WO2023184473A1 - 肽用于治疗神经退行性疾病或改善认知功能的用途 - Google Patents

肽用于治疗神经退行性疾病或改善认知功能的用途 Download PDF

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WO2023184473A1
WO2023184473A1 PCT/CN2022/084784 CN2022084784W WO2023184473A1 WO 2023184473 A1 WO2023184473 A1 WO 2023184473A1 CN 2022084784 W CN2022084784 W CN 2022084784W WO 2023184473 A1 WO2023184473 A1 WO 2023184473A1
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amino acid
peptide
disease
acid sequence
seq
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PCT/CN2022/084784
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English (en)
French (fr)
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刘承鑫
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首创生物技术有限公司
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Priority to PCT/CN2022/084784 priority Critical patent/WO2023184473A1/zh
Priority to TW112112599A priority patent/TW202340233A/zh
Priority to PCT/CN2023/085334 priority patent/WO2023186062A1/zh
Publication of WO2023184473A1 publication Critical patent/WO2023184473A1/zh

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • 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

Definitions

  • the present invention relates to the treatment of neurodegenerative diseases and cognitive impairment; and the improvement and improvement of cognitive function.
  • the present invention relates to the use of peptides for the prevention or treatment of neurodegenerative diseases and cognitive disorders, preferably Alzheimer's disease and Parkinson's disease.
  • the invention also relates to the use of peptides for improving or enhancing cognitive function in a subject.
  • Neurodegenerative disease or neurodegenerative disease is a disease directly caused by the gradual degeneration of neurons. Degenerative processes may involve progressive loss of neuronal structure, progressive loss of neuronal function, or progressive neuronal cell death. This progressive neurodegeneration often leads to physical disability and mental deterioration. Many neurodegenerative diseases are severe, progressive and ongoing diseases with few treatments. Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most important neurodegenerative diseases.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • Neurocognitive Disorder also known as Dementia
  • Dementia is a type of brain disease that causes long-term and gradual deterioration of thinking ability and memory, and affects a person's daily life activities.
  • the most common type of dementia is Alzheimer's disease, which accounts for 50 to 70 percent of all dementia patients.
  • Dementia affects 36 million people worldwide. About 10% of the population will develop the disease during their lifetime.
  • Dementia is closely related to age (aging), with approximately 3% of the population developing dementia between the ages of 65 and 74, another 19% between the ages of 75 and 84, and nearly half of the population over the age of 85 developing dementia. .
  • AD Alzheimer's disease
  • the most common early symptom is loss of short-term memory.
  • symptoms may gradually appear, including language difficulties, disorientation, mood swings, loss of motivation, inability to care for oneself, and many behavioral problems.
  • patients often become disconnected from family or society, gradually lose physical function, and eventually die.
  • the course of the disease varies from person to person, the average life expectancy after diagnosis is about three to nine years.
  • AD Alzheimer's disease
  • a ⁇ beta-amyloid
  • microglial activation is involved in the spread of tau tangles in the neocortex in Alzheimer's disease, which in turn leads to the occurrence of cognitive dysfunction in Alzheimer's disease patients (see Tharick A .Pascoal et al., Microglial activation and tau propagate jointly across Braak stages, Nature Medicine, 27, pages 1592–1599 (2021 August)).
  • Microglial activation is part of the immune response in the human brain and is a key factor associated with the development of Alzheimer's disease. Excessive activation of microglia is not only an epiphenomenon of inflammation, but also a key upstream mechanism that is crucial to the development of AD.
  • Parkinson's disease is a chronic neurodegenerative disease affecting the central nervous system, mainly affecting the motor nervous system. Its symptoms usually appear slowly over time. The most obvious early symptoms are tremor, limb stiffness, reduced motor function and abnormal gait. Cognitive and behavioral problems may also be present. Dementia is quite common in severely ill patients.
  • Selegiline is a monoamine oxidase (MAO)-B inhibitor (MAOI).
  • MAOI monoamine oxidase
  • WO2013/173941 and CN104321337A disclose an analgesic peptide with 11 to 14 amino acid residues, which matches the fragment of rabbit ⁇ 1-antiprotease through sequence alignment.
  • WO2016/165101 discloses that the peptide can effectively inhibit HCV replication.
  • WO2016/165102 and CN107847550A disclosed that the peptide is used to treat stroke (an acute cerebrovascular disease), verified that the peptide can pass through the brain-blood barrier, and verified the effect of the peptide on PC12 cells through in vitro cell experiments. Protection from glutamate- and hydrogen peroxide-induced cytotoxicity.
  • the PC12 cells tested in WO2016/165102 are a type of pheochromocytoma derived from rat adrenal medulla that exhibit certain characteristics of ganglion cells and are particularly useful in establishing a model of cellular hypoxic injury, thereby simulating acute cerebrovascular disease. rapid hypoxic and toxic states.
  • Alzheimer's disease and Parkinson's disease are chronic diseases that are not caused by acute cerebral hypoxia and toxicity caused by cerebral blood vessel embolism or rupture.
  • the prior art does not mention the possibility of using said peptides to treat AD or PD.
  • the object of the present invention includes providing drugs for treating neurodegenerative diseases and cognitive impairment, as well as providing active ingredients or compositions for improving or enhancing the cognitive function of subjects. It is also an object of the present invention to provide medicaments for the treatment of Alzheimer's disease and Parkinson's disease.
  • One of the technical problems of the present invention is solved by providing the peptides of the present invention and compositions comprising said peptides.
  • the present invention relates to a peptide having the amino acid sequence of SEQ ID NO: 1 or 2 or a variant or fragment thereof.
  • “Variant” and “fragment” mean a peptide that has certain amino acid residue changes compared to the peptide of the invention and substantially retains the same or similar biological function or activity as the peptide.
  • a variant of an amino acid sequence may be a variant of an amino acid sequence that has at least a certain percent identity with the amino acid sequence, or a variant of an amino acid sequence that has at least one or more amino acid mutations compared with the amino acid sequence.
  • the variant may be at least 70% identical to the amino acid sequence of SEQ ID NO: 1 or 2.
  • the variant may have 0 to 4 amino acid mutations in the amino acid sequence of SEQ ID NO: 1 or 2.
  • a fragment of an amino acid sequence may be an amino acid sequence having one or more deletions at the C-terminus and/or N-terminus compared to the amino acid sequence.
  • the deletions may be 1-5, such as 1, 2, 3, 4 or 5.
  • Fragments of the amino acid sequence may be 8 to 20 amino acids in length, such as 10 to 15 amino acids, such as 10, 11, 12, 13, 14 or 15 amino acids.
  • the peptide of the invention is selected from:
  • a peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).
  • the peptides of the invention comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 sexual amino acid sequence.
  • the peptide of the invention is comprised in or has 0 to 4 amino acid mutations relative to SEQ ID NO: 1, such as 0 to 3, such as 1 to 3, such as 0, 1, 2, Amino acid sequence with 3 or 4 amino acid mutations.
  • the peptides of the invention comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2 sexual amino acid sequence.
  • the peptide of the invention is comprised in or has 0 to 4 amino acid mutations relative to SEQ ID NO: 2, such as 0 to 3, such as 1 to 3, such as 0, 1, 2, Amino acid sequence with 3 or 4 amino acid mutations.
  • Amino acid mutations can be selected from additions, deletions or substitutions of amino acids.
  • the addition can be the insertion of an amino acid between two amino acid residues in the amino acid sequence, or the addition of an amino acid at the C-terminus or N-terminus of the amino acid sequence.
  • the deletion can be the deletion of an amino acid between two amino acid residues in the amino acid sequence, or the deletion of the C-terminus or N-terminus of the amino acid sequence.
  • the substitution may be of any amino acid residue in the amino acid sequence.
  • the addition, deletion or substitution of amino acids occurs at the C-terminus or N-terminus of the sequence. Substitutions may be conservative substitutions.
  • Conservative substitution refers to the substitution between amino acid molecules with similar properties, including the ionicity, hydrophobicity and molecular weight of the molecule.
  • Amino acids with similar properties can be classified as follows: aliphatic amino acids (such as glycine, alanine, valine, leucine, isoleucine); hydroxyl-containing or sulfur-containing amino acids (such as serine, cysteine, threonine).
  • amino acids methionine
  • cyclic amino acids such as proline
  • aromatic amino acids such as phenylalanine, tyrosine, tryptophan
  • basic amino acids such as histidine, lysine
  • arginine acidic or amide amino acids (such as aspartic acid, glutamic acid, asparagine, glutamine).
  • Amino acid mutations may be located at the C-terminus and/or N-terminus of the amino acid sequence.
  • the peptide may have 1, 2 or 3 amino acid additions at the C-terminus or N-terminus of SEQ ID NO: 1.
  • the peptide may have 1, 2 or 3 amino acid deletions at the C-terminus or N-terminus of SEQ ID NO:2.
  • the peptides of the invention may be from 8 to 20 amino acids in length, preferably from 10 to 18 amino acids in length, more preferably from 11 to 14 amino acids in length.
  • the peptide may be 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.
  • the peptides of the invention may be selected from
  • a peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 1; or
  • a peptide of the invention consists of the amino acid sequence of SEQ ID NO: 1.
  • the peptide of the invention consists of the amino acid sequence of SEQ ID NO:2.
  • the invention relates to the use of a peptide described herein in the preparation of a composition for preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof.
  • the invention relates to peptides for use in preventing, treating or ameliorating neurodegenerative diseases in a subject in need thereof.
  • the invention relates to a method of preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof, comprising administering an effective amount of a peptide to the subject in need thereof.
  • Neurodegenerative diseases can include cognitive impairment.
  • the neurodegenerative disease is a chronic neurodegenerative disease.
  • the invention relates to the use of a peptide described herein for the preparation of a composition for preventing, treating or ameliorating dementia in a subject in need thereof.
  • the invention relates to peptides for use in preventing, treating or ameliorating dementia in a subject in need thereof.
  • the invention relates to a method of preventing, treating or ameliorating dementia in a subject in need thereof, comprising administering an effective amount of a peptide to the subject in need thereof.
  • Dementia can be caused by neurodegenerative diseases. Alternatively, cognitive impairment can accompany neurodegenerative diseases.
  • the neurodegenerative disease or cognitive disorder is selected from Alzheimer's disease or Parkinson's disease. Therefore, the peptides of the present invention can be used to prevent, treat or improve Alzheimer's disease. In addition, the peptide of the present invention can be used to prevent, treat or improve Parkinson's disease.
  • Cognition can be the intelligent processing process by which the body recognizes and acquires knowledge, involving a series of random, psychological and social behaviors such as learning, memory, language, thinking, spirit, and emotion.
  • Cognitive impairment can include abnormalities in the brain's high-level intelligent processing related to the above-mentioned learning, memory, and thinking judgment, causing severe learning and memory impairments, accompanied by pathological processes such as aphasia, apraxia, agnosia, or aphasia.
  • the invention relates to the use of a peptide described herein in the preparation of a composition for improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof.
  • the invention relates to peptides for use in improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof.
  • the invention relates to a method of improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a peptide.
  • cognitive function or cognitive ability includes perceptual ability, logical thinking ability, memory ability, language ability, learning ability, emotional control ability, social ability or attention.
  • the subject has a cognitive function or cognitive ability decline, such as a cognitive function or cognitive ability decline caused by or accompanying aging.
  • the subject may suffer from cognitive impairment or a neurodegenerative disease, preferably a cognitive impairment caused by a neurodegenerative disease, more preferably Alzheimer's disease or Parkinson's disease.
  • dementia is characterized or manifested by communication difficulties, poor judgment, difficulty performing simple tasks, missing or misplacing objects, language impairment, personality changes, reduced behavioral abilities, confused sense of time and space, and reduced comprehension. , decreased problem-solving ability, decreased concentration, decreased social skills, sensory impairment, logical thinking disorder or memory impairment.
