WO2022218381A1 - 毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷治疗自身免疫性疾病的用途 - Google Patents

毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷治疗自身免疫性疾病的用途 Download PDF

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WO2022218381A1
WO2022218381A1 PCT/CN2022/086850 CN2022086850W WO2022218381A1 WO 2022218381 A1 WO2022218381 A1 WO 2022218381A1 CN 2022086850 W CN2022086850 W CN 2022086850W WO 2022218381 A1 WO2022218381 A1 WO 2022218381A1
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syndrome
glucoside
sjögren
mice
mullein
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PCT/CN2022/086850
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English (en)
French (fr)
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林响
俞苏岚
冯云
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香港大学
北京大学第三医院
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Priority to CN202280028478.8A priority Critical patent/CN117750961A/zh
Publication of WO2022218381A1 publication Critical patent/WO2022218381A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to a medicine, in particular to the application of a pharmaceutical composition comprising mullein and/or mullein glucoside in the preparation of medicines for preventing, relieving and treating autoimmune diseases. More specifically, the present invention relates to the use of vervasoflavones and/or versiflavone glucosides for the prevention, alleviation, treatment of multiple sclerosis, systemic lupus erythematosus or Sjögren's syndrome.
  • Autoimmune disease is a condition in which the body's abnormal immune response attacks normal cells. These diseases can occur in almost any part of the body. Common symptoms include fever and feeling tired. Symptoms are often intermittent.
  • Sjogren's syndrome ( Syndrome, SS) is the third most common systemic rheumatic disease in the world, after rheumatoid arthritis and systemic lupus erythematosus. Its clinical features include lymphocytic infiltration of exocrine glands, especially the salivary and lacrimal glands, resulting in dry mouth and eyes.
  • SS systemic rheumatic disease
  • the treatment of SS is mainly based on broad-spectrum non-specific immunosuppressive agents, and specific treatment plans still lack large-scale evidence-based medical data.
  • Targeted biopharmaceuticals are also in the early stage of clinical research and have not yet Large-scale testing showed ideal therapeutic effects. Therefore, it is urgent to find a specific drug for the treatment of Sjögren's syndrome.
  • Systemic lupus erythematosus is associated with localized cancers in various parts of the body, as well as lymphoproliferative cancers.
  • the disease affects multiple organ systems and is characterized by a widespread loss of immune tolerance in humans. Chronic inflammation throughout the body drives other cells to become malignant, increasing the risk of systemic and lymphoproliferative cancers.
  • Multiple Sclerosis is associated with a reduced risk of developing systemic cancers, but is associated with an increased risk of cancers of the central nervous system (mainly the brain).
  • Multiple sclerosis is a neurodegenerative disorder in which T cells, a specific type of immune cell, attack the important myelin sheath in the brain's neurons. The function of the nervous system is thus reduced, leading to inflammation and subsequent development of brain cancer.
  • the present invention proposes a pharmaceutical composition for treating or alleviating Sjögren's syndrome, wherein the composition comprises a pharmaceutically effective amount of mullein and/or mullein Flavonoid glucoside.
  • the medicine is a liquid oral agent, an injection agent, a capsule, a tablet, a powder, a water pill, and a plaster.
  • compositions for preventing Sjögren's syndrome comprising a pharmaceutically effective amount of mullein and/or mullein glucoside.
  • the medicine is a liquid oral agent, an injection agent, a capsule, a tablet, a powder, a water pill, and a plaster.
  • a method of using a composition for treating and alleviating Sjögren's syndrome comprising: administering the composition containing 0.2-10 mg of vervescoflavonoids and/or vervisoflavone glucosides per kilogram of body weight.
  • a method for use of a composition for preventing Sjögren's syndrome comprising: administering the composition containing 0.2-10 mg of velocinoin and/or velocinoin glucoside per kilogram of body weight; further, per kilogram of body weight (human ) containing 0.2 mg of the composition per administration of velocidone and/or versiflavone glucoside.
  • composition comprising mullein and/or mullein glucoside in the preparation of a medicament for preventing Sjögren's syndrome, wherein the content of the mullein and/or mullein glucoside is a pharmaceutically effective amount.
  • the present application discloses a medicine for preventing, relieving and treating Sjögren's syndrome, which not only has quick effect, but also has obvious therapeutic effect, and is suitable for the acute phase and the chronic phase of Sjögren's syndrome.
  • the present invention relates to a pharmaceutical composition for preventing, treating or alleviating autoimmune diseases in patients.
  • the composition comprises a pharmaceutically effective amount of mullein and/or mucus isoflavone glucoside.
  • the present invention relates to a method of preventing, treating or alleviating an autoimmune disease in a patient, comprising: administering to the patient a therapeutically effective amount of mullein and/or mullein glucoside.
  • the method may comprise administering to the patient 0.2-10 mg of mullesin and/or glucosinolate per kilogram of body weight.
  • the method may comprise administering to said patient (human) 0.2 mg per kilogram of body weight of mullein and/or mullein glucoside.
  • the method may comprise administering to the patient 1 time every 2 days of velocassone and/or velocinoin glucoside.
  • the present invention relates to the application of versiflavone and/or versiflavone glucoside in the preparation of a medicament for preventing, relieving and treating autoimmune diseases in patients.
  • Verisoflavones and/or verbasoflavone glucosides may be included in the composition.
  • the content of mullein and/or mullein glucoside is a pharmaceutically effective amount.
  • Autoimmune diseases can be chronic.
  • the autoimmune disease may be selected from multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, IgG4-related disease or systemic fibroinflammatory disease.
  • the autoimmune disease is multiple sclerosis, systemic lupus erythematosus or Sjögren's syndrome.
  • a patient can be a human.
  • Fig. 1A shows the relationship between the amount of saliva secretion and the level of autoantibody anti-SSA IgG in plasma and the time of immunization induction in immunization-induced ESS mice according to an embodiment of the present invention
  • Figure 1B is the OD value of autoantibodies in mouse peripheral blood serum according to an embodiment of the present invention.
  • 1C is a mouse salivary gland lymphocyte under an immunofluorescence microscope according to an embodiment of the present invention.
  • 2A is a control diagram of mouse saliva flow rate in different experimental groups according to an embodiment of the present invention.
  • Figure 2B is the mouse anti-SSA antibody (anti-SSA) level in different experimental groups according to an embodiment of the present invention.
  • 2C is a histopathological analysis of mouse glands in different experimental groups according to an embodiment of the present invention.
  • Fig. 3 is the preventive effect of mullein and mullein glucoside on Sjögren's syndrome in mice according to an embodiment of the present invention.
  • Figure 4 A shows the chemical structures of four similar isoflavones; B shows that only Caly and Cal-7 can inhibit murine T follicular helper (Tfh) cell responses; C shows that Caly inhibits IL-17A and IL-21 mRNA in culture expression effect.
  • Tfh murine T follicular helper
  • Figure 5 shows the therapeutic effect of Caly on Sjögren's syndrome mouse model.
