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WO2024179462A1 - Utilisation de bacteroides fragilis et de polysaccharide a capsulaire extrait de celui-ci dans la préparation d'un produit pour la prévention et le traitement de l'arthrite auto-immune - Google Patents

Utilisation de bacteroides fragilis et de polysaccharide a capsulaire extrait de celui-ci dans la préparation d'un produit pour la prévention et le traitement de l'arthrite auto-immune Download PDF

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Publication number
WO2024179462A1
WO2024179462A1 PCT/CN2024/078796 CN2024078796W WO2024179462A1 WO 2024179462 A1 WO2024179462 A1 WO 2024179462A1 CN 2024078796 W CN2024078796 W CN 2024078796W WO 2024179462 A1 WO2024179462 A1 WO 2024179462A1
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bacteroides fragilis
arthritis
rheumatoid arthritis
food
capsular polysaccharide
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Chinese (zh)
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郑丽君
蔡琰
王晔
李平
吴嘉棋
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Guangzhou Zhiyi Biotechnology Co Ltd
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Guangzhou Zhiyi Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of biomedicine, and specifically to the use of Bacteroides fragilis and zwitterionic capsular polysaccharides thereof in the preparation of drugs for treating autoimmune arthritis.
  • RA rheumatoid arthritis
  • synovial hyperplasia synovitis
  • bone and cartilage erosion in the joints, leading to joint swelling, symmetrical pain and stiffness, and ultimately joint deformity.
  • the patient's mobility is restricted and it is one of the main diseases causing loss of labor force and disability. Those with severe illness can shorten their life expectancy by 10-15 years, seriously damaging their quality of life.
  • the treatment drugs for autoimmune arthritis are mainly divided into four categories, namely glucocorticoids, nonsteroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs and biologics.
  • Glucocorticoids and nonsteroidal anti-inflammatory drugs are mainly used to control pain and inflammation.
  • the former mainly alleviates the disease by inhibiting inflammation, and the latter inhibits cyclooxygenase.
  • Disease-modifying anti-rheumatic drugs are commonly used drugs for the treatment of RA and are often used for new patients, including hydroxychloroquine, methotrexate, leflunomide and sulfasalazine.
  • DMARDs can inhibit the proliferation of immune cells, reduce the deposition of harmful factors in joints, reduce glutathione levels to reduce aerobic damage, and promote extracellular adenosine levels.
  • Biologics are a class of targeted inhibitory drugs for immune responses and/or cytokines, including TNF ⁇ inhibitors such as etanercept, infliximab and adalimumab, IL-1 ⁇ inhibitors such as anakinra and abatacept, and CD20 monoclonal antibodies such as rituximab.
  • TNF ⁇ inhibitors such as etanercept, infliximab and adalimumab
  • IL-1 ⁇ inhibitors such as anakinra and abatacept
  • CD20 monoclonal antibodies such as rituximab.
  • Non-steroidal anti-inflammatory drugs may cause blood system involvement, manifested as leukopenia and thrombocytopenia. Gastrointestinal lesions may also occur, such as gastric ulcers and gastrointestinal bleeding. Glucocorticoid drugs may have more serious side effects after long-term use, and long-term prescriptions are generally avoided as much as possible in clinical practice. Even short-term use can cause hormonal disorders in the human body, leading to obesity; it has an inhibitory effect on the immune system and causes infection; it is easy to cause adverse reactions such as gastrointestinal ulcers.
  • DMARDs are slow to take effect, and usually require more than one month of continuous medication to have a more obvious relief effect; adverse reactions include gastrointestinal discomfort, cardiovascular adverse reactions, infection, elevated liver transaminases, and interstitial pneumonia. Biological agents are more expensive, and adverse reactions include lung infection, herpes zoster, recurrence of tuberculosis, viral activity, and malignant tumors. In this context, finding a method or means to assist drug treatment has clinical application value.
  • RA immune abnormalities originate from other immune sites outside the joints, that is, the "mucosal origin hypothesis".
  • the mucosal immune system is an important part of the body's immune system. Mucosal-associated lymphoid tissues and mucosal immune cells form a relatively independent local immune system, including oral mucosa, lung mucosa, genital mucosa and intestinal mucosa. These mucosal sites play a barrier role.
  • the intestine is considered to be the largest immune organ, with a large number of microorganisms in the intestine. These microorganisms provide the body with necessary nutrients and energy, and also shape and "domesticate" the intestinal mucosal immune system. Intestinal microorganisms interact, respond and constrain the intestinal mucosa, including the activation or inhibition of the adaptive immune system and the induced immune system of mononuclear phagocytes. The process of mutual communication between the two is achieved through the intestinal mucosal barrier.
  • Rich intestinal microorganisms means that the intestine has the most antigenic substances, and changes in intestinal microorganisms can trigger changes in local and systemic immune responses.
