KR20210151400A - Composition for monitoring the condition of asthma control using EDN - Google Patents

Abstract

The present invention relates to a composition and the like for monitoring asthma control status. The present invention confirmed that the level of eosinophil-derived neurotoxin (EDN) was significantly increased in uncontrolled asthma patients compared with normal groups and controlled asthma patients, and at the same time that in comparison with blood eosinophil counts, EDN exhibited high accuracy as a marker for assessing asthma control status. Therefore, it is expected that EDN can be effectively used as a biomarker for the assessment of asthma control status.

Description

Composition for monitoring the condition of asthma control using EDN

The present invention relates to a composition and the like for monitoring asthma control status.

Asthma is a disease characterized by bronchial hyperresponsiveness to various stimuli, causing chronic airway inflammation, wheezing, dyspnea, Clinical symptoms such as cough appear, which can be reversibly improved either naturally or by treatment.

Severe eosinophilic asthma was reported as a characteristic feature of severe exacerbation, accompanying sinusitis, peripheral airway invasion, airway remodeling, and decreased lung function compared to the non-eosinophilic type. On the other hand, severe non-eosinophilic asthma is mainly characterized by neutrophil inflammation and severe asthma symptoms. Classification and approaches based on the eosinophilic inflammatory phenotype are expected to aid in the selection of asthma treatments.

On the other hand, the goal of asthma control is to keep asthma well controlled. According to the latest standards of the Global Initiative for Asthma (GINA), asthma control status is evaluated by combining the frequency of symptom exacerbation during the day or night and the presence or absence of restrictions in daily life. Other guidelines recommend including objective indicators reflecting airway inflammatory status in addition to these clinical symptoms. Although fractional exhaled nitric oxide (FeNO) and the number of eosinophils in induced sputum among objective indicators to determine the inflammatory state of the airways, it is not easy to perform an expiratory nitrogen concentration or induced sputum test, including lung function test, at every outpatient visit. not.

Asthma control has two areas: 1) symptom control, and 2) risk of future complications, which should always be evaluated. In addition, lung function is important to assess future risk and should be measured periodically at the start of treatment, 3-6 months after initiation of treatment, and thereafter for continued risk assessment.

Many previous studies have noted that there is a discrepancy between the patient's and the health care provider's assessment of the patient's level of control. In other words, it does not mean that patients overestimate their degree of control or underestimate the severity of their disease, but rather understand and use the meaning of asthma control differently from medical professionals. Therefore, when evaluating the degree of asthma control with the patient, the implications of this must be explained.

Journal of the Korean Society of Internal Medicine: Vol. 78, No. 6, 2010

The present inventors have completed the present invention as a result of efforts to develop a marker for evaluating asthma control status.

Accordingly, it is an object of the present invention to provide a composition for monitoring asthma control status, comprising as an active ingredient an agent for measuring EDN (Eosinophil-derived neurotoxin) protein or its mRNA expression level.

Another object of the present invention is to provide a method for monitoring asthma control status, comprising measuring the expression level of EDN protein or mRNA thereof in a sample of a subject.

Another object of the present invention is to provide a kit for monitoring asthma control status, which includes an agent for measuring the expression level of EDN protein or its mRNA as an active ingredient.

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

In order to solve the above problems, the present invention provides a composition for monitoring asthma control state comprising an EDN (Eosinophil-derived neurotoxin) protein or an agent for measuring the mRNA expression level thereof as an active ingredient.

In one embodiment of the present invention, the agent for measuring the mRNA expression level may be a probe or primer that specifically binds to the EDN mRNA, but is not limited thereto.

In another embodiment of the present invention, the agent for measuring the expression level of the protein may be an antibody specific for the EDN protein, but is not limited thereto.

In another embodiment of the present invention, the EDN protein may include an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the EDN mRNA may include a nucleotide sequence consisting of one sequence selected from the group consisting of SEQ ID NOs: 3 to 6, but is not limited thereto.

In another embodiment of the present invention, the asthma may be one or more selected from the group consisting of severe asthma and non-severe asthma, but is not limited thereto.

In addition, the present invention provides a method for monitoring asthma control, comprising measuring the expression level of EDN protein or mRNA thereof in a sample of a subject.

In one embodiment of the present invention, the sample may be blood, plasma, or serum, but is not limited thereto.

In another embodiment of the present invention, the method can evaluate that asthma is not well controlled when the expression level of EDN protein or its mRNA is increased compared to the control group, but is not limited thereto.

In another embodiment of the present invention, when the concentration of EDN in the sample is 48.5 ng/ml or more, it may be evaluated as uncontrolled asthma, but is not limited thereto.

In another embodiment of the present invention, the expression level of the EDN protein or its mRNA may not be correlated with one or more selected from the group consisting of sputum eosinophils and sputum neutrophils, but is not limited thereto.

In another embodiment of the present invention, the monitoring method may further include diagnosing the number of eosinophils in the blood, but is not limited thereto.

In addition, the present invention provides a kit for monitoring asthma control status comprising an agent for measuring the expression level of EDN protein or its mRNA as an active ingredient.

In addition, the present invention provides an information providing method for predicting asthma control status, comprising measuring the expression level of EDN protein or its mRNA in a sample of a subject.

In addition, the present invention provides a method for evaluating asthma control status, comprising measuring the expression level of EDN protein or its mRNA in a sample of a subject.

In addition, the present invention provides a use for evaluating asthma control status of a composition comprising an EDN protein or an agent for measuring the mRNA expression level thereof as an active ingredient.