  • A shows the salivary function of Caly-treated patients with experimental Sjogren's syndrome (ESS) mice;
  • B shows serum autoantibody levels;
  • C shows that Caly can target Th17 and Tfh cells, showing the expression of Tfh and Th17 Type;
  • D shows histological assessment of salivary glands in Caly-treated diseased ESS mice showing lymphocytic infiltration in ESS mice.
  • Figure 6 shows the therapeutic effect of Caly on a mouse model of multiple sclerosis.
  • A shows body weight changes in mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis, with or without Caly treatment.
  • B shows the clinical score.
  • C shows histological assessment of the spinal cord of EAE mice showing lymphocytic infiltration in EAE mice.
  • D shows Luxol Fast Blue staining of spinal cord myelin, showing demyelination in EAE mice.
  • Figure 7 shows the therapeutic effect of Caly on systemic lupus erythematosus.
  • A shows serum anti-double-stranded DNA IgG levels in a Caly-treated lupus mouse model.
  • B shows IgG deposition in kidney glomeruli and CD45+ lymphocyte infiltration in kidney.
  • Lupus lupus
  • Ctrl control; unprocessed.
  • Sjogren's syndrome or SS is a chronic autoimmune disease that invades exocrine glands, especially salivary and lacrimal glands. Mainly manifested as dry mouth and eyes, but also multi-organ and multi-system damage. A large number of lymphocytes were infiltrated in the affected organs, and a variety of autoantibodies were positive in the serum. This syndrome is also known as autoimmune exocrine gland disease, Sjogren's syndrome, and xerophthalmia syndrome. Often overlaps with other rheumatic or autoimmune diseases.
  • ESS The experimental SS model. This model can highly mimic the main clinical symptoms of SS, and its inflammatory progression is also highly similar to the patient's disease characteristics, which is in line with the European League against Rheumatism (EULAR) combined with the American College of Rheumatology (ACR). Consensus meeting on diagnostic criteria for Sjögren's syndrome.
  • EULAR European League against Rheumatism
  • ACR American College of Rheumatology
  • Anti-SSA antibodies which occur most frequently in Sjögren's syndrome, but can also occur in other autoimmune rheumatic diseases. In some countries, tissue biopsy of labial glands is considered necessary for the diagnosis of Sjögren's syndrome, but this test is difficult to implement in China. Later, the detection of anti-SSA antibodies in serum was used as the evidence for the diagnosis of Sjögren's syndrome.
  • IgG immunoglobulin G, immunoglobulin plays a role in activating complement and neutralizing a variety of toxins in the immune response.
  • High IgG levels in patients with Sjögren's syndrome indicate the presence of hyperimmunoglobulinemia, which is a manifestation of the systemic involvement of Sjögren's syndrome, mainly related to the basic pathological changes of the patients.
  • OD value is the abbreviation of optical density (optical density), which represents the optical density absorbed by the detected object, and is a proper noun in the detection method.
  • Ctrl-peptide an antibody whose expression level is not affected in wild-type mice and ESS mice subjected to immunization-induced disease in this application, and is a polypeptide.
  • M3R-peptide anti-muscarinic receptor 3 (M3) antibody, a cholinergic receptor distributed in exocrine glands and smooth muscle, and a G-protein coupled receptor that mediates glandular secretion .
  • M3 antibody anti-muscarinic receptor 3
  • cholinergic receptor distributed in exocrine glands and smooth muscle
  • G-protein coupled receptor that mediates glandular secretion .
  • Anti-ANA antinuclear antibody, which is elevated in patients with Sjögren's syndrome.
  • Calycosin (Calycosin, Caly): Its known pharmacological effects include enhancing antioxidant, anti-radiation and anti-cancer effects, protecting cardiovascular and cerebrovascular, protecting brain cells, relaxing vascular smooth muscle, hormone-like effects, antibacterial and antiviral effects.
  • Verasco isoflavone glucoside (Calycosin-7-O- ⁇ -Dglucoside, CG, Cal-7): also known as Verasco isoflavone glycoside, Verasco isoflavone-7-O- ⁇ -D glucoside.
  • the known pharmacological effects include anti-oxidation, anti-virus, inhibition of melanin formation, immunosuppressive effect, anti-tumor, and effect on cardiovascular and cerebrovascular diseases.
  • Sjogren's syndrome acute phase the acute onset of Sjögren's syndrome.
  • the middle drawing—ESS (5wk) of FIG. 1C of the present application is the lymphocyte infiltration of mice in the acute phase of Sjögren's syndrome after 5 weeks of immunization induction treatment.
  • Sjögren's syndrome chronic phase that is, with the progression of Sjögren's syndrome, the disease is more severe.
  • Figure 1C of the present application - ESS (30wk) is the lymphocyte infiltration of mice in the chronic phase of Sjögren's syndrome after 30 weeks of immune induction treatment.
  • 1A-C are experimental models of simulating SS onset in mice according to an embodiment of the present application.
  • 1A shows the relationship between the amount of saliva secretion and the level of autoantibody anti-SSA IgG in the plasma of ESS mice with immunization-induced disease according to an embodiment of the present invention and the immunization induction time; wherein, the abscissa represents the immunization induction time ( week), the left ordinate represents the salivary flow rate ( ⁇ l/g/15min), the right ordinate represents the OD value (450nm) of autoantibody anti-SSA IgG in plasma; the downward trend curve (green) is the saliva secretion curve , the upward trend curve (purple) is the OD value curve of autoantibody anti-SSA IgG in plasma;
  • Figure 1B is the OD value of autoantibody in mouse peripheral blood serum according to an embodiment of the present invention; wherein, the Ctrl of the abscissa is the wild-type mouse without immunization induction
  • Example 2 Therapeutic effect of mullein and mullein glucoside on acute phase of Sjögren's syndrome
  • Caly and Cal-7 can be made into oral liquid reagents or injection reagents, wherein the solvent can be edible oil, dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose (CMC- Na), double distilled water or MilliQ ultrapure water.
  • the solvent can be edible oil, dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose (CMC- Na), double distilled water or MilliQ ultrapure water.
  • the liquid reagent may also include auxiliary materials, wherein the auxiliary materials may be one or more of the following substances: ethanol, propylene glycol, glycerol, hydrochloric acid, lactic acid, sodium hydroxide, citric acid, sodium citrate , tartaric acid, sodium tartrate, sodium sulfite, sodium metabisulfite, sodium thiosulfate, disodium ethylenediaminetetraacetic acid (EDTA-NA2), phenol, benzyl alcohol, thimerosal, sodium chloride, glucose, etc.
  • the concentration of Caly or Cal-7 is 0.13-3.33 mg/mL
  • the concentration of Caly or Cal-7 is 0.04-0.8 mg/mL.
  • the present application does not limit the form of medicines containing either Caly or Cal-7, and Caly and Cal-7 can also be made into solid medicaments, including but not limited to capsules, tablets, powders, water pills, honey pills, and the like.
  • solid medicaments including but not limited to capsules, tablets, powders, water pills, honey pills, and the like.
  • other excipients may not be included in the drug.
