Use of peptides for treating neurodegenerative diseases or improving cognitive function

Abstract

The present invention relates to the use of peptides for treating neurodegenerative diseases or improving cognitive function. The present invention relates to the treatment of neurodegenerative diseases and cognitive disorders; and the cognitive function is improved and enhanced. In particular, 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 the peptides for improving or enhancing cognitive function in a subject.

Description

Use of peptides for treating neurodegenerative diseases or improving cognitive function Field of the Invention

[0001] The present invention relates to the treatment of neurodegenerative diseases and cognitive disorders, as well as the improvement and enhancement of cognitive function. Specifically, the present invention relates to the use of peptides for preventing or treating neurodegenerative diseases and cognitive disorders, preferably Alzheimer's disease and Parkinson's disease. The present invention also relates to the use of peptides for improving or enhancing cognitive function in a subject. Background Art

[0002] Neurodegenerative disease or neurodegenerative disease (Neurodegenerative Disorder or Neurodegenerative Disease) is a disease directly caused by the gradual degeneration of neurons. The degenerative process can involve the progressive loss of neuronal structure, the progressive loss of neuronal function or the progressive death of neuronal cells. This progressive neurodegeneration often leads to physical disability and mental deterioration. Many neurodegenerative diseases are serious progressive and uninterrupted diseases, and there are few treatments. Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most important neurodegenerative diseases.

[0003] Neurocognitive disorder, also known as dementia, is a type of brain disease that causes a long-term, gradual deterioration in thinking and memory, impacting a person's daily activities. The most common type of dementia is Alzheimer's disease, which accounts for 50% to 70% of all dementia cases. Dementia affects 36 million people worldwide. Approximately 10% of the population will develop the disease during their lifetime. Dementia is closely linked to aging, with approximately 3% of the population aged 65 to 74 developing dementia, another 19% between the ages of 75 and 84, and nearly half of the population over 85 developing dementia.

[0004] Alzheimer's disease (AD) is a chronic neurodegenerative disorder with a slow onset and progressive worsening over time. The most common early symptom is short-term memory loss. As the disease progresses, symptoms may gradually appear, including language impairment, disorientation, emotional instability, loss of motivation, inability to care for oneself, and numerous behavioral problems. As the disease worsens, patients often become disconnected from their families and society, gradually lose physical function, and ultimately die. While the course of the disease varies from person to person, the average life expectancy after diagnosis is approximately three to nine years. In AD, the hippocampus is the first area to be damaged, manifesting as memory loss and loss of orientation (see Yangling Mu et al., Adult hippocampal neurogenesis and its role in Alzheimer's disease, Mol Neurodegener, 2011;6:85). The progression of AD is associated with the deposition of β-amyloid (Aβ) plaques and Tau protein in the brain (see Clive Ballard et al., Alzheimer's disease, The Lancet. 2011 March, 377(9770): 1019–1031). Recent studies have found that neuroinflammation or microglial activation is involved in the spread of tau protein tangles in the neocortex in Alzheimer's disease, leading to the occurrence of cognitive dysfunction in patients with Alzheimer's disease (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 human brain's immune response and is a key factor associated with the development of Alzheimer's disease. Excessive microglial activation is not just an inflammatory epiphenomenon, but also a key upstream mechanism that is crucial to the development of AD.Studies have also found that neuroinflammation and certain inflammatory cytokines are involved in the pathology of AD (see Rishika Dhapola et al., Recent advances in molecular pathways and therapeutic implications targeting neuroinflammation for Alzheimer's disease, Inflammopharmacology volume 29, pages 1669–1681 (2021 November)). Therefore, reducing Aβ plaque deposition, alleviating neuroinflammation, and inhibiting microglial activation can be considered effective means to treat or alleviate AD.

[0005] Parkinson's disease (PD) is a chronic neurodegenerative disease affecting the central nervous system, primarily the motor system. Symptoms typically develop slowly over time, with the most obvious early symptoms being tremors, limb stiffness, decreased motor function, and abnormal gait. Cognitive and behavioral problems may also occur. Cognitive impairment is quite common in patients with severe disease. The primary pathological changes in Parkinson's disease occur in the substantia nigra pars compacta of the ventral midbrain, and the main symptoms are mostly due to the degeneration of dopamine neurons in the substantia nigra pars compacta (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, September 2008). Selegiline is a monoamine oxidase (MAO)-B inhibitor (MAOI). In 2006, the US FDA approved selegiline for the treatment of PD patients.

[0006] There is a need in the art for drugs that can effectively treat neurodegenerative diseases or cognitive impairment caused by neurodegenerative diseases.

[0007] WO2013 / 173941 and CN104321337A disclose an analgesic peptide having 11 to 14 amino acid residues, which matches a fragment of rabbit α1-antiprotease by sequence alignment. WO2016 / 165101 discloses that the peptide can effectively inhibit HCV replication. In addition, WO2016 / 165102 and CN107847550A disclose that the peptide is used to treat stroke (an acute cerebrovascular disease), verifying that the peptide can pass through the brain-blood barrier, and verifying the protective effect of the peptide on PC12 cells from glutamate and hydrogen peroxide-induced cytotoxicity through in vitro cell experiments. These documents are incorporated herein by reference. The PC12 cells tested in WO2016 / 165102 are a type of pheochromocytoma derived from the rat adrenal medulla that displays certain characteristics of ganglion cells, which are particularly used to establish a cell hypoxic injury model, thereby simulating the rapid hypoxia and toxicity state in acute cerebrovascular disease. However, Alzheimer's disease and Parkinson's disease are chronic diseases that are not caused by acute brain hypoxia and toxicity due to cerebral vascular embolism or rupture. The prior art has not mentioned the possibility of using the peptides to treat AD or PD.

[0008] Summary of the Invention

[0009] The present invention also provides a pharmaceutical composition for treating Alzheimer's disease and Parkinson's disease, and a pharmaceutical composition for treating neurodegenerative diseases and cognitive disorders.

[0010] One of the technical problems of the present invention is solved by providing the peptide of the present invention and a composition comprising the peptide.

[0011] The present invention relates to a peptide having an amino acid sequence of SEQ ID NO: 1 or 2 or a variant or fragment thereof. "Variant" and "fragment" mean a peptide having certain amino acid residue changes compared to the peptide of the present invention and substantially retaining 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 having at least a certain percentage of identity with the amino acid sequence, or a variant of an amino acid sequence having at least one or more amino acid mutations compared to the amino acid sequence. For example, the variant may have at least 70% identity with the amino acid sequence of SEQ ID NO: 1 or 2. Alternatively, 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, for example 1, 2, 3, 4 or 5. The length of an amino acid sequence fragment may be 8 to 20 amino acids, for example 10 to 15 amino acids, for example 10, 11, 12, 13, 14 or 15 amino acids.

[0012] In one aspect, the peptides of the invention are selected from:

[0013] a) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1);

[0014] b) a peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1);

[0015] c) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or

[0016] d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).

[0017] In one aspect, a peptide of the invention comprises an amino acid sequence that is 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.

[0018] In one aspect, the peptide of the invention comprises an amino acid sequence in or relative to SEQ ID NO: 1 having 0 to 4 amino acid mutations, such as 0 to 3, such as 1 to 3, for example 0, 1, 2, 3 or 4 amino acid mutations.

[0019] In one aspect, a peptide of the invention comprises an amino acid sequence that is 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.

[0020] In one aspect, the peptide of the invention comprises an amino acid sequence in or relative to SEQ ID NO: 2 having 0 to 4 amino acid mutations, such as 0 to 3, such as 1 to 3, for example 0, 1, 2, 3 or 4 amino acid mutations.

