WO2020004557A1 - Biomarqueurs de maladies pulmonaires obstructives - Google Patents

Biomarqueurs de maladies pulmonaires obstructives Download PDF

Info

Publication number
WO2020004557A1
WO2020004557A1 PCT/JP2019/025632 JP2019025632W WO2020004557A1 WO 2020004557 A1 WO2020004557 A1 WO 2020004557A1 JP 2019025632 W JP2019025632 W JP 2019025632W WO 2020004557 A1 WO2020004557 A1 WO 2020004557A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
subunit
ribosomal
factor
ribosomal protein
Prior art date
Application number
PCT/JP2019/025632
Other languages
English (en)
Japanese (ja)
Inventor
吉人 武田
淳 熊ノ郷
宗到 玄山
太郎 木庭
幸嗣 植田
Original Assignee
国立大学法人大阪大学
公益財団法人がん研究会
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人大阪大学, 公益財団法人がん研究会 filed Critical 国立大学法人大阪大学
Publication of WO2020004557A1 publication Critical patent/WO2020004557A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a biomarker for obstructive pulmonary disease, its use, and the like.
  • Bronchial asthma (BA) is estimated to reach 300 million patients, and its increasing prevalence is a social and economic burden.
  • the nature of bronchial asthma is chronic airway inflammation, but the key molecules controlling inflammation and airway remodeling are not fully understood.
  • Chronic obstructive pulmonary disease COPD
  • COPD chronic obstructive pulmonary disease
  • Obstructive pulmonary diseases such as bronchial asthma and COPD, develop on the basis of dysregulation of the cell molecular network formed by the interaction of individual genes and proteins, rather than the mutation itself forming a pathological condition. It is thought that. In fact, there are various phenotypes of asthma and COPD, which are characterized by airflow obstruction and inflammation, which may make it more difficult to elucidate the pathology of these diseases. Furthermore, although there is a high rate of overlap syndrome in which both diseases coexist, the identification method and detailed pathological condition are unknown.
  • PD COPD is estimated at 200 million patients worldwide and 5.3 million in Japan, of which only 5% are receiving treatment.
  • asthma which has an estimated 200 million patients worldwide, has been declining due to the spread of inhaled steroids, but the morbidity has continued to increase with aging.
  • ACOs with COPD and asthma have attracted attention because of their poor prognosis and an estimated 20% of COPD patients.
  • asthma has various phenotypes (phenotypes) and end types, so that individualized medical treatment is required.
  • therapeutic agents targeting molecules such as IL-5, ⁇ IL-13, and ⁇ IgE that induce Th2 type inflammation have been developed one after another.
  • therapeutic agents targeting Th2 type inflammation are being developed one after another overseas, almost no index (companion biomarker) for judging treatment selection or efficacy has been developed at present.
  • Serum is considered to be an ideal test sample because it can be collected repeatedly non-invasively, but more than 99% of the protein contained in it is a large amount of protein (such as albumin) that is considered a contaminant from the viewpoint of biomarkers. Therefore, its usefulness as a test data is low from the viewpoint of proteomics.
  • a recent paper on biomarkers that can distinguish between COPD and asthma attempted to analyze dozens of patients, but it was based on old mass spectrometry (MS) technology using two-dimensional electrophoresis.
  • MS mass spectrometry
  • serum containing a large amount of contaminants was used, the only biomarker found was serum contaminants containing prothrombin, and it was hard to say that it was a COPD-specific biomarker.
  • Extracellular vesicles are membrane vesicles secreted from cells. By transporting intracellular proteins, lipids, nucleic acids (mRNA, microRNA, etc.) to the outside of cells, local and systemic communication between cells is achieved. I am carrying it.
  • extracellular vesicles (exosomes) have attracted attention as a test sample for cancer. However, they have been used as test samples for obstructive pulmonary diseases such as inflammatory respiratory diseases, particularly bronchial asthma, which is a major obstructive pulmonary disease. The usefulness of is not known.
  • An object of the present invention is to provide a biomarker for obstructive pulmonary disease such as bronchial asthma and a method for using the same.
  • an object of the present invention is to provide a biomarker capable of distinguishing between bronchial asthma and chronic obstructive pulmonary disease (COPD) and a complication thereof, and a method of using the same.
  • COPD chronic obstructive pulmonary disease
  • the present inventors have conducted intensive studies in view of the above problems, and found that a specific protein group in extracellular vesicles or a blood sample of a body fluid collected from a subject is useful as a biomarker for obstructive pulmonary disease.
  • these biomarkers can also differentiate between bronchial asthma and chronic obstructive pulmonary disease (COPD) and their complications.
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • some biomarkers reflect the pathology of obstructive pulmonary disease.
  • the present invention includes the following embodiments.
  • Item 1 A method for testing for obstructive pulmonary disease, (1) At least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) in an extracellular vesicle or blood sample of a body fluid collected from a subject An inspection method including a step of detecting
  • step (2) (2a) the subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value;
  • the cut-off value is 2.5 to 6 times the value of the amount or concentration of the corresponding protein in an extracellular vesicle or blood sample of a body fluid collected from a subject not suffering from obstructive pulmonary disease, Item 4.
  • Item 5 The test method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  • Item 6. ⁇ The method according to any one of Items 1 to 5, wherein the subject is a human.
  • Item 7 A method for testing for obstructive pulmonary disease, (1) A test method comprising a step of detecting one or more proteins including Tripeptidyl-peptidase 2 in an extracellular vesicle or a blood sample of a body fluid collected from a subject.
  • a test agent for obstructive pulmonary disease comprising an agent for detecting at least one protein selected from the group consisting of the protein group (A), the protein group (B), and the protein group (C).
  • a preventive or therapeutic agent for obstructive pulmonary disease comprising an inhibitor of at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C).
  • Item 10 At least one selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C) in an extracellular vesicle or a blood sample of a body fluid collected from an animal treated with a test substance; A method for screening an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease, using the amount or concentration of a species protein as an index.
  • Item 11 When the value of the index is lower than the amount or concentration of the corresponding protein in the extracellular vesicles or blood sample of a body fluid collected from an animal that has not been treated with the test substance, the test substance is treated as an obstructive pulmonary disease.
  • Item 11 The screening method according to Item 10, comprising a step of selecting as an active ingredient of a prophylactic or therapeutic agent.
  • Item 12 At least one selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C) in an extracellular vesicle or a blood sample of a body fluid collected from an animal treated with a test substance; A method for evaluating the induction or malignancy of obstructive pulmonary disease using the amount or concentration of a species protein as an index.
  • a biomarker for obstructive pulmonary disease can be provided.
  • examination of obstructive pulmonary disease, determination of the degree of eosinophil infiltration, screening of an obstructive pulmonary disease model animal, and the like can be performed more easily, more efficiently, and at lower cost.
  • COPD chronic obstructive pulmonary disease