In addition, the present invention provides a method for predicting asthma control status, comprising measuring the expression level of EDN protein or its mRNA in a sample from an asthma patient.

In addition, the present invention provides a use for predicting asthma control status of a composition comprising an EDN protein or an agent for measuring the mRNA expression level thereof as an active ingredient.

The present inventors not only confirmed that EDN showed significantly increased levels in patients with uncontrolled asthma compared to the normal group and patients with controlled asthma, but also compared EDN with blood eosinophil levels, a marker for evaluating asthma control status As a result, it was confirmed that it has excellent accuracy.

Therefore, it is expected that EDN can be used as a useful biomarker for the evaluation of asthma control status.

1 shows the results of comparison of serum EDN levels in a healthy group, a controlled asthma group, and an uncontrolled asthma group. Mean serum EDN levels were higher in uncontrolled asthma (103.2 ± 60.2) than in controlled asthma (60.8 ± 49.7) and healthy controls (49.6 ± 28.3) groups.
Figure 2 shows the ROC curves of serum EDN and blood eosinophil count for predicting uncontrolled asthmatic status in all asthmatic patients. Comparison with blood eosinophil counts using the method derived by Hanley and McNeil (Hanley JA, McNeil BJ. A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 1983;148:839-43) One result showed that serum EDN levels were a better predictor of uncontrolled (p = 0.024).
3 shows the correlation between serum EDN and blood eosinophil count in all asthma patients.

The present invention relates to a composition for monitoring asthma control status, comprising an agent for measuring EDN (Eosinophil-derived neurotoxin) protein or its mRNA expression level as an active ingredient.

Hereinafter, the present invention will be described in detail.

As used herein, the term "protein" is used interchangeably with 'polypeptide' or 'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.

As used herein, "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included. In general, DNA consists of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA consists of uracil (U) instead of thymine (Uracil, U). has a In a double-stranded nucleic acid, A forms a hydrogen bond with T or U, and C forms a hydrogen bond with G bases, and the relationship between these bases is called 'complementary'.

On the other hand, 'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the base sequence of a specific gene to the ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through the transcription process.

As used herein, “Eosinophil-derived neurotoxin (EDN)” refers to a neurotoxin derived from eosinophils as a product of eosinophil degranulation. It can reduce the activity of ssRNA (single strand RNA) virus through enzymatic activity and plays a role in attracting immune cells.

In the present specification, the EDN protein may be derived from a mammal, preferably from a human. Most preferably, the EDN protein in the present invention is characterized in that it comprises the amino acid sequence of human EDN (AAA50284.1, AAG31585.1) represented by SEQ ID NO: 1 or SEQ ID NO: 2 (NCBI Genebank accession number in parentheses) .

The EDN mRNA is preferably human EDN mRNA ( M28129.1, M30510.1, X55988.1, X55987.1 ) represented by one sequence selected from the group consisting of SEQ ID NOs: 3 to 6 comprising a nucleotide sequence (NCBI Genebankaccession number in parentheses). Characteristics such as the coding region (exon) of the above-described human EDN mRNA transcript variants are confirmed from sequence information obtained by searching the NCBI database with the Genebank accession number listed in parentheses.

As used herein, the term "complementary" means that a targeting moiety in a nucleic acid molecule is selectively directed to a target (eg, an EDN gene) under certain hybridization or annealing conditions, specifically physiological conditions (in a cell). It means that it is sufficiently complementary to hybridize, it may have one or more mismatched nucleotide sequences, and has a meaning encompassing both substantially complementary and perfectly complementary. , more specifically meaning completely complementary.

As used herein, the term “detection” refers to measuring and confirming the presence (expression) of a target substance (marker protein, EDN in the present invention), or measuring a change in the presence level (expression level) of a target substance. And it is meant to include all of the confirmation. In the same context, measuring the expression level of the protein in the present invention means measuring the expression level (ie, measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. A specific protein level comparison method for each method is well known in the art. Therefore, the detection of the EDN protein is meant to include detecting the presence or absence of the EDN protein, or confirming the increase (up-regulation) or decrease (down-regulation) of the protein expression level.

As used herein, the term “diagnosis” refers to determining the susceptibility of a subject to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or having a specific disease or disorder. It is a concept that includes both determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information on treatment efficacy). Preferably, the diagnosis in the present invention may be to determine the presence or onset of asthma by measuring the expression level of EDN.

As used herein, the term “asthma control state” may be used in combination with “asthma control level” herein, and refers to the degree to which symptoms of asthma are observed in a patient or to a degree that has been reduced or eliminated by treatment. The level of asthma control is determined by the interaction of a patient's genetic background, disease course, treatment, environment, and psychosocial factors. As used herein, the asthma control level includes two areas: 1) symptom control, and 2) risk of future complications. Herein, the asthma control level was assessed by performing acute exacerbation, airway restriction assessment, adverse event assessment, and lung function tests.

As used herein, the definition of uncontrolled asthma may include, but is not limited to, one or more of the following conditions:

1) Low symptom control: Partial control status according to the Korean asthma treatment guidelines, or less than 20 points in the asthma control test

2) Frequent asthma exacerbations: 2 or more exacerbations of asthma that require systemic steroids for 3 days or more

3) Severe exacerbation: When asthma exacerbation requires hospitalization or mechanical ventilation

4) Airflow restriction: FEV1 < 80% after using bronchodilators.

