WO2019124666A2 - Composition pharmaceutique comprenant un inhibiteur de l'interleukine-17 et un inhibiteur du facteur de nécrose tumorale alpha en tant que principes actifs pour la prévention ou le traitement d'une maladie pulmonaire inflammatoire neutrophilique - Google Patents

Composition pharmaceutique comprenant un inhibiteur de l'interleukine-17 et un inhibiteur du facteur de nécrose tumorale alpha en tant que principes actifs pour la prévention ou le traitement d'une maladie pulmonaire inflammatoire neutrophilique Download PDF

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WO2019124666A2
WO2019124666A2 PCT/KR2018/008879 KR2018008879W WO2019124666A2 WO 2019124666 A2 WO2019124666 A2 WO 2019124666A2 KR 2018008879 W KR2018008879 W KR 2018008879W WO 2019124666 A2 WO2019124666 A2 WO 2019124666A2
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inhibitor
csf
tnf
pharmaceutical composition
bone marrow
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WO2019124666A3 (fr
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이승우
김영민
이승원
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포항공과대학교 산학협력단
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    • 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
    • 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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of a fungal constitutional pulmonary inflammatory disease comprising an interleukin-17 inhibitor and a tumor necrosis factor-alpha inhibitor as an active ingredient, and a method for screening for a therapeutic agent for fungal pulmonary inflammatory diseases.
  • Asthma is a typical allergic disease caused by the combination of genetic and environmental factors in a very sensitive state of the bronchial lung.
  • the chronic obstructive pulmonary disease (COPD) is caused by harmful particles or gas inhalation. It is a respiratory disease that causes pulmonary dysfunction and dyspnea due to abnormal inflammatory reaction.
  • asthma and COPD may be subdivided into eosinophilic and neutrophilic, depending on the type and invasion pattern of the immune cells that penetrate the airway or lung tissue.
  • Bone marrow is a major site of hematopoiesis that forms cellular components of blood. It is known to play an important role in the pathophysiology of inflammatory diseases in distal tissues by providing immune cells necessary for inflammation relief. Accordingly, immunological association between bone marrow and inflammatory tissue has been steadily proposed through the study of inflammatory diseases including infection, autoimmune diseases, cancer and allergy. However, despite the fact that bone marrow is the central site of granule cell formation, studies on asthma have focused primarily on the immunological response in the lungs, considering asthma as a local inflammatory response in airway.
  • bone marrow plays an important role in the pathophysiological aspects of fungal pulmonary inflammatory diseases including neutrophilic asthma, considering the inflammatory and biological characteristics of neutrophils, the most abundant immune cells produced in bone marrow And the effect of bone marrow on asthma was studied under the hypothesis that it would be important to study the hematopoiesis of the bone marrow.
  • hematopoietic stem and progenitor cells hematopoietic stem and progenitor cells
  • the present invention provides a pharmaceutical composition for the prevention or treatment of respiratory tract pulmonary inflammatory diseases, which comprises an interleukin-17 (IL-17) inhibitor and a tumor necrosis factor-alpha The purpose.
  • IL-17 interleukin-17
  • the present invention provides a pharmaceutical composition for preventing and treating pulmonary inflammatory diseases of the respiratory tract including an interleukin-17 (IL-17) inhibitor and a tumor necrosis factor-alpha Or a pharmaceutical composition for therapeutic use.
  • IL-17 interleukin-17
  • the fungal pulmonary inflammatory disease is selected from the group consisting of neutrophilic asthma, neutrophilic chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF) Acute lung injury (ALI), and acute respiratory distress syndrome (ARDS).
  • neutrophilic asthma neutrophilic chronic obstructive pulmonary disease
  • IPF idiopathic pulmonary fibrosis
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • the IL-17 inhibitor may be an anti-IL-17 antibody that specifically binds to the IL-17 protein.
  • the TNF-a inhibitor may be an anti-TNF-a antibody that specifically binds to the TNF-a protein.