  • molecular biology sequencing technology humans have a deep understanding of the correlation between intestinal microecology and human health. More and more studies have reported that the occurrence and development of autoimmune diseases such as RA are closely related to intestinal microecology.
  • Bacteroides fragilis (B. fragilis) is a Gram-negative, rod-shaped, darkly stained, capsule-free, non-spore-forming, non-motile obligate anaerobic bacterium. It is divided into enterotoxigenic (ETBF) and non-enterotoxigenic (NTBF). It is part of the normal intestinal flora of humans and animals, mainly found in the colon, and can also colonize and grow in the mucosa of the respiratory tract, gastrointestinal tract, and urogenital tract. The Bacteroides fragilis ZY-312 used in this application was sequenced and experimentally verified to be NTBF.
  • NTBF non-enterotoxigenic Bacteroides fragilis
  • PSA fragilis capsular polysaccharide A
  • Bacteroides fragilis is part of the normal intestinal flora of humans and animals, and its safety is more guaranteed.
  • the development of zwitterionic capsular polysaccharide A from Bacteroides fragilis extract for the treatment of autoimmune arthritis is promising and necessary.
  • the purpose of this application is to provide an application of zwitterionic capsular polysaccharide A of Bacteroides fragilis in the treatment of autoimmune arthritis.
  • PSA zwitterionic capsular polysaccharide A
  • 10685 can inhibit the secretion of TNF- ⁇ and IL-1 ⁇ , the core proinflammatory factors of rheumatoid arthritis immune response, by monocytes in vitro; in vivo, it can inhibit the inflammatory response by inhibiting the content of IL-1 ⁇ and IFN- ⁇ in serum, reduce the arthritis score and ankle thickness of the collagen-induced mouse arthritis model; reduce the arthritis score and paw solvent of the adjuvant-induced rat arthritis model. At the same time, it reduces the content of rheumatoid factor (RF) and C-reactive protein (CRP) in serum, alleviates the inflammatory response of the collagen antibody-induced arthritis mouse model, and the arthritis score and ankle thickness of each group of ZY-312 PSA are significantly reduced. It effectively relieves the symptoms of autoimmune arthritis and improves the quality of life of patients.
  • RF rheumatoid factor
  • CRP C-reactive protein
  • Bacteroides fragilis in the preparation of products for preventing and/or treating autoimmune arthritis, wherein the Bacteroides fragilis is Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685.
  • the Bacteroides fragilis is a live bacterium or a killed bacterium.
  • the Bacteroides fragilis is one or more of live Bacteroides fragilis, genetically recombinant, transformed or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis, Bacteroides fragilis lysate, and Bacteroides fragilis liquid culture supernatant.
  • the inactivation method includes one or more of thermal inactivation, pasteurization, dry heat inactivation, and short-wave ultraviolet (UVC) method.
  • thermal inactivation pasteurization
  • dry heat inactivation dry heat inactivation
  • UVC short-wave ultraviolet
  • the autoimmune arthritis is one or more of rheumatoid arthritis, inflammatory arthritis, juvenile idiopathic arthritis, osteoarthritis and psoriatic arthritis;
  • the rheumatoid arthritis may be collagen arthritis
  • the juvenile idiopathic arthritis is selected from oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis.
  • the rheumatoid arthritis is oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, or systemic rheumatoid arthritis.
  • the rheumatoid arthritis is collagen arthritis.
  • the medicine, food or health product is used to reduce joint swelling in rheumatoid arthritis.
  • the product is a food, a medicine or a health product.
  • the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.
  • the drug includes drugs for human use or drugs for animals.
  • the food comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereals.
  • the food can also be animal food, such as feed.
  • the second aspect of the present application provides a use of Bacteroides fragilis capsular polysaccharide A or a Bacteroides fragilis extract containing polysaccharide capsule A in the preparation of medicines, foods or health products for preventing and/or treating autoimmune arthritis.
  • the capsular polysaccharide A or the Bacteroides fragilis extract containing polysaccharide capsule A is extracted from Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685, Bacteroides fragilis NCTC 9343 or other non-enterotoxin-producing Bacteroides fragilis.
  • the content of capsular polysaccharide A lipid is less than 0.02 wt %.
  • the capsular polysaccharide A is a lipid-free capsular polysaccharide A
  • the structure of the capsular polysaccharide A is as follows:
  • the weight average molecular weight of the Bacteroides fragilis capsular polysaccharide A is 5-100kD; further preferably, the weight average molecular weight of the Bacteroides fragilis capsular polysaccharide A is 40-100kD; further preferably, the weight average molecular weight of the capsular polysaccharide A is 80-90kD; further preferably, the portion with a molecular weight distribution of 70KD-100KD accounts for 70-80% of the total.
  • the pharmaceutically effective in vivo dose of the Bacteroides fragilis capsular polysaccharide A is 5-45 mg/kg.
