vivo, and the GPCR19 agonist test substance is also the same as described above, the common contents between the two are described according to the repetition description. In order to avoid excessive complexity, the description thereof is omitted.
상기 세포 생리 활성 조절 물질 및 GPCR19 아고니스트 시험물질이 처리된 골수세포 집단 (bone marrow cell population)은 대퇴골 및 경골의 골수에서 유래된 세포 집단으로， 바람직하게는 조혈성세포 (hematopoietic) 또는 골수성세포 (myeloid eel Is)를 형성할 수 있는 세포이고, 보다 바람직하게는 B 세포, T 세포, NK 세포， 림프구 수지상 세포, 골수형 수지상 세포， 과립구, 마크로파지， 거핵세포 또는 적혈구 세포를 포함하는 조혈기관 계통의 모든 세포 유형으로 분화할 수 있는 클론원성 및 자가증식형 조혈성 세포， myeloid derived suppressor cell(MDSC)로 분화할 수 있는 공통 골수계 전구세포 (CMP, co讓 on myeloid precursor) 또는 공통 림프계 전구세포 (CLP, co隱 on lymphoid precursor)이고， 가장 바람직하게는 CD23hi 표면형질 마커를 발현하는 B 세포를 형성할 수 있는 세포 또는 Gr+CDllb+ 표면형질 마커를 발현하는 MDSC세포를 형성할 수 있는 세포이다. 본 발명의 방법에 있어서， 생체 내 생리적 활성 조절 물질을 골수세포 집단에 처리하는 것은 배양되는 골수세포의 배양액에 처리할 수 있으며， 생체 내 생리적 활성 조절 물질의 하나로 사용되는 리포폴리사카라이드 (LPS)의 농도로 바람직하게는 0.0001 mg/ l 내지 10 mg/m£이고， 보다 바람직하게는 0.0005 mg/mi 내지 1 mg/m£이고, 보다 더 바람직하게는 0.001 mg/mi 내지 0. 1 mg/ 이고 가장 바람직하게는 0.001 mg/i 를 처리할 수 있다. 또한 GPCR19 아고니스트 시험물질을 골수세포 집단에 처리하는 것은 배양되는 골수세포의 배양액에 처리할 수 있으며, GPCR19 아고니스트 시험물질의 하나로 사용되는 소듐 타우로데옥시콜레이드 (HY2191)의 농도로 바람직하게는 0.0001 mg/mt 내지 100 mg/mi 이고， 보다 바람직하게는 0.001 mg/ml 내지 10 mg/ml 이고， 보다 더 바람직하게는 0.01 mg/mi 내지 1 mg/ 이고， 가장 바람직하게는 0.05 mg/ 을 배양액에 처리한다. 단계 (b) : GPCR19-연관 질환 치료제로서의 GPCR19 아고니스트 시험물질 판정 Bone marrow cell population treated with the cell biological activity regulator and GPCR19 agonist test material is a cell population derived from the bone marrow of the femur and tibia, preferably hematopoietic or myeloid cells (myeloid). eel Is), more preferably all of the hematopoietic lineages, including B cells, T cells, NK cells, lymphocyte dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes or red blood cells Clonal and autologous hematopoietic cells that can differentiate into cell types, common myeloid precursor cells (CMP) or common lymphoid progenitor cells (CLP) that can differentiate into myeloid derived suppressor cells (MDSC) , co 隱 on lymphoid precursor), most preferably cells capable of forming B cells expressing a CD23 hi surface morphological marker or Gr +. Cells capable of forming MDSC cells expressing CDllb + surface markers. In the method of the present invention, the treatment of the physiologically active substance in vivo to the bone marrow cell population can be treated to the culture of bone marrow cells to be cultured, lipopolysaccharide (LPS) used as one of the physiologically active substance in vivo It is preferably in a concentration of 0.0001 mg / l to 10 mg / m £, more preferably 0.0005 mg / mi to 1 mg / m £, even more preferably 0.001 mg / mi to 0.1 mg / Most preferably 0.001 mg / i. In addition, the treatment of the bone marrow cell population with the GPCR19 agonist test substance may be treated with the culture medium of the cultured bone marrow cells, and preferably at a concentration of sodium taurodeoxycollide (HY2191) used as one of the GPCR19 agonist test substances. Is 0.0001 mg / mt to 100 mg / mi, more preferably 0.001 mg / ml to 10 mg / ml, even more preferably 0.01 mg / mi to 1 mg /, most preferably 0.05 mg / Treated in culture. Step (b): Determination of GPCR19 agonist test substance as GPCR19-associated disease treatment
GPCR19 아고니스트 시험물질 처리 결과, 시험물질을 처리하지 않은 대조군과 비교하여 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 시험물질을 처리한 골수세포 집단에서 상기 ( i ) 내지 ( iv)의 세포의 생성 정도가 2% 이상， 바람직하게는 1% 이상， 보다 바람직하게는 0.5% 이상 증가하면 시험물질은 GPCR19-연관 질환의 치료제로 판정한다.  As a result of the treatment of GPCR19 agonist test material, the production level of the cells of (i) to (iv) in the bone marrow cell population treated with the physiological activity modulator and GPCR19 agonist test material in vivo compared to the control group not treated with the test material. Is increased by at least 2%, preferably at least 1%, more preferably at least 0.5%, and the test substance is determined to be a treatment for GPCR19-associated disease.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 시험물질 처리로 인해 생성되는 상기 ( i ) 내지 ( iv)의 MDSC 및 면역조절 B 세포에서 고발현 또는 저발현되는 유전자 리스트 및 시험물질의 처리 방법은 앞서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서와 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. 본 발명의 또 다른 양태에 따르면， 본 발명은 다음 단계를 포함하는 GPCR19 아고니스트의 생체내 효능 분석 방법을 제공한다:  List of genes and methods of treatment of high or low expression genes in (i) to (iv) MDSC and immunoregulatory B cells produced by treatment of in vivo physiological activity modulators and GPCR19 agonist test substances are described above. Since the content is the same as the one, the content in common between the two is omitted in order to avoid excessive complexity and the present specification according to the repeated description. According to another aspect of the present invention, the present invention provides a method for analyzing the efficacy of a GPCR19 agonist in vivo, comprising the following steps:
(a) 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하는 단계 ; (b) 상기 포유동물에 GPCR19아고니스트를 투여하는 단계; 및 (a) administering an in vivo physiologically active modulator to a mammal other than a human; (b) administering a GPCR19 agonist to said mammal; And
(c) 상기 포유동물에서 (i) Gr-l+CDllb+ MDSC(Myeloid-der ived suppressor cell), (ii) Prok2, Osm, Ltf , Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2， Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110， Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 또는 v) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 , Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 1300014 I 06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7 , Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl , Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi , Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, lra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC100048346, Mx2, Tyki , Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, GprlW, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat , Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Rlf9, Pexlla, D14Ertd668e, Mlkl , Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 GPCR19 아고니스트를 주입하지 않은 대조군과 비교하여 상기 GPCR19 아고니스트를 주입한 포유동물에서 상기 ( i ) 내지 ( iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 GPCR19 아고니스트는 효능이 있는 것으로 판정한다. 본 발명의 방법에 사용되는 GPCR19 아고니스트의 생체내 효능 분석하는 방법을 각각의 단계 별로 상세하게 설명하면 다음과 같다: (c) (i) Gr-l + CDllb + Myeloid-derived suppressor cell (MDSC), (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, AdoracllO Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd863 Hi B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, MDnac3 or Dnasell3 A) CD23 hi immunomodulatory B cell or v) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Mybl2 If t 140, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 1300014 I 06 Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwga, Cr4, Arna Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbllv Pira Or expressing Actb gene or 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Srpb2pl, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC100048346, Mx2, Tyki, Hba-al, Lllm3bp Clf 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197 , GprlW, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Rlf9, Pexlla, Analyzing the production of immunoregulatory B cells that low express D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes; The GPCR19 agonist is effective when the degree of production of the cells of (i) to (iv) is increased in comparison with the control group in the mammal injected with the GPCR19 agonist compared to the control group not injected with the GPCR19 agonist. Determined to be. In vivo efficacy analysis of the GPCR19 agonist used in the method of the present invention will be described in detail for each step as follows:
단계 (a) : 생체 내 생리적 활성 조절 물질의 투여 Step (a): administration of physiologically active modulators in vivo
본 발명의 방법에 있어서， GPCR19 아고니스트의 효능 분석을 위하여 먼저 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하여 생체 내에 생리적 반웅을 유발한다. 단계 )에서 사용되는 생체 내 생리적 활성 조절 물질, 그의 투여하는 방법 및 포유동물은 앞에서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. 단계 (b) : GPCR19 아고니스트의 투여  In the method of the present invention, in order to analyze the efficacy of the GPCR19 agonist, a physiological reaction modulator in vivo is first administered to a mammal except a human to cause physiological reaction in vivo. Since the in vivo physiologically active substance used in step), the method of administration thereof, and the mammal are the same as described above, the common content between the two is to avoid excessive complexity of the present specification according to the repetitive description. Omit the description. Step (b): Administration of GPCR19 Agonist
본 발명의 방법에 있어서, 단계 (b)의 방법으로 생체 내 생리적 활성 조절 물질을 투여된 상기 포유동물에 GPCR19 아고니스트를 투여한다. 단계 (b)에서 GPCR19 아고니스트， 그의 투여하는 방법 및 포유동물은 앞에서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. 단계 (c) : GPCR19 아고니스트의 생체내 효능 판정  In the method of the present invention, GPCR19 agonist is administered to the mammal to which the physiologically active substance is administered in vivo by the method of step (b). In step (b), the GPCR19 agonist, the method of administration thereof and the mammal are the same as described above, so the common content between the two is omitted in order to avoid excessive complexity of the present specification according to the repeated description. do. Step (c): Determination of In Vivo Efficacy of GPCR19 Agonist
GPCR19 아고니스트 투여 결과， GPCR19 아고니스트를 투여하지 않은 대조군과 비교하여 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트를 투여한 포유동물에서 상기 ( i ) 내지 ( iv)의 세포의 생성 정도가 2% 이상， 바람직하게는 1% 이상， 보다 바람직하게는 0.5% 이상 증가하면 GPCR19 아고니스트는 효능이 있는 것으로 판정한다.  As a result of the GPCR19 agonist administration, the production level of the cells of (i) to (iv) was 2% or more in the mammal to which the physiologically active substance and GPCR19 agonist were administered in comparison with the control group to which the GPCR19 agonist was not administered. , Preferably at least 1%, more preferably at least 0.5%, determines that the GPCR19 agonist is efficacious.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 투여로 인해 생성되는 상기 ( i ) 내지 ( iv)의 MDSC 및 면역조절 B 세포에서 고발현 또는 저발현되는 유전자 리스트 및 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트의 투여 방법은 앞서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. 본 발명의 또 다른 양태에 따르면, 본 발명은 다음 단계를 포함하는 GPCR19 아고니스트의 시험관내 효능 분석 방법을 제공한다: High expression in the MDSC and immunoregulatory B cells of (i) to (iv) produced by administration of a physiologically active regulatory substance and GPCR19 agonist in vivo or Since the list of low-expressed genes and the method of administering the physiologically active regulatory substance and the GPCR19 agonist in vivo are the same as described above, the common content between the two is to avoid excessive complexity of the present specification according to the repetitive description. Omit the description. According to another aspect of the present invention, the present invention provides a method for analyzing in vitro potency of a GPCR19 agonist comprising the following steps:
(a) 세포 생리 활성 조절 물질 및 GPCR19 아고니스트를 골수세포 집단 (bone marrow cell population)에 처리하는 단계; 및  (a) treating the cell physiological activity modulator and GPCR19 agonist to a bone marrow cell population; And
(b) 상기 골수세포 집단에서 (0 Gr-l+CDllb+ MDSC(Myeloid-derived suppressor cell), (i i ) Prok2, Osm, Ltf , Kiral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 또는 (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg3 , Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 , Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C 100040592， B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dnwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Firm, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd^ Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4 Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC100048346 , Mx2, Tyki , Hba-al , Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprl , Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, lf9, Pexlla, D14Ertd668e, Mlkl , Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 GPCR19 아고니스트를 처리하지 않은 대조군과 비교하여 상기 GPCR19 아고니스트를 처리한 골수세포 집단에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 GPCR19 아고니스트는 효능이 있는 것으로 판정한다 단계 (a): 세포 생리 활성 조절 물질 및 GPCR19 아고니스트의 처리 (b) (0 Gr-l + CDllb + Myeloid-derived suppressor cell (MDSC), (ii) Prok2, Osm, Ltf, Kiral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h , Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2 , Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd861FflaRik Smp004, HiflaRik , D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 or MxlSC (iii) ) CD23 hi immunomodulatory B cells or (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg3, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4m ol, Cyp51, Sqle, L0C 100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dnwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwhga, Crm3, Fir , Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd4 Lmnbl, Pira Highly expressing the Blvrb or Actb gene or 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxaigo, Txndc5 Am Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp2in Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4 Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC100048346, Mx2, Tyki, Hba3alf Lgal , 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprl, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Coil Analyzing the production of immunoregulatory B cells that low express D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes; The GPCR19 agonist is more effective when the degree of production of the cells of (i) to (iv) is increased in comparison to the control group in the bone marrow cell treated with the GPCR19 agonist compared to the control group not treated with the GPCR19 agonist. Step (a): Treatment of cell bioactive modulators and GPCR19 agonists
본 발명의 방법에 있어서, GPCR19 아고니스트의 효능을 분석하기 위하여 먼저 생체 내 생리적 활성 조절 물질을 골수세포 집단 (bone marrow cell population)에 처리하여 골수세포의 생리적 변화를 유도한다. 단계 (a)에서 사용되는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트, 그의 처리하는 방법 및 골수세포 집단은 앞에서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. 단계 (b): GPCR19 아고니스트의 시험관내 효능 판정  In the method of the present invention, in order to analyze the efficacy of the GPCR19 agonist, a physiological change of bone marrow cells is first induced by treating the bone marrow cell population with a physiological activity regulator in vivo. In vivo physiologically active modulators and GPCR19 agonists, methods of treatment and bone marrow cell populations used in step (a) are identical to those described above, and therefore common content between the two is described herein according to the repetition. In order to avoid excessive complexity, the description is omitted. Step (b): In Vitro Efficacy Determination of GPCR19 Agonists
GPCR19 아고니스트 시험물질 처리 결과, 시험물질을 처리하지 않은 대조군과 비교하여 세포 생리 활성 조절 물질 및 GPCR19 아고니스트 시험물질을 처리한 골수세포 집단에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 이상， 바람직하게는 1% 이상， 보다 바람직하게는 0.5% 이상 증가하면 GPCR19 아고니스트는 효능이 있는 것으로 판정한다. 시험물질은 GPCR19—연관 질환의 치료제로 판정한다.  As a result of the GPCR19 agonist test material treatment, the production of the cells of (i) to (iv) in the bone marrow cell population treated with the cell bioactivity modulator and GPCR19 agonist test material was compared with the control group without the test material. Above, preferably at least 1%, more preferably at least 0.5%, the GPCR19 agonist is determined to be efficacious. The test substance is determined to be a treatment for GPCR19-associated disease.
세포 생리 ¾성 조절—물질 및 _GPCR19 -아고니스트ᅵ -시ᅭ험물질 처리로 인해 생성되는 상기 ( i ) 내지 ( iv)의 MDSC 및 면역조절 B 세포에서 고발현 또는 저발현되는 유전자 리스트 및 시험물질의 처리 방법은 앞서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 생략한다. 본 발명의 바람직한 구현 예에 따르면, 상기 포유동물은 마우스， 랫트， 개， 원숭이, 염소, 양 또는 토끼인 것을 특징으로 하는 방법이다. 본 발명의 방법에 사용이 가능한 포유동물은 인간을 제외한 포유동물에게 투여가 가능하며 바람직하게는 포유동물, 보다 바람직하게는 반려 동물 (예를 들어, 개， 고양이 또는 새)， 농장용 동물 (예를 들어, 소， 양， 돼지, 말 또는 닭) 또는 실험실용 동물 (예를 들어, 랫트, 마우스， 기니피그 또는 새)이고, 보다 더 바람직하게는 마우스, 랫트, 개, 원숭이， 염소， 양, 토끼이고 가장 바람직하게는 마우스이다. Regulation of Cell Physiological Properties—Materials and _GPCR1 9 -Agonists Since the list of genes and test substances that are high or low expression in the MDSC and immunoregulatory B cells of (i) to (iv) generated by the same method as described above is the same, the common contents between the two In order to avoid excessive complexity of the present specification according to the repeated description, the description is omitted. According to a preferred embodiment of the invention, the mammal is a method characterized in that the mouse, rat, dog, monkey, goat, sheep or rabbit. Mammals that can be used in the methods of the present invention can be administered to mammals other than humans and are preferably mammals, more preferably companion animals (eg dogs, cats or birds), farm animals (eg For example, cattle, sheep, pigs, horses or chickens or laboratory animals (eg, rats, mice, guinea pigs or birds), and more preferably mice, rats, dogs, monkeys, goats, sheep, rabbits. And most preferably a mouse.
본 발명의 바람직한 구현 예에 따르면, 본 발명의 방법에 사용되는 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질은 박테리아 유래 물질 진균 유래 물질， 바이러스 유래 물질, 자가 유래 물질, 또는 알러젠 (al lergen)인 것을 특징으로 하는 방법이다. 본 발명의 바람직한 구현예에 따르면， 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸이다.  According to a preferred embodiment of the present invention, the in vivo physiologically active regulatory substance or cell physiologically active substance used in the method of the present invention is a bacterial-derived fungal-derived, viral-derived, self-derived, or allergen It is a method characterized by the above. According to a preferred embodiment of the present invention, the physiological activity modulator or cell physiological activity modulator in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance.
본 발명의 바람직한 구현 예에 따르면, 본 발명의 방법에 사용되는 상기 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질은 GPCR19를 제외한 세포의 수용체에 작용하여 표적 세포의 염증 향진 작용을 촉진하는 물질이다. 이들은 앞서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 생략한다.  According to a preferred embodiment of the present invention, the in vivo physiologically active substance or cell physiological activity modulator used in the method of the present invention is a substance that promotes the inflammatory promoting action of target cells by acting on receptors of cells except GPCR19 . Since these are the same as the foregoing, the contents common between the two are omitted in order to avoid excessive complexity of the present specification according to the repetitive description.
