Use of chrysin in the preparation of a drug for preventing and / or treating myasthenia gravis

Abstract

The present application relates to the use of chicoric acid in the preparation of a medicament for preventing and / or treating myasthenia gravis. Specifically, the present application provides the use of chicoric acid and pharmaceutically acceptable salts, esters, solvates, polymorphs or prodrugs thereof in the preparation of a medicament for preventing and / or treating myasthenia gravis. The present application focuses on the use of the compound chicoric acid in correcting the core immune imbalance of myasthenia gravis, Th17 / Treg cell balance, intervening from the upstream of the disease, and is expected to achieve the fundamental treatment of myasthenia gravis and more persistent efficacy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of chicoric acid in the preparation of medicaments for the prevention and / or treatment of myasthenia gravis. Background Technology

[0002] Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies damaging acetylcholine receptors on the postsynaptic membrane of the neuromuscular junction. Clinical manifestations include partial or generalized skeletal muscle weakness and extreme fatigue. Typical symptoms are "worse in the evening and milder in the morning," worsening with activity and improving with rest. It is often accompanied by localized or systemic symptoms such as ptosis, diplopia, dysphagia, and limb weakness.

[0003] Currently, the clinical treatments for myocardial infarction (MG) include cholinesterase inhibitors, glucocorticoids, immunosuppressants, and intravenous human immunoglobulin. However, these drugs have several drawbacks, including significant individual differences in efficacy, a high risk of relapse after discontinuation, adverse reactions with long-term use (such as hormone-related osteoporosis and liver and kidney damage caused by immunosuppressants), and drug resistance in some patients. These issues make it difficult to meet the needs of clinical treatment.

[0004] Therefore, studying the pathogenesis of myocardial infarction (MG) and finding effective treatments remains a difficult and serious challenge facing the world today. Summary of the Invention

[0005] The purpose of this invention is to provide a drug that can effectively prevent and / or treat myasthenia gravis.

[0006] In a first aspect of the invention, the use of chicoric acid and pharmaceutically acceptable salts, esters, solvates, polymorphs or prodrugs thereof in the preparation of medicaments for the prevention and / or treatment of myasthenia gravis is provided.

[0007] In another preferred embodiment, the myasthenia gravis is acetylcholine receptor antibody positive, muscle-specific tyrosine kinase antibody positive, and / or serum antibody negative myasthenia gravis.

[0008] In another preferred embodiment, the drug is used to prevent and / or treat myasthenic crisis.

[0009] In another preferred embodiment, the drug is used to prevent and / or treat respiratory muscle weakness, dysphagia, or diplopia caused by myasthenia gravis.

[0010] In another preferred embodiment, the drug is used to modulate neuromuscular junction function and / or protect postsynaptic membrane structures.

[0011] In another preferred embodiment, the chicoric acid is derived from a natural plant extract selected from dandelion, chicory, echinacea, red chicory, beggar-ticks, lettuce, sow thistle, long elephant ear grass, horsetail, chicken foot ginseng, broken rice stalk, silk fern, filamentous needle algae, or a combination thereof.

[0012] In a second aspect of the invention, a pharmaceutical composition for the prevention and / or treatment of myasthenia gravis is provided, characterized in that it comprises a therapeutically effective amount of chicoric acid and one or more pharmaceutically acceptable carriers.

[0013] In another preferred embodiment, the dosage form of the pharmaceutical composition includes: oral solid dosage form, oral liquid dosage form, injection, inhalation, transdermal patch, or sublingual immediate-release dosage form.

[0014] In another preferred embodiment, the oral solid dosage form includes: enteric-coated tablets, sustained-release tablets, immediate-release capsules, or granules.

[0015] In another preferred embodiment, the concentration of chicoric acid in the pharmaceutical composition is from 0.1 mg / mL to 500 mg / mL, or from 0.2 wt% to 98 wt%.

[0016] In another preferred embodiment, chicoric acid is the sole active ingredient in the pharmaceutical composition.

[0017] In another preferred embodiment, the pharmaceutical composition further comprises one or more active ingredients selected from: cholinesterase inhibitors, glucocorticoids, immunosuppressants, monoclonal antibodies, immunoglobulins, or immunoadsorbents.

[0018] In another preferred embodiment, the immunosuppressant is selected from tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, methotrexate, or cyclophosphamide.