  • the use of the biomarkers may also enable prevention or treatment of obstructive pulmonary disease, screening of active ingredients of preventive or therapeutic agents for obstructive pulmonary disease, and the like.
  • 9 is a graph comparing the TPP2 concentration in serum (vertical axis) between a COPD subject and a healthy subject in Test Example 3. Each plot shows data for each subject. 9 is a graph comparing the TPP2 concentration in serum (vertical axis) between a COPD specimen and a healthy specimen for each stage in Test Example 3. Each plot shows data for each subject.
  • Test method of obstructive pulmonary disease is a method of testing obstructive pulmonary disease, (1) At least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C) in an extracellular vesicle or blood sample of a body fluid collected from a subject (Step 1) of the present invention, which is also referred to as “the method of the present invention for testing pulmonary obstructive pulmonary disease” in some cases.
  • Step 1 the method of the present invention for testing pulmonary obstructive pulmonary disease
  • the “obstructive pulmonary disease” to be tested is not particularly limited, but specific examples include bronchial asthma, COPD, diffuse panbronchiolitis, obstructive bronchiolitis, and the like.
  • the subject is a target organism of the test method of the present invention, and the species is not particularly limited.
  • the species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits, and preferably mice.
  • the state of the subject is not particularly limited.
  • a subject for example, a sample that is unknown whether or not suffering from obstructive pulmonary disease, a sample that has already been determined to be suffering from bronchial asthma, but is unknown whether or not suffering from COPD, Specimens that have already been determined to have COPD by another method, but are not known to have bronchial asthma, or have already been determined to have bronchial asthma and COPD by another method And specimens already determined to be free of obstructive pulmonary disease by another method, and specimens during treatment for obstructive pulmonary disease.
  • the detection sample in step (1) is preferably an extracellular vesicle of a body fluid.
  • the body fluid is not particularly limited.
  • the body fluid include whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and the like, and preferably whole blood, serum , Plasma and cerebrospinal fluid, and more preferably whole blood, serum and plasma.
  • the body fluid may be used alone or in a combination of two or more.
  • Body fluid can be collected from a subject by a method known to those skilled in the art.
  • whole blood can be collected by collecting blood using a syringe or the like.
  • Serum is a part obtained by removing blood cells and specific blood coagulation factors from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood.
  • Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not allow whole blood to coagulate.
  • blood sample blood itself such as whole blood, serum, and plasma or a sample derived from blood is referred to as “blood sample”.
  • Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells. Extracellular vesicles are usually defined as membrane vesicles that transport intracellular proteins and genetic information (mRNA, microRNA, etc.) extracellularly, thereby transmitting information between cells in the local and whole body. You. Examples of extracellular vesicles include exosomes, micro vesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles, and the like.
  • Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or according to known methods.
  • Methods for purifying, separating, concentrating, etc. extracellular vesicles include, for example, ultracentrifugation (eg, pellet down, sucrose cushion, density gradient centrifugation, etc.), methods using immunoaffinity carriers, gel filtration, and field filtration. Flow fractionation, FACS and the like can be mentioned. Purification, separation, concentration, etc. of extracellular vesicles can also be performed using a commercially available kit. These methods may be employed alone or in combination of two or more.
  • the detection target in the step (1) is at least one protein selected from the group consisting of a protein group (A), a protein group (B), and a protein group (C) (in this specification, these are collectively referred to as “ Sometimes referred to as "target protein").
  • Protein group (A) is (A) (A1) Putative high mobility group protein B1-like 1, Dynamin-1-like protein, Reticulon-3, Ig kappa chain VI region region Gal, Arginase-1, Guaneine nucleotide-binding protein G (k) subunit alpha, Keratin, type I ⁇ cytoskeletal 16, C-1-tetrahydrofolate synthase, cytoplasmic, Dynamin-2, P2X purinoceptor 1, Rho GDP-dissociation inhibitor 1, Brain acid soluble protein 1, 60S ribosomal protein, Solute carrier family 43 member 3, Reticulon-4, and (A2) D-3-phosphoglycerate dehydrogenase, vesicle-associated membrane membrane protein 5, Ig kappa chain V-III region NG9 Fragment, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit , Adenine ylphosphoribosyltransferase
  • Protein group (A) is a bronchial asthma-specific biomarker, and can be used as an index to distinguish bronchial asthma without chronic obstructive pulmonary disease.
  • the protein group (A1) is a protein extracted as a marker by a t-test.
  • Protein group (A2) is a protein extracted as a marker by Absent / Present search.
  • the protein group (A1) from the viewpoint that the ratio of the amount to the normal sample is higher, preferably Putative high mobility group protein B1-like 1, Ig kappa chain VI, region Gal, Arginase-1, Keratin, type I cytoskeletal 16 And C-1-tetrahydrofolate synthase, cytoplasmic, Dynamin-2, brain acid soluble protein 1, solute carrier family 43 member 3, etc., and more preferably C-1-tetrahydrofolate synthase, cytoplasmic.
  • Putative high mobility group protein B1-like 1, Ig kappa chain VI, region Gal, Arginase-1, Keratin, type I cytoskeletal 16 And C-1-tetrahydrofolate synthase, cytoplasmic, Dynamin-2, brain acid soluble protein 1, solute carrier family 43 member 3, etc. preferably C-1-tetrahydrofolate synthase, cytoplasmic.
  • the proteins (A2) from the viewpoint that the expression in normal samples tends to be lower, preferably D-3-phosphoglycerate dehydrogenase, Vesicle-associated membrane protein 5, Ig kappa chain V-III region NG9 (Fragment) , Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Adenine phosphoribosyltransferase, ATP-dependent 6-phosphofructokinase, muscle type, Alcohol dehydrogenase 4, Glutathione peroxidase 2, Liver carboxylesterase, ligase, germinase, ligase, ligase, germinalase, germinalase, germinalase, germline, germline, germinal, germinal, germinal, germinal, germ, and L15, 60S ribosomal protein L27, 60S ribosomal protein L32, T-complex protein 1 subunit beta
  • Protein group (B) is (B) (B1) Chloride intracellular channel protein 1, Tripeptidyl-peptidase 1, Nuclear factor of activated T-cells, cytoplasmic 1, Coagulation factor IX, Coagulation factor X, Ig kappa chain VI, region WEA, Fibrinogen beta chain, von Willebrand factor, Heat shock protein beta-1, Guineine nucleotide-binding protein G (i) subunit alpha-2, Sodium / potassium-transporting ATPase subunit alpha-1, Annexin A2, Receptor-type tyrosine-protein phosphatase C, Histone H2A.Z, Ras-related protein R-Ras, Heat shock cognate 71 kDa protein, Ras-related protein Ral-A, Coagulation factor V, Elongation factor 2, Ras-related C3 botulinum toxin substrate 2, Ezrin, Receptor -type tyrosine-protein phosphatase alpha, Peptidy
  • Protein group (B) is a biomarker specific to chronic obstructive pulmonary disease, and can be used as an indicator to distinguish chronic obstructive pulmonary disease without bronchial asthma.
  • the protein group (B1) is a protein extracted as a marker by a t-test.
  • Protein group (B2) is a protein extracted as a marker by Absent / Present search.
  • Tripeptidyl-peptidaseNucle1 Nuclear factor of activated T-cells, cytoplasmicag1, Coagulation factor IX, Ig kappa chain VI region WEA, Fibrinogen beta chain, von Willebrand factor, Heat shock protein beta-1, Sodium / potassium-transporting ATPase subunit alpha-1, Histone H2A.Z, Ras-related protein R-Ras, Elongation factor 2, Ezrin, Receptor-type tyrosine- protein phosphatase alpha, Peptidyl-prolyl cis-trans isomerase B, 40S ribosomal protein S3, Tripeptidyl-peptidase 2, Transgelin-2, 60S ribosomal protein L21, 40S ribosomal protein S9, F-actin-capping protein subunit acid-2Fatt synthase, CD81 antigen, 60S
