WO2016062242A1 - 增进神经元生长的胜肽及其应用 - Google Patents

增进神经元生长的胜肽及其应用 Download PDF

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WO2016062242A1
WO2016062242A1 PCT/CN2015/092336 CN2015092336W WO2016062242A1 WO 2016062242 A1 WO2016062242 A1 WO 2016062242A1 CN 2015092336 W CN2015092336 W CN 2015092336W WO 2016062242 A1 WO2016062242 A1 WO 2016062242A1
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peptide
amino acid
group
seq
rats
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PCT/CN2015/092336
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English (en)
French (fr)
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杨滢臻
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福又达生物科技股份有限公司
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Priority to US15/520,605 priority Critical patent/US10407465B2/en
Priority to CN201580056614.4A priority patent/CN107001427B/zh
Priority to JP2017522547A priority patent/JP6526807B2/ja
Priority to EP15853507.0A priority patent/EP3210996B1/en
Publication of WO2016062242A1 publication Critical patent/WO2016062242A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the present invention relates to a peptide which enhances the growth of neurons, particularly a peptide which can enhance the growth of neurites and its use.
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cell line-derived neurotrophic factor
  • IGF insulin-like growth factor
  • peptide drugs have the advantages of small dosage, low side effects, easy access to cells, etc., they have development advantages.
  • the present invention provides, in a first aspect, a peptide which enhances neuronal growth and comprises the following amino acid sequences:
  • R 1 is an amino acid sequence comprising from 1 to 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs.
  • R 2 in the above formula is an amino acid sequence comprising from 1 to 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs.
  • X 1 in the above formula is an amino acid selected from the group consisting of non-polar amino acids.
  • X 2 in the above formula is an amino acid selected from the group consisting of naturally occurring amino acids and amino acid analogs.
  • the indices a and b in the above formula are each independently selected and may be equal to 0 or 1.
  • the invention provides a polynucleotide encoding any of the above peptides.
  • the invention provides a pharmaceutical composition comprising at least one of the above-described peptides and a pharmaceutically acceptable carrier.
  • the invention provides a method of enhancing neuronal growth comprising contacting a neuronal cell with any of the above-described peptides sufficient to increase the amount of neuronal growth.
  • the present invention provides, in a fifth aspect, the use of any of the above peptides for the preparation of a medicament for ameliorating symptoms associated with damage or degeneration of neuronal cells in a subject.
  • Figure 1A to Figure 1I show the effect of C5 peptide (SEQ ID NO: 1) and C6 peptide (SEQ ID NO: 2) on the growth of primary hippocampus neurites.
  • PBS negative control group
  • Fig. 1A C5 peptide (10 -9 , 10 -12 , 10 -15 M)
  • Fig. 1B to Fig. 1D C6 peptide (10 -9 , 10 -12 , respectively.
  • 10 -15 M Fig. 1F to Fig. 1H
  • D-form C5 peptide (10 -9 M) Fig. 1E
  • D-form C6 peptide (10 -12 M) Fig.
  • FIG. 2A to 2G show P1 peptide (SEQ ID NO: 4), P2 peptide (SEQ ID NO: 5), P3 peptide (SEQ ID NO: 6), P4 peptide (SEQ ID NO: 7).
  • PBS negative control group
  • Fig. 2A 10 -9 M P1 peptide
  • FIG. 2C 10 -9 M P2 peptide
  • FIG. 2D 10 -9 M P3 peptide
  • Fig. 2E 10 -9 M P5 peptide
  • Figures 3A through 3E show statistical results of the number and length of branches of primary hippocampal gyrus axons after treatment with some exemplary peptides of the invention.
  • a schematic diagram of neuronal branching is shown in Figure 3A.
  • Primary hippocampal gyrus neurons (on day 3 of differentiation, DIV3) were treated with C5 peptide (10 -12 M) or C6 peptide (10 -12 M) for 3 days, followed by anti-Tau (green) antibody, anti- MAP2 (red) antibody and DAPI DNA fluorescent stain (blue) were immunostained and the number of axon branches (result as shown in Figure 3C) and axon length (results shown in Figure 3B) were calculated.
  • P1 peptide (10 -9 M), P2 peptide (10 -9 M), P3 peptide (10 -9 M), P4 peptide (10 -9 M), P5 peptide (10 -9) M) or P6 peptide (10 -9 M) was used to treat primary hippocampal gyrus neurons (on day 3 of differentiation, DIV3) for 3 days, followed by anti-Tau (green) antibodies, anti-MAP2 (red) antibodies, and DAPI DNA. Fluorescent stain (blue) was immunostained and the number of axon branches (result as shown in Figure 3E) and axon length (results shown in Figure 3D) were calculated. Analytical data was analyzed by Image J software and one-way ANOVA followed by Newman-Keuls comparison. The data is mean ⁇ SEM. ** indicates p ⁇ 0.01 compared to the control group.
  • Figure 4 shows the different concentrations of C5 peptide (10 -12 M, 10 -9 M, 10 -6 M, 10 -3 M) and C6 peptide (10 -12 M, 10 -9 ) by MTT assay.
  • M, 10 -6 M, 10 -3 M) Effect on the survival rate of primary hippocampal gyrus neurons. The data is shown as mean ⁇ SEM with 3 replicates per group.
  • Figure 5 shows the effect of C5 peptide and C6 peptide on neuronal axon regeneration.
  • the primary hippocampal gyrus neurons were treated with PBS (Fig. 5A), C5 peptide (10 -9 M) (Fig. 5B), and C6 peptide (10 -9 ) after treatment on scratch day (DIV5).
  • Figure 6 shows the effect of C5 peptide and C6 peptide on spatial learning and memory formation by water maze test.
  • SD rats were randomly divided into control group, C5 low dose (54 ⁇ g/kg) group, C5 high dose (270 ⁇ g/kg) group, C6 low dose (5.4 ⁇ g/kg) group, and C6 high dose (27 ⁇ g/kg) group.
  • the average escape time (in seconds) for each group of rats per day was recorded.
  • Two-way ANOVA was analyzed and then analyzed by Newman-Keuls. This data is mean ⁇ SEM. ** indicates p ⁇ 0.01 compared to the control group.
  • Figure 7 shows the effect of C5 peptide and C6 peptide on memory formation in a water maze by rats induced by scopolamine hydrochloride (Sco). Rats were randomly divided into control group, injection of purine hydrochloride group (Sco), injection of C5 peptide + Sco group and injection of C6 peptide + Sco group, and received water maze training. Record the average escape time (in seconds) for each day of study. Analysis data were analyzed by two-way ANOVA followed by Newman-Keuls comparison. This data is mean ⁇ SEM. * indicates p ⁇ 0.05 compared to the control group; ** indicates p ⁇ 0.01.
  • Figure 8 shows the effect of C5 peptide and C6 peptide on passive unidirectional inhibitory avoidance learning in rats with memory impairment caused by purine hydrochloride (Sco). Rats were randomly divided into control group, injection of purine hydrochloride group (Sco), C5 peptide (54 ⁇ g/kg) + Sco treatment group and C6 peptide (5.4 ⁇ g/kg) + Sco The group, and accepts passive one-way inhibitory avoidance learning experiments. There was no difference in the residence time of the rats in each group before the foot shock was given. The residence time in the bright box after 1 day and 7 days of giving the foot shock was recorded. One-way ANOVA was followed by a Newman-Keuls comparison assay. This data is mean ⁇ SEM. * indicates p ⁇ 0.05 compared to the control group; ** indicates p ⁇ 0.01.
  • Figures 9A through 9C show the effect of C5 peptide and C6 peptide on memory in a passive avoidance platform in rats with memory impairment caused by purine hydrochloride (Sco). Rats were randomly divided into control group, injection of purine hydrochloride group (Sco), C5+Sco treatment group and C6+Sco treatment group, and received passive avoidance platform memory test.
  • Figure 9A shows that there was no difference in the residence time of each group of rats on the platform prior to giving a foot shock.
  • Figure 9B shows the residence time of the rat on the platform 1 day after the electric shock was given to the foot.
  • Figure 9C shows the number of errors in which the rat jumped off the platform one day after the electric shock was given to the foot.
  • One-way ANOVA was followed by a Newman-Keuls comparison assay. This data is mean ⁇ SEM. ** indicates p ⁇ 0.01.
  • Figure 10 shows the effect of C5 peptide and C6 peptide on the novel object recognition test in rats with memory impairment caused by purine hydrochloride (Sco). Rats were randomly divided into control group, injection of purine hydrochloride group (Sco), injection C5+Sco group and injection C6+Sco group, and received novel object recognition test. Recording the rat's stay in the left object (LO), the right object (RO), and the novel object (NO) before the right object (RO) is replaced with a novel object, 3 hours later, 8 hours later, and 24 hours later. Time (seconds). Analysis data were analyzed by two-way ANOVA followed by Newman-Keuls comparison. This data is mean ⁇ SEM. * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01.
  • Figure 11A and Figure 11B show the effect of C5 peptide and C6 peptide on memory formation in a water maze by rats with aging-induced memory impairment. Rats were randomly divided into control group, C5 group, and C6 group, and received water maze training.
  • Figure 11A shows the average escape time (in seconds) for each group of rats per day prior to dosing.
  • Figure 11B shows the average escape time (in seconds) for each group of rats per day after 6 months of administration.
  • Analysis data were analyzed by two-way ANOVA followed by Newman-Keuls comparison. This data is mean ⁇ SEM. * indicates p ⁇ 0.05 compared to the control group; ** indicates p ⁇ 0.01.
  • Figure 12 shows the effect of C5 peptide and C6 peptide on the survival rate of aging rats.
  • the original number of each group was 6 and recorded every two months from 12 months of age (12 months, 14 months, 16 months, 18 months, 20 months, and 22 months). The results were expressed as the number of survivors in each group.
  • Figure 13 shows the C6 peptide for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) induced the effects of Parkinson's disease in rats.
  • Figure 14 shows the effect of C5 peptide and C6 peptide on memory formation in water maze induced by dextran galactose. Rats were randomly divided into control group, dextran galactose treatment group, injection of C5 peptide + dextran galactose treatment and injection of C6 peptide + dextran galactose, and received water maze training. Record the average escape time (in seconds) for each day of study. Two-way ANOVA and then Newman-Keuls compares the analytical data. This data is mean ⁇ SEM. ** indicates p ⁇ 0.01 compared to the control group.
  • the present invention provides a peptide which can enhance the growth of neurons, and comprises the following amino acid sequences:
  • R 1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs
  • R 2 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs
  • X 1 is selected from An amino acid of the group consisting of non-polar amino acids
  • X 2 is an amino acid selected from the group consisting of naturally occurring amino acids and amino acid analogs
  • a and b are each independently selected and Can be equal to 0 or 1.
  • the a and b are each equal to zero.
  • the amino acid sequence is NAIPQ (SEQ ID NO: 1).
  • the amino acid sequence is NPSPQ (SEQ ID NO: 2).
  • the amino acid sequence is NFEPQ (SEQ ID NO: 4).
  • the amino acid sequence is NMYPQ (SEQ ID NO: 5).
  • the amino acid sequence is NIKPQ (SEQ ID NO: 6).
  • the amino acid sequence is NLMPQ (SEQ ID NO: 7).
  • the amino acid sequence is NVAPQ (SEQ ID NO: 8).
  • the amino acid sequence is NWLPQ (SEQ ID NO: 9).
  • the above amino acid sequences are merely exemplary, and the peptides provided by the present invention which can enhance neuronal growth are not limited to the sequences set forth in SEQ ID NOs: 1, 2, 4-9.
  • X 1 is alanine (Ala, A)
  • X 2 may be alanine, Cysteine, Aspartic acid, glutamic acid ( Glutamic acid), Phenylalanine, Glycine, Histidine, Isoleucine, Lysine, Leucine, Methionine (Methionine), Asparagine, Proline, Glutamine, Arginine, Serine, Threonine, Proline ( Any of Valine), Tryptophan, and Tyrosine.
  • the peptide provided by the present invention which can enhance the growth of neurons, includes, but is not limited to, synthesized by a peptide synthesizer or by genetic selection.
  • the win The peptide is synthesized by a peptide synthesizer and is not limited to the configuration of D-form or L-form.
  • the amino acid residues of the peptides provided by the present invention may be composed of naturally occurring amino acids or unnatural amino acids known in the art, all L or all D, or combinations thereof.
  • the peptide can also be obtained by genetic selection.
  • the manner in which the peptide recombinant protein is obtained by genetic selection may be, but is not limited to, selecting a polynucleotide encoding any of the above peptides into a recombinant nucleic acid expression vector to form a vector containing any of the above-described peptides.
  • a recombinant nucleic acid expression vector of a polynucleotide which is then transfected into a biological expression host, and expressed by the protein to obtain a peptide which can enhance the growth of neurons disclosed in the present invention.
  • the present invention also provides a polynucleotide encoding any of the above-described peptides, and/or a recombinant nucleic acid expression vector comprising a polynucleotide encoding any of the above-described peptides, and/or a composition comprising any of The recombinant nucleic acid of the polynucleotide of the above peptide represents the host cell of the vector.
  • the polynucleotide encoding any of the above peptides of the present invention is derived from the amino acid sequence of the peptide of the present invention which enhances neuronal growth. Substituting each amino acid sequence of the amino acid sequence of the peptide of the present invention for enhancing neuronal growth into a nucleotide sequence encoding the amino acid listed in the genetic code table (including various degenerate codons)
  • the nucleotide sequence provided by the present invention can be obtained by codons, or synonymous codons.
  • the proline acid of the amino acid sequence of the peptide of the present invention which can enhance the growth of neurons can be encoded by nucleotide sequences such as CCA, CCC, CCG, CCT.
  • the invention further provides a composition comprising at least one of the above-described peptides which enhance neuronal growth and a pharmaceutically acceptable carrier.
  • the combination comprises more than one of the above peptides, such as, but not limited to, NAIPQ (SEQ ID NO: 1), NPSPQ (SEQ ID NO: 2), NFEPQ (SEQ ID NO: 4), NMYPQ (SEQ ID NO: 5), NIKPQ (SEQ ID NO: 6), NLMPQ (SEQ ID NO: 7), NVAPQ (SEQ ID NO: 8), NWLPQ (SEQ ID NO: 9) one of the peptides, or Its composition.
  • the composition contains, but need not be, a pharmaceutically effective amount of the above peptide.
  • the peptides provided by the present invention enhance the growth of neurons, particularly the number of axon branches and the increase in axon length.
  • a brain cell When a brain cell is to perform various activities (for example, learning, memory, repair, regeneration), it is necessary to form a new synapse.
  • the process of synaptogenesis is that presynaptic neuron first forms axonal terminal boutons and then attracts postsynaptic neuron to form dendritic protrusions. The two touch each other to form a synapse, so that the nerve cells can cross-talk, form a new connection, and brain development and remodeling can occur to perform various activities. .
