WO2023155938A2 - 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用 - Google Patents

脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用 Download PDF

Info

Publication number
WO2023155938A2
WO2023155938A2 PCT/CN2023/087988 CN2023087988W WO2023155938A2 WO 2023155938 A2 WO2023155938 A2 WO 2023155938A2 CN 2023087988 W CN2023087988 W CN 2023087988W WO 2023155938 A2 WO2023155938 A2 WO 2023155938A2
Authority
WO
WIPO (PCT)
Prior art keywords
capsular polysaccharide
acid
bacteroides fragilis
sodium
drug
Prior art date
Application number
PCT/CN2023/087988
Other languages
English (en)
French (fr)
Other versions
WO2023155938A3 (zh
Inventor
王薇
刘洋洋
王晔
智发朝
Original Assignee
广州知易生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州知易生物科技有限公司 filed Critical 广州知易生物科技有限公司
Publication of WO2023155938A2 publication Critical patent/WO2023155938A2/zh
Publication of WO2023155938A3 publication Critical patent/WO2023155938A3/zh

Links

Classifications

    • 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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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 invention relates to the technical field of biopharmaceuticals, in particular to the application of a capsular polysaccharide extract of Bacteroides fragilis in the preparation of drugs for preventing and treating Alzheimer's disease.
  • AD Alzheimer's disease
  • this neurological disease “Alzheimer's disease”. sick”.
  • a ⁇ ⁇ -amyloid
  • Plaque senile plaques
  • NFTs neurofibrillary tangles
  • NFTs neuronal Lost, accompanied by gliosis.
  • the pathogenesis and treatment of AD are still a worldwide problem.
  • AD Alzheimer's disease
  • the US FDA has approved six drugs for the treatment of AD, rivastigmine, galantamine, donepezil, memantine, memantine combined with donepezil and tacrine. Except for memantine, these drugs improve symptoms by temporarily increasing neurotransmitters in the brain, and memantine prevents excessive activation of NMDA (N-methyl-D-aspartic acid receptor) receptors in the brain from stimulating damage to nerve cells. Mannlute sodium, which was approved for marketing in China in 2019, can improve cognitive impairment by reshaping the balance of intestinal flora, reducing the accumulation of peripheral related metabolites phenylalanine/isoleucine, reducing neuroinflammation in the brain. These drugs relieve AD symptoms but are not effective in preventing AD. Therefore, there is an urgent need to develop drugs that can prevent and treat AD clinically.
  • the preservation number of the Bacteroides fragilis is CGMCC NO. Extract.
  • the content of the capsular polysaccharide A in the capsular polysaccharide extract is greater than 95wt%.
  • the weight average molecular weight of the capsular polysaccharide A is 70KD-90KD.
  • the weight average molecular weight of the capsular polysaccharide A is 80KD-90KD.
  • the lipid content in the capsular polysaccharide A extract is lower than 0.02 wt%.
  • the lipid content is less than 0.01 wt%.
  • the medicine comprises the capsular polysaccharide extract and pharmaceutically acceptable excipients.
  • the auxiliary materials include diluents, wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexes
  • diluents wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexes
  • solvents solubilizers, co-solvents
  • emulsifiers emulsifiers
  • antioxidants emulsifiers
  • metal complexes One or more of mixtures, inert gases, preservatives, local analgesics, pH regulators and isotonic or isotonic regulators.
  • the diluent is selected from at least one of starches, sugars, celluloses and inorganic salts.
  • the wetting agent is selected from at least one of water and ethanol.
  • the binder is selected from starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol at least one of diols.
  • the disintegrating agent is selected from dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant and At least one of effervescent disintegrants.
  • the lubricant is selected from at least one of talcum powder, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micronized silica gel and polyethylene glycol.
  • the color and flavor regulator is selected from at least one of pigments, spices, sweeteners, mucilages and odorants.
  • the solvent is at least one selected from water, oil, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil and ethyl acetate.
  • the solubilizing agent is selected from at least one of Tweens, mezes, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates.
  • the co-solvent is at least one selected from organic acids and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone and glycerin.
  • the emulsifier is selected from the group consisting of spanners, tweens, marzes, benzyls, glycerin fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, and western At least one of tragacanth gum, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide and bentonite.
  • the suspending agent is selected from glycerin, syrup, acacia gum, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol and At least one of the gums.
  • the antioxidant is selected from at least one of sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and esters thereof.
  • the metal complexing agent is selected from disodium edetate and polycarboxylic acid compounds
  • the inert gas is selected from one of nitrogen and carbon dioxide.
  • the preservative is at least one selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils.
  • the local analgesic is selected from at least one of benzyl alcohol, chlorobutanol, lidocaine and procaine.
  • the pH regulator is selected from hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate and at least one of the citrates.
  • the isotonic or isotonic regulator is selected from at least one of glucose, sodium chloride, sodium citrate, sorbitol and xylitol.
  • the dosage form of the drug is tablet or granule.
  • the dosage form of the drug is pill or powder.
  • the dosage form of the drug is capsule or injection.
  • the dosage form of the drug is oral liquid or tube feeding preparation.
  • the drug is administered by injection or oral administration.
  • the drug also includes an acetylcholinesterase inhibitor and/or an n-methyl-d-aspartate receptor antagonist, and the capsular polysaccharide A and the acetylcholinesterase inhibitor And/or the n-methyl-d-aspartate receptor antagonists are used in combination.
  • the acetylcholinesterase inhibitor comprises one or more of doneprazil, galantamine and rivastigmine.
  • the n-methyl-d-aspartate receptor antagonist comprises memantine.
  • Fig. 1 is the SEC-HPLC analysis result of capsular polysaccharide of Bacteroides fragilis
  • Figure 2 shows the results of SEC-MALS analysis of the capsular polysaccharide of Bacteroides fragilis.
  • the percentage content involved in this application refers to mass percentage for solid-liquid mixing and solid-solid phase mixing, and refers to volume percentage for liquid-liquid phase mixing.
  • the percentage concentration involved in this application refers to the final concentration.
  • the final concentration refers to the proportion of the added component in the system after the component is added.
  • the temperature parameters in this application are allowed to be treated at a constant temperature, and also allowed to be treated within a certain temperature range.
  • the isothermal treatment allows the temperature to fluctuate within the precision of the instrument control.
  • the prevention and treatment mentioned in this application include prevention and treatment.
  • Bacteroides fragilis belongs to Bacteroides, Bacteroidaceae, Bacteroides genus, Gram-negative non-spore-forming strict anaerobic bacilli; it normally resides in the human intestinal tract, oral cavity, and respiratory tract, and has a negative impact on the host's physiological functions. have far-reaching effects.
  • the Bacteroides fragilis capsular polysaccharide A involved in this application is a capsular polysaccharide prepared from Bacteroides fragilis ZY-312, and this application proves that the Bacteroides fragilis ZY-312 capsular polysaccharide A can improve the learning and memory ability of AD model animals , which can be used in the preparation of anti-AD drugs.
  • the present application finds that the capsular polysaccharide A of Bacteroides fragilis ZY-312 can effectively prevent and treat Alzheimer's disease, and the efficacy has been verified in an animal model by Morris water maze behavior experiments.
  • the medicine for preventing and treating Alzheimer's disease prepared by using the extract of capsular polysaccharide A of Bacteroides fragilis ZY-312 also has the advantage of no toxic and side effects.
  • Bacteroides fragilis used in this application is Bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312), which was deposited in the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microorganisms on April 2, 2015, and its preservation number is CGMCC No. 10685, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
  • the strain is a non-enterotoxigenic Bacteroides fragilis.
  • the capsular polysaccharide A content in the capsular polysaccharide extract is greater than 95wt%.
  • the weight average molecular weight of the capsular polysaccharide A is 70KD-90KD.
  • the weight average molecular weight of the capsular polysaccharide A is 70KD-90KD.
  • the weight average molecular weight of capsular polysaccharide A of the present application is 70KD, 71KD, 72KD, 73KD, 74KD, 75KD, 76KD, 77KD, 78KD, 79KD, 80KD, 81KD, 82KD, 83KD, 84KD, 85KD, 86KD, 87KD, 88KD, 89KD, 90KD.
  • the weight average molecular weight of the capsular polysaccharide A of the present application is 80KD-90KD.
  • the ratio of the weight-average molecular weight to the number-average molecular weight (Mw/Mn) of the capsular polysaccharide A is 1.0-1.3.
  • the portion of the extract of capsular polysaccharide A with a weight average molecular weight distribution of 70KD-100KD accounts for 70%-80% of the total extract weight
  • the weight content of bound lipid in the extract of capsular polysaccharide A is less than 0.02%.
  • the preparation of the extract of capsular polysaccharide A may, including but not limited to, include the following steps:
  • Bacteroides fragilis fermentation broth is centrifuged to collect bacterial sediment (sludge);
  • the above preparation process is mainly summarized as follows: anaerobic fermentation of Bacteroides fragilis ⁇ centrifugal collection of bacterial sediment ⁇ suspension of bacterial sludge ⁇ acid extraction ⁇ centrifugation to collect supernatant ⁇ ultrafiltration to remove small molecular impurities ⁇ ion exchange one-step chromatography ⁇ lyophilization process preparation .
  • the process is simpler, environmentally friendly, and the capsular polysaccharide has high purity and high yield.
  • the prepared capsular polysaccharide A can meet the following requirements: residual protein ⁇ 1%; 90KD, Mw/Mn ⁇ 1.3.
  • the medicine comprises the capsular polysaccharide extract and pharmaceutically acceptable excipients.
  • auxiliary materials are, for example, diluents, wetting agents, binders, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexes Agents, inert gases, preservatives, local analgesics, pH regulators, isotonic or isotonic regulators, etc., may contain one type, or two or more types.