As used herein, the uncontrolled asthma may include partially uncontrolled asthma and uncontrolled asthma. In one embodiment of the present invention, an experiment was conducted including partially controlled asthma and uncontrolled asthma according to the following GINA guidelines in the uncontrolled asthma group (see Example 1.2).

In the present specification, the asthma control status may be classified based on the table below, but is not limited thereto.

In the present specification, the asthma control status may be an asthma control status with respect to an asthma treatment agent, but is not limited thereto.

In the present specification, the asthma therapeutic agent is, for example, inhaled corticosteroids, formoterol, salmeterol, beta-2 agonists such as tolobeterol, xanthine-based drugs such as theophylline, parasympathetic nerve Blockers or leukotriene modulators such as montelukast, pranlukast, and zafirlukast, systemic steroids, etc., but are not limited thereto.

In the present specification, the asthma treatment agent may be at least one selected from the group consisting of inhaled corticosteroids (ICS), inhaled corticosteroid-long acting b agonist (ICS-LABA), and long acting muscarinic antagonist (ICS-LAMA), but is limited thereto. it is not going to be

In one embodiment of the present invention, it was confirmed that the asthma control status and EDN showed a significant correlation regardless of the asthma therapeutic agent and the dose of the therapeutic agent (see Table 1).

As used herein, the term “monitoring” may refer to monitoring the course of a disease for an individual who has been diagnosed with, is likely to be diagnosed with, or has been diagnosed with asthma. For example, it may monitor the worsening or improvement of the progression of asthma, and specifically monitor whether the asthma is controlled, partially controlled, or not controlled. The monitoring may be performed by observing the individual's symptoms, the number and dose of drug use, nighttime symptoms, sleep disturbance, aggravation, emergency room visits, side effects, and the like, but is not limited thereto.

In the present specification, if the concentration of EDN in the sample is 48.5 ng/ml or more, it may be evaluated as uncontrolled asthma, but is not limited thereto.

In the present specification, when the concentration of EDN in the sample is less than 48.5 ng/ml, it can be evaluated as controlled asthma, but is not limited thereto.

In the present specification, the meaning of 'increased expression (or high expression)' of a protein is that unexpressed is expressed (ie, undetected is detected) or is relatively overexpressed (ie, detected amount) means more). The meaning of opposite terms can be understood by those skilled in the art to have opposite meanings according to the above definition.

In the present specification, the agent for measuring the mRNA expression level may be a probe or primer that specifically binds to the EDN mRNA, but is not limited thereto.

In the present specification, the agent for measuring the expression level of the protein may be an antibody specific for the EDN protein, but is not limited thereto.

In the present specification, the EDN protein may include an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

In the present specification, the EDN mRNA may include a nucleotide sequence consisting of one sequence selected from the group consisting of SEQ ID NOs: 3 to 6, but is not limited thereto.

The 'primer' is a short single-stranded oligonucleotide serving as a starting point of DNA synthesis. The primer specifically binds to a polynucleotide as a template under suitable buffer and temperature conditions, and DNA polymerase adds a nucleoside triphosphate having a base complementary to the template DNA to the primer to link it. is synthesized A primer generally consists of a sequence of 15 to 30 bases, and the melting temperature (Tm) at which it binds to the template strand varies depending on the base composition and length. The sequence of the primer does not need to have a completely complementary sequence to a partial nucleotide sequence of the template, and it is sufficient if it hybridizes with the template and has sufficient complementarity within a range capable of performing an intrinsic function of the primer. Therefore, in the present invention, the primer for measuring the expression level of the EDN mRNA does not need to have a perfectly complementary sequence to each gene sequence, and is intended to measure the amount of mRNA by amplifying a specific section of mRNA or cDNA through DNA synthesis. It is sufficient if it has a length and complementarity suitable for the purpose. The primers for the amplification reaction are composed of a set (pair) that complementarily binds to the template (or sense) and the opposite side (antisense) of both ends of a specific section of the mRNA to be amplified. Primers can be easily designed by those skilled in the art by referring to the EDN mRNA or cDNA sequence.

The 'probe' refers to a fragment of a polynucleotide such as RNA or DNA having a length of several to several hundred base pairs, which can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. Meaning, it is labeled (labeling), so that the presence or absence of the target mRNA or cDNA to be bound, the amount of expression, etc. can be confirmed. For the purpose of the present invention, a probe complementary to EDN mRNA may be used for monitoring asthma control status by performing a hybridization reaction with a sample of a subject and measuring the expression level of EDN mRNA. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art. In the present invention, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with EDN mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.

The antibody refers to a specific protein molecule directed against an antigenic site. Antibodies for use in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab)2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, It may be a chimeric antibody or the like.

Since the EDN protein is a known protein, the antibody used in the present invention can be prepared by a conventional method widely known in the field of immunology using the protein as an antigen. The EDN protein used as the antigen of the antibody according to the present invention may be extracted from nature or synthesized, or it may be prepared by a recombinant method based on a DNA sequence. In the case of using genetic recombination technology, a nucleic acid encoding an EDN protein is inserted into an appropriate expression vector, and the EDN protein is recovered from the transformant after culturing the host cell so that the EDN protein is expressed in the transformant transformed with the recombinant expression vector. It can be obtained by

For example, polyclonal antibodies can be produced by injecting an EDN protein antigen into an animal and collecting blood from the animal to obtain a serum containing the antibody. Such antibodies can be prepared using various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

Monoclonal antibodies are also known by fusion methods (Kohler and Milstein, European J. Immmnunol. 6:511-519, 1976), recombinant DNA methods (US Pat. No. 4816567) and phage antibody libraries (Clackson et al., Nature, 352, 624-628, 1991; Marks et al., J. Mol. Biol. 222, 58:1-597, 1991).

