A Chinese medicine monomer composition for treating rheumatoid arthritis and its application

Abstract

The present invention provides a Chinese medicine monomer composition and its application for treating rheumatoid arthritis, belonging to the technical field of Chinese medicine preparations. The Chinese medicine monomer composition comprises triptolide and medipterolide. During implementation, it was unexpectedly discovered that triptolide and medipterolide exhibit a synergistic effect when used together at relatively low concentrations to inhibit osteoclast differentiation. This combined effect, revealed by the present invention's research results, suggests that triptolide and medipterolide, when used together, have the potential to be developed as a combination into a drug for treating rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparations, and in particular relates to a traditional Chinese medicine monomer composition for treating rheumatoid arthritis and its application. Background Art

[0002] Rheumatoid arthritis (RA) affects approximately 0.5% to 1% of the global population and is characterized by inflammation and bone destruction in affected joints, ultimately leading to joint deformity and loss of function. Excessive bone resorption by osteoclasts (OCs) is the direct cause of cortical bone destruction and damage in RA patients. The continued development of disease-modifying antirheumatic drugs (DMARDs) has achieved significant success in preventing and alleviating symptoms in RA patients. However, these drugs often fail to reverse existing joint destruction, and even with potent anti-inflammatory treatment strategies, such as blockade of TNF-α or IL-6R, there is only limited evidence of repair of bone erosion. Therefore, there is a need to optimize treatment options for bone destruction in RA.

[0003] Chinese patent CN 110638955 A discloses a Chinese medicine composition for treating arthritis. Its raw materials include Rhizoma Eupatorii, Radix Gentianae Macrophyllae, Radix Saposhnikoviae, Radix Puerariae, Radix Bupleuri, Rhizoma Cyperi with Vinegar, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Radix Astragali, Radix Codonopsis, Rhizoma Atractylodis Macrocephalae, Semen Coicis, Rhizoma Atractylodis Macrocephalae, Pericarpium Citri Reticulatae, Rhizoma Pinelliae Rhizoma, Flos Carthami, Rhizoma Prunellae, Sponge Fern, Twig of Cinnamomum, Radix Achyranthis Bidentatae and Radix Dipsaci. Among them, the herbal medicine is widely available, has the characteristics of low cost, easy preparation and remarkable therapeutic effect; the prescription does not contain medicinal materials with large toxic and side effects such as Tripterygium wilfordii and Semen Strychnos nux vomica, and long-term use has little irritation to the gastrointestinal tract; starting from the two aspects of replenishing qi and blood and promoting blood circulation and removing blood stasis, it can improve human immunity and prevent bone destruction. It has unique therapeutic effect for rheumatoid arthritis, compression fractures, periarthritis of shoulder and post-stroke limb pain of the syndrome differentiation belonging to qi deficiency and blood stasis type or liver and kidney deficiency type, and no obvious adverse reactions are observed, so it is suitable for promotion and application.

[0004] This existing technology increases the risk of significant side effects of Tripterygium wilfordii. However, studies have found that triptolide (Tp), one of the main active ingredients in Tripterygium wilfordii, can inhibit OC differentiation to alleviate RA. Although the therapeutic window of triptolide is very narrow and it can cause toxicity at slightly higher doses, its combination with other drugs can reduce the dose, improve the efficacy, and thus reduce side effects.

[0005] Chinese patent CN103735649A discloses a method for preparing a drug for treating articular cartilage cell destruction and inflammation. The drug is prepared according to the following preparation method: extracting snow lotus, Cistanche deserticola, Clematis chinensis, Herba Epimedii and Millettia reticulata with alcohol, and concentrating to obtain a thick paste; extracting volatile oil from Atractylodes macrocephala; extracting, concentrating and precipitating crude polysaccharides from Anazi root, Morus alba, Herba Lycopodii, Herba Polygoni Multiflori and the alcohol-extracted residues as well as the residues after extracting volatile oil from Atractylodes macrocephala; encapsulating the volatile oil to obtain an inclusion compound; mixing the thick paste and the crude polysaccharide, adding auxiliary materials, granulating and whole-granulating, and adding the inclusion compound to obtain the drug. The drug has low toxicity, has a good effect on articular cartilage cell destruction and inflammation, and has a certain analgesic effect, and can be used to treat articular cartilage cell destruction and inflammatory diseases.

