WO2022239764A1 - α－シヌクレインの凝集抑制剤、α－シヌクレイン凝集疾患用医薬組成物、およびその用途 - Google Patents

α－シヌクレインの凝集抑制剤、α－シヌクレイン凝集疾患用医薬組成物、およびその用途 Download PDF

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WO2022239764A1
WO2022239764A1 PCT/JP2022/019785 JP2022019785W WO2022239764A1 WO 2022239764 A1 WO2022239764 A1 WO 2022239764A1 JP 2022019785 W JP2022019785 W JP 2022019785W WO 2022239764 A1 WO2022239764 A1 WO 2022239764A1
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peptide
aggregation
amino acid
acid sequence
seq
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PCT/JP2022/019785
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English (en)
French (fr)
Japanese (ja)
Inventor
俊史 秋澤
源顕 齊藤
里菜 中村
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O Force
O Force Co Ltd
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O Force
O Force Co Ltd
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Priority to US18/560,252 priority Critical patent/US20240238366A1/en
Priority to EP22807464.7A priority patent/EP4338747A4/en
Priority to JP2023521203A priority patent/JPWO2022239764A1/ja
Publication of WO2022239764A1 publication Critical patent/WO2022239764A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to ⁇ -synuclein aggregation inhibitors, pharmaceutical compositions for ⁇ -synuclein aggregation diseases, and uses thereof.
  • Parkinson's disease is a disease in which movement of the body is impaired due to brain abnormalities. Since there is no fundamental treatment, there is a demand for the elucidation of the cause and the development of drugs that lead to the fundamental treatment.
  • an object of the present invention is to provide a new drug that suppresses aggregation of ⁇ -synuclein, which causes Parkinson's disease and the like.
  • the ⁇ -synuclein aggregation inhibitor of the present invention is characterized by containing the following peptide (A1), (A2), (B1) or (B2).
  • A1 Peptide RETLVYLTHLDYDDTE (SEQ ID NO: 1) consisting of an amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1
  • A2 Peptide consisting of an amino acid sequence in which 1-2 amino acids are deleted, substituted or added in the amino acid sequence of (A1)
  • B1 Peptide YSNFNTDY consisting of the amino acid sequence of SEQ ID NO: 4 (SEQ ID NO: 4)
  • B2 A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted or added in the amino acid sequence of (B1)
  • the present invention is a pharmaceutical composition for ⁇ -synuclein aggregation diseases, characterized by comprising the peptide (A1), (A2), (B1) or (B2).
  • the ⁇ -synuclein aggregation inhibitory method of the present invention is characterized by adding the ⁇ -synuclein aggregation inhibitor of the present invention to a subject.
  • the method for treating ⁇ -synuclein aggregation diseases of the present invention is characterized by administering the ⁇ -synuclein aggregation inhibitor of the present invention to a subject.
  • Syn ⁇ -synuclein (hereinafter also referred to as Syn) aggregation inhibitor of the present invention
  • aggregation due to intermolecular association of Syn can be suppressed.
  • prevention, progression control, improvement, etc. become possible.
  • FIG. 1 is a graph showing fluorescence intensity of a reaction solution to which an aggregating peptide was added in Example A1.
  • FIG. 2 is a graph showing the alternation behavior rate of mice to which an agglutinating peptide was administered in Example A1.
  • FIG. 3 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibiting peptide was added in Example B1.
  • FIG. 4 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibiting peptide was added in Example B2.
  • FIG. 5 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibiting peptide was added in Example B3.
  • FIG. 1 is a graph showing fluorescence intensity of a reaction solution to which an aggregating peptide was added in Example A1.
  • FIG. 2 is a graph showing the alternation behavior rate of mice to which an agglutinating peptide was administered in Example A1.
  • FIG. 6 is a graph showing the alternation behavior rate of mice to which aggregation-inhibiting peptides were added in Example B4.
  • FIG. 7 is a graph showing the Rota-Rod results of mice to which the aggregation-inhibiting peptide was added in Example B4.
  • FIG. 8 is a graph showing the alternation behavior rate of mice to which an aggregation-inhibiting peptide and an agglutinating peptide were simultaneously added in Example B5.