  • Brain-derived neurotrophic Factor is the dendritic arborization that occurs by stimulating neuronal cells to form new axonal neurite outgrowth to promote neuronal growth.
  • BDNF brain-derived neurotrophic Factor
  • the peptide provided by the present invention significantly enhances axonal growth, which acts similarly to brain-derived neurotrophic factor (BDNF) by increasing axon growth to promote neuronal growth. .
  • the peptide provided by the present invention promotes neurite branching by increasing axon growth, thereby reducing damage or degradation of neuronal cells caused by aging or other causes, and thus improving Symptoms associated with damage or degeneration of neuronal cells.
  • the present invention provides a method of enhancing neuronal growth comprising contacting a neuronal cell with any of the above-described peptides sufficient to increase the amount of neuronal growth.
  • the neuronal cell is a neuronal cell with normal function.
  • the neuronal cell is a degenerating neuronal cell.
  • the neuronal cell is a damaged neuronal cell.
  • Causes of damage or degradation of neuronal cells include, but are not limited to, physical damage (eg mechanical damage, contusion, severing), chemical damage (eg alcohol, scopolamine hydrochloride, 1 -Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), Amphetamine damage caused by amphetamine ), biological damage (such as: damage caused by cerebral hypoxia), aging or a combination thereof.
  • the present invention also provides a method of improving memory impairment in a subject.
  • the present invention also provides a method of improving a subject's intelligent decline.
  • the subject's memory impairment or mental decline is caused by aging.
  • the subject's memory impairment or intelligent decline is caused by mechanical damage.
  • the subject's memory disorder or mental decline is caused by a chemical agent.
  • the subject's memory disorder or mental decline is caused by purine base hydrochloride.
  • Scopolamine Hydrochloride (Sco) is an animal and human muscarinic receptor antagonist that causes learning and memory damage and is commonly used to establish animals with cognitive decline and memory impairment. model.
  • the present invention uses a memory-deleted animal model induced by purine base hydrochloride (Sco) to analyze the effect of the peptide disclosed herein on smart decay.
  • the subject's memory disorder or mental decline is caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP+ 1-methyl-4-phenylpyridinium
  • the invention uses an MPTP-induced Parkinson's disease animal model to analyze the effects of the peptides disclosed herein on the disease.
  • the subject's memory disorder or mental decline is caused by natural aging or by D-(+)-galactose.
  • dextran galactose is a physiologically available nutrient, but excessive intake of dextran galactose causes non-enzymatic glycation, making the protein or lipid molecule uncontrolled by enzymes. , attached to the sugar molecule.
  • Sugar and protein polymerize each other and undergo a series of reactions that produce non-reducible substances. These non-reducible substances are linked to other proteins into macromolecules, which cause abnormal protein metabolism, and form high concentrations of advanced glycation end products (AGEs) accumulated in the cells to produce a large number of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • the invention uses a naturally aged animal model to analyze the effects of the peptides disclosed herein on smart decay.
  • the invention uses an aging animal model induced by dextran galactose to analyze the effect of the peptide disclosed herein on smart decay.
  • the present invention also provides the use of any of the above-described peptides for the preparation of a medicament for ameliorating symptoms associated with damage or aging of neuronal cells in a subject.
  • the symptoms include, but are not limited to, memory impairment, mental decline, impaired motor coordination, decreased survival, central nervous system disorders, Parkinson's disease, Alzheimer's disease, affecting sensation Neuronal diseases, diseases of the cortical limb system, conditions associated with developmental delay and learning disabilities, Down's syndrome, oxidative stress-induced neuronal death, conditions caused by aging, conditions caused by chronic alcoholism, drugs Abuse of the resulting condition, pathological changes due to local trauma, and conditions resulting from the negative side effects of the therapeutic drug and treatment.
  • the symptoms are memory impairment, mental decline, impaired motor coordination, decreased survival, Parkinson's disease, and/or Alzheimer's disease. In some embodiments, the above symptoms are significantly improved after the subject has administered the peptide of the present invention for a period of time compared to the negative control group.
  • a means one or more than one (ie, at least one) item of grammar.
  • a component means one element or more than one element.
  • nucleotide is intended to include a monomer comprising a nitrogen base attached to a sugar phosphate, the sugar phosphate comprising a sugar, such as ribose or 2'-deoxyribose, attached to one or more Phosphoric acid group.
  • Polynucleotide and “nucleic acid” mean a polymer comprising more than one nucleomonomer, wherein the monomer is usually The sugar-phosphate bond of the sugar-phosphate backbone is linked. Polynucleotides do not have to include only one type of nucleomonomer.
  • a nucleotide comprising a given polynucleotide may be only a ribonucleotide, only a 2'-oxyribonucleotide, or both a ribonucleotide and a 2'-deoxyribonucleotide.
  • Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs comprising one or more non-naturally occurring monomers. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer.
  • the term "nucleic acid" generally refers to a large polynucleotide.
  • nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNA sequences (ie, A, U, G, C), where "U” replaces "T” ".
  • cDNA means a DNA that is complementary or identical to an mRNA, whether in single or double stranded form, but in which "T” is substituted for "U”.
  • recombinant nucleic acid means a polynucleotide or nucleic acid having sequences that are not naturally joined together. The recombinant nucleic acid can be in the form of a vector.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids.
  • Naturally occurring amino acids are those which are encoded by the genetic code and which are subsequently modified, for example, hydroxyproline, gamma carboxy glutamic acid, and O-phosphoric acid.
  • amino acid analog means a compound having the same basic chemical structure as a naturally occurring amino acid, that is, a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, the R The group is, for example, homoserine, orthraenic acid, thiomethionine sulfur oxide, methionine methylhydrazine. These analogs have a modified R group (eg, positive leucine) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acid.
  • amino acid mimetic refers to a compound having a structure different from the general chemical structure of an amino acid, but which acts in a manner similar to a naturally occurring amino acid.
  • non-polar amino acid refers to a hydrophobic alpha amino acid wherein the functional group is attached to the alpha carbon chain (ie, R at RCH(NH2)COOH).
  • the non-polar amino acids of the present invention comprise naturally occurring and synthetic non-polar amino acids, as well as non-polar amino acid analogs and non-polar amino acid mimetics that act in a manner similar to naturally occurring non-polar amino acids; for example, However, it is not limited to alanine, lysine, leucine, isoleucine, valine, phenylalanine, methylamine, tryptophan, or a-aminobutyric acid.
  • peptide refers to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are analogs of an analog or corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term "conservatively modified variation” applies to both amino acid and nucleic acid sequences.
  • Conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences for a particular nucleic acid sequence, or where the nucleic acid does not encode an amino acid sequence, to refer to substantially identical sequences.
  • substitution of degenerate codons can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is mixed with bases and/or Replaced with oxyinosine residues. Due to the degeneracy of the genetic code Sex, a large number of functionally identical nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG and GCU all encode alanine.
  • the codon can be altered to any of the corresponding codons described without altering the encoded multi-peptide.
  • Such nucleic acid variations are "silent variations" and are one of the conservatively modified variations.
  • Each of the nucleic acid sequences encoding a peptide herein also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan ) can be modified to produce molecules of the same function. Thus, each silent variation of a nucleic acid encoding a peptide is implicit in each of said sequences.
  • the term "vector” means a means by which a nucleic acid can be introduced into a host cell to transform the host cell and promote expression of the nucleic acid.
  • the vector may comprise a given target nucleotide sequence as well as a regulatory sequence.
  • a vector can be used to express the given nucleotide sequence or to maintain the given nucleotide sequence to replicate it, manipulate it, alter it, truncate it, expand it, and/or transfer between different positions. It is (for example, between different organisms or host cells or a combination thereof).
  • host cell refers to a prokaryotic or eukaryotic organism of a single cell, including but not limited to: actinomycetes, archaea, bacteria, and yeasts. Host cells can also be single cells, including but not limited to cultured cells - from higher order organisms such as plants and animals including, but not limited to, vertebrates such as mammals and invertebrates such as insects.
  • symptoms associated with damage or degeneration of neuronal cells refers to symptoms comprising neuropathy that cause neuronal cell death and/or are not fatal, including, for example:
  • Central nervous system disorders including degenerative diseases affecting the basal ganglia (eg, Huntington's disease, Wilson's disease, striatonigral degeneration, cortex Corticobasal ganglionic degeneration), Tourette's syndrome, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy (progressive) Bulbar palsy), hereditary spastic paraplegia, spinal muscular atrophy, amyotrophic lateral sclerosis, and variants thereof, dentate nucleus red globus pallidus Dentatorubral-pallidoluysian atrophy, olivopontocerebellar atrophy, paraneoplastic cerebellar degeneration, and dopamine toxicity;
  • basal ganglia eg, Huntington's disease, Wilson's disease, striatonigral degeneration, cortex Corticobasal ganglionic degeneration
  • Tourette's syndrome Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy (
  • cortical limb system for example, cerebral amyloid Angiopathy), Pick's atrophy, Retts syndrome; neurodegenerative diseases involving the nervous system and/or brainstem, including Alzheimer's disease, AIDS-related AIDS-related dementia, childhood Leigh's disease, diffuse Lewy body disease, epilepsy, multiple system atrophy, Guillain-Barre syndrome -Barre syndrome), lysosomal storage disorders, such as lipofuscinosis, late-degenerative stages of Down's syndrome, childhood spongiform encephalopathy (Alpers) Alper's disease, dizziness caused by degeneration of the central nervous system;
  • Conditions resulting from aging, chronic alcohol abuse, or drug abuse including, for example, degeneration of neurons in the anterior cerebral ventricle, cerebellum, and cholinergic basal brain due to alcoholism, cerebellar neurons and cortex due to aging Cognitive and motor impairment caused by neuronal degeneration, and motor damage caused by degeneration of neurons of the basal ganglia caused by long-term amphetamine abuse;
  • Pathological changes due to local trauma such as: stroke, ischemia, vascular insufficiency, hypoxic ischemic encephalopathy, hyperglycemia, hypoglycemia, closed head trauma, or direct trauma;
  • Conditions resulting from adverse side effects of therapeutic drugs and treatment eg, resistance to antagonists of glutamate receptors in response to N-methyl-D-aspartate (NMDA) Degeneration of the cingulate cortex and entorhinal cortex caused by sputum dose).
  • NMDA N-methyl-D-aspartate
  • the term "pharmaceutical composition” refers to any formulation in which the peptide of the invention can be formulated, stored, stored, altered, administered, or a combination thereof.
  • the formulations may include any of their pharmaceutically acceptable diluents, adjuvants, buffers, excipients, carriers, or combinations, as described below.
  • the compositional components of the formulation are selected based on the mode and route of administration, as well as standard pharmaceutical practice.
  • the term “pharmaceutically acceptable carrier” means that any substance or combination thereof may be physically or chemically mixed, dissolved, suspended, or otherwise combined with the peptide of the present invention to produce a pharmaceutical of the present invention. combination.
  • pharmaceutically effective amount means capable or sufficient to maintain or produce a desired physiological result including, but not limited to, treating, alleviating, eliminating, substantially preventing or preventing, or a combination thereof, a disease, a condition, or Its combination.
  • a pharmaceutically effective amount can include administering one or more doses sequentially or simultaneously.
  • the dosages of the present invention are adjusted to allow for various types of formulations including, but not limited to, sustained release formulations.
  • prevention means a composition that substantially prevents or prevents any aspect of a disease, disorder, or combination thereof.
  • therapeutic means capable Sufficient to treat, reduce, stop progression, slow progression, beneficially alter, eliminate, or a combination thereof, any aspect of the disease, condition, or a combination thereof.
  • the term "subject" means any individual that is administered to the present invention.
  • the subject can be, for example, a mammal.
  • the subject can be a human or veterinary animal, regardless of gender, age, or any combination thereof, and includes the fetus.
  • a subject can be selectively affected by, or at risk of, a particular disease, disorder, or combination thereof.
  • Formulations suitable for administration in accordance with the present invention may include, among other things well known to those skilled in the art: aqueous and non-aqueous solutions, antioxidants, bacteriostats, buffers, isotonic solutes, preservatives, Solubilizers, stabilizers, suspending agents, thickeners, or combinations thereof.
  • formulations suitable for administration of the present invention may include, among other things well known to those skilled in the art: gels, PEG such as PEG 400, propylene glycol, saline, sachets; Other suitable liquids known in the art, or a combination thereof.
  • formulations suitable for administration of the present invention may include, among other things well known to those skilled in the art: binders, buffers, calcium phosphate, cellulose, colloids, such as colloidal dioxide. Silicon, colorants, diluents, disintegrants, dyes, fillers, flavoring agents, gelatin, lactose, magnesium stearate, mannitol, microcrystalline gelatin, wetting agents, paraffin hydrocarbons, lozenges, polyethylene glycol, Preservatives, sorbitol, starches, such as corn starch, potato starch, or combinations thereof, stearic acid, sucrose, talc, triglycerides, or combinations thereof.
  • formulations suitable for administration in accordance with the present invention may include, among other things well known to those skilled in the art: alcohols such as benzyl alcohol or ethanol, benzalkonium chloride, buffers such as phosphate buffers. Agent, acetate buffer, citrate buffer, or a combination thereof, carboxymethyl cellulose or microcrystalline cellulose, cholesterol, glucose, fruit juice, such as grapefruit juice, milk, phospholipids such as lecithin, oils such as vegetable oil, Fish oil, or mineral oil, or a combination thereof; other pharmaceutically compatible carriers known in the art; or a combination thereof.
  • alcohols such as benzyl alcohol or ethanol
  • benzalkonium chloride buffers such as phosphate buffers.
  • Agent acetate buffer, citrate buffer, or a combination thereof, carboxymethyl cellulose or microcrystalline cellulose, cholesterol, glucose, fruit juice, such as grapefruit juice, milk, phospholipids such as lecithin, oils such as vegetable oil, Fish oil, or mineral oil, or a combination thereof;
  • formulations suitable for administration of the present invention may include, among other things well known to those skilled in the art: biodegradable, such as polylactic acid-polyglycolic acid (PLGA) polymers, Degradation products of other entities can be rapidly removed from a biological system, or a combination thereof.
  • biodegradable such as polylactic acid-polyglycolic acid (PLGA) polymers
  • PLGA polylactic acid-polyglycolic acid
  • the formulations of the invention may be administered in unit dosage form, in multiple dosage forms, or in a combination thereof. They can be packaged in unit dose containers, multi-dose containers, or combinations thereof.
  • the invention may be present in ampoules, capsules, capsules, granules, lozenges, powders, tablets, vials, emulsions including, but not limited to, gum arabic emulsions, suspensions, or combinations thereof.