  • proteins (B2) from the viewpoint that the expression in normal samples tends to be lower, preferably AP-3 complex subunit beta-1, 26S proteasome non-ATPase regulator regulator subunit 12, NADH-cytochrome b5 reductase 3, Adenine phosphoribosyltransferase, ATP-dependentphosph6-phosphofructokinase, muscle type, X-ray repairogencross-complementing protein 6, Heterogeneous nuclear ribonucleoprotein L, Eukaryotic peptide chain release factor GTP-binding subunit ERF3ACar, Carbonyl proteinasetic adenosine-3, Valine--tRNA ligase, Matrin-3, T-complex protein 1 subunit epsilon, Arginine--tRNA ligase, cytoplasmic, Methionine--tRNA ligase, cytoplasmic, 60S ribosomal protein L32, T-complex protein 1 subunit
  • AP-3 complex subunit beta-1 26S proteasome non-ATPase regulator subunit 12, NADH-cytochrome b5 reductase 3, Adenine phosphoribosyltransferase, ATP-dependent 6-phosphofructokinase, muscle type, X-ray repair cross-complementing Heterogeneous nuclear ribonucleoprotein L, Eukaryotic peptide chain release factor GTP-binding subunit ERF3A, Carbonyl reductase [NADPH] 1, Trifunctional purine biosynthetic protein adenosine-3, Valine-tRNA ligase, Matrin-3, T-complexArprotein --tRNA ligase, cytoplasmic, Methionine--tRNA ligase, cytoplasmic, 60S60ribosomal protein L32, T-complex protein 1 subunit beta, Eukaryotic translation initiation factor 3 subunit I, Eukaryotic translation initiation factor 3 subunit A, Phospho
  • the present invention relates to a method for examining obstructive pulmonary disease, comprising (1) Tripeptidyl-peptidase 2 in an extracellular vesicle or a blood sample of a body fluid collected from a subject.
  • An inspection method comprising a step of detecting one or more types of proteins.
  • the protein group (C) includes (C) Ferritin light chain, Ferritin heavy chain, Cathespin D, Trypsin-1, Neprilysin, X-ray repair cross-complementing protein 5, Pyruvate kinase PKM, NAD (P) H dehydrogenase quinone 1, Elongation factor 1-gamma, Keratin, type II cytoskeletal 2 epipidermal, Rho GDP-dissociation inhibitor 2, Eukaryotic initiation factor 4A-I, 40S ribosomal protein S16, 40S ribosomal protein S4, X isoform, GTP-binding Tubulin beta-4B chain, Heterogeneous nuclear nuclear ribonucleoprotein U, 60S ribosomal protein L18, ELAV-like protein 1, POTE ankyrin domain domain family member E, Synaptic vesicle membrane membrane protein VAT-1 homolog, and a protein group consisting of Retinoic acid receptors from protein groups. is there.
  • the protein group (C) is a biomarker specific to both bronchial asthma and chronic obstructive pulmonary disease, and by using this as an index, bronchial asthma with chronic obstructive pulmonary disease (or Chronic obstructive pulmonary disease).
  • the protein group (C) preferably Ferritin light chain, Ferritin heavy chain, Cathepsin D, Trypsin-1, Neprilysin, X-ray repair cross-complementing protein 5, NAD (P) H dehydrogenase [quinone] 1, Elongation factor 1-gamma, Rho GDP-dissociation inhibitor 2, Eukaryotic initiation factor 4A-I, 40S ribosomal protein S16, 40S ribosomal protein S4, X isoform, GTP-protein nuclear , Tubulin beta-4B chain, Heterogeneous nuclear ribonucleoprotein U, 60S ribosomal protein L18, ELAV-like protein 1, POTE ankyrin domain family member E, Synaptic vesicle membrane protein VAT-1 homolog, Retinoic acid receptor, etc.
  • NAD (P) H dehydrogenase [quinone] 1 1, Elongation factor 1-gamma, Rho GDP-dissociation inhibitor 2
  • Ferritin light chain More preferably Ferritin light chain, Ferritin heavy chain, Trypsin-1, X-ray repair cross-complementing protein 5, NAD (P) H dehydrogenase [quinone] 1, Elongatio n factor 1-gamma, Eukaryotic initiation factor 4A-I, 40S ribosomal protein S16, 40S ribosomal protein S4, X isoform, Tubulin beta-4B chain, Heterogeneous nuclear ribonucleoprotein U, 60S ribosomal protein L18 and the like.
  • NAD NAD
  • Proteins of the protein groups (A) to (C) are proteins identified by UniProtKB accession numbers shown in Tables 1 to 10 in the examples described later in the case of humans. In the case of other species, it is the ortholog of the protein identified by the UniProtKB accession number shown in Tables 1-10.
  • the number of target proteins in step (1) may be only one, or may be a combination of two or more. By combining more (eg, 2, 5, 10, 20, 40, 80, and 120 or more) targets for detection of obstructive pulmonary disease, bronchial asthma and chronic obstructive pulmonary disease , And the complications and the like can be more accurately performed.
  • Detection is usually performed by measuring the amount or concentration of the target protein.
  • concentration is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, or raw data measured to know the absolute concentration.
  • the method for detecting the target protein is not particularly limited as long as it can specifically detect a part or all of the target protein.
  • Specific examples of the detection method include, for example, mass spectrometry for detecting a peptide constituting the target protein, and immunological measurement using an antibody that specifically recognizes the target protein.
  • the amino acid sequence information of the target protein can be obtained by searching the EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) database based on the UniProtKB accession number.
  • immunological assay examples include immunohistochemical staining, ELISA, EIA, RIA, Western blotting, and the like.
  • Mass spectrometry is a method in which a peptide sample is converted into gaseous ions using an ion source (ionization).
  • the peptide sample is ionized by moving it in a vacuum and using electromagnetic force or by a time difference of flight.
  • a measurement method using a mass spectrometer that can be separated and detected according to the ratio, and ionization using an ion source includes EI method, CI method, FD method, FAB method, MALDI method, ESI method
  • the method of separating the ionized peptide sample in the analysis unit can be selected from magnetic field deflection type, quadrupole type, ion trap type, time-of-flight (TOF) type, Fourier transform
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry (MS / MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used.
  • the sample When the sample is a sample containing a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer. Further, peptides constituting the target protein can be separated and purified by liquid chromatography (LC) or HPLC to obtain a sample. Further, the detection unit and the data processing method can be appropriately selected.
  • a peptide constituting the target protein is detected and quantified by mass spectrometry using mass spectrometry, a peptide having the same amino acid sequence as the peptide and labeled with a stable isotope at a known concentration is used as an internal standard. can do.
  • Such a stable isotope-labeled peptide is a stable isotope-labeled peptide in which one or more of the amino acids in the peptide constituting the target protein to be detected contains one or more of 15 N, 13 C, 18 O, and 2 H. If so, the type, position, number, etc. of the amino acids can be appropriately selected.
  • Such a stable isotope-labeled peptide can be obtained by the F-moc method (Amblard., Et al. Methods) using an amino acid labeled with a stable isotope. Mol Biol.
  • the amount and / or concentration of the target protein which is a detection index for obstructive pulmonary disease
  • the test method of the present invention including the step (1) it is possible to provide the amount and / or concentration of the target protein as a detection index, thereby assisting the evaluation of the degree of eosinophil infiltration. You can also. Eosinophil infiltration is involved in the activity of bronchial asthma (sensitivity, reversibility, etc.) and acute exacerbation of COPD. It can be used for discrimination etc.
  • eosinophil infiltration is an important factor involved in obstructive disease, but how to evaluate it?
