KR20230026012A - Composition for treating or diagnosing osteoarthritis - Google Patents

Abstract

The present invention relates to a composition for treating or diagnosing degenerative osteoarthritis targeting Zmiz1 (Zinc finger MIZ domain-containing protein 1). More specifically, when Zmiz1 expression and activity increases during the onset of degenerative osteoarthritis and an increase in the expression of Mmp3 and Cox2, which induces cartilage destruction, is confirmed, Zmiz1 was provided as a biomarker for diagnosing the degenerative osteoarthritis. In addition, by confirming the change in the expression level of Zmiz1 and diagnosing the degenerative osteoarthritis early, the treatment effect for the degenerative osteoarthritis can be improved.

Description

Composition for treating or diagnosing degenerative arthritis {Composition for treating or diagnosing osteoarthritis}

The present invention relates to a composition for treating or diagnosing degenerative arthritis targeting Zmiz1 (Zinc finger MIZ domain-containing protein 1).

Osteoarthritis (OA) is a degenerative joint disease mainly caused by inhibition of cartilage ECM synthesis and promotion of cartilage destruction. Many etiologic risk factors and pathophysiological processes associated with aging contribute to the progression of osteoarthritis. Mechanical stress, including joint instability and injury, and aging-related factors that predispose to osteoarthritis are potential causes of degenerative arthritis. These factors are caused by activation of biochemical pathways in chondrocytes, a unique cell type that produces various catabolic and anabolic factors, leading to degradation of ECM (extracellular matrix) by Mmp (Matrix metalloproteinase), dedifferentiation of chondrocytes, and apoptosis. It results in the cessation of ECM synthesis through apoptosis. In particular, cartilage tissue constituting a joint does not normally regenerate in vivo once damaged. When the cartilage tissue of these joints is damaged, daily activities are restricted along with severe pain, and when chronic, it causes fatal degenerative arthritis, which interferes with normal life or professional activities.

On the other hand, in the case of rheumatoid arthritis (RA), unlike degenerative arthritis caused by the destruction of cartilage cells and cartilage tissue, the progression of the disease by an autoimmune reaction is known to be an important causative factor. Rheumatoid arthritis is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells, and unlike degenerative arthritis, osteoporosis and bone erosion of bones around joints occur. Rheumatoid arthritis progresses when inflammation of the synovial membrane spreads to the joint capsule, ligament, and tendon, and invades the bone. Therefore, degenerative arthritis and rheumatoid arthritis have completely different causes and progression stages, and treatment methods for them are also different.

So far, known therapeutic agents for rheumatoid arthritis include non-steroidal anti-inflammatory drugs (NSAIDs), penicillamine, steroid hormones, TNF inhibitors, interleukin inhibitors, JAK inhibitors, and anti-CD related inhibitors suitable for blocking the inflammatory mechanism. applicable NSAID drugs and steroid hormones are used in patients with degenerative arthritis for the purpose of relieving joint pain and inflammation, but since they only relieve symptoms rather than treating the disease itself, they cannot serve as a practical therapeutic agent for degenerative arthritis. In addition, since degenerative arthritis, which is mainly caused by destruction of cartilage cells and cartilage tissue, is distinctly different from rheumatoid arthritis, which is an inflammatory arthritis, in its cause and phenomenon, the treatment method for degenerative arthritis is also different from the treatment method for rheumatoid arthritis.

Therefore, in order to prevent or slow down the progression of degenerative arthritis, it is necessary to diagnose degenerative arthritis at an early stage, but until now, as a method of diagnosing degenerative arthritis, only methods such as orthopedic hospital visits or X-rays have been known.

Republic of Korea Patent Publication No. 10-2016-0104905 (2016.09.06. Publication)

The present invention provides a biomarker composition for early diagnosis of degenerative arthritis by confirming changes in the expression or activity level of Zmiz1 and a diagnostic kit capable of measuring the expression level of the biomarker, and diagnosing degenerative arthritis using the biomarker It is intended to provide a method and a method for screening a therapeutic agent for degenerative arthritis.

In addition, it is intended to provide a composition for preventing or treating degenerative arthritis containing a Zmiz1 expression or activity inhibitor as an active ingredient.

The present invention provides a biomarker composition for diagnosing degenerative arthritis, including Zmiz1 (Zinc finger MIZ domain-containing protein 1) gene or a protein encoded by the gene.

The present invention provides a kit for diagnosing degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of the Zmiz1 gene or a protein encoded by the gene.

The present invention provides a method for providing information for diagnosing degenerative arthritis comprising the step of confirming the expression or activity level of the Zmiz1 gene or a protein encoded by the gene from a sample isolated from the specimen.

