KR102499891B1 - biomarker composition for diagnosing inflammatory bowel disease and use thereof - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing inflammatory bowel disease and its use, based on the increased expression of Nsg-2 in tissues of patients with inflammatory bowel disease, inhibiting Nsg-2 to suppress intestinal atrophy and suppress enteritis In addition, it was confirmed that the expression of IL-17 and IFN-γ was significantly suppressed, and the fraction of CD4+ T cells was also reduced, indicating that Nsg-2 can be used as a therapeutic target and diagnostic biomarker for inflammatory bowel disease. show the possibilities

Description

Biomarker composition for diagnosing inflammatory bowel disease and use thereof {biomarker composition for diagnosing inflammatory bowel disease and use thereof}

The present invention provides a biomarker composition for diagnosing inflammatory bowel disease and its use.

Inflammatory bowel disease is a chronic, intractable disease, and the incidence of inflammatory bowel disease in Korea is rapidly increasing. Inflammatory bowel disease is prevalent in adolescence and in the 20s to 40s, reducing the labor force of young people who should be most active, and causing economic burdens on individuals and society due to the burden of treatment costs. As research on the treatment of inflammatory bowel disease is actively conducted, the development of biologics is rapidly increasing. According to a recent report by the Health Insurance Review and Assessment Service, the medical expenses paid for biologics reached about 15 billion won. However, even after the use of biologics, treatment often fails, and the reaction disappears over time.

Nsg-2 (neuronal vesicle trafficking-associated-2) is located in neuronal dendritic cells and is one of the proteins that play the most important role in nerve cell differentiation and synapse formation. It has been shown that Nsg-2 encodes AMPA-glutamate receptor (AMPARs) proteins that regulate excitatory neurotransmission. AMPA receptors are known to be activated by glutamate, one of the neurotransmitters secreted from nerve terminals, to cause electrical excitation. Previous studies have demonstrated the ability of glutamate to control enteritis in an ulcerative colitis mouse model, and in the case of Nsg-2 deficiency, as it suppresses the expression of AMPARs, it inhibits the binding between glutamate and AMPARs, thereby increasing the concentration of glutamate in the blood and improving enteritis. is thought to play a role in However, the role and regulatory mechanism of Nsg-2 in enteritis have not yet been elucidated. In this study, we analyzed the role of Nsg-2 in enteritis using animal models and human intestinal tissues.

Inflammatory bowel disease is known to have a two-fold higher risk of small bowel cancer and colorectal cancer compared to the general population, but drugs that can effectively prevent it have not been developed. The development of therapeutic agents through the discovery of targets is the most necessary condition.

1. US Patent Publication 20210132070

An object of the present invention is to provide a biomarker composition for diagnosing inflammatory bowel disease comprising Nsg-2 (Neuron-specific gene-2) protein or a gene encoding the protein as an active ingredient.

Another object of the present invention is to provide a composition for diagnosing inflammatory bowel disease containing, as an active ingredient, an agent capable of measuring the expression or activity level of Nsg-2 protein or the expression level of a gene encoding the protein. .

Another object of the present invention is to provide a kit for diagnosing inflammatory bowel disease comprising the composition.

Another object of the present invention is to measure the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein from a sample isolated from a patient with inflammatory bowel disease; comparing the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein with a control sample; and determining that inflammatory bowel disease is present when the expression level of the mRNA of the Nsg-2 gene or the expression level of the Nsg-2 protein is higher than that of the control sample. is in

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising an Nsg-2 expression or activity inhibitor as an active ingredient.

Another object of the present invention is to contact the test substance to inflammatory bowel disease cells; measuring the level of expression or activity of Nsg-2 protein in inflammatory bowel disease cells contacting the test substance; and selecting a test substance having a reduced expression or activity level of the Nsg-2 protein compared to a control sample.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing inflammatory bowel disease comprising Nsg-2 (Neuron-specific gene-2) protein or a gene encoding the protein as an active ingredient.

In addition, the present invention provides a composition for diagnosing inflammatory bowel disease containing, as an active ingredient, an agent capable of measuring the expression or activity level of Nsg-2 protein or the expression level of a gene encoding the protein.