[0019] In a third aspect of the invention, a drug delivery system is provided comprising nanoparticles, liposomes, microspheres, or cyclodextrin inclusions loaded with chicoric acid for the prevention and / or treatment of myasthenia gravis.

[0020] In a fourth aspect of the invention, the use of chicoric acid in the preparation of a medicament for regulating the imbalance of Th17 cells to Treg cells in the peripheral blood, spleen or lymph nodes of patients with myasthenia gravis is provided.

[0021] In another preferred embodiment, the drug is capable of reducing the proportion of Th17 cells by at least 10% and / or increasing the proportion of Treg cells by at least 10%.

[0022] In a fifth aspect of the invention, the use of chicoric acid in the preparation of a medicament for reducing the level of pathogenic autoantibodies in patients with myasthenia gravis is provided.

[0023] In a sixth aspect of the invention, a method for treating myasthenia gravis is provided, comprising: administering a therapeutically effective amount of chicoric acid to a subject diagnosed with myasthenia gravis.

[0024] In another preferred embodiment, the dosage of chicoric acid is from 5 mg to 3000 mg, or from 0.1 mg / kg to 60 mg / kg based on body weight.

[0025] In another preferred embodiment, the administration is oral.

[0026] In another preferred embodiment, the frequency of application includes once or twice daily for at least four weeks.

[0027] In another preferred embodiment, a second therapeutic agent, which is a cholinesterase inhibitor or an immunosuppressant, is administered in combination before, simultaneously with, or after the application of chicoric acid.

[0028] In another preferred embodiment, the object includes humans and non-human mammals.

[0029] In another preferred embodiment, the non-human mammals include rodents and non-human primates.

[0030] In another preferred embodiment, the rodents include rats and mice.

[0031] In another preferred embodiment, the object includes children, adults, or the elderly.

[0032] In a seventh aspect of the invention, a food, health food or dietary supplement containing chicoric acid is provided for the purpose of assisting in the prevention and / or improvement of symptoms of myasthenia gravis.

[0033] In an eighth aspect of the invention, a method for screening candidate compounds for treating myasthenia gravis is provided, comprising the steps of: testing the regulatory ability of the candidate compounds on Th17 / Treg cell balance; and selecting compounds that can significantly correct the Th17 / Treg cell imbalance; wherein chicoric acid is used as a positive control.

[0034] In a ninth aspect of the invention, a combination of biomarkers for evaluating the therapeutic effect of myasthenia gravis is provided, characterized in that the combination of biomarkers comprises at least two of the following: Th17 cell proportion, Treg cell proportion, and TCR-γδ cell proportion; wherein administration of chicoric acid causes the values ​​of the combination of biomarkers to recover to a healthy state.

[0035] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0036] Compared with the prior art, the present invention has the following technical effects:

[0037] (1) The chicoric acid provided by the present invention can effectively prevent and / or treat myasthenia gravis.

[0038] (2) This invention is the first to focus the application of the compound chicoric acid on correcting the core immune imbalance of myasthenia gravis—the balance of Th17 / Treg cells, and intervenes from the upstream of the disease, which is expected to achieve fundamental treatment and more lasting efficacy.

[0039] (3) Unlike traditional broad-spectrum immunosuppressants, the compound chicoric acid in this invention aims to "restore balance" rather than "completely suppress". Theoretically, it can significantly reduce the risk of serious side effects such as infection caused by excessive suppression of the immune system and has better tolerability.

[0040] (4) This invention provides novel drug candidates for patients who are ineffective, intolerant or relapsed by existing therapies, and has important clinical and social value.

[0041] (5) The compound chicoric acid of the present invention is a monomer with a known structure. Its chemical properties, basic pharmacology and safety data are relatively clear. Compared with a completely new molecular entity, the path of developing it into a new indication through "repurposing an old drug" is shorter and the success rate is higher. Attached Figure Description

[0042] Figure 1 The figure shown illustrates the effects of chicoric acid on the body weight (A), clinical score (B), cumulative score (C), highest score (D), macroscopic manifestations (E), and spleen pathology (F) scores of EAMG rats in this embodiment of the invention (*P<0.05, **P<0.01, ***P<0.001). Wherein, CFA represents the blank control group, EAMG represents the experimental autoimmune myasthenia gravis group, CA(H) represents the high-dose chicoric acid group (50 mg / kg), and CA(L) represents the low-dose chicoric acid group (25 mg / kg).