본 발명의 바람직한 구현예에 따르면, 본 발명의 방법에 사용되는 GPCR19 아고니스트는 하기 화학식 1의 화합물, 그의 염 또는 수화물이다: According to a preferred embodiment of the invention, the GPCR19 agonist used in the process of the invention is a compound of formula (1), a salt or a hydrate thereof:
Figure imgf000030_0001
Figure imgf000030_0001
상기 화학식에서 ^은 수소， 하이드록시， 치환 또는 비치환 알킬 또는 할로겐; R2는 수소 또는 α -하이드록시; R3은 하이드록시， NH(C¾)mS03H(m은 정수 0， 1， 2， 3 , 4 또는 5) 또는 NH(CH2)nC02H(n은 정수 0 , 1， 2 , 3， 4 또는 5) ; 는 수소， 알킬 또는 할로겐; R5는 수소， 치환 또는 비치환 알킬 또는 아릴이고; R6은 수소 또는 Rs 및 ¾가 부착된 탄소와 함께 3 , 4 , 5 또는 6개 원자 크기로 형성된 고리이고; R7은 수소, 하이드록시， 치환 또는 비치환 알킬; R8은 수소 또는 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; R10은 수소 또는 치환 또는 비치환 알킬; Ru은 수소 또는 치환 또는 비치환 알킬이다. ^ In the formula is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; R 2 is hydrogen or α-hydroxy; R 3 is hydroxy, NH (C¾) m S0 3 H (m is integer 0, 1, 2, 3, 4 or 5) or NH (CH 2 ) n C02H (n is integer 0, 1, 2, 3, 4 or 5); Is hydrogen, alkyl or halogen; R 5 is hydrogen, substituted or unsubstituted alkyl or aryl; R 6 is hydrogen or a ring formed with 3, 4, 5 or 6 atom size with Rs and ¾ attached carbon; R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl; R 8 is hydrogen or substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; R 10 is hydrogen or substituted or unsubstituted alkyl; R u is hydrogen or substituted or unsubstituted alkyl.
화학식 1의 화합물은 앞서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 본 발명의 바람직한 구현예에 따르면， 본 발명의 방법에 사용되는 GPCR19 아고니스트 시험물질， 생체 내 생리적 활성 조절 물질， 세포 생리 활성 조절 물질 및 GPCR19 아고니스트의 투여 또는 처리로 인해 생성된 MDSC에서 고발현 또는 저발현 되는 유전자는 앞서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 생략한다.  Since the compound of formula 1 is the same as described above, the common content between the two is to avoid the excessive complexity of the present specification according to the repetitive description, according to a preferred embodiment of the present invention, the method of the present invention Genes that are highly expressed or underexpressed in the MDCR produced by the administration or treatment of the GPCR19 agonist test substance, in vivo physiological activity modulator, cellular bioactivity modulator, and GPCR19 agonist are used in the same manner as described above. Therefore, in order to avoid excessive complexity of this specification according to repetitive description, the content common to both is abbreviate | omitted the description.
본 발명의 바람직한 구현 예에 따르면, 본 발명의 방법에 사용되는 GPC 19 아고니스트 시험물질， 생체 내 생리적 활성 조절 물질， 세포 생리 활성 조절 물질 및 GPCR19 아고니스트의 투여 또는 처리로 인해 생성된 면역조절 B 세포에서 고발현 또는 저발현 되는 유전자는 앞서 상술한 내용과 동일하기 때문에， 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다, 본 발명의 다른 양태에 따르면， 본 발명은 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성되고 Gr-l+CDllb+ 표면 형질형을 가지며 항염증 활성을 갖는 세포인 MDSC(Mye 1 o i d-der i ved suppressor cel l )를 제공한다. According to a preferred embodiment of the invention, an immunomodulatory B produced by the administration or treatment of a GPC 19 agonist test substance, an in vivo physiological activity modulator, a cell bioactivity modulator and a GPCR19 agonist for use in the method of the invention Since genes that are high or low in the cell are the same as those described above, the common content between the two is omitted to avoid excessive complexity of the present specification according to the repetitive description, According to another aspect of the present invention, the present invention relates to an MDSC which is a cell formed by in vivo physiological activity modulator or cell physiological activity modulator and GPCR19 agonist and has Gr-l + CDllb + surface morphology and has anti-inflammatory activity. Provides (Mye 1 oi d-der i ved suppressor cel l).
본 발명에서 사용된 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질의 처리는 앞서 상술한 방법과 동일하고, 생체 내 생리적 활성 조절 물질 또는 세포 생리 활성 조절 물질로 예컨대, 리포폴리사카라이드 (LPS)를 인간을 제외한 포유동물에게 투여할 경우 투여량으로 바람직하게는 0.001 mg/kg 내지 100 mg/kg이고， 보다 바람직하게는 0. 1 mg/kg 내지 50 mg/kg이고, 가장 바람직하게는 20 mg/kg를 투여한다.  The treatment of the physiologically active regulatory substance or cell physiologically active substance in vivo used in the present invention is the same as the method described above, and in vivo, for example lipopolysaccharide (LPS) When administered to mammals other than humans, the dosage is preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, and most preferably 20 mg. / kg is administered.
상기 리포폴리사카라이드 (LPS)를 골수세포 집단에 처리하는 경우 그 ^도로 바람직하게는 0.0001 mg/mi 내지 10 mg/m 이고, 보다 바람직하게는 0.0005 내지 1 mg/ 이고, 보다 더 바람직하게는 0.001 mg/mt 내지 0. 1 mg/ 이고 가장 바람직하게는 0.001 mg/ 을 배양액에 처리한다.  When the lipopolysaccharide (LPS) is treated to the bone marrow cell population, the degree is preferably 0.0001 mg / mi to 10 mg / m, more preferably 0.0005 to 1 mg /, even more preferably 0.001 mg / mt to 0.01 mg / and most preferably 0.001 mg / to the culture.
한편， GPCR19 아고니스트 또는 그 시험물질로서， 예컨대 소듬 타우로데옥시콜레이트를 인간을 제외한 포유동물에게 투여하는 경우에는 바람직하게는 0.01 mg/kg 내지 100 mg/kg이고， 보다 바람직하게는 0. 1 mg/kg 내지 10 mg/kg이고, 가장 바람직하게는 0.5 ~ 8 mg/kg을 투여한다. 상기 소듐 타우로데옥시콜레이트를 골수세포 집단에 처리하는 경우에는 바람직하게는 0.0001 mg/mt 내지 100 g/mi 이고， 보다 바람직하게는 0.001 mg/ml 내지 10 mg/mi 이고， 보다 더 바람직하게는 0.01 mg/mi 내지 1 mg/ml 이고， 가장 바람직하게는 0.05 mg/mC을 배양액에 처리한다.  On the other hand, when GPCR19 agonist or a test substance thereof is administered to mammals other than humans, for example, Soult taurodeoxycholate is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.1. mg / kg to 10 mg / kg, most preferably 0.5 to 8 mg / kg. When the sodium taurodeoxycholate is treated to the bone marrow cell population, it is preferably 0.0001 mg / mt to 100 g / mi, more preferably 0.001 mg / ml to 10 mg / mi, even more preferably 0.01 mg / mi to 1 mg / ml, most preferably 0.05 mg / mC is treated in the culture.
생체 내 생리적 활성 조절 물질로 예컨대， 리포폴리사카라이드 (LPS)를 처리하는 시간은 바람직하게는 6시간 내지 48시간이고, 가장 바람직하게는 18시간 처리한다. 한편， GPCR19 아고니스트로서 소듐 타우로데옥시콜레이트를 처리하는 시간은 바람직하게는 6시간 내지 96시간이고， 보다 바람직하게는 12시간 또는 72시간이며, 가장 바람직하게는 48시간이다.  The treatment time of, for example, lipopolysaccharide (LPS) with in vivo physiological activity modulators is preferably from 6 hours to 48 hours, most preferably 18 hours. On the other hand, the time for treating sodium taurodeoxycholate as GPCR19 agonist is preferably 6 hours to 96 hours, more preferably 12 hours or 72 hours, and most preferably 48 hours.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트의 처리 후에 MDSC (Myeloid— derived suppressor cell)는 Gr-l+CDllb+ 표면 형질형을 나타내는 세포로 분화한다. 분화된 MDSC 세포는 대식 세포 계열의 골수 세포 마커 CDllb 및 과립구에 대한 마커 Grl을 발현한다. Grl+CDllb+은 Grl+ 또는 CDllb+에 대한 항체를 이용하여 면역반웅을 유도하고, 유세포분석기를 사용하여 선별할 수 있다. Gr-l+CDllb+ 표면 형질을 갖는 MDSC 세포를 패혈증 마우스에 투여한 결과 대조군과 비교하여 현저하게 높은 생존율을 보였고, 이는 상기 MDSC세포가 항염증 활성을 나타낸다는 것을 제시한다, 본 발명의 바람직한 구현 예에 따르면， Grl+CDllb+ 표면 형질형을 갖는 MDSC는 Prok2, Osm, Ltf , Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl유전자를 저발현 한다. In Vivo Physiologically Active Modulators and After Treatment of GPCR19 Agonist Myeloid-derived suppressor cells (MDSCs) differentiate into cells exhibiting Gr-l + CDllb + surface phenotype. Differentiated MDSC cells express the myeloid cell marker CDllb of the macrophage lineage and the marker Grl for granulocytes. Grl + CDllb + can be induced using an antibody against Grl + or CDllb + and selected using flow cytometry. Administration of MDSC cells with Gr-l + CDllb + surface traits to sepsis mice showed significantly higher survival compared to the control group, suggesting that the MDSC cells exhibit anti-inflammatory activity, preferred embodiment of the invention By way of example, MDSCs with Grl + CDllb + surface traits are Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Highly expresses the Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2ml, Pnllaip6ml Save Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 or Mxl genes Expression.
본 발명의 바람직한 구현 예에 따르면, 본 발명에 사용되는 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질， 진균 유래 물질， 바이러스 유래 물질, 자가 유래 물질， 알러젠 (allergen) 또는 암 세포 유래 물질인 것을 특징으로 하여 생성되는 MDSC를 제공한다.  According to a preferred embodiment of the present invention, in vivo physiological activity control material used in the present invention is characterized in that the bacteria-derived material, fungal-derived material, virus-derived material, autologous material, allergen or cancer cell-derived material It provides MDSC generated by
본 발명의 바람직한 구현 예에 따르면， 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸이다. 본 발명의 바람직한 구현 예에 따르면, 생체 내 생리적 활성 조절 물질은 항원이다. 본 발명의 바람직한 구현 예에 따르면, 본 발명의 방법에서 상기 MDSC를 만들기 위하여 사용되는 GPCR19 아고니스트는 화학식 1의 화합물， 그의 염 또는 수화물이다. 본 발명의 또 다른 양태에 따르면, 본 발명은 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성되거나 또는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 MDSC 처리에 의해 형성되며 CD23hi 표면 형질형을 가지며 항염증 활성을 갖는 면역조절 B 세포 (Regulatory B cel l )를 제공한다. According to a preferred embodiment of the present invention, the physiologically active regulatory substance in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance. According to a preferred embodiment of the invention, the physiologically active modulator in vivo is an antigen. According to a preferred embodiment of the invention, the GPCR19 agonist used to make the MDSC in the process of the invention is a compound of formula 1, a salt or a hydrate thereof. According to another aspect of the invention, the invention is formed by treatment with a physiologically active regulatory substance in vivo and GPCR19 agonist or in vivo physiological Provided are immunoregulatory B cells (Regulatory B cel l) formed by MDSC treatment formed by treatment with an active modulator and GPCR19 agonist and having a CD23 hi surface trait and anti-inflammatory activity.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리하는 방법은 앞서 상술한 내용과 동일하기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 본 명세서의 과도한 복잡성을 피하기 위하여， 그 기재를 생략한다.  Since the in vivo physiologically active substance and GPCR19 agonist treatment method are the same as described above, the common content between the two is omitted in order to avoid excessive complexity of the present specification according to the repeated description.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 MDSC를 (H-MDSC) 포유동물에 투여할 경우， 투여하는 세포수로 바람직하게는 1 X 103 내지 1 X 108 세포수 이고， 보다 바람직하게는 1 X 105 내지 1 X 107 세포수 이고, 가장 바람직하게는 1 X 106세포수 이다. 상기 H-MDSC 투여로 인하여 CD23hi 표면형질 마커를 발현하는 면역조절 B세포가 생성되고 (예컨대， BHM 세포) , 이 세포를 패혈증에 걸린 마우스에 투여한 결과 대조군과 비교하여 현저하게 높은 생존율을 보였고, 이는 상기 면역조절 B 세포 (예컨대, BHM 세포)가 항염증 활성을 나타낸다는 것을 제시한다. When the in vivo physiologically active substance and GPCR19 agonist-treated MDSCs are administered to (H-MDSC) mammals, the number of cells to be administered is preferably 1 X 10 3 to 1 X 10 8 cells, more preferably. Preferably from 1 X 10 5 to 1 X 10 7 cells, most preferably from 1 X 10 6 cells. The H-MDSC administration resulted in immunoregulatory B cells expressing CD23 hi surface morphology markers (eg B HM cells), and administration of these cells to sepsis mice resulted in significantly higher survival compared to controls. It was shown that this immunoregulatory B cells (eg B HM cells) exhibit anti-inflammatory activity.
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트를 골수세포집단에 처리하는 경우는 앞서 상술한 내용과 동일하고， 이를 투여한 결과 CD23hi 표면형질 마커를 발현하는 면역조절 B 세포가 생성된다 (예컨대, 48H BH 세포) . 생성된 48H ¾ 세포를 패혈증 마우스에 투여한 결과 대조군과 비교하여 현저하게 높은 생존율을 보였고, 이는 상기 면역조절 B 세포 (예컨대, 48H BH 세포)가 항염증 활성을 나타낸다는 것을 제시한다. Treatment of the physiologically active substance in vivo and the GPCR19 agonist to the bone marrow cell population is the same as described above, and administration of the same results in the generation of immunoregulatory B cells expressing the CD23 hi surface morphology marker (eg, 48H). B H cells). Administration of the resulting 48H 3/4 cells to sepsis mice showed significantly higher survival compared to the control group, suggesting that the immunoregulatory B cells (eg 48H B H cells) exhibit anti-inflammatory activity.
GPCR19 아고니스트 또는 그 시험물질， 예컨대 소듐 타우로데옥시콜레이트를 골수세포 집단에 처리하는 것은 배양되는 골수세포의 배양액에 처리할 수 있으며， 바람직하게는 0.0001 mgM 내지 100 g/ t 이고， 보다 바람직하게는 0.001 mg/πιΓ내지 10 g/ml 이고， 보다 더 바람직하게는 0.01 g/ l 내지 1 mg/ 이고, 가장 바람직하게는 0.05 mg/ 을 배양액에 처리한다.  Treatment of a bone marrow cell population with a GPCR19 agonist or a test substance thereof, such as sodium taurodeoxycholate, may be treated with a culture medium of the cultured bone marrow cells, preferably 0.0001 mgM to 100 g / t, more preferably Is 0.001 mg / πιΓ to 10 g / ml, even more preferably 0.01 g / l to 1 mg /, most preferably 0.05 mg / is treated to the culture.
GPCR19 아고니스트 또는 그 시험물질의 처리 결과 생성되는 면역조절 Immunomodulation resulting from the treatment of GPCR19 agonists or their test substances
B 세포에서 발현하는 표현형질 마커인 CD23hi은 면역조절 B 세포 계통의 초기 마커인 전구체 (precursor) B 세포 표면에 성숙하는 기간 동안 나타나서 면역조절 B 세포가 살아있는 동안 남아있는 마커이다. CD23hi 면역조절 B 세포는 CD23+ 항체를 이용하여 염색한 후에, 유세포분석기를 사용하여 선별할 수 있다. CD23 hi , a phenotypic marker that is expressed in B cells, An early marker is a marker that appears during maturation on the surface of the precursor B cell and remains while the immunoregulatory B cells are alive. CD23 hi immunoregulatory B cells can be stained using CD23 + antibodies and then selected using a flow cytometer.
본 발명의 바람직한 구현 예에 따르면, CD23hi 표면형질 마커를 발현하는 면역조절 B세포는 Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 , Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll , Swap70, Ccbp2, Sc4mol , Cyp51, Sqle, L0C100040592 , B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi , Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, lrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4 Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346 , Mx2, Tyki , Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprl , Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b또는 Cd69유전자를 저발현 한다. According to a preferred embodiment of the present invention, immunoregulatory B cells expressing CD23 hi surface morphological markers are Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc , Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hspala, Srp, ksp , Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp80, C480 , Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 Lmnbl, Pira4, Blvrb or Actb genes, or 1190002H23Rik Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Sltl, Ly6cl, Tg His Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, lrel, Lgalsl, 60001 Ccl Sel ll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctlal Us8 , Mxl, L0C100048346, Mx2, Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprl, Trafdl, Cd3d, Ilpp, Rilpll, Lalpll , Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes.
본 발명의 바람직한 구현예에 따르면， CD23hi 표면형질 마커를 발현하는 면역조절 B세포는 CD21 및 CD23을 발현한다. According to a preferred embodiment of the present invention, immunoregulatory B cells expressing CD23 hi surface morphological markers express CD21 and CD23.
상기 면역조절 B 세포에서 발현하는 CD23(FCeRII)는 림프구 추적 수용체 (MEL-14) 및 내피 백혈구 부착 분자 -KELAM-1) 등을 포함하는 C-렉틴 군의 II형 분자이다. CD23(FCeRII)은 IgE에 대하여 친화력이 낮은 수용체이다. 포유동물의 수지상세포， B 세포， T 세포 및 거식세포를 포함하는 다양한 혈액 구성 세포타입들이 CD23을 세포 표면에 발현하고 있다. 또한 CD23 분자들은 생체액에 용해된 형태로 발견되기도 한다. 용해성 CD23(sCD23) 분자는 세포막 투과성 수용체의 단백질 절단에 의해 생성된다. 한편, CD21은 B 세포에서 발현하는 마커로써 , CD19 및 TAPA-1과 복합체를 형성하고 보체성분 3Cd와 결합하여 B 세포의 활성화 및 증식의 부자극 성분이다. CD23 (FCeRII) expressed in the immunoregulatory B cells is a type II molecule of the C-lectin group including lymphocyte tracking receptor (MEL-14) and endothelial leukocyte adhesion molecule -KELAM-1). CD23 (FCeRII) is a low affinity receptor for IgE. Dendritic cells, B cells, T cells and macrophages in mammals Various blood constituent cell types, including CD23, express CD23 on the cell surface. CD23 molecules are also found in dissolved form in biological fluids. Soluble CD23 (sCD23) molecules are produced by protein cleavage of cell membrane permeable receptors. On the other hand, CD21 is a marker expressed in B cells, complexes with CD19 and TAPA-1 and binds to the complement component 3Cd and is a negative component of activation and proliferation of B cells.
본 발명의 바람직한 구현 예에 따르면， 본 발명에 사용되는 생체 내 생리적 활성 조절 물질 또는 세포활성 조절물질은 박테리아 유래 물질, 진균 유래 물질， 바이러스 유래 물질， 자가 유래 물질， 또는 알러젠 (al lergen)인 것을 특징으로 하여 생성되는 면역조절 B 세포를 제공한다.  According to a preferred embodiment of the present invention, the physiologically active substance or cell activity modulator in vivo used in the present invention is a bacteria-derived, fungal-derived, virus-derived, autologous or allergen (al lergen) Characterized by the immunoregulatory B cells produced.
