KR102685706B1 - Composition for preventing or treating osteoarthritis comprising K-7174 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and health food for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient. More specifically, an increase in Zmiz1 expression was confirmed in joint tissue caused by degenerative arthritis, and the expression of Zmiz1 was confirmed to be increased in joint tissue caused by degenerative arthritis. As K-7174 binds to GATA, whose expression is induced by overexpression, collagenase activity and the expression of inflammation-inducing factors Mmp3 and Cox2 are suppressed, confirming the effect of reducing the progression of degenerative arthritis, the K- The aim is to provide a composition containing 7174 as an active ingredient as a treatment for degenerative arthritis.

Description

Composition for preventing or treating osteoarthritis comprising K-7174 as an active ingredient {Composition for preventing or treating osteoarthritis comprising K-7174}

The present invention relates to a pharmaceutical composition and health food for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient.

Osteoarthritis (OA) is a degenerative joint disease mainly caused by inhibiting cartilage formation (cartilage ECM synthesis) and promoting cartilage destruction (cartilage destruction). Many age-related etiological risk factors and pathophysiological processes contribute to the progression of osteoarthritis. Mechanical stress, including joint instability and damage, and age-related factors that predispose to degenerative arthritis are potential degenerative arthritis-causing mechanisms. These factors lead to the degradation of ECM (extracellular matrix) by Mmp (Matrix metalloproteinase) and the dedifferentiation and apoptosis of chondrocytes due to the activation of biochemical pathways within chondrocytes, a unique cell type that produces various catabolic and anabolic factors. It results in disruption of ECM synthesis through apoptosis. In particular, once the cartilage tissue forming the joint is damaged, it does not normally regenerate in vivo. If the cartilage tissue of these joints is damaged, daily activities will be restricted along with severe pain, and if it becomes chronic, it will cause fatal degenerative arthritis, interfering with normal life or occupational activities.

On the other hand, in the case of rheumatoid arthritis (RA), unlike degenerative arthritis caused by destruction of cartilage cells and cartilage tissue, disease progression due to 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 the bones around the joints occur. Rheumatoid arthritis progresses as inflammation of the synovial membrane spreads to the joint capsule, ligaments, and tendons, and invades the bones. Therefore, the causes and progression stages of degenerative arthritis and rheumatoid arthritis are completely different, and their treatment methods are also different.

The treatments for rheumatoid arthritis known so far include appropriate non-steroidal anti-inflammatory drugs (NSAIDs), penicillamine, steroid hormones, TNF inhibitors, interleukin inhibitors, JAK inhibitors, and anti-CD-related inhibitors to block the inflammatory mechanism. It applies. NSAID drugs and steroid hormones are used in patients with degenerative arthritis to relieve joint pain and inflammation, but since they only relieve symptoms rather than treating the disease itself, they cannot serve as actual treatments for degenerative arthritis. In addition, degenerative arthritis, which is mainly caused by destruction of cartilage cells and cartilage tissue, is completely different from rheumatoid arthritis, an inflammatory arthritis, in its causes and symptoms, so treatment methods for degenerative arthritis are also bound to be different from those for rheumatoid arthritis.

Republic of Korea Patent Publication No. 10-2016-0104905 (published on September 6, 2016)

The present invention seeks to provide a composition containing K-7174 as an active ingredient as a pharmaceutical composition for preventing or treating degenerative arthritis and as a health food for improving it.

The present invention provides a pharmaceutical composition for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient.

Additionally, the present invention provides a health food for preventing or improving degenerative arthritis disease containing K-7174 as an active ingredient.

According to the present invention, an increase in Zmiz1 expression was confirmed in joint tissue induced by degenerative arthritis, and as GATA and K-7174, whose expression is induced by overexpression of Zmiz1, bind, collagenase activity and Mmp3, an inflammation-inducing factor, are increased. As the effect of suppressing the progression of degenerative arthritis by suppressing the expression of Cox2 was confirmed, the composition containing K-7174 as an active ingredient can be provided as a treatment for degenerative arthritis.