[0021] Amino acid mutations can be selected from the group consisting of additions, deletions, and substitutions of amino acids. Additions can be insertions of amino acids between two amino acid residues in an amino acid sequence, or additions at the C-terminus or N-terminus of an amino acid sequence. Deletions can be deletions of amino acids between two amino acid residues in an amino acid sequence, or deletions at the C-terminus or N-terminus of an amino acid sequence. Substitutions can be substitutions of any amino acid residue in an amino acid sequence. Preferably, the addition, deletion, or substitution of amino acids occurs at the C-terminus or N-terminus of the sequence. Substitutions can be conservative substitutions. Conservative substitutions refer to substitutions between amino acid molecules with similar properties, including properties such as the ionicity, hydrophobicity, and molecular weight of the molecules. Amino acids with similar properties can be classified as follows: aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxyl- or sulfur-containing amino acids (e.g., serine, cysteine, threonine, methionine); cyclic amino acids (e.g., proline); aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan); basic amino acids (e.g., histidine, lysine, arginine); and acidic or amide amino acids (e.g., aspartic acid, glutamic acid, asparagine, glutamine).

[0022] Those skilled in the art will appreciate that the twenty common amino acids constituting peptides, polypeptides or proteins and their abbreviations are shown in Table 1. A skilled person can determine the amino acid residue sequence and composition of the peptides of the present invention, for example, based on Table 1 or common knowledge.

[0023] Table 1. Various amino acids and their abbreviations

[0024] The amino acid mutations can be located at the C-terminus and / or N-terminus of the amino acid sequence. For example, the peptide can have one, two, or three amino acid additions at the C-terminus or N-terminus of SEQ ID NO: 1. For example, the peptide can have one, two, or three amino acid deletions at the C-terminus or N-terminus of SEQ ID NO: 2.

[0025] In one aspect, the peptides of the invention can have a length of 8 to 20 amino acids, preferably 10 to 18 amino acids, more preferably 11 to 14 amino acids. For example, the peptide can be 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.

[0026] In one aspect, the peptides of the present invention can be selected from

[0027] 1) a peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 1; or

[0028] 2) A peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 2.

[0029] In one aspect, the peptide of the present invention consists of the amino acid sequence of SEQ ID NO: 1. Alternatively, the peptide of the present invention consists of the amino acid sequence of SEQ ID NO: 2.

[0030] In one aspect, the present invention relates to the use of the peptides described herein in the preparation of a composition for preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof. In one aspect, the present invention relates to peptides for preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof. In one aspect, the present invention relates to a method for preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof, the method comprising administering an effective amount of the peptide to a subject in need thereof. Neurodegenerative diseases may include cognitive impairment.

[0031] In one aspect, the neurodegenerative disease is a chronic neurodegenerative disease.

[0032] In one aspect, the present invention relates to the use of the peptides described herein in the preparation of a composition for preventing, treating or improving cognitive impairment in a subject in need thereof. In one aspect, the present invention relates to peptides for preventing, treating or improving cognitive impairment in a subject in need thereof. In one aspect, the present invention relates to a method for preventing, treating or improving cognitive impairment in a subject in need thereof, the method comprising administering an effective amount of a peptide to a subject in need thereof. Cognitive impairment may be caused by a neurodegenerative disease. Alternatively, cognitive impairment may be associated with a neurodegenerative disease.

[0033] In one aspect, the neurodegenerative disease or cognitive impairment 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 peptides of the present invention can be used to prevent, treat or improve Parkinson's disease.

[0034] Cognition is the intellectual processing by which an organism recognizes and acquires knowledge, encompassing a range of voluntary, psychological, and social behaviors, including learning, memory, language, thinking, spirit, and emotion. Cognitive impairment can include abnormalities in the brain's higher-level intellectual processing related to learning, memory, and judgment, leading to severe learning and memory impairments, accompanied by pathological changes such as aphasia, apraxia, agnosia, or apraxia.

[0035] In one aspect, the present invention relates to the use of peptides as described herein in the preparation of a composition for improving, enhancing or restoring cognitive function or cognitive ability in a subject in need. In one aspect, the present invention relates to peptides for improving, enhancing or restoring cognitive function or cognitive ability in a subject in need. In one aspect, the present invention relates to a method for improving, enhancing or restoring cognitive function or cognitive ability in a subject in need, the method comprising administering an effective amount of a peptide to a subject in need. In this aspect, cognitive function or cognitive ability include perceptual ability, logical thinking ability, memory ability, language ability, learning ability, emotional control ability, social ability or attention. In this aspect, the subject has cognitive function or cognitive ability decline, such as cognitive function or cognitive ability decline caused by aging or accompanied by aging. Alternatively, the subject may suffer from cognitive impairment or neurodegenerative disease, preferably cognitive impairment caused by neurodegenerative disease, more preferably Alzheimer's disease or Parkinson's disease.

[0036] In one aspect, dementia is characterized or manifested by communication impairment, poor judgment, difficulty performing simple tasks, missing or misplacing objects, language impairment, sudden personality changes, decreased behavioral ability, confusion about time and space, decreased comprehension, decreased problem-solving ability, decreased attention, decreased social skills, perceptual impairment, logical thinking impairment, or memory impairment.

[0037] In one aspect, neurodegenerative disease or cognitive impairment is Alzheimer's disease. The Alzheimer's disease is characterized by beta-amyloid protein (Aβ) deposition, neuroinflammation, and / or glial cell abnormality or activation in the brain, particularly in the hippocampus. The prevention, treatment or improvement of Alzheimer's disease is achieved by reducing or alleviating beta-amyloid protein (Aβ) deposition, neuroinflammation and / or glial cell abnormality or activation in the brain.

[0038] In one aspect, the present invention relates to the use of the peptides described herein in the preparation of a composition for reducing or alleviating beta-amyloid protein (Aβ) deposition, neuroinflammation and / or glial cell abnormality or activation in the brain, particularly the hippocampus, of a subject. The subject may be a patient with a neurodegenerative disease or a patient with cognitive impairment, preferably a patient with Alzheimer's disease.

[0039] The peptides described herein can effectively reduce or alleviate Aβ or Aβ plaque deposition in the subject's brain, such as the hippocampus. In addition, the peptides can reduce or alleviate the expression or level of inflammatory factors in the subject's brain, such as the hippocampus or cortex. The inflammatory factors can be selected from IL-1β, IL-6 or TNF-α. The peptides can also reduce or alleviate abnormalities or activation of glial cells in the hippocampus of the brain. Glial cells can also be called glial cells, and can be selected from astrocytes or microglia, preferably microglia.

[0040] In one aspect, the neurodegenerative disease or cognitive impairment is Parkinson's disease. The Parkinson's disease is characterized by abnormalities 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 abnormalities or damage to neurons in the substantia nigra of the brain.

[0041] In one aspect, the present invention relates to the use of the peptides described herein in the preparation of a composition for reducing or alleviating abnormalities or damage in substantia nigra neurons in a subject's brain. The subject may be a patient with a neurodegenerative disease or a cognitive disorder, preferably a patient with Parkinson's disease.

[0042] In one aspect, the subject or patient is a mammal. In one aspect, the subject or patient is a human, such as a middle-aged or elderly person, preferably an elderly person. A middle-aged person can be a person between 40 and 59 years old or a person between 45 and 59 years old. An elderly person can be a person over 60, over 65, over 70, over 75, or over 80 years old.

[0043] In one aspect, the subject can 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.

[0044] The compositions of the present invention comprise the peptides described herein. The compositions of the present invention may further comprise a pharmaceutical excipient, solvent, or carrier; or a pharmaceutical excipient, solvent, or carrier acceptable to humans. The compositions may be pharmaceutical compositions or nutritional compositions. Pharmaceutical compositions may be medicaments. Nutritional compositions may be in the form of nutritional supplements, nutritional supplements, dietary supplements, dietary supplements, health foods, health products, nutritional supplements, or health foods.

[0045] A "composition" as described herein generally may include a substance or formulation that improves or enhances the health of a subject (eg, a human) or treats a disease in the subject.

[0046] As used herein, a "nutritional composition" may refer to an ingestible substance or formulation that satisfies at least a portion of a subject's (e.g., a human) nutritional needs, regulates the subject's bodily functions, regulates the subject's physiological functions, and / or improves the subject's health. For example, a nutritional composition may be a health product, functional food, dietary supplement, nutraceutical, or health food that activates, regulates, or improves cognitive function or cognitive ability.

[0047] As used herein, a "pharmaceutical composition" may refer to an edible substance or formulation for preventing, treating, alleviating, ameliorating, regulating, or modulating a disease, disorder, symptom, or condition of a subject (e.g., a human). For example, a pharmaceutical composition may be in the form of a drug, including oral medications such as tablets, pills, or capsules.