본 발명의 바람직한 구현 예에 따르면， 생체 내 생리적 활성 조절 물질 또는 세포활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸이다. 본 발명의 바람직한 구현 예에 따르면， 본 발명의 방법에서 상기 MDSC 또는 면역조절 B 세포 (48H ¾ 세포) 또는 보다 구체적으로는 CD21+CD23+ B 세포를 만들기 위하여 사용되는 GPCR19 아고니스트는 화학식 1의 화합물， 그의 염 또는 수화물이다. 본 발명의 또 다른 양태에 따르면, 본 발명은 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr-l+CDllb+MDSC(H-MDSC)， 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr- 1+CDllb+MDSC 처리에 의해 형성된 면역조절 B 세포 (BHM 세포) , 보다 구체적으로는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr-1+CDllb+MDSC 처리에 의해 형성된 CD21+CD23+ B 세포, 또는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 면역조절 B 세포 (48H ¾ 세포) 또는 보다 구체적으로는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 CD21+CD23+ B 세포를 유효성분으로 포함하는 항염증 약제학적 조성물을 제공한다. According to a preferred embodiment of the present invention, the physiologically active regulatory substance or cell activity modulator in vivo is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria-derived substance. According to a preferred embodiment of the present invention, the GPCR19 agonist used to make the MDSC or immunoregulatory B cells (48H ¾ cells) or more specifically CD21 + CD23 + B cells in the method of the present invention is a compound of formula 1 Or salts or hydrates thereof. According to another aspect of the invention, the present invention is treated with in vivo physiological activity modulators and GPCR19 agonist treated Gr-1 + CDllb + MDSC (H-MDSC), in vivo physiological activity modulators and GPCR19 agonist treated immunomodulatory B cells (B HM cell) formed by the formed Gr- 1 + CDllb + MDSC treatment, more specifically, formed by the in vivo physiological activity regulatory substance and GPCR19 agonist is treated Gr-1 + CDllb + MDSC treatment formed CD21 + CD23 + B cells, or immunoregulatory B cells (48H ¾ cells) formed by treatment with physiologically active modulators and GPCR19 agonists in vivo (48H ¾ cells) or more specifically CD21 + formed by treatment with physiologically active modulators and GPCR19 agonists in vivo Provided is an anti-inflammatory pharmaceutical composition comprising CD23 + B cells as an active ingredient.
본 발명의 조성물의 유효성분으로 바람직하게는 Gr-l+CDllb DSC 또는 CD23hi 면역조절 B 세포 (48H ¾ 세포 또는 BHM 세포) 또는 B220+ CD21+CD23+ B 세포를 함유하는 항염증 약제학적 조성물이다. 본 발명의 바람직한 구현 예에 따르면, 본 발명의 방법으로 생성된 항염증 약제학적 조성물로 예방 또는 치료가 가능한 질환은 바람직하게는 패혈증， 자가면역질환， 염증성 질환, 류마티스 관절염， 아토괴성 피부염， 알쓰하이머 병, 궤양성 대장염, 제 1형 당뇨병, 크론병， 건선， 건선성 관절염, 강직성 척수염， 다발성 경화증， 캐슬만씨 병， 자가면역성 간염， 자가 면역성 포도막염， 중증 근무력증， 원형 탈모증 및 전신성 홍반성 낭창으로 구성된 군으로부터 선택되나 이에 한정되지 않고， 가장 바람직하게는 패혈증이다. An anti-inflammatory pharmaceutical composition containing preferably Gr-1 + CDllb DSC or CD23 hi immunomodulatory B cells (48H ¾ cells or B HM cells) or B220 + CD21 + CD23 + B cells as an active ingredient of the composition of the present invention to be. According to a preferred embodiment of the present invention, the disease that can be prevented or treated with the anti-inflammatory pharmaceutical composition produced by the method of the present invention is preferably sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease. Disease, ulcerative colitis, type 1 diabetes, Crohn's disease, psoriasis, psoriatic arthritis, ankylosing myelitis, multiple sclerosis, castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia areata and systemic lupus erythematosus It is selected from the group consisting of, but not limited to, most preferably sepsis.
본 발명의 조성물이 항염증 약제학적 조성물로 제조되는 경우， 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서， 락토스, 덱스트로스, 수크로스, 솔비를, 만니를， 전분， 아카시아 고무， 인산 칼슘, 알기네이트, 젤라틴， 규산 칼슘, 미세결정성 셀를로스， 폴리비닐피를리돈， 셀를로스， 물， 시럽， 메틸 셀를로스, 메틸히드록시벤조에이트， 프로필히드록시벤조에이트 , 활석， 스테아르산 마그네슘 및 미네랄 오일을 포함하나， 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제， 습윤제, 감미제， 향미제, 유화제， 현탁제, 보존제를 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington 's Pharmaceutical Sciencesi 19th ed . 1995 )에 상세히 기재되어 있다.  When the composition of the present invention is prepared as an anti-inflammatory pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, Contains gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil One is not limited thereto. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciencesi 19th ed. 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입， 피하 주입， 근육 주입, 관절내 주입 등으로 투여할 수 있다.  The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraarticular injection, or the like.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법， 투여 방식， 환자의 연령， 체중， 성， 병적 상태， 음식， 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 예시적인 투여량은 0.001-1000 nig/kg이다. 본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및 /또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액， 현탁액, 시럽제 또는 유화액 형태이거나 액스제， 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 본 발명의 또 다른 양태에 따르면, 본 발명은 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr-l+CDllb^DSCOl-MDSC) , 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr- 1+CDllb+MDSC 처리에 의해 형성된 면역조절 B 세포 (¾« 세포)， 보다 구체적으로는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 Gr-l+CDlll^iDSC 처리에 의해 형성된 CD21+CD23+ B 세포, 또는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 면역조절 B 세포 (48H BH 세포) 또는 보다 구체적으로는 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형성된 CD21+CD23+ B 세포를 유효성분으로 포함하는 포함하는 조성물의 약제학적 유효량을 객체 (Subject )에게 투여하는 단계를 포함하는, 염증의 예방 또는 치료 방법을 제공한다. Appropriate dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction sensitivity. Can be. Exemplary dosages of the pharmaceutical compositions of the invention are 0.001-1000 nig / kg. The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Can be prepared or incorporated into a multi-dose container. have. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of axes, powders, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer. According to another aspect of the invention, the present invention is Gr-l + CDllb ^ DSCOl-MDSC formed by in vivo physiological activity modulators and GPCR19 agonist treatment, Gr formed by treatment with physiologically active modulators and GPCR19 agonist in vivo - 1 + CDllb + immunoregulatory formed by MDSC treatment B cells (¾ «cells), and more particularly to CD21 + formed by the controlled in vivo physiological active substance and GPCR19 agonist is treated formed Gr-l + CDlll ^ iDSC treatment CD23 + B cells, or immunoregulatory B cells (48H B H cells) formed by treatment with physiologically active regulatory substances and GPCR19 agonists in vivo (48H B H cells) or more specifically CD21 + CD23 formed by treatment with physiologically active regulators and GPCR19 agonists in vivo + B cells comprising the step of administering a pharmaceutically effective amount of the composition to the object (Subject) comprising containing as an active ingredient, the prevention of inflammation, yet Provide treatment.
본 발명의 방법은 상술한 조성물을 이용하기 때문에, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.  Since the method of the present invention utilizes the composition described above, the common content between the two is omitted in order to avoid excessive complexity of the present specification.
【발명의 효과】 【Effects of the Invention】
본 발명의 특징 및 이점을 요약하면 다음과 같다:  The features and advantages of the present invention are summarized as follows:
(a) 본 발명 따르면 리포폴리사카라이드 (LPS)의 투여로 인해 염증이 유도된 마우스에서 소듐 타우로데옥시콜레이트 (HY2191)의 투여에 의하여 H- MDSC 세포의 수가 증가하고， 증가된 H-MDSC는 현저한 패혈증 치료 효과를 보인다.  (a) According to the present invention, the number of H-MDSC cells was increased by the administration of sodium taurodeoxycholate (HY2191) in the inflammation-induced mice due to the administration of lipopolysaccharide (LPS) and increased H-MDSC. Has a significant treatment effect on sepsis.
(b) 본 발명에 따르면 리포폴리사카라이드 (LPS)의 투여로 인해 염증이 유도된 마우스에서 소듐 타우로데옥시콜레이트 (HY2191)의 투여에 의하여 면역조절 B 세포 (48h ¾ 세포)의 수가 증가하고, 증가된 48h BH 세포 세포는 현저한 패혈증 치료 효과를 보인다. (b) According to the present invention, the number of immunoregulatory B cells (48h ¾ cells) is increased by administration of sodium taurodeoxycholate (HY2191) in mice induced with inflammation due to administration of lipopolysaccharide (LPS). , Increased 48h B H cell cells show significant sepsis treatment effect.
(c) 본 발명에 따르면 리포폴리사카라이드 (LPS)의 투여로 인해 염증이 유도된 마우스에서 소듐 타우로데옥시콜레이트 (HY2191)의 투여에 의하여 B220+CD21+CD23+ B 세포의 수가 증가하고, 증가된 B220+CD21+CD23+ B 세포는 현저한 패혈증 치료 효과를 보인다. (c) due to the administration of lipopolysaccharide (LPS) Administration of sodium taurodeoxycholate (HY2191) increased the number of B220 + CD21 + CD23 + B cells in the inflammation-induced mice, and the increased B220 + CD21 + CD23 + B cells showed a significant sepsis treatment effect.
(d) 본 발명에 따르면 리포폴리사카라이드 (LPS)의 투여로 염증이 유도된 마우스에서 H-MDSC의 투여에 의하여 BHM 세포의 수가 증가하고, 증가된 ¾M세포는 현저한 패혈증 치료 효과를 보인다.  (d) According to the present invention, the number of BHM cells is increased by administration of H-MDSC in inflammation-induced mice by administration of lipopolysaccharide (LPS), and the increased ¾M cells show a significant sepsis treatment effect.
(e) 본 발명에 따르면 리포폴리사카라이드 (LPS)의 투여로 염증이 유도된 마우스에서 H-MDSC의 투여에 의하여 B220+CD21+CD23+B 세포의 수가 증가하고, 증가된 B220+CD21+CD23+B 세포는 현저한 패혈증 치료 효과를 보인다.  (e) According to the present invention, the number of B220 + CD21 + CD23 + B cells increased and increased B220 + CD21 + CD23 by administration of H-MDSC in mice induced with inflammation by administration of lipopolysaccharide (LPS). + B cells show significant sepsis treatment effect.
( f ) 본 발명의 방법은 GPCR19-연관 질환의 치료제의 스크리닝 방법 또는 GPCR19 아고나스트의 효능 분석 방법을 제공하여, 항염증 약제학적 조성물을 제공한다. 【도면의 간단한 설명】  (f) The method of the present invention provides a method for screening a therapeutic agent for a GPCR19-associated disease or a method for analyzing the efficacy of a GPCR19 agonast to provide an anti-inflammatory pharmaceutical composition. [Brief Description of Drawings]
도 1A-1C는 소듐 타우로데옥시콜레이트 (HY2191)의 패혈증 치료효과를 보기위해 다양한 종류의 패혈증 유도 마우스에 소듐 타우로데옥시콜레이트 (HY2191)을 주입한 후 그 생존율을 도식화한 그래프이다. 실험방법을 도식화한 이미지를 그래프 상부에 첨부하였다. 도 1A는 HY2191을 LPS 유도 패혈증 마우스에 LPS처리 30분 및 24시간 후에 주입"하였을 때 생존율 Ϊ 나타내는 그래프이다, 도 1B는 HY2191올 CLP 유도 패혈증 마우스에 CLP 유도 2시간 후에 주입하였을 때 생존율을 도시하는 그래프이다. 도 1C는 HY2191가 B 세포 및 T 세포가 결핍된 마우스 (Rag2 K0)에서는 패혈증 치료효과를 나타내지 못함을 도시하는 그래프이다. 1A-1C are graphs showing the survival rate after injection of sodium taurodeoxycholate (HY2191) into various types of sepsis-induced mice to see the treatment of sepsis with sodium taurodeoxycholate (HY2191). An image showing the experimental method was attached at the top of the graph. Figure 1A is a graph showing the survival rate Ϊ when implanted "hayeoteul the HY2191 in LPS induced septic mice after LPS treatment for 30 minutes and 24 hours, and Figure 1B showing the survival rate hayeoteul injection after CLP induction two hours HY2191 all CLP induced sepsis mouse 1C is a graph showing that HY2191 does not show sepsis treatment effect in mice lacking B cells and T cells (Rag2 K0).
도 2A-2B는 HY2191에 의한 생체내 염증사이토카인 생성 억제를 도시하는 그래프이다. 도 2A는 HY2191가 LPS에 의해 유도된 혈중 염증사이토카인 농도를 감소시킴을 도시하는 그래프이다. 도 2B는 HY2191가 CLP에 의해 유도된 혈중 염증사이토카인 농도를 감소시킴을 도시하는 그래프이다.  2A-2B are graphs showing inhibition of inflammatory cytokine production in vivo by HY2191. 2A is a graph showing that HY2191 reduces inflammatory cytokine concentrations in blood induced by LPS. 2B is a graph showing that HY2191 decreases the inflammatory cytokine concentration in blood induced by CLP.
도 3A-3B는 HY2191처리후 패혈증에의한 저혈압증의 완화를 도시하는 그래프이다. 도 3A는 LPS에 의해 유도된 혈압 저하가 HY2191에 의해 정상화됨을 평균 혈압 (隨 Hg) 상승으로 나타낸 그래프이다. 도 3B는 LPS에 의해 유도된 신부전과 간기능 장애가 HY2191에 의해 완화됨을 각각 혈중 BUN과 AST 감소로 도시하는 그래프이다. 3A-3B are graphs showing relief of hypotension by sepsis after HY2191 treatment. Figure 3A shows that blood pressure drop induced by LPS was induced by HY2191. Normalized is a graph showing rise in mean blood pressure (隨 Hg). FIG. 3B is a graph showing blood BUN and AST reduction, respectively, that renal failure and liver dysfunction induced by LPS are alleviated by HY2191.
도 4는 LPS 유도 패혈증 마우스의 비장세포 특히 CDllb+세포 및 B220+ B 세포의 절대수를 도시한 그래프이다. 4 is a graph depicting the absolute numbers of splenocytes, in particular CDllb + cells and B220 + B cells, in LPS induced sepsis mice.
도 5는 LPS 유도 패혈증 마우스의 비장 CD4+Foxp3+ T cel l (regulatory T cel l , Treg)의 절대수를 도시한 그래프이다. 5 is a graph showing the absolute number of splenic CD4 + Foxp3 + T cel l (regulatory T cel l, Treg) of LPS-induced sepsis mice.
도 6는 LPS에 의한 비장 세포의 활성화가 HY2191에 의해 억제됨을 도시하는 그래프들이다. 도 6A에서 비장 CD11C+세포의 활성화를 CD86 활성화 마커의 평균 형광강도 (Mean f luoresense intensi ty, MFI )로 표시하였다. 도 6B에서 비장 B220+B 세포의 활성화를 CD86 활성화 마커의 평균 형광강도 (Mean f luoresense intensi ty, MFI )로 표시하였다. 도 6C는 비장 B220+ B 세포중 세포 활성화마커인 CD86을 과발현하는 B220+CD86+ B 세포의 상대적 비율 (%)로 나타내는 FACS폴롯이다 . 6 are graphs showing that activation of spleen cells by LPS is inhibited by HY2191. In FIG. 6A, activation of spleen CD11C + cells was expressed by the mean fluorescence intensity (Mean f luoresense intensi ty, MFI) of the CD86 activation marker. In FIG. 6B, activation of splenic B220 + B cells was expressed by the mean fluorescence intensity (Mean f luoresense intensi ty, MFI) of the CD86 activation marker. FIG. 6C is a FACS plot showing the relative percentage of B220 + CD86 + B cells overexpressing CD86, a cell activation marker in spleen B220 + B cells.
도 7은 HY2191를 처리한 LPS 유도 패혈증 마우스 생체 내에서 Gr- l+CDllb+ 세포 (H-MDSC)가 증가하고 이들이 패혈증 치료 효과를 보임을 도시하는 그래프들이다. 도 7A는 LPS 유도 패혈증 마우스의 비장에서의 Gr- l+CDllb+ MDSC 세포수가 HY2191 처리 군에서 24시간 및 48시간 후에 의미 있게 증가함을 도시한 그래프이다. 또한 HY2191가 B 세포 및 T 세포가 결핍된 마우스 (Rag2 K0)에서는 Gr-l+CDllb+ MDSC 세포수에 변화를 주지 않음을 도시하는 그래프이다. 도 7Bfe H-MDSC의 비장 세포내 상대적 비율 (%)을 도시한 FACS 플롯이다. 도 7C위는 H-MDSC의 미성숙도를 골수양세포 미성숙 마커인 CD31+ 세포의 비율 (%)로 나타낸 FACS 히스토그램이다. 도 7C아래는 H-MDSC가 Ly6G+Ly6CinterF4/80low 임을 보여주는 FACS 플롯이다. 도 7D는 H-MDSC와 L-MDSC의 발현 유전자를 비교하는 Heat-map이다. 붉은 색은 L-MDSC (HY2191을 처리하지 않은 LPS 유도 패혈증 마우스에서 수확한 Gr- l+CDllb+ MDSC)에 비하여 H-MDSC에서 과발현하는 유전자를, 초록색은 저발현하는 유전자를 각각 표시한다. 7 is graphs showing that Gr-1 + CDllb + cells (H-MDSC) increase in LPS induced sepsis mice treated with HY2191 and that they show a sepsis treatment effect. FIG. 7A is a graph showing significant increase in Gr-1 + CDllb + MDSC cell numbers in the spleen of LPS induced sepsis mice after 24 and 48 hours in the HY2191 treated group. It is also a graph showing that HY2191 does not change the Gr-1 + CDllb + MDSC cell numbers in mice lacking B cells and T cells (Rag2 K0). 7 is a FACS plot depicting the relative percentage (%) of splenic cells of Bfe H-MDSC. 7C is a FACS histogram showing the immaturity of H-MDSC as the percentage of CD31 + cells that are myeloid cell immature markers. 7C below is a FACS plot showing that H-MDSC is Ly6G + Ly6C inter F4 / 80 low . 7D is a heat-map comparing the expression genes of H-MDSC and L-MDSC. Red indicates genes overexpressing in H-MDSCs and green underexpressed genes compared to L-MDSCs (Gr-1 + CDllb + MDSCs harvested from LPS-induced sepsis mice not treated with HY2191).
도 8은 H-MDSC의 패혈증 치료효과를 보기위해 패혈증 유도 마우스에 여러 종류의 세포를 양자이입 (adopt ive transfer )한 후 그 생존율을 도식화한 그래프이다. LPS 유도 패혈증 마우스에서 H-MDSC 수확 및 투여 후 그 치료효과를 분석하기 위한 전반적인 실험방법을 도식화한 이미지를 그래프 상위에 첨부하였다. FIG. 8 is a graph illustrating the survival rate after adopting ive transfer of various types of cells in sepsis-induced mice to see the treatment effect of sepsis. After H-MDSC Harvest and Administration in LPS-Induced Sepsis Mice An image showing the overall experimental method for analyzing the therapeutic effect is attached to the upper part of the graph.