  • the neurodegenerative disease or cognitive disorder is Alzheimer's disease.
  • Alzheimer's disease is characterized by beta-amyloid (A ⁇ ) deposition, neuroinflammation, and/or glial cell abnormalities or activation in the brain, particularly the hippocampus.
  • a ⁇ beta-amyloid
  • Prevention, treatment or improvement of Alzheimer's disease is achieved by reducing or alleviating beta-amyloid (A ⁇ ) deposition, neuroinflammation and/or glial cell abnormalities or activation in the brain.
  • the invention relates to the use of the peptides described herein in the preparation of a method for reducing or alleviating beta-amyloid (A ⁇ ) deposition, neuroinflammation and/or glial abnormality or activation in the brain of a subject, particularly in the hippocampus.
  • a ⁇ beta-amyloid
  • the subject may be a neurodegenerative disease patient or a cognitive impairment patient, preferably an Alzheimer's disease patient.
  • the peptides described herein can be effective in reducing or alleviating A ⁇ or A ⁇ plaque deposition in a subject's brain, such as the hippocampus.
  • the peptide can reduce or alleviate the expression or levels of inflammatory factors in the subject's brain, such as the hippocampus or cortex.
  • the inflammatory factor can be selected from IL-1 ⁇ , IL-6 or TNF- ⁇ .
  • the peptides may also reduce or alleviate glial cell abnormalities or activation in the brain's hippocampus.
  • Glial cells also known as glial cells, can be selected from astrocytes or microglia, with microglia being preferred.
  • the neurodegenerative disease or cognitive disorder is Parkinson's disease.
  • Parkinson's disease is characterized by abnormality or damage to neurons in the substantia nigra of the brain.
  • the prevention, treatment or improvement of Parkinson's disease is achieved by reducing or alleviating neuronal abnormalities or damage in the substantia nigra of the brain.
  • the present invention relates to the use of a peptide described herein in the preparation of a composition for reducing or alleviating abnormality or damage to neurons in the substantia nigra of the brain of a subject.
  • the subject may be a neurodegenerative disease patient or a cognitive impairment patient, preferably a Parkinson's disease patient.
  • the subject or patient is a mammal.
  • the subject or patient is a human, such as a middle-aged or elderly person, preferably an elderly person.
  • Middle-aged people can be people between 40 and 59 years old or people between 45 and 59 years old.
  • An elderly person can be a person over 60, over 65, over 70, over 75 or over 80.
  • the subject may be a subject with cognitive function or cognitive ability decline, such as cognitive function or cognitive ability decline caused by or accompanying aging.
  • the decline generally refers to chronic decline.
  • compositions of the invention comprise peptides described herein.
  • the composition of the present invention may also contain pharmaceutical excipients, solvents or carriers; or pharmaceutical excipients, solvents or carriers acceptable to the human body.
  • the composition may be a pharmaceutical composition or a nutritional composition.
  • the pharmaceutical composition may be a drug.
  • the nutritional composition may be in the form of a nutritional supplement, nutritional supplement, dietary supplement, dietary supplement, nutraceutical, nutraceutical, nutraceutical or health food.
  • compositions as described herein may generally include substances or formulations that improve or enhance the health condition of a subject (eg, a human) or treat a disease in the subject.
  • Nutritional composition as described herein may refer to meeting at least part of the nutrient requirements of a subject (such as a human), regulating the body functions of the subject, regulating the physiological functions of the subject, and/or improving the health status of the subject Substances or formulations that may be taken.
  • the nutritional composition may be a nutraceutical, functional food, dietary supplement, nutraceutical or health food that activates, modulates or improves cognitive function or cognitive ability.
  • a "pharmaceutical composition” may refer to an ingestible substance or formulation for preventing, treating, alleviating, ameliorating, conditioning, or modulating a disease, disorder, symptom, or condition in a subject (eg, a human).
  • pharmaceutical compositions may be in the form of pharmaceuticals, including oral medications, such as tablets, pills, capsules, and the like.
  • compositions for oral administration may be formulated in dosages suitable for oral administration using carriers, vehicles or excipients known in the art.
  • Such carriers enable the compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by a subject.
  • polypeptide or composition of the present invention can be prepared as an injection, such as intramuscular injection, intraperitoneal injection, intraperitoneal injection, subcutaneous injection or intravenous injection.
  • injectables may be in the form of solutions, emulsions or suspensions, as well as powders or concentrated solutions for solution or suspension before use.
  • injections may also contain solvents such as water, isotonic agents, buffers, preservatives, co-solvents, solubilizers, suspending agents and emulsifiers.
  • the term "effective amount” or “therapeutically effective amount” refers to an amount of a peptide or composition of the invention that is effective in treating a disease or disorder or ameliorating or enhancing the health condition of a subject. Improving or improving one's health includes improving or improving cognitive functions or abilities.
  • the peptide of the present invention can be used at 0.01mg/kg to 10mg/kg, 0.02mg/kg to 8mg/kg, 0.04mg/kg to 5mg/kg, 0.05mg/kg to 1mg/kg, 0.1mg/kg to 0.8mg/kg. kg or 0.2 mg/kg to 0.5 mg/kg is administered to the subject.
  • the peptide of the invention can be administered to a subject in an amount of 0.04 mg/kg, 0.16 mg/kg or 0.64 mg/kg.
  • a therapeutically effective amount may be these doses.
  • the peptide or composition of the invention may be administered at once daily intervals for at least 1 week, for example 1 to 4 weeks.
  • compositions of the invention may comprise such combinations of peptides.
  • the peptide combination includes a first peptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof and a second peptide having the amino acid sequence of SEQ ID NO: 2 or a variant thereof.
  • FIG. 1 Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on short-term working memory, conditioned fear memory and social ability of APP/PS1 mice.
  • A Statistical chart showing the percentage of time mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups entered the novel arm in the Y maze experiment.
  • B In the new object recognition experiment, the statistical chart of the new object recognition cognitive index of the four groups of mice.
  • C Statistical chart of freezing time of mice in four groups during the conditioned fear memory experiment.
  • FIG. 1 Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on A ⁇ plaque deposition in the hippocampus and cortex of APP/PS1 mice.
  • A Fluorescence images of thioflavin S staining of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 200 ⁇ m.
  • B Immunofluorescence experiment shows fluorescence images of 6E10 expression in the cortex and hippocampus of four groups of mice. Scale bar is 200 ⁇ m.
  • C Quantitative statistical diagram of Thioflavin S-positive A ⁇ plaques in the cortex and hippocampus of four groups of mice.
  • FIG. 3 Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on glial cell activation around hippocampal A ⁇ plaques in APP/PS1 mice.
  • A Immunofluorescence experiment shows fluorescence images of Iba1, GFAP and 6E10 expression in the hippocampus of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 50 ⁇ m.
  • (B) Statistical chart of the percentage of positive expression areas of Iba1 and GFAP in the hippocampus of four groups of mice. The results are expressed as mean ⁇ standard error, n 4-5 per group, and the statistical graph uses one-way analysis of variance. Positive area of Iba1+: the positive area of Iba1+; Positive area of GFAP+: the positive area of GFAP+.
  • FIG. 4 Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on glial cell activation around cortical A ⁇ plaques in APP/PS1 mice.
  • A Immunofluorescence experiment shows fluorescence images of Iba1, GFAP and 6E10 expression in the cortex of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 50 ⁇ m.
  • (B) Statistical chart of the percentage of positive expression areas of Iba1 and GFAP in the cortex of four groups of mice. The results are expressed as mean ⁇ standard error, n 5 per group, and the statistical graph uses one-way analysis of variance. Positive area of Iba1+: the positive area of Iba1+; Positive area of GFAP+: the positive area of GFAP+.
  • FIG. 5 Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on the contents of IL-1 ⁇ , IL-6 and TNF- ⁇ in the hippocampus and cortex of APP/PS1 mice.
  • A ELISA detects the expression of inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the hippocampus of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups.
  • (B) ELISA detects the expression of inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the cortex of four groups of mice. The results are expressed as mean ⁇ standard error, n 5 per group, and the statistical graph uses one-way analysis of variance.
  • Cortext cortex; Hippocampus: hippocampus; Relative expression of IL-1 ⁇ : relative expression of IL-1 ⁇ ;; Relative expression of IL-6: relative expression of IL-6;; Relative expression of TNF- ⁇ : TNF- ⁇ Relative expression.
  • FIG. 7 Effects of PKT101 and PKT002 on the total movement distance of mice in the mine field experiment after the MPTP subacute model (1, 3, 7 and 14 days). *** p ⁇ 0.001, ** p ⁇ 0.01vs.Sham; ### p ⁇ 0.001, ## p ⁇ 0.01, # p ⁇ 0.05vs.MPTP.
  • FIG. 8 Effects of PKT101 and PKT002 on T-Turn in the pole climbing experiment of mice after the MPTP subacute model (1, 3, 7 and 14 days). ***p ⁇ 0.001,**p ⁇ 0.01vs.Sham; ###p ⁇ 0.001,##p ⁇ 0.01,#p ⁇ 0.05vs.MPTP.
  • FIG. 9 Effects of PKT101 and PKT002 on T-TLA in the pole climbing experiment of mice after the MPTP subacute model (1, 3, 7 and 14 days). ***p ⁇ 0.001vs.Sham; ###p ⁇ 0.001,##p ⁇ 0.01,#p ⁇ 0.05vs.MPTP.
  • FIG. 10 Effects of PKT101 and PKT002 on the latency period (1, 3, 7 and 14 days) of mice in the rotarod experiment after the subacute model of MPTP. ***p ⁇ 0.001vs.Sham; ###p ⁇ 0.001vs.MPTP.
  • Example 1 Study on the neuroprotective effect of the peptide of the present invention on the Alzheimer's disease model animal APP/PS1 mice
  • PKT101 peptide DEAQETAVSSHEQD(SEQ ID NO:2)
  • PKT002 peptide DEAQETAVSSH (SEQ ID NO:1)
  • mice Sixty 5-month-old APP/PS1 male mice were randomly divided into 4 groups, 15 in each group. They were intraperitoneally injected with normal saline, PKT101 (8mg/kg), PKT002 (8mg/kg) and PKT101/PKT002 ( 8 mg/kg) for 4 weeks. The final volume of intraperitoneal injection in each group was 80 ⁇ L. After weekly administration, 20-35 mice were randomly taken out and measured for weight measurement. The average weight was used as an important basis for the next week's dosage.
  • mice were tested for short-term learning and memory (Y maze and new object recognition), conditioned fear memory and social behavior, and the plaque deposition in the cerebral cortex and hippocampus was detected through thioflavin staining and 6E10 immunofluorescence staining; through immunofluorescence staining Observe the activation of microglia and astrocytes in the cortex and hippocampus; detect the inflammatory factors interleukin-1 ⁇ (IL-1 ⁇ ), interleukin-6 (IL-6) and tumors using enzyme-linked immunosorbent assay (ELISA) Necrosis factor (TNF- ⁇ ) content.
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin-6
  • ELISA enzyme-linked immunosorbent assay
  • the Y maze device used in this experiment has an angle of 120 degrees between the three arms, which are connected to each other.
  • the three arms are the same size, 29cm long, wide and high 29cm x 8cm x 15cm.
  • Different patterns are affixed to the inside of the arms to represent mice.
  • As visual markers name the starting arm, novelty arm and other arms respectively.
  • the experimental process is divided into two parts, the adaptation phase and the testing phase, with an interval of 1-2 hours. Adaptation is to separate the novel arms with baffles. Mice can only move freely in the starting arm and other arms.
  • the adaptation time of each mouse is 5 minutes.
  • the test phase is to open the novel arms after 1-2 hours. The mice can move freely within the three arms, and the test time for each mouse is 5 minutes.
  • Topscan software was used to record the time and number of times the mice entered the novel arm in real time. After each experiment, the experimental equipment must be cleaned with 75% alcohol to remove the mouse odor.
  • mice Before testing this device, the mice must be eliminated from any sense of strangeness and the mice must be stroked every day to avoid irritation to the mice during operation.
  • the mice In the first stage (familiarization period), put two identical objects (AB, make sure the objects have no smell and are not pushed) into the device. The objects are 10cm away from both sides of the walls. Put the mouse into the device from the middle of the two objects.
  • the mouse is put into the device from the middle of the two objects, and the number, time and distance of the mouse's exploration of the old and new objects within 5 minutes are recorded, that is, the number, time and distance of the mouse's activities around the old and new objects, and the cognitive status of the mouse is detected.
  • the experimental equipment needs to be cleaned with 75% alcohol. If the mouse has poor cognitive ability, there will be no difference in the exploration of new and old objects; if the mouse has normal cognitive ability, the exploration time of new objects will be longer than that of old objects.
