KR102060230B1 - Composition for preventing and treating arthritis comprising a nucleolar orophate receptor RORα expression and activity modulator - Google Patents

Abstract

The present invention provides a composition for preventing or treating osteoarthritis, a composition for diagnosis, a method for providing information for diagnosis, and a method for screening a therapeutic agent. The present invention is expected to be capable of diagnosing osteoarthritis, and is expected to be useful for developing osteoarthritis therapeutic agents by controlling the expression and activity of RORα, a marker of the present invention.

Description

Composition for preventing and treating arthritis comprising a nucleolar orophate receptor RORα expression and activity modulator

The present invention relates to a composition for preventing or treating osteoarthritis.

Osteoarthritis, also called osteoarthritis, is a disease in which inflammation and pain occur due to damage to bones and ligaments that form joints due to gradual damage or degenerative changes in cartilage that protects joints. Cartilage tissue is slow in regeneration of damaged areas because of lack of blood vessels and neural tissue, resulting in poor nutrition and oxygen supply. Thus, to date, osteoarthritis is a disease for which no fundamental treatment is established other than artificial joint replacement.

Recently, a treatment for inducing cartilage regeneration using stem cells or tissue engineering has been studied, but the treatment efficiency is inferior due to a lack of understanding of arthritis-induced mechanisms, and more essential arthritis control mechanisms and the discovery of regulatory factors are needed. to be. Recently, regulatory mechanisms regulating the expression of chondrocyte degrading enzymes such as HIF-2a or homeostasis of chondrocytes such as zinc-ZIP8-MTF1 axis have been identified, and methods of treating and preventing arthritis using the same have been developed. (Yang, S at al., Hypoxia-inducible factor-2α is a catabolic regulator of osteoarthritic cartilage destruction, Nat, Med. 16, 687-693 (2010); Kim, J.-H. at al., Regulation of the Catabolic Cascade in Osteoarthritis by the Zinc-ZIP8-MTF1 Axis, Cell, 156, 730-743 (2014)).

Retinoid related orphan receptor alpha (RORα) is a nuclear receptor known for its transcriptional activity as a ligand for cholesterol metabolites such as cholesterol and sulfided cholesterol (Kojetin, DJ at al., REV-ERB and ROR nuclear receptors as drug targets , Nat, Rev, Drug Discov. 13, 197-216 (2014)), known to regulate the circadian rhythm and metabolism.

The biological cycle is formed by repeated increase and decrease of expression of Bmal and Clock protein complexes, which are key factors of the cycle gene, and the increase and decrease of the Bmal / Clock complex is produced by RORα and Cryptochrome (Bmal). It is controlled by transcription factors such as Cryptochrome, Cry) and Period (Per). Among them, the Cry-Per complex acts as a repressor to form a negative feedback loop that suppresses Bmal expression, and RORα forms a positive feedback loop that is generated by Bmal and increases Bmal expression.

In addition, analysis of mutant mice of RORα shows that metabolic diseases induced by the high fat diet are reduced by mutations of RORα, which reduce the accumulation of lipids in adipocytes and inflammatory macrophages. Penetration and interleukin (IL) -6. It is believed to be due to decreased expression of Toll-like receptor 8 (TLR8), chemokine (C-C motif) ligand 8, and the like.

However, no studies have yet been conducted to investigate the function and role of RORα in osteoarthritis.

The inventors have sought to find bio-chemical substances that have an effect of preventing or treating osteoarthritis. The present inventors completed the present invention by confirming the osteoarthritis inhibitory effect by a substance that inhibits the expression or activity of RORα.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of osteoarthritis comprising an inhibitor of the expression or activity of Retinoid related orphan receptor alpha (RORα) as an active ingredient.

Another object of the present invention to provide a composition for diagnosing osteoarthritis comprising an agent for measuring the expression or activity level of RORα.

Still another object of the present invention is to provide an information providing method for diagnosing osteoarthritis.

It is another object of the present invention to provide a method for screening an osteoarthritis therapeutic agent.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

The present inventors confirmed the high expression of ROR α (Retinoid related orphan receptor alpha) in chondrocytes or tissues of osteoarthritis patients. In addition, the osteoarthritis inhibitory effect by RORα gene expression inhibition in the mouse animal model was confirmed, and based on this, the present invention was completed.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of osteoarthritis, comprising an inhibitor of the expression or activity of Retinoid related orphan receptor alpha (RORα) as an active ingredient.

