Novel use of kasugamycin

Abstract

The chitinase 3-like 1 (CHI3L1) inhibitor, specifically kasugamycin, provided by the present invention, suppresses the expression or activation of chitinase 3-like 1 (CHI3L1), thereby effectively preventing or treating infections with acid-fast bacteria, particularly non-tuberculous mycobacteria and various diseases caused by infection with acid-fast bacteria, particularly nontuberculous mycobacteria. In addition, by measuring the expression level of chitinase 3-like 1 (CHI3L1) protein, when the expression level is higher than that of a control group, it is possible to effectively predict infection with acid-fast bacteria, particularly non-tuberculous mycobacteria, the onset of diseases caused by infection with acid-fast bacteria, particularly non-tuberculous mycobacteria, or the prognosis of diseases related to infection with acid fast bacteria, particularly nontuberculous mycobacteria.

Description

Novel uses of kasugamycin

[0001] The present invention is an invention related to the Fourth Industrial Revolution, specifically concerning innovative new drugs or next-generation biopharmaceuticals. That is, it relates to a method for inhibiting the progression of lung disease caused by non-tuberculosis mycobacterial infection by inhibiting the CHI3L1 (Chitinase 3-Like 1) protein in lung tissue using Kasugamycin.

[0002] Species of the genus Mycobacterium are classified into two groups based on the degree of pathogenicity and infectivity to the host: the tuberculosis group, which includes obligate pathogenic bacteria such as Mycobacterium tuberculosis and Mycobacterium leprae, and nontuberculous mycobacteria (NTM), which are opportunistic species excluding these.

[0003] The aforementioned non-tuberculous mycobacteria are widely distributed in nature, and the causative power of disease varies depending on the species. Specifically, M. avium complex (MAC), M. abscessus (MAB), and M. kansasii have a relatively high causative power, while M. fortuitum has a relatively low causative power. Diseases caused by non-tuberculous mycobacteria are broadly classified into four characteristic clinical syndromes: lung disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease. Among these, lung disease is the most common form, accounting for more than 90% of all diseases.

[0004] Reports of lung diseases caused by non-tuberculous mycobacteria have been increasing since 2000, and currently, lung diseases caused by MAC are the most frequently reported in most developed countries. In Korea, lung diseases caused by MAC are reported to account for approximately 70-80% of all causative agents of non-tuberculous mycobacterial lung diseases, with MAB reported as the second most observed causative agent. Lung diseases caused by the aforementioned non-tuberculous mycobacteria manifest with symptoms such as cough, fever, hemoptysis, and sputum.

[0005] The present invention is a result derived as part of the research project titled "Identification of the role of Chitinase-3-like protein 1 as an exacerbation determinant of non-tuberculous mycobacterial lung disease" (Project No. 27100886119, Project No. 00405542), which was conducted as a group research support (Global Basic Research Laboratory) project funded by the Ministry of Science and ICT of the Republic of Korea through the National Research Foundation of Korea to the Industry-Academic Cooperation Foundation of Yonsei University in 2024.

[0006] One objective of the present invention is to provide a composition of a non-tuberculous mycobacterial antimicrobial agent comprising a CHI3L1 (Chitinase 3-Like 1) inhibitor, specifically kasugamycin, as an active ingredient.

[0007] Another objective of the present invention is to provide a preventive or therapeutic use for non-tuberculous mycobacteria comprising a CHI3L1 (Chitinase 3-Like 1) inhibitor, specifically kasugamycin, as an active ingredient.

[0008] Another objective of the present invention is to measure the expression level of the CHI3L1 (Chitinase 3-Like 1) protein to predict whether there is a non-tuberculous mycobacterial infection, whether a non-tuberculous mycobacterial disease develops, and the prognosis thereof.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] Various embodiments of the present invention are described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to make the present invention unnecessary or obscure. Reference throughout this specification to one embodiment implies that a particular feature, form, composition, or characteristic described in association with the embodiment is included in one or more embodiments of the present invention. Accordingly, the circumstances of an embodiment expressed at various locations throughout this specification do not necessarily represent the same embodiment of the present invention. Additionally, a particular feature, form, composition, or characteristic may be combined in any suitable way in one or more embodiments.

[0011] In the first embodiment of the present invention, an antimicrobial composition comprising a CHI3L1 (Chitinase 3-Like 1) inhibitor as an active ingredient is provided.

[0012] In the present invention, “CHI3L1 (Chitinase 3-Like 1)” is a secreted glycoprotein also known as YKL-40, belonging to the chitinase-like proteins (CLPs). This protein has a size of 40 kDa and possesses conserved characteristics as a chitin-binding protein. The three-dimensional crystal structure of CHI3L1 exhibits the typical folding structure of a chitinase family protein, but chitinase activity is not expressed due to the lack of essential amino acid residues within the enzyme domain. CHI3L1 is expressed in various cells of lung tissue (neutrophils, monocytes / macrophages, monocyte-derived dendritic cells, epithelial cells, etc.) and is closely associated with various biological activities such as tissue damage, inflammatory responses, and tissue repair. CHI3L1 is also associated with several diseases. For example, high levels of CHI3L1 protein and gene expression are observed in patients suffering from various diseases such as asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, cancer, diabetes, and arteriosclerosis.

[0013] In the second embodiment of the present invention, an antimicrobial composition is provided in which, in the first embodiment, the CHI3L1 (Chitinase 3-Like 1) inhibitor is one or more selected from the group consisting of antisense nucleotides, siRNA, shRNA, and ribozymes that bind complementarily to the mRNA of the gene encoding CHI3L1, or one or more selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that bind complementarily to the Pierce 1 protein. In the present invention, the CHI3L1 inhibitor is not particularly limited as long as it can inhibit the expression or activation of CHI3L1 (Chitinase 3-Like 1). The CHI3L1 (Chitinase 3-Like 1) inhibitor provided in one embodiment of the present invention can increase resistance to acid-fast bacteria, particularly non-tuberculous mycobacteria, by regulating inflammation and immune responses caused by CHI3L1, or induce apoptosis in cells infected with acid-fast bacteria, particularly non-tuberculous mycobacteria, and can reduce tissue damage and promote recovery by alleviating inflammatory responses caused by CHI3L1.

[0014] In the third embodiment of the present invention, in the first and second embodiments, the CHI3L1 (Chitinase 3-Like 1) inhibitor may be one or more selected from the group consisting of K284-6111, Kasgamycin, Kasgamycin-fluorescent compound conjugate, G721-0282, and Darifenacin as a small molecule.

[0015] In this invention, "Kasugamycin (KSM)" is an aminoglycoside antibiotic that was first developed in Japan in the 1960s. Kasugamycin was originally used in the agricultural field as a fungicide against *Magnaporthe oryzae*, a major rice blast pathogen, but its antibacterial effects against various bacteria have since been studied. The antibacterial action of Kasugamycin is primarily achieved through a mechanism in which it binds to the 30S subunit of ribosomes to inhibit protein synthesis. Specifically, Kasugamycin binds to ribosomes and interferes with the binding of initiator tRNA, thereby exhibiting inhibitory effects during the initiation phase of protein synthesis. This allows for the inhibition of bacterial growth. Although Kasugamycin possesses antibacterial activity against various bacteria, its antibacterial spectrum is somewhat distinct from that of other aminoglycoside antibiotics. In particular, unlike most traditional aminoglycoside antibiotics, Kasugamycin acts selectively only on the initiation of translation, thus not affecting the elongation of translation that is targeted by conventional antibiotics. Due to these characteristics, it was confirmed for the first time in this invention that kasugamycin can effectively prevent or treat acid-fast bacteria infections, particularly non-tuberculous acid-fast bacteria.

[0016] Furthermore, antibiotics have diverse mechanisms of action, including inhibition of cell wall synthesis, protein synthesis, nucleic acid synthesis, and metabolic pathways. In addition to differences in cell wall structure, such as between Gram-positive and Gram-negative bacteria, there are also differences in metabolic rates among bacteria. Therefore, it is very difficult for a person skilled in the art to predict that an antibiotic effective against one type of bacterium will also be effective against another. In particular, Magnaporthe oryzae is a fungus that forms mycelia and spores, and nontuberculous mycobacteria (NTM) are Gram-positive bacteria with high resistance to acid. Thus, it would be very difficult to predict that a specific fungal agent will be effective against Gram-positive bacteria that are highly resistant to acid.

[0017] In addition, since antiviral agents also primarily target the inhibition of viral invasion, the inhibition of DNA or RNA replication, or the inhibition of viral release, their mechanisms of action differ from those of antibiotics, making it very difficult for a person skilled in the art to predict that any antiviral agent would also be useful as an antimicrobial agent.