  • mice On the first day of this experiment, the mice were put into the box (the bottom of the box is a copper fence that can be powered). After adapting for 3 minutes, the mice were allowed to stay in the box for another 6 minutes. During this period, a single frequency sound was given at the same time every 2 minutes. Stimulation (1.0 KHZ, 70 db, 30 s) and inescapable foot shock (0.8 mA, 2 s) were performed three times in total. The total time of sound and electric shock-induced freezing behavior of the mice within 6 min was recorded, and then placed back in the cage. After each experiment, wipe the bottom of the box with 75% alcohol.
  • mice with established fear were placed in the original box, and the same intensity of sound stimulation (1.0KHZ, 70db, 30s) was immediately given, and the sound-induced freezing behavior of the mice was recorded within 3 minutes.
  • Catalepsy was defined as no movement other than breathing. Percentage of environmentally and sound-induced freezing time was recorded.
  • mice were placed in the behavioral testing room to adapt for 0.5 h, and the tested rectangular box was equally divided into three areas: Empty, Center and Stranger-1. Mice of the same sex, background, and age were placed into the metal cage in the Stranger-1 area, and the metal cage on the other side of the box was empty. Put the test mouse into the center box so that the test mouse can move freely in the three boxes for 5 minutes. Photograph and record relevant parameters: the duration of entering each box. When the mouse's head and four paws enter a box, it is considered to be in that box.
  • mice of the same sex and background and different ages were placed in an empty metal cage, and then recorded for 5 minutes to observe the time the test mice spent in the Stranger-1 and Stranger-2 areas.
  • the OCT resin-embedded brain tissue was sliced coronally using a Leica freezing microtome, with a thickness of 10 ⁇ m, and collected in PBS for immunofluorescence staining and A ⁇ histopathological analysis.
  • the sections were blocked with 10% fetal bovine serum PBS solution for 1 h at room temperature. Discard the blocking solution, add mouse-derived 6E10 antibody, chicken-derived glial fibrillary acidic protein antibody, and rabbit-derived calcium ion binding receptor molecule 1 (inized calcium binding adapter molecule 1, Iba1) antibody, and incubate overnight at 4°C. The next day, the primary antibody was washed away and washed 3 times with PBS for 5 minutes each time. Add secondary antibodies of corresponding species (goat anti-chicken IgG-488; donkey anti-rabbit IgG-488; donkey anti-mouse IgG-647) and incubate at room temperature in the dark for 1 hour.
  • Thioflavin S can mark A ⁇ core plaques on brain slices. Dewaxed and hydrated tissue sections can be stained with 1% thioflavin for 5 minutes, washed with running water for 1 minute, separated with 70% alcohol for 30 seconds, and then quenched with anti-fluorescence. The slides were sealed with agent and photographed under a fluorescence microscope.
  • Immunofluorescence-stained tissue sections were photographed using a Leica microscope, and ImageJ software was used to use grayscale threshold analysis to count the percentage of positive areas for GFAP, Iba1, 6E10, and thioflavin. There were 5 mice in each group, and 3 sections were selected for each mouse. , take its average value as the statistical result of this group.
  • mice were anesthetized, they were killed by decapitation, the skin on the neck and head was peeled off, the skull was exposed, the skull was cut along the sagittal suture, and the brain tissue was removed after the skull was separated. Put the cortical tissue and hippocampal tissue into marked centrifuge tubes, quickly freeze them in liquid nitrogen, and transfer them to a -80°C refrigerator for storage.
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin-6
  • TNF- ⁇ tumor necrosis factor- ⁇
  • 3 pro-inflammatory indicators add at least two wells to each sample, 45 ⁇ L of standard and sample in each well, then add 50 ⁇ L of biotinylated antibody working solution to the sample and standard wells, seal the reaction well plate with sealing paper, and place Incubate at room temperature for 2 hours (use micro shaker, frequency, 300 rpm). Prepare the enzyme-binding substrate 30 minutes in advance and store it at room temperature away from light. Discard the liquid in the wells and pat dry on absorbent paper.
  • Washing method absorb the reaction solution in the wells, add the washing solution to the well plate, and leave it for 2 minutes with slight shaking. Add 100 ⁇ L of enzyme-bound substrate working solution to each well except the blank well, and incubate at room temperature for 1 hour (use a micro shaker, frequency, 300 rpm). Discard the liquid in the wells and pat dry on absorbent paper. Wash 5 times. Add 100 ⁇ L of chromogenic substrate working solution to each well except the blank wells, and place at room temperature in the dark for 15 minutes. Add 100 ⁇ L of stop solution to each well except the blank well to stop the reaction, and measure the experimental results within 20 minutes. Detection at 450nm wavelength, absorption values of standard holes and test holes. A standard curve is drawn based on the standard wells and known concentrations, and then the actual concentration of the test wells is converted from the standard curve. Finally, the target protein amount per milligram of brain tissue is obtained based on the protein concentration.
  • the Y maze test was used to evaluate the short-term memory of mice in each group.
  • the results showed that compared with the control group APP/PS1 mice, the time for APP/PS1 mice in the PKT101 treatment group to enter the novel arm increased, while PKT002 and PKT101/PKT002
  • the time for APP/PS1 mice in the combination treatment group to enter the novel arm was significantly increased (Figure 1A).
  • the new object recognition test was used to evaluate the short-term memory of mice in each group.
  • the results showed that compared with the control group, the index of APP/PS1 mice in the above three treatment groups to recognize new objects increased significantly (Figure 1B).
  • Conditioned fear memory was used to evaluate the fear memory of mice in each group.
  • Thioflavin staining and 6E10 immunofluorescence staining were used to evaluate the fibrous and diffuse A ⁇ plaque deposition in the cortex and hippocampus of mice in each group.
  • the results of thioflavin staining showed that compared with the control group, PKT101, PKT002 and PKT101/PKT002 combined
  • the A ⁇ plaques in the hippocampus of APP/PS1 mice in the treatment group were significantly reduced, and there was no effect on the deposition of A ⁇ plaques in the cortex ( Figure 2A, C).
  • PKT101 and PKT002 treatment alone and PKT101/PKT002 combined treatment can improve short-term cognitive impairment, reduce hippocampal A ⁇ load deposition, glial cell activation and inflammatory factors in APP/PS1 mice to varying degrees.
  • PKT101 peptide DEAQETAVSSHEQD(SEQ ID NO:2)
  • PKT002 peptide DEAQETAVSSH (SEQ ID NO:1)
  • MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was used to establish a subacute Parkinson's disease mouse model. After the mice were weighed and recorded, 20 mg/kg MPTP was injected subcutaneously into the back of the neck once a day for 5 consecutive days. Following-up experiments were conducted on days 1, 3, 7 and 14 after the last administration.
  • Selegiline Selegiline, Imidopyr
  • mice in the MPTP model group were given an equal volume of physiological saline (0.1mL/10g).
  • the mice in the Sham group were only given an equal volume of physiological saline (0.1mL/10g).
  • the open field test was used to evaluate the voluntary movement ability of mice.
  • the open field device is made of opaque blue plastic and measures 60cm ⁇ 60cm ⁇ 45cm. During the test, each mouse was placed into the box from the central area, and at the same time, the movement trajectory of each mouse within 5 minutes was recorded, and the total movement distance was calculated using software (Clever Sys Inc., VA, USA). After each mouse test, use 75% ethanol to wipe the open area to prevent the smell of the previous mouse from affecting the next mouse.
  • the pole climbing test was used to evaluate the motor coordination function of mice.
  • the pole used has a diameter of 1 cm and a height of 50 cm.
  • a wooden ball with a diameter of 1.2 cm is fixed on the top of the pole.
  • the pole is wrapped with anti-slip tape to prevent mice from slipping. Place the mouse's head upward on the top of the pole.
  • start the timer Record the time it takes for the mouse to move over the top of the pole (T-Turn) and the time it takes for the mouse to climb to the bottom of the four paws.
  • Time to Landing T-TLA
  • each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required, and the shortest time is chosen.
  • the rotarod test was used to evaluate the motor balance ability and limb coordination of mice. Place the mouse on the drum of the rotarod tester, and set the rotation speed to increase from 5rpm/min to 25rpm/min at a constant speed within 2 minutes. When the mouse falls from the drum to the sensing area below, an infrared sensor will receive the signal and record it. Time for mice to move on the instrument roller. Before formal testing, each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required to calculate the average drop time (latency period).
  • mice from each group were taken, anesthetized with 4% chloral hydrate, and perfused with physiological saline + 4% PFA to obtain brain tissue.
  • Nissl bodies are basophilic substances in the cytoplasm that are widely found in various neurons and are used to count the number of brain neurons. Stain according to Nissl staining kit.
  • TH mainly labels dopaminergic neurons in the substantia nigra pars compacta, and TH-positive neurons in this brain area will be significantly reduced after MPTP modeling.
  • DAB diaminobenzidin
  • Immunohistochemistry-positive cells were counted using a Stereo Investigator (MBF bioscience) counting system to count the number of Nissl body-positive cells and TH + cells in the substantia nigra pars compacta. Cell counting was performed under a ⁇ 20 objective lens.
  • mice showed a decrease in the total movement distance in the open field test at 1, 3, 7, and 14 days after modeling, indicating a decrease in autonomous activity ability.
  • the positive control drug Selegiline can significantly improve MPTP-induced decrease in total exercise distance on days 1, 3, 7 and 14.
  • PKT101 and PKT002 also had significant improvement within 14 days.
  • the low, medium and high doses of PKT101 alone increased to 221.1%, 253.9% and 255.3% of the model group; PKT002 alone increased to 213.0% of the model group; the combined administration of PKT101 and PKT002 increased to the model group 214.8%.
  • mice showed prolonged T-Turn and T-TLA times in the pole climbing test at 1, 3, 7 and 14 days after modeling, indicating movement coordination. Decline in ability.
  • the positive control drug Selegiline can significantly improve MPTP-induced T-Turn and T-TLA time prolongation on days 1, 3, 7 and 14.
  • PKT101 and PKT002 also had significant improvement within 14 days.
  • mice showed a shortening of the falling latency in the rotarod test at 1, 3, 7 and 14 days after modeling, indicating a decrease in motor balance ability.
  • the positive control drug Selegiline can significantly improve the shortening of MPTP-induced latency time on days 1, 3, 7 and 14. Both PKT101 and PKT00 had significant improvement within 14 days.
  • the incubation period of low, medium and high doses of PKT101 alone was prolonged to 121.7, 119.9% and 118.3% of the model group respectively; the latency of PKT002 alone was prolonged to 120.3% of the model group; the combined administration of PKT101 and PKT002 was prolonged to 121.7%, 119.9% and 118.3% of the model group. 120.9% of the model group.
  • mice showed a decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta 7 and 14 days after modeling, indicating the death of neurons in this brain area.
  • the positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta on both 7 and 14 days.
  • PKT101 and PKT002 also had significant improvement effects at 7 and 14 days.
  • the number of Nissl-positive cells treated with low, medium and high doses of PKT101 alone increased to 120.9%, 124.0% and 130.9% of the model group, respectively; 124.5%; the number of Nissl-positive cells after combined administration of PKT101 and PKT002 increased to 128.0% of the model group.
  • the number of Nissl-positive cells treated with low, medium and high doses of PKT101 alone increased to 124.9%, 130.9% and 141.1% of the model group, respectively; 128.5%; the number of Nissl-positive cells after combined administration of PKT101 and PKT002 increased to 141.5% of the model group.
  • mice showed a decrease in the number of TH-positive neurons in the substantia nigra pars compacta 7 and 14 days after modeling, indicating the death of dopaminergic neurons in this brain area.
  • the positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of TH-stained positive neurons in the substantia nigra pars compacta on both 7 and 14 days.
  • PKT101 and PKT002 also had significant improvement effects at 7 and 14 days.
  • the number of TH-positive cells after single administration of low, medium and high doses of PKT101 increased to 114.5%, 124.9% and 129.7% of the model group respectively; the number of TH-positive cells after single administration of PKT002 increased to 129.9% of the model group. ;
  • the number of TH-positive cells treated with PKT101 and PKT002 combined increased to 130.3% of that in the model group.
  • the number of TH-positive cells after single administration of PKT101 at low, medium and high doses increased to 125.6%, 136.0% and 137.5% of the model group respectively; the number of TH-positive cells after single administration of PKT002 increased to 128.1% of the model group. ;
  • the number of TH-positive cells after combined administration of PKT101 and PKT002 increased to 137.7% of that in the model group.