The present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Confirming the expression level of Zmiz1 from the chondrocytes treated with the candidate substance; and a method of screening for a treatment for degenerative arthritis, wherein the Zmiz1 expression level is reduced compared to a group not treated with the candidate substance to provide a candidate substance as a treatment for degenerative arthritis.

The present invention provides a pharmaceutical composition for preventing or treating degenerative arthritis containing a Zmiz1 (Zinc finger MIZ domain-containing protein 1) expression or activity inhibitor as an active ingredient.

In addition, the present invention provides a health food for preventing or improving degenerative arthritis containing a Zmiz1 expression or activity inhibitor as an active ingredient.

According to the present invention, Zmiz1 expression and activity are increased during the onset of degenerative arthritis, and as a result, it is confirmed that the expression of Mmp3 and Cox2, which induce cartilage destruction, is increased, Zmiz1 is provided as a biomarker for diagnosing degenerative arthritis, and the expression level of the Zmiz1 is confirmed. By confirming the change and diagnosing degenerative arthritis at an early stage, the treatment effect of degenerative arthritis can be improved.

1 is a result confirming that Zmiz1 is expressed in joint tissues of degenerative arthritis patients and degenerative arthritis-induced mice.
2 is a result confirming that the expression of Zmiz1 is increased in aging mice.
3 is a result of confirming the correlation between the modulation of Zmiz1 expression and degenerative arthritis, and FIG. 3A is the result of overexpressing Zmiz1 in chondrocytes using an adenovirus delivery system, such as Mmp3 and Cox2. PCR analysis results confirming that catabolic factors are increased, FIG. 3B is a western blot result, and FIG. 3C is a result confirming that collagenase activity and PGE2 production are increased simultaneously with Zmiz1 overexpression.
Figure 4 is a result of confirming the cartilage destruction and degenerative arthritis development of the mouse knee joint injected with Ad-C and Ad-Zmiz1 into the joint. Figure 4A is safranin-O confirming that cartilage destruction is promoted when Zmiz1 is overexpressed (Safranin-O) staining results, and FIG. 4B is an immunohistochemical (IHC) analysis result confirming the increase in expression of Zmiz1, Mmp3, and Cox2.

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

The inventors of the present invention confirmed that the expression and activity of Zmiz1 increased in degenerative arthritis, and accordingly, the expression of Mmp3 and Cox2, which induce cartilage destruction, was increased. , The present invention was completed to improve the therapeutic effect of degenerative arthritis by regulating the expression or activity of Zmiz1.

The present invention can provide a biomarker composition for diagnosing degenerative arthritis, which includes a zinc finger MIZ domain-containing protein 1 (Zmiz1) gene or a protein encoded by the gene.

The Zmiz1 (Zinc finger MIZ domain-containing protein 1) has an NCBI assessment number. 57178 (Gene ID).

The present invention can provide a diagnostic kit for degenerative arthritis containing, as an active ingredient, an agent for detecting the expression or activity level of the Zmiz1 gene or a protein encoded by the gene.

The agent may be selected from the group consisting of a primer or probe that specifically binds to the Zmiz1 gene, an antibody, a peptide, an aptamer, or a compound that specifically binds to the Zmiz1 protein.

The present invention can provide a method for providing information for diagnosing degenerative arthritis, comprising the step of confirming the expression or activity level of the Zmiz1 gene or a protein encoded by the gene from a sample isolated from the specimen.

The sample may be selected from the group consisting of cells, tissues, blood, serum, urine and saliva.

A method of providing information for diagnosing degenerative arthritis may include diagnosing degenerative arthritis when the expression or activity level of the Zmiz1 gene or a protein encoded by the gene is higher than that of a normal control group.

The present invention comprises the steps of processing a candidate substance to chondrocytes isolated from a patient with degenerative arthritis; Confirming the expression level of Zmiz1 from the chondrocytes treated with the candidate substance; and a method of screening for a treatment for degenerative arthritis, wherein the Zmiz1 expression level is reduced compared to a group not treated with the candidate substance, and provides a candidate substance as a treatment for degenerative arthritis.

The "biomarker" of the present invention is a substance that can be diagnosed by distinguishing the tissue or cell of a subject suspected of having degenerative arthritis from the tissue or cell of a normal control group, and measures the increase or decrease in the tissue or cell of the subject compared to the normal control group. organic biomolecules such as visible proteins or nucleic acids, lipids, glycolipids, glycoproteins, and the like;

In the present invention, "diagnosis" in a broad sense means to judge the actual condition of a patient's disease in all aspects. The content of judgment is the name of the disease, etiology, type of disease, severity, detailed condition of the disease, and presence or absence of complications.

