WO2013111714A1 - Prophylactic or therapeutic agent for bronchial asthma and method for screening same - Google Patents

Prophylactic or therapeutic agent for bronchial asthma and method for screening same Download PDF

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WO2013111714A1
WO2013111714A1 PCT/JP2013/051120 JP2013051120W WO2013111714A1 WO 2013111714 A1 WO2013111714 A1 WO 2013111714A1 JP 2013051120 W JP2013051120 W JP 2013051120W WO 2013111714 A1 WO2013111714 A1 WO 2013111714A1
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heme peroxidase
peroxidase
heme
activity
bronchial asthma
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PCT/JP2013/051120
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French (fr)
Japanese (ja)
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出原　賢治
和彦 有馬
鈴木　章一
裕士 白石
昭一郎 太田
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国立大学法人佐賀大学
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Priority to JP2013555252A priority Critical patent/JP6143363B2/en
Publication of WO2013111714A1 publication Critical patent/WO2013111714A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • G01N33/5023—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 expression patterns

Definitions

  • the present invention relates to a preventive or therapeutic agent for bronchial asthma and a screening method thereof.
  • Bronchial asthma is a disease characterized by increased airway hypersensitivity to nonspecific stimulating substances, and is a disease that causes clinical symptoms such as cough, sputum, and dyspnea.
  • the therapeutic agents used in the treatment of bronchial asthma are mainly inhaled steroids, which are used in combination with leukotriene receptor antagonists, ⁇ 2 receptor stimulants, theophylline agents and the like.
  • inhaled steroids are highly effective for bronchial asthma, 10-15% of all bronchial asthma patients show steroid resistance, and patients with such steroid resistance are treated with drugs other than inhaled steroids Is required. In addition, in children with bronchial asthma, etc., the use of inhaled steroids may be refrained from the viewpoint of side effects.
  • Non-patent Document 1 thiamazole has been widely used as a therapeutic agent for hyperthyroidism, and its safety has been confirmed.
  • Non-Patent Document 2 agranulocytosis and polyarthritis are known as the main side effects of thiamazole, but the frequency is very low, less than 1%, and skin symptoms such as eczema and itch.
  • Non-Patent Document 3 describes that thiamazole does not cause chromosomal abnormalities in spermatogonia, spermatocytes, and bone marrow cells.
  • An object of the present invention is to provide a prophylactic or therapeutic agent for bronchial asthma that has a different mechanism of action from an inhaled steroid and can replace the inhaled steroid.
  • heme peroxidase in tracheal tissue is a factor that exacerbates bronchial asthma, and heme peroxidase inhibitors are useful for improving the pathology of bronchial asthma As a result, the present invention has been completed.
  • the present invention provides the following preventive or therapeutic agents for bronchial asthma, screening methods for preventive or therapeutic agents for bronchial asthma, and the like.
  • a prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor containing a heme peroxidase inhibitor.
  • the heme peroxidase inhibitor is at least one selected from the group consisting of the following (a) to (h): (a) a compound that inhibits the activity of heme peroxidase, (b) a compound that inhibits the expression of heme peroxidase, (c) an antibody that inhibits the activity of heme peroxidase, (d) siRNA or shRNA against a polynucleotide encoding heme peroxidase, (e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof, (f) a ribozyme against a polynucleotide encoding heme peroxidase, (g) a variant of heme peroxidase that acts dominant
  • the heme peroxidase inhibitor is at least one selected from the group consisting of the following (a) to (h): (a) a compound that inhibits the activity of heme peroxidase, (b) a compound that inhibits the expression of heme peroxidase, (c) an antibody that inhibits the activity of heme peroxidase, (d) siRNA or shRNA against a polynucleotide encoding heme peroxidase, (e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof, (f) a ribozyme against a polynucleotide encoding heme peroxidase, (g) a variant of heme peroxidase that acts dominantly negatively on heme peroxida
  • [3a] The method according to [1] above, wherein the heme peroxidase inhibitor is thiamazole.
  • [4a] The method according to any one of [1a] to [3a] above, wherein the bronchial asthma is steroid-resistant bronchial asthma.
  • [5] A method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with a cell expressing heme peroxidase and using the activity or expression level of heme peroxidase as an index.
  • a test compound in which the activity or expression level of heme peroxidase in the case of (i) is lower than the activity or expression level of heme peroxidase in the case of (ii) is a candidate compound for a prophylactic or therapeutic agent for bronchial asthma
  • the method according to [6] above, which is selected as [8] A method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with heme peroxidase and using the activity of heme peroxidase as an index.
  • a prophylactic or therapeutic agent for bronchial asthma that can improve the pathological condition of bronchial asthma by an action mechanism different from that of a steroid agent is provided.
  • the preventive or therapeutic agent for bronchial asthma according to a preferred embodiment of the present invention is also effective for steroid resistant bronchial asthma patients.
  • Example 1 The experimental result which investigated the effect
  • FIG. 6 shows the results of an experiment examining the effect of OSCN - ions on airway epithelial cells (Example 2). The experimental result which investigated the effect
  • Example 4 The result of another experiment which investigated the effect
  • the present invention provides a prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor.
  • the present invention also provides a method for screening a prophylactic or therapeutic agent for bronchial asthma using the activity or expression level of heme peroxidase as an index.
  • the present inventors considered that some anions which transmits Pendorin forms the asthmatic condition, SCN as such anions - focused on the ion.
  • the present inventors have found that during drinking asthma model mice SCN - since the asthma-like conditions to obtain a result that deterioration in mice was added ions (Fig. 1 (B)), SCN - ions to asthma It was revealed that it was an exacerbation factor.
  • the present inventors consider that SCN ⁇ ions undergo a reaction of SCN ⁇ + H 2 O 2 ⁇ OSCN ⁇ + H 2 O together with hydrogen peroxide (H 2 O 2 ) by heme peroxidase in airway tissues. It was.
  • the inventors of the present invention have obtained the result that the activity of NF- ⁇ B, which is a molecule important for inflammation, is increased by increasing the OSCN ⁇ ion in the cell in the cell culture system experiment (FIG. 2).
  • OSCN - ion was also found to be an exacerbation factor for asthma.
  • the present inventors have revealed that heme peroxidase in the airway tissue is a factor that exacerbates asthma.
  • the present inventor further revealed that inhibiting the activity of heme peroxidase, inhibiting the expression of heme peroxidase, and the like lead to improvement of the pathological condition of bronchial asthma.
  • thiamazole one of the heme peroxidase inhibitors and used as an anti-hyperthyroid drug, was administered to mice, it showed increased airway hypersensitivity and asthma-like conditions such as eosinophilic inflammation. It was able to be suppressed (FIGS. 3 (A) to (C)).
  • Prophylactic or therapeutic agent for bronchial asthma containing heme peroxidase inhibitor provides a prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor.
  • the present invention also provides a method for preventing or treating bronchial asthma, comprising administering a therapeutically effective amount of a heme peroxidase inhibitor to a patient in need of preventing or treating bronchial asthma.
  • Heme peroxidase inhibitor used in the present invention is a family of peroxidases and is a general term for peroxidases containing heme in the active site. In animals, myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), thyroid peroxidase (TPO) and the like are included in this family. These heme peroxidases catalyze an oxidation reaction of a substrate substance in a hydrogen peroxide-dependent manner.
  • MPO myeloperoxidase
  • EPO eosinophil peroxidase
  • LPO lactoperoxidase
  • TPO thyroid peroxidase
  • heme peroxidase is not particularly limited, and examples thereof include those derived from humans and those derived from mice.
  • the cDNA sequences and amino acid sequences of various heme peroxidases are as follows.
  • heme peroxidase is used in the sense of including its variants as long as it has substantially the same quality of activity.
  • the mutant include one to a plurality (for example, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 26) in the amino acid sequence of the above heme peroxidase. 25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1 15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1 (-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acids deleted, substituted, inserted, and / or added to the protein. . In general, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously.
  • the substantially homogeneous activity includes, for example, the activity of heme peroxidase.
  • Substantially homogeneous indicates that their activities are qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, it is preferable that the activity of heme peroxidase is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, the degree of these activities and the molecular weight of the protein Quantitative factors such as may be different.
  • the activity of heme peroxidase means an enzyme activity that catalyzes an oxidation reaction of a substance by peroxide.
  • Heme peroxidase inhibitor means a substance having an activity of inhibiting the activity of heme peroxidase or inhibiting the expression of heme peroxidase.
  • siRNA, shRNA, antibody, antisense, peptide, protein, enzyme and the like can be used in addition to a low molecular weight or high molecular compound.
  • “Inhibiting the activity of heme peroxidase” means inhibiting the enzyme activity of heme peroxidase.
  • ⁇ Inhibiting heme peroxidase expression '' means a series of events from gene encoding the protein to protein production, including inhibition of gene expression of the protein (e.g. transcription (production of mRNA), translation (protein Inhibiting the event of any of the above (including production) means inhibiting the production of the protein.
  • transcription production of mRNA
  • translation protein Inhibiting the event of any of the above (including production) means inhibiting the production of the protein.
  • heme peroxidase inhibitor used in the present invention a substance that inhibits the activity of heme peroxidase, a substance that inhibits the expression of heme peroxidase, or the like is used. Specifically, from the following (a) to (h) And at least one substance selected from the group.
  • “compound” includes low molecular weight compounds and high molecular weight compounds.
  • “Low molecular weight compound” means organic and inorganic substances having a molecular weight of 10,000 or less (preferably a molecular weight of 5,000 or less, more preferably a molecular weight of 2,000 or less, particularly preferably a molecular weight of 700 or less).
  • the “polymer compound” means an organic substance having a molecular weight of over 10,000 (preferably a molecular weight of 50,000 or more, more preferably a molecular weight of 100,000 or more).
  • the activity of heme peroxidase can be measured by a known method or a method analogous thereto.
  • the activity of heme peroxidase is, for example, a method using a color reaction generated by oxidation of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) (Thomas, EL et al, J Dent Res 73,544-555, 1994), etc. Can be measured.
  • TMB 5,5′-tetramethylbenzidine
  • the expression level of heme peroxidase can also be measured by a known method or a method analogous thereto.
  • the expression level of heme peroxidase is, for example, measuring the protein level of heme peroxidase (Free (Radic Biol Med, 49,1354-60,2010), measuring the amount of hemeperoxidase mRNA (J Immunol, 167, 1672- 1682, 2001), etc.
  • the compound (including salt form) that inhibits heme peroxidase activity is not particularly limited as long as it is a compound that can inhibit heme peroxidase activity. Examples thereof include a compound that binds to peroxidase and inhibits its enzyme activity, or a salt thereof.
  • Such a compound may be, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like.
  • the compound may be a novel compound or a known compound.
  • the salt form compounds include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. It is done.
  • the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like.
  • Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like.
  • Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like.
  • salts with basic amino acids include salts with arginine, lysine, ornithine and the like
  • salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
  • physiologically acceptable salts are preferred.
  • an inorganic salt such as an alkali metal salt (eg, sodium salt, potassium salt), an alkaline earth metal salt (eg, calcium salt, magnesium salt, barium salt)
  • an inorganic acid such as hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or acetic acid, phthalic acid, fumaric acid, oxalic acid
  • organic acids such as tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid and p-toluenesulfonic acid.
  • examples of the compound capable of inhibiting the activity of heme peroxidase include thiamazole, propylthiouracil, dapsone, azide and the like. Alternatively, such a compound can also be obtained by a screening method described later. These compounds that inhibit the activity of heme peroxidase can be used alone or in combination of two or more thereof as necessary.
  • the compound that inhibits heme peroxidase expression is not particularly limited as long as it can inhibit heme peroxidase expression.
  • a gene encoding heme peroxidase (DNA) encodes heme peroxidase.
  • examples thereof include compounds that inhibit transcription to mRNA, and (ii) compounds that inhibit translation from heme peroxidase-encoding mRNA into heme peroxidase.
  • any compound that inhibits transcription of heme peroxidase-encoding gene (DNA) into heme peroxidase-encoding mRNA any compound that inhibits transcription of heme peroxidase-encoding gene (DNA) into mRNA
  • a compound that binds to a factor involved in transcription from a gene (DNA) encoding heme peroxidase to mRNA and inhibits transcription can be mentioned.
  • any compound that inhibits translation from heme peroxidase-encoding mRNA into heme peroxidase is not particularly limited. Examples thereof include compounds that bind to a factor involved in translation from heme peroxidase-encoding mRNA into heme peroxidase and inhibit the translation.
  • Such a compound can be obtained, for example, by a screening method described later.
  • the compounds that inhibit the expression of heme peroxidase can be used alone or in combination of two or more thereof as necessary.
  • Antibody that inhibits the activity of heme peroxidase since the purpose is to inhibit the activity of heme peroxidase, any antibody that inhibits the activity of heme peroxidase can be used.
  • Such an antibody includes an antibody that can recognize heme peroxidase or a partial peptide thereof, and that inhibits the activity of heme peroxidase.
  • Such an antibody may be either a polyclonal antibody or a monoclonal antibody.
  • the antibody to heme peroxidase or a partial peptide thereof used in the present invention can be produced according to a known method for producing an antibody or antiserum using heme peroxidase or a partial peptide thereof as an antigen.
  • siRNA or shRNA against a polynucleotide encoding heme peroxidase Double-stranded RNA that has RNAi action on a polynucleotide encoding heme peroxidase (e.g., siRNA or shRNA against a polynucleotide encoding heme peroxidase) has low toxicity, and translates the gene encoding heme peroxidase. Since it can suppress and the expression of heme peroxidase can be suppressed, it can be used suitably as a substance which inhibits the expression of heme peroxidase.
  • Such double-stranded RNA having an RNAi action on a polynucleotide encoding heme peroxidase includes a double-stranded RNA containing a part of RNA encoding heme peroxidase (e.g., a polynucleotide encoding heme peroxidase).
  • SiRNA for nucleotides small (short) interfering RNA
  • shRNA small (short) hairpin RNA
  • Such double-stranded RNA is obtained by a known method (e.g., Nature, 411, 494, 2001; Special Tables 2002-516062; U.S. Patent Application Publication No. 2002/086356; Nature Genetics, 24). Volume, 180-183, 2000; Genesis, 26, 240-244, 2000; Nature, 407, 319-320, 2002; Genes & Dev., 16, 948-958, 2002 Proc. Natl. Acad. Sci. USA., 99, 5515-5520, 2002; Science, 296, 550-553, 2002; Proc. Natl. Acad. Sci.
  • the length of the double-stranded RNA having RNAi action used in the present invention is usually 17 to 30 bases, preferably 19 to 27 bases, more preferably 20 to 22 bases.
  • an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof, preferably a polynucleotide (preferably DNA) encoding heme peroxidase (hereinafter referred to as DNA)
  • DNA a polynucleotide encoding heme peroxidase
  • the base sequence substantially complementary to the DNA used in the present invention is, for example, the entire base sequence of the base sequence complementary to the DNA used in the present invention (that is, the complementary strand of the DNA used in the present invention) or Examples thereof include base sequences having homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a partial base sequence.
  • the base sequence of the portion encoding the N-terminal site of heme peroxidase for example, the start An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand (such as a base sequence near a codon)
  • an antisense polynucleotide directed to RNA degradation by RNaseH it is about 70% or more, preferably about 80% or more, more preferably about 80% or more with the complementary strand of the entire base sequence of DNA used in the present invention including introns.
  • Each of the antisense polynucleotides having 90% or more, most preferably about 95% or more homology is preferred.
  • the antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is changed to a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted.
  • the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base part (pyrimidine, purine) is also chemically modified. It may be any one as long as it hybridizes with DNA having the base sequence of the polynucleotide encoding heme peroxidase.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • the antisense polynucleotide of the present invention may be altered or contain modified sugars, bases and bonds, and may be provided in special forms such as liposomes, microspheres, or applied by gene therapy. Can be provided in an added form.
  • the additional form can be used as a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate group skeleton, a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acid ( Examples include hydrophobic ones such as phospholipid and cholesterol.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.).
  • nucleic acid can be attached via a base, sugar, intramolecular nucleoside bond.
  • the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • Ribozyme against a polynucleotide encoding heme peroxidase A polynucleotide having ribozyme activity against a polynucleotide encoding heme peroxidase can suppress the expression of heme peroxidase, and therefore inhibits the expression of heme peroxidase. Can be suitably used.
  • Such ribozymes are prepared by known methods (eg, TRENDS in Molecular Medicine, 7, 221, 2001; FEBS Lett., 228, 228, 1988; FEBS Lett., 239, 285, 1988). Nucl. Acids. Res., 17, 7059, 1989; Nature, 323, 349, 1986; Nucl. Acids.
  • RNA encoding heme peroxidase examples include a portion (RNA fragment) close to the cleavage site on the RNA encoding heme peroxidase that can be cleaved by a known ribozyme.
  • the ribozymes include large ribozymes such as group I introns and M1 RNA contained in RNaseP, and small ribozymes such as hammerhead and hairpin types (Protein Nucleic Acid Enzyme, 35, 2191, 1990).
  • hammerhead ribozymes include FEBS Lett., 228, 228, 1988; FEBS Lett., 239, 285, 1988; Protein Nucleic Acid Enzyme, 35, 2191, 1990; Nucl.
  • heme peroxidase that acts dominantly against heme peroxidase or a polynucleotide that encodes it
  • the term “mutant of a protein that acts dominantly against heme peroxidase” means a protein that has an action of inhibiting (eliminating or reducing) the activity of heme peroxidase when it is expressed. (See Taira Yoshikazu, edited by gene function inhibition experiment, Yodosha, pp. 26-32, 2001).
  • an aptamer for heme peroxidase can inhibit the activity and function of heme peroxidase, it can be suitably used as a substance that inhibits the activity of heme peroxidase.
  • Aptamers are obtained using a known method, for example, the SELEX (systematic evolution of ligands by exponential enrichment) method (Annual Review of Medicine 56, 555-583, 2005).
  • the structure of an aptamer can be determined using a known method, and an aptamer is produced according to a known method based on the structure.
  • a medicament containing the above-mentioned heme peroxidase inhibitor is used as a prophylactic or therapeutic agent for bronchial asthma. Since the heme peroxidase inhibitor of the preferred embodiment has a preventive / therapeutic effect on bronchial asthma, it may be administered for therapeutic purposes to patients with bronchial asthma, or for the purpose of prevention for patients who must consider prevention or recurrence of bronchial asthma Can also be administered. In a preferred embodiment of the invention, the heme peroxidase inhibitor is administered for therapeutic purposes.
