WO2006059637A1 - Agent for diagnosing, preventing and treating adhesion with the use of ccr8 inhibitor - Google Patents

Agent for diagnosing, preventing and treating adhesion with the use of ccr8 inhibitor Download PDF

Info

Publication number
WO2006059637A1
WO2006059637A1 PCT/JP2005/021980 JP2005021980W WO2006059637A1 WO 2006059637 A1 WO2006059637 A1 WO 2006059637A1 JP 2005021980 W JP2005021980 W JP 2005021980W WO 2006059637 A1 WO2006059637 A1 WO 2006059637A1
Authority
WO
WIPO (PCT)
Prior art keywords
ccr8
ccl1
adhesion
cells
ligand
Prior art date
Application number
PCT/JP2005/021980
Other languages
French (fr)
Japanese (ja)
Inventor
Taeko Dohi
Akiyoshi Hoshino
Kenji Yamamoto
Yuki Kawamura
Original Assignee
Japan Health Sciences Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Health Sciences Foundation filed Critical Japan Health Sciences Foundation
Publication of WO2006059637A1 publication Critical patent/WO2006059637A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5055—Cells of the immune system involving macrophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for diagnosing, preventing and treating adhesions, particularly peritoneal adhesion after intra-abdominal organ surgery, and an agent for preventing and treating adhesion diagnosis.
  • CCR8 is one of the chemokine receptors called ChemRl, CY6, CKR-L1 or TER1, and its gene exists in human chromosome 3p21-24. It is a protein-coupled 7-transmembrane CC chemokine receptor (Rucker et al., J Virol 1997, 71: 8999-9007) o CCR8 is expressed in the spleen, thymus, NK cells, monocytes It is also found in CD4 + T cells, and it is expressed steadily in peripheral blood leukocytes.
  • CCR8 is known as the co-receptor of many strains of human immunodeficiency virus-1 (HIV-1), M-oriented HIV-1, T-directed HIV-1, syncytium-directed HIV-1, brain cell-oriented It is considered to be one of the infection receptors for sex HIV-1.
  • HIV-1 human immunodeficiency virus-1
  • An endogenous ligand with high affinity for CCR8 is CCL I-3 09) (Tiffany et al., J Exp Med 1997, 186: 165-170; Roos et al., J Biol
  • TARC thymus and activation-regulated cytokine
  • MIP-1 18 macrophage inflammatory '-protein-1 beta
  • T cells In T cells, it is slightly expressed in the CD4 + CD8 + subset and is strongly expressed in CD4 + cells, but expression is lost in CD8 + cells.
  • CD4 + cells CCR8 expression increases upon Th2 cell activation (D'Ambrosio et al., J Immunol 1998, 161: 5111-511 5), and the ligand CCLl is a potent chemochemistry for Th2 cells. Acts as an attractant.
  • VMIP-I one of the chemokine homologs encoded by force-positive sarcoma virus, was found to be a high-affinity agonist of CCR 8 (Endres et al., J Exp Med 1999, 189: 1993-1998).
  • vMIP-1 also inhibits steroid-induced apoptosis in mouse lymphoma cells (Louahed et al "Eur J Immunol 2003, 33: 494-501), whereas it was encoded by human herpesvirus 8 MC148, encoded by vMIP-II and infectious mollusc virus, acts as an antagonist (Dairaghi et al., J Biol Chem 1999, 2 74: 21569-21574; Luttichau et al., J Exp Med 2000 , 191: 171-180), vMIP-II promotes Th2 site force-in secretion by nasal administration together with mouse-human CCL and also acts as an immune adjuvant (Singh et al "J Immunol 2004, 173: 5509-5516). Th2 cells secrete MDC and CCLl in a STA T6-dependent manner, facilitating further recruitment of Th2 cells (Zhang et al., J Immunol 2000,
  • human peripheral blood CD4 + CD25 + cells are said to have regulatory functions, but are characterized by the expression of CCR4 and CCR8, and are thought to be involved in the local migration of inhibitory T cells ( Ie Hem et al., J Exp Med 2001, 194: 847-853).
  • CD4 + CD25 + cells in human thymus inhibit CD4 + CD25—cell proliferation by allogeneic stimulation, but all contain CCR8 together with tumor necrosis factor type 2 receptor, cytoplasmic lymphocyte antigen— (CTLA) 4 It is expressed and responds to CCL1 secreted from thymic macrophages or epithelial cells (Annunziato et al., J Exp Med 2002, 196: 379-387).
  • CTL cytoplasmic lymphocyte antigen 4
  • CCR8 is expressed in microglial cells of the central nervous system by TNF stimulation, and CCR8 is involved in rapid-onset encephalitis by inducing the autoimmune encephalitis model in CCR8 knockout mice.
  • CCR8 is a monocyte
  • HAVECs human umbilical vein endothelial cells
  • VSMC vascular smooth muscle cells
  • CCR8 is expressed in peritoneal macrophages
  • CCR8 knockout mice are resistant to peritonitis-induced sepsis (cecal ligation nad pucture, CLP)
  • CCR8-deficient macrophages are more bactericidal and eliminate bacteria It has been reported that the ability is high (Akihiro Matsukawa, 24th Japanese Society of Inflammation and Regenerative Medicine, Kyoto, 2003, Nov. 26-27).
  • Non-patent literature l Becker et al, J. Am Coll Surg 1996, 183: 297-306
  • Non-Patent Document 2 Rucker et al "J Virol 1997, 71: 8999-9007
  • Non-Patent Document 3 Tiffany et al., J Exp Med 1997, 186: 165-170
  • Non-Patent Document 4 Roos et al., J Biol Chem 1997, 272: 17251-17254
  • Non-Patent Document 5 Horuk et al, J Biol Chem 1998, 273: 386-391
  • Non-Patent Document 6 D'Ambrosio et al., J Immunol 1998, 161: 5111-5115
  • Non-Patent Document 7 Endres et al., J Exp Med 1999, 189: 1993-1998
  • Non-Patent Document 8 Louahed et al., Eur J Immunol 2003, 33: 494-501
  • Non-patent literature 9 Daiairaghi et al., J Biol Chem 1999, 274: 21569-21574
  • Non-patent literature 10 Luttichau et al., J Exp Med 2000, 191: 171-180
  • Non-Patent Document ll Singh et al "J Immunol 2004, 173: 5509-5516
  • Non-Patent Document 12 Zhang et al "J Immunol 2000, 165: 10-14
  • Non-Patent Document 13 Chensue et al., J Exp Med 2001, 193: 573-584
  • Non-Patent Document 14 Chung et al., J Immunol 2003, 170: 581-587
  • Non-Patent Document 15 Goya et al., J Immunol 2003, 170: 2138-2146
  • Non-Patent Document 16 Bishop et al., J Immunol 2003, 170: 4810-4817
  • Non-Patent Document 17 Iellem et al "J Exp Med 2001, 194: 847-853
  • Non-Patent Document 18 Annunziato et al "J Exp Med 2002, 196: 379-387
  • Non-Patent Document 19 Murphy et al., J Immunol 2002, 169: 7054-7062
  • Non-Patent Document 20 Trebst et al., Am J Pathol 2003, 162: 427-438
  • Non-Patent Document 21 Haque et al., Blood 2004, 103: 1296-1304
  • Non-Patent Document 22 Akihiro Matsukawa, 24th Japan Inflammation 'Regenerative Medicine Society, Kyoto, 2003, Nov.26-2
  • An object of the present invention is to provide a drug for diagnosing, preventing and treating adhesions and a method for diagnosing, preventing and treating.
  • the present inventors have found that CCR8 and its ligand CCL1 are involved in the formation of adhesions, and that the formation of adhesions can be suppressed by administering an anti-CCL1 neutralizing antibody.
  • the present invention provides an adhesion comprising a CCR8 inhibitor as an active ingredient.
  • Pharmaceutical compositions for prevention or treatment are provided.
  • the CCR8 inhibitor comprises an antibody that binds to CCR8, a neutralizing antibody to CCR8 ligand CCL1, an aptamer to CCR8 and CCL 1, an antagonist of CCR8, and an antisense oligonucleotide to CCR8 and CCL1 gene and Selected from the group consisting of siRNA.
  • the present invention provides an adhesion detection method comprising detecting CCR8 or CCL1 gene expression in a test cell.
  • the present invention provides a method for detecting adhesions characterized by detecting the abundance of CCR8 or CCL1 in a test cell.
  • the present invention provides a diagnostic kit for adhesions comprising an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CCR8 or CCL1.
  • the present invention provides an adhesion diagnostic kit comprising an antibody or fragment thereof that binds to CCR8 or CCL1.
  • the present invention relates to a method for testing whether or not a test substance has an effect of preventing or treating adhesions, wherein the test substance inhibits binding between CCR8 and a ligand.
  • a method comprising measuring the ability to
  • the present invention provides a method for assaying whether a test substance has an effect of preventing or treating adhesions, and measuring the ability to inhibit the test substance force SCCR8 or CCL1 expression. To provide a method comprising:
  • the present invention provides an effect of preventing or treating adhesions comprising CCR8 or CCL1, or an oligonucleotide or polynucleotide encoding CCR8 or CCL1, or a cell expressing CCR8.
  • a kit for screening substances possessed is provided.
  • the present invention provides a method for testing whether a test substance has an effect of preventing or treating adhesions, wherein the test substance comprises peritoneal mesothelial cells and peritoneal macaques.
  • a method comprising measuring the ability of a mouth phage to inhibit the formation of aggregates is provided.
  • the present invention provides a method for measuring peritoneal mesothelial cells and peritoneal macrophages for the purpose of measuring the formation of aggregates between peritoneal mesothelial cells and peritoneal macrophages.
  • the pharmaceutical composition of the present invention is useful for preventing and treating adhesions, particularly peritoneal adhesions after intra-abdominal organ surgery. Further, according to the screening method of the present invention, a substance useful for preventing and treating adhesions can be screened.
  • FIG. 1 shows the difference in the expression of chemokine receptors between plaque peritoneal macrophages and unstimulated peritoneal macrophages (in two experiments, the results of each experiment are shown). Show).
  • Figure 2 shows the increase in CCR8 receptor mRNA expression in cultured peritoneal macrophages ( ⁇ ) by CCL8, an endogenous ligand of CCR8, and various immune / inflammatory stimulating factors, with a ratio of 1 before stimulation. (Average value). In addition, these stimulating factors have no or no effect on cultured bone marrow macrophages (BM ⁇ ).
  • BM ⁇ cultured bone marrow macrophages
  • FIG. 3 shows the increase in CCL1 mRNA expression in cultured peritoneal macrophages (PM ⁇ ) by CCL1 and various immunity / inflammatory stimulating factors, expressed as a ratio (average value) with 1 before stimulation.
  • FIG. 4 is a representative example of an image showing increased expression of CCR8 receptor in cultured peritoneal macrophages by CCL1 and LPS.
  • FIG. 5 is a representative example of an image showing increased expression of CD49d in cultured peritoneal macrophages by CCL1.
  • FIG. 6 shows the CCL1 mRNA expression level of cultured peritoneal mesothelial cells by CCL1 and various immunity / inflammatory stimulating factors in a ratio (mean value + standard deviation) with 1 being before stimulation.
  • the expression level of CC LI mRNA was compared with that of CCR8 and other genes.
  • FIG. 7 is a representative example of an image showing aggregates in co-cultured mesothelial cells and peritoneal macrophages.
  • CCL1 showed the effect of generating large agglomerates.
  • LPS had a weak aggregate formation effect up to 6 hours after stimulation.
  • FIG. 8 shows the aggregate area per visual field 24 hours after application of the stimulating factor in mesothelial cells and peritoneal macrophages co-cultured (mean value + standard deviation). CCL1 and LP s showed the effect of producing large agglomerates.
  • FIG. 9 shows that anti-CCL1 neutralizing antibodies suppressed the formation of aggregates after 24 hours of CCL1 stimulation in co-cultured mesothelial cells and peritoneal macrophages (average of aggregate area per field of view) Value + standard deviation).
  • FIG. 10 is an image showing that anti-CCL1 neutralizing antibody inhibited the accumulation of peritoneal macrophages in plaques 1 day after TNBS administration.
  • FIG. 11 shows that anti-CCL1 neutralizing antibody suppressed adhesion at the perforation of the large intestine occurring 4 days after TNBS administration (adhesion score of each mouse, and average value of each group).
  • FIG. 12 shows that anti-CCL1 neutralizing antibody suppressed adhesion of the peritoneal ischemia button site that occurred 7 days after the mouse laparotomy model (adhesion score of each mouse and the average value of each group). .
  • the pharmaceutical composition of the present invention comprises a CCR8 inhibitor as an active ingredient.
  • CCR8 is a protein-coupled seven-transmembrane CC chemokine receptor with 355 amino acid strength (Rucker et al., J Virol 1997, 71: 8999-9007), and the base sequence of the gene is known ( (GenBank: U45983, BC069067)
  • CCR8 inhibitors include CCR8 antagonists, CCR8 antagonists, aptamers to CCR8, and antisense oligonucleotides and siRNAs for the CCR8 gene.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • CCR8 endogenous ⁇ Gore - a strike CCL1 is a secreted protein also 96 amino acids forces the base sequence of the gene is Ru known der (GenBank: M57502, M57506) suppressing the activation of o CCR8 is This can be realized by inhibiting the binding of CCL1 to CCR8 or suppressing the expression of CCL1.
  • Substances that can inhibit CCR8 by this mechanism include neutralizing antibodies against CCL1, Abutaman against CCL1, and antisense oligonucleotides and siRNA against CCL1 gene.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the polyclonal antibody that binds to CCR8 of the present invention is obtained by immunizing an animal using CCR8 as a sensitizing antigen and collecting antiserum according to a method well known in the art. It can be obtained by taking.
  • the monoclonal antibody that binds to CCR8 of the present invention can be obtained by immunizing an animal using CCR8 as a sensitizing antigen according to a method well known in the art, taking out the resulting immune cells and fusing them with myeloma cells. It can be obtained by cloning a hyperpridoma that produces lysozyme and culturing this hyperidoma.
  • the monoclonal antibody of the present invention includes a gene recombinant antibody, a chimeric antibody, a CDR graft produced by a transformant transformed with an expression vector containing an antibody gene in addition to an antibody produced by a hyperidoma. Antibodies, and fragments of these antibodies are included.
  • Recombinant antibodies are obtained by cloning cDNA encoding an antibody from a hybridoma that produces a monoclonal antibody that binds to CCR8, inserting it into an expression vector, and transforming animal cells, plant cells, etc. This transformant can be produced by culturing.
  • a chimeric antibody is an antibody in which the heavy chain and light chain variable regions of an antibody derived from one animal and the heavy and light chain constant region forces of an antibody derived from another animal are also comprised. Examples of antibody fragments that can bind to CCR8 include Fab, F (ab ′) 2, Fab ′, scFv, and diabody.
  • the antibody against CCL1 of the present invention can be obtained according to the same well-known method in the art as the production of anti-CCR8 antibody using CCL1 as a sensitizing antigen. Whether the obtained antibody is a neutralizing antibody can be assayed by assaying whether it suppresses the physiological action of CCL1. Examples include, but are not limited to, binding of CCL1 to CCR8, migration of CCR8-expressing cells by CCL1 or increased expression of genes sensitive to intracellular Ca ++ increase or CCL1 stimulation, or CCL Measurement of the formation of aggregates of peritoneal mesothelial cells and peritoneal macrophages by 1 is mentioned.
  • Abutama is a nucleic acid ligand having a length of several tens of bases capable of binding to a protein.
  • Abutaman against various proteins has been identified.
  • Abutaman against vascular endothelial growth factor has been used for the treatment of age-related macular degeneration.
  • Abutama can be obtained by preparing a library having various nucleic acid chain strengths and selecting a nucleic acid chain that can bind to a target protein from the library.
  • the SELEX method described in Patent US5270163 is widely known.
  • vMIP-IIv viral macrophage inflammatory protein 2
  • vMCC-I viral macrophage inflammatory protein 2
  • MC148 WO2004 / 058709
  • Etc. are known. It can also be obtained by screening a substance that inhibits the binding between CCR8 and the ligand CCL1. Such antagonist screening methods are well known in the art.
  • Antisense oligonucleotides to CCR8 refer to mRNA that encodes CCR8. It is an oligonucleotide that can bind differently and inhibit its translation. Antisense oligonucleotides include antisense RNA and antisense DNA. siRNA is a double-stranded RNA that can cause RNA interference. RNA and DNA may be chemically modified. Various nucleic acid modifications are known to enhance the stability or cellular uptake of antisense oligonucleotides and siRNAs, any of which can be used in the present invention.
  • an antisense oligonucleotide or siRNA against CCR8 gene is used as a therapeutic agent for preventing adhesions, for example, the ability to administer antisense oligonucleotide or siRNA directly to a subject, or expression of an antisense oligonucleotide or siRNA Vectors can be prepared and these expression vectors can be administered. Methods for introducing anti-sense oligonucleotides, siRNAs or vectors expressing them are well known in the art.
  • composition of the present invention can be formulated by methods known to those skilled in the art.
  • a pharmaceutically acceptable carrier or medium specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It can be formulated by appropriately combining with agents, binders and the like.
  • the composition of the present invention is mixed with a pharmaceutically acceptable carrier well known in the art, thereby allowing tablets, pills, dragees, capsules, liquids, gels, mouthpieces.
  • a pharmaceutically acceptable carrier well known in the art, thereby allowing tablets, pills, dragees, capsules, liquids, gels, mouthpieces.
  • the carrier those conventionally known in the technical field can be widely used. For example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, kynic acid and the like can be used.
  • Forming agents water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polypyrrolidone, etc., dry starch, sodium alginate, agar powder , Laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc .; disintegration inhibitors such as sucrose, stearic cocoa butter, hydrogenated oil, etc.
  • the tablets can be made into tablets with ordinary coatings, if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets.
  • the pharmaceutical composition of the present invention can be formulated according to usual pharmaceutical practice using a pharmaceutically acceptable vehicle well known in the art.
  • Water-soluble vehicles for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-manntol, sodium chloride.
  • a suitable solubilizer such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80 (TM), HCO-50. May be.
  • oil-based vehicles examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer.
  • buffering agents such as a phosphate buffer solution, sodium acetate buffer solution, a soothing agent, for example, hydrochloric acid pro-power-in, a stabilizer, for example, benzyl alcohol, phenol, antioxidant.
  • the prepared injection is usually filled in a suitable ampoule.
  • Suitable administration routes for the pharmaceutical composition of the present invention include, but are not limited to, oral, rectal, transmucosal, or enteral administration, or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, Intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection is included.
  • the administration route and administration method can be appropriately selected depending on the age and symptoms of the patient.
  • a particularly preferred route and method of administration of the pharmaceutical composition of the present invention are as follows: a preparation containing the pharmaceutical composition of the present invention, preferably a sustained-release preparation, is administered during or after surgery to the affected area or the vicinity thereof, for example, intraperitoneally. It is to be.