  • the pharmaceutical form when it is solid, it may further include excipients, and the excipients may be selected from one or more of the following substances: starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, Microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, water, ethanol, hypromellose (HPMC), carboxymethyl cellulose (CMC), povidone (PVP), Starch syrup, syrup, dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrant, crospovidone, magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, Micropowder silica gel, titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, acrylic resin, cellulose acetate, etc.
  • the content of Caly or Cal-7 is 0.13-3.33 mg
  • the medicament containing Caly or Cal-7 of the present application can also be a topical medicament, such as a plaster and the like.
  • the adjuvant added to the plaster can be a medically known adjuvant that can be used for the plaster.
  • the medicament containing Caly or Cal-7 of the present application can also be in other pharmaceutically acceptable forms, including pharmaceutically acceptable carriers in various forms.
  • mice were divided into 4 groups, namely the control group, that is, the group of mice without any treatment; the model group, that is, the group of mice that were subjected to ESS mutagenesis but not treated; Cal-7 A treatment group, ie, a group of mice treated with Cal-7 after ESS mutagenesis for 2 weeks; and a Caly-treated group, ie, a group of mice treated with Caly after 2 weeks of ESS mutagenesis.
  • each group has 6 mice of similar age, all of which are about 15 weeks old.
  • the dose of Caly or Cal-7 administered to the mouse is 2-50 mg/kg, that is, the content of Caly or Cal-7 per kilogram of the drug administered to the mouse is 2-50 mg. Further, the dose of Caly or Cal-7 administered to the mice was 5 mg/kg.
  • the drug containing either Caly or Cal-7 was prepared as a liquid reagent for oral administration.
  • the acute phase of Sjögren's syndrome was administered to mice 2 weeks after the establishment of the ESS model. During the treatment period, the drug was administered once every 2 days, and the dose of each dose was 5 mg/kg. After continuous treatment for 2 weeks, the gland function and peripheral blood serological indexes of the mice were detected. The results are shown in Figures 2A and 2B.
  • 2A is a control diagram of mouse saliva flow rate in different experimental groups according to an embodiment of the present invention.
  • the abscissa represents 4 different experimental groups, and the left one (black) is the control group, that is, without immune induction treatment Wild-type mice; the second left (yellow) is the model group, that is, the mice that have been treated with immunization induction but not treated; the second right (blue) is the Cal-7 treatment group, that is, the mice have been treated with immunization induction and given Cal- 7.
  • mice treated with the drug is the Caly treatment group, that is, the mice treated by immune induction and given Caly drug treatment; the ordinate represents the mouse salivary flow rate ( ⁇ L/15min);
  • Figure 2B is based on this The mouse anti-SSA antibody (anti-SSA) levels in different experimental groups of an embodiment of the invention; wherein, the abscissa represents 4 different experimental groups, and the corresponding relationship is the same as in Figure 2A; the ordinate represents the OD of the anti-SSA antibody value (450nm);
  • Figure 2C is a histopathological analysis of mouse glands in different experimental groups according to an embodiment of the present invention; wherein, the abscissa represents 4 different experimental groups, and its corresponding relationship is the same as Figure 2A, the left one (Black) 0 indicates no pathological damage to the glandular tissue; the ordinate indicates the histopathological score, and the higher the score, the more severe the tissue infiltration in the glandular tissue.
  • Example 3 Therapeutic effect of mullein and mullein glucoside on chronic phase of Sjögren's syndrome
  • the medicines used are the same as those in Embodiment 2, and are not repeated here.
  • mice were divided into 4 groups, namely the control group, that is, the group of mice without any treatment; the model group, that is, the group of mice that were subjected to ESS mutagenesis but not treated; Cal-7 A treatment group, ie, a group of mice treated with Cal-7 after 10 weeks of ESS mutagenesis; and a Caly treated group, ie, a group of mice treated with Caly after 10 weeks of ESS mutagenesis.
  • each group has 6 mice of similar age, all of which are about 40 weeks old.
  • the drug containing either Caly or Cal-7 was prepared as a liquid reagent for oral administration.
  • the chronic phase of Sjögren's syndrome was administered to mice 10 weeks after the establishment of the ESS model, and the dose was 5 mg/kg every 2 days during the treatment period. After continuous treatment for 10 weeks, the pathological indicators of the mouse glands were detected, and the results are shown in Figure 2C.
  • Example 4 Preventive effect of mullein and mullein glucoside on Sjögren's syndrome
  • FIG. 3 is the preventive effect of mullein and mullein glucoside on Sjögren's syndrome in mice according to an embodiment of the present invention.
  • the figure on the left indicates the administration time and the induction time of immunization of mice: 3 days before immunization induction of mice, Caly and Cal-7 were given to mice in two groups of experimental groups, and equal amounts of PBS were given to mice in control group.
  • the experimental data was collected after 21 days of immunization induction;
  • the middle figure shows the salivary secretion flow rate of mice in different administration groups after the establishment of the mouse immunization induction model, in which the circle ( ⁇ ) represents the control given PBS Group mice, the square ( ⁇ ) represents the mice in the experimental group given Cal-7, the triangle ( ⁇ ) represents the mice in the experimental group given Caly;
  • the abscissa represents the time when the immune-induced mouse model was established (0 days and 21 days) , the ordinate represents the flow rate of saliva secretion in mice ( ⁇ L/15min);
  • the right picture shows the anti-SSA antibody IgG (anti-SSA IgG) levels of mice in different groups;
  • the abscissa represents 3 different groups of mice, the left (blue) is the mice in the control group given PBS, the middle (yellow) is the mice in the experimental group given Cal-7, and the right (red) is the mice in the experimental group given Caly;
  • the ordinate represents the amount of anti
  • mice 3 days before the establishment of immune-induced mice (ESS), the mice were treated with drug administration, wherein the applicant's prophylactic administration of Caly and Cal-7 to the mice Both were 5 mg/kg, to observe the preventive effect of muscarinone and glucosinolate on Sjögren's syndrome.
  • An experimental SS (ESS) model was induced in 8-week-old female mice. Briefly, proteins extracted from the bilateral salivary glands (SG) of normal mice were emulsified in an equal volume of Freund's complete adjuvant (Sigma-Aldrich) at a concentration of 2 mg/mL. To induce disease, on day 0, each mouse was injected subcutaneously with 0.2 mL of the emulsion on the back. On day 14, a booster injection was performed with 1 mg/mL SG protein emulsified in incomplete Freund's adjuvant (Sigma-Aldrich).
  • EAE Peripheral autoimmune encephalomyelitis
  • mice Female C57BL/6 mice were immunized to actively induce EAE [4]. Briefly, mice were injected subcutaneously with 200 ⁇ g MOG35-55 in complete Freund's adjuvant (5 mg/ml, Sigma-Aldrich). Pertussis toxin (200ng, List Biological Laboratories) was injected intravenously twice on days 0 and 2 (dpi.) post-immunization. Immunized mice were assessed daily with body weight measurements and clinical scores.
  • the disease severity was scored as follows: 0, normal; 1, tail paralysis; 2, hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb paralysis and forelimb paralysis or weakness; 5, dying/death.
