Use of dibenzazepine compounds in mistletoe for preparing a medicine for preventing or treating rheumatoid arthritis

Abstract

The application belongs to the technical field of natural medicinal chemistry, and relates to the use of a dibenzazepine compound in mistletoe in the preparation of a medicine for preventing or treating rheumatoid arthritis. In vitro experiments show that the use of the compound alone has different degrees of inhibitory effect on the proliferation of primary rat synovial fibroblasts, and the inhibitory effect is synergistically enhanced when the compound 2 is used in combination with mistletoe lectin. Animal experiments show that the use of the compound alone has different degrees of inhibitory effect on the joint swelling of a collagen-induced rat arthritis model, and the inhibitory effect of some dibenzazepine compounds is close to or better than that of a positive medicine, and the inhibitory effect of the compound 2 used in combination with mistletoe lectin on the joint swelling of the right hind foot of a rat is synergistically enhanced. In summary, the dibenzazepine compound in mistletoe has the potential to be developed into a medicine for treating rheumatoid arthritis, can provide more choices for clinical treatment, and has important social benefits and economic value.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to the use of diphenylheptane compounds from mistletoe in the preparation of drugs for the prevention or treatment of rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a common inflammatory autoimmune disease in clinical practice. As a chronic autoimmune disease, RA is characterized by persistent synovitis, systemic inflammatory response, and continuous production of autoantibodies. The condition is often protracted, recurrent, and difficult to cure. Most patients experience typical symptoms such as symmetrical pain, swelling, and dysfunction in the small joints of the hands and feet, often accompanied by morning stiffness, severely impacting daily activities. Pathologically, RA is characterized by inflammatory cell infiltration and abnormal synovial proliferation. The lesions can affect multiple joints throughout the body, and in advanced stages, joint deformities and even disability can occur, causing devastating damage to patients' quality of life.

[0003] The core goals of rheumatoid arthritis (RA) treatment are to control inflammatory activity, protect joint structure and function, prevent irreversible bone damage, and restore normal daily living abilities, ultimately maximizing the patient's quality of life. Currently, commonly used drugs for RA treatment mainly fall into three categories: glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and disease-modifying antirheumatic drugs (DMARDs). In recent years, with the rapid development of precision medicine, biologics and targeted drugs, with their precise targets, have brought new breakthroughs in RA treatment and have become an important direction for future treatment. However, as a chronic disease, most RA patients require long-term or even lifelong maintenance therapy. Long-term, high-dose medication may cause gastrointestinal adverse reactions and may also lead to liver and kidney damage, hematological abnormalities, and other toxic side effects. Therefore, developing new therapeutic drugs with proven efficacy and higher safety remains a core challenge that urgently needs to be addressed in the future prevention and treatment of RA.

[0004] Natural product monomers are monomeric components extracted and isolated from natural products. They may possess a variety of pharmacological activities, such as antioxidant, antitumor, antibacterial, and anti-inflammatory effects. Due to their single component and minimal side effects, they are a valuable resource for new drug development. Mistletoe is a plant belonging to the genus *Mistletoe* in the family Loranthaceae. Colored mistletoeThe leafy stems and branches of *Kom.* Nakai, also known as northern mistletoe, flying grass, willow mistletoe, and mistletoe shavings, are mainly distributed in Northeast and North China, where resources are abundant. Mistletoe primarily contains flavonoids, alkaloids, triterpenoids, sterols, and volatile oils. It has a bitter taste and neutral properties, and is believed to have effects such as dispelling wind and dampness, strengthening muscles and bones, tonifying the liver and kidneys, and calming the fetus. Modern pharmacological studies have shown that mistletoe also possesses anti-tumor, antioxidant, immunomodulatory, and anti-cardiovascular disease effects. Research on the monomeric compounds in mistletoe has been a hot topic, with its active ingredients mainly including mistletoe lectins, mistletoe flavonoids, and mistletoe toxins. Current research results show that quercetin, quercetin, and quercetin are the main antitumor components of mistletoe (e.g., see Huang Zhuqing et al., Isolation and identification of phenolic chemical components in mistletoe and their inhibitory activity on the proliferation of A549 cells, *Journal of Shandong University (Medical Edition)*, Vol. 55, No. 8, August 2017, pp. 35-41). In addition, CN115025207B also discloses that quercetin and quercetin both have therapeutic effects on rheumatoid arthritis.