As used herein, the term "prevention" means any action that inhibits or delays the development of osteoarthritis by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which symptoms for osteoarthritis are improved or beneficially altered by administration of the pharmaceutical composition according to the present invention.

Osteoarthritis, which is a disease to be prevented and treated by the composition of the present invention, is also called degenerative arthritis, and is caused by damage to bones and ligaments that form joints due to gradual damage or degenerative changes of cartilage protecting the joints. it means. The composition of the present invention provides osteoarthritis therapeutics that target RORα as part of gene target therapy.

In the present invention, RORα (Retinoid related orphan receptor alpha) gene, which is a target gene for the treatment of osteoarthritis, is known as a nuclear receptor known to have transcriptional activity using cholesterol metabolites such as cholesterol and sulfide cholesterol as a ligand, but the expression or activity of RORα There has been no report on the treatment of osteoarthritis through inhibition.

The nucleotide sequence of the gene is shown in SEQ ID NO: 1. In addition, the gene of the present invention will be readily available to those skilled in the art because the nucleotide sequence is registered in the gene bank.

In the present invention, the expression inhibitor of RORα is from the group consisting of antisense oligonucleotides, siRNA, shRNA, microRNA and ribozyme that specifically binds to RORα, the RORα activity inhibitor is a compound, peptide, protein, app that specifically binds to RORα Although it may include one or more selected from the group consisting of a tamer and an antibody, any agent capable of inhibiting the expression or activity of a gene may be included without limitation. Preferably SR3335 (CAS No.293753-05-6) which is an RORα activity inhibitor.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, it may further include a lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative and the like.

The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level may refer to a type of disease, severity, activity of a drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, since the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

In another aspect of the present invention, the present invention provides a method for treating osteoarthritis, comprising administering the pharmaceutical composition to a subject. As used herein, the term "individual" means a subject in need of treatment for a disease, and more specifically, a mammal, such as a primate, mouse, dog, cat, horse, and cow, which is human or non-human. .

According to another aspect of the present invention, the present invention provides a composition for diagnosing osteoarthritis, comprising an agent for measuring the level of expression or activity of Retinoid related orphan receptor alpha (RORα).

As used herein, the term "diagnosis" refers to the act of identifying the pathological condition, ie the presence or characteristics of osteoarthritis, by administration of a composition according to the invention.

In the present invention, the agent for measuring the expression or activity level of RORα may include one or more selected from the group consisting of primers, probes, proteins, and antibodies specifically binding to RORα, but the level of expression or activity of a gene Any agent capable of measuring can be included without limitation.

According to another aspect of the present invention, the present invention comprises the steps of measuring the expression or activity of RORα present in the biological sample; And it provides an information providing method for diagnosing osteoarthritis comprising the step of comparing the expression or activity of the measured RORα with the expression or activity of the normal control.

In the present invention, the degree of expression or activity of RORα present in a biological sample is measured, and the result of the measurement is compared with the level of expression or activity of a normal control sample. Can be.

The method of the invention comprises mammals, in particular humans. In this case, the human subject includes a person suspected of having osteoarthritis or a person not suspected of having osteoarthritis diagnosed.

In the present invention, the "biological sample" may be composed of tissue, cells, blood, serum, plasma, saliva and urine, but is not limited thereto.

The sample may be pretreated prior to use in detection, and may include, for example, filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

In the present invention, the step of measuring the expression or activity of RORα may be polymerase reaction (PCR), reverse transcriptase (RT-PCR), competitive reverse transcriptase (Competitive RT-PCR), real time reverse transcriptase ( Realtime RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blotting, enzyme linked immunosorbent assay (ELISA), Radioimmunoassay, RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, complement fixation assay, protein chip, and In It may be measured by one or more methods selected from the group consisting of in vitro kinase assays, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a method for screening a osteoarthritis therapeutic agent comprising the step of selecting a substance that reduces the level of expression or activity of RORα in a subject.