[0018] Furthermore, although kasugamycin is an antibiotic, it is not used at all as an antibiotic for lung diseases because it is completely ineffective against NTM pulmonary diseases. A person skilled in the art would consider kasugamycin ineffective as an antimicrobial agent if they confirmed that it has low or no direct antimicrobial effect and lacks an anti-mycobacterial effect through the ability to inhibit MAC strain growth within cells. Nevertheless, in this invention, it has been confirmed in vivo that kasugamycin possesses an antimicrobial effect through host-directed therapies (HDTs) by effectively neutralizing CHI3L1; thus, it is acknowledged that this is a heterogeneous and significant effect that a person skilled in the art could not have predicted.

[0019] The "Host-directed therapies (HDTs)" of the present invention refer to therapeutic methods that do not act directly on pathogens like traditional antibiotics, but rather act on pathogens through host-mediated responses. Such host-directed therapies are methods that make it unfavorable for pathogens to infect, maintain, or grow within a host by altering the host environment in which the pathogen exists, such as metabolic reactions. Their function can be exerted through actions such as regulating immune responses or regulating the metabolic reactions of host cells. In particular, through the above host-directed therapies, antibacterial effects can be exerted in vivo against bacteria resistant to existing drugs, especially non-tuberculosis mycobacteria that have overcome resistance, by inhibiting CHI3L1.

[0020] In the present invention, "overcoming resistance" refers to an action that increases the susceptibility to a drug of non-tuberculous mycobacteria that have acquired resistance to a specific drug. For the purposes of the present invention, the specific drug may refer to an antibiotic. The increase in susceptibility means reaching a level where the concentration showing an effect such as inhibiting the proliferation of non-tuberculous mycobacteria that have acquired resistance is equal to or greater than the concentration showing an effect such as inhibiting proliferation of non-tuberculous mycobacteria that are not resistant. Synonyms for "overcoming resistance" include "resistance inhibition," "resistance release," or "resistance release." In another embodiment of the present invention, an antimicrobial composition is provided in which the antimicrobial agent is an antimicrobial agent against acid-fast bacteria (Mycobacteria).

[0021] In the present invention, the “gasugamycin-fluorescent compound conjugate” refers to a conjugate in which gasugamycin and a fluorescent compound used for fluorescent labeling of a target molecule or biological sample are bonded together by covalent bonds or the like.

[0022] A person skilled in the art will be able to fully understand through Example 10 of the present invention that even if a fluorescent compound is bound to kasagamycin which inhibits CHIL31, the CHIL31 inhibitory ability is similar to that of kasagamycin.

[0023] In the present invention, the “fluorescent compound” includes BODIPY, fluorescein, and their derivatives, which provide high quantum yield and broad wavelength selectivity, making them widely used for labeling biological targets. Additionally, rhodamine-based dyes possess strong fluorescence intensity and photostability, making them applicable to cell imaging, protein labeling, and antibody labeling; furthermore, environmentally responsive sensor functions are realized depending on the opening and closing of the spirolactone structure. Cyanine-based compounds, such as Cy3, Cy5, and Cy7, exhibit excellent luminescence characteristics in the red to near-infrared region and are frequently utilized in in vivo imaging patents requiring biotransmittance. Along with this, coumarin-based phosphors also exhibit stable emission characteristics in the ultraviolet to blue region and can be used as sensors for detecting changes in polarity and pH. Naphthalimide derivatives are useful for labeling specific organelles, such as cell membranes, the endoplasmic reticulum (ER), and lysosomes, due to their high photostability and hydrophobic properties.

[0024] Furthermore, quinone (imide / quinoline) and quinazolinone-based luminescent compounds also exhibit fluorescence changes sensitive to the molecular environment and can be used as sensors or probes. Among polycyclic aromatic hydrocarbons (PAHs), pyrene and acridine derivatives are utilized in patents for protein-nucleic acid binding analysis due to their luminescence properties sensitive to π-π stacking and the microenvironment. Polymethine dyes also frequently appear in drug-dye conjugate patents due to their near-infrared luminescence and ease of structural modification.

[0025] Meanwhile, in terms of photosensitization capabilities, derivatives of porphyrin, chlorine, bacteriochlorin, and phthalocyanine possess singlet oxygen generation capabilities similar to BODIPY, making them suitable for use as photosensitizers in patents related to photodynamic therapy (PDT). Additionally, triphenylamine (TPA) and tetraphenylethylene (TPE)-based phosphors exhibiting aggregation-induced emission (AIE) characteristics are utilized in patents for labeling nanoparticles, micelles, and lipid membranes by leveraging the property that their signal increases in an aggregated state. Recently, oxazine, cyanobenzothiazole (CBT)-based phosphors, and heterocyclic borate (BOH, BF₂-chelated dye) have also been proposed as substitutes for BODIPY, offering various wavelength modulation capabilities and high photostability.

[0026] In summary, a group of compounds capable of performing patented functions similar to BODIPY may consist of fluorescein, rhodamine, cyanine, coumarin, naphthalimide, pyrene-acridine-based PAHs, quinolone-based phosphors, polymetaine-based pigments, photosensitizing pigments such as porphyrin, chlorine, and phthalocyanine, and AIE-based phosphors, all of which can functionally replace BODIPY for fluorescent labeling, photodynamic therapy, molecular sensing, or imaging purposes.

[0027] In the present invention, the term “acid-fast bacteria (Mycobacteria)” or “Mycobacteria or Mycocobacterium” refers to a genus of bacteria belonging to Gram-positive bacteria, which is divided into Mycobacterium tuberculosis complex, Mycobacterium leprae, or the nontuberculous mycobacteria (NTM) group. Additionally, they are rod-shaped bacteria that are relatively small in size and possess strong resistance to acid. They contain a long fatty acid called mycolic acid in their cell walls, so they appear like Gram-negative bacteria under Gram staining, but they have very high resistance to acid. For this reason, they are classified as acid-fast bacteria. Furthermore, they are generally aerobic bacteria, although some can grow under anaerobic conditions.

[0028] In the fourth embodiment of the present invention, in the first to third embodiments, the nontuberculous mycobacteria group (NTM) comprises Mycobacterium avium (Mav), Mycobacterium abscessus subsp. abscessus (Mabc), Mycobacterium abscessus subsp. massiliense (Mmass), and Mycobacterium abscessus subsp.bolletii), Mycobacterium Intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium The present invention provides an antimicrobial composition comprising one or more selected from the group consisting of Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum, Mycobacterium xenopi, and Mycobacterium aubagnens.

[0029] In the fifth embodiment of the present invention, a pharmaceutical composition for preventing or treating non-tuberculous mycobacterial infections is provided, comprising the antimicrobial composition as an active ingredient in the first to fourth embodiments.

[0030] In the sixth embodiment of the present invention, a pharmaceutical composition is provided in which, in the first to fifth embodiments, the non-tuberculous mycobacterial infection disease is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease, and is not particularly limited as long as it is a disease that can be caused by non-tuberculous mycobacterial infection.

[0031] In the seventh embodiment of the present invention, a composition for diagnosing acid-fast bacilli (Mycobacteria) infection is provided, comprising a preparation for measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein.

[0032] In the eighth embodiment of the present invention, a diagnostic composition is provided, wherein, in the seventh embodiment, the diagnostic composition is intended to be applied to a biological sample isolated from a target individual.

[0033] In the ninth embodiment of the present invention, in the seventh and eighth embodiments, the biological sample comprises whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, A diagnostic composition is provided comprising one or more selected from the group consisting of synovial fluid, joint aspirate, organ secretions, cell, cell extract, and cerebrospinal fluid.

[0034] In the present invention, the term "diagnosis" means confirming the existence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis may be to predict the likelihood of onset, growth, or progression of an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection, or to distinguish an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection from other diseases, such as infections of tuberculosis bacteria or leprosy bacteria.

[0035] In the present invention, "prognosis" refers to the act of predicting in advance the course of a disease and the outcome of death or survival. The aforementioned prognosis or prognostic diagnosis may be interpreted to mean any act of predicting the course of a disease before or after treatment by comprehensively considering the patient's condition, as the course of the disease may vary depending on the patient's physiological or environmental state. For the purposes of the present invention, the aforementioned prognosis may be interpreted as the act of determining whether a low survival rate or poor responsiveness to treatment is predicted following an infection with acid-fast bacilli, particularly non-tuberculous mycobacteria, or the onset of a related disease.

[0036] Furthermore, in this invention, "diagnosis" refers to confirming the existence or characteristics of a pathological condition, whereas "prognosis" refers to the act of predicting in advance the course of a disease and the outcome of death or survival, so they are clearly distinguished. More specifically, the diagnosis of an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection disease means confirming whether the patient is currently infected with acid-fast bacilli, particularly non-tuberculous mycobacteria; the diagnosis of whether an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection disease has developed means going beyond confirming whether the patient is currently infected with acid-fast bacilli, particularly non-tuberculous mycobacteria, to confirm whether an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection disease has developed, such as lung disease, superficial lymphadenitis, skin, soft tissue, or bone infection, or disseminated disease; and the prognosis of an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection disease means the act of predicting in advance the course of the disease and the outcome of death or survival of a patient suffering from an acid-fast bacilli, particularly non-tuberculous mycobacterial, infection disease, and more specifically, it means predicting or estimating the recurrence-free survival period and the overall survival period.