도 9는 LPS 패혈증 유도 마우스에 양자 이입된 H-MDSC가 유도하는 생체 내 면역 세포들의 변화를 절대수 및 상대적 비율 (%)로 도시화한 그래프들이다. 도 9A는 H-MDSC 양자 이입 24시간 후에 비장 B 세포의 상대적 및 절대수의 증가를 도시하는 FACS 플롯 이미지 (위)와 반복실험의 그래프 (아래)이다. 도 9B는 H-MDSC 양자 이입 24시간 후에 비장 및 골수 CD11B+ Gr-1+ F4/80 세포의 상대적 및 절대수의 증가를 도시하는 FACS 플롯 이미지 (위)와 반복실험의 그래프 (아래)이다. 도 9C는 H-MDSC 양자 이입 24시간 후에 비장 T 세포 (CD4+ 및 CD8+ T 세포)들의 상대적 및 절대수가 변화하지 않음을 도시하는 FACS 플롯 이미지 (위)와 그래프 (아래)이다. 도 9D는 H-MDSC 양자 이입 24시간 후에 비장 CD11C+ 수지상 세포 절대수의 의미 있는 변화가 없음을 도시하는 그래프이다. FIG. 9 is a graph depicting the absolute number and relative percentage (%) of in vivo immune cells induced by H-MDSC transduced in LPS sepsis induced mice. 9A is a graph of FACS plot images (top) and replicates (bottom) depicting an increase in the relative and absolute numbers of splenic B cells 24 h after H-MDSC transfection. 9B is a graph of FACS plot images (top) and replicates (bottom) depicting an increase in the relative and absolute numbers of spleen and bone marrow CD11B + Gr-1 + F4 / 80 cells after 24 hours of H-MDSC transfusion. FIG. 9C is a FACS plot image (top) and graph (bottom) showing that the relative and absolute numbers of splenic T cells (CD4 + and CD8 + T cells) do not change 24 hours after H-MDSC transfusion. FIG. 9D is a graph showing no significant change in splenic CD11C + dendritic cell absolute number after 24 hours of H-MDSC transfusion.
도 10은 LPS로 패혈증을 유도한 마우스에 양자 이입한 H-MDSC에 의해 증식된 비장 B 세포 (BHM 세포)의 패혈증 치료효과를 보기위해 패혈증 유도 마우스에 여러 종류의 B 세포를 양자이입 (adopt ive transfer)한 후 그 생존율을 도식화한 그래프이다. 10 shows the introduction of several types of B cells into sepsis-induced mice to see the effect of treating sepsis of splenic B cells (B HM cells) proliferated by H-MDSCs. The graph shows the survival rate after ive transfer.
도 11은 양자 이입된 ¾M세포가 B 세포 및 T 세포가 결핍된 마우스 (Rag2 K0)에세 i-MDSC 존재하에 패혈증 치료효과를 나타냄을 도시하는 그래프이다.  FIG. 11 is a graph showing the effect of sepsis treatment in the presence of i-MDSC in B- and T-cell deficient mice (Rag2 K0).
도 12A-C 는 LPS로 패혈증을 유도한 마우스에 양자 이입한 48h ¾ 세포가 유도하는 생체 내 비장 면역 세포들의 변화를 도시화한 그래프들이다. 도 12A는 48h ¾ 세포가 양자 이입 24시간 후에 LPS에 의한 비장 B 세포의 활성화를 도시화한 FACS 플롯 이미지이다. 도 12B는 48h ¾ 세포가 양자 이입 24시간 후에 LPS에 의한 비장 T 세포 (CD4+ 및 CD8+ T 세포)의 활성화를 도시화한 FACS 플롯 이미지이다. 도 12C는 48h ¾ 세포를 양자 이입 24시간 후에 비장에서 증가된 CDllb+ Ly6G+ Ly6C inter세포의 상대적 비율 (%)을 도시화한 FACS 플롯 이미지이다. 12A-C are graphs illustrating changes in in vivo splenic immune cells induced by 48h 3/4 cells transduced into sepsis-induced mice. 12A is a FACS plot image depicting the activation of splenic B cells by LPS at 24 h after 48 h ¾ cells transduction. 12B is a FACS plot image showing the activation of splenic T cells (CD4 + and CD8 + T cells) by LPS after 24 h of 48h 3 cells. FIG. 12C is a FACS plot image depicting the relative percentage of CDllb + Ly6G + Ly6C inter cells increased in the spleen 24 hours after transfection of 48h 3/4 cells.
도 13은 48h BH 세포의 표현형이 기존에 알려진 면역조적 B 세포와 차이가 있슴을 나타내는 FACS 플롯이다. 도 13A는 48h ¾세포가 Bla B 세포 특징적인 마커인 CDld 및 CD5를 발현을 도식화한 FACS 플롯이다. 도 13B는 48h BH 세포가 면역 조절 T2-MZP B 세포 특징적 마커인 CD93 발현을 도식화한 FACS 플롯이다. 도 13C는 LPS로 패혈증을 유도한 마우스 체내에서 관찰되는 B 세포에 비하여 48h BH세포가 성숙 B 세포의 특징적 마커인 IgM 발현을 도식화한 FACS플롯이다. FIG. 13 is a FACS plot showing that the phenotype of 48h B H cells differs from previously known immunoregulatory B cells. FIG. 13A is a FACS plot in which 48h 3 cells depict the expression of CDld and CD5, Bla B cell characteristic markers. 13B 48 h B H cells are FACS plots depicting CD93 expression, an immune regulatory T2-MZP B cell characteristic marker. FIG. 13C is a FACS plot in which 48h B H cells depict IgM expression, a characteristic marker of mature B cells, compared to B cells observed in mice induced with sepsis with LPS.
도 14A 는 L-MDSC (HY2191을 처리하지 않은 LPS 유도 패혈증 마우스에서 수확한 Gr-l+CDllb+ MDSC) 또는 H-MDSC (HY2191을 처리한 LPS 유도 패혈증 마우스에서 수확한 Gr-l+CDllb+ MDSC)를 양자 이입 24시간 후 증가된 비장 B220+CD23hiCD21int세포의 표현형 및 상대적 비율 (%)을 도식화한 FACS플롯이다. 14A is L-MDSC (harvested in the LPS induced septic mice not treated with HY2191 Gr-l + CDllb + MDSC) or H-MDSC (a Gr-l harvested in the LPS induced septic mice treated with HY2191 + CDllb + MDSC ) Is a FACS plot depicting the phenotype and relative percentage (%) of spleen B220 + CD23 hi CD21 int cells increased 24 hours after transfection.
도 14B는 LPS로 패혈증을 유도한 마우스에서 L-MDSC 또는 H-MDSC 양자 이입 24시간 후에 비장의 B220+CD23+세포 수 증가를 절대수로 도식화한 그래프이다. 14B is a graph plotting the absolute number of B220 + CD23 + cell count increase in the spleen after 24 hours of L-MDSC or H-MDSC transduction in mice induced with sepsis with LPS.
도 15A는 B220+CD23hiCD21hi세포 (CD23hi ¾ Cel l )가 LPS로 패혈증을 유도한 마우스에서 HY2191 주입 48시간 후에 CD23 발현을 나타내는 FACS 플롯이다. 도 15B는 B220+CD23hiCD21int세포 (CD23hi ¾ Cel l )의 패혈증 치료효과를 보기위해 패혈증 유도 마우스에 양자이입 (adopt ive transfer)한 후 마우스의 생존율을 도식화한 그래프이다. LPS로 패혈증을 유도한 마우스로 부터 CD23hi BH 세포를 수확한 시기와 투여 후 그 치료효과를 분석하기 위한 전반적인 실험방법을 그래프 상위에 도식화하였다. 15A is a FACS plot showing CD23 expression 48 hours after HY2191 injection in mice in which B220 + CD23 hi CD21 hi cells (CD23 hi ¾ Cel l) induced sepsis with LPS. 15B is a graph illustrating the survival rate of mice after adoptive transfer into sepsis-induced mice to see sepsis treatment effect of B220 + CD23 hi CD21 int cells (CD23 hi ¾ Cel l). The timing of harvesting CD23 hi B H cells from LPS-induced sepsis and the overall experimental method to analyze the therapeutic effect after administration are shown in the upper graph.
도 16은 LPS로 패혈증을 유도한 마우스에 양자 이입 (adopt ive transfer)한 CD23hi ¾ 세포가 마우스의 생체 내에서 B 세포 및 CDllb+ 세포 증식을 유도함을 도식화한 그래프이다. CD23hi BH 세포를 마우스에 양자 이입한 후 24시간 및 48시간 후의 비장의 B220+ B세포와 CDllb+ Gr-1+ 절대수를 대조군 (PBS) 및 CD2310 BH 세포와 비교하였다. FIG. 16 is a graph depicting the induction of B cell and CDllb + cell proliferation in vivo by mouse adoptive transfer of CD23 hi ¾ cells in mice induced with sepsis with LPS. CD23 hi B H cells were transfected into mice 24 and 48 hours after the spleen B220 + B cells and CDllb + Gr-1 + absolute numbers were compared with the control (PBS) and CD23 10 B H cells.
도 17A는 마우스 골수세포에 LPS (lmg/ml )과 HY2191 (50 mg/ml )을 17A shows LPS (lmg / ml) and HY2191 (50 mg / ml) in mouse bone marrow cells.
24h 동안 시험관내 처리한 후 Gr-l+CDllb+ 세포의 절대수가 증가함을 도식화한 그래프이다. LexMDSC (시험관내의 마우스 골수세포에 HY2191을 처리하지 않고 LPS만 단독으로 처리한 후 생성된 Gr-l+CDllb+ 세포) 또는 HexMDSC (시험관내의 마우스 골수세포에 HY2191와 LPS를 동시에 처리한 후 생성된 Gr-l+CDllb+ 세포)의 절대수를 도식화하였다. 도 17B는 도 17A의 HexMDSC, LexMDSC, 배지 (cel lgro, 음성대조군)의 패혈증 치료효과를 보기위하여 양자 이입후 생존한 LPS로 패혈증을 유도한 마우스의 생존율을 도식화한 그래프이다. It is a graph plotting the absolute number of Gr-1 + CDllb + cells increasing after in vitro treatment for 24h. LexMDSC (Gr-l + CDllb + cells generated after treatment with LPS alone without treatment with HY2191 in mouse bone marrow cells in vitro) or HexMDSC (treated with HY2191 and LPS in mouse bone marrow cells in vitro simultaneously The absolute numbers of Gr-1 + CDllb + cells) were plotted. 17B shows the sepsis treatment effect of HexMDSC, LexMDSC, medium (cel lgro, negative control) of FIG. 17A. For the sake of illustration, it is a graph illustrating the survival rate of mice inducing sepsis with LPS surviving after transfection.
【발명을 실시하기 위한 구체적인 내용】 [Specific contents to carry out invention]
이하， 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로， 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예  Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example
실시예 1. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium t aur odeoxy cho 1 at e； HY2191)의 효과 Example 1 Effect of Sodium Taurodeoxycholate (HY2191, sodium t aur odeoxy cho 1 at e; HY2191) in LPS-induced Sepsis Model
LPS(sigma( 미국)에 유도되는 패혈증 모델 설정을 위해 모든 실험에 8-12 주령의 C57BL/6 마우스 (SLC , 일본)를 사용하였다. 살모넬라 LPS(sigma, 미국) 20mg/kg (12 ,000 EU/g)를 마우스의 복강 내 투여한 지 30분 및 24시간 후에 꼬리정맥으로 대조 물질 (PBS, WelGENE, 한국) 또는 소듐 타우로데옥시콜레이트 [HY2191 , sodium taurodeoxycholate; HY2191(New Zealand Pharmaceut icals Ltd, 뉴질랜드) ]를 Medfusion2001(medex, 미국)을 사용하여 15분간 점적 주사하였다 (Medfusion2001 , medex, 미국) . 시간이 경과함에 따른 실험군 및 대조군의 생존율을 조사하였고 그 결과는 도 1A에 도시하였다. 도 1A에서 알 수 있듯이， 소듐 타우로데옥시콜레이트를 투여한 실험군의 마우스들은 대조군의 마우스들에 비하여 생존율이 30분후 투여한 경우에 4배, 24시간 후 투여한 경우에는 5배 높았다. 실시예 2. Cecal-l igation and Puncture (CLP) 패혈증 모델에서 소듬 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)의 효과 All experiments used 8-12 week old C57BL / 6 mice (SLC, Japan) to set up LPS (sigma ( US) -induced sepsis model.) Salmonella LPS (sigma, USA) 20 mg / kg (12,000 EU / g) 30 minutes and 24 hours after intraperitoneal administration of mice into the control vein (PBS, WelGENE, Korea) or sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was injected dropwise for 15 minutes using Medfusion2001 (medex, USA) (Medfusion2001, medex, USA) The survival rates of the experimental and control groups were examined over time and the results are shown in Figure 1A. As can be seen, the mice in the experimental group administered sodium taurodeoxycholate were 4 times higher in survival after 30 minutes and 5 times higher in 24 hours after the administration of sodium taurodeoxycholate. Cecal-l igation and pu Effects of Miniaturized Taurodeoxycholate (HY2191, Sodium Taurodeoxycholate; HY2191) in a Ncture (CLP) Sepsis Model
마우스를 인위적으로 맹장을 결찰 및 천공하여 복막염올 유발한후 시간 경과에 따라 패혈증이 유발되는 CLP 모델을 이용하여 소듐 타우로데옥시콜레이트의 약리 효과를 시험하였다. 20마리의 C57BL/6 마우스 (SLC, 일본)를 애버틴 (2,2,2-Tribromoethanol 25g + 2-Methyl-2- butanol 15.5ml로 제조된 stock용액을 PBS로 8배 희석하여 사용)으로 마취시킨 후, 복부 오른쪽 부위를 0.5 cm 길이로 절개하고 맹장을 노출시킨 다음 회맹부 관 (ileocecal valve) 아래부위 0.9 cm 하단부위를 결찰한 후 맹장에 23 게이지 바늘로 3개의 구멍을 내고， 복부를 다시 봉합하여 패혈증을 유발하였다. 봉합한지 2시간 후에 각 10마리의 C57BL/6 마우스에는 소듐 타우로데옥시콜레이트 [HY2191, sodium taurodeoxycholate; HY2191(New Zealand Pharmaceuticals Ltd, 뉴질랜드)]를 꼬리정맥으로 20분간 점적 투여하고 (실험군) 나머지 10마리의 C57BL/6 마우스에게는 PBS만을 동일한 방법으로 투여하였다 (대조군). 음성 대조군으로써 복부 피부와 복막만 인위적으로 절개한후 CLP 조치 없이 다시 수술로 복부 피부와 복막을 봉합하여 패혈증을 유도하지 않은 마우스를 샴 (sham)수술군으로 사용하였다. 시간이 경과함에 따른 실험군 및 대조군의 C57BL/6 마우스의 생존율을 조사하였다. 그 결과는 도 1B와 같으며, HY2191을 CLP 유도 패혈증 마우스에 2시간 후 주입 시 대조군에 비해 생존율이 7배 증가하는 치료 효과를 보여주었다. 실시예 3. LPS로 유도한 패혈증 모델에서 B세포 및 T세포의 중요성 분석 실시예 1에서와 동일한 LPS를 사용하여 패혈증을 유발하였으며, B세포 및 T 세포가 결핍된 마우스 (Rag2 K0)를 이용하여 소듐 타우로데옥시콜레이트 [HY2191, sodium taurodeoxycholate; HY2191(New Zealand Pharmaceuticals Ltd, 뉴질랜드)]의 효과를 실시예 1광 "일한 방법으로 시험하였다. 도 1C에서 보이는 바와 같이, 48시간 이내에 모든 개체가 폐사하여 실험군과 대조군에서 생존율의 변화가 없음을 확인하였다. 이는 HY2191가 패혈증 치료효과를 보이기 위하여 T 세포 또는 B세포를 필요로 함을 시사하는 소견이다. 실시예 4. LPS로 패혈증을 유도한 마우스 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)의 염증유발사이토카인 생성억제 효과 The pharmacological effects of sodium taurodeoxycholate were tested using a CLP model in which sepsis was induced over time after artificially ligation and perforation of the caecum to induce peritonitis. Twenty C57BL / 6 mice (SLC, Japan) were used as Avertin (stock solution prepared with 25 g of 2,2,2-Tribromoethanol + 15.5 ml of 2-Methyl-2-butanol diluted 8-fold with PBS). After anesthesia, the right part of the abdomen is cut in 0.5 cm length, the cecum is exposed, the ligation of the 0.9 cm lower part of the ileocecal valve is ligated and three holes are inserted into the cecum with 23 gauge needles. It was resealed to cause sepsis. Two hours after closure, each of the 10 C57BL / 6 mice contained sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand) was instilled in the tail vein for 20 minutes (experimental group) and the remaining 10 C57BL / 6 mice were administered only PBS in the same way (control). As a negative control, only the abdominal skin and the peritoneum were artificially dissected, and the abdominal skin and the peritoneum were sutured again without CLP, and mice that did not induce sepsis were used as the sham surgery group. Survival rates of C57BL / 6 mice in experimental and control groups were examined over time. The results are the same as in FIG. 1B, and when HY2191 was injected into CLP-induced sepsis mice after 2 hours, the survival rate was increased by 7 times compared to the control group. Example 3 Analysis of the Importance of B Cells and T Cells in LPS-Induced Sepsis Models The same LPS as in Example 1 was used to induce sepsis, and mice using B cells and T cells deficient (Rag2 K0) were used. Sodium taurodeoxycholate [HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was tested in Example 1 "light method. As shown in Figure 1C, all subjects died within 48 hours, confirming no change in survival in the experimental and control groups. This suggests that HY2191 requires T cells or B cells to treat sepsis Example 4. Example 4 Sodium taurodeoxycholate (HY2191, sodium taurodeoxycholate HY2191) Inhibitory Effect of Inflammatory Cytokine Production
실시예 1에서와 같이 C57BL/6 마우스에 LPS 20 mg/kg를 복강내 주사한 지 30분 후에 꼬리정맥으로 대조 물질 (PBS, Wei GENE, 한국) 또는 소듐 타우로데옥시콜레이트 [HY2191ᅳ sodium taurodeoxycholate; HY2191(New Zealand Pharmaceut icals Ltd, 뉴질랜드) ]를 점적 투여하였다. LPS 투여 후 6시간에 마우스 꼬리에서 채혈한 다음， 원심분리 (2000X g, 20분)하여 혈청만 얻어 CBA( cytometr ic bead array) kit (BD, 미국)를 사용하여 사이토카인 발현의 변화를 확인하였다. 30 minutes after intraperitoneal injection of LPS 20 mg / kg into C57BL / 6 mice as in Example 1, the control material (PBS, Wei GENE, Korea) or sodium taurodeoxycholate [HY2191 ᅳ sodium taurodeoxycholate] ; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand). Six hours after LPS administration, blood was collected from the tail of the mouse, and centrifuged (2000X g, 20 minutes) to obtain only serum. .
TNF-a 및 IFN-g, MCP-1 , IL-6 는 LPS 유발 6시간 경과 후 HY2191 투여에 의한 의미 있는 감소를 보였다 (도 2A) . 실시예 5. CLP 패혈증 모델에서 염증유발 사이토카인 변화에서의 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)의 효과 실시예 2에서와 같이 CLP 유도 시 나타나는 염증 사이토카인의 변화를 실시예 4의 방법과 동일한 방법으로 확인하였다. CLP 후 4， 24 및 48시간에 혈액을 채취하여 혈청을 분리한 다음 CBA( cytometric bead array) kit를 사용하여 사이토카인 발현의 변화를 확인하였다.  TNF-a and IFN-g, MCP-1, IL-6 showed a significant decrease by HY2191 administration 6 hours after LPS induction (FIG. 2A). Example 5 Effect of Sodium Taurodeoxycholate (HY2191, Sodium Taurodeoxycholate; HY2191) on Inflammatory Cytokine Changes in a CLP Sepsis Model Example 4 shows changes in inflammatory cytokines that occur upon CLP induction as in Example 2. It confirmed by the same method as the method of. Blood was collected at 4, 24, and 48 hours after CLP to separate serum, and then cytokine expression was confirmed using a cytometric bead array (CBA) kit.
