KR20190068387A - Screening Methods for the Therapeutic Candidate in Asthma using Cytokine and Claudins - Google Patents

Abstract

The present invention provides a method for screening candidates for asthma therapeutic agents by measuring the protein expression level of cytokine-associated tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-5 (IL-5), interleukin-13 (IL-13), interleukin-17 (IL-17), Claudin 3, Claudin 4, or Claudin 7.

Description

(Screening Methods for the Therapeutic Candidate in Asthma using Cytokine and Claudins) using Cytokine and Claudin < RTI ID = 0.0 >

The present invention provides a screening method for asthma therapeutic candidate substances.

Asthma is a lung disease characterized by chronic airway inflammation and airway hyperresponsiveness. It is caused by air pollutants, dust, allergens, and the like. In particular, asthma is an inflammatory respiratory disease causing dyspnea, and about 3 million people in Korea have asthma. Asthma is a disease characterized by colorless asthma, dyspnea, sneezing, severe cyanosis due to lack of oxygen, and chest pain. Recently, it has been rapidly increasing due to exposure to air pollution and various chemical substances and westernization of diet. Especially, And it is increasing with the change of eating habit and westernization. However, in asthmatic patients, the cause and mechanism of the disease are not clear. In this pathogenesis, Th2 (T helper type 2) type of immune response is exaggerated, resulting in increased secretion of interleukin-4, 5, and 13 (Liu et al., 2006; Williams et al. 2012), many inflammatory cells including eosinophils migrate and infiltrate into lung tissue in association with this reaction (Zhou et al., 2011). In addition, inflammatory cells release a variety of proinflammatory and chemoattractant factors, further exacerbating the inflammatory response, increasing mucus secretion in intratracheal goblet cells, and causing airway hyperresponsiveness (Chibana et al., 2008). Because of this series of reactions, patients with asthma exhibit clinical symptoms such as dyspnea, cyanosis, and chest pain.

Currently, steroids, bronchodilators, and anti-inflammatory drugs are mainly used in the treatment of asthma. Steroids and antiinflammatory agents are used for the treatment of asthma through immunity and inhibition of inflammatory responses, and bronchodilators are used to counteract clinical manifestations such as dyspnea. The most widely used inhaled corticosteroids have excellent therapeutic effect, but when used for a long period of time, adrenal suppression, bone density reduction, growth disorder, eye and skin complications, collagen synthesis And the like. In addition, persistent beta-2 agonists, such as salmeterol and formeterol, have been shown to be protective against asthma attacks, . And long-term use has been found to have side effects, limited use as a treatment for asthma.

Since the asthma treatment drugs developed so far have various side effects, it is urgent to find an asthma treatment agent without side effects.

Korean Patent Publication No. 10-2017-0057666

(TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin-13 (IL- 13) or interleukin- The present invention provides a method of screening for a therapeutic agent for asthma that controls the expression level of the protein of the present invention.

In order to achieve the above object, the present invention provides, as one embodiment, a " method for screening asthma therapeutic agent ".

The method for screening a therapeutic agent for asthma includes the steps of treating an asthma therapeutic candidate substance to an isolated cell, a separated tissue, or an animal other than a human; Interferon gamma (IFN-?), Interleukin-5 (IL-5), interleukin-13 (IL-13), or interleukin-17 (IL- 17). ≪ / RTI >

The term " asthma " in the present invention is a comprehensive disease name collectively referred to as various diseases characterized by inflammation of the airways leading to organs, bronchi, bronchioles, and alveoli, , Clin Exp Allergy 2005; 35: 1254-62). In particular, asthma is a condition in which the bronchus in the lung is very sensitive. Sometimes the bronchus narrows and the breathing, choking, and breathing sounds can be heard, causing severe coughing. Allergy caused by bronchial allergy inflammation Disease. The most common symptoms of asthma include dyspnea, cough, wheezing (wheezy breathing), a symptom reliever (bronchodilator) that alleviates the narrowed bronchus in a short time, or a disease modifier that prevents asthma attacks by inhibiting bronchial allergic inflammation Anti-inflammatory agents, and leukotriene modulators). In the present invention, the asthma may be, but not limited to, bronchial asthma, allergic asthma, atopic asthma, non-atopic asthma, exercise induced asthma, aspirin asthma, cardiogenic asthma or alveolar asthma.

The term "asthma therapeutic candidate substance " of the present invention may be an individual nucleic acid, protein, other extract or natural product, or compound, which is presumed to have the possibility of asthma treatment according to a conventional selection method or randomly selected .