  • FIG. 9 is a graph showing the Rota-Rod results of mice to which an aggregation-inhibiting peptide and an agglutinating peptide were simultaneously added in Example B5.
  • FIG. 9 is a graph showing the Rota-Rod results of mice to which an aggregation-inhibiting peptide and an agglutinating peptide were simultaneously added in Example B5.
  • FIG. 10 is a graph showing cell viability when a reaction solution containing an aggregation-inhibiting peptide and an agglutinating peptide was added to cells after incubation in Example B6.
  • FIG. 11 is a graph showing the fluorescence intensity of the reaction solution to which the aggregating peptide was added in Example A2.
  • Syn aggregation inhibitor of the present invention (hereinafter also referred to as a Syn aggregation inhibitor) is, as described above, the following (A1), (A2), (B1) or It is characterized by containing the peptide of (B2).
  • the peptide (A1), (A2), (B1) or (B2) is hereinafter referred to as an aggregation-inhibiting peptide, and the peptide (A1) or (A2) is combined with the peptide (A), or Hereinafter, it is also referred to as YS, and the peptide (B1) or (B2) is collectively referred to as the peptide (B), or hereinafter, LYZA-3.
  • (A1) Peptide RETLVYLTHLDYDDTE (SEQ ID NO: 1) consisting of an amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1
  • (A2) Peptide consisting of an amino acid sequence in which 1-2 amino acids are deleted, substituted or added in the amino acid sequence of (A1)
  • (B1) Peptide YSNFNTDY consisting of the amino acid sequence of SEQ ID NO: 4 (SEQ ID NO: 4)
  • B2 A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted or added in the amino acid sequence of (B1)
  • the peptide YS of (A1) may be an amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1, and its length is 6, 7, 8, 9, 10, 11, 12. , 13, 14, 15, or 16 residues.
  • the first amino acid residue of the peptide YS may be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11. .
  • the peptide YS of (A1) is, for example, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (hereinafter also referred to as YS-16), a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (hereinafter also referred to as YS-11), a sequence A peptide consisting of the amino acid sequence of number 3 (hereinafter also referred to as YS-6) can be exemplified, preferably YS-6.
  • YS-16 RETLVYLTHLDYDDTE (SEQ ID NO: 1) YS-11 RETLVYLTHLD (SEQ ID NO: 2) YS-6 DYDDTE (SEQ ID NO: 3)
  • the (A2) peptide is an amino acid sequence in which one or two amino acids are deleted, substituted, or added to the amino acid sequence of the (A1) peptide.
  • the peptide (A1) is the peptide YS-16, YS-11, or YS-6
  • the peptide (A2) is, for example, the amino acid of the peptide YS-16, YS-11, or YS-6
  • the peptide (B2) is an amino acid sequence in which one or two amino acids are deleted, substituted, or added to the amino acid sequence of the peptide (B1).
  • the Syn aggregation inhibitor of the present invention may contain, for example, only YS, only LYZA-3, or both as active ingredients. Further, the Syn aggregation inhibitor of the present invention may contain, as the YS, for example, only the YS-11, may contain only the YS-16, or may contain only the YS-6, Moreover, any two or more types may be included, and three or more types may be included.
  • the peptide YS and the peptide LYZA-3 may, for example, contain only the D-form, only the L-form, or both of the amino acid residues that are constituent units.
  • the peptide YS and the peptide LYZA-3 may be chemically modified peptide isosteres, for example.
  • the chemical modification may be, for example, all amino acid residues or part of the amino acid residues.
  • the type of chemical modification is not particularly limited. Examples include cyclization, modification of carboxyl groups and/or amino groups, amidation of terminal carboxyl groups, and the like.
  • the peptide YS and the peptide LYZA-3 can suppress aggregation of Syn or its fragment peptide (hereinafter also referred to as Syn fragment).
  • Syn fragment aggregation of Syn or its fragment peptide
  • the origin of Syn whose aggregation is suppressed by the peptide YS and the peptide LYZA-3 is not particularly limited, and examples thereof include humans and non-human animals described later, preferably humans.
  • the full-length amino acid sequence of human Syn is, for example, registered in a database (NCBI Reference Sequence) with accession number: NP_001139527 and represented by SEQ ID NO:5.