  • the use of the biomarker of the present invention makes it possible to evaluate eosinophil infiltration more simply and efficiently than in the past, without requiring such complicated work.
  • the test results of the test method of the present invention including the step (1) include elucidating the pathology of obstructive pulmonary disease, predicting the prognosis of obstructive disease (respiratory decline group, acute aggravation), stratifying subjects, treating It can be used for selection (individualized medicine, treatment responsiveness), refractory disease in obstructive pulmonary disease (particularly bronchial asthma), evaluation of remodeling, differentiation of histological type, phenotype, etc. of obstructive pulmonary disease.
  • the BMs that correlate with various eosinophil infiltration found by the present invention can be used to identify various phenotypes (clustering) and companion BMs of various molecular targeting drugs (Th2 targeting) currently under development. It can be.
  • the inspection method of the present invention further includes, as an aspect, (2) a step (step 2) of determining that the subject has obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is not less than a cutoff value Is preferred. According to the test method of the present invention including the step 2, it is possible to determine an obstructive pulmonary disease.
  • Step 2 is more specifically divided into, for example, the following (2a) to (2e).
  • (2a) the subject suffers from bronchial asthma when the amount or concentration of at least one protein selected from the group consisting of the protein group (A) and the protein group (C) is not less than a cutoff value;
  • Step (2) is more preferably at least one step selected from the group consisting of the above steps 2a to 2e.
  • Cut-off value, sensitivity, specificity, positive predictive value can be appropriately set by those skilled in the art from the viewpoint of negative predictive value, for example, collected from a subject not suffering from obstructive pulmonary disease
  • the value can be set to a predetermined value or a predetermined value in each case.
  • the cut-off value may be, for example, the amount and / or concentration of the protein of interest in extracellular vesicles or a blood sample of a bodily fluid collected from a subject not suffering from obstructive pulmonary disease. Value, median, etc.), for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2.5 to 6 times.
  • the amount or concentration of the protein detected in the step (1) is equal to or higher than the cutoff value by using a biomarker reflecting the pathology of the obstructive pulmonary disease.
  • a biomarker reflecting the pathology of the obstructive pulmonary disease it can be determined that the subject is suffering from a more severe obstructive pulmonary disease.
  • the cutoff value is set to a value based on the amount and / or concentration of the target protein in an extracellular vesicle or a blood sample of a body fluid collected from a subject suffering from obstructive pulmonary disease, for example. , It is possible to evaluate whether the disease is over a certain level.
  • the treatment is performed by setting the cutoff value to a value based on the amount and / or concentration of the target protein in a past sample of the same sample, for example.
  • the effect can be determined.
  • the amount or concentration of the protein detected in the step (1) can be used as an index.
  • the degree of acid sphere infiltration can be determined. More specifically, for example, a calibration curve is created in advance based on the correlation between the degree of eosinophil infiltration (the number of eosinophil infiltration in a certain area) and the amount or concentration of the biomarker. From the amount or concentration of eosinophils and the calibration curve, the degree of eosinophil infiltration can be determined.
  • the test method of the present invention further includes: By combining the steps of applying a diagnosis by a doctor for obstructive pulmonary disease, obstructive pulmonary disease can be diagnosed with higher accuracy.
  • the test method of the present invention can more accurately detect obstructive pulmonary disease, by combining the above-described steps with the test method of the present invention, it is possible to more efficiently and more accurately detect “obstructive pulmonary disease. Can be diagnosed.
  • test method of the present invention may further be used, or If the diagnosis of obstructive pulmonary disease is made as described in “Diagnosis of obstructive pulmonary disease with higher accuracy”, the combination of the test method of the present invention and the step of applying a diagnosis by a physician On the other hand, (3) treating a subject determined or diagnosed as suffering from obstructive pulmonary disease by performing a step of treating the disease, thereby treating the subject in the disease. It becomes possible.
  • test method of the present invention can more accurately detect obstructive pulmonary disease
  • the test method of the present invention or the combination of the test method of the present invention and the step of applying a diagnosis by a doctor can be used. By combining the three, a subject suffering from obstructive pulmonary disease can be treated more efficiently and more reliably.
  • the method for treating obstructive pulmonary disease is not particularly limited, but typically includes medication.
  • the medicament used for the drug treatment is not particularly limited.
  • anticholinergic agents such as Spiriva, Seebri, Enclasse, Ecrila, Atrovent, and Telshigan
  • ⁇ 2 stimulants such as Celevent, Ombres, Auxis, Saltanol, and Meptin
  • Ultibulo, Anoro And cholesterol / ⁇ 2 stimulants such as Spiolt
  • ⁇ ⁇ ⁇ ⁇ ⁇ steroid drugs such as Cuvar, Flutide, Pulmicort, Orbesco, Azmanex
  • steroid / ⁇ 2 stimulants such as Adair, Symbicort, Lervea and the like.
  • One, two, or three or more pharmaceuticals can be used in combination.
  • test method of the present invention it is possible to select an appropriate medicine to be used for drug treatment from medicines, for example, the medicines exemplified above.
  • test agent for obstructive pulmonary disease provides a reagent for detecting at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C).
  • the present invention also relates to a test agent for obstructive pulmonary disease (hereinafter, also referred to as “test agent of the present invention”). Hereinafter, this will be described.
  • the protein group (A), the protein group (B), the protein group (C), the obstructive pulmonary disease and the like are the same as defined in the above “1.
  • the detection agent is not particularly limited as long as it can specifically detect the target protein.
  • the detection agent include an antibody against the target protein.
  • the detection agent may be labeled. Labels include, for example, fluorescent substances, luminescent substances, dyes, enzymes, colloidal gold, radioisotopes, and the like. Further, the detection agent (particularly, antibody) may be in a state of being adsorbed on a substrate (for example, a plastic substrate such as a microwell plate).
  • the antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein.
  • “selectively (specifically) recognizes” means that a target protein can be specifically detected by, for example, a Western blotting method or an ELISA method. Any substance can be used as long as the substance can be determined to be derived from the target protein.
  • Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and some of the above-mentioned antibodies having antigen-binding properties, such as Fab fragments and fragments produced by Fab expression libraries.
  • Antibodies of the present invention also include an antibody having an antigen-binding property to a polypeptide that is at least contiguous, usually 8 amino acids, preferably 15 amino acids, and more preferably 20 amino acids in the amino acid sequence of the target protein.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current Protocols in Molecular Biology, Chapters 11.12 to 11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed and purified in Escherichia coli or the like according to a conventional method.