[0005] However, there are no reports of diphenylheptane compounds from mistletoe being used to treat rheumatoid arthritis. Summary of the Invention

[0006] The purpose of this invention is to address the current shortage of highly effective, safe, and inexpensive oral medications from natural sources for the treatment of rheumatoid arthritis available for clinical use. This invention aims to enable the in-depth development and utilization of natural products using modern pharmaceutical research methods, combined with extensive pharmacodynamic experiments, to provide the use of diphenylheptane compounds from mistletoe in the preparation of drugs for treating rheumatoid arthritis.

[0007] Specifically, the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides the use of diphenylheptane compounds from mistletoe in the preparation of medicaments for the prevention or treatment of rheumatoid arthritis.

[0008] Alternatively, in the above-described applications, the diphenylheptane compounds are selected from the following: .

[0009] The names of the diphenylheptane compounds mentioned above are shown in Table 1 below: Table 1 Alternatively, in the above-described uses, the medicament comprises a therapeutically effective amount of a diphenylheptane compound and a pharmaceutically acceptable carrier.

[0010] Alternatively, in the above-described uses, the medicine may also contain other natural products commonly used in the clinical treatment of rheumatoid arthritis.

[0011] Alternatively, in the above-described uses, the active ingredient in the drug consists of diphenylheptane compounds and other natural products derived from mistletoe.

[0012] Alternatively, in the above-described uses, the other natural product is mistletoe lectin.

[0013] Alternatively, in the above-described uses, the weight ratio of the diphenylheptane compound to the other natural products is 1:5 to 5:1.

[0014] Preferably, the weight ratio of the diphenylheptane compound to the other natural products is 1:3 to 3:1.

[0015] More preferably, the weight ratio of the diphenylheptane compound to the other natural product is 3:1, 2:1, 1:1, 1:2 or 1:3.

[0016] Alternatively, in the above-described uses, the drug may be in oral dosage form.

[0017] Alternatively, in the above-described uses, the oral dosage form is selected from oral liquids, tablets, powders, capsules, or granules.

[0018] Preferably, the above-mentioned dosage forms can be prepared according to conventional processes in the field of pharmaceutical formulation.

[0019] Alternatively, in the above-described uses, when the active ingredient in the drug consists of a diphenylheptane compound and other natural products derived from mistletoe, the dosage forms of the former and the latter can be the same or different. Furthermore, the former and the latter can be administered simultaneously or separately.

[0020] Alternatively, in the above-described uses, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0021] The fillers of this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; and the solvents include, but are not limited to, water, balanced salt solutions, etc.

[0022] In the applications described above, the timing, frequency, and duration of administration of "diphenylheptane compounds" and other active ingredients need to be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art. For example, when applying a treatment regimen for mice or rats to humans, the effective dose of all drugs in humans can be converted from the effective dose of the drug in mice or rats, which is easily achievable by those skilled in the art.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) Combining my country’s advantages in natural product research, this invention has for the first time discovered a new use of diphenylheptane compounds derived from mistletoe in the prevention or treatment of rheumatoid arthritis.

[0024] (2) In vitro experimental results showed that diphenylheptane compounds derived from mistletoe, when used alone, all had different degrees of inhibitory effects on the proliferation of primary rat synovial fibroblasts. In addition, it was found that compound 2 combined with mistletoe lectin could produce a synergistic effect in inhibiting primary rat synovial fibroblasts.

[0025] (3) Animal experiments showed that diphenylheptane compounds derived from mistletoe and mistletoe lectin alone had varying degrees of inhibitory effects on right hind paw joint swelling in a rat model of collagen-induced arthritis. Furthermore, some active ingredients showed inhibitory effects on right hind paw joint swelling that were close to or better than those of the positive control drug. Compound 2, in combination with mistletoe lectin, produced a significant synergistic effect in inhibiting right hind paw joint swelling in rats, with the combined group achieving an inhibition rate of 87.72%.