[0043] Figure 2 The figure shown is a graph illustrating the effect of chicoric acid on the proportions of Th17(A), Treg(B), and TCR(C) cells in the spleen and lymph nodes of rats with myasthenia gravis in this invention (*P<0.05, **P<0.01, ***P<0.001). Detailed Implementation

[0044] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] the term

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0048] Myasthenia gravis

[0049] Myasthenia gravis is an autoimmune disease caused by dysfunction of the neuromuscular junction. Its pathogenesis is complex, involving multi-level interactions of genetics, environment, immune imbalance, and neuromuscular junction dysfunction. Currently, it is generally believed that the functional imbalance between helper T cells 17 (Th17) and regulatory T cells (Treg) is one of the core immunological mechanisms driving the occurrence and progression of the disease, directly leading to the production of pathogenic autoantibodies, postsynaptic membrane damage, and signal transduction disorders.

[0050] To address the limitations of existing myasthenia gravis prevention and treatment methods, such as limited efficacy, significant side effects, or inability to fundamentally correct immune imbalance, this invention provides a novel therapeutic application for chicoric acid, a monomer derived from traditional Chinese medicine. This monomer can effectively regulate the balance of Th17 / Treg cell subsets, reversing the trend towards immune homeostasis; furthermore, it inhibits the production of pathogenic autoantibodies and directly or indirectly protects the structure and transmission function of the neuromuscular junction.

[0051] Cichoric acid (CA)

[0052] Chicoric acid is a caffeic acid compound isolated from plants in the Asteraceae family. It is widely distributed in the roots, leaves, and flowers of dandelion, accounting for 0.36% of the dandelion water extract (dried product). Chicoric acid has clear anti-inflammatory and immunomodulatory pharmacological activities, and exhibits good safety and tolerability.

[0053] This invention provides the application of chicoric acid in the preparation of drugs for the prevention and treatment of myasthenia gravis. It can regulate the balance of Th17 / Treg cell subsets, inhibit the production of pathogenic autoantibodies, and protect the neuromuscular junction's transmission function, opening up new treatment prospects for patients with myasthenia gravis. It can intervene in the disease process at the core of immune regulation, possessing multi-target and multi-level therapeutic advantages, and providing clinical practice with novel and more promising treatment strategies and drug options.

[0054] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0055] Experimental methods

[0056] 1. Establishment and treatment of an experimental autoimmune myasthenia gravis (EAMG) model.

[0057] Female Lewis rats were randomly divided into four groups (n=6): a blank control (CFA) group, an EAMG group, an EAMG + 25 mg / kg chicoric acid (CA, purchased from Chengdu Mansite Biotechnology Co., Ltd., product number: A0090) group (abbreviated as CA(L)), and an EAMG + 50 mg / kg CA group (abbreviated as CA(H)). EAMG rats were administered the medication by gavage from the start of initial immunization until sacrifice on day 49. Rats in the CA(L) group were administered CA (25 mg / kg / d) by gavage once daily, and rats in the CA(H) group were administered CA (50 mg / kg / d) by gavage once daily for 7 weeks. The therapeutic effect was observed. Rats in the CFA control group and the EAMG model group were administered an equal volume of physiological saline by gavage.

[0058] Rats were monitored for daily weight changes, and clinical disease scores were recorded using a double-blind assessment, with disease severity graded from 0 to 4.

[0059] Level 0, normal strength;

[0060] Level 1: Mild reduction in activity, weaker grip or crying, which is more pronounced at the end of the exercise;

[0061] Level 2: Symptoms of illness appeared before exercise (trembling, head down, hunched back, weak grip);

[0062] Level 3: Severe illness symptoms appear before exercise, with no grip strength and on the verge of death;

[0063] Level 4, death.

[0064] Mice exhibiting intermediate symptoms were scored as 0.5, 1.5, 2.5, or 3.5. Results are expressed as the average of the scores recorded for each animal at each time point.