  • the IL-17 inhibitor and the TNF-a inhibitor may inhibit the expression of a granulocyte colony-stimulating factor (G-CSF).
  • G-CSF granulocyte colony-stimulating factor
  • the pharmaceutical composition may further comprise a G-CSF inhibitor.
  • the G-CSF inhibitor may be an anti-G-CSF antibody that specifically binds to the G-CSF protein.
  • the pharmaceutical composition can normalize the balance change of hematopoietic action in bone marrow by G-CSF.
  • the pharmaceutical composition may reduce neutrophil infiltration of bronchoalveolar lavage fluid and lung tissue.
  • the pharmaceutical composition may reduce the activity of myeloperoxidase (MPO).
  • MPO myeloperoxidase
  • the pharmaceutical composition may reduce airway hypersensitivity.
  • the present invention provides a method for screening for a therapeutic agent for the respiratory tract inflammatory disease of the respiratory tract, comprising the following steps.
  • the therapeutic agent may inhibit the expression of granulocyte colony-stimulating factor (G-CSF).
  • G-CSF granulocyte colony-stimulating factor
  • the present invention provides a method of preventing or treating phosgenous pulmonary inflammatory disease, comprising administering the pharmaceutical composition to a subject.
  • the present invention provides the use of the pharmaceutical composition for the prevention or treatment of respiratory pulmonary inflammatory disease.
  • the inventors of the present invention studied a change in hematopoiesis in the bone marrow using a homocysteine-induced asthma mouse model in which a pulmonary inflammatory response was induced.
  • G-CSF a mediator of the bone marrow hematopoiesis
  • G-CSF was induced by the simultaneous stimulation of IL-17 and TNF- ⁇ in the cells.
  • the anti-G-CSF antibody alone or anti-IL-17 antibody and anti-TNF - ⁇ antibody to the bone marrow and to evaluate the therapeutic effect of the combined asthma.
  • the present invention provides a new therapeutic target for the respiratory tract pulmonary inflammatory disease, and the IL-17 inhibitor and the TNF-a inhibitor according to the present invention are expected to be a new therapeutic method for the respiratory tract pulmonary inflammatory disease without the conventional effective treatment .
  • FIG. 1A shows a process of producing a LPS / OVA-induced astaxanthoid mouse model to investigate the relationship between bone marrow and constituent asthma
  • FIGS. 1B to 1D show the results of the production of astrocyte-
  • LSK cells including hematopoietic stem cells (HSC) and pluripotent progenitor cells (MPP)
  • Fig. 1B hematopoietic stem cells
  • MPP pluripotent progenitor cells
  • GMPP granulocyte and monocyte progenitor cells
  • MEP megakaryocytes and erythroid precursor cells
  • B220 + lymphocyte reduction bone marrow lineage development through increased CD11b + myeloid cells and CD11b / B220 ratio
  • FIG. 2A shows the presence of a high level of G-CSF in the sera of mice in which asthma was induced
  • FIG. 2B to FIG. 2D show the results of systemic administration of recombinant mouse G-CSF (rmG-CSF)
  • rmG-CSF recombinant mouse G-CSF
  • FIG. 3 shows the results of analysis of genetic and functional changes of HSPC by G-CSF.
  • FIGS. 3A and 3B are graphs showing changes in the expression of HSPC in CD150 + LSK cells when rmG-CSF was administered to mice and mice induced asthma
  • FIG. 3C and FIG. 3D are graphs showing changes in the expression of myeloid-related genes and the expression of lymphoid-associated genes (lymphoid) LPS / OVA) and the increase in the ratio of GMP / MEP in CD150 + LSK cells and the increase in the ratio of myeloid / lymphoid in the spleen and peripheral blood to the myeloid lineage.
