KR20190073917A - Pharmaceutical composition for preventing or treating neutrophilic lung inflammatory diseases containing Inhibitors of IL-17 and TNF-α - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neutrophilic lung inflammatory disease by including an interleukin-17 inhibitor and a tumor necrosis factor-alpha inhibitor as active ingredients, and a screening method of a neutrophilic lung inflammatory disease treatment material. As study results of changes in hematopoiesis in bone marrow by using a neutrophilic asthmatic mouse model in which a neutrophilic lung inflammatory response was induced, inventors of the present invention: newly identified G-CSF which is a mediator of changing the hematopoietic function of bone marrow; furthermore, confirmed that G-CSF production was induced by simultaneous stimulation of IL-17 and TNF-α in lung epithelial cells; and experimentally confirmed the hematopoietic normalization of bone marrow and the treatment effect of neutrophilic asthma thereby according to the single injection of anti-G-CSF antibody or the simultaneous injection of anti-IL-17 antibody and anti-TNF-alpha antibody by using a mouse model. The present invention provides a new therapeutic target for neutrophilic lung inflammatory disease. According to the present invention, the IL-17 inhibitor and the TNF-α inhibitor are expected to be a new treatment method for the neutrophilic lung inflammatory disease which does not have effective treatment in conventional methods.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating respiratory-tract inflammatory disease, which comprises as an active ingredient an interleukin-17 inhibitor and a tumor necrosis factor-alpha inhibitor, α}

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 hereditary and environmental factors in a very sensitive state of the bronchi in the lungs and chronic obstructive pulmonary disease (COPD) is caused by inhalation of harmful particles or gases. Is a respiratory disease that causes pulmonary dysfunction and dyspnea due to abnormal inflammatory reaction.

These 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 area of granule cell production, studies on asthma have focused on immunologic responses in the lungs, primarily considering asthma as a local inflammatory response in airway. However, recent preclinical and clinical studies have shown systemic inflammatory responses in eosinophilic asthma due to elevated serum levels of IL-5 and increased eosinophil precursor in bone marrow (Am J Respir Crit Care Med 1998 Sep; (3): 951-7). These observations support a new concept of lung / bone marrow involvement in asthma.

On the other hand, other types of asthma are known to occur in about 25% of asthmatic patients, and they are highly associated with systemic inflammation and many have been reported to be resistant to steroids, a conventional asthma treatment. This group of asthmatic patients is only about 10% of the total patients, but it is difficult to treat and manage them, which accounts for about 50% of the socioeconomic costs of asthma treatment. Therefore, it is necessary to develop a new therapeutic agent that understands the immunological mechanism of asthma in Korea. However, the causes and mechanisms of asthma are still unknown.

Therefore, the present inventors have found that 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.

As a result of analyzing the hematopoietic effect of bone marrow asthma using the above-described hypothesis, a mouse model inducing a respiratory-induced pulmonary inflammatory response and inducing asthma in the respiratory tract was prepared, and the serum of the asthmatic mice CSF, and that the G-CSF changes the balance of hematopoiesis in the bone marrow by altering the genetic and functional characteristics of hematopoietic stem and progenitor cells (HSPCs). Based on these findings, it is possible to directly block the activity of the protein using an antibody against G-CSF, or simultaneously treat neutralizing antibodies against IL-17 and TNF-a, cytokines that induce the production of G-CSF, CSF, the hematopoietic effect in the bone marrow is normalized, and thus, the effect of improving airway inflammation in the airways and lungs is confirmed. Based on this, the present invention has been completed.

Accordingly, 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.

It is another object of the present invention to provide a method for screening a remedy for pulmonary inflammatory disease.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, 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.

In one embodiment of the present invention, 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).

In another embodiment of the invention, the IL-17 inhibitor may be an anti-IL-17 antibody that specifically binds to the IL-17 protein.

In another embodiment of the present invention, the TNF-a inhibitor may be an anti-TNF-a antibody that specifically binds to the TNF-a protein.

In another embodiment of the present invention, the IL-17 inhibitor and the TNF-a inhibitor may inhibit the expression of a granulocyte colony-stimulating factor (G-CSF).

In another embodiment of the present invention, the pharmaceutical composition may further comprise a G-CSF inhibitor.