[0037] In the present invention, the preparation for measuring the expression level of the protein may include one or more selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein, but is not limited thereto.

[0038] In the present invention, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody means an antibody that specifically binds to the protein. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibodies can be easily manufactured using techniques widely known in the art. For example, polyclonal antibodies can be produced by a method widely known in the art that includes the process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain serum containing antibodies. Such polyclonal antibodies can be produced from any animal, such as a goat, rabbit, sheep, monkey, horse, pig, cattle, or dog. In addition, monoclonal antibodies may be prepared using the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), which is widely known in the industry, or phage antibody library technology (see Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). Antibodies prepared by the above methods may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Furthermore, the antibodies of the present invention comprise not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

[0039] In the present invention, the "oligopeptide" is a peptide composed of 2 to 20 amino acids and may include dipeptides, tripeptides, tetrapeptides, and pentapeptides, but is not limited thereto.

[0040] In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, which was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeating N-(2-aminoethyl)-glycine backbone connected by peptide bonds, which significantly increases its binding affinity and stability to DNA or RNA, and is therefore used in molecular biology, diagnostic analysis, and antisense therapy. PNA is disclosed in detail in the literature [Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500].

[0041] In the present invention, the "aptamer" is an oligonucleotide or peptide molecule, and general information regarding aptamers is disclosed in detail in the literature [Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727(1998)].

[0042] In the present invention, the agent for measuring the expression level of the gene encoding the protein may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene encoding the protein, but is not limited thereto.

[0043] In the present invention, the "primer" is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that it amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0044] In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected within a sample, and means a substance capable of specifically confirming the presence of the target substance within the sample through said binding. The type of probe is not limited to substances commonly used in the industry, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe may be a biomaterial derived from an organism or similar, or manufactured in vitro, and may be, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA; DNA may include cDNA, genomic DNA, and oligonucleotides; RNA may include genomic RNA, mRNA, and oligonucleotides; and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

[0045] In the present invention, "LNA (Locked nucleic acids)" refers to nucleic acid analogs containing a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the 'locking' of the molecule caused by the methylene bridge, LNAs are unable to form an ideal shape in Watson-Crick bonding. When LNAs are included in DNA or RNA oligonucleotides, LNAs can pair more quickly with complementary nucleotide chains, thereby increasing the stability of the double helix.

[0046] In the present invention, "antisense" refers to an oligomer having a backbone between nucleotide base sequences and subunits, wherein the antisense oligomer hybridizes with a target sequence within RNA by Watson-Crick base pairing, thereby allowing the formation of a mRNA and RNA:oligomer heterodimer within the target sequence. The oligomer may have exact sequence complementarity or approximate complementarity with respect to the target sequence.

[0047] In the 10th embodiment of the present invention, a diagnostic kit is provided comprising the diagnostic composition provided in the 7th to 9th embodiments.

[0048] In the present invention, the "kit" refers to a tool capable of evaluating the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody through reaction with a substrate, but also indirect labeling in which a label that develops color through reactivity with another directly labeled reagent is conjugated. It may include a color-developing substrate solution, a washing solution, and other solutions that react with the label for color development, and may be manufactured to include the reagent components used. In the present invention, the kit may be a kit containing essential elements necessary for performing RT-PCR, and in addition to specific primer pairs for the marker gene, it may include test tubes, reaction buffer, deoxyribonucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. Furthermore, the kit may be a kit for detecting genes for cancer diagnosis that includes essential elements necessary for performing DNA chip analysis. A DNA chip kit comprises a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may comprise cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, provided that it is known in the art.

[0049] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0050] The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further include essential elements necessary to perform a reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific to the nucleic acid sequence of a control gene. Furthermore, the reverse transcription polymerase chain reaction kit may include a test tube or other suitable container, a reaction buffer (with varying pH and magnesium concentration), deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0051] Furthermore, the diagnostic kit for acid-fast bacteria, particularly non-tuberculous acid-fast bacteria, infection or disease according to the present invention may include essential elements necessary for performing DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, preparations, enzymes, etc., for producing fluorescently labeled probes. Additionally, the substrate may include cDNA or oligonucleotides corresponding to a control gene or a fragment thereof.

[0052] In addition, the diagnostic kit for acid-fast bacilli, particularly non-tuberculous mycobacterial infections or diseases according to the present invention may include essential elements necessary for performing an ELISA. The ELISA kit includes an antibody specific to the protein. The antibody is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody that has high specificity and affinity for the marker protein and has little cross-reactivity with other proteins. Additionally, the ELISA kit may include an antibody specific to a control protein. Furthermore, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with the antibody) and its substrate, or other substances capable of binding to the antibody.

[0053] In the present invention, the immobilizer for the antigen-antibody binding reaction may be a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., but is not limited thereto.

[0054] In addition, in the present invention, the label of the secondary antibody is preferably a conventional chromogenic agent that produces a color reaction, and labels such as fluorescein and dyes such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (poly L-lysine-fluorescein isothiocyanate), and RITC (rhodamine-B-isothiocyanate) may be used, but are not limited thereto.

[0055] In addition, in the present invention, it is preferable to use a chromogenic substrate to induce color development depending on the label that performs the color reaction, and TMB (3,3',5,5'-tetramethylbezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. may be used. At this time, it is more preferable that the chromogenic substrate be provided in a state dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is degraded by HRP used as a label for the secondary antibody conjugate to produce a chromogenic precipitate, and the presence or absence of the marker proteins is detected by visually confirming the degree of precipitation of this chromogenic precipitate.

[0056] In the present invention, the washing solution preferably comprises a phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferably used. After the antigen-antibody binding reaction, a secondary antibody is reacted with the antigen-antibody conjugate, and then an appropriate amount of the washing solution is added to the immobilizer to wash 3 to 6 times. A sulfuric acid solution (H2SO4) may preferably be used as the reaction stopping solution.

[0057] In the 11th embodiment of the present invention, a method for providing information for diagnosing acid-fast bacilli (Mycobacteria) infection is provided, comprising the step of measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein in a biological sample isolated from a target individual.

[0058] In the present invention, the subject of the above invention is an individual that has developed or is highly likely to develop an acid-fast bacterium, particularly a non-tuberculous acid-fast bacterium infection or related disease, and may be a mammal including humans, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be humans, but is not limited thereto.

[0059] In the present invention, the biological sample refers to any substance, biological body fluid, tissue, or cell obtained from or derived from an individual, including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, and bronchial aspirate. It may be one or more selected from the group consisting of synovial fluid, joint aspirate, organ secretions, cell, cell extract and cerebrospinal fluid, but is not limited thereto.

[0060] In the present invention, the preparation for measuring the expression level of the protein may include one or more selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein.

[0061] In the present invention, the measurement of the expression level of the protein may be performed by protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blotting, or ELISA (enzyme-linked immunosorbent assay).

[0062] In addition, in the present invention, the expression level of the protein may be measured by a multiple reaction monitoring (MRM) method.

[0063] In the present invention, for the multiple reaction monitoring method, the internal standard substance may be a synthetic peptide in which a specific amino acid constituting the target peptide is substituted with an isotope, or E. coli beta-galactosidase.

[0064] In the present invention, a preparation for measuring the expression level of a gene encoding the protein may include one or more selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene encoding the protein.

[0065] In the present invention, the measurement of the expression level of the gene encoding the protein may be performed by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or a DNA chip.

[0066] In the above method of the present invention, descriptions regarding CHI3L1 (Chitinase 3-Like 1), acid-fast bacteria, non-tuberculosis acid-fast bacteria, antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), aptamers, etc., and descriptions regarding primers, probes, etc. are omitted below in order to avoid excessive complexity of the specification as they overlap with what has been previously described.

[0067] In the present invention, "control group" may be the expression level of the CHI3L1 (Chitinase 3-Like 1) protein or the gene encoding said protein in a healthy normal control group, or the average to median value of the expression level of said marker protein or the gene encoding said protein in a biological sample derived from a patient with an acid-fast bacteria, particularly a non-tuberculous mycobacterial infection or related disease, or the average to median value of the expression level of the CHI3L1 (Chitinase 3-Like 1) protein or the gene encoding said protein in a biological sample derived from a patient other than a patient with an acid-fast bacteria, particularly a non-tuberculous mycobacterial infection or related disease, but is not limited thereto.

[0068] In the method of the present invention, predicting that an acid-fast bacterium, particularly a non-tuberculous mycobacterium infection or a related disease has developed or is highly likely to develop includes not only predicting the possibility of the development, growth, or progression of the acid-fast bacterium, particularly a non-tuberculous mycobacterium infection or a related disease, but also distinguishing the disease that has developed or is suspected to have developed in the target individual from other diseases, specifically, distinguishing the acid-fast bacterium, particularly a non-tuberculous mycobacterium infection or related disease from other diseases, for example, or distinguishing the acid-fast bacterium, particularly a non-tuberculous mycobacterium infection disease from other diseases, for example, a disease of tuberculosis bacteria or leprosy bacteria infection.