  • the effective concentration of the Bacteroides fragilis capsular polysaccharide A for inhibiting inflammatory factors in vitro is 0.1-10 ⁇ g/mL.
  • the autoimmune arthritis is one or more of rheumatoid arthritis, inflammatory arthritis, juvenile idiopathic arthritis, osteoarthritis and psoriatic arthritis;
  • the juvenile idiopathic arthritis is selected from oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis.
  • the rheumatoid arthritis is oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, or systemic rheumatoid arthritis.
  • the rheumatoid arthritis is collagen arthritis.
  • the medicine, food or health product is used to reduce joint swelling in rheumatoid arthritis.
  • the food is selected from any one of milk powder, cheese, curd, yogurt, ice cream, milk-based fermented food, and fermented cereal food; the food can also be animal food, such as feed, etc.; the food can also be baby food or pet food.
  • the medicament further comprises a pharmaceutically acceptable carrier.
  • the dosage form of the drug can be pills, tablets, granules, capsules, powders, suspensions, oral solutions or enema.
  • the drug can be administered orally, by enema or parenterally.
  • the drug can be administered intermittently, periodically, continuously or chronically.
  • the third aspect of the present application provides a composition for preventing and treating autoimmune arthritis, wherein the composition contains Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685 and/or a pharmaceutically effective dose of Bacteroides fragilis capsular polysaccharide A.
  • the capsular polysaccharide A is extracted from Bacteroides fragilis, Bacteroides fragilis with a deposit number of CGMCC No.10685. Bacteroides NCTC 9343 or other non-enterotoxigenic Bacteroides fragilis.
  • the capsular polysaccharide A is a lipid-free capsular polysaccharide A, or the lipid content of the capsular polysaccharide A is less than 0.02 wt %.
  • the weight average molecular weight of the capsular polysaccharide A is 5-100kD; further preferably, the weight average molecular weight of the Bacteroides fragilis capsular polysaccharide A is 40-100kD; further preferably, the weight average molecular weight of the capsular polysaccharide A is 80-90kD; further preferably, the portion with a molecular weight distribution of 70KD-100KD accounts for 70-80% of the total.
  • the pharmaceutically effective in vivo dose of the Bacteroides fragilis capsular polysaccharide A is 5-45 mg/kg.
  • the effective concentration of the Bacteroides fragilis capsular polysaccharide A for inhibiting inflammatory factors in vitro is 0.1-10 ⁇ g/mL.
  • the composition is a pharmaceutical composition, a health product composition or a probiotic composition.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in the form of pills, tablets, granules, capsules, powders, suspensions, oral solutions or enema. It may be administered orally, by enema or parenterally, and its administration cycle may be intermittent administration, periodic administration, continuous administration or long-term administration.
  • the method for preparing Bacteroides fragilis capsular polysaccharide A comprises the following steps:
  • step (1) comprises the following steps:
  • a single colony is selected and inoculated into plant-derived peptone broth for fermentation culture.
  • the obtained bacterial solution is centrifuged and precipitated, the supernatant is removed, and the precipitate is collected to obtain Bacteroides fragilis ZY-312 bacterial sludge; preferably, the fermentation culture conditions are 8 hours and 37°C, and/or the centrifugal precipitation conditions are a rotation speed of 3000r/min and centrifugation for 15min.
  • Bacteroides fragilis especially Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685 and its zwitterionic capsular polysaccharide A (PSA)
  • PSA zwitterionic capsular polysaccharide A
  • Non-steroidal anti-inflammatory drugs have relatively serious adverse reactions to the patient's blood system and digestive system, affecting the patient's compliance and thus affecting the treatment effect.
  • ZY-312 and its PSA not only effectively relieve the symptoms of autoimmune arthritis, but also have better safety than the current main treatment drugs for autoimmune arthritis, and are of great help in improving the quality of life of patients.
  • FIG1 is a colony characteristic diagram of Bacteroides fragilis ZY-312 of Example 1 of the present application.
  • FIG2 is a microscopic observation image of Bacteroides fragilis ZY-312 after Gram staining in Example 1 of the present application;
  • FIG3 is a nuclear magnetic resonance spectrometer analysis diagram of capsular polysaccharide A of Example 3 of the present application.
  • AE are respectively the 1H spectrum, 13C spectrum, COSY spectrum, HSQC spectrum of the capsular polysaccharide A of Example 3 of the present application analyzed by NMR spectrometer. spectrum, HMBC spectrum;
  • Figure 4 is the chemical structural formula of the structural unit of Bacteroides fragilis capsular polysaccharide A prepared in Example 3 of the present application.
  • the raw materials and reagents used in the following examples are commercially available. All cells were purchased from ATCC; all cell culture materials and trypsin were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; or they can be prepared by known methods.
  • the experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.
