Composition for treating rheumatoid arthritis and application thereof

Abstract

The invention discloses a composition for treating rheumatoid arthritis and application thereof, and belongs to the technical field of medicines. The technical problem to be solved is to provide the composition for treating rheumatoid arthritis, and the composition has a better effect of treating rheumatoid arthritis and is low in cost. The key point of the technical scheme is as follows: the composition for treating rheumatoid arthritis consists of cannabidiol and methotrexate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to compositions for treating rheumatoid arthritis and their applications. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of the synovial membrane of the joints. Pathological changes include synovial cell proliferation, inflammatory cell infiltration, and pannus formation, which gradually leads to destruction of articular cartilage and bone, eventually resulting in joint deformity and loss of function. To date, there is no specific treatment, which seriously affects patients' physical function and quality of life.

[0003] Methotrexate (MTX) is an antifolate-like antitumor drug that is also commonly used clinically to treat rheumatoid arthritis (RA), exerting anti-inflammatory, anti-swelling, and analgesic effects. However, studies have indicated that long-term use of methotrexate can produce significant adverse reactions, damaging liver and kidney function, and it also has the disadvantage of slow onset of action when taken orally. Therefore, finding safer and more effective drugs remains an urgent problem to be solved in the treatment of RA, and combination therapy with other drugs is one of the preferred strategies for treating RA.

[0004] Relevant patent application documents retrieved:

[0005] This document, published in China (CN118697792A) on September 27, 2024, discloses a composition for treating rheumatoid arthritis and its preparation method. The composition comprises the following raw materials in parts by weight: 2-4 parts methotrexate and 50-80 parts of a traditional Chinese medicine composition. The traditional Chinese medicine composition consists of Scutellaria baicalensis, Paeonia lactiflora, Trachelospermum jasminoides, and Sinomenium acutum in a weight ratio of 10-15:30-40:10-15:5-15. The invention provides a composition based on methotrexate; the addition of Paeonia lactiflora inhibits the nephrotoxicity of methotrexate. Sinomenium acutum, Scutellaria baicalensis, and Trachelospermum jasminoides have the effects of dispelling wind, clearing the meridians, cooling the blood, and reducing swelling. The combination of methotrexate, Paeonia lactiflora, Sinomenium acutum, Scutellaria baicalensis, and Trachelospermum jasminoides effectively treats rheumatoid arthritis.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The composition described in the aforementioned patent is not very effective in treating rheumatoid arthritis, and its complex composition results in high production costs. Relevant evidence includes: in mouse experiments, the experimental group in Table 1 showed a low swelling rate of only 12.82% and a relatively high arthritis score of 4.5.

[0007] Cannabidiol (CBD) is the non-addictive active ingredient in cannabis, possessing various pharmacological activities such as anticonvulsant, anti-inflammatory, and anti-anxiety effects. CBD has a good safety profile, avoiding the effects of tetrahydrocannabinol (THC) on the human nervous system. It has shown good antiemetic, anti-inflammatory, analgesic, and immunomodulatory effects in various diseases, including cancer, arthritis, epilepsy, and multiple sclerosis. Numerous studies have confirmed that CBD can inhibit the proliferation, invasion, and migration of tumor cells, and induce apoptosis and autophagy.

[0008] Current technology has not disclosed that methotrexate and cannabidiol can synergistically treat rheumatoid arthritis. Summary of the Invention

[0009] The purpose of this invention is to provide: Compositions for treating rheumatoid arthritis, and related technologies thereof, to solve technical problems such as providing a composition for treating rheumatoid arthritis that is more effective and less costly in treating rheumatoid arthritis, or combinations thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this invention have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures referenced in this invention are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this invention, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definitions of standard chemical terms can be found in the reference "Pharmacopoeia of the People's Republic of China (2020 Edition), China Medical Science and Technology Press, May 2020, 1st Edition".

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as testing methods for mouse paw swelling and cellular inflammatory factor levels, shall be used.

[0014] Unless specifically defined, the use of various commercially available products in this invention employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the reagent kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term "rheumatoid arthritis" as used in this invention refers to an autoimmune disease with synovitis as its pathological basis and which may eventually lead to joint deformity. Its pathological basis is synovial inflammation, which can also extend to connective tissues such as tendons. Finally, it erodes articular cartilage and bone tissue from the synovium, causing joint damage and affecting the patient's normal joint function, resulting in limited mobility.

