Application of ginkgolide A in treatment of rheumatoid arthritis
By using ginkgolactone A (GA) as an anti-inflammatory drug, the side effects, drug resistance and high cost in the treatment of rheumatoid arthritis were solved, and significant therapeutic effects and economic benefits were achieved.
Patent Information
- Application Number
- CN202510467471.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-06-17
AI Technical Summary
Existing rheumatoid arthritis (RA) treatments have serious side effects, drug resistance and high treatment costs.
Ginkgolactone A (GA) was used as a new Chinese medicine monomer to treat RA through anti-inflammatory effects, including verifying the anti-inflammatory effects of GA through in vivo animal experiments and in vitro cell experiments.
GA significantly inhibits joint swelling and inflammation, improves bone erosion, has good therapeutic effects, and has significant therapeutic cost advantages, and is suitable for primary medical promotion.
Smart Images
Figure CN120154597A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and provides the use of ginkgolide A as a new traditional Chinese medicine monomer in the treatment of rheumatoid arthritis. Background Art
[0002] At present, the drugs for treating rheumatoid arthritis (RA) clinically are mainly divided into three categories. The first category is traditional chemical drugs: including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying anti-rheumatic drugs (DMARDs, such as methotrexate). These drugs mainly act by inhibiting the inflammatory response or regulating the immune system, but long-term use is likely to cause side effects such as gastrointestinal damage, liver and kidney function damage, and bone marrow suppression. The second category is biological agents: such as tumor necrosis factor-α (TNF-α) inhibitors, interleukin 6 (IL-6) receptor antagonists, etc. Although these drugs have a rapid onset and strong targeting, there are problems such as high prices, increased risk of infection (such as tuberculosis, hepatitis recurrence), drug resistance, and increased risk of tumor occurrence. The third category is the application of plant extracts.
[0003] At present, the clinical treatment plan for RA still has obvious limitations, mainly including the following three aspects. The first is significant side effects: both traditional drugs and biological agents have serious side effects. For example, NSAIDs are prone to cause digestive tract ulcers, and biological agents increase the risks of infection and tumors. The fundamental reason lies in the insufficient drug targeting or excessive inhibition of the immune system function. The second is drug resistance and dependence: biological agents need to be used for a long time, and patients are prone to develop drug resistance; while traditional drugs are prone to relapse after discontinuation and cannot achieve disease-modifying treatment. The third is high treatment costs: the annual treatment cost of biological agents can reach tens of thousands of yuan, which is difficult to popularize in primary hospitals and limits the accessibility of patients.
[0004] Ginkgolide A (GA) is a natural terpene compound extracted from Ginkgo biloba leaves. As a plant extract, drugs related to ginkgolides have made certain research progress. In basic research, regarding its effects on the nervous system: GA has neuroprotective effects and can reduce neuronal damage through various mechanisms, such as inhibiting inflammatory responses and regulating apoptosis, and has certain research value for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Regarding its effects on the cardiovascular system: GA can dilate blood vessels, improve microcirculation, and reduce blood viscosity, and has certain therapeutic effects on cardiovascular diseases such as coronary heart disease and angina pectoris. In addition, GA also has effects such as antioxidant, anti-aging, and improving cognitive function. In clinical research, preparations such as ginkgolide injection are mainly used in the clinical treatment of stroke and can improve the neurological deficits of patients. In addition, it can also be combined with other drugs for the treatment of cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris, and cerebral infarction, showing a synergistic effect. The treatment of other diseases also includes diabetic peripheral neuropathy and tinnitus and dizziness, etc.
[0005] However, although Ginkgo biloba extracts (containing ginkgolide components) have been widely used in cardiovascular and neurological diseases, their research in the treatment of RA is less, and existing technologies mostly focus on crude extracts or mixed lactone components, without clarifying the mechanism of action of a single component (such as GA). Moreover, the clinical application of GA is still blank, mainly due to the lack of mechanism research: existing literature mostly focuses on the pharmacological effects of ginkgolide B, while the specific targets of GA in RA (such as PAF receptor antagonism and cytokine regulation) have not been clarified, lacking systematic mechanism research. Secondly, its indications are limited: the existing applications of ginkgolide components are concentrated in cardiovascular diseases, and their potential in autoimmune diseases (such as RA) has not been fully explored. Summary of the Invention
[0006] The present invention provides a use of GA for the treatment of RA. It is confirmed through in vivo animal experiments and in vitro cell experiments that GA can achieve the treatment of RA through its anti-inflammatory effect.