Figure 1 shows the results confirming that Zmiz1 is expressed in the joint tissues of patients with degenerative arthritis and mice suffering from degenerative arthritis.
Figure 2 shows the results confirming that modulation of Zmiz1 expression is correlated with degenerative arthritis. Figure 2A shows that when Zmiz1 is overexpressed in chondrocytes using an adenovirus delivery system, catabolic factors such as Mmp3 and Cox2 This is the result of PCR analysis confirming that (catabolic factor) is increased, Figure 2B is the Western blot result, and Figure 2C is the result confirming that collagenase activity and PGE2 production are also increased at the same time.
Figure 3 shows the results of analysis showing that the activity of Zmiz1 can be regulated by K-7174 by identifying the transcription factor regulated by Zmiz1 and simultaneously inhibiting the binding between Zmiz1 and the transcription factor. Figure 3A shows that the activity of Zmiz1 can be regulated by Zmiz1. After infecting chondrocytes with Ad-Zmiz1 to find regulated transcription factors, in order to analyze transcription factors regulated by Zmiz1, chondrocytes were first transfected with a Cignal 45-Pathway reporter (Qiagen, hilden, Germany) was transfected, followed by infection with Ad-Zmiz1 for 36 hours, and the transcription factors regulated by Zmiz1 were analyzed using the Dual-Glo Luciferase Assay system (Promega). Figures 3B and 3C show the results of Zmiz1 and This is a modeling result confirming how K-7174 inhibits the activity of Zmiz1 by combining GATA or Zmiz1. Figures 3D and 3E show that K-7174 binds to GATA and inhibits the activity of Mmp3 and cox2 regulated by Zmiz1. At the same time that expression was suppressed, Figure 3F shows the results confirming that Collegenase activity and PGE2 production, which measure the activities of MMP3 and Cox2, were also suppressed.
Figure 4 shows the results confirming that degenerative arthritis is suppressed by oral administration of K-7174 to an animal model inducing degenerative arthritis. Figure 4A shows the time and concentration of K-7174 administered after inducing degenerative arthritis by DMM. Figure 4B is a schematic diagram showing that K-7174 (10, 25, and 50 mg/kg) was orally administered every two days for 6 weeks in the manner shown in Figure 4A, and was administered to mice with degenerative arthritis induced by DMM. This result confirms through Safranin-O staining that the joints are not damaged when treated with 7174 at different concentrations (10, 25, and 50 mg/kg). At the same time, Figure 4C shows OARSI grade, SBP thickness, and Osteophyte formation based on these results. This is a result of numerical confirmation that K-7174 can suppress degenerative arthritis by separately measuring, and Figure 4D shows immunohistochemical analysis showing that the expression of MMP3 and COX2, which promote cartilage destruction, is also reduced by K-7174 administration. This is a result confirmed through, and Figure 4E shows the result of measuring the expression level of Zmiz1, MMP3, and COX2 per joint area as quantified in Figure 4D.
Figure 5 is a schematic diagram showing the molecular mechanism by which K-7174 inhibits degenerative arthritis.

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

The inventors of the present invention found that K-7174 binds to GATA, the expression of which is induced by Zmiz1 overexpressed in joint tissue caused by degenerative arthritis, and inhibits collagenase activity and the expression of Mmp3 and Cox2, which are inflammation-inducing factors, thereby preventing degenerative arthritis. The present invention was completed after confirming the effect of inhibiting the progression of arthritis.

The present invention can provide a pharmaceutical composition for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient.

The K-7174 may bind to GATA, the expression of which is induced by Zmiz1.

The K-7174 may inhibit MMP3 and COX2 expression induced by Zmiz1.