[0016] As used in this invention, the term "treatment" means: to achieve the desired pharmacological and / or physiological effect. The effect may be preventative, but not necessarily preventative, in relation to the prevention or partial prevention of a disease, symptom, or condition (such as dermatitis). The effect may be therapeutic in relation to the partial or complete cure of a disease, condition, symptom, or adverse reaction attributable to that disease, condition, or condition. Specifically, the term "treatment" can include any treatment of a subject (especially a person) for a relevant symptom or disease, and may include any one or more of the following: (a) prevention of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppression of the disease, i.e., prevention of its development; and (c) relief of the disease, i.e., reduction or improvement of the disease and / or its symptoms or condition. The term "treatment" can refer to therapeutic treatment alone, preventative treatment alone, or both therapeutic and preventative treatment. A person requiring treatment may include a person who already has a symptom and / or a person who needs to prevent a symptom.

[0017] In a first aspect, the present invention provides: The composition for treating rheumatoid arthritis consists of cannabidiol and methotrexate.

[0018] It contains: a ratio of cannabidiol and methotrexate.

[0019] In animal experiments, the mass ratio of cannabidiol to methotrexate was 6.25-25:1.

[0020] In animal experiments, the preferred mass ratio of cannabidiol to methotrexate is 6.25:1, 12.5:1, or 25:1.

[0021] In animal experiments, the mass ratio of cannabidiol to methotrexate is further preferably 6.25:1.

[0022] In the cell experiments, the concentration ratio of cannabidiol to methotrexate was 25-100:1.

[0023] In cell experiments, the preferred concentration ratio of cannabidiol to methotrexate is 25:1, 50:1, or 100:1.

[0024] In cell experiments, the concentration ratio of cannabidiol to methotrexate is further preferably 25:1.

[0025] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: In animal experiments, the mass ratio of cannabidiol to methotrexate is 6.25-25:1. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "this composition has better efficacy and lower cost in treating rheumatoid arthritis."

[0026] The second preferred embodiment: In cell experiments, the concentration ratio of cannabidiol to methotrexate is 25-100:1. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition having better efficacy and lower cost in treating rheumatoid arthritis."

[0027] Secondly, the present invention provides: The use of the above composition in the preparation of medicaments for the treatment and / or prevention of rheumatoid arthritis.

[0028] This includes: the efficacy of the composition; drugs, etc.

[0029] The composition can reduce the degree of knee swelling.

[0030] The composition can reduce the level of inflammatory factors in serum. It also includes inflammatory factors.

[0031] Among them, "inflammatory factors" include at least one of TNF-α, IL-1β and IL-6.

[0032] Drugs also include pharmaceutically acceptable excipients.

[0033] This also includes: excipients; drug dosage forms.

[0034] Among them, "auxiliaries" include one or more of the following: diluents, surfactants, humectants, lubricants, fillers, disintegrants, coating agents, and adhesives.

[0035] The dosage form of the drug is: tablet, capsule, granule, pill, emulsion, suspension, solution or syrup.

[0036] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: The first preferred embodiment: the composition can reduce the degree of knee joint swelling. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition is more effective and less costly in treating rheumatoid arthritis."

[0037] The second preferred embodiment: the composition can reduce the content of inflammatory factors in serum. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition has better efficacy and lower cost in treating rheumatoid arthritis."

[0038] The third preferred embodiment: the inflammatory factors include at least one of TNF-α, IL-1β, and IL-6. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition being more effective and less costly in treating rheumatoid arthritis."

[0039] The fourth preferred embodiment: the drug further includes pharmaceutically acceptable excipients. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition being more effective and less costly in treating rheumatoid arthritis."

[0040] The fifth preferred embodiment: the excipients include one or more of diluents, surfactants, humectants, lubricants, fillers, disintegrants, coating agents, and adhesives. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition has better efficacy and lower cost in treating rheumatoid arthritis."

[0041] The sixth preferred embodiment: the dosage form of the drug is tablets, capsules, granules, pills, emulsions, suspensions, solutions, or syrups. This technical solution, based on solving the technical problem of "a composition for treating rheumatoid arthritis," further solves the technical problem of "the composition has better efficacy and lower cost in treating rheumatoid arthritis."