  • Bronchial asthma is a disease characterized by increased airway hypersensitivity to nonspecific stimulating substances, and is a disease that causes clinical symptoms such as cough, sputum, and dyspnea.
  • the preventive or therapeutic agent of the present invention can prevent or treat at least one of these symptoms.
  • the preventive or therapeutic agent of the present invention can improve the pathological condition of bronchial asthma by an action mechanism different from that of an inhaled steroid. For this reason, the preventive or therapeutic agent of the preferable aspect of this invention is useful for the treatment of the patient resistant to a steroid.
  • the above-mentioned heme peroxidase inhibitor can be formulated according to conventional means and used as a prophylactic or therapeutic agent for bronchial asthma.
  • the heme peroxidase inhibitor is thiamazole.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like.
  • Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field.
  • a carrier for example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • compositions for parenteral administration for example, injections, suppositories, eye drops, nasal drops, spray inhalants, coating agents, patches and the like are used, and the injections are intravenous injections and subcutaneous injections.
  • dosage forms such as intradermal injection, intramuscular injection, infusion injection, intraarticular injection and the like.
  • Such an injection is prepared by a method known per se, for example, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily liquid usually used for injections.
  • aqueous solution for injection for example, isotonic solution containing physiological saline, glucose and other adjuvants, etc.
  • solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • Suppositories used for rectal administration are prepared by mixing the above active ingredients with a conventional suppository base. Eye drops, nasal drops, spray inhalants, suppositories, coating agents, patches and the like are also prepared according to methods known per se using the above active ingredients.
  • compositions may contain other active ingredients (for example, other prophylactic or therapeutic agents for bronchial asthma) as long as undesirable interactions are not caused by blending with the active ingredients. Further, it may be used in combination with other active ingredients (for example, other prophylactic or therapeutic agents for bronchial asthma).
  • active ingredients for example, other prophylactic or therapeutic agents for bronchial asthma
  • the dose of the active ingredient of the preventive or therapeutic agent of the present invention varies depending on its action, target disease, administration subject, symptom, administration route, etc., but for example, when administered orally, generally in adults (weight 60 kg)
  • the dosage of the active ingredient varies depending on the target disease, administration subject, symptom, administration route, etc., but when administered in the form of an injection, it is generally an adult (with a body weight of 60 kg).
  • the antisense polynucleotide can be formulated and administered according to a method known per se.
  • an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc.
  • a human or mammal eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.
  • the antisense polynucleotide can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
  • the above-mentioned antisense polynucleotide is formulated alone (injection) together with a carrier such as liposome and administered to veins, etc. Also good.
  • the double-stranded RNA, ribozyme, mutant of heme peroxidase that acts dominantly against the heme peroxidase or the polynucleotide encoding the same can be formulated and administered in the same manner as the antisense polynucleotide. it can.
  • the above antibodies, aptamers and the like can be administered per se or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such compositions are provided as dosage forms suitable for oral or parenteral administration (eg, intravenous injection). Preferably it is provided as an inhalant.
  • the present invention relates to a method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with a cell expressing heme peroxidase and using the heme peroxidase activity or heme peroxidase expression level as an index. Provide a way to do it.
  • the present invention also provides a method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with heme peroxidase and using the activity of heme peroxidase as an index.
  • a preferred embodiment of the screening method of the present invention is a method comprising evaluating a heme peroxidase inhibitory activity of a test compound and selecting a compound having a peroxidase inhibitory activity.
  • the selected compound having peroxidase inhibitory activity is a candidate compound for a prophylactic or therapeutic agent for bronchial asthma.
  • Heme peroxidase inhibitory activity means an activity that reduces or suppresses the activity of heme peroxidase or the expression level of heme peroxidase.
  • test compound examples include peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
  • the test compound may form a salt, and as the salt of the test compound, physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, bases And salts with acidic or acidic amino acids.
  • the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like.
  • Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like.
  • Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like.
  • salts with basic amino acids include salts with arginine, lysine, ornithine and the like
  • salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
  • a test compound in some methods for screening a prophylactic or therapeutic agent for bronchial asthma according to the present invention, (i) when a test compound is contacted with a cell expressing heme peroxidase, and (ii) the test compound expresses heme peroxidase Comparison of the activity or expression level of heme peroxidase in the cells when not in contact with the cells is performed.
  • a test compound is contacted with cells expressing heme peroxidase.
  • the origin of the “cell” used is not particularly limited, but is, for example, a cell derived from a human, a mouse or the like, and preferably a cell derived from a human.
  • Cells expressing heme peroxidase used in the screening method of the present invention are, for example, airway epithelial cells, neutrophils, eosinophils and the like.
  • the “cell expressing heme peroxidase” used in the screening method of the present invention can also be prepared by a general genetic engineering technique.
  • the activity or expression level of heme peroxidase is measured. Specifically, for example, in the cases (i) and (ii) above, the cells are cultured, and the activity or expression level of heme peroxidase in each case is measured.
  • the measurement of the activity or expression level of heme peroxidase can be performed, for example, by the above-described method for measuring the activity or expression level of heme peroxidase.
  • a compound that inhibits the activity or expression of heme peroxidase is selected as compared with the case where the test compound is not contacted (control).
  • a test compound that decreases by 50% or more can be selected as a compound that inhibits the activity of heme peroxidase.
  • test compound in which the expression level of heme peroxidase in the case of (i) is lower than the expression level of heme peroxidase in the case of (ii), in particular, 10% or more, 20% or more, 30% or more Test compounds that decrease by 40% or more, or 50% or more can be selected as compounds that inhibit heme peroxidase expression.
  • the activity of heme peroxidase is compared without using cells. That is, the activity of heme peroxidase is compared when (i) the test compound is contacted with heme peroxidase and (ii) when the test compound is not contacted with heme peroxidase.
  • the test compound is contacted with heme peroxidase.
  • a test compound is added to a reaction system that causes a reaction of SCN ⁇ + H 2 O 2 ⁇ OSCN ⁇ + H 2 O using heme peroxidase.
  • Heme peroxidase is as described above, but it is preferable to use one purified as heme peroxidase.
  • the reaction is preferably performed in a test tube.
  • the activity of heme peroxidase is measured. Specifically, for example, in the cases (i) and (ii) above, the activity of heme peroxidase in each case is measured.
  • the activity of heme peroxidase can be measured, for example, by the above-described method for measuring the activity of heme peroxidase.
  • a compound that inhibits the activity of heme peroxidase is selected as compared with the case where the test compound is not contacted (control).
  • a test compound that decreases by 50% or more can be selected as a compound that inhibits the activity of heme peroxidase.
  • the selected candidate compound is administered to an experimental animal (eg, mouse, rat, etc.), and the effect of the selected candidate compound on the prevention or treatment of bronchial asthma is confirmed.
  • an experimental animal eg, mouse, rat, etc.
  • the effect of the selected candidate compound on the prevention or treatment of bronchial asthma is confirmed.
  • FIG. 1 (A) shows the experimental protocol. As shown in the protocol of FIG. 1 (A), drinking water containing 20 mM thiocyanate ion (SCN ⁇ ) was taken daily from Day 0. Sensitize with allergen ovalbumin on days 0 and 12 (sensitization 1, 2), and ovalbumin exposure on days 22, 23, 24 (exposure 1-3), airway 24 hours after exposure 3 Hypersensitivity was measured. The group in which the experiment was conducted with this protocol is defined as an asthma group (+ SCN) or a sensitized group (+ SCN).
  • SCN ⁇ 20 mM thiocyanate ion
  • a non-asthma group (-SCN), a non-asthma group (+ SCN) group, and an asthma group (-SCN) were used.
  • the non-asthma group (-SCN) is indicated as the non-sensitized (-SCN) group in FIG. 1 (B), and sensitization with ovalbumin was not performed, and thiocyanate was used instead of drinking thiocyanate ion.
  • the experiment was performed using the same protocol as the sensitized group (+ SCN) except that drinking water containing no ions was administered.
  • the non-asthma group (+ SCN) group is shown as the non-sensitized (+ SCN) group in FIG.
  • non-asthmatic group (+ SCN) group is the same as the sensitized (+ SCN) group except that no sensitization with ovalbumin Experiments were performed using the same protocol.
  • the asthma group (-SCN) is labeled as a sensitized (-SCN) group in FIG. 1 (B), except that the thiocyanate ion-free drinking water was administered instead of the thiocyanate ion drinking water, Experiments were performed using the same protocol as the sensitized (+ SCN) group.
  • Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine (purchased from sigma) was diluted with physiological saline and used at the concentration shown in FIG. 1 (B).
  • BUXCO unrestrained whole body plethysmography
  • FIG. 1 (B) The results are shown in FIG. 1 (B). Increased airway hyperresponsiveness was observed in the sensitized group compared to the non-sensitized group. Then, in the sensitization Yes group, SCN - in drinking water Yes group (with sensitization (+ SCN) group), SCN - further enhancement of airway hyperresponsiveness compared to drinking water without the group (with sensitization (-SCN) group) was recognized. From the above results, it was revealed that SCN - ion is an exacerbation factor for asthma.
  • Example 2 Effect of OSCN - ion on airway epithelial cells
  • the following reaction was caused in a cell culture medium.
  • Airway epithelial cells (NCI-H292 cells) were suspended in RPMI1640 / 5% FCS / 50 mM HEPES medium, and 1.8 ⁇ 10 6 cells were seeded in a 6-well plate. After overnight culture, NaSCN (final concentration 1 mM), GOX (final concentration 9 mUnits / ml), and LPO (final concentration 40 U / ml) were added to the cell culture medium. After further incubation for 5 hours, a nuclear extract was prepared using the method of Schreiber et al (Nucleic Acids Res.
  • FIG. 3 (A) shows the experimental protocol. As shown in the protocol of FIG. 3 (A), sensitization was performed with ovalbumin, an allergen, on day 0 and 12 (sensitization 1, 2), and ovalbumin exposure was performed on days 22, 26, 30 ( Exposure 1-3). From Day 20, I took drinking water containing 0.2 mg / ml thiamazole every day.
  • asthma group (with thiamazole).
  • the asthma group (with thiamazole) is represented as a thiamazole administration group in FIGS. 3 (B) and 3 (C).
  • a non-asthma group and an asthma group were used.
  • the non-asthma group is described as a control group in FIGS. 3 (B) and 3 (C), and sensitization with ovalbumin was not performed, and drinking water containing no thiamazole was administered instead of drinking thiamazole. Except for this, this group was tested using the same protocol as the asthma group (with thiamazole).
  • the asthma group (without thiamazole) is labeled as the asthma group in FIGS. 3 (B) and (C), and the asthma group (thiamazole) was administered except that thiazazole-free drinking was administered instead of thiamazole drinking. This is a group in which an experiment was conducted with the same protocol as (Yes).
  • BALB / c mice (6 weeks old, male) obtained from Japan SLC were used.
  • Drinking water containing thiamazole was prepared by dissolving 20 mg thiamazole (Wako Pure Chemical Industries) in 100 ml sterile distilled water (final concentration 0.2 mg / ml).
  • Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine was diluted with physiological saline and used at the concentration shown in FIG. 3 (B).
  • BUXCO unrestrained whole body plethysmography
  • Bronchoalveolar lavage fluid was collected and analyzed according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, bronchoalveolar lavage fluid was prepared using 1.5 ml of physiological saline, and the total number of cells in this lavage fluid was measured with a hemocytometer (CDA500; Sysmex), and then the cells in the lavage fluid were cytospun and Diff-Quik The number of eosinophils was measured by staining with (Sysmex).
  • FIG. 3 (B) The measurement results of airway hypersensitivity are shown in FIG. 3 (B).
  • suppression of airway hypersensitivity was observed compared to the group not receiving thiamazole (asthma group).
  • FIG. 3 (C) The analysis result of bronchoalveolar lavage fluid is shown in FIG. 3 (C).
  • suppression of the number of eosinophils in the bronchoalveolar lavage fluid was observed compared to the group not receiving thiamazole (asthma group).
  • Example 4 Comparison of effects of thiamazole and steroids in bronchial asthma model mice
  • inhibition of heme peroxidase activity, inhibition of heme peroxidase expression, and the like are related to the steroid drug dexamethasone (DEX) Even when compared with the administration of, it has been confirmed that it exhibits an excellent preventive or therapeutic effect on bronchial asthma.
  • FIG. 4 (A) shows the experimental protocol. As shown in the protocol of FIG. 4 (A), sensitization was performed with ovalbumin, an allergen, on Day 0 and 12 (sensitization 1, 2), and exposure to ovalbumin was performed on Day 22, 23, 24 ( Exposure 1-3).
  • a non-asthma group to which no drug was administered and an asthma group to which no drug was administered were used.
  • the non-asthmatic group that did not receive the drug did not receive sensitization with ovalbumin, and the same protocol as the asthmatic group (thiamazole), except that it received drinking water that did not contain thiamazole instead of drinking thiamazole.
  • the asthma group to which no drug is administered is a group in which an experiment was conducted according to the same protocol as the asthma group (thiamazole), except that drinking water containing no thiamazole was administered instead of drinking thiamazole.
  • BALB / c mice (6 weeks old, female) obtained from Japan SLC were used for the experiment.
  • Drinking water containing thiamazole was prepared by dissolving 20 mg thiamazole (Wako Pure Chemical Industries) in 100 ml sterile distilled water (final concentration 0.2 mg / ml).
  • the physiological saline containing DEX was prepared and administered as follows. Water-soluble DEX (cat No. D2915, Sigma-Aldrich Japan) was dissolved in physiological saline to a concentration of 25 ⁇ g / ml or 75 ⁇ g / ml, and 200 ⁇ l of each was intraperitoneally administered.
  • the method of preparation, sensitization, and exposure of ovalbumin was performed according to the method described in Example 3.
  • Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine was diluted with physiological saline and used at the concentration shown in FIG. 4 (B).
  • BUXCO unrestrained whole body plethysmography
  • Bronchoalveolar lavage fluid was collected and analyzed according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, bronchoalveolar lavage fluid was prepared using 1.5 ml of physiological saline, and the total number of cells in the lavage fluid was measured with a hemocytometer (CDA500; Sysmex). Furthermore, the cells in the washing solution were treated with respective antibodies against Ly-6G antigen (neutrophil marker), siglec-F antigen (eosinophil marker), CD3 antigen (T cell marker), and F4 / 80 antigen (macrophage marker).
  • Ly-6G antigen neutral marker
  • siglec-F antigen eosinophil marker
  • CD3 antigen T cell marker
  • F4 / 80 antigen microphage marker
  • FIG. 4 (B) The measurement results of airway hypersensitivity are shown in FIG. 4 (B).
  • the asthma group (thiamazole) further suppression of airway hypersensitivity was observed compared to the asthma group (DEX 5 ⁇ g) and the asthma group (DEX 15 ⁇ g).
  • the analysis results of bronchoalveolar lavage fluid are shown in FIGS. 4 (C) and 4 (D).
  • the number of cells in the bronchoalveolar lavage fluid eosinophil count, neutrophil count, macrophage count, And suppression of T cell count. From the above, inhibiting heme peroxidase activity, inhibiting heme peroxidase expression, etc. may be more effective in preventing or treating bronchial asthma than when administering a steroid. It could be confirmed.
  • [SEQ ID NO: 1] This is the cDNA sequence of human lactoperoxidase.
  • [SEQ ID NO: 2] This is the amino acid sequence of human lactoperoxidase.
  • [SEQ ID NO: 3] This is the cDNA sequence of mouse lactoperoxidase.
  • [SEQ ID NO: 4] This is the amino acid sequence of mouse lactoperoxidase.
  • [SEQ ID NO: 5] This is the cDNA sequence of human myeloperoxidase.
  • [SEQ ID NO: 6] is an amino acid sequence of human myeloperoxidase.
  • [SEQ ID NO: 7] This is the cDNA sequence of mouse myeloperoxidase.
  • [SEQ ID NO: 8] This is the amino acid sequence of mouse myeloperoxidase.
  • [SEQ ID NO: 9] This is the cDNA sequence of human eosinophil peroxidase.
  • [SEQ ID NO: 10] This is the amino acid sequence of human eosinophil peroxidase.
  • [SEQ ID NO: 11] This is the cDNA sequence of mouse eosinophil peroxidase.
  • [SEQ ID NO: 12] This is the amino acid sequence of mouse eosinophil peroxidase.
  • [SEQ ID NO: 13] This is the cDNA sequence of human thyroid peroxidase.
  • [SEQ ID NO: 14] This is the amino acid sequence of human thyroid peroxidase.
  • [SEQ ID NO: 15] This is the cDNA sequence of mouse thyroid peroxidase.
  • [SEQ ID NO: 16] Amino acid sequence of mouse thyroid peroxidase.

Abstract

The present invention provides a prophylactic or therapeutic agent for bronchial asthma, said agent comprising a heme peroxidase inhibitor. Thus, a prophylactic or therapeutic agent for bronchial asthma, which has a function mechanism different from the function mechanism of inhaled steroids and is usable as a substitute for inhaled steroids, is provided.

Description

気管支喘息の予防又は治療薬及びそのスクリーニング方法Prophylactic or therapeutic agent for bronchial asthma and screening method thereof
　本発明は、気管支喘息の予防又は治療薬及びそのスクリーニング方法などに関する。 The present invention relates to a preventive or therapeutic agent for bronchial asthma and a screening method thereof.
　気管支喘息は、非特異的な刺激物質に対して気道過敏性の亢進を示すことを特徴とした疾患であり、咳嗽、喀痰、呼吸困難などの臨床症状を引き起こす疾患である。気管支喘息の治療で用いられる治療薬は吸入ステロイド剤が中心であり、これにロイコトリエン受容体拮抗薬、β2受容体刺激薬、テオフィリン剤などが併用して用いられる。 Bronchial asthma is a disease characterized by increased airway hypersensitivity to nonspecific stimulating substances, and is a disease that causes clinical symptoms such as cough, sputum, and dyspnea. The therapeutic agents used in the treatment of bronchial asthma are mainly inhaled steroids, which are used in combination with leukotriene receptor antagonists, β2 receptor stimulants, theophylline agents and the like.