[0006] The existing technology uses Millettia reticulata as one of the medicinal ingredients, but direct extraction of traditional Chinese medicine for use as medicine has certain toxic side effects. Therefore, in recent years, combined use of drugs to act on multiple targets to enhance pharmacological activity and reduce toxic side effects has become a new strategy. Medipterocarpin (Med) is one of the flavonoid active ingredients derived from the traditional Chinese medicine Millettia reticulata. It has anti-inflammatory, anti-osteoclastogenesis, and anti-angiogenesis effects. It can be combined with triptolide to relieve and treat rheumatoid arthritis, and can enhance pharmacological activity and reduce toxic side effects. There are no reports of related research in the existing technology. Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention aims to provide a Chinese herbal monomer composition and its application for the treatment of rheumatoid arthritis. During implementation, it was unexpectedly discovered that triptolide and medipterocarbone exhibit a synergistic effect when used together at relatively low concentrations to inhibit osteoclast differentiation. This combined effect, revealed by the present invention's research results, suggests that triptolide and medipterocarbone, when used together, may be a promising combination for the treatment of rheumatoid arthritis.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a traditional Chinese medicine monomer composition for treating rheumatoid arthritis. The traditional Chinese medicine monomer composition consists of triptolide and meditarol.

[0010] The molar concentration ratio of triptolide to medipterostilbene is 1:500-2000.

[0011] Further preferably, the molar concentration ratio of triptolide to medipterostilbene is 1:1000.

[0012] The concentration of triptolide is 5-20 nM, and the concentration of meditartin is 5-20 μM.

[0013] Preferably, the concentration of triptolide is 10-20 nM, and the concentration of meditartin is 10-20 μM.

[0014] As some preferred embodiments, the concentration of triptolide is 10 nM, and the concentration of meditartin is 10 μM;

[0015] Or, the concentration of triptolide is 10 nM, and the concentration of meditartin is 20 μM;

[0016] Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 5 μM;

[0017] Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 10 μM;

[0018] Alternatively, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

[0019] The present invention also provides use of the above-mentioned traditional Chinese medicine monomer composition in preparing a medicine for treating rheumatoid arthritis.

[0020] A medicine for treating rheumatoid arthritis, comprising the above-mentioned traditional Chinese medicine monomer composition and pharmaceutically acceptable excipients.

[0021] "Pharmaceutically acceptable excipients" refer to excipients and additives used in the production of pharmaceuticals and formulations. These substances, in addition to the active ingredient, have been reasonably evaluated for safety and are included in pharmaceutical preparations. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different effects and uses. Pharmaceutical excipients added to the drugs provided herein can serve as excipients, carriers, or enhance stability. Furthermore, they can have important functions such as solubilization, dissolution-enhancing, or sustained-release.

[0022] Typical but non-limiting excipients include: one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

[0023] Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked carboxymethyl cellulose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone,

[0024] Microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: saccharin sodium, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0025] The pharmaceutical dosage forms of the present invention include tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release preparations, controlled-release preparations or targeted preparations.

[0026] Preferably, the pharmaceutical dosage form is a tablet

[0027] Pharmaceutical excipients used in the preparation of tablets include but are not limited to:

[0028] (1) Fillers or diluents, which are used to increase the weight or volume of tablets to facilitate tablet compression, including starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, inorganic salts, and mannitol;

[0029] (2) Wetting agent or binder. Wetting agent refers to an excipient that is non-viscous but can induce the viscosity of the soft material drug, thereby achieving the purpose of granulation and tableting, including distilled water and ethanol; binder refers to an excipient that gives viscosity to non-viscous or insufficiently viscous materials, thereby agglomerating the materials into granules, including starch slurry, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, ethyl cellulose, povidone, gelatin, polyethylene glycol, 50%-70% sucrose solution, and sodium alginate solution;

[0030] (3) Disintegrants, which refer to excipients that promote the rapid disintegration of tablets into fine particles in gastrointestinal fluid, include dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and cross-linked polyvinylpyrrolidone;

[0031] (4) Lubricant, which is a general term for glidants, anti-adherents and lubricants (in a narrow sense). Flow aids refer to auxiliary materials that reduce friction between particles, thereby improving powder fluidity and reducing weight differences; anti-adherents refer to auxiliary materials that prevent materials from adhering to the punch and die surfaces during tableting to ensure smooth tableting operations and smooth tablet surfaces; lubricants (in a narrow sense) refer to auxiliary materials that reduce friction between tablets and the die wall during tableting and ejection to ensure uniform stress distribution during tableting and prevent tablet cracking; lubricants include: magnesium stearate, micropowder silica gel,

[0032] Talc, hydrogenated vegetable oil, polyethylene glycols such as PEG4000, PEG6000, sodium lauryl sulfate.

