WO2023232083A1 - β2-微球蛋白封闭肽、其药物组合物及用途 - Google Patents

β2-微球蛋白封闭肽、其药物组合物及用途 Download PDF

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WO2023232083A1
WO2023232083A1 PCT/CN2023/097492 CN2023097492W WO2023232083A1 WO 2023232083 A1 WO2023232083 A1 WO 2023232083A1 CN 2023097492 W CN2023097492 W CN 2023097492W WO 2023232083 A1 WO2023232083 A1 WO 2023232083A1
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peptide
seq
brain
isolated polypeptide
induced
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French (fr)
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王鑫
赵依妮
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厦门大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the invention belongs to the field of biomedicine and relates to ⁇ 2-microglobulin blocking peptide, its pharmaceutical composition and uses.
  • AD Alzheimer's disease
  • a ⁇ amyloid deposition formed by the oligomerization of ⁇ -amyloid (A ⁇ ) produced by the cleavage of amyloid precursor protein (APP) in the brain, and abnormal phosphorylation of the intracellular microtubule-binding protein tau.
  • NFTs Neurofibrillary tangles formed after aggregation, neuronal loss and excessive neuroinflammation.
  • AD Alzheimer's disease
  • AD Since the discovery of AD in 1901, there have been many studies on its pathogenesis, but there is still no clear conclusion.
  • the "A ⁇ cascade hypothesis” is one of the mainstream theories of AD pathogenesis. This theory believes that the neurotoxic A ⁇ 1-40/42 produced by abnormal shearing of APP plays a central role in the pathogenesis of AD and is induced by various reasons. Common pathways in AD. A series of neurotoxic reactions caused by A ⁇ oligomers can stimulate neuroinflammation, lead to nerve cell dysfunction and neuron loss, and ultimately cause dementia. Therefore, reducing the production of A ⁇ in the brain, promoting A ⁇ clearance, inhibiting A ⁇ aggregation, and reducing its neurotoxicity have become one of the main measures to treat AD.
  • B2M ⁇ 2-microglobulin
  • MHC-I Major histocompatibility complex I
  • B2M protein exists in the form of soluble monomers, but under the influence of some pathological factors, B2M will aggregate and deposit. These pathological factors include aging, long-term renal dysfunction, and inflammation.
  • B2M amyloid deposition is mainly found in bone and joint areas and eventually leads to severe arthritis, fractures and carpal tunnel syndrome.
  • the levels of B2M in serum and plasma increase. What is particularly noteworthy is that the levels of B2M in the plasma and cerebrospinal fluid of AD patients are significantly higher than those of normal controls of the same age.
  • Stereotaxic brain injection of B2M inhibits nerves B2m deletion can promote neuron regeneration and reverse aging-related cognitive function defects.
  • B2M has a direct or indirect impact on the occurrence and development of AD.
  • One aspect of the invention relates to an isolated polypeptide, which is the polypeptide shown in SEQ ID NO:3 or a truncated fragment of the polypeptide shown in SEQ ID NO:3, wherein the truncated fragment comprises SEQ ID NO:7 Or the polypeptide shown in SEQ ID NO:8.
  • the isolated polypeptide, wherein the truncated fragment does not include the N-terminal 6 histidines of the polypeptide shown in SEQ ID NO:3 (for example, as shown in SEQ ID NO: 3 below The polypeptide represented by any sequence in NOs: 16-25).
  • the isolated polypeptide is a polypeptide represented by any one of SEQ ID NOs: 7-8.
  • the isolated polypeptide is a polypeptide represented by any one of SEQ ID NOs: 11-15:
  • the isolated polypeptide is a polypeptide represented by any one of SEQ ID NOs: 16-25:
  • Another aspect of the invention relates to an isolated polynucleotide encoding an isolated polypeptide according to any one of the invention.
  • Yet another aspect of the invention relates to a recombinant expression vector comprising an isolated polynucleotide of the invention.
  • Yet another aspect of the invention relates to a transformed cell comprising the recombinant expression vector of the invention.
  • a further aspect of the invention relates to a pharmaceutical composition comprising an isolated polypeptide according to any one of the invention.
  • the pharmaceutical composition also contains one or more pharmaceutically acceptable auxiliary materials, such as carriers or excipients; specifically, it is an injection, oral liquid, capsule, Tablets, granules, pills or extract remix dosage forms.
  • pharmaceutically acceptable auxiliary materials such as carriers or excipients; specifically, it is an injection, oral liquid, capsule, Tablets, granules, pills or extract remix dosage forms.
  • Conventional processing methods can be used to produce injections, oral liquids, capsules, tablets, granules, pills, extracts and other dosage forms containing the polypeptide of the present invention and excipients and then mixed.
  • the pharmaceutical composition of the present invention contains 0.1-90% by weight of active ingredients.
  • Pharmaceutical compositions can be prepared according to methods known in the art.
  • the active ingredient may be combined, if necessary, with one or more solid or liquid pharmaceutical excipients and/or auxiliaries to prepare a suitable administration form or dosage form for human use.
  • the pharmaceutical composition of the present invention can be administered in unit dosage form, and the administration route can be intestinal or parenteral, such as oral, intramuscular, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum, etc.
  • Dosage forms such as tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and freeze-dried powder injections wait. It can be ordinary preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems.
  • To formulate unit dosage forms into tablets a wide variety of carriers known in the art may be used.
  • carriers are, for example, diluents and absorbing agents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, Sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and adhesives, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin , syrup, honey, glucose solution, arabic slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, Agar powder, fucoid starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.; disintegr
  • Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets.
  • a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as gum arabic, gum tragacanth, and gelatin.
  • disintegrating agents such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.
  • a wide variety of carriers known in the art can be used.
  • the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.
  • the active ingredient is mixed with the various carriers mentioned above, and the resulting mixture is placed in a hard capsule or soft capsule.
  • the active ingredients can also be made into microcapsules and suspended in an aqueous medium to form a suspension. They can also be packed into hard capsules or made into injections.
  • injection preparations such as solutions, emulsions, lyophilized powders for injection and suspensions
  • all diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1,3 -Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation.
  • conventional co-solvents, buffers, pH adjusters, etc. can also be added.
  • colorants if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations.
  • Another aspect of the present invention relates to the use of the isolated polypeptide according to any one of the present invention in the preparation of a medicament for treating or preventing Alzheimer's disease.
  • a further aspect of the present invention relates to the use of the isolated polypeptide according to any one of the present invention in the preparation of the following medicaments:
  • Drugs that inhibit B2M-induced A ⁇ 1-42 oligomerization, drugs that inhibit B2M-induced ⁇ -amyloid plaque formation drugs or drugs that inhibit the neurotoxicity of A ⁇ 1-42 are included in the formulation.
  • the present invention has discovered for the first time that the expression of B2M in the brain tissue of AD patients is significantly increased, and there is a significant positive correlation between the expression level of B2M in the brain and the content of A ⁇ 1-42 .
  • the AD mouse model (5 ⁇ FAD) B2M content in the brain can increase ⁇ -amyloid plaque deposition in the brain of 5 ⁇ FAD mice. Further, the inventor found that B2M has a direct interaction with A ⁇ 1-42 .
  • Using the truncated B2M amino acid sequence as a blocking peptide can inhibit the aggregation of B2M and A ⁇ 1-42 and inhibit the neurotoxicity of B2M and A ⁇ 1-42 .
  • the isolated polypeptide according to any one of the present invention is used to treat or prevent Alzheimer's disease.
  • Yet another aspect of the invention relates to a method of treating or preventing Alzheimer's disease, comprising the step of administering to a subject in need thereof an effective amount of an isolated polypeptide according to any one of the invention.
  • Yet another aspect of the present invention relates to a method of inhibiting B2M-induced A ⁇ 1-42 oligomerization or B2M-induced ⁇ -amyloid plaques or inhibiting A ⁇ 1-42 neurotoxicity, comprising administering to a subject in need or with an effective amount of an isolated polypeptide of any one of the invention.
  • the dosage of the polypeptide or pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of Alzheimer's disease to be prevented or treated, the gender, age, weight and individual response of the patient or animal, the route of administration and Number of doses, etc.
  • the above dosage may be administered as a single dosage or divided into several, for example two, three or four dosages. Dosage levels must be selected based on the specific route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it is common practice in the art to administer doses starting at levels lower than those required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.
  • the total daily dosage of the polypeptide or pharmaceutical composition of the present invention must be determined by the attending physician within the scope of reliable medical judgment.
  • the specific therapeutically effective dosage level for any particular patient will be determined by a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the patient's age, weight, general health Condition, sex, and diet; time of administration, route of administration, and excretion rate; duration of treatment; drugs used in combination or concomitantly; and similar factors known in the medical field. For example, it is common practice in the art to administer doses starting at levels lower than those required to achieve the desired therapeutic effect and gradually increasing the dose. Amount until you get the desired effect.