In another embodiment of the present invention, the G-CSF inhibitor may be an anti-G-CSF antibody that specifically binds to the G-CSF protein.

In another embodiment of the present invention, the pharmaceutical composition can normalize the balance change of hematopoietic action in bone marrow by G-CSF.

In another embodiment of the present invention, the pharmaceutical composition may reduce neutrophil infiltration of bronchoalveolar lavage fluid and lung tissue.

In another embodiment of the present invention, the pharmaceutical composition may reduce myeloperoxidase (MPO) activity.

In another embodiment of the present invention, the pharmaceutical composition may reduce airway hypersensitivity.

In addition, 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.

(a) treating candidate substances in lung cells in vitro;

(b) measuring the expression or activity of interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-a) in the lung cells; And

(c) selecting a substance that reduces the expression or activity of IL-17 and TNF-? compared to the candidate substance-untreated group as a therapeutic substance for constitutive pulmonary inflammatory disease.

In one embodiment of the present invention, the therapeutic agent may inhibit the expression of granulocyte colony-stimulating factor (G-CSF).

In addition, the present invention provides a method of preventing or treating phosgenous pulmonary inflammatory disease, comprising administering the pharmaceutical composition to a subject.

In addition, 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. As a result, G-CSF, a mediator of the bone marrow hematopoiesis, G-CSF was induced by the simultaneous stimulation of IL-17 and TNF-α in the cells. In the mouse model, the anti-G-CSF antibody alone or anti-IL-17 antibody and anti-TNF -α antibody to the bone marrow and to evaluate the therapeutic effect of the combined asthma. Accordingly, 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- As a result of the analysis of hematopoietic changes, the increase of LSK cells including hematopoietic stem cells (HSC) and pluripotent progenitor cells (MPP) (Fig. 1B), granulocyte and monocyte progenitor cells (GMP) / megakaryocytes and erythroid precursor cells MEP) (Fig. 1C), and B220 + lymphocyte reduction, bone marrow lineage development through increased CD11b + myeloid cells and CD11b / B220 ratio (Fig. 1d).
FIG. 2A shows the presence of a high level of G-CSF in the sera of mice in which asthma was induced, and FIG. 2B to FIG. 2D show the results of systemic administration of recombinant mouse G-CSF (rmG-CSF) As a result of analysis of the changes in post-hematopoietic action, it was found that the increase of LSK cells (Fig. 2B), the hematopoietic balance toward bone marrow formation by increasing the ratio of GMP / MEP (Fig. 2C), and the decrease of B220 + lymphocytes, (Fig. 2d) of bone marrow cells through an increase in CD11b / B220 ratio.
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.
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-α in lung epithelial cells, and 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.
FIG. 6 shows the results of analysis of the hematopoietic effect of IL-17 and TNF-α (IL17 + TNF-α) on bone marrow in mice. As a result, the increase of LSK cells (FIG. 6A) 6b), and a change in hematopoietic balance towards myeloid cell formation through B220 + lymphocyte reduction, CD11b + myeloid cells and CD11b / B220 + ratio increase (Fig. 6c).
FIG. 7 is a graph showing the results of the normalization of the hematopoietic action by neutralization of IL-17 and TNF-α 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-α 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-α 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 (α-IL-17), TNF-α alone (α-TNF-α) (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.

Accordingly, 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.

In the present invention, 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.

In one embodiment of the present invention, 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).

In another embodiment of the present invention, G-CSF circulating in the body functions as a lineage factor of hematopoietic stem cells and progenitor cells (HSPCs). As a result, it was confirmed that recombinant mouse G- CSF in the systemically administered mice also showed that the genetic profile of CD150 + LSK cells was programmed into the myeloid cell generation, thereby changing the intramolecular hematopoiesis to the myeloid cell line (see Example 3).

In another embodiment of the present invention, when a neutralizing antibody that specifically binds to G-CSF and blocks its activity is administered to a mouse in which asthma is induced, the inflammatory reaction in the airways and lungs is alleviated, And the change in bone marrow hematopoiesis was normalized (see Example 4).

In another embodiment of the present invention, 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).