The isolated cells may be primary cells isolated from the human body and include human normal bronchial epithelial cells (NHBE), human fetal lung fibroblast cells (MRC-5 ), Adenocarcinomic human alveolar basal epithelial cells (A549), human umbilical vein endothelial cells (EA.hy926), or Ad12-SV40 2B-transformed bronchial epithelial cells (Bronchial Epithelial cells transformed with Ad12-SV40 2B, BEAS-2B) may also be used.

The term "control group" of the present invention means a separate cell, a separate tissue or human being in a side-by-side relationship with the group treated with the candidate substance, Means an animal other than animals.

The " method for screening asthma therapeutic agents " of the present invention is designed in such a manner that the therapeutic agent candidates are compared with control cells that have not been treated, by confirming the expression of cytokines and Claudins in asthma.

In the present invention, the cytokine is selected from the group consisting of tumor necrosis factor alpha (TNF-a), interferon gamma (IFN-?), Interleukin-5 (IL-5), interleukin- 17).

In one embodiment of the present invention, an asthma animal model was prepared by sensitizing mice with ovalbumin (OVA). The OVA-sensitized mice showed increased airway hyperresponsiveness (AHR) Bronchoalveolar lavage fluid (BALF) increased the number of total inflammatory cells.

In addition, tumor necrosis factor alpha (TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin-13 (IL- 17 (IL-17) expression was confirmed by Western blot and immunohistochemical staining. In addition, when the mouse was treated with the candidate substance Hederacoside C, tumor necrosis factor alpha (TNF-a), interferon gamma (IFN-y), interleukin-5 (IL- IL-13) and interleukin-17 (IL-17). Thus, the expression of the TNF-a, interferon gamma, interleukin-5, interleukin-13, Of asthma in children with asthma.

(TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-6) in isolated cells, isolated tissues or animals other than humans in the absence of candidate substances capable of preventing or treating asthma. (IL-13) or interleukin-17 (IL-17) in the presence of the candidate substance and measuring the level of the protein of the tumor necrosis factor alpha (TNF-?), Interferon gamma IFN-?), Interleukin-5 (IL-5), interleukin-13 (IL-13), or interleukin-17 (IL-17) protein in the presence of the candidate substance (TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin-13 (IL-13), or interleukin-17 A substance which is lower than the level under the absence of the candidate substance can be predicted by an agent for preventing or treating asthma.

(IL-5), interleukin-13 (IL-13) or interleukin-17 (IL-13) -17) expression level of the protein is lower than that of the control group not treated with the candidate substance, the candidate substance may be selected as an asthma treatment agent.

In addition, in one embodiment of the present invention, when OVA sensitization was performed on an asthma model mouse, the expression of Claudin 3 was decreased and the expression of Claudin 4 and Claudin 7 was increased. On the other hand, when Paeoniflorin or Hederacoside C was treated with the candidate substance, expression of Claudin 3 was increased and expression of Claudin 4 and Claudin 7 was decreased.

Therefore, it was found that a substance that increases the expression of Claudin 3 or reduces the expression of Claudin 4 or Claudin 7 can be used as a therapeutic agent for asthma.

Accordingly, the level of the protein of Claudin 3 in isolated cells, isolated tissues or animals other than humans is measured in the absence of candidate substances capable of preventing or treating asthma, and also the level of Claudin 3 in the presence of the candidate substance The level of the protein is measured and compared with each other, and then a substance which increases the level of the protein of Claudin 3 when the candidate substance is present in the absence of the candidate substance is predicted as a prophylactic or therapeutic agent for asthma.

In addition, the level of the protein of Claudin 4 or 7 in an isolated cell, isolated tissue or an animal other than human is measured in the absence of a candidate substance capable of preventing or treating asthma, and the level of Claudin 4 Or 7, and comparing the two with each other, the substance that reduces the level of the protein of Claudin 4 or 7 when the candidate substance is present to a level lower than that of the candidate substance in the absence of the candidate substance is used as a prophylactic or therapeutic agent for asthma .

Accordingly, in the screening method of the present invention, the expression level of the protein of Claudin 3 is further measured in the candidate substance treatment group, and the expression level of the protein of Claudin 3 is measured when the expression level of the protein of Claudin 3 is higher than that of the control substance And selecting the candidate substance as the therapeutic agent for asthma.