[0026] (4) Diphenylheptane compounds derived from mistletoe have the potential to be developed into oral drugs for the treatment of rheumatoid arthritis, providing more treatment options for the clinical treatment of rheumatoid arthritis and having significant social and economic benefits. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0030] Unless otherwise stated, all percentages and parts in this invention refer to weight percentages and weight parts.

[0031] Example 1: Effects of diphenylheptane compounds on primary synovial fibroblasts In Example 1, the effects of various diphenylheptane compounds derived from mistletoe on primary synovial fibroblasts were mainly investigated. Additionally, a combination of some diphenylheptane compounds with good inhibitory effects on primary synovial fibroblasts was explored with other monomeric components of mistletoe that have therapeutic effects on rheumatoid arthritis (e.g., mistletoe lectins) to preliminarily investigate the combined effect.

[0032] The experimental methods used in this part of the study mainly refer to the relevant methods in CN119157878B.

[0033] 1.1 Experimental Methods: Primary rat synovial fibroblast culture: Synovial tissue from the hind foot of rats was digested with type II collagenase at 37 ℃ for 2 h. The supernatant was filtered through a filter and centrifuged at 1000×g for 2 min to collect synovial fibroblasts. The cells were cultured in DMEM complete medium (DMEM high glucose medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin) under the following conditions: 37 ℃, 5% CO2 and 95% humidity.

[0034] MTT assay: Synovial fibroblasts were seeded in 48-well plates at a density of 2 × 10⁶ cells / well. 4After 12 h of growth in an incubator, control and drug-treated groups were randomly assigned to four wells. The control group received no treatment, while the drug-treated groups were treated with various test natural product monomers at a concentration of 5 μM. After 24 h of drug treatment, the original culture medium in the 48-well plate was carefully aspirated, and DMEM high-glucose medium containing 50 μg / mL MTT was added. The plates were incubated at 37 ℃, 5% CO2, and 95% humidity in the dark for 4 h. The culture medium in the 48-well plate was carefully aspirated, and 350 μL of DMSO was added to dissolve the purple crystals. The absorbance of the samples was detected using a multi-mode microplate reader at a wavelength of 570 nm. Cell viability = (absorbance of drug-treated group - blank) / (absorbance of control group - blank) × 100%.

[0035] The synergy index is determined using the Jin Zhengjun q-value method, and the q-value is obtained by the following formula: q = P A+B / (P A +P B -P A ×P B In the formula, P A P B and P A+B The treatment improvement rates are categorized into drug A group, drug B group, and the combination of the two drugs, respectively. q < 1 indicates an antagonistic effect after the two drugs are used together; q > 1 indicates a synergistic effect after the two drugs are used together; and q = 1 indicates an additive effect after the two drugs are used together.

[0036] 1.2 Experimental Results: The experimental results are shown in Table 2 below. The cell survival rate results in the table are the average of three independent experiments.

[0037] Table 2: Survival rate of synovial fibroblasts in each drug group (%) Experimental results showed that diphenylheptane compounds (compounds 1 to 7) derived from mistletoe, when used alone, all had varying degrees of inhibitory effects on the proliferation of primary rat synovial fibroblasts, among which compounds 1, 2, 5 and 6 showed relatively strong inhibitory effects.

[0038] When the above-mentioned compounds with relatively good activity were combined with mistletoe lectin, which has been reported to have therapeutic effects on rheumatoid arthritis, it was found that the combination of compound 2 and mistletoe lectin could produce a synergistic effect in inhibiting primary rat synovial fibroblasts.

[0039] Therefore, the next step will be to further validate the efficacy of diphenylheptane compounds, both alone and in combination, as well as the combination of diphenylheptane compounds and mistletoe lectins, in a collagen-induced rat arthritis model for the treatment of rheumatoid arthritis.

[0040] Example 2: The therapeutic effect of diphenylheptane compounds on a collagen-induced rat model of arthritis 2.1 Experimental Methods: Wistar rats, male, 180-200 g, provided by the Animal Center of Heilongjiang University of Traditional Chinese Medicine, SPF grade, divided into 9 groups.