In another embodiment of the present invention, mice immunized with the aerosolized asthma were simultaneously administered with antibodies against IL-17 and TNF-alpha, respectively, to block their activity. As a result, 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).

Thus, 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.

In the present invention, the IL-17 may preferably be IL-17A, and 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).

In the present invention, the TNF-α inhibitor may be an anti-TNF-α antibody that specifically binds to the TNF-α protein (NCBI accession #. CAA68530), but is not limited thereto.

In the present invention, 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.

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. Such 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. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. 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. In the present invention, the term "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.

Specifically, 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. However, 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.

In another aspect of the present invention, 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.

(a) treating candidate substances in lung cells in vitro;

(b) measuring the expression or activity of interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-a) in the lung cells; And

(c) selecting a substance that reduces the expression or activity of IL-17 and TNF-? compared to the candidate substance-untreated group as a therapeutic substance for constitutive pulmonary inflammatory disease.

In the present invention, 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.

In another aspect of the present invention, 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.

The term " individual " as used herein 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.

In another aspect of the present invention, the present invention provides the use of the pharmaceutical composition for the prevention or treatment of a respiratory tract inflammatory disease.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Experimental animal

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.

1-2. In vitro  Cytokine stimulation

Mouse lung epithelial cells (MLE-12) 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). Next, the cultured cells (1 × 10 ⁵ ) were seeded in a 24-well plate and stimulated with 10 ng / ml mouse IL-17A (ebioscience) and / or 20 ng / ml mouse TNF-a (Biolegend). After 24 hours, the cell culture was recovered and the level of G-CSF protein was measured by ELISA.

1-3. Analysis of Asthma Animal Model and Treatment Effect

In order to prepare a homogenous asthmatic mouse model induced by LPS (lipopolysaccharide) / OVA (ovalbumin), 20 μl of LPS sterilized on days 0, 1, 2 and 7 and 10 μg LPS and 75 μg of OVA Sigma) were mixed and sensitized by nasal aspiration. Then, starting from day 14, 50 μg of OVA was repeatedly administered to 20 μl of sterile PBS twice a week for 4 weeks. All intranasal injections were carried out after anesthetizing mice by intraperitoneally administering 120 μl of a ketamine / xylazine solution.

On the other hand, 100 μg 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. For neutralization of IL-17 and TNF-alpha (BioXcell), 50 ug of anti-IL-17 and / or anti-TNF- or isotype antibodies were intraperitoneally injected intraperitoneally Lt; / RTI > Next, mice were sacrificed at 6, 12, 24 and 48 hours after the last OVA administration to perform cytokine and bone marrow analysis. In addition, BAL fluid was collected by tracheal catheterization, and mouse lungs were collected by perfusion and stored in 4% paraformaldehyde (PFA) for histological analysis .

1-4. Recombinant mouse G-CSF, IL-17 and TNF-alpha administration

The 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. In addition, 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 .

1-5. Cell separation

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. On the other hand, to collect bone marrow cells, leg bone (one tibia and one femur) was washed with RPMI1640 medium (Welgene) supplemented with 2% calf serum, and then debris was removed using a 40-μm strainer to obtain a single cell suspension Respectively. The BAL fluid was centrifuged, and the pellet was resuspended in 100 μl of PBS containing 2% FBS and the number of cells was measured.

1-6. Cytokine level measurement

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.

1-7. Analysis of myeloperoxidase (MPO) activity in bone marrow cells

The BAL fluid of 10 ㎕ at 37 ℃ with TMB solution of 80 ㎕ 88 mM H ₂ O ₂ and 110 of ㎕ and incubated for 5 minutes. After stopping the reaction by treating the 50 ㎕ of 1N H ₂ SO ₄ and the absorbance was measured at 450 nm.