On the other hand, the present invention provides a kit for monitoring asthma control state comprising an agent for measuring the expression level of EDN protein or its mRNA as an active ingredient.

The kit of the present invention may further include tools and/or reagents known in the art for use in immunological analysis in addition to the protein antibody of EDN.

In the above immunological analysis, any method capable of measuring antigen-antibody binding may be included. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, Enzyme Linked Immunoabsorbent assay (ELISA), immunoblotting, Parar assay ( Farr assay), immunoprecipitation, latex aggregation, hemagglutination, nephrocytometry, immunodiffusion, counter-current electrophoresis, single-radical immunodiffusion, protein chip and immunofluorescence.

Tools and/or reagents used in immunological assays include suitable carriers or supports, labels capable of producing a detectable signal, solubilizing agents, and detergents. In addition, when the labeling material is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The EDN included in the kit of the present invention may be immobilized on a suitable carrier or support using various methods as disclosed in the literature (Antibodies: A Labotory Manual, Harlow &Lane; Cold Spring Harbor, 1988), and a suitable carrier Or examples of the support include agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterin, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, glass , polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, cups, flat packs, and the like. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).

Labels capable of generating a detectable signal enable qualitative or quantitative measurement of the formation of antigen-antibody complexes, such as enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive substances. Isotopes and the like can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydrogenase, glucose-6 -Phosphate dehydrogenase, invertase, etc. can be used. As the fluorescent material, fluorescein, isothiocyanate, rhodamine, phycoerythrine, phycocyanin, allophycocyanin, fluorescein isothioxyanate, and the like can be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. Microparticles include colloidal gold and colored latex, and redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, and the like. However, in addition to those exemplified above, any one that can be used in an immunological assay may be used.

In addition, the present invention relates to a method for monitoring asthma control, comprising measuring the expression level of EDN protein or mRNA thereof in a sample of a subject.

In the present invention, the sample can be used without limitation as long as it is collected from a subject to evaluate or predict asthma control status, for example, cells or tissues, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, obtained by biopsy, etc. It may be various secretions, urine, feces, and the like. Preferably, it may be selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, ascites, vaginal secretion and urine, and preferably blood, plasma, or serum. The sample may be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

When the expression level of EDN protein or its mRNA in the subject measured by the above method is increased compared to a control measured by the same method, the subject is evaluated as having asthma uncontrolled. can That is, when the expression level of EDN protein or its mRNA is increased compared to the control group, it can be evaluated that asthma is not well controlled.

In the present specification, mRNA expression level measurement is RT-PCR, quantitative or semi-quantitative RT-PCR (Quantitative or semi-Quantitative RT-PCR), quantitative or semi-quantitative real-time RT-PCR (Quantitative or semi-Quantitative real-time RT-PCR), Northern blot, and may be measured using one or more methods selected from the group consisting of DNA or RNA chips, but is not limited thereto.

In the present specification, the protein expression level measurement is Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, complement fixation analysis , may be measured using one or more methods selected from the group consisting of FACS and protein chip, but is not limited thereto.

When the concentration of EDN in the sample is 48.5 ng/ml or more, it may be evaluated as uncontrolled asthma, but is not limited thereto.

When the concentration of EDN in the sample is less than 48.5 ng/ml, it may be evaluated as controlled asthma, but is not limited thereto.

The expression level of the EDN protein or its mRNA may be one or more selected from the group consisting of sputum eosinophils and sputum neutrophils, but is not limited thereto.

The composition, monitoring method and kit of the present invention may be characterized in that the AUC is 0.6 or more, 0.65 or more, 0.7 or more, 0.72 or more, 0.75 or more, but is not limited thereto. The AUC is all samples in which the EDN level measured in samples isolated from the controlled asthma group and the uncontrolled asthma group, respectively, is set as a standard for monitoring asthma control status, the X-axis is 1-specificity, and the Y-axis is the sensitivity. When a graph (ROC) connecting the points of is drawn, it means the area of the graph.

In addition, the compositions, monitoring methods and kits of the present invention have a sensitivity of 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or about 100%. It may be characterized, but is not limited thereto.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

<Example 1. Experimental method>

1.1 Target

From January 2005 to December 2018, 131 asthma patients with available serum samples and medical records were enrolled at Asan Medical Center. The inclusion criteria were: (1) over 18 years of age; (2) symptoms such as shortness of breath, wheezing, or cough for more than 3 months; and (3) airway hypersensitivity indicated by a 20% reduction in forced expiratory volume in 1 second (FEV1) at a methacholine dose of 6 mg/mL (PC 20) by a provocation test; Airway reversibility at FEV1 > 12% (and at least 200 mL) after inhalation of short-acting β-agonists.

Patients with a history of severe lung injury, bronchiectasis, or lung resection were excluded. Sputum samples were obtained by inhalation of nebulized sterile saline solution followed by filtering of cough and airway secretions. In addition, serum EDN levels of 43 healthy controls without allergic diseases were measured at Asan Hospital. This study was approved by the Institutional Review Board of Asan Hospital (2019-0376) and all patients provided written informed consent.