[0033] The excipients may also contain colorants and flavoring agents. Colorants are excipients that impart color to the drug, including pharmaceutical or food-grade pigments such as chlorophyll, caramel, amaranth, tartrazine, carmine, and indigo, with their content in the pharmaceutical composition not exceeding 0.05%. Flavoring agents are pharmaceutical excipients used in drugs to improve or mask the drug's unpleasant odor and taste, making the drug's strong bitterness (or other unpleasant flavors such as pungent or irritating) less noticeable to patients. These include sucrose, citrus glycosides, saccharin sodium, proteose, sorbitol, aspartame, and acesulfame-K.

[0034] The preparation method of the above-mentioned drug of the present invention is not strictly limited. For example, it can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Drugs such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions.

[0035] Dosage levels of the active compounds in the medicaments contemplated by this invention will depend on the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated.

[0036] The administration routes of the drug of the present invention include, but are not limited to, the following: administration via the gastrointestinal tract, such as oral administration; and administration without the gastrointestinal tract, such as intramuscular injection, intravenous injection, or transdermal administration.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] During the implementation of the present invention, it was unexpectedly discovered that triptolide and medipterostilbene have a synergistic effect when used in combination at lower concentrations to inhibit osteoclast differentiation. The combined effect revealed by the research results of the present invention makes triptolide and medipterostilbene have the application prospect of being developed into a drug for treating rheumatoid arthritis in the form of a combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The combination of triptolide and meditarostemonin inhibits the generation and differentiation of osteoclasts;

[0040] Figure symbols: A is TRAP staining for osteoclast formation, scale bar 200 μm; B is the quantification of the number of osteoclasts in each group; C is the CI value of the drug inhibition rate on TRAP-positive cells;

[0041] Compared with the model group, ***p<0.001, **p<0.01, *p<0.05; compared with the combination group, ###p<0.001, ##p<0.01, #p<0.05.

[0042] Figure 2 The combination of triptolide and meditarol inhibits the resorption function of osteoclasts;

[0043] Figure symbols: A is the formation of F-actin rings in osteoclasts stained with phalloidin, scale bar 100 μm; B is the area ratio of F-actin rings in each group; C is a representative scanning electron microscopy image of bone resorption pits in bone slices, scale bar 21 mm; D is the area ratio of bone resorption pits.

[0044] Compared with the model group, ***p<0.001, **p<0.01, *p<0.05; compared with the combination group, ###p<0.001, ##p<0.01, #p<0.05.

[0045] Figure 3 Effects of the combination of triptolide and meditartin on osteoclastogenesis, differentiation, and absorption-related mRNA;

[0046] Figure symbols: A represents the effect on Nfatc1 mRNA; B represents the effect on C-fos mRNA; C represents the effect on Atp6v0d2 mRNA; D represents the effect on Dc-stamp mRNA; and E represents the effect on Ctsk mRNA. DETAILED DESCRIPTION

[0047] In order to facilitate understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0048] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0050] Among them: triptolide (CAS No. 38748-32-2) and medipterostilbene (CAS No. 73340-41-7).

[0051] The present invention discloses a traditional Chinese medicine monomer composition for treating rheumatoid arthritis. The traditional Chinese medicine monomer composition consists of triptolide and medipterocarpin; the molar concentration ratio of the triptolide to medipterocarpin is 1:500-2000.

[0052] Further preferably, the molar concentration ratio of triptolide to medipterostilbene is 1:1000.

[0053] Preferably, the concentration of triptolide is 10-20 nM, and the concentration of meditartin is 10-20 μM.

[0054] As some preferred embodiments, the concentration of triptolide is 10 nM, and the concentration of meditartin is 10 μM;

[0055] Or, the concentration of triptolide is 10 nM, and the concentration of meditartin is 20 μM;

[0056] Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 5 μM;

[0057] Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 10 μM;

[0058] Alternatively, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

[0059] When triptolide and medipterostilbene are used in combination at the above lower concentrations to inhibit osteoclast differentiation, the combined effect revealed by the research results of the present invention enables triptolide and medipterostilbene to be used as a drug for treating rheumatoid arthritis in the form of a composition.

[0060] Example 1: Effect of Chinese herbal monomer composition on primary osteoclast differentiation

[0061] Primary monocytes from the bone marrow of C57 mice were extracted and seeded into 96-well plates. Different concentrations of drugs (as shown in Table 1) were added for treatment for 4-6 days. The prepared TRAP working solution was added and incubated for 1 hour. The number of osteoclasts in each group was counted. The results are shown in Tables 1 and Figure 1 .