  • the aggregation inhibitor of the present invention can be used, for example, to treat diseases caused by aggregation of Syn or Syn fragments (hereinafter also referred to as Syn aggregation diseases).
  • treatment includes, for example, prevention, suppression of progression, and amelioration (alleviation).
  • Said prevention also includes, for example, the prevention of recurrence.
  • the aggregation inhibitor of the present invention for example, can suppress the formation of the aggregate itself, so it is useful for prevention and suppression of progress, and because it can dissociate the aggregate, it suppresses progression and improves ( mitigation) is also useful.
  • the Syn aggregation disease is not particularly limited and is a disease caused by the aggregation of Syn, and specific examples thereof include Parkinson's disease, motor dysfunction, and memory disorder.
  • dissociation of the aggregate means, for example, that the aggregate is loosened and separated into Syn that constitutes the aggregate, or separated into Syn fragments that constitute the aggregate. means that Since the peptide YS and the peptide LYZA-3 do not have hydrolytic activity against Syn or Syn fragments, the aggregation inhibitor of the present invention does not cause the degradation of Syn or Syn fragments, but the aggregates diverged.
  • the aggregation inhibitor of the present invention is also referred to as the aggregation inhibitor composition of the present invention.
  • the aggregation inhibitor of the present invention contains at least one of the peptide YS and the peptide LYZA-3 as an active ingredient.
  • the active ingredient contained in the aggregation inhibitor of the present invention may be the peptide YS alone, the peptide LYZA-3 alone, or both the peptide YS and the peptide LYZA-3.
  • the aggregation inhibitor of the present invention may be, for example, a composition consisting only of the active ingredient, or a composition containing the active ingredient and other additive ingredients.
  • the additive component is not particularly limited, and examples thereof include pharmacologically acceptable components. With regard to the additive component, for example, the description of the pharmaceutical composition described later can be used.
  • the aggregation inhibitor of the present invention can be used, for example, in an environment where Syn or a Syn fragment exists, or is presumed to exist.
  • the aggregation inhibitor of the present invention can be added to a subject, for example.
  • the subject may be, for example, a non-biological subject that does not contain cells or the like, or a biological subject such as cells such as brain cells, tissues such as brain, or a living body.
  • a biological subject such as cells such as brain cells, tissues such as brain, or a living body.
  • said addition can be performed, for example, in vivo or in vitro.
  • the cells and tissues may be, for example, human-derived or non-human animal-derived, and the living body may be, for example, human or non-human animal. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cows and camels.
  • suppression of aggregation is, for example, suppression of aggregation of Syn or Syn fragments, or suppression by dissociation of already aggregated Syn aggregates or dissociation of Syn fragment aggregates.
  • the peptide YS and the peptide LYZA-3 in the present invention can, for example, dissociate Syn aggregates or Syn fragment aggregates, and can suppress aggregation itself.
  • the dissociation of the Syn aggregate means, for example, dissociating the aggregate into single Syn fragments
  • the dissociation of the Syn fragment aggregate means, for example, the aggregate to a single Syn fragment. It means solving.
  • the aggregation inhibitor of the present invention can also be said to be, for example, an aggregation dissociation agent.
  • Syn aggregates can be read as, for example, Syn fragment aggregates.
  • the aggregation inhibitor of the present invention may have, for example, an additional peptide in addition to the peptide YS and/or the peptide LYZA-3.
  • additional peptide include a form bound to peptide YS or peptide LYZA-3.
  • additional peptide include those having DDS ability to sites where Syn, Syn fragments, Syn aggregates, or Syn fragment aggregates are present.
  • the additional peptide is, for example, bound to the peptide YS or the peptide LYZA-3 at the time of administration to the living body, and after being administered to the body contents, the peptide YS or the peptide LYZA-3 is activated by enzymes in the body.
  • a signal peptide or the like that is cleaved off from 3 may also be used.
  • the aggregation inhibitor of the present invention can also be used, for example, as a pharmaceutical composition for Syn aggregation diseases of the present invention, which will be described later.