In the present invention, the expression or activity of RORα in the subject is measured, and when the candidate substance for osteoarthritis treatment is reduced, the candidate substance may be used as a therapeutic agent for osteoarthritis when the expression or activity of RORα is reduced compared to the conventional one.

It may measure the expression or activity of RORα in the subject before treatment to the subject, and compare and measure it after treatment, or compare the expression or activity with the comparison group by treating the subject to some subjects among several individuals. have.

In the present invention, the step of measuring the expression or activity of RORα may be polymerase reaction (PCR), reverse transcriptase (RT-PCR), competitive reverse transcriptase (Competitive RT-PCR), real time reverse transcriptase ( Realtime RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blotting, enzyme linked immunosorbent assay (ELISA), Radioimmunoassay, RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, complement fixation assay, protein chip, and In It may be measured by one or more methods selected from the group consisting of in vitro kinase assays, but is not limited thereto.

The method of treating the subject substance with the subject is not particularly limited, and may be treated with a biological sample obtained from the subject, for example. The biological sample may be a sample as described above.

In the screening method of the present invention, "subject material" means an unknown substance used in screening to test whether the degree of expression or activity of RORα of the present invention is affected. The substance of interest includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing or treating osteoarthritis, a composition for diagnosis, a method for providing information for diagnosis, and a method for screening a therapeutic agent.

(b) The present invention is expected to be capable of diagnosing osteoarthritis, and is expected to be useful for developing osteoarthritis therapeutic agents by regulating the expression and activity of RORα, a marker of the present invention.

1 is a diagram showing the increase in the expression and activity of RORα in arthritis cartilage tissue and chondrocytes compared to normal cartilage tissue and chondrocytes.
2 shows that the onset of arthritis occurs when the expression of RORα is artificially increased through analysis of cartilage tissue, and through the analysis of chondrocytes, the expression of cartilage stromatase is directly expressed in chondrocytes by RORα. Figure showing increase and decrease in the expression of cartilage matrix.
3 is a diagram showing that the progression of arthritis in the cartilage tissue of RORα-mutant mice is inhibited, and the effect of increasing the expression of cartilage matrix degrading enzyme by IL-1b in the chondrocytes is attenuated.
4 is a diagram showing that the inhibition of RORα activity (SR3335) inhibits the progression of arthritis in cartilage tissue, and attenuates the effect of IL-1b inducing increased expression of cartilage matrixase in chondrocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Throughout this specification, unless otherwise indicated, “%” used to indicate the concentration of a particular substance is solid / solid (weight / weight)%, solid / liquid (weight / volume)%, and Liquid / liquid is (volume / volume)%.

Preparation Example 1 Induction of Arthritis in Mice

Arthritis was induced artificially by destabilization of medial meniscus (DMM) surgery in 9-week-old mice. DMM surgery is a method of artificially inducing cartilage damage by inducing mechanical instability of the joint tissue by dissecting the ligaments holding the medial meniscus present in the joint tissue of the mouse.

Preparation Example 2 Primer Sequence Information

Primer sequence information used in the following experiments is shown in Table 1.