[0062] Table 1 Analysis of synergistic effect of triptolide and medipterostilbene

[0063]

[0064] According to Table 1 and Figure 1 The results showed that triptolide or medipterostilbene alone could inhibit RANKL-induced osteoclastogenesis and reduce the number of osteoclasts (OC) in a dose-dependent manner. Notably, the combination of 10 nM Tp and 10 μM Med, 10 nM Tp and 20 μM Med, 20 nM Tp and 5 μM Med, 20 nM Tp and 10 μM Med, and 20 nM Tp and 20 μM Med had the best effect on reducing the number of osteoclasts (OC), with synergistic combination index (CI) values ​​less than 1.

[0065] Example 2: Effect of Chinese herbal monomer composition on primary osteoclast bone resorption

[0066] Primary monocytes from the bone marrow of C57 mice were extracted and seeded into 96-well plates. Different concentrations of drugs (as shown in Table 1) were added for treatment for 4-6 days. The prepared phalloidin working solution was added and the F-actin ring area of ​​osteoclasts in each group was calculated. The results are shown in Figure 1. Figure 2 .

[0067] according to Figure 2 The results showed that triptolide or medipterostilbene alone inhibited RANKL-induced osteoclast F-actin ring formation and reduced the F-actin ring area in a dose-dependent manner. Notably, the combined triptolide and medipterostilbene group showed a statistically significant difference in the extent of inhibition of osteoclast F-actin ring area compared to the single-dose group. Bone slice analysis showed that the combined group exhibited a more pronounced inhibitory effect on bone resorption than the single-dose group.

[0068] Example 3: Effects of Chinese herbal monomer composition on osteoclast differentiation and absorption-related mRNA

[0069] according to Figure 1 and 2 Results: 10nM triptolide and 10μM medipterostilbene were selected as the combination drug group, and RNA was extracted from osteoclasts. RT-qPCR was used to detect the effects of Nfatc1, C-fos, Atp6v0d2, Dc-stamp, and Ctsk mRNA. The results were as follows: Figure 3 .

[0070] The specific groups are:

[0071] Normal group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL;

[0072] Model group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor kappa-B ligand (RANKL) 50 ng / mL;

[0073] Triptolide group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor kappa-B ligand (RANKL) 50 ng / mL, triptolide 10 nM;

[0074] Medipterocarbone group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor kappa-B ligand (RANKL) 50 ng / mL, and Medipterocarbone 10 μM;

[0075] Chinese medicine monomer composition group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor κ-B ligand (RANKL) 50 ng / mL, triptolide 10 nM, and meditartin 10 μM.

[0076] according to Figure 3 The results showed that triptolide and medipterone used alone or in combination had an inhibitory effect on Nfatc1, C-fos, Atp6v0d2, Dc-stamp, and Ctsk mRNA in osteoclasts, and the inhibitory effect of the Chinese medicine monomer combination on Nfatc1, Atp6v0d2, Dc-stamp, and CtskmRNA was significantly better than that of the single drug group.

[0077] The above studies show that triptolide and medipterostilbene have a synergistic effect when used together at lower concentrations to inhibit osteoclast differentiation. The combined effect makes triptolide and medipterostilbene have the application prospect of being developed into a drug for the treatment of rheumatoid arthritis in the form of a combination.

[0078] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A Chinese medicine monomer composition for treating rheumatoid arthritis, characterized in that: The traditional Chinese medicine monomer composition consists of triptolide and medipterocarpin; the molar ratio of triptolide to medipterocarpin is 1:500-2000.

2. The Chinese medicine monomer composition according to claim 1, characterized in that: The molar ratio of triptolide to medipterostilbene is 1:1000.

3. The Chinese medicine monomer composition according to claim 1, characterized in that: The concentration of triptolide is 5-20 nM, and the concentration of meditartin is 5-20 μM.

4. The Chinese medicine monomer composition according to claim 3, characterized in that: The concentration of triptolide is 10-20 nM, and the concentration of meditartin is 10-20 μM.

5. The Chinese medicine monomer composition according to claim 4, characterized in that: The concentration of triptolide is 10 nM, and the concentration of meditartin is 10 μM; Or, the concentration of triptolide is 10 nM, and the concentration of meditartin is 20 μM; Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 10 μM; Alternatively, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

6. Use of the traditional Chinese medicine monomer composition according to any one of claims 1 to 5 in the preparation of a medicament for treating rheumatoid arthritis.

7. A drug for treating rheumatoid arthritis, characterized in that: The medicine comprises the traditional Chinese medicine monomer composition according to any one of claims 1 to 5 and pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that: The excipients are one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

Images