  • autoimmune arthritis includes, but is not limited to, one or more of rheumatoid arthritis, inflammatory arthritis, juvenile idiopathic arthritis, osteoarthritis, and psoriatic arthritis. In some embodiments, autoimmune arthritis may include other inflammatory conditions associated with arthritis. In some embodiments, juvenile idiopathic arthritis is selected from oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and systemic onset juvenile rheumatoid arthritis.
  • rheumatoid arthritis is oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, and systemic onset rheumatoid arthritis. In some embodiments, rheumatoid arthritis is collagen arthritis.
  • Bacteroides fragilis strain was streaked and inoculated on a blood plate and cultured anaerobically for 48 hours. The colony morphology, staining characteristics, size, club shape, and distribution were observed. Colony characteristics: After culturing Bacteroides fragilis ZY-312 on a blood plate for 48 hours, it was round, slightly convex, translucent, white, with a smooth surface, and non-hemolytic. The colony diameter was between 1-3 mm, see Figure 1.
  • Gram staining of Bacteroides fragilis ZY-312 showed that it was a Gram-negative bacterium with a typical rod shape, blunt and darkly stained ends, and a non-stained part in the middle of the bacteria that looked like a vacuole, see Figure 2.
  • Bacteroides fragilis strain was streaked and inoculated on a blood plate and cultured anaerobically for 48 hours. The colony morphology, staining characteristics, size, club shape, and distribution were observed. Colony characteristics: After culturing Bacteroides fragilis ZY-312 on a blood plate for 48 hours, it was round, slightly convex, translucent, white, with a smooth surface, and non-hemolytic. The colony diameter was between 1-3 mm, see Figure 1.
  • Gram staining of Bacteroides fragilis ZY-312 showed that it was a Gram-negative bacterium with a typical rod shape, blunt and darkly stained ends, and a non-stained part in the middle of the bacteria that looked like a vacuole, see Figure 2.
  • a single colony was taken from the culture medium and inoculated into a plant-derived peptone liquid culture medium for fermentation for 8 hours (temperature was 37°C) to obtain a live bacterial solution of Bacteroides fragilis.
  • the obtained bacterial solution was centrifuged at 3000 r/min for 15 min, the supernatant was removed, and the precipitate was collected to obtain bacterial slurries of Bacteroides fragilis ZY-312 and NCTC 9343, respectively.
  • step (3) adding excipients to the inactivated bacteria sludge collected in step (2) to make the total weight consistent with the weight of the bacteria solution before inactivation, stirring to completely dissolve, and obtaining an inactivated bacteria powder stock solution.
  • step (3) The inactivated bacterial powder stock solution obtained in step (3) is subjected to vacuum freeze drying, pre-frozen at -40 ⁇ 2°C for 1 to 3 hours, pre-frozen at -20 ⁇ 2°C for 0.5 to 1 hour, and finally pre-frozen at -40 ⁇ 2°C for another 0.5 to 2 hours, and subjected to primary drying (at -5 ⁇ 2°C and 0 ⁇ 2°C) and analytical drying (at 35 ⁇ 2°C) under a vacuum degree of 0.25 mbar to prepare inactivated bacterial powder, wherein the bacterial cell count of the bacterial powder reaches more than 1 ⁇ 10 11 Cells/g.
  • Example 1 (1) 50 g of the bacterial sludge prepared in Example 1 was taken, 300 g of purified water was added to resuspend the bacterial sludge, the pH was adjusted to 3.5 with 1 mol/L hydrochloric acid solution, extracted at 100° C. for 1.5 h, cooled to room temperature, centrifuged at 12000 g for 10 min at room temperature, and the supernatant was taken to obtain a crude sugar solution;
  • the prepared capsular polysaccharide A is degraded, and the degradation method includes but is not limited to chemical degradation, physical degradation and biodegradation.
  • an ultrasonic method is used to obtain capsular polysaccharide A with different molecular weights.
  • the ultrasonic method is to treat the capsular polysaccharide A at 195kHz and 20°C for 3 hours, 2 hours and 0 hours, respectively, and collect capsular polysaccharide A with molecular weights of 2kDa, 5kDa, 40kDa and original molecular weight, respectively. They are respectively recorded as: ZY-312 PSA_1, ZY-312 PSA_2, ZY-312 PSA_3 and ZY-312 PSA.
  • Example 4 Bacteroides fragilis capsular polysaccharide A inhibits LPS-induced upregulation of inflammatory factors in vitro
  • This example uses human monocytic THP-1 cell line for endotoxin tolerance test. Pretreatment with ZY-312 extract PSA of different molecular weights was performed for 24 hours. Subsequently, the cells were washed with fresh medium and 10 ng/mL of LPS (Sigma, L2886) was prepared to stimulate the cells for 24 hours. The cell culture supernatant was collected and the levels of cytokines TNF- ⁇ (R&D Systems, DTA00D) and cytokines IL-1 ⁇ (R&D Systems, QK201) in the supernatant were detected.
