Application of eupatilin in medicine for inhibiting proliferation and invasion ability of synovial fibroblast-like cells

Abstract

The invention belongs to the technical field of medicines, particularly relates to application of eupatilin in treatment of synovial arthropathy (such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and the like) and other joint diseases characterized by synovial pathology hyperplasia, and provides novel application of eupatilin in preparation of medicines for resisting synovial arthropathy. Particularly, the compound has a good inhibition effect on abnormal behaviors such as proliferation and invasion of synovial fibroblasts; eupatilin is provided as an effective medicine component for treating joint deformation, and the main action mechanism is that clinical symptoms are relieved by inhibiting proliferation and invasion of RA-FLS; the innovation of the invention lies in providing a therapy based on eupatilin, and through application of a specific dosage, joint inflammation of patients can be effectively relieved and joint functions can be improved.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and specifically to the use of a pharmaceutical composition containing a natural flavonoid compound, eupatilin, as an active ingredient in the preparation of a drug for inhibiting the proliferation and invasion of synovial fibroblasts. The pharmaceutical composition is particularly suitable for treating joint diseases characterized by pathological synovial hyperplasia, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), and psoriatic arthritis (PsA). Background Art

[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by persistent inflammation and aggressive proliferation of the synovium. Its core pathological manifestation is the abnormal activation of synovial fibroblasts (FLS), which leads to: (1) pathological proliferation and invasion, forming pannus that erode cartilage and bone tissue; and (2) excessive secretion of proinflammatory cytokines such as IL-6 and TNF-α, which amplifies the inflammatory cascade and ultimately leads to cartilage and bone tissue destruction and joint dysfunction. Therefore, directly inhibiting the proliferation and invasion ability of FLS is a key target for blocking the progression of RA.

[0003] Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and immunosuppressants (such as methotrexate and leflunomide) are commonly used clinically to control pain, swelling, and systemic inflammatory responses. However, these drugs are mostly symptomatic and are difficult to directly inhibit synovial proliferation and invasion. NSAIDs (such as ibuprofen) only relieve symptoms but cannot inhibit synovial invasion; glucocorticoids (such as prednisone): long-term use leads to osteoporosis and increased risk of infection; immunosuppressants (such as methotrexate): although they can delay joint destruction, they have side effects such as hepatotoxicity and bone marrow suppression, and their direct inhibitory effect on FLS is limited. At the same time, long-term use of these drugs can easily cause gastrointestinal, cardiovascular, and immune adverse reactions, and the economic burden on patients is also heavy.

[0004] Traditional Chinese medicine has been used to treat rheumatoid arthritis for thousands of years, with moxa, moxa patches, and moxibustion therapy, derived from Artemisia argyi extract, being the most widely used. Eupatilin, the primary active flavonoid found in Artemisia argyi plants, possesses diverse biological activities, including anti-inflammatory, antioxidant, and anti-fibrotic activities. Modern research has demonstrated that eupatilin exhibits promising therapeutic effects in various inflammatory and fibrotic disease models. However, its direct inhibitory effects on the proliferation and invasion of RA-FLS have not been systematically reported, and no drug development programs are available. Summary of the Invention

[0005] In response to the problem that the existing technology has not yet effectively inhibited RA synovial hyperplasia and joint invasion, the present invention provides a use and pharmaceutical preparation of isoeupretinoin, which can specifically inhibit the proliferation, migration / invasion ability of RA-FLS, thereby delaying the progression of synovial pathology and improving joint function.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides the use of isoeupretinoic acid in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts. The isoeupretinoic acid inhibits the proliferation activity and invasion / migration ability of synovial fibroblasts, thereby delaying the progression of synovial lesions, improving joint function, and treating rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis.

[0008] Furthermore, the effective concentration of isoeutin on synovial fibroblasts is 12.5-50 μM, preferably 50 μM, with an effective inhibitory effect duration of 24-48 hours. It can significantly reduce the proliferation rate (inhibition rate >50%, P <0.001) and invasion ability (inhibition rate >80%, P <0.01) of RA-FLS, and has no significant toxicity to normal fibroblasts (HFF).

[0009] Furthermore, the dosage form of the drug includes oral preparations, topical preparations or injection preparations.

[0010] Furthermore, the oral preparation includes tablets and capsules, and the content of isoeuphenin in the oral preparation is 5-100 mg / unit.