  • the dosage of the pharmaceutical composition of the present invention for mammals, especially humans, calculated based on the active ingredients can be between 0.001-1000 mg/kg body weight/day, for example, between 0.01-800 mg/kg body weight/day, for example, between 0.01-500mg/kg body weight/day.
  • amino acid sequence of the ⁇ 2-microglobulin is shown in SEQ ID NO: 9.
  • amino acid sequence (N-terminus to C-terminus) of human B2M protein is as follows:
  • the amino acid sequence of ⁇ 2-microglobulin when referring to the amino acid sequence of ⁇ 2-microglobulin (B2M), it includes the full length of ⁇ 2-microglobulin and also includes its fusion protein.
  • B2M amino acid sequence of ⁇ 2-microglobulin
  • mutations or variations can occur naturally or be artificially introduced without affecting its biological function.
  • the ⁇ 2-microglobulin is human ⁇ 2-microglobulin.
  • the amino acid sequence of ⁇ 2-microglobulin is shown in SEQ ID NO: 9.
  • the term "host cell” refers to the cell into which the vector is introduced, including many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, yeast cells or fungal cells such as Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or animal cells such as human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • yeast cells or fungal cells such as Aspergillus, such as S2 Drosophila cells or Insect cells such as Sf9, or fibroblast cells
  • CHO cells COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells
  • animal cells such as human cells.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a certain protein can be inserted.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage and animal viruses, etc.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses.
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses.
  • Viruses such as SV40
  • a vector may contain multiple elements that control expression.
  • disease and/or disorder refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.
  • subject may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and/or alleviate the disease or condition described in the present invention. , horses, etc.
  • blocking peptide refers to a polypeptide that can competitively bind to A ⁇ with the full-length B2M protein, thereby inhibiting the biological effect of B2M binding to A ⁇ that promotes A ⁇ oligomerization.
  • the concentration unit ⁇ M represents ⁇ mol/L
  • mM represents mmol/L
  • nM represents nmol/L.
  • amino acid sequence of A ⁇ 1-42 is as follows:
  • the present invention has achieved one or more of the following technical effects (1)-(4):
  • the polypeptide of the present invention can effectively prevent and/or treat Alzheimer's disease.
  • polypeptide of the present invention can effectively inhibit B2M-induced A ⁇ 1-42 oligomerization.
  • polypeptide of the present invention can effectively inhibit the formation of ⁇ -amyloid plaques induced by B2M.
  • the polypeptide of the present invention can effectively inhibit the neurotoxicity of A ⁇ 1-42 .
  • FIG. 1A to Figure 1B B2M expression levels are significantly increased in the brains of AD patients. in:
  • Figure 1A Western blot detection results of B2M expression in cerebral cortex tissue of AD patients.
  • Figure 1C The A ⁇ 1-42 levels in the above 29 human brain tissues were detected by A ⁇ 1-42 ELISA. Pearson correlation analysis found that there was a significant difference between the A ⁇ 1-42 levels in the human brain and the B2M expression level in the human brain. positive correlation.
  • Figure 1D to Figure 1F Increasing B2M levels in the brains of 5 ⁇ FAD mice can promote amyloid plaque deposition in the brain.
  • Figure 1E Statistical results of the number of ⁇ -amyloid plaques (Number of A ⁇ depositis) in the hippocampal DG area in Figure 1D. Data were statistically analyzed using Paired t test. ns, no significant difference, P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • Figure 1F Statistical results of the area of ⁇ -amyloid plaques (Area of A ⁇ depositis) in the hippocampal DG area in Figure 1D. Data were statistically analyzed using Paired t test. ns, no significant difference, P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 1 Co-immunoprecipitation (co-IP) experimental results of the interaction between in vitro purified B2M protein and purified A ⁇ 1-42 protein. in:
  • FIG. 1A Immunoprecipitation of A ⁇ 1-42 and B2M using anti-A ⁇ antibodies.
  • FIG. 2B Immunoprecipitation of B2M and A ⁇ 1-42 using anti-B2M antibodies.
  • Figure 2C The results of co-IP experiments on the interaction between B2M and A ⁇ 1-42 in vivo using brain tissue of 12-month-old wild type (WT) and 5 ⁇ FAD mice.
  • the antibodies used were anti-A ⁇ antibodies.
  • Figure 2D Continuous detection results of fluorescence microplate reader on the promotion of A ⁇ 1-42 oligomerization after incubation of A ⁇ 1-42 with thioflavin-T indicated in vitro B2M.
  • the full-length B2M protein of different concentrations was incubated with A ⁇ 1-42 .
  • FIG. 2E Thioflavin-T fluorescence detection results of corresponding concentrations of B2M protein under the same experimental conditions as Figure 2D. Data represent the average of absolute fluorescence signal intensity.
  • Figure 3A Schematic diagram of truncating the full-length B2M amino acid sequence into four short peptides without overlapping sequences.
  • FIG. 3B Thioflavin-T experimental results of truncated B2M small peptide inhibiting B2M-promoted A ⁇ 1-42 oligomerization.
  • the truncated B2M amino acid sequence was used as a blocking peptide and was pre-incubated with A ⁇ 1-42 at 37°C for 3 hours.
  • the control group was pre-incubated with the nonsense peptide sequence (NS) and A ⁇ 1-42 , and then full-length B2M was added.
  • the protein continues to incubate A ⁇ 1-42 , and the blocking peptide's inhibitory effect on B2M's promotion of A ⁇ 1-42 oligomerization is analyzed by analyzing changes in thioflavin-T fluorescence signal intensity. fruit. Data represent the average of absolute fluorescence signal intensity.
  • Figure 3C Based on the above-mentioned thioflavin-T experiment, the inhibitory effect of different concentrations of B2M-3 blocking peptide on B2M-promoted A ⁇ 1-42 oligomerization was detected. Data represent the average of absolute fluorescence signal intensity.
  • Figure 3D Immunoprecipitation of A ⁇ bound to B2M-3 peptide with anti-His antibody.
  • Figure 3E Detection of equilibrium dissociation constant K D between B2M-3 peptide and A ⁇ 1-42 using surface plasmon resonance technology.
  • Figure 3F Detection of equilibrium dissociation constant K D between NS peptide and A ⁇ 1-42 using surface plasmon resonance technology.
  • Figure 3G Detection of the morphological effects of B2M-3 blocking peptide on the fibrils formed by A ⁇ 1-42 by preventing B2M from promoting A ⁇ 1-42 oligomerization through transmission electron microscopy (TEM) technology. Scale bar, 1 ⁇ m.
  • TEM transmission electron microscopy
  • Figure 3H In order to further narrow the scope of the amino acid sequence that exerts the inhibitory effect, the inventor further truncated the B2M-3 blocking peptide into three short peptides without overlapping sequences.
  • Figure 3I to Figure 3K Perform the above-mentioned thioflavin-T experiment on three further truncated small peptides to analyze the inhibitory effect of different concentrations of small peptide sequences on B2M's promotion of A ⁇ 1-42 oligomerization. Data represent the average of absolute fluorescence signal intensity. in:
  • FIG. 3I Three different concentrations of the first B2M-3 truncated peptide (B2M-3-1) were pre-incubated with A ⁇ 1-42 for 3 hours, and then the full-length B2M protein was added, and sulfur was continuously detected based on a fluorescent microplate reader. Changes in fluorescence signal of phospho-T.
  • Figure 3J Three different concentrations of the second B2M-3 truncated peptide (B2M-3-2) were pre-incubated with A ⁇ 1-42 for 3 hours, and then the full-length B2M protein was added, and sulfur was continuously detected based on a fluorescent microplate reader. Changes in fluorescence signal of phospho-T.
  • Figure 3K Three different concentrations of the third B2M-3 truncated peptide (B2M-3-3) were pre-incubated with A ⁇ 1-42 for 3 hours, and then the full-length B2M protein was added, and sulfur was continuously detected based on a fluorescent microplate reader. Changes in fluorescence signal of phospho-T.
  • Figure 4A to Figure 4B In vivo Golgi experimental results of the inhibitory effect of B2M-3 blocking peptide on the neurotoxicity produced by B2M promoting A ⁇ 1-42 oligomerization. Pre-incubate A ⁇ 1-42 with B2M-3 blocking peptide or NS peptide for 3 hours at 37°C, then add full-length B2M protein and continue incubating for 72 hours to obtain two oligomerized A ⁇ with different degrees of oligomerization. 1-42 products.