  • the auxiliary material can be selected from, including but not limited to, as follows: the diluent is starch, sugar, cellulose and inorganic salts; the wetting agent is selected from at least one of water and ethanol; the The binding agent is selected from at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone and polyethylene glycol; the disintegrant is selected from dry starch, sodium carboxymethyl starch, low Substitute at least one of hydroxypropyl cellulose, croscarmellose sodium, crospovidone, surfactant and effervescent disintegrant; the lubricant is selected from talcum powder, calcium stearate At least one of , magnesium stearate, magnesium lauryl sulfate, micropowder silica gel and polyethylene glycol; the color and flavor regulator is selected from pigments, spices, sweeteners, glue and odorants at least one of; the solvent is selected from at least one of water, oil, ethanol
  • the drug can be prepared into appropriate dosage forms according to clinical needs, such as tablets, granules, pills, powders, capsules, injections, oral liquids, tube feeding preparations and the like.
  • the drug can be administered through an appropriate route of administration according to clinical needs, such as injection administration, oral administration and the like.
  • the drug also includes an acetylcholinesterase inhibitor and/or an n-methyl-d-aspartate receptor antagonist, and the capsular polysaccharide A is combined with the acetylcholinesterase inhibitor and /or the n-methyl-d-aspartate receptor antagonists are used in combination.
  • the acetylcholinesterase inhibitor comprises one or more of doneprazil, galantamine and rivastigmine.
  • the n-methyl-d-aspartate receptor antagonist comprises memantine.
  • Bacteroides fragilis fermentation broth was centrifuged to collect the sludge.
  • Table 1 shows the comparison results of B. fragilis capsular polysaccharide obtained by the two preparation methods.
  • S1-PSA S2-PSA
  • S3-PSA S4-PSA
  • Grouping of animals 90 SD rats, half male and half female, were divided into 9 groups according to the random number table method, each group had 5 male and female rats, respectively sham operation group, model group, S1-PSA low-, medium-, and high-dose groups, S3-PSA low, medium and high dose groups, donepezil hydrochloride group (positive drug group), see Table 2 for details.
  • Rats in 9 groups were anesthetized by intraperitoneal injection of 10% chloral hydrate, the local hair on the top of the brain was shaved, and the flat head of the rat was fixed on the brain stereotaxic instrument.
  • the stereotaxic atlas of the rat brain make a 2.0 mm incision on the left and right sides of the midline of the brain, and 3.0 mm behind the bregma on both sides with no Bacteria dental drill drilling, drilling depth of 3mm, to avoid intracranial hemorrhage.
  • rats in each group were slowly injected with 5 ⁇ L of condensed A ⁇ 1-40 ( ⁇ -amyloid, 1 ⁇ L/min), with a needle depth of 4.0 mm.
  • the sham operation group was injected with an equal volume of normal saline solution, and the needle was kept for 5 minutes after the injection, after which the wound was sutured, and amikacin was sprayed on the wound to prevent wound infection. Routine feeding for 1 week after operation. One week later, all rats were subjected to the water maze test. Compared with the sham operation group, the escape latency of the modeled rats was longer and significantly different, and the model was determined to be successful, and finally all the models were successfully built.
  • the amount of the experimental drug is converted according to the ratio of body surface area, and the S1-PSA high, medium and low dose groups and the S3-PSA high, medium and low dose groups are respectively configured for oral administration at 5 mg/kg, 3 mg/kg and 1 mg/kg.
  • the donepezil hydrochloride group the donepezil hydrochloride tablets were ground into powder, made into a suspension and administered orally at 1.02 mg ⁇ kg -1 , equivalent to 6 times the daily dose for adults.
  • Rats in the sham operation group and the model group were intragastrically administered with normal saline daily.
  • the volume of intragastric administration in each group was 10mL/Kg, and the intragastric administration was continued for 10 weeks. See Table 2 for details.
  • Morris water maze is an experimental method designed by the British psychologist Morris in the early 1980s (1981) and applied to the study of brain learning and memory mechanisms. It is widely used in AD research.
  • the test program mainly includes two parts: positioning navigation test and space exploration test. Among them, the positioning navigation test lasted for several days, and the rats were put into the water several times from 4 water entry points facing the pool wall every day, and the time it took to find the platform hidden under the water surface (escape latency period) was recorded. Generally, the more severe AD, the longer the escape latency of rats.
  • the space exploration test is to remove the platform after the navigation test, and then choose a water entry point to put the rat into the pool, record its swimming track within a certain period of time, and examine the memory of the rat on the original platform.
  • the more severe the AD the less times the rats cross the platform, the shorter the stay time in the effective area, and the shorter the movement distance in the effective area.
  • the Morris water maze behavior test after 10 weeks of continuous gavage, the Morris water maze behavior test was performed. The experiment continued for 5 days and was divided into positioning navigation and space exploration experiments. The positioning navigation experiment lasted for 4 days, and the time required for animals to find and climb up the platform from being released into the water was recorded, that is, the escape latency period. If no platform is found within 120s, the incubation period will be recorded as 120s. On the 5th day, the space search experiment was carried out, the original platform was removed, and the number of times the rats crossed the platform within 120 s, the residence time in the effective area (2 times the diameter of the platform) and the swimming distance in the effective area were recorded.
  • the escape latency of male and female rats in each administration group in the water maze shortened with the increase of training time and times.
  • the escape latency of the male and female rats in the model group was significantly prolonged (P ⁇ 0.01), indicating that the modeling was successful;
  • the escape latency was significantly shortened (P ⁇ 0.05, P ⁇ 0.01), indicating that capsular polysaccharide A of Bacteroides fragilis can improve AD symptoms in SD rats induced by A ⁇ .
  • capsular polysaccharide A of Bacteroides fragilis provided by the present application can prevent and treat AD.
  • Animal grouping 110 SD rats, half male and half female, were divided into 11 groups according to the random number table method, with 5 male and female rats in each group, respectively sham operation group, model group, S2-PSA medium dose group, and S2-PSA low dose group , medium and high dose combined donepezil group, S4-PSA medium dose group, S4-PSA low, medium and high dose donepezil group, donepezil hydrochloride group (positive drug group), see Table 5 for details.
  • AD model preparation 11 groups of rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the local hair on the top of the brain was shaved off. Make a longitudinal incision with a scalpel at the midline of the scalp, about 1.5 cm in length, to fully expose the bregma. According to the stereotaxic atlas of the rat brain, make a 2.0 mm incision on the left and right sides of the midline of the brain, and 3.0 mm behind the bregma on both sides with no Bacteria dental drill drilling, drilling depth of 3mm, to avoid intracranial hemorrhage.
  • rats in each group were slowly injected with 5 ⁇ L of condensed A ⁇ 1-40 ( ⁇ -amyloid, 1 ⁇ L/min), with a needle depth of 4.0 mm.
  • the sham operation group was injected with an equal volume of normal saline solution, and the needle was kept for 5 minutes after the injection, after which the wound was sutured, and amikacin was sprayed on the wound to prevent wound infection. Routine feeding for 1 week after operation. One week later, all rats were subjected to the water maze test. Compared with the sham operation group, the escape latency of the modeled rats was longer and significantly different, and the model was determined to be successful, and finally all the models were successfully built.
  • the amount of the experimental drug is converted according to the ratio of body surface area, S2-PSA medium dose group, S2-PSA high, medium and low dose combined with donepezil group, S4-PSA medium dose group, S4-PSA high, medium and low dose combined with donepezil Group, high, middle and low doses of PSA were configured to be administered orally at 5 mg/kg, 3 mg/kg, and 1 mg/kg, respectively.
  • the donepezil hydrochloride group the donepezil hydrochloride tablets were ground into powder, made into a suspension and administered orally at 1.02 mg ⁇ kg -1 , equivalent to 6 times the daily dose for adults.
  • Rats in the sham operation group and the model group were intragastrically administered with normal saline daily.
  • the volume of intragastric administration in each group was 10mL/Kg, and the intragastric administration was continued for 10 weeks. See Table 5 for details.
  • the test program mainly includes two parts: positioning navigation test and space exploration test.
  • the positioning navigation test lasted for several days. Every day, the rats were put into the water from 4 water entry points facing the pool wall for several times, and it was recorded that they found the fish hidden on the water surface. Time to get off the platform (escape latency).
  • the space exploration test is to remove the platform after the navigation test, and then choose a water entry point to put the rat into the pool, record its swimming track within a certain period of time, and examine the memory of the rat on the original platform.
  • the more severe the AD the less times the rats cross the platform, the shorter the stay time in the effective area, and the shorter the movement distance in the effective area.
  • the Morris water maze behavior test after 10 weeks of continuous gavage, the Morris water maze behavior test was performed. The experiment continued for 5 days and was divided into positioning navigation and space exploration experiments. The positioning navigation experiment lasted for 4 days, and the time required for animals to find and climb up the platform from being released into the water was recorded, that is, the escape latency period. If no platform is found within 120s, the incubation period will be recorded as 120s. On the 5th day, the space search experiment was carried out, the original platform was removed, and the number of times the rats crossed the platform within 120 s, the residence time in the effective area (2 times the diameter of the platform) and the swimming distance in the effective area were recorded.
  • the escape latency of male and female rats in each administration group shortened with the increase of training time and times in the water maze.
  • the escape latency of the male and female rats in the model group was significantly prolonged (P ⁇ 0.05, P ⁇ 0.01), indicating that the modeling was successful;
  • the escape latency was significantly shortened (P ⁇ 0.01), and the combination group had a shorter escape latency than the single group (P ⁇ 0.001).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本申请涉及一种脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC NO.10685，所述荚膜多糖提取物包含荚膜多糖A。