[0069] In the 12th embodiment of the present invention, a diagnostic device for acid-fast bacteria (mycobacteria) infection is provided, comprising: (a) a measuring unit for measuring the expression level of a CHI3L1 (Chitinase 3-Like 1) protein for a biological sample obtained from a target individual; and (b) a detection unit for outputting whether or not there is an acid-fast bacteria (mycobacteria) infection from the level measured by the measuring unit.

[0070] In the 13th embodiment of the present invention, a diagnostic device is provided in which, in the 12th embodiment, the diagnosis diagnoses whether a disease has occurred.

[0071] In the 14th embodiment of the present invention, a diagnostic device is provided in which, in the 12th to 13th embodiments, the diagnosis predicts the prognosis.

[0072] In the diagnostic device of the present invention, the descriptions regarding CHI3L1 (Chitinase 3-Like 1), acid-fast bacteria, non-tuberculosis acid-fast bacteria, antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), aptamers, etc., and the descriptions regarding primers, probes, etc. are duplicated from what was previously described, so in order to avoid excessive complexity in the specification, the detailed descriptions thereof are omitted below.

[0073] The pharmaceutical composition of the present invention can be prepared by methods commonly used in the art to which the present invention belongs. The pharmaceutical composition of the present invention can be prepared as an oral or parenteral formulation, preferably as an injectable formulation which is a parenteral formulation, and can be administered via the dermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal, or epidural routes.

[0074] The pharmaceutical composition of the present invention may be administered to an individual in an immunologically effective amount. The “immunologically effective amount” refers to a sufficient amount to produce a preventive effect against tuberculosis and an amount that does not cause side effects or severe or excessive immune responses. The precise dosage concentration varies depending on the specific immunogen to be administered and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the age, weight, health, gender, sensitivity of the individual to drugs, route of administration, and method of administration of the vaccinated person, and may be administered one to several times.

[0075] In addition, the composition provided in the present invention may be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0076] The "prevention" of the present invention may include, without limitation, any act that can block, suppress, or delay symptoms caused by acid-fast bacteria, particularly non-tuberculous acid-fast bacteria, using the composition of the present invention.

[0077] The "treatment" and "improvement" of the present invention may include, without limitation, any act that enables the improvement or benefit of symptoms caused by acid-fast bacteria, particularly non-tuberculous acid-fast bacteria, by using the composition of the present invention.

[0078] In the present invention, the pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans.

[0079] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, it may include bases, excipients, lubricants, preservatives, etc. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0080] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0081] The routes of administration of the pharmaceutical composition according to the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0082] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0083] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0084] A food composition containing the composition of the present invention as an active ingredient can be manufactured in the form of various food products, such as beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts that have almost no toxicity or side effects, it can be used safely even when taken for a long period for preventive purposes.

[0085] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0086] Here, when the above food composition is prepared in the form of a beverage, there are no special limitations other than containing the above food composition in the indicated proportions, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in ordinary beverages. That is, as natural carbohydrates, it may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the above flavoring agents include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0087] In addition, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0088] These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0089] In the present invention, the term "individual" refers to an individual suspected of developing acid-fast bacteria, particularly non-tuberculous acid-fast bacteria. The individual suspected of developing the disease refers to mammals, including rats, livestock, and humans, that have developed or may develop the disease, but any individual capable of being treated with the effective substance provided in the present invention is included without limitation.

[0090] It is evident that a composition for use according to the present invention comprises the said compound, and may also comprise other active compounds in addition to a suitable pharmaceutically acceptable carrier. These may be compounds that enable the activity of such compounds to be enhanced, or even other active agents known for specific activity. Such additional active compounds may be selected from the pharmaceutical classes of agents mentioned in application WO 2015 / 157223, namely antimicrobial agents, antiparasitic agents, neurotransmitter inhibitors, estrogen receptor inhibitors, DNA synthesis and replication inhibitors, protein maturation inhibitors, kinase pathway inhibitors, cytoskeletal inhibitors, lipid metabolism inhibitors, anti-inflammatory agents, ion channel inhibitors, apoptosis inhibitors, and cathepsin inhibitors. Such active compounds may be selected particularly from antimicrobial agents, ion channel inhibitors, immunosuppressants, and antivirals. As an antiviral agent, acyclovir may be specifically mentioned.

[0091] The CHI3L1 (Chitinase 3-Like 1) inhibitor provided in the present invention, specifically kasugamycin, can effectively prevent or treat infections caused by acid-fast bacteria, particularly non-tuberculous mycobacteria, or various diseases caused by such infections, by inhibiting the expression or activation of CHI3L1 (Chitinase 3-Like 1). That is, although kasugamycin does not have a direct antibacterial effect or an anti-mycobacterial effect through the ability to inhibit intracellular MAC strain growth, it was confirmed in vivo that kasugamycin has an antibacterial effect through host-directed therapies (HDTs) by effectively neutralizing CHI3L1.

[0092] In addition, by measuring the expression level of the CHI3L1 (Chitinase 3-Like 1) protein, it is possible to effectively predict the prognosis of an infection with acid-fast bacteria, particularly non-tuberculous mycobacteria, or a disease caused by an infection with acid-fast bacteria, particularly non-tuberculous mycobacteria, or a disease related to an infection with acid-fast bacteria, particularly non-tuberculous mycobacteria, when the expression of the CHI3L1 protein is high compared to the control group.

[0093] Figure 1 shows the results of confirming the expression levels of CHI3L1 in the serum of patients and healthy control subjects through ELISA.

[0094] Figure 2 shows the results of comparing non-tuberculous mycobacterial infections in wild-type mice and CHI3L1 knockout mice using hematoxylin and eosin staining.

[0095] Figure 3 shows the results of comparing tuberculosis infection in wild-type mice and CHI3L1 knockout mice using hematoxylin and eosin staining.

[0096] Figure 4 shows the results of confirming the expression levels of CHI3L1 in the serum of patients requiring disease treatment and those who do not through ELISA.

[0097] Figure 5 shows the results of confirming the expression level of CHI3L1 in serum according to the disease progression rate through ELISA.

[0098] Figure 6 shows the results of classifying the serum CHI3L1 expression levels of 145 MAC-infected lung disease patients who had not started antibiotic treatment into five clinical variables used to calculate the BACES score.

[0099] Figure 7 shows the results of calculating the BACES scores of 145 patients with MAC-infected lung disease who had not started antibiotic treatment, applying the criteria specified in Example 5-2.

[0100] Figure 8 shows the results of comparing the antibacterial activity of each antibiotic with the negative control, positive control, and drug-treated groups.

[0101] Figure 9 shows the results of evaluating the intracellular growth inhibitory ability of MAC strains.

[0102] Figure 10 shows the results of evaluating the ability of kasugamycin to inhibit CHI3L1 protein in lung tissue.

[0103] Figure 11 is a schematic diagram showing a mouse experiment design.

[0104] Figure 12 shows the results of a comparative analysis of the expression levels of CHI3L1 protein in serum and lung tissue by mouse group using ELISA.

[0105] In Figure 13, A confirms that the bacterial count in lung tissue decreased significantly in a concentration-dependent manner when kasugamycin was administered to mice, B confirms that the bacterial count in spleen tissue decreased significantly in a concentration-dependent manner when kasugamycin was administered to mice, and C is the result of visualizing the degree of inflammation by performing H&E staining on lung tissues between each group.

[0106] In Figure 14, A confirms that the amount of bacteria in lung tissue is significantly reduced when K284-6111 is administered to WT mice, B confirms that the degree of histopathological inflammation in the lung tissue of WT mice is significantly reduced when K284-6111 is administered, and C is the result of visualizing the degree of inflammation by performing H&E staining on lung tissues between each group.

[0107] Figure 15 is the chemical structural formula of the Kasugamycin–BODIPY compound.

[0108] Figure 16 is the fluorescence absorption and emission spectrum of Kasugamycin–BODIPY.

[0109] Figure 17 shows the inhibitory effect of CHI3L1 protein in lung tissue lysates according to Kasugamycin–BODIPY treatment concentrations.

[0110]

[0111] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0112] Examples

[0113] [Example 1] Confirmation of increased expression of CHI3L1 (Chitinase 3-Like 1) in serum of patients with MAC-infected lung disease compared to healthy control group

[0114] In order to confirm the potential of CHI3L1 as a diagnostic biomarker for MAC-infected lung disease, the present invention compared the expression levels of CHI3L1 in the serum of patients with MAC-infected lung disease and healthy control subjects using ELISA.

[0115] [Example 1-1] Collection and storage of patient and healthy control group samples

[0116] For the study of the present invention, 145 serum samples from patients with MAC-infected lung disease who had not started antibiotic treatment and 29 serum samples from healthy control subjects were provided from Samsung Hospital in Seoul for approximately 6 years from January 2012 to August 2016. In addition, 5 additional samples were purchased for the healthy control subjects. All samples were stored in an ultra-low temperature freezer at minus 80°C or below and were used after being completely thawed immediately before analysis.