[0011] Furthermore, the topical preparation includes gel and ointment, and the mass fraction of isoeuphenin in the topical preparation is 0.1%-5%.

[0012] Furthermore, the injection preparation includes a microneedle, and the concentration of isoeufonin in the injection preparation is 100-200 μg / mL.

[0013] The present invention also provides a pharmaceutical composition for inhibiting the proliferation and invasion of synovial fibroblasts. The pharmaceutical composition comprises isoeupretinoic acid as the sole active ingredient and further comprises pharmaceutically acceptable excipients. Isoeupretinoic acid is derived from an extract of an Artemisia plant. The pharmaceutical composition is used to inhibit the proliferation and invasion of RA-FLS, thereby delaying the progression of synovial lesions and improving joint function.

[0014] Furthermore, the pharmaceutical composition, including but not limited to tablets, capsules, ointments, gels and transdermal microneedle dosage forms, is suitable for oral administration, topical use or local injection to maintain blood drug concentration or local concentration within the above-mentioned effective range, and is used for systemic administration to inhibit RA-FLS proliferation and synovial invasion. For local treatment, it can be made into an external preparation and applied to the affected joint to inhibit joint pain and local inflammatory reactions caused by abnormal proliferation and invasion of the synovial membrane.

[0015] Isoeupretinoin inhibits the proliferation and invasion of RA-FLS cells, thereby regulating the pathological progression of the synovial membrane in rheumatoid arthritis and improving patients' joint function and quality of life. The present invention verified the anti-proliferative and anti-invasive effects of isoeupretinoin using the CCK-8 assay and the xCELLigence RTCA system.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides isoeupretinoin which has the function of directly targeting synovial fibroblast-like cells, specifically inhibiting pathologically activated RA-FLS, inhibiting the pathological proliferation and invasion thereof, and controlling the progression of synovial joint lesions.

[0018] (1) Inhibition of RA-FLS proliferation: CCK-8 assay confirmed that it can significantly inhibit cell proliferation in the concentration range of 12.5-50 μM (P < 0.001). At 50 μM, the inhibition rate is > 50% and there is no cytotoxicity, controlling abnormal synovial proliferation.

[0019] (2) Blocking RA-FLS invasion: As detected by the xCELLigence RTCA system, the cell invasion ability decreased by >80% after 24 hours of treatment with 50 μM (P < 0.01), inhibiting the cartilage infiltration and erosion ability of synovial cells;

[0020] (3) High safety and flexible dosing: The innovation of this invention lies in providing a therapy based on isoeupretinoic acid, which can effectively relieve patients' joint inflammation and improve joint function. It can be administered in a variety of dosage forms, including oral, injection, or topical. In addition, isoeupretinoic acid is derived from the traditional Chinese medicine Artemisia argyi plant, has a low extraction cost, and is suitable for chronic, long-term use, reducing the financial burden on patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the experimental results of cytotoxicity of isoeliaflavin to human dermal fibroblasts (HFF);

[0022] Figure 2 is the inhibitory effect curve of isoeupretinoin on RA-FLS proliferation;

[0023] Figure 3is the inhibitory effect curve of isoeupretinoin on RA-FLS invasion;

[0024] Figure 4 This is an experiment to inhibit the invasion of synovial fibroblasts by isoeutaxanthin. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] Experimental study on the safety of isoeutin on human dermal fibroblasts

[0028] 1. Experimental Conditions

[0029] (1) Cell source: human dermal fibroblasts (HFF, ATCC CRL-4026);

[0030] (2) Culture conditions: DMEM medium (containing 10% FBS), 37°C, 10% CO2 constant temperature incubator;

[0031] (3) Detection method: Real Time Cell Analysis (RTCA) E-plate, cytotoxicity assay;

[0032] (4) Seeding density: 5000 cells / well.

[0033] 2. Drug treatment

[0034] (1) Isozepin concentration: set up 7 concentration groups (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM) and a blank control;

[0035] (2) Intervention / detection time of isoelastin: After HFF cells entered the logarithmic growth phase, they were continuously tested for 48 hours after drug treatment.

[0036] 3. Real-time monitoring

[0037] (1) Detection method: The Agilent xCELLigence RTCA system was used to dynamically monitor the cell index (CI) using impedance spectroscopy.