  • mice 8-week-old female C57BL/6 mice were subcutaneously immunized with chromatin extracted from splenocytes [6]. Briefly, nuclear chromatin samples were prepared from splenocytes stimulated with 5 ng/mL concanavalin A for 72 hours. On days 14 and 28 after the first immunization, mice were immunized with 200 ⁇ g of chromatin emulsified in complete Freund's adjuvant (CFA; InvivoGen, CA, USA) and 100 ⁇ g in incomplete Freund's adjuvant. Chromatin booster in IFA (IFA; InvivoGen). Mice immunized with PBS emulsified in adjuvant served as controls.
  • CFA complete Freund's adjuvant
  • IFA InvivoGen
  • FIG. 4 shows that calycosin (Caly) significantly inhibits Th17 and T follicular helper cell responses.
  • Caly was previously reported as a metabolite of Cal-7.
  • the IC50 (half-maximal inhibitory concentration) of Caly in inhibiting IL-17A and IL-21 was measured by culturing with T cells for 24 hours, and the IC50 was 8.65 ⁇ M and 10.65 ⁇ M, respectively, detected by quantitative polymerase chain reaction (qPCR) .
  • Figure 5 shows the therapeutic effect of Caly on a mouse model of Sjögren's syndrome.
  • ESS salivary gland-derived antigens
  • salivary gland-derived antigens to induce experimental Sjögren's syndrome [1].
  • the main features of disease pathology include hyposalivation (checked by measuring salivary secretion capacity), elevated serum anti-SSA IgG levels (checked by ELISA) and salivary gland histopathological changes (checked by H&E staining), as in lymphatic As revealed by cellular infiltration, it was clearly observed in ESS mice.
  • Treatment with Caly (5 mg/kg) significantly improved disease pathology in mice.
  • T effector cell subsets (termed IL-17-producing Th17 cells and PD-1+ICOS+T follicular helper (Tfh) cells) in the pathogenesis of ESS (through flow cytometry). cytometry) was also significantly reduced after Caly treatment [1-3].
  • Th17 cells play a key role in the pathogenesis of multiple sclerosis and Tfh cell-mediated humoral autoimmunity is critical in systemic lupus erythematosus, the following are examples of the therapeutic effects of Caly in the treatment of these mouse models .
  • Figure 6 shows the therapeutic effect of Caly on a mouse model of multiple sclerosis.
  • mice Female C57BL/6 mice were immunized to actively induce experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis [4]. Following disease induction, mice were treated with PBS or Caly (5 mg/kg) until the peak time of EAE progression (16 days). Caly treatment improved daily and cumulative weight loss and decreased clinical scores in EAE mice [5].
  • H&E analysis showed that EAE mice in the PBS group had characteristic inflammatory foci in the white matter of lumbar spinal cord lesions, whereas Caly-treated EAE mice had significantly reduced inflammatory infiltration and lower infiltration fractions. Demyelination was also checked by LFB staining (blue). A smaller area of demyelination was observed in spinal cord injury in Caly-treated mice than in PBS-group EAE mice, quantified and presented by a lower demyelination fraction.
  • Figure 7 shows the therapeutic effect of Caly on a mouse model of systemic lupus erythematosus.
  • mice C57BL/6 mice were immunized with apoptotic chromatin for experimental systemic lupus erythematosus [6]. After disease induction, mice were treated with PBS or Caly (5 mg/kg) for 8 weeks. Lupus mice clearly showed increased serum levels of anti-dsDNA IgG autoantibodies (checked by ELISA). This was exemplified by severe renal inflammation, as marked IgG deposition and CD45+ lymphocyte infiltration (by confocal microscopy) were observed in the kidneys of lupus mice. However, Caly treatment significantly improved disease pathology, showing lower serum levels of anti-dsDNA IgG, milder lymphocytic infiltration, and reduced IgG deposition in the kidneys.