Abstract

本发明涉及肽用于治疗神经退行性疾病或改善认知功能的用途。本发明涉及神经退行性疾病和认知障碍症的治疗；以及认知功能的改善和提高。具体而言，本发明涉及肽用于预防或治疗神经退行性疾病和认知障碍症，优选阿尔茨海默病和帕金森病的用途。本发明还涉及肽用于改善或提高受试者认知功能的用途。

Description

肽用于治疗神经退行性疾病或改善认知功能的用途 发明领域
本发明涉及神经退行性疾病和认知障碍症的治疗；以及认知功能的改善和提高。具体而言，本发明涉及肽用于预防或治疗神经退行性疾病和认知障碍症，优选阿尔茨海默病和帕金森病的用途。本发明还涉及肽用于改善或提高受试者认知功能的用途。
背景技术
神经退行性疾病或神经退化性疾病(Neurodegenerative Disorder或Neurodegenerative Disease)是神经元逐渐退化所直接导致的疾病。退行性过程可涉及神经元结构的进行性损失、神经元功能的进行性损失、或进行性神经元细胞死亡。这种进行性神经退行经常导致身体残疾和精神恶化。许多神经退行性疾病是严重的进行性和不间断疾病，并且治疗方法很少。阿尔茨海默病(AD)和帕金森病(PD)是最主要的两种神经退行性疾病。
认知障碍症(Neurocognitive Disorder)也称为痴呆症(Dementia)，是脑部疾病的其中一类，此症导致思考能力和记忆力长期而逐渐地退化，并使个人日常生活活动受到影响。最常见的痴呆症类型是阿尔茨海默病，阿尔茨海默病患者占所有痴呆症患者人数的50％到70％。痴呆症影响全球三千六百万人口。大约10％的人口，会在有生之年中发病。痴呆症与年龄(老化)息息相关，约3％的人口在65到74岁之间得到痴呆症，另外19％的人口则在75到84岁之间，而将近一半的人口超过85岁得到痴呆症。
阿尔茨海默病(Alzheimer's Disease,AD)是一种发病进程缓慢、随着时间不断恶化的慢性神经退行性疾病。最常见的早期症状为丧失短期记忆，当疾病逐渐进展，症状可能逐渐出现，包括语言障碍、定向障碍、情绪不稳、丧失动机、无法自理和许多行为问题。当情况恶化时，患者往往会因此和家庭或社会脱节，并逐渐丧失身体机能，最终导致死亡。虽然疾程因人而异，但诊断后的平均余命约为三到九年。在AD中，海马体(hippocampus)是首先受到损伤的区域，表现症状为记忆力衰退以及方向知觉的丧失(见Yangling Mu et al.,Adult hippocampal neurogenesis and its role in Alzheimer's disease,Mol Neurodegener,2011；6:85)。AD的进程与大脑中的β-淀粉样蛋白(Aβ)斑块沉积和Tau蛋白有关(见Clive Ballard  et al.,Alzheimer's disease,The Lancet.2011 March,377(9770):1019–1031)。最近研究发现，神经炎症或小胶质细胞的激活参与了阿尔茨海默病中tau蛋白缠结在新皮质中的扩散，进而导致阿尔茨海默病患者认知功能障碍的发生(见Tharick A.Pascoal et al.,Microglial activation and tau propagate jointly across Braak stages,Nature Medicine,27,pages1592–1599(2021 August))。小胶质细胞激活是人类大脑免疫反应的一部分，是与阿尔兹海默病发展相关的关键因素。小胶质细胞的过度激活不仅仅是一种炎症附带现象，更是一种关键的上游机制，对AD的发展至关重要。研究还发现，神经炎症和某些炎症细胞因子参与AD的病理学(见Rishika Dhapola et al.,Recent advances in molecular pathways and therapeutic implications targeting neuroinflammation for Alzheimer’s disease,Inflammopharmacology volume 29,pages1669–1681(2021 November))。因此，减少Aβ斑块沉积、缓解神经炎症以及抑制小胶质细胞的激活可视为治疗或缓解AD的有效手段。
帕金森病(Parkinson's disease，PD)是一种影响中枢神经系统的慢性神经退行性疾病，主要影响运动神经系统。它的症状通常随时间缓慢出现，早期最明显的症状为颤抖、肢体僵硬、运动功能减退和步态异常，也可能有认知和行为问题。认知障碍症在病情严重的患者中相当常见。帕金森病的主要病理变化发生在中脑黑质腹侧的致密部，主要症状大多数因为黑质致密部的多巴胺性神经元退化(Jose A Obeso et al.,Functional organization of the basal ganglia:therapeutic implications for Parkinson's disease,Movement Disorders,Volume 23,Issue S3 p.S548-S559,Sept.2008)。司来吉兰是一种单胺氧化酶(MAO)-B抑制剂(MAOI)，2006年美国FDA批准司来吉兰用于治疗PD患者。
本领域中存在有效治疗神经退行性疾病或者由神经退行性疾病引起的认知障碍症的药物的需求。
WO2013/173941和CN104321337A公开了一种具有11至14个氨基酸残基的镇痛肽，经序列比对匹配兔α1-抗蛋白酶的片段。WO2016/165101公开了所述肽可以有效抑制HCV复制。此外，WO2016/165102和CN107847550A公开了该肽用于治疗脑卒中(一种急性的脑血管疾病)，验证了所述肽能够通过脑血屏障，并通过体外细胞实验验证了所述肽对PC12细胞免受谷氨酸和过氧化氢诱导的细胞毒性的保护作用。这些文件通过引用结合到本文中。WO2016/165102中测试的PC12细胞是一种衍生自显示神经节细胞的某些特征的大鼠肾上腺髓质的嗜铬细胞瘤，其特别用于建立细胞缺氧损伤模型，从而模拟急性脑血管疾病中的迅速缺氧和毒性状态。然而，阿尔茨海默病和帕金森病属于慢性疾病，它们并非由脑血管栓塞或破裂而导致的急性大脑缺氧和毒性引起。现有技术尚未提及所述肽用于治疗AD或PD的可能性。
发明内容
本发明目的包括提供用于治疗神经退行性疾病和认知障碍症的药物，以及提供用于改善或提高受试者认知功能的活性成分或组合物。本发明目的还包括提供用于治疗阿尔茨海默病和帕金森病的药物。
本发明的技术问题之一通过提供本发明的肽以及包含所述肽的组合物来解决。
本发明涉及一种肽，其具有SEQ ID NO:1或2的氨基酸序列或其变体或片段。“变体”和“片段”意指与本发明肽相比具有一定的氨基酸残基变化并且基本上保留与所述肽的相同或类似的生物功能或活性的肽。氨基酸序列的变体可以是与所述氨基酸序列具有至少某个百分比同一性的氨基酸序列的变体，或者与所述氨基酸序列相比具有至少一个或多个氨基酸突变的氨基酸序列的变体。例如，所述变体可以与SEQ ID NO:1或2的氨基酸序列具有至少70％的同一性。或者，所述所述变体可以在SEQ ID NO:1或2的氨基酸序列中具有0至4个氨基酸突变。氨基酸序列的片段可以是与所述氨基酸序列相比在C-端和/N-端具有一个或多个缺失的氨基酸序列。所述缺失可以是1-5个，例如1、2、3、4或5个。氨基酸序列的片段的长度可以是8至20个氨基酸，例如10至15个氨基酸，例如10、11、12、13、14或15个氨基酸。
在一个方面中，本发明的肽选自：
a)包含与DEAQETAVSSH(SEQ ID NO:1)具有至少70％同一性的氨基酸序列的肽；
b)包含在DEAQETAVSSH(SEQ ID NO:1)中具有0至4个氨基酸突变的氨基酸序列的肽；
c)包含与DEAQETAVSSHEQD(SEQ ID NO:2)具有至少70％同一性的氨基酸序列的肽；或
d)包含在DEAQETAVSSHEQD(SEQ ID NO:2)中具有0至4个氨基酸突变的氨基酸序列的肽。
在一个方面，本发明肽包含与SEQ ID NO:1具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％或者100％同一性的氨基酸序列。
在一个方面，本发明肽包含在SEQ ID NO:1中或相对于SEQ ID NO:1具有0至4个氨基酸突变，例如0至3个，例如1至3个，例如0、1、2、3或4个氨基酸突变的氨基酸序列。
在一个方面，本发明肽包含与SEQ ID NO:2具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％或者100％同一 性的氨基酸序列。
在一个方面，本发明肽包含在SEQ ID NO:2中或相对于SEQ ID NO:2具有0至4个氨基酸突变，例如0至3个，例如1至3个，例如0、1、2、3或4个氨基酸突变的氨基酸序列。
氨基酸突变可以选自氨基酸的添加、缺失或取代。添加可以是氨基酸序列中两个氨基酸残基之间插入氨基酸，或者是在氨基酸序列C-端或者N-端的添加。缺失可以是氨基酸序列中两个氨基酸残基之间删除氨基酸，或者是氨基酸序列C-端或者N-端的缺失。取代可以是氨基酸序列中任意氨基酸残基的取代。优选地，氨基酸的添加、缺失或取代发生在序列的C-端或者N-端。取代可以是保守取代。保守取代是指性状相近的氨基酸分子之间的取代，其中的性状包含分子的离子性、疏水性和分子量等。性状相近的氨基酸可以按以下分类：脂肪族氨基酸(例如甘氨酸,丙氨酸,缬氨酸,亮氨酸,异亮氨酸)；含羟基或含硫氨基酸(例如丝氨酸,半胱氨酸,苏氨酸,甲硫氨酸)；环状氨基酸(例如脯氨酸)；芳香族氨基酸(例如苯丙氨酸,酪氨酸,色氨酸)；碱性氨基酸(例如组氨酸,赖胺酸,精氨酸)和酸性或酰胺类氨基酸(例如天冬氨酸,谷氨酸,天冬酰胺,谷氨酰胺)。
本领域技术人员理解，构成肽、多肽或蛋白的二十种常见的氨基酸及其字母缩写如下表1所示。技术人员可以例如根据下表1或者公知常识确定本发明肽的氨基酸残基顺序和组成。
表1.各种氨基酸及其字母缩写
中文名 三字母缩写 单字母缩写
丙氨酸 Ala A
精氨酸 Arg R
天冬酰胺 Asn N
天冬氨酸 Asp D
半胱氨酸 Cys C
谷氨酰胺 Gln Q
谷氨酸 Glu E
甘氨酸 Gly G
组氨酸 His H
异亮氨酸 Ile I
亮氨酸 Leu L
赖氨酸 Lys K
甲硫氨酸 Met M
苯丙氨酸 Phe F
脯氨酸 Pro P
丝氨酸 Ser S
苏氨酸 Thr T
色氨酸 Trp W
酪氨酸 Tyr Y
缬氨酸 Val V
氨基酸突变可以位于氨基酸序列的C-端和/或N-端。例如，所述肽可以在SEQ ID NO:1的C-端或者N-端具有1个、2个或3个氨基酸添加。例如，所述肽可以在SEQ ID NO:2的C-端或者N-端具有1个、2个或3个氨基酸缺失。
在一个方面，本发明的肽可以具有8至20个氨基酸的长度，优选10至18个氨基酸长度，更优选11至14个氨基酸长度。例如，所述肽的长度可以为10、11、12、13、14、15、16、17或18个氨基酸。
在一个方面，本发明的肽可以选自
1)包含SEQ ID NO:1的氨基酸序列、基本上由SEQ ID NO:1的氨基酸序列组成或者由SEQ ID NO:1的氨基酸序列表示的肽；或者
2)包含SEQ ID NO:2的氨基酸序列、基本上由SEQ ID NO:2的氨基酸序列组成或者由SEQ ID NO:2的氨基酸序列表示的肽。
在一个方面，本发明的肽由SEQ ID NO:1的氨基酸序列组成。或者，本发明的肽由SEQ ID NO:2的氨基酸序列组成。
在一个方面，本发明涉及本文所述的肽在制备用于预防、治疗或改善有需要的受试者中神经退行性疾病的组合物中的用途。在一个方面，本发明涉及肽，其用于预防、治疗或改善有需要的受试者中神经退行性疾病。在一个方面，本发明涉及一种预防、治疗或改善有需要的受试者中神经退行性疾病的方法，所述方法包括将有效量的肽给予需要的受试者。神经退行性疾病可包括认知障碍症。
在一个方面，神经退行性疾病是慢性神经退行性疾病。
在一个方面，本发明涉及本文所述的肽在制备用于预防、治疗或改善有需要的受试者中认知障碍症的组合物中的用途。在一个方面，本发明涉及肽，其用于预防、治疗或改善有需要的受试者中认知障碍症。在一个方面，本发明涉及一种预防、治疗或改善有需要的受试者中认知障碍症的方法，所述方法包括将有效量的肽给予需要的受试者。认知障碍症可以由神经退行性疾病引起。或者，认知 障碍症可以伴随神经退行性疾病。