The present invention can provide a pharmaceutical composition for preventing or treating degenerative arthritis, which contains a Zmiz1 (Zinc finger MIZ domain-containing protein 1) expression or activity inhibitor as an active ingredient.

The Zmiz1 expression inhibitor may be one or more selected from the group consisting of antisense nucleotides, siRNAs, shRNAs, microRNAs, and ribozymes that specifically bind to Zmiz1 gene or mRNA of a gene that promotes Zmiz1 expression.

The Zmiz1 activity inhibitor may be one or more selected from the group consisting of a protein that promotes Zmiz1 activity or a compound that specifically binds to the Zmiz1 protein, a peptide, a peptidomimetic, a protein, an aptamer, and an antibody.

In one embodiment of the present invention, the pharmaceutical composition is any one selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops or liquids according to conventional methods. formulations can be used.

In another embodiment of the present invention, a pharmaceutical composition for preventing or treating degenerative arthritis containing a Zmiz1 expression or activity inhibitor as an active ingredient is suitable carriers, excipients, disintegrants, sweeteners, coating agents, It may further include one or more additives selected from the group consisting of an expanding agent, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binder, and a lubricant.

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 suspending agents. 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 administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

A preferred dosage of the Zmiz1 expression or activity inhibitor may vary depending on the condition and body weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention can provide a health food for preventing or improving degenerative arthritis containing a Zmiz1 expression or activity inhibitor as an active ingredient.

The health food may be used together with other foods or food additives in addition to the Zmiz1 expression or activity inhibitor, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the health food may be used according to the effective dose of the therapeutic agent, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. Since there is no problem in terms of safety, it is certain that the components can be used in an amount greater than the above range.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes; and the like.

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

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Preparation of human osteoarthritis samples and experimental osteoarthritis mouse models

Human cartilage samples were obtained from individuals aged 63 to 80 years who underwent total knee arthroplasty (Table 1). All patients gave written informed consent, and sample collection was approved by the IRB of The Catholic University of Korea (UC14CNSI0150).

To produce the degenerative arthritis model, 12-week-old male mice were subjected to surgical Destabilization of the medial meniscus (DMM) and sacrificed 10 weeks after surgery. Female mice were excluded from use because of the effect of female hormones on the pathogenesis of degenerative arthritis. Adenoviruses for intra-articular injection were purchased from Vector Biolabs (Malvern, USA): Ad-C (1060), Ad-Zmiz1 (ADV-277085). Wild-type mice were injected with adenovirus (1 × 10 ⁹ PFUs/10 μL) twice weekly into the knee joint and sacrificed 3 weeks after the first adenovirus injection.

2. Isolation and cell culture of primary mouse articular chondrocytes

Mouse articular chondrocytes were isolated from cartilage of ICR mice on day 5 after birth, enzymatically digested with proteolytic enzyme and collagenase, and then treated with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. This was maintained in supplemented DMEM (Capricorn science GmbH; Hessen, Germany). On day 3, cells (4.25 × 10 ⁵ cells/well) were infected with adenovirus or treated with recombinant protein.

3. Reagents

Antibodies of Zmiz1, COX2 and MMP3 were purchased from Abcam (Cambridge, UK).

4. Western blotting

Chondrocytes were lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% Tween 20, 0.2% NP-40 and protease inhibitors. Equal amounts of cell extracts were separated by SDS-PAGE (6% stacking gel and 10% running gel) and analyzed by immunoblotting.

5. Histology and Immunohistochemistry

Human degenerative arthritic cartilage and mouse knee joints were fixed in 4% paraformaldehyde and embedded in paraffin. Mouse knee joints were calcified in 0.5 M EDTA (pH 7.4) for 2 weeks. Paraffin-embedded samples were stained with Safranin-O or Alcian blue or immunostaining. Representative safranin-O staining images used to measure cartilage destruction were selected from the most advanced lesions of each section.

6. Statistical analysis

All experiments were performed independently at least 4 times. Two independent groups were compared using Shapiro-Wilk normality test, Levene's homogeneity of variance test, and two-tailed independent t-test. Multiple comparisons were performed using the Shapiro-Wilk test, Levene's test, and one-way analysis of variance with Bonferroni's post-hoc test. Data based on an ordinal grading system were analyzed using non-parametric Mann-Whitney U tests. A P value of less than 0.05 was considered statistically significant.

<Example 1> Confirmation of correlation between changes in gene expression patterns due to Zmiz1 overexpression and degenerative arthritis

To explore the possible role of Zmiz1 (Zinc finger MIZ domain-containing protein 1) in osteoarthritis, we checked Zmiz1 expression in both human and mouse degenerative arthritis intact and damaged cartilage samples.