In addition, the present invention provides a kit for diagnosing inflammatory bowel disease comprising the composition.

In addition, the present invention comprises the steps of measuring the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein from a sample isolated from a patient with inflammatory bowel disease; comparing the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein with a control sample; and determining that the sample has inflammatory bowel disease when the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein is higher than that of a control sample.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising an Nsg-2 expression or activity inhibitor as an active ingredient.

In addition, the present invention comprises the steps of contacting the test substance to the inflammatory bowel disease cells; measuring the level of expression or activity of Nsg-2 protein in inflammatory bowel disease cells contacting the test substance; and selecting a test substance having a reduced expression or activity level of the Nsg-2 protein compared to a control sample.

The present invention relates to a biomarker composition for diagnosing inflammatory bowel disease and its use, based on the increased expression of Nsg-2 in inflammatory bowel disease patient tissue, inhibiting Nsg-2 to inhibit IL-17 and IFN-γ It was confirmed that the expression was significantly suppressed and the fraction of CD4+ T cells was also reduced, so that Nsg-2 can be used as a therapeutic target and diagnostic biomarker for inflammatory bowel disease.

1 is a result confirming comparison of body weight and intestinal atrophy between control mice and Nsg-2 gene deficient mice.
Figure 2 is a result confirmed by comparing the enteritis inhibitory effect of control mice and Nsg-2 gene deficient mice.
Figure 3 is a result confirming that CD4+ T cells are reduced among lamina propria monocytes of control mice and Nsg-2 gene deficient mice.
4 is a result of confirming the expression of Nsg-2 in intestinal tissues of patients with ulcerative colitis.

Hereinafter, the present invention will be described in more detail.

The present invention provides a biomarker composition for diagnosing inflammatory bowel disease comprising Nsg-2 (Neuron-specific gene-2) protein or a gene encoding the protein as an active ingredient.

The inflammatory bowel disease may be selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, converting colitis, and Behcet's syndrome.

The "biomarker" of the present invention is a substance that can be diagnosed by distinguishing tissues or cells of a subject with inflammatory bowel disease from tissues or cells of a normal control group, and increases or decreases in tissues or cells of a subject with a disease compared to a normal control group. It includes organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, etc., which show a decreasing pattern.

"Diagnosis" of the present invention refers to determining the susceptibility of a subject to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or determining an individual suffering from a specific disease or disorder determining the prognosis of, or therametrics (eg, monitoring the condition of an individual to provide information about the efficacy of a treatment).

In addition, the present invention provides a composition for diagnosing inflammatory bowel disease containing, as an active ingredient, an agent capable of measuring the expression or activity level of Nsg-2 protein or the expression level of a gene encoding the protein.

The agent capable of measuring the expression level of Nsg-2 may be a primer or probe that specifically binds to the Nsg-2 gene, an antibody, a peptide, an aptamer, or a compound that specifically binds to the Nsg-2 protein. there is.

As used herein, the term "primer" is a short gene sequence that is the starting point of DNA synthesis, and refers to an oligonucleotide synthesized for the purpose of diagnosis, DNA sequencing, and the like. The primers may be synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, or the like by a known method.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA of several to several hundred bases in length prepared through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be confirmed by labeling a radioactive isotope or an enzyme, and it can be designed and modified using a known method.

As used herein, the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to NSG-2 of the present invention, and the antibody can be prepared according to a conventional method in the art. The type of antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant in its complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

The term "peptide" used in the present invention has the advantage of high binding ability to a target substance, and does not undergo denaturation even during heat/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

As used herein, the term “aptamer” is a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. It means a kind of composed polynucleotide. As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are more stable than proteins, have a simple structure, and are composed of polynucleotides that are easy to synthesize, so they can be used instead of antibodies. can

In addition, the present invention provides a kit for diagnosing inflammatory bowel disease comprising the composition.