1.2 Definition of asthma control status and phenotype

Asthma control status was assessed by the clinician at the time of enrollment and the patient was classified as presenting either well-controlled or poorly controlled disease. The definition of asthma is derived from the treatment goals of the World Asthma Organization/National Institutes of Health guidelines and is a composite measure that includes the following outcomes: symptoms, rescue medication use, nighttime awakenings, exacerbations, emergency visits and adverse events. The uncontrolled group included patients with partially controlled or uncontrolled and exacerbated asthmatic conditions.

For subgroup analysis, patients were assigned severe eosinophilic asthma (SEA), non-severe eosinophilic asthma (NSEA) and severe non-eosinophilic asthma (SNEA) according to their severity and eosinophilic asthma phenotype. -eosinophilic asthma) group. Severe asthma was determined according to the guidelines of the International European Asthma and Respiratory Association/American Thoracic Society guidelines. Severe asthma was defined as eosinophilic inflammation at enrollment and blood eosinophil counts greater than 300/μL.

Therefore, the SEA group included patients with severe asthma and an eosinophil count ≥300/μL, and the SNEA group included the group with a blood eosinophil count <300/μL. Finally, the NSEA group included patients with non-severe asthma and patients with eosinophil counts >300/μL.

1.3 Measurement of Serum Eosinophil-derived Neurotoxin (EDN) Levels

Serum EDN concentrations were measured using a K ^® EDN ELISA kit (SKIMS-BIO Co., Seoul, Korea), and the results were expressed in nanograms per milliliter (ng/mL). ELISA detects human EDN with minimum and maximum detection limits of 6.0 and 400 ng/mL, respectively, and has cross-reactivity with Eosinophil cationic protein (ECP).

A more specific ELISA method is [Kim CK, Callaway Z, Park JS, Kwon E. Utility of serum eosinophil-derived neurotoxin (EDN) measurement by ELISA in young children with asthma. Allergol Int 2017;66:70-4.].

1.4 Statistical Analysis

Student's t-test and Pearson chi-square test were used to determine between-group differences for continuous and dichotomous variables. One-way analysis of variance (ANOVA) was performed to confirm the significance of the differences between the three groups (SEA, NSEA, SNEA). Dunn's multiple comparison post-hoc test was performed to confirm the significance of each difference between the two groups. Logistic regression analysis was used to determine whether EDN was an independent parameter for determining asthma control status. Significant univariate variables (p <0.05) were included for multivariate analysis. The diagnostic performance of EDN and blood eosinophil counts to predict uncontrolled status was evaluated using area under the receiver operating characteristic curve (AUC, ROC). The AUC of each variable was compared using a method derived from Hanley and McNeil. Incremental values of significant variables to improve predictability were compared to net reclassification improvement (NRI) indices using the R package. Correlation between continuous variables was confirmed using Spearman rank correlation analysis. All calculations were performed using R (version 3.5.2) and MedCalc (version 15.5) statistical software. P <0.05 was considered significant.

<Example 2. Experimental Results>

2.1 Clinical characteristics of subjects according to asthma control status

The baseline and clinical characteristics of 131 asthmatic patients are summarized in Table 1. Patients were divided into two groups according to their asthma control status, and 56 and 75 patients were included in the controlled and uncontrolled asthma groups, respectively. There were no statistically significant differences in age, sex, body mass index (BMI), asthma duration, and smoking history between the control and uncontrolled asthma groups.

In addition, no differences were observed in prevalence according to atopic dermatitis, allergic rhinitis, exacerbation events in the previous year, and inhaled corticosteroid (ICS) dose at enrollment. Compared with controlled asthma, patients with uncontrolled asthma had asthma control test (ACT ) scores (15.4±4.2 vs. 21.3±3.8, p <0.001) and baseline FEV1 levels (67.4±20.0 vs. 80.4±16.4, p < 0.001) was low.

Although the sputum eosinophil and neutrophil ratios were not significantly different, blood eosinophil counts were higher in the uncontrolled asthma group than in the controlled asthma group (658.6±524.0 versus 554.0±783.7/μL, p = 0.001).

That is, as shown in FIG. 1 , the serum EDN level was significantly higher in the uncontrolled asthma group than in the controlled asthma group (103.2±60.2 vs. 60.8±49.7 ng/ml, p <0.001).

In addition, the cut-off value of the ROC curve for controlled asthma and uncontrolled asthma was 48.5 ng/ml. If the concentration of EDN in the sample is 48.5 ng/ml or higher, it can be evaluated as uncontrolled asthma. was confirmed.

Total
(n=131) Controlled asthma
(n=56) uncontrolled asthma
(n=75) P age (yr) 51.9 ± 15.7 53.3 ± 15.7 50.9 ± 15.7 0.387 Gender (female, %) 72 (55.0) 30 (53.6) 42 (56.0) 0.921 BMI 25.4 ± 4.1 25.7 ± 3.7 25.2 ± 4.4 0.457 Asthma duration (yr) 3.5 ± 4.6 3.9 ± 5.1 3.1 ± 4.2 0.485 smoking (%) 0.280 non-smoking 73 (55.7) 34 (60.7) 39 (52.0) past 39 (29.8) 17 (30.4) 22 (29.3) Now 19 (14.5) 5 (8.9) 14 (18.7) atopy (%)* 32 (46.4) 15 (41.7) 17 (51.5) 0.563 Allergic rhinitis (%) 61 (81.3) 27 (87.1) 34 (77.3) 0.439 Worsening (Year) (%) 58 (87.9) 28 (87.5) 30 (88.2) 1.000 ICS dose (%) 0.124 Low 8 (11.9) 3 (7.0) 5 (20.8) Moderate to high 59 (88.1) 40 (93.0) 19 (79.2) ACT score 18.0 ± 5.0 21.3 ± 3.8 15.4 ± 4.2 <0.001 Total IgE (kU/L) 416.4 ± 639.8 399.9 ± 574.7 475.6 ± 870.3 0.745 FEV1, pred, pre-BD (%) 73.6 ± 19.6 80.4 ± 16.7 67.4 ± 20.0 <0.001 FEV1/FVC (%) 69.1 ± 15.4 70.2 ± 13.3 68.2 ± 17.2 0.488 Sputum Eosinophils (%)† 28.73 ± 28.27 2 30.6 ± 28.3 0.346 Sputum Neutrophils (%)† 55.6 ± 27.2 82 52.2 ± 26.9 0.338 blood eosinophils, absolute count (/uL) 613.9 ± 647.2 554.0 ± 783.7 658.6 ± 524.0 0.001 Serum EDN (ng/ml) 85.1 ± 59.6 60.8 ± 49.7 103.2 ± 60.2 <0.001