[0065] 2. HE staining

[0066] Spleen samples were fixed in 4% paraformaldehyde solution for 24 hours. They were then treated sequentially with 70% ethanol for 30 minutes, 80% ethanol for 45 minutes, 95% ethanol for 30 minutes, 95% ethanol again for 40 minutes, 100% ethanol for 30 minutes, and finally 100% ethanol for 40 minutes. Next, they were treated sequentially with xylene I and II for 10 minutes each, followed by treatment with soft paraffin at 65°C for 2 hours and hard paraffin for 3 hours. The specimens were placed in the molten paraffin in the embedding frame and allowed to solidify. Coronal serial sections of 5 μm thickness were prepared using a paraffin microtome. The sections were unfolded in a 40°C water bath and adhered to glass slides, then dried in a 37°C oven. Finally, they were treated sequentially with xylene I for 20 minutes, xylene II for 20 minutes, 100% ethanol for 10 minutes, 95% ethanol for 10 minutes, 80% ethanol for 10 minutes, and 70% ethanol for 10 minutes, and rinsed with distilled water for 2 minutes. Cell nuclei were stained with Hanys hematoxylin for 3 minutes, followed by rinsing with distilled water, treatment with 1% hydrochloric acid alcohol for 15 seconds, rinsing with running water, and finally counterstaining the cytoplasm with 5% eosin alcohol solution for 3-5 minutes. Dehydration was performed using a gradient of alcohols, followed by clearing with xylene, and finally mounting with neutral resin.

[0067] 3. Preparation of mononuclear cells

[0068] Under aseptic conditions, spleen and lymph nodes were harvested and placed in culture dishes. A 70 μm cell filter sieve was placed on the culture dish, and the spleen and lymph node tissues were minced using ophthalmic scissors. The minced tissues were then placed on the cell filter sieve and ground using the plunger of a syringe. After grinding, the filter sieve was rinsed with RPMI 1640 to collect the cell suspension, which was then collected into 15 mL centrifuge tubes. The cells were centrifuged at 1200 xg for 5 minutes, and the supernatant was discarded. Red blood cells in the spleen cell suspension were lysed on ice for 5 minutes using RBC lysis buffer (BioGems, 64,010-00). Finally, after three washes, the cell concentration was adjusted to 2 × 10⁶ cells / mL in complete culture medium containing 1% (v / v) penicillin / streptomycin, 10% (v / v) fetal bovine serum (FBS), and 89% RPMI 1640. 6 / ml, to prepare for subsequent experiments.

[0069] 4. Flow cytometry analysis

[0070] Following standard experimental procedures, the cells were stained with the following antibodies:

[0071] BV605-Zombie (Biolgend, 423,103), FITC-CD4 (Biolgend, 201,505), APC-IL-17A (eBioscience, 17-7177-81), PE-CD25 (Biolgend, 202,105), AF647-FOXP3 (Biolgend, 320,014), FITC-CD3 (Biolgend, 100,204), PE-TCR-γ / δ (Biolgend, 118,116).

[0072] For intracellular cytokine staining, cells were stimulated at 37°C for 5 hours using a cell stimulation mixture (eBioscience, 00-975-03), followed by FITC-CD4 surface staining (Biolgend, 201,505) at 4°C for 30 minutes.

[0073] Cells were then fixed at 4°C for 20 minutes with a fixation and permeabilization solution (BD Biosciences, 554,723), and permeabilized after treatment with Perm / Wash buffer (BD Biosciences, 554,722).

[0074] When detecting helper T cell (Th) expression, APC-IL-17A (eBioscience, 17-7177-81) was used for staining at 4°C for 30 minutes.

[0075] To detect the expression of regulatory T cells (Tregs), FITC-CD4 (Biolgend, 201,505) and PE-CD25 (Biolgend,202,105) staining was performed first. Then, the cells were fixed overnight at 4°C in the dark using a dedicated fixation and permeabilization buffer. After washing and centrifugation, the cells were stained with AF647-FOXP3 (Biolgend, 320,014) at 4°C in the dark for 30 minutes. The samples were then analyzed by flow cytometry.

[0076] The expression of T cell receptor (TCR) was detected by staining with FITC-CD3 (Biolgend, 100, 204) and PE-TCR-γ / δ (Biolgend, 118, 116) at 4°C for 30 minutes.

[0077] Example 1: Chicoric acid (CA) improves clinical symptoms of EAMG

[0078] To investigate the therapeutic effect of chicoric acid (CA) on EAMG rats, an EAMG animal model was constructed based on the above experimental methods. From day 1 to day 49, the model rats were given different doses of CA (25 mg / kg, 50 mg / kg) by gavage daily.