  • a particularly suitable method of administration is to bind the pharmaceutical composition of the present invention to a polymer film and leave it in the affected area after surgery.
  • the polymer film for example, Seprafilm (registered trademark), Mesophor (registered trademark) and the like used for prevention of adhesion after surgery can be used.
  • the dosage of the pharmaceutical composition of the present invention can be selected, for example, in the range of 0.0001 mg to lOOOmg per kg of body weight per time. Or, for example, 0.001 per patient
  • the ability to select a dose in the range of ⁇ 100,000 mg / body is not necessarily limited to these values.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art.
  • the present invention features a method for diagnosing adhesions by detecting the ability to detect CCR8 gene expression in a test cell or the amount of CCR8 present in a test cell. And According to the present invention, it is possible to diagnose whether or not adhesion has occurred using as an index the abundance of CCR8 or the expression level of the CCR8 gene in a tissue suspected of causing adhesion.
  • the present invention features a method of diagnosing adhesions by detecting the abundance of CCL1 in a test cell. According to the present invention, whether or not adhesion has occurred can be diagnosed by using the abundance of CCL1 in a tissue suspected of having adhesion or a body fluid such as ascites as an index.
  • Methods for measuring the abundance of CCR8 or CCL1 or the expression level of CCR8 or CCL1 gene include, for example, the ability to collect ascites from a drain tube installed to drain ascites after surgery or a small amount of ascites Collecting and measuring the expression level of CCR8 or CCL1 in macrophages or mesothelial cells. Another example is measuring the macrophage migration ability collected by CCR8 agonists such as CCL1.
  • an anti-CCR8 antibody or an anti-CCL1 antibody, an aptamer for CCR8 or an aptamer for CCL1, or a CCR8 ligand is labeled, and this is administered to a patient and measured by image analysis.
  • labels include radioisotopes, stable isotopes, magnetic substances, and foaming agents.
  • Image analysis methods include positron emission tomography (PET), single photon emission computer tomography (SPECT), and nuclear magnetic resonance. Examples include diagnostic imaging (MRI) or ultrasonic diagnostic imaging.
  • the expression level of the CCR8 gene can be quantified by measuring the abundance of mRNA encoding CCR8 in the specimen.
  • the abundance of mRNA encoding CCR8 can be measured using a gene analysis technique well known in the art using a probe or primer designed based on a known CCR8 gene sequence. So Examples of such techniques include Northern blotting, DNA microarray, RT-PC R, and the like.
  • the expression level of CCL1 gene can be measured by the same technique as that for CCR8 gene.
  • the abundance of CCR8 or CCL1 in a specimen can be measured using protein analysis techniques well known in the art. Examples of such techniques include Western blotting, immunoprecipitation, ELISA, and tissue immunostaining using an antibody or fragment thereof that binds to CCR8 or CCL1.
  • the invention features a diagnostic kit for adhesions.
  • the adhesion diagnosis kit of the present invention is an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CCR8 of a subject, or a subject as a reagent for measuring the expression level of CCR8 gene in a sample collected from the subject. Antibody or a fragment thereof that binds to CCR8.
  • the adhesion diagnosis kit of the present invention further comprises an oligonucleotide or a polynucleotide that can hybridize to a subject's CCL1-encoding nucleic acid, or a reagent for measuring the expression level of the CCR8 gene in a sample collected from a subject, or It may contain an antibody or a fragment thereof that binds to CCL1 of the subject.
  • the adhesion diagnosis kit of the present invention may further comprise a reagent for quantifying the expression product of CCR8 or CCL1, or the CCR8 gene or CCL1 gene, and a method of use.
  • the present invention is characterized by a method for screening a substance useful as a component of an agent for preventing or treating adhesions.
  • the test substance can prevent adhesion or prevent adhesion by measuring the ability of the test substance to inhibit the binding between CCR8 and a ligand, or the test substance can inhibit the expression of CCR8. It is possible to test whether the force has a therapeutic effect. Inhibition of CCR8 gene expression includes inhibition of both transcription and translation of the CCR8 gene.
  • the screening method of the present invention measures the inhibition of binding between CCR8 and its ligand, for example, CCL1, by measuring the amount of transcription and Z or translation of CCR8 gene in the presence and absence of a candidate substance.
  • the present invention provides a method for screening a substance useful as a component of an adhesion prevention or treatment agent, wherein peritoneal mesothelial cells and peritoneal macrophages are mixed and cultured, and these cells are subjected to, for example,
  • an in vitro test method characterized by a method using a reaction in which these cells form an aggregate by giving a stimulus such as holding CCL1 in a culture dish.
  • the preventive or therapeutic effect of adhesion can be assayed depending on whether the test substance suppresses this aggregate formation reaction.
  • the effect of preventing or treating adhesions can be assayed based on whether or not the test substance has the ability to reduce aggregates formed.
  • Digested tissues are transferred to collagen-coated Falcon culture dishes (Becton Dickinson, Franklin Lakes, NJ, USA), 20% fetal calf serum (FCS), 5 ⁇ g / mL mouse recombinant epidermal growth factor, and penicillin streptomycin Incubate with DMEM containing amphotericin B. Incubate 5 to 6 times and incubate for 14 days.
  • peritoneal macrophages of animals are cultured according to a general procedure for culturing macrophages collected from animals.
  • a more specific example is the following procedure.
  • the mouse peritoneal fluid is collected, and the cells contained in it are incubated in DMEM medium containing 2% FCS for 45 minutes, and the adhered cells are collected to obtain a peritoneal macrophage.
  • these cells may be labeled.
  • peritoneal macrophages can be labeled with a quantum dot showing fluorescent coloration by the method of Hoshino et al. (Biochem Biophys Res Commun 2004, 314: 46-53).
  • Peritoneal macrophages are co-cultured with mesothelial cells to form cell clusters.
  • peritoneal macrophages are transferred to 10% FCS-DMEM medium and added to a culture dish in which monolayer culture of mesothelial cells is performed by spotting. Next, this peritoneal phage and mesothelial cells are mixed and cultured, and a reaction is formed in the culture dish by applying a stimulus to the culture dish.
  • An example of the specific method is the addition of CCL1 as a stimulant.
  • the concentration of CCL1 is preferably 0.1 ng / mL to 10 ng / mL, and more preferably 5 ng / mL.
  • a stimulating substance that activates the CCL1 / CCR8 receptor system siRNAring may be added, or a gene having the same function may be introduced.
  • stimulating substances include lipopolysaccharides, bacteria or peptide glycans derived from bacteria, oligodeoxynucleotides, and flammable substances such as cytodynamic force in.
  • a test substance can be added to a culture dish in which peritoneal macrophages and mesothelial cells are mixed and cultured before, simultaneously with, after stimulation, and after formation of a cell mass.
  • Cell mass can be observed and measured using a microscope. It is also possible to use labeled cell labels for easy observation and measurement.
  • An image processing apparatus may be used for observation and measurement. It is also possible to remove the cell mass with a dropper or tweezers and measure the weight instead of the volume. Compare the degree of cell clump formation in the culture dish to which the test substance has been added without adding the test substance. ⁇ Compare the degree of cell clump formation before and after the addition of the stimulating substance. Thus, the effect of the test substance can be determined.
  • the present invention features a kit for screening a substance useful as a component of an agent for preventing or treating adhesions.
  • the kit of the present invention comprises CCR8, or an oligonucleotide or polynucleotide encoding CCR8, or a cell expressing CCR8, or CCL1, or an oligonucleotide or polynucleotide encoding CCL1, and is used for these screening.
  • the kit can further include the reagents and solutions required for the assembly and instructions for use.
  • the kit of the present invention is intended to measure that peritoneal mesothelial cells and peritoneal macrophages form aggregates, and includes research materials suitable for carrying out this measurement.
  • kit of the present invention may further contain reagents and solutions necessary for the assembly and directions for use.
  • research materials suitable for carrying out this measurement include peritoneal phages or peritoneal mesothelial cells, ie organs such as the digestive tract, omentum, peritoneum or ascites.
  • the substance found by the screening method of the present invention is useful as an agent for preventing or treating adhesions. It is considered that these adhesion prevention and treatment agents can exert the effect of preventing or treating adhesions by inhibiting the expression of CCR8 gene or CCL1 gene, or by inhibiting the function of CCR8.
  • C57BL / 6 mouse peritoneal macrophages are labeled with quantum dots (Q dot) by conventional methods (Hoshino et al., Biochem Biophys Res Commun 2004, 314: 46-53) and transplanted intraperitoneally into allogeneic mice
  • Q dot quantum dots
  • TNBS enteritis he induced enteritis due to high dose (loogZg body weight) tri-oral benzenesulfonic acid enema.
  • the large intestine site due to full-thickness ulcer was collected, frozen sections were prepared, and macrophages labeled with Qdot were observed with a fluorescence microscope. Further, 24 hours after induction of TNBS enteritis, the large intestine was taken out, cut longitudinally, and washed with phosphate buffered saline (PBS). The cells accumulated on the serosa surface and the plaque portion formed on the ulcer perforation were removed. The colon specimen was treated with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 30 minutes to remove epithelial cells.
  • EDTA ethylenediaminetetraacetic acid
  • FIG. 1 shows changes in chemokine receptor expression.
  • the X-axis shows the ratio to the expression in unstimulated peritoneal macrophages
  • Expl and 2 show the results of experiments conducted independently.
  • peritoneal macrophages administered to the abdominal cavity were accumulated in the plaque, which was the pre-adhesion stage, but did not migrate into the colon wall.
  • peritoneal macrophages accumulated in plaques showed less expression of other chemokine receptors with high CCR8 expression.
  • peritoneal macrophages accumulated in the plaque in Example 1 showed an activity that increased CCR8 expression by stimulation such as inflammation, in this example, CCL1, an endogenous ligand of CCR8, was cultured peritoneally. The effect was examined by applying to macrophages. Peritoneal macrophages from which C57BL / 6 mouse force was also collected and bone marrow macrophages in which bone marrow was separated using macrophage colony stimulating factor (M-CSF) by a conventional method were cultured.
  • M-CSF macrophage colony stimulating factor
  • CCL8 ligand CCL1 or a control Lipopolysaccharide (LPS; Sigma-Aldrich) derived from Escherichia coli 055B5 (100 ng / mL) or peptidoglycan derived from Staphylococcus aureus (PGN; Fluk a, Buchs ⁇ Switzerland) (1 ⁇ g / mL) or an unmethylated cytosine-guanine sequence oligodeoxynucleotide (CpG) (Takara Bio, Tokyo, Japan) (5)-TCCATGACGTTCCTGA TGCT-3 '(SEQ ID NO: 1) (1) ⁇ g / mL), is a site force-in that induces inflammation, tumor necrosis factor-a (TNF-a) (1 g / mL) or interleukin-1 ⁇ (IL-1 j8) (10 ⁇ g / mL) was applied.
  • LPS Lipopolysaccharide
  • FIG. 2 shows the relative values of the CCR8 mRNA expression level 2 hours after application of each stimulation factor, with the mRNA expression level before application of the stimulation being 1.
  • CCL1 10 ng / mL
  • the effects of CCL1 (10 ng / mL) are LPS (1 00 ng / mL), PGN (1 ⁇ g / mL), CpG (1 g / mL), TNF— ⁇ (1 ⁇ g / mL), IL- 1 ⁇ (10 ⁇ g / mL) was more potent than the effect of increasing CCR8 mRNA expression.
  • CCL1 and other stimulating factors were also not effective in significantly increasing the expression level of CCR8 mRNA.
  • FIG. 3 shows the relative value of the CCL1 mRNA expression level 2 hours after application of each stimulation factor, with the mRNA expression level before application of the stimulation being 1.
  • CCL1 (10 ng / mL) stimulation increased CCL1 mRNA expression in peritoneal macrophages ( ⁇ ).
  • the effect of CCL1 (10 ng / mL) stimulation on CCL1 expression was weaker than that of LPS (100 ng / mL), which significantly increased CCL1 expression, and PGN (1 g / mL) and CpG (1 g / mL).
  • CCR8 was expressed on a part of the cell surface. Also abdominal cavity 48 hours after stimulation with CCL1 (10 ng / mL) in macrophages, immunohistochemical staining using an antibody against CD-I lb and an anti-CD49d antibody revealed that CD49d expression was increased. It was revealed.
  • Figure 5 shows a typical example. The white line shows the shape of the cells detected by anti-CDllb antibody staining, and shows the white region force CD49d positive staining region.
  • CD49d is a protein that binds to VCAM1 expressed in peritoneal mesothelial cells, which are cells that cover the peritoneum and organs in the peritoneal cavity. Therefore, it was shown that the result force CCL1ZCCR8 system of this example may be involved in adhesion between peritoneal macrophages and mesothelial cells during adhesion formation.
  • Example 2 it was shown that the CCL1 / CCR8 system may be involved in the adhesion between peritoneal macrophages and peritoneal mesothelial cells. Therefore, we examined CCL1 production by peritoneal mesothelial cells.
  • the purity of the cells was confirmed by immunohistochemical staining using an anti-mouse pancytokeratin antibody.
  • a 24-well culture dish with collagen-coated mesothelial cells Transfer and culture until confluent. Add each stimulating factor and incubate for 6 hours, extract mRNA from cells, and use quantitative RT-PCR to determine mRNA expression levels of CCR8, CCL1, IL-1 ⁇ , IL-6, and TNF-a. It was measured.
  • FIG. 6 shows the relative values of the CCL1 mRNA expression level after application of each stimulation factor, with the unstimulated mRNA expression level being 1.
  • Mesothelial cells significantly enhance CCL1 mRNA expression by stimulation with LPS (100 ng / mL), PGN (100 ng / mL), and TNF-a (1 g / mL)! ] The increase was selective for CC L1.
  • IL-1 ⁇ (10 ⁇ g / mL) stimulation showed an increase in CCL1 mRNA and TNF-a.
  • CpG (1 ⁇ g / mL) did not increase CCL1 mRNA expression.
  • CCL1 (5 ng / mL) stimulation significantly increased CCL1, IL-1 ⁇ , and CCR8 mRNA expression.
  • the released peritoneal macrophages accumulate at the site and bind to mesothelial cells to form adhesions.
  • adhesion can be diagnosed by detecting CCL1 production by mesothelial cells or CCL1 released to the outside of the cells.
  • Fig. 7 shows representative examples of Q dot fluorescence images 1 hour, 3 hours, and 6 hours after addition of peritoneal macrophage labeled with Q dot. In the absence of stimulation (Fig. 7a), agglutination of peritoneal macrophages hardly occurred.
  • CCL1 (10 ng / mL) was added to this mixed culture, aggregates increased, and aggregates exceeding 100 m in diameter were formed after 3 or 6 hours.
  • peritoneal macrophages or bone marrow macrophages labeled with Q dots were added to cultured mesothelial cells, and 24 hours after no stimulation or various concentrations of CCL1 stimulation were applied, the aggregate area in one visual field was calculated.
  • CCL1 increased the aggregate area of peritoneal macrophages (PM ⁇ ) and mesothelial cells. The effect was strongest at a concentration of 5 ng / mL.
  • bone marrow macrophages ( ⁇ ) and mesothelial cells were mixed and cultured instead of peritoneal macrophages, no significant increase in the aggregate area due to the application of CCL1 was observed.
  • LPS 100 ng / mL
  • LPS did not show a strong effect until 6 hours after stimulation, and was shown to significantly increase the aggregate area after 24 hours of force stimulation. This suggests that aggregates were formed as a result of the release of CCL1 from peritoneal macrophages or mesothelial cells by LPS stimulation.
  • a test was carried out to suppress the formation of agglomerates of peritoneal macrophages and mesothelial cells by stimulation with CCL1 with a rat anti-mouse CCL1 neutralizing monoclonal antibody (R & D Systems, Minneapolis, MN, USA).
  • FIG. 9 shows the aggregate area in one visual field 24 hours after application of CCL1 (5 ng / mL) and anti-CCL1 neutralizing antibody. Anti-CCL1 neutralizing antibody suppressed the formation of aggregates in a concentration-dependent manner.
  • mice were administered anti-CCL1 neutralizing antibody in a TNBS colitis model in which adhesion occurs.
  • TNBS enteritis was induced in the same manner as in Example 1.
  • mice administered with rat IgG instead of anti-CCL1 antibody were used. Tests were performed to analyze the image of peritoneal macrophage dynamics and to measure adhesion formation.
  • peritoneal macrophages (2.5 x 10 5 cells) labeled with Q dot were injected intraperitoneally the day before TNBS administration.
  • TNBS injection the large intestine was taken out, and the Q dot fluorescence image was superimposed on the visible light image using an image analyzer (Relion, Tokyo, Japan).
  • Figure 10a shows images of labeled peritoneal macrophage accumulation in the entire large intestine of each mouse for 3 patients in each group.
  • the bright area is the area where Q dot-labeled peritoneal macrophages are accumulated.
  • FIG. 10b shows a representative example of an image obtained by preparing a section of the large intestine where Q dots are strongly colored and observing it using a fluorescence microscope. The bright area is the area where Q dot-labeled abdominal cavity macrophages are accumulated. Lines indicate the shape of the large intestine and perforation. The arrow indicates the plaque on the perforated part.
  • peritoneal macrophages were accumulated in plaques in the perforated area where adhesion occurred later.
  • peritoneal macrophages labeled with Q dots and injected into the peritoneal cavity were scattered in the lamina intestinal and mucosa. From these results and the results of Examples 1 and 2, anti-CCL1 antibody showed that peritoneal macrophages acted to increase CCR8 receptor expression and inhibited peritoneal macrophage accumulation at the perforation site. Indicated.
  • Adhesion scores were as follows: no adhesion (0 points), thin membrane adhesion S1 location (1 point), thin thin film adhesions 2 locations (2 points), 1 location thick There are adhesions (3 points), thick adhesion with the bottom surface or thick adhesions at 2 or more locations (4 points), very thick adhesion with angiogenesis, or thickness with 2 or more bottom surfaces, adhesion Yes (5 points).
  • FIG. 12 shows the adhesion score of each mouse as a square, and the average value of each group as a cross symbol.
  • adhesion occurred in all 9 patients, with an average adhesion score of 2.89 points.
  • the adhesion score averaged 0.78 points, indicating that the anti-CCL1 antibody administration was effective in suppressing adhesions.
  • the pharmaceutical composition of the present invention is useful for diagnosing, preventing and treating adhesions.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Surgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