[0042] The beneficial effects of this invention are as follows: This invention improves the synergistic effect between the components by controlling the specific ratio of methotrexate and cannabidiol, thereby enhancing the therapeutic effect on rheumatoid arthritis. Animal experiments show that the composition of this invention can significantly reduce paw swelling in mice and effectively lower serum levels of pro-inflammatory factors. Cellular experiments show that the composition can significantly reduce lipopolysaccharide-induced cellular inflammatory factor levels. Attached Figure Description

[0043] Figure 1 The degree of paw swelling in each group of mice.

[0044] Figure 2 The thickness of the paws of mice in each group was measured.

[0045] Figure 3 Arthritis index scores were assigned to each group of mice.

[0046] Figure 4 The TNF-α levels in each group of mice are shown.

[0047] Figure 5 The dose-response curves for the combined use of the drugs and methotrexate alone on the TNF-α levels in mice are shown.

[0048] Figure 6 The values ​​represent the IL-1β levels in each group of mice.

[0049] Figure 7 The dose-response curves for the combined use of the drugs and methotrexate alone on the IL-1β levels in mice are shown.

[0050] Figure 8 The values ​​represent the IL-6 levels in each group of mice.

[0051] Figure 9 The dose-response curves for the combined use of the drugs and methotrexate alone on the IL-6 levels in mice are shown.

[0052] Figure 10 The IL-6 content of cells in each group.

[0053] Figure 11 Dose-response curves of combined drug therapy and methotrexate alone on IL-6 levels in cells.

[0054] Figure 12 The TNF-α content of cells in each group.

[0055] Figure 13 Dose-response curves of combined drug therapy and methotrexate alone on TNF-α levels in cells.

[0056] Figure 14 The relative expression levels of IL-6 mRNA in each group of cells.

[0057] Figure 15 The relative expression levels of TNF-α mRNA in each group of cells are shown.

[0058] Note: Figure 2 , Figure 3 On day 15, each group was compared with the MC group. ### indicates P<0.001.

[0059] Figure 4In the mean group, compared with the MC group, ### indicates P<0.001; compared with the MTX-L group, ￥￥￥ indicates P<0.01; compared with the MTX-M group, $$￥ indicates P<0.001; compared with the MTX-H group, $$$ indicates P<0.001.

[0060] Figure 6 In the comparison with the MC group, ### indicates P<0.001; compared with the MTX-M group, ¥ indicates P<0.05; and compared with the MTX-H group, $$$ indicates P<0.001.

[0061] Figure 8 In the comparison with the MC group, ### indicates P<0.001; compared with the MTX-M group, ￥￥￥ indicates P<0.001; compared with the MTX-H group, $$$ indicates P<0.001.

[0062] Figure 10 In the mean group, compared with the MC group, ### indicates P<0.001; compared with the MTX-L group, ￥￥￥ indicates P<0.001; compared with the MTX-M group, $$￥ indicates P<0.001; compared with the MTX-H group, $$$ indicates P<0.001.

[0063] Figure 12 In the mean group, compared with the MC group, ### indicates P<0.001; compared with the MTX-L group, ￥￥￥ indicates P<0.01; compared with the MTX-M group, $$￥ indicates P<0.001; compared with the MTX-H group, $$$ indicates P<0.001.

[0064] Figure 5 , Figure 7 , Figure 9 , Figure 11 and Figure 13 In comparison to the MTX group, This indicates that P < 0.05. This indicates that P < 0.01. Detailed Implementation

[0065] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0066] The present invention will be further described below by way of specific embodiments. All kinds of instruments, devices, equipment, reagents, products, etc. used in the embodiments of the present invention are obtained through conventional commercial channels unless otherwise specified.

[0067] Example 1 Synergistic effect of cannabidiol combined with methotrexate in treating a mouse model of rheumatoid arthritis I. Experimental materials 1. Drugs: Cannabidiol (CBD, molecular formula C 21 H 30 O2, CAS: 13956 - 29 - 1, purity 99.90%), purchased from MCE, product number HY - 118641. Methotrexate (MTX, molecular formula C 20 H 22 N8O5, CAS: 59 - 05 - 2, purity 99.79%), purchased from Selleck, product number S1210.

[0068] 2. Kits: TNF - α ELISA detection kit, IL - 6 ELISA detection kit, and IL - 1β ELISA detection kit were all purchased from Elabscience.

[0069] 3. Experimental animals: SPF - level male C57BL6J mice at 4 - 6 weeks old, weighing 18 - 22 g, provided by Beijing Speyford Biotechnology Co., Ltd., animal production license number: SCXK (Beijing) 2019 - 0010, entered the experimental period after 7 days of adaptive feeding.