[0048] The polypeptide or composition of the present invention can be administered orally. Compositions for oral administration can be formulated using carriers, solvents, or excipients known in the art in dosages suitable for oral administration. Such carriers enable the composition to be formulated into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like suitable for ingestion by a subject.

[0049] In addition, the polypeptide or composition of the present invention can be prepared as an injection, such as an intramuscular injection, an intraperitoneal injection, an intraperitoneal injection, a subcutaneous injection or an intravenous injection. The injection can be in the form of a solution, an emulsion or a suspension, as well as a powder or a concentrated solution that is prepared into a solution or suspension before use. It will be understood by those skilled in the art that the injection may also contain a solvent such as water, an isotonic agent, a buffer, a preservative, a cosolvent, a solubilizing agent, a suspending agent and an emulsifier.

[0050] The term "effective amount" or "therapeutically effective amount" refers to the amount of the peptide or composition of the present invention that effectively treats a disease or condition of a subject or improves or enhances their health status. Improving or enhancing their health status includes enhancing or improving cognitive function or ability. The peptide of the present invention can be administered to a subject in an amount of 0.01 mg / kg to 10 mg / kg, 0.02 mg / kg to 8 mg / kg, 0.04 mg / kg to 5 mg / kg, 0.05 mg / kg to 1 mg / kg, 0.1 mg / kg to 0.8 mg / kg or 0.2 mg / kg to 0.5 mg / kg. For example, the peptide of the present invention can be administered to a subject in an amount of 0.04 mg / kg, 0.16 mg / kg or 0.64 mg / kg. The therapeutically effective amount can be these dosages. The peptide or composition of the present invention can be administered at intervals of once a day for at least 1 week, for example, 1 to 4 weeks.

[0051] The peptides of the present invention can be administered to a subject in the form of a peptide combination or prepared as a composition. The composition of the present invention may include such a peptide combination. For example, 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.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1. Effects of PKT101, PKT002, and the combined PKT101 / PKT002 on short-term working memory, conditioned fear memory, and social ability in APP / PS1 mice. (A) Statistical graph of the percentage of time spent in the novel arm of the control (Con), PKT101, PKT002, and PKT101 / PKT002 combined groups in the Y-maze test. (B) Statistical graph of the novel object recognition cognitive index of the four groups of mice in the novel object recognition test. (C) Statistical graph of the freezing time of the four groups of mice in the conditioned fear memory test. (D) Statistical graph of the percentage of time spent in the empty and stranger-1 chambers in the social preference and social memory test (left); (right) () Statistical graph of the percentage of time spent in the stranger-1 and stranger-2 chambers in the social preference and social memory test. Results are presented as mean ± standard error, n = 13-15 per group. In Figure D, the percentage of time spent in the Empty and Stranger-1 chambers, as well as the percentage of time spent in the Stranger-1 and Stranger-2 chambers, was compared using a T-test. All other statistical graphs were analyzed using one-way ANOVA. Enter number: number of entries; Recognition index: cognitive index; Time spent: time spent; % of freezing: percentage of freezing time.

[0054] Figure 2. Effects of PKT101, PKT002, and the combined PKT101 / PKT002 on Aβ plaque deposition in the hippocampus and cortex of APP / PS1 mice. (A) Fluorescence images of thioflavin S staining in mice treated with the control group (Con), PKT101, PKT002, and the combined PKT101 / PKT002 group. Scale bar, 200 μm. (B) Fluorescence images of 6E10 expression in the cortex and hippocampus of the four groups of mice using immunofluorescence. Scale bar, 200 μm. (C) Quantification of thioflavin S-positive Aβ plaques in the cortex and hippocampus of the four groups of mice. (D) Statistical graph showing the percentage of 6E10-positive area in the cortex and hippocampus of the four groups of mice. Results are presented as mean ± standard error (SEM), n = 4-5 per group. Statistical graphs were analyzed using one-way analysis of variance. Cortext: cortex; Hippocampus: hippocampus; Relative Density: relative density; Positive area of ​​6E10+: 6E10+ positive area.

[0055] Figure 3. Effects of PKT101, PKT002, and the combined PKT101 / PKT002 therapy on glial activation surrounding Aβ plaques in the hippocampus of APP / PS1 mice. (A) Immunofluorescence analysis showing Iba1, GFAP, and 6E10 expression in the hippocampus of mice treated with the control group (Con), PKT101, PKT002, and the combined PKT101 / PKT002 therapy. Scale bar, 50 μm. (B) Statistical analysis of the percentage of hippocampal Iba1 and GFAP-positive areas in the four groups of mice. Results are presented as mean ± standard error (SEM), n = 4-5 per group. Statistical analysis was performed using one-way ANOVA. Positive area of ​​Iba1+: Iba1+ positive area; positive area of ​​GFAP+: GFAP+ positive area.

[0056] Figure 4. Effects of PKT101, PKT002, and the combined PKT101 / PKT002 therapy on glial activation surrounding Aβ plaques in the cortex of APP / PS1 mice. (A) Immunofluorescence analysis showing the expression of Iba1, GFAP, and 6E10 in the cortex of mice treated with the control group (Con), PKT101, PKT002, and the combined PKT101 / PKT002 therapy. Scale bar, 50 μm. (B) Statistical analysis of the percentage of Iba1 and GFAP-positive areas in the cortex of the four groups of mice. Results are presented as mean ± standard error (SEM), n = 5 per group. Statistical analysis was performed using one-way ANOVA. Positive area of ​​Iba1+: Iba1+ positive area; Positive area of ​​GFAP+: GFAP+ positive area.

[0057] Figure 5. Effects of PKT101, PKT002, and the combined PKT101 / PKT002 on IL-1β, IL-6, and TNF-α levels in the hippocampus and cortex of APP / PS1 mice. (A) ELISA assay for the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus of mice treated with the control group (Con), PKT101, PKT002, and the combined PKT101 / PKT002 group. (B) ELISA assay for the expression of inflammatory factors IL-1β, IL-6, and TNF-α in the cortex of the four groups of mice. Results are presented as mean ± standard error (SE), n = 5 per group. Statistical graphs were analyzed using one-way ANOVA. Cortex: cortex; Hippocampus: hippocampus; Relative expression of IL-1β: relative expression of IL-1β; Relative expression of IL-6: relative expression of IL-6; Relative expression of TNF-α: relative expression of TNF-α.

[0058] Figure 6. Effects of PKT101 and PKT002 on body weight changes in a subacute MPTP model in mice. Days 0-7, n = 20 or 28; Day 14, n = 10 or 14. Sham: normal control group; Selegiline: selegiline-treated group.

[0059] Figure 7. Effects of PKT101 and PKT002 on the total distance traveled in the mine field test in mice after subacute MPTP model (days 1, 3, 7, and 14). *** p<0.001, ** p<0.01vs.Sham; ### p<0.001, ## p<0.01, # p<0.05vs.MPTP.

[0060] Figure 8. Effects of PKT101 and PKT002 on T-turn in the pole climbing test in mice after subacute MPTP model (days 1, 3, 7, and 14). ***p<0.001, **p<0.01 vs. Sham; ###p<0.001, ##p<0.01, #p<0.05 vs. MPTP.

[0061] Figure 9. Effects of PKT101 and PKT002 on T-TLA in the pole climbing test in mice after subacute MPTP model (days 1, 3, 7, and 14). ***p<0.001 vs. Sham; ###p<0.001, ##p<0.01, #p<0.05 vs. MPTP.

[0062] Figure 10. Effects of PKT101 and PKT002 on the latency of mice in the rotarod test after subacute MPTP model (days 1, 3, 7, and 14). ***p<0.001 vs. Sham; ###p<0.001 vs. MPTP.

[0063] Figure 11. Effects of PKT101 and PKT002 on the number of Nissl-positive neurons in the substantia nigra pars compacta of mice after subacute MPTP model (7 and 14 days). ***p<0.001 vs. Sham; ##p<0.01, #p<0.05 vs. MPTP; scale bar, 250 μm.