  • Example 1 Effect of C5 peptide, C6 peptide, P1 peptide, P2 peptide, P3 peptide, P4 peptide, P5 peptide, P6 peptide on neuron growth
  • the C5, C6, P1, P2, P3, P4, P5, and P6 peptides of D-form and L-form were synthesized by a synthesizer (Neogene Biomedicals, Taipei, Taiwan).
  • Synthetic C5 peptide (SEQ ID NO: 1), C6 peptide (SEQ ID NO: 2), P1 peptide (SEQ ID NO: 4), P2 peptide (SEQ ID NO: 5), P3 peptide (SEQ ID NO: 6), P4 peptide (SEQ ID NO: 7), P5 peptide (SEQ ID NO: 8), P6 peptide (SEQ ID NO: 9) were dissolved in DMSO and PBS, respectively.
  • the medium was then replaced with 2% B27-neurobasal medium (Invitrogen, Carlsbad, CA, USA) containing 0.5 mM glutamine and 12.5 mM glutamate.
  • Embryonic primary hippocampal neurons were cultured at 37 ° C, 5% CO 2 for further testing.
  • Embryonic primary hippocampal neurons were cultured on day 3 in vitro (days in vitro 3, DIV3), C5 peptide (C-peptide) or C6 peptide at concentrations of 10 -9 M, 10 -12 M, 10 -15 M (L-form), 10 -9 M concentration of each of P1, P2, P3, P4, P5, P6 peptides (L-form), 10 -9 M concentration of C5-peptide (D-form) and 10-12
  • the concentration of C6 peptide (D-form) was treated in the primary hippocampal neurons for 3 days, followed by immunocytochemical analysis.
  • the primary hippocampal gyrus of the embryo in the negative control group was added to PBS. Each treatment condition was repeated 3 times.
  • the primary cultured hippocampal gyrus neurons were fixed with 4% paraformaldehyde and penetrated at room temperature (RT) with 0.1% Triton X-100.
  • Primary antibodies against rabbit anti-Tau protein (selective marker for axon, Millipore, MA, USA) and primary antibody against mouse anti-MAP2 protein (microtubule-binding protein-2, dendritic) were added at room temperature Selective marker, Millipore, Massachusetts, USA) for 1 hour. After washing, the cells are Alexa 488 goat anti-rabbit secondary antibody (green fluorescent, Abcam, UK) with Alexa 594 goat anti-mouse secondary antibody (red fluorescent, Abcam, UK) was incubated for 1 hour at room temperature.
  • DAPI 4',6-diamidino-2-phenylindole
  • Test data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls multiple comparison assays.
  • the number of branches of the axons is calculated and the length of each branch is measured, and the statistical results are shown in FIGS. 3B to 3E.
  • the axon coming out of the cell body is called the primary branch; it is divided into the secondary branch by the first branch; the third branch is divided by the secondary branch, and so on (as shown in Figure 3A). ). Axons longer than 10 ⁇ m are included in the calculation.
  • C5 peptide and C6 peptide increased the number of third-order branches of axons relative to the number of axon three-stage branches in the untreated negative control group (0.7 third-order branches) (3 The third branch of the branch and the 4.9 branch of the branch) had significant differences (p ⁇ 0.01).
  • the number of secondary branches of axons (6.5 secondary branches) was increased by the number of secondary branches of axons (4.3 secondary branches) relative to the untreated negative control group, There was a significant difference (p ⁇ 0.01).
  • the P6 peptide (10 -9 M) increased the length of the axon triad branch of hippocampal gyrus cells with significant differences relative to the axon three-stage branch length of the untreated negative control group ( p ⁇ 0.05).
  • the number of branches of the axons is shown in Figure 3E. P6 peptide increased the number of axon secondary branches (4.2 secondary branches) relative to the number of axon secondary branches in the untreated negative control group (2.5 secondary branches), and Significant difference (p ⁇ 0.01).
  • the method for obtaining the C5 peptide, the C6 peptide and the embryonic primary hippocampal gyrus is the same as in the first embodiment.
  • the primary embryonic hippocampal neurons were treated on day 1 of in vitro culture (1DIV), and the embryos were treated with C5 peptide or C6 peptide at concentrations of 10 -3 M, 10 -6 M, 10 -9 M, 10 -12 M, respectively.
  • the hippocampus returned neurons for 24 hours, followed by MTT assay to measure cell viability.
  • MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) assay for cell viability.
  • the cells to be tested were co-cultured with 0.5 mg/mL MTT reagent (Sigma-Aldrich, USA) at 37 ° C for 1 hour.
  • the absorbance values at 570 nm and 630 nm were measured using an ELISA disk reader (SpectraMax M2 Microplate Readers, Molecular Devices, Inc., Sunnyvale, CA, USA) to quantify the MTT formazan product.
  • Test data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls multiple comparison assays.
  • C5 peptide and C6 peptide are not neurotoxic to primary neurons.
  • the results of the MTT analysis are shown in Figure 4.
  • Primary cortical neurons were treated with C5 peptide or C6 peptide at concentrations of 10 -3 M, 10 -6 M, 10 -9 M, 10 -12 M for 24 hours.
  • the results showed that, regardless of the untreated negative control group, neither the C5 peptide nor the C6 peptide affected the survival rate of the hippocampal gyrus neurons in the primary embryo (p>0.05).
  • the method for obtaining the C5 peptide, the C6 peptide and the embryonic primary hippocampal gyrus is the same as in the first embodiment.
  • C5 peptide and C6 peptide were analyzed by scratch injury test.
  • a scratch injury test was performed. Draw a scratch on the cell culture plate with a micropipette to damage the primary hippocampal neurons of the cultured embryo, and then add a medium containing 10 -9 M C5 peptide or 10 -9 M C6 peptide to be scratched.
  • the embryonic primary hippocampus returned neurons for 72 hours. Immunofluorescence staining is then performed.
  • the immunocytochemical analysis method is the same as in the first embodiment.
  • the primary hippocampal gyrus neurons were treated with a primary antibody against rabbit anti-Tau protein (a selective marker for axon, Millipore), followed by Alexa. 488 goat anti-rabbit secondary antibody (green fluorescent, Abcam) stained.
  • the nuclei were then stained with DAPI DNA stain (blue fluorescent, Vector Laboratories). Images were observed using an Axio Observer D1 microscope (Zeiss) and images were analyzed as ImageJ software (Inage Processing and Analysis in Java, National Institutes of Health, USA).
  • Test data were analyzed by one-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls multiple comparison assays.
  • Mature male Sprague-Dawley (SD) rats (300-400 g) were purchased from the National Laboratory Animal Center (National Laboratory Animal Center, Taiwan). Rats were housed in the Animal Experimental Center of the National Yilan University; 1 cage per 2 rats, at controlled temperature (22-24 ° C) and humidity (50% - 60%), 12 hours photoperiod / 12 hours dark cycle, feed Raised in an environment where water can be obtained at will. All animals had a week to adapt to the environment before any experiment was conducted. All experimental procedures are in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH), NIH Publication No. 8023, revised in 1978, and Experiments were conducted by personnel who had received appropriate training from the National Laboratory Animal Center. All experiments were approved by the Yilan University Animal Experiment Ethics Committee.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • Twenty-five untreated male SD rats were randomly divided into 5 groups, 5 rats in each group. Each group of rats was intraperitoneally injected with low dose C5 peptide (54 ⁇ g/kg) and high dose C5 peptide (270 ⁇ g). /kg), low-dose C6 peptide (5.4 ⁇ g/kg), high-dose C6 peptide (27 ⁇ g/kg), 0.5% (v/v) DMSO/PBS (negative control), and the injection volume per rat was 1 ⁇ L/g body weight, each group of rats was injected 14 days before the start of the test, once a day, and continued for injection during the test.
  • a plastic circular cylinder with a diameter of 183 cm was used as a pool (water temperature 25 ⁇ 2 ° C), and a circular platform was placed at a specific position on the edge of the pool and below the water surface. Add a non-toxic dye to the pool to make the water turbid. Special visual cues are placed on the pool wall. For spatial learning, the test animals received 3 trials per day, the training program lasted 4 days, and a total of 12 trials were performed separately. Rats were placed at different starting points around the circumference of the pool at equal intervals in random order. The rats were allowed to swim in the pool for 60 seconds. If a rat could not find the platform, it would be guided to the platform and allowed to Stay on the platform for 20 seconds.
  • test data was analyzed by The data were analyzed with two-way ANOVA and then analyzed by the post-Newman-Keuls multiple comparison test.
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • Scopolamine hydrochloride (Sco) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Sco was dissolved in physiological saline to a final concentration of 1.5 mg/mL for use.
  • Rats were randomly divided into control group, purine hydrochloride treatment group (Sco), C5+Sco treatment group and C6+Sco treatment group.
  • the vehicle was injected daily before the start of the behavioral test (control group and purine hydrochloride treatment group), C5 peptide (54 ⁇ g/kg/day), C6 peptide (5.4 ⁇ g/kg/day), continuous injection 14 Days, then each group of rats were injected intraperitoneally with vehicle (control group), vehicle plus saponin hydrochloride (1.5 mg/kg/day) (scopolamine treated group), C5 peptide (54 ⁇ g/ Kg/day) plus Sco (1.5mg/kg/day) (C5+Sco treatment group), C6 peptide (5.4 ⁇ g/kg/day) plus Sco (1.5mg/kg/day) (C6+Sco treatment group), continuous injection for 7 days, after the start of the behavioral experiment, the injection was continued during the experiment.
  • Each rat was injected at a volume of 1 ⁇ L/g body weight, and the injection of purine hydrochloride, vehicle, C5 peptide or C6 peptide was injected at 5 pm the previous day 30 minutes before the start of the morning behavior test.
  • the method is the same as that in the fourth embodiment.
  • Test data were analyzed by two-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • Both C5 peptide and C6 peptide can improve spatial learning and memory damage caused by Sco.
  • This example uses a memory-deleted animal model induced by purine base hydrochloride (Sco) to analyze the effects of C5 peptides and C6 peptides on aging and intelligent decline.
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • the preparation and preparation of the purine base hydrochloride (Sco) is the same as in the fifth embodiment.
  • the drug treatment method of the rat is the same as that in the fifth embodiment.
  • the device includes a slotted path separated by a sliding door to separate a secure clear box from a black box.
  • a current-generating electric shocker was attached to the floor of the dark compartment (UGO Basile, Comerio VA, Italy). The behavioral test was recorded from 8 am to 6 pm, including training and testing procedures. Prior to this test, rats were placed in a dark room for 1 hour to accommodate the environment. During training, the rat is placed in the clear box away from the farthest end of the sliding door. After the rat turned into the dark box, after closing the door, the foot was given a shock of 1 mA/s twice. The rat is then removed from the trail and returned to its cage. A retention test was given at different time points (1 day and 7 days later) after training.
  • the stop test is the same except that no electric shock is received.
  • the test was terminated when the rat entered the dark box, or after 600 seconds after the rat had not entered the dark box. Rats that did not enter the dark box and reached the upper limit of 600 seconds were removed from the path and designated as rats with good memory. Animals that were placed in a dark box and directly received a foot shock (1 mA/s for 2 seconds) were designated as a control group with only foot shocks.
  • Test data were analyzed by one-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • Both C5 peptide and C6 peptide can improve memory impairment caused by Sco. Rats were randomly divided into control group, scopolamine treatment group (Sco), C5+Sco treatment group and C6+Sco treatment group. C5 peptide (54 ⁇ g/kg/day), C6 peptide (5.4 ⁇ g/kg/day), 0.5% DMSO/saline (negative control group) were injected daily for 14 days before the start of the behavioral test.
  • Rats in each group were intraperitoneally injected with Sco (1.5 mg/kg/day), C5 peptide (54 ⁇ g/kg/day) plus Sco (1.5 mg/kg/day) (C5+Sco group), C6 peptide ( 5.4 ⁇ g/kg/day) plus Sco (1.5mg/kg/day) (C6+Sco group), 0.5% DMSO/saline (negative control group), continuous injection for 7 days, after the start of behavioral experiment, during the experiment Continuous injection. Each rat was injected at a volume of 1 ⁇ L/g body weight, and Sco was injected 30 minutes before the start of the morning behavior test, and the C5 peptide or C6 peptide was injected at 5 pm the previous day.
  • the time taken for all groups of rats to enter the dark box from the clear box was approximately the same (p>0.05) before the foot shock training.
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • the preparation and preparation of the purine base hydrochloride (Sco) is the same as in the fifth embodiment.
  • the drug treatment method of the rat is the same as that in the fifth embodiment.
  • mice should be familiar with the test equipment 24 hours before training.
  • the rats were placed on a high platform the next day.
  • the platform was placed in the center of the floor of the passive avoidance test chamber and the time the rats jumped off the platform was recorded.
  • the rats were immediately subjected to a slight electric shock (3V for 3 seconds, DC) through the grid floor as they jumped off the platform, and then returned to their cages.
  • the rats were placed on the platform again the next day (24 hour retention interval) and no electric shock was given. Record the latency of the rat jumping off the platform. If the rat stayed on the platform for 5 minutes, the rat scored the highest score of 300 seconds.
  • Test data were analyzed by one-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • Both C5 peptide and C6 peptide can improve memory impairment caused by scopolamine hydrochloride (Sco). Rats were randomly divided into control group, purine hydrochloride treatment group (Sco), C5+Sco treatment group and C6+Sco treatment group.
  • the vehicle was injected daily before the start of the behavioral test (control group and purine hydrochloride treatment group), C5 peptide (54 ⁇ g/kg/day), C6 peptide (5.4 ⁇ g/kg/day), continuous injection 14 Days, then each group of rats were injected intraperitoneally with vehicle (control group), vehicle plus saponin hydrochloride (1.5 mg/kg/day) (hydrochloride treatment group), C5 Peptide (54 ⁇ g/kg/day) plus Sco (1.5mg/kg/day) (C5+Sco treatment group), C6 peptide (5.4 ⁇ g/kg/day) plus Sco (1.5mg/kg/day) (C6+Sco treatment group), continuous After 7 days of injection, after the start of the behavioral experiment, the injection was continued during the experiment.
  • Each rat was injected at a volume of 1 ⁇ L/g body weight, and the injection of purine hydrochloride, vehicle, C5 peptide or C6 peptide was injected at 5 pm the previous day 30 minutes before the start of the morning behavior test.
  • Figure 9A the time it took for all groups of rats to jump off the platform was approximately the same (p > 0.05) before being subjected to a foot shock.
  • Example 8 Effect of C5 peptide and C6 peptide on novel object recognition and memory impairment induced by purine hydrochloride - Rat novel object recognition learning
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • the preparation and preparation of the purine base hydrochloride (Sco) is the same as in the fifth embodiment.
  • the drug treatment method of the rat is the same as that in the fifth embodiment.