A medicinal composition for preventing or treating adhesion which contains a CCR8 inhibitor as the active ingredient. The CCR8 inhibitor is preferably a member selected from the group consisting of an antibody binding to CCR8, a neutralizing antibody against a CCR8 ligand CCL1, aptamers to CCR8 and CCL1, an antagonist to CCR8, antisense oligonucleotides to CCR8 and CCL1 genes and siRNAs thereto. A method of diagnosing adhesion which comprises detecting the expression of a CCR8 gene or a CCL1 gene in test cells or detecting the content of CCR8 or CCL1 in the test cells. A method of screening a substance which is useful in preventing and treating adhesion.

Description

明 細 書  Specification
CCR8阻害剤を用いる癒着の診断、予防および治療剤  Diagnosis, prevention and treatment of adhesions using CCR8 inhibitors
技術分野  Technical field
[0001] 本発明は、癒着、特に腹腔内臓器手術後の腹膜癒着を診断、予防および治療する 方法、ならびに癒着の診断 '予防および治療剤に関する。  [0001] The present invention relates to a method for diagnosing, preventing and treating adhesions, particularly peritoneal adhesion after intra-abdominal organ surgery, and an agent for preventing and treating adhesion diagnosis.
背景技術  Background art
[0002] 腹腔内臓器手術後や消化管炎症における腹膜の炎症及び癒着は、不妊、再手術 の原因となるなど、長期にわたる深刻な後遺症を残すだけでなぐ腹痛、便秘などの ために患者の QOLを著しく損なう。  [0002] Peritoneal inflammation and adhesion after intra-abdominal organ surgery and gastrointestinal inflammation may cause infertility, re-operation, and so on. Is significantly impaired.
[0003] その防止のために、現在とられている措置は、組織面との間に一時的物理的隔離 を設けることである。ジェンザィム社が開発した生体吸収性の膜セプラフイルムを手術 部位に貼付する。この方法で術後癒着は約半数に減少したが（Becker et al, J. Am Coll Surg 1996, 183: 297-306)、まだ、半数に術後の癒着が起こっている。 また、癒着を診断する方法はこれまでに確立されて 、な 、。  [0003] To prevent this, the current action is to provide temporary physical isolation from the organizational surface. A bioabsorbable membrane seprafilm developed by Genzym is applied to the surgical site. Although this method reduced postoperative adhesion by about half (Becker et al, J. Am Coll Surg 1996, 183: 297-306), postoperative adhesions still occur in half. In addition, a method for diagnosing adhesions has been established so far.
[0004] CCR8は ChemRl、 CY6、 CKR-L1または TER1と呼ばれていたケモカイン受容体の 一つで、ヒトクロモゾーム 3p21-24に遺伝子が存在する。 355アミノ酸力もなり、蛋白共 役型 7回膜貫通型の CCケモカインレセプターである（Rucker et al., J Virol 1997 , 71:8999-9007) o CCR8の発現は脾臓、胸腺、 NK細胞、単球及び CD4+T細胞にみ られ、末梢血白血球でも低いながら定常的に発現している。 CCR8はヒト免疫不全ゥ ィルス- 1 (HIV- 1)の多くの株のコレセプターとして知られており M指向性 HIV-1, T 指向性 HIV-1, シンシチウム指向性 HIV-1、脳細胞指向性 HIV-1の感染受容体の一 つであるとされている。 CCR8に対して高親和性を持つ内因性リガンドには CCL I-3 09)があり（Tiffany et al., J Exp Med 1997, 186:165-170; Roos et al., J Biol [0004] CCR8 is one of the chemokine receptors called ChemRl, CY6, CKR-L1 or TER1, and its gene exists in human chromosome 3p21-24. It is a protein-coupled 7-transmembrane CC chemokine receptor (Rucker et al., J Virol 1997, 71: 8999-9007) o CCR8 is expressed in the spleen, thymus, NK cells, monocytes It is also found in CD4 + T cells, and it is expressed steadily in peripheral blood leukocytes. CCR8 is known as the co-receptor of many strains of human immunodeficiency virus-1 (HIV-1), M-oriented HIV-1, T-directed HIV-1, syncytium-directed HIV-1, brain cell-oriented It is considered to be one of the infection receptors for sex HIV-1. An endogenous ligand with high affinity for CCR8 is CCL I-3 09) (Tiffany et al., J Exp Med 1997, 186: 165-170; Roos et al., J Biol
Chem 1997, 272:17251-17254)、 CCL1は HIV-1による細胞融合と感染の成立を 効率的に阻害することができた（Horuk et al., J Biol Chem 1998, 273:386-391 ；)。 TARC (thymus and activation-regulated cytokine)及びマクロファージ炎症'性 蛋白質- 1 ベータ (MIP-1 18 )も CCRと結合するという報告もあった力 結局否定された 。マウス CCR8はヒト CCR8と 71%のホモロジ一を示し、 T細胞、 B細胞、マクロファージな ど様々な細胞に発現して!/、る。 Chem 1997, 272: 17251-17254), and CCL1 was able to efficiently inhibit cell fusion and infection by HIV-1 (Horuk et al., J Biol Chem 1998, 273: 386-391;) . TARC (thymus and activation-regulated cytokine) and macrophage inflammatory '-protein-1 beta (MIP-1 18) were also reported to bind to CCR. . Mouse CCR8 shows 71% homology with human CCR8 and is expressed on various cells such as T cells, B cells, and macrophages!
[0005] T細胞の中では CD4+CD8+サブセットにわずかに発現しており、 CD4+細胞で強く発 現するが、 CD8+細胞では発現が失われる。 CD4+細胞のうち CCR8の発現は Th2細 胞の活性化に際して上昇し（D'Ambrosio et al.， J Immunol 1998, 161:5111-511 5)、リガンドの CCLlは活性ィ匕 Th2細胞に対する強力なケモアトラクタントとして作用す る。力ポジ肉腫ウィルスがコードするケモカインホモログの一つである vMIP-Iは、 CCR 8の高親和性ァゴニストであることが明らかになり、力ポジ肉腫内にみられる顕著な Th 2細胞浸潤のメカニズムとされている（Endres et al.， J Exp Med 1999, 189:1993 -1998)。 CCL-1, vMIP- 1はまたマウスリンパ腫細胞におけるステロイド誘導性アポト 一シスを阻害する（Louahed et al" Eur J Immunol 2003, 33:494- 501)。一方、 ヒトヘルぺスウィルス 8にコードされた vMIP-IIや、伝染性軟属種ウィルス由来のコード する MC148はアンタゴ-ストとして作用し（Dairaghi et al., J Biol Chem 1999, 2 74:21569-21574; Luttichau et al., J Exp Med 2000, 191:171— 180)、 vMIP— II はマウス ·ヒト CCLとともに点鼻投与により Th2サイト力イン分泌を促し免疫アジュバント の作用も持つ（Singh et al" J Immunol 2004, 173:5509- 5516)。 Th2細胞は STA T6依存性に MDCと、 CCLlを分泌し、さらなる Th2細胞のリクルートメントを促す (Zhan g et al., J Immunol 2000, 165:10—14)。  [0005] In T cells, it is slightly expressed in the CD4 + CD8 + subset and is strongly expressed in CD4 + cells, but expression is lost in CD8 + cells. Among CD4 + cells, CCR8 expression increases upon Th2 cell activation (D'Ambrosio et al., J Immunol 1998, 161: 5111-511 5), and the ligand CCLl is a potent chemochemistry for Th2 cells. Acts as an attractant. VMIP-I, one of the chemokine homologs encoded by force-positive sarcoma virus, was found to be a high-affinity agonist of CCR 8 (Endres et al., J Exp Med 1999, 189: 1993-1998). CCL-1, vMIP-1 also inhibits steroid-induced apoptosis in mouse lymphoma cells (Louahed et al "Eur J Immunol 2003, 33: 494-501), whereas it was encoded by human herpesvirus 8 MC148, encoded by vMIP-II and infectious mollusc virus, acts as an antagonist (Dairaghi et al., J Biol Chem 1999, 2 74: 21569-21574; Luttichau et al., J Exp Med 2000 , 191: 171-180), vMIP-II promotes Th2 site force-in secretion by nasal administration together with mouse-human CCL and also acts as an immune adjuvant (Singh et al "J Immunol 2004, 173: 5509-5516). Th2 cells secrete MDC and CCLl in a STA T6-dependent manner, facilitating further recruitment of Th2 cells (Zhang et al., J Immunol 2000, 165: 10-14).
[0006] CCR8ノックアウトマウスを用いた実験では、始めにマンソン住血吸虫可溶性卵抗原 による肉芽腫形成、卵白アルブミン及びゴキブリ抗原によるアレルギー性気道炎症が V、ずれも減弱しており、 Th2型サイト力インの産生不全と好酸球浸潤の減少が特徴で あつたという報告があった（Chensue et al., J Exp Med 2001, 193:573- 584)。こ のグループによると、ノックアウトマウスでは Th2細胞の発達には欠陥はないが、好酸 球の動員が十分でないとされた。し力しながら、その後に 2つのグループからこれを 否定する結果が報告され CCL1の発現上昇がアレルギー性気道炎症モデルでみら れるものの、 CCR8ノックアウトマウスでは炎症'好酸球浸潤に影響はなぐ CCL1中和 抗体の投与にも反応しなかったとしている（Chung et al., J Immunol 2003, 170:5 81-587; Goya et al., J Immunol 2003, 170:2138- 2146)。さらに別の報告では C CL-1中和抗体は好酸球浸潤を減少させたが、 Th2細胞の動員に影響はなぐ気道 の炎症の重症度、 Th2サイト力インの分泌にも影響は見られなかったとしている（Bisho p et al.， J Immunol 2003, 170:4810- 4817)。彼らは、 CCL1の吸入により、 Th2細 胞ではなく好酸球の肺への動員を観察したことから、アレルギー性気道炎症モデル においては、 Th2細胞より、好酸球への作用の方が重要なのではないかと結論づけ ている。 [0006] In an experiment using CCR8 knockout mice, granuloma formation by Schistosoma mansoni soluble egg antigen, allergic airway inflammation by ovalbumin and cockroach antigens were first reduced, and Th2 type site force Have been reported to be characterized by poor production of erythrocytes and reduced eosinophil infiltration (Chensue et al., J Exp Med 2001, 193: 573-584). According to this group, knockout mice were not defective in Th2 cell development, but eosinophil recruitment was inadequate. However, two groups later reported that this was denied and CCL1 expression increased in the allergic airway inflammation model, but CCR8 knockout mice had no effect on eosinophil infiltration. It is said that it did not respond to the administration of the neutralizing antibody (Chung et al., J Immunol 2003, 170: 5 81-587; Goya et al., J Immunol 2003, 170: 2138-2146). Yet another report is C Although CL-1 neutralizing antibody decreased eosinophil infiltration, it did not affect the severity of airway inflammation and Th2 site force-in secretion, which had no effect on Th2 cell recruitment (Bisho). p et al., J Immunol 2003, 170: 4810-4817). Since they observed the recruitment of eosinophils, not Th2 cells, to the lungs by inhalation of CCL1, the action on eosinophils is more important than the Th2 cells in the allergic airway inflammation model. It is concluded that there is no.
[0007] 一方、ヒト末梢血 CD4+CD25+細胞は制御性機能を持つとされるが CCR4と CCR8の 発現を特徴としており、抑制性 T細胞の局所への遊走に関与していると考えられる (Ie Hem et al.， J Exp Med 2001, 194:847- 853)。ヒト胸腺の CD4+CD25+細胞は、 C D4+CD25—細胞の同種異系刺激による増殖を抑制するが、すべてが腫瘍壊死因子タ イブ 2受容体、細胞質リンパ球抗原— (CTLA) 4とともに CCR8を発現しており、胸腺 マクロファージまたは上皮細胞から分泌される CCL1に応答する（Annunziato et al.， J Exp Med 2002, 196:379-387)。  [0007] On the other hand, human peripheral blood CD4 + CD25 + cells are said to have regulatory functions, but are characterized by the expression of CCR4 and CCR8, and are thought to be involved in the local migration of inhibitory T cells ( Ie Hem et al., J Exp Med 2001, 194: 847-853). CD4 + CD25 + cells in human thymus inhibit CD4 + CD25—cell proliferation by allogeneic stimulation, but all contain CCR8 together with tumor necrosis factor type 2 receptor, cytoplasmic lymphocyte antigen— (CTLA) 4 It is expressed and responds to CCL1 secreted from thymic macrophages or epithelial cells (Annunziato et al., J Exp Med 2002, 196: 379-387).
[0008] さらに、 CCR8は TNF刺激により中枢神経のミクログリア細胞に発現しており、自己免 疫性脳炎モデルを CCR8ノックアウトマウスに誘導することによって、急性開始 (rapid- onset)脳炎への CCR8の関与が示された（Murphy et al.， J Immunol 2002, 169:7 054-7062)。 CCR8のミクログリアでの発現は脳梗塞や進行性多発性白質脳症など貧 食細胞の関与する病態にみられた（Trebst et al.， Am J Pathol 2003, 162:427- 438) o CCR8は単球だけでなくヒト臍帯静脈内皮細胞 (HUVECs)や、血管平滑筋細胞 (VSMC)に発現しており， CCL1による VSMCのケモタクシスが観察されたことから血 管病変への関与も示唆されている（Haque et al.， Blood 2004, 103:1296-1304)。  [0008] Furthermore, CCR8 is expressed in microglial cells of the central nervous system by TNF stimulation, and CCR8 is involved in rapid-onset encephalitis by inducing the autoimmune encephalitis model in CCR8 knockout mice. (Murphy et al., J Immunol 2002, 169: 7 054-7062). Expression of CCR8 in microglia was observed in pathologies involving phagocytic cells such as cerebral infarction and progressive multiple leukoencephalopathy (Trebst et al., Am J Pathol 2003, 162: 427-438) o CCR8 is a monocyte In addition, it was expressed in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC), and the VSMC chemotaxis by CCL1 was observed, suggesting its involvement in vascular lesions (Haque et al., Blood 2004, 103: 1296-1304).
[0009] 腹腔マクロファージにおいて CCR8が発現しており、 CCR8ノックアウトマウスは腹膜 炎誘発性敗血症（cecal ligation nad pucture, CLP)に抵抗性であり CCR8欠損マ クロファージは野生型に比べて殺菌能、細菌排除能も高いと学会報告されている (松 川昭博、第 24回日本炎症 ·再生医学会， 京都， 2003, Nov.26- 27)。  [0009] CCR8 is expressed in peritoneal macrophages, CCR8 knockout mice are resistant to peritonitis-induced sepsis (cecal ligation nad pucture, CLP), and CCR8-deficient macrophages are more bactericidal and eliminate bacteria It has been reported that the ability is high (Akihiro Matsukawa, 24th Japanese Society of Inflammation and Regenerative Medicine, Kyoto, 2003, Nov. 26-27).
[0010] 非特許文献 l : Becker et al, J. Am Coll Surg 1996, 183: 297-306  [0010] Non-patent literature l: Becker et al, J. Am Coll Surg 1996, 183: 297-306
非特許文献 2 : Rucker et al" J Virol 1997, 71:8999-9007  Non-Patent Document 2: Rucker et al "J Virol 1997, 71: 8999-9007
非特許文献 3 :Tiffany et al., J Exp Med 1997, 186:165-170 非特許文献 4:Roos et al.， J Biol Chem 1997, 272:17251-17254 非特許文献 5:Horuk et al, J Biol Chem 1998, 273:386-391 Non-Patent Document 3: Tiffany et al., J Exp Med 1997, 186: 165-170 Non-Patent Document 4: Roos et al., J Biol Chem 1997, 272: 17251-17254 Non-Patent Document 5: Horuk et al, J Biol Chem 1998, 273: 386-391
非特許文献 6:D'Ambrosio et al., J Immunol 1998, 161:5111-5115  Non-Patent Document 6: D'Ambrosio et al., J Immunol 1998, 161: 5111-5115
非特許文献 7:Endres et al., J Exp Med 1999, 189:1993-1998  Non-Patent Document 7: Endres et al., J Exp Med 1999, 189: 1993-1998
非特許文献 8:Louahed et al., Eur J Immunol 2003, 33:494-501  Non-Patent Document 8: Louahed et al., Eur J Immunol 2003, 33: 494-501
非特許文献 9:Dairaghi et al., J Biol Chem 1999, 274:21569-21574 非特許文献 10:Luttichau et al., J Exp Med 2000, 191:171-180  Non-patent literature 9: Daiairaghi et al., J Biol Chem 1999, 274: 21569-21574 Non-patent literature 10: Luttichau et al., J Exp Med 2000, 191: 171-180
非特許文献 ll:Singh et al" J Immunol 2004, 173:5509-5516  Non-Patent Document ll: Singh et al "J Immunol 2004, 173: 5509-5516
非特許文献 12:Zhang et al" J Immunol 2000, 165:10-14  Non-Patent Document 12: Zhang et al "J Immunol 2000, 165: 10-14
非特許文献 13:Chensue et al., J Exp Med 2001, 193:573-584  Non-Patent Document 13: Chensue et al., J Exp Med 2001, 193: 573-584
非特許文献 14:Chung et al., J Immunol 2003, 170:581-587  Non-Patent Document 14: Chung et al., J Immunol 2003, 170: 581-587
非特許文献 15: Goya et al., J Immunol 2003, 170:2138-2146  Non-Patent Document 15: Goya et al., J Immunol 2003, 170: 2138-2146
非特許文献 16: Bishop et al., J Immunol 2003, 170:4810-4817  Non-Patent Document 16: Bishop et al., J Immunol 2003, 170: 4810-4817
非特許文献 17:Iellem et al" J Exp Med 2001, 194:847-853  Non-Patent Document 17: Iellem et al "J Exp Med 2001, 194: 847-853
非特許文献 18:Annunziato et al" J Exp Med 2002, 196:379-387  Non-Patent Document 18: Annunziato et al "J Exp Med 2002, 196: 379-387
非特許文献 19: Murphy et al., J Immunol 2002, 169:7054-7062  Non-Patent Document 19: Murphy et al., J Immunol 2002, 169: 7054-7062
非特許文献 20:Trebst et al., Am J Pathol 2003, 162:427-438  Non-Patent Document 20: Trebst et al., Am J Pathol 2003, 162: 427-438
非特許文献 21:Haque et al., Blood 2004, 103:1296-1304  Non-Patent Document 21: Haque et al., Blood 2004, 103: 1296-1304
非特許文献 22:松川昭博、第 24回日本炎症'再生医学会， 京都， 2003, Nov.26-2 Non-Patent Document 22: Akihiro Matsukawa, 24th Japan Inflammation 'Regenerative Medicine Society, Kyoto, 2003, Nov.26-2
7 7
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0011] 本発明は、癒着を診断、予防および治療するための薬剤および診断、予防および 治療方法を提供することを目的とする。 [0011] An object of the present invention is to provide a drug for diagnosing, preventing and treating adhesions and a method for diagnosing, preventing and treating.
課題を解決するための手段  Means for solving the problem
[0012] 本発明者らは、 CCR8およびそのリガンド CCL1が癒着の形成に関与すること、およ び、抗 CCL1中和抗体を投与することにより癒着の形成を抑制しうることを見いだして 、本発明を完成させた。すなわち本発明は、 CCR8阻害剤を有効成分とする、癒着を 予防または治療するための医薬組成物を提供する。好ましくは、 CCR8阻害剤は、 C CR8に結合する抗体、 CCR8のリガンド CCL1に対する中和抗体、 CCR8ならびに CCL 1に対するァプタマ一、 CCR8のアンタゴ-スト、および CCR8ならびに CCL1遺伝子に 対するアンチセンスオリゴヌクレオチドおよび siRNAからなる群より選択される。 [0012] The present inventors have found that CCR8 and its ligand CCL1 are involved in the formation of adhesions, and that the formation of adhesions can be suppressed by administering an anti-CCL1 neutralizing antibody. Completed the invention. That is, the present invention provides an adhesion comprising a CCR8 inhibitor as an active ingredient. Pharmaceutical compositions for prevention or treatment are provided. Preferably, the CCR8 inhibitor comprises an antibody that binds to CCR8, a neutralizing antibody to CCR8 ligand CCL1, an aptamer to CCR8 and CCL 1, an antagonist of CCR8, and an antisense oligonucleotide to CCR8 and CCL1 gene and Selected from the group consisting of siRNA.
[0013] 別の観点においては、本発明は、被検細胞における CCR8または CCL1遺伝子の 発現を検出することを特徴とする癒着の検出方法を提供する。別の態様においては 、本発明は、被検細胞における CCR8または CCL1の存在量を検出することを特徴と する癒着の検出方法を提供する。  [0013] In another aspect, the present invention provides an adhesion detection method comprising detecting CCR8 or CCL1 gene expression in a test cell. In another aspect, the present invention provides a method for detecting adhesions characterized by detecting the abundance of CCR8 or CCL1 in a test cell.
[0014] さらに別の観点においては、本発明は、 CCR8または CCL1をコードする核酸にハイ ブリダィズし得るオリゴヌクレオチドまたはポリヌクレオチドを含む、癒着の診断用キッ トを提供する。別の態様においては、本発明は、 CCR8または CCL1に結合する抗体 またはその断片を含む、癒着の診断用キットを提供する。  [0014] In yet another aspect, the present invention provides a diagnostic kit for adhesions comprising an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CCR8 or CCL1. In another aspect, the present invention provides an adhesion diagnostic kit comprising an antibody or fragment thereof that binds to CCR8 or CCL1.
[0015] さらに別の観点においては、本発明は、被検物質が癒着の予防または治療効果を 有する力否かを検定する方法であって、前記被検物質が CCR8とリガンドとの結合を 阻害する能力を測定することを含む方法を提供する。別の態様においては、本発明 は、被検物質が癒着の予防または治療効果を有するか否かを検定する方法であつ て、前記被検物質力 SCCR8または CCL1の発現を阻害する能力を測定することを含む 方法を提供する。  [0015] In still another aspect, the present invention relates to a method for testing whether or not a test substance has an effect of preventing or treating adhesions, wherein the test substance inhibits binding between CCR8 and a ligand. A method comprising measuring the ability to In another embodiment, the present invention provides a method for assaying whether a test substance has an effect of preventing or treating adhesions, and measuring the ability to inhibit the test substance force SCCR8 or CCL1 expression. To provide a method comprising:
[0016] さらに別の観点においては、本発明は、 CCR8あるいは CCL1、または CCR8あるい は CCL1をコードするオリゴヌクレオチドまたはポリヌクレオチド、または CCR8を発現す る細胞を含む、癒着の予防または治療効果を有する物質をスクリーニングするための キットを提供する。  [0016] In still another aspect, the present invention provides an effect of preventing or treating adhesions comprising CCR8 or CCL1, or an oligonucleotide or polynucleotide encoding CCR8 or CCL1, or a cell expressing CCR8. A kit for screening substances possessed is provided.