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Abstract

本发明涉及毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷治疗自身免疫性疾病的用途。本发明涉及一种包含毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的组合物在制备缓解、治疗干燥综合征的药物方面的应用，其中，所述毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的含量为药物学上的有效量。本申请还公开了预防、缓解及治疗干燥综合征的药物，其不仅见效快，并且治疗效果明显，适用于干燥综合征急性期及慢性期。

Description

毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷治疗自身免疫性疾病的用途 技术领域
本发明涉及一种药物，特别地涉及一种包含毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的药物组合物在制备预防、缓解、治疗自身免疫性疾病方面的药物中的应用。更具体而言，本发明涉及毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷用于预防、缓解、治疗多发性硬化症、系统性红斑狼疮或干燥综合征的用途。
背景技术
自身免疫性疾病(Autoimmune disease)，是指人体异常的免疫反应攻击正常细胞所产生的状况。这类疾病几乎可发生在人体的任何部位。常见症状包括发热、感到疲劳。症状经常是断续式的发作。
干燥综合征(
Figure PCTCN2022086850-appb-000001
syndrome，SS)是世界排名第三的系统性风湿性疾病，仅次于类风湿性关节炎和系统性红斑狼疮。其临床特征包括外分泌腺特别是唾液腺和泪腺的淋巴细胞浸润，从而导致口干和眼干。现阶段SS的治疗主要以广谱非特异性的免疫抑制剂为主，而特异性的治疗方案还缺乏大规模的循证医学资料，靶向性生物制药也处于临床研究的初期阶段，还未在大规模测试中显示出理想的治疗效果。因此寻找一种治疗干燥综合征的特效药物为现阶段的当务之急。
系统性红斑狼疮(Systemic lupus erythematosus)与身体各部的局灶性癌症，以及淋巴球增生性癌症有关联。这种病对多个器官系统发生影响，特征是人体免疫耐受性广泛的丧失。遍布全身的慢性发炎会促使其他细胞转化为恶性，从而提高罹患系统性和淋巴球增生性癌症的风险。
多发性硬化症(Multiple Sclerosis)与降低罹患全身性癌症的风险有关联，但与中枢神经系统癌症(主要是大脑)的风险增加有关。多发性硬化症是一种神经退化障碍性疾病，其中T细胞(一种特定类型的免疫细胞)攻击大脑神经元中重要的髓鞘。神经系统的功能因此降低，导致发炎和随后的脑部癌症发生。
发明内容
针对现有技术中存在的技术问题，本发明提出了一种用于治疗或缓解干燥综合征的药物组合物，其中，所述组合物中包含药物学上的有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
如上所述的组合物，所述药物为液体口服试剂、注射试剂、胶囊、片剂、粉末、水丸、膏药。
一种用于预防干燥综合征的药物组合物，其中，所述组合物中包含药物学上的有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
如上所述的组合物，所述药物为液体口服试剂、注射试剂、胶囊、片剂、粉末、水丸、膏药。
一种用于治疗、缓解干燥综合征的组合物的使用方法，包括：每千克体重每次给予含有0.2-10mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物。
如上所述的方法，进一步包括：每千克体重(人)每次给予含有0.2mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物。
如上任一所述的方法，进一步包括，每2日给予1次所述组合物。
一种用于预防干燥综合征的组合物的使用方法，包括：每千克体重每次给予含有0.2-10mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物；进一步地，每千克体重(人)每次给予含有0.2mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物。
一种包含毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的组合物在制备缓解、治 疗干燥综合征的药物方面的应用，其中，所述组合物中毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的含量为药物学上的有效量。
一种包含毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的组合物在制备预防干燥综合征的药物方面的应用，其中，所述毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的含量为药物学上的有效量。
本申请公开了预防、缓解及治疗干燥综合征的药物，其不仅见效快，并且治疗效果明显，适用于干燥综合征急性期及慢性期。
本发明涉及一种用于预防、治疗或缓解患者中自身免疫性疾病的药物组合物。所述组合物包含药物学上的有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
本发明涉及一种预防、治疗或缓解患者中自身免疫性疾病的方法，包括：向所述患者给予治疗有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。该方法可以包括向所述患者给予每千克体重0.2-10mg的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。该方法可以包括向所述患者(人)给予每千克体重0.2mg的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。该方法可以包括向所述患者每2日给予1次毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
本发明涉及毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷在制备用于在患者中预防、缓解、治疗自身免疫性疾病的药物中的应用。毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷可以包含在组合物中。在所述组合物中，毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的含量为药物学上的有效量。
自身免疫性疾病可以是慢性的。自身免疫性疾病可以选自多发性硬化症、系统性红斑狼疮、干燥综合征、IgG4相关性疾病或系统性炎症伴纤维化疾病(systemic fibroinflammatory disease)。优选的是，自身免疫性疾病是多发性硬化症、系统性红斑狼疮或干燥综合征。
患者可以是人。
附图说明
下面，将结合附图对本发明的优选实施方式进行进一步详细的说明，其中：
图1A是根据本发明的一个实施例的免疫诱导发病的ESS小鼠体内唾液分泌量及血浆中自身抗体anti-SSA IgG水平与免疫诱导时间的关系；
图1B是根据本发明的一个实施例的小鼠外周血血清中自身抗体OD值；
图1C是根据本发明的一个实施例的免疫荧光显微镜下的小鼠唾液腺淋巴细胞；
图2A是根据本发明的一个实施例的不同实验组中小鼠唾液流速对照图；
图2B是根据本发明的一个实施例的不同实验组中小鼠抗SSA抗体(anti-SSA)水平；
图2C是根据本发明的一个实施例的不同实验组中小鼠腺体组织病理学分析；以及
