Application of BAY-069 in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of BAY-069 in preparation of a medicine for treating rheumatoid arthritis. Through metabonomics research, the inventor finds that the metabolic enzyme BCAT2 is remarkably highly expressed in synovial tissues of an active-stage RA patient and is relatively low in expression in synovial tissues of a remission-stage RA patient. The high expression of the metabolic enzyme BCAT2 is closely related to synovitis and bone destruction. The BAY-069 as a BCAT2 inhibitor can significantly reduce the score of joint inflammation, reduce synovitis and bone destruction, and effectively reduce bone loss. The invention provides an innovative RA metabolism targeted treatment strategy which has high safety and strong targeting property.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of BAY-069 in the preparation of drugs for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease mainly characterized by synovial inflammation and bone destruction. It is a chronic systemic inflammatory disease with an incompletely understood etiology. In the early stage, it is mainly manifested as joint swelling and pain, and the affected joints are mostly small joints such as both hands, wrists, and feet, showing a symmetrical distribution. Morning stiffness is one of the hallmark symptoms of RA. Patients experience joint stiffness and limited mobility when waking up in the morning, and the duration usually exceeds 1 hour. In the late stage, joint stiffness and deformity occur, resulting in loss of function and disability. Some patients may present with systemic symptoms such as fever, dry mouth and eyes, anemia, subcutaneous nodules, and vasculitis. The pathogenesis of RA is complex, involving multiple links such as immune cells (T cells, B cells, macrophages), inflammatory factors (TNF-α, IL-6, IL-1β), and metabolic disorders.

[0003] Currently, the main treatment drugs for RA include: 1) Non-steroidal anti-inflammatory drugs (NSAIDs), which are mostly used to initially relieve joint swelling and pain symptoms. They can relieve symptoms but cannot control the disease progression; 2) Immunosuppressants (such as methotrexate): They improve the disease by inhibiting the immune response, but long-term use may cause serious side effects; 3) Biological agents (such as TNF-α inhibitors, IL-6 inhibitors), which have significant efficacy but are expensive, and some patients are resistant to them; 4) JAK inhibitors, which act by inhibiting cell signaling pathways but have a relatively high risk of infection.

[0004] Although existing drugs have improved the treatment effect of RA to a certain extent, there are still significant defects, especially in the aspect of simultaneously controlling inflammation and bone destruction. Metabolic abnormalities are an important link in the pathology of RA, but there is currently no treatment strategy targeting metabolic targets.

[0005] The main problems existing in current drug treatments are as follows: 1) Limitations in target selection. Existing RA treatment drugs (such as methotrexate, TNF-α inhibitors, JAK inhibitors, etc.) mainly target inflammatory factors or cell signaling pathways, and fail to address the key pathological link of immune cell metabolic disorders in the pathogenesis of RA, resulting in limited efficacy; 2) Drug safety and drug resistance problems. Existing drugs generally have serious side effects (such as infection risk, liver and kidney damage, etc.), and some patients develop drug resistance. There is an urgent need in clinical practice to develop RA treatment drugs with new mechanisms of action and higher safety; 3) Insufficient treatment effect: Existing drugs are difficult to effectively inhibit bone destruction while controlling inflammation.

[0006] Disorders of branched-chain amino acid (BCAA) metabolism play important roles in various metabolic diseases (such as obesity, insulin resistance, and type 2 diabetes) and cancer. BCAA metabolic disorders lead to the accumulation of their metabolites (such as branched-chain keto acids, BCKAs), activate the mTORC1 signaling pathway, and exacerbate oxidative stress and inflammation. The key steps of BCAA metabolism are mediated by branched-chain amino acid transaminases (BCAT1 and BCAT2), which initiate the metabolic process by catalyzing the conversion of BCAA to BCKAs. High expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) is associated with the invasive phenotypes of various cancers. High expression of BCATs promotes tumor proliferation by providing metabolic precursors and activating the mTORC1 signaling pathway, and inhibition of BCATs can slow down tumor progression. The roles of BCAA and BCATs in the field of rheumatoid arthritis are unknown.

[0007] BCAT2 is significantly highly expressed in certain diseases (such as tumors) and promotes disease progression by regulating cell metabolism. It has been found that BCAT2 inhibitors (such as BAY-069) can inhibit the activity of BCAT2.

[0008]

[0009] BAY-069 is an effective inhibitor of branched-chain amino acid transaminase 1 and 2 (BCAT1, BCAT2), and is currently used as a chemical probe and also in anti-cancer research.