Description

脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用
相关申请
本申请要求2022年02月16日申请的，申请号为2022101413434，名称为“脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本发明涉及生物制药技术领域，特别涉及一种脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用。
背景技术
阿尔茨海默病(Alzheimer’s disease，AD)又称老年痴呆，是一种神经退行性脑部疾病。1906年Alois Alzheimer博士利用脑切片染色法发现老年痴呆患者脑内神经纤维缠结，伴有神经元细胞退化，并且大脑皮层出现很多斑块，于是将这种神经系统疾病命名为“阿尔茨海默病”。随着现代医学的发展，人们发现AD特征性病理变化：大脑β淀粉样蛋白(β-amyloid，Aβ)沉积形成老年斑(Plaque)，Tau蛋白过度磷酸化造成神经纤维缠结(NFTs)以及神经元丢失，并伴随胶质细胞增生。然而，AD的发病机理以及治疗依然是世界难题。
AD发病率呈逐年上升趋势，成为仅次于心血管病和癌症的高发性疾病。每三秒就有一个人患上痴呆症，目前痴呆症的年成本估计为1万亿美元，到2030年这一数字将翻一番，这对全球来说是极重的医疗负担。然而，目前关于AD药物的研发几乎没有任何突破性进展。由于AD临床表现为记忆能力、定向能力、思维和判断能力减退，以及日常生活能力降低，甚至出现异常精神行为症状等，使患者护理难度较大，给社会和家庭带来沉重负担。目前世界上没有一种药物能够减缓或停止AD导致的神经元损伤。美国FDA批准了六种治疗AD的药物，卡巴拉汀、加兰他敏、多奈哌齐、美金刚、美金刚联合多奈哌齐和他克林。除了美金刚外，这些药物都是通过暂时增加大脑中神经递质来改善症状，美金刚则阻止大脑中NMDA(N-methyl-D-aspartic acid receptor)受体过度激活对神经细胞的刺激损伤。2019年在中国获批上市的甘露特钠通过重塑肠道菌群平衡、降低外周相关代谢产物苯丙氨酸/异亮氨酸的积累，减轻脑内神经炎症，进而改善认知障碍。这些药物能缓解AD症状，但不能有效预防AD。因此，临床上迫切需要研发对AD具有预防和治疗作用的药物。
发明内容
基于此，根据本申请的各种实施例，提供一种脆弱拟杆菌的荚膜多糖A提取物的新应用，技术方案为：
脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC NO.10685，所述荚膜多糖提取物为荚膜多糖A的提取物。
在其中一些实施例中，所述荚膜多糖提取物中所述荚膜多糖A的含量大于95wt％。
在其中一些实施例中，所述荚膜多糖A的重均分子量为70KD-90KD。
在其中一些实施例中，所述荚膜多糖A的重均分子量为80KD-90KD。
在其中一些实施例中，所述荚膜多糖A的提取物中脂质含量低于0.02wt％。
在其中一些实施例中，所述脂质含量低于0.01wt％。
在其中一些实施例中，所述药物包含所述荚膜多糖提取物和药学上可以接受的辅料。
在其中一些实施例中，所述辅料包含稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂和等渗或等张调节剂中的一种或者多种。
在其中一些实施例中，所述稀释剂选自淀粉类、糖类、纤维素类以及无机盐类中的至少一种。
在其中一些实施例中，所述润湿剂选自水以及乙醇中的至少一种。
在其中一些实施例中，所述黏合剂选自淀粉浆、糊精、糖、纤维素衍生物、明胶、聚维酮以及聚乙 二醇中的至少一种。
在其中一些实施例中，所述崩解剂选自干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联羧甲基纤维素钠、交联聚维酮、表面活性剂以及泡腾崩解剂中的至少一种。
在其中一些实施例中，所述润滑剂选自滑石粉、硬脂酸钙、硬脂酸镁、十二烷基硫酸镁、微粉硅胶以及聚乙二醇中的至少一种。
在其中一些实施例中，所述色香味调节剂选自色素、香料、甜味剂、胶浆剂以及矫臭剂中的至少一种。
在其中一些实施例中，所述溶剂选自水、油、乙醇、甘油、丙二醇、聚乙二醇、二甲基亚砜、液体石蜡、脂肪油以及乙酸乙酯中的至少一种。
在其中一些实施例中，所述增溶剂选自吐温类、卖泽类、聚氧乙烯脂肪醇醚类、肥皂类、硫酸化物、以及磺酸化物中的至少一种。
在其中一些实施例中，所述助溶剂选自有机酸及其盐类、酰胺及胺类化合物、无机盐、聚乙二醇、聚维酮以及甘油中的至少一种。
在其中一些实施例中，所述乳化剂选自司盘类、吐温类、卖泽类、苄泽类、甘油脂肪酸酯、高级脂肪酸盐、硫酸化物、磺酸化物、阿拉伯胶、西黄耆胶、明胶、果胶、磷脂、琼脂、海藻酸钠、氢氧化物、二氧化硅以及皂土中的至少一种。
在其中一些实施例中，所述助悬剂选自甘油、糖浆、阿拉伯胶、西黄耆胶、琼脂、海藻酸钠、纤维素衍生物、聚维酮、卡波普、聚乙烯醇以及触变胶中的至少一种。
在其中一些实施例中，所述抗氧剂选自亚硫酸盐、焦亚硫酸盐、亚硫酸氢盐、抗坏血酸、没食子酸及其酯类中的至少一种。
在其中一些实施例中，所述金属络合剂选自乙二胺四乙酸二钠以及多羧酸化合物中的一种
在其中一些实施例中，所述惰性气体选自氮气以及二氧化碳中的一种。
在其中一些实施例中，所述防腐剂选自尼泊金类、有机酸及其盐、季铵类化合物、醋酸氯己定、醇类、酚类以及挥发油中的至少一种。
在其中一些实施例中，所述局部止痛剂选自苯甲醇、三氯叔丁醇、利多卡因以及普鲁卡因中的至少一种。
在其中一些实施例中，所述pH调节剂选择盐酸、硫酸、磷酸、枸橼酸、酒石酸、醋酸、氢氧化钠、碳酸氢钠、乙二胺、葡甲胺、磷酸盐、醋酸盐以及枸橼酸盐中的至少一种。
在其中一些实施例中，所述等渗或等张调节剂选自葡萄糖、氯化钠、枸橼酸钠、山梨醇以及木糖醇中的至少一种。
在其中一些实施例中，所述药物的剂型为片剂或者颗粒剂。
在其中一些实施例中，所述药物的剂型为丸剂或者粉剂。
在其中一些实施例中，所述药物的剂型为胶囊剂或者注射剂。
在其中一些实施例中，所述药物的剂型为口服液或者管饲制剂。
在其中一些实施例中，所述药物的给药途径为注射给药或者口服给药。