1-8. Flow cytometry

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. Then, 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? (Clone RAM34), FcrRII / III-PE (CD16 / 32, clone 93), IL-7Ra-PerCP / cy5.5 (CD127, clone A7R34) MMP (LIN-SCA-1 + c- Kit + CD34- Respectively. For the neutrophils (TER119-CD11b + Gr-1 +), eosinophils (TER119-CD11bintsiglecF + Gr-1low), B cells (TER119-Gr-1-CD11b- B220 +) and alveolar macrophages (FSChighSSChighTER119-CD11blowsiglecFhighGr- Adult immune cells from bone marrow, lung, and BAL were identified as TER119-FITC, CD11b-PE / Cy7, B220-PB, siglecF-PE and Gr-1-APC. In addition, epithelial cells (TER119-CD45-CD31-EpCAM +) and endothelial cells (TER119-CD45-CD31 + EpCAM-) were identified as TER119-FITC, CD45-FITC, EpCAM- CD31-PE. Expression of cytokine receptors in lung epithelial cells was measured using IL-17RA-PE, IL-17RC-biotin, TNFR1-biotin, TNFR2-PE and streptavidin-APC.

Example 2. Confirmation of hematopoietic effect on circulating G-CSF by bone marrow

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.

As a result, as shown in FIG. 1B, LSK cells containing hematopoietic stem cells (HSC) and pluripotent progenitor cells (MPP) were cultured in the inflammation-induced mice as compared with the control mice As shown in FIG. 1C, granulocyte and monocyte progenitors (GMP) were increased in the progenitor cells, while megakaryocytes and erythrocyte progenitors (MEPs) were increased But rather 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 + .

Based on the above results, 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). As a result, as shown in FIG. 2A, it was surprisingly confirmed that only granulocyte colony stimulating factor (G-CSF) was present in a large amount in the serum, and that the serum concentration of G-CSF decreased with time.

Next, to investigate the role of G-CSF as a hematopoietic factor based on the above results, recombinant mouse G-CSF (rmG-CSF) was systemically administered to mice and the change in hematopoietic activity was analyzed. As a result, an increase in the HSPC population and an increase in the number of myeloid cells by administration of G-CSF was confirmed, similar to the above results, as shown in Figs. 2B to 2D. These results indicate that the change in hematopoietic activity in the bone marrow of asthmatic mice is induced by circulating G-CSF.

Example 3. Confirmation of Genetic and Functional Changes of HPSC by G-CSF

As reported in many studies, inflammatory mediators can determine the phylogeny of hematopoietic stem and progenitor cells (HSPCs) by modulating the genetic program associated with phylogeny. Therefore, in order to confirm the effect of G-CSF as a systemic determinant, the genetic and functional changes of HSPCs in the asthmatic mouse model of the present invention were analyzed.

As a result, as shown in FIG. 3A, the expression of myeloid-associated genes was increased in the CD150 + LSK cells of the mice induced asthma, while the lymphoid- Affected genes were slightly decreased. Similar to these results, 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.

Furthermore, in order to determine whether this genetic reprogramming reflects functional changes in the phylogenetic selection of HSPCs in vivo, the number of CD150 + LSK cells in the inflammatory mouse (LPS / OVA) compared to control mice (PBS / OVA) The potential for differentiation of the system was compared. As a result, HSPCs of inflammatory mice tend to progress to the myeloid cell line through the increase of the ratio of GMP / MEP in the spleen and the increase of the myeloid / lymphatic ratio in the spleen and peripheral blood as shown in FIGS. 3C and 3D . Thus, it has been confirmed that airway G-CSF induced by phagocytic inflammation circulates through the blood and induces genetic and functional changes of HSPC, thereby changing the hematopoietic action toward the myeloid cell lineage.

Example 4. Normalization of hematopoietic action by blocking G-CSF and consequent formation of airborne airways.

In order to further study the role of 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.

As a result, 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) In the case of one group, HSPCs increased, whereas G-CSF was neutralized, and the increase in the proportion of myeloid / lymphoid cells was normalized to a level similar to that of the normal control, . Also, observing the lung tissues in a manner compatible with the above results, 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) As shown in Fig. 4h, neutrophil infiltration and MPO activity of BAL fluid were decreased, and the number of neutrophils in lung tissue was decreased.

These results demonstrate the importance of G-CSF as a signal mediator between the lung and bone marrow and the importance of bone marrow in asthma.

Example 5. Generation of G-CSF by IL-17 and TNF-α and the Change of Bone Marrow Hematopoiesis

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. As a result, it was confirmed that 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.