[0017] さらに別の観点においては、本発明は、被検物質が癒着の予防または治療効果を 有する力否かを検定する方法であって、前記被検物質が、腹膜中皮細胞と腹腔マク 口ファージが凝集塊を形成することを抑制する能力を測定することを含む方法を提供 する。  [0017] In still another aspect, the present invention provides a method for testing whether a test substance has an effect of preventing or treating adhesions, wherein the test substance comprises peritoneal mesothelial cells and peritoneal macaques. A method comprising measuring the ability of a mouth phage to inhibit the formation of aggregates is provided.
[0018] さらに別の観点においては、本発明は、腹膜中皮細胞と腹腔マクロファージが凝集 塊を形成することの測定を実施することを目的とした、腹膜中皮細胞、腹腔マクロファ ージおよび CCL1の少なくとも 2つを含む、癒着の予防または治療効果を有する物質 をスクリーニングするためのキットを提供する。 発明の効果 [0018] In still another aspect, the present invention provides a method for measuring peritoneal mesothelial cells and peritoneal macrophages for the purpose of measuring the formation of aggregates between peritoneal mesothelial cells and peritoneal macrophages. And a kit for screening a substance having an effect of preventing or treating adhesions, comprising at least two of CZ1 and CCL1. The invention's effect
[0019] 本発明の医薬組成物は、癒着、特に腹腔内臓器手術後の腹膜癒着を予防および 治療するのに有用である。また、本発明のスクリーニング方法にしたがえば、癒着を 予防および治療するのに有用な物質をスクリーニングすることができる。  [0019] The pharmaceutical composition of the present invention is useful for preventing and treating adhesions, particularly peritoneal adhesions after intra-abdominal organ surgery. Further, according to the screening method of the present invention, a substance useful for preventing and treating adhesions can be screened.
図面の簡単な説明  Brief Description of Drawings
[0020] [図 1]図 1は、プラークの腹腔マクロファージと、無刺激の腹腔マクロファージのケモカ イン受容体の発現の相違を比率で示す (2回の実験にっ 、てそれぞれの実験の結果 を示す)。  [0020] [FIG. 1] FIG. 1 shows the difference in the expression of chemokine receptors between plaque peritoneal macrophages and unstimulated peritoneal macrophages (in two experiments, the results of each experiment are shown). Show).
[図 2]図 2は、 CCR8の内因性リガンドである CCL1および各種の免疫 ·炎症刺激因子 による、培養腹腔マクロファージ (ΡΜΦ)の CCR8受容体 mRNA発現増加を、刺激前 を 1とした比率で示す (平均値)。また、これらの刺激因子は、培養骨髄マクロファージ (BM Φ )では全く効果がな 、か、または効果が弱力つたことを示す。  [Figure 2] Figure 2 shows the increase in CCR8 receptor mRNA expression in cultured peritoneal macrophages (ΡΜΦ) by CCL8, an endogenous ligand of CCR8, and various immune / inflammatory stimulating factors, with a ratio of 1 before stimulation. (Average value). In addition, these stimulating factors have no or no effect on cultured bone marrow macrophages (BMΦ).
[図 3]図 3は、 CCL1および各種の免疫 ·炎症刺激因子による培養腹腔マクロファージ (PM Φ )の CCL1 mRNAの発現増加を、刺激前を 1とした比率で示す（平均値)。  [FIG. 3] FIG. 3 shows the increase in CCL1 mRNA expression in cultured peritoneal macrophages (PMΦ) by CCL1 and various immunity / inflammatory stimulating factors, expressed as a ratio (average value) with 1 before stimulation.
[図 4]図 4は、 CCL1および LPSによる培養腹腔マクロファージの CCR8受容体の発現 増加を示す画像の代表例である。  FIG. 4 is a representative example of an image showing increased expression of CCR8 receptor in cultured peritoneal macrophages by CCL1 and LPS.
[図 5]図 5は、 CCL1による培養腹腔マクロファージの CD49dの発現増加を示す画像 の代表例である。  FIG. 5 is a representative example of an image showing increased expression of CD49d in cultured peritoneal macrophages by CCL1.
[図 6]図 6は、 CCL1および各種の免疫 ·炎症刺激因子による培養腹腔中皮細胞の C CL1 mRNA発現量を、刺激前を 1とした比率で示す (平均値 +標準偏差)。また、 CC LI mRNA発現量を CCR8および他の遺伝子の mRNA 発現量と比較した。  [FIG. 6] FIG. 6 shows the CCL1 mRNA expression level of cultured peritoneal mesothelial cells by CCL1 and various immunity / inflammatory stimulating factors in a ratio (mean value + standard deviation) with 1 being before stimulation. In addition, the expression level of CC LI mRNA was compared with that of CCR8 and other genes.
[図 7]図 7は、同時培養した中皮細胞と腹腔マクロファージにおける凝集塊を示す画 像の代表例である。 CCL1は大きな凝集塊を生じさせる効果を示した。 LPSは刺激後 6 時間までの凝集塊形成効果は弱力つた。  FIG. 7 is a representative example of an image showing aggregates in co-cultured mesothelial cells and peritoneal macrophages. CCL1 showed the effect of generating large agglomerates. LPS had a weak aggregate formation effect up to 6 hours after stimulation.
[図 8]図 8は、同時培養した中皮細胞と腹腔マクロファージにおける、刺激因子適用 2 4 時間後の 1視野あたりの凝集塊面積を示す (平均値 +標準偏差)。 CCL1および LP sは大きな凝集塊を生じさせる効果を示した。 [FIG. 8] FIG. 8 shows the aggregate area per visual field 24 hours after application of the stimulating factor in mesothelial cells and peritoneal macrophages co-cultured (mean value + standard deviation). CCL1 and LP s showed the effect of producing large agglomerates.
[図 9]図 9は、同時培養した中皮細胞と腹腔マクロファージにおける、 CCL1刺激 24 時間後における凝集塊形成を抗 CCL1中和抗体が抑制したことを示す（1視野あたり の凝集塊面積の平均値 +標準偏差)。  [FIG. 9] FIG. 9 shows that anti-CCL1 neutralizing antibodies suppressed the formation of aggregates after 24 hours of CCL1 stimulation in co-cultured mesothelial cells and peritoneal macrophages (average of aggregate area per field of view) Value + standard deviation).
[図 10]図 10は、 TNBS投与 1 日後における腹腔マクロファージのプラークへの集積を 、抗 CCL1中和抗体が抑制したことを示す画像である。  FIG. 10 is an image showing that anti-CCL1 neutralizing antibody inhibited the accumulation of peritoneal macrophages in plaques 1 day after TNBS administration.
[図 11]図 11は、 TNBS投与 4 日後に生じる大腸穿孔部の癒着を抗 CCL1中和抗体が 抑制したことを示す (各マウスの癒着スコア、および各群の平均値)。  [FIG. 11] FIG. 11 shows that anti-CCL1 neutralizing antibody suppressed adhesion at the perforation of the large intestine occurring 4 days after TNBS administration (adhesion score of each mouse, and average value of each group).
[図 12]図 12は、マウス開腹手術モデル 7 日後に生じる腹膜虚血ボタン部位の癒着 を抗 CCL1中和抗体が抑制したことを示す (各マウスの癒着スコア、および各群の平 均値)。  [FIG. 12] FIG. 12 shows that anti-CCL1 neutralizing antibody suppressed adhesion of the peritoneal ischemia button site that occurred 7 days after the mouse laparotomy model (adhesion score of each mouse and the average value of each group). .
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0021] 本発明の医薬組成物は、 CCR8阻害剤を有効成分とすることを特徴とする。 CCR8と は、 355アミノ酸力もなり、蛋白共役型 7回膜貫通型の CCケモカインレセプターであり (Rucker et al., J Virol 1997, 71:8999-9007)、その遺伝子の塩基配列は公知で ある（GenBank: U45983、 BC069067) o CCR8阻害剤としては、 CCR8に結合する抗 体、 CCR8のアンタゴ-スト、 CCR8に対するァプタマ一、および CCR8遺伝子に対す るアンチセンスオリゴヌクレオチドおよび siRNAを挙げることができる。抗体はポリクロ ーナル抗体でもモノクローナル抗体でもよい。 CCR8の内因性ァゴ-ストである CCL1 は、 96アミノ酸力もなる分泌性タンパク質であり、その遺伝子の塩基配列は公知であ る（GenBank: M57502, M57506) o CCR8の活性化を抑制することは、 CCL1力 CCR8 に結合することを阻害したり CCL1の発現を抑制したりすることによって実現できる。こ のメカニズムにより CCR8を阻害しうる物質としては、 CCL1に対する中和抗体、 CCL1 に対するアブタマ一、および CCL1遺伝子に対するアンチセンスオリゴヌクレオチドお よび siRNAを挙げることができる。抗体はポリクローナル抗体でもモノクローナル抗体 でもよい。 [0021] The pharmaceutical composition of the present invention comprises a CCR8 inhibitor as an active ingredient. CCR8 is a protein-coupled seven-transmembrane CC chemokine receptor with 355 amino acid strength (Rucker et al., J Virol 1997, 71: 8999-9007), and the base sequence of the gene is known ( (GenBank: U45983, BC069067) o CCR8 inhibitors include CCR8 antagonists, CCR8 antagonists, aptamers to CCR8, and antisense oligonucleotides and siRNAs for the CCR8 gene. The antibody may be a polyclonal antibody or a monoclonal antibody. CCR8 endogenous § Gore - a strike CCL1 is a secreted protein also 96 amino acids forces the base sequence of the gene is Ru known der (GenBank: M57502, M57506) suppressing the activation of o CCR8 is This can be realized by inhibiting the binding of CCL1 to CCR8 or suppressing the expression of CCL1. Substances that can inhibit CCR8 by this mechanism include neutralizing antibodies against CCL1, Abutaman against CCL1, and antisense oligonucleotides and siRNA against CCL1 gene. The antibody may be a polyclonal antibody or a monoclonal antibody.
[0022] 本発明の CCR8に結合するポリクローナル抗体は， 当該技術分野においてよく知ら れる方法にしたがって， CCR8を感作抗原として用いて動物を免疫して，抗血清を採 取することにより得ることができる。本発明の CCR8に結合するモノクローナル抗体は， 当該技術分野においてよく知られる方法にしたがって， CCR8を感作抗原として用い て動物を免疫し，得られる免疫細胞を取り出して骨髄腫細胞と融合させ，抗体を産生 するハイプリドーマをクローユングし，このハイプリドーマを培養することにより得ること ができる。 [0022] The polyclonal antibody that binds to CCR8 of the present invention is obtained by immunizing an animal using CCR8 as a sensitizing antigen and collecting antiserum according to a method well known in the art. It can be obtained by taking. The monoclonal antibody that binds to CCR8 of the present invention can be obtained by immunizing an animal using CCR8 as a sensitizing antigen according to a method well known in the art, taking out the resulting immune cells and fusing them with myeloma cells. It can be obtained by cloning a hyperpridoma that produces lysozyme and culturing this hyperidoma.
[0023] 本発明のモノクローナル抗体には，ハイプリドーマにより産生される抗体にカ卩えて， 抗体遺伝子を含む発現ベクターで形質転換した形質転換体により生産される遺伝子 組換え抗体，キメラ抗体， CDR移植抗体，およびこれらの抗体の断片等が含まれる。  [0023] The monoclonal antibody of the present invention includes a gene recombinant antibody, a chimeric antibody, a CDR graft produced by a transformant transformed with an expression vector containing an antibody gene in addition to an antibody produced by a hyperidoma. Antibodies, and fragments of these antibodies are included.
[0024] 遺伝子組み換え抗体は， CCR8に結合するモノクローナル抗体を生産するハイプリ ドーマから抗体をコードする cDNAをクローユングし，これを発現ベクター中に挿入し て，動物細胞，植物細胞などを形質転換し，この形質転換体を培養することにより製 造することができる。キメラ抗体とは，ある動物に由来する抗体の重鎖可変領域およ び軽鎖可変領域と，他の動物に由来する抗体の重鎖定常領域および軽鎖定常領域 力も構成される抗体である。また、 CCR8に結合しうる抗体断片としては， Fab, F(ab')2 , Fab', scFv,ディアボディー等が挙げられる。  [0024] Recombinant antibodies are obtained by cloning cDNA encoding an antibody from a hybridoma that produces a monoclonal antibody that binds to CCR8, inserting it into an expression vector, and transforming animal cells, plant cells, etc. This transformant can be produced by culturing. A chimeric antibody is an antibody in which the heavy chain and light chain variable regions of an antibody derived from one animal and the heavy and light chain constant region forces of an antibody derived from another animal are also comprised. Examples of antibody fragments that can bind to CCR8 include Fab, F (ab ′) 2, Fab ′, scFv, and diabody.
[0025] 同様に本発明の CCL1に対する抗体は、 CCL1を感作抗原として用い、抗 CCR8抗 体作製と同じ当該技術分野においてよく知られる方法にしたがって得ることができる 。得られた抗体が中和抗体である力否かの検定は、 CCL1の生理作用を抑制するか 否かを検定することにより行うことができる。例としては、これらに限定されるものでは ないが、 CCL1の CCR8への結合、あるいは CCL1による CCR8発現細胞の遊走または 細胞内 Ca++増加または CCL1刺激に感受性のある遺伝子の発現変動、あるいは CCL 1による腹膜中皮細胞と腹腔マクロファージの凝集塊形成などを測定することが挙げ られる。 Similarly, the antibody against CCL1 of the present invention can be obtained according to the same well-known method in the art as the production of anti-CCR8 antibody using CCL1 as a sensitizing antigen. Whether the obtained antibody is a neutralizing antibody can be assayed by assaying whether it suppresses the physiological action of CCL1. Examples include, but are not limited to, binding of CCL1 to CCR8, migration of CCR8-expressing cells by CCL1 or increased expression of genes sensitive to intracellular Ca ++ increase or CCL1 stimulation, or CCL Measurement of the formation of aggregates of peritoneal mesothelial cells and peritoneal macrophages by 1 is mentioned.
[0026] アブタマ一はタンパク質に結合しうる数十塩基の長さの核酸リガンドである。これま で種々のタンパク質に対するアブタマ一が同定されており、例えば、血管内皮増殖 因子に対するアブタマ一は加齢性黄斑変性症治療に用いられて 、る。アブタマ一は 、多種の核酸鎖力もなるライブラリを製造し、その中から標的タンパク質に結合しうる 核酸鎖を選びだすことによって得ることができる。その具体的な方法の例としては、例 えば、特許 US5270163に記載される SELEX法が広く知られている。 [0026] Abutama is a nucleic acid ligand having a length of several tens of bases capable of binding to a protein. To date, Abutaman against various proteins has been identified. For example, Abutaman against vascular endothelial growth factor has been used for the treatment of age-related macular degeneration. Abutama can be obtained by preparing a library having various nucleic acid chain strengths and selecting a nucleic acid chain that can bind to a target protein from the library. As an example of the specific method, For example, the SELEX method described in Patent US5270163 is widely known.
CCR8のアンタゴ-ストとしては、例えば、 vMIP— IIv(viral macrophage inflammat ory protein 2)、 vMCC— I、 MC148、 WO2004/058709に記載される式（I)の化合 物：  As an antagonist of CCR8, for example, a compound of formula (I) described in vMIP-IIv (viral macrophage inflammatory protein 2), vMCC-I, MC148, WO2004 / 058709:
[化 1]
Figure imgf000011_0001
[Chemical 1]
Figure imgf000011_0001
(I).  (I).
WO2004/058736に記載される式（I)の化合物：  Compounds of formula (I) described in WO2004 / 058736:
[化 2] [Chemical 2]
Figure imgf000011_0002
Figure imgf000011_0002
WO2004/073619および WO2004/074438に記載される式（I)の化合物： Compounds of formula (I) described in WO2004 / 073619 and WO2004 / 074438:
[化 3] [Chemical 3]
Figure imgf000011_0003
等が知られている。また、 CCR8とリガンド CCL1との結合を阻害する物質をスクリー- ングすること〖こよっても得ることができる。そのようなアンタゴニストのスクリーニング方 法は当該技術分野においてよく知られている。
Figure imgf000011_0003
Etc. are known. It can also be obtained by screening a substance that inhibits the binding between CCR8 and the ligand CCL1. Such antagonist screening methods are well known in the art.
CCR8に対するアンチセンスオリゴヌクレオチドとは、 CCR8をコードする mRNAに特 異的に結合してその翻訳を阻害しうるオリゴヌクレオチドである。アンチセンスオリゴヌ クレオチドには、アンチセンス RNAおよびアンチセンス DNAが含まれる。 siRNAとは 、 RNA干渉を引き起こすことができる二本鎖 RNAである。 RNAおよび DNAは化学 的に修飾されて 、てもよ 、。アンチセンスオリゴヌクレオチドおよび siRNAの安定性ま たは細胞取り込みを増強するための種々の核酸修飾が知られており、本発明におい ては、そのいずれをも用いることができる。 Antisense oligonucleotides to CCR8 refer to mRNA that encodes CCR8. It is an oligonucleotide that can bind differently and inhibit its translation. Antisense oligonucleotides include antisense RNA and antisense DNA. siRNA is a double-stranded RNA that can cause RNA interference. RNA and DNA may be chemically modified. Various nucleic acid modifications are known to enhance the stability or cellular uptake of antisense oligonucleotides and siRNAs, any of which can be used in the present invention.
[0029] CCR8遺伝子に対するアンチセンスオリゴヌクレオチドまたは siRNAを癒着の予防' 治療剤として利用する場合には、例えばアンチセンスオリゴヌクレオチドまたは siRNA を被験者に直接投与する力 または、アンチセンスオリゴヌクレオチドまたは siRNAを 発現するベクターを作製し、これらの発現ベクターを投与することができる。アンチセ ンスオリゴヌクレオチド、 siRNAまたはこれらを発現するベクターを導入する方法は、 当該技術分野にぉ 、てよく知られて 、る。  [0029] When an antisense oligonucleotide or siRNA against CCR8 gene is used as a therapeutic agent for preventing adhesions, for example, the ability to administer antisense oligonucleotide or siRNA directly to a subject, or expression of an antisense oligonucleotide or siRNA Vectors can be prepared and these expression vectors can be administered. Methods for introducing anti-sense oligonucleotides, siRNAs or vectors expressing them are well known in the art.
[0030] 本発明の医薬組成物は、当業者に公知の方法で製剤化することができる。例えば 、薬学的に許容しうる担体もしくは媒体、具体的には、滅菌水や生理食塩水、植物油 、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、賦形剤、べヒクル、防腐剤、結合 剤などと適宜組み合わせて製剤化することができる。  [0030] The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. For example, a pharmaceutically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It can be formulated by appropriately combining with agents, binders and the like.
[0031] 経口投与用には、本発明の組成物を当該技術分野においてよく知られる薬学的に 許容しうる担体と混合することにより、錠剤、丸薬、糖衣剤、カプセル、液体、ゲル、シ 口ップ、スラリー、懸濁液等として処方することができる。担体としては、当該技術分野 において従来公知のものを広く使用することができ、例えば、乳糖、白糖、塩化ナトリ ゥム、グルコース、尿素、澱粉、炭酸カルシウム、カオリン、結晶セルロース、ケィ酸等 の賦形剤；水、エタノール、プロパノール、単シロップ、グルコース液、澱粉液、ゼラチ ン溶液、カルボキシメチルセルロース、セラック、メチルセルロース、リン酸カリウム、ポ リビュルピロリドン等の結合剤、乾燥澱粉、アルギン酸ナトリウム、寒天末、ラミナラン 末、炭酸水素ナトリウム、炭酸カルシウム、ポリオキシエチレンソルビタン脂肪酸エス テル、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、澱粉、乳糖等の崩壊剤；白 糖、ステアリンカカオバター、水素添加油等の崩壊抑制剤;第 4級アンモ-ゥム塩類、 ラウリル硫酸ナトリウム等の吸収促進剤;グリセリン、澱粉等の保湿剤;澱粉、乳糖、力 ォリン、ベントナイト、コロイド状ケィ酸等の吸着剤；精製タルク、ステアリン酸塩、ホウ 酸末、ポリエチレングリコール等の潤沢剤等を用いることができる。さらに錠剤は、必 要に応じ、通常の剤皮を施した錠剤、例えば、糖衣錠、ゼラチン被包錠、腸溶被錠、 フィルムコーティング錠、あるいは二重錠、多層錠とすることができる。 [0031] For oral administration, the composition of the present invention is mixed with a pharmaceutically acceptable carrier well known in the art, thereby allowing tablets, pills, dragees, capsules, liquids, gels, mouthpieces. Can be formulated as a cup, slurry, suspension or the like. As the carrier, those conventionally known in the technical field can be widely used. For example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, kynic acid and the like can be used. Forming agents: water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polypyrrolidone, etc., dry starch, sodium alginate, agar powder , Laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc .; disintegration inhibitors such as sucrose, stearic cocoa butter, hydrogenated oil, etc. ; Quaternary ammonia salt, Absorption promoters such as sodium lauryl sulfate; humectants such as glycerin and starch; starch, lactose, strength Adsorbents such as olin, bentonite and colloidal carboxylic acid; refined talc, stearate, boric acid powder, and lubricants such as polyethylene glycol can be used. Furthermore, the tablets can be made into tablets with ordinary coatings, if necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets.
[0032] 非経口投与用には、本発明の医薬組成物を当該技術分野においてよく知られる薬 学的に許容しうるべヒクルを用いて通常の製剤実施に従って処方することができる。  [0032] For parenteral administration, the pharmaceutical composition of the present invention can be formulated according to usual pharmaceutical practice using a pharmaceutically acceptable vehicle well known in the art.
[0033] 注射剤用の水溶性べヒクルとしては、例えば生理食塩水、ブドウ糖やその他の補助 薬を含む等張液、例えば D-ソルビトール、 D-マンノース、 D-マン-トール、塩化ナトリ ゥムが挙げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポ リアルコール、例えばプロピレングリコール、ポリエチレングリコール、非イオン性界面 活性剤、例えばポリソルベート 80 (TM)、 HCO- 50と併用してもよい。  [0033] Water-soluble vehicles for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-manntol, sodium chloride. In combination with a suitable solubilizer such as alcohol, specifically ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80 (TM), HCO-50. May be.