In addition, the screening method of the present invention further comprises the step of measuring the expression level of the protein of Claudin 4 or 7 in the candidate substance treatment group and the level of expression of the protein of Claudin 4 or 7 measured in the candidate substance treatment group And selecting the candidate substance as the therapeutic agent for asthma if it is decreased.

The term "Claudin 3" of the present invention is also known as CLDN3, and the specific base sequence and protein information of the gene encoding Claudin 3 is known from NCBI (Genebank: NM_009902.4 or NM_001306.3).

The term "Claudin 4" of the present invention is also known as CLDN4, and the specific base sequence and protein information of the gene encoding Claudin 4 is known from NCBI (Genebank: NM_001305.4 or NM_009903.2).

The term "Claudin 7" of the present invention is also known as CLDN7, and the specific base sequence and protein information of the gene encoding Claudin 7 are known in NCBI (Genebank: NM_001193619.1. Or NM_016887.6.).

The term "measurement of protein expression levels" as used herein refers to the determination of the expression levels of TNF-a, interferon gamma (IFN-gamma), and IFN-gamma in the absence of candidate substances and in separate cells, Expression of proteins using antibodies specifically binding to interleukin-5 (IL-5), interleukin-13 (IL-13), interleukin-17 (IL-17), Claudin 3, Claudin 4 or Claudin 7 It is the process of confirming the degree.

Specifically, the protein expression level of the present invention can be measured and compared using an antibody that specifically binds to the protein. The antibody and the protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting the antibody-antibody complex is used.

The term "antibody" as used herein refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, the antibody may be an antibody that specifically binds to the TNF-a, interferon gamma (IFN-y), interleukin-5 (IL- 5), interleukin- -17), Claudin 3, Claudin 4 or Claudin 7, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. The production of the antibody can be easily carried out using techniques well known in the art.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

As used herein, the term "antigen-antibody complex" refers to a conjugate of a corresponding protein antigen in a biological sample and an antibody recognizing it. The detection of the antigen-antibody complex can be detected using methods such as those known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorbance, chemical and other methods.

Since the information on the protein is known by Genebank et al., A person skilled in the art can design an antibody specifically binding to the protein based on the above sequence.

As the protein level measurement or comparative assay, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Immunohistochemistry analysis, immunocytochemistry analysis, complement fixation assay, two-dimensional electrophoresis, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, Analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting and enzyme linked immunosorbent assay (ELISA) But is not limited to.

In one embodiment of the invention, the tumor necrosis factor alpha (TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin- -17), immunohistochemistry and western blot were used to measure and compare the expression levels of Claudin 3, Claudin 4 or Claudin 7 proteins themselves.

As described above, the present invention provides a screening method for asthma treatment agent by measuring and comparing the level of protein expression that is changed as compared with a control group in which the candidate substance is not treated, by treating the asthma treatment agent candidate substance.

1 shows an experimental design in which a candidate agent for treating a therapeutic agent is administered to a mouse induced to induce airway hyperresponsiveness by induction with ovalbumin (OVA) in a mouse.
FIG. 2A shows airway hyperresponsiveness (AHR) of a mouse that has been sensitized with OVA to induce airway hyperresponsiveness in a mouse and a mouse that has been treated with asthma treatment candidate substance.
FIG. 2B shows the number of inflammatory cells increased in Bronchoalveolar lavage fluid (BALF) of mouse treated with OVA-sensitized mouse and asthma therapeutic candidate.
FIG. 3 is a graph showing the results of the Western blot analysis of asthma-related cells in asthma using OVA-stimulated asthmatic mice and lung tissues of mice treated with asthma treatment candidate Paeoniflorin and Hederacoside C (TNF-α), interferon gamma (IFN_γ), interleukin-5 (IL-5), interleukin-13 (IL-13) and interleukin-17 And standardized with beta-actin. * P <0.05 compared with the control group, and # P <0.05 compared to the OVA stimulated group.
FIG. 4A shows the results of Western blot analysis using Claudin 3, Claudin 4, and Claudin 7 using asthmatic mice stimulated with OVA, and lung tissues of mice treated with Paeoniflorin and Hederacoside C, (Claudin 7), and standardized with beta-actin.
FIG. 4B shows the expression of Claudin 3, Claudin 4 and Claudin 7 by immunohistochemical staining using OVA-stimulated asthmatic mice and lung tissues of mice treated with Paeoniflorin and Hederacoside C, which are candidates for asthma treatment . * P <0.05 compared with the control group, # P <0.05 compared with the OVA stimulated group

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

A. Experimental Method

Example 1 Preparation of Asthma Animal Model

Ovalbumin (OVA) was intraperitoneally injected into female female BALB / c mice at 6 weeks of age on days 1 and 14, and mice were intravenously injected with OVA intranasally (aerosol) at 21 to 23 days, (OVA group).