在一个方面，神经退行性疾病或认知障碍症选自阿尔茨海默病或帕金森病。因此，本发明的肽可以用于预防、治疗或改善阿尔茨海默病。此外，本发明的肽可以用于预防、治疗或改善帕金森病。
认知可以是机体认识和获取知识的智能加工过程，涉及学习、记忆、语言、思维、精神、情感等一系列随意、心理和社会行为。认知障碍可以包括与上述学习记忆以及思维判断有关的大脑高级智能加工过程出现异常，从而引起严重学习、记忆障碍，同时伴有失语或失用或失认或失行等改变的病理过程。
在一个方面，本发明涉及本文所述的肽在制备用于改善、增强或恢复有需要的受试者中认知功能或认知能力的组合物中的用途。在一个方面，本发明涉及肽，其用于改善、增强或恢复有需要的受试者中认知功能或认知能力。在一个方面，本发明涉及一种改善、增强或恢复有需要的受试者中认知功能或认知能力的方法，所述方法包括将有效量的肽给予需要的受试者。在该方面中，认知功能或认知能力包括感知能力、思维逻辑能力、记忆能力、语言能力、学习能力、情绪控制能力、社交能力或注意力。在该方面中，所述受试者具有认知功能或认知能力衰退，例如年龄老化而引致的或伴随年龄老化的认知功能或认知能力衰退。或者，所述受试者可以患有认知障碍症或神经退行性疾病引起，优选由神经退行性疾病引起的认知障碍症，更优选阿尔茨海默病或帕金森病。
在一个方面，认知障碍症特征在于或表现在于沟通障碍、判断力差、难以进行简单任务、遗漏或放错物件、语言障碍、性格突变、行为能力下降、时间及空间感混乱、理解能力下降、问题解决能力下降、注意力下降、社交能力下降、感知障碍、思维逻辑障碍或记忆障碍。
在一个方面，神经退行性疾病或认知障碍症为阿尔茨海默病。所述阿尔茨海默病特征在于在大脑特别是海马体中β-淀粉样蛋白(Aβ)沉积、神经炎症、和/或胶质细胞异常或活化。阿尔茨海默病的预防、治疗或改善通过减少或缓解在大脑中β-淀粉样蛋白(Aβ)沉积、神经炎症和/或胶质细胞异常或活化来实现。
在一个方面，本发明涉及本文所述的肽在制备用于减少或缓解受试者大脑特别是海马体中β-淀粉样蛋白(Aβ)沉积、神经炎症和/或胶质细胞异常或活化的组合物中的用途。所述受试者可以是神经退行性疾病患者或认知障碍症患者，优选阿尔茨海默病患者。
本文所述的肽可以有效减少或缓解受试者大脑例如海马体中的Aβ或Aβ斑块沉积。此外，所述肽可以减少或缓解受试者大脑例如海马体或者皮层中炎症因子的表达或水平。炎症因子可以选自IL-1β、IL-6或TNF-α。所述肽还可以减少或缓解大脑海马体胶质细胞异常或活化。胶质细胞也可称为神经胶质细胞，可以 选自星形胶质细胞或小胶质细胞，优选小胶质细胞。
在一个方面，神经退行性疾病或认知障碍症为帕金森病。所述帕金森病特征在于大脑黑质神经元异常或损伤。帕金森病的预防、治疗或改善通过减少或缓解大脑黑质神经元异常或损伤来实现。
在一个方面，本发明涉及本文所述的肽在制备用于减少或缓解受试者大脑黑质神经元异常或损伤的组合物中的用途。所述受试者可以是神经退行性疾病患者或认知障碍症患者，优选帕金森病患者。
在一个方面，受试者或患者是哺乳动物。在一个方面，受试者或患者是人，例如中年人或老年人，优选老年人。中年人可以是40至59岁的人或者45至59岁的人。老年人可以是60岁以上、65岁以上、70岁以上、75岁以上或80岁以上的人。
在一个方面，受试者可以是认知功能或认知能力衰退的受试者，例如年龄老化而引致的或伴随年龄老化的认知功能或认知能力衰退。所述衰退通常是指慢性衰退。
本发明的组合物包含本文所述的肽。本发明的组合物还可以包含药用辅料、溶媒或载体；或者人体可接受的药用辅料、溶媒或载体。组合物可以是药物组合物或者营养组合物。药物组合物可以是药物。营养组合物可为营养补充剂、营养补充品、膳食补充剂、饮食补充剂、保健食品、保健品、营养品或健康食品的形式。
本文所述的“组合物”总体上可以包括改善或提高受试者(例如人)的健康况状或治疗所述受试者的疾病的物质或配方。
本文所述的“营养组合物”可以指满足受试者(例如人)至少部分营养物需求、调节所述受试者身体机能、调理受试者生理功能和/或改善受试者健康状态的可服用的物质或配方。例如，营养组合物可以是激活、调节或改善认知功能或认知能力的保健品、功能性食品、膳食补充剂、营养品或健康食品。
本文所述的“药物组合物”可以指用于预防、治疗、缓解、改善、调理或调节受试者(例如人)的疾病、病症、症状或病状的可服用的物质或配方。例如，药物组合物可以是药物的形式，包括口服药物，例如药片、药丸或胶囊等。
本发明的多肽或组合物的服用可以是口服形式。用于口服的组合物可以使用本领域已知的载体、溶媒或赋形剂以适合于口服施用的剂量进行配制。这样的载体使得组合物能够配制成适合于受试者摄入的片剂、丸剂、胶囊、液体、凝胶、糖浆、浆液、混悬剂等。
此外，本发明的多肽或组合物可被制备成注射剂，例如肌肉注射剂、腹膜内注射剂、腹腔注射剂、皮下注射剂或者静脉注射剂。注射剂可以呈溶液、乳浊液 或混悬液形式，以及临用前配成溶液或混悬液的粉末或浓溶液。本领域技术人员理解，注射剂中还可包含溶剂例如水、等渗剂、缓冲剂、防腐剂、助溶剂、增溶剂、助悬剂和乳化剂等。
术语“有效量”或“治疗有效量”是指本发明的肽或组合物有效治疗受试者的疾病或病症或者改善或提高其健康状况的量。改善或提高其健康状况包括提高或改善认知功能或能力。本发明的肽可以按0.01mg/kg至10mg/kg、0.02mg/kg至8mg/kg、0.04mg/kg至5mg/kg、0.05mg/kg至1mg/kg、0.1mg/kg至0.8mg/kg或0.2mg/kg至0.5mg/kg的量给予受试者。例如，本发明的肽可以按0.04mg/kg、0.16mg/kg或0.64mg/kg的量给予受试者。治疗有效量可以是这些剂量。本发明的肽或组合物可以按一天一次的间隔给药，持续至少1周，例如1至4周。
本发明的肽可以按肽组合的形式给予受试者或制备成组合物。本发明的组合物可包含这样的肽组合。例如，所述肽组合包含具有SEQ ID NO:1的氨基酸序列或其变体的第一肽以及具有SEQ ID NO:2的氨基酸序列或其变体的第二肽。
附图简述
图1.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠短期工作记忆、条件恐惧记忆和社交能力的影响。(A)Y迷宫实验中对照组(Con)，PKT101，PKT002和PKT101/PKT002联合治疗组小鼠进入新奇臂时间百分比的统计图。(B)新物体识别实验中，四组小鼠新物体识别认知指数统计图。(C)条件恐惧记忆实验中，四组小鼠僵直不动时间统计图。(D)社交偏好和社会记忆实验中，(左)小鼠进入Empty室和Stranger-1室的时间百分比统计图；(右)小鼠进入Stranger-1室和Stranger-2室的时间百分比统计图。结果以均值±标准误的方式表示，n＝13-15每组。数据在D图中，四组小鼠进入Empty室和Stranger-1的时间百分的比较以及四组小鼠进入Stranger-1室和Stranger-2室的时间百分比的比较使用T检验，其他统计图使用单因素方差分析。Enter number:进入次数；Recognition index:认知指数；Time spent:耗时；％of freezing:僵直时间百分比。
图2.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠海马和皮层Aβ斑块沉积的影响。(A)对照组(Con)、PKT101、PKT002和PKT101/PKT002联合治疗组小鼠硫磺素S染色的荧光图。标尺为200μm。(B)免疫荧光实验显示四组小鼠皮层和海马6E10表达的荧光图。标尺为200μm。(C)四组小鼠皮层和海马硫磺素S阳性表达Aβ斑块定量统计图。(D)四组小鼠皮层和海马6E10阳性表达面积百分比的统计图。结果以均值±标准误的方式表示，n＝4-5每组，统计图使用单因素方差分析。Cortext：皮层；Hippocampus：海马体；Relative Density：相对密度；Positive area of 6E10+：6E10+的阳性区域。
图3.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠海马Aβ斑块周围胶质细胞活化的影响。(A)免疫荧光实验显示对照组(Con)、PKT101、PKT002和PKT101/PKT002联合治疗组组小鼠海马Iba1，GFAP和6E10表达的荧光图。标尺为50μm。(B)四组小鼠海马Iba1和GFAP阳性表达面积百分比统计图。结果以均值±标准误的方式表示，n＝4-5每组，统计图使用单因素方差分析。Positive area of Iba1+：Iba1+的阳性区域；Positive area of GFAP+：GFAP+的阳性区域。
图4.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠皮层Aβ斑块周围胶质细胞活化的影响。(A)免疫荧光实验显示对照组(Con)、PKT101、PKT002和PKT101/PKT002联合治疗组小鼠皮层Iba1，GFAP和6E10表达的荧光图。标尺为50μm。(B)四组小鼠皮层Iba1和GFAP阳性表达面积百分比统计图。结果以均值±标准误的方式表示，n＝5每组，统计图使用单因素方差分析。Positive area of Iba1+：Iba1+的阳性区域；Positive area of GFAP+：GFAP+的阳性区域。
图5.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠海马和皮层区IL-1β，IL-6和TNF-α含量的影响。(A)ELISA检测对照组(Con)、PKT101、PKT002和PKT101/PKT002联合治疗组小鼠海马中炎症因子IL-1β，IL-6和TNF-α的表达。(B)ELISA检测四组小鼠皮层中炎症因子IL-1β，IL-6和TNF-α的表达。结果以均值±标准误的方式表示，n＝5每组，统计图使用单因素方差分析。Cortext：皮层；Hippocampus：海马体；Relative expression of IL-1β：IL-1β的相对表达；；Relative expression of IL-6：IL-6的相对表达；；Relative expression of TNF-α：TNF-α的相对表达。
图6.PKT101和PKT002对小鼠MPTP亚急性模型后体重改变的影响。0～7天时，n＝20或28；14天时，n＝10或14。Sham：正常对照组；Selegiline：司来吉兰治疗组。
图7.PKT101和PKT002对MPTP亚急性模型后小鼠在矿场实验中总运动路程的影响(1、3、7和14天)。 ***p<0.001, **p<0.01vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
图8.PKT101和PKT002对MPTP亚急性模型后小鼠在爬杆实验中T-Turn的影响(1、3、7和14天)。***p<0.001,**p<0.01vs.Sham；###p<0.001,##p<0.01,#p<0.05vs.MPTP。
图9.PKT101和PKT002对MPTP亚急性模型后小鼠在爬杆实验中T-TLA的影响(1、3、7和14天)。***p<0.001vs.Sham；###p<0.001,##p<0.01,#p<0.05vs.MPTP。
图10.PKT101和PKT002对MPTP亚急性模型后小鼠在转棒实验中潜伏期的影响(1、3、7和14天)。***p<0.001vs.Sham；###p<0.001vs.MPTP。
图11.PKT101和PKT002对MPTP亚急性模型后小鼠黑质致密部尼氏阳性神经元数量的影响(7和14天)。***p<0.001vs.Sham；##p<0.01,#p<0.05vs.MPTP；标尺为250μm。
图12.PKT101和PKT002对MPTP亚急性模型后小鼠黑质致密部TH阳性神经元数量的影响(7和14天)。*p<0.05vs.Sham；###p<0.001,##p<0.01vs.MPTP；标尺为250μm。
具体实施方式
除非另有说明，否则本文使用的所有科学术语都和本领域一般技术人员通常理解的含义一致。下文描述了示例方法和材料，可以使用其等同物。本文提及的所有出版物和其它参考文献都通过引用整体结合到本文中。
提供以下的实施例是为了进一步阐述本发明。以下实施例无意以任何理由限制本发明的范围。
实施例
实施例1–本发明肽对阿尔茨海默病模式动物APP/PS1小鼠神经保护作用的研究