Aggrecan (mouse) S 5'-GAAGACGACATCACCATCCAG-3 ' RT-PCR AS 5'-CTGTCTTTGTCACCCACACAT-3 ' Adamts4 (mouse) S 5'-CATCCGAAACCCTGTCAACTTG-3 ' RT-PCR AS 5'-GCCCATCATCTTCCACAATAGC-3 ' Adamts4 ChIP # 1 (mouse) S 5'-ACC AGG CTG GAT AGG AAG GAC-3 ' ChIP assay AS 5'-AAA TCC CAC CAT CAG CCC TCA-3 ' Adamts 5 (mouse) S 5'-GCCATTGTAATAACCCTGCACC-3 ' RT-PCR AS 5'-TCAGTCCCATCCGTAACCTTTG-3 ' Adamts5 ChIP # 1 (mouse) S 5'-TGTAAGTGTGCCTGCTCTCCTCT-3 ' ChIP assay AS 5'-CCTCCATCCTTTGTTTCCTCTAAT-3 ' Col2a1 (mouse) S 5'-CACACTGGTAAGTGGGGCAAGA-3 ' RT-PCR AS 5'-GGATTGTGTTGTTTCAGGGTTCG-3 ' MMP3 (mouse) S 5'-TCCTGATGTTGGTGGCTTCAG-3 ' RT-PCR AS 5'-TGTCTTGGCAAATCCGGTGTA-3 ' MMP3 ChIP # 1 (mouse) S 5'-AGCCAGTCCTATGATTTTACTTTGAG-3 ' ChIP assay AS 5'-CTTTCCTTTCACCGTGTTAATCC-3 ' MMP3 ChIP # 2 (mouse) S 5'-ATTTTAAGTGTGTGCTATATGTCAAG-3 ' ChIP assay AS 5'-CGTTAGACTTCCCTGAGCA-3 ' MMP9 (mouse) S 5'-ACCACATCGAACTTCGA-3 ' RT-PCR AS 5'-CGACCATACAGATACTG-3 ' MMP12 (mouse) S 5'-CCCAGAGGTCAAGATGGATG-3 ' RT-PCR AS 5'-GGCTCCATAGAGGGACTGAA-3 ' MMP12 ChIP # 1 (mouse) S 5'-TGGCTACTGTGTTATCGATGTT-3 ' ChIP assay AS 5'-GACTCACTGTTGATGTCATCCC-3 ' MMP13 (mouse) S 5'-TGATGGACCTTCTGGTCTTCTGG-3 ' RT-PCR AS 5'-CATCCACATGGTTGGGAAGTTCT-3 ' MMP13 ChIP # 1 (mouse) S 5'-GCCACATCACCTCCAATAAGA-3 ' ChIP assay AS 5'-ACCAGGAACATGTAAATAAGAAAAG-3 ' MMP13 ChIP # 2 (mouse) S 5'-TCTATCTGTTTTGCTTTCTCTTAT-3 ' ChIP assay AS 5 ''-CCATATCTCAAAATCTACCTCC-3 ' Nos2 ChIP # 1 (mouse) S 5'-TGTCTTTACCTCCCCAGAACTTATT-3 ' ChIP assay AS 5'-GGTTGGTGAGATAAGGAATTTACACA-3 ' ROR? (human, mouse) S 5'-GCAGATAACGTGGCAGACCT-3 ' RT-PCR AS 5'-GCGATCCGCTGACATCAGTA-3 ' SOX9 (mouse) S 5? -CACTGGCAGTTACGGCATCAG-3? RT-PCR AS 5? -CATGTAAGTGAAGGTGGAGTAGAGC-3?

Example 1.RORα Identification in Mouse / Patient Cartilage Tissue

1-1. Mouse Sample Preparation

First, to analyze the cartilage tissue of the mouse, the joint tissues were taken at 4, 8, and 10 weeks after DMM surgery, and the joint tissues were fixed with 4% paraformaldehyde, and then soaked in 0.5 M EDTA solution for 3 days. Decalcification was performed. The tissues were then immersed in 30%, 50%, 70%, 90%, 95% and 100% ethanol, respectively, followed by xylene and paraffin infiltration. The tissue was then cut to a thickness of 6 um and stored on a slide.

Next, hematoxylin was rehydrated in order of xylene, 100%, 95%, 90%, 70%, 50% and 30% ethanol and water to determine the degree of arthritis. Immerse in 0.1% Fast Green, 0.1% Safranin O solution, dye and soak in 70%, 90%, 95% and 100% ethanol, xylene, and then sprayed with fixative to fix. The degree of safranin O staining of the tissue sections was examined through a microscope to analyze the extent of cartilage damage, the thickness of the subchondral bone plate, and the degree of osteophyte formation.

1-2. Arthritis Patient Sample Preparation

To determine the degree of cartilage damage in the cartilage tissue of arthritis patients, first, the damaged and damaged mechanical strength of the cartilage tissue of arthritis patients (Undamaged) and undamaged parts (undamaged) are cut out with a surgical knife. , OCT compound (Leica / Cat. No. 3801480) was stored frozen at -70 ℃. Frozen cartilage tissue was cut to a thickness of 10 um and placed on a slide for freezing at -20 ° C.

Next, the tissue sections are dried at room temperature for 30 minutes, washed with water to remove the OCT compound and dried, and the tissues are fixed by drying with cold acetone solution for 2 minutes, and then stained for 10 minutes with 1% Alcian blue solution. The tissue sections were then washed for 10 seconds with 0.1 N HCl solution. Tissue sections were washed with water and fixed, followed by microscopic staining to determine the degree of cartilage damage.