　　気管支喘息に対する吸入ステロイド剤の有効性は高いが、気管支喘息患者全体の10～15%の患者はステロイド抵抗性を示し、そのようなステロイド抵抗性を示す患者については吸入ステロイド剤以外の薬剤による治療が必要となる。また、小児の気管支喘息患者などにおいては、副作用の観点から吸入ステロイド剤の使用が控えられる場合がある。 Although inhaled steroids are highly effective for bronchial asthma, 10-15% of all bronchial asthma patients show steroid resistance, and patients with such steroid resistance are treated with drugs other than inhaled steroids Is required. In addition, in children with bronchial asthma, etc., the use of inhaled steroids may be refrained from the viewpoint of side effects.
　こうしたことから、吸入ステロイド剤の薬理学的効果とは全く異なり、吸入ステロイド剤に代わりうる気管支喘息治療薬の開発が望まれている。 For these reasons, it is completely different from the pharmacological effects of inhaled steroids, and the development of bronchial asthma drugs that can replace inhaled steroids is desired.
　ところで、チアマゾールは、これまで甲状腺機能亢進症に対する治療薬として広く用いられてきており、その安全性が確認されている(非特許文献1)。 By the way, thiamazole has been widely used as a therapeutic agent for hyperthyroidism, and its safety has been confirmed (Non-patent Document 1).
　また、非特許文献2には、チアマゾールの主な副作用として無顆粒球症、多発性関節炎が知られているが、その頻度は非常に低く1%未満であり、また湿疹や痒みなどの皮膚症状が現れたとしても軽度であることが記載されており、非特許文献3には、チアマゾールは精原細胞や***細胞、ならびに骨髄細胞の染色体異常を引き起こさないことが記載されている。 In Non-Patent Document 2, agranulocytosis and polyarthritis are known as the main side effects of thiamazole, but the frequency is very low, less than 1%, and skin symptoms such as eczema and itch. However, Non-Patent Document 3 describes that thiamazole does not cause chromosomal abnormalities in spermatogonia, spermatocytes, and bone marrow cells.
　本発明は、吸入ステロイド剤と作用機序が異なり、吸入ステロイド剤に代わり得る気管支喘息の予防又は治療薬を提供することを目的とする。 An object of the present invention is to provide a prophylactic or therapeutic agent for bronchial asthma that has a different mechanism of action from an inhaled steroid and can replace the inhaled steroid.
　本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、気管組織内のヘムペルオキシダーゼは気管支喘息を増悪させる因子であること、ヘムペルオキシダーゼ阻害薬が気管支喘息の病態の改善に有用であることなどを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that heme peroxidase in tracheal tissue is a factor that exacerbates bronchial asthma, and heme peroxidase inhibitors are useful for improving the pathology of bronchial asthma As a result, the present invention has been completed.
　すなわち、本発明は、以下に示す、気管支喘息の予防又は治療薬、気管支喘息の予防又は治療薬のスクリーニング方法などを提供する。
[1] ヘムペルオキシダーゼ阻害薬を含有する、気管支喘息の予防又は治療薬。
[2] ヘムペルオキシダーゼ阻害薬が、以下の(a)～(h)からなる群から選択される少なくとも１つである上記[1]に記載の気管支喘息の予防又は治療薬：
(a)ヘムペルオキシダーゼの活性を阻害する化合物、
(b)ヘムペルオキシダーゼの発現を阻害する化合物、
(c)ヘムペルオキシダーゼの活性を阻害する抗体、
(d)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNA、
(e)ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列に相補的若しくは実質的に相補的な塩基配列又はその一部を含有するアンチセンスポリヌクレオチド、
(f)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するリボザイム、
(g)ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチド、及び
(h)ヘムペルオキシダーゼに対するアプタマー。
[3] ヘムペルオキシダーゼ阻害薬がチアマゾールである、上記[1]に記載の気管支喘息の予防又は治療薬。
[4] 気管支喘息が、ステロイド抵抗性の気管支喘息である、上記[1]～[3]のいずれか1項に記載の気管支喘息の予防又は治療薬。
[1a] 気管支喘息の予防または治療を必要とする患者に治療的有効量のヘムペルオキシダーゼ阻害薬を投与することを含む、気管支喘息の予防又は治療方法。
[2a] ヘムペルオキシダーゼ阻害薬が、以下の(a)～(h)からなる群から選択される少なくとも１つである上記[1a]に記載の方法：
(a)ヘムペルオキシダーゼの活性を阻害する化合物、
(b)ヘムペルオキシダーゼの発現を阻害する化合物、
(c)ヘムペルオキシダーゼの活性を阻害する抗体、
(d)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNA、
(e)ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列に相補的若しくは実質的に相補的な塩基配列又はその一部を含有するアンチセンスポリヌクレオチド、
(f)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するリボザイム、
(g)ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチド、及び
(h)ヘムペルオキシダーゼに対するアプタマー。
[3a] ヘムペルオキシダーゼ阻害薬がチアマゾールである、上記[1]に記載の方法。
[4a] 気管支喘息が、ステロイド抵抗性の気管支喘息である、上記[1a]～[3a]のいずれか1項に記載の方法。
[5] 試験化合物をヘムペルオキシダーゼを発現する細胞と接触させ、ヘムペルオキシダーゼの活性又は発現量を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法。
[6] (i)試験化合物をヘムペルオキシダーゼを発現する細胞と接触させた場合と、(ii) 試験化合物をヘムペルオキシダーゼを発現する細胞と接触させない場合の、該細胞におけるヘムペルオキシダーゼの活性又は発現量の比較を行う上記[5]に記載の方法。
[7] 前記(i)の場合におけるヘムペルオキシダーゼの活性又は発現量が、前記(ii)の場合のヘムペルオキシダーゼの活性又は発現量より低くなる試験化合物を、気管支喘息の予防又は治療薬の候補化合物として選択する上記[6]に記載の方法。
[8] 試験化合物をヘムペルオキシダーゼと接触させ、ヘムペルオキシダーゼの活性を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法。
[9] (i)試験化合物をヘムペルオキシダーゼと接触させた場合と、(ii) 試験化合物をヘムペルオキシダーゼと接触させない場合の、ヘムペルオキシダーゼの活性の比較を行う上記[8]に記載の方法。
[10] 前記(i)の場合におけるヘムペルオキシダーゼの活性が、前記(ii)の場合のヘムペルオキシダーゼの活性より低くなる試験化合物を、気管支喘息の予防又は治療薬の候補化合物として選択する上記[9]に記載の方法。 That is, the present invention provides the following preventive or therapeutic agents for bronchial asthma, screening methods for preventive or therapeutic agents for bronchial asthma, and the like.
[1] A prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor.
[2] The prophylactic or therapeutic agent for bronchial asthma according to the above [1], wherein the heme peroxidase inhibitor is at least one selected from the group consisting of the following (a) to (h):
(a) a compound that inhibits the activity of heme peroxidase,
(b) a compound that inhibits the expression of heme peroxidase,
(c) an antibody that inhibits the activity of heme peroxidase,
(d) siRNA or shRNA against a polynucleotide encoding heme peroxidase,
(e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof,
(f) a ribozyme against a polynucleotide encoding heme peroxidase,
(g) a variant of heme peroxidase that acts dominantly negatively on heme peroxidase or a polynucleotide encoding the same, and
(h) Aptamer to heme peroxidase.
[3] The prophylactic or therapeutic agent for bronchial asthma according to [1] above, wherein the heme peroxidase inhibitor is thiamazole.
[4] The agent for preventing or treating bronchial asthma according to any one of [1] to [3] above, wherein the bronchial asthma is steroid-resistant bronchial asthma.
[1a] A method for preventing or treating bronchial asthma, comprising administering a therapeutically effective amount of a heme peroxidase inhibitor to a patient in need of preventing or treating bronchial asthma.
[2a] The method according to [1a] above, wherein the heme peroxidase inhibitor is at least one selected from the group consisting of the following (a) to (h):
(a) a compound that inhibits the activity of heme peroxidase,
(b) a compound that inhibits the expression of heme peroxidase,
(c) an antibody that inhibits the activity of heme peroxidase,
(d) siRNA or shRNA against a polynucleotide encoding heme peroxidase,
(e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof,
(f) a ribozyme against a polynucleotide encoding heme peroxidase,
(g) a variant of heme peroxidase that acts dominantly negatively on heme peroxidase or a polynucleotide encoding the same, and
(h) Aptamer to heme peroxidase.
[3a] The method according to [1] above, wherein the heme peroxidase inhibitor is thiamazole.
[4a] The method according to any one of [1a] to [3a] above, wherein the bronchial asthma is steroid-resistant bronchial asthma.
[5] A method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with a cell expressing heme peroxidase and using the activity or expression level of heme peroxidase as an index.
[6] Activity or expression level of heme peroxidase in the cell when (i) the test compound is contacted with a cell expressing heme peroxidase and (ii) when the test compound is not contacted with a cell expressing heme peroxidase The method according to [5] above, wherein the comparison is performed.
[7] A test compound in which the activity or expression level of heme peroxidase in the case of (i) is lower than the activity or expression level of heme peroxidase in the case of (ii) is a candidate compound for a prophylactic or therapeutic agent for bronchial asthma The method according to [6] above, which is selected as
[8] A method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with heme peroxidase and using the activity of heme peroxidase as an index.
[9] The method according to [8] above, wherein the activity of heme peroxidase is compared when (i) the test compound is contacted with heme peroxidase and (ii) when the test compound is not contacted with heme peroxidase.
[10] The test compound in which the activity of heme peroxidase in the case of (i) is lower than the activity of heme peroxidase in the case of (ii) is selected as a candidate compound for a prophylactic or therapeutic agent for bronchial asthma [9] ] Method.
　本発明によれば、ステロイド剤とは異なる作用機序により気管支喘息の病態を改善することができる気管支喘息の予防又は治療薬が提供される。本発明の好ましい態様の気管支喘息の予防又は治療薬は、ステロイド抵抗性の気管支喘息患者に対しても有効性を示す。 According to the present invention, a prophylactic or therapeutic agent for bronchial asthma that can improve the pathological condition of bronchial asthma by an action mechanism different from that of a steroid agent is provided. The preventive or therapeutic agent for bronchial asthma according to a preferred embodiment of the present invention is also effective for steroid resistant bronchial asthma patients.
気管支喘息モデルマウスに対するSCN－イオンの作用を調べた実験結果を示す(実施例１)。実験のプロトコルを示す図である。The experimental result which investigated the effect | action of SCN - ion with respect to a bronchial asthma model mouse is shown (Example 1). It is a figure which shows the protocol of experiment. 気管支喘息モデルマウスに対するSCN－イオンの作用を調べた実験結果を示す(実施例１)。メサコリン吸入により誘発した気道過敏性の測定結果を示す図である。The experimental result which investigated the effect | action of SCN - ion with respect to a bronchial asthma model mouse is shown (Example 1). It is a figure which shows the measurement result of the airway hypersensitivity induced by the inhalation of mesacolin. 気道上皮細胞に対するOSCN－イオンの作用を調べた実験結果を示す図である（実施例２）。FIG. 6 shows the results of an experiment examining the effect of OSCN - ions on airway epithelial cells (Example 2). 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた実験結果を示す(実施例３)。実験のプロトコルを示す図である。The experimental result which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 3). It is a figure which shows the protocol of experiment. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた実験結果を示す(実施例３)。メサコリン吸入により誘発した気道過敏性の測定結果を示す図である。The experimental result which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 3). It is a figure which shows the measurement result of the airway hypersensitivity induced by the inhalation of mesacolin. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた実験結果を示す(実施例３)。気管支肺胞洗浄液中の好酸球数を示す図である。The experimental result which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 3). It is a figure which shows the number of eosinophils in bronchoalveolar lavage fluid. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた別の実験結果を示す(実施例４)。実験のプロトコルを示す図である。The result of another experiment which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 4). It is a figure which shows the protocol of experiment. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた別の実験結果を示す(実施例４)。メサコリン吸入により誘発した気道過敏性の測定結果を示す図である。The result of another experiment which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 4). It is a figure which shows the measurement result of the airway hypersensitivity induced by the inhalation of mesacolin. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた別の実験結果を示す(実施例４)。気管支肺胞洗浄液中の好酸球数、好中球数、マクロファージ数、及びＴ細胞数を示す図である。The result of another experiment which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 4). It is a figure which shows the number of eosinophils in a bronchoalveolar lavage fluid, the number of neutrophils, the number of macrophages, and the number of T cells. 気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用を調べた別の実験結果を示す(実施例４)。気管支肺胞洗浄液中の総細胞数を示す図である。The result of another experiment which investigated the effect | action of heme peroxidase with respect to a bronchial asthma model mouse is shown (Example 4). It is a figure which shows the total cell number in a bronchoalveolar lavage fluid.
　以下、本発明について詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施し得る。なお、本明細書に記載した全ての文献及び刊行物は、その目的にかかわらず参照によりその全体を本明細書に組み込むものとする。また、本明細書は、本願の優先権主張の基礎となる日本国特許出願である特願2012-11838号(2012年1月24日出願)の特許請求の範囲、明細書、および図面の開示内容を包含する。 Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these explanations, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. It should be noted that all documents and publications described in this specification are incorporated herein by reference in their entirety regardless of their purposes. In addition, this specification is a disclosure of claims, description, and drawings of Japanese Patent Application No. 2012-11838 (filed on Jan. 24, 2012), which is a Japanese patent application that is the basis of the priority claim of the present application. Includes content.
1. 本発明の概要
　本発明は、ヘムペルオキシダーゼ阻害薬を含有する、気管支喘息の予防又は治療薬を提供する。また、本発明は、ヘムペルオキシダーゼの活性又は発現量を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法を提供する。 1. Summary of the Present Invention The present invention provides a prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor. The present invention also provides a method for screening a prophylactic or therapeutic agent for bronchial asthma using the activity or expression level of heme peroxidase as an index.
　以前、本発明者らは、陰イオンチャネルであるペンドリンの発現が喘息モデルマウスの肺組織において増強しており、強制的にペンドリンをマウス気道上皮細胞に発現させると、気道過敏性の亢進や粘液産生といった喘息様の病態が形成されることを見出した(Nakao I et al., J Immunol, vol.180, 6262－6269, 2008)。 Previously, the present inventors have shown that the expression of pendrin, an anion channel, is enhanced in the lung tissue of asthma model mice, and when pendrin is forcibly expressed in mouse airway epithelial cells, airway hypersensitivity and mucus are increased. It was found that an asthma-like condition such as production was formed (Nakao I et al., J Immunol, vol.180, 6262-6269, 2008).
　今回、本発明者らは、ペンドリンを透過する何らかの陰イオンがこの喘息様病態を形成していると考え、そのような陰イオンとしてSCN－イオンに着目した。本発明者らは、喘息モデルマウスの飲水中にSCN－イオンを加えたところマウスにおける喘息様病態が悪化するという結果を得たことから(図1(B))、SCN－イオンが喘息に対して増悪因子であることを明らかにした。 This time, the present inventors considered that some anions which transmits Pendorin forms the asthmatic condition, SCN as such anions - focused on the ion. The present inventors have found that during drinking asthma model mice SCN - since the asthma-like conditions to obtain a result that deterioration in mice was added ions (Fig. 1 (B)), SCN - ions to asthma It was revealed that it was an exacerbation factor.
　さらに、本発明者らは、SCN－イオンは気道組織内のヘムペルオキシダーゼによって過酸化水素(H2O2)とともに、SCN－ + H2O2 → OSCN－ + H2Oの反応を受けると考えた。本発明者らは、実際に細胞培養系実験において細胞内でOSCN－イオンを増加させると炎症に重要な分子であるNF－κBの活性が上昇するという結果を得たことから(図2)、OSCN－イオンも喘息に対する増悪因子であることを明らかにした。 Furthermore, the present inventors consider that SCN − ions undergo a reaction of SCN − + H 2 O 2 → OSCN − + H 2 O together with hydrogen peroxide (H 2 O 2 ) by heme peroxidase in airway tissues. It was. The inventors of the present invention have obtained the result that the activity of NF-κB, which is a molecule important for inflammation, is increased by increasing the OSCN − ion in the cell in the cell culture system experiment (FIG. 2). OSCN - ion was also found to be an exacerbation factor for asthma.
　これらのことより、本発明者らは、気道組織内のヘムペルオキシダーゼは喘息を増悪させる因子であることを明らかにした。本発明者は、さらに、ヘムペルオキシダーゼの活性を阻害すること、ヘムペルオキシダーゼの発現を阻害することなどが、気管支喘息の病態の改善につながることを明らかにした。実際に、ヘムペルオキシダーゼ阻害薬の1つであり、抗甲状腺亢進症薬として用いられているチアマゾールをマウスに投与したところ、気道過敏性の亢進、好酸球性炎症などの喘息様病態の発現を抑制することができた(図3(A)～(C))。 From these facts, the present inventors have revealed that heme peroxidase in the airway tissue is a factor that exacerbates asthma. The present inventor further revealed that inhibiting the activity of heme peroxidase, inhibiting the expression of heme peroxidase, and the like lead to improvement of the pathological condition of bronchial asthma. In fact, when thiamazole, one of the heme peroxidase inhibitors and used as an anti-hyperthyroid drug, was administered to mice, it showed increased airway hypersensitivity and asthma-like conditions such as eosinophilic inflammation. It was able to be suppressed (FIGS. 3 (A) to (C)).
2. ヘムペルオキシダーゼ阻害薬を含有する気管支喘息の予防又は治療薬
　本発明は、ヘムペルオキシダーゼ阻害薬を含有する気管支喘息の予防又は治療薬を提供する。また、本発明は、気管支喘息の予防又は治療を必要とする患者に治療的有効量のヘムペルオキシダーゼ阻害薬を投与することを含む気管支喘息の予防又は治療方法を提供する。 2. Prophylactic or therapeutic agent for bronchial asthma containing heme peroxidase inhibitor The present invention provides a prophylactic or therapeutic agent for bronchial asthma containing a heme peroxidase inhibitor. The present invention also provides a method for preventing or treating bronchial asthma, comprising administering a therapeutically effective amount of a heme peroxidase inhibitor to a patient in need of preventing or treating bronchial asthma.
2.1. 本発明で用いられるヘムペルオキシダーゼ阻害薬
　「ヘムペルオキシダーゼ」は、ペルオキダーゼのファミリーであり、活性部位にヘムを含むペルオキシダーゼの総称である。動物ではミエロペルオキシダーゼ（MPO）、好酸球ペルオキシダーゼ(EPO)、ラクトペルオキシダーゼ(LPO)、甲状腺ペルオキシダーゼ(TPO)などがこのファミリーに含まれる。これらのヘムペルオキダーゼは過酸化水素依存的に、基質となる物質の酸化反応を触媒する。 2.1. Heme peroxidase inhibitor used in the present invention "Heme peroxidase" is a family of peroxidases and is a general term for peroxidases containing heme in the active site. In animals, myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), thyroid peroxidase (TPO) and the like are included in this family. These heme peroxidases catalyze an oxidation reaction of a substrate substance in a hydrogen peroxide-dependent manner.