The pharmaceutical composition may contain 0.1 to 10 parts by weight of K-7174 based on a total of 100 parts by weight of the composition.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient is administered in the form of injections, granules, powders, tablets, pills, capsules, suppositories, gels, etc. according to conventional methods. Any one formulation selected from the group consisting of suspension, emulsion, drops, or liquid can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient includes appropriate carriers, excipients, disintegrants, sweeteners, coating agents, and bulking agents commonly used in the preparation of pharmaceutical compositions. , it may further include one or more additives selected from the group consisting of lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants.

Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, and microcrystalline. Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agents, etc., and such solid preparations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc can also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered to the subject.

The preferred dosage of K-7174 may vary depending on the subject's condition and weight, type and degree of disease, drug form, administration route and period, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, but is 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 may be 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 humans, but is not limited to these examples.

The present invention can provide a health food for preventing or improving degenerative arthritis disease containing K-7174 as an active ingredient.

The health food can be used with other foods or food additives in addition to K-7174, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, for example, prevention, health, or therapeutic treatment.

The effective dose of the compound contained in the health food may be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, and the effective dose may be less than the above range. Since the ingredient poses no safety issues, it is certain that it can be used in amounts exceeding the above range.

There are no particular restrictions on the types of health foods, and examples include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Examples include drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate 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 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. Production of human osteoarthritis samples and experimental osteoarthritis mouse model

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

Male C57BL/6 and SNCG-/- mice (008843-B6.129P2-SNCGtm1vlb/J, Jackson laboratory, USA) were grown at Ajou University Laboratory Animal Research Center in accordance with the guidelines of the Institutional Animal Care and Use Committee, which approved all animal procedures. It was maintained accordingly.

To produce an experimental 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 due to the influence 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 × ¹⁰⁹ PFUs/10 μL) into the knee joint twice weekly 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 the cartilage of ICR mice at postnatal day 5, enzymatically digested with proteolytic enzymes and collagenase, and then incubated with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. were 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 proteins.

3. Reagents

Antibodies were purchased from Abcam (Cambridge, UK; Zmiz1, COX2, MMP3), and K-7174 was purchased from Cayman (Michigan, USA; 32773).

4. Histology and immunohistochemistry

Human degenerative arthritis cartilage and mouse knee joint 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. Cartilage destruction was assessed in experimental osteoarthritis mouse models by three observers blinded to experimental grouping and scored according to the OARSI (Occupation for the Study of Osteoarthritis International) grading system (grade 0–6). OARSI scores were presented as the average maximum score for each mouse. Representative Safranin-O staining images were selected from the highest advanced lesions in each section and osteophyte maturity was quantified as previously described. Subchondral bone sclerosis was determined by measuring the thickness of the subchondral bone plate. Immunohistochemical staining was performed on human and mouse cartilage sections using MMP3, COX2, and Zmiz1 antibodies.

5. Western blotting

Chondrocytes were lysed in lysis buffer consisting of 50mM Tris-HCl (pH 7.5), 150mM NaCl, 50mM 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.

6. Transcription factor analysis

Chondrocytes were first transfected with the Cignal 45-Pathway reporter (Qiagen, hilden, Germany), which can identify 45 transcription factors, and then infected with Ad-Zmiz1 for 36 hours to transform the transcription factors regulated by Zmiz1 into Dual-Glo. Analysis was performed using the Luciferase Assay system (Promega).

7. Discovery studio analysis

Discovery studio predicted the binding site where GATA and K-7174 can bind.

8. Statistical analysis

All experiments were performed independently at least four times. Two independent groups were compared using the 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 pattern due to Zmiz1 overexpression and degenerative arthritis

To determine the possible role of Zmiz1 in osteoarthritis, Zmiz1 expression was examined in intact and damaged cartilage samples from human and mouse osteoarthritis.

As a result, as shown in Figure 1, it was confirmed that Zmiz1 expression was significantly higher in damaged degenerative arthritis cartilage compared to undamaged samples (Figure 1A; human, 1B; mouse).