[0064] Figure 12. Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta of mice after subacute MPTP model (7 and 14 days). *p<0.05 vs. Sham; ###p<0.001, ##p<0.01 vs. MPTP; scale bar, 250 μm.

[0065] Figure 13. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of GL261 cells.

[0066] FIG14 . Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis in LN229 cells.

[0067] Figure 15. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis in WERI-RB-1 cells.

[0068] Figure 16. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis in N2A cells.

[0069] Figure 17. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis in HT22 cells.

[0070] Figure 18. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis in SK-N-SH cells. DETAILED DESCRIPTION

[0071] Unless otherwise indicated, all scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Exemplary methods and materials are described below, and their equivalents may be used. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0072] The following examples are provided to further illustrate the present invention. The following examples are not intended to limit the scope of the present invention for any reason.

[0073] Example

[0074] Example 1 - Study on the neuroprotective effect of the peptide of the present invention on APP / PS1 mice, a model animal of Alzheimer's disease

[0075] PKT101 peptide: DEAQETAVSSHEQD (SEQ ID NO: 2)

[0076] PKT002 peptide: DEAQETAVSSH (SEQ ID NO: 1)

[0077] The above peptides can be obtained by the methods described in WO2013 / 173941 and WO2016 / 165102.

[0078] 1. Materials and Methods

[0079] Sixty five-month-old male APP / PS1 mice were randomly divided into four groups of 15 mice each and received daily intraperitoneal injections of saline, PKT101 (8 mg / kg), PKT002 (8 mg / kg), or a combination of PKT101 / PKT002 (8 mg / kg) for four weeks. The final intraperitoneal injection volume for each group was 80 μL. After each week of dosing, 20-35 mice were randomly removed and weighed. The average weight served as the basis for the next week's dosing.

[0080] The mice were tested for short-term learning and memory (Y-maze and novel object recognition), conditioned fear memory, and social behavior. Thioflavin staining and 6E10 immunofluorescence staining were used to detect plaque deposition in the cerebral cortex and hippocampus. Immunofluorescence staining was used to observe the activation of microglia and astrocytes in the cortex and hippocampus. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α).

[0081] 1.1. Behavioral testing

[0082] 1.1.1. Short-term learning and memory

[0083] 1.1.1.1.Y Maze

[0084] The Y-maze apparatus used in this experiment had three interconnected arms with an angle of 120 degrees between them. All three arms were identical in size, measuring 29 cm × 8 cm × 15 cm. Different patterns were applied to the inside of the arms as visual markers for the mice, designated as the start arm, novel arm, and other arms. The experimental procedure consisted of two phases: an acclimation phase and a test phase, with a 1-2 hour interval between each phase. During the acclimation phase, the novel arm was separated by a barrier, allowing mice to move freely only within the start arm and the other arms. Each mouse was allowed to acclimate for 5 minutes. After 1-2 hours, the test phase opened the novel arm, allowing mice to move freely within the three arms. Each mouse was tested for 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 apparatus was cleaned with 75% alcohol to remove mouse odor.

[0085] 1.1.1.2. Novel Object Recognition

[0086] Before testing the device, mice were familiarized with the device and handled daily to avoid irritation during handling. In the first phase (familiarization phase), two identical objects (A and B, ensuring they were odorless and could not be pushed) were placed in the device, 10 cm from the two walls. The mouse was placed between the two objects. A camera and software were used to record the amount of time the mouse explored each object (exploration was counted as touching the object with its mouth or nose or moving within 2-3 cm of the object). Over a 5-minute period (many experiments have demonstrated that animals exhibit a preference for novel objects after 2 minutes of familiarization, and this preference becomes more pronounced after 3 minutes of familiarization), the number of times, duration, and distance the animal explored each object were measured. In the second phase (test phase), one hour after the completion of phase 1, memory was tested. One of the two identical objects was replaced with a different object (AC or BC) placed in the device. The mouse was again placed between the two objects. The number of times, duration, and distance the mouse explored each object was recorded over a 5-minute period. This was used to assess the mouse's cognitive abilities. After each experiment, the experimental apparatus was cleaned with 75% alcohol. Mice with poor cognitive abilities will explore the new object in the same way as the old one. Mice with normal cognitive abilities will explore the new object longer than the old one. The recognition index (RI) is calculated as: RI = new object / (new object + old object) × 100%.

[0087] 1.1.2. Conditioned fear memory

[0088] On the first day of this experiment, mice were placed in a box (with copper bars at the bottom and electricity). After acclimation for 3 minutes, the mice were allowed to stay in the box for another 6 minutes. During this period, a single frequency sound stimulus (1.0KHZ, 70db, 30s) and an inescapable foot shock (0.8mA, 2s) were given every 2 minutes for a total of three times. The total time of sound and electric shock-induced rigidity in the mice within 6 minutes was recorded, and then the mice were returned to the breeding cage. After each experiment, the bottom of the box was wiped with 75% alcohol. After 24 hours, the mice with established conditioned fear were placed in the original box and immediately given sound stimulation of the same intensity (1.0KHZ, 70db, 30s). The sound-induced rigidity behavior of the mice was recorded within 3 minutes. Rigidity behavior is defined as the absence of other movement behaviors except breathing. The percentage of rigidity time induced by environment and sound was recorded.

[0089] 1.1.3. Social Behavior

[0090] 1.1.3.1. Social preference stage

[0091] Before the experiment began, the mice were placed in the behavioral testing room to adapt for 0.5 hours. The rectangular boxes to be tested were divided into three areas: Empty, Center, and Stranger-1. Mice of the same sex, background, and age were placed in the metal cages in the Stranger-1 area, and the metal cages in the boxes on the other side were empty. The test mice were placed in the box in the middle (Center) so that the test mice could move freely in the three boxes for 5 minutes. The relevant parameters were photographed and recorded: the duration of entering each box, and when the mouse's head and four paws entered a box, it was considered to be in that box.

[0092] 1.1.3.2. Social Memory Stage

[0093] In the social memory phase of the experiment, mice of the same sex, background, and age (Stranger-2) were placed in an empty metal cage, and then recorded for 5 minutes to observe the time the test mice spent active in the Stranger-1 and Stranger-2 areas.

[0094] 1.2. Brain tissue collection and slice preparation

[0095] 1.2.1. Collection of materials

[0096] After behavioral testing, mice were promptly sampled. Blood was collected from the eyeballs after anesthesia. Saline was perfused through the left ventricle of the heart until the liver turned a khaki color. The solution was then replaced with 4% paraformaldehyde and perfused for 5 minutes. The brain was then removed and fixed in 4% paraformaldehyde overnight at 4°C. The hippocampus and cortex were excised and dehydrated using a sucrose gradient.

[0097] 1.2.2. Frozen Section Preparation

[0098] The brain tissue embedded in OCT resin was sectioned coronally with a thickness of 10 μm using a Leica cryostat and collected in PBS for immunofluorescence staining and Aβ histopathological analysis.

[0099] Immunofluorescence staining

[0100] Sections were blocked with 10% fetal bovine serum in PBS for 1 hour at room temperature. The blocking solution was discarded, and antibodies against mouse 6E10, chicken glial fibrillary acidic protein, and rabbit calcium binding adapter molecule 1 (Iba1) were added and incubated overnight at 4°C. The primary antibodies were removed the next day, and the sections were washed three times with PBS for 5 minutes each. Secondary antibodies of the corresponding species (goat anti-chicken IgG-488; donkey anti-rabbit IgG-488; donkey anti-mouse IgG-647) were added and incubated at room temperature in the dark for 1 hour. The secondary antibodies were discarded, and the sections were washed three times with PBS for 5 minutes each. DAPI staining was performed for 10 minutes at room temperature, the DAPI was discarded, and the sections were washed three times with PBS. Slides were mounted with anti-quencher and photographed under a fluorescence microscope.

[0101] Thioflavin S staining

[0102] Thioflavin S can mark Aβ core plaques on brain slices. Tissue sections can be stained after dewaxing and hydration. Use 1% thioflavin staining for 5 minutes, rinse with running water for 1 minute, separate with 70% alcohol for 30 seconds, then seal with anti-fluorescence quencher and photograph under a fluorescence microscope.