  • the rats were allowed to explore 2 identical objects in an open field box (90 x 70 x 60 cm) for 5 minutes.
  • the criterion used to judge the exploration is that the distance between the rat and the object is less than 1.5 cm, or the rat is in direct contact with the object.
  • the rats were placed back into the same box during the retention test, but one of the familiar objects had been replaced with a novel object of approximately the same size.
  • the time each mouse was used to explore the 2 objects during the 5 minute period was recorded. Rats placed in a box with an open space without any objects were designated as non-training groups.
  • Test data were analyzed by two-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • Both C5 peptide and C6 peptide can improve memory impairment caused by scopolamine hydrochloride (Sco).
  • rats were randomly divided into control group, purine hydrochloride treatment group (Sco), C5+Sco treatment group and C6+Sco treatment group.
  • the vehicle was injected daily before the start of the behavioral test (control group and purine hydrochloride treatment group), C5 peptide (54 ⁇ g/kg/day), C6 peptide (5.4 ⁇ g/kg/day), continuous injection 14 Days, then each group of rats were injected intraperitoneally with vehicle (control group), vehicle plus saponin hydrochloride (1.5 mg/kg/day) (hydrochloride treatment group), C5 Peptide (54 ⁇ g/kg/day) plus Sco (1.5mg/kg/day) (C5+Sco treatment group), C6 peptide (5.4 ⁇ g/kg/day) plus purine base hydrochloride (Sco) (1.5 mg/kg/day) (C6+Sco treatment group), continuous injection for 7 days, after the start of the behavioral experiment, the injection was also continued during the experiment.
  • Each rat was injected at a volume of 1 ⁇ L/g body weight, and the injection of purine hydrochloride, vehicle, C5 peptide or C6 peptide was injected at 5 pm the previous day 30 minutes before the start of the morning behavior test.
  • the test animals had no preference for either the left object (LO) or the right object (RO) during the recognition training (all p > 0.05).
  • the right object (RO) was replaced with a novel object (NO) for 3, 8, and 24 hours, the control group, the (C5+Sco) treatment group, and the (C6+Sco) treatment group showed a novel object (NO).
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • the method is the same as that in the fourth embodiment.
  • Test data were analyzed by two-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • Both C5 peptides and C6 peptides can improve memory impairment caused by aging.
  • the 12-month-old rats were randomly divided into 4 groups and subjected to water maze training. The results are shown in Fig. 11A.
  • the average escape time of each group was similar, and the rats increased with the number of days of training. The time to escape the dehydration maze is reduced.
  • the 18-month-old aged rats were subjected to a water maze test, and the results are shown in Fig. 11B.
  • Rats receiving C5 peptide (54 ⁇ g/kg) and C6 peptide (5.4 ⁇ g/kg) were found to have a hidden platform faster than the negative control group, and there was a significant difference on the first day of the experiment. (p ⁇ 0.01). This result indicates that the C5 peptide and the C6 peptide can improve the decline in spatial learning ability caused by aging and the damage of memory.
  • the survival rate of rats was recorded every two months (12 months, 14 months, 16 months, 18 months, 20 months, and 22 months) from the beginning of December.
  • the survival rate results of aged rats are shown in Fig. 12. Compared with the aging rats of the negative control group, the aged rats administered C5 peptide had higher survival rate; while the aged rats administered C6 peptide had the survival rate as high as 100% at 22 months of age. This result indicates that C5 peptide and C6 peptide can prolong the life of rats.
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C6 peptide is the same as in the first embodiment.
  • 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was purchased from Sigma-Aldrich ( St. Louis, Missouri, USA) and dissolved in DMSO.
  • mice Five-week-old male C57BL/6 mice were divided into a negative control group (PBS), an MPTP-treated group (30 mg/kg), and a C6 peptide (10.7 ⁇ g/kg) + MPTP group.
  • the intraperitoneal injection was performed according to the daily weight of the mice, and the C6 peptide was pre-injected for three weeks, and then the MPTP and C6 peptides were injected for one week, and then the treadmill test was performed.
  • mice The motor coordination ability of the mice was tested with a treadmill.
  • the mice were first acclimated on a treadmill at a fixed speed of 2 rpm for 2 minutes, accelerated (5 minutes added to the highest speed) to 20 rpm for 15 minutes, and the time the mice were dropped from the treadmill during the recording period.
  • Test data were analyzed by one-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.
  • the C6 peptide can improve the motor coordination ability of mice infected with Parkinson's disease induced by MPTP.
  • This example uses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce Parkinson's disease in mice, and this model mouse is used to analyze C6 peptide pairs.
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • Figure 13 shows the time each group of mice stayed on the treadmill, and the longer they stayed, the better their motor coordination.
  • the negative control group injected with PBS stayed on the treadmill for an average of 778.5 seconds, and the Parkinson's disease in the MPTP-treated group had an average residence time of 159.8 seconds, while the C6 peptide + MPTP group had an average residence time. It is 261 seconds.
  • the animal source and care are the same as in the fourth embodiment.
  • the method for obtaining the C5 peptide and the C6 peptide is the same as in the first embodiment.
  • D-(+)-galactose Purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and dissolved in 0.9% NaCl solution for later use.
  • Seven-week-old rats were randomly divided into control group (0.9% NaCl), dextran galactose treatment group, C5+ dextran galactose treatment group and C6+ dextran galactose treatment group.
  • each group of rats were injected intraperitoneally with vehicle (control group), vehicle plus dextran galactose (150 mg/kg/day) (dextran galactose treatment group), C5 peptide (54 ⁇ g) /kg/day) plus dextran galactose (150 mg/kg/day) (C5+ dextran galactose treatment group), C6 peptide (5.4 ⁇ g/kg/day) plus dextran galactose (150 mg/kg/ Day) (C6+ dextran galactose treatment group), after 9 weeks of continuous injection, water maze and platform test were performed.
  • the injection volume per rat was 1 ⁇ L/g body weight.
  • the method is the same as that in the fourth embodiment.
  • Test data were analyzed by two-way ANOVA followed by post hoc Newman-Keuls multiple comparison assays.

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Abstract

本发明提供了增进神经元生长的胜肽，特别是可以增进神经突（neurite）生长的胜肽及其应用。

Description

增进神经元生长的胜肽及其应用 技术领域
本发明系关于增进神经元生长的胜肽，特别是可以增进神经突(neurite)生长的胜肽及其应用。
背景技术