TNF-a 및 IL-lb, MCP-1는 CLP 유도 후 24h에서 HY2191 투여에 의한 현저한 감소를 보였다 (도 2B) . 실시예 6. 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)를사용한 패혈증 치료후 혈압 저하 정상화 효과  TNF-a, IL-lb, MCP-1 showed a significant decrease by HY2191 administration at 24 h after CLP induction (FIG. 2B). Example 6 Normalization of Blood Pressure Lowering after Sepsis Treatment with Sodium Taurodeoxycholate (HY2191, HY2191)
실시예 1과 동일한 방법으로 처치한 C57BL/6 마우스들의 꼬리에서 LPS 유도 후 1， 2， 4 및 6시간에 비침습적 혈압측정기 (non-invasive blood pressure, NIBP, CODA, 미국)를 사용하여 혈압을 측정하였다. LPS 투여 1시간 후부터 평균 혈압이 두 군 모두에서 감소하기 시작하였으며, HY2191을 처리한 군의 평균 혈압이 LPS 유도 후 4시간부터 PBS를 처리한 대조군의 혈압과 비교하여 유의하게 상승하였다 (도 3A) . 실시예 7. 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의한신장 및 간기능 보호 효과  Blood pressure was measured using a non-invasive blood pressure (NIBP, CODA, USA) at 1, 2, 4 and 6 hours after LPS induction in the tails of C57BL / 6 mice treated in the same manner as in Example 1. Measured. After 1 hour of LPS administration, mean blood pressure began to decrease in both groups, and the mean blood pressure of HY2191 treated group was significantly increased compared to the blood pressure of PBS treated control group 4 hours after LPS induction (FIG. 3A). . Example 7 Protective Effect of Kidney and Hepatic Function by Sodium Taurodeoxycholate (HY2191, HY2191) in Sepsis Model
LPS로 패혈증 유도 시 나타나는 신장 및 간 기능 손상을 관찰하고 HY2191 투여에 따른 장기 손상 정도 변화를 혈액 내 요소 질소 (blood urea nitrogen, BUN) 및 아스파르테이트 아미노트랜스퍼레이즈 (aspartate aminotransferase, AST) 농도를 측정하여 확인하였다 (도 3B) . LPS 유발 30분 후 HY2191 또는 PBS를 꼬리에 점적 투여하고 24시간 및 48시간에 혈액을 채취하여 혈청을 분리하였다. 분리한 혈청에서 BUN을 측정하여 신기능의 손상 정도를 관찰하였고 AST를 측정하여 간기능의 손상 정도를 관찰하였다. HY2191은 LPS 유도 48시간에 신장의 손상을 나타내는 BUN의 상승을 현저히 감소시킴을 확인하였다 (도 3B 왼쪽) . 또한 HY2191은 24시간 48시간 동안 관찰한 모두에서 패혈증으로 인한 AST 농도의 상승을 현저히 억제하였다. 이는 HY2191이 패혈증에 의한 간 손상을 치료하는 효과가 있슴을 나타내는 소견이다. (도 3B오른쪽) . 실시예 8. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의해 증가되는 비장세포 확인 Observe renal and hepatic impairment during induction of sepsis with LPS, and measure the changes in organ urea nitrogen (BUN) and aspartate aminotransferase (AST) levels in the blood according to the change in organ damage following HY2191 administration. It was confirmed by (Fig. 3B). LPS induced After 30 minutes, HY2191 or PBS was instilled into the tail and blood was collected at 24 and 48 hours to separate serum. BUN was measured in the isolated serum to observe renal impairment, and AST was measured to evaluate liver impairment. HY2191 was found to significantly reduce the elevation of BUN, indicating kidney damage at 48 hours of LPS induction (FIG. 3B left). In addition, HY2191 significantly suppressed the increase in AST concentration due to sepsis in all observed for 24 hours 48 hours. This suggests that HY2191 is effective in treating liver damage caused by sepsis. (Figure 3B right). Example 8 Identification of Splenocytes Increased by Sodium Taurodeoxycholate (HY2191, HY2191) in LPS-Induced Sepsis Model
실시예 1과 같이， LPS로 패혈증을 유도한 마우스 모델에서 HY2191 및 PBS 대조군을 처치한 후 6시간， 24시간 및 48시간에 비장세포의 변화를 확인하였다. 전체 비장세포의 수는 LPS 유도 48시간 후에 PBS 대조군에서 감소를 보이는 것과 달리 HY2191 투여 군에서 의미 있게 증가함을 보였다. 유세포 분석기를 이용하여， CDllb+ 세포와 B220+ 세포의 절대수가 전체 비장세포 중에서 현저히 증가한 것임을 확인하였다 (도 4) . 실시예 9. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의해 증가되는 T조절세포 확인 As in Example 1, the change of splenocytes was confirmed at 6 hours, 24 hours and 48 hours after treatment with HY2191 and PBS in the mouse model induced sepsis with LPS. The total number of splenocytes was significantly increased in the HY2191-administered group, unlike the decrease in the PBS control 48 hours after LPS induction. Using a flow cytometer, it was confirmed that the absolute number of CDllb + cells and B220 + cells was significantly increased among all splenocytes (FIG. 4). Example 9 Identification of T Regulatory Cells Increased by Sodium Taurodeoxycholate (HY2191, HY2191) in LPS-Induced Sepsis Model
실시예 8과 동일한 방법으로 실험하여， 비장세포 중에서 T 조절세포인 CD4+Foxp3+ T 세포의 절대수가 HY2191에 의해 LPS 유도 48시간 후에 유의하게 증가함을 확인하였다 (도 5) , 실시예 10. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의한 수지상세포 활성화 억제 효과 Experiment in the same manner as in Example 8, it was confirmed that the absolute number of T regulatory cells CD4 + Foxp3 + T cells in splenocytes significantly increased 48 hours after LPS induction by HY2191 (Fig. 5), Example 10. Inhibitory Effect of Sodium Taurodeoxycholate (HY2191, HY2191) on Dendritic Cell Activation in LPS-induced Sepsis Model
실시예 8과 동일한 방법으로 실험하여, LPS 유도 후 비장세포 중에서도 CDllc+ 수지상세포의 활성도 (CD86 발현)를 확인하였다. 도 6A에 나타난 바와 같이, 살아있는 수지상세포만을 분석하기 위해In the same manner as in Example 8, after the LPS induction, the activity of CDllc + dendritic cells (CD86 expression) was confirmed among splenocytes. In Figure 6A As shown, to analyze only live dendritic cells
CDllc(+)/7AAD (-) 세포만을 선별한 후， 수지상세포 공자극분자 중에서 CD86 발현능을 분석하였다. 평균 형광강도 (Mean f luoresence intensi ty, MFI )로 CD86 발현능을 표시하였으며 LPS 유도 후 24시간이후 부터 HY2191에 의해 유의하게 발현이 억제됨을 확인하였다. 실시예 11. LPS로 패혈증을 유도한 마우스 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의한 B 세포 활성화 억제 효과 Only CDllc (+) / 7AAD (−) cells were selected and analyzed for CD86 expression in dendritic cell costimulatory molecules. The mean fluorescence intensity (Mean f luoresence intensi ty, MFI) expressed CD86 expression ability, and it was confirmed that expression was significantly inhibited by HY2191 after 24 hours after LPS induction. Example 11. Effect of Inhibiting B Cell Activation by Sodium Taurodeoxycholate (HY2191, HY2191) in Mouse Model Induced Sepsis with LPS
실시예 8과 동일한 방법으로 실험하여 , LPS유도 후 비장세포 증에서 Experiment in the same manner as in Example 8, in the splenocytes after LPS induction
B220+인 B 세포의 활성도 (CD86 발현)를 확인하였다. 살아있는 B 세포만을 분석하기 위해 B220(+)/7MD (-) 세포만올 선별한 후， B 세포 공자극분자 중에서 CD86 발현올 분석하였다. 평균 형광강도 (Mean f luoresence intensity, MFI )로 CD86 발현을 표시하였으며 LPS 유도 후 6시간과 24시간에 HY2191에 의해 유의하게 억제됨을 확인하였다 (도 6B, 도 6C) . 실시예 12. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의하여 증가되는 H-MDSC의 패혈증 치료 효과 The activity (CD86 expression) of B cells that were B220 + was confirmed. In order to analyze only live B cells, only B220 (+) / 7MD (−) cells were selected, followed by analysis of CD86 expression in B cell co-stimulatory molecules. The mean fluorescence intensity (Mean f luoresence intensity, MFI) was expressed CD86 expression was confirmed that significantly inhibited by HY2191 6 hours and 24 hours after LPS induction (Fig. 6B, 6C). Example 12 Sepsis Treatment Effect of H-MDSC Increased by Sodium Taurodeoxycholate (HY2191, HY2191) in LPS-Induced Sepsis Model
실시예 1과 같이 LPS로 패혈증을 유도한 마우스에 소듐 타우로데옥시콜레이트를 투여한 지 24시간이 지난 뒤 각각의 마우스들을 희생시켜 비장을 적출하여 비장세포에서 CDllb가 발현되면서 동시에 Gr-1이 높게 발현되는 세포들 (골수유래억제세포, MDSC)을 분석하였다 (도 7A, 도 7B) . 소듐 타우로데옥시콜레이트를 투여하지 않고 LPS만을 투여한 마우스들을 회생시켜 비장을 적출하여 비장세포에서 CDllb가 발현되면서 동시에 Gr-1이 높게 발현되는 세포들 (이하 L-MDSC)에 비하여 패혈증 마우스에 소듐 타우로데옥시콜레이트를 투여한 지 24시간이 지난 뒤에 각각의 마우스들을 회생시켜 확보한 비장세포에서 CDllb가 발현되면서 동시에 Gr-1이 높게 발현되는 세포들 (이하 H-MDSC)의 수가 의미 있게 증가하였다 (도 7A, 도 7B) . L-MDSC 및 H-MDSC 세포들의 미성숙도를 골수양세포 미성숙 마커인 CD31을 이용하여 확인하였다 (도 7C 위) . 두 가지 세포에서의 CD31+ 발현 세포의 비율은 의미있는 차이를 관찰할 수 없었다. L-MDSC 및 H-MDSC는 F4/80loGr-l+이면서 Ly6g+ Ly6c+표현형을 나타내었다 (도 7C 아래). 이러한 표현형을 가지는 MDSC는 소듐 타우로데옥시콜레이트를 투여한 패혈증 마우스에서 의미 있는 증가를 보였다. 24 hours after the administration of sodium taurodeoxycholate to LPS-induced sepsis as in Example 1, each mouse was sacrificed to extract the spleen and CDllb was expressed in the splenocytes. Highly expressed cells (myeloid derived suppressor cells, MDSC) were analyzed (FIG. 7A, FIG. 7B). Mice treated with LPS alone without sodium taurodeoxycholate were regenerated to extract spleen and CDllb expression in splenocytes, and at the same time to sepsis mice compared to cells with high Gr-1 expression (hereinafter referred to as L-MDSC). 24 hours after administration of sodium taurodeoxycholate, CDllb was expressed in splenocytes obtained by regeneration of each mouse, and at the same time, the number of cells with high Gr-1 expression (hereinafter referred to as H-MDSC) was significant. Increased (FIG. 7A, FIG. 7B). Immaturity of L-MDSC and H-MDSC cells was confirmed using CD31, a myeloid cell immature marker (upper FIG. 7C). CD31 + Expression in Two Cells No significant difference in cell proportions was observed. L-MDSC and H-MDSC were F4 / 80 lo Gr-1 + with Ly6g + Ly6c + phenotype (below Figure 7C). MDSC with this phenotype showed a significant increase in sepsis mice administered sodium taurodeoxycholate.
QIAGEN RNeasy Mini KITCQiagen, 미국)를 이용해 MDSC 에서 total Total in MDSC using QIAGEN RNeasy Mini KITCQiagen (USA)
RNA를 추출하였다. 패혈증을 유도한 마우스에 HY2191을 처리하지 않고 분리선별한 Grl+CDllb+ MDSC (L-MDSC)와 비교하여 패혈증을 유도한 마우스에 HY2191을 처리하고 분리선별한 Grl+CDllb+ MDSC (H-MDSC)에서 특이적으로 발현이 증가되는 유전자들과 발현이 감소되는 유전자를 관찰하였다 (도 8B, 표 1， 표 2). 유전자들의 발현 차이를 IUumina Sentrix MouseRef-8(vl.1) BeadChip arrays(Ilhimina, 미국)로 비교하였고,
Figure imgf000047_0001
0.05 미만이며 fold change가 2 이상 또는 -2 이하인 유전자를 선별하였고， 목록은 다음 표 1 내지 표 2와 같다: RNA was extracted. Specific to Grl + CDllb + MDSC (H-MDSC) treated with HY2191 in sepsis-induced mice compared to Grl + CDllb + MDSC (L-MDSC) isolated without treatment with HY2191 in sepsis-induced mice As a result, genes with increased expression and genes with reduced expression were observed (FIG. 8B, Table 1, and Table 2). The expression differences of genes were compared with IUumina Sentrix MouseRef-8 (vl.1) BeadChip arrays (Ilhimina, USA).
Figure imgf000047_0001
Genes with less than 0.05 and a fold change of 2 or more or -2 or less were selected and the list is shown in Tables 1 to 2 below:
【표 1】  Table 1
L-MDSC에 비해 H-MDSC에서 고발현하는 유전자 Genes Highly Expressed in H-MDSC Compared to L-MDSC
Figure imgf000047_0002
Figure imgf000047_0002
Figure imgf000048_0001
Figure imgf000048_0001
【표 2】  Table 2
L-MDSC에 비해 H-MDSC에서 저발현하는 유전자
Figure imgf000048_0002
Genes Low-expressing in H-MDSC Compared to L-MDSC
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000051_0001
CDllb 및 Gr-1의 발현 정도에 따라 세포를 크게 세군으로 나누고 이를 FACS sorter로 분리 선별하였다 (도 8) . 분리한 세포들의 세포 수를 헤모사이토미터로 측정하여 살아있는 세포수가 1 X 105 이 되게 PBS에 동량으로 섞어 패혈증이 유도된 마우스에 꼬리정맥으로 주입하였다 (도 8 위) . 이 때 세포를 주사하기 24시간 전에 마우스들은 LPS를 치사량 (20mg/kg, 12,000 EU/g)으로 복강 내 투여하였다. PBS 그룹 및 L-MDSC, H-MDSC 그룹에서 각각 마우스를 5마리씩 사용하였으며， Gr-lint 세포를 주사한 군에서는 3마리， Gr-Γ세포를 주사한 군에서는 4마리를 사용하였다. H- MDSC를 투여한 마우스들에서 L-MDSC(40% 생존) 및 다른 표면 표현형의 세포들에 비해 현저히 증가된 생존율 (80% 생존)을 보였다. H-MDSC에 의한 치료효과의 작용기전을 확인하기 위하여 세포가 주사된 마우스의 비장 세포 변화를 관찰 하였다 (도 9) . LPS 투여한 지 24시간이 지난 마우스에 H— MDSC 및 L-MDSC를 양자 이입하였다. 세포 양자 이입후 24시간이 지난 마우스의 비장세포를 수확하였다. 도 9A (위: 대표적 FACS 플롯， 아래: 반복실험 그래프)와 같이 H-MDSC를 양자 이입한 경우에 B220+ 세포가 유의하게 증가함을 확인하였다. 또한， 도 9B (위: 대표적 FACS 플롯， 아래: 반복실험 그래프)에서 보여주는 바와같이 H— MDSC를 양자이입한 경우에 L-MDSC에서 보다 골수 및 비장에서 CDllb+Gr-l+세포가 증가함을 확인하였다. 그러나 도 9C 및 9D에서 확인할 수 있듯이， CD4+ T 세포, CD8+ T 세포 및 CDllc+ DC 수의 변화는 없었다. 실시예 13. LPS로 유도된 패혈증 모델에서 소듐 타우로데옥시콜레이트에 의하여 증가되는 48h BH세포의 패혈증 치료 효과 Cells were divided into three groups according to the expression level of CDllb and Gr-1, which were separated and selected by FACS sorter (FIG. 8). The number of cells of the isolated cells was measured by a hemocytometer and mixed in the same amount in PBS so that the number of living cells was 1 X 10 5 and injected into the sepsis-induced tail vein (Fig. 8). At this time, mice were intraperitoneally administered LPS at a lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before cell injection. Five mice were used in the PBS group, the L-MDSC, and the H-MDSC group, respectively, three in the group injected with Gr-l int cells and four in the group injected with Gr-Γ cells. Mice receiving H-MDSC showed significantly increased survival (80% survival) compared to L-MDSC (40% survival) and other surface phenotype cells. In order to confirm the mechanism of action of the therapeutic effect by H-MDSC, splenic cell changes were observed in mice injected with cells (FIG. 9). H—MDSC in mice 24 hours after LPS administration And L-MDSC were protonated. Splenocytes of mice were harvested 24 hours after cell proliferation. As shown in FIG. 9A (top: representative FACS plot, bottom: replicate graph), B220 + cells were significantly increased when H-MDSC was protonated. Furthermore, as shown in FIG. 9B (top: representative FACS plot, bottom: replicate graph), CDllb + Gr-l + cells increased in bone marrow and spleen than in L-MDSC when H—MDSC was protonated. Confirmed. However, as can be seen in Figure 9C and 9D, there was no change in the number of CD4 + T cells, CD8 + T cells and CDllc + DC. Example 13. Sepsis Treatment Effect of 48h B H Cells Increased by Sodium Taurodeoxycholate in LPS-Induced Sepsis Model