  • the aggregation inhibitor of the present invention can be used, for example, in the method for inhibiting Syn aggregation and the method for treating Syn aggregation diseases of the present invention, which will be described later.
  • the aggregation inhibitor of the present invention can refer to the explanations of the pharmaceutical composition for Syn aggregation disease, the method for inhibiting Syn aggregation, and the treatment method for Syn aggregation disease of the present invention, which will be described later.
  • composition for Syn Aggregation Diseases comprises, as described above, the peptide YS of (A) and the peptide YS of (B) and at least one of the peptide LYZA-3 of
  • the pharmaceutical composition of the present invention is characterized by containing at least one of said peptide YS and said peptide LYZA-3 capable of suppressing aggregation of Syn or Syn fragments, and other configurations are not particularly limited.
  • the peptide YS and the peptide LYZA-3 the description of the aggregation inhibitor of the present invention can be used.
  • the pharmaceutical composition of the present invention contains at least one of the peptide YS and the peptide LYZA-3 as an active ingredient.
  • the active ingredient contained in the pharmaceutical composition of the present invention may be the peptide YS alone, the peptide LYZA-3 alone, or both the peptide YS and the peptide LYZA-3. It may contain other active ingredients against Syn aggregation diseases.
  • the other active ingredient may be, for example, an active ingredient that suppresses the formation of aggregates or an active ingredient that decomposes the formed aggregates, like the peptide YS and peptide LYZA-3.
  • the decomposition of the aggregate may be, for example, decomposition by cleaving the aggregate by hydrolytic activity or the like, or the aggregate may be broken down into molecules (Syn or Syn fragment ) may be decomposed.
  • the pharmaceutical composition of the present invention may be, for example, a composition consisting only of the active ingredient, or a composition containing the active ingredient and other additive ingredients.
  • the additive component is not particularly limited, and examples thereof include pharmacologically acceptable components.
  • the above-mentioned additive components can be appropriately set according to, for example, the administration method, administration site, and dosage form of the pharmaceutical composition of the present invention.
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited, and includes parenteral administration, oral administration, and the like.
  • parenteral administration methods include affected area administration, intravenous administration, subcutaneous administration, intradermal administration, nasal administration, and transdermal administration.
  • administration site may be, for example, direct administration to the treatment site or indirect administration to the treatment site. In the latter case, for example, it is the site that can deliver the active ingredient of the pharmaceutical composition of the present invention to the treatment site.
  • Syn aggregation diseases are often caused by the occurrence of Syn aggregates in the brain, such as the aforementioned Parkinson's disease and memory disorders. Therefore, the site to be treated is, for example, the brain, and the method of administration thereof is preferably direct administration to the brain by injection or the like, intranasal administration, or the like.
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately set depending on the administration method.
  • the dosage form for administration of the pharmaceutical composition of the present invention is, for example, liquid, cream, gel, powder, and the like.
  • the dosage form before administration of the pharmaceutical composition of the present invention, specifically the dosage form in the distribution process may be the same as or different from the dosage form at the time of administration. , it may be in a dosage form that can be prepared by a pharmacist, a nurse, a doctor, or the like.
  • Examples of the dosage form before administration include solids such as powders and granules, and concentrated liquids.
  • the additive component can be appropriately set according to the administration method, dosage form, etc., as described above, and examples thereof include solvents, diluents, excipients, carriers, and the like.
  • the solvent include aqueous solvents such as water, physiological saline, isotonic solutions, and buffer solutions, oil solvents such as soybean oil, and emulsifying solvents that are mixtures of the aqueous solvent and the oil solvent.
  • Said peptide YS and said peptide LYZA-3 in the present invention are soluble in said aqueous solvent, for example.
  • the pharmaceutical composition of the present invention may contain, for example, alcohol, polyalcohol, surfactant, etc. as the additive component.
  • the pharmaceutical composition of the present invention may also contain, for example, a DDS agent for effectively delivering the active ingredient to the treatment site.
  • the pharmaceutical composition of the present invention may be, for example, in a form containing a carrier encapsulating the active ingredient.
  • the carrier include nanoparticles such as polymers.
  • the pharmaceutical composition of the present invention is preferably used for administration by intravenous injection or the like.