[0038] (2) Parameter Settings: Cells were seeded at a density of 5,000 cells per well in an E-plate, which was placed on the RTCA platform in an incubator at 37°C and 10% CO2. Cell index was recorded every 15 minutes. When cells reached the logarithmic growth phase, the test compound was introduced, and proliferation dynamics were monitored continuously for 48 hours.

[0039] 4. Data Processing Methods

[0040] (1) Time dependence curve analysis ( Figure 1 , A): CI(t) = (Z(t) - Z(blank)) / (Z(max) - Z(blank)) × 1000; where Z is the transwell impedance value and Z(blank) is the blank well baseline value;

[0041] (2) Calculation of normalized cell index ( Figure 1 , B): Normalized CI = control group CI / treatment group CI × 100%, CI_norm(t) = [CI(t)-CI(0)] / [CI(24h)-CI(0)]×100%, the 24h and 48h time points were selected for inter-group comparison;

[0042] (3) Calculation of proliferation rate ( Figure 1 , C): Proliferation Rate = ΔCIpre / ΔCIpost, normalized comparison of the proliferation rate in the 24-hour interval before drug treatment and the proliferation rate 48 hours after drug treatment.

[0043] 5. Experimental Results

[0044] The experimental results showed that compared with the control group, isoetzatin showed no significant cytotoxicity to HFF at concentrations ≤50 μM.

[0045] Example 2

[0046] Experimental study on the inhibition of proliferation of synoviocyte-like fibroblasts (RA-FLS) by isoeutin

[0047] 1. Experimental Conditions

[0048] (1) Cell source: Primary isolated synovial fibroblasts from human rheumatoid arthritis patients;

[0049] (2) Culture conditions: DMEM medium (containing 15% FBS), 37°C, 10% CO2 constant temperature incubator;

[0050] (3) Detection methods: Real Time Cell Analysis (RTCA) E-plate; CCK-8 cell viability detection kit (CCK8, Elabscience);

[0051] (4) Seeding density: 2500 cells / well.

[0052] 2. Drug treatment

[0053] (1) Isozepin concentration: set up four concentration groups (6.25 μM, 12.5 μM, 25 μM, 50 μM) and a blank control;

[0054] (2) Intervention / detection time of isoelastin: After the cells enter the logarithmic growth phase, the cells are tested continuously for 48 hours after drug treatment.

[0055] 3. Real-time monitoring

[0056] (1) RTCA Cell Proliferation Assay: Real-time cell proliferation was monitored using the Agilent xCELLigence RTCADPlus system. Cells were seeded at a density of 2,500 cells per well in an E-plate, which was placed on the RTCA platform in an incubator at 37°C and 10% CO2. The cell index was recorded every 15 minutes. When the cells reached the logarithmic growth phase, the test compound was introduced, and the proliferation dynamics were monitored continuously for 48 hours.

[0057] (2) CCK-8 cell viability assay: Cell viability was assessed using the Cell Counting Kit-8. Cells in the logarithmic growth phase were counted at 5×10 ^4 The cells were inoculated into a 96-well plate at a density of 100 μL / well at 48 h. After 48 h of incubation, the test compound was added to the test wells and incubated for another 48 h. Subsequently, 10 μL of CCK8 reagent was added to each well and incubated at 37°C, 10% CO₂ for 3 h. The optical density (OD) was measured at 450 nm using a microplate reader (CLARIOstar Plus, BMGLABTECH).

[0058] 4. Data Processing Methods

[0059] (1) CCK-8 cell activity assay ( Figure 2 , A): Cell survival rate (%) = (average OD value of experimental group / average OD value of control group) × 100%

[0060] (2) RTCA time dependence curve analysis ( Figure 2, B): CI(t) = (Z(t) - Z(blank)) / (Z(max) - Z(blank)) × 1000; where Z is the transwell impedance value and Z(blank) is the blank well baseline value;

[0061] (3) Calculation of normalized cell index ( Figure 2 , C): Normalized CI = control group CI / treatment group CI × 100%, CI_norm(t) = [CI(t)-CI(0)] / [CI(24h)-CI(0)]×100%, the 24h and 48h time points were selected for inter-group comparison;

[0062] (4) Calculation of proliferation rate ( Figure 2 , D): Proliferation Rate = ΔCIpre / ΔCIpost, normalized comparison of the proliferation rate in the 24-hour interval before drug treatment and the proliferation rate 48 hours after drug treatment.