在其中一些实施例中，所述药物还包括乙酰胆碱酯酶抑制剂和/或n-甲基-d-天冬氨酸受体拮抗剂，所述荚膜多糖A与所述乙酰胆碱酯酶抑制剂和/或所述n-甲基-d-天冬氨酸受体拮抗剂联合使用。
在其中一些实施例中，所述乙酰胆碱酯酶抑制剂包含多奈呱齐、加兰他敏和卡巴拉汀中的一种或者多种。
在其中一些实施例中，所述n-甲基-d-天冬氨酸受体拮抗剂包含美金刚。
本申请的一个或多个实施例细节在下面的描述中提出，本申请的其他特征、目的和优点将从说明书及其权利要求书变得明显。
附图说明
为了更清楚地说明本申请实施例或传统技术中的技术方案，下面将对实施例或传统技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据公开的附图获得其他的附图。
图1为脆弱拟杆菌荚膜多糖SEC-HPLC分析结果；
图2为脆弱拟杆菌荚膜多糖SEC-MALS分析结果。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，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，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
本申请中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本申请中，涉及到数值区间，如无特别说明，则包括数值区间的两个端点。
本申请中涉及的百分比含量，如无特别说明，对于固液混合和固相-固相混合均指质量百分比，对于液相-液相混合指体积百分比。
本申请中涉及的百分比浓度，如无特别说明，均指终浓度。所述终浓度，指添加成分在添加该成分后的体系中的占比。
本申请中的温度参数，如无特别限定，既允许为恒温处理，也允许在一定温度区间内进行处理。所述的恒温处理允许温度在仪器控制的精度范围内进行波动。
本申请所述的防治，包括预防和治疗。
脆弱拟杆菌(B.fragilis)属于拟杆菌门，拟杆菌科，拟杆菌属，革兰氏阴性非生孢的严格厌氧杆菌；正常寄居于人体肠道、口腔、呼吸道，对宿主的生理机能有深远影响。
本申请涉及的脆弱拟杆菌荚膜多糖A是从脆弱拟杆菌ZY-312制备得到的荚膜多糖，本申请证实所涉脆弱拟杆菌ZY-312荚膜多糖A可改善AD模型动物的学习记忆能力，可在制备防治AD药物中应用。脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC NO.10685，所述荚膜多糖提取物包含荚膜多糖A的提取物。
本申请发现脆弱拟杆菌ZY-312的荚膜多糖A能有效预防和治疗阿尔茨海默病，且该药效在经Morris水迷宫行为实验在动物模型得以验证。采用脆弱拟杆菌ZY-312的荚膜多糖A的提取物制备防治阿尔茨海默病的药物还具有无毒副作用的优势。
本申请所用的脆弱拟杆菌为脆弱拟杆菌ZY-312(bacteroides fragilis ZY-312)，于2015年4月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，其保藏编号为CGMCC No.10685，保藏地址为北京市朝阳区北辰西路1号院3号。该菌株是一株非产肠毒脆弱拟杆菌。
在其中一个示例中，所述荚膜多糖提取中所述荚膜多糖A的含量大于95wt％。
在其中一个示例中，所述荚膜多糖A的重均分子量为70KD-90KD。所述荚膜多糖A的重均分子量为70KD-90KD。本申请的荚膜多糖A的重均分子量为70KD、71KD、72KD、73KD、74KD、75KD、76KD、77KD、78KD、79KD、80KD、81KD、82KD、83KD、84KD、85KD、86KD、87KD、88KD、89KD、90KD。较优地，本申请的荚膜多糖A的重均分子量为80KD-90KD。
在其中一个示例中，所述荚膜多糖A的重均分子量和数均分子量(Mw/Mn)的比值为1.0-1.3。
在其中一个示例中，所述荚膜多糖A的提取物中重均分子量分布于70KD-100KD的部分占总提取物重量的70％-80％；
在其中一个示例中，所述荚膜多糖A的提取物中结合脂质的重量含量＜0.02％。
本申请上述应用中，荚膜多糖A的提取物的制备，可以，包括但不限于，包括以下步骤：
(1)脆弱拟杆菌发酵液经过离心，收集细菌沉淀(菌泥)；
(2)菌泥中加入纯化水，充分搅拌，使细菌重悬；
(3)调节细菌重悬液的pH至酸性；
(4)细菌重悬液转移至提取容器中，加热提取，离心取上清；
(5)超滤除小分子杂质，收集截留液；
(6)柱交换层析；
(7)柱层析收集液经超滤除盐、浓缩后，冷冻干燥，密封保存。
上述制备工艺主要概括为：脆弱拟杆菌厌氧发酵→离心收集菌体沉淀→菌泥混悬→酸提→离心收集上清→超滤除小分子杂质→离子交换一步层析→冻干工艺制备。该工艺更简单、环保、荚膜多糖纯度高且得率高。制备的荚膜多糖A可满足：蛋白残留＜1％；核酸残留＜0.05％；结合脂质＜0.02％；不含脂多糖或其他污染性多糖组分；分子量分布均一，重均分子量为70KD-90KD，Mw/Mn＜1.3。
在其中一个示例中，所述药物包含所述荚膜多糖提取物和药学上可以接受的辅料。
可以理解得是，所述辅料例如为稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂、等渗或等张调节剂等，可以含一种，也可以含两种或者两种以上。