[0103] Image Analysis

[0104] Immunofluorescence-stained tissue sections were photographed using a Leica microscope, and the grayscale threshold analysis method was used with ImageJ software to calculate the percentage of positive areas for GFAP, Iba1, 6E10, and thioflavin. There were five mice in each group, and three sections were selected for each mouse. The average value was taken as the statistical result for the group.

[0105] 1.6. Enzyme-linked immunosorbent assay (ELISA)

[0106] 1.6.1. Collection of materials

[0107] After anesthesia, mice were decapitated and the skin of the neck and head was peeled off to expose the skull. The skull was cut open along the sagittal suture, and the brain tissue was removed after separation. The cortical and hippocampal tissues were placed in separately labeled centrifuge tubes, quickly frozen in liquid nitrogen, and stored in a -80°C freezer.

[0108] Sample preparation

[0109] Remove approximately 10-20 mg of cortical and hippocampal tissue from an ice box, weigh it into a 1.5 mL centrifuge tube, and add RIPA lysis buffer at a mass-to-volume ratio of 1:10. Return the remaining tissue to the original tube and snap-freeze in liquid nitrogen. Add two steel balls to the centrifuge tube, balance it in a homogenizer, and homogenize and lyse it. Shake on ice for 30 minutes. Remove the steel balls, centrifuge at 12,000 rpm, 4°C, for 15 minutes, aspirate the supernatant thoroughly, and transfer it to a new 1.5 mL centrifuge tube. Quantify protein concentration using a protein quantification method.

[0110] 1.6.3.ELISA

[0111] Dilute the sample to the appropriate concentration in diluent and add the diluted sample to the enzyme-labeled reaction wells for testing three pro-inflammatory markers: interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). At least two wells should be prepared for each sample, with 45 μL of standard and sample per well. Then, add 50 μL of biotinylated antibody working solution to the sample and standard wells. Seal the reaction wells with sealing paper and incubate at room temperature for 2 hours (using a microplate shaker at 300 rpm). Prepare the enzyme-conjugated substrate 30 minutes in advance and store at room temperature in the dark. Discard the liquid from the wells, pat dry on absorbent paper, and wash five times. Washing method: Aspirate the reaction solution, add the wash solution to the wells, and incubate for 2 minutes with gentle agitation. Add 100 μL of enzyme-conjugated substrate working solution to each well, except for the blank well, and incubate at room temperature for 1 hour (using a microplate shaker at 300 rpm). After discarding the liquid from the wells, pat dry on absorbent paper. Wash five times. Add 100 μL of chromogenic substrate working solution to each well except the blank well and incubate at room temperature in the dark for 15 minutes. Terminate the reaction by adding 100 μL of stop solution to each well except the blank well. Measure the results within 20 minutes. Measure the absorbance of the standard and test wells at 450 nm. Draw a standard curve based on the standard wells and known concentrations. Calculate the actual concentration of the test wells from the standard curve. Finally, calculate the target protein mass per milligram of brain tissue based on the protein concentration.

[0112] 2. Effects of PKT101, PKT002, and PKT101 / PKT002 combined administration on cognitive behavior in APP / PS1 mice

[0113] Short-term memory was assessed in each group using a Y-maze test. Results showed that compared to the APP / PS1 control group, the APP / PS1 mice in the PKT101-treated group spent more time in the novel arm, while the APP / PS1 mice in the PKT002 and PKT101 / PKT002 combined treatment groups spent significantly more time in the novel arm (Figure 1A). Short-term memory was assessed in each group using a novel object recognition test. Results showed that compared to the control group, the APP / PS1 mice in the three treatment groups had significantly increased novel object recognition indices (Figure 1B). Conditioned fear memory was used to assess fear memory in each group of mice. Results showed that the percentage of fear time in APP / PS1 mice treated with PKT101, PKT002, and the PKT101 / PKT002 combination group did not change significantly, but decreased compared to the control group (Figure 1C). A three-chamber test was used to assess the sociability of each group of mice. Results showed that during the social preference phase, the PKT101 group showed a preference for a same-sex mouse (Stranger1) (Figure 1D, left), suggesting improved social learning ability. During the social memory phase, the PKT101 group still showed a certain preference for Stranger1 (Figure 1D, right). The experimental results are also shown in Table 2 below.

[0114] 3. Effects of PKT101, PKT002, and PKT101 / PKT002 combined administration on Aβ deposition in APP / PS1 mice

[0115] Thioflavin staining and 6E10 immunofluorescence staining were used to assess the accumulation of fibrillar and diffuse Aβ plaques in the cortex and hippocampus of each group of mice. Thioflavin staining revealed that compared with the control group, treatment with PKT101, PKT002, and the combined PKT101 / PKT002 treatment significantly reduced Aβ plaques in the hippocampus of APP / PS1 mice, while having no effect on Aβ plaque accumulation in the cortex (Figures 2A, C). 6E10 staining also revealed that treatment with PKT101, PKT002, and the combined PKT101 / PKT002 treatment significantly reduced Aβ accumulation in the hippocampus of APP / PS1 mice, while having no effect on Aβ accumulation in the cortex (Figures 2B, D). The experimental results are also shown in Table 3.

[0116] Table 3. Effects of PKT101, PKT002, and the combined use of PKT101 / PKT002 on Aβ plaque deposition in the hippocampus and cortex of APP / PS1 mice

[0117] *p<0.05, vs. control group.

[0118] 4. PKT101, PKT002, and PKT101 / PKT002 combined administration improve glial cell activation around Aβ plaques in the hippocampus of APP / PS1 mice

[0119] 6E10, GFAP, and Iba1 co-labeling staining were used to assess microglial and astrocyte activation surrounding Aβ plaques in the hippocampus and cortex of APP / PS1 mice. The results showed that compared with the control group, the degree of microglial activation in the hippocampus of mice treated with PKT101, PKT002, and the PKT101 / PKT002 combination treatment group was significantly reduced (Figures 3A-B). PKT002 and the PKT101 / PKT002 combination treatment group significantly reduced astrocyte activation in the hippocampus of mice (Figures 3A, C). Compared with the control group, no significant improvement was observed in glial cell activation in the cerebral cortex of mice treated with PKT101, PKT002, or the PKT101 / PKT002 combination treatment group (Figures 4A-B). The experimental results are also shown in Table 4.

[0120] Table 4. Effects of PKT101, PKT002, and the combined use of PKT101 / PKT002 on glial cell activation surrounding Aβ plaques in the cortex and hippocampus of APP / PS1 mice

[0121] *p<0.05, vs. control group.

[0122] Effects of PKT101, PKT002, and PKT101 / PKT002 combined administration on IL-1β, IL-6, and TNF-α levels in the hippocampus and cerebral cortex of APP / PS1 mice

[0123] ELISA was used to measure the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus and cerebral cortex of each group of mice. The results showed that compared with the control group, the levels of inflammatory factors IL-6 and TNF-α in the hippocampus of mice treated with PKT101, PKT002, and the PKT101 / PKT002 combination were significantly reduced. IL-1β levels were significantly reduced in the PKT002 and PKT101 / PKT002 combination treatment groups (Figure 5A). However, IL-6 expression in the cerebral cortex of the three groups of mice was significantly reduced. No significant differences were observed in the expression of other inflammatory factors, IL-1β and TNF-α (Figure 5B). The experimental results are also shown in Table 5.

[0124] 6. Conclusion

[0125] PKT101 and PKT002 alone, as well as PKT101 / PKT002 combined treatment, can improve short-term cognitive impairment in APP / PS1 mice to varying degrees, reduce hippocampal Aβ deposition, glial cell activation, and the content of inflammatory factors.

[0126] Example 2 - Pharmacodynamics of the peptide of the present invention on the MPTP subacute Parkinson's disease mouse model

[0127] PKT101 peptide: DEAQETAVSSHEQD (SEQ ID NO: 2)

[0128] PKT002 peptide: DEAQETAVSSH (SEQ ID NO: 1)

[0129] The above peptides can be obtained by the methods described in WO2013 / 173941 and WO2016 / 165102.

[0130] 1. Materials and Methods

[0131] A subacute Parkinson's disease mouse model was established using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Mice were weighed and recorded, and then injected subcutaneously at the back of the neck with 20 mg / kg of MPTP once daily for five consecutive days. Subsequent experiments were performed on days 1, 3, 7, and 14 after the last dose.