  • oA ⁇ oligomerized A ⁇ 1-42
  • the inventors injected the above two A ⁇ products with different oligomerization states (referred to as oA ⁇ ) into the lateral ventricles of 2-month-old wild-type mice and continued to raise them.
  • brain tissue was collected for Golgi staining experiments to detect the effect of oA ⁇ on dendritic spines in the hippocampus of the brain.
  • the data in Figure 4B were statistically analyzed using one-way ANOVA. ns, no significant difference, P>0.05;*P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001;****P ⁇ 0.001. in:
  • Figure 4A Representative images of Golgi staining in the hippocampus of different treatment groups, scale bar, 10 ⁇ m.
  • Figure 4C to Figure 4D In vitro electrophysiological experimental results of the inhibitory effect of B2M-3 blocking peptide on the neurotoxicity produced by B2M promoting A ⁇ 1-42 oligomerization. Pre-incubate A ⁇ 1-42 with B2M-3 blocking peptide or NS peptide for 3 hours at 37°C, then add full-length B2M protein and continue incubating for 12 hours to obtain two oligomerized A ⁇ with different degrees of oligomerization. 1-42 products.
  • oligomerized A ⁇ 1-42 Based on the neurotoxicity of oligomerized A ⁇ 1-42 , the inventors incubated the above two A ⁇ products with different oligomerization states (abbreviated as oA ⁇ ) in isolated brain slices of 2-month-old wild-type mice for 1.5 Hours later, electrophysiological long-term potentiation (LTP) experiments were performed to detect the effect of oA ⁇ on synaptic plasticity of neural circuits in isolated brain slices. Among them, the data in Figure 4D were statistically analyzed using one-way ANOVA. ns, no significant difference, P>0.05;*P ⁇ 0.05;**P ⁇ 0.01;***P ⁇ 0.001;****P ⁇ 0.001. in:
  • Figure 4D Statistical analysis results of fEPSP amplitude in the last 10 minutes of the LTP recording results in Figure 4A. The n value of each group is the same as in Figure 4C.
  • Figure 4E Statistical analysis results of spontaneous rotation frequency in the Y maze test of 5 ⁇ FAD mice injected with B2M-3 blocking peptide or nonsense peptide.
  • Figure 4F Statistical analysis results of the time to first reach the platform in the water maze test for 5 ⁇ FAD mice injected with B2M-3 blocking peptide or nonsense peptide.
  • Figure 4G Statistical analysis results of the number of platform shuttles in the water maze test of 5 ⁇ FAD mice injected with B2M-3 blocking peptide or nonsense peptide.
  • Figure 4H Statistical analysis results of the time spent in the target quadrant in the water maze test for 5 ⁇ FAD mice injected with B2M-3 blocking peptide or nonsense peptide.
  • Figure 4I to Figure 4K Intracerebral injection of B2M-3 blocking peptide in 5 ⁇ FAD mice significantly reduced the number and area of ⁇ -amyloid plaque deposition in the brains of AD mice.
  • the data in Figures 4J and 4K were analyzed statistically using the Paired t test. ns, no significant difference, P>0.05; *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001. in:
  • Figure 4I ⁇ -deposited in the hippocampal DG area of 5 ⁇ FAD mice injected with B2M-3 blocking peptide or nonsense peptide. Immunofluorescence staining results of amyloid plaques.
  • DAPI 4',6-diamidino-2-phenylindole
  • Scale bar 300 ⁇ m.
  • n 6 mice per group.
  • Figure 4J Statistical analysis results of the number of ⁇ -amyloid plaques in the hippocampal DG area in Figure 4I.
  • Figure 4K Statistical analysis results of ⁇ -amyloid plaque area in the hippocampal DG area in Figure 4I.
  • the 5 ⁇ FAD mice used are transgenic model mice of AD disease. Such mice will show the unique pathological characteristics of ⁇ -amyloid plaques in the brain of AD; 5 ⁇ FAD mice were purchased from Jackson Laboratory (Ellsworth, ME, USA), No. 34840-JAX.
  • the buffer used in all ThT experiments is the same.
  • the specific formula is: 50mM sodium phosphate buffer (pH 7.4), 50mM NaCl, 10 ⁇ M ThT (a dye) and 0.01% Sodium azide.
  • Example 1 The expression of B2M is increased in the brain tissue of patients with Alzheimer's disease, and the content of A ⁇ 1-42 in the brain There is a significant positive correlation with B2M expression
  • the cerebral cortex tissue of AD patients and the brain tissue of non-AD-like dementia controls of the same age were collected respectively (obtained from the National Human Brain Tissue Resource Bank in Health and Disease (Zhejiang University) and the Neurodegeneration Research Center of the University of Science and Technology of China. Before the brain tissue samples were stored in the library, Informed consent has been obtained), the total protein was extracted after tissue grinding and lysis with RIPA protein lysate, BCA concentration was measured and samples were prepared, and then Western blotting was performed.
  • a ⁇ 1-42 ELISA detection is mainly used to detect the content of A ⁇ 1-42 in the brain. This method can quantitatively detect A ⁇ 1-42 per unit weight of brain tissue.
  • a ⁇ 1-42 ELISA was used to test Kit (Thermo Fisher Scientific, Cat. No. KHB3441) was used to measure A ⁇ 1-42 content. Total protein concentration was determined by BCA method.
  • a Based on the standard material provided by the kit, make a standard curve after gradient dilution. Dilute the sample and detect it together with the standard. After the ELISA reaction, use a microplate reader to detect the OD value of each sample, and then calculate the A ⁇ value of each sample based on the standard curve.
  • Example 2 Increasing B2M content in the brain can increase beta-amyloid plaque deposition in the brain
  • a cannula was embedded in the hippocampus area of the brain of 3-month-old 5 ⁇ FAD mice using brain stereotaxy technology, and then the mice were injected with 1 ⁇ l (1 ⁇ g/ ⁇ l) of purified B2M protein or PBS through the cannula every 7 days. Inject continuously for 2 months. After the injection, the mice were anesthetized with 5% chloral hydrate, and cardiac perfusion was performed using phosphate buffer. The brain tissue was removed, fixed in 4% paraformaldehyde overnight, dehydrated with 25% and 30% sucrose solutions, and the brain tissue was analyzed using OCT. After embedding and frozen sections, immunofluorescence staining was performed.
  • DAPI dye 4',6-diamidino-2-phenylindole
  • the purified B2M protein was co-incubated with purified A ⁇ 1-42 protein, and anti-A ⁇ antibody (BioLegend, 6E10) or anti-B2M antibody (abcam, #75853) was used for co-immunoprecipitation, and then immunoprecipitated Blot analysis was used to determine whether there is a direct interaction between the two in vitro.
  • Example 4 The interaction between B2M and A ⁇ 1-42 can promote the oligomerization of A ⁇ 1-42
  • Thioflavin-T is a fluorescent dye that can bind to ⁇ -sheet-rich proteins. After binding, the fluorescence intensity of thioflavin-T will be enhanced, and it is an effective indicator for detecting fibrin formation.
  • the inventor conducted a separate thioflavin-T experiment on the purified B2M protein under the same experimental conditions.
  • Example 5 B2M truncated peptide can be used as a blocking peptide to prevent B2M from binding to A ⁇ 1-42 and inhibit B2M induction.
  • the B2M truncated peptide is pre-incubated with A ⁇ 1-42 .
  • the effective binding of the small peptide can hinder the binding of the full-length B2M protein to A ⁇ 1-42 , thereby inhibiting B2M-induced A ⁇ 1-42 oligomerization.
  • the inventor pre-incubated 10 ⁇ M of the four truncated peptides and the meaningless control peptide with 10 ⁇ M of A ⁇ 1-42 at 37°C for 3 hours, and then added 1 ⁇ M of the full-length B2M protein for thioflavin-T detection.
  • the inventors pre-incubated the B2M-3 peptide with different concentrations (5 ⁇ M, 10 ⁇ M, 20 ⁇ M) and 10 ⁇ M A ⁇ 1-42 at 37°C for 3 hours, then added 1 ⁇ M full-length B2M and then performed thioflavin- T-test.
  • B2M-3 peptide inhibits B2M-promoted A ⁇ oligomerization
  • the inventors used (0.55 ⁇ M) B2M-3 peptide or NS peptide (with His tag) and (1.39 ⁇ M) A ⁇ 1-42 in vitro at 4°C. After incubation under the same conditions for 24 hours, co-immunoprecipitation was performed with an antibody against His-tagged protein, and then Western blotting was used to analyze whether there is a direct interaction between the two in vitro.