  • cytokines TNF- ⁇ R&D Systems, DTA00D
  • cytokines IL-1 ⁇ R&D Systems, QK201
  • THP-1 cells were grown in DMEM medium containing 10% FBS, 1% penicillin/streptomycin, and cultured in an incubator at 37°C, 5% CO 2 , and saturated humidity. The medium was changed every two days.
  • a) Take out the cell line from liquid nitrogen and thaw it quickly in a 37°C constant temperature water bath. Open the cryovial under sterile conditions, transfer the liquid to a 15mL centrifuge tube, resuspend the cells with 2-3mL of DMEM complete medium, and centrifuge at 1000rpm for 5min. After discarding the supernatant, add 5mL of DMEM complete medium, inoculate the cells into a culture flask, and culture in an incubator at 37°C and 5% CO2 for 48h.
  • ZY-312 extract PSA and products of different molecular weights were prepared.
  • ZY-312 PSA inhibits LPS-induced upregulation of inflammatory factors in THP-1 cells
  • THP-1 cells 10 6 cells
  • 2 mL of 10 ⁇ g/mL original molecular weight ZY-312 extract PSA ZY-312 PSA
  • the other three groups were pretreated for 24 h by adding 2 mL of 10 ⁇ g/mL ZY-312 PSA_1, ZY-312 PSA_2, and ZY-312 PSA_3.
  • the cells were then washed three times with fresh culture medium and stimulated with 10 ng/mL LPS for 24 h.
  • the cell culture supernatant was collected.
  • Cytokines play an important role in regulating the immune response of rheumatoid arthritis.
  • TNF- ⁇ and IL-1 ⁇ play a key role in synovitis and bone destruction. Both are important pro-inflammatory cytokines, which are positively correlated with the condition of rheumatoid arthritis to a certain extent, thus reflecting the degree of inflammatory activity. Inhibiting the amount of TNF- ⁇ and IL-1 ⁇ in the body can inhibit the symptoms of autoimmune arthritis.
  • the amount of inflammatory cytokines TNF- ⁇ and IL-1 ⁇ secreted by THP-1 cells in the model group was significantly upregulated.
  • Pretreatment with the original molecular weight PSA of Bacteroides fragilis ZY-312 can inhibit the secretion of TNF- ⁇ and IL-1 ⁇ by THP-1 cells under inflammatory conditions, and both are statistically significant compared with the model group.
  • the LPS tolerance experiment was performed using PSAs of different molecular weights, ZY-312 PSA_1, ZY-312 PSA_2, and ZY-312 PSA_3.
  • PSA with a molecular weight ⁇ 5kDa has the ability to tolerate LPS and significantly inhibits the secretion of TNF- ⁇ and IL-1 ⁇ compared with the model group, but compared with the original molecular weight PSA treatment group, the function of the small molecular weight ZY-312 PSA_2 and ZY-312 PSA_3 in inhibiting the secretion of TNF- ⁇ and IL-1 ⁇ induced by LPS is weaker than that of the original molecular weight ZY-312 PSA.
  • Bacteroides fragilis ZY-312 PSA can treat autoimmune arthritis by inhibiting the secretion of TNF- ⁇ and IL-1 ⁇ by monocytes, and the original molecular weight has the best effect.
  • mice SPF grade 6-week-old DBA/1 male mice were purchased. Animal quarantine and adaptive feeding were performed for 5 days, and group experiments were performed. Except for 10 animals in the normal group, the rest of the animals were used for model construction. Type II collagen (Chondrex, 190354) and complete Freund's adjuvant (Sigma, SLBR3877V) were mixed and emulsified in equal volumes, and 0.1 mL was injected intradermally at the base of the tail of each mouse to cause inflammation. On the 20th day, 0.1 mL of the emulsion was injected intraperitoneally as a provocative injection.
  • Type II collagen Choondrex, 190354
  • complete Freund's adjuvant Sigma, SLBR3877V
  • the modeling mice were randomly divided into a model group, a ZY-312 PSA low-, medium-, and high-dose group, and a celecoxib group (Shandong Qilu Pharmaceutical Co., Ltd.) according to the clinical score of arthritis.
  • the ankle thickness (mm) of the left and right hind legs of the mice was measured on the 32nd, 36th, 40th, 44th, and 46th days, and the clinical score of arthritis of the four paws of the mice was performed.
  • the mice in each group were killed and the serum was frozen in a -80°C refrigerator for the detection of cytokines, etc.
  • CFA complete Freund's adjuvant
  • mice were randomly divided into 5 groups according to the clinical score of arthritis on the 32nd day, with 10 mice in each group: model group, ZY-312 PSA low (10 mg/kg), medium (20 mg/kg), and high (30 mg/kg) dose groups, and celecoxib group (80 mg/kg).