As a result, as shown in FIG. 1, it was confirmed that Zmiz1 expression was significantly higher in the damaged degenerative arthritic cartilage than in the undamaged sample (FIG. 1A; human, 1B; mouse).

In addition, since degenerative arthritis is a representative geriatric disease, the expression pattern of Zmiz1 was confirmed in aged mice.

As a result, as shown in FIG. 2, it was confirmed that the expression of Mmp3, Cox2, and Col10, which can induce degenerative arthritis in aging mice, was increased, and at the same time, it was confirmed that the expression of Sox-9 and Col2a1, which are important for joint ECM formation, was decreased. could At the same time, it was confirmed that the expression of Zmiz1 also increased when the expression of Mmp3, Cox2, and Col10, which promote cartilage destruction, increased.

<Example 2> Confirmation of regulation of degenerative arthritis by Zmiz1

MMP3 is known to play an important role in the pathogenesis of degenerative arthritis, and Cox2 is mainly involved in inflammation and induces cartilage matrix degradation by activating collagenase and aggrecanase.

Accordingly, in order to confirm the association between the elevated Zmiz1 expression confirmed in the previous experiment and the pathogenesis of degenerative arthritis, articular cartilage cells were injected into chondrocytes using adenovirus Zmiz1.

As a result, it was confirmed that up-regulated catabolic factors (MMP3 and COX2) were expressed, as shown in FIGS. 3A and 3B. In addition, consistent with the results confirmed in chondrocytes, it was confirmed that collagenase activity (FIG. 3C; top) and PGE2 production (FIG. 3C; bottom) were increased, as shown in FIG. 3C.

<Example 3> Confirmation of regulation of degenerative arthritis by Zmiz1 in vivo

Ad-Zmiz1 and Ad-C were injected into the knee by intra-articular injection using an adenoviral delivery system, and safranin-O staining and immunohistochemistry were performed.

As a result, as shown in FIG. 4A, it was confirmed that severe cartilage destruction and degenerative arthritis symptoms were induced in the joint tissues of WT mice injected with Ad-Zmiz1, and at the same time, when Ad-Zmiz1 was overexpressed as shown in FIG. It was confirmed that the expression of Cox2 was also increased.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

A biomarker composition for diagnosing degenerative arthritis comprising a Zmiz1 (Zinc finger MIZ domain-containing protein 1) gene or a protein encoded by the gene. A kit for diagnosing degenerative arthritis comprising, as an active ingredient, an agent for detecting the expression or activity level of a Zmiz1 gene or a protein encoded by the gene. The kit for diagnosis of degenerative arthritis according to claim 2, wherein the agent is selected from the group consisting of a primer or probe that specifically binds to the Zmiz1 gene, an antibody, a peptide, an aptamer, or a compound that specifically binds to the Zmiz1 protein. . A method for providing information for diagnosing degenerative arthritis comprising the step of confirming the expression or activity level of the Zmiz1 gene or a protein encoded by the gene from a sample separated from the specimen. The method of claim 4, wherein the sample is selected from the group consisting of cells, tissues, blood, serum, urine and saliva. The method according to claim 4, wherein the method for providing information for diagnosing degenerative arthritis is characterized by diagnosing degenerative arthritis when the expression or activity level of the Zmiz1 gene or a protein encoded by the gene is higher than that of a normal control group. How to provide information for. Treating chondrocytes isolated from a patient with degenerative arthritis with a candidate substance;
Confirming the expression level of Zmiz1 from the chondrocytes treated with the candidate substance; and
A method for screening a treatment for degenerative arthritis, wherein the Zmiz1 expression level is reduced compared to a group not treated with the candidate substance to provide a candidate substance as a treatment for degenerative arthritis. A pharmaceutical composition for preventing or treating degenerative arthritis, comprising a Zmiz1 (Zinc finger MIZ domain-containing protein 1) expression or activity inhibitor as an active ingredient. The method according to claim 8, wherein the Zmiz1 expression inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA, shRNA, microRNA, and ribozymes that specifically bind to the Zmiz1 gene or the mRNA of a gene that promotes the expression of Zmiz1. A pharmaceutical composition for preventing or treating degenerative arthritis. The method according to claim 8, wherein the Zmiz1 activity inhibitor is at least one selected from the group consisting of a protein that promotes Zmiz1 activity or a compound that specifically binds to the Zmiz1 protein, a peptide, a peptidomimetic, a protein, an aptamer, and an antibody. A pharmaceutical composition for preventing or treating degenerative arthritis. A health food for preventing or improving degenerative arthritis containing a Zmiz1 expression or activity inhibitor as an active ingredient.

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