[0070] SPF - level NOD / ShiLtJ mice at 4 - 6 weeks old, 18 - 22 g, provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd. KRN + mice were donated by Soochow University. Among them, KRN + mice and NOD / ShiLtJ mice were used to obtain K / BxN mice, so as to prepare serum for inducing rheumatoid arthritis (i.e., K / BxN mouse serum). K / BxN mice can spontaneously develop severe inflammatory arthritis, and its core mechanism lies in that the serum is rich in high - level anti - 6 - phosphoglucose isomerase (GPI) autoantibodies, and this antibody can induce rheumatoid arthritis in C57BL / 6J mice by serum transfer.

[0071] All experimental mice were housed in a standard SPF - level laboratory environment, and the feeding conditions were strictly controlled as follows: temperature 23 ± 1°C, relative humidity 55 ± 5%, using a 12 h light / 12 h dark day - night cycle mode, with free access to food and water.

[0072] II. Animal model establishment On day 1 of the experiment, each mouse was injected intraperitoneally with 0.2 mL of K / BxN mouse serum. To enhance the immune induction effect, a booster injection was given on day 3, with the same injection method and dosage as on day 1, thus obtaining a mouse model of rheumatoid arthritis.

[0073] The serum from K / BxN mice was obtained as follows: (1) Breeding cage setup: Breeding cages are set up with a breeding ratio of 1 male and 2 females, and each cage contains 1 KRN that is 6 weeks old or older. + Male mice and two NOD / ShiLtJ female mice older than 6 weeks.

[0074] (2) Screening of mice with arthritis: The offspring mice obtained by breeding (6-7 weeks old) were identified for arthritis by visual observation. The screening criteria were: obvious redness, swelling and other inflammatory manifestations in the joints.

[0075] (3) Serum collection: Arthritis mice were fed until 9 weeks of age, and blood was collected by ocular blood collection method. Blood contamination was avoided during the operation.

[0076] (4) Serum separation and storage: The collected blood was allowed to stand at room temperature for 30-60 min before centrifugation (centrifuge parameters were set to 500×g, 4℃, and centrifugation time for 5 min). After centrifugation, the supernatant serum was carefully aspirated, aliquoted, and stored at -80℃ for later use.

[0077] III. Grouping and Administration 1. Animal grouping: Forty SPF-grade male C57BL / 6J mice were randomly divided into 8 groups of 5 mice each, ensuring no statistically significant differences in baseline indicators such as body weight and paw thickness among the groups. Specific groups were: normal control group (NC), model control group (MC), cannabidiol and low-dose methotrexate combination group (CBD+MTX-L), cannabidiol and medium-dose methotrexate combination group (CBD+MTX-M), cannabidiol and high-dose methotrexate combination group (CBD+MTX-H), low-dose methotrexate group (MTX-L), medium-dose methotrexate group (MTX-M), and high-dose methotrexate group (MTX-H).

[0078] Except for the normal control group, animal models were established in all other groups.

[0079] 2. Administration: First, dissolve cannabidiol in olive oil (98% concentration) and methotrexate in 1xPBS buffer (Biosharp, catalog number BL302A). Administration begins on day 1 after successful model establishment and continues for 12 days, with dosages as shown in Table 1.

[0080] Specifically: the group receiving cannabidiol (CBD) and low-dose methotrexate (CTX-L) was given 12.5 mg / kg of CBD via gavage daily, and methotrexate 0.5 mg / kg was administered intraperitoneally every 3 days; the group receiving CBD and medium-dose methotrexate (CBD+MTX-M) was given 12.5 mg / kg of CBD via gavage daily, and methotrexate 1 mg / kg was administered intraperitoneally every 3 days; the group receiving CBD and high-dose methotrexate (CBD+MTX-H) was given 12.5 mg / kg of CBD via gavage daily, and methotrexate 2 mg / kg was administered intraperitoneally every 3 days; the low-dose methotrexate group (MTX-L) received methotrexate 0.5 mg / kg intraperitoneally every 3 days; and the medium-dose methotrexate group (MTX-M) received methotrexate 1 mg / kg intraperitoneally every 3 days. mg / kg; the high-dose methotrexate group (MTX-H) received an intraperitoneal injection of methotrexate 2 mg / kg every 3 days; the normal control group and the model control group received an equal volume of physiological saline by gavage daily, and an equal volume of PBS buffer by intraperitoneal injection every 3 days.