[0079] The experimental results showed that, compared with the EAMG group, chicoric acid (CA) reduced the weight loss of rats in the EAMG group. On day 49, the body weight score of rats in the EAMG group was significantly lower than that in the low-dose CA group. Figure 1 A). Furthermore, compared to the EAMG group, rats in the chicoric acid CA-treated group showed significantly improved grip strength, reduced limb tremors, and decreased head lift. Figure 1 E); In rats treated with CA, the clinical scores were significantly lower than those in the EAMG model group. On day 49, compared with the blank control group (CFA), the clinical scores of rats in the EAMG group were significantly higher; while chicoric acid CA significantly reduced the clinical scores of rats in the EAMG model group ( Figure 1 B); Compared with the blank control group (CFA), the cumulative score and peak score of EAMG group rats were significantly increased, while chicoric acid (CA) could also reduce the cumulative score and peak score of EAMG model rats (B); Figure 1 CD. H&E staining showed that CA intervention reduced splenic nodule enlargement in EAMG group rats ( Figure 1 F), suggesting that CA has a therapeutic effect on EAMG rats.

[0080] Example 2: Chicoric acid (CA) regulates the imbalance of Th17, Treg, and TCR cell ratios in EAMG.

[0081] To clarify CA in CD4 + Regulatory functions in T cells: Based on the above experimental methods, this invention used flow cytometry to evaluate Th17 (CD4) cells in the spleen and lymph nodes of rats. + IL-17 + ), Treg(CD4) + CD25 + FOXP3 + ) and TCR (CD3) + γδ + ) level.

[0082] The results showed that, compared with the control group, the levels of Th17 and TCR cells in the spleen and lymph nodes of rats in the EAMG group were significantly increased. Figure 2 A- Figure 2 C), Treg cell levels were significantly reduced ( Figure 2 B), the levels of Th17 and TCR cells in the spleen and lymph nodes of rats in the CA group were reduced ( Figure 2 A- Figure 2C), Treg cell levels were significantly increased ( Figure 2 B).

[0083] The above data indicate that chicoric acid (CA) improves myasthenia gravis EAMG by regulating the imbalance of Th17, Treg, and TCR cells in EAMG rats.

[0084] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. Use of chicoric acid and its pharmaceutically acceptable salts, esters, solvates, polymorphs or prodrugs in the preparation of medicaments for the prevention and / or treatment of myasthenia gravis.

2. The use according to claim 1, characterized in that, The myasthenia gravis referred to is the acetylcholine receptor antibody positive type, muscle-specific tyrosine kinase antibody positive type, and / or serum antibody negative type of myasthenia gravis.

3. A pharmaceutical composition for the prevention and / or treatment of myasthenia gravis, characterized in that, It contains a therapeutically effective amount of chicoric acid and one or more pharmaceutically acceptable carriers.

4. The pharmaceutical composition according to claim 2, characterized in that, The dosage forms of the pharmaceutical composition include: oral solid dosage forms, oral liquid dosage forms, injections, inhalers, transdermal patches, or sublingual immediate-release formulations.

5. The pharmaceutical composition according to claim 2, characterized in that, The concentration of chicoric acid in the pharmaceutical composition is from 0.1 mg / mL to 500 mg / mL, or from 0.2 wt% to 98 wt%.

6. The pharmaceutical composition according to claim 2, characterized in that, Chicoric acid is the sole active ingredient in the pharmaceutical composition.

7. A drug delivery system comprising nanoparticles, liposomes, microspheres, or cyclodextrin inclusions loaded with chicoric acid for the prevention and / or treatment of myasthenia gravis.

8. Use of chicoric acid in the preparation of drugs for regulating the imbalance of Th17 cells to Treg cells in peripheral blood, spleen or lymph nodes of patients with myasthenia gravis.

9. Use of chicoric acid in the preparation of drugs for reducing the level of pathogenic autoantibodies in patients with myasthenia gravis.

10. A method for screening candidate compounds for treating myasthenia gravis, comprising the following steps: The ability of the candidate compounds to regulate Th17 / Treg cell homeostasis was tested. And select compounds that can significantly correct the Th17 / Treg cell imbalance; chicoric acid was used as a positive control.

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