[0034] 油性べヒクルとしてはゴマ油、大豆油があげられ、溶解補助剤として安息香酸ベン ジル、ベンジルアルコールと併用してもよい。また、緩衝剤、例えばリン酸塩緩衝液、 酢酸ナトリウム緩衝液、無痛化剤、例えば、塩酸プロ力イン、安定剤、例えばべンジル アルコール、フエノール、酸化防止剤と配合してもよい。調製された注射液は通常、 適当なアンプルに充填させる。  [0034] Examples of oil-based vehicles include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Moreover, you may mix | blend with buffering agents, such as a phosphate buffer solution, sodium acetate buffer solution, a soothing agent, for example, hydrochloric acid pro-power-in, a stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection is usually filled in a suitable ampoule.
[0035] 本発明の医薬組成物の適当な投与経路には、限定されないが、経口、直腸内、経 粘膜、または腸内投与、または筋肉内、皮下、骨髄内、鞘内、直接心室内、静脈内、 硝子体内、腹腔内、鼻腔内、または眼内注射が含まれる。投与経路および投与方法 は、患者の年齢、症状により適宜選択することができる。  [0035] Suitable administration routes for the pharmaceutical composition of the present invention include, but are not limited to, oral, rectal, transmucosal, or enteral administration, or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, Intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection is included. The administration route and administration method can be appropriately selected depending on the age and symptoms of the patient.
[0036] 本発明の医薬組成物の特に好ましい投与経路および投与方法は、本発明の医薬 組成物を含む製剤、好ましくは徐放製剤を術中もしくは術後に患部またはその近傍、 例えば腹腔内に投与することである。特に適した投与方法は、ポリマーフィルムに本 発明の医薬組成物を結合させて、手術後に患部に留置することである。ポリマーフィ ルムとしては、例えば、術後の癒着予防用に用いられているセプラフイルム (登録商 標）、メソフオル (登録商標)等を用いることができる。  [0036] A particularly preferred route and method of administration of the pharmaceutical composition of the present invention are as follows: a preparation containing the pharmaceutical composition of the present invention, preferably a sustained-release preparation, is administered during or after surgery to the affected area or the vicinity thereof, for example, intraperitoneally. It is to be. A particularly suitable method of administration is to bind the pharmaceutical composition of the present invention to a polymer film and leave it in the affected area after surgery. As the polymer film, for example, Seprafilm (registered trademark), Mesophor (registered trademark) and the like used for prevention of adhesion after surgery can be used.
[0037] 本発明の医薬組成物の投与量としては、例えば、一回につき体重 lkgあたり 0.0001 mgから lOOOmgの範囲で選ぶことが可能である。あるいは、例えば、患者あたり 0.001 〜100000mg/bodyの範囲で投与量を選ぶことができる力 これらの数値に必ずしも制 限されるものではない。投与量、投与方法は、患者の体重や年齢、症状などにより変 動するが、当業者であれば適宜選択することが可能である。 [0037] The dosage of the pharmaceutical composition of the present invention can be selected, for example, in the range of 0.0001 mg to lOOOmg per kg of body weight per time. Or, for example, 0.001 per patient The ability to select a dose in the range of ~ 100,000 mg / body is not necessarily limited to these values. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., but can be appropriately selected by those skilled in the art.
[0038] 別の観点においては、本発明は、被検細胞における CCR8遺伝子の発現を検出す る力、または被検細胞における CCR8の存在量を検出することにより、癒着を診断す る方法を特徴とする。本発明にしたがえば、癒着が生じていると疑われる組織におけ る CCR8の存在量または CCR8遺伝子の発現レベルを指標として、癒着が生じて 、る か否かを診断することができる。  [0038] In another aspect, the present invention features a method for diagnosing adhesions by detecting the ability to detect CCR8 gene expression in a test cell or the amount of CCR8 present in a test cell. And According to the present invention, it is possible to diagnose whether or not adhesion has occurred using as an index the abundance of CCR8 or the expression level of the CCR8 gene in a tissue suspected of causing adhesion.
[0039] さらに別の観点においては、本発明は、被検細胞における CCL1の存在量を検出 することにより、癒着を診断する方法を特徴とする。本発明にしたがえば、癒着が生じ ていると疑われる組織あるいは腹水などの体液における CCL1の存在量を指標として 、癒着が生じているか否かを診断することができる。  [0039] In still another aspect, the present invention features a method of diagnosing adhesions by detecting the abundance of CCL1 in a test cell. According to the present invention, whether or not adhesion has occurred can be diagnosed by using the abundance of CCL1 in a tissue suspected of having adhesion or a body fluid such as ascites as an index.
[0040] CCR8あるいは CCL1の存在量または CCR8あるいは CCL1遺伝子の発現レベルを 測定する方法としては、例えば、手術後に腹水を排出するために設置されるドレイン 管から出る腹水を回収する力または腹水を少量採取して、これに含まれるマクロファ ージまたは中皮細胞の CCR8発現量または CCL1の発現量を測定することが挙げられ る。また、 CCL1などの CCR8ァゴニスト刺激による採取したマクロファージ遊走能を測 定することが挙げられる。さらに、抗 CCR8抗体もしくは抗 CCL1抗体、または CCR8に 対するァプタマ一もしくは CCL1に対するァプタマ一、または CCR8リガンドに標識を 行い、これを患者に投与して画像解析により測定するなどの方法が挙げられる。標識 としては放射性同位体、安定同位体、磁性体または発泡剤等が挙げられ、画像解析 の方法としては、ポジトロン放射トモグラフィー（PET)、シングルフォトンェミッションコ ンピュータートモグラフィー（SPECT)、核磁気共鳴画像診断 (MRI)または超音波 画像診断等が挙げられる。  [0040] Methods for measuring the abundance of CCR8 or CCL1 or the expression level of CCR8 or CCL1 gene include, for example, the ability to collect ascites from a drain tube installed to drain ascites after surgery or a small amount of ascites Collecting and measuring the expression level of CCR8 or CCL1 in macrophages or mesothelial cells. Another example is measuring the macrophage migration ability collected by CCR8 agonists such as CCL1. In addition, there may be mentioned a method in which an anti-CCR8 antibody or an anti-CCL1 antibody, an aptamer for CCR8 or an aptamer for CCL1, or a CCR8 ligand is labeled, and this is administered to a patient and measured by image analysis. Examples of labels include radioisotopes, stable isotopes, magnetic substances, and foaming agents. Image analysis methods include positron emission tomography (PET), single photon emission computer tomography (SPECT), and nuclear magnetic resonance. Examples include diagnostic imaging (MRI) or ultrasonic diagnostic imaging.
[0041] CCR8遺伝子の発現レベルは、検体における CCR8をコードする mRNAの存在量を 測定することにより定量することができる。 CCR8をコードする mRNAの存在量は、公 知の CCR8遺伝子配列に基づ 、て設計したプローブまたはプライマーを用いて、当 該技術分野においてよく知られる遺伝子解析技術を用いて測定することができる。そ のような技術としては、例えば、ノーザンブロット法、 DNAマイクロアレイ法、 RT-PC R等が挙げられる。 CCL1遺伝子の発現レベルは、 CCR8遺伝子に対するものと同様 の技術により測定することができる。 [0041] The expression level of the CCR8 gene can be quantified by measuring the abundance of mRNA encoding CCR8 in the specimen. The abundance of mRNA encoding CCR8 can be measured using a gene analysis technique well known in the art using a probe or primer designed based on a known CCR8 gene sequence. So Examples of such techniques include Northern blotting, DNA microarray, RT-PC R, and the like. The expression level of CCL1 gene can be measured by the same technique as that for CCR8 gene.
[0042] 検体における CCR8または CCL1の存在量は、当該技術分野においてよく知られる タンパク質解析技術を用いて測定することができる。そのような技術としては、例えば 、 CCR8または CCL1に結合する抗体またはその断片を利用したウェスタンブロッティ ング法、免疫沈降法、 ELISA、組織免疫染色法等が挙げられる。  [0042] The abundance of CCR8 or CCL1 in a specimen can be measured using protein analysis techniques well known in the art. Examples of such techniques include Western blotting, immunoprecipitation, ELISA, and tissue immunostaining using an antibody or fragment thereof that binds to CCR8 or CCL1.
[0043] さらに別の観点においては、本発明は、癒着の診断用キットを特徴とする。本発明 の癒着の診断用キットは、被験者力 採取した検体における CCR8遺伝子の発現レ ベルを測定するための試薬として、被験者の CCR8をコードする核酸にハイブリダィズ し得るオリゴヌクレオチドまたはポリヌクレオチド、あるいは、被験者の CCR8に結合す る抗体またはその断片を含む。本発明の癒着の診断用キットはさらに、被験者から採 取した検体における CCR8遺伝子の発現レベルを測定するための試薬として、被験 者の CCL1をコードする核酸にハイブリダィズし得るオリゴヌクレオチドまたはポリヌク レオチド、あるいは、被験者の CCL1に結合する抗体またはその断片を含むものでも 良い。本発明の癒着の診断用キットはさらに、 CCR8または CCL1、または CCR8遺伝 子または CCL1遺伝子の発現産物を定量するための試薬、および使用方法の指針を 含むことができる。  [0043] In yet another aspect, the invention features a diagnostic kit for adhesions. The adhesion diagnosis kit of the present invention is an oligonucleotide or polynucleotide that can hybridize to a nucleic acid encoding CCR8 of a subject, or a subject as a reagent for measuring the expression level of CCR8 gene in a sample collected from the subject. Antibody or a fragment thereof that binds to CCR8. The adhesion diagnosis kit of the present invention further comprises an oligonucleotide or a polynucleotide that can hybridize to a subject's CCL1-encoding nucleic acid, or a reagent for measuring the expression level of the CCR8 gene in a sample collected from a subject, or It may contain an antibody or a fragment thereof that binds to CCL1 of the subject. The adhesion diagnosis kit of the present invention may further comprise a reagent for quantifying the expression product of CCR8 or CCL1, or the CCR8 gene or CCL1 gene, and a method of use.
[0044] さらに別の観点においては、本発明は、癒着の予防または治療剤の成分として有 用な物質をスクリーニングする方法を特徴とする。本発明の方法にしたがえば、被検 物質が CCR8とリガンドとの結合を阻害する能力、または被検物質が CCR8の発現を 阻害する能力を測定することにより、被検物質が癒着の予防または治療効果を有す る力否かを検定することができる。 CCR8遺伝子の発現の阻害には、 CCR8遺伝子の 転写および翻訳のいずれの阻害も含まれる。本発明のスクリーニング方法は、候補 物質の存在下および非存在下において、 CCR8遺伝子の転写および Zまたは翻訳 量を測定することにより、 CCR8とそのリガンド、例えば CCL1との結合の阻害を測定す ることにより、 CCR8発現マクロファージを CCL1で刺激したときの遊走能を調べること により、または CCR8を CCL1で刺激したときの Ca+ +の変動を測定することにより、また は CCL1遺伝子の転写および Zまたは翻訳量を測定することにより、または CCL1の 細胞からの放出量を測定することにより、または腹膜中皮細胞と腹腔マクロファージ が凝集塊を形成することを測定することにより容易に実施することができる。 [0044] In still another aspect, the present invention is characterized by a method for screening a substance useful as a component of an agent for preventing or treating adhesions. According to the method of the present invention, the test substance can prevent adhesion or prevent adhesion by measuring the ability of the test substance to inhibit the binding between CCR8 and a ligand, or the test substance can inhibit the expression of CCR8. It is possible to test whether the force has a therapeutic effect. Inhibition of CCR8 gene expression includes inhibition of both transcription and translation of the CCR8 gene. The screening method of the present invention measures the inhibition of binding between CCR8 and its ligand, for example, CCL1, by measuring the amount of transcription and Z or translation of CCR8 gene in the presence and absence of a candidate substance. By examining the migration ability of CCR8-expressing macrophages when stimulated with CCL1, or by measuring changes in Ca ++ when CCR8 was stimulated with CCL1, By measuring the amount of transcription and Z or translation of the CCL1 gene, by measuring the amount of CCL1 released from cells, or by measuring the formation of aggregates between peritoneal mesothelial cells and peritoneal macrophages It can be easily implemented.
[0045] さらに別の観点においては、本発明は、癒着の予防または治療薬の成分として有 用な物質をスクリーニングする方法として、腹膜中皮細胞と腹腔マクロファージを混合 培養し、これらの細胞に例えば CCL1を培養皿中にカ卩えるなどの刺激を与えることに よりこれらの細胞が凝集塊を形成する反応を利用する方法を特徴とするインビトロの 試験方法を提供する。被検物質がこの凝集塊形成反応を抑制するか否かによって 癒着の予防または治療効果を検定することができる。あるいは、被検物質が形成され た凝集塊を小さくする力否かによって癒着の予防または治療効果を検定することが できる。 [0045] In still another aspect, the present invention provides a method for screening a substance useful as a component of an adhesion prevention or treatment agent, wherein peritoneal mesothelial cells and peritoneal macrophages are mixed and cultured, and these cells are subjected to, for example, There is provided an in vitro test method characterized by a method using a reaction in which these cells form an aggregate by giving a stimulus such as holding CCL1 in a culture dish. The preventive or therapeutic effect of adhesion can be assayed depending on whether the test substance suppresses this aggregate formation reaction. Alternatively, the effect of preventing or treating adhesions can be assayed based on whether or not the test substance has the ability to reduce aggregates formed.
[0046] 以下に、限定されるものではないが、その方法の一例を示す。動物臓器から採取し た細胞の培養を行う一般的な手順にした力 Sい、動物 (ヒトを含む)の腹腔内の膜から 中皮細胞の単層培養を作成する。より具体的な例としては、次の手順による。マウス の大網を採取し、小片に切った後に 0.01%コラゲナーゼを含む DMEM (Dulbecco m odified Eagle medium)により 37°Cにおいて 30 分間消化する。消化した組織はコラ 一ゲンコートした Falcon培養皿（Becton Dickinson, Franklin Lakes, NJ, USA)に 移し、 20% 牛胎児血清 (FCS)、5 ^ g/mL マウスリコンビナント上皮成長因子、およ びペニシリン ストレプトマイシンならびにアンホテリシン Bを含む DMEMにより培養す る。継代を 5から 6回行い 14日間培養する。  [0046] An example of the method is shown below without being limited thereto. Create a monolayer culture of mesothelial cells from the peritoneal membranes of animals (including humans) using the general procedure for culturing cells collected from animal organs. A more specific example is according to the following procedure. The greater omentum of a mouse is collected, cut into small pieces, and then digested with DMEM (Dulbec modified eagle medium) containing 0.01% collagenase for 30 minutes at 37 ° C. Digested tissues are transferred to collagen-coated Falcon culture dishes (Becton Dickinson, Franklin Lakes, NJ, USA), 20% fetal calf serum (FCS), 5 ^ g / mL mouse recombinant epidermal growth factor, and penicillin streptomycin Incubate with DMEM containing amphotericin B. Incubate 5 to 6 times and incubate for 14 days.
[0047] 同時に、動物から採取したマクロファージの培養を行う一般的な手順にしたがい、 動物（ヒトを含む）の腹腔マクロファージを培養する。より具体的な例としては、次の手 順〖こよる。マウス腹腔液を採取し、それに含まれる細胞を 2% FCSを含む DMEM培地 中で 45 分間インキュベーションし、付着した細胞を採取することで腹腔マクロファー ジを得る。なお、その後の細胞塊の測定を容易にするために、これらの細胞に標識を 施しておいても良い。その具体的な例としては、腹腔マクロファージに対して Hoshino ら（Biochem Biophys Res Commun 2004, 314:46-53)の方法により、蛍光発色 を示すカンタムドットで標識することを行うことが挙げられる。 [0048] 細胞塊を形成させるために腹腔マクロファージを中皮細胞と共培養する。その具体 的な方法の例として、腹腔マクロファージを 10%FCS-DMEM培地に移し、これをスポィ トによって中皮細胞の単層培養を行っている培養皿に添加する。次に、この腹腔マク 口ファージと中皮細胞を混合培養して 、る培養皿中に刺激を加えることよって細胞塊 を形成する反応を生じさせる。その具体的な方法の例として、刺激剤として CCL1を 添加することが挙げられる。 CCL1の濃度は 0.1 ng/mLから 10 ng/mLが望ましぐさ らに望ましくは 5 ng/mLが良い。また、 CCL1の代わりに CCL1/CCR8受容体系のシ ダナリングを活性ィ匕する刺激物質を添加することや同じ機能を示す遺伝子導入を行 つても良い。そのような刺激物質の例として、リポ多糖、細菌あるいは細菌由来のぺ プチドグリカン、オリゴデォキシヌクレオチド、サイト力イン等の起炎物質が挙げられる [0047] At the same time, peritoneal macrophages of animals (including humans) are cultured according to a general procedure for culturing macrophages collected from animals. A more specific example is the following procedure. The mouse peritoneal fluid is collected, and the cells contained in it are incubated in DMEM medium containing 2% FCS for 45 minutes, and the adhered cells are collected to obtain a peritoneal macrophage. In order to facilitate the subsequent measurement of cell mass, these cells may be labeled. As a specific example, peritoneal macrophages can be labeled with a quantum dot showing fluorescent coloration by the method of Hoshino et al. (Biochem Biophys Res Commun 2004, 314: 46-53). [0048] Peritoneal macrophages are co-cultured with mesothelial cells to form cell clusters. As an example of the specific method, peritoneal macrophages are transferred to 10% FCS-DMEM medium and added to a culture dish in which monolayer culture of mesothelial cells is performed by spotting. Next, this peritoneal phage and mesothelial cells are mixed and cultured, and a reaction is formed in the culture dish by applying a stimulus to the culture dish. An example of the specific method is the addition of CCL1 as a stimulant. The concentration of CCL1 is preferably 0.1 ng / mL to 10 ng / mL, and more preferably 5 ng / mL. Instead of CCL1, a stimulating substance that activates the CCL1 / CCR8 receptor system siRNAring may be added, or a gene having the same function may be introduced. Examples of such stimulating substances include lipopolysaccharides, bacteria or peptide glycans derived from bacteria, oligodeoxynucleotides, and flammable substances such as cytodynamic force in.
[0049] 被検物質は、腹腔マクロファージと中皮細胞を混合培養して ヽる培養皿中に刺激 前、刺激と同時、刺激後、細胞塊の形成後に添加することができる。細胞塊は顕微鏡 を用いて観察および測定することができる。観察および測定を容易にするために標 識した細胞の標識を用いることも良い。観察および測定の際に画像処理装置を用い ても良い。また、細胞塊をスポイトやピンセットなどにより取り出して体積の代わりに重 量を測定しても良 ヽ。被験物質を添加した培養皿における細胞塊形成の程度を被験 物質を添加しな ヽ細胞皿における細胞塊形成の程度と比較すること、ある ヽは刺激 物質添加前後の細胞塊形成の程度を比較することによって、被験物質の効果を判定 することができる。 [0049] A test substance can be added to a culture dish in which peritoneal macrophages and mesothelial cells are mixed and cultured before, simultaneously with, after stimulation, and after formation of a cell mass. Cell mass can be observed and measured using a microscope. It is also possible to use labeled cell labels for easy observation and measurement. An image processing apparatus may be used for observation and measurement. It is also possible to remove the cell mass with a dropper or tweezers and measure the weight instead of the volume. Compare the degree of cell clump formation in the culture dish to which the test substance has been added without adding the test substance. ヽ Compare the degree of cell clump formation before and after the addition of the stimulating substance. Thus, the effect of the test substance can be determined.
[0050] さらに別の観点においては、本発明は、癒着の予防または治療剤の成分として有 用な物質をスクリーニングするためのキットを特徴とする。本発明のキットは、 CCR8、 または CCR8をコードするオリゴヌクレオチドまたはポリヌクレオチド、または CCR8を発 現する細胞、または CCL1、または CCL1をコードするオリゴヌクレオチドまたはポリヌク レオチドを含むものであり、これらのスクリーニング用キットはさらに、アツセィに必要な 試薬および溶液、ならびに使用方法の指針を含むことができる。あるいは、本発明の キットは、腹膜中皮細胞と腹腔マクロファージが凝集塊を形成することを測定すること を目的としたものであり、この測定を実施することに適した研究材料を含むものであり 、本発明のキットはさらにアツセィに必要な試薬および溶液ならびに使用方法の指針 を含むことができる。この測定を実施することに適した研究材料の例として、腹腔マク 口ファージまたは、腹膜中皮細胞、すなわち消化管などの臓器、大網、腹膜あるいは 腹水由来の中皮細胞が挙げられる。 [0050] In still another aspect, the present invention features a kit for screening a substance useful as a component of an agent for preventing or treating adhesions. The kit of the present invention comprises CCR8, or an oligonucleotide or polynucleotide encoding CCR8, or a cell expressing CCR8, or CCL1, or an oligonucleotide or polynucleotide encoding CCL1, and is used for these screening. The kit can further include the reagents and solutions required for the assembly and instructions for use. Alternatively, the kit of the present invention is intended to measure that peritoneal mesothelial cells and peritoneal macrophages form aggregates, and includes research materials suitable for carrying out this measurement. In addition, the kit of the present invention may further contain reagents and solutions necessary for the assembly and directions for use. Examples of research materials suitable for carrying out this measurement include peritoneal phages or peritoneal mesothelial cells, ie organs such as the digestive tract, omentum, peritoneum or ascites.