  • a non-human animal such as a rabbit can be immunized and obtained from the serum of the immunized animal according to a conventional method.
  • a target protein expressed and purified in E in the case of a monoclonal antibody, a target protein expressed and purified in E.
  • an oligopeptide having a partial amino acid sequence of the target protein is immunized to a non-human animal such as a mouse, and the obtained spleen cells and It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.1.4 to 11.11).
  • the target protein used as an immunizing antigen for the production of an antibody is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and recovery of the protein from the culture. Can be obtained. These operations are performed according to methods known to those skilled in the art, or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL Press (1985)), and the like. Can be done.
  • a recombinant DNA capable of expressing a gene encoding a target protein in a desired host cell is prepared, and this is introduced into a host cell, transformed, and the transformant is cultured.
  • a protein as an immunizing antigen for producing the antibody of the present invention can be obtained.
  • the partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as the target protein.
  • An oligo (poly) peptide preferably having this immunogenic property and comprising at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
  • Production of an antibody against such an oligo (poly) peptide can also be carried out by increasing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances include human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium-Parvum.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other components as necessary.
  • Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and fragrances. And chelating agents.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain, besides the above-mentioned detection agent or the above-mentioned composition containing the same, those which can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject or a blood sample.
  • Specific examples of such substances include various reagents (eg, secondary antibodies, buffers, etc.), instruments (eg, instruments for purification, separation, and concentration of extracellular vesicles (eg, columns)).
  • the present invention relates to suppression of at least one protein selected from the group consisting of protein group (A), protein group (B), and protein group (C).
  • the present invention relates to a prophylactic or therapeutic agent for obstructive pulmonary disease, which comprises an agent (hereinafter, may be referred to as "the agent of the present invention”). Hereinafter, this will be described.
  • the protein group (A), the protein group (B), the protein group (C), etc. are the same as defined in the above “1. Method for testing obstructive pulmonary disease”.
  • the inhibitor preferably includes an expression inhibitor of the target protein.
  • the expression inhibitor of the target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, and the like.
  • the gene-specific small interfering RNA (siRNA) of the target protein the gene specificity of the target protein MicroRNAs (miRNA), gene-specific antisense nucleic acids of the target protein, their expression vectors; ⁇ ⁇ ⁇ gene-specific ribozymes of the target protein; ⁇ gene gene editing agents for the target protein by the CRISPR / Cas system.
  • expression suppression refers to the target protein, its mRNA expression level, for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100 , 1/200, 1/300, 1/500, 1/1000, 1 / 10,000 or less, including setting the expression level to 0.
  • the gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein.
  • the siRNA preferably has a length of, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more.
  • the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. It is assumed that the upper limit and the lower limit of the length of the siRNA described here are arbitrarily combined.
  • the lower limit is 18 bases
  • the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases
  • the lower limit is 19 bases
  • the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases.
  • a length with a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases, and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases A combination of lengths that are bases is envisioned.
  • SiRNA may be shRNA (small hairpin RNA).
  • shRNAs can be designed such that a portion forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand such that sequence a, spacer, sequence b And can be designed to have a total length of 45 to 60 bases.
  • the sequence a is a sequence of a partial region of a base sequence encoding a target protein to be targeted.
  • the target region is not particularly limited, and any region can be a candidate.
  • the length of sequence a is 19 to 25 bases, preferably 19 to 21 bases.
  • the gene-specific siRNA of the target protein may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually about 2 to 4 bases.
  • the additional base may be DNA or RNA, but using DNA may improve the stability of the nucleic acid in some cases.
  • Such additional base sequences include, for example, ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 ', Uuuuu-3' and the like, but are not limited thereto.
  • the siRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specific examples include those to which dTdT (dT represents deoxythymidine) has been added. In addition, blunt ends without addition of terminals (blunt ends) may be used.
  • the sense strand and the antisense strand may have different numbers of bases. For example, “asymmetrical interfering RNA” in which the antisense strand has a protruding portion sequence (overhang) at the 3 ′ end and the 5 ′ end. aiRNA)].
  • a typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand.
  • the position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, in one embodiment, up to about 50 bases from the 5′-UTR and the start codon, and from a region other than the 3′-UTR, It is desirable to select a sequence.
  • BLAST http://www.ncbi.nlm.nih.gov/BLAST/ )
  • a sense strand having a TT or UU 3 ′ terminal overhang at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 base and TT or A double-stranded RNA consisting of an antisense strand having an overhang at the 3 'end of UU may be designed as siRNA.
  • shRNA which is a precursor of siRNA, appropriately selects an arbitrary linker sequence (for example, about 5 to 25 bases) capable of forming a loop structure, and connects the sense strand and the antisense strand via the linker sequence. It can be designed by connecting.
  • siRNA and / or shRNA can be searched using search software provided free of charge on various websites.
  • Such sites include, for example, the following. Ambion's siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) pSilencer (registered trademark) Insert Vector Insert Design Tool (http://www.ambion.com/ jp / techlib / misc / psilencer_converter.html) GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.
  • the siRNA is obtained by synthesizing the sense strand and the antisense strand of the target sequence on the mRNA with a DNA / RNA automatic synthesizer and denaturing them in a suitable annealing buffer at about 90 to about 95 ° C. for about 1 minute. It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing shRNA which is a precursor of siRNA and cleaving it using an RNA-cleaving protein dicer.
  • the gene-specific miRNA of the target protein is optional as long as it inhibits the translation of the gene encoding the target protein.
  • the miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation.
  • the miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA.
  • the length of the miRNA is not particularly limited, the length of the pri-miRNA is usually several hundred to several thousand bases, the length of the pre-miRNA is usually 50 to 80 bases, and the length of the mature miRNA is usually 18 ⁇ 30 bases.
  • the gene-specific miRNA of the protein of interest is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA.
  • a gene-specific miRNA of the target protein may be synthesized by a known technique, or may be purchased from a company that provides synthetic RNA.