  • lymphoid-associated genes lymphoid-associated genes
  • FIG. 4A is a graph showing an experimental protocol using a mouse model in which asthma is induced in the respiratory tract
  • FIG. 4A is a graph showing an experimental protocol for normalizing hematopoiesis by G-CSF neutralization
  • Figure 4d shows the reduction of HSPCs (Figure 4b), the reduction of the ratio of increased GMP / MEP ( Figure 4c), and the development of myeloid lineage cells, following administration of G-CSF neutralizing antibody in the asthma- (Fig. 4d).
  • Figs. 4e to 4h show the reduction of neutrophil infiltration (Fig. 4e), reduction of MPO activity (Fig. 4d) of BAL fluid following administration of G-CSF neutralizing antibody (Fig. 4f), and decreased neutrophil counts of lung tissue (Fig. 4g, 4h).
  • FIG. 5 shows the results of confirming the production of G-CSF by IL-17 and TNF- ⁇ in lung epithelial cells
  • FIG. 5a shows the results of G-CSF production in lung epithelial cells in response to the respiratory component airway inflammation (LPS / OVA) CSF is expressed at a high level
  • FIG. 5B shows that G-CSF production is induced by simultaneous stimulation of IL-17A and TNF-a with mouse lung epithelial cell line (MLE-12) at the in vitro level 5c and 5d show G-CSF production (FIG. 5c) and G-CSF circulation increase (FIG. 5d) in the lung epithelial cells when the two cytokines were administered to the mouse nasal cavity.
  • MLE-12 mouse lung epithelial cell line
  • FIG. 6 shows the results of analysis of the hematopoietic effect of IL-17 and TNF- ⁇ (IL17 + TNF- ⁇ ) on bone marrow in mice.
  • FIG. 6A LSK cells
  • FIG. 6c shows the results of analysis of the hematopoietic balance towards myeloid cell formation through B220 + lymphocyte reduction, CD11b + myeloid cells and CD11b / B220 + ratio increase.
  • FIG. 7 is a graph showing the results of the normalization of the hematopoietic action by neutralization of IL-17 and TNF- ⁇ and the effect of improving airway inflammation in the respiratory tract
  • FIG. 7A is an illustration of an experimental protocol using a mouse model in which airway- 7b to 7f show that neutrophil infiltration reduction (FIG. 7b) and remarkable reduction of MPO activity in the BAL fluid (FIG. 7c) were observed after administration of the neutralizing antibody of IL-17 and TNF- ), Decrease in neutrophils in lung tissues (Fig. 7d, 7e), and decrease in airway hyperresponsiveness (Fig. 7f).
  • FIG. 8A is a graph showing changes in BAL fluid, lung lysate (TNF-alpha), and TNF-alpha in the case of administration of each IL-17 and TNF-alpha neutralizing antibody ) And serum (Serum), and FIGS. 8b to 8d show the decrease of the increased HSPC due to the administration of the IL-17 and TNF- ⁇ neutralizing antibodies (FIG. 8b) / MEP ( Figure 8c), and a reduction in the development of myeloid lineage cells (Figure 8d).
  • FIG. 9 is a graph showing the superiority of mixed administration by confirming the effect of treatment of asthma by the single administration and the mixed administration of IL-17 or TNF- ⁇ neutralizing antibody, respectively.
  • FIGS. 9A to 9D show the results Neutrophil infiltration was reduced in BAL fluid when the control antibody (Iso), IL-17 antibody alone ( ⁇ -IL-17), TNF- ⁇ alone ( ⁇ -TNF- ⁇ ) (Fig. 9A), a decrease in MPO activity (Fig. 9B), and a reduction in neutrophils in lung tissue (Fig. 9C and Fig. 9D).
  • Fig. 9E shows the results of comparing BAL fluid, lung lysate and serum (Serum), which is the result of comparing the concentration of G-CSF.