  • the inventors used surface plasmon resonance (SPR) technology to flow B2M-3 peptide or NS peptide of different concentrations (5 ⁇ M, 4 ⁇ M, 3 ⁇ M, 2 ⁇ M, 1 ⁇ M) through the A ⁇ 1- 42 protein CM5 chip surface, detect the equilibrium dissociation constant (K D ) of B2M-3 peptide or NS peptide and A ⁇ 1-42 protein at room temperature.
  • SPR surface plasmon resonance
  • a ⁇ 1-42 oligomerizes to form fibrillar oligomers, which are then deposited to form amyloid plaques.
  • 10 ⁇ M A ⁇ 1-42 polypeptide was pre-incubated with 10 ⁇ M B2M-3 peptide at 37°C for 3 hours, and then co-incubated with 1 ⁇ M purified B2M protein at 37°C for 72 hours, and then the sample was spotted on the carbon coating.
  • the samples were stained with 1% uranyl acetate on a layer grid, and images were collected using a Hitachi HT-7800 transmission electron microscope (Hitachi High-Tech, Japan).
  • the experimental results are shown in Figure 3G. The results showed that compared with the control group, pre-incubation with B2M-3 peptide could effectively inhibit the formation of fibrillar oligomers by A ⁇ 1-42 induced by B2M full-length protein.
  • Example 6 B2M-3 blocking peptide blocks B2M-induced oligomerization of A ⁇ 1-42 , thereby inhibiting the neuronal development of A ⁇ 1-42 toxicity
  • Oligomerized A ⁇ 1-42 is highly neurotoxic, and B2M promotes the oligomerization of A ⁇ 1-42 , thereby further enhancing its neurotoxicity. Since B2M-3 blocking peptide can inhibit B2M-induced oligomerization of A ⁇ 1-42 , it is necessary to study whether B2M-3 blocking peptide can inhibit the neurotoxicity of B2M-induced oligomerization of A ⁇ 1-42 .
  • FD Rapid Golgi Stain Kit FD Neuro Technologies, PK401
  • the dendritic spines of neurons are the basic unit of nerve signal transduction.
  • B2M-3 blocking peptide weakens the neurotoxicity of B2M and A ⁇ copolymer
  • the inventors used electrophysiological experiments to detect the specific nerve signal transduction function.
  • the brain tissue was quickly removed and cooled in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF), and then transferred to an oscillating microtome for coronal sectioning.
  • the thickness of the brain slices was 400 ⁇ m.
  • the brain slices were incubated in oxygen-saturated ACSF at 32°C for 1 hour, and then transferred to room temperature for 1 hour.
  • Oligomeric A ⁇ 1-42 was diluted to 200 nM with ACSF and the brain slices were incubated at room temperature for 1.5 hours. Then the brain slice was transferred to the recording slot, the recording electrode was placed in the CA1 area radial layer of the Schaffer collateral-commissural pathway, and the stimulating electrode was placed in the CA3 area.
  • the stimulation intensity is 30% of the maximum value of the excitatory postsynaptic potential (fEPSP).
  • FAS high-frequency stimulation
  • LTP two series of stimulations, Each stimulation train contains 100 stimulation pulses, and each stimulation train is separated by 30 seconds), and the recording lasts for 60 minutes.
  • pre-incubation of A ⁇ 1-42 with B2M-3 blocking peptide can inhibit the neurotoxicity caused by B2M's promotion of A ⁇ 1-42 oligomerization, significantly reduce the loss of dendritic spines in the brain, and allow the related electrophysiological signaling functions to proceed normally. .
  • Examples 5-6 respectively prove that the B2M-3 blocking peptide can inhibit B2M-induced oligomerization of A ⁇ 1-42 under in vivo and in vitro conditions, thereby attenuating the neurotoxicity of oligomerized A ⁇ 1-42.
  • Example 7 B2M-3 blocking peptide can prevent cognitive function defects in 5 ⁇ FAD mice in the mouse brain
  • Example 8 B2M-3 blocking peptide can prevent B2M-induced ⁇ -amyloid plaque formation in mouse brain
  • mice were anesthetized with 5% chloral hydrate, and cardiac perfusion was performed using phosphate buffer.
  • the brain tissue was removed, fixed in 4% paraformaldehyde overnight, dehydrated with 25% and 30% sucrose solutions, and the brain tissue was analyzed using OCT. After embedding and frozen sections, immunofluorescence staining was performed.
  • DAPI dye 4',6-diamidino-2-phenylindole