실시예 1과 동일한 C57BL/6 마우스를 사용하였다. PBS 그룹, 정상 마우스 B 세포 투여군, 48h BL 세포 투여군， 24h ¾ 세포 투여군， 48h ¾ 세포 투여군 및 BHM 세포 투여군에서 각각 5마리씩 사용하였다. BHM 세포를 확보하기 위하여 LPS와 소듐 타우로데옥시콜레이트를 투여한 지 24시간이 지난 뒤에 마우스를 희생시켜 비장을 적출하였으며， 이 비장세포에서 CDllb와 Gr-1 분자를 높게 발현하는 MDSC 들을 FACS sorter로 분리 선별하였다 (도 10 위 왼쪽) . 살아있는 (7ΑΑΕΓ) CDllb+ Gr-1+ 세포 1 X 105 개를 PBS에 부유하여 24시간 전에 패혈증이 유도된 B6마우스에 꼬리정맥으로 주사하였다, 이 마우스로부터 BHM 세포를 확보하기 위하여 CDllb+ Gr-1+ 세포를 투여하고 나서 24시간이 지난 뒤에 마우스를 희생시켜 비장을 적출하였으며, 이 비장세포에서 B220분자를 높게 발현하는 B 세포들을 FACS sorter로 분리 선별하였다 (BHM 세포， 도 10 위 왼쪽) . 24h 또는 48h BH 세포를 확보하기 위하여 LPS와 소듐 타우로데옥시콜레이트를 투여한 지 24 시간 또는 48시간이 지난 뒤에 마우스를 회생시켜 비장을 적출하였으며, 이 비장세포에서 B220분자를 높게 발현하는 B 세포들을 FACS sorter로 분리 선별하였다 (24h ¾ 세포 또는 48h ¾ 세포， 도 10 위 가운데) . 48h BL 세포를 확보하기 위하여 LPS를 투여한 지 48시간이 지난 뒤에 마우스를 회생시켜 비장을 적출하였으며， 이 비장세포에서 B220분자를 높게 발현하는 B 세포들을 FACS sorter로 분리 선별하였다 (48h BL 세포， 도 10 위 오른쪽) . 살아있는 (7ΜΕΓ) 세포 1 X 106 개를 PBS에 부유하여 패혈증이 유도된 B6마우스의 꼬리정맥에 이 세포들을 주사하였다. 이 때 세포를 양자 입양 받은 마우스들은 B 세포를 주사하기 24시간 전에 LPS(20mg/kg, 12 ,000 EU/g)를 복강 내 투여하여 패혈증을 유도하였다. 정상 마우스의 B 세포를 주사한 군 (0% 생존) 및 24h BH 세포 주사군 (20% 생존)， 48h BL 세포 (40% 생존)에 비해 48h BH 세포를 투여한 마우스들에서 현저히 증가된 생존율 (80% 생존)을 보였다. BHM 세포를 투여한 마우스들에서 가장 좋은 생존율 ( 100% 생존)을 보였다. 실시예 14. LPS로 유도된 패혈증 모델에서 소듐 타우로데옥시콜레이트에 의하여 증가되는 48h BH세포 및 H-MDSC의 중요성 확인 The same C57BL / 6 mice as in Example 1 were used. Five mice were used in the PBS group, the normal mouse B cell group, the 48h B L cell group, the 24h ¾ cell group, the 48h ¾ cell group, and the B HM cell group. 24 hours after LPS and sodium taurodeoxycholate were administered to secure B HM cells, the mice were sacrificed and spleens were extracted, and MDSCs expressing high CDllb and Gr-1 molecules in these splenocytes were FACS. Sorting and sorting by sorter (upper left in Figure 10). 1 × 10 5 live (7ΑΑΕΓ) CDllb + Gr-1 + cells were suspended in PBS and injected into the sepsis-induced sepsis-induced B6 mice 24 hours ago by CDVbb + Gr to secure B HM cells from these mice. At 24 hours after administration of −1 + cells, mice were sacrificed to harvest the spleen, and B cells expressing high B220 molecules were separated and selected by FACS sorter (B HM cells, left side of FIG. 10). . To secure 24h or 48h B H cells, mice were regenerated 24 hours or 48 hours after administration of LPS and sodium taurodeoxycholate, and the spleen cells expressed high B220 molecules in the splenocytes. Cells were isolated and sorted by FACS sorter (24h ¾ cells or 48h ¾ cells, middle in FIG. 10). To secure 48h B L cells, 48 hours after LPS administration, mice were regenerated to extract spleen, and B cells expressing high B220 molecules were separated and selected by FACS sorter (48h B L Cells, right above FIG. 10). 1 x 10 6 (7ΜΕΓ) living cells were suspended in PBS to get sepsis These cells were injected into the tail vein of induced B6 mice. At this time, the mice adopting the cells were induced sepsis by intraperitoneally administering LPS (20 mg / kg, 12,000 EU / g) 24 hours before B cell injection. Significantly increased in mice receiving 48h B H cells compared to B cells injected in normal mice (0% survival) and 24h B H cell injection groups (20% survival) and 48h B L cells (40% survival). Survival rate (80% survival). Mice administered with B HM cells showed the best survival rate (100% survival). Example 14 Confirmation of Importance of 48h B H Cells and H-MDSC Increased by Sodium Taurodeoxycholate in LPS-Induced Sepsis Model
실시예 12와 13에서 처럼, C57BL/6 마우스에서 만들어진 H-MDSC와 BHM 세포를 B 세포와 T 세포가 없는 Rag2K0 마우스에 LPS 유도 24시간 후에 양자이입하여 패혈증 모델에서의 효과를 확인하였다. PBS군， BHM 세포 ( 1 X 106 , 5 X 106)개 및 BHM 세포 (1 X 106， 5 x 106)와 48h H-MDSC ( 1 x 105)군에서 2마리씩 사용하였다. 실시예 3에서처럼 Rag2K0 마우스에 LPS(20mg/kg, 12 ,000 EU/g)를 복강 내 투여하였을 시 24시간 후에 PBS 또는 BHM 세포 1 X 106 개를 꼬리 정맥으로 양자 이입한 경우에 48시간 이내에 모든 개체가 폐사하여 실험군과 대조군에서 생존율의 변화가 없음을 확인하였다. BHM 세포 1 X 106개와 48h H-M)SC를 양자이입한 경우에 72시간 이내에 모든 개체가 폐사하였고, ¾M 세포 5 x 106개를 양자이입한 경우에 96시간 이내에 모든 개체가 폐사하였다. 다른 군들과는 달리 BHM 세포 5 X 106개와 48h H-MDSC 1 x 105개를 함께 양자이입한 군에서만 120시간이상 지속적으로 생존함을 확인하였다. BHM 세포를 양자이입한 경우에는 세포 수에 관계없이 PBS군에 비해서 생존기간이 연장됨을 확인할 수 있어 효과를 보였다. 실시예 15. LPS로 유도한 패혈증 모델에서 소듐 타우로데옥시콜레이트 (HY2191, sodium taurodeoxycholate; HY2191)에 의하여 증가되는 ¾세포 양자 이입 후 비장세포에서의 변화 확인 As in Examples 12 and 13, H-MDSC and BHM cells produced in C57BL / 6 mice were transfused into Rag2K0 mice without B cells and T cells 24 hours after LPS induction to confirm the effect in the sepsis model. PBS group, B HM cells (1 × 10 6 , 5 × 10 6 ), B HM cells (1 × 10 6 , 5 × 10 6 ) and 48h H-MDSC (1 × 10 5 ) groups were used two by one. . Within 48 hours when 1 × 10 6 PBS or BHM cells were transfused into the tail vein 24 hours after intraperitoneal administration of LPS (20 mg / kg, 12,000 EU / g) to Rag2K0 mice as in Example 3 It was confirmed that all the individuals died and there was no change in survival rate in the experimental and control groups. All subjects died within 72 hours when 1 X 10 6 B HM cells and 48 h HM) SC were injected, and all individuals died within 96 hours when 5 x 10 6 ¾M cells were protonated. Unlike other groups, B HM cells 5 x 10 6 and 48 h H-MDSC 1 x 10 5 only in the group transfected lasting more than 120 hours was confirmed that. The proliferation of B HM cells showed an effect of prolonging survival compared to the PBS group regardless of the number of cells. Example 15 Confirmation of Changes in Splenocytes After ¾ Cell Transduction Increased by Sodium Taurodeoxycholate (HY2191, HY2191) in LPS-Induced Sepsis Model
실시예 13과 동일한 방법으로 48h ¾ 세포를 제조하여, LPS 유도 후 48h ¾ cells were prepared in the same manner as in Example 13, after LPS induction.
24시간 째 48h BH 세포 1 X 106개를 양자이입한 후 24시간 지난 마우스를 회생시켜 비장세포 중에서 B220+인 B 세포의 활성도 (CD86 발현)를 확인하였다. 살아있는 B 세포만을 분석하기 위해 B220(+)/7AAD(-) 세포만을 선별한 후， B 세포 공자극분자 중에서 CD86 발현을 분석하였다. 24시간째 대조물질인 PBS만 투여한 군과 비교 시 ¾ 세포를 양자이입한 경우에 CD86 발현이 감소됨을 보였다 (도 12A) . 도 12B에서는 비장세포 중에서도 CD4+ T 세포 또는 CD8+ T 세포에서 LPS 투여시의 CD69hiCD62L10 T 세포의가 감소됨을 확인하였다. 도 12C는 48h BH 세포를 양자 이입 24시간 후에 비장에서 증가된 CDllb+ Ly6G+ Ly6C inter세포의 상대적 비율 ( >)을 도시화한 FACS 플롯 이미지로 CDllb+세포만을 gat ing한 후 분석하였다. PBS군과 비교시 48h ¾ 세포를 양자 이입한 경우에 CDllb+ Ly6G+ Ly6C inter세포의 상대적 비율이 58%에서 82%로 증가함을 확인하였다. 실시예 16. BHM세포， BL 및 ¾세포 표현형 분석 Mice were left 24 h after protonation of 1 × 10 6 48h B H cells at 24 h. Regeneration was performed to confirm the activity (CD86 expression) of B cells that are B220 + in splenocytes. In order to analyze only live B cells, only B220 (+) / 7AAD (−) cells were selected, and then CD86 expression was analyzed in B cell co-stimulatory molecules. The expression of CD86 was decreased when ¾ cells were protonated compared to the control group treated with PBS at 24 hours (FIG. 12A). In FIG. 12B, it was confirmed that CD69 hi CD62L 10 T cells in LPS administration were reduced in CD4 + T cells or CD8 + T cells among splenocytes. FIG. 12C analyzed 48h B H cells after gat ing only CDllb + cells with FACS plot images depicting the relative proportion (>) of increased CDllb + Ly6G + Ly6C inter cells in the spleen after 24 hours of transfection. In comparison with the PBS group, the relative proportion of CDllb + Ly6G + Ly6C inter cells increased from 58% to 82% when 48h ¾ cells were protonated. Example 16. BHM Cell, B L and ¾ Cell Phenotyping Assay
BHM 세포와 유사한 특징을 가지며, BHM 세포의 제조 방법보다 수월하고 효율적으로 제조할 수 있는 48h BH 세포를 제조하여 두 세포를 비교 분석하였다ᅳ 8주에서 12주 사이의 정상 C57BL/6 마우스 비장을 2장의 프로스티드 슬라이드 (frosted sl ide)로 갈아서 단일 세포 부양액으로 만든 후 염색여백부위 차단용 완충액 [blocking buffer , puri f ied 2.4 G2(rat IgG2b against FC Y RI I , eBioscience, 미국) ]과 불활성화시킨 10 )의 마우스， 랫트 및 염소 혈청을 함유한 인산완충용액 (PBS)을 흔합하여 4°C에서 30분간 배양하였다. 비장 세포들은 항-마우스 CD11MM1/70) , 항-마우스 B220(RA3- 6B2) , 항-마우스 Gr-1(RB6-8C5) , 항-마우스 F4/80 CI :Α3-1) , 항-마우스 CD23CB3B4) , 항-마우스 CD21(7G6) 및 항—마우스 CD93(M4.1) , 기타 모든 항체를 이용하여 15분간 염색하였다. 세척 조건 염색을 마친 세포들은 살아있는 세포만을 확인할 수 있게 7-아미노 액티노마이신 D(7 D, 7-amino act inomycin D)를 10분간 염색한 다음， FACS Canto I I (BD, 미국)를 이용하여 측정하였다. 자료들은 Flowjo software(Tree Star , Inc . , 미국)를 이용하여 분석하였다. ΒΗΜ 세포와 48h ¾ 세포는 기존의 T2-MZP 조절 B 세포와 유사하게 CD21 , CD23 은 높게 발현 하지만 (도 14， 도 15A CD5, CDld 및 AA4(CD93)는 발현하지 않았다 (도 13A-B) . IgM의 발현이 48h BL 세포에 비하여 48h BH세포가 고르게 높았으며 이는 성숙 B 세포의 특징을 지니고 있음을 보여주었다 (도 13C) . 실시예 17. LPS로 유도된 패혈증 모델에서 소듐 타우로데옥시콜레이트 처치후 증가되는 CD23hi ¾세포의 패혈증 치료 효과 48 h B H cells with similar characteristics to BHM cells, which are easier and more efficient than the method for producing B HM cells, were prepared and compared between the two cells. Normal C57BL / 6 mouse spleen between 8 and 12 weeks. Was prepared into single cell suspension by grinding into two frosted slides and then used as a buffer for blocking the staining margin (blocking buffer, puri f ied 2.4 G2 (rat IgG2b against FC Y RI I, eBioscience, USA)). Phosphate buffer solution (PBS) containing inactivated 10) mice, rats and goat serum was mixed and incubated at 4 ° C for 30 minutes. Spleen cells are anti-mouse CD11MM1 / 70), anti-mouse B220 (RA3- 6B2), anti-mouse Gr-1 (RB6-8C5), anti-mouse F4 / 80 CI: A3-1), anti-mouse CD23CB3B4 ), Anti-mouse CD21 (7G6) and anti-mouse CD93 (M4.1), and all other antibodies were stained for 15 minutes. After washing, stained cells were stained with 7-amino actinomycin D (7 D) for 10 minutes to identify only living cells and measured using FACS Canto II (BD, USA). It was. Data were analyzed using Flowjo software (Tree Star, Inc., USA). Β ΗΜ cells and 48h ¾ cells expressed high CD21 and CD23 similarly to conventional T2-MZP regulatory B cells (FIG. 14, FIG. 15A but did not express CD5, CDld and AA4 (CD93) (FIGS. 13A-B). The expression of IgM was evenly higher in 48h B H cells compared to 48h B L cells. It has been shown (FIG. 13C). Example 17. Sepsis Treatment Effect of CD23 hi ¾ Cells Increased After Sodium Taurodeoxycholate Treatment in LPS-Induced Sepsis Model
LPS와 소듐 타우로데옥시콜레이트를 투여한 지 48시간이 지난 뒤에 마우스를 희생시켜 비장을 적출하였다. 비장세포에서 B220+CD23hiCD21hi Gr-Γ CDllb—의 표현형을 나타내는 CD23hi BH 세포들을 FACS sorter로 분리 선별하였다. 동시에 B220+CD23loCD21hiGr-rCDllb 표현형을 나타내는 CD2310 BH 세포들도 같은 방법으로 선별하여 대조군으로 사용하였다. 살아있는 (7ΑΑΕΓ) 세포 1 X 105 및 0.5 X 105 개를 PBS에 부유한 후 마우스의 꼬리정맥으로 주입하였다. 이 때 마우스들은 세포를 주사하기 24시간 전에 LPS를 치사량 (20mg/kg, 12,000 EU/g)으로 복강 내 투여하였다. 1 x 105및 0.5 X 105 의 CD23hi BH 세포를 투여한 패혈증 마우스들 (100% 및 75% 생존)에서 CD2310 ¾세포를 투여한 패혈증 마우스 (각각 75% 및 25% 생존)에 비해 현저히 증가된 생존율을 보였다. PBS만 주입한 마우스는 0%의 생존율을 보였다. (도 15B) . PBS 투여군 3마리, CD2310 ¾ 세포 투여군 및 CD23hi ¾세포 투여군에서 두 가지 용량 군 별로 ( 1 X 105 및 0.5 X 105) 각각 4마리씩 사용하였다, QIAGEN R easy Mini KITCQiagen, 미국)를 이용해: B 세포에서 total RNA를 추출하였다. 패혈증을 유도한 마우스에 HY2191을 처리하지 않고 48시간 후에 분리선별한 B220+ B 세포 (48h BL cel l ) 또는 패혈증 마우스에서 HY2191 처리 48시간 후에 분리선별한 CD23loCD21+B220+ B세포 (CD2310 ¾ cel l )와 비교하여 패혈증 마우스에서 HY2191 처리 48시간 후 분리선별한 B220+ B 세포 (48h ¾ cel l ) 또는 CD23hiCD21+B220+ B세포 (CD23hi BH cel l )에서 특이적으로 발현이 증가 또는 감소되는 유전자들의 발현 차이를 I l lumina Sentrix MouseRef— 8(vl .1) BeadChi arraysd 1 lumina, 미국)로 비교하였고,
Figure imgf000055_0001
0.05 미만이며 fold change가 2 이상 또는 -2 이하인 유전자를 선별하였고, 목톡은 다음 표 3 내지 표 8과 같다. 48 hours after administration of LPS and sodium taurodeoxycholate, the mice were sacrificed and the spleens were extracted. CD23 hi B H cells expressing the phenotype of B220 + CD23 hi CD21 hi Gr-Γ CDllb— in splenocytes were isolated and selected by FACS sorter. At the same time, CD23 10 B H cells exhibiting B220 + CD23 lo CD21 hi Gr-rCDllb phenotype were also selected and used as controls. 1 × 10 5 and 0.5 × 10 5 live (7ΑΑΕΓ) cells were suspended in PBS and injected into the tail vein of the mouse. At this time, mice were intraperitoneally administered LPS at a lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before cell injection. Sepsis mice receiving 1 × 10 5 and 0.5 × 10 5 CD23 hi B H cells (100% and 75% survival) compared to sepsis mice receiving CD23 10 ¾ cells (75% and 25% survival, respectively). Significantly increased survival. Mice injected with PBS alone showed 0% survival. (FIG. 15B). Three PBS-treated, CD23 10 ¾-cell and CD23 hi ¾-cell administered groups were used in each of two dose groups (1 × 10 5 and 0.5 × 10 5 ), using QIAGEN R easy Mini KITCQiagen, USA. Total RNA was extracted from B cells. B220 + B cells (48 h B L cel l) isolated after 48 hours without treatment with HY2191 in sepsis-induced mice or CD23 lo CD21 + B220 + B cells (CD23 isolated after 48 hours with HY2191 treatment in sepsis mice) 10 ¾ cel l) specifically in B220 + B cells (48h ¾ cel l) or CD23 hi CD21 + B220 + B cells (CD23 hi B H cel l) isolated after 48 hours of HY2191 treatment in sepsis mice Differences in expression of genes with increased or decreased expression were compared to I lumina Sentrix MouseRef-8 (vl .1) BeadChi arraysd 1 lumina, USA.
Figure imgf000055_0001
Genes with less than 0.05 and a fold change of 2 or less or -2 or less were selected, and Moktok are shown in Tables 3 to 8 below.