图3是根据本发明的一个实施例的毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对小鼠干燥综合征的预防作用。
图4：A显示4种类似的异黄酮的化学结构；B显示只有Caly和Cal-7可以抑制鼠T滤泡辅助(Tfh)细胞反应；C显示Caly抑制培养物中IL-17A和IL-21mRNA表达的效果。
图5显示Caly对干燥综合征小鼠模型的治疗效果。A显示Caly治疗的患病实验性干燥综合征(ESS，experimental Sjogren’s syndrome)小鼠的唾液功能；B显示血清自身抗体水平；C显示Caly可以靶向Th17和Tfh细胞，展示了Tfh和Th17的表型；D显示Caly治疗的患病ESS小鼠唾液腺的组织学评估，显示ESS小鼠中的淋巴细胞浸润。
图6显示Caly对多发性硬化症小鼠模型的治疗效果。A显示患病实验性自身免疫性脑脊髓炎(EAE，Experimental autoimmune encephalomyelitis)小鼠 的体重变化，EAE是一种人类多发性硬化症的小鼠模型，有或没有Caly治疗。B显示临床评分。C显示EAE小鼠脊髓的组织学评估，显示EAE小鼠中的淋巴细胞浸润。D显示脊髓髓鞘的Luxol Fast Blue染色，显示EAE小鼠的脱髓鞘。
图7显示Caly对系统性红斑狼疮的治疗效果。A显示Caly治疗的狼疮小鼠模型的血清抗双链DNA IgG水平。B显示在肾脏肾小球中IgG沉积和肾脏中CD45+淋巴细胞浸润。C显示每个肾小球IgG沉积的平均荧光强度(每组n＝100)。Lupus:狼疮；Ctrl：对照；
Figure PCTCN2022086850-appb-000002
未经处理的。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在以下的详细描述中，可以参看作为本申请一部分用来说明本申请的特定实施例的各个说明书附图。在附图中，相似的附图标记在不同图式中描述大体上类似的组件。本申请的各个特定实施例在以下进行了足够详细的描述，使得具备本领域相关知识和技术的普通技术人员能够实施本申请的技术方案。应当理解，还可以利用其它实施例或者对本申请的实施例进行结构、逻辑或者电性的改变。
本申请中提及的专有名词具有如下释义：
干燥综合征或SS：即Sjogren syndrome，是一种侵犯外分泌腺体尤以侵犯唾液腺和泪腺为主的慢性自身免疫性疾病。主要表现为口、眼干燥，也可有多器官、多系统损害。受累器官中有大量淋巴细胞浸润，血清中多种自身抗体阳 性。本综合征也称为自身免疫性外分泌腺病(autoimmune exocrine gland disease)、斯约格伦综合征、口眼干燥关节炎综合征。常与其他风湿病或自身免疫性疾病重叠。
ESS：即实验性SS模型。该模型能高度模仿SS的主要临床症状，其炎症进展也与病人的疾病特征高度类似，符合欧洲抗风湿病联盟(European League Against Rheumatism,EULAR)联合美国风湿病学会(American College of Rheumatology,ACR)共识会议对干燥综合征的诊断标准。
anti-SSA：即抗SSA抗体，在干燥综合症中出现率最高，但也可以出现在其他自身免疫性风湿病。一些国家认为唇腺体组织活检是诊断干燥综合征的必要检查，但在中国这项检查难以实施，后来以检测血清中抗SSA抗体作为诊断干燥综合征的证据。
IgG：(immunoglobulin G,免疫球蛋白)在免疫应答中起着激活补体，中和多种毒素的作用。干燥综合征患者IgG偏高，提示着存在高免疫球蛋白血症，它是干燥综合征系统性受累的一种表现，主要是与患者的基本病理改变有关系。
OD值：OD值是optical density(光密度)的缩写，表示被检测物吸收掉的光密度，是检测方法里的专有名词。
Ctrl-peptide：为野生型小鼠和本申请中进行免疫诱导发病的ESS小鼠体内表达量不受影响的抗体，为一种多肽。
M3R-peptide：即抗毒蕈碱受体3(M3)抗体，是一种分布于外分泌腺及平滑肌的胆碱受体，也是一种G-蛋白耦联受体，可介导腺体的分泌。目前发现，SS患者免疫功能异常可导致抗M3抗体的产生，该抗体是一种对SS诊断较为特异的标记性抗体之一。
Anti-ANA：即抗核抗体，干燥综合征患者体内该抗体含量升高。
毛蕊异黄酮(calycosin,Caly)：其已知的药理作用有增强抗氧化，抗辐射 和抗癌作用，保护心脑血管，保护脑细胞，舒张血管平滑肌，激素样作用，抗菌及抑制病毒作用。
毛蕊异黄酮葡萄糖苷(Calycosin-7-O-β-Dglucoside,CG,Cal-7)：又名毛蕊异黄酮苷、毛蕊异黄酮-7-O-β-D葡萄糖苷。现已知的药理作用包括抗氧化、抗病毒、抑制黑色素形成、免疫抑制作用、抗肿瘤、对心脑血管疾病的作用等。
干燥综合征急性期：即干燥综合征急性发病时期。例如，本申请附图1C的中间附图—ESS(5wk)即为经过免疫诱导处理5周后的，处于干燥综合征急性期的小鼠的淋巴细胞浸润情况。
干燥综合征慢性期：即随干燥综合征病程进展而处于疾病更严重的时期。例如，本申请附图1C的右侧附图—ESS(30wk)即为经过免疫诱导处理30周后的，处于干燥综合征慢性期的小鼠的淋巴细胞浸润情况。
申请人近期的研究结果发现黄酮类单体毛蕊异黄酮葡萄糖苷，以及其代谢产物毛蕊异黄酮具有良好的免疫调节作用，能治疗实验性干燥综合征小鼠模型。
实施例1：小鼠干燥综合征模型建立
为了获得更理想的研究工具，申请人构建了一种在普通小鼠上诱导的实验性SS模型(experimental
Figure PCTCN2022086850-appb-000003
syndrome,ESS)，构建方法可见于Lin X,Rui K,Deng J,et al.,Th17 cells play a critical role in the development of experimental Sjogren's syndrome.Ann Rheum Dis,2015.74(6):p.1302-10.。
图1A-C为根据本申请一个实施例的小鼠模拟SS发病实验模型。其中，图1A是根据本发明的一个实施例的免疫诱导发病的ESS小鼠体内唾液分泌量及血浆中自身抗体anti-SSA IgG水平与免疫诱导时间的关系；其中，横坐标表示免疫诱导时间(星期)，左侧纵坐标表示唾液流速(μl/g/15min)，右侧纵坐标表示血浆中自身抗体anti-SSA IgG的OD值(450nm)；呈下降趋势曲线(绿色)为唾液分泌量曲线，呈上升趋势曲线(紫色)为血浆中自身抗体anti-SSA  IgG的OD值曲线；图1B是根据本发明的一个实施例的小鼠外周血血清中自身抗体OD值；其中，横坐标的Ctrl为未经免疫诱导处理的野生型小鼠，ESS为经过免疫诱导处理的ESS小鼠；纵坐标表示外周血血清中自身抗体OD值(450nm)；白色柱形图表示Ctrl-peptide的含量，即对照抗体的含量；黑色柱形图表示M3R-peptide的含量，即抗M3胆碱受体抗体的含量；灰色柱形图表示Anti-ANA的含量，即抗核抗体的含量；图1C是根据本发明的一个实施例的免疫荧光显微镜下的小鼠唾液腺淋巴细胞；其中，黑框中细胞为红框中细胞的局部放大图；左侧Ctrl为未经免疫诱导处理的野生型小鼠，中间ESS(5wk)为经过免疫诱导处理5周后的ESS小鼠，右侧ESS(30wk)为经过免疫诱导处理30周后的ESS小鼠。
如图1A-C所示，申请人观察到免疫诱导发病的ESS小鼠中唾液分泌量不断减少及血浆中自身抗体anti-SSA IgG水平不断增加(A)。外周血中也伴随有抗核抗体和抗M3胆碱受体抗体等特征性自身抗体(B)。此外，ESS小鼠的唾液腺出现明显的淋巴细胞浸润，且随着病程进展逐渐增多(C)。
实施例2：毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对干燥综合征急性期的治疗作用
为方便小鼠对给药，可将Caly和Cal-7制成口服液体试剂或者注射试剂，其中溶剂可以为食用油、二甲基亚砜(DMSO)、羧羧甲基纤维素钠(CMC-Na)、双蒸水或者MilliQ超纯水。在一些实施例中，液体试剂中还可以包括辅料，其中辅料可以是下列物质中的一种或者多种：乙醇、丙二醇、甘油、盐酸、乳酸、氢氧化钠、枸橼酸、枸橼酸钠、酒石酸、酒石酸钠、亚硫酸钠、焦亚硫酸钠、硫代硫酸钠、乙二胺四乙酸二钠(EDTA-NA2)、苯酚、苯甲醇、硫柳汞、氯化钠、葡萄糖等。在一些实施例中，口服液体试剂药物中，Caly或者Cal-7的浓度为0.13-3.33mg/mL；注射液体试剂药物中，Caly或者Cal-7的浓度为0.04-0.8mg/mL。