[0007] In the first aspect of the present invention, there is provided an application of GA, its crystal form, its pharmaceutically acceptable salt, or a combination thereof as an active ingredient for the preparation of a drug for the treatment of RA.
[0008] As an optional solution of the present invention, the drug further includes a pharmaceutically acceptable carrier or excipient.
[0009] As an optional solution of the present invention, the dosage form of the drug includes liquid preparation or solid preparation.
[0010] As an optional solution of the present invention, the dosage form of the drug includes injection, powder for injection, tablet, capsule, oral liquid, granule, dripping pill or suppository.
[0011] As an alternative embodiment of the present invention, the treatment includes reducing the expression level of local joint inflammatory factors in RA individuals.
[0012] As an alternative embodiment of the present invention, the treatment includes alleviating the degree of joint synovial inflammation and / or bone erosion in RA individuals.
[0013] As an alternative embodiment of the present invention, the treatment includes preventing an individual from developing RA symptoms, or reducing the risk or frequency of onset of rheumatoid arthritis in the individual.
[0014] The technical solution of the present invention reveals and verifies that GA has good anti-inflammatory effects and can be applied to the treatment of RA, with significantly improved curative effects compared to conventional regimens. Specifically, the research in the examples of the present invention proves that it can significantly inhibit joint swelling and joint inflammation in mice, and has good anti-inflammatory effects; GA can also significantly improve bone erosion: MicroCT scans show that bone destruction in the treatment group is significantly reduced compared to the model group. Moreover, applying GA to prepare drugs for the treatment of RA has significant advantages in clinical application and social and economic benefits. Specifically, GA has significant cost advantages in treatment. Its estimated treatment cost is much lower than that of biological agents, and it does not require cold chain transportation, making it suitable for promotion in primary medical care. In addition, GA also has the potential to expand clinical indications. Basic mechanism research confirms that GA can inhibit the expression of inflammatory factors and has the potential to be extended to other autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 Results of the degree of swelling and incidence of arthritis in GA-treated arthritis model mice.
[0016] Figure 2 Results of ELISA and fluorescence quantitative PCR for detecting the levels of inflammatory factors in the sera and joint tissues of mice, respectively.
[0017] Figure 3 Results of histopathological examination of synovial inflammation and bone erosion in mice of each group.
[0018] Figure 4 Results of Micro-CT examination of joint bone erosion in mice of each group.
[0019] Figure 5 Results of the inhibition of the expression of related inflammatory factors in synovial fibroblasts induced by GA and TNF-α. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0020] The present invention discloses the use of GA in the treatment of rheumatoid arthritis. As a new traditional Chinese medicine monomer, GA can be used as an active ingredient to prepare a drug or a pharmaceutical composition for treating RA. More specifically, the present invention discloses the application of ginkgolide A, its crystal form, its pharmaceutically acceptable salts, or a combination thereof as an active ingredient for preparing a drug for treating RA.
[0021] GA can be extracted from Ginkgo biloba leaves. Typical extraction methods include extraction, chromatography, column extraction, or column chromatography, etc. GA can also directly obtain finished products through public channels. For example, its CAS# is 15291-75-5.
[0022] The "crystal form" herein refers to various crystal structures formed by the compound ginkgolide A of the present invention under different crystallization conditions. The chemical compositions of different crystal forms are exactly the same, but the molecular arrangement or crystal morphology may vary due to conditions such as temperature, solvent, and pressure. The "pharmaceutically acceptable salts" refer to salts formed by the compound ginkgolide A of the present invention with acids or bases that are suitable for use as drugs, including inorganic salts or organic salts.