[0132] One hundred and sixty-eight healthy adult C57BL / 6J male mice were randomly divided into eight groups according to body weight: a normal control (Sham) group (n=20), an MPTP model group (n=28), a low-dose PKT101 (0.5 mg / kg / d) treatment group (n=20), a medium-dose PKT101 (2 mg / kg / d) treatment group (n=20), a high-dose PKT101 (8 mg / kg / d) treatment group (n=20), a PKT002 (8 mg / kg / d) + PKT002 (8 mg / kg / d) combined treatment group (n=20), and a positive control drug, selegiline (0.5 mg / kg / d) treatment group (n=20). Treatment was performed by intraperitoneal injection of the corresponding drug, starting 1 hour after the first MPTP administration, once daily for 7 or 14 days. The mice in the MPTP model group were given an equal volume of normal saline (0.1 mL / 10 g), while the mice in the sham group were only given an equal volume of normal saline (0.1 mL / 10 g).

[0133] Behavioral changes in the mice were assessed using open field, pole climbing, and rotarod tests on days 1, 3, 7, and 14 after the last MPTP administration. Nissl and TH staining of the midbrain was performed on days 7 and 14 after the last MPTP administration, and the number of Nissl- and TH-positive cells in the substantia nigra pars compacta was counted to assess the extent of dopaminergic neuronal damage in the corresponding brain regions.

[0134] 2. Behavioral evaluation

[0135] Behavioral assessments were performed on days 1, 3, 7, and 14 after the last administration of MPTP.

[0136] 2.1. Open field test

[0137] The open field test is used to assess the locomotor ability of mice. The open field apparatus is made of opaque blue plastic and measures 60 cm × 60 cm × 45 cm. During the test, each mouse is placed into the chamber from the center area. The movement trajectory of each mouse is simultaneously recorded over a 5-minute period, and the total distance traveled is calculated using software (Clever Sys Inc., VA, USA). After each mouse is tested, the open field area is wiped with 75% ethanol to prevent the odor of the previous mouse from affecting the next mouse.

[0138] 2.2. Pole climbing test

[0139] The pole climbing test is used to evaluate the motor coordination function of mice. The pole used is 1 cm in diameter and 50 cm in height. A wooden ball with a diameter of 1.2 cm is fixed to the top of the pole. The pole is wrapped with anti-slip tape to prevent the mouse from slipping. The mouse is placed head-up on the top of the pole. When the mouse starts to climb up, the timer is started. The time required for the mouse to cross the top of the pole (T-Turn) and the time from the start of movement to the landing of the four paws at the bottom (T-TLA) are recorded. Before the formal test, each mouse needs to undergo three consecutive training sessions. During the test, three consecutive measurements are required, and the shortest time is taken.

[0140] Rotarod test

[0141] The rotarod test assesses the motor balance and limb coordination of mice. The mouse is placed on the drum of the rotarod tester, and the speed is set to increase uniformly from 5 rpm / min to 25 rpm / min over 2 minutes. When the mouse falls from the drum to the sensing area below, an infrared sensor receives a signal and records the time the mouse spends on the instrument's drum. Before the formal test, each mouse undergoes three consecutive training sessions. During the test, three consecutive measurements are taken, and the average time to fall (latency) is taken.

[0142] 3. Immunohistochemistry

[0143] On days 7 and 14 after the last MPTP administration, half of the mice in each group were anesthetized with 4% chloral hydrate and perfused with saline + 4% PFA to obtain brain tissue. After dehydration for three days each through a gradient of 20% (v / v) and 30% (v / v) sucrose solutions, the brain tissue was embedded in OCT gel and frozen. Serial tissue sections (20 μm) were cut using a Leica cryostat. Slices from the relevant regions were collected and placed in cryopreservative solution (glycerol:0.01 M PBS = 1:1) and stored at -80°C until use.

[0144] 3.1. Nissl staining

[0145] Nissl bodies are basophilic substances found in the cytoplasm of various neurons and are used to count brain neurons. Staining is performed using a Nissl staining kit.

[0146] TH (Tyrosine hydroxylase) staining

[0147] TH primarily labels dopaminergic neurons in the substantia nigra pars compacta. The number of TH-positive neurons in this brain region is significantly reduced after MPTP modeling. Midbrain slices were removed and washed once with 0.01M PBS for 10 minutes. They were then incubated with 3% H₂O₂ for 15 minutes to eliminate endogenous peroxidase activity. The H₂O₂ was then washed away with 0.01M PBS for 10 minutes × 3. The slices were then blocked with PBS containing 5% BSA and 0.3% Triton X-100 for 1 hour at room temperature. The slices were then incubated with a mouse anti-TH primary antibody (T2928, Sigma, USA) at 4°C overnight, followed by incubation with a secondary antibody at room temperature for 1 hour. The slices were washed with PBS for 5 minutes × 3, and then developed with diaminobenzidin (DAB) in the dark.

[0148] 4. Cell Counting and Data Processing and Analysis

[0149] Immunohistochemical positive cells were counted using a stereoinvestigator system (MBF bioscience) to count Nissl body-positive cells and TH positive cells in the substantia nigra pars compacta. + Cell counts were performed under a ×20 objective lens.

[0150] All data were expressed as mean ± SEM and statistically analyzed using GraphPad Prism 8.0 statistical analysis software. One-way ANOVA combined with Dunnett's test was used to analyze the differences between each group and the model group. P < 0.05 indicated a statistically significant difference.

[0151] 5. Experimental Results

[0152] Effects of PKT101 and PKT002 on survival in mice after subacute MPTP model

[0153] Subacute administration of MPTP caused mice to become listless, anorexic, lose weight, and develop a tail erection reaction after modeling. However, no mice died in this experiment, and no mice in the drug-treated groups died.

[0154] Effects of PKT101 and PKT002 on Body Weight Changes in the Subacute MPTP Model in Mice

[0155] Subacute administration of MPTP resulted in a decrease in the mice's food intake after modeling, leading to a slight decrease in weight within 7 days. After 14 days, their food intake gradually increased, and their weight gradually recovered. There was no significant effect on the mice's weight after any of the treatments. The results are shown in Figure 6.

[0156] Effects of PKT101 and PKT002 on locomotor activity in a mining experiment in mice after a subacute MPTP model

[0157] As shown in Table 6 and Figure 7, in the subacute MPTP model, mice showed a decrease in total locomotor distance in the open field test at 1, 3, 7, and 14 days after modeling, indicating a decline in locomotor activity. The positive control drug, selegiline, significantly improved the MPTP-induced decrease in total locomotor distance at 1, 3, 7, and 14 days. Both PKT101 and PKT002 also showed significant improvements within 14 days. At 14 days, the increase in total locomotor distance after administration of PKT101 alone at low, medium, and high doses was 221.1%, 253.9%, and 255.3% of the model group, respectively; the increase after administration of PKT002 alone was 213.0% of the model group; and the increase after administration of PKT101 and PKT002 combined was 214.8% of the model group.

[0158] Table 6. Effects of PKT101 and PKT002 on the total distance traveled by mice in the mining experiment after subacute MPTP model

[0159] *** p<0.001, ** p<0.01vs.Sham; ### p<0.001, ## p<0.01, # p<0.05 vs. MPTP

[0160] Effects of PKT101 and PKT002 on motor coordination in the pole climbing test after subacute MPTP in mice

[0161] As shown in Tables 7-8 and Figures 8-9, in the subacute MPTP model, mice exhibited prolonged T-Turn and T-TLA times in the pole climbing test at 1, 3, 7, and 14 days after modeling, indicating decreased motor coordination. The positive control drug, selegiline, significantly ameliorated MPTP-induced prolonged T-Turn and T-TLA times at 1, 3, 7, and 14 days. Both PKT101 and PKT002 also demonstrated significant improvements within 14 days. Regarding T-Turn, at 14 days, administration of PKT101 alone at low, medium, and high doses reduced T-Turn to 26.1%, 24.6%, and 26.0% of the model group, respectively; administration of PKT002 alone reduced T-Turn to 24.4% of the model group; and combined administration of PKT101 and PKT002 reduced T-Turn to 21.6% of the model group. For T-TLA, at 14 days, low, medium and high doses of PKT101 alone reduced the incidence of T-TLA to 68.8%, 65.1% and 38.6% of the model group, respectively; single PKT002 administration reduced the incidence of T-TLA to 46.0% of the model group; and combined administration of PKT101 and PKT002 reduced the incidence of T-TLA to 46.3% of the model group.