[0010] There is currently no literature or research reporting the pathological role of BCAT2 in RA, nor any drug targeting BCAT2 for the treatment of RA. Summary of the Invention

[0011] To solve the above problems, the present invention provides the use of BAY-069 in the preparation of a drug for treating rheumatoid arthritis. BAY-069 can significantly improve inflammation and bone destruction, and at the same time has advantages such as strong targeting and low side effects.

[0012] Use of BAY-069 in the preparation of a drug for regulating the inflammatory state of joint synovium.

[0013] Use of BAY-069 in the preparation of a drug for reducing synovial hyperplasia and bone destruction.

[0014] Preferably, the rheumatoid arthritis is active rheumatoid arthritis.

[0015] Use of BAY-069 in the preparation of a drug for treating rheumatoid arthritis.

[0016] The dosage concentration of the BAY-069 is 1 - 10 μM, preferably 10 μM.

[0017] Through metabolomics research, the inventors of the present application found that the metabolic enzyme BCAT2 was significantly highly expressed in the synovial tissues of RA patients in the active stage, while its expression was lower in the synovial tissues of RA patients in the remission stage.

[0018] According to one embodiment of the present invention, further analysis showed that the high expression of the metabolic enzyme BCAT2 was closely related to synovial inflammation and bone destruction.

[0019] Verification of the effect of BAY-069: In animal models of CIA (collagen-induced arthritis) and CAIA (antibody-induced arthritis), BAY-069 could significantly reduce the joint inflammation score and decrease synovial inflammation and bone destruction.

[0020] Micro-CT analysis showed that BAY-069 could effectively reduce bone loss.

[0021] Advantages of the present invention:

[0022] The research of the present invention found that BCAT2 was highly expressed in the synovial tissues of RA patients in the active stage and was closely related to inflammation and bone destruction. The present invention first proposed that BAY-069 could be used as a BCAT2 inhibitor to regulate the inflammatory state of synovial macrophages by inhibiting the BCAA (branched-chain amino acid) metabolic pathway, thereby inhibiting the RA inflammatory response and reducing synovial hyperplasia and bone destruction.

[0023] Through targeted metabolomics analysis, the present invention found that, compared with RA patients in the remission stage and normal control populations, the BCAA metabolism of RA patients in the active stage was disordered, especially the accumulation of BCKA (branched-chain keto acid) mediated by BCAT2, which was closely related to the progression of inflammation. Animal experiments proved that a high-BCAA diet could exacerbate RA inflammation, while BCAT2 inhibition could relieve synovial inflammation and bone destruction. In addition, BAY-069 could effectively inhibit BCAT2 activity and reduce the progression of RA inflammation caused by BCAA metabolism disorder. The present invention verified the efficacy of BAY-069 in treating RA and its metabolic regulation mechanism, and proposed an innovative metabolic-targeted treatment strategy for RA, which has high safety and strong targeting.

[0024] The specific effects are as follows:

[0025] 1) New target and new mechanism: For the first time, the metabolic enzyme BCAT2 was used as a therapeutic target for RA, and a treatment strategy based on metabolic regulation was proposed. And through immunomics research, it was first found that the metabolic enzyme BCAT2 was highly expressed in the synovium of RA patients in the active stage, suggesting its important role in the pathogenesis of RA, and it was closely related to inflammation and bone destruction.

[0026] 2) Verify the significant efficacy of BAY-069: BAY-069 was first applied to the treatment of RA. Through animal model experiments, BAY-069 showed significant anti-inflammatory and anti-bone destruction effects in animal models, and its efficacy was verified in animal models. Moreover, through in vitro experiments, it was found that BAY-069 significantly reduced the expression of inflammatory factors (such as TNF-α, IL-6), verifying the significant efficacy of BAY-069 as a metabolic enzyme A inhibitor in the treatment of RA.

[0027] 3) High safety: BAY-069 is a known drug with high safety and can be rapidly advanced to clinical application. The present invention provides a novel RA treatment strategy based on metabolic enzyme A inhibitors, which has high efficiency and safety.