* Atopic dermatitis was defined as a positive skin prick test response to one or more common aeroallergens or a history of atopic dermatitis or allergic rhinitis.

†Sputum samples suitable for analysis include patients with controlled (n=1) and uncontrolled asthma (n=14).

2.2 Confirming the predictability of EDN levels for asthma control status

The predictability of EDN for asthma control status was analyzed by univariate and multivariate logistic regression analysis (see Table 2). The variance inflation factor (VIF) was calculated to investigate the collinearity between EDN and blood eosinophil counts and was found to be 1.35. Since the VIF was <4, no collinearity was considered between EDN and blood eosinophil counts.

Univariate analysis indicated that serum EDN levels and baseline FEV1 were important clinical parameters for predicting uncontrolled asthmatic status (p = 0.001 and p <0.001, respectively). In addition, they were confirmed to be significant independent indicators for predicting uncontrolled asthmatic status in multivariate analysis (p = 0.001 and p <0.001, respectively).

Clinical variables OR 95% CI P Univariate analysis age (yr) 0.990 0.968-1.012 0.384 Asthma duration (yr) 0.962 0.816-1.126 0.629 atopy (%) 1.488 0.576-3.897 0.413 Allergic rhinitis (%) 0.504 0.127-1.690 0.288 Worsening (Year) (%) 1.071 0.233-4.927 0.927 Total IgE (kU/L) 1.000 0.999-1.001 0.739 FEV1, pred, pre-BD (%) 0.962 0.939-0.983 0.001 FEV1/FVC (%) 0.422 0.033-4.705 0.486 sputum eosinophils (%) 1.966 1.051-34.379 0.349 sputum neutrophils (%) 0.913 0.639-1.030 0.329 Blood eosinophils, absolute count (/μL) 1.000 0.999-1.000 0.368 Serum EDN (ng/mL) 1.015 1.008-1.023 <0.001 Multivariate analysis FEV1, pred, pre-BD (%) 0.960 0.935-0.983 0.001 Serum EDN (ng/mL) 1.019 1.010-1.031 <0.001

2.3 Comparison of the Effects of EDN and Blood Eosinophil Counts to Predict Uncontrolled Asthma

ROC curves of serum EDN levels and blood eosinophil counts were analyzed to compare their predictiveness for uncontrolled asthmatic status.

As a result, as shown in FIG. 2 , the serum EDN level had a significantly higher AUC than the blood eosinophil count (AUC = 0.726, p <0.001). Serum EDN had a sensitivity of 81.3% and a specificity of 57.1% for predicting uncontrolled asthmatic status. The AUC from ROC curve analysis of blood eosinophil counts was 0.628 with 64.0% sensitivity and 64.3% specificity (p=0.011). That is, it was confirmed that the serum EDN level could better predict the uncontrolled state compared with the blood eosinophil count (AUC, 0.726 vs. 0.628, p=0.024). Also, in the NRI test for predicting the uncontrolled state, the model including EDN had an NRI score of 0.4925 (95% CI, 0.1523-0.8327; p=0.005), indicating that the performance was remarkably good.

2.4 Confirmation of Correlation Between Serum EDN Levels and Clinical Parameters

As shown in FIG. 3 , serum EDN levels were significantly correlated with blood eosinophil counts.

However, serum EDN levels did not correlate with sputum eosinophil and neutrophil ratios. Regarding lung function, FEV1 (% Predicted), FEV1/forced vital capacity (FEV1/FVC) (%) and PC20 did not correlate with serum EDN levels. In addition, no significant correlation was observed between serum EDN levels and the inflammatory parameter, C-reactive protein (CRP).