学习与记忆是人类及其他哺乳类动物不可或缺的能力。经由探索而获得知识或经验，再透过大脑储存并统合后使心境及行为转变，让生物体足以藉此适应变化万千的环境。记忆力退化主要原因为脑神经细胞持续退化或是死亡，使讯息无法顺利的传递所导致。成年人的突触具有可塑性，透过学习及环境的刺激可改变神经突的生长，加强突触的化学讯息传递能力，进而形成记忆并巩固之。因此，如果能促进脑部神经可塑性，理论上将可强化记忆的形成。许多神经滋养因子，如：脑源性神经滋养因子(brain-derived neurotrophic factor,BDNF)、胶细胞源性神经滋养因子(glial cell line-derived neurotrophic factor,GDNF)、类胰岛素生长因子(insulin-like growth factors,IGF)等，已经被证实可以促进神经细胞可塑性，进而改善记忆。然而，这类大分子蛋白，往往具有体内结构不稳定、半衰期短、不易通过血脑屏障等问题。由于胜肽药物具有药用剂量小、副作用低、易于进入细胞内等优点，因此具有开发优势。
发明内容
本发明的目的在于提供一种增进神经元生长的胜肽及其应用。
本发明于第一方面提供一种可以增进神经元生长之胜肽，包含下列氨基酸序列：
(R1)a-Asn-X1-X2-Pro-Gln-(R2)b(SEQ ID NO:3)
及其保守性修饰变异。在上述式中，R1为一包含由1至40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成之群组。如同R1，在上述式中的R2为一包含由1至40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成之群组。在上述式中的X1，系为选自于由非极性氨基酸所组成之群组的一个氨基酸。在上述式中的X2，系为选自于由自然产生的氨基酸及氨基酸类似物所组成之群组 的一个氨基酸。在上述式中的指数a与b，系各自独立选于且可等于0或1。
本发明于第二方面提供一种编码任何上述的胜肽的多核苷酸。
本发明于第三方面提供一种药学组合物，包含至少一个上述的胜肽以及一药学上可接受之载剂。
本发明于第四方面提供一种增进神经元生长的方法，包含使一神经元细胞与一足够增进神经元生长量的任何上述的胜肽接触。
本发明于第五方面提供一种任何上述的胜肽在制备一种用以在一受试者体内改善与神经元细胞受损或退化有关之症状的药物的用途。
本发明系以下面的实施例予以示范阐明，但本发明不受下述实施例所限制。
附图说明
前面的概述，以及本发明以下的详细描述，在结合附图一起阅读时将可以被更好地理解。为了说明本发明的目的，所附图式示出了一些，但不是所有的，可替代的具体实施例。然而，应该理解的是，本发明并不限于所示的精确安排和手段。这些图式，其被并入并构成说明书的一部分，有助于解释本发明的原理。
图1A至图1I所示为C5胜肽(SEQ ID NO:1)及C6胜肽(SEQ ID NO:2)对初代海马回神经突(neurites)生长的影响。分别以PBS(负对照组)(图1A)、C5胜肽(10-9,10-12,10-15M)(图1B至图1D)、C6胜肽(10-9,10-12,10-15M)(图1F至图1H)、D-form C5胜肽(10-9M)(图1E)，以及D-form C6胜肽(10-12M)(图1I)处理初代海马回神经元(于体外培养第3天，DIV3)连续3天，接着以抗-Tau抗体(轴突标记，绿色萤光)、抗MAP-2抗体(树突标记，红色萤光)以及4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI)DNA萤光染剂(蓝色)于DIV6进行免疫染色。比例尺长度：50μm。
图2A至图2G所示为P1胜肽(SEQ ID NO:4)、P2胜肽(SEQ ID NO:5)、P3胜肽(SEQ ID NO:6)、P4胜肽(SEQ ID NO:7)、P5胜肽(SEQ ID NO:8)，以及P6胜肽(SEQ ID NO:9)对初代海马回神经突(neurites)生长的影响。分别以PBS(负对照组)(图2A)、10-9M P1胜肽(图2B)、10-9M P2胜肽(图2C)、10-9M P3胜肽(图2D)、10-9M P4胜肽(图2E)、10-9M P5胜肽(图2F)，以及10-9M P6胜肽(图2G)处理初代海马回神经元(于分化第3天，DIV3)3天，接着以抗-Tau抗体(轴突标记，绿色萤光)、抗MAP-2抗体(树突标记，红色萤光)以及4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI)DNA萤光染剂(蓝色)进行免疫染色。比例尺长度：50μm。
图3A至图3E所示为以本发明一些示例性的胜肽处理后，初代海马回神经轴突的分枝数目及长度的统计结果。神经元分枝示意图如图3A所示。以C5胜肽(10-12 M)或C6胜肽(10-12M)处理初代海马回神经元(于分化第3天，DIV3)3天，接着以抗-Tau(绿色)抗体、抗-MAP2(红色)抗体以及DAPI DNA萤光染剂(蓝色)进行免疫染色并计算轴突分枝数目(结果如图3C所示)及轴突长度(结果如图3B所示)。另外，以P1胜肽(10-9M)、P2胜肽(10-9M)、P3胜肽(10-9M)、P4胜肽(10-9M)、P5胜肽(10-9M)或P6胜肽(10-9M)处理初代海马回神经元(于分化第3天，DIV3)3天，接着以抗-Tau(绿色)抗体、抗-MAP2(红色)抗体以及DAPI DNA萤光染剂(蓝色)进行免疫染色并计算轴突分枝数目(结果如图3E所示)及轴突长度(结果如图3D所示)。以Image J软体及单因子变异数分析(One-way ANOVA)并接着以Newman-Keuls比较检定分析数据。数据为mean±SEM。相较于对照组，**表示p<0.01。
图4所示为以MTT分析法评估不同浓度的C5胜肽(10-12M,10-9M,10-6M,10-3M)及C6胜肽(10-12M,10-9M,10-6M,10-3M)对初代海马回神经元存活率的影响。该数据所示为mean±SEM，每组3重复。
图5所示为C5胜肽与C6胜肽对神经元轴突再生的影响。初代海马回神经元于体外培养第5天时(DIV5)以刮痕损伤处理后，分别以PBS(图5A)、C5胜肽(10-9M)(图5B)、C6胜肽(10-9M)(图5C)处理被刮伤的初代海马回神经元96小时，接着以抗-Tau抗体(轴突标记，绿色萤光)以及4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole，DAPI)DNA萤光染剂(蓝色)进行免疫染色。比例尺长度：100μm。
图6所示为以水迷宫试验分析C5胜肽与C6胜肽对空间学习与记忆形成的影响。SD大鼠随机被分为对照组、C5低剂量(54μg/kg)组、C5高剂量(270μg/kg)组、C6低剂量(5.4μg/kg)组、C6高剂量(27μg/kg)组。记录每组大鼠每天学习的平均逃脱时间(秒)。以二因子重复量数变异数分析(Two-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。相较于对照组，**表示p<0.01。
图7所示为C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐(Sco)诱发记忆障碍的大鼠在水迷宫里记忆形成的影响。大鼠随机分为对照组、注射莨菪碱氢氯酸盐组(Sco)、注射C5胜肽+Sco组以及注射C6胜肽+Sco组，并接受水迷宫训练。记录每天学习的平均逃脱时间(秒)。以二因子变异数分析(Two-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。相较于对照组，*表示p<0.05；**表示p<0.01。
图8所示为C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐(Sco)引起的记忆障碍的大鼠在被动单向抑制性回避学习的影响。大鼠随机分为对照组、注射莨菪碱氢氯酸盐组(Sco)、C5胜肽(54μg/kg)+Sco处理组以及C6胜肽(5.4μg/kg)+Sco处 理组，并接受被动单向抑制性回避学习试验。在给予足部电击前，各组大鼠在明箱的停留时间并无差异。记录给予足部电击1天与7天之后在明箱中的停留时间。以单因子变异数分析(One-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。相较于对照组，*表示p<0.05；**表示p<0.01。
图9A至图9C所示为C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐(Sco)引起的记忆障碍的大鼠在被动回避平台记忆的影响。大鼠随机分为对照组、注射莨菪碱氢氯酸盐组(Sco)、C5+Sco处理组以及C6+Sco处理组，并接受被动回避平台记忆试验。图9A所示为在给予足部电击前，各组大鼠在平台上的停留时间并无差异。图9B所示为给予足部电击后1天，大鼠在平台上的停留时间。图9C所示为给予足部电击后1天，大鼠跳下平台的错误次数。以单因子变异数分析(One-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。**表示p<0.01。
图10所示为C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐(Sco)引起的记忆障碍的大鼠在新颖物体识别试验的影响。大鼠随机分为对照组、注射莨菪碱氢氯酸盐组(Sco)、注射C5+Sco组以及注射C6+Sco组，并接受新颖物体识别试验。记录大鼠在右边物体(RO)被置换为新颖物体前、3小时后、8小时后与24小时后，花费在探索左边物体(LO)、右边物体(RO)与新颖物体(NO)的停留时间(秒)。以二因子变异数分析(Two-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。*表示p<0.05；**表示p<0.01。
图11A与图11B所示为C5胜肽与C6胜肽对于由老化诱发记忆障碍的大鼠在水迷宫里记忆形成的影响。大鼠随机被分为对照组、C5组、以及C6组，并接受水迷宫训练。图11A所示为给药前每组大鼠每天学习的平均逃脱时间(秒)。图11B所示为给药6个月后，每组大鼠每天学习的平均逃脱时间(秒)。以二因子变异数分析(Two-way ANOVA)并接着以Newman-Keuls比较检定分析数据。该数据为mean±SEM。相较于对照组，*表示p<0.05；**表示p<0.01。
图12所示为C5胜肽与C6胜肽对于由老化大鼠的存活率的影响。各组原始只数皆为6只，从12月龄开始每隔两个月记录一次(12月龄、14月龄、16月龄、18月龄、20月龄及22月龄)。结果资料以各组存活只数表示。
图13所示为C6胜肽对于由1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森氏症大鼠的影响。
图14所示为C5胜肽与C6胜肽对于由右旋半乳糖诱导的老化大鼠在水迷宫里记忆形成的影响。大鼠随机分为对照组、右旋半乳糖处理组、注射C5胜肽+右旋半乳糖处理以及注射C6胜肽+右旋半乳糖处理，并接受水迷宫训练。记录每天学习的平均逃脱时间(秒)。以二因子变异数分析(Two-way ANOVA)并接着以 Newman-Keuls比较检定分析数据。该数据为mean±SEM。相较于对照组，**表示p<0.01。
具体实施方式
应当理解的是，前述一般描述和下面的详细描述都是示例性和说明性的，但并非用以限制本发明所请之权利。本发明的一个或多个实施例的某些细节阐述于以下说明中。从以下代表性实施例的非穷举的列表中，亦从所附的权利要求中，本发明的其它特征或优点将是显而易见的。
本发明提供一种可以增进神经元生长的胜肽，包含下列氨基酸序列：
(R1)a-Asn-X1-X2-Pro-Gln-(R2)b       (SEQ ID NO:3)
及其保守性修饰变异；其中，R1为一包含由1至约40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成之群组；R2为一包含由1至约40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成之群组；X1系为选自于由非极性氨基酸所组成之群组的一个氨基酸；X2，系为选自于由自然产生的氨基酸及氨基酸类似物所组成之群组的一个氨基酸；a与b，系各自独立选于且可等于0或1。
在一具体实施例中，该a与b各自等于0。在一具体实施例中，该氨基酸序列为NAIPQ(SEQ ID NO:1)。在一具体实施例中，该氨基酸序列为NPSPQ(SEQ ID NO:2)。在一具体实施例中，该氨基酸序列为NFEPQ(SEQ ID NO:4)。在一具体实施例中，该氨基酸序列为NMYPQ(SEQ ID NO:5)。在一具体实施例中，该氨基酸序列为NIKPQ(SEQ ID NO:6)。在一具体实施例中，该氨基酸序列为NLMPQ(SEQ ID NO:7)。在一具体实施例中，该氨基酸序列为NVAPQ(SEQ ID NO:8)。在一具体实施例中，该氨基酸序列为NWLPQ(SEQ ID NO:9)。然而，上述之氨基酸序列仅为示例性的，本发明所提供的可以增进神经元生长的胜肽并不限于SEQ ID NOs:1,2,4-9所列的序列。例如，但不限于，当X1为丙胺酸(Ala,A)时，X2可为丙氨酸(Alanine)、胱胺酸(Cysteine)、天门冬胺酸(Aspartic acid)、麸胺酸(Glutamic acid)、苯丙胺酸(Phenylalanine)、甘胺酸(Glycine)、组胺酸(Histidine)、异白胺酸(Isoleucine)、离胺酸(Lysine)、白胺酸(Leucine)、甲硫胺酸(Methionine)、天冬酰胺(Asparagine)、脯胺酸(Proline)、谷胺酰胺(Glutamine)、精胺酸(Arginine)、丝胺酸(Serine)、苏胺酸(Threonine)、缬胺酸(Valine)、色胺酸(Tryptophan)、酪胺酸(Tyrosine)中的任一个。
本发明所提供之可以增进神经元生长的胜肽包含但不限于以胜肽合成仪(peptide synthesizer)合成由或以基因选殖方式而得。在一具体实施例中，该胜 肽是以胜肽合成仪合成的，且不限于D-form或L-form的构型。本发明所提供的胜肽的氨基酸残基可能是由天然存在的氨基酸或本领域中已知的非天然氨基酸，全L型或全D型，或其组合所组成。在另一实例中，该胜肽亦可以基因选殖的方式获得。以基因选殖方式获得该胜肽重组蛋白的方式可为，但不限于：将编码任何上述的胜肽的多核苷酸选殖到重组核酸表现载体中，以形成含有编码任何上述的胜肽的多核苷酸的重组核酸表现载体，再将该表现载体转殖到生物表现宿主中，经蛋白质表现后而得到本发明所揭露的可以增进神经元生长的胜肽。
因此，本发明并提供一种编码任何上述的胜肽的多核苷酸，及/或一种包含编码任何上述的胜肽的多核苷酸的重组核酸表现载体，及/或一种包含具有编码任何上述的胜肽的多核苷酸的重组核酸表现载体的宿主细胞。
该编码本发明任何上述的胜肽的多核苷酸，是由本发明之可以增进神经元生长的胜肽的氨基酸序列衍生而来。将本发明之可以增进神经元生长的胜肽的氨基酸序列上的各个氨基酸置换为遗传密码表(genetic code table)所列之编码该氨基酸的核苷酸序列(包含各种简并密码子(degenerate codons，或称同义密码子，synonymous codons)，即可得到本发明所提供之该核苷酸序列。例如，本发明之可以增进神经元生长的胜肽的氨基酸序列上的脯胺酸(Proline)可由CCA、CCC、CCG、CCT等核苷酸序列所编码。
本发明进一步提供一种组合物，包含至少一个上述的可以增进神经元生长的胜肽以及一药学上可接受之载剂。在一些具体实施例中，该组合包含一个以上的上述胜肽，例如，但不限于NAIPQ(SEQ ID NO:1)、NPSPQ(SEQ ID NO:2)、NFEPQ(SEQ ID NO:4)、NMYPQ(SEQ ID NO:5)、NIKPQ(SEQ ID NO:6)、NLMPQ(SEQ ID NO:7)、NVAPQ(SEQ ID NO:8)、NWLPQ(SEQ ID NO:9)其中之一个胜肽，或其组合物。在一些具体实施例中，该组合物含有，但不必要为，药学有效量的上述胜肽。