  • Model group Normal saline 10mL/kg, once a day, continuous gavage for 33-46 days.
  • Capsular polysaccharide A (PSA) from Bacteroides fragilis extract was prepared with normal saline at 1, 2, and 3 mg/mL, respectively, and administered by oral gavage at 10 mL/kg, once a day, for 33-46 consecutive days.
  • Celecoxib group The positive control drug was celecoxib. 200 mg, twice a day, i.e. 400 mg/day, the dosage for mice was 80 mg/kg after conversion by body surface area. Celecoxib was prepared into a suspension of 8 mg/mL with normal saline and administered by gavage at 10 mL/kg, once a day.
  • the four paws of the mice were observed on days 32, 36, 40, 44, and 46, and clinical scores were performed according to the severity of arthritis.
  • the sub-score ranges from 0 to 3 points, and the total sum of the scores of the four paws of each mouse is the clinical score.
  • the scoring criteria are:
  • the ankle joint thickness (mm) of the left and right hind legs of the mice was measured with a vernier caliper with a precision of 0.01 mm on days 32, 36, 40, 44, and 46, and the average value was calculated.
  • the thickest part of the ankle joint and the most severely inflamed and edematous part were measured.
  • Elisa was used to detect the levels of IFN- ⁇ (R&D Systems, DY485) and IL-1 ⁇ (R&D Systems, MLB00C) cytokines in mouse serum.
  • the arthritis scores of the ZY-312 PSA groups were reduced, and there were statistical differences at 40, 44, and 46 days; the PSA low-dose group had statistical differences at 44 and 46 days.
  • the positive drug celecoxib can also treat collagen-induced arthritis in mice, but there is no significant difference with ZY-312 PSA. This shows that in the collagen-induced mouse arthritis model, ZY-312 PSA can effectively relieve inflammation and swelling in mouse joints.
  • the ankle thickness of the model group mice gradually increased from 32 to 46 days, reaching a peak at 44 days.
  • the Bacteroides fragilis ZY-312 PSA low-dose group had statistical differences at 44 and 46 days; the PSA medium and high-dose groups had statistical differences at 40, 44, and 46 days, and there was no significant difference in efficacy with the positive drug celecoxib.
  • Cytokine IL-1 ⁇ plays a key role in synovitis and bone destruction. It is positively correlated with the condition of rheumatoid arthritis to a certain extent, thus reflecting the degree of inflammatory activity. IFN- ⁇ is also closely related to the onset of rheumatoid arthritis. It is an interferon with a general immunomodulatory effect and a signature Th1-type cytokine. As shown in Table 4, the serum IL-1 ⁇ and IFN- ⁇ levels in the model group increased significantly.
  • capsular polysaccharide A from Bacteroides fragilis ZY-312 extract can effectively treat collagen-induced arthritis in mice.
  • Wistar rats were purchased and after passing the quarantine, 100 rats were randomly divided into 10 groups, with 10 rats in each group: normal group (normal saline), model group (normal saline), ZY-312 PSA low (5 mg/kg), medium (15 mg/kg), and high (45 mg/kg) dose groups, ZY-312 group (10 9 CFU/rat), inactivated ZY-312 group (10 9 Cells/rat), NCTC-9343 group (10 9 CFU/rat), NCTC-9343 PSA (45 mg/kg), and meloxicam group (Suzhou Wilson Pharmaceutical, 0.5 mg/kg). Except for the 10 animals in the normal group, the rest of the animals were used to construct the model.
  • the modeling reagent CFA was first prepared, and liquid paraffin and anhydrous lanolin were mixed in a ratio of 2:1 to form a 1 mL mixed solution. After high-pressure sterilization, attenuated BCG inactivated at 80°C for 1h was added. The CFA was fully emulsified and mixed to a final concentration of 10 g/L and injected subcutaneously into the hind feet of the rats at a dose of 0.1 mL/side/time. The rats were orally administered once a day at the designed dose from day 8 to day 27.
  • One hundred rats were randomly divided into 10 groups, with 10 rats in each group: normal group, model group, ZY-312 PSA low (5 mg/kg), medium (15 mg/kg), and high (45 mg/kg) dose groups, ZY-312 group (10 9 CFU/rat), inactivated ZY-312 group (10 9 Cells/rat), NCTC-9343 group (10 9 CFU/rat), NCTC-9343 PSA (45 mg/kg), and meloxicam group (0.5 mg/kg).
  • Model group Normal saline 10mL/kg, once a day, continuous gavage for 8-27 days.
  • PSA Bacteroides fragilis capsular polysaccharide A
  • ZY-312, inactivated ZY-312 and NCTC-9343 groups The three groups of Bacteroides fragilis were administered with normal saline to a concentration of 5 ⁇ 10 8 CFU/mL, 2 mL/animal by oral gavage, once a day, for 8-27 days.