An animal model (OVA + Paeoniflorin group) was also prepared by orally administering 50 mg / kg of Paeoniflorin dissolved in DPBS to the OVA group mice continuously for 21 days from 21 days. An animal model was also prepared (OVA + Hederacoside C group) by orally administering 50 mg / kg Hederacoside C dissolved in DPBS to the OVA group mice continuously for 21 days from 21 days. Control mice were given saline (sham group).

Airway hyperreactivity (AHR) was assessed after administering 0, 5, 20, or 100 mg / ml of methacholine (Sigma-Aldrich) On the following day, anesthetized with 2.5 mg / kg tiletamine and xylazine (Zolethyl and lumpum; Bayer Korea Co., Seoul, Korea), and bronchoalveolar lavage fluid (BALF) Were evaluated.

Example 2 Evaluation of lung tissue

The paraffin of the mouse lung slice was removed and rehydrated with an ethanol series. The sections were stained with hematoxylin and eosin solutions.

&Lt; Example 3 > Western blot

Mouse lung tissue was suspended in distilled water in a solution containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5 mM ethylenediaminetetraacetic acid Methylsulfonyl fluoride (PMSF), centrifuged at 20,000 g for 30 minutes at 4 ° C, and then the soluble material was collected. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with Tris buffered saline (TBS) containing 0.1% Tween-20 containing 5% bovine serum albumin (BSA) for 2 hours at 21 ° C and anti-tumor necrosis factor-alpha (anti-IL-5), anti-IL-13, anti-interleukin-17 -IL-17), anti-Claudin 3, anti-Claudin 4 or anti-claudin 7 antibody (4 ° C overnight) and then incubated with HRP (horseradish peroxidase) -binding secondary antibody. WEST-ZOL and Western blot detection system (iNtRon, SungNam, Korea). Relative protein levels were determined by quantitative concentration method and data were normalized to beta-actin (Sigma-Aldrich).

&Lt; Example 4 > Statistical analysis

Data were expressed as mean ± standard error (SEM) and Mann-Whitney U test was used to determine significant differences between the two groups. p <0.05 was considered to mean statistical significance.

B. Experimental Results

Experimental Example 1: Increased AHR and inflammatory cells in an asthmatic animal model after treatment with a candidate substance

FIG. 1 shows an experimental design in which a candidate agent for treating a therapeutic agent is administered to a mouse that induces sensitization with OVA in a mouse to induce airway hyperresponsiveness.

FIG. 2A shows airway hyperresponsiveness (AHR) of mouse treated with asthma treatment agent and a mouse induced to sensitize with OVA to induce airway hyperresponsiveness. FIG. 2B shows the number of inflammatory cells increased in bronchoalveolar lavage fluid (BALF) of mice sensitized with OVA and asthma treatment candidate candidate mice.

As shown in Fig. 2A, mice sensitized with OVA showed increased airway hyperresponsiveness than those treated with saline. On the other hand, mice treated with Hederacoside C and Paeoniflorin as candidates for asthma treatment with OVA-sensitized mice showed a marked decrease in airway hyperresponsiveness compared with OVA-sensitized mice

As shown in FIG. 2B, OVA-sensitized mice showed an increase in total inflammatory cells of bronchoalveolar lavage fluid compared to sham mice, and the numbers of macrophages, eosinophils and neutrophils were significantly increased.

On the other hand, in mice treated with Hederacoside C or Paeoniflorin, the total inflammatory cells of the alveolar lavage fluid decreased as compared with mice sensitized with OVA, and the numbers of macrophages, eosinophils and neutrophils were significantly decreased.

EXPERIMENTAL EXAMPLE 2 Expression Pattern of Cytokines in Asthma Models Treated with Asthma Therapeutics

Next, the expression of cytokines known to be involved in asthma was analyzed using Western blot. FIG. 3 is a graph showing the expression of TNF-.alpha. (TNF-.alpha.), Which is known to be involved in asthma by western blotting using lung proteins of OVA-stimulated asthma mice and mice treated with Paeoniflorin and Hederacoside C, Protein levels of interferon gamma (IFN-γ), interleukin-5 (IL-5), interleukin-13 (IL-13) and interleukin-17 (IL-17).