[0028] 4) The drug of the BCAT2 inhibitor based on metabolic enzyme BCAT2 of the present invention can be prepared into an oral preparation, which is convenient for treatment and the patient compliance is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Expression of BCAT1 and BCAT2 in synovium of joints of RA patients in active and remission phases

[0030] The left figure is a typical figure of the expression of BCAT1 and BCAT2 in synovium of joints of RA patients in active and remission phases; the right figure is a statistical chart of the positive area of the expression of BCAT1 and BCAT2;

[0031] Wherein: ACT: synovial tissue of knee joints of RA patients in active phase (n = 20); REM: synovial tissue of knee joints of RA patients in remission phase (n = 20); BCAT1: branched-chain amino acid transaminase 1; BCAT2 branched-chain amino acid transaminase 2; statistical method: T test.

[0032] Figure 2 To detect the change trend of BCAA and its metabolites (BCKAs) by targeted metabolomics;

[0033] ACT: synovial fluid of knee joints of RA patients in active phase (n = 10); REM: synovial fluid of knee joints of RA patients in remission phase (n = 10); statistical method: T test.

[0034] Figure 3 High BCAA diet exacerbates joint swelling and bone destruction in CAIA model;

[0035] a, Schematic diagram of animal experiment: After sterile mice were given different diets (LAA diet, HBCAA diet) for 4 weeks, the existing diet was maintained, and CAIA modeling was performed or not;

[0036] b-c, Arthritis score and bilateral posterior ankle swelling degree score of each group;

[0037] d, Representative HE and safranin-O staining images of the hind ankle joints of mice in each group, and representative micro-CT images;

[0038] e, Histopathological scores of the hind ankle joints of mice in each group (upper), and scores of the bone destruction index BS / BV (lower);

[0039] Among them, NC: blank group; LAA, control diet group; HBCAA, diet with 200% more BCAA than LAA, with calorie and nitrogen balance; CAIA, RA animal model.

[0040] Figure 4 Conditional knockout of BCAT2 alleviates joint inflammation and bone destruction in CAIA;

[0041] Among them,

[0042] a, Arthritis scores (upper) and average paw thickness (lower) of wild-type (WT) and BCAT1 knockout (BCAT1-KO) mice in the CAIA model;

[0043] b, Arthritis scores (upper) and average paw thickness (lower) of wild-type (WT) and BCAT2 conditional knockout (BCAT2-cKO) mice in the CAIA model;

[0044] c, Representative H&E staining images of joint tissue sections, showing histological changes in wild-type, BCAT1-KO, and BCAT2-cKO mice in the CAIA model;

[0045] d-e, Histopathological scores of joint inflammation, comparing wild-type with BCAT1-KO (d) and wild-type with BCAT2-cKO (e) mice;

[0046] f, Representative micro-CT images, showing the joint structure of wild-type and BCAT2-cKO mice;

[0047] g, Quantification of the bone surface area / bone volume ratio (BS / BV) of wild-type and BCAT2-cKO mice;

[0048] Error bars represent mean ± standard error of the mean (mean±s.e.m.). Statistical significance was analyzed using unpaired two-tailed Student's t-test (d, e, g).

[0049] Figure 5 SPR results of BAY-069 and BCAT2.

[0050] Figure 6 BAY-069 improves joint inflammation and bone destruction in the CAIA model;

[0051] a, Swelling scores of joints in the blank control group (NC), model group (CAIA), and BAY-069 group; b, Comparison of joint pathology among groups based on HE staining and safranin-fast green staining; c, Comparison of joint destruction scores among groups based on micro-CT; d, Representative HE staining and safranin-fast green staining images of each group, and representative micro-CT images of each group. Detailed implementation manners

[0052] Next, the technical solutions in the embodiments of the present application will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0053] Example 1: Immunohistochemical comparison of the expression of BCAT2 in synovial tissues of knee joints of patients with active and remission RA

[0054] Experimental method: Synovium of 20 patients with active and remission RA who underwent knee joint replacement surgery in the Department of Orthopaedics of Wuxi Hospital Affiliated to Nanjing University of Chinese Medicine was collected, and immunohistochemical staining of BCAT1 and BCAT2 was performed; Antibody information: BCAT1, cat. NBP2-01826, purchased from NOVUS; BCAT2, cat. ab309514, purchased from Abcam.

[0055] Experimental results: As can be seen from Figure 1 : Immunohistochemical staining was performed on synovial tissues of 20 knee joints of patients with active rheumatoid arthritis (poorly controlled by drugs) and 20 knee joints of patients with remission rheumatoid arthritis (patients who underwent knee joint replacement). It was found that BCAT2 was highly expressed in synovial tissues of knee joints of patients with active rheumatoid arthritis, while there was no difference in BCAT1.

[0056] Example 2: Targeted metabolomics detection of the changing trends of BCAA and its metabolites (BCKAs) in synovial fluid of patients with active and remission RA Experimental method: Synovial fluid of 10 patients with active and remission RA who underwent knee joint replacement surgery in the Department of Orthopaedics of Wuxi Hospital Affiliated to Nanjing University of Chinese Medicine was collected, and targeted metabolomics detection was performed. That is, the changes of BCAA and its metabolites (BCKAs) in synovial fluid of patients with active RA and remission RA were compared by targeted metabolomics.

[0057] Experimental results: As can be seen from Figure 2It can be seen that: targeted metabolomics was used to detect branched-chain amino acids (BCAAs) and their branched-chain keto acids in the knee joint synovial fluid of patients with active RA (n = 10); REM: knee joint synovial fluid of patients with remission-phase RA (n = 10), and it was found that the levels of two branched-chain keto acids increased in the knee joint synovial fluid of patients with active RA (n = 10).

[0058] Example 3: Animal experiment to reveal the exacerbating effect of high-BCAA diet on CAIA model Experimental protocol: In this study, a collagen antibody-induced arthritis (CAIA) mouse model was used to evaluate the effects of a high branched-chain amino acid diet on inflammation and bone destruction in a rheumatoid arthritis (RA) model.

[0059] 1. Experimental animals

[0060] · Source of animals: 6-week-old male C57BL / 6 mice;

[0061] · Breeding conditions:

[0062] ο Temperature: 22 ± 2°C, humidity 50% ± 10%, 12-hour light-dark cycle.

[0063] ο Fed a standard experimental animal diet and had free access to water.

[0064] ο After 7 days of adaptive feeding, the experiment was conducted.

[0065] 2. Experimental protocol Modeling method

[0066] Experimental protocol: Five mice in each group were given an LAA control diet (2 groups) and an HBCAA diet (2 groups) starting from Day 0

[0067] (1) Collagen antibody injection (Day 30)

[0068] · A mouse anti-type II collagen antibody mixture (CII Antibody Cocktail, Chondrex) was used to induce an autoimmune response.

[0069] · Administration method: Intravenous injection via the tail vein (4 mg / kg).

[0070] (2) LPS-induced inflammation (Day 33 & Day 41)

[0071] · On Day 3: Intraperitoneal injection of lipopolysaccharide (LPS, 50 μg / mouse) to promote the onset of arthritis.

[0072] · On Day 11: Second LPS injection to enhance the inflammatory response and promote the development of acute arthritis.

[0073] (4) Terminal experiment (Day 60)

[0074] · Sacrifice the mice (CO euthanasia) and collect:

[0075] ο Left ankle joint (after decalcification, embedding, and H&E staining).

[0076] ο Right ankle joint (assess bone loss by Micro-CT).

[0077] 3. Statistical analysis

[0078] · The arthritis scores were compared between groups using two-way ANOVA with repeated measures (RM-ANOVA).

[0079] · Other data (qPCR, ELISA, Micro-CT) were analyzed using one-way ANOVA followed by Bonferroni post hoc test.

[0080] · Statistical software: GraphPad Prism 10. A significance level of P < 0.05 was considered statistically significant.

[0081] 4. Research results

[0082] Combined with Figure 3 , conclusion: A high branched-chain amino acid diet (HBCAA) exacerbates joint inflammation and bone destruction in the CAIA model.

[0083] Example 4: Animal experiments reveal that targeting BCAT2 alleviates CAIA joint inflammation and bone destruction

[0084] Combined with attached Figure 4 , where:

[0085] a. Global knockout of BCAT1 in C57 mice did not reduce joint inflammation and bilateral ankle swelling in CAIA model mice;

[0086] b. Conditional knockout of BCAT2 in C57 mice (Bcat2-flox / flox; Lyz2-Cre) alleviated joint inflammation and bilateral ankle swelling in CAIA model mice;

[0087] C. Conditional knockout of BCAT2 in C57 mice (Bcat2-flox / flox; Lyz2-Cre) alleviated synovial inflammation in CAIA model mice;

[0088] d. Based on HE staining and pathological score statistics, it was shown that BCAT2-cKO

[0089] (Bcat2-flox / flox; Lyz2-Cre) could significantly improve the pathological score;

[0090] e.g., in the typical CT images, the statistical results of BS / BV showed that BCAT2-cKO could significantly reduce bone erosion.

[0091] Example 5: Experimental discovery of the inhibitory effect of BAY-069 on BCAT2 through the NADH light absorption detection system

[0092] To detect the effect of BAY-069 on the enzyme activity of BCAT2 (branched-chain amino acid transaminase 2), the researchers established a NADH light absorption detection system to measure the rate of NADH consumption during the conversion of branched-chain amino acids (BCAAs) to branched-chain keto acids (BCKAs) catalyzed by BCAT2.

[0093] 1. Experimental system:

[0094] ο Substrate: L-Leucine (10 mM) served as the main acceptor substrate for BCAT2.

[0095] ο Coenzyme: α-Ketoglutarate (5 mM), acting as the amino acceptor.

[0096] ο Enzyme source: BCAT2 protein recombinantly expressed and purified from E. coli (final concentration 5 μM).

[0097] ο Detection system:

[0098] ■ The reaction system contained 100 mM potassium phosphate buffer (pH 7.4), 5 mM dithiothreitol (DTT), and 5 μM pyridoxal-5'-phosphate (PLP).

[0099] ■ The reaction system also contained excess leucine and α-ketoglutarate to ensure that the reaction rate was limited by enzyme activity.

[0100] ο Enzymatic reaction:

[0101] ■ BCAT2 catalyzed the conversion of BCAA (leucine) to BCKA (α-ketoisocaproic acid, KIC), while α-ketoglutarate was reduced to glutamate.

[0102] ■ This process generated NADH, which could be further catalyzed by NADH-dependent leucine dehydrogenase (LeuDH) to consume NADH.

[0103] ο Light absorption detection:

[0104] ■ The decrease in NADH absorbance was detected at a wavelength of 340 nm to indirectly reflect the rate of the BCAT2-catalyzed reaction.

[0105] ■ The absorbance (OD340 nm) was read using a 96-well microplate reader to monitor the change in NADH absorption from 0 to 30 minutes, and the enzyme activity was calculated.

[0106] 2. Experimental design:

[0107] ο Control group (Control): Without BAY-069, containing only BCAT2, substrate, and coenzyme.

[0108] ο Experimental group (treated with BAY-069):

[0109] ■ Different concentrations of BAY-069 (0.1 μM, 1 μM, 10 μM) were set.

[0110] ■ After pre-incubation for 30 minutes, the reaction was initiated by adding the substrate.

[0111] 3. Data processing:

[0112] ο Calculate the rate of change in NADH absorbance (ΔOD / min) as an index of the catalytic rate of BCAT2.

[0113] The results are shown in Table 1:

[0114] Table 1: Experimental findings on the inhibitory effect of BAY-069 on BCAT2 in the NADH light absorption detection system

[0115]

[0116]

[0117] Data interpretation:

[0118] · Control group (without BAY-069): The absorbance of NADH decreased rapidly over time, indicating that BCAT2 was normally catalyzing BCAA metabolism.

[0119] · BAY-069-treated group:

[0120] ο BAY-069 0.1 μM: The rate of NADH degradation slowed slightly, indicating that low-concentration BAY-069 slightly inhibited BCAT2 activity.

[0121] ο BAY-069 1 μM: The rate of NADH degradation further slowed, and the inhibitory effect was enhanced.

[0122] ο BAY-069 10 μM: The absorbance of NADH remained almost unchanged, indicating that high-concentration BAY-069 almost completely inhibited BCAT2 enzyme activity;

[0123] Example 6: SPR results confirmed that BAY-069 can bind to human BCAT2 with high affinity

[0124] Experimental method:

[0125] Experimental equipment: SPR analysis was performed using a Biacore T200 (Cytiva).

[0126] Chip type: CM5 sensor chip (carboxymethylated dextran surface).

[0127] Immobilization method: The recombinant human BCAT2 protein (His-tagged) was immobilized on the chip surface using the amine coupling method (Amine Coupling).

[0128] Experimental buffer: PBS (pH 7.4) + 0.05% Tween-20

[0129] BAY-069 was used as the analyte and was prepared at different concentrations and flowed over the chip surface.

[0130] Binding and dissociation phases:

[0131] · Association phase: BAY-069 flowed over the chip surface for 120 s at different concentrations.

[0132] · Dissociation phase: The chip was washed with buffer for 300 s.

[0133] Data analysis:

[0134] · Fitting was performed using a 1:1 binding kinetic model.

[0135] · The dissociation constant (Kd), association rate constant (ka), and dissociation rate constant (kd) were calculated.

[0136] Result analysis:

[0137] Binding appendix Figure 5 , BAY-069 was able to directly bind to BCAT2, and Kd = 336 nM, indicating that the two have medium affinity.

[0138] Association rate (Ka = 1.12×10 5 M -1 s -1 ) was relatively fast, indicating that BAY-069 could rapidly bind to BCAT2.

[0139] Dissociation rate (Kd = 3.75×10 -1 s -1 ) was relatively high, indicating that BAY-069 still had a relatively fast dissociation trend after binding to BCAT2, and the binding was relatively reversible.

[0140] Example 7: Animal experiments reveal that BAY-069 targets BCAT2 to improve joint inflammation and bone destruction in the CAIA model

[0141] Method for establishing a mouse model of collagen antibody-induced arthritis (CAIA)

[0142] In this study, a mouse model of collagen antibody-induced arthritis (CAIA) was used to evaluate the effects of BAY-069 on rheumatoid arthritis (RA)-related inflammation and bone destruction.

[0143] 1. Experimental animals

[0144] · Source of animals: Male C57BL / 6 mice, 6 - 8 weeks old;

[0145] · Breeding conditions:

[0146] ο Temperature: 22 ± 2°C, humidity 50% ± 10%, 12-hour light-dark cycle.

[0147] ο Fed with standard experimental animal feed, free access to water.

[0148] ο Adaptively bred for 7 days before the experiment.

[0149] 2. Modeling method

[0150] (1) Collagen antibody injection (Day 0)

[0151] · A mouse anti-type II collagen antibody mixture (CII Antibody Cocktail, Chondrex) was used to induce an autoimmune reaction.

[0152] · Administration method: Intravenous injection via the tail vein (4 mg / kg).

[0153] (2) LPS-induced inflammation (Day 3 & Day 11)

[0154] · Day 3: Intraperitoneal injection of lipopolysaccharide (LPS, 50 μg / mouse) to promote the onset of arthritis.

[0155] · Day 11: Second LPS injection to enhance the inflammatory response and promote the development of acute arthritis.

[0156] (3) Treatment groups (Day 4)

[0157] · Mice were randomly divided into groups (n = 8 / group):

[0158] 1. Normal control group (Control): Not induced with CAIA, only gavaged with PBS.

[0159] 2. CAIA group (CAIA + Vehicle): Received CAIA induction without drug intervention.

[0160] 3. BAY - 069 group (BAY - 069): CAIA + BAY - 069 4.5 mg / kg / d, administered by gavage

[0161] (4) Endpoint experiment (Day 31)

[0162] · Mice were sacrificed (euthanized by CO), and the following were collected:

[0163] ο Left ankle joint (after decalcification, embedding, and H&E staining).

[0164] ο Right ankle joint (assessed for bone loss by Micro - CT).

[0165] 3. Statistical analysis

[0166] · Arthritis scores were compared between groups using two - way ANOVA with repeated measures (Two - way ANOVA, RM - ANOVA).

[0167] · Other data (qPCR, ELISA, Micro - CT) were analyzed using one - way ANOVA (One - way ANOVA) with Bonferroni post - hoc test.

[0168] · Statistical software: GraphPad Prism 10. A significance level of P < 0.05 was considered statistically significant.

[0169] 4. Research results

[0170] Combined with the appendix Figure 6 , BAY - 069 can significantly improve joint inflammation and bone destruction in the CAIA model.

[0171] The above - mentioned is only the preferred specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any person skilled in the art within the technical scope disclosed by this application, according to the technical solution and its concept of this application, makes equivalent substitutions or changes, and all should be covered within the protection scope of this application.

Claims

1. Use of BAY-069 in the preparation of a drug for regulating the inflammatory state of articular synovium.

2. Use of BAY-069 in the preparation of a drug for reducing synovial hyperplasia and bone destruction.

3. Use of BAY-069 in the preparation of a drug for treating rheumatoid arthritis.

4. The application according to claim 3, wherein: The rheumatoid arthritis is active rheumatoid arthritis.

5. Use of BAY-069 in the preparation of a drug for reducing bone loss.

6. The application according to any one of claims 1-5, characterized in that: The dosage concentration of BAY-069 is 1 - 10 μM.

7. The application according to claim 6, wherein: The dosage concentration of BAY-069 is 10 μM.

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