The “kit” of the present invention includes an antibody that specifically binds to a biomarker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with a substrate, a color-developing substrate solution that will react with the label, and a washing solution. and an enzyme reaction stop solution, etc., and may be manufactured in a plurality of separate packaging or compartments containing reagent components to be used.

In addition, the present invention comprises the steps of measuring the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein from a sample isolated from a patient with inflammatory bowel disease; comparing the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein with a control sample; and determining that the sample has inflammatory bowel disease when the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein is higher than that of a control sample.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising an Nsg-2 expression or activity inhibitor as an active ingredient.

The Nsg-2 expression inhibitor may be selected from the group consisting of antisense nucleotides complementary to mRNA of the Nsg-2 gene, small interfering RNA (siRNA), and short hairpin RNA (shRNA). there is.

The Nsg-2 activity inhibitor may be selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the Nsg-2 protein.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, One or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants may be further included. Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used. According to one embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route. The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug type, the route and duration of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg. /kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, and more preferably 0.1 mg/kg to 100 mg/kg. Administration may be administered once a day or divided into several times, and the scope of the present invention is not limited thereby.

In addition, the present invention comprises the steps of contacting the test substance to the inflammatory bowel disease cells; measuring the level of expression or activity of Nsg-2 protein in inflammatory bowel disease cells contacting the test substance; and selecting a test substance having a reduced expression or activity level of the Nsg-2 protein compared to a control sample.

The Nsg-2 expression level is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry ), microarray, western blotting, and flow cytometry (FACS).

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention and the like are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Dextran sulfate sodium (DSS) induced acute enteritis model creation

7-week-old wild type (hereinafter referred to as WT) mice (n=5, C57BL/6, Orientbio, Seongnam, Korea) and Nsg-2 gene deficient mice (n=5, Nsg2-/-, National Mouse Phenotyping Agency) ) was used, and feed and water were allowed to be freely consumed, and 5 mice were used for each group. Colitis was induced by mixing DSS (MP Biochemicals, CA, USA) with drinking water to make a 3% DSS solution and orally administering it for 7 days.

<Example 2> Confirmation of changes in body weight and intestinal atrophy in Nsg-2 gene deficient mice

To investigate changes in body weight and intestinal atrophy in Nsg-2 gene-deficient mice, 3% DSS was administered to WT mice and Nsg-2 gene-deficient mice for 7 days. evaluated. A disease activity index evaluated by weight change, diarrhea, and bloody stool was obtained. After colitis was induced, the mice were sacrificed, the large intestine was removed, opened longitudinally along the mesentery, washed with PBS, and then ulcers, erosions, redness, etc. were visually evaluated.

As a result, according to FIG. 1, WT mice showed a weight loss of about 20% compared to the conventional weight loss, but weight loss was suppressed in Nsg-2 gene deficient mice. When the excised colon was observed, severe intestinal atrophy and bloody stools were observed in the wt mice, but intestinal atrophy was suppressed and bloody stools were not evident in the Nsg-2 gene-deficient mice. (DAI 3.6 vs 6.0 p=0.048, gut length 6.7 vs 6.4 p>0.05)

<Example 3> Evaluation of severity of enteritis in Nsg-2 gene deficient mice

H&E staining was performed using the excised colon, and the microscopic severity of inflammation was evaluated. For evaluation, a 2 cm size colon tissue was collected from the rectum and colon, fixed in a formalin solution, and paraffin-embedded tissue sections were made for histological evaluation. After that, the colon tissue sections were stained with H&E as follows. First, paraffin was removed using xylene, and then xylene was removed using ethanol (EtOH). After that, it was stained with hematoxylin, then destained with Hcl, neutralized with ammonia, and stained with eosin. Finally, it was dehydrated with ethanol (EtOH) and ethanol (EtOH) was removed with xylene. After staining, the degree and extent of inflammation, intestinal gland damage, and mucosal damage were evaluated by converting them into scores, and the scores for each item were summed and calculated.