　本発明において、ヘムペルオキシダーゼは、特に限定されないが、例えば、ヒト由来のもの、マウス由来のものなどが挙げられる。各種ヘムペルオキシダーゼのcDNA配列とアミノ酸配列は下記の通りである。 In the present invention, heme peroxidase is not particularly limited, and examples thereof include those derived from humans and those derived from mice. The cDNA sequences and amino acid sequences of various heme peroxidases are as follows.
ヒトラクトペルオキシダーゼ(cDNA配列: 配列番号:1); アミノ酸配列: Accession No. NP_006142.1 (配列番号:2))
マウスラクトペルオキシダーゼ(cDNA配列: 配列番号:3; アミノ酸配列: Accession No. NP_536345(配列番号:4)) Human lactoperoxidase (cDNA sequence: SEQ ID NO: 1); amino acid sequence: Accession No. NP_006142.1 (SEQ ID NO: 2))
Mouse lactoperoxidase (cDNA sequence: SEQ ID NO: 3; amino acid sequence: Accession No. NP_536345 (SEQ ID NO: 4))
ヒトミエロペルオキシダーゼ (cDNA配列: 配列番号:5; アミノ酸配列: Accession No. NP_000241.1(配列番号:6))
マウスミエロペルオキシダーゼ (cDNA配列: 配列番号:7; アミノ酸配列: Accession No. NP_034954.2(配列番号:8)) Human myeloperoxidase (cDNA sequence: SEQ ID NO: 5; amino acid sequence: Accession No. NP_000241.1 (SEQ ID NO: 6))
Mouse myeloperoxidase (cDNA sequence: SEQ ID NO: 7; amino acid sequence: Accession No. NP_034954.2 (SEQ ID NO: 8))
ヒト好酸球ペルオキダーゼ(cDNA配列: 配列番号:9; アミノ酸配列: Accession No. NP_000493(配列番号:10))
マウス好酸球ペルオキダーゼ(cDNA配列: 配列番号:11; アミノ酸配列: Accession No. NP_031972.2(配列番号:12)) Human eosinophil peroxidase (cDNA sequence: SEQ ID NO: 9; amino acid sequence: Accession No. NP_000493 (SEQ ID NO: 10))
Mouse eosinophil peroxidase (cDNA sequence: SEQ ID NO: 11; amino acid sequence: Accession No. NP_031972.2 (SEQ ID NO: 12))
ヒト甲状腺ペルオキシダーゼ(cDNA配列: 配列番号:13; アミノ酸配列: Accession No. NP_000538.3 (配列番号:14))
マウス甲状腺ペルオキシダーゼ(cDNA配列: 配列番号:15; アミノ酸配列: Accession No. NP_033443.1(配列番号:16)) Human thyroid peroxidase (cDNA sequence: SEQ ID NO: 13; amino acid sequence: Accession No. NP_000538.3 (SEQ ID NO: 14))
Mouse thyroid peroxidase (cDNA sequence: SEQ ID NO: 15; amino acid sequence: Accession No. NP_033443.1 (SEQ ID NO: 16))
　本明細書中では、ヘムペルオキシダーゼは、それらと実質的に同質の活性を有する限り、その変異体をも包含する意味で用いられる。該変異体としては、例えば、上記ヘムペルオキシダーゼのアミノ酸配列中の1～複数個(例えば、1～30個、1～29個、1～28個、1～27個、1～26個、1～25個、1～24個、1～23個、1～22個、1～21個、1～20個、1～19個、1～18個、1～17個、1～16個、1～15個、1～14個、1～13個、1～12個、1～11個、1～10個、1～9個(1～数個)、1～8個、1～7個、1～6個、1～5個、1～4個、1～3個、1～2個、または1個)のアミノ酸が欠失、置換、挿入および／または付加したアミノ酸配列を有するタンパク質が挙げられる。欠失、置換、挿入もしくは付加したアミノ酸の数は、一般的に少ないほど好ましい。上記アミノ酸残基の欠失、置換、挿入および付加のうち2種以上が同時に生じてもよい。 In the present specification, heme peroxidase is used in the sense of including its variants as long as it has substantially the same quality of activity. Examples of the mutant include one to a plurality (for example, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 26) in the amino acid sequence of the above heme peroxidase. 25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1 15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1 (-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acids deleted, substituted, inserted, and / or added to the protein. . In general, the smaller the number of amino acids deleted, substituted, inserted or added, the better. Two or more of the amino acid residue deletions, substitutions, insertions and additions may occur simultaneously.
　実質的に同質の活性としては、例えば、ヘムペルオキシダーゼの活性が挙げられる。実質的に同質とは、それらの活性が性質的に(例、生理学的に、または薬理学的に)同等であることを示す。したがって、ヘムペルオキシダーゼの活性等が同等（例、約0.01～100倍、好ましくは約0.1～10倍、より好ましくは0.5～2倍）であることが好ましいが、これらの活性の程度、タンパク質の分子量などの量的要素は異なっていてもよい。 The substantially homogeneous activity includes, for example, the activity of heme peroxidase. Substantially homogeneous indicates that their activities are qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, it is preferable that the activity of heme peroxidase is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, the degree of these activities and the molecular weight of the protein Quantitative factors such as may be different.
　「ヘムペルオキシダーゼの活性」は、過酸化物による物質の酸化反応を触媒する酵素活性を意味する。 “The activity of heme peroxidase” means an enzyme activity that catalyzes an oxidation reaction of a substance by peroxide.
　「ヘムペルオキシダーゼ阻害薬」は、ヘムペルオキシダーゼの活性を阻害する又はヘムペルオキシダーゼの発現を阻害する活性を有する物質を意味する。かかる物質としては、低分子若しくは高分子化合物のほか、siRNA、shRNA、抗体、アンチセンス、ペプチド、タンパク質、酵素などを用いることができる。 “Heme peroxidase inhibitor” means a substance having an activity of inhibiting the activity of heme peroxidase or inhibiting the expression of heme peroxidase. As such a substance, siRNA, shRNA, antibody, antisense, peptide, protein, enzyme and the like can be used in addition to a low molecular weight or high molecular compound.
「ヘムペルオキシダーゼの活性を阻害する」とは、ヘムペルオキシダーゼの酵素活性を阻害することを意味する。 “Inhibiting the activity of heme peroxidase” means inhibiting the enzyme activity of heme peroxidase.
　「ヘムペルオキシダーゼの発現を阻害する」とは、当該タンパク質の遺伝子の発現阻害を含む、当該タンパク質をコードする遺伝子からタンパク質生成までの一連の事象(例えば、転写(mRNAの生成)、翻訳(タンパク質の生成)を含む)のうちのいずれかの事象を阻害することによって、当該タンパク質の生成を阻害することを意味する。 `` Inhibiting heme peroxidase expression '' means a series of events from gene encoding the protein to protein production, including inhibition of gene expression of the protein (e.g. transcription (production of mRNA), translation (protein Inhibiting the event of any of the above (including production) means inhibiting the production of the protein.
　本発明で用いられるヘムペルオキシダーゼ阻害薬としては、ヘムペルオキシダーゼの活性を阻害する物質又はヘムペルオキシダーゼの発現を阻害する物質等が用いられるが、具体的には、以下の(a)～(h)からなる群から選択される少なくとも１つの物質等が挙げられる。 As the heme peroxidase inhibitor used in the present invention, a substance that inhibits the activity of heme peroxidase, a substance that inhibits the expression of heme peroxidase, or the like is used. Specifically, from the following (a) to (h) And at least one substance selected from the group.
(a) ヘムペルオキシダーゼの活性を阻害する化合物、
(b) ヘムペルオキシダーゼの発現を阻害する化合物、
(c) ヘムペルオキシダーゼの活性を阻害する抗体、
(d) ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNA、
(e) ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列に相補的若しくは実質的に相補的な塩基配列又はその一部を含有するアンチセンスポリヌクレオチド、
(f) ヘムペルオキシダーゼをコードするポリヌクレオチドに対するリボザイム、
(g) ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチド、及び
(h) ヘムペルオキシダーゼに対するアプタマー。 (a) a compound that inhibits the activity of heme peroxidase,
(b) a compound that inhibits the expression of heme peroxidase,
(c) an antibody that inhibits the activity of heme peroxidase,
(d) siRNA or shRNA against a polynucleotide encoding heme peroxidase,
(e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof,
(f) a ribozyme against a polynucleotide encoding heme peroxidase,
(g) a variant of heme peroxidase that acts dominantly negatively on heme peroxidase or a polynucleotide encoding the same, and
(h) Aptamer to heme peroxidase.
　ここで、「化合物」には低分子化合物と高分子化合物が含まれる。「低分子化合物」とは、分子量10，000以下(好ましくは、分子量5，000以下、より好ましくは分子量2,000以下、特に好ましくは分子量700以下)の有機および無機物質を意味する。また「高分子化合物」とは、分子量10，000超(好ましくは、分子量50，000以上、より好ましくは分子量100,000以上)の有機物質を意味する。 Here, “compound” includes low molecular weight compounds and high molecular weight compounds. “Low molecular weight compound” means organic and inorganic substances having a molecular weight of 10,000 or less (preferably a molecular weight of 5,000 or less, more preferably a molecular weight of 2,000 or less, particularly preferably a molecular weight of 700 or less). The “polymer compound” means an organic substance having a molecular weight of over 10,000 (preferably a molecular weight of 50,000 or more, more preferably a molecular weight of 100,000 or more).
　ヘムペルオキシダーゼの活性は、公知の方法又はそれに準ずる方法にて測定することができる。ヘムペルオキシダーゼの活性は、例えば、3,3',5,5'-tetramethylbenzidine (TMB)の酸化により生じる呈色反応を利用した方法（Thomas,EL et al, J Dent Res 73,544-555,1994）などにより測定することができる。 The activity of heme peroxidase can be measured by a known method or a method analogous thereto. The activity of heme peroxidase is, for example, a method using a color reaction generated by oxidation of 3,3 ′, 5,5′-tetramethylbenzidine (TMB) (Thomas, EL et al, J Dent Res 73,544-555, 1994), etc. Can be measured.
　ヘムペルオキシダーゼの発現量も、公知の方法又はそれに準ずる方法にて測定することができる。ヘムペルオキシダーゼの発現量は、例えば、ヘムペルオキシダーゼのタンパク質量を測定すること(Free Radic Biol Med,49,1354-60,2010)、ヘムペルオキシダーゼのmRNA量を測定すること(J Immunol,167, 1672-1682, 2001)、などにより測定することができる。 The expression level of heme peroxidase can also be measured by a known method or a method analogous thereto. The expression level of heme peroxidase is, for example, measuring the protein level of heme peroxidase (Free (Radic Biol Med, 49,1354-60,2010), measuring the amount of hemeperoxidase mRNA (J Immunol, 167, 1672- 1682, 2001), etc.
(a) ヘムペルオキシダーゼの活性を阻害する化合物
　ヘムペルオキシダーゼの活性を阻害する化合物(塩形態を含む)としては、ヘムペルオキシダーゼの活性を阻害しうる化合物であればよく特に制限はないが、例えば、ヘムペルオキシダーゼに結合してその酵素活性を阻害する化合物又はその塩などが挙げられる。 (a) Compound that inhibits heme peroxidase activity The compound (including salt form) that inhibits heme peroxidase activity is not particularly limited as long as it is a compound that can inhibit heme peroxidase activity. Examples thereof include a compound that binds to peroxidase and inhibits its enzyme activity, or a salt thereof.
　このような化合物は、例えば、ペプチド、タンパク質、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などから選ばれた化合物であってもよい。該化合物は、新規な化合物であってもよいし、公知の化合物であってもよい。塩形態の化合物としては、例えば、生理学的に許容される金属塩、アンモニウム塩、有機塩基との塩、無機酸との塩、有機酸との塩、塩基性又は酸性アミノ酸との塩などが挙げられる。金属塩の好適な例としては、例えばナトリウム塩、カリウム塩などのアルカリ金属塩；カルシウム塩、マグネシウム塩、バリウム塩などのアルカリ土類金属塩；アルミニウム塩などが挙げられる。有機塩基との塩の好適な例としては、例えばトリメチルアミン、トリエチルアミン、ピリジン、ピコリン、2，6－ルチジン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、シクロヘキシルアミン、ジシクロヘキシルアミン、N，N'－ジベンジルエチレンジアミンなどとの塩が挙げられる。無機酸との塩の好適な例としては、例えば塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。有機酸との塩の好適な例としては、例えばギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、フタル酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、安息香酸、ベンゼンスルホン酸、p－トルエンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩の好適な例としては、例えばアルギニン、リジン、オルニチンなどとの塩が挙げられ、酸性アミノ酸との塩の好適な例としては、例えばアスパラギン酸、グルタミン酸などとの塩が挙げられる。 Such a compound may be, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. The compound may be a novel compound or a known compound. Examples of the salt form compounds include physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. It is done. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
　このうち、生理学的に許容し得る塩が好ましい。例えば、化合物内に酸性官能基を有する場合にはアルカリ金属塩(例、ナトリウム塩，カリウム塩など)、アルカリ土類金属塩(例、カルシウム塩，マグネシウム塩，バリウム塩など)などの無機塩、アンモニウム塩など、また、化合物内に塩基性官能基を有する場合には、例えば臭化水素酸、硝酸、硫酸、リン酸など無機酸との塩、又は酢酸、フタル酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、メタンスルホン酸、p－トルエンスルホン酸などの有機酸との塩が挙げられる。 Of these, physiologically acceptable salts are preferred. For example, when the compound has an acidic functional group, an inorganic salt such as an alkali metal salt (eg, sodium salt, potassium salt), an alkaline earth metal salt (eg, calcium salt, magnesium salt, barium salt), When the compound has a basic functional group, for example, a salt with an inorganic acid such as hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or acetic acid, phthalic acid, fumaric acid, oxalic acid, Examples thereof include salts with organic acids such as tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid and p-toluenesulfonic acid.
　ヘムペルオキシダーゼの活性を阻害し得る化合物としては、より具体的には、チアマゾール、プロピルチオウラシル、ダプソン、アザイドなどが挙げられる。あるいは、このような化合物は、後述するスクリーニング方法によっても得ることができる。これらのヘムペルオキシダーゼの活性を阻害する化合物は、単独で、又は必要に応じてそれらの2種以上を組み合わせて用いることができる。 More specifically, examples of the compound capable of inhibiting the activity of heme peroxidase include thiamazole, propylthiouracil, dapsone, azide and the like. Alternatively, such a compound can also be obtained by a screening method described later. These compounds that inhibit the activity of heme peroxidase can be used alone or in combination of two or more thereof as necessary.
(b) ヘムペルオキシダーゼの発現を阻害する化合物
　ヘムペルオキシダーゼの発現を阻害する化合物(塩形態を含む)は、ヘムペルオキシダーゼの発現を抑制することができるので、ヘムペルオキシダーゼの発現を阻害する物質として好適に使用することができる。 (b) Compounds that inhibit heme peroxidase expression Compounds (including salt forms) that inhibit heme peroxidase expression can suppress heme peroxidase expression and are therefore suitable as substances that inhibit heme peroxidase expression. Can be used.
　ヘムペルオキシダーゼの発現を阻害する化合物としては、ヘムペルオキシダーゼの発現を阻害しうるものであればよく特に制限はないが、例えば、(i) ヘムペルオキシダーゼをコードする遺伝子(DNA)からヘムペルオキシダーゼをコードするmRNAへの転写を阻害する化合物、(ii) ヘムペルオキシダーゼをコードするmRNAからヘムペルオキシダーゼへの翻訳を阻害する化合物などが挙げられる。(i) ヘムペルオキシダーゼをコードする遺伝子(DNA)からヘムペルオキシダーゼをコードするmRNAへの転写を阻害する化合物としては、ヘムペルオキシダーゼをコードする遺伝子(DNA)からmRNAへの転写を阻害するものであればよく特に制限はないが、例えば、ヘムペルオキシダーゼをコードする遺伝子(DNA)からmRNAへの転写に関与する因子に結合し、転写を阻害する化合物などが挙げられる。(ii) ヘムペルオキシダーゼをコードするmRNAからヘムペルオキシダーゼへの翻訳を阻害する化合物としては、ヘムペルオキシダーゼをコードするmRNAからヘムペルオキシダーゼへの翻訳を阻害するものであればよく特に制限はないが、例えば、ヘムペルオキシダーゼをコードするmRNAからヘムペルオキシダーゼへの翻訳に関与する因子に結合し、翻訳を阻害する化合物などが挙げられる。 The compound that inhibits heme peroxidase expression is not particularly limited as long as it can inhibit heme peroxidase expression. For example, (i) a gene encoding heme peroxidase (DNA) encodes heme peroxidase. Examples thereof include compounds that inhibit transcription to mRNA, and (ii) compounds that inhibit translation from heme peroxidase-encoding mRNA into heme peroxidase. (i) As a compound that inhibits the transcription of heme peroxidase-encoding gene (DNA) into heme peroxidase-encoding mRNA, any compound that inhibits transcription of heme peroxidase-encoding gene (DNA) into mRNA Although there is no particular limitation, for example, a compound that binds to a factor involved in transcription from a gene (DNA) encoding heme peroxidase to mRNA and inhibits transcription can be mentioned. (ii) As a compound that inhibits translation of mRNA encoding heme peroxidase into heme peroxidase, any compound that inhibits translation from heme peroxidase-encoding mRNA into heme peroxidase is not particularly limited. Examples thereof include compounds that bind to a factor involved in translation from heme peroxidase-encoding mRNA into heme peroxidase and inhibit the translation.
　このような化合物は、例えば、後述するスクリーニング方法によって得ることができる。ヘムペルオキシダーゼの発現を阻害する化合物は、単独で、又は必要に応じてそれらの2種以上を組み合わせて用いることができる。 Such a compound can be obtained, for example, by a screening method described later. The compounds that inhibit the expression of heme peroxidase can be used alone or in combination of two or more thereof as necessary.