[0023] In addition, for the above-mentioned drug for treating RA, in addition to the active ingredient GA, it may also include pharmaceutically acceptable excipients or carriers. For a drug containing GA as an active ingredient, the dosage form of the drug includes liquid preparations or solid preparations. More specifically, the dosage forms of the drug include injections, powder injections, tablets, capsules, oral liquids, granules, dripping pills, or suppositories. The drugs of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
[0024] The present invention provides a drug development plan for treating RA based on GA. It is confirmed through in vivo animal experiments and in vitro cell experiments that GA achieves the treatment of RA through an anti-inflammatory effect, including: (1) In vivo experiment: Anti-inflammatory effect of GA on mice with arthritis models A collagen-induced arthritis (CIA) mouse model was constructed. Different groups of mice were intraperitoneally injected with GA. The anti-inflammatory effect of GA on CIA mice was comprehensively evaluated from systemic inflammation and local inflammation through methods such as mouse body weight, joint swelling, arthritis index scoring, arthritis incidence, histopathology, Micro-CT imaging, and serological detection.
[0025] (2) In vitro experiment: Effect of GA on RA synovial fibroblasts Through in vitro experiments, the effects of GA on the inflammation, apoptosis, proliferation, migration, and invasion abilities of the RA synovial fibroblast cell line (MH7A) were observed respectively.
[0026] More specific examples and experimental results based on the above experimental protocol are shown in Examples 1-4 below.
[0027] Example 1 Study on the effect of GA on the arthritis inflammation index in CIA model mice Healthy DBA / 1 mice aged 8-10 weeks (male, body weight 18-22 g), 6-8 mice in each group, were divided into the following 3 groups: ① Normal control group: Arthritis was not induced, and only solvent was given. ② CIA model group: Solvent was given after arthritis was induced. ③ GA treatment group: GA was intraperitoneally injected (5 mg / kg) after the second immunization, administered daily for 28 consecutive days. GA was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (CAS#15291-75-5), in powder form, and dissolved with 0.1% DMSO.
[0028] The following steps were for constructing the CIA mouse model: Bovine type II collagen was mixed with complete Freund's adjuvant (CFA) in equal volume and emulsified on ice until a stable emulsion was formed. For the primary immunization, 100 μL of the emulsion was subcutaneously injected at the base of the tail of each mouse; for the secondary immunization, on the 21st day after the primary immunization, it was emulsified with incomplete Freund's adjuvant (IFA) and type II collagen, and the injection was repeated. Model verification: After the secondary immunization, the status of the mice, paw swelling and redness, joint deformation, and body weight were observed daily. The mice were sacrificed on the 49th day, and peripheral blood and paws were taken.
[0029] The joint inflammation score of CIA mice was usually quantified based on the lesion degree of the four limbs (front paws and hind paws), and the specific criteria were as follows: 0 points: No erythema or swelling, and the joint structure is normal.
[0030] 1 point: Erythema or mild swelling, only limited to the middle part of the foot or the ankle joint.
[0031] 2 points: Erythema and swelling spread from the ankle joint to the middle part of the foot, but did not involve the entire foot.
[0032] 3 points: Erythema and severe swelling cover the ankle, foot, and toes, and may be accompanied by mild joint dysfunction.
[0033] 4 points: Severe swelling of the entire foot, accompanied by obvious joint deformity or dysfunction (such as stiffness, limited movement).
[0034] The results were as Figure 1 shown, indicating that GA alleviated the swelling degree and incidence of arthritis in CIA mice.
[0035] Example 2 Study on the effect of GA on the expression of local inflammatory factors in the joints of CIA model mice The levels of serum inflammatory cytokines (TNF-α and IL-1β) were detected by ELISA. RNA was extracted from the mouse paws, and fluorescence quantitative PCR was performed to detect the expression of inflammatory factors (TNF-α and IL-1β).
[0036] The results were as follows Figure 2 , showing that GA has significant anti-inflammatory effects on the whole body and local joints of CIA mice, can reduce joint inflammation and the expression levels of local TNF-α and IL-1β in model mice, and has the strongest inhibitory effect on the expression of TNF-α.