As a result, according to FIG. 2, as a result of analyzing the severity of enteritis through H&E staining, WT mice were accompanied by severe inflammatory findings such as inflammatory cell infiltration and loss of crypts, but these findings were alleviated in Nsg-2 gene deficient mice and statistically significant. It also showed a significant inhibitory effect on enteritis. (Nsg-KO 6.8, wt 9.6 p=0.028)

<Example 4> Confirmation of CD4+ cell changes in Nsg-2 gene deficient mice

Lamina propria mononuclear cells were isolated from the excised colon using Percoll, and then prepared for FACS analysis as follows. Cell lysis was performed using PharMlyse (BD Bioscience, NJ, USA). Thereafter, vortexing and centrifugation were performed to make the pellet have a cell concentration of 2x10 ⁷ cells/ml. Thereafter, 50 μl of CD 45, CD3, and CD 4 monoclonal antibodies at optimal concentrations and 50 μl of cells (10 ⁶ cells) were mixed well, followed by incubation. Thereafter, the cells were washed and prepared using a washing buffer, and data were obtained using BD FACS Aria (BD biosciences, NJ, USA) and analyzed with FlowJo (BD biosciences, NJ, USA) to determine the fraction of CD4 T cells.

As a result, according to FIG. 3, when FACS analysis was performed, CD4 cells among lamina propria monocytes were decreased in Nsg-2 gene deficient mice compared to WT mice.

<Example 5> Confirmation of Nsg-2 gene expression in patients with ulcerative colitis

Expression of Nsg-2 in intestinal tissues (rectum) of ulcerative colitis patients (n = 1) and normal controls (n = 1) to determine the level of Nsg-2 gene expression in ulcerative colitis patients was analyzed through the following immunohistochemical staining process (IRB No. 1104-066-358). After the slides were immersed in citrate buffer (pH 6.0) in a polyethylene chamber, they were heated for 10 minutes and cooled for 10-20 minutes. After washing with PBS, anti-Nsg-2 antibody (Abcam, Cambridge, UK) was stained using an automated staining system (Tech Mate 1000 TM) and left at room temperature for 1 hour. Then, the slide was washed three times with PBS and treated with a secondary antibody (Abcam, Cambridge, UK).

As a result, according to FIG. 4, although the expression of Nsg-2 was clearly observed centering on epithelial cells in the intestinal tissues of patients with ulcerative colitis, this finding could not be observed in the normal control group.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (11)

A biomarker composition for diagnosing inflammatory bowel disease comprising Nsg-2 (Neuron-specific gene-2) protein or a gene encoding the protein as an active ingredient. The method of claim 1,
The inflammatory bowel disease is a biomarker composition for diagnosing inflammatory bowel disease, characterized in that selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, converting colitis and Behcet's syndrome. A composition for diagnosing inflammatory bowel disease containing, as an active ingredient, an agent capable of measuring the expression or activity level of Nsg-2 protein or the expression level of a gene encoding the protein. The method of claim 3,
The agent capable of measuring the expression level of Nsg-2 is a primer or probe specifically binding to the Nsg-2 gene, an antibody, peptide, aptamer or compound binding specifically to the Nsg-2 protein. A composition for diagnosing inflammatory bowel disease, characterized in that A kit for diagnosing inflammatory bowel disease comprising the composition of claim 3 or claim 4. (1) measuring the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein from a sample isolated from a patient with inflammatory bowel disease;
(2) comparing the mRNA expression level of the Nsg-2 gene or the expression level of the Nsg-2 protein with a control sample; and
(3) A method for providing information necessary for diagnosing inflammatory bowel disease, comprising determining that inflammatory bowel disease is present when the expression level of the Nsg-2 gene mRNA or the expression level of the Nsg-2 protein is higher than that of a control sample. A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising an Nsg-2 expression or activity inhibitor as an active ingredient. The method of claim 7,
The Nsg-2 expression inhibitor is any one selected from the group consisting of an antisense nucleotide, small interfering RNA (siRNA), and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the Nsg-2 gene. A pharmaceutical composition for preventing or treating inflammatory bowel disease, characterized in that. The method of claim 8,
The Nsg-2 activity inhibitor is any one selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to Nsg-2 protein. Prevention of inflammatory bowel disease or Therapeutic pharmaceutical composition. delete delete

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