  • the administration target (subject) of the pharmaceutical composition of the present invention includes, for example, humans and non-human animals.
  • the administration conditions of the pharmaceutical composition of the present invention are not particularly limited, and can be appropriately determined according to species, age, body weight, sex, presence or absence of Syn aggregation disease, progress, and the like.
  • the administration conditions for the pharmaceutical composition of the present invention are, for example, the total amount of the peptide YS and/or the peptide LYZA-3 per administration is 0.002 to 400 mg.
  • the administration frequency is 1 to 3 times a day, and the interval is every 1 to 10 days.
  • treatment includes, for example, prevention, suppression of progression, and amelioration (mitigation), as described above.
  • the pharmaceutical composition of the present invention may be used for any one purpose, or may be used for two or more purposes, for example.
  • the method for inhibiting Syn aggregation of the present invention is characterized by adding the Syn aggregation inhibitor of the present invention to a subject, as described above.
  • the suppression method of the present invention is characterized by using the aggregation inhibitor of the present invention, and other steps and conditions are not limited at all.
  • the method for inhibiting aggregation of the present invention preferably further includes an incubation step after the addition step of adding the aggregation inhibitor of the present invention to the subject, for example.
  • the incubation temperature is room temperature to 37° C.
  • the incubation time is 4-72 hours
  • the pH is 6.5-8.
  • the incubation temperature is room temperature to 37° C.
  • the incubation time is 1 to 7 days
  • the pH is 6.5 to 8.
  • the method for treating Syn aggregation diseases of the present invention is characterized by administering the Syn aggregation inhibitor of the present invention to a subject, as described above.
  • the therapeutic method of the present invention is characterized by using the pharmaceutical composition of the present invention, and other steps and conditions are not limited at all.
  • the subject (subject) to which the Syn aggregation inhibitor of the present invention is administered includes humans and non-human animals, preferably humans.
  • the description of the aggregation inhibitor and pharmaceutical composition of the present invention can be cited.
  • the peptide of the present invention is the peptide (A1), (A2), (B1) or (B2) for use in inhibiting aggregation of ⁇ -synuclein (Syn). Also, the peptide of the present invention is the peptide (A1), (A2), (B1) or (B2) for use in treating Syn aggregation diseases caused by Syn aggregation.
  • the peptide of the present invention is the peptide (A1), (A2), (B1) or (B2) for use in the production of a Syn aggregation inhibitor. Further, the peptide of the present invention is the peptide (A1), (A2), (B1) or (B2) for use in the production of a drug for Syn aggregation diseases caused by Syn aggregation.
  • ⁇ -Synuclein Fragment Peptides The present invention relating to ⁇ -synuclein fragment peptides (also referred to as Syn fragments) is presented below.
  • the object of the present invention is to provide a soluble flocculant that can serve as a substitute for the poorly soluble full-length Syn.
  • the flocculant of the present invention is characterized by containing the following peptide (C1) or (C2).
  • C1 Peptide EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV consisting of an amino acid sequence of 16 to 26 consecutive amino acid residues containing the 10th to 24th regions in the amino acid sequence of SEQ ID NO: 6 (SEQ ID NO: 6)
  • C2 A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted or added in the amino acid sequence of (C1)
  • the above (C1) or (C2) peptide is also collectively referred to as (C) peptide.
  • the (C2) peptide is an amino acid sequence obtained by deleting, substituting or adding one or two amino acids in the amino acid sequence of the (C1) peptide.
  • the method for evaluating aggregation-inhibiting ability of the present invention comprises a detection step of detecting aggregation of the aggregating agent by allowing the aggregating agent of the present invention or an aggregate of the aggregating agent and a test substance to coexist, and The anti-aggregation ability of the test substance is evaluated based on the degree of aggregation of the aggregating agent in the presence of the substance.
  • the aggregation inhibitor screening method of the present invention comprises a detection step of detecting aggregation of the agglutinating agent by coexisting the aggregating agent of the present invention or an aggregate of the aggregating agent and a test substance, and and a selection step of selecting the test substance as an aggregation inhibitor when aggregation is suppressed.
  • the method for producing a Syn aggregation disease model animal of the present invention is characterized by administering the aggregating agent of the present invention into the brain of a non-human animal.