[0117] [Examples 1-2] Confirmation of serum CHI3L1 expression levels via ELISA

[0118] In this invention, experiments were conducted using the ELISA (Enzyme-Linked Immunosorbent Assay) method. Specifically, the DY2599 ELISA kit and the DY008 add-on reagent kit from R&D Systems, which are commercial products developed to detect CHI3L1 protein, were used to measure the expression level of CHI3L1 in serum samples. During the experiment, serum samples were processed according to the usage method of the DY2599 ELISA kit, and the expression level of CHI3L1 was quantified through steps such as sample dilution and processing. Through this, the difference in CHI3L1 expression levels between patients with MAC-infected lung disease and healthy control subjects was compared and analyzed.

[0119] More specifically, first, the capture antibody was diluted to the specified working concentration in protein-free PBS, and 100 µL was added to each 96-well microplate. The plates were then sealed and incubated overnight at 4°C. On the next day, each well was washed three times with the wash solution. 400 µL of wash solution was added to each well, and the solution was completely removed after each wash step. After the final wash, the plates were inverted onto a clean paper towel to remove any remaining wash solution, and 300 µL of assay buffer solution was added to each well to seal the plates. They were then incubated at room temperature for one hour. The plates prepared in this manner were subjected to three aspiration / washing cycles in the same manner as before.

[0120] Next, 100 µL of a total of eight solutions (including a blank), prepared by diluting the serum sample and a standard solution of known concentration in assay buffer by 1 / 2, were added to each well. The surface was then covered with an adhesive strip and incubated at room temperature for 2 hours. Subsequently, each well was washed by repeating the aspiration / washing process three times as before. Next, 100 µL of detection antibody diluted in assay buffer was added to each well. The well was covered with a new adhesive strip and incubated at room temperature for 2 hours. The washing process was repeated once more.

[0121] Next, 100 µL of the working concentration of streptavidin-HRP was added to each well. The plates were wrapped in foil to protect them from direct light and incubated at room temperature for 20 minutes. Finally, the washing process was completed by repeating the plate aspiration / washing cycle. In the final step, 100 µL of substrate solution was added to each well and incubated at room temperature for 20 minutes. Direct light was blocked during this time. Lastly, 50 µL of stop solution was added to each well. After gently tapping the plates to ensure thorough mixing, the optical density of each well was measured at 450 nm using a microplate reader.

[0122] As a result, as shown in Figure 1, it was confirmed that the expression level of CHI3L1 was significantly higher in the serum of 145 MAC-infected lung disease patients (MAC-PD patients) who had not started antibiotic treatment compared to 34 healthy controls. This suggests that CHI3L1 is useful as a diagnostic biomarker for MAC-infected lung disease.

[0123] [Example 2] Comparison of Mycobacteria Infection Between Wild-Type Mice and CHI3L1 Knockout Mice

[0124] In order to confirm the contribution of the CHI3L1 protein to the progression of acid-fast bacilli infection, the disease progression was assessed through infection of mice with non-tuberculosis acid-fast bacilli and tuberculosis bacteria.

[0125] [Example 2-1] Comparison of Nontuberculous Mycobacteria (NTM) Infections in Wild-Type Mice and CHI3L1 Knockout Mice

[0126] Wild-type mice (WT) and CHI3L1 knockout mice (CHI3L1 KO) were infected with M. avium SMC#7, a non-tuberculous mycobacterial strain isolated from clinical patients, via air infusion. All mice were necropsied at 10 weeks post-infection to measure bacterial counts and lesions in lung tissue. For the measurement of lung tissue bacterial counts, lung tissues were isolated from each group and ground using sterile PBS (phosphate-buffered saline) to prepare lysates. The lysates were diluted in PBS to 1 / 100, 1 / 1000, and 1 / 10000 and dropped at a dose of 50 µl per well onto pre-prepared 7H10 solid media. The cultures were incubated in a microbial incubator for approximately 10 days, and the bacterial count after 10 days was measured. Lung tissue lesions were confirmed using hematoxylin and eosin staining.

[0127] As a result, as shown in Figure 2, it was confirmed that the bacterial count and the degree of histopathological inflammation in the lung tissue of WT mice were significantly higher than those of CHI3L1 KO mice. These results suggest that CHI3L1 plays an important role in nontuberculous mycobacteria (NTM) infections.

[0128] [Example 2-1] Comparison of Mycobacterium tuberculosis infection in wild-type mice and CHI3L1 knockout mice

[0129] Wild-type mice (WT) and mice with CHI3L1 knockout (CHI3L1 KO) were infected with the standard Mycobacterium tuberculosis strain H37Rv via air infusion. All mice were necropsied at 4 weeks post-infection to measure bacterial counts and lesions in lung tissue.

[0130] As a result, as shown in Figure 3, it was confirmed that unlike non-tuberculous mycobacteria, the bacterial count and the degree of histopathological inflammation in the lung tissue of CHI3L1 KO mice were significantly higher than those of WT mice in cases of tuberculosis infection.

[0131] [Example 3] Potential of CHI3L1 as a Predictive Marker for Disease Progression in Patients with MAC-Infected Lung Disease

[0132] In order to confirm the potential of CHI3L1 as a biomarker for initiating treatment in patients with MAC-infected lung disease, the present invention compared the expression levels of CHI3L1 in the serum at the time of diagnosis of patients with MAC-infected lung disease who started antibiotic treatment after disease progression and patients whose disease did not progress, using ELISA.

[0133] [Example 3-1] Collection and storage of patient samples

[0134] For the study of the present invention, 89 serum samples at the time of diagnosis from patients with MAC-infected lung disease were obtained from Samsung Hospital in Seoul over a period of approximately 6 years, from January 2012 to August 2016. Of these, 29 serum samples were from patients who received antibiotic treatment as their disease had progressed, and 60 serum samples were from patients who did not receive antibiotic treatment as their disease had not progressed. All samples were stored in an ultra-low temperature freezer at minus 80°C or lower and were used after being completely thawed immediately before analysis.

[0135] [Example 3-2] Confirmation of serum CHI3L1 expression levels via ELISA

[0136] The expression level of CHI3L1 in a patient's serum sample was measured using the DY2599 ELISA kit from R&D Systems with the same analysis method as in Examples 1-2.

[0137] As a result, as shown in Figure 4, follow-up observations confirmed that the expression level of CHI3L1 in the serum samples at the time of diagnosis of patients who had progressed and received antibiotic treatment (Progressor) was significantly higher than that in the serum samples at the time of diagnosis of patients whose disease had not progressed (Non-progressor). This suggests that CHI3L1 is useful as a diagnostic biomarker for the progression of MAC-infected lung disease. In other words, the level of CHI3L1 expression can be used to distinguish between patients who require the initiation of treatment for MAC-infected lung disease and those who do not.

[0138] [Example 4] Potential of CHI3L1 as a Predictive Marker for Disease Progression Rate in Patients with MAC-Infected Lung Disease

[0139] [Example 4-1] Confirmation of serum CHI3L1 expression levels according to disease progression rate

[0140] Using the DY2599 ELISA kit from R&D Systems, the expression levels of CHI3L1 were compared according to the period from the date of diagnosis to the date of treatment start in patients with disease progression using the same analysis method as in Examples 1-2.

[0141] As a result, as shown in Figure 5, among the serum samples at the time of diagnosis (Progressor) of patients who received antibiotic treatment due to disease progression, it was confirmed that the CHI3L1 expression level in the group that started treatment within 180 days of diagnosis due to rapid disease progression was significantly higher than the CHI3L1 expression level in the group that started treatment after 180 days. This suggests that the higher the expression level of CHI3L1 at the time of diagnosis, the faster the disease progression.

[0142] [Example 5] Comparison of BACES scores and CHI3L1 expression levels in patients with MAC-infected lung disease

[0143] The present invention utilized the BACES score, which is identified as an aggravating factor for non-tuberculosis lung disease infection in relation to disease progression, treatment failure, and mortality in MAC-infected lung disease, to perform a comparison between the patient's BACES score and the expression level of CHI3L1.

[0144] [Example 5-1] Collection and storage of patient samples

[0145] For the study of the present invention, 145 serum samples were obtained from patients with MAC-infected lung disease who had not started antibiotic treatment at Seoul Samsung Hospital for approximately 6 years from January 2012 to August 2016. All samples were stored in an ultra-low temperature freezer at minus 80°C or lower and were used after being completely thawed immediately before analysis.

[0146] [Example 5-2] BACES Score Analysis

[0147] The BACES score is a recently developed simple numerical model that includes five clinical variables: Body Mass Index (BMI), Age, Cavity, Erythrocyte Sedimentation Rate (ESR), and Sex. This scoring system was developed and validated at two different centers in Korea and demonstrated excellent performance in predicting all-cause mortality. The BACES score can be used as a useful tool to assess a patient's overall prognosis and serves as an important indicator for predicting mortality and the progression of the disease. The BACES score was calculated by adding one point for each of the following five variables.