[0041] Animal groups: normal control group, 9 animals / group; model group, 9 animals / group; positive control group (methotrexate, 1 mg / kg / 2d), 9 animals / group; compound 1 group (20 mg / kg / d), 9 animals / group; compound 2 group (20 mg / kg / d), 9 animals / group; compound 5 group (20 mg / kg / d), 9 animals / group; compound 6 group (20 mg / kg / d), 9 animals / group; mistletoe lectin group (10 mg / kg / d), 9 animals / group; combination group (compound 2 20 mg / kg / d, mistletoe lectin 10 mg / kg / d).

[0042] The joint volume of the right hind paw of all rats was determined using the water volume method and recorded as the pre-inflammatory volume.

[0043] Emulsify a 10 mg / mL bovine type II collagen acetate solution with an equal volume of complete adjuvant to obtain a suspension. Intradermal injection of the suspension into the right hind paw pad of rats in the model group, positive drug group, compound 1 group, compound 2 group, compound 5 group, compound 6 group, mistletoe lectin group, and combination group, with an injection volume of 0.1 mL for each group, followed by a booster injection at the base of the tail 7 days later. The blank group was injected with 0.1 mL of autoclaved liquid paraffin.

[0044] Four hours after the first bovine type II collagen injection, the animals were administered the treatment drug via gavage according to the above protocol. The normal control group and the model group received physiological saline. The drug was administered once daily for three consecutive weeks. Four hours after administration on day 21, the volume of the right hind paw joint of the rats was measured using the water volume method. The swelling rate (%) and the inhibition rate (%) of the right hind paw joint swelling were calculated. Specific experimental results are shown in Table 3. Swelling rate of the right hind paw joint in rats (%) = (post-inflammatory joint volume - pre-inflammatory joint volume) / pre-inflammatory joint volume × 100%; Rat paw joint swelling inhibition rate (%) = (model group paw joint volume - experimental group paw joint volume) / model group paw joint volume × 100%.

[0045] The experimental data were statistically analyzed using software. The method was to use a t-test under one-way ANOVA to compare whether there was a significant difference in the mean of each column. A p < 0.05 was considered to be a significant difference.

[0046] 2.2 Experimental Results: The experimental results are shown in Table 3 below.

[0047] Table 3: Joint swelling rate (%) in each drug group being tested Note: Compared with the model group: p <0.05, p <0.01, p <0.001.

[0048] Similar to the in vitro experimental results in Example 1, the experimental results in Table 3 show that diphenylheptane compounds derived from mistletoe (compounds 1, 2, 5, and 6) and mistletoe lectin alone have varying degrees of inhibitory effects on right hind paw joint swelling in a rat model of collagen-induced arthritis. Furthermore, the inhibitory effects of some active ingredients (e.g., compounds 1, 2, and 6) on right hind paw joint swelling in rats are close to or better than those of the positive control drug.

[0049] When compound 2 was used in combination with mistletoe lectin, similar results as in the in vitro experiment in Example 1 were obtained. Specifically, the combination of compound 2 and mistletoe lectin produced a significant synergistic effect in inhibiting swelling of the right hind paw joint in rats, with the swelling inhibition rate of the rat paw joint in the combination group reaching 87.72%.

[0050] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of diphenylheptane compounds from mistletoe in the preparation of drugs for the prevention or treatment of rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The diphenylheptane compounds are selected from the following: 。 3. The use according to claim 1 or claim 2, characterized in that: The drug comprises a therapeutically effective amount of a diphenylheptane compound and a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The drug also contains other natural products commonly used in clinical treatment of rheumatoid arthritis.

5. The use according to claim 4, characterized in that: The active ingredient in the drug consists of diphenylheptane compounds and other natural products derived from mistletoe.

6. The use according to claim 5, characterized in that: The other natural product mentioned is mistletoe lectin.

7. The use according to claim 5, characterized in that: The weight ratio of the diphenylheptane compound to the other natural products is 1:5 to 5:

1.

8. The use according to claim 7, characterized in that: The weight ratio of the diphenylheptane compound to the other natural products is 1:3 to 3:

1.

9. The use according to any one of claims 1 to 8, characterized in that: The drug is an oral dosage form.

10. The use according to claim 9, characterized in that: The oral dosage form is selected from oral liquid, tablets, powder, capsules or granules.

Images