[0063] 5. Experimental Results

[0064] The experimental results showed that compared with the control group, 12.5μM, 25μM, and 50μM concentrations of isoelastin all showed significant proliferation inhibitory effects on RA-FLS cells, among which 50μM had the strongest proliferation inhibition ability, with an inhibition rate greater than 50% (P<0.001).

[0065] Example 3

[0066] Inhibitory effect of isoeutin on the invasion of synovial fibroblasts

[0067] 1. Experimental Conditions

[0068] (1) Cell source: Primary isolated synovial fibroblasts from human rheumatoid arthritis patients;

[0069] (2) Culture conditions: DMEM medium (containing 15% FBS), 37°C, 10% CO2 constant temperature incubator;

[0070] (3) Detection method: Agilent's xCELLigence RTCADPlus system CIM-plate.

[0071] 2. Drug treatment

[0072] (3) Isozepin concentration: 50 μM and blank control;

[0073] (4) Intervention / detection time of isoelastin: After the cells entered the logarithmic growth phase, they were pretreated with drugs for 24 hours, and then the invasion experiment was performed. The invasion detection process continued with drug administration for 24 hours.

[0074] 3. Invasion Inhibition Assay

[0075] (1) Coating of CIM invasion chambers: Dilute Corning's Matrigel concentrate (HC type) with serum-free culture medium at a ratio of 1:100, take 20 μL of the mixture and evenly spread it on the CIM upper chamber, and incubate at 37°C for 12 hours to allow the Matrigel to solidify.

[0076] (2) Drug pretreatment: As described in Section 2, RA-FLS cells in the logarithmic growth phase were pretreated with 50 μM isoelastin for 24 h. The cells were collected and the density was adjusted to 2 × 10 5 / ml;

[0077] (3) Cell seeding: 20,000 cells per well were seeded in the upper chamber of the CIM-plate (containing serum-free medium with the same concentration of the test compound), and the lower chamber was supplemented with serum-containing medium to establish a gradient. Real-time detection was continued by incubation for 48 hours.

[0078] (4) Real-time detection of invasive ability ( Figure 3 A) The CIM-plate was placed in the RTCA system and monitored in real time for 24 hours. Cells were seeded in parallel in 96-well plates for microscopic nuclei counting. To eliminate potential differences in cell seeding density, cells were seeded in parallel in 96-well plates and counted to normalize the invasion cell index. Cells were then fixed and stained for nuclei with H33342. Cell number was quantified by counting nuclei using a Zeiss (Germany) Cell Discoverer 7 imaging system.

[0079] 4. Data Processing Methods

[0080] (1) RTCA invasion data were quantified using Delta-CI ( Figure 3 ,B): Delta-CI(t)=CI(t)-CI(0); the Delta-CI values ​​at 0h and 24h were selected to compare the changes between the treatment group and the control group;

[0081] (2) Calculation of normalized relative cell index ( Figure 3 , C): The cell nuclei were stained with H33342, and the number of cell nuclei was counted under a microscope. The normalized invasion rate (%) was calculated as [number of cell nuclei in the treatment group / number of cell nuclei in the control group] × 100%.

[0082] 5. Experimental Results

[0083] Experimental results ( Figure 3) showed that compared with the control group, 50 μM isoelastin concentration had a significant inhibitory effect on the invasion ability of RA-FLS cells, and the invasion ability of cells decreased by more than 80% within 24 hours (P<0.01).

[0084] Example 4

[0085] Interventional experiment of isoelastin injection in CFA arthritis model rats

[0086] 1. Experimental Animals and Grouping Labeling

[0087] The experimental animals were Wistar rats, weighing 180-220 g, of either sex. The animals were sourced in accordance with the Standards for Laboratory Animal Care and were acclimated for 7 days prior to the experiment. The rats were divided into the following two groups (n = 8 each): a normal control group and an isoepiphyseal-treated group:

[0088] 2. CFA modeling operation

[0089] (1) Weigh the animal, calculate the injection dose of complete Freund's adjuvant (CFA) (0.75 mg / kg), and dispense the drug using a 1 mL disposable syringe;

[0090] (2) Start the isoflurane anesthesia machine, set the anesthetic concentration to 1.5%, and the anesthetic pressure to 1, and operate two rats at a time;

[0091] (3) After the rat's limbs relax and lose consciousness, remove it and place it on the operating table. Disinfect the plantar part of the left hind paw with a 75% alcohol cotton ball.