  • B2M-3 blocking peptide can effectively inhibit B2M-induced ⁇ -amyloid plaque deposition in the brain of 5 ⁇ FAD mice.

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Abstract

本发明属于生物医药领域，涉及β2-微球蛋白封闭肽、其药物组合物及用途。具体地，本发明涉及一种分离的多肽，其为SEQ ID NO:3所示的多肽或SEQ ID NO:3所示多肽的截短片段，其中，所述截短片段包含SEQ ID NO:8所示的多肽。本发明的分离的多肽能够有效地防治AD或者AD所致认知损伤，具有良好的应用前景。

Description

β2-微球蛋白封闭肽、其药物组合物及用途 技术领域
本发明属于生物医药领域，涉及β2-微球蛋白封闭肽、其药物组合物及用途。
背景技术
阿尔兹海默病(Alzheimer’s disease，AD)是人类最常见的神经退行性疾病之一，根据世界阿尔茨海默病2018年报告，2018年全球有5000万人患有AD，到2050年，这一数字将增至1.52亿。该病的主要病理特征包括脑内淀粉样前体蛋白(Amyloid precursor protein,APP)切割产生的β-淀粉样蛋白(Aβ)寡聚化形成的淀粉样沉积、胞内微管结合蛋白tau异常磷酸化后聚集形成的神经纤维缠结(Neurofibrillary tangles,NFTs)、神经元丢失和过度的神经炎症等。AD患者的临床表现主要是记忆力的衰退和认知障碍，并且这种衰退随着病程的发展而恶化，最终丧失一切记忆和生活自理能力直至死亡。鉴于全球老龄化的到来，加之AD发病率的持续升高，以及目前尚未发现有效的治疗措施，致使AD成为严重威胁人类健康的疾病之一。
自1901年发现AD以来，有关其发病机制的研究颇多，然而目前仍未有明确定论。“Aβ级联假说”是AD发病机制的主流学说之一，该学说认为APP异常剪切产生的具有神经毒性的Aβ1-40/42在AD发病过程中发挥核心作用，并且是各种原因诱发AD的共同通路。Aβ寡聚物引起的一系列神经毒性反应能够激发神经炎症，导致神经细胞功能紊乱和神经元丢失，最终引发痴呆。因此减少Aβ在脑内的产生、促进Aβ清除、抑制Aβ聚集以及降低其神经毒性已成为治疗AD的主要措施之一。
β2-微球蛋白(β2-microglobulin，B2M)是近年来逐渐受到广泛关注的促衰老因子，B2M是主要组织相容性复合体I(Major histocompatibility complex I，MHC-I)的组成亚基，由人的15号染色体基因编码，包含119个氨基酸。由于B2M不是通过跨膜结构域锚定在细胞膜上，因此B2M可以从MHC-I复合体上游离下来进入细胞间隙。正常情况下，B2M蛋白以可溶性的单体形式存在，但是在一些病理因素作用下，B2M会发生聚集、沉积。这些病理因素包括衰老、长期的肾功能障碍和炎症等。B2M淀粉样沉积主要存在于骨关节区域，并最终导致严重的关节炎、骨折及腕管综合征。另外，在很多疾病状态下，血清和血浆中B2M的含量均有增加，其中尤为值得关注的是，AD病人血浆和脑脊液中B2M含量均显著高于正常同龄对照组。脑立体定位注射B2M会抑制神经 元再生并损伤小鼠认知功能，而B2m缺失可以促进神经元再生、逆转衰老相关的认知功能缺陷。然而，B2M是否对AD疾病的发生发展产生直接或间接的影响则未见研究报道。
发明内容
本发明人经过深入的研究和创造性的劳动，发现了B2M在AD发生发展过程中的作用，本发明人惊奇地发现，阻碍B2M与Aβ1-42结合的封闭肽具有作为防治AD特别是AD所致认知损伤的药物的潜力。由此提供了下述发明：
本发明的一个方面涉及一种分离的多肽，其为SEQ ID NO:3所示的多肽或SEQ ID NO:3所示多肽的截短片段，其中，所述截短片段包含SEQ ID NO:7或SEQ ID NO:8所示的多肽。
在本发明的一些实施方式中，所述的分离的多肽，其中，所述截短片段不包含SEQ ID NO:3所示多肽的N末端的6个组氨酸(例如，如下面的SEQ ID NOs:16-25中任一序列所示的多肽)。
在本发明的一些实施方式中，所述的分离的多肽，其为SEQ ID NOs:7-8中任一序列所示的多肽。
在本发明的一些实施方式中，所述的分离的多肽，其为SEQ ID NOs:11-15中任一序列所示的多肽：
在本发明的一些实施方式中，所述的分离的多肽，其为SEQ ID NOs:16-25中任一序列所示的多肽：

本发明的另一方面涉及一种分离的多核苷酸，其编码本发明中任一项所述的分离的多肽。
本发明的再一方面涉及一种重组表达载体，其包含本发明的分离的多核苷酸。
本发明的再一方面涉及一种转化的细胞，其包含本发明的重组表达载体。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的分离的多肽。
在本发明的一些实施方式中，所述的药物组合物，其还包含一种或多种药学上可接受的辅料，例如载体或赋形剂；具体地，其为注射剂、口服液、胶囊、片剂、颗粒剂、丸剂或提取物再混合剂型。
可采用常规加工方法，制成包含本发明多肽和辅料的注射液、口服液、胶囊、片剂、颗粒剂、丸剂、提取物再混合等剂型。
通常本发明药物组合物含有0.1－90重量％的有效成分。药物组合物可根据本领域已知的方法制备。用于此目的时，如果需要，可将有效成分与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人用的适当的施用形式或剂量形式。
本发明的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。给药剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、 氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将给药单元制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将给药单元制成胶囊，将有效成分与上述的各种载体混合，并将由此得到的混合物置于硬的明明胶囊或软胶囊中。也可将有效成分制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中或制成注射剂应用。为了将给药单元制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
本发明的再一方面涉及本发明中任一项所述的分离的多肽在制备治疗或预防阿尔兹海默病的药物中的用途。
本发明的再一方面涉及本发明中任一项所述的分离的多肽在制备如下药物中的用途：
抑制B2M诱导的Aβ1-42寡聚化的药物、抑制B2M诱导的β-淀粉样斑块的形成的药 物或者抑制Aβ1-42的神经毒性的药物。
本发明首次发现AD病人脑组织内B2M表达显著升高，并且脑内B2M表达水平与Aβ1-42含量之间存在显著的正相关性，通过脑立体定位注射进一步增加AD小鼠模型(5×FAD)脑中B2M含量，能够增加5×FAD小鼠脑内β-淀粉样斑沉积。进一步地，本发明人发现B2M与Aβ1-42存在直接的相互作用，使用截短的B2M氨基酸序列作为封闭肽，能够抑制B2M和Aβ1-42聚集，抑制B2M和Aβ1-42神经毒性，体内实验表明封闭肽能够显著逆转5×FAD小鼠的认知功能损伤、抑制脑内B2M对Aβ1-42聚集的促进作用，进而减少脑内β-淀粉样斑沉积。本发现为AD的临床治疗提供了一个潜在的药物靶点以及基于该靶点的新的治疗方式。
根据本发明中任一项所述的分离的多肽，其用于治疗或预防阿尔兹海默病。
根据本发明中任一项所述的分离的多肽，其用于抑制B2M诱导的Aβ1-42寡聚化、抑制B2M诱导的β-淀粉样斑块的形成或者用于抑制Aβ1-42的神经毒性。
本发明的再一方面涉及一种治疗或预防阿尔兹海默病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的分离的多肽的步骤。
本发明的再一方面涉及一种抑制B2M诱导的Aβ1-42寡聚化或者抑制B2M诱导的β-淀粉样斑块或者抑制Aβ1-42的神经毒性的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的分离的多肽的步骤。
本发明的多肽或药物组合物的给药剂量取决于许多因素，例如所要预防或治疗阿尔兹海默病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。剂量水平须根据具体的给药途径、所治疗病况的严重程度以及待治疗患者的病况和既往病史等来选定。但是，本领域的做法是，给药剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
但应认识到，本发明的多肽或药物组合物的总日用量须由主诊医师在可靠的医学判断范围内作出决定。对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；给药时间、给药途径和排泄率；治疗持续时间；与组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，给药的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂 量，直到得到所需的效果。一般说来，本发明的药物组合物以有效成分计算用于哺乳动物特别是人的剂量可以介于0.001－1000mg/kg体重/天，例如介于0.01－800mg/kg体重/天，例如介于0.01－500mg/kg体重/天。
在本发明的一些实施方式中，所述β2-微球蛋白的氨基酸序列如SEQ ID NO:9所示。