  • the present inventors have now found that the use of a neutralizing antibody against G-CSF directly blocks the activity or neutralizes the neutralizing antibody against IL-17 and TNF-a, which are cytokines that induce the production of G-CSF, It was confirmed that the hematopoietic effect of the bone marrow was normalized, and that the inflammation of the respiratory tract was improved in the airways and lungs. Based on these findings, the present invention was completed.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of respiratory tract pulmonary inflammatory diseases, which comprises an interleukin-17 (IL-17) inhibitor and a tumor necrosis factor-alpha (TNF-?) Inhibitor as an active ingredient.
  • IL-17 interleukin-17
  • TNF- tumor necrosis factor-alpha
  • the respiratory tract pulmonary inflammatory disease which is a disease to be prevented or treated, includes all diseases caused by inflammatory reaction caused by infiltration of neutrophils into lung tissue, preferably neutrophilic asthma, Acute pulmonary fibrosis (IPF), acute lung injury (ALI), and acute respiratory distress syndrome (ARDS), as well as chronic obstructive pulmonary disease (COPD) And more preferably, it can be, but is not limited to, asthma or arthritic constitutional chronic obstructive pulmonary disease.
  • the present inventors produced a mouse model mimicking asthma by using a method of administering a lipopolysaccharide (LPS) and ovalbumin (OVA), and by using the example, the bone marrow, which is a main site for producing neutrophils, Hypothesis that it is likely to be related to the inflammatory response.
  • LPS lipopolysaccharide
  • OVA ovalbumin
  • the hematopoietic activity of the bone marrow in the asthmatic mouse model of the fowl was analyzed. As a result, it was confirmed that the hematopoietic balance toward the myeloid cell formation was changed. It was confirmed that G-CSF is present at a remarkably high concentration in the serum. Furthermore, in order to test whether G-CSF directly influences the hematopoietic action of bone marrow, systemic administration of recombinant mouse G-CSF to mice induced the change in bone marrow hematopoiesis in the same manner as above (See Example 2).
  • G-CSF circulating in the body functions as a lineage factor of hematopoietic stem cells and progenitor cells (HSPCs).
  • HSPCs progenitor cells
  • G-CSF is induced by simultaneous stimulation of IL-17 and TNF-a in pulmonary epithelial cells, , And it was confirmed that when the two cytokines were simultaneously administered to the mouse, the change in the hematopoietic effect of the bone marrow was induced as in the case of administration of G-CSF (see Example 5).
  • mice immunized with the aerosolized asthma were simultaneously administered with antibodies against IL-17 and TNF-alpha, respectively, to block their activity.
  • BAL fluid, lung tissue lysate, G-CSF concentration was significantly decreased and as in the case of treatment with anti-G-CSF antibody, the inflammatory response in the airway and lungs was alleviated and the hematopoietic effect of the bone marrow was normalized.
  • This effect was also observed when the IL-17 and TNF-? Antibodies were treated simultaneously, and it was confirmed that a limited therapeutic effect was obtained when each treatment was effected (see Example 6).
  • the present inventors have found IL-17 and TNF-a that induce G-CSF and its production as novel therapeutic targets of asthma in the respiratory tract through this example, and simultaneously inhibit IL-17 and TNF- In the case of lung and airway tissue, it was confirmed that the inflammatory reaction of the respiratory tract was effectively alleviated and its mechanism of action was confirmed.
  • the IL-17 may preferably be IL-17A
  • the IL-17 inhibitor may be an anti-IL-17 antibody that specifically binds to the IL-17 protein, IL-17A antibody that specifically binds to the IL-17A protein (NCBI accession #. NP_034682).
  • the TNF- ⁇ inhibitor may be an anti-TNF- ⁇ antibody that specifically binds to the TNF- ⁇ protein (NCBI accession #. CAA68530), but is not limited thereto.
  • the pharmaceutical composition is characterized in that it comprises both the IL-17 inhibitor and the TNF-alpha inhibitor, more preferably the anti-IL-17 antibody and the anti-TNF- CSF inhibitor may further comprise a granulocyte colony-stimulating factor (G-CSF) inhibitor, wherein the G-CSF inhibitor may be an anti-G-CSF antibody that specifically binds to a G-CSF protein, But is not limited thereto.