[0162] Table 7. Effects of PKT101 and PKT002 on T-turn in the pole climbing test in mice after subacute MPTP model

[0163] *** p<0.001, ** p<0.01vs.Sham; ### p<0.001, ## p<0.01, # p<0.05 vs. MPTP

[0164] Table 8. Effects of PKT101 and PKT002 on T-TLA in the pole climbing test in mice after subacute MPTP model

[0165] *** p<0.001vs.Sham; ### p<0.001, ## p<0.01, # p<0.05 vs. MPTP

[0166] Effects of PKT101 and PKT002 on motor balance in the rotarod test in mice after subacute MPTP

[0167] As shown in Table 9 and Figure 10, in the subacute MPTP model, mice showed a shortened latency to fall 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 significantly improved the MPTP-induced shortening of the latency at 1, 3, 7, and 14 days. PKT101 and PKT00 both had significant improvement effects within 14 days. At 14 days, the latency of low, medium, and high doses of PKT101 alone was extended to 121.7%, 119.9%, and 118.3% of the model group, respectively; the latency of PKT002 alone was extended to 120.3% of the model group; and the combined administration of PKT101 and PKT002 was extended to 120.9% of the model group.

[0168] Table 9. Effects of PKT101 and PKT002 on the latency of mice in the rotarod test after subacute MPTP model

[0169] *** p<0.001vs.Sham; ### p<0.001 vs. MPTP

[0170] 5.6. Effect of Lizaishi on the number of Nissl-positive neurons in the substantia nigra pars compacta after subacute MPTP in mice

[0171] As shown in Table 10 and Figure 11, in the subacute MPTP model, mice showed a decrease in the number of Nissl-positive neurons in the substantia nigra pars compacta at 7 and 14 days after modeling, indicating neuronal death in this brain region. The positive control drug selegiline significantly improved the MPTP-induced decrease in the number of Nissl-positive neurons in the substantia nigra pars compacta at both 7 and 14 days. PKT101 and PKT002 also had significant improvement effects at 7 and 14 days. At 7 days, the number of Nissl-positive cells in the low, medium, and high doses of PKT101 alone increased to 120.9%, 124.0%, and 130.9% of the model group, respectively; the number of Nissl-positive cells in the PKT002 alone increased to 124.5% of the model group; and the number of Nissl-positive cells in the combined PKT101 and PKT002 group increased to 128.0% of the model group. At 14 days, the number of Nissl-positive cells in the low, medium and high doses of PKT101 alone increased to 124.9%, 130.9% and 141.1% of the model group, respectively; the number of Nissl-positive cells in the PKT002 alone increased to 128.5% of the model group; and the number of Nissl-positive cells in the combined administration of PKT101 and PKT002 increased to 141.5% of the model group.

[0172] Table 10. Effects of PKT101 and PKT002 on the number of Nissl-positive neurons in the substantia nigra pars compacta of mice after subacute MPTP model

[0173] *** p<0.001vs.Sham; ## p<0.01, # p<0.05 vs. MPTP

[0174] Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta after subacute MPTP in mice

[0175] As shown in Table 11 and Figure 12, in the subacute MPTP model, mice showed a decrease in the number of TH-positive neurons in the substantia nigra pars compacta at 7 and 14 days after modeling, indicating the death of dopaminergic neurons in this brain region. The positive control drug selegiline significantly improved the MPTP-induced decrease in the number of TH-positive neurons in the substantia nigra pars compacta at both 7 and 14 days. PKT101 and PKT002 also had significant improvement effects at 7 and 14 days. At 7 days, the number of TH-positive cells increased to 114.5%, 124.9% and 129.7% of the model group with low, medium and high doses of PKT101 alone, respectively; the number of TH-positive cells increased to 129.9% of the model group with PKT002 alone; and the number of TH-positive cells increased to 130.3% of the model group with the combined administration of PKT101 and PKT002. At 14 days, the number of TH-positive cells increased to 125.6%, 136.0% and 137.5% of the model group after administration of low, medium and high doses of PKT101 alone, respectively; the number of TH-positive cells increased to 128.1% of the model group after administration of PKT002 alone; and the number of TH-positive cells increased to 137.7% of the model group after combined administration of PKT101 and PKT002.

[0176] Table 11. Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta of mice after subacute MPTP model

[0177] * p<0.05vs.Sham; ### p<0.001, ## p<0.01, # p<0.05vs.MPTP.

[0178] 6. Conclusion

[0179] Treatment with the peptides PKT101 and PKT002 for 7 and 14 days significantly improved behavioral impairments in mice following the subacute MPTP model, increasing total distance traveled in the open field test, shortening the T-turn time and T-TLA time in the pole climbing test, and prolonging the latency in the rotarod test, although no dose-dependent relationship was evident between the groups. Furthermore, both PKT101 and PKT002 alleviated the MPTP-induced decrease in the number of Nissl-positive and TH-positive neurons in the substantia nigra pars compacta. These results suggest that both PKT101 and PKT002 possess neuroprotective effects in the subacute MPTP model, with no significant difference observed between the combined administration of PKT101 and PKT002 and either alone.

[0180] Example 3 - Preliminary study on the protective effect of the peptides of the present invention on glial cell and neuronal damage in neurodegenerative disease models

[0181] 1. Purpose of the experiment

[0182] At the cellular level, we preliminarily explored the protective effects of PKT001 and PKT002 against damage to glial cells and neurons, and discussed the general mechanisms for the treatment of neurodegenerative diseases.

[0183] 2. Experimental Plan

[0184] Mouse or human glioma cells and neuronal cells were pretreated with 1, 3, and 9 μg / ml PKT001 and PKT002 in the experimental and control groups for 24 hours, and then induced with lipopolysaccharide (LPS, 1 μg / ml) for 48 hours. The proportion of apoptotic cells was analyzed by flow cytometry using Annexin V / propidium iodide (PI) double staining to detect the effects of PKT001 and PKT002 on apoptosis in various neural cell lines.

[0185] 3. Experimental Materials

[0186] cell lines

[0187] GL261 mouse glioma cells, culture medium: DMEM + 10% FBS + 1% P / S;

[0188] LN229 human glioma cells, culture medium: DMEM + 10% FBS + 1% P / S;

[0189] WERI-RB-1 human retinal glioma cells, culture medium RPMI-1640 + 10% FBS + 1% P / S;

[0190] SK-N-SH human neuroblastoma cells, culture medium: MEM (containing NEAA) + 10% FBS + 1% P / S;

[0191] HT22 mouse hippocampal neuronal cells, culture medium: DMEM + 10% FBS + 1% P / S;

[0192] neuro-2a (N2A) mouse brain neuroma cells, culture medium: MEM (containing NEAA) + 10% FBS + 1% P / S;

[0193] Culture conditions: 37°C, 5% CO2.

[0194] All cells used in the experiment were in the logarithmic growth phase.

[0195] 4. Experimental Procedure

[0196] Effects of PKT001 and PKT002 on apoptosis of neural cells

[0197] (1) Cell subculture and plating: Take the cells in normal culture, remove the original culture medium, add PBS to wash, and then add trypsin to digest for 1-3 minutes to stop the digestion. Use a pipette to blow into single cells, centrifuge the cell suspension at 200g for 5 minutes, resuspend in culture medium, take 20μl of cell suspension and add 20μl trypan blue to count, 3*10 5 The cells were plated at 1000 cells / well (6-well plate), replicated in triplicate, and cultured overnight at 37°C with 5% CO2.