(c) ヘムペルオキシダーゼの活性を阻害する抗体
　本発明においては、ヘムペルオキシダーゼの活性を阻害することが目的であるから、ヘムペルオキシダーゼの活性を阻害する抗体であればいかなる抗体をも用いることができる。そのような抗体は、ヘムペルオキシダーゼ又はその部分ペプチドを認識し得る抗体であって、ヘムペルオキシダーゼの活性を阻害する抗体などを含む。このような抗体としては、ポリクローナル抗体、モノクローナル抗体の何れであってもよい。 (c) Antibody that inhibits the activity of heme peroxidase In the present invention, since the purpose is to inhibit the activity of heme peroxidase, any antibody that inhibits the activity of heme peroxidase can be used. Such an antibody includes an antibody that can recognize heme peroxidase or a partial peptide thereof, and that inhibits the activity of heme peroxidase. Such an antibody may be either a polyclonal antibody or a monoclonal antibody.
　本発明で用いられるヘムペルオキシダーゼ若しくはその部分ペプチドに対する抗体は、ヘムペルオキシダーゼ若しくはその部分ペプチドを抗原として用い、自体公知の抗体又は抗血清の製造法に従って製造することができる。 The antibody to heme peroxidase or a partial peptide thereof used in the present invention can be produced according to a known method for producing an antibody or antiserum using heme peroxidase or a partial peptide thereof as an antigen.
(d) ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNA
　ヘムペルオキシダーゼをコードするポリヌクレオチドに対してRNAi作用を有する二重鎖RNA(例、ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNAなど)は、低毒性であり、ヘムペルオキシダーゼをコードする遺伝子の翻訳を抑制することができ、ヘムペルオキシダーゼの発現を抑制することができるので、ヘムペルオキシダーゼの発現を阻害する物質として好適に使用することができる。このような、ヘムペルオキシダーゼをコードするポリヌクレオチドに対してRNAi作用を有する二重鎖RNAとしては、ヘムペルオキシダーゼをコードするRNAの一部を含有する二重鎖RNA(例、ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA(small (short) interfering RNA)、shRNA(small (short) hairpin RNA)など)などが挙げられる。 (d) siRNA or shRNA against a polynucleotide encoding heme peroxidase
Double-stranded RNA that has RNAi action on a polynucleotide encoding heme peroxidase (e.g., siRNA or shRNA against a polynucleotide encoding heme peroxidase) has low toxicity, and translates the gene encoding heme peroxidase. Since it can suppress and the expression of heme peroxidase can be suppressed, it can be used suitably as a substance which inhibits the expression of heme peroxidase. Such double-stranded RNA having an RNAi action on a polynucleotide encoding heme peroxidase includes a double-stranded RNA containing a part of RNA encoding heme peroxidase (e.g., a polynucleotide encoding heme peroxidase). SiRNA for nucleotides (small (short) interfering RNA), shRNA (small (short) hairpin RNA) and the like.
　このような二重鎖RNAは、公知の方法(例、Nature, 411巻, 494頁, 2001年; 特表2002-516062号公報; 米国特許出願公開第2002/086356号明細書; Nature Genetics, 24巻, 180-183頁, 2000年; Genesis, 26巻, 240-244頁, 2000年； Nature, 407巻, 319-320頁, 2002年; Genes & Dev.,16巻, 948-958頁, 2002年; Proc. Natl. Acad. Sci. USA., 99巻, 5515-5520頁, 2002年; Science, 296巻, 550-553頁, 2002年; Proc. Natl. Acad. Sci. USA, 99巻, 6047-6052頁, 2002年; Nature Biotechnology, 20巻, 497-500頁, 2002年; Nature Biotechnology, 20巻, 500-505頁, 2002年; Nucleic Acids Res., 30巻, e46, 2002年)に準じて、ヘムペルオキシダーゼをコードするポリヌクレオチドの配列を基に設計して製造することができる。 Such double-stranded RNA is obtained by a known method (e.g., Nature, 411, 494, 2001; Special Tables 2002-516062; U.S. Patent Application Publication No. 2002/086356; Nature Genetics, 24). Volume, 180-183, 2000; Genesis, 26, 240-244, 2000; Nature, 407, 319-320, 2002; Genes & Dev., 16, 948-958, 2002 Proc. Natl. Acad. Sci. USA., 99, 5515-5520, 2002; Science, 296, 550-553, 2002; Proc. Natl. Acad. Sci. USA, 99, 6047-6052, 2002; Nature Biotechnology, 20, 497-500, 2002; Nature Biotechnology, 20, 500-505, 2002; Nucleic Acids Res., 30, e46, 2002) Accordingly, it can be designed and produced based on the sequence of the polynucleotide encoding heme peroxidase.
　本発明で用いられるRNAi作用を有する二重鎖RNAの長さは、通常、17～30塩基、好ましくは19～27塩基、より好ましくは20～22塩基である。 The length of the double-stranded RNA having RNAi action used in the present invention is usually 17 to 30 bases, preferably 19 to 27 bases, more preferably 20 to 22 bases.
(e) ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列に相補的若しくは実質的に相補的な塩基配列又はその一部を含有するアンチセンスポリヌクレオチド
　ヘムペルオキシダーゼをコードするポリヌクレオチド(好ましくはDNA)(以下、アンチセンスポリヌクレオチドの説明においては、これらのDNAを「本発明で用いられるDNA」と略記する場合がある)の塩基配列に相補的な、又は実質的に相補的な塩基配列又はその一部を有するアンチセンスポリヌクレオチドとしては、本発明で用いられるDNAの塩基配列に相補的な、又は実質的に相補的な塩基配列又はその一部を有し、該DNAの発現を抑制し得る作用を有するものであれば、いずれのアンチセンスポリヌクレオチドであってもよいが、アンチセンスDNAが好ましい。 (e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof, preferably a polynucleotide (preferably DNA) encoding heme peroxidase (hereinafter referred to as DNA) In the description of the antisense polynucleotide, these DNAs may be abbreviated as “DNA used in the present invention”), or a base sequence complementary to or substantially complementary to the base sequence. The antisense polynucleotide having a nucleotide sequence that is complementary to or substantially complementary to the nucleotide sequence of the DNA used in the present invention and has an action capable of suppressing the expression of the DNA. Any antisense polynucleotide can be used, but antisense DNA is preferred.
　本発明で用いられるDNAに実質的に相補的な塩基配列とは、例えば、本発明で用いられるDNAに相補的な塩基配列(すなわち、本発明で用いられるDNAの相補鎖)の全塩基配列あるいは部分塩基配列と約70％以上、好ましくは約80％以上、より好ましくは約90％以上、最も好ましくは約95％以上の相同性を有する塩基配列などが挙げられる。特に、本発明で用いられるDNAの相補鎖の全塩基配列うち、(i)翻訳阻害を指向したアンチセンスポリヌクレオチドの場合は、ヘムペルオキシダーゼのN末端部位をコードする部分の塩基配列(例えば、開始コドン付近の塩基配列など)の相補鎖と約70％以上、好ましくは約80％以上、より好ましくは約90％以上、最も好ましくは約95％以上の相同性を有するアンチセンスポリヌクレオチドが、(ii)RNaseHによるRNA分解を指向するアンチセンスポリヌクレオチドの場合は、イントロンを含む本発明で用いられるDNAの全塩基配列の相補鎖と約70％以上、好ましくは約80％以上、より好ましくは約90％以上、最も好ましくは約95％以上の相同性を有するアンチセンスポリヌクレオチドがそれぞれ好適である。 The base sequence substantially complementary to the DNA used in the present invention is, for example, the entire base sequence of the base sequence complementary to the DNA used in the present invention (that is, the complementary strand of the DNA used in the present invention) or Examples thereof include base sequences having homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a partial base sequence. In particular, of the entire base sequence of the complementary strand of DNA used in the present invention, (i) in the case of an antisense polynucleotide directed to translation inhibition, the base sequence of the portion encoding the N-terminal site of heme peroxidase (for example, the start An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand (such as a base sequence near a codon) ii) In the case of an antisense polynucleotide directed to RNA degradation by RNaseH, it is about 70% or more, preferably about 80% or more, more preferably about 80% or more with the complementary strand of the entire base sequence of DNA used in the present invention including introns. Each of the antisense polynucleotides having 90% or more, most preferably about 95% or more homology is preferred.
　アンチセンスポリヌクレオチドは通常、10～40個程度、好ましくは15～30個程度の塩基から構成される。 The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
　ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンスDNAを構成する各ヌクレオチドのりん酸残基(ホスフェート)は、例えば、ホスホロチオエート、メチルホスホネート、ホスホロジチオネートなどの化学修飾りん酸残基に置換されていてもよい。また、各ヌクレオチドの糖(デオキシリボース)は、2’－O－メチル化などの化学修飾糖構造に置換されていてもよいし、塩基部分(ピリミジン、プリン)も化学修飾を受けたものであってもよく、ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列を有するDNAにハイブリダイズするものであればいずれのものでもよい。これらのアンチセンスポリヌクレオチドは、公知のDNA合成装置などを用いて製造することができる。 In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is changed to a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base part (pyrimidine, purine) is also chemically modified. It may be any one as long as it hybridizes with DNA having the base sequence of the polynucleotide encoding heme peroxidase. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
　本発明のアンチセンスポリヌクレオチドは、変化せしめられたり、修飾された糖、塩基、結合を含有していて良く、リポゾーム、ミクロスフェアのような特殊な形態で供与されたり、遺伝子治療により適用されたり、付加された形態で与えられることができうる。こうして付加形態で用いられるものとしては、リン酸基骨格の電荷を中和するように働くポリリジンのようなポリカチオン体、細胞膜との相互作用を高めたり、核酸の取込みを増大せしめるような脂質(例、ホスホリピド、コレステロールなど)などの疎水性のものが挙げられる。付加するのに好ましい脂質としては、コレステロールやその誘導体(例、コレステリルクロロホルメート、コール酸など)が挙げられる。こうしたものは、核酸の3’端又は5’端に付着させることができ、塩基、糖、分子内ヌクレオシド結合を介して付着させることができうる。その他の基としては、核酸の3’端又は5’端に特異的に配置されたキャップ用の基で、エキソヌクレアーゼ、RNaseなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。こうしたキャップ用の基としては、ポリエチレングリコール、テトラエチレングリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護基が挙げられるが、それに限定されるものではない。 The antisense polynucleotide of the present invention may be altered or contain modified sugars, bases and bonds, and may be provided in special forms such as liposomes, microspheres, or applied by gene therapy. Can be provided in an added form. In this way, the additional form can be used as a polycationic substance such as polylysine that acts to neutralize the charge of the phosphate group skeleton, a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acid ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
(f) ヘムペルオキシダーゼをコードするポリヌクレオチドに対するリボザイム
　ヘムペルオキシダーゼをコードするポリヌクレオチドに対してリボザイム活性を有するポリヌクレオチドは、ヘムペルオキシダーゼの発現を抑制することができるので、ヘムペルオキシダーゼの発現を阻害する物質として好適に使用することができる。このようなリボザイムは、公知の方法(例、TRENDS in Molecular Medicine, 7巻, 221頁, 2001年; FEBS Lett., 228巻, 228頁, 1988年； FEBS Lett., 239巻, 285頁, 1988年; Nucl. Acids. Res., 17巻, 7059頁, 1989年; Nature, 323巻, 349頁, 1986年; Nucl. Acids. Res., 19巻, 6751頁, 1991年; Protein Eng. 3巻, 733頁, 1990年; Nucl. Acids Res., 19巻, 3875頁, 1991年; Nucl. Acids Res., 19巻, 5125頁, 1991年; Biochem. Biophys. Res. Commun., 186巻, 1271頁, 1992年など参照)に準じて、ヘムペルオキシダーゼをコードするポリヌクレオチドの配列を基に設計して製造することができる。例えば、ヘムペルオキシダーゼをコードするRNAの一部に公知のリボザイムを連結することによって製造することができる。ヘムペルオキシダーゼをコードするRNAの一部としては、公知のリボザイムによって切断され得るヘムペルオキシダーゼをコードするRNA上の切断部位に近接した部分(RNA断片)が挙げられる。上記リボザイムには、グループIイントロン型やRNasePに含まれるM1 RNAなどのラージリボザイム、ハンマーヘッド型やヘアピン型などのスモールリボザイムなどが含まれる(タンパク質核酸酵素， 35巻， 2191頁, 1990年)。ハンマーヘッド型リボザイムについては、例えば、FEBS Lett., 228巻, 228頁,  1988年; FEBS Lett., 239巻, 285頁, 1988年; タンパク質核酸酵素, 35巻, 2191頁, 1990年; Nucl. Acids Res., 17巻, 7059頁, 1989年などを参照することができる。また、ヘアピン型リボザイムについては、例えば、Nature, 323巻, 349頁, 1986年; Nucl. Acids Res., 19巻,  6751頁, 1991年; 化学と生物, 30巻, 112頁, 1992年などを参照することができる。 (f) Ribozyme against a polynucleotide encoding heme peroxidase A polynucleotide having ribozyme activity against a polynucleotide encoding heme peroxidase can suppress the expression of heme peroxidase, and therefore inhibits the expression of heme peroxidase. Can be suitably used. Such ribozymes are prepared by known methods (eg, TRENDS in Molecular Medicine, 7, 221, 2001; FEBS Lett., 228, 228, 1988; FEBS Lett., 239, 285, 1988). Nucl. Acids. Res., 17, 7059, 1989; Nature, 323, 349, 1986; Nucl. Acids. Res., 19, 6751, 1991; Protein Eng. 3 , 733, 1990; Nucl. Acids Res., 19, 3875, 1991; Nucl. Acids Res., 19, 5125, 1991; Biochem. Biophys. Res. Commun., 186, 1271 Page, 1992, etc.) and can be designed and produced based on the sequence of the polynucleotide encoding heme peroxidase. For example, it can be produced by linking a known ribozyme to a part of RNA encoding heme peroxidase. Examples of the RNA encoding heme peroxidase include a portion (RNA fragment) close to the cleavage site on the RNA encoding heme peroxidase that can be cleaved by a known ribozyme. The ribozymes include large ribozymes such as group I introns and M1 RNA contained in RNaseP, and small ribozymes such as hammerhead and hairpin types (Protein Nucleic Acid Enzyme, 35, 2191, 1990). Examples of hammerhead ribozymes include FEBS Lett., 228, 228, 1988; FEBS Lett., 239, 285, 1988; Protein Nucleic Acid Enzyme, 35, 2191, 1990; Nucl. Acids Res., 17, 7059, 1989, etc. can be referred to. For hairpin ribozymes, see, for example, Nature, 323, 349, 1986; Nucl. Acids Res., 19, 6751, 1991; Chemistry and Biology, 30, 112, 1992, etc. You can refer to it.
(g) ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチド
　ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチドは、ヘムペルオキシダーゼの活性を阻害することができるので、ヘムペルオキシダーゼの活性を阻害する物質として好適に使用することができる。本明細書において、「ヘムペルオキシダーゼに対してドミナントネガティブに作用するタンパク質の変異体」とは、それが発現することによって、ヘムペルオキシダーゼの活性を阻害(消失若しくは低下)させる作用を有するタンパク質を意味する(多比良和誠編, 遺伝子の機能阻害実験法, 羊土社, 26～32頁, 2001年など参照)。 (g) A variant of heme peroxidase that acts dominantly against heme peroxidase or a polynucleotide that encodes it A variant of heme peroxidase that acts dominantly negatively on heme peroxidase or a polynucleotide that encodes it Since the activity of peroxidase can be inhibited, it can be suitably used as a substance that inhibits the activity of heme peroxidase. In the present specification, the term “mutant of a protein that acts dominantly against heme peroxidase” means a protein that has an action of inhibiting (eliminating or reducing) the activity of heme peroxidase when it is expressed. (See Taira Yoshikazu, edited by gene function inhibition experiment, Yodosha, pp. 26-32, 2001).
(h) ヘムペルオキシダーゼに対するアプタマー
　ヘムペルオキシダーゼに対するアプタマーは、ヘムペルオキシダーゼの活性や機能を阻害することができるので、ヘムペルオキシダーゼの活性を阻害する物質として好適に使用することができる。アプタマーは、公知の方法、例えばSELEX(systematic evolution of ligands by exponential enrichment)法(Annual Review of Medicine 56巻，555-583頁，2005年)を用いて取得する。アプタマーの構造は、公知の方法を用いて決定することができ、その構造を基に公知の方法に従いアプタマーを製造する。 (h) Aptamer for heme peroxidase Since an aptamer for heme peroxidase can inhibit the activity and function of heme peroxidase, it can be suitably used as a substance that inhibits the activity of heme peroxidase. Aptamers are obtained using a known method, for example, the SELEX (systematic evolution of ligands by exponential enrichment) method (Annual Review of Medicine 56, 555-583, 2005). The structure of an aptamer can be determined using a known method, and an aptamer is produced according to a known method based on the structure.
2.2. 本発明が対象とする疾患
　本発明においては、上述したヘムペルオキシダーゼ阻害薬を含有する医薬を、気管支喘息の予防又は治療薬として用いる。好ましい態様のヘムペルオキシダーゼ阻害薬は、気管支喘息に対する予防・治療効果を有するので、気管支喘息患者に治療目的で投与してよく、あるいは、気管支喘息の予防や再発を考慮しなければならない患者に予防目的で投与することもできる。本発明の好ましい態様においては、ヘムペルオキシダーゼ阻害薬は、治療目的で投与される。 2.2. Diseases targeted by the present invention In the present invention, a medicament containing the above-mentioned heme peroxidase inhibitor is used as a prophylactic or therapeutic agent for bronchial asthma. Since the heme peroxidase inhibitor of the preferred embodiment has a preventive / therapeutic effect on bronchial asthma, it may be administered for therapeutic purposes to patients with bronchial asthma, or for the purpose of prevention for patients who must consider prevention or recurrence of bronchial asthma Can also be administered. In a preferred embodiment of the invention, the heme peroxidase inhibitor is administered for therapeutic purposes.
　「気管支喘息」は、非特異的な刺激物質に対して気道過敏性の亢進を示すことを特徴とした疾患であり、咳嗽、喀痰、呼吸困難などの臨床症状を引き起こす疾患である。本発明の予防又は治療薬により、これらの症状の少なくとも1つの症状を予防又は治療することができる。 “Bronchial asthma” is a disease characterized by increased airway hypersensitivity to nonspecific stimulating substances, and is a disease that causes clinical symptoms such as cough, sputum, and dyspnea. The preventive or therapeutic agent of the present invention can prevent or treat at least one of these symptoms.