当然，本申请并不限定含有Caly或Cal-7任一成分药物的形式，也可将Caly和Cal-7制成固体药剂，包括但不限于胶囊、片剂、粉末、水丸、蜜丸等。在一些实施例中，当药物形式为固体时，药物中可以不包括其他辅料。在另一些实施例中，当药物形式为固体时，可以进一步包括辅料，辅料可以选自下列物质中的一种或者多种：淀粉、预胶化淀粉、糊精、蔗糖、乳糖、甘露醇、微晶纤维素、硫酸钙、磷酸氢钙、轻质氧化镁、碳酸钙、水、乙醇、羟丙甲纤维素(HPMC)、羧羧甲基纤维素(CMC)、聚维酮(PVP)、淀粉浆、糖浆、干淀粉、羟甲基淀粉钠、低取代羟丙基纤维素、泡腾崩解剂、交联聚维酮、硬脂酸镁、滑石粉、氢化植物油、聚乙二醇、微粉硅胶、二氧化钛、日落黄、亚甲蓝、药用氧化铁红、丙烯酸树脂、纤维醋法酯等。在一些实施例中，固体试剂药物中，Caly或者Cal-7的含量为0.13-3.33mg/g。
在另一些实施例中，本申请的含有Caly或Cal-7的药剂还可以为外用药剂，如膏药等。膏药中添加辅料可以为医学上已知可用于膏药的辅料。
在一些实施例中，本申请的含有Caly或Cal-7的药剂还可以为药物学上可接受的其他形式，包括药物学上可接受的各种形式载剂。
在本实施例中，将小鼠分成4各组，分别为对照组，即不进行任何处理的小鼠组；模型组，即进行ESS诱变处理，但不治疗的小鼠组；Cal-7治疗组，即进行ESS诱变2周后，使用Cal-7对其治疗的小鼠组；以及Caly治疗组，即进行ESS诱变2周后，使用Caly对其治疗的小鼠组。在本实施例中，每个组有6只年龄相近的小鼠，其年龄均为15周左右。
在一些实施例中，对小鼠的Caly或Cal-7的给药量为2-50mg/kg，即每千克小鼠服用的药物中Caly或Cal-7的含量为2-50mg。进一步地，对小鼠的Caly或Cal-7的给药量为5mg/kg。
在本实施例中，将含有Caly或Cal-7任一成分的药物制成口服的液体试剂。干燥综合征急性期给药治疗于ESS模型建立后2周的小鼠，治疗期间每2日给 药一次，每次给药量为5mg/kg。连续治疗2周，后检测小鼠腺体功能及外周血血清学指标，结果如图2A及2B所示。
其中，图2A是根据本发明的一个实施例的不同实验组中小鼠唾液流速对照图；其中，横坐标表示4个不同实验组，左一(黑色)为对照组，即未经免疫诱导处理的野生型小鼠；左二(黄色)为模型组，即经免疫诱导处理，但未治疗的小鼠；右二(蓝色)为Cal-7治疗组，即经免疫诱导处理，并给予Cal-7药物治疗的小鼠；右一(红色)为Caly治疗组，即经免疫诱导处理，并给予Caly药物治疗的小鼠；纵坐标表示小鼠唾液流速(μL/15min)；图2B是根据本发明的一个实施例的不同实验组中小鼠抗SSA抗体(anti-SSA)水平；其中，横坐标表示4个不同的实验组，其对应关系与图2A中相同；纵坐标表示抗SSA抗体的OD值(450nm)；图2C是根据本发明的一个实施例的不同实验组中小鼠腺体组织病理学分析；其中，横坐标表示4个不同的实验组，其对应关系与图2A相同，左一(黑色)为0表示其腺体组织无病理学损伤；纵坐标表示组织病理学评分，评分越高则表示腺体组织内组织浸润越严重。
如图2A-C所示，毛蕊异黄酮葡萄糖苷(Cal-7)和毛蕊异黄酮(Caly)小鼠干燥综合征的治疗效果明显(A)，能在建立模型后的发病期保护ESS小鼠的唾液腺体功能，维持其唾液分泌量。同时，血浆中抗SSA IgG自身抗体水平显著降低(B)。进一步地，组织病理学分析显示，给予Cal-7或者Caly的小鼠，其腺体组织损伤明显好于未经治疗的小鼠。值得注意的是，Caly的治疗效果略好于Cal-7。
实施例3：毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对干燥综合征慢性期的治疗作用
在本实施例中，使用的药物与实施例2中相同，此处不再赘述。
在本实施例中，将小鼠分成4各组，分别为对照组，即不进行任何处理的小鼠组；模型组，即进行ESS诱变处理，但不治疗的小鼠组；Cal-7治疗组， 即进行ESS诱变10周后，使用Cal-7对其治疗的小鼠组；以及Caly治疗组，即进行ESS诱变10周后，使用Caly对其治疗的小鼠组。在本实施例中，每个组有6只年龄相近的小鼠，其年龄均为40周左右。
在本实施例中，将含有Caly或Cal-7任一成分的药物制成口服的液体试剂。干燥综合征慢性期给药治疗于ESS模型建立后10周的小鼠，治疗期间每2日给药一次，每次给药量为5mg/kg。连续治疗10周，后检测小鼠腺体病理学指标，结果如图2C所示。
如图2C所示，通过在ESS小鼠疾病慢性期给药，申请人发现Cal-7和Caly均能显著降低ESS小鼠的唾液腺病理损伤，减少其腺体内组织浸润(C)。值得注意的是，Caly的治疗效果略好于Cal-7。
实施例4：毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对干燥综合征的预防作用
图3为根据本发明的一个实施例的毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对小鼠干燥综合征的预防作用。其中，左侧图指示给药时间及对小鼠免疫诱导时间：对小鼠免疫诱导前3天，分别对2组实验组小鼠给予Caly和Cal-7，对对照组小鼠给予等计量PBS(图中未示出)，免疫诱导21天后搜集实验数据；中间附图为小鼠免疫诱导模型建立后，不同给药组小鼠的唾液分泌流速，其中圆点(●)表示给予PBS的对照组小鼠，方形(■)表示给予Cal-7的实验组小鼠，三角形(▲)表示给予Caly的实验组小鼠；横坐标表示免疫诱导小鼠模型建立的时间(0天和21天)，纵坐标表示小鼠唾液分泌流速(μL/15min)；右侧图为不同组小鼠抗SSA抗体IgG(anti-SSA IgG)水平；其中，横坐标表示3个不同的小鼠分组，左侧(蓝色)为给予PBS的对照组小鼠，中间(黄色)为给予Cal-7的实验组小鼠，右侧(红色)为给予Caly的实验组小鼠；纵坐标表示抗SSA抗体IgG的OD值(450nm)
如图3所示，在本实施例中，在建立免疫诱导小鼠(ESS)前3天，对小鼠进行给药处理，其中，申请人对小鼠的预防性给药Caly和Cal-7均为5mg/kg， 以观察毛蕊异黄酮及毛蕊异黄酮葡萄糖苷对干燥综合征的预防作用。
实验结果表明，在免疫诱导0和21天检测唾液分泌流速发现Caly和Cal-7均能有效保护腺体功能预防疾病发展，而外周血抗体水平(抗SSA抗体IgG)亦显著性低于ESS模型组(n＝6)。
实施例5
1.方法_
1.1小鼠中ESS诱导
在8周大的雌性小鼠中诱导了实验性SS(ESS)模型。简而言之，从正常小鼠的双侧唾液腺(SG)中提取的蛋白质在等体积的弗氏完全佐剂(Sigma-Aldrich)中乳化，浓度为2mg/mL。为了诱导疾病，在第0天，每只小鼠在背部皮下注射0.2mL乳液。在第14天，用弗氏不完全佐剂(Sigma-Aldrich)乳化的1mg/mL SG蛋白进行加强注射。
1.2外周自身免疫性脑脊髓炎(EAE)模型
对雌性C57BL/6小鼠进行免疫以主动诱导EAE[4]。简而言之，小鼠皮下注射含200μg MOG35-55的完全弗氏佐剂(5mg/ml，Sigma-Aldrich)。百日咳毒素(200ng，List Biological Laboratories)在免疫后第0天和第2天(dpi.)静脉内注射两次。每天用体重测量和临床评分评估免疫接种的小鼠。
临床评分标准
疾病严重程度评分如下：0，正常；1、尾巴瘫痪；2、后肢无力；3、后肢瘫痪；4、后肢麻痹和前肢麻痹或无力；5、奄奄一息/死亡。
1.3系统性红斑狼疮(SLE)模型
对于染色质免疫的狼疮模型，用从脾细胞中提取的染色质皮下免疫8周龄的雌性C57BL/6小鼠[6]。简而言之，核染色质样品是从用5ng/mL伴刀豆球蛋白A刺激72小时的脾细胞制备。在第一次免疫后第14天和第28天，用200μg在完全弗氏佐剂(CFA；InvivoGen，CA，USA)中乳化的染色质对小鼠 进行免疫，并用100μg在弗氏不完全佐剂(IFA；InvivoGen)中的染色质加强免疫。用在佐剂中乳化的PBS免疫的小鼠作为对照。
2.结果
图4显示毛蕊异黄酮(calycosin,Caly)显着抑制Th17和T滤泡辅助细胞反应。