<Example 2> Control of the onset of degenerative arthritis by Zmiz1

MMP3 is known to play an important role in the development of degenerative arthritis, and Cox2 is mainly involved in inflammation and causes cartilage matrix degradation by activating collagenase and aggrecanase. Accordingly, to investigate the relationship between elevated Zmiz1 expression and the pathogenesis of degenerative arthritis, adenovirus Zmiz1 was injected into articular cartilage cells.

As a result, it was confirmed that up-regulated catabolic factors (MMP3 and COX2) were expressed, as shown in Figures 2A and 2B. Additionally, consistent with what was confirmed in chondrocytes as shown in Figure 2C, it was confirmed that collagenase activity (Figure 2C; top) and PGE2 production (Figure 2C; bottom) were increased.

<Example 3> Identification of transcription factors related to Zmiz1

The adenovirus Zmiz1 was overexpressed in chondrocytes using a 45-Pathway reporter (Qiagen, hilden, Germany) to identify transcription factors regulated by Zmiz1.

As a result, it was confirmed that GATA was most regulated by Zmiz1, as shown in Figure 3A.

<Example 4> Confirmation of binding site between GATA and K-7174 and binding site of Zmiz1 and K-7174 using Discovery Studio

As a result of molecular docking analysis using Discovery studio, it was found that binding is possible due to the binding between the GATA amino acid sequence and Zmiz1 amino acid sequence, as shown in Figure 3B, and at the same time, Zmiz1 and K-7174 can bind as shown in Figure 3C. I was able to confirm that it was there.

From the above results, it was confirmed that K-7174 binds to Zmiz1, thereby inhibiting the binding of Zmiz1 and GATA, thereby suppressing the expression of MMP3 and Cox2 by Zmiz1.

<Example 5> Confirmation of inhibition of Zmiz1-mediated MMP3 and COX2 expression by K-7174

We confirmed whether K-7174 could inhibit Zmiz1-mediated degenerative arthritis pathogenesis. Chondrocytes were infected with Ad-Zmiz1 to overexpress Zmiz1. To determine whether K-7174 could suppress the increased expression of MMP3 and Cox2, the chondrocytes were treated with K-7174 to increase the expression of MMP3 and Cox2. It was confirmed by PCR, qRT-PCR, Western blot, and densitometry analysis.

As a result, as shown in Figure 3D and Figure 3E, it was confirmed that MMP3 and COX2 expression due to Zmiz1 overexpression was decreased in a K-7174 dose-dependent manner. At the same time, as shown in Figure 3F, collagenase activity increased by MMP3 and PGE2 production increased by Cox2 expression were also confirmed to be reduced by K-7174.

<Example 6> Confirmation of inhibition of degenerative arthritis induced by K-7174

K-7174 (10, 25, and 50 mg/kg) was administered orally three times for 6 weeks in an animal model in which degenerative arthritis was induced by DMM through the same procedure as shown in Figure 4A.

As a result, it was confirmed that joint destruction caused by degenerative arthritis was suppressed, as shown in Figures 4B and 4C. At the same time, as shown in Figures 4D and 4E, the expression of MMP3 and COX2, which causes cartilage destruction, was also decreased by K-7174 administration. The decrease was confirmed through immunohistochemical analysis.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

A pharmaceutical composition for preventing or treating degenerative arthritis disease containing K-7174 as an active ingredient, wherein the pharmaceutical composition is intended for administration to patients with increased Zmiz1 expression or activity compared to normal. Pharmaceutical composition. The pharmaceutical composition for preventing or treating degenerative arthritis disease according to claim 1, wherein K-7174 binds to GATA whose expression is induced by Zmiz1. The pharmaceutical composition for preventing or treating degenerative arthritis disease according to claim 1, wherein K-7174 inhibits the expression of MMP3 and COX2 induced by Zmiz1. The pharmaceutical composition for preventing or treating degenerative arthritis disease according to claim 1, wherein the pharmaceutical composition contains 0.1 to 10 parts by weight of K-7174 based on a total of 100 parts by weight of the composition. delete