在一些具体实施例中，本发明所提供的胜肽增进了神经元的生长，特别是轴突分枝数目与轴突长度的增加。当脑细胞要执行各种活动时(例如，学习、记忆、修复、再生)都需要形成新的突触(synapse)。突触形成(synaptogenesis)的过程为，突触前神经元(presynaptic neuron)先形成轴突末端突出(axonal terminal boutons)，然后吸引突触后神经元(postsynaptic neuron)形成树突突起(dendritic protrusion)，两者互相接触形成突触，使得神经细胞可以交流沟通(cross-talk),形成新的连结(connectivity)，大脑发育及重塑(brain development and remodel ing)才会发生，以执行各种活动。因此，要先刺激神经元的轴突生长，后续才会发生树突分枝(dendritic arborization)，形成突触以执行学习、记忆等活动。脑源性神经滋养因子(Brain-derived neurotrophic  factor，BDNF)便是藉由先刺激神经元细胞形成新的轴突生长(axonal neurite outgrowth)，才发生树突分枝(dendritic arborization)，以促进神经元生长。当加入神经滋养因子受体的抑制剂时，就会抑制神经元轴突的生长，导致记忆无法被储存。在一些具体实施例中，本发明所提供的胜肽显着地增进了轴突的生长，其作用类似于脑源性神经滋养因子(BDNF)，系藉由增加轴突的生长以促进神经元生长。
此外，由于成年后哺乳类动物的脑中神经元细胞数目几乎不会增加，神经元细胞只会不断的退化、死亡，数量减少，因此成年或老年后需要靠增加神经突分枝(neurite arborization)才能维持或促进神经间的交流沟通。在一些具体实施例中，本发明所提供的胜肽藉由增加轴突的生长以促进神经突分枝，进而减少因老化或其他原因而造成的与神经元细胞受损或退化，并因此改善与神经元细胞受损或退化有关之症状。
是以，本发明提供一种增进神经元生长的方法，包含使一神经元细胞与一足够增进神经元生长量的任何上述的胜肽接触。在一些具体实施例中，该神经元细胞为具有正常功能的神经元细胞。在一些具体实施例中，该神经元细胞为退化的神经元细胞。在一些具体实施例中，该神经元细胞为受损的神经元细胞。造成神经元细胞受损或退化的原因包括，但不限于，物理性伤害(如：机械损伤、挫伤、切断)、化学性伤害(如：酒精、莨菪碱氢氯酸盐(scopolamine hydrochloride)、1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)、安非他命(Amphetamine)所造成的损害)、生物性伤害(如：脑缺氧所造成的损害)、老化或其组合。
因此，本发明并提供一种改善一受试者的记忆障碍的方法。本发明亦提供一种改善一受试者智能衰退的方法。在一些实施例中，该受试者的记忆障碍或智能衰退是由老化所引起。在一些实施例中，该受试者的记忆障碍或智能衰退是由机械损伤所引起。在一些实施例中，该受试者的记忆障碍或智能衰退是由化学药剂所引起。在一些实施例中，该受试者的记忆障碍或智能衰退是由莨菪碱氢氯酸盐所引起。莨菪碱氢氯酸盐(Sco)是一种动物与人类的蕈毒碱性受体拮抗剂(muscarinic receptor antagonist)，会造成学习与记忆的损伤，常用于建立认知功能减退及记忆障碍之动物模型。在一些实施例中，本发明使用由莨菪碱氢氯酸盐(Sco)诱导的记忆缺失动物模式来分析本发明揭露的胜肽对智能衰退的影响。
在一些实施例中，该受试者的记忆障碍或智能衰退是由1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)所引起。1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)是1-甲基-4-苯基吡啶(1-methyl-4-phenylpyridinium,MPP+)的一种神经毒素前驱物， 其透过破坏在大脑黑质内的产生多巴胺的神经元而造成永久性帕金森氏症。在一些实施例中，本发明使用由MPTP诱导的帕金森氏症动物模式来分析本发明揭露的胜肽对该疾病的影响。
在一些实施例中，该受试者的记忆障碍或智能衰退是由自然老化或由右旋半乳糖(D-(+)-galactose)所引起。在正常情况下，右旋半乳糖为生理上可利用的养分，但摄取过量的右旋半乳糖会造成非酵素型的醣化作用(glycation)，使蛋白质或脂质分子在不受酶的控制下，附加上醣类分子。醣与蛋白质相互聚合并经过一系列的反应后，会产生不可还原之物质。而这些不可还原物质会与其他蛋白质连成大分子，使蛋白质代谢异常，形成高浓度的醣化终末产物(advanced glycation end product,AGEs)累积在细胞中产生大量活性氧物种(reactive oxygen species,ROS)，最后引起氧化压力、神经炎症，使记忆及突触传导受阻碍，神经变性后导致记忆功能赤字。这种非酵素型的醣化作用是一种促进氧化性伤害及老化相关性疾病的途径。无论是从生理或病理的情况上分析来看，投与右旋半乳糖的大鼠的老化情形会相当于16-24月龄的老化大鼠。在一些实施例中，本发明使用自然老化的动物模式来分析本发明揭露的胜肽对智能衰退的影响。在一些实施例中，本发明使用由右旋半乳糖诱导的老化动物模式来分析本发明揭露的胜肽对智能衰退的影响。
本发明并提供一种任何上述的胜肽在制备一种用以在一受试者体内改善与神经元细胞受损或老化有关之症状的药物的用途。于一些具体实施例中，该症状包含，但不限于，记忆受损、智能衰退、运动协调能力受损、存活率下降、中枢神经系统病变、帕金森氏症、阿滋海默症、影响感觉神经元的疾病、皮质边缘系统的疾病、与发育迟缓及学习障碍相关的病症、唐氏症、氧化压力诱导的神经元死亡、因老化所产生的病症、因慢性酗酒所产生的病症、因药物滥用所产生的病症、因局部创伤造成的病理改变，以及因治疗药物及治疗的负面副作用所产生的病症。于一些具体实施例中，该症状为记忆受损、智能衰退、运动协调能力受损、存活率下降、帕金森氏症及/或阿滋海默症。在一些具体实施例中，相较于负对照组，受试者施用本发明之胜肽一段时间后，上述症状获得了显着地改善。
除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域中的技术人员所通常理解相同的含义。
如本文所用，冠词「一」、「一个」以及「任何」是指一个或多于一个(即至少一个)的物品的文法物品。例如，「一个元件」意指一个元件或多于一个元件。
如本文所用，术语「核苷酸」意指包括含连接到糖磷酸盐的氮碱基的单体，该糖磷酸盐包括糖，如核糖或2'-去氧核糖，连接到一个或多个磷酸基团。「多核苷酸」与「核酸」意指包括超过一个的核苷酸单体的聚合物，其中该单体通常被 糖-磷酸主链的糖-磷酸键所连接。多核苷酸不必只包括一个类型的核苷酸单体。例如，包含一个给定的多核苷酸的核苷酸可以仅为核糖核苷酸，仅为2’-氧核糖核苷酸，或核糖核苷酸和2’-去氧核糖核苷酸二者的组合。多核苷酸包括天然存在的核酸，例如去氧核糖核酸(DNA)和核糖核酸(RNA)，以及包含一种或多种非天然存在的单体的核酸类似物。多核苷酸可以被合成，例如，使用自动化DNA合成仪。术语「核酸」通常是指大的多核苷酸。将会理解的是，当核苷酸序列由DNA序列(即A、T、G、C)所表示，这还包括RNA序列(即A、U、G、C)，其中“U”取代“T”。术语「cDNA」意指一种与一mRNA互补或相同的DNA，不论是以单链或双链形式，但在其中的“T”取代“U”。术语「重组核酸」意指具有非天然接合在一起的序列的多核苷酸或核酸。重组核酸可以存在于载体的形式。
如本文所用，术语「氨基酸」是指天然存在的与合成的氨基酸，以及氨基酸类似物与以类似于天然存在的氨基酸的方式作用的氨基酸模拟物。天然存在的氨基酸是那些由遗传密码所编码的，以及后来被修饰的那些氨基酸，例如，羟脯氨酸、γ羧基麸胺酸，以及O-磷丝胺酸。针对本发明的目的，术语「氨基酸类似物」是指具有与天然存在的氨基酸相同的基本化学结构的化合物，亦即，一个与氢、羧基、氨基，以及一个R基团结合的碳，该R基团例如，高丝氨酸、正白胺酸、甲硫胺酸硫氧化物、甲硫氨酸甲基锍。这些类似物具有修饰的R基团(例如正白胺酸)或修饰的胜肽骨架，但保留了与天然存在的氨基酸相同的基本化学结构。针对本发明的目的，术语「氨基酸模拟物」是指一种具有一个与氨基酸的一般化学结构不同的结构的化合物，但是，其以类似于天然存在的氨基酸的方式作用。
如本文所用，术语「非极性氨基酸」是指一具有疏水性的α氨基酸，其中该官能基团贴附于该α碳链(即在RCH(NH2)COOH的R)上。本发明之非极性氨基酸包含天然存在的与合成的非极性氨基酸，以及非极性氨基酸类似物与以类似于天然存在的非极性氨基酸的方式作用的非极性氨基酸模拟物；例如，但不限于，丙胺酸、缬胺酸、白胺酸、异白胺酸、脯胺酸、苯丙胺酸、甲基胺酸、色氨酸、α-胺基丁酸。
如本文所用，术语「胜肽」是指氨基酸残基的聚合物。该术语适用于氨基酸聚合物，其中一个或多个氨基酸残基是一个类似物或相应的天然存在的氨基酸的模拟物，以及适用于天然存在的氨基酸聚合物。
如本文所用，术语「保守性修饰变异」适用于氨基酸与核酸序列。针对特定核酸序列，保守性修饰变异是指那些编码相同或基本相同的氨基酸序列的核酸，或其中该核酸不编码氨基酸序列时，是指基本相同的序列。具体而言，简并密码子的取代可透过产生序列来实现，该序列其中的一个或多个所选的(或所有的)密码子的第三位置被以混合的碱基及/或去氧肌苷残基所取代。由于遗传密码的简并 性，大量功能相同的核酸编码任何给定蛋白质。例如，密码子GCA，GCC，GCG和GCU都编码丙胺酸。因此，在编码为丙胺酸的每个位置上，该密码子可改变成任何所述的相应密码子而不改变所编码的多胜肽。这样的核酸变异为「沉默变异」，是保守性修饰变异的一种。本文中编码一胜肽的每个核酸序列还描述了该核酸的每种可能的沉默变异。本发明所属技术领域之技艺者将认识到，核酸中的每个密码子(除了AUG，其通常是甲硫胺酸的唯一密码子，以及TGG，其通常是色胺酸的唯一密码子之外)可以被修饰以产生功能相同的分子。因此，编码一胜肽的一个核酸的每个沉默变异都隐含在每个所述的序列中。
如本文所用，术语「载体」意指通过一核酸可以被引入宿主细胞，以转型该宿主细胞，并促进该核酸的表达的手段。载体可包含一给定的标的核苷酸序列以及一调节序列。载体可被用于表达该给定核苷酸序列或维持该给定的核苷酸序列，以复制它，操纵它，改变它，截断它，扩展它，及/或在不同的位置之间转移它(例如，在不同的生物体或宿主细胞或其组合之间)。
如本文所用，术语「宿主细胞」是指单细胞的原核或真核生物，包括但不限于：放线菌，古细菌，细菌和酵母菌。宿主细胞还可以是单细胞，包括但不限于培养细胞-来自高阶生物如植物和动物，包括但不限于脊椎动物，如哺乳动物和无脊椎动物如昆虫。
如本文所用，术语「与神经元细胞受损或退化有关之症状」是指包含导致神经元细胞死亡及/或尚不致死的神经病变的症状，包括，例如：
中枢神经系统病变，包括影响基底神经节的退化性疾病(例如：杭丁顿氏舞蹈症(Huntington's disease)、威尔森氏症(Wilson's disease)、纹状体黑质变性(striatonigral degeneration)、皮质基底神经节退化(corticobasal ganglionic degeneration))、妥瑞氏症(Tourette's syndrome)、帕金森氏症(Parkinson's disease)、进行性核上眼神经麻痹症(progressive supranuclear palsy)、进行性延髓麻痹症(progressive bulbar palsy)、遗传痉挛性截瘫(hereditary spastic paraplegia)、脊髓性肌肉萎缩症(spinal muscular atrophy)、肌萎缩脊椎侧索硬化症(amyotrophic lateral sclerosis)，及其变体、齿状核红核苍白球丘脑底核萎缩(dentatorubral-pallidoluysian atrophy)、橄榄体桥脑小脑萎缩(olivopontocerebellar atrophy)、副肿瘤性小脑变性(paraneoplastic cerebellar degeneration)，和多巴胺毒性(dopamine toxicity)；
影响感觉神经元的疾病，例如弗里德赖希共济失调(Friedreich's ataxia)、糖尿病，周围神经病变(peripheral neuropathy)、视网膜神经元退化(retinal neuronal degeneration)；
皮质边缘系统的疾病，例如，大脑类淀粉血管病变(cerebral amyloid  angiopathy)、皮克氏萎缩症(Pick's atrophy)、雷特氏症候群(Retts syndrome)；涉及神经系统及/或脑干的神经退化性病变，包括阿滋海默症(Alzheimer's disease)、爱滋病相关的失智症(AIDS-related dementia)、童年期脑脊髓病变(Leigh's disease)、扩散性路易体症(diffuse Lewy body disease)、癫痫、多系统萎缩(multiple system atrophy)、格林-巴利症候群(Guillain-Barre syndrome)、溶酶体储积病(lysosomal storage disorders)，如褐脂素储积病(lipofuscinosis)，唐氏症后期退化阶段(late-degenerative stages of Down's syndrome)、幼儿海绵状脑病(阿尔珀斯病)(Alper's disease)、中枢神经系统退化所造成的晕眩；
与发育迟缓及学习障碍相关的病症、唐氏症，以及氧化压力诱导的神经元死亡；
因老化、慢性酗酒或药物滥用所产生的病症，包括，例如，因酗酒造成的在蓝斑核、小脑、胆碱能基底前脑内的神经元的退化、因老化造成的小脑神经元与皮层神经元的退化而导致的认知与运动损伤、因长期安非他命滥用造成的基底神经节的神经元的退化而导致的运动损伤；
因局部创伤造成的病理改变，例如：中风、局部缺血、血管供血不足、缺氧缺血性脑病变、高血糖、低血糖、闭锁式头部外伤(closed head trauma)、或直接外伤；以及
因治疗药物及治疗的负面副作用所产生的病症(例如，回应N-甲基-D-天门冬胺酸(N-methyl-D-aspartate,NMDA)类的麸胺酸受体的拮抗剂的抗痉挛剂量而造成的扣带皮质与内嗅皮质的退化)。
如本文所用，术语「药学组合物」是指任何制剂，其中本发明的胜肽可以被配制、存储、保存、改变、给药，或其组合。如下所述，该制剂可以包括它们的任何药学上可接受的稀释剂、佐剂、缓冲剂、赋形剂、载体或组合。在一般情况下，该制剂的组成分系基于给药的方式和途径，以及标准药学实行而被选择的。如本文所用，术语「药学上可接受之载剂」是指任何物质或其组合与本发明的胜肽可以是物理或化学的混合、溶解、悬浮，或以其它方式组合以产生本发明的药学组合物。
如本文所用，术语「药学有效量」意指能够或足以维持或产生所期望的生理结果，包括但不限于，治疗、减轻、消除、基本上防止或预防，或其组合，疾病、病症，或其组合。药学有效量可以包括依序地或同时地施用一个或多个剂量。本发明技术领域的技术人员将知道调节本发明的剂量，以考虑到各种类型的制剂，包括但不限于缓释制剂。如本文所用，术语「预防」意指能基本上防止或预防疾病、病症，或其组合的任何方面的组合物。如本文所用，术语「治疗的」意指能 够治疗、减少、停止进展、减缓进展、有利地改变、消除，或其组合，疾病、病症的任何方面，或其组合。
如本文所用，术语「受试者」意指任何施与针对本发明的个体。受试者可以是，例如，哺乳动物。受试者可以是人或兽医动物，不考虑性别、年龄，或他们的任意组合，并且包括胎儿。受试者可以选择性地受特定疾病、病症或其组合的影响，或具有风险，或其组合。
适用于本发明给药的制剂可包括，可能在本领域技术人员公知的其他事情之中：水性和非水性溶液、抗氧化剂、抑菌剂、缓冲液、影响等渗性的溶质、防腐剂、增溶剂、稳定剂、悬浮剂、增稠剂，或其组合。
此外或在替代方案中，适用于本发明给药的制剂可以包括，可能在本领域技术人员公知的其他事情之中：凝胶、PEG如PEG 400、丙二醇、盐水、香囊；、水、本领域中已知的其他适当的液体，或其组合。
此外或在替代方案中，适用于本发明给药的制剂可以包括，可能在本领域技术人员公知的其他事情之中：粘合剂、缓冲剂、磷酸钙、纤维素、胶体，如胶体二氧化硅、着色剂、稀释剂、崩解剂、染料、填料、调味剂、明胶、乳糖、硬脂酸镁、甘露醇、微晶明胶、润湿剂、石蜡烃、锭剂、聚乙二醇、防腐剂、山梨糖醇、淀粉，如玉米淀粉、马铃薯淀粉，或其组合，硬脂酸、蔗糖、滑石、甘油三酯，或其组合。
此外或在替代方案中，适用于本发明给药的制剂可以包括，可能在本领域技术人员公知的其他事情之中：醇，如苯甲醇或乙醇、苯扎氯铵、缓冲剂如磷酸盐缓冲剂、乙酸盐缓冲剂、柠檬酸盐缓冲剂，或其组合，羧甲基纤维素或微晶纤维素、胆固醇、葡萄糖、果汁，如柚子汁，牛奶、磷脂如卵磷脂，油如植物油、鱼油、或矿物油，或其组合；本领域中已知的其它药学上相容的载体；或其组合。
此外或在替代方案中，适用于本发明给药的制剂可以包括，可能在本领域技术人员公知的其他事情之中：可生物降解，例如聚乳酸-聚乙二醇酸(PLGA)聚合物，其它实体的降解产物可以迅速地从一个生物系统，或其组合被清除。
本发明的制剂可以单位剂量形式，多剂量形式，或其组合方式来施用。它们可以被包装在单位剂量容器中、多剂量容器，或其组合。本发明可能存在于安瓿、小胶囊、胶囊、颗粒、含片、粉剂、片剂、小瓶、乳剂，包括但不限于阿拉伯胶乳剂、悬浮液，或其组合。