1-3. Expression of RORα Protein in Mouse / Patient Cartilage Tissues

In order to confirm the expression of the RORα protein in cartilage tissue was tested as follows.

Mouse cartilage tissue sections undergoing rehydration in the order of xylene, 100%, 95%, 90%, 70%, 50% and 30% ethanol, water (the tissues of arthritis patients were frozen to make tissue sections. The procedure was omitted.) Soaking in 3% H2O2 solution for 5 minutes inhibited peroxidase activity in the tissue, and soaking antigen in 0.1% trypsin and 0.1% tryptone X-100 solution for 5 minutes respectively. After soaking in 1% BSA (bovine serum albumin) solution for 1 hour blocking, and reacted with RORα primary antibody (Sigma aldrich) for 2 hours. After washing, the solution was reacted with the secondary antibody-streptavidin-HRP solution for 30 minutes, and reacted with the DAB + substrate-chromogen solution for 10 minutes to develop color. After washing, fixation was performed to analyze the expression of RORα under a microscope.

As a result, as shown in FIG. 1A, the expression of RORα protein is increased in cartilage tissue of mice (DMM) and arthritis patients (Damaged) with cartilage damage as compared to normal cartilage tissue (mouse and arthritis patients, Sham and Undamaged, respectively). It was confirmed.

1-4. Expression of RORα Gene mRNA in Mouse / Patient Cartilage Tissues

In order to confirm the expression of RORα gene mRNA in cartilage tissue was tested as follows.

Cartilage tissue sections were immersed in trizol solution and then tissue and cells were disrupted with a homogenizer. Thereafter, the chloroform solution was mixed and centrifuged to separate the protein in the tissue, and the isopropanol was mixed with the aqueous solution in which mRNA was dissolved and centrifuged to remove water. 75% ethanol was added to the precipitate from which the water was removed, and the remaining organic solvent was dissolved by removing it by centrifugation, dried in air to evaporate ethanol, and RNA was obtained. CDNA was obtained by adding and reacting reverse transcriptase to the obtained RNA. SYBR and RORα primers (Forward: 5'-GCAGATAACGTGGCAGACCT-3 ', Reverse: 5'-GCGATCCGCTGACATCAGTA-3') were added to the obtained cDNA, followed by quantitative real-time PCR (qRT-PCR), followed by quantification of RORα expression. It was.

As a result, as shown in FIG. 1B, the expression of RORα mRNA was increased in cartilage tissue of mice (DMM) and arthritis patients (Damaged) that had cartilage damage as compared to normal cartilage tissue (mouse and arthritis patients, Sham and Undamaged, respectively). It was confirmed.

Example 2. RORα Identification in Mouse Chondrocytes

2-1. Mouse Sample Preparation

First, in order to secure cartilage cells of the mouse, the knee tissues of 5 day old mice are collected and treated with 0.2% type 2 collagenase (Sigma aldrich) and 0.02% trypsin for 3 hours to remove skin and muscles of the knee tissue. Removed. After washing well with PBS, only cartilage tissue was isolated, tissue was dissolved with 0.2% type 2 collagenase to secure chondrocytes, and the cartilage cells were cultured at a concentration of 3 × 105 per dish. On the second day, the DMEM medium was replaced, and then treated with IL-1b, TNF-a or infected with RORα adenovirus (Ad-N) for 2 hours, and 36 hours later, the cells were washed with PBS.

2-2. Expression of RORα Gene mRNA in Mouse Chondrocytes

In order to analyze the expression of RORα mRNA in chondrocytes of mice treated with IL-1b and TNF-a, chloroform was treated 10 minutes after treatment with trizol in a dish that had undergone all the procedures of Example 2-1. Thereafter, after securing the cDNA in the same manner as in Example 1-4, qRT-PCR was performed by adding SYBR and RORα primers.

As a result, as shown in Figure 2a, it was confirmed that the expression of RORα in chondrocytes increased by IL-1b and TNF-a.

2-3. Confirmation of RORα Activity in Mouse Chondrocytes

Next, experiments were performed as follows to analyze the activity of RORα in mouse chondrocytes.