  • G-CSF granulocyte colony-stimulating factor
  • the pharmaceutical composition according to the present invention comprises the IL-17 inhibitor and the TNF-alpha inhibitor as an active ingredient, and may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed.
  • Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference.
  • the pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method, but preferably can be administered orally, Depends on the condition and the weight of the patient, the degree of the disease, the type of the drug, the administration route and time, but can be appropriately selected by those skilled in the art.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level will depend on the type of disease, severity, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and the excretion rate, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or one to three divided doses per day.
  • the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.
  • the present invention provides a method of screening for a therapeutic agent for the respiratory tract inflammatory disease of the respiratory tract, comprising the following steps.
  • the lung cell is more preferably a pulmonary tissue-derived cell in which a lung-constituting lung inflammation disease is induced, but is not limited thereto.
  • the therapeutic agent may inhibit the expression of G-CSF, but is not limited thereto.
  • the candidate substance may be selected from the group consisting of a compound, a microorganism culture or extract, a natural product extract, a nucleic acid, and a peptide.
  • the present invention provides a method for the prevention or treatment of respiratory tract pulmonary inflammatory disease, comprising the step of administering the pharmaceutical composition to a subject.
  • &quot refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.
  • the present invention provides the use of the pharmaceutical composition for the prevention or treatment of a respiratory tract inflammatory disease.
  • Wild-type C57BL / 6 (B6, CD45.1 and CD45.2) mice, 6 to 8 weeks old, were used in this example. All mice were housed in POSTECH's non-pathogenic animal facilities and the experiments were performed according to POSTECH's animal guidelines.
  • Mouse lung epithelial cells were cultured in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS), 1x ITS solution (sigma), 10 nM hydrocortisone, 10 nM b-estradiol, And cultured in DMEM: Ham's F12, 50:50 medium (Welgene) supplemented with glutamine (L-glutamine).
  • FBS fetal bovine serum
  • 1x ITS solution sigma
  • 10 nM hydrocortisone 10 nM b-estradiol
  • DMEM Ham's F12, 50:50 medium (Welgene) supplemented with glutamine (L-glutamine).
  • the cultured cells 1 x 10 5
  • the cell culture was recovered and the level of
  • 100 ⁇ g of anti-G-CSF (ebioscience) or isotype antibody was administered intraperitoneally on the 28th day after the mice were sensitized through the nasal cavity to conduct the G-CSF neutralization experiment.
  • 50 ug of anti-IL-17 and / or anti-TNF- or isotype antibodies were intraperitoneally injected intraperitoneally Lt; / RTI >
  • BAL fluid was collected by tracheal catheterization, and mouse lungs were collected by perfusion and stored in 4% paraformaldehyde (PFA) for histological analysis .
  • PFA paraformaldehyde
  • mice were injected with 2.5 [mu] g of recombinant mouse G-CSF (Biolegend) for 2 consecutive days for 2 consecutive days and mice were sacrificed 48 hours later to evaluate the bone marrow cellularity.
  • 500 ng of recombinant IL-17 and TNF- [alpha] were mixed in 30 [mu] l PBS and injected into the mouse by nasal aspiration, and then the mice were sacrificed 6 hours for cytokine analysis and 48 hours for bone marrow cell analysis .
  • mice In order to prepare mouse-derived lung cells, the right lung tissue of the mice was excised and cultured in collagenase (Roche) and DNase (Roche) solution for 15 minutes at 37 ° C with vigorous stirring, Lt; / RTI > to obtain a single cell suspension.
  • leg bone one tibia and one femur
  • RPMI1640 medium Welgene
  • debris was removed using a 40- ⁇ m strainer to obtain a single cell suspension Respectively.
  • the BAL fluid was centrifuged, and the pellet was resuspended in 100 ⁇ l of PBS containing 2% FBS and the number of cells was measured.