本发明通过下列的实施例进一步说明，其提供了用于示范而非限制的目的。本领域中的技术人员应，在根据本公开内容的，应当理解，许多变化可以在所公开的特定具体实施例中产生，且仍然获得相同或类似的结果而不脱离本发明的精神和范围。
实施例
实施例一C5胜肽、C6胜肽、P1胜肽、P2胜肽、P3胜肽、P4胜肽、P5胜肽、P6胜肽对神经元生长的影响
1.胜肽的制备
以合成仪合成D-form及L-form的C5、C6、P1、P2、P3、P4、P5、P6胜肽(Neogene Biomedicals公司，台北市，台湾)。将合成的C5胜肽(SEQ ID NO:1)、C6胜肽(SEQ ID NO:2)、P1胜肽(SEQ ID NO:4)、P2胜肽(SEQ ID NO:5)、P3胜肽(SEQ ID NO:6)、P4胜肽(SEQ ID NO:7)、P5胜肽(SEQ ID NO:8)、P6胜肽(SEQ ID NO:9)分别溶解于DMSO及PBS中。
2.细胞培养
为了培养胚胎初代海马回神经元，自国家实验动物中心(台北市，台湾)购买怀孕的Sprague-Dawley(SD)大鼠。来自SD大鼠(怀孕19天，E19)胚胎的海马回组织系以酵素分离，且将其种植于涂覆有聚离胺酸(poly-L-lysine)的小圆玻片上，培养于含有5％胎牛血清、5％马血清，以及50ng/mL胰岛素转铁蛋白硒酸盐(insulin-transferrin-selenite，Sigma-Aldrich公司，圣路易市，密苏里州，美国)的最低基本培养基(minimal essential medium，MEM)中。之后将培养基置换为含有0.5mM麸酰胺酸(glutamine)与12.5mM麸胺酸(glutamate)的2％B27-neurobasal培养基(Invitrogen公司，卡尔斯巴市，加州，美国)。于37℃、5％CO2下培养胚胎初代海马回神经元以待进一步试验。
3.神经元生长试验
胚胎初代海马回神经元于体外培养第3天时(days in vitro 3，DIV3)，以10-9M、10-12M、10-15M浓度的C5胜肽(L-form)或C6胜肽(L-form)、各10-9M浓度的P1、P2、P3、P4、P5、P6胜肽(L-form)、10-9M浓度的C5胜肽(D-form)以及10-12浓度的C6胜肽(D-form)分别处理该初代海马回神经元3天，接着进行免疫细胞化学分析。负对照组中的胚胎初代海马回神经元则加入PBS。每个处理条件进行3重复。
4.免疫细胞化学分析
以4％多聚甲醛(paraformaldehyde)固定上述培养的初代海马回神经元，并以0.1％Triton X-100在室温(RT)下进行穿透。在室温下分别加入兔抗-Tau蛋白的 初级抗体(轴突的选择性标记，Millipore公司，麻州，美国)与小鼠抗-MAP2蛋白的初级抗体(微小管结合蛋白-2，树突的选择性标记，Millipore公司，麻州，美国)作用1小时。清洗后，将细胞以Alexa
Figure PCTCN2015092336-appb-000001
488山羊抗-兔次级抗体(绿色萤光，Abcam公司，英国)与Alexa
Figure PCTCN2015092336-appb-000002
594山羊抗-小鼠次级抗体(红色萤光，Abcam公司，英国)在室温下培养1小时。接着以4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)DNA染剂(蓝色萤光，Vector Laboratories公司，美国)对细胞核进行染色。以盖玻片封片后，使用Axio Observer D1显微镜(Zeiss公司，Jena，德国)观察影像，并以ImageJ软体(Inage Processing and Analysis in Java,National Institutes of Health,USA)分析影像。长于10μm的神经突出物被定义为神经突(neurites)。
5.统计分析
试验数据以单因子变异数分析法(one-way analysis of variance，ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽、C6胜肽、P1胜肽、P2胜肽、P3胜肽、P4胜肽、P5胜肽或P6胜肽会促进神经突的增长与分枝。免疫细胞化学分析的结果分别如图1与图2所示。如图1A至图1I所示，相较于负对照组(图1A)，10-9M、10-12M、10-15M浓度的C5胜肽(L-form)(图1B至图1D)与C6胜肽(L-form)(图1F至图1H)皆会促进神经突的生长。此外，自然界氨基酸以L-form形式存在，而改以合成的D-form的C5胜肽(10-9M)与C6胜肽(10-12M)处理初代神经元亦可促进神经突生长，且效果与L-form的C5胜肽及L-form的C6胜肽差不多(分别如图1E及1I所示)。此外，如图2A至图2G所示，相较于负对照组(图2A)，10-9M浓度的P1胜肽、P2胜肽、P3胜肽、P4胜肽、P5胜肽或P6胜肽皆会促进神经突的生长(分别如图2B至图2G所示)。
此外，计算轴突各级分枝的数目并且测量各级分枝的长度，统计结果如图3B至图3E所示。细胞本体出来的轴突，称为一级分枝；由一级分枝再分出去成为二级分枝；由二级分枝分出去算三级分枝，以此类推(如图3A所示)。长于10μm之轴突才列入计算。
以C5胜肽及C6胜肽处理初代海马回神经元后，轴突各层分枝的长度计算结果如图3B所示。相对于未处理的负对照组的轴突一级分枝长度，C5胜肽(10-9M)与C6胜肽(10-9M)皆增加了海马回神经细胞的轴突一级分枝的长度，且皆具有显着差异(p<0.01)。另外，轴突各层分枝的数目如图3C所示。C5胜肽(10-9M)与C6 胜肽(10-9M)两者皆会促进轴突形成分枝。相对于未处理的负对照组的轴突三级分枝的数目(0.7枝三级分枝)，C5胜肽与C6胜肽皆增加了轴突之第三级分枝的数目(分别为3枝三级分枝与4.9枝三级分枝)，且具有显着差异(p<0.01)。相对于未处理的负对照组的轴突二级分枝的数目(4.3枝二级分枝)，C6胜肽增加了轴突之第二级分枝的数目(6.5枝二级分枝)，且具有显着差异(p<0.01)。
以P1至P6胜肽处理初代海马回神经元后，轴突各层分枝的长度计算结果如图3D所示。相对于未处理的负对照组的轴突一级分枝及二级分枝长度，P1至P6胜肽(10-9M)全部都可以增加海马回神经细胞的轴突一级分枝及二级分枝的长度，且皆具有显着差异(p<0.01或p<0.05)。此外，相对于未处理的负对照组的轴突三级分枝长度，P6胜肽(10-9M)可以增加海马回神经细胞的轴突三级分枝的长度，且具有显着差异(p<0.05)。另外，轴突各层分枝的数目如图3E所示。相对于未处理的负对照组的轴突二级分枝的数目(2.5枝二级分枝)，P6胜肽增加了轴突二级分枝的数目(4.2枝二级分枝)，且具有显着差异(p<0.01)。
实施例二C5胜肽与C6胜肽对初代神经元的神经毒性分析
1.试验物质
C5胜肽、C6胜肽以及胚胎初代海马回神经元取得之方法同实施例一。
2.细胞处理
胚胎初代海马回神经元于体外培养第1天时(1DIV)，分别以10-3M、10-6M、10-9M、10-12M浓度的C5胜肽或C6胜肽处理该胚胎初代海马回神经元24小时，接着进行MTT分析测量细胞存活率。
3.细胞存活率分析
以MTT(3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四氮唑鎓溴化物，3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)分析法评估细胞存活率。待测细胞于37℃下与0.5mg/mL MTT试剂(Sigma-Aldrich公司，美国)共同培养1小时。使用ELISA盘测读仪(SpectraMax M2Microplate Readers,Molecular Devices公司，森尼威尔市，加州，美国)测量波长570nm与630nm的吸光值以定量MTT甲瓒(formazan)产物。
4.统计分析
试验数据以单因子变异数分析法(one-way analysis of variance，ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
5.结果
C5胜肽与C6胜肽对初代神经元不具神经毒性。MTT分析结果如图4所示。分别以10-3M、10-6M、10-9M、10-12M浓度的C5胜肽或C6胜肽处理初代皮质神经元24小时。结果显示，相对于未处理的负对照组，不论是C5胜肽或C6胜肽，皆不影响胚胎初代海马回神经元的存活率(p>0.05)。
实施例三C5胜肽与C6胜肽对初代海马回神经元再生的影响
1.试验物质
C5胜肽、C6胜肽以及胚胎初代海马回神经元取得之方法同实施例一。
2.刮痕损伤试验
以刮痕损伤试验分析C5胜肽与C6胜肽对神经元再生的影响。胚胎初代海马回神经元于体外培养5天后，进行刮痕损伤试验。以微量滴管在细胞培养盘上画出一道刮痕以损伤培养的胚胎初代海马回神经元，接着加入含有10-9M C5胜肽或10-9M C6胜肽的培养基培养被刮伤的胚胎初代海马回神经元72小时。接着进行免疫萤光染色。
3.免疫细胞化学分析
免疫细胞化学分析方法同实施例一。初代海马回神经元以兔抗-Tau蛋白的初级抗体(轴突的选择性标记，Millipore公司)作用后，再以Alexa
Figure PCTCN2015092336-appb-000003
488山羊抗-兔次级抗体(绿色萤光，Abcam公司)染色。接着以DAPI DNA染剂(蓝色萤光，Vector Laboratories公司)对细胞核进行染色。使用Axio Observer D1显微镜(Zeiss公司)观察影像，并以ImageJ软体(Inage Processing and Analysis in Java,National Institutes of Health,USA)分析影像。
4.统计分析
试验数据以单因子变异数分析法(one-way analysis of variance，ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
5.结果
C5胜肽与C6胜肽促进神经元轴突的再生。刮痕损伤分析的结果如图5所示。相较于处理PBS的负对照组只有少数的轴突长出跨越刮痕的边界(如图5A所示)，处理C5胜肽与C6胜肽的被刮伤的初代皮质神经元具有较多且较长的轴突长出， 并跨越刮痕的边界(如图5B、5C所示)。该结果显示，C5胜肽与C6胜肽两者皆可促进受损的神经元长出新的轴突。
实施例四C5胜肽与C6胜肽对空间学习与记忆形成的影响–大鼠水迷宫试验
1.试验动物
成熟雄性Sprague-Dawley(SD)大鼠(300-400克)系购自国家实验动物中心(National Laboratory Animal Center，台湾)。大鼠系饲养于国立宜兰大学动物实验中心；每2只大鼠1笼，于控制温度(22-24℃)与湿度(50％-60％)、12小时光周期/12小时暗周期、饲料与水可随意取得的环境下饲养。在任何实验进行前，所有动物皆有一周时间适应环境。所有的实验流程皆遵守美国国家卫生研究院(National Institutes of Health,NIH)出版的实验动物照护与使用指南(the Guide for the Care and Use of Laboratory Animals，NIH出版号8023，1978年修改)，且由已经接受过国家实验动物中心适当训练的人员进行实验。所有的实验皆由宜兰大学动物实验道德委员会所许可。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。
3.药物处理
将25只未做过试验的成熟雄性SD大鼠随机分为5组，每组5只，各组大鼠分别以腹腔注射低剂量C5胜肽(54μg/kg)、高剂量C5胜肽(270μg/kg)、低剂量C6胜肽(5.4μg/kg)、高剂量C6胜肽(27μg/kg)、0.5％(v/v)DMSO/PBS(负对照组)，每只大鼠注射体积为1μL/g体重，自行为试验开始前14天开始对各组大鼠进行注射，每天注射1次，并持续在试验期间进行注射。
4.水迷宫学习模式
取直径183cm的塑胶圆形筒作为水池(水温25±2℃)，一圆形平台系放置于水池边缘的特定位置，且位于水面下。添加无毒染剂到水池中，使水变混浊。特殊的视觉线索系设置在水池墙上。针对空间学习，试验动物每天接受3次试验，训练程序持续4天，且共有12项试验分别进行。大鼠被放置在以随机顺序等间距围绕水池圆周的不同起始点，试验大鼠有60秒钟可在泳池游泳，若一大鼠无法发现该平台，其会被引导至该平台，且允许其停留在该平台上20秒钟。将每只动物到达该平台所花费的时间记录作为脱逃时间(escape latency)。在试验第5天时进行60秒钟的探索试验(probe trial)以测试其记忆的能力，将大鼠放置于移走 平台的水池内，且记录其在每个象限(象限1、2、3、4)中花费的时间，在目标象限(第4象限)内停留时间越久代表记忆越好。最终，大鼠需接受可见平台学习。可见平台学习是指将一标帜安装于该平台上，且将该平台升起至水面上，纪录大鼠找寻到平台的时间。此外，水池内不添加染剂，因此该动物可以从水中看见该平台的位置。
5.统计分析
试验数据以二因子变异数分析法(The data were analyzed with two-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可提升大鼠的空间学习能力与记忆。图6显示每组大鼠每天在水迷宫学习的平均逃脱时间。与负对照组大鼠相较，接受C5胜肽(54μg/kg、270μg/kg)与C6胜肽(5.4μg/kg、27μg/kg)注射的大鼠较快找到隐藏的平台[F(4,20)＝15.168,p<0.01]。此结果表示C5胜肽及C6胜肽可提升大鼠的空间学习能力与记忆。
实施例五C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐诱发的空间学习与记忆障碍的影响–大鼠水迷宫试验
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。莨菪碱氢氯酸盐(scopolamine hydrochloride，Sco)系购自Sigma-Aldrich公司(圣路易市，密苏里州，美国)。将Sco溶解于生理食盐水中至终浓度为1.5mg/mL备用。
3.药物处理
将大鼠随机分为对照组、莨菪碱氢氯酸盐处理组(Sco)、C5+Sco处理组以及C6+Sco处理组。于行为试验开始前先每天注射载剂(对照组及莨菪碱氢氯酸盐处理组)、C5胜肽(54μg/kg/day)、C6胜肽(5.4μg/kg/day)，连续注射14天，接着再对各组大鼠分别以腹腔注射载剂(对照组)、载剂加上莨菪碱氢氯酸盐(1.5mg/kg/day)(scopolamine处理组)、C5胜肽(54μg/kg/day)加上Sco(1.5mg/kg/day)(C5+Sco处理组)、C6胜肽(5.4μg/kg/day)加上Sco(1.5mg/kg/day) (C6+Sco处理组)，连续注射7天，行为实验开始后，在实验期间也持续注射。每只大鼠注射体积为1μL/g体重，于每天上午行为试验开始前30分钟注射莨菪碱氢氯酸盐，载剂、C5胜肽或C6胜肽于前一天下午5时进行注射。
4.水迷宫学习模式
方法同实施例四。
5.统计分析
试验数据以二因子变异数分析法(two-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由Sco引起的空间学习及记忆的损伤。本实施例使用由莨菪碱氢氯酸盐(Sco)诱导的记忆缺失动物模式来分析C5胜肽与C6胜肽对老化与智能衰退的影响。图7显示每组大鼠每天在水迷宫学习的平均逃脱时间。注射莨菪碱氢氯酸盐(Sco)的大鼠需要较长的逃脱时间去找到隐藏的平台(F3,20＝10.36,P<0.01)。与注射莨菪碱氢氯酸盐(Sco)的大鼠相较，C5+Sco组与C6+Sco组的大鼠自试验第3天起，找到隐藏平台的时间显着减少(q＝6.58and 6.77,bothP<0.01)，甚至比未处理莨菪碱氢氯酸盐(Sco)的大鼠更快找到隐藏的平台。此结果表示C5胜肽及C6胜肽改善了由莨菪碱氢氯酸盐(Sco)引起的记忆障碍大鼠的空间学习能力。
实施例六C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐诱发的记忆障碍的影响–大鼠抑制性回避试验
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。莨菪碱氢氯酸盐(Sco)的取得与制备同实施例五。
3.药物处理
大鼠的药物处理方法同实施例五。
4.抑制性回避学习任务