Summarizing the above examples, it was confirmed that the control status of asthma can be predicted by measuring the serum EDN level. In addition, it was confirmed that EDN is an independent biomarker for predicting asthma control status.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Composition for monitoring the condition of asthma control using EDN <130> MP20-068 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 1 Met Val Pro Lys Leu Phe Thr Ser Gln Ile Cys Leu Leu Leu Leu Leu 1 5 10 15 Gly Leu Leu Ala Val Glu Gly Ser Leu His Val Lys Pro Pro Gln Phe 20 25 30 Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln 35 40 45 Gln Cys Thr Asn Ala Met Gln Val Ile Asn Asn Tyr Gln Arg Arg Cys 50 55 60 Lys Asn Gln Asn Thr Phe Leu Leu Thr Thr Phe Ala Asn Val Val Asn 65 70 75 80 Val Cys Gly Asn Pro Asn Met Thr Cys Pro Ser Asn Lys Thr Arg Lys 85 90 95 Asn Cys His His Ser Gly Ser Gln Val Pro Leu Ile His Cys Asn Leu 100 105 110 Thr Thr Pro Ser Pro Gln Asn Ile Ser Asn Cys Arg Tyr Ala Gln Thr 115 120 125 Pro Ala Asn Met Phe Tyr Ile Val Ala Cys Asp Asn Arg Asp Gln Arg 130 135 140 Arg Asp Pro Pro Gln Tyr Pro Val Val Pro Val His Leu Asp Arg Ile 145 150 155 160 Ile <210> 2 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 2 Met Val Pro Lys Leu Phe Thr Ser Gln Ile Cys Leu Leu Leu Leu Leu 1 5 10 15 Gly Leu Leu Ala Val Glu Gly Ser Leu His Val Lys Pro Pro Gln Phe 20 25 30 Thr Trp Ala Gln Trp Phe Glu Thr Gln His Ile Asn Met Thr Ser Gln 35 40 45 Gln Cys Thr Asn Ala Met Gln Val Ile Asn Asn Tyr Gln Arg Arg Cys 50 55 60 Lys Asn Gln Asn Thr Phe Leu Leu Thr Thr Phe Ala Asn Val Val Asn 65 70 75 80 Val Cys Gly Asn Pro Asn Met Thr Cys Pro Ser Asn Lys Thr Arg Lys 85 90 95 Asn Cys His His Ser Gly Ser Gln Val Pro Leu Ile His Cys Asn Leu 100 105 110 Thr Thr Pro Ser Pro Gln Asn Ile Ser Asn Cys Arg Tyr Ala Gln Thr 115 120 125 Pro Ala Asn Met Phe Tyr Ile Val Ala Cys Asp Asn Arg Asp Gln Arg 130 135 140 Arg Asp Pro Pro Gln Tyr Pro Val Val Pro Val Asn Leu Asp Arg Ile 145 150 155 160 Ile <210> 3 <211> 718 <212> DNA <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 3 ggcagctgga tcagttctca caggagctac agcgcggaga ctgggaaaca tggttccaaa 60 actgttcact tcccaaattt gtctgcttct tctgttgggg cttctggctg tggagggctc 120 actccatgtc aaacctccac agtttacctg ggctcaatgg tttgaaaccc agcacatcaa 180 tatgacctcc cagcaatgca ccaatgcaat gcaggtcatt aacaattatc aacggcgatg 240 caaaaaccaa aatactttcc ttcttacaac ttttgctaac gtagttaatg tttgtggtaa 300 cccaaatatg acctgtccta gtaacaaaac tcgcaaaaat tgtcaccaca gtggaagcca 360 ggtgccttta atccactgta acctcacaac tccaagtcca cagaatattt caaactgcag 420 gtatgcgcag acaccagcaa acatgttcta tatagttgca tgtgacaaca gagatcaacg 480 acgagaccct ccacagtatc cggtggttcc agttcacctg gatagaatca tctaagctcc 540 tgtatcagca ctcctcatca tcactcatct gccaagctcc tcaatcatag ccaagatccc 600 atctctccat atactttggg tatcagcatc tgtcctcatc agtctccata ccccttcagc 660 tttcctgagc tgaagtgcct tgtgaaccct gcaataaact gctttgcaaa ttcaaaaa 718 <210> 4 <211> 694 <212> DNA <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 4 cacaggagct acagcgcgga gactggaaac atggttccaa aactgttcac ttcccaaatt 60 tgtctgcttc ttctgttggg gcttctggct gtggagggct cactccatgt caaacctcca 120 cagtttacct gggctcaatg gtttgaaacc cagcacatca atatgacctc ccagcaatgc 180 accaatgcaa tgcaggtcat taacaattat caacggcgat gcaaaaacca aaatactttc 240 cttcttacaa cttttgctaa cgtagttaat gtttgtggta acccaaatat gacctgtcct 300 agtaacaaaa ctcgcaaaaa ttgtcaccac agtggaagcc aggtgccttt aatccactgt 360 aacctcacaa ctccaagtcc acagaatatt tcaaactgca ggtatgcgca gacaccagca 420 aacatgttct atatagttgc atgtgacaac agagatcaac gacgagaccc tccacagtat 480 ccggtggttc cagttcacct ggatagaatc atctaagctc ctgtatcagc actcctcatc 540 atcactcatc tgccaagctc ctcaatcata gccaagatcc catctctcca tatactttgg 600 gtatcagcat ctgtcctcat cagtctccat accccttcag ctttcctgag ctgaagtgcc 660 ttgtgaaccc tgcaataaac tgctttgcaa attc 694 <210> 5 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 5 gctgcccctg aaccccagaa caaccagctg gatcagttct cacaggagct acaggccgga 60 gactgggaaa catggttcca aaactgttca cttcccaaat ttgtctgctt cttctgttgg 120 ggcttctggc tgtggagggc tcactccatg tcaaacctcc acagtttacc tgggctcaat 180 ggtttgaaac ccagcacatc aatatgacct cccagcaatg caccaatgca atgcaggtca 240 ttaacaatta tcaacggcga tgcaaaaacc aaaatacttt ccttcttaca acttttgcta 300 acgtagttaa tgtttgtggt aacccaaata tgacctgtcc tagtaacaaa actcgcaaaa 360 attgtcacca cagtggaagc caggtgcctt taatccactg taacctcaca actccaagtc 420 cacagaatat ttcaaactgc aggtatgcgc agacaccagc aaacatgttc tatatagttg 480 catgtgacaa cagagatcaa cgacgagacc ctccacagta tccggtggtt ccagttcacc 540 tggatagaat catctaagct cctgtatcag cactcctcat catcactcat ctgccaagct 600 cctcaatcat agccaagatc ccatctctcc atatactttg ggtatcagca tctgtcctca 660 tcagtctcca taccccttca gctttcctga gctgaagtgc cttgtgaacc ctgcaataaa 720 ctgctttgca aattc 735 <210> 6 <211> 1501 <212> DNA <213> Artificial Sequence <220> <223> EDN (Eosinophil-derived neurotoxin) <400> 6 gaatatcacc caccacgtct gcagcaggca tatagttttc atccagagtt tggatctaac 60 cagcaaaact ctgtcttaca caggatgact tggaattaga gtccttatag cagaaagagc 120 agcagggctg tccttgggta tccgttgctc agccaagtca tcaaataaaa aggatgattg 180 cacaagtgac tatgtaccaa tctgtgggtt tctcgatcgc aagagccaga ccctcctctg 240 cgtctgctgg cccaacccac caagggatgc tttatttaaa cagttccaag taggggagac 300 cagctgcccc tgaaccccag aacaaccagc tggatcagtt ctcacaggag ctacagcgcg 360 gagactgggt aagtcaacga tccccagagc tgggacagaa ggggcagcaa tggggcagca 420 actgagggag aagagagctg acgttagtgc ttaggagacg ttgcacactt tgcagacagg 480 aagtaaagga aatgggaccc cagagtggcc gcagaggggc tgtggggtaa gacactacag 540 tgtgtgtcat aaccaagacc cgatcaggag tagttacttc tcttcttttc ttacaggaaa 600 catggttcca aaactgttca cttcccaaat ttgtctgctt cttctgttgg ggcttctggc 660 tgtggagggc tcactccatg tcaaacctcc acagtttacc tgggctcaat ggtttgaaac 720 ccagcacatc aatatgacct cccagcaatg caccaatgca atgcaggtca ttaacaatta 780 tcaacggcga tgcaaaaacc aaaatacttt ccttcttaca acttttgcta acgtagttaa 840 tgtttgtggt aacccaaata tgacctgtcc tagtaacaaa actcgcaaaa attgtcacca 900 cagtggaagc caggtgcctt taatccactg taacctcaca actccaagtc cacagaatat 960 ttcaaactgc aggtatgcgc agacaccagc aaacatgttc tatatagttg catgtgacaa 1020 cagagatcaa cgacgagacc ctccacagta tccggtggtt ccagttcacc tggatagaat 1080 catctaagct cctgtatcag cactcctcat catcactcat ctgccaagct cctcaatcat 1140 agccaagatc ccatctctcc atatactttg ggtatcagca tctgtcctca tcagtctcca 1200 taccccttca gctttcctga gctgaagtgc cttgtgaacc ctgcaataaa ctgctttgca 1260 aattcatctg aaagtgtctg tgtgtcttca ttagccgctc tgctgtcatt tagtgacaat 1320 ctactctaga gattttttct tcctctaacc tgagacttcc gggaaacaga gagatttgaa 1380 gataagagat gctttctgtc atgaaacagc acagtcttat cctctccctg ctttaggctg 1440 agaagctgag gtctcaaccg atatctagca actgtcgaag catcttgctt tgatcaagct 1500 t 1501