  • the gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the gene encoding the target protein or a part thereof, and is specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming a target and stable double strand and binding.
  • the antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera.
  • the antisense nucleic acid is DNA
  • an RNA DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA.
  • the target sequence may be not only the sequence in the mRNA but also the sequence of the intron region in the initial translation product of the gene of the target protein.
  • the intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the gene of the target protein using a homology search program such as BLAST or FASTA.
  • the length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the hybridization of the antisense nucleic acid results in the inhibition of translation into the target protein.
  • the gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein.
  • oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but not limited thereto.
  • a 3′-end hairpin loop or the like may be selected as a preferable target region of the antisense nucleic acid, but is not limited thereto.
  • the gene-specific antisense nucleic acid of the target protein not only hybridizes to the mRNA and early transcript of the gene of the target protein and inhibits translation into the protein, but also binds to these double-stranded DNA genes. Those which can form a triplex (tripplex) and inhibit transcription to RNA (antigene) may be used.
  • the nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein have stability (chemical and / or counterpart enzyme) and specific activity ( In order to improve the affinity for RNA), various chemical modifications may be included.
  • a phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is replaced with, for example, phosphorothioate (PS), methylphosphonate (methylphosphonate), phosphorodithioate. It can be substituted with a chemically modified phosphate residue such as a phosphorodithioate.
  • the base moiety pyrimidine, purine
  • some of the nucleotide molecules constituting the siRNA and the miRNA may be replaced with a natural type DNA.
  • the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the target protein gene. It can be prepared by determining the sequence and synthesizing a sequence complementary thereto using a commercially available automatic DNA / RNA synthesizer. In addition, antisense nucleic acids containing the various modifications described above can be chemically synthesized by any known method.
  • the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, or a coding sequence of a gene-specific antisense nucleic acid of the protein of interest (optionally, (A transcription termination signal sequence), and other sequences as necessary.
  • the promoter is not particularly limited, for example, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ actin promoter, RNA polymerase II (polII) promoter such as CAG promoter; mouse and human U6-snRNA promoter, Human H1-RNase P RNA promoter, human valine-tRNA promoter and other RNA polymerase III (polIII) promoters and the like. Of these, from the viewpoint that short RNA can be accurately transcribed, the polIII promoter is preferable.
  • the other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be employed. Examples of such a sequence include, for example, an origin of replication, a drug resistance gene, and the like. The types of drug resistance genes and types of vectors can be those described above.
  • RNA-specific ribozyme of the target protein includes a gene-specific ribozyme of the target protein.
  • "Ribozyme” in a narrow sense means RNA having an enzymatic activity for cleaving a nucleic acid, but in the present application, it includes DNA as long as it has a sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and viruses, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzymatic activity at about 40 bases, and the bases at both ends adjacent to the hammerhead structure (about 10 bases in total) are converted into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Since this type of ribozyme nucleic acid uses only RNA as a substrate, it has the advantage that it does not attack genomic DNA. When the mRNA of the gene of the target protein has a double-stranded structure by itself, the target sequence can be single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase. [Proc. Natl.
  • ribozyme when used in the form of an expression vector containing the DNA encoding the ribozyme, a hybrid ribozyme in which a tRNA-modified sequence is further linked in order to promote the transfer of the transcript to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • the application target of the agent of the present invention is not particularly limited, and includes, for example, various mammals such as human, monkey, mouse, rat, dog, cat, rabbit, pig, horse, cow, sheep, goat, deer and the like. .
  • the form of the agent of the present invention is not particularly limited, and may take a form usually used in each application depending on the use of the agent of the present invention.
  • the form is a drug, a health enhancer, a nutritional supplement (such as a supplement), and the like, for example, a tablet (including an intraorally disintegrating tablet, a chewable tablet, an effervescent tablet, a troche, a jelly-like drop, etc.) ), Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions and syrups), and preparations suitable for oral ingestion such as jellies Dosage forms (oral dosage forms), nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, creams, etc. Oral formulation).
  • a tablet including an intraorally disintegrating tablet, a chewable tablet, an effervescent tablet, a troche, a jelly-like drop, etc.
  • Pills granules, fine granules, powders, hard capsules, soft capsules
  • a liquid, gel or solid food for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, powdered milk for childcare , Cake mixes, powdered or liquid dairy products, breads, cookies and the like.
  • liquids for example, liquids (solutions, emulsions, suspensions, etc.), semi-solids (gels, creams, pastes, etc.), solids (tablets, particulates, capsules, Films, kneaded materials, molten solids, waxy solids, elastic solids, etc.), more specifically, dentifrices (toothpaste, liquid dentifrice, liquid dentifrice, powder dentifrice, etc.), mouthwashes, Coating agents, patches, mouth fresheners, foods (eg, chewing gum, tablet confectionery, candy, gummy, film, troche, etc.) and the like can be mentioned.
  • liquids solutions, emulsions, suspensions, etc.
  • semi-solids gels, creams, pastes, etc.
  • solids tablets, particulates, capsules, Films, kneaded materials, molten solids, waxy solids, elastic solids, etc.
  • dentifrices teethpaste
  • the agent of the present invention may further contain other components as necessary.
  • Other components are not particularly limited as long as they can be blended in, for example, a medicine, a food composition, an oral composition, a health enhancer, a nutritional supplement (such as a supplement), and the like.
  • a carrier a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a humectant, a colorant, a flavor, and a chelating agent.
  • the content of the inhibitor of the target protein in the agent of the present invention depends on the type, use, use mode, application object, state of the application object, and the like of the inhibitor, and is not limited. For example, 0.0001 to 100 % By weight, preferably 0.001 to 50% by weight.
  • the amount of application (eg, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is generally 0.1 to 1000 per day as the weight of the active ingredient. mg / kg body weight. It is preferable to administer the above dose once or twice or three times a day, and it can be appropriately increased or decreased according to age, disease state and symptoms.
  • the present invention relates to a method for screening an active ingredient of an agent for preventing or treating obstructive pulmonary disease.
  • the present invention provides a method for screening a protein group ( Screening for an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease using as an index the amount or concentration of at least one protein selected from the group consisting of A), protein group (B), and protein group (C)