  • BAL fluid, lung tissue lysate, and serum G-CSF levels were measured using a DuoSet ELISA kit (R & D systems) according to the manufacturer's instructions.
  • MPO myeloperoxidase
  • Flow cytometry analysis was performed using monoclonal antibodies and phenotypic markers to analyze hematopoietic cells in bone marrow, lung, and BAL. Mature phylogenetic cells for HSPC analysis in bone marrow were excluded using APC junctional lineage markers such as TER119 (clone TER-119), CD11b, Gr-1, CD3e, NK1.1, MHCII and B220.
  • APC junctional lineage markers such as TER119 (clone TER-119), CD11b, Gr-1, CD3e, NK1.1, MHCII and B220.
  • c-Kit-PE / Cy7 (clone 2B8) was added to the LSK cells (LIN-SCA-1 + c-Kit +), LSK CD150 + CD48-, LSK CD150-CD48-, LSK CD150- SCA-1 + c-Kit + SCA-1-Brilliant Violet (clone D7), CD150-PerCP / eFlour710 (clone mShad150) and CD48- SCA-1, CD34-FITC (LIN-IL-7R?), And CD34 + Fc?
  • TER119-CD11b + Gr-1 + neutrophils
  • eosinophils TER119-CD11bintsiglecF + Gr-1low
  • B cells TER119-Gr-1-CD11b- B220 +
  • alveolar macrophages FSChighSSChighTER119-CD11blowsiglecFhighGr-
  • TER119-FITC CD11b-PE / Cy7, B220-PB, siglecF-PE and Gr-1-APC.
  • epithelial cells TER119-CD45-CD31-EpCAM +
  • endothelial cells TER119-CD45-CD31 + EpCAM-
  • TER119-FITC TER119-FITC
  • CD45-FITC TER119-FITC
  • EpCAM- CD31-PE epithelial cells
  • Expression of cytokine receptors in lung epithelial cells was measured using IL-17RA-PE, IL-17RC-biotin, TNFR1-biotin, TNFR2-PE and streptavidin-APC.
  • the present inventors In order to test the hypothesis that bone marrow plays an important role in the pathophysiology of asthma, the present inventors firstly investigated whether the hematopoietic effect of bone marrow changes in the asthmatic mouse model. To this end, a LPS / OVA-induced asthmatic mouse model was prepared according to the procedures shown in Examples 1-3 and FIG. 1 a, and whether or not the hematopoietic effect of the bone marrow was changed in the above- Respectively.
  • the LSK cells including hematopoietic stem cells (HSC) and multipotent progenitor cells (MPP) were significantly As shown in FIG. 1C, granulocyte and monocyte progenitors (GMP) were increased in the progenitor cells whereas megakaryocyte and erythrocyte progenitors (MEP) were decreased .
  • the change in the hematopoietic balance toward the formation of myeloid cells reflected by the increase in the GMP / MEP ratio as described above is due to the decrease of B220 + lymphocytes, the development of myeloid cells by the increase of CD11b + myeloid cells and the ratio of CD11b / B220 + .
  • the present inventors investigated the kinetics of the inflammatory mediators circulating at 6, 12, 24, and 48 hours after the last OVA administration in the phosgenous mouse model to investigate the mediator of the inflammatory signal to the bone marrow kinetics).
  • G-CSF granulocyte colony stimulating factor
  • HSPCs hematopoietic stem and progenitor cells
  • mice treated with rmG-CSF as shown in FIG. 3B also showed increased expression of myeloid cell-related genes and decreased expression of lymphoid-related genes in CD150 + LSK cells The genetic profile was reprogrammed toward more myeloid cell formation.
  • Example 4 Normalization of hematopoietic action by blocking G-CSF and consequent formation of airborne airways.
  • G-CSF As a modulator of bone marrow function and hematopoiesis in asthma, the present inventors conducted periodic administration of OVA in the asthmatic mouse model according to the procedure shown in FIG. 4A GCSF antibody was injected to neutralize G-CSF and analyzed for cellularity in the bone marrow and lung.