【표 3】 Table 3
자
Figure imgf000055_0002
character
Figure imgf000055_0002
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000058_0001
NM_009382.3 Thyl 2.48 NM_009382.3 Thyl 2.48
Figure imgf000059_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000060_0001
M_009174.3 Siah2 2.07 M_009174.3 Siah2 2.07
Figure imgf000061_0001
Figure imgf000061_0001
【표 4】 Table 4
24h BH세포와 비교하여 48h ¾세포에서 저발현하는 Low expression in 48h ¾ cells compared to 24h B H cells
Figure imgf000061_0002
Figure imgf000061_0002
Figure imgf000062_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000064_0001
【표 5]  [Table 5]
48h BL세포와 비교하여 48h ¾세포에서 고발현하는 유전자
Figure imgf000064_0002
Highly expressed genes in 48h ¾ cells compared to 48h B L cells
Figure imgf000064_0002
Figure imgf000065_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000066_0001
XM_131538.1 Dhcr24 1.52 XM_131538.1 Dhcr24 1.52
Figure imgf000067_0001
Figure imgf000067_0001
【표 6】  Table 6
48h ¾세포와 비교하여 48h ¾세포에서 저발현하는 유전자
Figure imgf000067_0002
Low expression genes in 48h ¾ cells compared to 48h ¾ cells
Figure imgf000067_0002
Figure imgf000068_0001
Figure imgf000068_0001
M_010654.2 Klrdl -2.29
Figure imgf000069_0001
M_010654.2 Klrdl -2.29
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000070_0001
【표 7】  Table 7
48h CD2310 ¾세포와 비교하여 48h CD23hi ¾세포에서 고발현하는 유전자
Figure imgf000070_0002
Highly expressed genes in 48h CD23 hi ¾ cells compared to 48h CD23 10 ¾ cells
Figure imgf000070_0002
Figure imgf000071_0001
Figure imgf000071_0001
【표 8】  Table 8
48h CD2310 ¾세포와 비교하여 48h CD23hi ¾세포에서 저발현하는 유전자
Figure imgf000071_0002
Low expression genes in 48h CD23 hi ¾ cells compared to 48h CD23 10 ¾ cells
Figure imgf000071_0002
Figure imgf000072_0001
Figure imgf000072_0001
NM_011261.2 Rein -2.52 NM_011261.2 Rein -2.52
Figure imgf000073_0001
실시예 18. LPS로 유도된 패혈증 모델에서 소듐 타우로데옥시콜레이트 처치후 증가되는 CD23hi BH 세포를 양자 이입 하였을 때 늘어나는 세포 아형 분석
Figure imgf000073_0001
Example 18 Analysis of Cell Subtypes Increased by Protonation of CD23 hi B H Cells Increased After Sodium Taurodeoxycholate Treatment in LPS-Induced Sepsis Model
실시예 17에서， CD23hi 면역조절 ¾ 세포를 양자이입한 경우 CDllb+ In Example 17, CDllb + when adopting CD23 hi immunomodulatory ¾ cells
Gr-1+세포 및 면역조절 B 세포의 증가를 확인할 수 있었다 (도 16) . 위 실시 예에사 사용한 수용 마우스 수는 PBS 그룹 3마리， CD2310 면역조절 ¾ 세포 그룹 및 CD23hi 면역조절 ¾ 세포 그룹에서 1 X 105 개씩 양자이입한 3마리씩 사용하였다. LPS 투여 후 24시간과 48시간에 각각 마우스들을 회생시켜 B세포와 CDllb+ Gr-1+세포에서 CD23hi ¾ 세포를 양자이입한 경우에 CD2310 BH 세포를 양자이입한 경우에 의미있는 세포 수의 증가를 보였다, 실시예 19. LPS로 유도된 패혈증 모델에서 소듐 타우로데옥시콜레이트 처치후 증가되는 H-MDSC 세포를 양자 이입 하였을 때 늘어나는 B세포 아형 분석 An increase in Gr-1 + cells and immunoregulatory B cells could be seen (FIG. 16). The number of recipient mice used in the above examples was used by three PBS groups, three x23 10 immunoregulatory ¾ cell groups, and one CD10 hi immunoregulatory ¾ cell group, each transfused with 1 X 10 5 . Mice were treated 24 and 48 hours after LPS administration, respectively. Regeneration of B cells and CDllb + Gr-1 + cells by transfusion of CD23 hi ¾ cells showed significant increase in the number of CD23 10 B H cells when transfused, Example 19. LPS-induced Analysis of B-cell Subtypes after Proliferation of Increased H-MDSC Cells after Treatment with Sodium Taurodeoxycholate in Isolated Sepsis Models
실시예 12에서와 같이 만들어진 L-MDSC 및 H-MDSC를 양자이입한 경우 도 14A에서 볼 수 있듯이 H-MDSC를 양자이입한 후 24시간째에 B세포에서 GD23 high 부분이 확연히 증가됨을 FACS 플롯 이미지로 확인하였다. 도 14B는 H-MDSC를 양자이입한 경우에 B 세포에서 CD23 high 부분이 L-MDSC의 경우에서 보다 유의한 증가를 보임을 확인하였다. 실시예 20. LPS와 소듐 타우로데옥시콜레이트를 이용하여 시험관에서의 H- MDSC 제조 및 이들 세포의 패혈증 치료효과분석  In the case of protonation of L-MDSC and H-MDSC prepared as in Example 12, as shown in FIG. 14A, the GD23 high portion of B cells was significantly increased at 24 hours after H-MDSC was protonated. It was confirmed as. 14B confirmed that the CD23 high portion of B cells showed a significant increase in L-MDSC when H-MDSC was protonated. Example 20 Preparation of H-MDSC in Vitro Using LPS and Sodium Taurodeoxycholate and Analysis of Sepsis Treatment Effect of These Cells
8주에서 12주 사이의 정상 C57BL/6 마우스를 회생시켜 대퇴골 및 경골의 골수에서 세포를 채취한 후 셀그로 (Cel lgro) 배지 (Cel lGenix, 독일)에 세포 (1 X 106 /ml )를 부유하였다. 1 X 106 /wel l의 세포에 1 mg/mi 의 LPS와 50 mg/m의 HY2191 을 흔합하여 24시간 배양하였다. 8 weeks to regenerate a normal C57BL / 6 mice to 12 weeks in the cells (1 X 10 6 / ml) to the femur, and then harvesting the cells from the bone marrow of the tibia thereby cell (Cel lgro) medium (Cel lGenix, Germany) Wealthy 1 mg / mi LPS and 50 mg / m HY2191 were mixed in 1 × 10 6 / wel l cells and cultured for 24 hours.
골수세포에 LPS만을 시험관내 처리하여 수확한 MDSC (LexMDSC)과 LPS와 HY2191를 동시에 시험관내에서 골수세포에 처리한 후 수확한 MDSC (HexMDSC) 사이에 CDllb+Gr-l+ MDSC 분화 정도를 비교하였다. 24시간 배양 시, 대조군에 비하여 HY2191 처리군에서 CDllb+Gr-l+세포의 절대수가 각각 36% 및 34% 증가한 것을 유세포 분석법 (FASC)으로 확인하였다 (도 17A) . 24시간 배양을 완료한 LexMDSC및 HexMDSC를 LPS 10 mg/kg을 24시간전에 주사한 C57BL/6 마우스에 정맥으로 양자이입였다. 그 중 HexMDSC를 주사한 마우스만이 생존하였고， PBS만 처리한 군, LexMDSC 투여군 및 Media 투여한 군 모두 폐사하였다 (도 17B) . 이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서， 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. The degree of CDllb + Gr-l + MDSC differentiation was compared between MDSC (LexMDSC) harvested by treating only bone marrow cells in vitro with LPS and MDSC (HexMDSC) harvested after treating LPS and HY2191 with bone marrow cells simultaneously. . When cultured for 24 hours, the absolute number of CDllb + Gr-1 + cells increased 36% and 34% in the HY2191 treated group compared to the control group, respectively, by flow cytometry (FASC) (FIG. 17A). LexMDSC and HexMDSC, which had completed 24-hour incubation, were intravenously injected into C57BL / 6 mice injected with LPS 10 mg / kg 24 hours ago. Among them, only mice injected with HexMDSC survived, and all of the groups treated with PBS, LexMDSC-treated group and Media-treated group died (FIG. 17B). As described above in detail a specific part of the present invention, for those of ordinary skill in the art such specific technology is only a preferred embodiment, which is not limited to the scope of the present invention It is obvious. Accordingly, the substantial scope of the invention will be defined by the appended claims and equivalents thereof.

Claims

【특허청구범위】 [Patent Claims]
【청구항 1】  [Claim 1]
다음 단계를 포함하는 GPCR19-연관 질환의 치료제의 스크리닝 방법: Screening methods for the treatment of GPCR19-related diseases comprising the following steps:
(a) 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하는 단계 ; (a) administering an in vivo physiologically active modulator to a mammal other than a human;
(b) 상기 포유동물에 GPCR19(G-protein cou led receptor 19) 아고니스트 시험물질을 투여하는 단계; 및  (b) administering a G-protein cou led receptor 19 agonist test substance to said mammal; And
(c) 상기 포유동물에서 (i) Gr-l+CDllb+ MDSC(Myelo id-derived suppressor cell), (i i ) Prok2, Osm, Ltf , Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 (Regulatory B cell) 또는 (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71， Igsf9, Gypc, Clqbp, 1(13， I14il, Gbpl, Cd83, Mybl2, If 1140, Snxll, Swap70, Ccbp2, Sc4mol , Cyp51, Sqle, L0C100040592 , B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C 100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, Oasl2, I17r, Uspl8, Mxl, L0C100048346 , Mx2 Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, GprlW, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 시험물질을 주입하지 않은 대조군과 비교하여 상기 시험물질을 주입한 포유동물에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 증가하면 상기 시험물질은 GPCR19-연관 질환의 치료제로 판정한다. (c) (i) Gr-l + CDllb + MDSC (Myelo id-derived suppressor cell), (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h , Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2 , Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd861FflaRik Smp004, HiflaRik , D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 or MxlSC (iii) ) CD23 hi Regulatory B cell or (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, 1 (13 ， I14il, Gbpl, Cd83, Mybl2, If 1140, Snxll, Sw ap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tawdl, Cr2, Arn Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C 100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 Highly expressing Lmnbl, Pira4, Blvrb or Actb genes or 1190002H23 Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7m Anxa2 , Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, Cxcasl2, Cla I17r, Uspl8, Mxl, L0C100048346, Mx2 Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, GprlW, Trafdl, Cp3d, Apd3d Analyzing the generation of immunoregulatory B cells that underexpress Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes; When the production level of the cells of (i) to (iv) is increased in the mammal to which the test substance is injected compared with the control group not injected with the test substance, the test substance is determined as a therapeutic agent for GPCR19-associated disease.
【청구항 2] [Claim 2]
다음 단계를 포함하는 GPCR19-연관 질환의 치료제의 스크리닝 방법 : (a) 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트 시험물질을 골수세포 집단 (bone marrow cell population)에 처리하는 단계;  A method for screening a therapeutic agent for a GPCR19-associated disease comprising the steps of: (a) treating a bone marrow cell population with an in vivo physiological activity modulator and a GPCR19 agonist test substance;
(b) 상기 골수세포 집단에서 (i) Gr-l+CDllb+ MDSC(Myeloid— derived suppressor cell), (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 (Regulatory B cell) 또는 (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg3U, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Iftl40, Snxll, S ap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC 100040592 , B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855 , Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik, BhlhbS, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5 Tmem66, Amigo2, Klra4, LOC 100038894 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC 100048346 , Mx2 Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat , Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pe lla, D14Ertd668e, Mlkl , Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 시험물질을 처리하지 않은 대조군과 비교하여 상기 시험물질을 처리한 골수세포 집단에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 시험물질은 GPCR19-연관 질환의 치료제로 판정한다 (b) (i) Gr-l + CDllb + MDSC (Myeloid-derived suppressor cell) in the myeloid cell population, (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h , Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2 , Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd861FflaRik Smp004, HiflaRik , D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 or MxlSC (iii) ) CD23 hi Regulatory B cell or (iv) Fcer2a, Grm6, AU019823, Marcksll, Bcl2alc, Bcl2ald, Afg3U, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Iftl40, Snxll, S ap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC 100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafldl, Crnn, Arhgap4, Pira3, Flna Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Ca l, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr2 Highly expressing Lmnbl, Pira4, Blvrb or Actb genes or 1190002H23 Rik, BhlhbS, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa , Klra4, LOC 100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Tcol2 Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, LOC 100048346, Mx2 Tyki, Hba3al, Lba3al Ifitm3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, F cerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pe lla, D14Ertd668e, Mlkl, Itk, Cpin696 Analyzing the production of low-expressing immunoregulatory B cells; When the degree of production of the cells of (i) to (iv) in the bone marrow cell population treated with the test substance was increased compared to the control group, the test substance was associated with GPCR19-associated disease. We judge with treatment
【청구항 3】 [Claim 3]
다음 단계를 포함하는 GPCR19아고니스트의 효능 분석 방법 :  Method for analyzing efficacy of GPCR19 agonist, including the following steps:
(a) 생체 내 생리적 활성 조절 물질을 인간을 제외한 포유동물에 투여하는 단계 ;  (a) administering an in vivo physiologically active modulator to a mammal other than a human;
(b) 상기 포유동물에 GPCR19 아고니스트를 투여하는 단계 ; 및  (b) administering a GPCR19 agonist to the mammal; And
(c) 상기 포유동물에서 (0 Gr-l+CDllb+ MDSC(Myek) id-derived suppressor cell), (ii) Prok2, Osm, Ltf , lral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 (Regulatory B cell) 또는 (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 , Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Iftl40, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, ffdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb유전자를 고발현하거나 또는 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, lra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, TrP53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki, Hba-al , Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, Gprll4, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 GPCR19 아고니스트를 주입하지 않은 대조군과 비교하여 상기 GPCR19 아고니스트를 주입한 포유동물에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 GPCR19 아고니스트는 효능이 있는 것으로 판정한다. 【청구항 4】 (c) (0 Gr-l + CDllb + MDSC (Myek) id-derived suppressor cell) in the mammal, (ii) Prok2, Osm, Ltf, lral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pil6, Dfna5h , Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Highly expresses Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Cd274 , Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2112, Ccl3, Tgm2, Rcl, Tap , MDSC, which expresses Sdc4, Clec4e, Klhll5, Dnasell3 or Mxl genes, (iii) CD23 hi immunoregulatory B cells or (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Iftl40, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 13000dp Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrpsy, Map Caps Lta, Rbm4, Cdl80, Cfp , Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, ffdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb or Actb genes or 1190002H23Rik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rsphl, Rsphl Ly6cl, Histlhlc, Tg, lra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrl, 001Mlgal, Xbplik Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Tr P 53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2x Slfnl Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA46497 Traf Generation of immunomodulatory B cells that underexpress Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Serpinb6b, or Cd69 genes Analyzing; The GPCR19 agonist has an efficacy when the degree of production of the cells of (i) to (i v ) is increased in comparison with the control group in the mammal injected with the GPCR19 agonist compared to the control group not injected with the GPCR19 agonist. It is determined that there is. [Claim 4]
다음 단계를 포함하는 GPCR19 아고니스트의 효능 분석 방법 :  Method for analyzing efficacy of GPCR19 agonist, including the following steps:
(a) 생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트를 골수세포 집단 (bone marrow cell population)에 처리하는 단계; 및  (a) treating the bone marrow cell population with a physiologically active regulatory substance and a GPCR19 agonist in vivo; And
(b) 상기 골수세포 집단에서 (i) Gr-l+CDllb+ MDSC(Myeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf , Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110， Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3 또는 Mxl 유전자를 저발현하는 MDSC, (iii) CD23hi 면역조절 B 세포 또는 (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Iftl40, Snxll, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592 , B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna, Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2: Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23Rik: Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi , Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki , Hba~al , Lgals3bp, Ifitra3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA467197, GprlH, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, lf9, Pexlla, D14Ertd668e, Mlkl , Itk, Ser inb6b 또는 Cd69 유전자를 저발현하는 면역조절 B 세포의 생성 여부를 분석하는 단계; 상기 GPCR19 아고니스트를 처리하지 않은 대조군과 비교하여 상기 GPCR19 아고니스트를 처리한 골수세포 집단에서 상기 (i) 내지 (iv)의 세포의 생성 정도가 대조군과 비교하여 증가하면 상기 GPCR19 아고니스트는 효능이 있는 것으로 판정한다. (b) (i) Gr-1 + CDllb + Myeloid-derived suppressor cells (MDSC), (ii) Prok2, Osm, Ltf, Klral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcll genes or Ilia, Ednl, AdoracllO Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd863 Hi B930041F14Rik, D330014H01Rik, Tnfaip2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Ta l, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, MDnac3 or Dnasell3 iii) CD23 hi immunomodulatory B cells or (iv) Fcer2a, Grm6, AUO 19823, Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71, Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83 , Mybl2, Iftl40, Snxll, Swap70, Ccbp2, Sc4m ol, Cyp51, Sqle, L0C100040592, B3gnt8, 13000 14 Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2 : Lmnbllv, Prbd2 Highly expressing the Actb gene or 1190002H23Rik : Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi, Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem661000 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssrb2, Rein , F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcllO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki, Hba ~ al, Lgals3bp, Ifitra3, Cfll, 0as2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, Fcerlg, Ifng, AA46713d, Gdl CdHd Analysis of the production of immunoregulatory B cells that underexpress Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, lf9, Pexlla, D14Ertd668e, Mlkl, Itk, Ser inb6b or Cd69 genes Making; The GPCR19 agonist is more effective when the degree of production of the cells of (i) to (iv) is increased in comparison to the control group in the bone marrow cell treated with the GPCR19 agonist compared to the control group not treated with the GPCR19 agonist. It is determined that there is.
【청구항 5] [Claim 5]
제 1 항또는 제 3 항에 있어세 상기 포유동물은 마우스， 랫트, 개， 원승이， 염소， 양 또는 토끼인 것을 특징으로 하는 방법. 【청구항 6】  4. A method according to claim 1 or 3, wherein said mammal is a mouse, rat, dog, animal, goat, sheep or rabbit. [Claim 6]
제 1 항 내지 제 4 항 중 어느 한 항에 있어서， 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질 진균 유래 물질， 바이러스 유래 물질, 자가 유래 물질, 알러젠 (allergen) 또는 암 세포 유래 물질인 것을 록징으로 하는 방법 .  The method according to any one of claims 1 to 4, wherein the in vivo physiological activity modulator is a bacterium derived material, a virus derived material, an autologous material, an allergen or a cancer cell derived material. How to .
【청구항 7】 [Claim 7]
제 6 항에 있어서， 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸인 것을 특징으로 하는 방법 .  7. The method of claim 6, wherein the in vivo physiological activity modulator is a bacteria derived material, lipopolysaccharide (LPS) or peptidoglycan.
【청구항 8】 [Claim 8]
제 1 항 내지 제 4 항 중 어느 한 항에 있어서， 상기 GPCR19 아고나스트는 하가 화학식 1의 화합물， 그의 염 또는 수화물인 것을 특징으로 하는 방법 :  The method of any one of claims 1 to 4, wherein the GPCR19 agonast is a compound of Formula 1, a salt or a hydrate thereof:
화학식 1 Formula 1
Figure imgf000082_0001
Figure imgf000082_0001
상기 화학식에서 ¾은 수소， 하이드록시， 치환 또는 비치환 알킬 ¾ in the above formula is hydrogen, hydroxy, substituted or unsubstituted alkyl
^는 할로겐; R2는 수소 또는 α-하이드록시; ¾은 하이드록시, Nk(C¾)mS03H(m은 정수 0, 1, 2, 3, 4 또는 5) 또는 NH(CH2)nC02H(n은 정수 0, 1, 2, 3, 4 또는 5); ,R4는 수소, 알킬 또는 할로겐; ¾는 수소， 치환 또는 ti]치환 알킬 또는 아릴이고; ¾은 수소 또는 ¾ 및 R6가 부착된 탄소와 함께^ Halogen; R 2 is hydrogen or α-hydroxy; ¾ is hydroxy, Nk (C¾) m S0 3 H (m is integer 0, 1, 2, 3, 4 or 5) or NH (CH 2 ) n C02H (n is integer 0, 1, 2, 3, 4 Or 5); R4 is hydrogen, alkyl or halogen; ¾ is hydrogen, substituted or ti] substituted alkyl or aryl; ¾ together with hydrogen or carbon with ¾ and R 6 attached
3, 4, 5 또는 6개 원자 크기로 형성된 고리이고; R7은 수소， 하이드록시, 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; ¾은 수소 B는 치환 또는 비치환 알킬; R10은 수소 또는 치환 또는 비치환 알킬; Ru은 수소 또는 치환 또는 비치환 알킬이다. A ring formed of 3, 4, 5 or 6 atomic sizes; R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; ¾ is hydrogen B is substituted or unsubstituted alkyl; R 10 is hydrogen or substituted or unsubstituted alkyl; R u is hydrogen or substituted or unsubstituted alkyl.