　本発明の予防又は治療薬は、吸入ステロイド剤とは異なる作用機序により、気管支喘息の病態を改善することができる。このため、本発明の好ましい態様の予防又は治療薬は、ステロイド剤抵抗性の患者の治療に有用である。 The preventive or therapeutic agent of the present invention can improve the pathological condition of bronchial asthma by an action mechanism different from that of an inhaled steroid. For this reason, the preventive or therapeutic agent of the preferable aspect of this invention is useful for the treatment of the patient resistant to a steroid.
2．3．ヘムペルオキシダーゼ阻害薬を含有する製剤
　本発明においては、上述のヘムペルオキシダーゼ阻害薬を常套手段に従って、製剤化し、気管支喘息の予防又は治療薬として用いることができる。本発明のいくつかの態様の気管支喘息の予防又は治療薬では、ヘムペルオキシダーゼ阻害薬はチアマゾールである。 2.3. Formulation containing heme peroxidase inhibitor In the present invention, the above-mentioned heme peroxidase inhibitor can be formulated according to conventional means and used as a prophylactic or therapeutic agent for bronchial asthma. In the preventive or therapeutic agent for bronchial asthma according to some embodiments of the present invention, the heme peroxidase inhibitor is thiamazole.
　例えば、経口投与のための組成物としては、固体又は液体の剤形、具体的には錠剤(糖衣錠、フィルムコーティング錠を含む)、丸剤、顆粒剤、散剤、カプセル剤(ソフトカプセル剤を含む)、シロップ剤、乳剤、懸濁剤などがあげられる。かかる組成物は自体公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤若しくは賦形剤を含有するものである。例えば、錠剤用の担体、賦形剤としては、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムなどが用いられる。 For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
　非経口投与のための組成物としては、例えば、注射剤、坐剤、点眼剤、点鼻剤、噴霧吸入剤、塗布剤、貼付剤などが用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤、関節内注射剤などの剤形を包含する。かかる注射剤は、自体公知の方法に従って、例えば、上記活性成分を通常注射剤に用いられる無菌の水性若しくは油性液に溶解、懸濁又は乳化することによって調製する。注射用の水性液としては、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などが用いられ、適当な溶解補助剤、例えば、アルコール(例、エタノール)、ポリアルコール(例、プロピレングリコール、ポリエチレングリコール)、非イオン界面活性剤〔例、ポリソルベート80、HCO－50(polyoxyethylene(50mol)adduct of hydrogenated castor oil)〕などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、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などを併用してもよい。調製された注射液は、通常、適当なアンプルに充填される。直腸投与に用いられる坐剤は、上記活性成分を通常の坐薬用基剤に混合することによって調製される。点眼剤、点鼻剤、噴霧吸入剤、坐剤、塗布剤、貼付剤等も、上記活性成分を用いて自体公知の方法に従って調製される。 As a composition for parenteral administration, for example, injections, suppositories, eye drops, nasal drops, spray inhalants, coating agents, patches and the like are used, and the injections are intravenous injections and subcutaneous injections. And dosage forms such as intradermal injection, intramuscular injection, infusion injection, intraarticular injection and the like. Such an injection is prepared by a method known per se, for example, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, isotonic solution containing physiological saline, glucose and other adjuvants, etc. are used, and appropriate solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration are prepared by mixing the above active ingredients with a conventional suppository base. Eye drops, nasal drops, spray inhalants, suppositories, coating agents, patches and the like are also prepared according to methods known per se using the above active ingredients.
　なお前記した各組成物は、上記活性成分との配合により好ましくない相互作用を生じない限り他の活性成分(例えば、他の気管支喘息の予防又は治療薬など)を含有してもよい。また、他の活性成分(例えば、他の気管支喘息の予防又は治療薬など)と併用してもよい。 Each of the above-described compositions may contain other active ingredients (for example, other prophylactic or therapeutic agents for bronchial asthma) as long as undesirable interactions are not caused by blending with the active ingredients. Further, it may be used in combination with other active ingredients (for example, other prophylactic or therapeutic agents for bronchial asthma).
　本発明の予防又は治療薬の活性成分の投与量は、その作用、対象疾患、投与対象、症状、投与ルートなどによっても異なるが、例えば経口投与する場合、一般的に成人(体重60kgとして)においては、一日につき該活性成分を、約0.1～100mg、好ましくは約1．0～50mg、より好ましくは約1．0～20mg投与する。非経口的に投与する場合は、該活性成分の投与量は、対象疾患、投与対象、症状、投与ルートなどによっても異なるが、注射剤の形で投与する場合、一般的に成人(体重60kgとして)においては、一日につき該活性成分を、約0．01～30mg、好ましくは約0．1～20mg、より好ましくは約0．1～10mgを静脈注射により投与するのが好都合である。他の動物の場合も、体重60kg当たりに換算した量を投与することができる。 The dose of the active ingredient of the preventive or therapeutic agent of the present invention varies depending on its action, target disease, administration subject, symptom, administration route, etc., but for example, when administered orally, generally in adults (weight 60 kg) Administers the active ingredient from about 0.1 to 100 mg, preferably from about 1.0 to 50 mg, more preferably from about 1.0 to 20 mg per day. When administered parenterally, the dosage of the active ingredient varies depending on the target disease, administration subject, symptom, administration route, etc., but when administered in the form of an injection, it is generally an adult (with a body weight of 60 kg). ), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg of the active ingredient per day by intravenous injection. In the case of other animals, an amount converted per 60 kg body weight can be administered.
　上記アンチセンスポリヌクレオチドは、自体公知の方法に従って製剤化し、投与することができる。また、例えば、前記のアンチセンスポリヌクレオチドを単独あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当なベクターに挿入した後、常套手段に従って、ヒト又は哺乳動物(例、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど)に対して経口的又は非経口的に投与することができる。該アンチセンスポリヌクレオチドは、そのままで、あるいは摂取促進のために補助剤などの生理学的に認められる担体とともに製剤化し、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与できる。あるいは、エアロゾル化して吸入剤として気管内に局所投与することもできる。さらに、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記のアンチセンスポリヌクレオチドを単独又はリポゾームなどの担体とともに製剤(注射剤)化し、静脈等に投与してもよい。上記二重鎖RNA、リボザイム、上記ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチドなどは、上記アンチセンスポリヌクレオチドと同様にして製剤化し、投与することができる。 The antisense polynucleotide can be formulated and administered according to a method known per se. In addition, for example, after inserting the above-described antisense polynucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., according to conventional means, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) orally or parenterally. The antisense polynucleotide can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant. Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving cellular uptake efficiency, the above-mentioned antisense polynucleotide is formulated alone (injection) together with a carrier such as liposome and administered to veins, etc. Also good. The double-stranded RNA, ribozyme, mutant of heme peroxidase that acts dominantly against the heme peroxidase or the polynucleotide encoding the same can be formulated and administered in the same manner as the antisense polynucleotide. it can.
　上記抗体、アプタマーなどは、それ自体又は適当な医薬組成物として投与することができる。上記投与に用いられる医薬組成物は、上記抗体又はその塩と薬理学的に許容され得る担体、希釈剤若しくは賦形剤とを含むものである。かかる組成物は、経口又は非経口投与(例、静脈注射)に適する剤形として提供される。好ましくは吸入剤として提供される。 The above antibodies, aptamers and the like can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration (eg, intravenous injection). Preferably it is provided as an inhalant.
3．気管支喘息の予防又は治療薬のスクリーニング方法
　本発明は、試験化合物をヘムペルオキシダーゼを発現する細胞と接触させ、ヘムペルオキシダーゼの活性又はヘムペルオキシダーゼの発現量を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法を提供する。また、本発明は、試験化合物をヘムペルオキシダーゼと接触させ、ヘムペルオキシダーゼの活性を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法を提供する。 3． The present invention relates to a method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with a cell expressing heme peroxidase and using the heme peroxidase activity or heme peroxidase expression level as an index. Provide a way to do it. The present invention also provides a method for screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with heme peroxidase and using the activity of heme peroxidase as an index.
　本発明のスクリーニング方法の好ましい態様は、試験化合物のヘムペルオキシダーゼ阻害活性を評価し、ペルオキシダーゼ阻害活性を有する化合物を選択することを含む方法である。選択されたペルオキシダーゼ阻害活性を有する化合物は、気管支喘息の予防または治療薬のための候補化合物となる。 A preferred embodiment of the screening method of the present invention is a method comprising evaluating a heme peroxidase inhibitory activity of a test compound and selecting a compound having a peroxidase inhibitory activity. The selected compound having peroxidase inhibitory activity is a candidate compound for a prophylactic or therapeutic agent for bronchial asthma.
　「ヘムペルオキシダーゼ阻害活性」とは、ヘムペルオキシダーゼの活性又はヘムペルオキシダーゼの発現量を低下させ又は抑制する活性を意味する。 “Heme peroxidase inhibitory activity” means an activity that reduces or suppresses the activity of heme peroxidase or the expression level of heme peroxidase.
　試験化合物としては、例えば、ペプチド、タンパク質、抗体、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液、血漿などが挙げられ、これら化合物は新規な化合物であってもよいし、公知の化合物であってもよい。試験化合物は塩を形成していてもよく、試験化合物の塩としては、生理学的に許容される金属塩、アンモニウム塩、有機塩基との塩、無機酸との塩、有機酸との塩、塩基性または酸性アミノ酸との塩などが挙げられる。金属塩の好適な例としては、例えばナトリウム塩、カリウム塩などのアルカリ金属塩；カルシウム塩、マグネシウム塩、バリウム塩などのアルカリ土類金属塩；アルミニウム塩などが挙げられる。有機塩基との塩の好適な例としては、例えばトリメチルアミン、トリエチルアミン、ピリジン、ピコリン、2，6－ルチジン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、シクロヘキシルアミン、ジシクロヘキシルアミン、N，N'－ジベンジルエチレンジアミンなどとの塩が挙げられる。無機酸との塩の好適な例としては、例えば塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。有機酸との塩の好適な例としては、例えばギ酸、酢酸、トリフルオロ酢酸、プロピオン酸、フタル酸、フマル酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、安息香酸、ベンゼンスルホン酸、p－トルエンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩の好適な例としては、例えばアルギニン、リジン、オルニチンなどとの塩が挙げられ、酸性アミノ酸との塩の好適な例としては、例えばアスパラギン酸、グルタミン酸などとの塩が挙げられる。 Examples of the test compound include peptides, proteins, antibodies, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound. The test compound may form a salt, and as the salt of the test compound, physiologically acceptable metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, bases And salts with acidic or acidic amino acids. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid And salts with benzoic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
　本発明のいくつかの態様の気管支喘息の予防または治療薬のスクリーニング方法では、 (i)試験化合物をヘムペルオキシダーゼを発現する細胞と接触させた場合と、(ii)試験化合物をヘムペルオキシダーゼを発現する細胞に接触させない場合の、該細胞におけるヘムペルオキシダーゼの活性又は発現量の比較を行う。
　本方法においては、ヘムペルオキシダーゼを発現する細胞に、試験化合物を接触させる。用いられる「細胞」の由来としては、特に制限されないが、例えば、ヒト、マウスなど由来の細胞であり、好ましくは、ヒト由来の細胞である。本発明のスクリーニング方法で用いられる「ヘムペルオキシダーゼを発現する細胞」は、例えば、気道上皮細胞、好中球、好酸球等である。本発明のスクリーニング方法で用いられる「ヘムペルオキシダーゼを発現する細胞」は、一般的な遺伝子工学技術によって作製することもできる。 In some methods for screening a prophylactic or therapeutic agent for bronchial asthma according to the present invention, (i) when a test compound is contacted with a cell expressing heme peroxidase, and (ii) the test compound expresses heme peroxidase Comparison of the activity or expression level of heme peroxidase in the cells when not in contact with the cells is performed.
In this method, a test compound is contacted with cells expressing heme peroxidase. The origin of the “cell” used is not particularly limited, but is, for example, a cell derived from a human, a mouse or the like, and preferably a cell derived from a human. “Cells expressing heme peroxidase” used in the screening method of the present invention are, for example, airway epithelial cells, neutrophils, eosinophils and the like. The “cell expressing heme peroxidase” used in the screening method of the present invention can also be prepared by a general genetic engineering technique.
　次に、ヘムペルオキシダーゼの活性又は発現量を測定する。具体的には、例えば、上記（i）と（ii）の場合において、上記細胞を培養し、それぞれの場合のヘムペルオキシダーゼの活性又は発現量を測定する。ヘムペルオキシダーゼの活性又は発現量の測定は、例えば、前述のヘムペルオキシダーゼの活性又は発現量の測定方法により行うことができる。 Next, the activity or expression level of heme peroxidase is measured. Specifically, for example, in the cases (i) and (ii) above, the cells are cultured, and the activity or expression level of heme peroxidase in each case is measured. The measurement of the activity or expression level of heme peroxidase can be performed, for example, by the above-described method for measuring the activity or expression level of heme peroxidase.
　次いで、試験化合物を接触させない場合（コントロール）と比較して、ヘムペルオキシダーゼの活性又は発現を阻害する化合物を選択する。例えば、上記(i)の場合におけるヘムペルオキシダーゼの活性が、上記(ii)の場合のヘムペルオキシダーゼの活性より低くなる試験化合物、特には、10％以上、20％以上、30％以上、40％以上、又は50％以上低くなる試験化合物を、ヘムペルオキシダーゼの活性を阻害する化合物として選択することができる。あるいは、例えば、上記(i)の場合におけるヘムペルオキシダーゼの発現量が、上記(ii)の場合のヘムペルオキシダーゼの発現量より低くなる試験化合物、特には、10％以上、20％以上、30％以上、40％以上、又は50％以上低くなる試験化合物を、ヘムペルオキシダーゼの発現を阻害する化合物として選択することができる。 Next, a compound that inhibits the activity or expression of heme peroxidase is selected as compared with the case where the test compound is not contacted (control). For example, test compounds in which the activity of heme peroxidase in the case of (i) is lower than the activity of heme peroxidase in the case of (ii), particularly 10% or more, 20% or more, 30% or more, 40% or more Alternatively, a test compound that decreases by 50% or more can be selected as a compound that inhibits the activity of heme peroxidase. Alternatively, for example, a test compound in which the expression level of heme peroxidase in the case of (i) is lower than the expression level of heme peroxidase in the case of (ii), in particular, 10% or more, 20% or more, 30% or more Test compounds that decrease by 40% or more, or 50% or more can be selected as compounds that inhibit heme peroxidase expression.
　本発明の別のいくつかの態様の気管支喘息の予防又は治療薬のスクリーニング方法では、細胞を用いずに、ヘムペルオキシダーゼの活性の比較を行う。すなわち、(i)試験化合物をヘムペルオキシダーゼと接触させた場合と、(ii)試験化合物をヘムペルオキシダーゼに接触させない場合の、ヘムペルオキシダーゼの活性の比較を行う。
　本方法においては、試験化合物をヘムペルオキシダーゼと接触させる。具体的には、ヘムペルオキシダーゼを用いてSCN－ + H2O2 → OSCN－ + H2Oの反応を起こす反応系に、試験化合物を添加する。ヘムペルオキシダーゼは前記の通りであるが、ヘムペルオキシダーゼとして精製したものを用いるのが好ましい。また、反応は、試験管内で行うのが好ましい。 In the screening method for a prophylactic or therapeutic agent for bronchial asthma according to another aspect of the present invention, the activity of heme peroxidase is compared without using cells. That is, the activity of heme peroxidase is compared when (i) the test compound is contacted with heme peroxidase and (ii) when the test compound is not contacted with heme peroxidase.
In this method, the test compound is contacted with heme peroxidase. Specifically, a test compound is added to a reaction system that causes a reaction of SCN − + H 2 O 2 → OSCN − + H 2 O using heme peroxidase. Heme peroxidase is as described above, but it is preferable to use one purified as heme peroxidase. The reaction is preferably performed in a test tube.
　次に、ヘムペルオキシダーゼの活性を測定する。具体的には、例えば、上記（i）と（ii）の場合において、それぞれの場合のヘムペルオキシダーゼの活性を測定する。ヘムペルオキシダーゼの活性の測定は、例えば、前述のヘムペルオキシダーゼの活性の測定方法により行うことができる。 Next, the activity of heme peroxidase is measured. Specifically, for example, in the cases (i) and (ii) above, the activity of heme peroxidase in each case is measured. The activity of heme peroxidase can be measured, for example, by the above-described method for measuring the activity of heme peroxidase.
　次いで、試験化合物を接触させない場合（コントロール）と比較して、ヘムペルオキシダーゼの活性を阻害する化合物を選択する。例えば、上記(i)の場合におけるヘムペルオキシダーゼの活性が、上記(ii)の場合のヘムペルオキシダーゼの活性より低くなる試験化合物、特には、10％以上、20％以上、30％以上、40％以上、又は50％以上低くなる試験化合物を、ヘムペルオキシダーゼの活性を阻害する化合物として選択することができる。 Next, a compound that inhibits the activity of heme peroxidase is selected as compared with the case where the test compound is not contacted (control). For example, test compounds in which the activity of heme peroxidase in the case of (i) is lower than the activity of heme peroxidase in the case of (ii), particularly 10% or more, 20% or more, 30% or more, 40% or more Alternatively, a test compound that decreases by 50% or more can be selected as a compound that inhibits the activity of heme peroxidase.
　さらに、選択された候補化合物を実験動物(例、マウス、ラットなど)に投与して、選択された候補化合物の気管支喘息予防又は治療効果を確認する。気管支喘息予防又は治療効果を確認するための試験法としては、後述の実施例に記載の方法のように、気管支喘息モデルマウスを用いて気道過敏性を測定すること、気管支肺胞洗浄液を解析することなどが挙げられる。 Further, the selected candidate compound is administered to an experimental animal (eg, mouse, rat, etc.), and the effect of the selected candidate compound on the prevention or treatment of bronchial asthma is confirmed. As a test method for confirming the effect of preventing or treating bronchial asthma, as in the method described in the examples below, measuring airway hypersensitivity using a bronchial asthma model mouse and analyzing bronchoalveolar lavage fluid And so on.