  • the peptide (C) in the agglutinating agent of the present invention is soluble in a water-soluble solvent and causes aggregation similar to full-length Syn. Therefore, according to the agglutinating agent of the present invention, for example, as a substitute for poorly soluble full-length Syn, it is possible to construct a reaction system for confirming the suppression of Syn aggregation, and to administer Syn agglutination to non-human animals. It becomes possible to create model animals of Therefore, it can be said that the present invention is extremely useful, for example, in elucidating Syn aggregation diseases such as Parkinson's disease and in developing drugs for such diseases.
  • the peptide (C) can be used for aggregation as a substitute for full-length Syn as described above, it is hereinafter also referred to as an aggregating peptide (C).
  • the flocculant of the present invention is characterized by containing the peptide (C), ie, the peptide (C1) or (C2) below.
  • C1 Peptide EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV consisting of an amino acid sequence of 16 to 26 consecutive amino acid residues containing the 10th to 24th regions in the amino acid sequence of SEQ ID NO: 6 (SEQ ID NO: 6)
  • C2 A peptide consisting of an amino acid sequence in which 1 to 2 amino acids are deleted, substituted or added in the amino acid sequence of (C1)
  • the full-length sequence Syn is poorly soluble in aqueous solvents, but the aggregating peptide (C) in the aggregating agent of the present invention is soluble in aqueous solvents. Furthermore, among the fragments of Syn, the agglutinating peptide (C) of the present invention exhibits agglutinating properties similar to full-length Syn. Therefore, for example, by dissolving the aggregating peptide (C) of the present invention in an aqueous solvent, a liquid agglutinating reagent can be prepared. Further, according to the liquid agglutinant reagent, for example, addition to a reaction system in vitro, administration to animals in vivo, and the like are possible.
  • the agglutinating agent of the present invention is also referred to as a Syn-like agglutinating agent or a Syn-like agglutinating reagent because it produces full-length Syn-like agglutination as a substitute for full-length Syn.
  • “solubility” can be judged to be “poorly soluble” when, for example, precipitation occurs visually when mixed with an aqueous solvent, and when precipitation does not occur visually when mixed with an aqueous solvent. It can be judged as "soluble or soluble”.
  • 1 mmol of full-length Syn is mixed with 1 L of an aqueous solvent (eg, water) at room temperature (20 to 25° C.) (1 mmol/L), and the mixture is heated to room temperature (20 to 25° C.). When the solution is allowed to stand for 5 to 10 minutes at room temperature, a precipitate is visually observed, so it can be determined that the solution is poorly soluble.
  • the state in which the peptide is dissolved in an aqueous solvent may be, for example, a true solution or a colloidal solution, and a true solution is preferred.
  • the aggregating peptide (C) exhibits aggregating properties similar to full-length Syn.
  • the aggregation peptide (C) can simulate, for example, the behavior of full-length ⁇ -synuclein (Syn) aggregation in the body. That is, the aggregating peptide (C) can be aggregated, for example, by incubation in an aqueous solvent under temperature conditions comparable to those in the body.
  • the temperature conditions similar to body temperature are, for example, 30 to 38°C and 35 to 38°C. is preferred.
  • the concentration of the aggregation peptide (C) in the reaction system is not particularly limited.
  • the lower limit of the concentration of the aggregation peptide (C) in the reaction system is, for example, 0.5 ⁇ mol/L, 1 ⁇ mol/L, 5 ⁇ mol/L, 0.01 mmol/L, 0.02 mmol/L, 0.05 mmol/L.
  • 0.1 mmol/L, 0.2 mmol/L, etc. preferably 50 ⁇ mol/L, 0.1 mmol/L, 0.2 mmol/L, and the upper limit is not particularly limited, for example, 0.1 mmol /L, 1 mmol/L, 2 mmol/L, 5 mmol/L, and the ranges are, for example, 0.5 ⁇ mol/L to 5 mmol/L, 1 ⁇ mol/L to 5 mmol/L, 0.05 to 5 mmol/L, 0 0.05-2 mmol/L, 0.01-0.1 mmol/L, 0.02-1 mmol/L, 0.1-1 mmol/L, 0.5-1 mmol/L.