【청구항 9】 [Claim 9]
제 1 항 내지 제 4 항 중 어느 한 항에 있어서, 상기 MDSC에서 고발현 되는 유절자는 Prok2, Osm, Ltf, lral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm, P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 및 Xcll로 구성된 군으로부터 선택되는 최소 1개 이상의 유전자인 '것을 특:징으로 하는 방법 . 【청구항 10】  The method according to any one of claims 1 to 4, wherein the decanter high in MDSC is Prok2, Osm, Ltf, lral7, Slc40al, Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm , At least one gene selected from the group consisting of P2ry6, Fosb, Cdl77, Sortl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 and Xcll. [Claim 10]
제 1 항 내지 제 4 항 중 어느 한 항에 있어서， 상기 MDSC에서 ^발현 되는 유졌자는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, 18 The method according to any one of claims 1 to 4, wherein the retainer expressed in the MDSC is Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Serpina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, Mefv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, 18
'm 'm ' m m
Figure imgf000083_0001
'm' m 'mm
Figure imgf000083_0001
'S^PIS '9P3 '30dy 'TldUH 'djSl 'PSP3 'T JBJX ' Χ-ΐ¾ οε 'S ^ PIS' 9P3 '30dy' TldUH 'djSl' PSP3 'T JBJX' Χ-ΐ¾ οε
'L6lL9m
Figure imgf000083_0002
'gsBO 'L6lL9m
Figure imgf000083_0002
'gsBO
'sisBo ' '^ηο 'ΟΪΡΧ 'TOIS 'ζ^.η 'ιιη ':οζεζ99 σι 졔 'sis B o''^ηο' ΟΪΡΧ 'TOIS' ζ ^ .η 'ιιη': οζεζ99 σι 졔
'6P3 0 'Ssoos dsnQ 'ITISS 'SPO '09ΐΡ3 '^ '^ΙΗ60Ι¾ΐ000ΐ02 32 'TSlBSl 'T9JI¾ '[dqx 'i JDl '찌 쎄 'φο 'q pdedd 'εΐ· d ' 39< '6P3 0' Ssoos dsn Q 'ITI S S' SPO '09 ΐΡ3 '^' ^ ΙΗ60Ι¾ΐ000ΐ02 32 'TSlBSl' T9JI¾ '[dqx' i JDl 'Ci Chi' φο 'q pdedd' εΐ · d '39 <
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s s
•^i¾극^ 百 늘륭^
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opjjy 'TdBX' ΒζΐΠ 'go' giuSl
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op jj y
S9C800/CT0ZaM/X3d 9LL£0/S10Z OAV Serpinb6b 및 Cd69로 구성된 군으로부터 선택되는 최소 1개 이상의 유전자인 것을 특징으로 하는 방법. S9C800 / CT0ZaM / X3d 9LL £ 0 / S10Z OAV At least one gene selected from the group consisting of Serpinb6b and Cd69.
【청구항 13】 [Claim 13]
생체 내 생리적 활성 조절 물질 및 GPCR19 아고니스트를 처리하여 In vivo treatment with physiologically active modulators and GPCR19 agonists
Gr-l+CD.llb+표면형질형을 가지며 항염증 활성을 갖는 MDSC(Myeloid-derived suppressor cell). Myeloid-derived suppressor cells (MDSCs) with Gr-l + CD.llb + surface morphology and anti-inflammatory activity.
【청구항 14】 [Claim 14]
제 13 항에 있 ^서, 상기 MDSC는 Prok2, Osm, Ltf, Klral7, Slc40al, The method of claim 13, wherein the MDSC is Prok2, Osm, Ltf, Klral7, Slc40al,
Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm P2ry6, Fosb, Cdl77, Sprtl, Gent 2， Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 또는 Xcll 유전자를 고발현하거나 또는 Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Ser ina3f , Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, M fv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik D330014H01Rik, Tnfai 2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mtl, Rbak, Rundc3b, Sdc4, Clec4e, Klhll5, Dnasell3또는 Mxl 유전자를 저발현하는 것을 특징으로 하는 MDSC. Fcer2a, Hspala, I18rb, Lyz2, Pi 16, Dfna5h, Mtusl, 4732429D16Rik, Myadm P2ry6, Fosb, Cdl77, Sprtl, Gent 2, Chad, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, or the Tacll gene. Or Ilia, Ednl, CxcllO, Adora2b, Cxcl9, Ser ina3f, Cxcll, Ptx3, Ifng, Slc7all, Batf2, Gbp5, Cfll, M fv, Bcl2111, Gbp2, Sphkl, Cd274, Icaml, 1110, Ahnak, Gbp3, Cirbp Gpr84, Bcl2alc, Tgtp, Cd86, Hifla, Smpdl3b, B930041F14Rik D330014H01Rik, Tnfai 2, Pexlla, Arrdc4, St6galnac4, Irgml, Pde4d, Marcksll, Tgm2, Ccl3, 1112a, Tapl, Mdc, Rbak, Dlc MDSC characterized by low expression of Dnasell3 or Mxl gene.
【청구항 15】 [Claim 15]
제 13항에 있어서， 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질, 진균 유래 물질, 바이러스 유래 물질, 자가 유래 물질， 또는알러젠 (allergen)인 것을 특징으로 하여 생성되는 MDSC.  14. The MDSC of claim 13, wherein the physiologically active regulatory substance in vivo is a bacterium-derived, fungal-derived, virus-derived, autologous, or allergen.
【청구항 16】 [Claim 16]
제 15항에 있어서, 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸인 것을 특징으로 하여 생성되는 MDSC.  16. The MDSC of claim 15, wherein said in vivo physiological activity modulator is a lipopolysaccharide (LPS) or peptidoglycan as a bacteria derived material.
【청구항 17】 제 13 항에 있어서, 상기 GPCR19 아고니스트는 하기 화학식 화합물, 그의 염 또는 수화물인 것을 특징으로 하여 생성되는 MDSC: [Claim 17] 14. The MDSC of claim 13, wherein said GPCR19 agonist is a compound of formula:
화학식 1  Formula 1
Figure imgf000085_0001
Figure imgf000085_0001
상기 화학식에서. ^은 수소, 하이드록시, 치환 또는 비치환 알킬 또는 할로겐; ¾는 수소 또는 α-하이드록시; ¾은 하이드록시， ΝΗθ¾)^03Η(ηι은 장수 0， 1, 2, 3, 4또는 5) 또는 NH(CH2)nC02H(n은 정수 0 1, 2， 3, 4 또는 5); R4는 수소， 알킬 또는 할로겐; R5는 수소, 치환 또는 비치환 알킬 또는 아릴이고; ¾은 수소 또는 R5 및 ¾가 부착된 탄소와 함께 3, 4, 5 또는 6개 원자 크기로 형성된 고리이고; R7은 수소， 하이드록시, 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; ¾은 수소 또는 ¾환 또는 비치환 알킬; R10은 수소 또는 치환 또는 비치환 알킬; Ru은 수소 또는 치환 또는 비^환 알킬이다. 【청구항 18】 In the above formula. ^ Is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; ¾ is hydrogen or α-hydroxy; ¾ is hydroxy, ΝΗθ¾) ^ 0 3 Η (ηι is longevity 0, 1, 2, 3, 4 or 5) or NH (CH 2 ) n C0 2 H (n is an integer 0 1, 2, 3, 4 or 5); R4 is hydrogen, alkyl or halogen; R 5 is hydrogen, substituted or unsubstituted alkyl or aryl; ¾ is hydrogen or a ring formed in 3, 4, 5 or 6 atom sizes with R 5 and ¾ attached carbon; R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; ¾ is hydrogen or ¾ ring or unsubstituted alkyl; R 10 is hydrogen or substituted or unsubstituted alkyl; R u is hydrogen or substituted or unsubstituted alkyl. [Claim 18]
생체 내 생리적, 활성 조절 물질 및 GPCR19 아고니스트 처리되어 형질전환 또는 중폭되거나 또는 상기 제 13항의 MDSC 처리에 의해 형질전환 또는 ^폭되어 CD23hi표면 형질형을 가지며 항염증 활성을 갖는 면역조절 B 세.포 (Regulatory B cell,). In vivo physiological, activity modulators and GPCR19 agonists treated to transform or heavy or transformed or expanded by the MDSC treatment of claim 13 to have a CD23 hi surface morphology and have anti-inflammatory activity. P (Regulatory B cell,).
【청구항 19] [Claim 19]
제 18항에 있어서, 상기 면역조절 B세포는 Fcer2a, Grm6, AU019823 Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, If t 140, Snxll, S ap70, Ccbp2, Sc4mol, Cyp51 , Sqle, L0C100040592, B3gnt8, 1300014106Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafl5, Ddxl7, Cnn2, Brwdl, Cr2, Arhgap4, Pira3, Flna Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnbl, Pira4, Blvrb 또는 Actb 유전자를 고발현하거나 또는 1190002H23fik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndc5 Tmem66, Ami o2, Klr.a4, L0C100038894 , Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inpl, Ssr2, Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcUO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346 , Mx2 Tyki, Hba-al, Lgals3bp, Ifitm3, Cfll, Oas2, Serpina3f , Emb, P2ryl4, Cirbp, Dhx58, sFcerlg, Ifng, AA467197, Gprl , Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat , Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl , Itk, Serpinb6b 또는 Cd69 유전자를 저발현하는 것을 특장으로 하는 면역조절 B세포. 【청구항 20】 The method according to claim 18, wherein the immunomodulatory B cells are Fcer2a, Grm6, AU019823 Marcksll, Bcl2alc, Bcl2ald, Afg311, Cdk5rl, Cd40, Rrplb, Slamfl, Luc71 Igsf9, Gypc, Clqbp, Id3, I14il, Gbpl, Cd83, Mybl2, Mybl2 If t 140, Snxll, S ap70, Ccbp2, Sc4mol, Cyp51, Sqle, L0C100040592, B3gnt8, 1300014106 Rik, Fdps, Ctsa, Dmwd, Sfrs7, Hbb-bhl, Hspala, Srpk3, Hhex, Insigl, Cbfa2t3h, Tafldl, Cr2, Dhl, Cr2, Arn , Pira3, Flna Cdk5rapl, Cugbpl, Ankrd37, Cd44, Mrps7, Mapkll, Pycard, Capl, Lta, Rbm4, Cdl80, Cfp, Cmah, L0C100046855, Srebfl, Ciita, Ryrl, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Pcbd Highly expresses Pira4, Blvrb or Actb genes or 1190002H23fik, Bhlhb8, Mistl, Fkbpll, Cacnalh, Rsphl, Rbm47, Slpi Ly6cl, Histlhlc, Tg, Klra7, Creb312, Prg2, Creld2, Nkg7, Anxa2, Txndcmi Klr.a4, L0C100038894, Speer3, Derl3, Ppapdclb, Ckb, Thyl, Pqlc3, Klrdl, Xbpl, Klrel, Lgalsl, 2010001M09Rik, Ctsw, Cdl60, Ccl5, Selll, Dusp4, Socs2, Ccl4, Cd9, Mr2n Rein, Tubb2b, Akp2, F2r, H13, Cd8bl, Ifit3, Rsad2, L0C667370, Irf7, Ifit2, Slfnl, CxcUO, Ctla4, Rgsl, 0asl2, I17r, Uspl8, Mxl, L0C100048346, Mx2, Tyki Lba3 , Cfll, Oas2, Serpina3f, Emb, P2ryl4, Cirbp, Dhx58, sFcer lg, Ifng, AA467197, Gprl, Trafdl, Cd3d, Igtp, Rilpll, Apoe, Lat, Sh2d2a, Cd6, Slcl5a2, Gbp2, Oasll, Spoil, Ctse, Klf9, Pexlla, D14Ertd668e, Mlkl, Itk, Ced69b Immunoregulatory B cell which is featured by expressing. [Claim 20]
제 18 에 있 서:, 상기 면역조절 B 세포는 CD21 및 CD23을 발현하는 것을 특징으로 하는 면역조절 B세포.  19. The immunoregulatory B cell of claim 18, wherein said immunoregulatory B cells express CD21 and CD23.
【청구항 21】 [Claim 21]
제 18항에 있 서 , 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질, 진균 유래 물질， 바이러스 유래 물질, 자가 유래 물질, 알러젠 (allergen;) 또는 암 세포 유래 물질인 것을 특징으로 하여 생성되는 역조절 B세포.. 【청구항 22】  19. The method of claim 18, wherein the physiologically active regulatory substance in vivo is a bacterium-derived material, a fungal-derived material, a virus-derived material, an autologous material, an allergen, or a cancer cell-derived material. B cell .. [claim 22]
제 21 항에 있엇서, 상기 생체 내 생리적 활성 조절 물질은 박테리아 유래 물질로서 리포폴리사카라이드 (LPS) 또는 펩티도글리칸인 것을 특징으로 하여 생성되는 면역조절 B세포. 22. The method of claim 21, wherein the biophysiologically active substance in vivo is bacteria An immunomodulatory B cell produced as a derived material is lipopolysaccharide (LPS) or peptidoglycan.
【청구항 23】 [Claim 23]
제 21 항에 있어서， 상기 GPCR19 아고니스트는 하기 화학식 1의 화합물， 그의 염 또는 수화물인 것을 특징으로 하는 면역조절 B 세포:  The immunomodulatory B cell of claim 21, wherein the GPCR19 agonist is a compound of Formula 1, a salt thereof, or a hydrate thereof:
화학식 1  Formula 1
Figure imgf000087_0001
Figure imgf000087_0001
상기 화학식에서. ^은 수소, 하이드록시， 치환 또는 비치환 알킬 또는 할로겐; R2는 수소 또는 α -하이드록시; ¾은 하이드톡시, KC¾)mS03H(m은 정수 0， 1 , 2 , 3 , 4 또는 5) 또는 NH(CH2)nC02H(n은 정수 0, 1， 2 , 3, 4 또는 5) ; 는 수소， 알킬 또는 할로겐; R5는 수소， 치환 또는 비치환 알킬 또는 아릴이고;. ¾은 수소 또는 R5 및 ¾가 부착된 탈소와 함께 3, 4, 5 또는 6.71] 원자 크기로 형성된 고리이고; R7은 수소， 하이드록시， 치환 또는 비치환 알킬; ¾은 수소 또는 치환 또는 비치환 알킬; ¾은 수소 또는 치 ^ 또는 비치환 알킬; R10은 수소 또는 치환 또는 비치환 알킬; Rii은 수소 또는 치환또는 비^환 알킬이다. In the above formula . ^ Is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; R2 is hydrogen or α-hydroxy; ¾ is hydroxy, KC¾) m S0 3 H (m is an integer 0, 1, 2, 3, 4 or 5) or NH (CH 2 ) n C0 2 H (n is an integer 0, 1, 2, 3, 4 Or 5); Is hydrogen, alkyl or halogen; R 5 is hydrogen, substituted or unsubstituted alkyl or aryl; ¾ is hydrogen or a ring formed at 3, 4, 5 or 6.71 atomic size with R 5 and ¾ attached; R 7 is hydrogen, hydroxy, substituted or unsubstituted alkyl; ¾ is hydrogen or substituted or unsubstituted alkyl; ¾ is hydrogen or chi ^ or unsubstituted alkyl; R 10 is hydrogen or substituted or unsubstituted alkyl; Rii is hydrogen or substituted or unsubstituted alkyl.
【청구항 24】 [Claim 24]
상기 제 13항 내지 제 17항 중 어느 한 항의 MDSC 또는 상기 제 18항 내지 제 23항 중 어느 한 항의 면역조절 B 세포를 유효성분으로 포함하는 항염증 약제학적 조성물.  An anti-inflammatory pharmaceutical composition comprising the MDSC of any one of claims 13 to 17 or the immunoregulatory B cells of any one of claims 18 to 23 as an active ingredient.
【청구항 25】 제 24 항에 있어서， 상기 조성물은 패혈증, 자가면역질환, 염증성 질환， 류마티스 관절염， 아토피성 피부염， 알 2하이머 병， 궤양성 대장염, 제 1형 당뇨병， 크론병, 건선， 건선성 관절염， 강직성 척수염, 다발성 경화증, 캐슬만씨 병， 자가면역성 간염， 자가 면역성 포도막염, 중증 근무력증， 원형 탈보증 및 전신성 홍반성 낭창으로 구성된 군으로부터 선택되는 어느 하나의 염증 질환에 대하여 예방 또는 치료 효능이 있는 것을 특징으로 하는 항염증 약제학적 조성물. [Claim 25] 25. The method of claim 24, wherein the composition is sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Al 2 Haimer disease, ulcerative colitis, type 1 diabetes, Crohn's disease, psoriasis, psoriatic arthritis, ankylosing myelitis , Prophylactic or therapeutic efficacy against any one inflammatory disease selected from the group consisting of multiple sclerosis, Castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, aberrant decompensation and systemic lupus erythematosus Anti-inflammatory pharmaceutical composition.
【청구항 26】 [Claim 26]
상기 제 13항 내지 제 17항 중 어느 한 항의 DSC 또는 상기 제 18항 내지 제 23항 중 어느 한 항의 면역조절 B 세포를 유효성분으로 포함하는 조성물의 약제학적 유효,량을 객체 (Subject )에게 투여하는 단계를 포함하는， 염증의 예방 또는 치료 방 [청구항 27】  A pharmaceutically effective amount of a composition comprising the DSC of any one of claims 13 to 17 or the immunoregulatory B cells of any one of claims 18 to 23 as an active ingredient is administered to a subject. A method for preventing or treating inflammation, comprising the step of claim [claim 27]
제 26 항쎄 있어서, 상기 조성물은 패혈증， 자가면역질환， 염증성 환, 류마티스 관절염， 아토피성 피부염, 알 S하이머 병， 궤양성 대장염， 제 1형 당뇨병, a론병， 건선， 건선성 관절염, 강직성 척수염, 다발성 경화증 캐슬만씨 병' 자가면역성 간염， 자가 면역성 포도막염, 중증 근무력증， 원형 탈 S증. 및 전신성 흥반성 낭창으로 구성된 군으로부터 선택되는 어느 하나의 염종 질환에 대하여 예방 또는 치료 효능이 있는 것을 특징으로 하는 방법 .  27. The composition of claim 26, wherein the composition is sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Al Sheimer's disease, ulcerative colitis, type 1 diabetes, ahron disease, psoriasis, psoriatic arthritis, ankylosing myelitis, Multiple sclerosis Castleman's disease 'autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia de novo syndrome. And a prophylactic or therapeutic effect against any one hydrochloride disease selected from the group consisting of systemic lupus erythematosus.
PCT/KR2013/008365 2013-09-16 2013-09-16 In vivo and ex vivo amplification of myeloid-derived immunoregulatory cell and immunoregulatory b lymphocyte by activation of gpcr19 pathway WO2015037764A1 (en)

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