[0051] 本発明のスクリーニング方法によって見いだされた物質は、癒着の予防 ·治療剤と して有用である。これらの癒着の予防 '治療剤は、 CCR8遺伝子または CCL1遺伝子 の発現を阻害することにより、あるいは、 CCR8の機能を阻害することにより、癒着の予 防，治療効果を発揮しうると考えられる。  [0051] The substance found by the screening method of the present invention is useful as an agent for preventing or treating adhesions. It is considered that these adhesion prevention and treatment agents can exert the effect of preventing or treating adhesions by inhibiting the expression of CCR8 gene or CCL1 gene, or by inhibiting the function of CCR8.
[0052] 本明細書において明示的に引用される全ての特許および参考文献の内容は全て 本明細書の一部としてここに引用する。また，本出願が有する優先権主張の基礎とな る出願である日本特許出願 2004-345267号および 2005-264160号の明細書および図 面に記載の内容は全て本明細書の一部としてここに引用する。  [0052] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specifications and drawings of Japanese Patent Applications Nos. 2004-345267 and 2005-264160, which are the applications on which the priority of the present application is based, are incorporated herein as a part of this specification. Quote.
[0053] 以下に実施例により本発明をより詳細に説明するが，これらの実施例は本発明の 範囲を制限するものではな 、。  [0053] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples do not limit the scope of the present invention.
[0054] 実施例 1  [0054] Example 1
マウス大腸炎モデルの 孔部位における腹腔マクロファージの奪穑 CCR8の 現 Abduction of peritoneal macrophages in the pore region of mouse colitis model
C57BL/6マウスの腹腔マクロファージを慣用法（Hoshino et al.， Biochem Biophy s Res Commun 2004, 314:46- 53)によりカンタムドット（quantum dots; Q dot)で ラベルし、同種マウス腹腔内に移植するとともに、高用量（loo gZg体重）のトリ-ト 口ベンゼンスルホン酸注腸による腸炎 (TNBS腸炎）を誘導した。 C57BL / 6 mouse peritoneal macrophages are labeled with quantum dots (Q dot) by conventional methods (Hoshino et al., Biochem Biophys Res Commun 2004, 314: 46-53) and transplanted intraperitoneally into allogeneic mice In addition, he induced enteritis (TNBS enteritis) due to high dose (loogZg body weight) tri-oral benzenesulfonic acid enema.
[0055] 24時間後、全層性潰瘍による大腸の部位を採取して凍結切片を作成し、 Qdot蛍 光標識したマクロファージを蛍光顕微鏡を用いて観察した。また、 TNBS腸炎誘導 24 時間後に大腸を取り出し、縦に切開しリン酸緩衝生理食塩水（PBS)にて洗浄した。 漿膜表面および潰瘍穿孔部に生じたプラーク部分に集積した細胞を剥した。大腸標 本は 30 分間 2 mMエチレンジアミンテトラ酢酸（EDTA) PBS処置を施し上皮細胞 を除去した。残った部分に対し、洗浄およびタイプ Fコラゲナーゼ (Sigma-Aldrich, M 0, USA)を用いて 20 分間消化する過程を 2回繰り返し、粘膜固有層単核球 (LPMC )を得た。プラーク部分に集積した細胞サンプルおよび大腸 LPMCサンプルについて 、フローサイトメトリを用いて蛍光標識した抗 CD1 lb抗体陽性細胞と Q dot 蛍光標識 したマクロファージを測定した。 [0055] After 24 hours, the large intestine site due to full-thickness ulcer was collected, frozen sections were prepared, and macrophages labeled with Qdot were observed with a fluorescence microscope. Further, 24 hours after induction of TNBS enteritis, the large intestine was taken out, cut longitudinally, and washed with phosphate buffered saline (PBS). The cells accumulated on the serosa surface and the plaque portion formed on the ulcer perforation were removed. The colon specimen was treated with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 30 minutes to remove epithelial cells. The remaining part was washed twice and digested for 20 minutes with type F collagenase (Sigma-Aldrich, M0, USA) twice to obtain lamina propria mononuclear cells (LPMC). Cell samples and colon LPMC samples accumulated in the plaque Then, anti-CD1 lb antibody-positive cells labeled with fluorescence and macrophages labeled with Q dot fluorescence were measured using flow cytometry.
[0056] 大腸壁内に多数の CD1 lb陽性のマクロファージ浸潤がみられたにもかかわらず、 Q dot 蛍光標識した腹腔マクロファージは穿孔部位のプラークのみに集積し、大腸 壁内への浸潤が全く認められな力つた。 [0056] Despite numerous CD1 lb-positive macrophage infiltration in the colon wall, Q dot fluorescently labeled peritoneal macrophages accumulate only in the plaques at the perforated site, and no infiltration into the colon wall is observed. I was helped.
[0057] 次に、プラーク部分に集積した細胞をマイクロダイセクションによって切り取り、 mRN[0057] Next, the cells accumulated in the plaque portion are cut out by microdissection, and mRN
Aを抽出し、ケモカイン受容体の発現を無刺激の腹腔マクロファージと、定量 RT-PCExtract A and peritoneal macrophages unstimulated for expression of chemokine receptors and quantitative RT-PC
Rにより比較した。ケモカイン受容体の発現の変化を図 1に示す。図中、 X軸は無刺激 腹腔マクロファージでの発現に対する比を示し、 Expl及び 2は独立して行った実験 結果を示す。 Comparison by R. Figure 1 shows changes in chemokine receptor expression. In the figure, the X-axis shows the ratio to the expression in unstimulated peritoneal macrophages, and Expl and 2 show the results of experiments conducted independently.
[0058] また、マウス抗 CCR8抗体により凍結切片を蛍光抗体法で染色したところ、プラーク 部分及び大腸漿膜側の細胞が陽性を示した。  [0058] Further, when the frozen section was stained with the mouse anti-CCR8 antibody by the fluorescent antibody method, the plaque portion and the colon serosa-side cells were positive.
[0059] 本実施例において、腹腔に投与した腹腔マクロファージは、癒着の前段階であるプ ラークには集積していたが、大腸壁内には移行しないことが明らかとなった。本実施 例においてプラークに集積した腹腔マクロファージでは、 CCR8の発現量が高ぐ他 のケモカイン受容体の発現は少なくなることが明らかになった。この結果から、ブラー クに集積した腹腔マクロファージにおけるケモカインに関する主要なシステムは、リガ ンド CCL1および受容体 CCR8による情報伝達システムであることが示された。また、 腹腔マクロファージの CCR8の存在量または発現レベルを測定することにより、その時 点およびその後の癒着の有無を検出しうることを示唆する。  [0059] In this example, it was revealed that peritoneal macrophages administered to the abdominal cavity were accumulated in the plaque, which was the pre-adhesion stage, but did not migrate into the colon wall. In the present example, it was revealed that peritoneal macrophages accumulated in plaques showed less expression of other chemokine receptors with high CCR8 expression. These results indicate that the main system for chemokines in peritoneal macrophages accumulated in black is a signal transduction system using ligand CCL1 and receptor CCR8. In addition, it is suggested that by measuring the abundance or expression level of CCR8 in peritoneal macrophages, it is possible to detect the time point and the presence or absence of subsequent adhesion.
[0060] 実施例 2  [0060] Example 2
CCL1による腹腔マクロファージの CCR8、 CCL1および CD49dの発現増加  Increased expression of CCR8, CCL1 and CD49d in peritoneal macrophages by CCL1
実施例 1においてプラークに集積した腹腔マクロファージは、炎症などの刺激により CCR8発現が高くなる活性ィ匕をしたことが示されたので、本実施例では、 CCR8の内因 性リガンドである CCL1を培養腹腔マクロファージに適用して、その効果を検討した。 C57BL/6マウス力も採取した腹腔マクロファージ、および定法により骨髄をマクロファ ージコロニー刺激因子 (M-CSF)を用いて分ィ匕させた骨髄マクロファージを培養した 。これらの細胞の培地に、刺激剤として、 CCL8のリガンドである CCL1、あるいは対照 として免疫反応を誘発する細菌成分である大腸菌 055B5由来のリポ多糖 (LPS; Sig ma-Aldrich) (100 ng/mL)または黄色ブドウ球菌由来のペプチドグリカン（PGN; Fluk a、 Buchsゝ Switzerland) (1 μ g/mL)または塩基配列が 5， - TCCATGACGTTCCTGA TGCT-3' (配列番号 1)である非メチル化シトシンーグァニン配列オリゴデォキシヌク レオチド (CpG) (タカラバイオ、東京、日本)（1 μ g/mL),ある 、は炎症を誘発するサ イト力インである腫瘍壊死因子一 a (TNF- a ) (1 g/mL)またはインターロイキン一 1 β (IL-1 j8 ) (10 μ g/mL)を適用した。腹腔マクロファージカも mRNAを抽出し、 CCR 8 mRNA発現量あるいは CCL1 mRNA発現量を定量 RT-PCRにより測定した。 mRN A発現量は GAPDHの mRNA量により標準化した。 Since the peritoneal macrophages accumulated in the plaque in Example 1 showed an activity that increased CCR8 expression by stimulation such as inflammation, in this example, CCL1, an endogenous ligand of CCR8, was cultured peritoneally. The effect was examined by applying to macrophages. Peritoneal macrophages from which C57BL / 6 mouse force was also collected and bone marrow macrophages in which bone marrow was separated using macrophage colony stimulating factor (M-CSF) by a conventional method were cultured. In these cell media, as a stimulator, CCL8 ligand CCL1, or a control Lipopolysaccharide (LPS; Sigma-Aldrich) derived from Escherichia coli 055B5 (100 ng / mL) or peptidoglycan derived from Staphylococcus aureus (PGN; Fluk a, Buchs ゝ Switzerland) (1 μ g / mL) or an unmethylated cytosine-guanine sequence oligodeoxynucleotide (CpG) (Takara Bio, Tokyo, Japan) (5)-TCCATGACGTTCCTGA TGCT-3 '(SEQ ID NO: 1) (1) μg / mL), is a site force-in that induces inflammation, tumor necrosis factor-a (TNF-a) (1 g / mL) or interleukin-1 β (IL-1 j8) (10 μ g / mL) was applied. Peritoneal macrophages also extracted mRNA, and CCR 8 mRNA expression level or CCL1 mRNA expression level was measured by quantitative RT-PCR. The mRNA expression level was normalized by the amount of GAPDH mRNA.
[0061] 各刺激因子適用 2 時間後の CCR8 mRNA発現量の、刺激適用前の mRNA発現量 を 1とした相対値を図 2に示す。腹腔マクロファージ (ΡΜΦ)において、 CCL 10 ng/ mL)刺激により CCR8 mRNA発現が増加した。 CCL1 (10 ng/mL)の効果は、 LPS (1 00 ng/mL), PGN (1 μ g/mL), CpG (1 g/mL)、 TNF— α (1 μ g/mL), IL-1 β (10 μ g/mL)による CCR8 mRNA発現増加効果に比べて強力つた。一方、骨髄マ クロファージ（ΒΜΦ)においては、 CCL1および他の刺激因子も CCR8 mRNAの発現 量を顕著に増カロさせる効果は示さな力つた。  [0061] FIG. 2 shows the relative values of the CCR8 mRNA expression level 2 hours after application of each stimulation factor, with the mRNA expression level before application of the stimulation being 1. In peritoneal macrophages (ΡΜΦ), CCR8 mRNA expression was increased by CCL 10 ng / mL) stimulation. The effects of CCL1 (10 ng / mL) are LPS (1 00 ng / mL), PGN (1 μg / mL), CpG (1 g / mL), TNF—α (1 μg / mL), IL- 1 β (10 μg / mL) was more potent than the effect of increasing CCR8 mRNA expression. On the other hand, in bone marrow macrophages (ΒΜΦ), CCL1 and other stimulating factors were also not effective in significantly increasing the expression level of CCR8 mRNA.
[0062] 次に、各刺激因子適用 2 時間後の CCL1 mRNA発現量の、刺激適用前の mRNA 発現量を 1とした相対値を図 3に示す。 CCL1 (10 ng/mL)刺激により腹腔マクロファ ージ（ΡΜΦ)の CCL1 mRNA発現が増加した。 CCL1 (10 ng/mL)刺激の CCL1 発 現増加効果は、顕著に CCL1発現量を増加させた LPS (100 ng/mL)の効果よりも弱 く、 PGN (1 g/mL)および CpG (1 g/mL)と同程度であった。  [0062] Next, FIG. 3 shows the relative value of the CCL1 mRNA expression level 2 hours after application of each stimulation factor, with the mRNA expression level before application of the stimulation being 1. CCL1 (10 ng / mL) stimulation increased CCL1 mRNA expression in peritoneal macrophages (ΡΜΦ). The effect of CCL1 (10 ng / mL) stimulation on CCL1 expression was weaker than that of LPS (100 ng / mL), which significantly increased CCL1 expression, and PGN (1 g / mL) and CpG (1 g / mL).
[0063] さらに、腹腔マクロファージによる CCR8受容体タンパク質の発現を確認するために 、マクロファージの表面抗原である CD-I lbに対する抗体および抗 CCR8抗体を用い た免疫組織ィ匕学染色による検討を行った。腹腔マクロファージに CCL1 (10 ng/mL) あるいは LPS 100 ng/mL)を適用した 12時間後に染色を実施した。その結果、 CCL1 および LPSが CCR8タンパク質の発現を増カロさせることが示された。図 4にその代表例 を示す。白線は抗 CD-I lb抗体染色で検出した細胞の形を示し、白色の領域が CCR 8陽性の染色領域を示す。 CCR8は細胞の表面の一部分に発現していた。また、腹腔 マクロファージに CCL1 (10 ng/mL)刺激を行った 48時間後に CD-I lbに対する抗体 および抗 CD49d抗体を用いた免疫組織化学染色による検討を行った結果、 CD49d の発現が増加していることが明らかになった。図 5にその代表例を示す。白線は抗 C D-l lb抗体染色で検出した細胞の形を示し、白色の領域力CD49d陽性の染色領域 を示す。 [0063] Furthermore, in order to confirm the expression of CCR8 receptor protein by peritoneal macrophages, examination was conducted by immunohistochemical staining using antibodies against CD-I lb, which is a surface antigen of macrophages, and anti-CCR8 antibodies. . Staining was performed 12 hours after CCL1 (10 ng / mL) or LPS 100 ng / mL) was applied to peritoneal macrophages. As a result, it was shown that CCL1 and LPS increase the expression of CCR8 protein. Figure 4 shows a typical example. The white line indicates the shape of the cells detected by anti-CD-I lb antibody staining, and the white area indicates the CCR 8-positive staining area. CCR8 was expressed on a part of the cell surface. Also abdominal cavity 48 hours after stimulation with CCL1 (10 ng / mL) in macrophages, immunohistochemical staining using an antibody against CD-I lb and an anti-CD49d antibody revealed that CD49d expression was increased. It was revealed. Figure 5 shows a typical example. The white line shows the shape of the cells detected by anti-CDllb antibody staining, and shows the white region force CD49d positive staining region.
[0064] 以上の結果は、腹腔マクロファージが刺激を受けると CCR8を発現させる反応は、骨 髄マクロファージには見られない、腹腔マクロファージに特徴的な性質であることを示 す。また、腹腔マクロファージにおいては、受容体 CCR8のリガンドである CCL1が受 容体 CCR8および CCL1自身の発現を増加させる、ポジティブフィードバックの機構が あることが示された。この結果から、急速に大量の腹腔マクロファージが障害部位に 集積することに CCL1ZCCR8系が関与している可能性が示唆された。本実施例では さらに、 CCL1刺激により腹腔マクロファージが CD49d (インテグリン α 4 |8 1 ;VLA4) を発現することが示された。 CD49dは、腹膜や腹腔内の臓器を覆う細胞である腹膜中 皮細胞に発現する VCAM1と結合するタンパク質である。したがって、本実施例の結 果力 CCL1ZCCR8系が癒着形成時に腹腔マクロファージと中皮細胞が接着するこ とに関与している可能性が示された。  [0064] The above results indicate that the response that expresses CCR8 when peritoneal macrophages are stimulated is a characteristic property of peritoneal macrophages that is not seen in bone marrow macrophages. It was also shown that in peritoneal macrophages, there is a positive feedback mechanism in which the receptor CCR8 ligand CCL1 increases the expression of the receptors CCR8 and CCL1 itself. This result suggests that the CCL1ZCCR8 system may be involved in the rapid accumulation of large numbers of peritoneal macrophages at the site of injury. In this example, it was further shown that peritoneal macrophages expressed CD49d (integrin α 4 | 8 1; VLA4) by CCL1 stimulation. CD49d is a protein that binds to VCAM1 expressed in peritoneal mesothelial cells, which are cells that cover the peritoneum and organs in the peritoneal cavity. Therefore, it was shown that the result force CCL1ZCCR8 system of this example may be involved in adhesion between peritoneal macrophages and mesothelial cells during adhesion formation.
[0065] 実飾 13  [0065] Decoration 13
f^flf巾 細qによる CCT 1の  f ^ flf width
実施例 2で腹腔マクロファージと腹膜中皮細胞の接着に CCL1/CCR8系が関与して いる可能性が示された。そこで、腹膜中皮細胞による CCL1産生について検討した。  In Example 2, it was shown that the CCL1 / CCR8 system may be involved in the adhesion between peritoneal macrophages and peritoneal mesothelial cells. Therefore, we examined CCL1 production by peritoneal mesothelial cells.
[0066] C57BL/6マウスの大網由来の中皮細胞を次の方法を用いて培養した。小片に切つ た大網組織を、 0.01%コラゲナーゼを含む DMEM (Dulbecco modified Eagle mediu m)により 37°Cにおいて 30 分間消化した。消化した組織はコラーゲンコートした Falco n培養皿（Becton Dickinson, Franklin Lakes, NJ, USA)に移し、 20% 牛胎児血清 (FCS)、5 μ g/mL マウスリコンビナント上皮成長因子、およびペニシリン ストレプ トマイシンならびにアンホテリシン Bを含む DMEMにより培養した。継代を 5から 6回行 い 14日間培養した。細胞の純度は抗マウスパンサイトケラチン抗体を用いた免疫組 織ィ匕学染色によって確認した。中皮細胞をコラーゲンコートした 24ゥエルの培養皿に 移し、コンフルェントになるまで培養した。各刺激因子を添加して 6 時間培養し、細 胞から mRNAを抽出し、定量 RT- PCRを用いて、 CCR8、 CCL1、 IL- 1 β、 IL- 6、および TNF- aの mRNA発現量を測定した。 [0066] Mesothelial cells derived from the greater omentum of C57BL / 6 mice were cultured using the following method. The omentum tissue cut into small pieces was digested with DMEM (Dulbecco modified Eagle medium) containing 0.01% collagenase at 37 ° C for 30 minutes. The digested tissue is transferred to a collagen-coated Falcon culture dish (Becton Dickinson, Franklin Lakes, NJ, USA), 20% fetal calf serum (FCS), 5 μg / mL mouse recombinant epidermal growth factor, and penicillin streptomycin and The cells were cultured in DMEM containing amphotericin B. The passage was performed 5 to 6 times and cultured for 14 days. The purity of the cells was confirmed by immunohistochemical staining using an anti-mouse pancytokeratin antibody. In a 24-well culture dish with collagen-coated mesothelial cells Transfer and culture until confluent. Add each stimulating factor and incubate for 6 hours, extract mRNA from cells, and use quantitative RT-PCR to determine mRNA expression levels of CCR8, CCL1, IL-1β, IL-6, and TNF-a. It was measured.
[0067] 各刺激因子適用後の CCL1 mRNA発現量の、無刺激での mRNA発現量を 1とした 相対値を図 6に示す。中皮細胞は、 LPS (100 ng/mL)、 PGN (100 ng/mL)、 TNF- a (1 g/mL)刺激により、 CCL1の mRNA発現を著しく増力！]させた。その増大は CC L1に選択的であった。また、 IL-1 β (10 μ g/mL)刺激により、 CCL1 mRNA,およ び TNF- aの増加が示された。 CpG (1 μ g/mL)は CCL1 mRNA発現を増加させなか つた。中皮細胞には CCR8の発現が認められたが、これらの刺激因子を適用した場合 には CCR8 mRNAの発現増加は示されなかった。一方、 CCL1 (5ng/mL)刺激により 、 CCL1、 IL-1 β、および CCR8の mRNAの発現が顕著に増加した。  [0067] FIG. 6 shows the relative values of the CCL1 mRNA expression level after application of each stimulation factor, with the unstimulated mRNA expression level being 1. Mesothelial cells significantly enhance CCL1 mRNA expression by stimulation with LPS (100 ng / mL), PGN (100 ng / mL), and TNF-a (1 g / mL)! ] The increase was selective for CC L1. In addition, IL-1 β (10 μg / mL) stimulation showed an increase in CCL1 mRNA and TNF-a. CpG (1 μg / mL) did not increase CCL1 mRNA expression. Although mesothelial cells showed CCR8 expression, application of these stimulating factors did not show increased CCR8 mRNA expression. On the other hand, CCL1 (5 ng / mL) stimulation significantly increased CCL1, IL-1β, and CCR8 mRNA expression.
[0068] 以上の結果は、腹膜中皮細胞が炎症反応などによる刺激を受けた際に CCL1を大 量に放出するという機構が存在することを示す。また腹膜中皮細胞においても、 CCL 1が受容体である CCR8および CCL1自身の発現を高めるポジティブフィードバック機 構が存在することが明らかとなった。 CCL1は、腹腔マクロファージの遊走を高め、実 施例 2で示されたように接着因子 CD49dを発現させる作用を持つ。したがって、本実 施例の結果は次のような過程があることを示すものである： 傷害部位の腹膜や臓器 表面の膜に存在する中皮細胞が、傷害による刺激を受けて腹腔内 CCL1を放出し、 その部位に活性ィ匕した腹腔マクロファージが集積して、中皮細胞と結合して癒着が 形成されて行く。また、本実施例の結果は、中皮細胞による CCL1の産生または細胞 外に放出された CCL1を検出することで、癒着の診断ができることを示す。  [0068] The above results indicate that a mechanism exists in which peritoneal mesothelial cells release a large amount of CCL1 when stimulated by an inflammatory reaction or the like. It was also clarified that there is a positive feedback mechanism that enhances the expression of CCR8, which is a receptor for CCL1, and CCL1 itself in peritoneal mesothelial cells. CCL1 enhances the migration of peritoneal macrophages and has the effect of expressing the adhesion factor CD49d as shown in Example 2. Therefore, the results of this example show that the following processes are observed: Mesothelial cells present in the peritoneum at the site of injury or the membrane on the surface of the organ receive intraperitoneal CCL1 upon stimulation by the injury. The released peritoneal macrophages accumulate at the site and bind to mesothelial cells to form adhesions. In addition, the results of this example indicate that adhesion can be diagnosed by detecting CCL1 production by mesothelial cells or CCL1 released to the outside of the cells.