[0081] Table 1 Dosage Table

[0082] IV. Research Indicators: 1. Foot swelling degree and arthritis index score Starting from day 1 of modeling, the mice were assessed for arthritis every other day. The assessment indicators included paw thickness measurement and arthritis index score to dynamically monitor the occurrence and development of arthritis.

[0083] 1.1 Paw Thickness Measurement: The thickness of the hind paws of mice was measured using digital vernier calipers at the bulbus metatarsus (the most prominent part of the paw). During measurement, ensure that the inner measuring surface of the vernier caliper is in complete contact with the skin of the paw and remains perpendicular to it, avoiding forceful squeezing that could cause measurement errors. Each mouse's hind paws were measured three times, and the average value was taken as the final measurement result.

[0084] 1.2 Arthritis Index Score: Using the paws of both hind limbs of mice as the assessment subject, a scoring standard of 0-4 points was established based on the degree of redness, swelling, and deformity of the joints, as follows: 0 points: No redness, swelling, or any other symptoms in any joint of both hind limbs; 1 point: Redness and / or swelling in one joint of both hind limbs; 2 points: Redness and / or swelling in multiple joints of both hind limbs; 3 points: Redness and / or swelling in the entire paw of both hind limbs; 4 points: Severe deformity and / or joint stiffness in the paw of both hind limbs.

[0085] The scores of both hind limbs were added together to obtain the total Arthritis Index (AI) score for each mouse. The higher the total score, the more severe the arthritis condition.

[0086] 2. Serum TNF-α, IL-1β and IL-6 levels in mice Serum from each group of mice was collected, and the levels of TNF-α, IL-1β, and IL-6 in the serum of each group of mice were detected using a detection kit.

[0087] Reference: The article "Several Calculation Methods for Drug Synergistic Effects" by Luo Fei, Han Nianlin, and Han Jisheng, *Progress in Physiological Sciences*, Vol. 26, No. 3, 1995, demonstrates the synergistic therapeutic effect of combined prescriptions on serum metastatic arthritis. Specifically: Step 1: Combine drug DI and drug DII, keep the drug DI dose constant (a dose that causes a low effect), vary the drug DII dose, and statistically analyze the changes in efficacy to obtain the first curve; Step 2: Plot the dose-response curve for drug DII alone as the second curve; Step 3: Result interpretation: Perform a two-factor repeated design ANOVA to determine whether there is an interaction between the DII dose and the drug combination.

[0088] There are three possible scenarios: (1) The interaction between the two curves is not significant: additive effect; (2) The two curves move further apart as the dosage increases: synergistic effect; (3) The two curves approach or cross each other as the dose increases: antagonistic effect.

[0089] V. Experimental Results 1. The degree of paw swelling in each group of mice is as follows: Figure 1 As shown, the thickness of the claw is measured as follows: Figure 2 As shown, the Arthritis Index score is as follows: Figure 3 As shown.

[0090] from Figure 1 It can be seen that after 12 days of continuous drug treatment, the ankle joints of mice in the model control group (MC) showed extremely severe diffuse redness, swelling, and deformity, accompanied by significant limitation of movement. In contrast, the joint inflammation symptoms of mice in each drug treatment group were improved to varying degrees. Among them, the combination treatment group of cannabidiol and methotrexate showed a trend of improvement in joint appearance compared with the same dose of methotrexate monotherapy group. Especially at medium and high doses of methotrexate, the ankle joint morphology of mice in the combination treatment group was close to that of the normal control group (NC).

[0091] Furthermore, from Figures 2-3As can be seen, after drug intervention, the arthritis scores and paw thickness curves of all treatment groups were significantly lower than those of the model control group (MC) (P<0.001), indicating that each treatment regimen had clear anti-inflammatory activity. Both the methotrexate monotherapy group and the cannabidiol and methotrexate combination therapy group effectively reduced joint inflammation in rheumatoid arthritis mice, and the efficacy increased with increasing dosage.

[0092] 2. TNF-α levels in each group of mice were as follows: Figure 4 As shown, the dose-response curves for the combined use of cannabidiol and methotrexate versus methotrexate alone are as follows: Figure 5 As shown.