PKT101肽：DEAQETAVSSHEQD(SEQ ID NO:2)
PKT002肽：DEAQETAVSSH(SEQ ID NO:1)
可通过WO2013/173941和WO2016/165102中描述的方法获得上述肽。
1.材料和方法
5月龄的APP/PS1雄性小鼠60只，随机分为4组，每组15只，分别每日腹腔注射生理盐水、PKT101(8mg/kg)、PKT002(8mg/kg)和PKT101/PKT002(8mg/kg)联合给药，为期4周。每组腹腔注射终体积为80μL，每周给药结束后，随机取出20-35只小鼠测量体重，体重平均数作为下周给药剂量的重要依据。
对小鼠进行短期学习记忆(Y迷宫和新物体识别)、条件性恐惧记忆和社交行为学检测，并通过硫磺素染色和6E10免疫荧光染色检测大脑皮层和海马斑块沉积情况；通过免疫荧光染色观察皮质和海马脑区小胶质细胞和星形胶质细胞的活化情况；酶联免疫吸附实验(ELISA)检测炎症因子白介素-1β(IL-1β)、白介素-6(IL-6)和肿瘤坏死因子(TNF-α)的含量。
1.1.行为学检测
1.1.1.短期学习记忆
1.1.1.1.Y迷宫
本实验所使用的Y迷宫装置，三个臂之间夹角为120度，相互联通，三个臂尺寸相同，均为长宽高29cm×8cm×15cm，在臂内侧贴上不同图案为小鼠作为视觉标记，分别命名为起始臂，新奇臂和其他臂。实验流程分为两部分，适应阶段和测试阶段，两次间隔1-2h。适应是将新奇臂用挡板隔开，小鼠只能在起始臂和其他臂内自由活动，每只小鼠适应时间为5min，测试阶段是在1-2h后，将新奇臂打开，小鼠可以在三个臂内自由活动，每只小鼠测试时间为5min。实验过程中用Topscan软件实时记录小鼠进入新奇臂的时间与次数。每次实验结束后都需用75％酒精清理实验器具，清除小鼠气味。
1.1.1.2.新物体识别
本装置在进行测试前，要对小鼠消除陌生感，每天抚摸小鼠，以免操作时对小鼠产生刺激。第1阶段(熟悉期)，在装置中放入2个相同的物体(AB，确保物体没有气味，不被推动)，物体距离两侧壁10cm，将小鼠从两物体中间放入装置中，用摄像头及软件来记录小鼠在每个物体上的探索时间(以嘴或者鼻子接触到物体和凑近物体约2-3cm范围都算对物体的探索)，在5min内(许多实验已经证实熟悉期2min时，动物对新奇事物已经有偏好，而多熟悉3min则偏好更加明显)测定动物探索每个物体的次数、时间和距离。第2阶段(测试期)，第1阶段完成后的1h作为检测记忆的时间间隔，将两个相同物体中的一个物体替换成一个不同的物体放入装置中(AC或BC)，同样将小鼠从两个物体中间放入装置中，记录5min内小鼠对新旧物体探索的次数、时间和距离，即小鼠在新旧物体周围活动的次数、时间和距离，检测小鼠的认知情况。每次实验结束后都需用75％酒精清理实验器具。若小鼠认知能力差，则在新旧物体的探索无差异；若小鼠认知能力正常，则对新事物的探索时间较旧事物长。认知指数(recognition index，RI)计算公式为：RI＝新物体/(新物体+旧物体)×100％。
1.1.2.条件性恐惧记忆
本实验第1天将小鼠放入箱内(箱子底部为铜条栅栏，可通电)，适应3min后，让小鼠在箱内继续停留6min，在此期间，每2min同时给予一次单一频率声音刺激(1.0KHZ，70db，30s)以及不可逃避足底电击(0.8mA，2s)，共三次，记录小鼠6min内声音和电击诱发僵直行为的总时间，随后放回饲养笼。每次实验结束后用75％酒精擦拭箱底。24h后将已建立条件恐惧的小鼠放入原来的箱内，立即给予相同强度的声音刺激(1.0KHZ，70db，30s)，记录小鼠3min内声音诱发僵直行为。僵直行为定义为除呼吸外无其他运动行为。记录环境和声音诱发僵直时间百分比。
1.1.3.社交行为
1.1.3.1.社交偏好阶段
实验开始前，将小鼠放在行为测试室适应0.5h，用所测试的长方形箱子等分为Empty、Center和Stranger-1三个区域。将同性别同背景相同月龄的小鼠放进Stranger-1区域里的金属笼子里，另外一侧箱子的金属笼子空着。将测试小鼠从中间(Center)的箱子里放入，使测试小鼠可以在三个箱子中自由活动5min。拍摄并记录相关参数：进入每个箱子持续的时间，当小鼠的头和四爪都进入一个箱子就认为它处在那个箱子中。
1.1.3.2.社交记忆阶段
在社交记忆阶段实验中空着的金属笼子中放入同性别同背景不同月龄的小鼠(Stranger-2)，然后记录5min，观察测试小鼠在Stranger-1和Stranger-2区域活动的时间。
1.2.脑组织取材和切片制备
1.2.1.取材
在小鼠行为学检测结束后及时取材。麻醉后经眼球取血。用生理盐水由心脏左心室灌流，灌注至肝脏呈土黄色，更换4％多聚甲醛溶液灌注5min，取脑置入4％多聚甲醛，4℃固定过夜。切取海马和皮层部分的脑组织，进行蔗糖梯度脱水。
1.2.2.冰冻切片制备
应用莱卡冰冻切片机对OCT树脂包埋的脑组织，进行冠状切片，片厚10μm，收集于PBS中用于免疫荧光染色和Aβ病理组织学分析。
1.3.免疫荧光染色
切片用10％胎牛血清PBS溶液室温下封闭1h。弃去封闭液，加入鼠源性6E10抗体、鸡源性胶质纤维酸性蛋白抗体和兔源性钙离子结合受体分子1(inized calcium binding adapter molecule 1，Iba1)抗体，4℃孵育过夜。次日洗去一抗，PBS洗3遍，每次5min。加入相应种属的二抗(山羊抗鸡IgG-488；驴抗兔IgG-488；驴抗鼠IgG-647)，室温避光孵育1h。弃去二抗PBS洗3次，每次5min，DAPI染色室温10min，弃去DAPI，PBS洗3遍。抗荧光淬灭剂封片，荧光显微镜下摄片。
1.4.硫磺素S染色
硫磺素S可以标记脑片上的Aβ核心斑块，脱蜡水化后的组织切片即可染色，采用1％硫磺素染色5min，流水冲洗1min，70％酒精分色30s，再用抗荧光淬灭剂封片，荧光显微镜下摄片。
1.5.图像分析
采用Leica显微镜拍摄免疫荧光染色的组织切片，应用ImageJ软件采用灰度阈值分析方法，统计GFAP、Iba1、6E10、硫磺素的阳性面积百分比，每组5只小鼠，每只小鼠选择3张切片，取其平均值作为该组的统计结果。
1.6.酶联免疫吸附实验(ELISA)
1.6.1.取材
小鼠麻醉后，断头处死，剥离颈部和头部皮肤，暴露颅骨，沿矢状缝剪开颅骨，分离颅骨后取出脑组织。将皮层组织和海马组织分别放入标记好的离心管中，迅速在液氮中冷冻，转移到-80℃冰箱保存。
1.6.2.样品准备
冰盒上取皮层和海马组织10-20mg左右，放入1.5mL离心管中称量后按质量体积比1:10加入RIPA裂解液，剩余组织放回原来的管子，用液氮速冻。同时离心管中加入两枚钢珠，放入匀浆机配平后匀浆裂解，然后冰上摇晃30min。取出钢珠，12000rpm，4℃，15min离心，充分吸取上清，转移至新的1.5mL离心管中。采用蛋白定量法定量蛋白浓度。
1.6.3.ELISA
样本加入稀释液稀释到适当浓度，将稀释好的样品加入酶标反应孔，用于测试白介素-1β(IL-1β)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)3种促炎指标，每样品至少加双孔，标准品和样本每孔45μL，随后在样本和标准品孔中加入50μL生物素化抗体工作液，用封板纸封住反应孔板，置室温孵育2h(使用微量振荡器，频率，300rpm)。提前30min制备酶结合底物，室温避光放置。弃去孔中液体后在吸水纸上拍干，洗涤次数5次，洗涤方法：吸干孔内反应液，将洗涤液加入孔板，放置2min略作摇动。除空白孔外的每孔加100μL酶结合底物工作液，置室温孵育1h(使用微量振荡器，频率，300rpm)。弃去孔中液体后在吸水纸上拍干，洗涤次数5次，除空白孔外的每孔加100μL显色底物工作液，室温避光放置15min。除空白孔外的每孔加入终止液100μL终止反应，于20min内测定实验结果。450nm波长下检测，标准孔和测试孔的吸收值。根据标准孔和已知浓度绘制标准曲线，后由标准曲线换算出测试孔的的实际浓度，最后根据蛋白浓度得出每毫克脑组织中含有的目标蛋白质量。
2.PKT101、PKT002和PKT101/PKT002联合给药后对APP/PS1小鼠认知行为的影响
应用Y迷宫实验评价各组小鼠的短期记忆，结果显示：较对照组APP/PS1小鼠相比，PKT101治疗组APP/PS1小鼠进入新奇臂的时间有所增加，而PKT002和PKT101/PKT002联合治疗组APP/PS1小鼠进入新奇臂的时间显著性增加(图1A)。应用新物体识别实验评价各组小鼠的短期记忆，结果显示：较对照组相 比，上述三种治疗组APP/PS1小鼠识别新物体的指数显著性增加(图1B)。应用条件恐惧记忆评价各组小鼠的恐惧记忆，结果显示：PKT101、PKT002和PKT101/PKT002联合治疗组APP/PS1小鼠恐惧时间百分比均未出现明显的变化，但相对于对照组有所减少(图1C)；应用三箱实验评价各组小鼠的社交能力，结果显示：在社会偏爱阶段，PKT101表现出对同性别小鼠(Stranger1)偏爱(图1D左)，提示社会学习能力有所提高；在社会记忆阶段，PKT101组中对Stranger1仍有一定偏爱(图1D右)。实验结果还见下表2。
Figure PCTCN2022084784-appb-000001
3.PKT101、PKT002和PKT101/PKT002联合给药后对APP/PS1小鼠Aβ沉积的影响
应用硫磺素染色和6E10免疫荧光染色，分别评价各组小鼠皮层和海马纤维性和弥散性Aβ斑块沉积情况，硫磺素染色结果显示：与对照组相比，PKT101、PKT002和PKT101/PKT002联合治疗组APP/PS1小鼠海马Aβ斑块均显著减少，另对皮层中沉积Aβ斑块没有影响(图2A，C)。6E10染色结果显示，PKT101、PKT002和PKT101/PKT002联合给药后对APP/PS1小鼠海马Aβ沉积显著性降低，而对皮层中Aβ沉积没有影响(图2B，D)。实验结果还见表3。
表3.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠海马和皮层Aβ斑块沉积的影响
Figure PCTCN2022084784-appb-000002
*p<0.05，vs.对照组。
4.PKT101、PKT002和PKT101/PKT002联合给药后改善APP/PS1小鼠海马Aβ斑块周围胶质细胞活化
通过6E10、GFAP和Iba1共标染色分别评价APP/PS1小鼠海马和皮层Aβ斑块周围小胶质细胞和星形胶质细胞活化情况，结果显示：与对照组相比，PKT101、PKT002和PKT101/PKT002联合治疗组小鼠海马小胶质细胞活化程度均明显降低(图3A-B)。PKT002和PKT101/PKT002联合给药治疗组显著降低小鼠海马星形胶质细胞活化(图3A，C)。与对照组相比，PKT101、PKT002和PKT101/PKT002联合给药治疗组小鼠大脑皮层胶质细胞活化均未见明显改善(图4A-B)。实验结果还见表4。
表4.PKT101、PKT002和PKT101/PKT002联合服用对APP/PS1小鼠皮层和海马Aβ斑块周围胶质细胞活化的影响
Figure PCTCN2022084784-appb-000003
*p<0.05，vs.对照组。
5.PKT101、PKT002和PKT101/PKT002联合给药后对APP/PS1小鼠海马和大脑皮层IL-1β、IL-6和TNF-α含量的影响
应用ELISA检测各组小鼠海马和大脑皮层炎症因子IL-1β、IL-6和TNF-α的含量，结果显示：与对照组相比，PKT101、PKT002和PKT101/PKT002联合治疗小鼠海马炎症因子IL-6和TNF-α的含量显著性降低，PKT002和PKT101/PKT002联合治疗组IL-1β的含量显著性降低(图5A)，而上述三组小鼠大脑皮层IL-6的表达显著性降低，其他炎症因子IL-1β和TNF-α表达均未见显著性差异(图5B)。实验结果还见表5。
6.结论
PKT101和PKT002单独治疗以及PKT101/PKT002联合治疗均能不同程度改善APP/PS1小鼠短期认知障碍、减低海马Aβ负荷沉积、胶质细胞活化和炎症因子的含量。
Figure PCTCN2022084784-appb-000004
实施例2–本发明肽对MPTP亚急性帕金森病小鼠模型药效学实验研究
PKT101肽：DEAQETAVSSHEQD(SEQ ID NO:2)
PKT002肽：DEAQETAVSSH(SEQ ID NO:1)