所述的辅料可以选自，包括但不限于，如下：稀释剂为淀粉类、糖类、纤维素类以及无机盐类；所述润湿剂选自水以及乙醇中的至少一种；所述黏合剂选自淀粉浆、糊精、糖、纤维素衍生物、明胶、聚维酮以及聚乙二醇中的至少一种；所述崩解剂选自干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联羧甲基纤维素钠、交联聚维酮、表面活性剂以及泡腾崩解剂中的至少一种；所述润滑剂选自滑石粉、硬脂酸钙、硬脂酸镁、十二烷基硫酸镁、微粉硅胶以及聚乙二醇中的至少一种；所述色香味调节剂选自色素、香料、甜味剂、胶浆剂以及矫臭剂中的至少一种；所述溶剂选自水、油、乙醇、甘油、丙二醇、聚乙二醇、二甲基亚砜、液体石蜡、脂肪油以及乙酸乙酯中的至少一种；所述增溶剂选自吐温类、卖泽类、聚氧乙烯脂肪醇醚类、肥皂类、硫酸化物、以及磺酸化物中的至少一种；所述助溶剂选自有机酸及其盐类、酰胺及胺类化合物、无机盐、聚乙二醇、聚维酮以及甘油中的至少一种；所述乳化剂选自司盘类、吐温类、卖泽类、苄泽类、甘油脂肪酸酯、高级脂肪酸盐、硫酸化物、磺酸化物、阿拉伯胶、西黄耆胶、明胶、果胶、磷脂、琼脂、海藻酸钠、氢氧化物、二氧化硅以及皂土中的至少一种；所述助悬剂选自甘油、糖浆、阿拉伯胶、西黄耆胶、琼脂、海藻酸钠、纤维素衍生物、聚维酮、卡波普、聚乙烯醇以及触变胶中的至少一种；所述抗氧剂选自亚硫酸盐、焦亚硫酸盐、亚硫酸氢盐、抗坏血酸、没食子酸及其酯类中的至少一种；所述金属络合剂选自乙二胺四乙酸二钠以及多羧酸化合物中的一种；所述惰性气体选自氮气以及二氧化碳中的一种；所述防腐剂选自尼泊金类、有机酸及其盐、季铵类化合物、醋酸氯己定、醇类、酚类以及挥发油中的至少一种；所述局部止痛剂选自苯甲醇、三氯叔丁醇、利多卡因以及普鲁卡因中的至少一种；所述pH调节剂选择盐酸、硫酸、磷酸、枸橼酸、酒石酸、醋酸、氢氧化钠、碳酸氢钠、乙二胺、葡甲胺、磷酸盐、醋酸盐以及枸橼酸盐中的至少一种；所述等渗或等张调节剂选自葡萄糖、氯化钠、枸橼酸钠、山梨醇以及木糖醇中的至少一种。
可以理解得是，所述药物可以根据临床需求制备成合适的剂型，例如片剂、颗粒剂、丸剂、粉剂、胶囊剂、注射剂、口服液、管饲制剂等。
可以理解得是，所述药物可以根据临床需求通过合适的给药途径进行给药，例如注射给药、口服给药等。
在其中一个示例中，所述药物还包括乙酰胆碱酯酶抑制剂和/或n-甲基-d-天冬氨酸受体拮抗剂，所述荚膜多糖A与所述乙酰胆碱酯酶抑制剂和/或所述n-甲基-d-天冬氨酸受体拮抗剂联合使用。
在其中一个示例中，所述乙酰胆碱酯酶抑制剂包含多奈呱齐、加兰他敏和卡巴拉汀中的一种或者多种。
在其中一个示例中，所述n-甲基-d-天冬氨酸受体拮抗剂包含美金刚。
实施例1、脆弱拟杆菌荚膜多糖A的制备
脆弱拟杆菌发酵液离心，收集菌泥。
(1)方法1：酸提取法制备脆弱拟杆菌荚膜多糖A
1)取50g菌泥，加入300g纯化水使菌体重悬，用1mol/L盐酸溶液调节其pH至4.5，90℃提取1.5h，冷却至室温，12000g常温离心10min，取上清，得到粗糖溶液。
2)粗糖溶液经10KD超滤膜超滤浓缩，收集截流液。
3)截流液中加入等体积40mmol/L Tris-HCl(pH8.5)缓冲溶液转盐；DEAE Sepharose Fast Flow离子交换柱层析(16mm×200mm)，流速20mL/min，20mmol/L Tris-HCl(pH8.5，含0.2mol/L NaCl)缓 冲溶液梯度洗脱25个柱体积，分段收集，100mL/瓶(组分)，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，10KD超滤膜超滤，加入纯化水反复超滤，至电导率稳定，收集回流液，冻干；平行2次试验，得到脆弱拟杆菌荚膜多糖分别命名为“S1”和“S2”，检测结果参见图1和图2。
(2)方法2：苯酚/水法制备脆弱拟杆菌荚膜多糖A
①取50g菌泥，加入0.15mol/L氯化钠溶液200mL，洗涤后12000g常温离心20min，收集菌泥。
②加入750mL纯化水，使菌体重悬，再加入等体积75％苯酚水溶液(m/m)，68℃搅拌提取30min，16000g室温离心30min，去沉淀。
③上清用等体积乙醚萃取3次，收集水相；水相经旋转蒸发仪浓缩，透析后冻干。
④上述样品用0.1mol/L乙酸钠溶液(含有10mmol/L CaCl2和10mmol/L MgCl2)溶解，加入2mg脱氧核糖核酸酶和10mg核糖核酸酶，37℃搅拌2h，再次加入2mg脱氧核糖核酸酶和10mg核糖核酸酶，37℃搅拌过夜；调节pH至7.0，加入20mg链霉蛋白酶，37℃搅拌2h，再次加入20mg链霉蛋白酶，37℃搅拌过夜；加入无水乙醇至终浓度为80％，4℃过夜，12000g离心10min，取沉淀。
⑤沉淀中加入5％乙酸溶液复溶，100℃水解1h，透析除盐后，DEAE Sepharose Fast Flow离子交换柱层析，分段收集，SEC-HPLC跟踪监测，合并206nm吸收峰为单一、对称峰的组分，超滤除盐、冻干；平行2次试验，得到脆弱拟杆菌荚膜多糖A分别命名为“S3”和“S4”。
(3)实验结果
两种制备方法所得脆弱拟杆菌荚膜多糖的比较结果见表1。
表1、两种制备方法所得脆弱拟杆菌荚膜多糖A的质量属性比较
上述样品分别记为：S1-PSA、S2-PSA、S3-PSA、S4-PSA。
实施例2、脆弱拟杆菌荚膜多糖A防治AD的效果验证
一、实验方法
动物：本实验选取SPF级SD大鼠90只，8周龄，雌雄各半，体质量200g-240g。
动物分组：90只SD大鼠，雌雄各半，按照随机数字表法分为9组，每组雌雄各5只，分别为假手术组，模型组，S1-PSA低、中、高剂量组，S3-PSA低、中、高剂量组，盐酸多奈哌齐组(阳性药组)，详见表2。
AD模型制备：9组大鼠均用10％水合氯醛腹腔注射麻醉，剃去脑顶局部毛发，把大鼠平颅头部固定在脑立体定位仪上，碘伏常规皮肤消毒，用无菌手术刀在头皮正中线位置切一纵行切口，长约1.5cm，充分暴露前卤，根据大鼠脑立体定位图谱在脑正中线左右旁开2.0mm，前囟后3.0mm两侧分别用无菌牙科钻钻孔，钻孔深度为3mm，避免颅脑内出血。除假手术组，各组大鼠均缓慢注射5μL凝聚态Aβ1-40(β-淀粉样蛋白，1μL/min)，进针深度4.0mm。假手术组注射等体积生理盐水溶液，注射完毕均留针5min，之后缝合伤口，并于伤口处使用阿米卡星喷雾，预防伤口感染。术后常规饲养1周。1周后，所有大鼠进行水迷宫试验，造模大鼠逃避潜伏期与假手术组相比逃避潜伏期长且有明显差异，则判定造模成功，最终全部造模成功。
给药：实验药物用量按体表面积比率换算，S1-PSA高、中、低剂量组和S3-PSA高、中、低剂量组分别配置以5mg/kg、3mg/kg、1mg/kg灌胃。盐酸多奈哌齐组将盐酸多奈哌齐片研碎成粉末，制成混悬液以1.02mg·kg-1灌胃，相当于成人日用剂量的6倍。假手术组与模型组大鼠每日予生理盐水灌胃。各组灌胃容积为10mL/Kg，连续灌胃10周，详见表2。
表2、动物分组及给药