  • the present invention also relates to a method (hereinafter, also referred to as “the active ingredient screening method of the present invention”). Hereinafter, this will be described.
  • Animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits.
  • any of naturally occurring compounds or artificially produced compounds can be widely used.
  • a purified compound but also a composition in which various kinds of compounds are mixed, or an extract of animals and plants can be used.
  • the compound is not limited to a low molecular compound, but also includes a high molecular compound such as a protein, a nucleic acid, and a polysaccharide.
  • the active ingredient screening method of the present invention is characterized in that the value of the index is the amount or concentration of the corresponding protein in an extracellular vesicle or blood sample of a body fluid collected from an animal not treated with the test substance. Selecting the test substance as an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease (or a candidate substance of an active ingredient of a preventive or therapeutic agent for obstructive pulmonary disease) when the value is lower than (control value) including.
  • the corresponding protein means the same protein as the target protein used as an index.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • the present invention relates to a protein group (A) in an extracellular vesicle or a blood sample of a body fluid collected from an animal treated with a test substance.
  • a protein group (B), and a protein group (C) the method of evaluating the induction or malignancy of obstructive pulmonary disease using the amount or concentration of at least one protein selected from the group consisting of
  • the method may be referred to as “toxicity evaluation method of the present invention”.
  • this will be described.
  • obstructive pulmonary disease About body fluid, extracellular vesicle, protein group (A), protein group (B), protein group (C), obstructive pulmonary disease, measurement of target protein amount or concentration, animal species, test substance, etc. This is the same as the definition in “1. Method for testing obstructive pulmonary disease” and “7. Method for screening active ingredient of preventive or therapeutic agent for obstructive pulmonary disease”.
  • the value of the above-mentioned index is the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance (control value). If the test substance is higher than the threshold, the test substance is determined to be inducible or malignant in obstructive pulmonary disease.
  • the corresponding protein means the same protein as the target protein used as an index.
  • “High” means that, for example, the index value is twice, five times, ten times, twenty times, fifty times, and one hundred times the control value.
  • Test example 1 Preparation of extracellular vesicle fraction Serum of each of human subjects (8) diagnosed with bronchial asthma, each of the human subjects (7) diagnosed with chronic obstructive pulmonary disease (COPD) Extracellular vesicle fractions were prepared from serum and serum from each of healthy human subjects (8 subjects). The preparation of the extracellular vesicle fraction was performed using an extracellular vesicle purification column (EV-Second, manufactured by GL Sciences) with the serum volume of each specimen being equalized.
  • E-Second extracellular vesicle purification column
  • Test example 2 Proteomics analysis (non-label, LC-MS / MS) The protein in the extracellular vesicle fraction was quantified by LC-MS / MS analysis (non-label method). Specifically, it was performed as follows.
  • sample preparation The extracellular vesicle fraction was reduced with 5 mM TCEP for 30 minutes at 37 ° C. and alkylated with 25 mM iodoacetamide for 45 minutes at room temperature. Thereafter, the sample was diluted 7-fold with 50 mM ammonium bicarbonate, placed in a 96-well filter plate, and digested by shaking with 5 ⁇ L of immobilized trypsin (Thermo Fisher Scientific) at 37 ° C. for 6 hours. The trypsin digest was desalted using an Oasis HLB 96-well ⁇ Elution Plate (Waters Corporation, USA) and subjected to LC-MS / MS analysis.
  • Oasis HLB 96-well ⁇ Elution Plate Waters Corporation, USA
  • the eluted peptide was ionized at a spray voltage of 2000 V, and MS data was obtained by a data-dependent fragmentation method.
  • the measurement scan (survey scan) was performed at m / z 400 to 1600, resolution 60,000, and AGC target value 1.0 ⁇ 10 6 ion count.
  • the top 20 intensities of precursor ions in each measurement scan were subjected to low-resolution MS / MS acquisition using a normal CID scan mode with an AGC target value of 5000 ion counts in a linear ion trap.
  • the proteins significantly higher than the control group only in the bronchial asthma group were Putative high mobility group protein B1-like 1, Dynamin-1-like protein, Reticulon-3, Ig kappa chain, VI region gal, and arginase.
  • bronchial asthma group has more than twice the protein of the control group Putative high mobility group protein B1-like 1 and Ig kappa chain VI region Gal, Arginase-1, Keratin, type I cytoskeletal 16, C-1-tetrahydrofolate synthase, cytoplasmic, Dynamin-2, Brain acid soluble protein 1, Solute carrier family 43 member 3 Of these five times more protein bronchial asthma group control group C-1-tetrahydrofolate synthase, it was cytoplasmic.
  • the proteins significantly higher in the chronic obstructive pulmonary disease group alone than in the control group were Chloride intracellular channel protein 1, Tripeptidyl-peptidase 1, Nuclear factor of activated T-cells, cytoplasmic factor 1, Coagulation factor IX, Coagulation factor X, Ig kappa chain VI region WEA, Fibrinogen beta chain, von Willebrand factor, Heat shock protein beta-1, Guanine nucleotide-binding protein G (i) subunit alpha-2, Sodium / potassium-transporting ATPase subunit alpha -1, Annexin A2, Receptor-type tyrosine-protein phosphatase C, Histone H2A.Z, Ras-related protein R-Ras, Heat shock cognate 71 kDa protein, Ras-related protein Ral-A, Coagulation factor V, Elongation factor 2 , Ras-related C3 botulinum toxin substrate 2, Ezrin, Receptor
  • the proteins significantly higher in both the bronchial asthma group and the chronic obstructive pulmonary disease group than in the control group were Ferritin light chain, Ferritin heavy chain, Cathepsin D, Trypsin-1, Trypsin-1, Neprilysin, and X-.
  • Test example 3 Analysis of usefulness as a serum biomarker Human subjects diagnosed as stage 1-2 by GOLD stage classification (25 subjects), human subjects diagnosed as stage 3-4 by GOLD stage classification (13 subjects), and Blood was collected from each of the healthy human specimens (24) to obtain serum. Using serum as a sample, Tripeptidyl-peptidase 2 (TPP2) was detected by ELISA. Specifically, a sandwich ELISA was performed using a Human tripeptidyl peptidase II ELISA Kit (manufactured by MyBioSource, MBS9337216).
  • AUC was calculated from the results of ELISA. Specifically, it is as follows. JMP v. 8.0 (SAS Institute, Cary, NC, USA) was used. Sensitivity is plotted on the vertical axis, and (1-specificity) is plotted on the horizontal axis. For the ROC curve of the multi-marker, a logistic regression analysis was performed on TPP2 to calculate a prediction probability, and an ROC curve was similarly drawn from the prediction probability.
  • TPP2 was found to be also useful as a serum marker.
  • AUC was 0.82.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Pulmonology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des biomarqueurs pour des maladies pulmonaires obstructives telles que l'asthme bronchique, et un procédé d'utilisation de ces biomarqueurs. Les biomarqueurs sont des groupes protéiques (A)-(C) dans un échantillon de sang ou des vésicules extracellulaires dans un liquide corporel prélevé chez un sujet.
PCT/JP2019/025632 2018-06-27 2019-06-27 Biomarqueurs de maladies pulmonaires obstructives WO2020004557A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-121888 2018-06-27
JP2018121888A JP2021156577A (ja) 2018-06-27 2018-06-27 閉塞性肺疾患バイオマーカー