Furthermore, in order to investigate the factors that induce G-CSF production in lung epithelial cells, inflammatory cytokines were measured in the lungs, and then the cytokines were individually or mixed in vitro to generate mouse lung epithelial cell line (MLE-12) And the level of G-CSF was measured. As a result, as shown in FIG. 5B, it was confirmed that G-CSF production was induced by co-treatment of IL-17A and TNF-a, rather than a single cytokine, as reported in previous studies. In addition, when the two cytokines were administered to the nasal passages of the mice, as shown in FIGS. 5C and 5D, 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.

These results demonstrate that the coexistence of IL-17 and TNF- [alpha] in pulmonary-induced lung-induced lungs stimulates lung epithelial cells to produce G-CSF, resulting in reprogramming of the hematopoiesis in the bone marrow.

Example 6. Normalization of hematopoietic action by IL-17 and TNF-a blockade, and thus, improvement of airway inflammation

In order to reaffirm the role of IL-17 / TNF- [alpha] in inducing G-CSF production and to identify the molecular mechanism for development of targeted therapies in asthma, 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. As a result, as shown in FIGS. 7B and 7F, when the two antibodies were administered simultaneously (α-IL-17 + α-TNF-α) 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.

In addition, when the antibodies against both cytokines were simultaneously treated as described above, the concentration of G-CSF was remarkably decreased in BAL fluid, lung tissue lysate and serum as shown in FIG. 8A, and FIG. 8B As shown in FIGS. 8A to 8D, it was confirmed that the phenomenon occurring in the bone marrow of the mice induced by the phosgenation-induced inflammation such as the increase of HSPC in the bone marrow and the unbalance of bone marrow / lymphatic development was normalized.

Further, the therapeutic effect of the above-mentioned 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 (Α-IL-17, α-TNF-α) showed limited therapeutic effects in terms of airway and lung inflammation and G-CSF induction.

The 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-α 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.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (14)

A pharmaceutical composition for the prevention or treatment of respiratory pulmonary inflammatory disease, comprising an interleukin-17 (IL-17) inhibitor and a tumor necrosis factor-alpha (TNF-a) inhibitor as an active ingredient.
The method according to claim 1,
The common pulmonary inflammatory diseases include neutrophilic asthma, neutrophilic chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI) ), And acute respiratory distress syndrome (ARDS). ≪ RTI ID = 0.0 > 21. < / RTI >
The method according to claim 1,
Wherein the IL-17 inhibitor is an anti-IL-17 antibody that specifically binds to the IL-17 protein.
The method according to claim 1,
Wherein the TNF-a inhibitor is an anti-TNF-? Antibody that specifically binds to the TNF-alpha protein.
The method according to claim 1,
Wherein the IL-17 inhibitor and the TNF-a inhibitor inhibit the expression of a granulocyte colony-stimulating factor (G-CSF).
The method according to claim 1,
Wherein said pharmaceutical composition further comprises a G-CSF inhibitor.
The method according to claim 6,
Wherein the G-CSF inhibitor is an anti-G-CSF antibody that specifically binds to the G-CSF protein.
The method according to claim 1,
Wherein said pharmaceutical composition normalizes the balance change of hematopoietic action in bone marrow by G-CSF.
The method according to claim 1,
Wherein said pharmaceutical composition reduces neutrophil infiltration of bronchoalveolar lavage fluid and lung tissue.
The method according to claim 1,
Wherein the pharmaceutical composition reduces myeloperoxidase (MPO) activity.
The method according to claim 1,
Wherein said pharmaceutical composition reduces airway hypersensitivity.
A method of screening for a therapeutic agent for the respiratory tract inflammatory disease of the respiratory tract comprising the steps of:
(a) treating candidate substances in lung cells in vitro ;
(b) measuring the expression or activity of interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-a) in the lung cells; And
(c) selecting a substance that reduces the expression or activity of IL-17 and TNF-? compared to the candidate substance-untreated group as a therapeutic substance for constitutive pulmonary inflammatory disease.
13. The method of claim 12,
The above-mentioned pulmonary inflammatory diseases include neutrophilic asthma or neutrophilic chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI) ), And acute respiratory distress syndrome (ARDS). ≪ Desc / Clms Page number 13 >
13. The method of claim 12,
Wherein the therapeutic agent inhibits the expression of granulocyte colony-stimulating factor (G-CSF).

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