人B2M蛋白的氨基酸序列(N端至C端)如下：
在本发明中，当提及β2-微球蛋白(B2M)的氨基酸序列时，其包括β2-微球蛋白的全长，还包括其融合蛋白。然而，本领域技术人员理解，在β2-微球蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。在本发明的一个实施方案中，β2-微球蛋白为人β2-微球蛋白。在本发明的一个实施方案中，β2-微球蛋白的氨基酸序列如SEQ ID NO:9所示。
在本发明中，术语“宿主细胞”指的是导入载体的细胞，包括如下许多细胞类型，如大肠杆菌或枯草菌等原核细胞，如酵母细胞或曲霉菌等真菌细胞，如S2果蝇细胞或Sf9等昆虫细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞，或动物细胞例如人细胞。
在本发明中，术语“载体”指的是，可将抑制某蛋白的多核苷酸插入其中的一种核酸运载工具。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件。
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。
术语“受试者”可以指患者或者其它接受本发明药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
术语“封闭肽”是指这样一种多肽，其能够与全长B2M蛋白竞争性结合Aβ，从而抑制B2M结合Aβ后促进Aβ寡聚化的生物学效应。
本发明中，如果没有特别说明，浓度单位μM表示μmol/L，mM表示mmol/L，nM表示nmol/L。
本发明中，提到细胞中的加药量时，如果没有特别说明，一般是指加药后药物的终浓度。
Aβ1-42的氨基酸序列如下：
发明的有益效果
本发明取得了如下的技术效果(1)-(4)项中的一项或多项：
(1)本发明的多肽能够有效地预防和/或治疗阿尔兹海默病。
(2)本发明的多肽能够有效地抑制B2M诱导的Aβ1-42寡聚化。
(3)本发明的多肽能够有效地抑制B2M诱导的β-淀粉样斑块的形成。
(4)本发明的多肽能够有效地抑制Aβ1-42的神经毒性。
附图说明
图1A至图1B：AD病人脑内B2M表达水平显著增加。其中：
图1A：AD病人大脑皮层组织中B2M表达免疫印迹检测结果。
图1B：Image J分析图1A中B2M表达水平，对照组n＝8个人脑组织，AD组n＝21个人脑组织。数据采用student t test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。
图1C：通过Aβ1-42ELISA检测了上述29个人脑组织中的Aβ1-42水平，通过Pearson相关性分析发现人脑内的Aβ1-42水平与人脑内B2M表达水平之间存在显著的正相关性。
图1D至图1F:增加5×FAD小鼠脑内B2M水平能够促进脑内淀粉样斑块沉积。通过脑立体定位注射向4月龄5×FAD小鼠大脑海马内注射1μl(1μg/μl)的纯化的B2M蛋白或1μl PBS(每只小鼠自身大脑左右对照)，注射2个月后用5％水合氯醛麻醉小鼠，使用磷酸盐缓冲液进行心脏灌注，取脑组织，于4％多聚甲醛固定过夜，经25％ 和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，冰冻切片后，进行免疫荧光染色，染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)标记细胞核、抗体6E10标记Aβ，通过激光共聚焦荧光显微镜采集图像。标尺，300μm。数据代表平均值±标准误。其中：
图1D：5×FAD小鼠脑组织淀粉样斑免疫荧光染色结果。每组n＝6只小鼠。其中，虚线框指征海马DG区域范围，也代表统计的斑块所在的脑组织区域。
图1E：图1D中海马DG区β-淀粉样斑数量(Number of Aβdepositis)统计结果。数据采用Paired t test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。
图1F：图1D中海马DG区β-淀粉样斑面积(Area of Aβdepositis)统计结果。数据采用Paired t test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。
图2A至图2B：体外纯化的B2M蛋白与纯化的Aβ1-42蛋白之间相互作用的免疫共沉淀(co-IP)实验结果。其中：
图2A：用抗Aβ的抗体免疫沉淀Aβ1-42与B2M。
图2B：用抗B2M的抗体免疫沉淀B2M与Aβ1-42。
图2C：用12月龄野生型(wild type，WT)和5×FAD小鼠脑组织做体内B2M与Aβ1-42相互作用的co-IP实验结果，所用抗体为抗Aβ的抗体。
图2D：用硫黄素-T指征体外B2M孵育Aβ1-42后促进Aβ1-42寡聚化的荧光酶标仪连续检测结果。在37℃条件下，将不同浓度的全长B2M蛋白与Aβ1-42共孵育，同时在实验体系内加入硫黄素-T，用荧光酶标仪在激发光波长λ＝448nm和吸收光波长λ＝488nm的条件下每5min检测一次硫黄素-T荧光信号值的变化情况。试验结束后绘制荧光信号值随时间变化的曲线图。数据代表绝对荧光信号强度的平均值。
图2E：与图2D相同实验条件下，对应浓度的B2M蛋白的硫黄素-T荧光检测结果。数据代表绝对荧光信号强度的平均值。
图3A：将B2M氨基酸全长序列截短为4条无重叠序列的短肽的节段示意图。
图3B：截短的B2M小肽抑制B2M促进Aβ1-42寡聚化的硫黄素-T实验结果。将截短的B2M氨基酸序列作为封闭肽与Aβ1-42在37℃条件下预孵育3小时，对照组用无意义肽序列(NS)与Aβ1-42预孵育，然后再加入全长的B2M蛋白继续孵育Aβ1-42，通过分析硫黄素-T荧光信号强度的变化分析封闭肽对B2M促进Aβ1-42寡聚化抑制效 果。数据代表绝对荧光信号强度的平均值。
图3C：基于上述的硫黄素-T实验检测不同浓度B2M-3封闭肽对B2M促进Aβ1-42寡聚化的阻抑效果。数据代表绝对荧光信号强度的平均值。
图3D：用抗His的抗体免疫沉淀与B2M-3肽结合的Aβ。
图3E：用表面等离子共振技术检测B2M-3肽与Aβ1-42之间的平衡解离常数KD。
图3F：用表面等离子共振技术检测NS肽与Aβ1-42之间的平衡解离常数KD。
图3G：通过透射电镜(TEM)技术检测B2M-3封闭肽阻碍B2M促进Aβ1-42寡聚化对Aβ1-42形成的纤维丝的形态影响。标尺，1μm。
图3H：为了进一步缩小发挥阻抑效果的氨基酸序列范围，本发明人将B2M-3封闭肽进一步截短为3条无重叠序列的短肽的节段示意图。
图3I至图3K：针对3个进一步截短的小肽做上述的硫黄素-T实验，分析不同浓度的小肽序列对B2M促进Aβ1-42寡聚化的抑制效果。数据代表绝对荧光信号强度的平均值。其中：
图3I：三个不同浓度的第一段B2M-3截短小肽(B2M-3-1)预孵育Aβ1-42 3小时，然后再加入全长的B2M蛋白，基于荧光酶标仪连续检测硫黄素-T荧光信号的变化情况。
图3J：三个不同浓度的第二段B2M-3截短小肽(B2M-3-2)预孵育Aβ1-42 3小时，然后再加入全长的B2M蛋白，基于荧光酶标仪连续检测硫黄素-T荧光信号的变化情况。
图3K：三个不同浓度的第三段B2M-3截短小肽(B2M-3-3)预孵育Aβ1-42 3小时，然后再加入全长的B2M蛋白，基于荧光酶标仪连续检测硫黄素-T荧光信号的变化情况。
图4A至图4B：B2M-3封闭肽对B2M促进Aβ1-42寡聚化所产生的神经毒性的抑制效果的体内高尔基实验结果。在37℃条件下，用B2M-3封闭肽或NS肽预孵育Aβ1-423小时，然后再加入全长的B2M蛋白继续孵育72小时，得到两种不同寡聚化程度的寡聚化Aβ1-42产物。基于寡聚化Aβ1-42具有神经毒性，本发明人将上述两种不同寡聚化状态的Aβ产物(简称为oAβ)分别注射到2月龄的野生型小鼠的侧脑室内，继续饲养14天后收脑组织做高尔基染色实验，检测oAβ对脑内海马区树突棘的影响。其中，图4B数据采用one-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。其中：
图4A：不同处理组海马区高尔基染色代表图，标尺，10μm。
图4B：图4A中高尔基染色的统计分析结果。每组n＝4只小鼠，每组统计神经突数目见图上标注。
图4C至图4D：B2M-3封闭肽对B2M促进Aβ1-42寡聚化所产生的神经毒性的抑制效果的体外电生理实验结果。在37℃条件下，用B2M-3封闭肽或NS肽预孵育Aβ1-423小时，然后再加入全长的B2M蛋白继续孵育12小时，得到两种不同寡聚化程度的寡聚化Aβ1-42产物。基于寡聚化Aβ1-42具有神经毒性，本发明人将上述两种不同寡聚化状态的Aβ产物(简称为oAβ)分别孵育2月龄的野生型小鼠的离体脑片，孵育1.5小时后做电生理的长时程增强(LTP)实验，检测oAβ对离体脑片神经环路的突触可塑性的影响。其中，图4D数据采用one-way ANOVA进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001。其中：
图4C：离体脑片CA1区LTP记录分析结果。oAβ处理组每组n＝4只小鼠，每组记录脑片数n＝8。单纯肽处理的对照组每组n＝4只小鼠，每组记录脑片数n＝7。
图4D：图4A中LTP记录结果最后10min fEPSP振幅统计分析结果。每组n值同图4C。
图4E至图4H：5×FAD小鼠脑内注射B2M-3封闭肽后显著逆转AD小鼠认知行为损伤。每组n＝8只小鼠。数据采用Paired t test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01。其中：