  • NCTC-9343 PSA group Capsular polysaccharide A (PSA) extracted from the standard strain of Bacteroides fragilis was prepared with normal saline to 4.5 mg/mL and administered by oral gavage at 10 mL/kg, once a day for 8-27 consecutive days.
  • PSA Capsular polysaccharide A
  • Meloxicam group The positive control drug is meloxicam.
  • the dosage for rats is 0.5 mg/kg according to the body surface area conversion.
  • Meloxicam is prepared into a 0.05 mg/mL solution with normal saline and administered by gavage at 10 mL/kg, once a day.
  • the four paws of the rats were observed on the 8th, 14th, 20th and 26th days, and clinical scores were performed according to the severity of arthritis, with each paw scored 0-4 points, and the total score of the four paws of each rat was added up to form the clinical score.
  • the scoring criteria are:
  • the paw volume was measured by the water displacement method on the 8th, 14th, 20th, and 26th days. First, a circle was drawn about 1 cm above the ankle joint of the rat to make a mark, and a 10 mL glass graduated tube was filled with water. The rat's foot was vertically inserted into the water to the marked position and then removed. Then, a syringe filled with water was used to add water to the original water level. The remaining water in the syringe was recorded, and the difference was the amount of water discharged by the paw. Each measurement was performed by a dedicated person, and each paw was measured twice each time to take the average value to minimize the error.
  • Elisa was used to detect the levels of cytokines such as IFN- ⁇ (R&D Systems, RIF00) and IL-1 ⁇ (R&D Systems, RLB00) in rat serum.
  • cytokines such as IFN- ⁇ (R&D Systems, RIF00) and IL-1 ⁇ (R&D Systems, RLB00) in rat serum.
  • arthritis and paw volume of rats gradually increased from 8 to 27 days, reaching a peak at 20 days.
  • arthritis scores and paw volumes of the medium and high dose groups of ZY-312PSA decreased, and there were statistical differences at 14, 20, and 26 days; the low dose group of PSA, ZY-312 group, inactivated ZY-312 group, NCTC-9343 group and NCTC-9343 PSA had statistical differences at 20 and 26 days.
  • the positive drug meloxicam can also treat adjuvant-induced arthritis in rats, but there is no significant difference in efficacy with ZY-312 PSA.
  • the statistical results at 20 and 26 days showed that the arthritis scores and paw volume results of the ZY-312 live bacteria and inactivated bacteria administration groups were statistically different from those of the model group, and were better than those of the NCTC-9343 group.
  • the arthritis scores and paw volumes of the ZY-312 live bacteria group at 26 days were significantly lower than those of the NCTC-9343 group (P ⁇ 0.05); at the same dose, the therapeutic effect of the ZY-312 PSA administration group was better than that of the standard strain PSA, and there was a statistical difference at 20 days. This shows that in the collagen-induced rat arthritis model, ZY-312 PSA and live and inactivated bacteria can effectively relieve rat joint inflammation and swelling and reduce paw volume.
  • the P value between the two groups was calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P ⁇ 0.05; ** indicates extremely significant difference P ⁇ 0.01.
  • the P value between the ZY-312 PSA high and NCTC-9343 PSA groups was calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P ⁇ 0.05; the P value between the ZY-312 and NCTC-9343 groups was calculated according to the unpaired t-test (two-tailed) method, and @ indicates significant difference P ⁇ 0.05.
  • the P value between the two groups was calculated according to the unpaired t-test (two-tailed) method. * indicates significant difference P ⁇ 0.05; ** indicates extremely significant difference P ⁇ 0.01.
  • the P value between the ZY-312 PSA high and NCTC-9343 PSA groups was calculated according to the unpaired t-test (two-tailed) method. # indicates significant difference P ⁇ 0.05; the P value between the ZY-312 and NCTC-9343 groups was calculated according to the unpaired t-test (two-tailed) method, and @ indicates significant difference P ⁇ 0.05.
  • Cytokine IL-1 ⁇ plays a key role in synovitis and bone destruction. To a certain extent, it is positively correlated with the condition of rheumatoid arthritis, thus reflecting the degree of inflammatory activity. IFN- ⁇ is also closely related to the onset of rheumatoid arthritis. It is an interferon with a general immunomodulatory effect and a signature Th1-type cytokine. As shown in Table 7, the serum IL-1 ⁇ and IFN- ⁇ levels in the model group increased significantly.
  • the serum IL-1 ⁇ and IFN- ⁇ levels in the low, medium, and high dose groups of Bacteroides fragilis ZY-312 PSA, ZY-312 group, inactivated ZY-312 group, NCTC-9343 group, and NCTC-9343 PSA were statistically different.
  • the ZY-312 PSA administration group had a better effect on downregulating IL-1 ⁇ and IFN- ⁇ inflammatory factors than the standard strain PSA, and there was a statistical difference.