RORα-renilla reporter gene construct and b-gal expression vector were transfected into the chondrocytes cultured according to the procedure of Example 2-1. After 36 hours after treatment with IL-1b, TNF-a or overexpression of RORα, chondrocyte proteins were isolated and renilla substrate and b-gal substrate were added thereto to measure RORα activity.

As a result, as shown in Figure 2b, it was confirmed that IL-1b and TNF-a increased the activity of RORα as in the case of overexpressing RORα in chondrocytes (Ad-R).

Example 3. Confirmation of the role of RORα in cartilage tissue and chondrocytes

To confirm the role of RORα in cartilage tissue and chondrocytes, RORα was overexpressed using adenovirus, immunohistostaining, safranin O staining, qRT-PCR, Western blot and ChIP analysis were performed.

3-1. Cartilage tissue

First, RORα adenovirus was injected into the knee of 8-week-old mice three times a week, 1x109 PFU (10 ul), and 5 weeks later, the knee tissue was secured and immunohistostained as described above. Overexpression was confirmed. In addition, safranin O staining was performed in the same manner as in Example 1-1, and damage to cartilage tissue, generation of bone muscle and thickness of subchondral bone were measured.

As a result, as shown in Figure 3a, it was confirmed that RORα overexpression occurred in the cartilage tissue by RORα adenovirus injection (RORα and Ad-RORα), and by RORα overexpression (Ad-RORα and Ad-R) It was confirmed that arthritis develops by measuring damage to cartilage tissues, increasing osteomyelitis and subchondral bone thickness (OARSI grade, Osteophyte maturity and SBP thickness).

3-2. Cartilage cells

Next, after culturing the chondrocytes of the mouse through the procedure of Example 2-1, RORα adenovirus was infected for 2 hours on day 3, and cDNA was obtained from the chondrocytes after 36 hours. Thereafter, the genes were amplified by qRT-PCR using primers of RORα, Mmp3, 9, 12, 13, Adamts4, 5, Sox9, Col2a1, and Acan.

As a result, as shown in Figure 3b, the expression of cartilage matrix degrading enzymes Mmp3, 9, 12, 13 and Adamts4, 5 increased with overexpression of RORα in chondrocytes, type II collagen (Col2a1) ) And aggrecan (Acan) and the expression of Sox9, a cartilage matrix expression inducing factor, was reduced.

In addition, the protein was isolated from chondrocytes overexpressing RORα and subjected to SDS-PAGE (sodium dodesyl sulfate polyacrylamide gel electrophoresis), which was then delivered to nitrocellulose (NC) membrane for 30 minutes with 5% skim milk. After blocking for 2 hours, antibodies of RORα, Mmp3, Mmp13, Sox9, and Col-II were reacted for 2 hours, and then HRP-tagged secondary antibody was reacted. Thereafter, the ECL solution was sprayed and then exposed to X-ray films to measure the expression level of the protein (Western blotting).

As a result, as shown in Figure 3c, it was confirmed that the expression of Mmp3, 13 and the expression of Sox9 and Col2a1 decreased with overexpression of RORα in chondrocytes.

Finally, chondrocytes overexpressing RORα were fixed with paraformaldehyde and crushed with an ultrasonic grinder to separate nuclear proteins to obtain protein-DNA complexes. Then, protein A magnetic beads and RORα antibody (Sigma-Aldrich) were added and reacted for 12 hours, followed by immunoprecipitation. Magnetic bead-RORα-DNA complexes were separated using a metal separator, followed by proteinase. RORα was digested with K, and DNA was isolated using spin-column containing silicon filter. After securing a DNA promoter site that binds to RORα through this process, PCR was performed using a primer that amplifies a region (RORe motif) to which RORα binds in the promoters of Mmp3, Mmp12, Mmp13, Adamts4, and Adamts5.

As a result, as shown in Figure 3d, it was confirmed that RORα overexpressed in chondrocytes directly binds to the promoters of Mmp3, 12, 13, Adamts4, 5 to regulate its expression.