  • G-CSF was neutralized by administering an antibody (? -G-CSF) that specifically binds to G-CSF as shown in FIGS. 4B to 4D, and as a result, an isotype antibody (Iso)
  • an antibody ? -G-CSF
  • Iso an isotype antibody
  • HSPCs increased
  • G-CSF was neutralized
  • the increase in the proportion of myeloid / lymphoid cells was normalized to a level similar to that of the normal control, .
  • the inflammation of the lungs was alleviated through administration of G-CSF neutralizing antibody (? -G-CSF) unlike the case of administration of isopyte antibody (Iso)
  • neutrophil infiltration and MPO activity of BAL fluid were decreased, and the number of neutrophils in lung tissue was decreased.
  • G-CSF is known to be produced by several types of cells, including macrophages, epithelial cells, endothelial cells, bone marrow stromal cells and fibroblasts, in response to a variety of inflammatory stimuli such as LPS or cytokines. Therefore, in order to investigate the main cells producing G-CSF in the lung, the present inventors isolated the hematopoietic and non-hematopoietic compartments of the lung and analyzed the level of G-CSF mRNA by RT-PCR.
  • the epithelial cells express G-CSF mRNA at a significantly higher level than CD45 + hematopoietic cells and endothelial cells in response to phagocytic inflammation (LPS / OVA) as shown in FIG. 5a Respectively.
  • G-CSF was produced at a significantly higher level in the lung epithelial cells than in the case of administration alone, G-CSF circulation was increased. It should be noted that when the above two cytokines were simultaneously administered to mice, an increase in HSPCs and a change in the bone marrow hematopoiesis were reproduced as shown in FIGS. 6A to 6C.
  • Example 6 Normalization of hematopoietic action by IL-17 and TNF-a blockade, and thus, improvement of airway inflammation
  • IL-17 and anti-TNF [alpha] neutralizing antibodies were simultaneously administered to mouse models to block IL-17 and TNF- [alpha] at the same time.
  • FIGS. 7B and 7F when the two antibodies were administered simultaneously ( ⁇ -IL-17 + ⁇ -TNF- ⁇ ) as compared with the case of administration of the isotype antibody (Iso) And MPO activity were significantly decreased, neutrophil increase was decreased in lung tissue and airway hypersensitivity was decreased.
  • phosgenous inflammation was effective only when IL-17 and TNF- [alpha] cytokine were simultaneously removed as shown in Figs. 9A to 9E, and the antibody against each cytokine alone was administered to mice ( ⁇ -IL-17, ⁇ -TNF- ⁇ ) showed limited therapeutic effects in terms of airway and lung inflammation and G-CSF induction.
  • results of the present invention show that co-treatment of IL-17 and TNF- [alpha] neutralizing antibody, which induces the production of C-GSF neutralizing antibody or G-CSF, G-CSF and IL-17 / TNF- ⁇ as targets for the development of therapeutic agents for asthma, as well as the normalization of bone marrow hematopoiesis by inhibition of G-CSF expression or activity.
  • the pharmaceutical composition comprising the IL-17 inhibitor and the TNF-? Inhibitor according to the present invention as an active ingredient or the pharmaceutical composition further comprising an anti-G-CSF inhibitor in the composition can be used for normalizing the hematopoiesis of excellent bone marrow, Suggesting a new therapeutic target for the respiratory tract inflammatory disease of the respiratory tract without the effective treatment, and may be usefully used in the field of the development of therapeutic agents for the above diseases.