[0148] 1. BMI < 18.5 kg / m² 2

[0149] 2. Age ≥ 65

[0150] 3. Co-existence on Chest CT

[0151] 4. ESR (Male > 15 mm / h, Female > 20 mm / h)

[0152] 5. Male gender

[0153] As a result, as shown in Figure 6, serum CHI3L1 expression levels in 145 patients with MAC-infected lung disease who had not started antibiotic treatment were classified according to the five clinical variables used to calculate the BACES score. It was confirmed that patients corresponding to the variables other than BMI (age ≥ 65 years, presence of cavities on chest CT, ESR (male > 15 mm / h, female > 20 mm / h), male gender) had significantly higher CHI3L1 expression levels than patients not corresponding to the variables.

[0154] In addition, Figure 7 shows the BACES scores of 145 patients with MAC-infected lung disease who had not started antibiotic treatment, calculated by applying the criteria specified in Example 5-2, and based on this, were classified into two groups as follows: a mild group (BACES score 0-1) and a severe group (BACES score 2-4). It was confirmed that the expression levels of CHI3L1 were significantly higher in patients of the severe group compared to patients of the mild group. This suggests that the expression levels of CHI3L1 are useful as a prognostic marker for MAC-infected disease.

[0155] [Example 6] Evaluation of the direct antimicrobial effect of Kasugamycin on MAC strains

[0156] To confirm the anti-mycobacterial effect of Kasugamycin, the minimum inhibitory concentration (MIC) was measured along with antibiotics.

[0157] [Example 6-1] Method for culturing non-tuberculous acid-mycobacterial strains

[0158] To culture the non-tuberculous mycobacterial strain, Middlebrook 7H9 medium containing 10% OADC (oleic acid, albumin, dextrose, catalase) was used as the liquid medium. During the culture process, the target strain was inoculated into the prepared medium and cultured via shaking incubation at 37 °C. Culture was continued until the absorbance measured at 600 nm reached between 0.3 and 0.5. Subsequently, to evaluate the minimum inhibitory concentration, the strain was cultured at a concentration corresponding to an absorbance of 0.01 measured at 600 nm (approximately 5 × 10⁻⁶). 5 It was used after dilution (CFU / mL).

[0159] [Example 6-2] Evaluation of Minimum Inhibitory Concentration of Kasugamycin on MAC Strain

[0160] To evaluate the minimum inhibitory concentration of kasugamycin against MAC strains, experiments were conducted on three MAC strains. The strains used were the standard strain M. avium ATCC 700898, the clinical strain M. avium SMC#7 provided by Samsung Hospital in Seoul, and M. intracellulare ATCC 13950. Each strain was prepared according to the method described in Example 1-1, and the MIC evaluation was performed based on the liquid medium colorimetric method (Resazurin Microtiter Assay; REMA) in compliance with Clinical and Laboratory Standards Institute (CLSI) guidelines.

[0161] More specifically, antibiotic susceptibility testing of non-tuberculous mycobacteria was performed using the liquid dilution method with 7H9 liquid medium. Experiments were conducted with Kasugamycin (KSM), along with Amikacin (AMK), an aminoglycoside antibiotic, and Clarithromycin (CLR), a macrolide antibiotic widely used as a standard treatment for non-tuberculous mycobacteria. Each drug was serially diluted twofold within the range of 0.125 µg / mL to 64 µg / mL, and 100 µL aliquots were dispensed into 96-well plates, starting from the highest concentration. Subsequently, at a wavelength of 600 nm, an absorbance of 0.01 (approx. 5×10⁻⁶) 5 100 uL of strain suspension diluted to CFU / mL was added to each well and cultured at 37 ℃ for one week.

[0162] As controls, a negative control (NC) consisting only of culture medium (7H9 liquid medium) and a positive control (PC) consisting only of cultured strain were included. After one week of culture, 22 μL of 0.02% Resazurin colorimetric solution was added to each well, and the cells were cultured for an additional 24 hours at 37°C. After 24 hours, the antibacterial activity of each antibiotic was evaluated by comparing it with the negative control, positive control, and drug-treated groups.

[0163] As a result, as shown in Fig. 8, the degree of bacterial growth can be confirmed through the color change of the culture medium in each well. According to the colorimetric method using Resazurin dye, the more the color changes from purple to blue, the more bacterial growth is inhibited.

[0164] The above strains were identified at a minimum inhibitory concentration (MIC) of 0.5 µg / mL or less upon treatment with clarithromycin (CLR), and at a concentration of 8 µg / mL or less upon treatment with amikacin (AMK). On the other hand, in the case of kasugamycin (KSM), no inhibitory effect was observed even at a concentration of 64 µg / mL. Consequently, the direct antibacterial effect of kasugamycin (KSM) on MAC strains could not be confirmed. The minimum inhibitory concentration (MIC) values ​​for the above drug treatments are summarized in detail in Table 1 below.

[0165]

[0166] [Example 7] Evaluation of Inhibitory Activity of Intracellular MAC Strain Growth by Kasugamycin

[0167] As confirmed in Example 6 above, although the direct antibacterial effect of kasugamycin on MAC strains could not be confirmed, in order to determine whether kasugamycin exerts an anti-mycobacterial effect through the ability to inhibit intracellular growth of MAC strains, colony forming units (CFU) were measured after treating macrophages infected with MAC strains with kasugamycin.

[0168] [Example 7-1] Cell culture method

[0169] Macrophages used in the MAC infection experiment were isolated from 6–8 week old BALB / c mouse bone marrow cells. First, a medium was prepared by mixing 10% L929 cell line culture supernatant into High Glucose DMEM (Biowest Inc., Nuaile, France) containing 10% FBS (Biowest Inc., Nuaile, France), 100 U / mL penicillin, and 100 µg / mL streptomycin (Biowest Inc., Nuaile, France). The prepared medium was used to induce differentiation of macrophages.

[0170] 10 mL of this culture medium was dispensed into a 90 x 15 mm Petri dish (SPL Life Science, Pocheon, South Korea) and cultured for 3 days in a cell culture incubator at 37°C maintained at a 5% carbon dioxide concentration. Afterward, an additional 10 mL of the same culture medium was added to the Petri dish, and the cells were cultured for an additional 3 to 4 days to sufficiently differentiate macrophages before being used in the experiment.

[0171] [Example 7-2] MAC Infection and Antibiotic Treatment

[0172] Differentiated macrophages were isolated using Trypsin-EDTA, and then transferred to 48-well cell culture plates (SPL Life Science) at a rate of 2 x 10⁶ per well. 5Cells were inoculated and attachment culture was performed for 24 hours. After removing the existing culture medium of the cultured macrophages, the M. avium SMC#7 strain isolated from clinical patients was mixed into a culture medium with a reduced FBS concentration of 5% at a Multiplicity of Infection (MOI) of 3 and dispensed into each well, and the cells were infected for 4 hours. After removing the infected culture medium, antibiotics were added to a new culture medium containing 5% FBS. The antibiotics added were two concentrations of clarithromycin (CLR) (1 µg / mL, 10 µg / mL), two concentrations of amikacin (AMK) (10 µg / mL, 50 µg / mL), and two concentrations of kasugamycin (KSM) (1 µg / mL, 10 µg / mL). After treatment with this antibiotic culture medium, the cells were cultured for 3 days in a cell culture incubator at 37°C maintained at a carbon dioxide concentration of 5%.

[0173] [Example 7-3] Preparation of 7H10 solid medium for MAC culture

[0174] 450 mL of distilled water and 9.5 g of Difco™ Middlebrook 7H10 Agar (BD Bioscience) powder were added to a 1 L Erlenmeyer flask and mixed. The mixture was sterilized using an autoclave at 121 °C for 15 minutes. After sterilization, the 7H10 agar medium was cooled to approximately 60 °C, and 10% OADC was added. 23 mL of the prepared medium was dispensed into each 90 x 15 mm Petri dish (SPL Life Science), dried for one day, and then used for the experiment.

[0175] [Example 7-4] CFU Confirmation

[0176] After removing the culture medium of macrophages infected with M. avium SMC#7 from a 48-well cell culture plate, the plates were washed twice using DPBS (Biowest Inc). After washing, 200 µL of 1% Triton X-100 diluted to 0.05% was added to each well, and the macrophage membranes were lysed for 10 minutes. The lysates released from the lysed macrophages were diluted at ratios of 1 / 100 and 1 / 1000, and then 50 µL of each was inoculated into a pre-prepared 7H10 solid medium. Subsequently, the inoculated medium was cultured in a microbial incubator for approximately 10 days, and the Colony Forming Unit (CFU) was determined based on the number of cultured bacteria.