在抗CD3和抗CD28 mAb预包被3天的培养板中，在存在或不存在毛蕊异黄酮(Calycosin，Caly)、刺芒柄花素(Formonetin，For)、毛蕊异黄酮-7-O-葡萄糖苷(Calyconsin-7-O-glucoside，Cal-7)和芒柄花苷(Ononin，Ono)(20μM)的情况下，在T滤泡辅助细胞极化条件(重组IL-6、IL-21和抗IFN-γ/IL-4抗体)下从正常C57BL/6小鼠中纯化鼠CD4+T细胞。流式细胞术检测Caly和Cal-7显着抑制Tfh细胞分化，虽然Ono和For与其在结构上相似。Caly以前曾被报道为Cal-7的代谢物。通过与T细胞一起培养24小时来测量Caly在抑制IL-17A和IL-21中的IC50(半最大抑制浓度)，通过定量聚合酶链式反应(qPCR)分别检测到IC50为8.65μM和10.65μM。
图5显示Caly对干燥综合征小鼠模型的治疗作用。
用源自唾液腺的抗原对C57BL/6小鼠进行免疫以诱导实验性干燥综合征(ESS)，这是一种建立的小鼠模型以概括人类原发性干燥综合征[1]。根据诊断标准，疾病病理学的主要特征包括唾液功能减退(通过测量唾液分泌能力检查)，血清抗SSA IgG水平升高(通过ELISA检查)和唾液腺组织病理学变化(通过H&E染色检查)，正如淋巴细胞浸润所揭示的，在ESS小鼠中可以明显观察到。Caly(5mg/kg)的治疗显着改善了小鼠的疾病病理学。之前的研究首先确定了T效应细胞亚群(称为产生IL-17的Th17细胞和PD-1+ICOS+T滤泡辅助(Tfh)细胞)在ESS发病机制中不可或缺的作用(通过流式细胞术检查)在Caly治疗后也显着降低[1-3]。
由于Th17细胞在多发性硬化症的发病机制中起关键作用，而Tfh细胞介 导的体液自身免疫在系统性红斑狼疮中至关重要，接下来举例说明Caly在治疗这些小鼠模型中的治疗效果。
图6显示Caly对多发性硬化小鼠模型的治疗作用。
对雌性C57BL/6小鼠进行免疫以主动诱导实验性自身免疫性脑脊髓炎(EAE)，一种人类多发性硬化症的小鼠模型[4]。在疾病诱导后，用PBS或Caly(5mg/kg)治疗小鼠直至EAE进展的高峰时间(16天)。Caly治疗改善了EAE小鼠每日和累积的体重减轻并降低了临床评分[5]。在组织学研究中，H&E分析表明，PBS组的EAE小鼠在腰脊髓病变的白质中具有特征性炎症灶，而Caly治疗的EAE小鼠的炎症浸润显着减少且渗透分数较低。还通过LFB染色(蓝色)检查脱髓鞘。在Caly治疗的小鼠的脊髓损伤中观察到比PBS组EAE小鼠更小的脱髓鞘区域，通过较低的脱髓鞘分数来量化和呈现。
图7显示Caly对系统性红斑狼疮小鼠模型的治疗作用。
针对实验性系统性红斑狼疮，用凋亡染色质免疫C57BL/6小鼠[6]。在疾病诱导后，用PBS或Caly(5mg/kg)处理小鼠8周。狼疮小鼠清楚地显示抗dsDNA IgG自身抗体的血清水平增加(通过ELISA检查)。这通过严重的肾脏炎症例证，因为在狼疮小鼠的肾脏中观察到显着的IgG沉积和CD45+淋巴细胞浸润(通过共聚焦显微镜检查)。然而，Caly治疗显着改善了疾病病理学，显示抗dsDNA IgG的血清水平较低，淋巴细胞浸润较温和，肾脏中IgG沉积减少。
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2.Fu,W.,et al.,Deficiency in T follicular regulatory cells promotes autoimmunity.J Exp Med,2018.215(3):p.815-825.
3.Lin,X.,et al.,IL-10-producing regulatory B cells restrain the T follicular helper cell response in primary Sjogren's syndrome.Cell Mol Immunol,2019.16(12):p.921-931.
4.Li,W.,et al.,Acteoside ameliorates experimental autoimmune encephalomyelitis through inhibiting peroxynitrite-mediated mitophagy activation.Free Radic Biol Med,2020.146:p.79-91.
5.Stromnes,I.M.and J.M.Goverman,Active induction of experimental allergic encephalomyelitis.Nat Protoc,2006.1(4):p.1810-9.
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上述实施例仅供说明本发明之用，而并非是对本发明的限制，有关技术领域的普通技术人员，在不脱离本发明范围的情况下，还可以做出各种变化和变型，因此，所有等同的技术方案也应属于本发明公开的范畴。

Claims (15)

  1. 一种用于预防、治疗或缓解干燥综合征的药物组合物，其中，所述组合物中包含药物学上的有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  2. 一种用于预防、治疗或缓解患者中自身免疫性疾病的药物组合物，其中，所述组合物包含药物学上的有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  3. 根据权利要求2所述的组合物，其中，所述自身免疫性疾病选自多发性硬化症、系统性红斑狼疮、干燥综合征、IgG4相关性疾病或系统性炎症伴纤维化疾病(systemic fibroinflammatory disease)。
  4. 根据权利要求1-3中任一项所述的组合物，所述组合物包括液体口服试剂、注射试剂、胶囊、片剂、粉末、水丸、膏药。
  5. 一种用于预防、治疗或缓解干燥综合征的组合物的使用方法，包括：每千克体重每次给予含有0.2-10mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物。
  6. 根据权利要求5所述的方法，进一步包括：每千克体重(人)每次给予含有0.2mg毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的所述组合物。
  7. 根据权利要求5-6任一所述的方法，进一步包括，每2日给予1次所述组合物。
  8. 一种预防、治疗或缓解患者中自身免疫性疾病的方法，包括：向所述患者给予治疗有效量的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  9. 根据权利要求8所述的方法，其中，所述自身免疫性疾病选自多发性硬化症、系统性红斑狼疮、干燥综合征、IgG4相关性疾病或系统性炎症伴纤维化疾病(systemic fibroinflammatory disease)。
  10. 根据权利要求8或9所述的方法，包括：向所述患者给予每千克体重0.2-10mg的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  11. 根据权利要求8或9所述的方法，包括：向所述患者(人)给予每千克体重0.2mg的毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  12. 根据权利要求8或9所述的方法，包括：向所述患者每2日给予1次毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷。
  13. 一种包含毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的组合物在制备预防、缓解、治疗干燥综合征的药物方面的应用，其中，所述组合物中毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷的含量为药物学上的有效量。
  14. 毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷在制备用于在患者中预防、缓解、治疗自身免疫性疾病的药物中的应用。
  15. 根据权利要求14所述的应用，其中，所述自身免疫性疾病选自多发性硬化症、系统性红斑狼疮、干燥综合征、IgG4相关性疾病或系统性炎症伴纤维化疾病(systemic fibroinflammatory disease)。
PCT/CN2022/086850 2021-04-15 2022-04-14 毛蕊异黄酮和/或毛蕊异黄酮葡萄糖苷治疗自身免疫性疾病的用途 WO2022218381A1 (zh)

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