可通过WO2013/173941和WO2016/165102中描述的方法获得上述肽。
1.材料和方法
应用MPTP(1-甲基-4-苯基-1,2,3,6-四氢吡啶)建立亚急性帕金森病小鼠模型。小鼠称重后记录，按照20mg/kg颈后部皮下注射MPTP，每天一次，连续注射5天，最后一次给药后的第1、3、7和14天时进行后续实验。
将168只健健康成年C57BL/6J雄性小鼠，按体重随机分为八组，为正常对照(Sham)组(n＝20)、MPTP模型组(n＝28)、PKT101低剂量(0.5mg/kg/d)治疗组(n＝20)、PKT101中剂量(2mg/kg/d)治疗组(n＝20)、PKT101高剂量(8mg/kg/d)治疗组(n＝20)、PKT002(8mg/kg/d)治疗组(n＝20)、PKT101(8mg/kg/d)+PKT002(8mg/kg/d)合并治疗组(n＝20)和阳性对照药司来吉兰(Selegiline，咪多吡)(0.5mg/kg/d)治疗组(n＝20)。从第一次MPTP给药后1小时开始腹腔注射相应药物进行治疗，持续7或14天，每天一次。MPTP模型组小鼠给予等体积生理盐水(0.1mL/10g)。Sham组小鼠仅给予等体积生理盐水(0.1mL/10g)。
在MPTP最后一次给药后的第1、3、7和14天时进行旷场、爬杆和转棒实验，评价小鼠的行为学改变。在MPTP最后一次给药后的第7和14天时对中脑进行尼氏和TH染色，计数黑质致密部尼氏和TH阳性细胞数量，评价相应脑区多巴胺能神经元损伤程度。
2.行为学评价
分别在MPTP最后一次给药后的第1、3、7和14天时进行行为学评价。
2.1.旷场试验
旷场试验用于评价小鼠的自主运动能力。开放式旷场装置由不透明的蓝色塑料制成，大小为60cm×60cm×45cm。测试时，将每只小鼠从中心区域放入箱内，同时开始记录每只小鼠在5min内的运动轨迹，使用软件(Clever Sys Inc.，VA，USA)计算总运动路程。每只小鼠测试结束后，使用75％乙醇擦拭旷场区域，防止上一只小鼠的气味对下一只鼠造成影响。
2.2.爬杆试验
爬杆试验用于评估小鼠的运动协调功能。所用杆子直径1cm，高度50cm，立杆顶端固定有直径为1.2cm的木球，将立杆用防滑胶带裹住以防止小鼠滑落。 将小鼠头部向上置于杆顶，小鼠开始向上爬时启动计时器，记下小鼠从开始运动到越过杆顶所需时间(T-Turn)和从开始运动到爬至底部四爪落地时间(T-TLA)。正式测试前，需对每只小鼠进行三次连续训练。测试期间，需连续测量三次，取其中所用最短时间。
2.3.转棒测试
使用转棒试验评估小鼠的运动平衡能力和肢体协调性。将小鼠放于rotarod测试仪滚筒上，将转速设成2分钟内从5rpm/min匀速增加到25rpm/min，小鼠从滚筒上掉落至下方感应区域时会有红外感应器接收信号，记录小鼠在仪器滚筒上运动的时间。正式测试前，需对每只小鼠进行三次连续训练。测试期间，需连续测量三次，取平均掉落时间(潜伏期)。
3.免疫组化研究
分别在MPTP最后一次给药后的第7和14天取每组一半的小鼠，用4％水合氯醛麻醉、生理盐水+4％PFA灌注取脑组织。后经由20％(v/v)和30％(v/v)的蔗糖溶液梯度脱水各3天后，将小鼠脑组织用O.C.T.胶包埋后冰冻，冰冻切片机(Leica)连续组织切片(20μm)，收集相关区域脑片置于冻存液(甘油：0.01M PBS＝1:1)中，于-80℃保存待用。
3.1.尼氏染色
尼氏体是胞质内的一种嗜碱性物质，广泛见于各种神经元，用于计算脑神经元数量。按照尼氏染色试剂盒进行染色。
3.2.TH(Tyrosine hydroxylase，酪氨酸羟化酶)染色
TH主要标记黑质致密部多巴胺能神经元，MPTP造模后该脑区TH阳性神经元会显著减少。取出中脑脑片，0.01M PBS 10min洗片一次后，以3％H 2O 2孵育15min以去除内源性过氧化物酶活性，0.01M PBS 10min×3洗去H 2O 2，然后用含5％BSA和0.3％Triton X-100的PBS于室温下封闭1小时，继而孵育小鼠抗TH一抗(T2928，Sigma，USA)4℃过夜，二抗室温孵育1小时，PBS洗涤5min×3次后二氨基联苯氨(diaminobenzidin，DAB)避光显色。
4.细胞计数和数据处理及分析
免疫组织化学阳性细胞计数采用体式学计数系统(Stereo Investigator，MBF bioscience)计算黑质致密部的尼氏小体阳性细胞和TH +细胞数，细胞计数在×20物镜下进行。
所有数据均采用Mean±S.E.M的表示，采用GraphPad Prism 8.0统计分析软件进行统计学处理。计量数据采用One-way ANOVA合并Dunnett’s test分析各组与造模组之间的差异，p<0.05表示差异具有统计学意义。
5.实验结果
5.1.PKT101和PKT002对小鼠MPTP亚急性模型后生存率的影响
MPTP亚急性给药使得小鼠在造模后精神萎靡、厌食、体重下降，并出现竖尾反应，但本次实验中未出现小鼠的死亡，药物治疗各组小鼠也未出现死亡现象。
5.2.PKT101和PKT002对小鼠MPTP亚急性模型后体重改变的影响
MPTP亚急性给药使得小鼠在造模后进食量小，7天内小鼠体重略降低，14天后进食逐渐增加，体重逐渐恢复。各组处理对小鼠体重无显著影响。结果如图6所示。
5.3.PKT101和PKT002对小鼠MPTP亚急性模型后在矿场实验中自主运动能力的影响
如表6和图7所示，MPTP亚急性模型中，小鼠在造模后1、3、7和14天时出现旷场试验中总运动路程的减少，表明自主活动能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的总运动路程减低。PKT101和PKT002在14天内也均有显著的改善作用。在14天时，单PKT101给药低、中和高剂量增加为模型组的221.1％、253.9％和255.3％；单PKT002给药增加为模型组的213.0％；PKT101和PKT002合并给药增加为模型组的214.8％。
表6.PKT101和PKT002对MPTP亚急性模型后小鼠在矿场实验中总运动路程的影响
Figure PCTCN2022084784-appb-000005
***p<0.001, **p<0.01vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP
5.4.PKT101和PKT002对小鼠MPTP亚急性模型后在爬杆实验中运动协调能力的影响
如表7～8和图8～9，MPTP亚急性模型中，小鼠在造模后1、3、7和14天时出现爬杆实验中T-Turn和T-TLA时间的延长，表明运动协调能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的T-Turn 和T-TLA时间延长。PKT101和PKT002在14天内也均有显著的改善作用。对T-Turn，在14天时，单PKT101给药低、中和高剂量分别减少为模型组的26.1％、24.6％和26.0％；单PKT002给药减少为模型组的24.4％；PKT101和PKT002合并给药减少为模型组的21.6％。对T-TLA，在14天时，单PKT101给药低、中和高剂量分别减少为模型组的68.8％、65.1％和38.6％；单PKT002给药减少为模型组的46.0％；PKT101和PKT002合并给药减少为模型组的46.3％。
表7.PKT101和PKT002对MPTP亚急性模型后小鼠在爬杆实验中T-Turn的影响
Figure PCTCN2022084784-appb-000006
***p<0.001, **p<0.01vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP
表8.PKT101和PKT002对MPTP亚急性模型后小鼠在爬杆实验中T-TLA的影响
Figure PCTCN2022084784-appb-000007
***p<0.001vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP
5.5.PKT101和PKT002对小鼠MPTP亚急性模型后在转棒实验中运动平衡能力的影响
如表9和图10，MPTP亚急性模型中，小鼠在造模后1、3、7和14天时出 现转棒实验中掉落潜伏期的缩短，表明运动平衡能力的下降。阳性对照药司来吉兰(Selegiline)在1、3、7和14天均能显著改善MPTP诱导的潜伏期时间缩短。PKT101和PKT00在14天内均有显著的改善作用。在14天时，单PKT101给药低、中和高剂量的潜伏期分别延长为模型组的121.7、119.9％和118.3％；单PKT002给药延长为模型组的120.3％；PKT101和PKT002合并给药延长为为模型组的120.9％。
表9.PKT101和PKT002对MPTP亚急性模型后小鼠在转棒实验中潜伏期的影响
Figure PCTCN2022084784-appb-000008
***p<0.001vs.Sham； ###p<0.001vs.MPTP
5.6.立再适对小鼠MPTP亚急性模型后黑质致密部尼氏阳性染色神经元数量的影响
如表10和图11，MPTP亚急性模型中，小鼠在造模后7和14天时出现黑质致密部尼氏染色阳性神经元数量的减少，表明该脑区神经元的死亡。阳性对照药司来吉兰(Selegiline)在7和14天均能显著改善MPTP诱导的黑质致密部尼氏染色阳性神经元数量的减少。PKT101和PKT002在7和14天时也均有显著的改善作用。在7天时，单PKT101给药低、中和高剂量的尼氏阳性细胞数量分别增加为模型组的120.9％、124.0％和130.9％；单PKT002给药的尼氏阳性细胞数量增加为模型组的124.5％；PKT101和PKT002合并给药的尼氏阳性细胞数量增加为模型组的128.0％。在14天时，单PKT101给药低、中和高剂量的尼氏阳性细胞数量分别增加为模型组的124.9％、130.9％和141.1％；单PKT002给药的尼氏阳性细胞数量增加为模型组的128.5％；PKT101和PKT002合并给药的尼氏阳性细胞数量增加为模型组的141.5％。
表10.PKT101和PKT002对MPTP亚急性模型后小鼠黑质致密部尼氏阳性神经元数量的影响
Figure PCTCN2022084784-appb-000009
***p<0.001vs.Sham； ##p<0.01, #p<0.05vs.MPTP
5.7.PKT101和PKT002对小鼠MPTP亚急性模型后黑质致密部TH阳性染色神经元数量的影响
如表11和图12，MPTP亚急性模型中，小鼠在造模后7和14天时出现黑质致密部TH阳性神经元数量的减少，表明该脑区多巴胺能神经元的死亡。阳性对照药司来吉兰(Selegiline)在7和14天均能显著改善MPTP诱导的黑质致密部TH染色阳性神经元数量的减少。PKT101和PKT002在7和14天时也均有显著的改善作用。在7天时，单PKT101给药低、中和高剂量的TH阳性细胞数量分别增加为模型组的114.5％、124.9％和129.7％；单PKT002给药的TH阳性细胞数量增加为模型组的129.9％；PKT101和PKT002合并给药的TH阳性细胞数量增加为模型组的130.3％。在14天时，单PKT101给药低、中和高剂量的TH阳性细胞数量分别增加为模型组的125.6％、136.0％和137.5％；单PKT002给药的TH阳性细胞数量增加为模型组的128.1％；PKT101和PKT002合并给药的TH阳性细胞数量增加为模型组的137.7％。
表11.PKT101和PKT002对MPTP亚急性模型后小鼠黑质致密部TH阳性神经元数量的影响
Figure PCTCN2022084784-appb-000010
Figure PCTCN2022084784-appb-000011
*p<0.05vs.Sham； ###p<0.001, ##p<0.01, #p<0.05vs.MPTP。
6.结论
多肽PKT101和PKT002持续7天和14天治疗均能显著改善MPTP亚急性模型后小鼠行为学障碍，可增加旷场试验中总运动路程，缩短爬杆实验T-Turn时间和T-TLA时间，延长转棒实验潜伏期，但各组间剂量依赖关系不明显。同时，PKT101和PKT002均能缓解MPTP诱导的黑质致密部尼氏阳性和TH阳性神经元数量减少。这些结果表明PKT101和PKT002均具有对MPTP亚急性模型的神经保护作用，PKT101和PKT002合并给药与分别单独给药相比，无明显差别。