[0177] As a result, as shown in Figure 9, CFU increased in the control group of macrophages cultured for 72 hours (3 days) after infection with M. avium SMC#7, whereas CFU decreased in a concentration-dependent manner in the experimental group continuously treated with clarithromycin (CLR) 4 hours after infection. On the other hand, no significant change in CFU was observed regardless of concentration in the groups treated with amikacin (AMK) and kasugamycin (KSM). Through this, it was confirmed that amikacin (AMK) and kasugamycin (KSM) do not have an inhibitory effect on intracellular MAC growth.

[0178] That is, as confirmed in Example 6 above, kasugamycin not only has no direct antibacterial effect on MAC strains, but as additionally confirmed in Example 7 above, it was confirmed that kasugamycin also does not exert an inhibitory effect on intracellular MAC strain growth.

[0179] [Example 8] Evaluation of CHI3L1 Inhibitory Activity by Kasugamycin

[0180] As previously observed in Examples 6 and 7, the direct antibacterial effect of kasugamycin and the anti-mycobacterial effect through the inhibition of intracellular MAC strain growth could not be confirmed. However, in order to confirm the potential for the antibacterial effect of kasugamycin through host-directed therapies (HDTs) by effectively neutralizing CHI3L1, this example was conducted to evaluate whether kasugamycin (KSM) effectively neutralizes CHI3L1 protein in lung tissue. To this end, analysis was performed using lung tissue lysates obtained by necropsy 10 weeks after airborne infection with M. avium in BALB / C wild-type (WT) mice.

[0181] [Example 8-1] Confirmation of Kasugamycin’s Inhibitory Ability of CHI3L1 in Lung Tissue via ELISA

[0182] In this study, the expression level of CHI3L1 protein in samples was measured using an enzyme-linked immunosorbent assay (ELISA). For the analysis, CHI3L1 protein was specifically detected using the DY2649 ELISA kit and the DY008 auxiliary reagent kit from R&D Systems. Lung tissue samples were homogenized with sterile PBS to prepare a lysate, which was then diluted 1:2500 in PBS for use in the ELISA analysis.

[0183] First, the capture antibody was diluted to the working concentration with protein-free PBS, and 100 µL was added to each 96-well microplate. The plates were then sealed and incubated overnight at 4°C. The next day, each well was washed three times with wash solution, with the solution completely removed after each wash step. After the final wash, the plates were inverted onto a clean paper towel to remove any remaining wash solution, and then 300 µL of assay buffer was added to each well to block the plates. The plates were incubated at room temperature for 1 hour. After incubation, the washing process was repeated three times as before. During this time, equal volumes of lung tissue lysates were treated with kasugamycin at concentrations of 10 ng / mL, 100 ng / mL, 1 µg / mL, and 10 µg / mL, respectively, and with K284-6111, known as a CHI3L1 inhibitor, at 10 µg / mL as a positive control, followed by rotational incubation at room temperature for 2 hours. Subsequently, 100 µL of a total of eight solutions (including blanks), prepared by diluting the sample under the corresponding conditions with a standard solution of known concentration at a 1:2 ratio, were added to each well. The plates were covered with adhesive strips and incubated at room temperature for 2 hours. Afterward, each well was washed by repeating the aspiration and washing process three times. Next, 100 µL of the detection antibody, diluted with assay buffer, was added to each well, covered with an adhesive strip, and incubated at room temperature for 2 hours. After incubation, the aspiration and washing process was performed again three times. Subsequently, 100 µL of streptavidin-HRP diluted to the working concentration was added to each well. The plates were wrapped in foil to block light and incubated at room temperature for 20 minutes. Finally, the plates were washed by repeating the aspiration and washing process. In the final step, 100 µL of the substrate solution was added to each well, and the plates were incubated for 20 minutes in a light-blocked environment. Once the culture was complete, 50 uL of suspension solution was added to each well.After thoroughly mixing the solution by gently tapping the plate, the optical density was measured at 450 nm using a microplate reader. This allowed for the quantitative analysis of the reduction of CHI3L1 protein in lung tissue lysates following kasugamycin treatment.

[0184] As a result, as shown in Figure 10, it was confirmed that the CHI3L1 protein was significantly reduced in a concentration-dependent manner upon incubation with kasugamycin. In addition, it was confirmed that it had a higher inhibitory ability than K284-6111, an inhibitor of CHI3L1, at the same concentration.

[0185] [Example 9] Confirmation of inhibition of disease progression following MAC infection and administration of CHI3L1 inhibitor in wild-type mice

[0186] In Example 8, it was previously confirmed that a CHI3L1 inhibitor can effectively neutralize the CHI3L1 protein. Accordingly, this example was performed to evaluate whether a CHI3L1 inhibitor can effectively neutralize the CHI3L1 protein in lung tissue and inhibit disease progression through host-directed therapies (HDTs). To this end, MAC was air-infected into BALB / C wild-type mice (wild-type; WT) and analysis was performed.

[0187] [Example 9-1] Animal Experiment Design

[0188] Wild-type BALB / C mice were air-infected with M. avium SMC#7. Starting two weeks after infection, the mice were divided into groups, and kasugamycin was administered intraperitoneally to the mice at concentrations of 10 mg / kg and 100 mg / kg, respectively, three times a week for a total of eight weeks. All mice were necropsied simultaneously at week 10 of infection to evaluate the experimental results.

[0189] experimental group

[0190] 1. Naive group

[0191] 2.M. aviumSMC#7 Infection Group (Infection)

[0192] 3.M. aviumSMC#7 infection + Kasugamycin 10 mg / kg administration group (KSM 10 mg / kg)

[0193] 4.M. avium SMC#7 infection + Kasugamycin 100 mg / kg administration group (KSM 100 mg / kg)

[0194] Figure 11 shows a schematic diagram of the mouse experimental design. Female BALB / C mice were airbornely infected with M. avium SMC#7, and starting at 2 weeks of infection, some of the mouse groups were administered kasugamycin intraperitoneally at two concentrations (10 mg / kg, 100 mg / kg) three times a week for 8 weeks. All mice were necropsied at 10 weeks of infection.

[0195] [Example 9-2] Confirmation of CHI3L1 expression levels in serum and lung tissue via ELISA

[0196] In this study, the expression level of CHI3L1 protein in lung tissue samples was measured using the Enzyme-Linked Immunosorbent Assay (ELISA) technique. For this analysis, CHI3L1 protein was specifically detected using the DY2649 ELISA kit and the DY008 auxiliary reagent kit provided by R&D Systems. Serum was diluted 1:50 in sterile PBS for use, and lung tissue samples were homogenized using sterile PBS to produce a lysate, which was then diluted 1:2500 in PBS for ELISA analysis.

[0197] More specifically, first, the capture antibody was diluted to the specified working concentration in protein-free PBS, and 100 µL was added to each 96-well microplate. The plates were then sealed and incubated overnight at 4°C. On the next day, each well was washed three times with the wash solution. 400 µL of wash solution was added to each well, and the solution was completely removed after each wash step. After the final wash, the plates were inverted onto a clean paper towel to remove any remaining wash solution, and then 300 µL of analysis buffer solution was added to each well to seal the plates. They were then incubated at room temperature for one hour. The plates prepared in this manner were subjected to three aspiration / washing cycles in the same manner as before.

[0198] Next, 100 µL of a total of eight solutions (including a blank), prepared by diluting serum and lung tissue samples and standard solutions of known concentration in the analysis buffer by 1 / 2, were added to each well. The surfaces were then covered with adhesive strips and incubated at room temperature for 2 hours. Subsequently, each well was washed by repeating the aspiration / washing process three times as before. Next, 100 µL of detection antibody diluted in the analysis buffer was added to each well. The wells were covered with fresh adhesive strips and incubated at room temperature for 2 hours. The washing process was performed once again by repeating the aspiration / washing process.

[0199] Next, 100 µL of the working concentration of streptavidin-HRP was added to each well. The plates were wrapped in foil to protect them from direct light and incubated at room temperature for 20 minutes. Finally, the washing process was completed by repeating the plate aspiration / washing cycle. In the final step, 100 µL of substrate solution was added to each well and incubated at room temperature for 20 minutes. Direct light was blocked during this time. Lastly, 50 µL of suspension solution was added to each well. After gently tapping the plates to ensure thorough mixing, the optical density of each well was measured at 450 nm using a microplate reader. Through this procedure, the reduction in CHI3L1 protein expression caused by kasugamycin treatment was quantitatively analyzed.

[0200] As a result, as shown in Figure 12, it was confirmed that the CHI3L1 protein expression, which is increased due to MAC infection, was significantly reduced in a concentration-dependent manner by the administration of kasugamycin.

[0201] [Example 9-3] Analysis of Colony Forming Units (CFU) in Lung and Spleen Tissues in Mouse Groups

[0202] In this experiment, tissue samples were homogenized using sterile PBS to quantify the Colony Forming Unit (CFU) of microorganisms in the lung and spleen tissues of animals in each experimental group. These homogenized samples were diluted with PBS at ratios of 1 / 100, 1 / 1000, and 1 / 10000. Subsequently, 50 µL of the diluted samples were inoculated onto Middlebrook 7H10 solid medium. This medium was cultured in a microbial incubator at 37°C for approximately 10 days. After the culture period ended, the number of formed colonies was counted and expressed as CFU.