　以下に、実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
実施例1：気管支喘息モデルマウスに対するSCN － イオンの作用
　図1(A)に実験のプロトコルを示す。図1(A)のプロトコルに示したように、Day 0より毎日20 mMのチオシアネートイオン(SCN－)を含んだ飲み水を服用させた。Day0と12にアレルゲンである卵白アルブミンで感作を行い(感作1, 2)、Day 22, 23, 24で卵白アルブミンの曝露を行って(曝露1～3)、曝露3の24時間後に気道過敏性を測定した。このプロトコルにて実験を行った群を、喘息群（＋SCN）又は感作あり（＋SCN）群とする。 Example 1: Action of SCN - ion on bronchial asthma model mice FIG. 1 (A) shows the experimental protocol. As shown in the protocol of FIG. 1 (A), drinking water containing 20 mM thiocyanate ion (SCN − ) was taken daily from Day 0. Sensitize with allergen ovalbumin on days 0 and 12 (sensitization 1, 2), and ovalbumin exposure on days 22, 23, 24 (exposure 1-3), airway 24 hours after exposure 3 Hypersensitivity was measured. The group in which the experiment was conducted with this protocol is defined as an asthma group (+ SCN) or a sensitized group (+ SCN).
　対照として、非喘息群（－SCN）、非喘息群（＋SCN）群、及び喘息群（－SCN）を用いた。
　非喘息群（－SCN）は、図1(B)では感作なし（－SCN）群と表記しており、卵白アルブミンによる感作を行わなかったこと、並びにチオシアネートイオンの飲水投与の代わりにチオシアネートイオンを含有しない飲水を投与したことを除いて、感作あり（＋SCN）群と同様のプロトコルにて実験を行った。
　非喘息群（＋SCN）群は、図1(B)では感作なし（＋SCN）群と表記しており、卵白アルブミンによる感作を行わなかったことを除いて、感作あり（＋SCN）群と同様のプロトコルにて実験を行った。
　喘息群（－SCN）は、図１(B)では感作あり（－SCN）群と表記しており、チオシアネートイオンの飲水投与の代わりにチオシアネートイオンを含有しない飲水を投与したことを除いて、感作あり（＋SCN）群と同様のプロトコルにて実験を行った。 As controls, a non-asthma group (-SCN), a non-asthma group (+ SCN) group, and an asthma group (-SCN) were used.
The non-asthma group (-SCN) is indicated as the non-sensitized (-SCN) group in FIG. 1 (B), and sensitization with ovalbumin was not performed, and thiocyanate was used instead of drinking thiocyanate ion. The experiment was performed using the same protocol as the sensitized group (+ SCN) except that drinking water containing no ions was administered.
The non-asthma group (+ SCN) group is shown as the non-sensitized (+ SCN) group in FIG. 1 (B), and the non-asthmatic group (+ SCN) group is the same as the sensitized (+ SCN) group except that no sensitization with ovalbumin Experiments were performed using the same protocol.
The asthma group (-SCN) is labeled as a sensitized (-SCN) group in FIG. 1 (B), except that the thiocyanate ion-free drinking water was administered instead of the thiocyanate ion drinking water, Experiments were performed using the same protocol as the sensitized (+ SCN) group.
　実験には、日本SLCから入手したBALB/c (6週齢、メス)マウスを用いた。
　チオシアネートイオン(SCN－)を含んだ飲み水を、次のように作製した。すなわち、1.94gのチオシアン酸カリウム（分子量97.18、和光純薬から購入）を1Lの滅菌蒸留水に溶かして調製した。 For the experiment, BALB / c (6 weeks old, female) mice obtained from Japan SLC were used.
Drinking water containing thiocyanate ion (SCN − ) was prepared as follows. That is, 1.94 g of potassium thiocyanate (molecular weight 97.18, purchased from Wako Pure Chemical Industries, Ltd.) was dissolved in 1 L of sterile distilled water.
　　気管支喘息モデルマウスを作製するための、卵白アルブミンの調製、感作、及び曝露は、Nakao I et al., J Immunol, vol.180, 6262－6269, 2008に記載の方法に従って行った。具体的には、生理食塩水に水酸化アルミニウム(ALUM, LSL CO., LTDから購入)と卵白アルブミン(OVA, sigmaから購入)を濃度がそれぞれ2mg/mlと0.1mg/mlになるように加えてALUM/ OVA溶液を調製した。調製したALUM/ OVA溶液を、1匹あたり500μlのALUM/ OVA溶液をマウスの腹腔内に投与することにより、感作を行った。曝露はOVAを1g/mlの濃度で生理食塩水に溶かし、このOVA溶液を密閉容器に入れたマウスに超音波霧化器を用いて噴霧し吸引させることにより行った。 Preparation, sensitization, and exposure of ovalbumin for producing bronchial asthma model mice were performed according to the methods described in Nakao I et al., J Immunol, vol.180, 6262-6269, 2008. Specifically, aluminum hydroxide (purchased from ALUM, LSL か ら CO., LTD) and ovalbumin (purchased from OVA, sigma) were added to physiological saline to a concentration of 2 mg / ml and 0.1 mg / ml, respectively. The ALUM / OVA solution was prepared. Sensitization was performed by administering 500 μl of ALUM / 500OVA solution per mouse intraperitoneally to the prepared ALUM / OVA solution. The exposure was performed by dissolving OVA in physiological saline at a concentration of 1 g / ml, and spraying and sucking the OVA solution in a sealed container using an ultrasonic atomizer.
　気道過敏性の測定方法は、Matsushita, H. et al. Int Immunol 22, 739-747, 2010に記載の方法に従って行った。具体的には、最終曝露から24時間後、非拘束式全身プレチスモグラフィー(BUXCO)を用いてメサコリンに対する気道過敏性を測定した。メサコリン(sigmaから購入)は生理食塩水で希釈し図1(B)に示した濃度で用いた。 Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine (purchased from sigma) was diluted with physiological saline and used at the concentration shown in FIG. 1 (B).
　結果を図1(B)に示す。感作あり群では、感作なし群に比べて気道過敏性の亢進が認められた。そして、感作あり群の中で、SCN－飲水あり群(感作あり(＋SCN)群)では、SCN－飲水なし群(感作あり(－SCN)群)に比べて気道過敏性のさらなる亢進が認められた。
　以上の結果より、SCN－イオンが喘息に対して増悪因子であることが明らかになった。 The results are shown in FIG. 1 (B). Increased airway hyperresponsiveness was observed in the sensitized group compared to the non-sensitized group. Then, in the sensitization Yes group, SCN - in drinking water Yes group (with sensitization (+ SCN) group), SCN - further enhancement of airway hyperresponsiveness compared to drinking water without the group (with sensitization (-SCN) group) Was recognized.
From the above results, it was revealed that SCN - ion is an exacerbation factor for asthma.
実施例2：気道上皮細胞に対するOSCN － イオンの作用
　この実験では、細胞培養液中において次のような反応を起こさせた。 Example 2: Effect of OSCN - ion on airway epithelial cells In this experiment, the following reaction was caused in a cell culture medium.
Figure JPOXMLDOC01-appb-C000001
  
Figure JPOXMLDOC01-appb-C000001
  
　つまり、GOXとLPOの両方が存在する場合にはOSCN－が、GOXのみが存在する場合にはH2O2が存在することになる。 That is, if there are both GOX and LPO is OSCN - is, there will be H 2 O 2 in the case where only the GOX is present.
　具体的には、気道上皮細胞に対するOSCN－イオンの作用を次のようにして調べた。気道上皮細胞（NCI-H292細胞）をRPMI1640/5%FCS/50mM HEPES培地に懸濁し、1.8x106個の細胞を6穴プレートに蒔いた。一晩培養後、細胞培養液にNaSCN(終濃度1mM)、GOX(終濃度9 mUnits/ml)、LPO(終濃度40U/ml)を添加した。さらに５時間培養後、Schreiber et alの方法(Nucleic Acids Res. 17, 6419,1989)を用いて核抽出液を調製し、Midwinteretal と同様の方法（Biochem. J.396, 71-78, 2006）でNF-κBの活性化の状態を解析した。NaSCNはsigmaから、GOXは和光純薬から、LPOはCALZYME Laboratoryから購入した。 Specifically, the effect of OSCN - ion on airway epithelial cells was examined as follows. Airway epithelial cells (NCI-H292 cells) were suspended in RPMI1640 / 5% FCS / 50 mM HEPES medium, and 1.8 × 10 6 cells were seeded in a 6-well plate. After overnight culture, NaSCN (final concentration 1 mM), GOX (final concentration 9 mUnits / ml), and LPO (final concentration 40 U / ml) were added to the cell culture medium. After further incubation for 5 hours, a nuclear extract was prepared using the method of Schreiber et al (Nucleic Acids Res. 17, 6419, 1989) and the same method as Midwinteretal (Biochem. J.396, 71-78, 2006). Was used to analyze the activation state of NF-κB. NaSCN was purchased from sigma, GOX was purchased from Wako Pure Chemicals, and LPO was purchased from CALZYME Laboratory.
　その結果を図2に示す。GOXとLPOの両方が存在する場合、つまりOSCN－が存在する場合にのみ炎症反応に重要なNF－κBの活性化が認められた。
　以上の結果より、OSCN－イオンも喘息に対する増悪因子であることを明らかになった。
　そして、気道組織内のヘムペルオキシダーゼは喘息を増悪させる因子であることが明らかになった。 The result is shown in FIG. Activation of NF-κB, which is important for the inflammatory response, was observed only in the presence of both GOX and LPO, ie in the presence of OSCN − .
The above results revealed that OSCN - ion is also an exacerbation factor for asthma.
Heme peroxidase in the airway tissue was found to be a factor that exacerbates asthma.
実施例3：気管支喘息モデルマウスに対するヘムペルオキシダーゼの作用
　本実施例では、さらに、ヘムペルオキシダーゼの活性を阻害すること、ヘムペルオキシダーゼの発現を阻害することなどが、気管支喘息の病態の改善につながることを確認した。
　図3(A)に実験のプロトコルを示す。図3(A)のプロトコルに示したように、Day0と12にアレルゲンである卵白アルブミンで感作を行い(感作1, 2)、Day 22, 26, 30で卵白アルブミンの曝露を行った(曝露1～3)。Day 20より毎日0.2 mg/mlのチアマゾールを含んだ飲み水を服用させた。3回目の曝露の24時間後に気道過敏性を測定し、48時間後に気管支肺胞洗浄液の解析を行った。このプロトコルにて実験を行った群を、喘息群(チアマゾールあり)とする。尚、喘息群(チアマゾールあり)は、図3(B), (C)ではチアマゾール投与群と表記されている。 Example 3: Effect of heme peroxidase on bronchial asthma model mice In this example, it was further confirmed that inhibition of heme peroxidase activity, inhibition of heme peroxidase expression, etc., led to improvement of the pathology of bronchial asthma. confirmed.
FIG. 3 (A) shows the experimental protocol. As shown in the protocol of FIG. 3 (A), sensitization was performed with ovalbumin, an allergen, on day 0 and 12 (sensitization 1, 2), and ovalbumin exposure was performed on days 22, 26, 30 ( Exposure 1-3). From Day 20, I took drinking water containing 0.2 mg / ml thiamazole every day. Airway hyperresponsiveness was measured 24 hours after the third exposure, and bronchoalveolar lavage fluid was analyzed 48 hours later. The group for which the experiment was conducted using this protocol is referred to as an asthma group (with thiamazole). Note that the asthma group (with thiamazole) is represented as a thiamazole administration group in FIGS. 3 (B) and 3 (C).
　対照として、非喘息群、及び喘息群(チアマゾールなし)を用いた。
　非喘息群は、図3(B),(C)ではコントロール群と表記されており、卵白アルブミンによる感作を行わなかったこと、並びにチアマゾールの飲水投与の代わりにチアマゾールを含有しない飲水を投与したことを除いて、喘息群(チアマゾールあり)と同様のプロトコルにて実験を行った群である。
　喘息群(チアマゾールなし)は、図3(B),(C)では喘息群と表記されており、チアマゾールの飲水投与の代わりにチアマゾールを含有しない飲水を投与したことを除いて、喘息群(チアマゾールあり)と同様のプロトコルにて実験を行った群である。 As controls, a non-asthma group and an asthma group (without thiamazole) were used.
The non-asthma group is described as a control group in FIGS. 3 (B) and 3 (C), and sensitization with ovalbumin was not performed, and drinking water containing no thiamazole was administered instead of drinking thiamazole. Except for this, this group was tested using the same protocol as the asthma group (with thiamazole).
The asthma group (without thiamazole) is labeled as the asthma group in FIGS. 3 (B) and (C), and the asthma group (thiamazole) was administered except that thiazazole-free drinking was administered instead of thiamazole drinking. This is a group in which an experiment was conducted with the same protocol as (Yes).
　ここで、実験には、日本SLCから入手したBALB/c マウス(6週齢、オス)を用いた。
　チアマゾールを含んだ飲み水は、20mgのチアマゾール(和光純薬)を100mlの滅菌蒸留水に溶かすことで調製した（終濃度は0.2mg/ml）。 Here, for the experiment, BALB / c mice (6 weeks old, male) obtained from Japan SLC were used.
Drinking water containing thiamazole was prepared by dissolving 20 mg thiamazole (Wako Pure Chemical Industries) in 100 ml sterile distilled water (final concentration 0.2 mg / ml).
　　気管支喘息モデルマウスを作製するための、卵白アルブミンの調製、感作、及び曝露は、Nakao I et al., J Immunol, vol.180, 6262－6269, 2008に記載の方法に従って行った。具体的には、生理食塩水に水酸化アルミニウム(ALUM, LSL CO., LTDから購入)と卵白アルブミン(OVA, sigmaから購入)を濃度がそれぞれ2mg/mlと0.1mg/mlになるように加えてALUM/ OVA溶液を調製した。調製したALUM/ OVA溶液を、1匹あたり500μlのALUM/ OVA溶液をマウスの腹腔内に投与することで感作を行った。曝露はOVAを1g/mlの濃度で生理食塩水に溶かし、このOVA溶液を密閉容器に入れたマウスに超音波霧化器を用いて噴霧し吸引させることで行った。 Preparation, sensitization, and exposure of ovalbumin for producing bronchial asthma model mice were performed according to the methods described in Nakao I et al., J Immunol, vol.180, 6262-6269, 2008. Specifically, aluminum hydroxide (purchased from ALUM, LSL か ら CO., LTD) and ovalbumin (purchased from OVA, sigma) were added to physiological saline to a concentration of 2 mg / ml and 0.1 mg / ml, respectively. The ALUM / OVA solution was prepared. Sensitization was performed by administering 500 μl of ALUM / VAOVA solution per mouse intraperitoneally to the prepared ALUM / 匹 OVA solution. The exposure was performed by dissolving OVA in physiological saline at a concentration of 1 g / ml, and spraying and sucking the OVA solution in a sealed container using an ultrasonic atomizer.
　気道過敏性の測定方法は、Matsushita, H. et al. Int Immunol 22, 739-747, 2010に記載の方法に従って行った。具体的には、最終曝露から24時間後、非拘束式全身プレチスモグラフィー(BUXCO)を用いてメサコリンに対する気道過敏性を測定した。メサコリンは生理食塩水で希釈し図3(B)に示した濃度で用いた。 Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine was diluted with physiological saline and used at the concentration shown in FIG. 3 (B).
　気管支肺胞洗浄液の採取及び解析はMatsushita, H. et al. Int Immunol 22, 739-747, 2010に記載の方法に従って行った。具体的には、1.5mlの生理食塩水を用いて気管支肺胞洗浄液を調製し、この洗浄液中の細胞総数をhemocytometer (CDA500; Sysmex)で測定後、洗浄液中の細胞をサイトスピンし Diff-Quik (Sysmex)で染色して好酸球の数を測定した。 Bronchoalveolar lavage fluid was collected and analyzed according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, bronchoalveolar lavage fluid was prepared using 1.5 ml of physiological saline, and the total number of cells in this lavage fluid was measured with a hemocytometer (CDA500; Sysmex), and then the cells in the lavage fluid were cytospun and Diff-Quik The number of eosinophils was measured by staining with (Sysmex).
　気道過敏性の測定結果を図3(B)に示す。チアマゾール投与群では、チアマゾール投与を行っていない群(喘息群)に比べて気道過敏性の抑制が認められた。
　また、気管支肺胞洗浄液の解析結果を図3(C)に示す。チアマゾール投与群では、チアマゾール投与を行っていない群(喘息群)に比べて気管支肺胞洗浄液中の好酸球数の抑制が認められた。 The measurement results of airway hypersensitivity are shown in FIG. 3 (B). In the thiamazole-administered group, suppression of airway hypersensitivity was observed compared to the group not receiving thiamazole (asthma group).
The analysis result of bronchoalveolar lavage fluid is shown in FIG. 3 (C). In the thiamazole administration group, suppression of the number of eosinophils in the bronchoalveolar lavage fluid was observed compared to the group not receiving thiamazole (asthma group).
　以上のことから、ヘムペルオキシダーゼの活性を阻害すること、ヘムペルオキシダーゼの発現を阻害することなどが、気管支喘息の病態の改善につながることが明らかになった。
　また、ヘムペルオキシダーゼの活性又は発現量を指標として、気管支喘息の予防又は治療薬をスクリーニングすることができることが明らかとなった。 From the above, it has been clarified that inhibiting the activity of heme peroxidase, inhibiting the expression of heme peroxidase, and the like lead to improvement of the pathological condition of bronchial asthma.
Moreover, it became clear that the prophylactic or therapeutic agent for bronchial asthma can be screened using the activity or expression level of heme peroxidase as an index.
実施例4：気管支喘息モデルマウスにおけるチアマゾールとステロイド剤の効果の比較
　本実施例では、ヘムペルオキシダーゼの活性を阻害すること、ヘムペルオキシダーゼの発現を阻害することなどが、ステロイド剤であるデキサメタゾン(DEX)を投与したときと比較しても、優れた気管支喘息の予防又は治療効果を示すことを確認した。
　図4(A)に実験のプロトコルを示す。図4(A)のプロトコルに示したように、Day0と12にアレルゲンである卵白アルブミンで感作を行い(感作1, 2)、Day 22, 23, 24で卵白アルブミンの曝露を行った(曝露1～3)。喘息群（チアマゾール）では、Day 20より毎日0.2 mg/mlのチアマゾールを含んだ飲み水を服用させた。喘息群（DEX(5μg)）と喘息群（DEX(15μg)）では、チアマゾールを含有しない飲水を投与する一方で、DEXを生理食塩水に溶かし、マウス１匹あたり5μgまたは15μgを、1回目の曝露の2時間前と、各曝露の2時間前に腹腔内に投与した（Reber et al., J Immunol 2012; 188:3478-3487に準じた方法）。3回目の曝露の24時間後に気道過敏性を測定し、48時間後に気管支肺胞洗浄液の解析を行った。 Example 4: Comparison of effects of thiamazole and steroids in bronchial asthma model mice In this example, inhibition of heme peroxidase activity, inhibition of heme peroxidase expression, and the like are related to the steroid drug dexamethasone (DEX) Even when compared with the administration of, it has been confirmed that it exhibits an excellent preventive or therapeutic effect on bronchial asthma.