Claims (15)

  1. 肽在制备用于预防、治疗或改善有需要的受试者中神经退行性疾病或认知障碍症的组合物中的用途，所述肽选自：
    a)包含与DEAQETAVSSH(SEQ ID NO:1)具有至少70％同一性的氨基酸序列的肽；
    b)包含在DEAQETAVSSH(SEQ ID NO:1)中具有0至4个氨基酸突变的氨基酸序列的肽；
    c)包含与DEAQETAVSSHEQD(SEQ ID NO:2)具有至少70％同一性的氨基酸序列的肽；或
    d)包含在DEAQETAVSSHEQD(SEQ ID NO:2)中具有0至4个氨基酸突变的氨基酸序列的肽。
  2. 权利要求1的用途，其中所述认知障碍症由神经退行性疾病引起。
  3. 权利要求1或2的用途，其中神经退行性疾病或认知障碍症选自阿尔茨海默病或帕金森病。
  4. 肽在制备用于改善、增强或恢复有需要的受试者中认知功能或认知能力，优选包括感知能力、思维逻辑能力、记忆能力、语言能力、学习能力、情绪控制能力、社交能力或注意力的组合物中的用途，所述肽选自：
    a)包含与DEAQETAVSSH(SEQ ID NO:1)具有至少70％同一性的氨基酸序列的肽；
    b)包含在DEAQETAVSSH(SEQ ID NO:1)中具有0至4个氨基酸突变的氨基酸序列的肽；
    c)包含与DEAQETAVSSHEQD(SEQ ID NO:2)具有至少70％同一性的氨基酸序列的肽；或
    d)包含在DEAQETAVSSHEQD(SEQ ID NO:2)中具有0至4个氨基酸突变的氨基酸序列的肽。
  5. 权利要求4的用途，其中
    所述受试者具有认知功能或认知能力衰退，例如年龄老化而引致的或伴随年龄老化的认知功能或认知能力衰退；或者
    所述受试者患有认知障碍症或神经退行性疾病，优选由神经退行性疾病引起的认知障碍症，更优选阿尔茨海默病或帕金森病。
  6. 权利要求1-3和5中任一项的用途，其中认知障碍症特征在于沟通障碍、判断力差、难以进行简单任务、遗漏或放错物件、语言障碍、性格突变、行为能力下降、时间及空间感混乱、理解能力下降、问题解决能力下降、注意力下降、社交能力下降、感知障碍、思维逻辑障碍或记忆障碍。
  7. 前述权利要求任一项中的用途，其中所述神经退行性疾病或认知障碍症为阿尔茨海默病，其中
    a)所述阿尔茨海默病特征在于在大脑中β-淀粉样蛋白(Aβ)沉积、神经炎症、和/或胶质细胞异常或活化；或者
    b)阿尔茨海默病的预防、治疗或改善通过减少或缓解在大脑中β-淀粉样蛋白(Aβ)沉积、神经炎症和/或胶质细胞异常或活化来实现。
  8. 前述权利要求任一项中的用途，其中所述神经退行性疾病或认知障碍症为帕金森病，其中
    a)所述帕金森病特征在于大脑黑质神经元异常或损伤；或者
    b)帕金森病的预防、治疗或改善通过减少或缓解大脑黑质神经元异常或损伤来实现。
  9. 前述权利要求任一项中的用途，所述受试者是人，优选老年人。
  10. 前述权利要求任一项中的用途，所述组合物为药物组合物或营养组合物，优选所述药物组合物或营养组合物包含所述肽和药用辅料、溶媒或载体。
  11. 前述权利要求任一项中的用途，所述肽选自：
    a)包含与DEAQETAVSSH(SEQ ID NO:1)具有至少80％同一性的氨基酸序列的肽；
    b)包含在DEAQETAVSSH(SEQ ID NO:1)中具有0至3个氨基酸突变的氨基酸序列的肽；
    c)包含与DEAQETAVSSHEQD(SEQ ID NO:2)具有至少80％同一性的氨基酸序列的肽；或
    d)包含在DEAQETAVSSHEQD(SEQ ID NO:2)中具有0至3个氨基酸突变 的氨基酸序列的肽。
  12. 前述权利要求任一项中的用途，所述氨基酸突变选自氨基酸的添加、缺失或取代。
  13. 前述权利要求任一项中的用途，所述氨基酸突变位于氨基酸序列的C-端和/或N-端，优选是氨基酸序列的C-端和/或N-端的添加或缺失。
  14. 前述权利要求任一项中的用途，所述肽具有8至20个氨基酸的长度，优选10至18个氨基酸长度，更优选11至14个氨基酸长度。
  15. 前述权利要求任一项中的用途，所述肽选自
    1)包含SEQ ID NO:1的氨基酸序列、基本上由SEQ ID NO:1的氨基酸序列组成或者由SEQ ID NO:1的氨基酸序列表示的肽；或者
    2)包含SEQ ID NO:2的氨基酸序列、基本上由SEQ ID NO:2的氨基酸序列组成或者由SEQ ID NO:2的氨基酸序列表示的肽。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321337A (zh) * 2012-05-25 2015-01-28 永林有限公司 肽及其用途
TW201711695A (zh) * 2016-08-19 2017-04-01 首創生物藥品發展有限公司 用於腦血管疾病的治療肽
CN107847550A (zh) * 2015-04-16 2018-03-27 首创生物药品发展有限公司 用于脑血管疾病的治疗肽
JP2020063311A (ja) * 2020-01-31 2020-04-23 プライム・バイオ‐ドラッグ・ディヴェロップメント・リミテッドＰｒｉｍｅ Ｂｉｏ‐Ｄｒｕｇ Ｄｅｖｅｌｏｐｍｅｎｔ Ｌｉｍｉｔｅｄ 脳血管疾患用治療ペプチド

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090232910A1 (en) * 2004-07-22 2009-09-17 Duke University Biomarkers and therapeutics targets for cognitive decline
WO2008031190A1 (en) * 2006-09-15 2008-03-20 Osta Biotechnologies Inc. Alpha-1-antitrypsin as a diagnostic/prognostic indicator for neurodegenerative diseases
ES2332645B1 (es) * 2009-06-30 2010-10-18 Grifols, S.A. Utilizacion de alfa-1-antitripsina para la preparacion de medicamentos para el tratamiento del sindrome de fatiga cronica.
WO2012029061A2 (en) * 2010-08-31 2012-03-08 Uri Wormser POLYPEPTIDES DERIVED FROM α-1 ANTITRYPSIN AND METHODS OF USE THEREOF
KR20170137184A (ko) * 2015-04-16 2017-12-12 이노 바이오-드러그 디벨롭먼트 리미티드 인간 지방 유래 줄기 세포 및 간세포에서 c형 간염 바이러스의 복제를 저해할 수 있는 펩타이드 및 그 유도체
WO2021009288A1 (en) * 2019-07-16 2021-01-21 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Combination comprising alpha-1 antitrypsin for use in treating ischaemia in a subject

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321337A (zh) * 2012-05-25 2015-01-28 永林有限公司 肽及其用途
CN107847550A (zh) * 2015-04-16 2018-03-27 首创生物药品发展有限公司 用于脑血管疾病的治疗肽
CN113893327A (zh) * 2015-04-16 2022-01-07 首创生物药品发展有限公司 用于脑血管疾病的治疗肽
TW201711695A (zh) * 2016-08-19 2017-04-01 首創生物藥品發展有限公司 用於腦血管疾病的治療肽
JP2020063311A (ja) * 2020-01-31 2020-04-23 プライム・バイオ‐ドラッグ・ディヴェロップメント・リミテッドＰｒｉｍｅ Ｂｉｏ‐Ｄｒｕｇ Ｄｅｖｅｌｏｐｍｅｎｔ Ｌｉｍｉｔｅｄ 脳血管疾患用治療ペプチド

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"New Progress in The Diagnosis and Treatment of Common Diseases in Neurology", 31 August 2019, SCIENTIFIC AND TECHNICAL DOCUMENTATION PRESS, CN, ISBN: 978-7-5189-6037-8, article ZHANG, HONGMEI ET AL.: "Passage; New Progress in The Diagnosis and Treatment of Common Diseases in Neurology", pages: 154 - 155, XP009550172 *

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