Example 4. Confirmation of Arthritis Control of RORα in RORα Mutant Mice

Staggerer (Sg) mice, which are RORα mutant mice, produce a RORα whose activity is inhibited by eliminating ligand binding sites due to a defect in the RORα gene. Homozygote Sg mice have been shown to have problems with the cerebellar development process, have a marked decrease in motor capacity, and are known to die before they enter maturity. These features are known to be similar in homozygote RORα KO mice that chemically induced RORα knockout (KO), indicating that Sg mutations play a similar role as RORα KO.

Therefore, in the present invention, arthritis was induced by DMM surgery on heterozygote Sg (Sg / +) mice in which a normal phenotype appears, and then joint tissues were analyzed by safranin O staining. Gene expression patterns of chondrocytes of Homozygote Sg (Sg / Sg) mice were analyzed by qRT-PCR.

4-1. Cartilage tissue

First, 10-week-old normal mice (WT) and Sg / + mice were induced arthritis through DMM surgery, and after 8 weeks, safranin O staining was performed in the same manner as in Example 1-1, and cartilage tissue damage, Osteotomy and subchondral bone thickness were measured.

As a result, as shown in Figure 4a, it was confirmed that the cartilage damage, bone root formation and subchondral bone thickness induced by DMM in Sg / + mice is inhibited compared to normal mice.

4-2. Cartilage cells

Next, the knee joint tissues of 5 days old normal mice and Sg / Sg mice were collected, and the chondrocytes were cultured in the same manner as in Example 2-1, and IL-1b 2 ng / ml was treated on the third day of culture. After 36 hours, cDNA was obtained from each chondrocyte. Using the obtained cDNA, qRT-PCR for Mmp3, 9, 12, 13 and Adamts4, 5 and Sox9, Col2a1, and Acan were performed.

As a result, as shown in Figure 4b, it was confirmed that the increased expression of cartilage matrixases Mmp3, 9, 12, 13 and Adamts4, 5 induced by IL-1b in the chondrocytes of Sg / Sg mice attenuated, It was confirmed that the expression-reducing effects of cartilage matrix Col2a1, Acan induced by IL-1b and cartilage matrix expression regulator Sox9 were not attenuated.

Example 5. Confirmation of arthritis control ability of RORα by treatment with RORα activity inhibitor (SR3335)

SR3335 (CAS No.293753-05-6) is known as a RORα specific activity inhibitor. Thus, intraarticular injection of adenovirus overexpressed RORα or DMM surgery, and SR3335 was injected into the knees of arthritis-induced mice, followed by safranin O staining to analyze joint tissues and treatment of IL-1b. One chondrocyte was treated with SR3335 and gene expression patterns were analyzed by qRT-PCR.

5-1. Cartilage tissue

First, RORα adenovirus 1 × 10 ⁹ PFU (10 ul) and SR3335 (7.5 mg / kg body weight) were injected three times a week in the knees of 10-week old mice, and knee tissue was secured after 5 weeks. In addition, arthritis was induced by DMM surgery in 10-week-old mice, and SR3335 (7.5 mg / kg body weight) was injected into the knee three times a week, and knee tissue was secured 8 weeks after the surgery. Safranin O staining was performed on the two kinds of knee tissues secured in the same manner as in Example 1-1, and the damage to cartilage tissue, the generation of the bone muscle and the thickness of the subchondral bone were measured.

As a result, as shown in Figure 5a, it was confirmed that the intraarticular injection of SR3335 inhibited cartilage damage, bone formation and subchondral bone thickness induced by RORα overexpression and DMM.

5-2. Cartilage cells

Next, in order to confirm the inhibition of RORα activity by SR3335, the knee joint tissues of 5 day old mice were collected, and the chondrocytes were cultured in the same manner as in Example 2-1, and on the third day of culture, the RORα-renilla reporter gene construct Was transfected into the b-gal expression vector. After pretreatment with SR3335 (25 uM), 2 ng / ml of IL-1b after 2 hours, and chondrocytes were separated after 36 hours, and renilla substrate and b-gal substrate were added thereto to enhance RORα activity. Measured.

As a result, as shown in Figure 5b, it was confirmed that SR3335 inhibited the activity of RORα increased by IL-1b in chondrocytes.

In addition, cDNA was secured in the chondrocytes of Example 5-2, and qRT-PCR was performed for Mmp3, 9, 12, 13, and Adamts5 using the obtained cDNA.