  • the amino acid sequence of SEQ ID NO: 6 is a region of 35 residues from 61st to 95th in the amino acid sequence of full-length ⁇ -synuclein (Syn) (SEQ ID NO: 5 described above).
  • the agglutinating peptide (C1) in the agglutinating agent of the present invention is a fragment peptide thereof, and includes the underlined region from 10th to 24th in the amino acid sequence of SEQ ID NO: 6, and its length are consecutive 16-26 amino acid residues in SEQ ID NO:6.
  • 61 EQVTNVGGA VVTGVTAVAQKTVEG AGSIAAATGFV 95 (SEQ ID NO: 6)
  • the aggregating peptide (C1) is not particularly limited, and examples thereof include a peptide consisting of the amino acid sequence of SEQ ID NO:7.
  • the underlined portion is the 10th to 24th regions of SEQ ID NO:6. Since the amino acid sequence of SEQ ID NO: 7 is the 61st to 84th regions in the amino acid sequence of full-length Syn described above, it is hereinafter also referred to as Syn61-84.
  • EQVTNVGGA VVTGVTAVAQKTVEG SEQ ID NO: 7
  • the aggregating peptide (C1) is, for example, a peptide consisting of the amino acid sequence of SEQ ID NO:8.
  • the underlined portion is the 10th to 24th regions of SEQ ID NO: 6 described above. Since the amino acid sequence of SEQ ID NO: 8 is the 70th to 95th region in the amino acid sequence of full-length Syn described above, it is hereinafter also referred to as Syn70-95.
  • VVTGVTAVAQKTVEG AGSIAAATGFV SEQ ID NO: 8
  • the aggregating peptide (C) may be, for example, one type or two or more types.
  • the peptide (C1) is particularly preferable as the aggregation peptide (C), and the peptide (C1) may be, for example, one type or two or more types.
  • the flocculant of the present invention may contain only the aggregating peptide (C), or may further contain other additives.
  • the additive include aqueous solvents such as water, physiological saline, and buffer solutions.
  • the flocculant of the present invention for example, when used in an in vitro or in vivo reaction system as described above, can be a liquid flocculant containing the aggregating peptide (C) and the aqueous solvent.
  • the aggregating agent of the present invention may be, for example, a ready-to-use agent that is prepared as a liquid containing the aggregating peptide (C) and the aqueous solvent at the time of use.
  • the concentration of the aggregating peptide (C) is not particularly limited, and the lower limits are, for example, 1 ⁇ mol/L, 5 ⁇ mol/L, and 5 ⁇ mol/L.
  • L 0.01 mmol/L, 0.05 mmol/L, 0.1 mmol/L, 0.2 mmol/L, 1 mmol/L, 2 mmol/L, and the upper limit is, for example, 10 mmol/L, 5 mmol/L.
  • ranges are, for example, 1 ⁇ mol/L to 10 mmol/L, 0.05 to 10 mmol/L, 0.05 to 5 mmol/L, 1 to 5 mmol/L, 2 to 5 mmol/L.
  • the method of using the aggregating agent of the present invention is not particularly limited, and it can be used, for example, in the method for evaluating aggregation-inhibiting ability, screening method, method for producing model animals, and the like, which will be described later.
  • the evaluation method of the present invention comprises the coexistence of the agglutinating agent of the present invention or aggregates of the aggregating agent and a test substance, and detecting aggregation of the aggregating agent. and evaluating the ability of the test substance to inhibit aggregation based on the degree of aggregation of the aggregating agent in the presence of the test substance.
  • the aggregating peptide (C) in the aggregating agent of the present invention exhibits aggregation properties like full-length Syn, and is soluble in an aqueous solvent unlike full-length Syn. Therefore, for example, the aggregating peptide (C) dissolved in an aqueous solvent or an aggregate thereof and the test substance can coexist in vivo or in vitro. By detecting aggregation of the aggregating peptide (C) in the presence of the test substance, it is possible to evaluate the aggregation-inhibiting ability of the test substance.