Morris水迷宫简介：Morris水迷宫是英国心理学家Morris于20世纪80年(1981)代初设计并应用于脑学习记忆机制研究的一种实验手段，其在AD研究中的应用非常普遍。较为经典的Morris水迷宫，测试程序主要包括定位航行试验和空间探索试验两个部分。其中定位航行试验历时数天，每天将大鼠面向池壁分别从4个入水点放入水中若干次，记录其寻找到隐藏在水面下平台的时间(逃避潜伏期)。一般AD越严重，大鼠的逃避潜伏期越长。空间探索试验是在定位航行试验后去除平台，然后任选一个入水点将大鼠放入水池中，记录其在一定时间内的游泳轨迹，考察大鼠对原平台的记忆。一般AD越严重，大鼠穿越平台次数越少，有效区域停留时间越短，有效区域运动路程越短。
Morris水迷宫行为实验：连续灌胃10周结束后，进行Morris水迷宫行为实验。实验连续5天，分为进行定位航行和空间探索实验。定位航行实验历时4天，记录动物从投放入水至寻找并爬上站台所需时间，即逃避潜伏期。如120s内未找到站台，潜伏期记录为120s。第5天进行空间搜索实验，撤下原有站台，记录大鼠120s内穿越平台次数、有效区(站台直径2倍范围)停留时间及有效区游泳路程。
统计学分析：数据以表示，采用SPSS 23.0进行统计分析。组间比较采用单因素方差分析(ANOVA)，方差齐时，组间均值两两比较采用SNK法；若方差不齐，改用邓尼特T3(Dunnett T3)检验，以P＜0.05表示差异有统计学意义。
二、结果与分析
口服脆弱拟杆菌荚膜多糖A对Aβ诱导的SD大鼠的影响结果见表3和表4。
表3、荚膜多糖A对Aβ诱导的SD大鼠在水迷宫测试中逃避潜伏期的影响

注:与假手术组比较，*P＜0.05，**P＜0.01；与模型组比较#P＜0.05，##P＜0.01。
如表3所示，各给药组雌雄大鼠在水迷宫的逃避潜伏期随着训练时间及次数的增加而缩短。第4天，与假手术组比较，模型组雌雄大鼠的逃避潜伏期显著延长(P＜0.01)，说明造模成功；与模型组比较，脆弱拟杆菌荚膜多糖A给药组雌雄大鼠的逃避潜伏期明显缩短(P＜0.05，P＜0.01)，说明脆弱拟杆菌的荚膜多糖A可以改善Aβ诱导的SD大鼠的AD症状。
表4、荚膜多糖A对Aβ诱导的SD大鼠在水迷宫测试中空间探索的影响