[0037] Example 3 Study on the effects of GA on arthritis synovial destruction and bone erosion in CIA model mice The steps for joint tissue pathological analysis were as follows: The hind limb joints were taken for fixation, decalcification, paraffin embedding and sectioning. HE staining was used to observe synovial hyperplasia, inflammatory cell infiltration and cartilage destruction. Immunohistochemical staining was used to detect the protein expression of joint inflammatory factors TNF-α, IL-1β and IL-6 in mice. The results are shown in Figure 3 . The degree of bone erosion was evaluated by imaging. Micro-CT scanning was used to analyze the degree of bone erosion and bone destruction ( Figure 4 ). The above results of histopathology and imaging studies showed that GA exhibited significant protective effects in the CIA mouse model, especially in reducing local joint inflammation and bone erosion.
[0038] Example 4 Study on the effects of GA on the expression of inflammatory factors in rheumatoid arthritis synovial fibroblast cell line (MH7A) Cell viability detection (CCK-8 method) was used to verify the toxicity of GA to MH7A: Cells were seeded in 96-well cell culture plates. After 48 hours of drug treatment, CCK-8 reagent was added, and the absorbance at 450 nm was measured to calculate the cell survival rate and screen the effective dose of the drug. The results were as follows Figure 5 A.
[0039] Cells were seeded in 24-well cell culture plates. After stimulation with TNF-a (10 ng / ml) for 1 hour, GA (100 μM) was added and the cells were treated for 48 hours. RNA was extracted, and the mRNA expression changes of inflammatory factors TNF-a, IL-1β and IL-6 were detected by fluorescence quantitative PCR. The internal reference gene was β-actin, and the 2 ^（-ΔΔCt） method was used to analyze the relative expression level. The results were as follows Figure 5 B.
[0040] Cells were seeded in 6-well cell culture plates. Cells were treated with drugs by the above method. The protein expression of inflammatory factors was detected by Western blotting (WB). The results were analyzed using Image J software to analyze the gray values of the target band and the internal reference band, and calculate the relative expression level of the target protein (gray value of the target protein / gray value of the internal reference). The results were as follows Figure 5 C.
[0041] The results of PCR and WB showed that GA could inhibit the expression levels of TNF-α-induced inflammatory factors (TNF-α, IL-1β, and IL-6) in MH7A. This finding is consistent with other research results, which indicate that certain compounds and natural products can inhibit the expression of inflammatory factors by regulating inflammatory signaling pathways. For example, Foenumoside B has been shown to reduce the expression of LPS-induced inflammatory factors by inhibiting the transcriptional activities of NF-κB and AP-1, thereby increasing the survival rate in a mouse sepsis model.
[0042] In summary, GA has the effect of reducing joint inflammation and bone erosion in the CIA mouse model. In vitro experiments showed that GA could inhibit the expression of inflammatory factors in TNF-α-induced RA synovial fibroblasts. These experimental results verified the therapeutic effect of GA on joint inflammation in the CIA model. More importantly, they predicted the prospect of GA in the treatment of RA, providing a potential theoretical basis and research direction for the development of new anti-inflammatory treatment strategies.
Claims
1. Use of Ginkgolide A, its crystal form, its pharmaceutically acceptable salt or a combination thereof as an active ingredient for preparing a drug for treating rheumatoid arthritis.
2. The use according to claim 1, characterized in that: The medicament further includes a pharmaceutically acceptable carrier or excipient.
3. The use according to claim 1, characterized in that: The dosage form of the drug includes a liquid preparation or a solid preparation.
4. The use according to claim 1, characterized in that: The dosage form of the drug includes injection, powder injection, tablet, capsule, oral liquid, granule, pill or suppository.
5. The use according to claim 1, characterized in that: The treatment includes reducing the expression of local inflammatory factors in the joints of individuals with rheumatoid arthritis.
6. The use according to claim 1, characterized in that: The treatment includes reducing the extent of synovial inflammation and / or bone erosion in a subject with rheumatoid arthritis.
7. The use according to claim 1, characterized in that: The treatment includes preventing a subject from developing symptoms of rheumatoid arthritis, or reducing the risk or frequency of rheumatoid arthritis in a subject.