Publications (1)

Publication Number Publication Date
WO2020004557A1 true WO2020004557A1 (fr) 2020-01-02

Family

ID=68984543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/025632 WO2020004557A1 (fr) 2018-06-27 2019-06-27 Biomarqueurs de maladies pulmonaires obstructives

Country Status (2)

Country Link
JP (1) JP2021156577A (fr)
WO (1) WO2020004557A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023153496A1 (fr) * 2022-02-10 2023-08-17 国立大学法人大阪大学 Biomarqueur de l'asthme éosinophilique ou bronchique

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008520552A (ja) * 2004-10-22 2008-06-19 メディミューン，インコーポレーテッド Ｈｍｇｂ１に対する高親和性の抗体およびその使用法
JP2012508577A (ja) * 2008-11-12 2012-04-12 カリス ライフ サイエンシズ ルクセンブルク ホールディングス 表現型を決定するためのエキソソームの使用方法およびそのシステム
US20160033516A1 (en) * 2014-07-31 2016-02-04 Salk Institute For Biological Studies Use of polyclonal and monoclonal antibodies specific for 3-phosphohistidine
WO2016130543A1 (fr) * 2015-02-09 2016-08-18 Abogen, Inc. Dispositifs, solutions et procédés destinés à des applications, une analyse et un diagnostic associés à un prélèvement d'échantillons

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008520552A (ja) * 2004-10-22 2008-06-19 メディミューン，インコーポレーテッド Ｈｍｇｂ１に対する高親和性の抗体およびその使用法
JP2012508577A (ja) * 2008-11-12 2012-04-12 カリス ライフ サイエンシズ ルクセンブルク ホールディングス 表現型を決定するためのエキソソームの使用方法およびそのシステム
US20160033516A1 (en) * 2014-07-31 2016-02-04 Salk Institute For Biological Studies Use of polyclonal and monoclonal antibodies specific for 3-phosphohistidine
WO2016130543A1 (fr) * 2015-02-09 2016-08-18 Abogen, Inc. Dispositifs, solutions et procédés destinés à des applications, une analyse et un diagnostic associés à un prélèvement d'échantillons

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KO, H. K.: "High expression of high-mobility group box 1 in the blood and lungs is associated with the development of chronic obstructive pulmonary disease in smokers", RESPIROLOGY, vol. 19, no. 2, February 2014 (2014-02-01), pages 253 - 261 *
KOBA, TARO ET AL.: "Proteomic profiling of peripheral exosomes to identify novel biomarker candidates for emphysema), non-official translation", PROGRAM AND ABSTRACT OF ANNUAL MEETING OF JAPANESE PROTEOMICS SOCIETY, 2017, pages 202 *
POUWELS, S. D.: "Increased serum levels of LL37, HMGB1 and S 100A9 during exacerbation in COPD patients", EUR RESPIR J., vol. 45, no. 5, May 2015 (2015-05-01), pages 1482 - 1485 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023153496A1 (fr) * 2022-02-10 2023-08-17 国立大学法人大阪大学 Biomarqueur de l'asthme éosinophilique ou bronchique

Also Published As

Publication number Publication date
JP2021156577A (ja) 2021-10-07

Similar Documents

Publication Publication Date Title
US11333665B2 (en) Tissue-derived extracellular vesicles and their use as diagnostics
EP3082840B1 (fr) Méthodes et dosages biologiques se rapportant à des cellules tumorales circulantes
WO2019173799A1 (fr) Sondes oligonucléotidiques et leurs utilisations
Wang et al. MicroRNA microarray-based identification of involvement of miR-155 and miR-19a in development of oral lichen planus (OLP) by modulating Th1/Th2 balance via targeting eNOS and toll-like receptor 2 (TLR2)
Schwartz et al. Insights into epithelial cell senescence from transcriptome and secretome analysis of human oral keratinocytes
JP2024010173A (ja) 尿路上皮がんを検査する方法
WO2020004557A1 (fr) Biomarqueurs de maladies pulmonaires obstructives
KR102015982B1 (ko) 고밀도 지단백 기능과 관련된 심혈관 질환 치료 표적 및 이를 이용하여 심혈관 질환을 진단하는 방법
WO2018168769A1 (fr) Biomarqueur de fibrose tissulaire
WO2018145095A1 (fr) Procédés de prédiction de la réactivité d'un cancer à un agent immunothérapeutique et procédés de traitement du cancer
JP2010014689A (ja) プロテオミクス解析を用いたメラノーママーカーの同定
JP2018151276A (ja) 老化バイオマーカー
Cinque et al. The Generation of Cancer-Specific Ribosomes by the lncRNA LISR Suppresses Melanoma Anti-Tumour Immunity
WO2018168779A1 (fr) Biomarqueur de la bronchopneumopathie chronique obstructive
JP2019158753A (ja) リンパ脈管筋腫症バイオマーカー
WO2020162441A1 (fr) Biomarqueur de maladie granulomateuse
WO2023027186A1 (fr) Biomarqueur de fibrose pulmonaire
WO2023153496A1 (fr) Biomarqueur de l'asthme éosinophilique ou bronchique
WO2018168768A1 (fr) Biomarqueur des maladies respiratoires obstructives
WO2022085687A1 (fr) Agent de prévention ou de traitement de maladies des voies respiratoires, procédé de détection, agent de détection, procédé de criblage de principe actif d'agent de prévention ou de traitement, et procédé d'évaluation d'inductibilité ou de risques d'aggravation
JP2023000812A (ja) 悪性胸膜中皮腫バイオマーカー
Zhang et al. The XPO1 inhibitor selinexor ameliorates bleomycin-induced pulmonary fibrosis in mice via GBP5/NLRP3 inflammasome signaling
EP3133400B1 (fr) Utilisation de protéines associées à la fécondité masculine ak6 et gpx5 ou de leur combinaison
US11874277B2 (en) Systems and methods for Barrett's esophagus pathogenesis and esophageal adenocarcinoma progression revealing markers
KR102202120B1 (ko) 알츠하이머 질환의 진단 또는 치료를 위한 Ｕｂｅ２ｈ의 용도

Legal Events

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

Ref document number: 19825183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19825183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP