Application of BCAT2 as target spot in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of BCAT2 as a target spot 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 BCAT2 inhibitor (telmisartan) can significantly reduce joint inflammation score, 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 specifically relates to the application of BCAT2 as a target 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, and is a chronic systemic inflammatory disease with incompletely clarified etiology. The peak incidence age is 45 - 60 years old, but it can occur at any age. 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 have 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. 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 for initially relieving 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 play a role by inhibiting cell signaling pathways, but have a relatively high risk of infection.

[0004] Although the 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 the current drug treatment are: 1) Limitations in target selection. The existing RA treatment drugs (such as methotrexate, TNF-α inhibitors, JAK inhibitors, etc.) mainly target inflammatory factors or cell signaling pathways, and fail to solve 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. The existing drugs generally have serious side effects (such as infection risk, liver and kidney damage, etc.), and some patients show 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: While the existing drugs control inflammation, it is difficult to effectively inhibit bone destruction.

[0006] Disorders of branched-chain amino acid (BCAA) metabolism play an important role 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 ketoacids, 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 phenotype of various cancers. High expression of BCATs promotes tumor proliferation by providing metabolic precursors and activating the mTORC1 signaling, and inhibiting BCATs can slow down tumor progression. The role of BCAA and BCATs in the field of rheumatoid arthritis is 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 telmisartan, BAY-069) can inhibit the activity of BCAT2.

[0008] Telmisartan is a specific angiotensin II receptor (type ATⅠ) antagonist and is currently used for the treatment of primary hypertension in adults.

[0009] 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

[0010] To solve the above problems, the present invention provides the application of BCAT2 as a target in the treatment of rheumatoid arthritis. BCAT2 inhibitors can significantly improve inflammation and bone destruction, and at the same time have advantages such as strong targeting and low side effects.

[0011] The application of BCAT2 as a target in the preparation of a drug for regulating the inflammatory state of synovium.

[0012] The application of BCAT2 as a target in the preparation of a drug for simultaneously controlling inflammation and bone destruction.

[0013] The application of BCAT2 as a target in the preparation of a drug for the treatment of rheumatoid arthritis.

[0014] The application of BCAT2 inhibitors in the preparation of a drug for regulating the inflammatory state of synovium.

[0015] The application of BCAT2 inhibitors in the preparation of a drug for reducing synovial hyperplasia and bone destruction.

[0016] The application of BCAT2 inhibitors in the preparation of a drug for the treatment of rheumatoid arthritis.

[0017] Preferably, the rheumatoid arthritis is active rheumatoid arthritis.

[0018] Preferably, the BCAT2 inhibitor is telmisartan.

[0019] Through metabolomics research, the inventors of the present application found that the metabolic enzyme BCAT2 was significantly highly expressed in the synovial tissues of patients with active RA, while its expression was lower in the synovial tissues of patients with remission RA.

[0020] According to an 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.

[0021] Verification of the effect of telmisartan: In the CIA (collagen-induced arthritis) and CAIA (antibody-induced arthritis) animal models, telmisartan could significantly reduce the joint inflammation score and reduce synovial inflammation and bone destruction.

[0022] Micro-CT analysis showed that telmisartan could effectively reduce bone loss.

[0023] Advantages of the present invention:

[0024] The research of the present invention found that BCAT2 was highly expressed in the synovial tissues of patients with active RA and was closely related to inflammation and bone destruction. The present invention first proposed that telmisartan 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.

[0025] Through targeted metabolomics analysis, the present invention found that compared with patients with remission RA and normal control populations, the BCAA metabolism of patients with active RA 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, telmisartan could effectively inhibit BCAT2 activity and reduce the progression of RA inflammation caused by BCAA metabolic disorder. The present invention verified the efficacy of telmisartan in treating RA and its metabolic regulation mechanism, and proposed an innovative RA metabolic targeted treatment strategy with high safety and strong targeting.

[0026] The specific effects are as follows:

[0027] 1) New target, new mechanism: For the first time, the metabolic enzyme BCAT2 was used as a treatment 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 patients with active RA, suggesting its important role in the pathogenesis of RA, and it was closely related to inflammation and bone destruction.

[0028] 2) Verify that the antihypertensive drug telmisartan has a significant efficacy: For the first time, the antihypertensive drug telmisartan is applied to the treatment of RA. Through animal model experiments, the antihypertensive drug telmisartan shows significant anti-inflammatory and anti-bone destruction effects in animal models, and its efficacy is verified in animal models. Moreover, through in vitro experiments, it is found that the antihypertensive drug telmisartan significantly reduces the expression of inflammatory factors (such as TNF-α, IL-6), verifying the significant efficacy of the antihypertensive drug telmisartan as a metabolic enzyme A inhibitor in the treatment of RA.

[0029] 3) High safety: The antihypertensive drug telmisartan is a known drug with high safety and can be quickly promoted to clinical application. The present invention provides a novel RA treatment strategy based on a metabolic enzyme A inhibitor, which has high efficiency and safety.

[0030] 4) The drugs of the BCAT2 inhibitors (such as telmisartan, BAY-069) based on BCAT2 of the present invention can be prepared into oral preparations, which are convenient for treatment and the patient compliance is better. Description of the Drawings

[0031] Figure 1 Expression of BCAT1 and BCAT2 in synovium of joints of RA patients in active and remission phases

[0032] 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;

[0033] 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.

[0034] Figure 2 To detect the change trend of BCAA and its metabolites (BCKAs) by targeted metabolomics;

[0035] 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.

[0036] Figure 3 High BCAA diet exacerbates joint swelling and bone destruction in CAIA model;

[0037] a, Schematic diagram of animal experiment: After sterile mice are given different diets (LAA diet, HBCAA diet) for 4 weeks, the existing diet is maintained, and CAIA modeling is carried out or not;

[0038] b-c, Arthritis scores of each group and swelling degree scores of bilateral posterior ankles;

[0039] d, Representative HE and Safranin-O staining images and micro-CT images of the posterior ankle joints of mice in each group;

[0040] e, Pathological scores of the posterior ankle joints of mice in each group (upper), Bone destruction index BS / BV scores (lower);

[0041] Among them, NC: Blank group; LAA, Control diet group; HBCAA, BCAA increased by 200% compared with LAA, calorie and nitrogen balance diet; CAIA, RA animal model.

[0042] Figure 4 Conditional knockout of BCAT2 reduces CAIA joint inflammation and bone destruction;

[0043] Among them,

[0044] a, Arthritis scores (upper) and average paw thickness (lower) of wild-type (WT) and BCAT1 knockout (BCAT1-KO) mice in the CAIA model;

[0045] b, Arthritis scores (upper) and average paw thickness (lower) of wild-type (WT) and BCAT2 conditional knockout (BCAT2-cKO) mice in the CAIA model;

[0046] c, Representative H&E staining images of joint tissue sections showing histological changes of wild-type, BCAT1-KO and BCAT2-cKO mice in the CAIA model;

[0047] d-e, Histopathological scores of joint inflammation, comparing wild-type with BCAT1-KO (d) and wild-type with BCAT2-cKO (e) mice;

[0048] f, Representative micro-CT images showing joint structures of wild-type and BCAT2-cKO mice;

[0049] g, Quantification of bone surface area / bone volume ratio (BS / BV) of wild-type and BCAT2-cKO mice;

[0050] 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).

[0051] Figure 5 It is the SPR result of telmisartan and BCAT2.

[0052] Figure 6To investigate the effect of telmisartan on improving joint inflammation and bone destruction in the CAIA model;

[0053] TEL, telmisartan; a, joint swelling scores of the blank group (NC), model group (CAIA), and telmisartan group (TEL); 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, typical images of HE staining and safranin-fast green staining for each group; e, typical micro-CT images for each group.

[0054] Figure 7 SPR results of BAY-069 and BCAT2.

[0055] Figure 8 To investigate the effect of BAY-069 on improving joint inflammation and bone destruction in the CAIA model;

[0056] a, joint swelling scores of the blank 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, typical images of HE staining and safranin-fast green staining for each group and typical micro-CT images for each group. Specific implementation manners

[0057] The technical solutions in the embodiments of the present application will be clearly and completely described below 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0058] Example 1: Immunohistochemical comparison of the expression of BCAT2 in the synovial tissues of the knee joints of patients with active and remission RA

[0059] Experimental method: Synovium was collected from 20 patients with active and remission RA who underwent knee joint replacement surgery in the Department of Orthopedics of Wuxi Hospital Affiliated to Nanjing University of Chinese Medicine, and immunohistochemical staining of BCAT1 and BCAT2 was performed; Antibody information: BCAT1, cat. NBP2-01826, purchased from NOVUS; BCAT2, cat. ab309514, purchased from Abcam.

[0060] Experimental results: Combined with Figure 1It can be seen from the following: Immunohistochemical staining was performed on the knee joint synovial tissues of 20 patients with active rheumatoid arthritis (poorly controlled by drugs) and 20 patients with remission rheumatoid arthritis (patients who received knee joint replacement). It was found that BCAT2 was highly expressed in the knee joint synovial tissues of patients with active rheumatoid arthritis, while there was no difference in BCAT1.

[0061] Example 2: Targeted metabolomics to detect the changing trends of BCAA and its metabolites (BCKAs) in synovial fluid of patients with active and remission RA Experimental method: For 10 patients with active and remission RA who underwent knee joint replacement surgery in the Department of Orthopedics of Wuxi Hospital Affiliated to Nanjing University of Chinese Medicine, knee joint synovial fluid was collected for targeted metabolomics detection. That is, the changes in BCAA and its metabolites (BCKAs) in the joint synovial fluid of patients with active RA and remission RA were compared through targeted metabolomics.

[0062] Experimental results: Combined with Figure 2 It can be seen from the following: Targeted metabolomics detected BCAA and its 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 RA (n = 10). It was found that the 2 branched-chain keto acids increased in the knee joint synovial fluid of patients with active RA (n = 10).

[0063] Example 3: Animal experiment reveals 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 the related inflammation and bone destruction in a rheumatoid arthritis (RA) model.

[0064] 1. Experimental animals

[0065] · Source of animals: 6-week-old male C57BL / 6 mice;

[0066] · Breeding conditions:

[0067] ο Temperature: 22 ± 2 °C, humidity 50% ± 10%, 12-hour light-dark cycle.

[0068] ο Fed with standard experimental animal feed and allowed free access to water.

[0069] ο After 7 days of adaptive feeding, the experiment was carried out.

[0070] 2. Experimental protocol Modeling method

[0071] Experimental protocol: Each group had 5 animals. Starting from Day 0, LAA control diet (2 groups) and HBCAA diet (2 groups) were given respectively.

[0072] (1) Collagen antibody injection (Day 30)

[0073] · Induce an autoimmune response using a mouse anti-type II collagen antibody mixture (CII Antibody Cocktail, Chondrex).

[0074] · Administration method: Intravenous injection into the tail vein (4 mg / kg).

[0075] (2) LPS-induced inflammation (Day 33 & Day 41)

[0076] · Day 3: Intraperitoneal injection of lipopolysaccharide (LPS, 50 μg / mouse) to promote the onset of arthritis.

[0077] · Day 11: Second LPS injection to enhance the inflammatory response and promote the development of acute arthritis.

[0078] (4) Endpoint experiment (Day 60)

[0079] · Sacrifice the mice (euthanize by CO), and collect:

[0080] ο The left ankle joint (after decalcification, embedding, and H&E staining).

[0081] ο The right ankle joint (assess bone loss by Micro-CT).

[0082] 3. Statistical analysis

[0083] · Compare the groups for arthritis scores using two-way analysis of variance with repeated measures (Two-way ANOVA, RM-ANOVA).

[0084] · Analyze other data (qPCR, ELISA, Micro-CT) using one-way analysis of variance (One-way ANOVA) with Bonferroni post hoc test.

[0085] · Statistical software: GraphPad Prism 10. A significance level of P < 0.05 is considered statistically significant.

[0086] 4. Research results

[0087] Combined with Figure 3 , conclusion: A high branched-chain amino acid diet (HBCAA) exacerbates joint inflammation and bone destruction in the CAIA model.

[0088] Example 4: Animal experiments reveal that targeting BCAT2 alleviates CAIA joint inflammation and bone destruction

[0089] Combined with the appendix Figure 4 , where:

[0090] a. In C57 mice with global knockout of BCAT1, the joint inflammation and swelling of the double ankle joints in CAIA model mice could not be alleviated;

[0091] b. In C57 mice with conditional knockout of BCAT2 (Bcat2-flox / flox; Lyz2-Cre), the joint inflammation and swelling of the double ankle joints in CAIA model mice were alleviated;

[0092] C. In C57 mice with conditional knockout of BCAT2 (Bcat2-flox / flox; Lyz2-Cre), the synovial inflammation of the joints in CAIA model mice was alleviated;

[0093] d. Based on HE staining and pathological score statistics, it was shown that BCAT2-cKO (Bcat2-flox / flox; Lyz2-Cre) could significantly improve the pathological score;

[0094] e-g. The typical CT images and the statistical results of BS / BV showed that BCAT2-cKO could significantly reduce bone erosion.

[0095] Example 5: Experimental discovery of the inhibitory effect of telmisartan on BCAT2 by NADH light absorption detection system

[0096] To detect the effect of telmisartan (TEL) on the enzyme activity of BCAT2 (branched-chain amino acid aminotransferase 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.

[0097] 1. Experimental system:

[0098] ο Substrate: L-leucine (10 mM) was used as the main acceptor substrate of BCAT2.

[0099] ο Coenzyme: α-ketoglutarate (5 mM), which served as the amino acceptor.

[0100] ο Enzyme source: BCAT2 protein recombinantly expressed and purified from E. coli (final concentration 5 μM).

[0101] ο Detection system:

[0102] ■ The reaction system contained 100 mM potassium phosphate buffer (pH 7.4), 5 mM dithiothreitol (DTT), and 5 μM pyridoxal-5'-phosphate (PLP).

[0103] ■ The reaction system also contains an excess of leucine and α-ketoglutaric acid to ensure that the reaction rate is limited by enzyme activity.

[0104] ο Enzymatic reaction:

[0105] ■ BCAT2 catalyzes the conversion of BCAA (leucine) to BCKA (α-ketoisocaproic acid, KIC), while α-ketoglutaric acid is reduced to glutamic acid.

[0106] · This process generates NADH, which can be further catalyzed by NADH-dependent leucine dehydrogenase (LeuDH), consuming NADH.

[0107] ο Light absorption detection:

[0108] ■ The decrease in the absorbance of NADH is detected at a wavelength of 340 nm, indirectly reflecting the rate of the BCAT2-catalyzed reaction.

[0109] ■ The absorbance (OD340 nm) is read using a 96-well microplate reader, and the change in NADH absorption from 0 to 30 minutes is monitored to calculate the enzyme activity.

[0110] 2. Experimental design:

[0111] ο Control group: Without TEL, containing only BCAT2, substrate, and coenzyme.

[0112] ο Experimental group (TEL treatment):

[0113] ■ Different concentrations of TEL (0.1 μM, 1 μM, 10 μM) are set.

[0114] ■ After pre-incubation for 30 minutes, the substrate is added to initiate the reaction.

[0115] 3. Data processing:

[0116] ο Calculate the rate of change in NADH absorbance (ΔOD / min) as an indicator of the BCAT2 catalytic rate.

[0117] The results are shown in Table 1:

[0118] Table 1: Inhibitory effect of telmisartan on BCAT2 found in the NADH light absorption detection system

[0119]

[0120]

[0121] Data interpretation:

[0122] · Control group (without TEL): The absorbance of NADH decreased rapidly with time, indicating that BCAT2 normally catalyzes BCAA metabolism.

[0123] · TEL treatment group:

[0124] ο TEL 0.1 μM: The degradation rate of NADH slowed down slightly, indicating that low-concentration TEL slightly inhibited BCAT2 activity.

[0125] ο TEL 1 μM: The degradation rate of NADH further slowed down, and the inhibitory effect was enhanced.

[0126] ο TEL 10 μM: The absorbance of NADH remained almost unchanged, indicating that high-concentration TEL almost completely inhibited BCAT2 enzyme activity;

[0127] Example 6: SPR results confirmed that telmisartan could bind to human BCAT2 with high affinity

[0128] Experimental method:

[0129] Experimental equipment: SPR analysis was performed using Biacore T200 (Cytiva).

[0130] Chip type: CM5 sensor chip (carboxymethylated dextran surface).

[0131] Immobilization method: The recombinant human BCAT2 protein (His-tag) was immobilized on the chip surface by amine coupling method.

[0132] Experimental buffer: PBS (pH 7.4) + 0.05% Tween-20.

[0133] TEL was used as the analyte and different concentrations were prepared and flowed through the chip surface.

[0134] Binding and dissociation phases:

[0135] · Association phase: TEL flowed through the chip surface for 120 s at different concentrations.

[0136] · Dissociation phase: The chip was rinsed with buffer for 300 s.

[0137] Data analysis:

[0138] · A 1:1 binding kinetic model was used for fitting.

[0139] · The dissociation constant (Kd), association rate constant (ka) and dissociation rate constant (kd) were calculated.

[0140] Result analysis:

[0141] Combined with the attached Figure 5 , telmisartan (TEL) can directly bind to BCAT2, and Kd = 632 nM, indicating that the two have medium affinity.

[0142] Binding rate (Ka = 6.06×10 5 M -1 s -1 ) is relatively fast, indicating that TEL can quickly bind to BCAT2.

[0143] Dissociation rate (Kd = 3.83×10 -1 s -1 ) is relatively high, indicating that TEL still has a relatively fast dissociation trend after binding to BCAT2, and the binding is relatively reversible.

[0144] Example 7: Animal experiments reveal that telmisartan targets BCAT2 to improve joint inflammation and bone destruction in the CAIA model

[0145] Method for establishing a collagen antibody-induced arthritis (CAIA) mouse model

[0146] In this study, a collagen antibody-induced arthritis (CAIA) mouse model was used to evaluate the effects of telmisartan (TEL) on rheumatoid arthritis (RA)-related inflammation and bone destruction.

[0147] 1. Experimental animals

[0148] · Source of animals: 6-8-week-old male C57BL / 6 mice;

[0149] · Breeding conditions:

[0150] ο Temperature: 22±2°C, humidity 50%±10%, 12-hour light-dark cycle.

[0151] ο Feed standard experimental animal feed and drink water freely.

[0152] ο Conduct experiments after 7 days of adaptive feeding.

[0153] 2. Modeling method

[0154] (1) Collagen antibody injection (Day 0)

[0155] · Use a mouse anti-type II collagen antibody mixture (CII Antibody Cocktail, Chondrex) to induce an autoimmune reaction.

[0156] · Administration method: Intravenous injection into the tail vein (4 mg / kg).

[0157] (2) LPS-induced inflammation (Day 3 & Day 11)

[0158] · Day 3: Intraperitoneal injection of lipopolysaccharide (LPS, 50 μg / mouse) to promote the onset of arthritis.

[0159] · Day 11: Second LPS injection to enhance the inflammatory response and promote the development of acute arthritis.

[0160] (3) Treatment groups (Day 4)

[0161] · Mice were randomly divided into groups (n = 8 / group):

[0162] 1. Normal control group (Control): Not subjected to CAIA induction, only gavaged with PBS.

[0163] 2. CAIA group (CAIA + Vehicle): Subjected to CAIA induction but without drug intervention.

[0164] 3. Negative control group (irbesartan, IRB): CAIA + IRB 5 mg / kg / d, gavaged)

[0165] 4. TEL group (telmisartan, TEL): CAIA + TEL 5 mg / kg / d, gavaged)

[0166] (4) Endpoint experiment (Day 31)

[0167] · Mice were sacrificed (euthanized by CO), and the following were collected:

[0168] ο Left ankle joint (decalcified, embedded, and subjected to H&E staining).

[0169] ο Right ankle joint (assessed for bone loss by Micro-CT).

[0170] 3. Statistical analysis

[0171] · Arthritis scores were compared between groups using two-way analysis of variance with repeated measures (Two-way ANOVA, RM-ANOVA).

[0172] · Other data (qPCR, ELISA, Micro-CT) were analyzed using one-way analysis of variance (One-way ANOVA) with Bonferroni post hoc test.

[0173] · Statistical software: GraphPad Prism 10, and a significance level of P < 0.05 was considered statistically significant.