[0092] (4) 0.1 mL of CFA solution was slowly injected subcutaneously through the sole of the left hind foot. The needle was advanced from the heel along the sole to the midfoot. The needle tip was faintly visible through the skin during injection. After confirming that there was no local blood backflow, the injection was slowly injected to avoid tissue rupture.

[0093] (5) After the injection, the rats were returned to a clean cage and the inflammatory response was continuously observed.

[0094] 3. Local intervention with isoetzatin

[0095] 48 hours after the model was established, significant swelling and redness of the ankles were confirmed, and drug injection intervention was performed on the soles of the feet of the rats in the isoeuphenin group:

[0096] (1) Prepare isoelastin injection at a concentration of 1 mg / μL by dissolving it in a small amount of DMSO and then diluting it in sterile PBS to a final concentration of DMSO < 1%;

[0097] (2) Under aseptic operation, the skin surface of the left ankle joint was disinfected again with 75% alcohol, and a 1 mL syringe was used to puncture the posterior edge of the medial malleolus. Each rat was injected with 100 μL (100 μg) of isoepin solution once;

[0098] (3) The control group was injected with an equal amount of PBS solution.

[0099] 4. Efficacy evaluation indicators

[0100] (1) Ankle joint redness and swelling score ( Figure 4 A): Record the degree of ankle redness and swelling every day according to the swelling grade (0-4 points) to evaluate the progression and relief trend of inflammation;

[0101] (2) Ankle thickness measurement Figure 4 , B): The vertical diameter of the thickest part of the ankle joint was measured daily with a vernier caliper, and the data were recorded for 7 consecutive days to assess the degree of inflammation and swelling;

[0102] (3) Synovial tissue sampling and HE staining Figure 4 , C): After treatment, the animals were sacrificed, and the synovial tissue of the left ankle joint was fixed, sectioned in paraffin, and stained with HE to evaluate the degree of synovial proliferation, pannus formation, and inflammatory cell infiltration;

[0103] 5. Experimental Results

[0104] Results showed that ankle swelling was significantly reduced in the isoeupretinoic acid-treated group, with the ankle diameter decreasing by more than 50% (P < 0.01). Inflammatory cell infiltration in the synovial tissue was also significantly reduced, suggesting that isoeupretinoic acid significantly inhibits synovial pathology. HE staining showed that it effectively blocked synovial hyperplasia and neovascularization, protecting joint structural integrity.

[0105] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0106] It should be noted that the above content merely illustrates the technical idea of ​​the present invention and cannot be used to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. Use of isoeupretinoic acid in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The isoeupretinoin delays the progression of synovial lesions and improves joint function by inhibiting the proliferation activity and invasion / migration ability of synovial fibroblasts.

2. The use of isoeupretinoic acid according to claim 1 in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The effective concentration of the isoeupretinoin acting on synovial fibroblasts is 12.5-50 μM, and the effective inhibitory effect time is 24-48 hours.

3. Use of isoeupretinoic acid according to claim 1 or 2 in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The dosage forms of the drug include oral preparations, topical preparations or injection preparations.

4. Use of isoeupretinoic acid according to claim 3 in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The oral preparation includes tablets and capsules, and the content of isoeuphenin in the oral preparation is 5-100 mg / unit.

5. Use of isoeupretinoic acid according to claim 3 in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The topical preparation includes gel and ointment. The mass content of isoeuphenin in the topical preparation is 0.1%-5%.

6. Use of isoeupretinoic acid according to claim 3 in the preparation of a medicament for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The injection preparation comprises a microneedle, and the concentration of isoeuphenin in the injection preparation is 100-200 μg / mL.

7. A pharmaceutical composition for inhibiting the proliferation and invasion of synovial fibroblasts, characterized in that: The pharmaceutical composition is a pharmaceutical composition with isoeupretinoic acid as an active ingredient, and further comprises pharmaceutically acceptable excipients. The isoeupretinoic acid in the pharmaceutical composition is derived from an extract of an Artemisia plant.

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