图4E：注射B2M-3封闭肽或无意义肽的5×FAD小鼠在Y迷宫测试中自发轮转频率的统计分析结果。
图4F：注射B2M-3封闭肽或无意义肽的5×FAD小鼠在水迷宫测试中到首次到达平台的时间的统计分析结果。
图4G：注射B2M-3封闭肽或无意义肽的5×FAD小鼠在水迷宫测试中在平台穿梭次数的统计分析结果。
图4H：注射B2M-3封闭肽或无意义肽的5×FAD小鼠在水迷宫测试中在目标象限停留时间的统计分析结果。
图4I至图4K：5×FAD小鼠脑内注射B2M-3封闭肽后显著减少AD小鼠脑内β-淀粉样斑沉积数量和斑块沉积面积。其中，图4J和4K数据采用Paired t test进行统计分析。ns，无显著性差异，P>0.05；*P<0.05；**P<0.01；***P<0.001。其中：
图4I：注射B2M-3封闭肽或无意义肽的5×FAD小鼠脑内海马DG区沉积的β- 淀粉样斑免疫荧光染色结果。染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)标记细胞核、抗体6E10标记Aβ。标尺，300μm。每组n＝6只小鼠。
图4J：图4I中海马DG区β-淀粉样斑数量统计分析结果。
图4K：图4I中海马DG区β-淀粉样斑面积的统计分析结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
下面的实施例中，用到的5×FAD小鼠是AD疾病的转基因模型小鼠，这种小鼠脑内会表现出AD特有的β-淀粉样斑病理特征；5×FAD小鼠购买于Jackson Laboratory(Ellsworth,ME,USA)，编号34840-JAX。
下面的实施例中，所有ThT实验(硫黄素-T检测)用的buffer均相同，具体的配方为：50mM磷酸钠缓冲液(pH 7.4)、50mM NaCl、10μM ThT(一种染料)和0.01％叠氮化钠。
实施例1：B2M在阿尔兹海默氏病患者脑组织中表达量增加，且脑内Aβ1-42含量 与B2M表达量存在显著正相关性
分别取AD患者大脑皮层组织和同龄非AD样痴呆对照人脑组织(从国家健康和疾病人脑组织资源库(浙大)和中国科学技术大学神经退行性研究中心获得，在脑组织样品入库前已获得知情同意书)，经组织研磨、RIPA蛋白裂解液裂解后提取总蛋白、BCA测浓度制样，然后进行免疫印迹检测。
结果如图1A至图1B所示。结果表明，相对于对照组，AD患者大脑皮层中B2M蛋白显著升高。同时检测的突触前囊泡蛋白(synaptophysin)在AD患者脑组织内的表达量显著低于对照组水平。
Aβ1-42ELISA检测主要用于检测脑内Aβ1-42含量，该方法可以对单位重量脑组织内的Aβ1-42进行定量检测。针对上述免疫印迹实验所用人脑组织，用Aβ1-42ELISA试 剂盒(Thermo Fisher Scientific公司，货号KHB3441)测定Aβ1-42含量。BCA法测定总蛋白浓度。
将人脑内的Aβ1-42含量与相对B2M表达量之间做Pearson相关性分析。结果如图1C所示。结果表明，人脑组织内的Aβ1-42含量a与脑内相对B2M表达量b之间存在显著的正相关性。
a：基于试剂盒提供的标准品，梯度稀释后做标准曲线。将样品稀释后与标准品一同检测，ELISA反应结束后用酶标仪检测每个样品的OD值，然后基于标准曲线，计算每个样品的Aβ值。本次实验标准曲线为y＝154.21x+5.4233，R2＝0.9932，y值为Aβ含量，x值为OD值，对照样品检测前稀释40倍，AD人脑样品检测前稀释200倍。
b：这里的相对表达量结果基于图1A的胶图，数据来源于图1B的统计结果。以对照组人脑内B2M表达量(蛋白条带灰度值)的平均值为基础值，每个样品均与基础值相比，得到的值即为相对表达量变化倍数。
实施例2：增加脑内B2M含量能够增加脑内的β-淀粉样斑块沉积
通过脑立体定位技术给3月龄的5×FAD小鼠大脑的海马区埋置套管，然后每隔7天通过套管给小鼠注射1μl(1μg/μl)的纯化的B2M蛋白或PBS，连续注射2个月。注射结束后用5％水合氯醛麻醉小鼠，使用磷酸盐缓冲液进行心脏灌注，取脑组织，于4％多聚甲醛固定过夜，经25％和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，冰冻切片后，进行免疫荧光染色，染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)标记细胞核、抗体6E10(Biolegend公司，货号803016)标记Aβ，通过激光共聚焦荧光显微镜采集图像。
结果如图1D至图1F所示，与注射PBS相比，注射B2M蛋白能够显著增加脑内β-淀粉样斑块的沉积。
结果表明，AD小鼠脑内B2M蛋白水平升高，且增加脑内B2M含量能够进一步增加AD小鼠脑内β-淀粉样斑块的沉积。
实施例3：B2M与Aβ1-42之间存在相互作用
在体外条件下，将纯化的B2M蛋白与纯化的Aβ1-42蛋白共孵育，用抗Aβ的抗体(BioLegend，6E10)或抗B2M的抗体(abcam，#75853)做免疫共沉淀，然后经免疫印迹分析二者之间在体外是否存在直接的相互作用情况。
实验结果如图2A至图2B所示。结果显示，用抗Aβ的抗体(6E10)或者用抗B2M的抗体做免疫共沉淀均能将Aβ1-42和B2M两种纯化蛋白一起沉淀下来。
进一步地，取12月龄的WT和5×FAD小鼠大脑皮层组织，经组织裂解后，用抗Aβ的抗体(6E10)做免疫共沉淀，然后经免疫印迹分析二者之间在体内是否存在直接的相互作用情况。
实验结果如图2C所示。结果显示，用抗Aβ的抗体(6E10)做免疫共沉淀能将5×FAD小鼠脑内的Aβ1-42和B2M一起沉淀下来。
结果表明，在体内、体外两种条件下，B2M与Aβ1-42之间均存在相互作用。
实施例4：B2M与Aβ1-42之间的相互作用能够促进Aβ1-42的寡聚化
硫黄素-T是一种荧光染料，可以与富含β折叠的蛋白结合，结合后，硫黄素T的荧光强度会增强，是一个用来检测纤维原形成的有效指标。在存在有硫黄素-T的情况下，将纯化的B2M与Aβ1-42两种蛋白在37℃条件下共孵育，同时用荧光酶标仪每隔5min读取一次荧光值(激发光λ＝448nm，吸收光λ＝488nm)，连续检测12-14小时。实验结束后基于荧光值随时间的变化情况绘制曲线图，用于反应体系内Aβ1-42原纤维的形成情况。
实验结果如图2D所示。结果显示，B2M蛋白孵育Aβ1-42后，能够促进Aβ1-42原纤维的形成，并且表现出一定的浓度依赖性。
另外，由于考虑到B2M蛋白其自身也具有一定的寡聚化能力，本发明人在相同的实验条件下，将纯化的B2M蛋白单独做硫黄素-T实验。
实验结果如图2E所示。结果显示，相同实验条件下，单独的B2M蛋白并不会发生寡聚化，不产生原纤维，不会干扰图2D中的实验结果。
结果表明，B2M与Aβ1-42之间的相互作用能够促进Aβ1-42的寡聚化。
实施例5：B2M截短肽可以作为封闭肽，阻碍B2M与Aβ1-42结合，抑制B2M诱 导的Aβ1-42寡聚化
为了明确B2M与Aβ1-42发生相互作用的氨基酸区域，如图3A所示，本发明人将全长的人B2M序列截短为4个无重叠序列的小肽，分别命名为B2M-1、B2M-2、B2M-3、B2M-4(详细序列见下面的表1)。合成表1中的多肽。
表1
用B2M截短肽与Aβ1-42预孵育，发生有效结合的小肽能够阻碍全长的B2M蛋白与Aβ1-42的结合，进而抑制B2M诱导的Aβ1-42寡聚化。本发明人将10μM的四种截短肽和无意义对照肽分别与10μM的Aβ1-42在37℃条件下预孵育3小时，然后加入1μM全长B2M蛋白做硫黄素-T检测。
实验结果如图3B所示，结果显示，只有第三条B2M-3肽能够有效阻碍B2M与Aβ1-42结合，抑制B2M诱导的Aβ1-42寡聚化。
进一步地，本发明人用不同浓度(5μM、10μM、20μM)的B2M-3肽与10μM的Aβ1-42在37℃条件下预孵育3小时，然后加入1μM全长B2M后再次做硫黄素-T检测。
实验结果如图3C所示。结果显示，B2M-3肽能够有效阻碍B2M诱导的Aβ1-42寡聚化，并且表现出一定的浓度依赖性。
为了明确B2M-3肽抑制B2M促进Aβ寡聚化的具体机制，本发明人用(0.55μM)B2M-3肽或NS肽(带His标签)与(1.39μM)Aβ1-42在体外4℃条件下共孵育24小时，然后通过抗His标签蛋白的抗体做免疫共沉淀，然后经免疫印迹分析二者之间在体外是否存在直接的相互作用。
实验结果如图3D所示。结果显示，用His标签的抗体做免疫共沉淀能够将B2M-3肽与Aβ1-42一起沉淀下来。
进一步地，本发明人采用表面等离子共振(Surface Plasmon Resonance，SPR)技术，将不同浓度(5μM、4μM、3μM、2μM、1μM)的B2M-3肽或NS肽分别流过包被有Aβ1-42蛋白的CM5芯片表面，检测在室温条件下B2M-3肽或NS肽与Aβ1-42蛋白的平衡解离常数(KD)。
实验结果如图3E至图3F所示。结果显示，B2M-3肽与Aβ1-42蛋白的KD值约为0.542±0.067μM，而NS肽与Aβ1-42蛋白则无结合。
Aβ1-42通过寡聚化形成纤维状的寡聚物，继而沉积形成淀粉样斑。将10μM的Aβ1-42多肽与10μM B2M-3肽在37℃条件下预孵育3小时，然后再与纯化的1μM的B2M蛋白在37℃条件下共孵育72小时，然后将样品点在碳涂层网格上，再用1％醋酸铀酰对样品进行染色，通过Hitachi HT-7800透射电镜(日立高新技术，日本)采集图像。实验结果如图3G所示。结果显示，与对照组相比较，B2M-3肽预孵育后，能够有效抑制B2M全长蛋白诱导Aβ1-42形成纤维状寡聚物。
如图3H所示，在B2M-3肽的基础上，本发明人进一步缩短氨基酸序列以缩小有效结合范围。B2M-3截短的详细序列见下面的表2。
表2