[0069] 実施例 4  [0069] Example 4
CCL1による朥腔マクロファージ 朥腔中皮細胞の凝暴塊,の形成  Formation of acupuncture macrophages by the CCL1
実施例 1から 3の知見から、 CCL1が腹腔マクロファージが集積し中皮細胞と結合し て癒着を形成してゆく過程に深く関与していることが明らかとなった。そこで、新たに 、癒着形成過程を生じさせる培養細胞を用いたインビトロのモデル系を作製した。  From the findings in Examples 1 to 3, it has been clarified that CCL1 is deeply involved in the process in which peritoneal macrophages accumulate and bind to mesothelial cells to form adhesions. Therefore, an in vitro model system using cultured cells that cause an adhesion formation process was newly created.
[0070] C57BL/6マウスの大網由来の中皮細胞を次の方法を用いて培養した。小片に切つ た大網組織を、 0.01%コラゲナーゼを含む DMEM (Dulbecco modified Eagle mediu m)により 37°Cにおいて 30 分間消化した。消化した組織はコラーゲンコートした Falco n培養皿（Becton Dickinson, Franklin Lakes, NJ, USA)に移し、 20% 牛胎児血清 (FCS)、 5 μ g/mL マウスリコンビナント上皮成長因子、およびペニシリン ストレプ トマイシンならびにアンホテリシン Bを含む DMEMにより培養した。継代を 5から 6回行 い 14日間培養した。細胞の純度は抗マウスパンサイトケラチン抗体を用いた免疫組 織ィ匕学染色によって確認した。 [0070] Mesothelial cells derived from omentum of C57BL / 6 mice were cultured using the following method. The omentum tissue cut into small pieces is treated with DMEM (Dulbecco modified Eagle mediu) containing 0.01% collagenase. m) for 30 minutes at 37 ° C. The digested tissue is transferred to a collagen-coated Falcon culture dish (Becton Dickinson, Franklin Lakes, NJ, USA), 20% fetal calf serum (FCS), 5 μg / mL mouse recombinant epidermal growth factor, and penicillin streptomycin and The cells were cultured in DMEM containing amphotericin B. The passage was performed 5 to 6 times and cultured for 14 days. The purity of the cells was confirmed by immunohistochemical staining using an anti-mouse pancytokeratin antibody.
[0071] 中皮細胞をコラーゲンコートした 24ゥエルの培養皿に移し、コンフルェントになるま で培養した。中皮細胞は単層を形成した力 この上に Q dotでラベルした腹腔マクロ ファージ 1 X 105個/ゥエルを含む 10%FCS- DMEMを添カ卩し、 CCL1あるいは他の刺激 因子をカ卩え、 37°Cで培養した。 Q dotによる蛍光の発色は蛍光顕微鏡を用いて観察 した。また、評価尺度として、画像解析ソフトウェア（NIH image J、 National Instituti on of Health、 Bethesda、 MD、 USA)を用いて、面積 10— 1Q m2 以上の大きさの蛍光 発色領域の面積の総和力 S i視野に占める割合を算出し、 1視野中の凝集塊面積とし た。 [0071] Mesothelial cells were transferred to a 24-well culture dish coated with collagen and cultured until confluent. Mesothelial cells have the power to form a monolayer on top of this. Add 10% FCS-DMEM containing peritoneal macrophages labeled with Q dot 1 X 10 5 cells / well to cover CCL1 or other stimulating factors. It was cultured at 37 ° C. The color development of the fluorescence by Q dot was observed using a fluorescence microscope. In addition, using the image analysis software (NIH image J, National Institute of Health, Bethesda, MD, USA) as an evaluation scale, the total power of the area of fluorescent color development area with an area of 10-1Q m 2 or more S The percentage of the i field of view was calculated and used as the aggregate area in one field of view.
[0072] 培養腹腔中皮細胞上に添加した Q dotでラベルした腹腔マクロファージは、中皮 細胞にゆるく結合し、丸い形態を保っていた。図 7に Q dotでラベルした腹腔マクロフ ァージ添加 1 時間、 3 時間、 6 時間後の Q dot蛍光の画像の代表例を示す。刺激 を与えない場合（図 7a)には腹腔マクロファージの凝集はほとんど生じな力つた。この 混合培養に CCL1 (10 ng/mL)を加えると凝集塊が大きくなり、 3 時間あるいは 6時 間後には直径 100 mを超える凝集塊が形成された。図 7bに示した CCL1添加後の 画像の代表例に関しては、 6 時間後において直径 100 mを超える大きな凝集塊 が認められる。 LPS (100 ng/mL)刺激によっても凝集塊が形成されたが、適用 6 時 間後までの LPSの促進効果は CCL1の効果に比べて弱 、ものであった（図 7c)。  [0072] Peritoneal macrophages labeled with Q dots added on cultured peritoneal mesothelial cells were loosely bound to mesothelial cells and maintained a round shape. Fig. 7 shows representative examples of Q dot fluorescence images 1 hour, 3 hours, and 6 hours after addition of peritoneal macrophage labeled with Q dot. In the absence of stimulation (Fig. 7a), agglutination of peritoneal macrophages hardly occurred. When CCL1 (10 ng / mL) was added to this mixed culture, aggregates increased, and aggregates exceeding 100 m in diameter were formed after 3 or 6 hours. In the representative example of the image after CCL1 addition shown in Fig. 7b, a large aggregate exceeding 100 m in diameter is observed after 6 hours. Aggregates were also formed by stimulation with LPS (100 ng / mL), but the effect of promoting LPS until 6 hours after application was weaker than that of CCL1 (Fig. 7c).
[0073] CCL1刺激下で形成された大きな凝集塊の凍結切片を作製して免疫組織ィ匕学染色 を行ったところ、中皮細胞のマーカーである抗サイトケラチン抗体陽性細胞が存在し た。 CCL1を適用後に小さな凝集塊が大きくなつて行く過程では、凝集塊が中皮細胞 の層の上を転がって行くこと、および凝集塊が中皮細胞上を通った際には中皮細胞 が培養皿力 はがれて凝集塊に集まって行くことが観察された。なお、培養中皮細胞 に腹腔マクロファージを添加しない場合には、 CCL1を適用しても中皮細胞が培養皿 力もはがれることはなかった。 [0073] When frozen sections of large aggregates formed under CCL1 stimulation were prepared and immunohistochemical staining was performed, anti-cytokeratin antibody positive cells, which are markers of mesothelial cells, were present. In the process where small clumps grow larger after CCL1 is applied, the clumps roll on the mesothelial cell layer, and when the clumps pass over the mesothelial cells, the mesothelial cells are cultured. It was observed that the dish force peeled off and gathered into agglomerates. Cultured mesothelial cells When peritoneal macrophages were not added to the cells, mesothelial cells did not lose their culture dish strength even when CCL1 was applied.
[0074] また、 Q dotでラベルした腹腔マクロファージあるいは骨髄マクロファージを培養中 皮細胞に添加し、無刺激あるいは各種濃度の CCL1刺激を加えた 24 時間後に、 1視 野中の凝集塊面積を算出した。結果は図 8に示す。 CCL1は腹腔マクロファージ (PM Φ)と中皮細胞の凝集塊面積を増加させた。その効果は濃度が 5 ng/mLの場合に最 も強かった。一方、腹腔マクロファージの代わりに骨髄マクロファージ（ΒΜΦ)と中皮 細胞を混合培養した場合には、 CCL1を適用による顕著な凝集塊面積の増大は認め られな力つた。 LPS 100 ng/mL)は、刺激 6 時間 後までは強い効果を示さなかった 力 刺激 24 時間後においては顕著に凝集塊面積を増大させることが示された。この ことは、 LPS刺激によって腹腔マクロファージあるいは中皮細胞力も CCL1が放出され た結果凝集塊が形成されたことを示唆する。次に、 CCL1刺激による腹腔マクロファー ジと中皮細胞の凝集塊の形成を、ラット抗マウス CCL1中和モノクローナル抗体 (R&D Systems, Minneapolis, MN、 USA)により抑制する試験を実施した。 CCL1 (5 ng/m L)および抗 CCL1中和抗体適用 24 時間後の 1視野中の凝集塊面積を図 9に示す 。抗 CCL1中和抗体は濃度依存的に凝集塊の形成を抑制した。  [0074] In addition, peritoneal macrophages or bone marrow macrophages labeled with Q dots were added to cultured mesothelial cells, and 24 hours after no stimulation or various concentrations of CCL1 stimulation were applied, the aggregate area in one visual field was calculated. The results are shown in FIG. CCL1 increased the aggregate area of peritoneal macrophages (PM Φ) and mesothelial cells. The effect was strongest at a concentration of 5 ng / mL. On the other hand, when bone marrow macrophages (ΒΜΦ) and mesothelial cells were mixed and cultured instead of peritoneal macrophages, no significant increase in the aggregate area due to the application of CCL1 was observed. LPS (100 ng / mL) did not show a strong effect until 6 hours after stimulation, and was shown to significantly increase the aggregate area after 24 hours of force stimulation. This suggests that aggregates were formed as a result of the release of CCL1 from peritoneal macrophages or mesothelial cells by LPS stimulation. Next, a test was carried out to suppress the formation of agglomerates of peritoneal macrophages and mesothelial cells by stimulation with CCL1 with a rat anti-mouse CCL1 neutralizing monoclonal antibody (R & D Systems, Minneapolis, MN, USA). FIG. 9 shows the aggregate area in one visual field 24 hours after application of CCL1 (5 ng / mL) and anti-CCL1 neutralizing antibody. Anti-CCL1 neutralizing antibody suppressed the formation of aggregates in a concentration-dependent manner.
[0075] 以上の結果は、 CCR8受容体のリガンドである CCL1、および腹腔マクロファージの C CR8受容体が癒着の形成に深く関与していることを示す。また、この癒着形成過程を 生じさせるインビトロのモデル系により癒着を抑制する物質のスクリーニングを行うこと ができることを示す。  [0075] The above results indicate that CCL1, which is a ligand of CCR8 receptor, and CCR8 receptor of peritoneal macrophages are deeply involved in adhesion formation. In addition, we show that it is possible to screen for substances that suppress adhesion using an in vitro model system that generates this adhesion formation process.
[0076] 実施例 5  [0076] Example 5
CCLl ^禾ロ 体によるマウス JE モデル 孔部 における 开 の 葡1 Opening of the mouse JE model hole by CCLl ^ 2
In vivoにおいて CCL1ZCCL8系を阻害することにより癒着を抑制することを示すた め、癒着が生じる TNBS大腸炎モデルにおいて、マウスに抗 CCL1中和抗体投与を行 つた。 C57BL/6マウスに実施例 4で用いた抗 CCL1抗体（中和抗体） 150 μ gを腹腔 内投与した 3 時間後に、実施例 1と同様にして TNBS腸炎を誘導した。対照としては 、抗 CCL1抗体の代わりにラット IgGを投与したマウスを用いた。腹腔マクロファージの 動態に関する画像解析を行う試験、および癒着形成を測定する試験を行った。 [0077] 腹腔マクロファージの動態に関する試験では、 Q dotでラベルした腹腔マクロファ ージ（2.5 X 105個）を TNBS投与の前日に腹腔内に注入した。 TNBS注入 1 日後に大 腸を取り出し、画像解析装置 (リライオン、東京、 日本)を用いて Q dotの蛍光発色像 を可視光線画像に重ねあわせて観察した。図 10aは各群 3例についてそれぞれのマ ウスの大腸全体における標識腹腔マクロファージ集積に関する画像を示す。明るい 領域が Q dot標識した腹腔マクロファージが集積した領域を示す。対照のラット IgG 投与群では、 TNBS投与後に腹腔マクロファージが腸漿膜上の 1箇所力 数箇所に 集積していた。一方、抗 CCL1抗体投与群では、腹腔マクロファージの集積が対照よ りも少なかった。図 10bには、大腸の Q dotの発色が強い部分の切片を作製して蛍 光顕微鏡を用いて観察した画像の代表例を示す。明るい領域が Q dot標識した腹 腔マクロファージが集積した領域を示す。線は大腸および穿孔部の形を示す。矢印 は穿孔部のプラークを示す。対照のラット IgG投与群では、腹腔マクロファージが後 に癒着を起こす穿孔部のプラークに集積していた。これに対して、抗 CCL1抗体投与 群では、 Q dotでラベルして腹腔に注入した腹腔マクロファージが固有層および粘 膜層に散在していた。これらの結果と実施例 1および 2の結果から、抗 CCL1抗体が、 腹腔マクロファージが CCR8受容体発現を増カロさせる活性ィ匕をすること、および腹腔 マクロファージの穿孔部位への集積を抑制したことが示された。 In order to demonstrate that adhesion is suppressed by inhibiting the CCL1ZCCL8 system in vivo, mice were administered anti-CCL1 neutralizing antibody in a TNBS colitis model in which adhesion occurs. Three hours after intraperitoneal administration of 150 μg of the anti-CCL1 antibody (neutralizing antibody) used in Example 4 to C57BL / 6 mice, TNBS enteritis was induced in the same manner as in Example 1. As a control, mice administered with rat IgG instead of anti-CCL1 antibody were used. Tests were performed to analyze the image of peritoneal macrophage dynamics and to measure adhesion formation. [0077] In the study on the dynamics of peritoneal macrophages, peritoneal macrophages (2.5 x 10 5 cells) labeled with Q dot were injected intraperitoneally the day before TNBS administration. One day after TNBS injection, the large intestine was taken out, and the Q dot fluorescence image was superimposed on the visible light image using an image analyzer (Relion, Tokyo, Japan). Figure 10a shows images of labeled peritoneal macrophage accumulation in the entire large intestine of each mouse for 3 patients in each group. The bright area is the area where Q dot-labeled peritoneal macrophages are accumulated. In the control rat IgG-administered group, peritoneal macrophages accumulated at one place on the serosa after TNBS administration. On the other hand, in the anti-CCL1 antibody administration group, the accumulation of peritoneal macrophages was less than that of the control. Figure 10b shows a representative example of an image obtained by preparing a section of the large intestine where Q dots are strongly colored and observing it using a fluorescence microscope. The bright area is the area where Q dot-labeled abdominal cavity macrophages are accumulated. Lines indicate the shape of the large intestine and perforation. The arrow indicates the plaque on the perforated part. In the control rat IgG-administered group, peritoneal macrophages were accumulated in plaques in the perforated area where adhesion occurred later. In contrast, in the anti-CCL1 antibody administration group, peritoneal macrophages labeled with Q dots and injected into the peritoneal cavity were scattered in the lamina propria and mucosa. From these results and the results of Examples 1 and 2, anti-CCL1 antibody showed that peritoneal macrophages acted to increase CCR8 receptor expression and inhibited peritoneal macrophage accumulation at the perforation site. Indicated.
[0078] 大腸の癒着は TNBS注入 4 日後に開腹して観察し、スコア化した。癒着スコアは次 の通りとした:癒着がない (0点）、薄い膜状の癒着力 S1箇所ある（1点）、薄い薄膜状の 癒着が 2箇所以上ある（2点）、 1箇所に厚い癒着がある（3点）、底面との厚い癒着ま たは 2箇所以上の厚い癒着がある (4点）、非常に厚く血管新生を伴う癒着または 2箇 所以上の底面との厚 、癒着がある（5点)。  [0078] Colonic adhesions were observed by laparotomy 4 days after TNBS injection and scored. Adhesion scores were as follows: no adhesion (0 points), thin membrane adhesion S1 location (1 point), thin thin film adhesions 2 locations (2 points), 1 location thick There are adhesions (3 points), thick adhesion with the bottom surface or thick adhesions at 2 or more locations (4 points), very thick adhesion with angiogenesis, or thickness with 2 or more bottom surfaces, adhesion Yes (5 points).
[0079] 抗 CCL1抗体投与群において、対照群に比較して TNBSによる炎症や傷害が悪化し たり、体重を指標とした全身状態が悪ィ匕したりすることはな力 た。抗 CCL1抗体投与 により癒着の形成は顕著に抑制された。図 11に、各マウスの癒着スコアを四角で、各 群の平均値を十字記号で示す。対照のラット IgG投与群では、 5例全例にスコア 4点 または 5点の癒着が生じていた力 抗 CCL1抗体投与群の癒着スコアは明らかに少な かった。 [0080] これらの結果は、 CCL1ZCCR8系が癒着形成に深く関与していることを示す。また 、内因性の CCL1が受容体 CCR8に結合するシグナリングの経路を、抗 CCL1中和抗 体や CCR8受容体拮抗薬などを用いて阻害することで、腹腔内臓器の癒着形成が抑 制できることを示す。 [0079] In the anti-CCL1 antibody administration group, inflammation and injury due to TNBS were not worsened, and the general condition using body weight as an index was worse than the control group. The formation of adhesions was significantly suppressed by the administration of anti-CCL1 antibody. Figure 11 shows the adhesion score for each mouse as a square and the average value for each group as a cross symbol. In the control rat IgG administration group, the adhesion score of the anti-CCL1 antibody administration group was clearly lower, in which all 5 cases had 4 or 5 score adhesions. [0080] These results indicate that the CCL1ZCCR8 system is deeply involved in adhesion formation. In addition, by inhibiting the signaling pathway of endogenous CCL1 binding to the receptor CCR8 using anti-CCL1 neutralizing antibodies, CCR8 receptor antagonists, etc., it is possible to suppress the formation of adhesions in the abdominal organs. Show.
[0081] 実施例 6  [0081] Example 6
杭 CCL1中和杭体によるマウス開腹手術モデルにおける瘉着形成の抑制  Suppression of adhesion formation in a mouse laparotomy model with a pile of CCL1 neutralization
ラットにおける開腹手術後の腹膜癒着モデル（Reed et al.， Proc Natl Acad Sci U S A. 2004 , 101:9115-9120)を応用して、 C57BL/6マウスを用いた癒着形成 モデルを作製し、抗 CCL1中和抗体の癒着形成抑制効果を示した。マウスに麻酔を かけ、正中線に沿って開腹手術を行った。腹膜を止血鉗子でつまんで根元を 4-0絹 糸で結紮することにより、いぼ状の虚血ボタンを作成した。虚血ボタンを左右の腹膜 に 1箇所ずつ作成した後に閉腹した。実施例 4および 5で用いた抗 CCL1抗体（中和 抗体) 150 gを、開腹手術終了直後およびその 3 日後に腹腔内投与した。対照と したマウスには抗 CCL1抗体の代わりにラット IgGを投与した。開腹手術の 7 日後に 開腹して癒着を観察した。癒着部位にはマクロファージの集積が認められた。癒着の 評価尺度として、左右の虚血ボタンそれぞれについてスコア化し、左右のスコアを合 計した。癒着スコアは次の通りとした:癒着がない (0点）、薄い膜状の癒着がある（1 点）、厚く癒着している（2点)。  By applying the peritoneal adhesion model after laparotomy in rats (Reed et al., Proc Natl Acad Sci US A. 2004, 101: 9115-9120) The anti-adhesion effect of CCL1 neutralizing antibody was shown. The mice were anesthetized and laparotomy was performed along the midline. A wart-shaped ischemic button was created by pinching the peritoneum with hemostatic forceps and ligating the root with 4-0 silk. After creating ischemic buttons in the left and right peritoneum, the abdomen was closed. 150 g of the anti-CCL1 antibody (neutralizing antibody) used in Examples 4 and 5 was intraperitoneally administered immediately after the completion of the laparotomy and 3 days later. Control mice received rat IgG instead of anti-CCL1 antibody. Seven days after the laparotomy, the abdomen was opened and adhesions were observed. Accumulation of macrophages was observed at the adhesion site. As a scale for evaluating adhesions, the left and right ischemic buttons were scored, and the left and right scores were combined. The adhesion score was as follows: no adhesion (0 points), thin membrane adhesion (1 point), thick adhesion (2 points).
[0082] 図 12に各マウスの癒着スコアを四角で、各群の平均値を十字記号で示す。対照の ラット IgG投与群では、全 9例に癒着が生じ、癒着スコアは平均 2. 89点であった。抗 CCL1抗体投与群では、 9例中 5例では癒着が認められず、癒着スコアは平均 0. 78 点であり、抗 CCL1抗体投与による癒着抑制効果が示された。  FIG. 12 shows the adhesion score of each mouse as a square, and the average value of each group as a cross symbol. In the control rat IgG administration group, adhesion occurred in all 9 patients, with an average adhesion score of 2.89 points. In the anti-CCL1 antibody administration group, no adhesion was observed in 5 out of 9 cases, and the adhesion score averaged 0.78 points, indicating that the anti-CCL1 antibody administration was effective in suppressing adhesions.
[0083] 本実施例の結果は、開腹手術後の癒着形成に CCL1ZCCR8系が深く関与してい ることを示す。また、内因性の CCL1が受容体 CCR8に結合することによるシグナリング の経路を、抗 CCL1中和抗体や CCR8受容体拮抗薬などを用いて阻害することで、癒 着形成が抑制できることを示す。  [0083] The results of this example indicate that the CCL1ZCCR8 system is deeply involved in adhesion formation after laparotomy. We also show that adhesion formation can be suppressed by inhibiting the signaling pathway of endogenous CCL1 binding to the receptor CCR8 using anti-CCL1 neutralizing antibodies, CCR8 receptor antagonists, and the like.
産業上の利用可能性  Industrial applicability
[0084] 本発明の医薬組成物は、癒着を診断、予防および治療するために有用である。  [0084] The pharmaceutical composition of the present invention is useful for diagnosing, preventing and treating adhesions.