注:与假手术组比较，*P＜0.05，**P＜0.01；与模型组比较#P＜0.05，##P＜0.01。
从表4结果可以看出，与假手术组比较，模型组雌雄大鼠穿越平台次数、有效区域停留时间及运动路程均显著减少(P＜0.01)；与模型组比较，给药多奈哌齐和脆弱拟杆菌荚膜多糖A均能提高大鼠穿越平台次数、有效区域停留时间及运动路程，其中脆弱拟杆菌荚膜多糖A组雌雄大鼠穿越平台次数、有效区域停留时间及运动路程增加更显著(P＜0.05，P＜0.01)。
由以上实验结果可见，本申请提供的脆弱拟杆菌荚膜多糖A可以防治AD。
实施例3、脆弱拟杆菌荚膜多糖A联合多奈哌齐防治AD的效果验证
一、实验方法
动物：本实验选取SPF级SD大鼠90只，8周龄，雌雄各半，体质量200g-240g。
动物分组：110只SD大鼠，雌雄各半，按照随机数字表法分为11组，每组雌雄各5只，分别为假手术组，模型组，S2-PSA中剂量组，S2-PSA低、中、高剂量联合多奈哌齐组，S4-PSA中剂量组，S4-PSA低、中、高剂量多奈哌齐组，盐酸多奈哌齐组(阳性药组)，详见表5。
AD模型制备：11组大鼠均用10％水合氯醛腹腔注射麻醉，剃去脑顶局部毛发，把大鼠平颅头部固定在脑立体定位仪上，碘伏常规皮肤消毒，用无菌手术刀在头皮正中线位置切一纵行切口，长约1.5cm，充分暴露前卤，根据大鼠脑立体定位图谱在脑正中线左右旁开2.0mm，前囟后3.0mm两侧分别用无菌牙科钻钻孔，钻孔深度为3mm，避免颅脑内出血。除假手术组，各组大鼠均缓慢注射5μL凝聚态Aβ1-40(β-淀粉样蛋白，1μL/min)，进针深度4.0mm。假手术组注射等体积生理盐水溶液，注射完毕均留针5min，之后缝合伤口，并于伤口处使用阿米卡星喷雾，预防伤口感染。术后常规饲养1周。1周后，所有大鼠进行水迷宫试验，造模大鼠逃避潜伏期与假手术组相比逃避潜伏期长且有明显差异，则判定造模成功，最终全部造模成功。
给药：实验药物用量按体表面积比率换算，S2-PSA中剂量组，S2-PSA高、中、低剂量联合多奈哌齐组，S4-PSA中剂量组，S4-PSA高、中、低剂量联合多奈哌齐组，高中低剂量PSA分别配置以5mg/kg、3mg/kg、1mg/kg灌胃。盐酸多奈哌齐组将盐酸多奈哌齐片研碎成粉末，制成混悬液以1.02mg·kg-1灌胃，相当于成人日用剂量的6倍。假手术组与模型组大鼠每日予生理盐水灌胃。各组灌胃容积为10mL/Kg，连续灌胃10周，详见表5。
表5、动物分组及给药
较为经典的Morris水迷宫，测试程序主要包括定位航行试验和空间探索试验两个部分。其中定位航行试验历时数天，每天将大鼠面向池壁分别从4个入水点放入水中若干次，记录其寻找到隐藏在水面 下平台的时间(逃避潜伏期)。一般AD越严重，大鼠的逃避潜伏期越长。空间探索试验是在定位航行试验后去除平台，然后任选一个入水点将大鼠放入水池中，记录其在一定时间内的游泳轨迹，考察大鼠对原平台的记忆。一般AD越严重，大鼠穿越平台次数越少，有效区域停留时间越短，有效区域运动路程越短。
Morris水迷宫行为实验：连续灌胃10周结束后，进行Morris水迷宫行为实验。实验连续5天，分为进行定位航行和空间探索实验。定位航行实验历时4天，记录动物从投放入水至寻找并爬上站台所需时间，即逃避潜伏期。如120s内未找到站台，潜伏期记录为120s。第5天进行空间搜索实验，撤下原有站台，记录大鼠120s内穿越平台次数、有效区(站台直径2倍范围)停留时间及有效区游泳路程。
统计学分析：数据以表示，采用SPSS 23.0进行统计分析。组间比较采用单因素方差分析(ANOVA)，方差齐时，组间均值两两比较采用SNK法；若方差不齐，改用邓尼特T3(Dunnett T3)检验，以P＜0.05表示差异有统计学意义。
二、结果与分析
表6、荚膜多糖A联用多奈哌齐对Aβ诱导的SD大鼠在水迷宫测试中逃避潜伏期的影响

注:与假手术组比较，*P＜0.05，**P＜0.01；与模型组比较#P＜0.05，##P＜0.01，###P＜0.001。
如表6所示，各给药组雌雄大鼠在水迷宫的逃避潜伏期随着训练时间及次数的增加而缩短。与假手术组比较，模型组雌雄大鼠的逃避潜伏期显著延长(P＜0.05，P＜0.01)，说明造模成功；与模型组比较，脆弱拟杆菌荚膜多糖A组和多奈哌齐的雌雄大鼠的逃避潜伏期明显缩短(P＜0.01)，联用组比单用组具有更短的逃避潜伏期(P＜0.001)。
表7、荚膜多糖A联用多奈哌齐对Aβ诱导的SD大鼠在水迷宫测试中空间探索的影响