Claims (13)

EDN (Eosinophil-derived neurotoxin) protein or a composition for monitoring asthma control status comprising an agent for measuring the mRNA expression level thereof as an active ingredient.
According to claim 1,
The agent for measuring the mRNA expression level is a probe or primer that specifically binds to the EDN mRNA, the composition for monitoring asthma control status.
According to claim 1,
The agent for measuring the expression level of the protein is characterized in that the antibody specific to the EDN protein, asthma control status monitoring composition.
According to claim 1,
The EDN protein is characterized in that it comprises an amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2, asthma control status monitoring composition.
According to claim 1,
The EDN mRNA is SEQ ID NO: 3 to SEQ ID NO: 6, characterized in that it comprises a nucleotide sequence consisting of one sequence selected from the group consisting of, asthma control status monitoring composition.
According to claim 1,
The asthma is at least one selected from the group consisting of severe asthma and non-severe asthma, the composition for monitoring asthma control status.
A method for monitoring asthma control status, comprising measuring the expression level of EDN protein or mRNA thereof in a sample of a subject.
8. The method of claim 7,
The method, characterized in that the sample is blood, plasma or serum.
8. The method of claim 7,
A method, characterized in that when the expression level of EDN protein or its mRNA is increased compared to a control, asthma is evaluated as not well controlled.
8. The method of claim 7,
When the concentration of EDN in the sample is 48.5 ng/ml or more, characterized in that it is evaluated as uncontrolled asthma, the method.
8. The method of claim 7,
The method, characterized in that the expression level of the EDN protein or its mRNA is not correlated with at least one selected from the group consisting of a sputum eosinophil count and a sputum neutrophil count.
8. The method of claim 7,
The method of monitoring, characterized in that it further comprises the step of diagnosing the number of eosinophils in the blood.
A kit for monitoring asthma control status, comprising an agent for measuring the expression level of EDN protein or its mRNA as an active ingredient.

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