合成小肽后，采用上述同样的实验方法，本发明人将不同浓度(1μM、5μM、10μM)的B2M-3截短的三条小肽序列(B2M-3-1，B2M-3-2和B2M-3-3)分别与10μM的Aβ1-42预孵育，然后加入1μM的B2M全长蛋白做硫黄素-T检测。实验结果如图3I至图3K所示。结果显示，B2M-3-2肽和B2M-3-3肽均能够有效抑制B2M诱导的Aβ1-42寡聚化，并具有一定的浓度依赖性。
结果表明，B2M与Aβ1-42相互作用的氨基酸区域主要位于B2M蛋白的第三段截短范围内，并且B2M-3肽通过与Aβ1-42的直接结合抑制B2M与Aβ的共聚集。进一步的实验研究结果表明，将B2M-3肽、B2M-3-2肽或B2M-3-3肽作为封闭肽均能够有效抑制B2M诱导的Aβ1-42寡聚化。
实施例6：B2M-3封闭肽阻碍B2M诱导的Aβ1-42寡聚化，进而抑制Aβ1-42的神经 毒性
寡聚化的Aβ1-42具有很强的神经毒性，而B2M促进Aβ1-42的寡聚化，从而进一步增强其神经毒性。基于B2M-3封闭肽能够抑制B2M诱导的Aβ1-42寡聚化，因此有必要研究B2M-3封闭肽是否能够抑制B2M诱导的寡聚化的Aβ1-42的神经毒性。
分别用10μM的B2M-3肽或无意义对照肽与10μM的Aβ1-42在37℃条件下预孵育3小时，然后加入1μM的全长B2M蛋白继续孵育12小时，分别得到两种Aβ1-42寡聚化程度不同的孵育产物。
2-3月WT小鼠麻醉后，经脑立体定位注射将两种Aβ1-42寡聚化程度不同的孵育产物以及无Aβ1-42的对照组蛋白孵育产物分别注射到小鼠侧脑室内(每组n＝4只小鼠)，随后缝合创口继续饲养。注射后第14天，用异氟烷快速麻醉小鼠，颈椎脱臼后快速收取脑组织，用商品化的高尔基染色试剂盒(FD Rapid Golgi Stain Kit,FD Neuro Technologies,PK401)做高尔基染色，通过激光共聚焦荧光显微镜采集图像。
实验结果如图4A至图4B所示。结果显示，与单纯的Aβ寡聚物相比，共孵育了NS肽和B2M产生的Aβ寡聚物会导致海马内树突棘丢失更多，即NS肽未能阻碍B2M增强Aβ神经毒性；相反，在注射了与B2M-3封闭肽和B2M共孵育产生的Aβ寡聚物的海马内，其树突棘丢失显著减少，也即B2M-3封闭肽显著降低B2M与Aβ共聚物的神经毒性。
神经元的树突棘是神经信号转导的基础单位，为了进一步验证B2M-3封闭肽削弱B2M与Aβ共聚物神经毒性，本发明人采用电生理实验检测具体的神经信号传导功能。
2月龄WT小鼠经麻醉后，快速取出脑组织置于冰冷且通氧的人工脑脊液(ACSF)中冷却，随后转至振荡切片机进行冠状切片，脑片厚度为400μm。将脑片置于32℃氧饱和的ACSF中孵育1小时，之后转移至室温孵育1小时。将寡聚化的Aβ1-42用ACSF稀释到200nM，在室温条件下孵育脑片1.5小时。然后将脑片转移至记录槽，将记录电极放置在Schaffer collateral-commissural通路的CA1区辐射层，刺激电极放置在CA3区。刺激强度为兴奋性突触后场电位(field excitatory postsynaptic potential，fEPSP)最大值的30％，fEPSP基线稳定记录20分钟后，高频刺激(HFS)诱导长时程增强即LTP(2串刺激，每串刺激包含100个刺激脉冲，每串刺激间隔30秒)，持续记录60分钟。
实验结果如图4C至图4D所示。结果显示，相对于用单纯小肽孵育的脑片相比， 用无意义肽预孵育产生的寡聚化的Aβ1-42会显著损伤WT小鼠的海马CA3区至CA1区Schaffer collateral-commissural通路的LTP，而用B2M-3封闭肽预孵育产生的寡聚化的Aβ1-42对WT小鼠的LTP无明显的损伤作用。
综上，用B2M-3封闭肽预孵Aβ1-42能够抑制B2M促进Aβ1-42寡聚化产生的神经毒性，脑内树突棘丢失显著减少，相关的电生理信号传导功能得以正常进行。
实施例5-6的结果分别证明在体内、体外条件下B2M-3封闭肽能够抑制B2M诱导的Aβ1-42寡聚化，进而减弱寡聚化Aβ1-42的神经毒性。
实施例7：B2M-3封闭肽能够在小鼠脑内阻碍5×FAD小鼠认知功能缺陷
为了进一步验证B2M-3封闭肽在体内对5×FAD小鼠认知功能损伤的抑制效果，本发明人通过脑立体定位技术给B2M人源化的5×FAD小鼠大脑的海马区域埋置套管，随后每周通过套管给小鼠的海马分别注射2μl(5μg/μl)的无意义肽或B2M-3封闭肽(每组n＝8只小鼠)，连续注射2个月。注射结束后对小鼠进行认知功能相关的行为学测试，包括Y迷宫和Morris water maze水迷宫实验。行为学测试图像的采集和记录采用Noldus EthoVision XT(Ugo Basile,Italy)软件系统进行。
实验结果如图4E至图4H所示。结果显示，与注射无意义肽相比，注射B2M-3封闭肽能够显著逆转5×FAD小鼠的认知功能损伤。
实施例8：B2M-3封闭肽能够在小鼠脑内阻碍B2M诱导的β-淀粉样斑块的形成
为了进一步验证B2M-3封闭肽在体内对B2M诱导β-淀粉样斑块形成的抑制效果，本发明人通过脑立体定位技术给B2M人源化的5×FAD小鼠大脑的海马区域埋置套管，随后每周通过套管给小鼠的左右海马分别注射1μl(5μg/μl)的无意义肽或B2M-3封闭肽(n＝5只小鼠，每只小鼠左右脑自身对比)，连续注射3个月。注射结束后用5％水合氯醛麻醉小鼠，使用磷酸盐缓冲液进行心脏灌注，取脑组织，于4％多聚甲醛固定过夜，经25％和30％蔗糖溶液脱水，使用OCT进行脑组织包埋，冰冻切片后，进行免疫荧光染色，染料4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)标记细胞核、抗体6E10(Biolegend公司，货号803016)标记Aβ1-42，通过激光共聚焦荧光显微镜采集图像。
实验结果如图4I至图4K所示。结果显示，与注射无意义肽相比，注射B2M-3封闭肽能够显著减少脑内β-淀粉样斑块的沉积。
综上，B2M-3封闭肽在5×FAD小鼠脑内能够有效抑制B2M诱导的β-淀粉样斑块的沉积。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (14)

  1. 分离的多肽，其为SEQ ID NO:3所示的多肽或SEQ ID NO:3所示多肽的截短片段，其中，所述截短片段包含SEQ ID NO:7或SEQ ID NO:8所示的多肽。
  2. 根据权利要求1所述的分离的多肽，其中，所述截短片段不包含SEQ ID NO:3所示多肽的N末端的6个组氨酸。
  3. 根据权利要求1或2所述的分离的多肽，其为SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:11至SEQ ID NO:25中任一序列所示的多肽。
  4. 分离的多核苷酸，其编码权利要求1至3中任一权利要求所述的分离的多肽。
  5. 一种重组表达载体，其包含权利要求4所述的分离的多核苷酸。
  6. 一种转化的细胞，其包含权利要求5所述的重组表达载体。
  7. 一种药物组合物，其包含权利要求1至3中任一权利要求所述的分离的多肽。
  8. 根据权利要求7所述的药物组合物，其还包含一种或多种药学上可接受的辅料。
  9. 权利要求1至3中任一权利要求所述的分离的多肽在制备治疗或预防阿尔兹海默病的药物中的用途。
  10. 权利要求1至3中任一权利要求所述的分离的多肽在制备如下药物中的用途：
    抑制B2M诱导的Aβ1-42寡聚化的药物、抑制B2M诱导的β-淀粉样斑块的形成的药物或者抑制Aβ1-42的神经毒性的药物。
  11. 根据权利要求1至3中任一权利要求所述的分离的多肽，其用于治疗或预防阿尔兹海默病。
  12. 根据权利要求1至3中任一权利要求所述的分离的多肽，其用于抑制B2M诱导的Aβ1-42寡聚化、抑制B2M诱导的β-淀粉样斑块的形成或者用于抑制Aβ1-42的神经毒性。
  13. 一种治疗或预防阿尔兹海默病的方法，包括给予有需求的受试者以有效量的权利要求1至3中任一权利要求所述的分离的多肽的步骤。
  14. 一种抑制B2M诱导的Aβ1-42寡聚化或者抑制B2M诱导的β-淀粉样斑块或者抑制Aβ1-42的神经毒性的方法，包括给予有需求的受试者以有效量的权利要求1至3中任一权利要求所述的分离的多肽的步骤。
PCT/CN2023/097492 2022-05-31 2023-05-31 β2-微球蛋白封闭肽、其药物组合物及用途 WO2023232083A1 (zh)

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STEFAN SCHAUB; JOHN A. WILKINS; MIHAELA ANTONOVICI; OLEG KROKHIN; TRACEY WEILER; DAVID RUSH; PETER NICKERSON: "Proteomic‐Based Identification of Cleaved Urinary β2‐microglobulin as a Potential Marker for Acute Tubular Injury in Renal Allografts", AMERICAN JOURNAL OF TRANSPLANTATION, BLACKWELL MUNKSGAARD, DK, vol. 5, no. 4, 10 March 2005 (2005-03-10), DK , pages 729 - 738, XP072340692, ISSN: 1600-6135, DOI: 10.1111/j.1600-6143.2005.00766.x *

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