注:与假手术组比较，*P＜0.05，**P＜0.01；与模型组比较#P＜0.05，##P＜0.01，###P＜0.001。
从表7结果可以看出，与假手术组比较，模型组雌雄大鼠穿越平台次数、有效区域停留时间及运动路程均显著减少(P＜0.01)；与模型组比较，脆弱拟杆菌荚膜多糖A单用组和多奈哌齐单用组的雌雄大鼠穿越平台次数、有效区域停留时间及运动路程均增加(P＜0.01)，脆弱拟杆菌荚膜多糖A与多奈哌齐联用组的大鼠穿越平台次数、有效区域停留时间及运动路程具有更明显的增加(P＜0.01，P＜0.001)。
由以上实验结果可见，本申请提供的脆弱拟杆菌荚膜多糖A联用多奈哌齐具有更显著的防治AD效果。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用，所述脆弱拟杆菌的保藏编号为CGMCC NO.10685，所述荚膜多糖提取物包含荚膜多糖A。
  2. 根据权利要求1所述的应用，其特征在于，所述荚膜多糖提取物中所述荚膜多糖A的含量约大于95wt％。
  3. 根据权利要求1或者2所述的应用，其特征在于，所述荚膜多糖A的重均分子量约为70KD-90KD；优选的，所述荚膜多糖A的重均分子量约为80KD-90KD。
  4. 根据权利要求1至3任一项所述的应用，其特征在于，所述荚膜多糖A中脂质的含量约低于0.02wt％。
  5. 根据权利要求1至4任一项所述的应用，其特征在于，所述荚膜多糖A中脂质含量约低于0.01wt％。
  6. 根据权利要求1至5任一项所述的应用，其特征在于，所述药物包含所述荚膜多糖提取物和药学上可以接受的辅料。
  7. 根据权利要求6所述的应用，其特征在于，所述辅料包含稀释剂、润湿剂、黏合剂、崩解剂、润滑剂、色香味调节剂、溶剂、增溶剂、助溶剂、乳化剂、抗氧剂、金属络合剂、惰性气体、防腐剂、局部止痛剂、pH调节剂和等渗或等张调节剂中的一种或者多种。
  8. 根据权利要求7所述的应用，其特征在于，所述辅料满足如下(1)至(18)项所示条件中的一种或者多种：
    (1)所述稀释剂选自淀粉类、糖类、纤维素类以及无机盐类中的至少一种；
    (2)所述润湿剂选自水以及乙醇中的至少一种；
    (3)所述黏合剂选自淀粉浆、糊精、糖、纤维素衍生物、明胶、聚维酮以及聚乙二醇中的至少一种；
    (4)所述崩解剂选自干淀粉、羧甲基淀粉钠、低取代羟丙基纤维素、交联羧甲基纤维素钠、交联聚维酮、表面活性剂以及泡腾崩解剂中的至少一种；
    (5)所述润滑剂选自滑石粉、硬脂酸钙、硬脂酸镁、十二烷基硫酸镁、微粉硅胶以及聚乙二醇中的至少一种；
    (6)所述色香味调节剂选自色素、香料、甜味剂、胶浆剂以及矫臭剂中的至少一种；
    (7)所述溶剂选自水、油、乙醇、甘油、丙二醇、聚乙二醇、二甲基亚砜、液体石蜡、脂肪油以及乙酸乙酯中的至少一种；
    (8)所述增溶剂选自吐温类、卖泽类、聚氧乙烯脂肪醇醚类、肥皂类、硫酸化物、以及磺酸化物中的至少一种；
    (9)所述助溶剂选自有机酸及其盐类、酰胺及胺类化合物、无机盐、聚乙二醇、聚维酮以及甘油中的至少一种；
    (10)所述乳化剂选自司盘类、吐温类、卖泽类、苄泽类、甘油脂肪酸酯、高级脂肪酸盐、硫酸化物、磺酸化物、阿拉伯胶、西黄耆胶、明胶、果胶、磷脂、琼脂、海藻酸钠、氢氧化物、二氧化硅以及皂土中的至少一种；
    (11)所述助悬剂选自甘油、糖浆、阿拉伯胶、西黄耆胶、琼脂、海藻酸钠、纤维素衍生物、聚维酮、卡波普、聚乙烯醇以及触变胶中的至少一种；
    (12)所述抗氧剂选自亚硫酸盐、焦亚硫酸盐、亚硫酸氢盐、抗坏血酸、没食子酸及其酯类中的至少一种；
    (13)所述金属络合剂选自乙二胺四乙酸二钠以及多羧酸化合物中的一种；
    (14)所述惰性气体选自氮气以及二氧化碳中的一种；
    (15)所述防腐剂选自尼泊金类、有机酸及其盐、季铵类化合物、醋酸氯己定、醇类、酚类以及挥发油中的至少一种；
    (16)所述局部止痛剂选自苯甲醇、三氯叔丁醇、利多卡因以及普鲁卡因中的至少一种；
    (17)所述pH调节剂选择盐酸、硫酸、磷酸、枸橼酸、酒石酸、醋酸、氢氧化钠、碳酸氢钠、乙 二胺、葡甲胺、磷酸盐、醋酸盐以及枸橼酸盐中的至少一种；
    (18)所述等渗或等张调节剂选自葡萄糖、氯化钠、枸橼酸钠、山梨醇以及木糖醇中的至少一种。
  9. 根据权利要求1至8任一项所述的应用，其特征在于，所述药物的剂型为片剂、颗粒剂、丸剂、粉剂、胶囊剂、注射剂、口服液或者管饲制剂；或/和，所述药物的给药途径为注射给药或者口服给药。
  10. 根据权利要求1至9任一项所述的应用，其特征在于，所述药物还包括乙酰胆碱酯酶抑制剂和/或n-甲基-d-天冬氨酸受体拮抗剂，所述荚膜多糖A与所述乙酰胆碱酯酶抑制剂和/或所述n-甲基-d-天冬氨酸受体拮抗剂联合使用；
    优选的，所述乙酰胆碱酯酶抑制剂包含多奈呱齐、加兰他敏和卡巴拉汀中的一种或者多种；
    优选的，所述n-甲基-d-天冬氨酸受体拮抗剂包含美金刚。
PCT/CN2023/087988 2022-02-16 2023-04-13 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用 WO2023155938A2 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210141343.4 2022-02-16
CN202210141343.4A CN114699422B (zh) 2022-02-16 2022-02-16 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用

Publications (2)

Publication Number Publication Date
WO2023155938A2 true WO2023155938A2 (zh) 2023-08-24
WO2023155938A3 WO2023155938A3 (zh) 2023-10-12

Family

ID=82166694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/087988 WO2023155938A2 (zh) 2022-02-16 2023-04-13 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用

Country Status (2)

Country Link
CN (1) CN114699422B (zh)
WO (1) WO2023155938A2 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114699422B (zh) * 2022-02-16 2023-07-18 广州知易生物科技有限公司 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109481473B (zh) * 2017-09-11 2023-03-21 广州知易生物科技有限公司 脆弱拟杆菌提取物在制备防治肠易激综合征的药物中的应用
CN112587552B (zh) * 2020-09-17 2023-09-12 大连图腾生命科学发展有限公司 脆弱拟杆菌839在制备治疗或辅助治疗免疫相关疾病的药物中的应用
CN113234770B (zh) * 2021-06-15 2024-06-14 广州知易生物科技有限公司 脆弱拟杆菌荚膜多糖a的制备方法
CN114699422B (zh) * 2022-02-16 2023-07-18 广州知易生物科技有限公司 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用

Also Published As

Publication number Publication date
WO2023155938A3 (zh) 2023-10-12
CN114699422A (zh) 2022-07-05
CN114699422B (zh) 2023-07-18

Similar Documents

Publication Publication Date Title
CN104662007B (zh) 胱硫醚‑γ‑裂合酶(CSE)抑制剂
JP7066679B2 (ja) 認知症の処置
WO2023155938A2 (zh) 脆弱拟杆菌的荚膜多糖提取物在制备防治阿尔茨海默病的药物中的应用
EA030093B1 (ru) Лекарственная форма пирфенидона в виде капсул с фармацевтически приемлемыми эксципиентами
JP2009537565A (ja) Ｒ（＋）およびｓ（−）プラミペキソール組成とそれを利用する方法
JPH02215719A (ja) 線維化病変組織の修復並びに線維化病変の阻止剤
TWI226829B (en) Pharmaceutical compositions for treatment of partial responders or refractory depression
WO2011019845A1 (en) Use of 4-aminopyridine to improve neuro-cognitive and/or neuro-psychiatric impairment in patients with demyelinating and other nervous system conditions
JP2587441B2 (ja) 製薬学的組成物
JP2002500651A (ja) 精神分裂病及び精神病の治療方法
JP2002522479A (ja) 視覚障害および記憶障害のためのｎ−複素環式カルボン酸またはそのイソスターのｎ−結合スルホンアミド
WO2023155937A2 (zh) 脆弱拟杆菌的荚膜多糖提取物在制备防治精神分裂症的药物中的应用
WO2023155936A2 (zh) 脆弱拟杆菌两性离子荚膜多糖或/和改性的两性离子荚膜多糖的新应用
WO2024040935A1 (zh) 沙库巴曲缬沙坦类物质在制备药物成瘾和复吸药物的应用
JPH11189533A (ja) 点眼薬
JPH0152363B2 (zh)
US20230248726A1 (en) Pharmaceutical composition and application thereof
WO2016047662A1 (ja) 脳卒中の治療剤
JP5389471B2 (ja) 鼻閉抑制剤
TW589182B (en) Use of 1-(aminoalkyl)-3-quinoxaline-2-on derivatives for the preparation of compositions acting as antioxidants
CN111317746B (zh) 一种富勒烯结构在制备治疗阿尔茨海默病的药物中的应用
US6486169B1 (en) Botanical drug for treatment and prevention of alzhimer's disease
JPH11116477A (ja) アレルギー性結膜疾患の処置剤
US20070054940A1 (en) Remedy for down's syndrome
JP2010047566A (ja) 医薬組成物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23755938

Country of ref document: EP

Kind code of ref document: A2