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Abstract

La présente invention concerne une composition pharmaceutique comprenant un inhibiteur de l'interleukine-17 et un inhibiteur du facteur de nécrose tumorale alpha en tant que principes actifs pour la prévention ou le traitement d'une maladie pulmonaire inflammatoire neutrophilique, ou un procédé de recherche d'une substance thérapeutique contre une maladie pulmonaire inflammatoire neutrophilique. Suite à des recherches portant sur la modification de l'hématopoïèse dans la moelle osseuse d'un modèle de souris souffrant d'asthme neutrophilique chez lequel une réponse pulmonaire inflammatoire neutrophilique a été induite, les présents inventeurs ont récemment révélé que le G-CSF est un médiateur modifiant l'hématopoïèse de la moelle osseuse. Ils ont, en outre, identifié une production de G-CSF induite par co-stimulation par l'IL-17 et le TNF-α dans les cellules épithéliales pulmonaires et déterminé expérimentalement que l'hématopoïèse de la moelle osseuse a été normalisée par l'administration d'un anticorps anti-G-CSF seul ou par l'administration d'une combinaison associant un anticorps anti-IL-17 et un anticorps anti-TNF-α chez le modèle de souris, avec un effet thérapeutique résultant sut l'asthme neutrophilique. Par conséquent, la présente invention suggère une nouvelle cible thérapeutique en cas de maladie inflammatoire pulmonaire neutrophilique, et l'on s'attend à ce que l'inhibiteur de l'IL-17 et l'inhibiteur du TNF-α selon la présente invention constituent un nouveau traitement contre une maladie pulmonaire inflammatoire neutrophilique n'ayant pas été efficacement traitée par les procédés classiques.
PCT/KR2018/008879 2017-12-19 2018-08-06 Composition pharmaceutique comprenant un inhibiteur de l'interleukine-17 et un inhibiteur du facteur de nécrose tumorale alpha en tant que principes actifs pour la prévention ou le traitement d'une maladie pulmonaire inflammatoire neutrophilique WO2019124666A2 (fr)

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WO2021243424A1 (fr) * 2020-06-04 2021-12-09 CSL Innovation Pty Ltd Procédé de traitement ou de prévention du syndrome de détresse respiratoire aiguë
CN115804821A (zh) * 2022-12-19 2023-03-17 新疆维吾尔药业有限责任公司 寒喘祖帕颗粒在制备治疗激素抵抗型哮喘药物中的应用

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EP4268835A1 (fr) * 2020-12-28 2023-11-01 MD Healthcare Inc. Composition pour la prévention ou le traitement d'une maladie pulmonaire neutrophile comprenant des vésicules extracellulaires dérivées de micrococcus luteus

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JP4272254B1 (ja) * 2008-08-27 2009-06-03 ゆき江 中村 帽子にも使えるパレオ
KR101858346B1 (ko) * 2015-07-07 2018-05-16 가톨릭대학교 산학협력단 Ripk 억제제를 유효성분으로 포함하는 면역질환의 예방 또는 치료용 조성물
KR101760512B1 (ko) * 2015-07-31 2017-07-21 일동제약(주) 혼합 생약 추출물을 포함하는 만성 염증성 질환의 예방, 치료 또는 개선용 조성물
US20170218092A1 (en) * 2016-01-28 2017-08-03 Janssen Biotech, Inc. Bispecific Anti-TNF-Alpha/IL17A Antibodies and Anti-TNF-Alpha Antibodies and Methods of Their Use

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WO2021243424A1 (fr) * 2020-06-04 2021-12-09 CSL Innovation Pty Ltd Procédé de traitement ou de prévention du syndrome de détresse respiratoire aiguë
CN112106705A (zh) * 2020-08-31 2020-12-22 南京新环检测科技有限公司 一种评价药物预防病毒性肺炎效果的方法
CN112106705B (zh) * 2020-08-31 2022-04-08 南京新环检测科技有限公司 一种评价药物预防病毒性肺炎效果的方法
CN115804821A (zh) * 2022-12-19 2023-03-17 新疆维吾尔药业有限责任公司 寒喘祖帕颗粒在制备治疗激素抵抗型哮喘药物中的应用
CN115804821B (zh) * 2022-12-19 2024-05-14 新疆维吾尔药业有限责任公司 寒喘祖帕颗粒在制备治疗激素抵抗型哮喘药物中的应用

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