As a result, as shown in Figure 5c, it was confirmed that the cartilage cells of Sg / Sg mice attenuate the increased expression of cartilage matrix lyase Mmp3, 9, 12, 13 and Adamts5 induced by IL-1b.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Composition for preventing and treating arthritis comprising a          nucleolar orophate receptor RORa expression and activity          modulator <130> PN170319 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1572 <212> DNA <213> Retinoid related orphan receptor alpha <220> <223> Retinoid related orphan receptor alpha <400> 1 atggagtcag ctccggcagc ccccgacccc gccgccagcg aaccgggcag cagcggctcg 60 gaggcggccg cgggctccag ggagaccccg ctgacccagg acacgggccg caagagcgag 120 gcaccgggcg cggggcgcag gcagagctat gcgagctcca gccgaggtat ctcagtcacg 180 aagaagacac acacatctca aattgaaatt attccatgca agatctgtgg agacaaatcg 240 tcaggaatcc attatggtgt cattacgtgt gaaggctgca agggcttttt caggagaagt 300 cagcagagca atgccaccta ctcctgtcct cgtcagaaga actgtttgat tgatcggacc 360 agcagaaacc gctgccagca ttgtcggctg cagaaatgcc tggccgtggg gatgtctcga 420 gatgctgtca agtttggtcg gatgtccaag aagcagagag acagcttgta cgccgaggtg 480 cagaagcacc ggatgcagca gcagcagcga gaccaccagc agcagcctgg ggaggcggag 540 ccgctgacgc ccacctacaa catctcagcc aatgggctga cggaactgca tgatgacctc 600 agcacctata tggatgggca cacccccgag ggcagcaagg ccgactcagc cgtcagcagc 660 ttctacctgg acatccagcc ctccccagac cagtcgggat tggacatcaa tgggatcaaa 720 cccgaaccca tatgtgacta cacaccagca tctggcttct tcccctactg ttccttcacc 780 aacggagaga cttccccaac cgtgtccatg gcagaactag aacaccttgc ccagaacata 840 tccaaatccc acctggaaac ctgccagtac ttgcgggaag agctccagca gataacgtgg 900 cagaccttcc tgcaggagga gattgaaaac taccagaaca agcagagaga ggtgatgtgg 960 cagctgtgtg ccatcaagat tacagaagct atccagtatg tggtggagtt tgccaaacgc 1020 attgatggat ttatggagct gtgtcaaaat gatcaaattg tgcttctaaa agcaggctcg 1080 ctagaggtgg tgtttattag gatgtgccgt gcctttgact ctcagaacaa caccgtgtac 1140 tttgacggga agtatgcgag ccccgatgtc ttcaagtccc taggttgtga agacttcatc 1200 agctttgtgt ttgaatttgg gaagagtttg tgttctatgc acctgaccga agacgaaatc 1260 gcgttatttt ctgcattcgt actgatgtca gcggatcgct cgtggcttca ggaaaaggta 1320 aaaatagaaa agctgcaaca gaaaattcag ctggcccttc agcacgtcct acagaagaac 1380 caccgagaag atggaattct aaccaagcta atatgcaagg tgtctacgtt aagagcccta 1440 tgtggacgac atacggaaaa gctaatggca tttaaagcaa tatacccaga cattgtgcga 1500 ctccattttc ctccattata caaggaattg ttcacttcag aatttgagcc agccatgcaa 1560 atcgatgggt aa 1572

Claims (9)

At least one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs, microRNAs, ribozymes, compounds, peptides, aptamers, proteins and antibodies that specifically bind to RORα (Retinoid related orphan receptor alpha) as an active ingredient Pharmaceutical composition for the prevention or treatment of osteoarthritis comprising.
delete delete delete Osteoarthritis diagnostic composition comprising one or more selected from the group consisting of primers, probes, proteins and antibodies that specifically bind to RORα.
delete Providing information for diagnosing osteoarthritis, which includes the following steps:
(a) measuring the expression or activity of RORα present in the biological sample; And
(b) comparing the level of expression or activity of the measured RORα with the level of expression or activity of the normal control.
The method of claim 7, wherein the biological sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.
A method for screening a therapeutic agent for osteoarthritis, comprising selecting a substance that reduces the level of expression or activity of RORα in a subject.

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