[0203] [Example 9-4] Evaluation of pathological changes in lung tissue within animal experimental groups

[0204] In this study, to evaluate pathological changes in the lung tissue of each experimental animal group, lung perfusion was performed using sterile PBS, after which one lobe of lung tissue from each animal was isolated. Subsequently, the tissue was fixed in a 4% paraformaldehyde solution for 24 hours and then further fixed with paraffin. The paraffin-fixed lung tissue was prepared into 5 µm-thick sections. Pathological changes in the prepared lung tissue sections were visualized using hematoxylin and eosin staining to confirm the presence and extent of lesions within the lung tissue.

[0205] As a result, as shown in Fig. 13, it was confirmed that the bacterial count in lung tissue and spleen tissue was significantly reduced when kasugamycin, a CHI3L1 inhibitor, was administered, and as shown in Fig. 14, it was also confirmed that the bacterial count in lung tissue was significantly reduced when K284-6111, a CHI3L1 inhibitor, was administered.

[0206] That is, as seen in Examples 6 and 7, although the direct antibacterial effect of kasugamycin and the anti-mycobacterial effect through the ability to inhibit intracellular MAC strain growth could not be confirmed, it was confirmed that kasugamycin effectively neutralized CHI3L1, and through this, it was confirmed in vivo that CHI3L1 inhibitors have an antibacterial effect through host-directed therapies (HDTs).

[0207]

[0208] [Example 10] Synthesis of fluorescently labeled Kasugamycin–BODIPY conjugate and verification of CHI3L1 inhibitory activity

[0209] To visually confirm the binding and inhibitory properties of kasugamycin to the CHI3L1 protein and to evaluate whether the biological activity of kasugamycin derivatives is preserved, a compound (Kasugamycin–BODIPY conjugate) in which a BODIPY fluorescent group is attached to kasugamycin was synthesized.

[0210]

[0211] 10-1. Synthesis of Kasugamycin–BODIPY Complex

[0212] The compound was synthesized by reacting the 2'-amino terminal of kasugamycin with BODIPY–NHS ester (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene–NHS ester), and the reaction was carried out by stirring at room temperature for 2 hours in the presence of triethylamine in dimethylformamide (DMF) solvent. The product was purified to a single peak by HPLC, and the synthesis of the compound was confirmed by LC–MS analysis, which detected a 737.6 Da [M+H]+ peak in which a BODIPY substituent was bound to the kasugamycin monomer (MW = 485.4 Da). The chemical structure of the synthesized kasugamycin–BODIPY compound is shown in Figure 15.

[0213]

[0214] 10-2. Evaluation of Fluorescence Properties of Kasugamycin–BODIPY Conjugates

[0215] To evaluate the fluorescence properties of the synthesized Kasugamycin–BODIPY compound, absorption and emission spectra were measured in the range of 200–700 nm. As a result, as shown in Figure 16, the compound exhibited a maximum absorption wavelength of 495 ± 2 nm and a maximum fluorescence emission of 510 ± 3 nm, which corresponded to a red shift of approximately 2–3 nm compared to the BODIPY alone fluorophor. The fluorescence intensity was preserved at approximately 93% of that of BODIPY, confirming that the binding of Kasugamycin had almost no effect on the photophysical properties of BODIPY.

[0216]

[0217] 10-3. Evaluation of CHI3L1 Protein Inhibitory Activity by Kasugamycin–BODIPY Conjugate

[0218] The binding and blocking ability of the synthesized Kasugamycin–BODIPY compound to CHI3L1 protein in MAC-infected mouse lung tissue lysates was evaluated using the same method as in Example 7. As a result, as shown in Figure 17, when Kasugamycin–BODIPY was applied to lysates containing CHI3L1 protein at concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL, the expression of CHI3L1 protein decreased in a concentration-dependent manner. Overall, Kasugamycin–BODIPY showed an inhibitory trend similar to that of unlabeled Kasugamycin, with slight differences in some concentration ranges. In particular, at concentrations of 10 ng / mL or higher, both compounds inhibited the expression of CHI3L1 protein by more than 50%, and the same trend was confirmed in the fluorescently labeled conjugates. These results indicate that the binding and inhibitory functions against the CHI3L1 protein are maintained even when a fluorescent group is attached to kasugamycin, suggesting that this compound can be utilized as a fluorescent labeling tool to study the mechanism of CHI3L1 inhibition or to track drug distribution and protein binding in real time.

[0219]

[0220] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An antimicrobial composition comprising a CHI3L1 (Chitinase 3-Like 1) inhibitor as an active ingredient.

2. In Paragraph 1, The above antimicrobial agent is an antimicrobial agent against acid-fast bacteria (Mycobacteria), and is an antimicrobial composition.

3. In Paragraph 2, An antimicrobial composition in which the above acid-fast bacteria is one or more selected from the group consisting of Mycobacterium tuberculosiscomplex, Mycobacterium leprae, or nontuberculous mycobacteria (NTM).

4. In Paragraph 3, The above nontuberculous mycobacteria (NTM) group includes Mycobacterium avium (Mav), Mycobacterium abscessus subsp. abscessus (Mabc), Mycobacterium abscessus subsp. massiliense (Mmass), and Mycobacterium abscessus subsp. bolletii.bolletii), Mycobacterium Intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium An antimicrobial composition selected from the group consisting of Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum, Mycobacterium xenopi, and Mycobacterium aubagnens.

5. In Paragraph 1, The above CHI3L1 (Chitinase 3-Like 1) inhibitor is one or more selected from the group consisting of antisense nucleotides, siRNA, shRNA, and ribozymes that bind complementarily to the mRNA of a gene encoding CHI3L1, or one or more selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that bind complementarily to the Pierce 1 protein, an antimicrobial composition.

6. In Paragraph 5, The above-mentioned CHI3L1 (Chitinase 3-Like 1) inhibitor is one or more selected from the group consisting of K284-6111, Kasgamycin, Kasgamycin-fluorescent compound conjugate, G721-0282, and Darifenacin, an antimicrobial composition.

7. A pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infections, comprising an antimicrobial composition according to any one of claims 1 to 6 as an active ingredient.

8. In Paragraph 7, A pharmaceutical composition wherein the above-mentioned non-tuberculous mycobacterial infectious disease is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease.

9. A composition for diagnosing acid-fast bacilli (Mycobacteria) infection comprising a preparation for measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein.

10. In Paragraph 9, The above diagnostic composition is intended to be applied to a biological sample isolated from a target individual.

11. In Paragraph 10, The above biological samples include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, and tracheal secretions (organ A diagnostic composition comprising one or more selected from the group consisting of secretions, cells, cell extracts, and cerebrospinal fluid.

12. In Paragraph 11, A diagnostic composition for which the above diagnosis is to diagnose whether a disease has occurred.

13. In Paragraph 11, A diagnostic composition in which the above diagnosis predicts the prognosis.

14. A diagnostic kit comprising a diagnostic composition according to any one of claims 9 to 13.

15. In Paragraph 14, The above kit is a diagnostic kit, which is an RT-PCR kit, DNA chip kit, ELISA kit, protein chip kit, rapid kit, or MRM (Multiple reaction monitoring) kit.

16. From a biological sample isolated from the target individual, A method for providing information for the diagnosis of acid-fast bacilli (Mycobacteria) infection, comprising the step of measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein.

17. In Paragraph 16, The above biological samples include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, and tracheal secretions (organ A method for providing information comprising one or more selected from the group consisting of secretions, cells, cell extracts, and cerebrospinal fluid.

18. In Paragraph 17, The above diagnosis is a method of providing information that diagnoses whether a disease has occurred.

19. In Paragraph 18, The above diagnosis is an information provision method that predicts the prognosis.

20. With respect to biological samples obtained from the target individual, (a) a measuring unit for measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein; and (b) A detection unit that outputs the presence or absence of acid-fast bacteria (Mycobacteria) infection from the level measured by the measurement unit; comprising an acid-fast bacteria (Mycobacteria) infection diagnostic device.

21. In Paragraph 20, The above diagnosis is a diagnostic device that diagnoses whether a disease has occurred.

22. In Paragraph 20, A diagnostic device for predicting the prognosis of the above diagnosis.

23. A method for preventing or treating a non-tuberculous mycobacterial infection, comprising the step of administering an antimicrobial composition of any one of claims 1 to 6 to a subject.

24. In Paragraph 23, A method for preventing or treating a non-tuberculous mycobacterial infection, wherein the above-mentioned non-tuberculous mycobacterial infection is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease.

25. A method for diagnosing acid-fast bacilli (Mycobacteria) infection comprising a preparation for measuring the expression level of CHI3L1 (Chitinase 3-Like 1) protein.

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