  • ZY-312 had better IL-1 ⁇ and IFN- ⁇ inhibitory effects than the NCTC-9343 group, and the difference was statistically significant.
  • Bacteroides fragilis ZY-312 and its extract capsular polysaccharide A can effectively treat adjuvant-induced arthritis in rats.
  • Example 7 Efficacy test of Bacteroides fragilis capsular polysaccharide A in treating collagen antibody-induced arthritis in mice
  • mice Seven-week-old male DBA/1 mice were purchased and passed the quarantine. Except for 10 normal group animals, the rest of the animals were used for model construction. On day 0, mice were intraperitoneally injected with 5 mg of anti-collagen antibody mixture (Chondrex), and on day 3, mice were intraperitoneally injected with 50 ⁇ g LPS. 60 mice were randomly divided into 6 groups, with 10 mice in each group: normal group (normal saline), model group (normal saline), ZY-312 PSA low (10 mg/kg), medium (20 mg/kg), and high (30 mg/kg) dose groups, and meloxicam group (0.5 mg/kg). From day 1 to day 15, the mice were orally administered once a day according to the designed dose.
  • normal group normal saline
  • model group normal saline
  • ZY-312 PSA low low (10 mg/kg
  • high (30 mg/kg) dose groups meloxicam group (0.5 mg/kg). From day 1 to day 15, the mice were orally administered once
  • mice 60 mice were randomly divided into 6 groups, with 10 mice in each group: normal group, model group, ZY-312 PSA low (10 mg/kg), medium (20 mg/kg), and high (30 mg/kg) dose groups, and meloxicam group (0.5 mg/kg).
  • Model group 10 mL/kg normal saline, once a day, continuous gavage for 1-15 days;
  • Capsular polysaccharide A (PSA) from Bacteroides fragilis extract was prepared with normal saline at 1, 2, and 3 mg/mL, respectively, and administered by oral gavage at 10 mL/kg, once a day, for 1-15 consecutive days.
  • Meloxicam group The positive control drug was meloxicam. The dosage for mice was 0.5 mg/kg according to the body surface area. Celecoxib was prepared into a 0.05 mg/mL suspension with normal saline and administered by gavage at 10 mL/kg, once a day, for 1-15 days.
  • the four paws of the mice were observed on the 7th, 11th and 15th days respectively, and clinical scores were performed according to the severity of arthritis, with each paw scored 0-4 points, and the total score of the four paws of each mouse was the clinical score.
  • the scoring criteria are:
  • the ankle thickness (mm) of the left and right hind legs of the mice was measured with a vernier caliper with a precision of 0.01 mm on the 7th, 11th and 15th day, and the average value was calculated.
  • the thickest part of the ankle joint and the most severely inflamed and edematous part were measured.
  • the mouse serum biochemical index detection procedure was selected, and the levels of rheumatoid factor (RF) and C-reactive protein (CRP) in serum were determined using a biochemical analyzer.
  • RF rheumatoid factor
  • CRP C-reactive protein
  • CRP and RF are the most commonly used biochemical indicators for clinical diagnosis and evaluation of rheumatoid arthritis.
  • the increase of CRP is positively correlated with the severity and activity of the disease.
  • Table 10 compared with the normal control group, the serum CRP and RF levels in the model group increased significantly.
  • the serum RF and CRP levels in the medium and high dose groups of Bacteroides fragilis ZY-312 PSA were reduced, and both were statistically significant.
  • the serum RF and CRP levels in the low dose group of PSA were reduced, and only RF was statistically significant compared with the model group.
  • capsular polysaccharide A of Bacteroides fragilis ZY-312 extract can effectively treat collagen antibody-induced arthritis in mice.

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Abstract

L'invention concerne l'utilisation de Bacteroides fragilis, d'un polysaccharide A capsulaire de Bacteroides fragilis ou d'un extrait de Bacteroides fragilis contenant un polysaccharide A capsulaire dans la préparation de médicaments, d'aliments et de produits de soins de santé pour prévenir et/ou traiter l'arthrite auto-immune. Le Bacteroides fragilis est Bacteroides fragilis ZY-312 ayant un numéro de conservation CGMCC NO. 10685, et le polysaccharide A capsulaire ou l'extrait de Bacteroides fragilis contenant le polysaccharide A capsulaire est extrait du Bacteroides fragilis ZY-312 ayant le numéro de conservation CGMCC NO. 10685, de Bacteroides fragilis NCTC 9343 ou d'un autre Bacteroides fragilis non entérotoxinogène.
PCT/CN2024/078796 2023-02-27 2024-02-27 Utilisation de bacteroides fragilis et de polysaccharide a capsulaire extrait de celui-ci dans la préparation d'un produit pour la prévention et le traitement de l'arthrite auto-immune Ceased WO2024179462A1 (fr)

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