[0093] IL-1β levels such as Figure 6 As shown, the dose-response curves for the combined use of cannabidiol and methotrexate versus methotrexate alone are as follows: Figure 7 As shown.

[0094] IL-6 levels such as Figure 8 As shown, the dose-response curves for the combined use of cannabidiol and methotrexate versus methotrexate alone are as follows: Figure 9 As shown.

[0095] from Figure 4 , Figure 6 and Figure 8 As can be seen, the serum concentrations of TNF-α, IL-1β, and IL-6 in the normal control group (NC) mice remained at low baseline levels. In contrast, the expression levels of these three pro-inflammatory factors were significantly increased in the model control group (MC) (P<0.001). After drug intervention, the serum concentrations of pro-inflammatory factors in each treatment group were significantly lower than those in the model control group (MC) (P<0.001), confirming that methotrexate monotherapy and combination therapy can effectively inhibit the release of inflammatory mediators.

[0096] Further comparative analysis revealed the significant synergistic effect of cannabidiol and methotrexate in inhibiting the secretion of inflammatory factors.

[0097] Compared with methotrexate monotherapy, the combination therapy with cannabidiol (CBD) showed stronger anti-inflammatory activity at all dose levels. At low and medium dose levels, the serum TNF-α, IL-1β, and IL-6 concentrations in the CBD and methotrexate combination group were significantly lower than those in the methotrexate monotherapy group at the same dose, with statistically significant differences (P<0.05). With increasing dosage, the advantages of the combination therapy further expanded. At high doses, the CBD and methotrexate combination group showed significantly better inhibitory effects on pro-inflammatory factors than the methotrexate monotherapy group at the same dose (P<0.001), and its serum factor levels recovered to or even approached the physiological state of the normal control group. Interaction tests, two-way ANOVA, and interaction plots were used to assess the synergistic effect. Figure 5 , Figure 7 , Figure 9 This further confirmed the nature of this enhancing effect. Statistical results showed a significant interaction between treatment method (single vs. combination therapy) and drug dosage. The addition of cannabidiol significantly altered the dose-response curve of methotrexate, resulting in a significantly higher rate of efficacy enhancement in inhibiting pro-inflammatory cytokine secretion in the combination therapy group compared to the single-drug group. This result strongly demonstrates that cannabidiol can significantly enhance the control of systemic inflammatory responses by methotrexate through a synergistic mechanism, and this synergistic effect is statistically significant across the entire dose range.

[0098] In summary, the combined use of cannabidiol and methotrexate can synergistically downregulate the serum levels of TNF-α, IL-1β, and IL-6 in a mouse model of rheumatoid arthritis.

[0099] Example 2: The effect of combined cannabidiol and methotrexate in treating various inflammatory factors I. Experimental Materials Cannabidiol (CBD, molecular formula C) 21 H 30 O2 (CAS: 13956-29-1), purchased from MCE, catalog number HY-118641; Methotrexate (MTX, molecular formula C) 20 H 22 N8O5 (CAS: 59-05-2), purchased from Selleck, catalog number S1210; dimethyl sulfoxide (cell culture grade), purchased from Solarbio.

[0100] The RAW264.7 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0101] Both the TNF-α ELISA kit and the IL-6 ELISA kit were purchased from Elabscience.

[0102] II. Grouping and Administration 1. Grouping: RAW264.7 cell lines (with a plating density of approximately 80%) were randomly divided into 8 groups: normal control group (NC), model control group (MC), cannabidiol and low-dose methotrexate combination group (CBD+MTX-L), cannabidiol and medium-dose methotrexate combination group (CBD+MTX-M), cannabidiol and high-dose methotrexate combination group (CBD+MTX-H), low-dose methotrexate group (MTX-L), medium-dose methotrexate group (MTX-M), and high-dose methotrexate group (MTX-H).

[0103] 2. Dosage: Cannabidiol and methotrexate were dissolved in DMSO to prepare appropriate concentrations. After 24 h of stimulation with lipopolysaccharide (LPS 1 μg / mL, purchased from Sigma) and intervention with the corresponding drugs, the levels of TNF-α and IL-6 in the cell culture supernatant of each group were detected using a assay kit. The dosages are shown in Table 2.

[0104] Table 2 Dosage Table

[0105] III. Experimental Results IL-6 levels in each group of cells are as follows Figure 10 As shown, the dose-response curves for the combined use of cannabidiol and methotrexate versus methotrexate alone are as follows: Figure 11 As shown.