FIG. 4 (A) shows the experimental protocol. As shown in the protocol of FIG. 4 (A), sensitization was performed with ovalbumin, an allergen, on Day 0 and 12 (sensitization 1, 2), and exposure to ovalbumin was performed on Day 22, 23, 24 ( Exposure 1-3). In the asthma group (thiamazole), from Day 20, daily drinking water containing 0.2 mg / ml thiamazole was taken. In the asthma group (DEX (5 μg)) and the asthma group (DEX (15 μg)), drinking water that does not contain thiamazole, while dissolving DEX in physiological saline, 5 μg or 15 μg per mouse It was administered intraperitoneally 2 hours before exposure and 2 hours before each exposure (method according to Reber et al., J Immunol 2012; 188: 3478-3487). Airway hyperresponsiveness was measured 24 hours after the third exposure, and bronchoalveolar lavage fluid was analyzed 48 hours later.
　対照として、薬剤を投与しない非喘息群、及び薬剤を投与しない喘息群を用いた。
　薬剤を投与しない非喘息群は、卵白アルブミンによる感作を行わなかったこと、並びにチアマゾールの飲水投与の代わりにチアマゾールを含有しない飲水を投与したことを除いて、喘息群(チアマゾール)と同様のプロトコルにて実験を行った群である。
　薬剤を投与しない喘息群は、チアマゾールの飲水投与の代わりにチアマゾールを含有しない飲水を投与したことを除いて、喘息群(チアマゾール)と同様のプロトコルにて実験を行った群である。 As controls, a non-asthma group to which no drug was administered and an asthma group to which no drug was administered were used.
The non-asthmatic group that did not receive the drug did not receive sensitization with ovalbumin, and the same protocol as the asthmatic group (thiamazole), except that it received drinking water that did not contain thiamazole instead of drinking thiamazole. This is a group in which experiments were conducted.
The asthma group to which no drug is administered is a group in which an experiment was conducted according to the same protocol as the asthma group (thiamazole), except that drinking water containing no thiamazole was administered instead of drinking thiamazole.
　ここで、実験には、日本SLCから入手したBALB/c マウス(6週齢、メス)を用いた。
　チアマゾールを含んだ飲み水は、20mgのチアマゾール(和光純薬)を100mlの滅菌蒸留水に溶かすことで調製した（終濃度は0.2mg/ml）。
　DEXを含む生理食塩水は、具体的には次のようにして調製し、投与した。水可溶性のDEX(cat No.D2915,シグマアルドリッチ ジャパン)を25μg/mlまたは75μg/mlの濃度になるように生理食塩水で溶かし、各200μlを腹腔内に投与した。
　卵白アルブミンの調製、感作、及び曝露の方法は、実施例３に記載の方法に準じて行った。 Here, BALB / c mice (6 weeks old, female) obtained from Japan SLC were used for the experiment.
Drinking water containing thiamazole was prepared by dissolving 20 mg thiamazole (Wako Pure Chemical Industries) in 100 ml sterile distilled water (final concentration 0.2 mg / ml).
Specifically, the physiological saline containing DEX was prepared and administered as follows. Water-soluble DEX (cat No. D2915, Sigma-Aldrich Japan) was dissolved in physiological saline to a concentration of 25 μg / ml or 75 μg / ml, and 200 μl of each was intraperitoneally administered.
The method of preparation, sensitization, and exposure of ovalbumin was performed according to the method described in Example 3.
　気道過敏性の測定方法は、Matsushita, H. et al. Int Immunol 22, 739-747, 2010に記載の方法に従って行った。具体的には、最終曝露から24時間後、非拘束式全身プレチスモグラフィー(BUXCO)を用いてメサコリンに対する気道過敏性を測定した。メサコリンは生理食塩水で希釈し図4(B)に示した濃度で用いた。 Airway hypersensitivity was measured according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, airway hypersensitivity to mesacholine was measured using unrestrained whole body plethysmography (BUXCO) 24 hours after the final exposure. Mesacholine was diluted with physiological saline and used at the concentration shown in FIG. 4 (B).
　気管支肺胞洗浄液の採取及び解析はMatsushita, H. et al. Int Immunol 22, 739-747, 2010に記載の方法に従って行った。具体的には、1.5mlの生理食塩水を用いて気管支肺胞洗浄液を調製し、この洗浄液中の細胞総数をhemocytometer (CDA500; Sysmex)で測定した。さらに、洗浄液中の細胞をLy-6G抗原（好中球マーカー）、siglec-F抗原（好酸球マーカー）、CD3抗原(T細胞マーカー)、F4/80 抗原（マクロファージ　マーカー）に対するそれぞれの抗体で染色後、フローサイトメーター(FACSCaliburTM； BD Biosciences)で解析し、各種細胞の割合と数を測定した。抗siglec-F抗体はBD Pharmingenから、これ以外の抗体はeBioscienceから購入した。 Bronchoalveolar lavage fluid was collected and analyzed according to the method described in Matsushita, H. et al. Int Immunol 22, 739-747, 2010. Specifically, bronchoalveolar lavage fluid was prepared using 1.5 ml of physiological saline, and the total number of cells in the lavage fluid was measured with a hemocytometer (CDA500; Sysmex). Furthermore, the cells in the washing solution were treated with respective antibodies against Ly-6G antigen (neutrophil marker), siglec-F antigen (eosinophil marker), CD3 antigen (T cell marker), and F4 / 80 antigen (macrophage marker). After staining, analysis was performed with a flow cytometer (FACSCalibur ™; BD Biosciences), and the ratio and number of various cells were measured. The anti-siglec-F antibody was purchased from BD Pharmingen, and the other antibodies were purchased from eBioscience.
　気道過敏性の測定結果を図4(B)に示す。喘息群（チアマゾール）では、喘息群（DEX 5μg）や喘息群（DEX 15μg）と比較して、より一層の気道過敏性の抑制が認められた。
　また、気管支肺胞洗浄液の解析結果を図4(C), (D)に示す。喘息群（チアマゾール）では、喘息群（DEX 5μg）や喘息群（DEX 15μg）と比較して、より一層の気管支肺胞洗浄液中の細胞数（好酸球数、好中球数、マクロファージ数、およびＴ細胞数）の抑制が認められた。
　以上のことから、ヘムペルオキシダーゼの活性を阻害すること、ヘムペルオキシダーゼの発現を阻害することなどが、ステロイド剤を投与する場合と比較しても、気管支喘息の予防又は治療により効果的であることが確認できた。
  The measurement results of airway hypersensitivity are shown in FIG. 4 (B). In the asthma group (thiamazole), further suppression of airway hypersensitivity was observed compared to the asthma group (DEX 5 μg) and the asthma group (DEX 15 μg).
The analysis results of bronchoalveolar lavage fluid are shown in FIGS. 4 (C) and 4 (D). In the asthma group (thiamazole), compared to the asthma group (DEX 5 μg) and the asthma group (DEX 15 μg), the number of cells in the bronchoalveolar lavage fluid (eosinophil count, neutrophil count, macrophage count, And suppression of T cell count).
From the above, inhibiting heme peroxidase activity, inhibiting heme peroxidase expression, etc. may be more effective in preventing or treating bronchial asthma than when administering a steroid. It could be confirmed.
[配列番号：1] ヒトラクトペルオキシダーゼのcDNA配列である。
[配列番号：2] ヒトラクトペルオキシダーゼのアミノ酸配列である。
[配列番号：3] マウスラクトペルオキシダーゼのcDNA配列である。
[配列番号：4] マウスラクトペルオキシダーゼのアミノ酸配列である。
[配列番号：5] ヒトミエロペルオキシダーゼのcDNA配列である。
[配列番号：6] ヒトミエロペルオキシダーゼのアミノ酸配列である。
[配列番号：7] マウスミエロペルオキシダーゼのcDNA配列である。
[配列番号：8] マウスミエロペルオキシダーゼのアミノ酸配列である。
[配列番号：9] ヒト好酸球ペルオキダーゼのcDNA配列である。
[配列番号：10] ヒト好酸球ペルオキダーゼのアミノ酸配列である。
[配列番号：11] マウス好酸球ペルオキダーゼのcDNA配列である。
[配列番号：12] マウス好酸球ペルオキダーゼのアミノ酸配列である。
[配列番号：13] ヒト甲状腺ペルオキシダーゼのcDNA配列である。
[配列番号：14] ヒト甲状腺ペルオキシダーゼのアミノ酸配列である。
[配列番号：15] マウス甲状腺ペルオキシダーゼのcDNA配列である。
[配列番号：16] マウス甲状腺ペルオキシダーゼのアミノ酸配列である。 [SEQ ID NO: 1] This is the cDNA sequence of human lactoperoxidase.
[SEQ ID NO: 2] This is the amino acid sequence of human lactoperoxidase.
[SEQ ID NO: 3] This is the cDNA sequence of mouse lactoperoxidase.
[SEQ ID NO: 4] This is the amino acid sequence of mouse lactoperoxidase.
[SEQ ID NO: 5] This is the cDNA sequence of human myeloperoxidase.
[SEQ ID NO: 6] is an amino acid sequence of human myeloperoxidase.
[SEQ ID NO: 7] This is the cDNA sequence of mouse myeloperoxidase.
[SEQ ID NO: 8] This is the amino acid sequence of mouse myeloperoxidase.
[SEQ ID NO: 9] This is the cDNA sequence of human eosinophil peroxidase.
[SEQ ID NO: 10] This is the amino acid sequence of human eosinophil peroxidase.
[SEQ ID NO: 11] This is the cDNA sequence of mouse eosinophil peroxidase.
[SEQ ID NO: 12] This is the amino acid sequence of mouse eosinophil peroxidase.
[SEQ ID NO: 13] This is the cDNA sequence of human thyroid peroxidase.
[SEQ ID NO: 14] This is the amino acid sequence of human thyroid peroxidase.
[SEQ ID NO: 15] This is the cDNA sequence of mouse thyroid peroxidase.
[SEQ ID NO: 16] Amino acid sequence of mouse thyroid peroxidase.

Claims (10)

  1. 　ヘムペルオキシダーゼ阻害薬を含有する、気管支喘息の予防又は治療薬。 ¡Preventive or therapeutic agent for bronchial asthma containing heme peroxidase inhibitor.
  2. 　ヘムペルオキシダーゼ阻害薬が、以下の(a)～(h)からなる群から選択される少なくとも１つである請求項1に記載の気管支喘息の予防又は治療薬：
    (a)ヘムペルオキシダーゼの活性を阻害する化合物、
    (b)ヘムペルオキシダーゼの発現を阻害する化合物、
    (c)ヘムペルオキシダーゼの活性を阻害する抗体、
    (d)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するsiRNA又はshRNA、
    (e)ヘムペルオキシダーゼをコードするポリヌクレオチドの塩基配列に相補的若しくは実質的に相補的な塩基配列又はその一部を含有するアンチセンスポリヌクレオチド、
    (f)ヘムペルオキシダーゼをコードするポリヌクレオチドに対するリボザイム、
    (g)ヘムペルオキシダーゼに対してドミナントネガティブに作用するヘムペルオキシダーゼの変異体若しくはそれをコードするポリヌクレオチド、及び
    (h)ヘムペルオキシダーゼに対するアプタマー。     The prophylactic or therapeutic agent for bronchial asthma according to claim 1, wherein the heme peroxidase inhibitor is at least one selected from the group consisting of the following (a) to (h):
    (a) a compound that inhibits the activity of heme peroxidase,
    (b) a compound that inhibits the expression of heme peroxidase,
    (c) an antibody that inhibits the activity of heme peroxidase,
    (d) siRNA or shRNA against a polynucleotide encoding heme peroxidase,
    (e) an antisense polynucleotide containing a base sequence complementary to or substantially complementary to the base sequence of a polynucleotide encoding heme peroxidase, or a part thereof,
    (f) a ribozyme against a polynucleotide encoding heme peroxidase,
    (g) a variant of heme peroxidase that acts dominantly negatively on heme peroxidase or a polynucleotide encoding the same, and
    (h) Aptamer to heme peroxidase.
  3. 　ヘムペルオキシダーゼ阻害薬がチアマゾールである、請求項1に記載の気管支喘息の予防又は治療薬。 2. The preventive or therapeutic agent for bronchial asthma according to claim 1, wherein the heme peroxidase inhibitor is thiamazole.
  4. 　気管支喘息が、ステロイド抵抗性の気管支喘息である、請求項1～3のいずれか1項に記載の気管支喘息の予防又は治療薬。 The preventive or therapeutic agent for bronchial asthma according to any one of claims 1 to 3, wherein the bronchial asthma is steroid-resistant bronchial asthma.
  5. 　試験化合物をヘムペルオキシダーゼを発現する細胞と接触させ、ヘムペルオキシダーゼの活性又は発現量を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法。 A method of screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with a cell expressing heme peroxidase and using the activity or expression level of heme peroxidase as an index.
  6. 　(i)試験化合物をヘムペルオキシダーゼを発現する細胞と接触させた場合と、(ii) 試験化合物をヘムペルオキシダーゼを発現する細胞と接触させない場合の、該細胞におけるヘムペルオキシダーゼの活性又は発現量の比較を行う、請求項5に記載の方法。 Comparison of the activity or expression level of heme peroxidase in the cells when (i) the test compound is contacted with cells expressing heme peroxidase and (ii) the test compound is not contacted with cells expressing heme peroxidase 6. The method of claim 5, wherein the method is performed.
  7. 　前記(i)の場合におけるヘムペルオキシダーゼの活性又は発現量が、前記(ii)の場合のヘムペルオキシダーゼの活性又は発現量より低くなる試験化合物を、気管支喘息の予防又は治療薬の候補化合物として選択する、請求項6に記載の方法。 The test compound in which the activity or expression level of heme peroxidase in the case of (i) is lower than the activity or expression level of heme peroxidase in the case of (ii) is selected as a candidate compound for the prevention or treatment of bronchial asthma The method according to claim 6.
  8. 　試験化合物をヘムペルオキシダーゼと接触させ、ヘムペルオキシダーゼの活性を指標として、気管支喘息の予防又は治療薬をスクリーニングする方法。 A method of screening a prophylactic or therapeutic agent for bronchial asthma by contacting a test compound with heme peroxidase and using the activity of heme peroxidase as an index.
  9. 　(i)試験化合物をヘムペルオキシダーゼと接触させた場合と、(ii) 試験化合物をヘムペルオキシダーゼと接触させない場合の、ヘムペルオキシダーゼの活性の比較を行う、請求項8に記載の方法。 9. The method according to claim 8, wherein the activity of heme peroxidase is compared when (i) the test compound is contacted with heme peroxidase and (ii) when the test compound is not contacted with heme peroxidase.
  10. 　前記(i)の場合におけるヘムペルオキシダーゼの活性が、前記(ii)の場合のヘムペルオキシダーゼの活性より低くなる試験化合物を、気管支喘息の予防又は治療薬の候補化合物として選択する、請求項9に記載の方法。 The test compound in which the activity of heme peroxidase in the case of (i) is lower than the activity of heme peroxidase in the case of (ii) is selected as a candidate compound for the prevention or treatment of bronchial asthma. the method of.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099523A1 (en) * 2009-02-27 2010-09-02 University Of Cincinnati Method of treating reactive airway disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099523A1 (en) * 2009-02-27 2010-09-02 University Of Cincinnati Method of treating reactive airway disease

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CAPRON,M. ET AL.: "Inhibitory effects of lodoxamide on eosinophil activation", INT. ARCH. ALLERGY. IMMUNOL., vol. 116, no. 2, 1998, pages 140 - 6 *
DATABASE DATABASE MEDLINE U.S. NATIONAL LIBRARY OF MEDICINE(NLM); 5 March 2013 (2013-03-05), accession no. 988225346 *
DATABASE DATABASE MEDLINE U.S. NATIONAL LIBRARY OF MEDICINE(NLM); 5 March 2013 (2013-03-05), accession no. 998314415 *
EZEAMUZIE,C.I. ET AL.: "Effect of wortmannin on human eosinophil responses in vitro and on bronchial inflammation and airway hyperresponsiveness in Guinea pigs in vivo", AM. J. RESPIR. CRIT. CARE. MED., vol. 164, no. 9, 2001, pages 1633 - 9, XP055081544 *
EZEAMUZIE,C.I.: "Involvement of A3 receptors in the potentiation by adenosine of the inhibitory effect of theophylline on human eosinophil degranulation: possible novel mechanism of the anti-inflammatory action of theophylline", BIOCHEM. PHARMACOL., vol. 61, no. 12, 2001, pages 1551 - 9, XP055081541 *
HIDEMASA UCHIMURA: "Emergency Jissen Guide IX. Naibunpitsu Taisha Kojosen Crisis", NAIKA, vol. 103, no. 6, 2009, pages 1701 - 1705 *
KAWAJI,K. ET AL.: "Inhibition of eosinophil peroxidase release by chymotrypsin type protease inhibitors from activated human eosinophils", ARERUGI, vol. 42, no. 10, 1993, pages 1591 - 9 *
LEE,C.M. ET AL.: "Lycopene suppresses ovalbumin- induced airway inflammation in a murine model of asthma", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 374, no. 2, 2008, pages 248 - 52, XP023521464 *
MEHTA,A.K. ET AL.: "Effect of choline chloride in allergen-induced mouse model of airway inflammation", EUR. RESPIR. J., vol. 30, no. 4, 2007, pages 662 - 71, XP055081543 *
PETRY,T.W. ET AL.: "The mechanism for the inhibition of prostaglandin H synthase- catalyzed xenobiotic oxidation by methimazole. Reaction with free radical oxidation products", J. BIOL. CHEM., vol. 262, no. 29, 1987, pages 14112 - 8, XP055081546 *
PINCEMAIL,J. ET AL.: "Human myeloperoxidase activity is inhibited in vitro by quercetin. Comparison with three related compounds", EXPERIENTIA, vol. 44, no. 5, 1988, pages 450 - 3 *

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