(TNF-a), interferon gamma (IFN-?), Interleukin-5 (IL-5), interleukin-13 (IL-13), and interleukin-13 in asthmatic mice induced by OVA, And the expression of interleukin-17 (IL-17) was increased. The expression of these cytokines was not decreased when Paeoniflorin was treated with asthma as a candidate agent for asthma treatment in mice induced by OVA. On the other hand, the group treated with the candidate agent for asthma treatment Hederacoside C confirmed the decrease in the expression of these cytokines.

In this way, mice that were stimulated with OVA by Hederacoside C treatment decreased the number of airway hyperresponsiveness and inflammatory cells, and TNF-α, interferon gamma (IFN-γ), interleukin-5 (IL- (TNF-α), interferon gamma (IFN-γ), and interleukin-17 (IL-17) (IL-5), interleukin-13 (IL-13), or interleukin-17 (IL-17).

EXPERIMENTAL EXAMPLE 3 Expression Pattern of Claudins in Asthma Model Treated with Asthma Therapeutic Candidate

Expression of Claudins protein in mice and sham mice was confirmed by treatment with OVA-sensitized mice and further asthma treatment candidates. Expression levels of Claudin 3, Claudin 4, and Claudin 7 in OVA, Paeoniflorin, OVA, Hederacoside, OVA, and saline controls were analyzed.

FIG. 3 shows the protein levels of Claudin 3, Claudin 4, and Claudin 7 in OVA and Paeoniflorin treated groups, OVA and Hederacoside treated groups, OVA treated groups, and saline control lung tissues by western blotting and standardized with beta-actin

FIG. 4 shows the expression of Claudin 3, Claudin 4, and Claudin 7 in OVA and Paeoniflorin treated groups, OVA and Hederacoside treated groups, OVA treated groups, and saline control lung tissues by Western blot and immunohistochemical staining.

 As shown in FIGS. 3 and 4, expression of Claudin 3 was decreased and expression of Claudin 4 and Claudin 7 was increased in the lung tissue of OAV-only group. On the other hand, the expression of Claudin 3 and Claudin 4 and Claudin 7 were decreased in lung tissue of Paeoniflorin or Hederacoside C treated with OAV.

As shown above, in the mice stimulated with OAV by treatment with Paeoniflorin or Hederacoside C, the number of airway hyperresponsiveness and inflammatory cells were decreased, and the expression of Claudin 3 was increased and the expression of Claudin 4 and Claudin 7 was decreased. Of Claudin 3 or of Claudin 4 or of Claudin 7 in asthma can be developed as a therapeutic agent for asthma.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (6)

Treating the asthma therapeutic candidate substance to an isolated cell, an isolated tissue, or an animal other than a human; And
Interferon gamma (IFN-?), Interleukin-5 (IL-5), interleukin-13 (IL-13), or interleukin-17 (IL-17 ) &Lt; / RTI &gt; of the protein of the present invention. The method according to claim 1,
(TNF-a), interferon gamma (IFN-y), interleukin-5 (IL-5), interleukin-13 (IL-13), or interleukin- The method further comprises selecting the candidate substance as an asthma treatment agent when the expression level of the candidate substance is lower than that of the control group not treated with the candidate substance. The method according to claim 1,
Measuring an expression level of the protein of Claudin 3 in the candidate substance treatment group; And
Further comprising the step of selecting the candidate substance as an asthma treatment agent when the measured expression level of the protein of Claudin 3 is higher than that of the control substance not treated with the candidate substance. The method according to claim 1,
Measuring an expression level of a protein of Claudin 4 or Claudin 7 in the candidate substance treatment group; And
Wherein the candidate substance is selected as an asthma treatment agent when the expression level of the measured protein of Claudin 4 or Claudin 7 is lower than that of the control substance not treated with the candidate substance, Screening method. The method according to claim 1,
The cells were cultured in the presence of normal human bronchial epithelial cells (NHBE), human fetal lung fibroblast cells (MRC-5), adenocarcinomic human alveolar basal epithelial cells (A549 ), A human endothelial cell line (EA.hy926), or a transformed bronchial epithelial cell line (BEAS-2B). The method according to claim 1,
The protein expression level can be measured by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulfate Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) Immunocytochemistry, immunocytochemistry, complement fixation, two-dimensional electrophoresis, liquid chromatography, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, (LC-MS), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assays Wherein the asthma treatment agent is administered orally or parenterally.

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