[0198] (2) Dosing treatment:

[0199] ①Cell;

[0200] ②Cell+LPS (1 μg / ml);

[0201] ③Cell+PKT001(1μg / ml)+LPS(1μg / ml);

[0202] ④Cell+PKT001(3μg / ml)+LPS(1μg / ml);

[0203] ⑤Cell+PKT001(9μg / ml)+LPS(1μg / ml);

[0204] ⑥Cell+PKT002(1μg / ml)+LPS(1μg / ml);

[0205] ⑦Cell+PKT002(3μg / ml)+LPS(1μg / ml);

[0206] ⑧Cell+PKT002(9μg / ml)+LPS(1μg / ml);

[0207] There were 8 groups in total. The drugs were administered for 24 hours before pretreatment, and then LPS was induced for 48 hours, and then relevant tests were performed.

[0208] (3) Apoptosis detection:

[0209] ① Collect cells: Centrifuge at 200g for 5 min at 4°C to collect cells.

[0210] ② Wash the cells twice with pre-cooled PBS, centrifuge at 200g each time for 5 minutes at 4℃.

[0211] ③ Aspirate and discard PBS, add 100 μL 1× Binding Buffer to resuspend the cells.

[0212] ④Add 5μL Annexin FITC and 2μL PI and mix gently.

[0213] ⑤Avoid light and react at room temperature for 15 minutes.

[0214] ⑥Add 300 μL 1× Binding Buffer, mix well and place on ice. The sample should be detected by flow cytometry within 1 hour.

[0215] 5. Experimental Results

[0216] Effects of PKT001 and PKT002 on LPS-induced apoptosis in GL261 mouse glioma cells

[0217] Annexin V-FITC / PI double-staining flow cytometry analysis revealed that, compared with the LPS control group, pretreatment with 1-9 μg / ml PKT001 significantly reduced the proportion of GL261 cells undergoing apoptosis 48 hours after LPS induction. Pretreatment with 1-9 μg / ml PKT002 also significantly reduced the proportion of GL261 cells undergoing apoptosis 48 hours after LPS induction. The results are shown in Figure 13.

[0218] Effects of PKT001 and PKT002 on LPS-induced apoptosis in LN229 human glioma cells

[0219] Compared to the LPS control group, pretreatment with 1 μg / ml PKT001 did not significantly change the apoptotic rate of LN229 cells 48 hours after LPS induction. However, pretreatment with 3 μg / ml and 9 μg / ml PKT001 significantly reduced the apoptotic rate of LN229 cells 48 hours after LPS induction. Pretreatment with 1-9 μg / ml PKT002 also significantly reduced the apoptotic rate of LN229 cells 48 hours after LPS induction. The results are shown in Figure 14.

[0220] Effects of PKT001 and PKT002 on LPS-induced apoptosis in WERI-RB-1 human retinal glioma cells

[0221] Compared with the LPS control group, pretreatment with 1-9 μg / ml PKT001 significantly reduced the apoptosis rate of WERI-RB-1 cells induced by LPS for 48 hours; pretreatment with 1-9 μg / ml PKT002 also significantly reduced the apoptosis rate of WERI-RB-1 cells induced by LPS for 48 hours. The results are shown in Figure 15.

[0222] Effects of PKT001 and PKT002 on LPS-induced apoptosis of neuro-2a (N2A) mouse brain neuroma cells

[0223] Compared to the LPS control group, pretreatment with 1 μg / ml and 3 μg / ml PKT001 did not significantly change the percentage of N2A cells undergoing apoptosis 48 hours after LPS induction. However, pretreatment with 9 μg / ml PKT001 significantly reduced the percentage of N2A cells undergoing apoptosis 48 hours after LPS induction. Pretreatment with 1-9 μg / ml PKT002 also significantly reduced the percentage of N2A cells undergoing apoptosis 48 hours after LPS induction. The results are shown in Figure 16.

[0224] Effects of PKT001 and PKT002 on LPS-induced apoptosis of hippocampal neurons in HT22 mice

[0225] The experimental results showed that compared with the LPS control group, pretreatment with 1-9 μg / ml PKT001 significantly reduced the proportion of HT22 cells undergoing apoptosis 48 hours after LPS induction; and pretreatment with 1-9 μg / ml PKT002 significantly reduced the proportion of HT22 cells undergoing apoptosis 48 hours after LPS induction. The results are shown in Figure 17.

[0226] Effects of PKT001 and PKT002 on LPS-induced apoptosis in SK-N-SH human neuroblastoma cells

[0227] Compared to the LPS control group, pretreatment with 1 μg / ml PKT001 did not significantly alter the apoptotic rate of SK-N-SH cells 48 hours after LPS induction. However, pretreatment with 3 μg / ml and 9 μg / ml PKT001 significantly reduced the apoptotic rate of SK-N-SH cells 48 hours after LPS induction. Pretreatment with 1-9 μg / ml PKT002 also significantly reduced the apoptotic rate of SK-N-SH cells 48 hours after LPS induction. The results are shown in Figure 18.

Claims

1. Use of a peptide selected from the group consisting of: a) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1); b) a peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1); c) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).

2. The method according to claim 1, wherein the cognitive impairment is caused by a neurodegenerative disease.

3. The use according to claim 1 or 2, wherein the neurodegenerative disease or cognitive disorder is selected from Alzheimer's disease or Parkinson's disease.

4. Use of a peptide selected from the group consisting of: a) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1); b) a peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1); c) a peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).

5. The method of claim 4, wherein The subject has a decline in cognitive function or cognitive ability, such as a decline in cognitive function or cognitive ability caused by or accompanying aging; or The subject suffers from cognitive impairment or a neurodegenerative disease, preferably cognitive impairment caused by a neurodegenerative disease, more preferably Alzheimer's disease or Parkinson's disease.

6. The use according to any one of claims 1 to 3 and 5, wherein the dementia is characterized by communication impairment, poor judgment, difficulty performing simple tasks, missing or misplacing objects, language impairment, sudden personality changes, decreased behavioral ability, confusion about time and space, decreased comprehension ability, decreased problem-solving ability, decreased attention, decreased social skills, perceptual impairment, logical thinking disorder or memory impairment.

7. The method according to claim 1, wherein the neurodegenerative disease or cognitive disorder is Alzheimer's disease, wherein a) the Alzheimer's disease is characterized by β-amyloid (Aβ) deposition, neuroinflammation, and / or glial cell abnormalities or activation in the brain; or b) Prevention, treatment or improvement of Alzheimer's disease is achieved by reducing or alleviating β-amyloid protein (Aβ) deposition, neuroinflammation and / or glial cell abnormality or activation in the brain.

8. The method according to claim 1, wherein the neurodegenerative disease or cognitive disorder is Parkinson's disease, wherein a) the Parkinson's disease is characterized by abnormalities or damage to neurons in the substantia nigra of the brain; or b) Prevention, treatment or improvement of Parkinson's disease is achieved by reducing or alleviating abnormalities or damage to neurons in the substantia nigra of the brain.

9. Use according to any one of the preceding claims, wherein the subject is a human, preferably an elderly person.

10. The use according to any one of the preceding claims, wherein the composition is a pharmaceutical composition or a nutritional composition, preferably the pharmaceutical composition or nutritional composition comprises the peptide and a pharmaceutically acceptable excipient, solvent or carrier.

11. The use according to any one of the preceding claims, wherein the peptide is selected from the group consisting of: a) a peptide comprising an amino acid sequence that is at least 80% identical to DEAQETAVSSH (SEQ ID NO: 1); b) a peptide comprising an amino acid sequence having 0 to 3 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1); c) a peptide comprising an amino acid sequence that is at least 80% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or d) comprising 0 to 3 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2) amino acid sequence of a peptide.

12. Use according to any one of the preceding claims, wherein the amino acid mutation is selected from the group consisting of addition, deletion or substitution of amino acids.

13. The use according to any one of the preceding claims, wherein the amino acid mutation is located at the C-terminus and / or N-terminus of the amino acid sequence, preferably an addition or deletion at the C-terminus and / or N-terminus of the amino acid sequence.

14. Use according to any one of the preceding claims, wherein the peptide has a length of 8 to 20 amino acids, preferably a length of 10 to 18 amino acids, more preferably a length of 11 to 14 amino acids.

15. The use according to any one of the preceding claims, wherein the peptide is selected from 1) a peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 1; or 2) A peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 2.

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