[0174] 4. Research results

[0175] Combined with the attached Figure 6 Compared with irbesartan, telmisartan can significantly improve joint inflammation and bone destruction in the CAIA model.

[0176] Example 8: Inhibition of BCAT2 by BAY-069 found in the NADH light absorption detection system experiment

[0177] 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.

[0178] 4. Experimental system:

[0179] ο Substrate: L-Leucine (10 mM) as the main acceptor substrate for BCAT2.

[0180] ο Coenzyme: α-Ketoglutarate (5 mM), as the amino acceptor.

[0181] ο Enzyme source: BCAT2 protein recombinantly expressed and purified from E. coli (final concentration 5 μM).

[0182] ο Detection system:

[0183] ■ The reaction system contains 100 mM potassium phosphate buffer (pH 7.4), 5 mM dithiothreitol (DTT), and 5 μM pyridoxal-5'-phosphate (PLP).

[0184] ■ The reaction system also contains excess leucine and α-ketoglutarate to ensure that the reaction rate is limited by enzyme activity.

[0185] ο Enzymatic reaction:

[0186] ■ BCAT2 catalyzes the conversion of BCAA (leucine) to BCKA (α-ketoisocaproic acid, KIC), while α-ketoglutarate is reduced to glutamate.

[0187] ■ This process generates NADH, which can be further catalyzed by NADH-dependent leucine dehydrogenase (LeuDH) to consume NADH.

[0188] ο Light absorption detection:

[0189] ■ The decrease in NADH absorbance is detected at a wavelength of 340 nm, indirectly reflecting the rate of the BCAT2-catalyzed reaction.

[0190] ■ 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.

[0191] 5. Experimental design:

[0192] ο Control group: Without BAY-069, containing only BCAT2, substrate, and coenzyme.

[0193] ο Experimental group (treated with BAY-069):

[0194] ■ Different concentrations of BAY-069 (0.1 μM, 1 μM, 10 μM) were set.

[0195] ■ After pre-incubation for 30 minutes, the reaction was initiated by adding the substrate.

[0196] 6. Data processing:

[0197] ο Calculate the rate of change in NADH absorbance (ΔOD / min) as an indicator of the catalytic rate of BCAT2.

[0198] The results are shown in Table 2:

[0199] Table 2: Experimental findings on the inhibitory effect of BAY-069 on BCAT2 in the NADH light absorption detection system

[0200]

[0201] Data interpretation:

[0202] · Control group (without BAY-069): The absorbance of NADH decreased rapidly over time, indicating that BCAT2 was normally catalyzing BCAA metabolism.

[0203] · BAY-069-treated group:

[0204] ο BAY-069 0.1 μM: The degradation rate of NADH slowed slightly, indicating that low-concentration BAY-069 slightly inhibited BCAT2 activity.

[0205] ο BAY-069 1 μM: The degradation rate of NADH further slowed, and the inhibitory effect was enhanced.

[0206] ο BAY-069 10 μM: The absorbance of NADH remained almost unchanged, indicating that high-concentration BAY-069 almost completely inhibited BCAT2 enzyme activity;

[0207] Example 9: SPR results confirmed that BAY-069 can bind to human BCAT2 with high affinity

[0208] Experimental method:

[0209] Experimental equipment: SPR analysis was performed using a Biacore T200 (Cytiva).

[0210] Chip type: CM5 sensor chip (carboxymethylated dextran surface).

[0211] Immobilization method: The recombinant human BCAT2 protein (His-tagged) was immobilized on the chip surface using the amine coupling method (Amine Coupling).

[0212] Experimental buffer: PBS (pH 7.4) + 0.05% Tween-20

[0213] BAY-069 was used as the analyte and different concentrations were prepared and flowed over the chip surface.

[0214] Binding and dissociation phases:

[0215] · Association phase: BAY-069 flowed over the chip surface for 120 s at different concentrations.

[0216] · Dissociation phase: Flushed with buffer for 300 s.

[0217] Data analysis:

[0218] · Fitted using a 1:1 binding kinetic model.

[0219] · Calculate the dissociation constant (Kd), association rate constant (ka), and dissociation rate constant (kd).