[0106] The TNF-α content of cells in each group is as follows: Figure 12 As shown, the dose-response curves for the combined use of cannabidiol and methotrexate versus methotrexate alone are as follows: Figure 13 As shown.

[0107] Compared with the control group, LPS stimulation significantly induced RAW264.7 cells to secrete large amounts of TNF-α and IL-6. Although methotrexate monotherapy showed a dose-dependent inhibitory effect, at the same methotrexate dose, the combined administration of 5 μM cannabidiol significantly enhanced the inhibitory effect on TNF-α and IL-6. Two-way ANOVA and interaction plots further confirmed that cannabidiol and methotrexate showed a significant synergistic effect in reducing pro-inflammatory cytokine levels, rather than a simple additive effect.

[0108] Example 3: The effect of cannabidiol combined with different drugs in the treatment of various inflammatory factors I. Experimental Materials Cannabidiol (CBD, molecular formula C) 21 H 30O2 (CAS: 13956-29-1), purchased from MCE, catalog number HY-118641; Methotrexate (MTX, molecular formula C) 20 H 22 N8O5 (CAS: 59-05-2), purchased from Selleck, product number S1210; cyclophosphamide (CTX, molecular formula C7H) 15 Cl2N2O2P (CAS: 50-18-0), purchased from MCE, catalog number HY-17420; 5-fluorouracil (5-FU, C4H3FN2O2, CAS: 51-21-8), purchased from MCE, catalog number HY-90006.

[0109] Dimethyl sulfoxide (DMSO) (cell culture grade) was purchased from Solarbio.

[0110] The RAW264.7 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0111] The SYBR Green SupTaq HS premixed qPCR kit (containing low Rox) was purchased from Akerbio.

[0112] II. Grouping and Administration 1. Grouping: RAW264.7 cell lines (with a plating density of about 80%) were randomly divided into 5 groups: normal control group (NC), model control group (MC), cannabidiol and methotrexate combined group (CBD+MTX), cannabidiol and cyclophosphamide combined group (CBD+CTX), and cannabidiol and 5-fluorouracil combined group (CBD+5-FU).

[0113] 2. Dosage: Cannabidiol, methotrexate, 5-fluorouracil, and cyclophosphamide were dissolved in DMSO to prepare corresponding concentrations. RAW264.7 cells were stimulated with lipopolysaccharide (LPS 1 μg / mL, purchased from Sigma) and treated with the corresponding drugs for 24 h. The relative expression levels of target genes (IL-6, TNF-α) were measured by RT-qPCR. The dosages are shown in Table 3. Table 3 Dosage Table

[0114] III. Experimental Results The relative expression levels of IL-6 mRNA in each group of cells are as follows: Figure 14 As shown, the relative expression levels of TNF-α mRNA are as follows: Figure 15As shown, compared with the normal control group (NC), the relative expression levels of IL-6 and TNF-α mRNA in RAW264.7 cells were significantly increased after LPS stimulation. Among them, the combination of cannabidiol and methotrexate (CBD+MTX) was superior to the combination of cannabidiol and cyclophosphamide (CBD+CTX) and the combination of cannabidiol and 5-fluorouracil (CBD+5-FU) in reducing the relative expression levels of pro-inflammatory cytokine mRNA.

[0115] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for treating rheumatoid arthritis, characterized in that, It is composed of cannabidiol and methotrexate.

2. The composition according to claim 1, characterized in that, In animal experiments, the mass ratio of cannabidiol to methotrexate was 6.25-25:

1.

3. The composition according to claim 2, characterized in that, In cell experiments, the concentration ratio of cannabidiol to methotrexate is 25-100:

1.

4. The use of a composition according to any one of claims 1-3 in the preparation of a medicament for treating and / or preventing rheumatoid arthritis.

5. The application according to claim 4, characterized in that, The composition can reduce the degree of swelling in the knee joint.

6. The application according to claim 4, characterized in that, The composition can reduce the level of inflammatory factors in serum.

7. The application according to claim 6, characterized in that, The inflammatory factors include at least one of TNF-α, IL-1β and IL-6.

8. The application according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients include one or more of the following: diluents, surfactants, humectants, lubricants, fillers, disintegrants, coating agents, and adhesives.

10. The application according to claim 4, characterized in that, The dosage form of the drug is tablets, capsules, granules, pills, emulsions, suspensions, solutions, or syrups.

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