Claims

請求の範囲  The scope of the claims
[I] CCR8阻害剤を有効成分とする、癒着を予防または治療するための医薬組成物。  [I] A pharmaceutical composition for preventing or treating adhesions comprising a CCR8 inhibitor as an active ingredient.
[2] CCR8阻害剤が CCR8に結合する抗体または CCR8のリガンド CCL1に対する中和抗 体である、請求項 1記載の医薬組成物。 [2] The pharmaceutical composition according to claim 1, wherein the CCR8 inhibitor is an antibody that binds to CCR8 or a neutralizing antibody against the ligand CCL1 of CCR8.
[3] CCR8阻害剤が CCR8に対するァプタマ一または CCR8のリガンド CCL1に対するァプ タマ一である、請求項 1記載の医薬組成物。 [3] The pharmaceutical composition according to claim 1, wherein the CCR8 inhibitor is an aptamer for CCR8 or an aptamer for ligand CCL1 of CCR8.
[4] CCR8阻害剤が CCR8のアンタゴ-ストである、請求項 1記載の医薬組成物。 [4] The pharmaceutical composition according to claim 1, wherein the CCR8 inhibitor is an antagonist of CCR8.
[5] CCR8阻害剤が CCR8遺伝子に対するアンチセンスオリゴヌクレオチドまたは siRNA、 あるいは CCR8のリガンド CCLlに対するアンチセンスオリゴヌクレオチドまたは siRNA である請求項 1記載の医薬組成物。 [5] The pharmaceutical composition according to claim 1, wherein the CCR8 inhibitor is an antisense oligonucleotide or siRNA against the CCR8 gene, or an antisense oligonucleotide or siRNA against the ligand CCLl of CCR8.
[6] 被検細胞における CCR8遺伝子または CCR8のリガンド CCL1の発現を検出することを 含む、癒着の検出方法。 [6] A method for detecting adhesion, comprising detecting expression of a CCR8 gene or a CCR8 ligand CCL1 in a test cell.
[7] 被検細胞における CCR8または CCR8のリガンド CCL1の存在量を検出することを含む[7] including detecting the abundance of CCR8 or the CCR8 ligand CCL1 in the test cells
、癒着の検出方法。 , Adhesion detection method.
[8] CCR8または CCR8のリガンド CCL1をコードする核酸にハイブリダィズし得るオリゴヌク レオチドまたはポリヌクレオチドを含む、癒着の診断用キット。  [8] A kit for diagnosis of adhesion comprising an oligonucleotide or polynucleotide capable of hybridizing to a nucleic acid encoding CCR8 or CCR8 ligand CCL1.
[9] CCR8または CCR8のリガンド CCL1に結合する抗体またはその断片を含む、癒着の 診断用キット。 [9] A diagnostic kit for adhesion comprising an antibody or fragment thereof that binds to CCR8 or CCR8 ligand CCL1.
[10] 被検物質が癒着の予防または治療効果を有する力否かを検定する方法であって、 前記被検物質が CCR8とリガンドとの結合を阻害する能力を測定することを含む方法  [10] A method for testing whether or not a test substance has an effect of preventing or treating adhesions, comprising measuring the ability of the test substance to inhibit the binding between CCR8 and a ligand
[II] 被検物質が癒着の予防または治療効果を有するか否かを検定する方法であって、 前記被検物質が CCR8または CCL1の発現を阻害する能力を測定することを含む方 法。 [II] A method for testing whether a test substance has an effect of preventing or treating adhesions, comprising measuring the ability of the test substance to inhibit the expression of CCR8 or CCL1.
[12] CCR8あるいは CCL1、または CCR8あるいは CCLlをコードするオリゴヌクレオチドまた はポリヌクレオチド、または CCR8を発現する細胞を含む、癒着の予防または治療効 果を有する物質をスクリーニングするためのキット。  [12] A kit for screening a substance having a preventive or therapeutic effect on adhesions, including an oligonucleotide or polynucleotide encoding CCR8 or CCL1, or CCR8 or CCLl, or a cell expressing CCR8.
[13] 被検物質が癒着の予防または治療効果を有するか否かを検定する方法であって、 前記被検物質が腹膜中皮細胞と腹腔マクロファージが凝集塊を形成することを抑制 する能力を測定することを含む方法。 [13] A method for testing whether a test substance has an effect of preventing or treating adhesions, A method comprising measuring the ability of the test substance to inhibit peritoneal mesothelial cells and peritoneal macrophages from forming aggregates.
腹膜中皮細胞、腹腔マクロファージおよび CCL1の少なくとも 2つを含む、癒着の予防 または治療効果を有する物質をスクリーニングするためのキット。 A kit for screening a substance having an effect of preventing or treating adhesions, comprising at least two of peritoneal mesothelial cells, peritoneal macrophages and CCL1.
PCT/JP2005/021980 2004-11-30 2005-11-30 Agent for diagnosing, preventing and treating adhesion with the use of ccr8 inhibitor WO2006059637A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2004-345267 2004-11-30
JP2004345267 2004-11-30
JP2005-264160 2005-09-12
JP2005264160A JP2006182759A (en) 2004-11-30 2005-09-12 Agent for diagnosing, preventing and treating adhesion with use of ccr8 inhibitor

Publications (1)

Publication Number Publication Date
WO2006059637A1 true WO2006059637A1 (en) 2006-06-08

Family

ID=36565071

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/021980 WO2006059637A1 (en) 2004-11-30 2005-11-30 Agent for diagnosing, preventing and treating adhesion with the use of ccr8 inhibitor

Country Status (2)

Country Link
JP (1) JP2006182759A (en)
WO (1) WO2006059637A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244400A (en) * 2021-05-21 2021-08-13 华北理工大学 Application of C5aRA in preparation of product for treating and/or preventing abdominal cavity adhesion

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0478210A (en) * 1990-07-18 1992-03-12 Miharu Tsushin Kk Automatic frequency control method for notch filter or band eliminate filter

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMARI MASAO.: "Kyomaku Yuchaku no Shoki Byohen ni okeru Chuhi Saibo to Taishoku Saibo no Sogo Sayo.", DAI 44 KAI THE JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY SOKAI, GAKUJUTSU SHUKAI, KOEN PROGRAM YOKOSHU., 2003, pages 71, XP002996725 *
HAQUE N. S. ET AL: "Chemokine receptor-8 (CCR8) mediates human vascular smooth muscle cell chemotaxis and metalloproteinase-2 secretion.", BLOOD., vol. 103, no. 4, 15 February 2004 (2004-02-15), pages 1296 - 1304, XP002988211 *
TREBST C.: "CC Chemokine Receptor 8 in the Central Nervous System Is Associated with phagocytic Macrophages.", AMERICAN JOURNAL OF PATHOLOGY., vol. 162, no. 2, February 2003 (2003-02-01), pages 427 - 438, XP009023102 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244400A (en) * 2021-05-21 2021-08-13 华北理工大学 Application of C5aRA in preparation of product for treating and/or preventing abdominal cavity adhesion

Also Published As

Publication number Publication date
JP2006182759A (en) 2006-07-13

Similar Documents

Publication Publication Date Title
US20170226182A1 (en) Pd-1 modulation and uses thereof for modulating hiv replication
AU2012307336B2 (en) Methods for monitoring responsiveness to anti-SMAD7 therapy
JP6999417B2 (en) Therapeutic targets and biomarkers in IBD
JP6437467B2 (en) Molecular targets and compounds useful in the treatment of fibrotic diseases and methods for identifying them
KR101820572B1 (en) Method for providing the information for chronic myeloid leukemia
KR20170031245A (en) Methods and compositions for diagnosing and treating inflammatory bowel disorders
KR102157768B1 (en) Method for diagnosing a molecular phenotype of a patient suffering from an illness accompanied by chronic inflammation
KR20230155405A (en) Biomarkers for diagnosing or predicting prognosis of SARS-CoV-2-induced sepsis
WO2006059637A1 (en) Agent for diagnosing, preventing and treating adhesion with the use of ccr8 inhibitor
WO2021011799A1 (en) Inflammatory bowel diseases therapy involving disrupting ccr9:drd5 heteromer assembly
EP2795337B1 (en) Screening methods to identify compounds useful in the prevention and/or treatment of inflammatory conditions
TW202110436A (en) Gpcr heteromer inhibitors and uses thereof
US9709552B2 (en) Use of inhibitors of leukotriene B4 receptor BLT2 for treating asthma
JPWO2005046724A1 (en) Treatment / prevention agent for vascular disorder and hypertension, and screening method thereof
CN109071661A (en) The antiviral immunity therapy connected by membrane receptor
US20220364184A1 (en) Compositions and methods for treatment of a poor prognosis subtype of colorectal cancer
EP1542016A1 (en) PUMA, phosphorylated p53 and p38 map kinase as markers for HIV-1-induced apoptosis in circulating T cells of infected patient
WO2022073915A1 (en) Gdf3 as biomarker and biotarget in post-ischemic cardiac remodeling
KR20200112069A (en) Use of Jmjd2b inhibitors for preventing or treating of bone diseases
KR20200030444A (en) Composition for preventing or treating behcet's diseases containing cd83 inhibitor
WO2015182121A1 (en) Method for examining allergy-inducing substance and diagnosis and treatment of allergy
AU2002356400A1 (en) Functional inactivation of cxcr4-mediated responses in growth hormone transgenic mice through socs3 upregulation
Martin Molecular and cellular mechanisms of increased angiogenesis in multiple myeloma: a role for CXCL12.
NZ622067B2 (en) Methods for monitoring responsiveness to anti-smad7 therapy
KR20100104316A (en) Pharmaceutical composition for treating and preventing bony erosion and osteoporosis comprising interleukin-32 inhibitor

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05811570

Country of ref document: EP

Kind code of ref document: A1