[0220] Result analysis:

[0221] Binding appendix Figure 7 , BAY-069 was able to directly bind to BCAT2, and Kd = 336 nM, indicating a medium affinity between the two.

[0222] Association rate (Ka = 1.12×10 5 M -1 s -1 ) was relatively fast, indicating that BAY-069 could quickly bind to BCAT2.

[0223] 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.

[0224] Example 10: Animal experiments revealed that BAY-069 targets BCAT2 to improve joint inflammation and bone destruction in the CAIA model

[0225] Method for Establishing a Mouse Model of Collagen Antibody-Induced Arthritis (CAIA)

[0226] 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)-associated inflammation and bone destruction.

[0227] 1. Experimental Animals

[0228] · Source of animals: Male C57BL / 6 mice, 6 - 8 weeks old;

[0229] · Breeding conditions:

[0230] ο Temperature: 22 ± 2°C, humidity 50% ± 10%, 12-hour light-dark cycle.

[0231] ο Fed with standard experimental animal feed, free access to water.

[0232] ο Adaptively bred for 7 days before the experiment.

[0233] 2. Modeling Method

[0234] (1) Collagen Antibody Injection (Day 0)

[0235] · Induce an autoimmune response using a mouse anti-type II collagen antibody mixture (CII Antibody Cocktail, Chondrex).

[0236] · Administration method: Intravenous injection via the tail vein (4 mg / kg).

[0237] (2) LPS-Induced Inflammation (Day 3 & Day 11)

[0238] · Day 3: Intraperitoneal injection of lipopolysaccharide (LPS, 50 μg / mouse) to promote the onset of arthritis.

[0239] · Day 11: Second LPS injection to enhance the inflammatory response and promote the development of acute arthritis.

[0240] (3) Treatment Grouping (Day 4)

[0241] · Randomly divide the mice into groups (n = 8 / group):

[0242] 1. Normal Control Group (Control): Not subjected to CAIA induction, only gavaged with PBS.

[0243] 2. CAIA group (CAIA + Vehicle): Received CAIA induction without drug intervention.

[0244] 3. BAY-069 group (BAY-069): CAIA + BAY-069 at 4.5 mg / kg / d, by gavage

[0245] (4) Endpoint experiment (Day 31)

[0246] · Mice were sacrificed (euthanized by CO), and the following were collected:

[0247] ο Left ankle joint (decalcified, embedded, and stained with H&E).

[0248] ο Right ankle joint (assessed for bone loss by Micro-CT).

[0249] 3. Statistical analysis

[0250] · The arthritis scores were compared between groups using two-way analysis of variance with repeated measures (Two-way ANOVA, RM-ANOVA).

[0251] · Other data (qPCR, ELISA, Micro-CT) were analyzed using one-way analysis of variance (One-way ANOVA) with Bonferroni post hoc test.

[0252] · Statistical software: GraphPad Prism 10. A significance level of P < 0.05 was considered statistically significant.

[0253] 4. Research results

[0254] Combined with Appendix Figure 8 , BAY-069 can significantly improve joint inflammation and bone destruction in the CAIA model.

[0255] As described above, the above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application, according to the technical solutions and concepts of the present application, making equivalent substitutions or changes, should be covered within the protection scope of the present application.

Claims

1. Use of BCAT2 as a target in the preparation of a drug for regulating the inflammatory state of joint synovium.

2. Use of BCAT2 as a target in the preparation of a drug for simultaneously controlling inflammation and bone destruction.

3. Use of BCAT2 as a target in the preparation of a drug for treating rheumatoid arthritis.

4. Use of BCAT2 as a target in the preparation of a drug for reducing bone loss.

5. Use of a BCAT2 inhibitor in the preparation of a drug for regulating the inflammatory state of joint synovium.

6. Use of a BCAT2 inhibitor in the preparation of a drug for reducing synovial hyperplasia and bone destruction.

7. Use of a BCAT2 inhibitor in the preparation of a drug for treating rheumatoid arthritis.

8. Use of a BCAT2 inhibitor in the preparation of a drug for reducing bone loss.

9. The application according to claim 3 or 7, characterized in that: The rheumatoid arthritis is active rheumatoid arthritis.

10. The application according to any one of claims 5-9, characterized in that: The BCAT2 inhibitor is telmisartan.

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