该装置包含一个由拉门分隔的槽形小径，以隔开一安全的明箱与一暗箱。将一产生电流的电击器连接到该黑暗隔间的地板上(UGO Basile公司，Comerio VA，义大利)。于上午8时至下午6时记录该行为试验，包含训练与测试程序。在该试验之前，大鼠先置于一暗室中1小时，以适应该环境。在训练期间，大鼠被放置于该明箱中远离该拉门的最远端。当大鼠转身时进入暗箱后，关上拉门之后，给予1mA/s足部电击两次。然后将该大鼠移出该小径并返回其笼中。在训练后不同时间点(1天与7天后)给予停留测试(retention test)。停留测试除了未接受电击之外，其他方式皆相同。当该大鼠进入暗箱时，或经过600秒钟后大鼠仍未进入暗箱时，终止该测试。将未进入该暗箱且达到600秒钟上限的大鼠移出该小径，并指定为具有良好记忆的大鼠。被置于暗箱中直接接受足部电击(1mA/s持续2秒钟)的动物则指定为仅有足部电击的对照组。
5.统计分析
试验数据以单因子变异数分析法(one-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由Sco引起的记忆障碍。将大鼠随机分为对照组、scopolamine处理组(Sco)、C5+Sco处理组以及C6+Sco处理组。于行为试验开始前先每天注射C5胜肽(54μg/kg/day)、C6胜肽(5.4μg/kg/day)、0.5％DMSO/盐水(负对照组)，连续注射14天，接着再对各组大鼠分别以腹腔注射Sco(1.5mg/kg/day)、C5胜肽(54μg/kg/day)加上Sco(1.5mg/kg/day)(C5+Sco组)、C6胜肽(5.4μg/kg/day)加上Sco(1.5mg/kg/day)(C6+Sco组)、0.5％DMSO/盐水(负对照组)，连续注射7天，行为实验开始后，在实验期间也持续注射。每只大鼠注射体积为1μL/g体重，于每天上午行为试验开始前30分钟注射Sco，C5胜肽或C6胜肽于前一天下午5时进行注射。如图8所示，在尚未受到足部电击训练前，所有组别的大鼠自明箱进入暗箱所花费的时间大致相同(p>0.05)。在足部电击后7天，Sco处理组的大鼠在明箱中停留的时间较短(q＝4.14,P<0.05)(在第7天时比较对照组与Sco处理组)。在足部电击后7天，(C5+Sco)处理组大鼠在明箱中停留的时间较长(q＝4.84,P<0.05)(在第7天时比较Sco处理组与(C5+Sco)处理组)。在足部电击后7天，(C6+Sco)处理组大鼠在明箱中停留的时间较长(q＝4.84,P<0.05)(在第7天时比较Sco处理组与(C6+Sco)处理组)。此结果表示C5胜肽及C6胜肽改善了由Sco引起记忆障碍的大鼠在被动单向抑制性回避试验中的表 现。
实施例七C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐诱发的记忆障碍的影响–大鼠被动回避平台试验
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。莨菪碱氢氯酸盐(Sco)的取得与制备同实施例五。
3.药物处理
大鼠的药物处理方法同实施例五。
4.被动回避平台试验(跳台法)
在受训前24小时，先让动物熟悉测试用具。隔天将大鼠置于一架高的平台上，该平台系设置在被动回避试验箱的地板中心，并记录大鼠跳下平台的时间。在该试验的第3天时，当大鼠跳下平台时通过网格地板立刻受到轻微的电击(3V，持续3秒钟，直流电)，然后回到其笼子内。隔天(24小时保留间隔)大鼠再度被放置在该平台上，且不给予电击。记录大鼠跳下平台的潜伏时间。若该大鼠停留在该平台5分钟，则该大鼠得到300秒钟的最高分。
5.统计分析
试验数据以单因子变异数分析法(one-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由莨菪碱氢氯酸盐(Sco)引起的记忆障碍。大鼠随机分为对照组、莨菪碱氢氯酸盐处理组(Sco)、C5+Sco处理组以及C6+Sco处理组。于行为试验开始前先每天注射载剂(对照组及莨菪碱氢氯酸盐处理组)、C5胜肽(54μg/kg/day)、C6胜肽(5.4μg/kg/day)，连续注射14天，接着再对各组大鼠分别以腹腔注射载剂(对照组)、载剂加上莨菪碱氢氯酸盐(1.5mg/kg/day)(莨菪碱氢氯酸盐处理组)、C5胜肽(54μg/kg/day)加上Sco(1.5mg/kg/day)(C5+Sco处理组)、C6胜肽(5.4μg/kg/day)加上Sco(1.5mg/kg/day)(C6+Sco处理组)，连续 注射7天，行为实验开始后，在实验期间也持续注射。每只大鼠注射体积为1μL/g体重，于每天上午行为试验开始前30分钟注射莨菪碱氢氯酸盐，载剂、C5胜肽或C6胜肽于前一天下午5时进行注射。如图9A所示，在受到足部电击前，所有组别的大鼠跳下平台所花费的时间大致相同(p>0.05)。如图9B及图9C所示，在足部电击后1天，莨菪碱氢氯酸盐处理组(Sco)的大鼠在平台上停留的时间较短，且跳下的次数显着较多(q＝4.4,P<0.05)(在第1天时比较对照组与Sco处理组)。在足部电击后1天，相较于莨菪碱氢氯酸盐处理组(Sco)，(C5+Sco)处理组以及(C6+Sco)处理组的大鼠在平台上停留的时间皆较长，且跳下的次数显着较少(C5+Sco)处理组以及(C6+Sco)处理组的q值皆为4.4,P<0.05)。此结果表示C5胜肽及C6胜肽改善了由莨菪碱氢氯酸盐(Sco)引起记忆障碍的大鼠在被动回避平台试验中的表现。
实施例八C5胜肽与C6胜肽对于由莨菪碱氢氯酸盐诱发的新颖物体辨识与记忆障碍的影响–大鼠新颖物体识别学习
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。莨菪碱氢氯酸盐(Sco)的取得与制备同实实施例五。
3.药物处理
大鼠的药物处理方法同实施例五。
4.新颖物体识别学习
在熟悉期间，允许大鼠在一个开放场地的箱子(90×70×60cm)中探索2个相同的物体5分钟。用来判断探索的标准是该大鼠与该物体之间的距离少于1.5cm，或是该大鼠直接接触该物体。3小时，8小时及24小时后，在给予停留测试(retention test)期间，将该大鼠放回相同的箱子中，但其中一个熟悉的物体已被置换为一个尺寸大约相同的新颖物体。记录每只大鼠在5分钟期间内用于探索该2物体的时间。而被置于一个没有任何物体之开放场地的箱子内的大鼠则被指定为非训练组。
5.统计分析
试验数据以二因子变异数分析法(two-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由莨菪碱氢氯酸盐(Sco)引起的记忆障碍。在新颖物体识别试验中，将大鼠随机分为对照组、莨菪碱氢氯酸盐处理组(Sco)、C5+Sco处理组以及C6+Sco处理组。于行为试验开始前先每天注射载剂(对照组及莨菪碱氢氯酸盐处理组)、C5胜肽(54μg/kg/day)、C6胜肽(5.4μg/kg/day)，连续注射14天，接着再对各组大鼠分别以腹腔注射载剂(对照组)、载剂加上莨菪碱氢氯酸盐(1.5mg/kg/day)(莨菪碱氢氯酸盐处理组)、C5胜肽(54μg/kg/day)加上Sco(1.5mg/kg/day)(C5+Sco处理组)、C6胜肽(5.4μg/kg/day)加上莨菪碱氢氯酸盐(Sco)(1.5mg/kg/day)(C6+Sco处理组)，连续注射7天，行为实验开始后，在实验期间也持续注射。每只大鼠注射体积为1μL/g体重，于每天上午行为试验开始前30分钟注射莨菪碱氢氯酸盐，载剂、C5胜肽或C6胜肽于前一天下午5时进行注射。如图10所示，在识别训练期间，试验动物对于左边物体(LO)或右边物体(RO)皆无偏好(所有的p>0.05)。将右边物体(RO)以新颖物体(NO)置换后3、8、24小时，对照组、(C5+Sco)处理组、(C6+Sco)处理组大鼠显现出对新颖物体(NO)的偏好(q＝3.87,P<0.05；q＝3.57,P<0.05)。注射Sco的大鼠在探索新颖物体(NO)所花费的时间则与探索原来的右边物体(RO)所花费的时间无差异(p>0.05)。此结果表示C5胜肽及C6胜肽改善了由莨菪碱氢氯酸盐(Sco)引起记忆障碍的大鼠在新颖物体识别学习中的表现。
实施例九C5胜肽与C6胜肽对老化大鼠的空间学习与记忆形成的影响–大鼠水迷宫试验
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。
3.药物处理
在药物处理前，将12个月大的大鼠随机分为4组，并接受水迷宫训练。接着，各组大鼠分别以腹腔注射PBS(负对照组)、C5胜肽(54μg/kg/day)以及C6胜肽(5.4μg/kg/day)(各组N＝6)。注射体积为1μL/g体重。每天注射一次，持续注 射6个月。
4.水迷宫学习模式
方法同实施例四。
5.统计分析
试验数据以二因子变异数分析法(two-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由老化引起的记忆障碍。在药物处理前，将12个月大的大鼠随机分为4组，并接受水迷宫训练，结果如图11A所示，各组平均逃脱的时间相似，且随着训练的天数增加，大鼠逃脱水迷宫的时间减少。以药物处理6个月后，再对18个月龄的老化大鼠进行水迷宫试验，结果如图11B所示。与负对照组大鼠相较，接受C5胜肽(54μg/kg)与C6胜肽(5.4μg/kg)注射的大鼠较快找到隐藏的平台，且在试验第一天即具有显着差异(p<0.01)。此结果表示C5胜肽及C6胜肽可改善由老化引起的空间学习能力的衰退以及记忆的损伤。
此外，从十二月龄开始每隔两个月(12月龄、14月龄、16月龄、18月龄、20月龄及22月龄)记录一次大鼠存活率。老化大鼠的存活率结果如图12所示。相较于负对照组的老化大鼠，施用C5胜肽的老化大鼠具有较高的存活率；而施用C6胜肽的老化大鼠到22月龄时，存活率高达100％。此结果表示，C5胜肽及C6胜肽可延长大鼠之寿命。
实施例十C6胜肽对于由1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森氏症小鼠的影响
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C6胜肽取得之方法同实施例一。1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)系购自Sigma-Aldrich公司(圣路易市，密苏里州，美国)，并溶于DMSO中。
3.药物处理
使用五周龄雄性C57BL/6小鼠，分为负对照组(PBS)、MPTP处理组(30mg/kg)，以及C6胜肽(10.7μg/kg)+MPTP组。依照小鼠每天的体重换算注射量后进行腹腔注射，预先注射C6胜肽持续三周后，同时注射MPTP与C6胜肽持续一周后，再进行滚筒跑步机试验。
4.滚筒跑步机试验
以滚筒跑步机来测试小鼠的运动协调能力。先让小鼠在跑步机上以固定速度2rpm适应2分钟后，开始加速(5分钟内会加到最高速)到20rpm持续15分钟，并记录时间内小鼠自跑步机上掉落的时间。
5.统计分析
试验数据以单因子变异数分析法(one-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C6胜肽可以改善由MPTP诱导产生帕金森氏症的小鼠的运动协调能力。本实施例使用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)来诱导小鼠产生帕金森氏症，并以此模式小鼠来分析C6胜肽对帕金森氏症的影响。图13显示每组小鼠在滚筒跑步机上停留的时间，停留越久表示其运动协调能力越好。注射PBS的负对照组在滚筒跑步机上停留的时间平均为778.5秒，MPTP处理组的帕金森氏症小鼠的停留时间平均为159.8秒，而C6胜肽+MPTP组小鼠的停留时间则平均为261秒。各组相较于负对照组皆有显着差异(q＝24.12,23.42,20.18,p＜0.01)。而相较于MPTP处理组，C6胜肽+MPTP组小鼠停留在滚筒跑步机上的时间较久，并且具有显着差异(q＝4.19,3.45,p＜0.05)。此结果表示C6胜肽改善了由MPTP诱导产生帕金森氏症的小鼠的运动协调能力。
实施例十一C5胜肽与C6胜肽对于由右旋半乳糖诱发的老化大鼠的空间学习与记忆的影响–大鼠水迷宫试验
1.试验动物
动物来源与照护同实施例四。
2.试验药物
C5胜肽、C6胜肽取得之方法同实施例一。右旋半乳糖(D-(+)-galactose)系 购自Sigma-Aldrich公司(圣路易市，密苏里州，美国)，并且被溶解于0.9％NaCl溶液中备用。
3.药物处理
将七周龄大鼠随机分为对照组(0.9％NaCl)、右旋半乳糖处理组、C5+右旋半乳糖处理组以及C6+右旋半乳糖处理组。于行为试验开始前先每天注射0.9％NaCl载剂(对照组及右旋半乳糖处理组)、C5胜肽(54μg/kg/day)、C6胜肽(5.4μg/kg/day)，连续注射7天，接着再对各组大鼠分别以腹腔注射载剂(对照组)、载剂加上右旋半乳糖(150mg/kg/day)(右旋半乳糖处理组)、C5胜肽(54μg/kg/day)加上右旋半乳糖(150mg/kg/day)(C5+右旋半乳糖处理组)、C6胜肽(5.4μg/kg/day)加上右旋半乳糖(150mg/kg/day)(C6+右旋半乳糖处理组)，连续注射9周后，再进行水迷宫及跳台实验。每只大鼠注射体积为1μL/g体重。
4.水迷宫学习模式
方法同实施例四。
5.统计分析
试验数据以二因子变异数分析法(two-way ANOVA)分析，接着以事后Newman-Keuls多重比较检定来分析。
6.结果
C5胜肽及C6胜肽皆可改善由半乳糖诱导的老化大鼠的空间学习及记忆的能力。图14所示为每组大鼠每天在水迷宫学习的平均逃脱时间。注射右旋半乳糖的大鼠需要较长的逃脱时间去找到隐藏的平台(平均53.06秒)。与注射右旋半乳糖的大鼠相较，C5+右旋半乳糖组与C6+右旋半乳糖组的大鼠自试验第2天起，找到隐藏平台的时间显着减少(q＝5.34,4.37,p<0.01)，但与对照组相比则无统计上的显着差异(p>0.05)。此结果表示C5胜肽及C6胜肽改善了由右旋半乳糖诱导的老化大鼠的空间学习能力。
上列详细说明系针对本发明之一可行实施例的具体说明，惟该实施例并非用以限制本发明的专利范围，凡未脱离本发明技艺精神所为的等效实施或变更，均应包含于本案的专利范围中。

Claims (10)

  1. 一种增进神经元生长的胜肽，其特征在于包含下列氨基酸序列：
    (R1)a-Asn-X1-X2-Pro-Gln-(R2)b(SEQ ID NO:3)
    其中，
    R1是一包含由1至40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成的群组；
    R2是一包含由1至40个氨基酸的氨基酸序列，其中每个氨基酸系各自独立地选自于由自然产生的氨基酸及氨基酸类似物所组成的群组；
    X1，为选自于由非极性氨基酸所组成的群组的一个氨基酸；
    X2，为选自于由自然产生的氨基酸及氨基酸类似物所组成的群组的一个氨基酸；以及a与b是各自独立选于且可等于0或1。
  2. 如权利要求1所述的胜肽，其特征在于所述a与b各自等于0。
  3. 如权利要求1所述的胜肽，其特征在于所述氨基酸序列包含至少下列之一序列：NAIPQ(SEQ ID NO:1)、NPSPQ(SEQ ID NO:2)、NFEPQ(SEQ ID NO:4)、NMYPQ(SEQ ID NO:5)、NIKPQ(SEQ ID NO:6)、NLMPQ(SEQ ID NO:7)、NVAPQ(SEQ ID NO:8)，以及NWLPQ(SEQ ID NO:9)。
  4. 一种编码如权利要求1所述的胜肽的多核苷酸。
  5. 一种药学组合物，其特征在于包含如权利要求1所述的胜肽以及一药学上可接受的载剂。
  6. 一种增进神经元生长的方法，其特征在于包含使一神经元细胞与一足够增进神经元生长量的如权利要求1所述的胜肽接触。
  7. 如权利要求6的方法，其特征在于所述神经元细胞为正常或受损或退化的神经元细胞。
  8. 一种如权利要求1所述的胜肽在制备一种用以在一受试者体内改善与神经元细胞受损或退化有关之症状的药物的用途。
  9. 如权利要求8所述的用途，其特征在于所述症状是选自于下列所组成之群组：记忆受损、智能衰退、运动协调能力受损、存活率下降、中枢神经系统病变、帕金森氏症、阿滋海默症、影响感觉神经元的疾病、皮质边缘系统的疾病、与发育迟缓及学习障碍相关的病症、唐氏症、氧化压力诱导的神经元死亡、因老化所产生的病症、因慢性酗酒所产生的病症、因药物滥用所产生的病症、因局部创伤造成的病理改变，以及因治疗药物及治疗的负面副作用所产生的病症。
  10. 一种如权利要求1所述的胜肽在制备一种用以促进健康个体记忆或学习能 力的组合物的用途。
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