  • the aggregating peptide (C) has the same agglutinating properties as full-length Syn, according to the present invention, for example, the aggregation-inhibiting ability of the aggregating peptide (C) can be directly evaluated, In addition, it is possible to indirectly evaluate the ability of full-length Syn to suppress aggregation.
  • the flocculant is preferably used as a liquid flocculant dissolved in an aqueous solvent, for example, as described above.
  • the aggregation-inhibiting ability for example, the aggregation-inhibiting ability of suppressing the formation of aggregates can be evaluated. Performance assessment can also be performed.
  • the former is exemplified as form 1 and the latter as form 2 below.
  • the evaluation method of the present invention allows the coexistence of the agglutinating agent and the test substance in the detection step, and the generation of aggregates from the aggregating agent can be detected.
  • the ability of the test substance to suppress aggregation that is, the ability to suppress the formation of aggregates of the agglutinating peptide (C) depends on the degree of formation of the aggregate in the presence of the test substance. can be evaluated.
  • the degree of formation of aggregates can be determined, for example, by comparison with evaluation criteria.
  • the evaluation criterion is, for example, the amount (P 0 ) of aggregates produced from the aggregating agent in the absence of the test substance. If the amount of aggregates produced from the flocculant in the presence of the test substance (P 1 ) is less than the production amount (P 0 ) of the evaluation criteria, the test substance is the flocculant, That is, it can be said that it has an aggregation-inhibiting ability against the aggregating peptide (C), and it can also be said that it has an aggregation-inhibiting ability against full-length Syn. In addition, it can be said that the smaller the amount of production (P 1 ) compared to the amount of production (P 0 ) of the evaluation criteria, the higher the aggregation suppressing ability.
  • the coexistence method of the aggregating agent and the test substance is not particularly limited, and may be, for example, in vitro or in vivo.
  • the coexistence When the coexistence is in vitro, it may be coexisted in a cell-free reaction system or in a reaction system containing cells or tissues, for example, in a container such as a well or tube.
  • the types of the cells and tissues are not particularly limited, and the origin thereof may be, for example, human or non-human animal.
  • the final concentration of the agglutinating peptide (C) is 0.01 to 0.1 mmol/L (eg, 0.1 mmol/L), and the concentration of the test substance is The final concentration is 0.1 ⁇ mol/L to 100 ⁇ mol/L.
  • the aggregating peptide (C) is added as a liquid aggregating agent dissolved in an aqueous solvent
  • the test substance is added as a test solution dissolved in a solvent such as an aqueous solvent. .
  • the aggregating agent and the test substance may be coexisted by administering them to an animal.
  • the animal is not particularly limited, and examples thereof include non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cows and camels.
  • the agglutinating agent and the test substance are administered to the non-human animal.
  • the aggregating agent is administered, for example, by dissolving the aggregating peptide (C) in an aqueous solvent and administering this solution (liquid aggregating agent).
  • the concentration of the aggregating peptide (C) in the solution is, for example, 0.1-1 mmol/L, 0.5-1 mmol/L.
  • the administration conditions of the aggregating agent are not particularly limited. , 4 ⁇ L).
  • the dosage per administration is, for example, 2 to 4 nmol, and the number of administrations of the flocculant is not particularly limited, and is, for example, once.
  • the test substance is preferably added as a test solution dissolved in a solvent such as an aqueous solvent, and the dose of the test substance is not particularly limited. Then, for example, the agglutinating peptide and the test subject are administered, and after leaving for 1 to 2 weeks, whether or not the aggregating peptide is aggregated may be confirmed.
  • the evaluation method of the present invention is characterized in that, in the detection step, the aggregate of the agglutinating agent (that is, the aggregate of the aggregating peptide (C)) and the test substance are allowed to coexist, and the aggregate Decrease can be detected.
  • the ability of the test substance to suppress aggregation that is, the dissociation ability of the agglutinating peptide (C) to dissociate the aggregate, is evaluated by the degree of reduction of the aggregate in the presence of the test substance. can do.
  • the degree of reduction of aggregates can be determined, for example, by comparison with evaluation criteria.
  • the evaluation criterion is, for example, the amount (Q 0 ) of the aggregate in the absence of the test substance. If the amount of aggregates (Q 1 ) in the presence of the test substance is less than the production amount (Q 0 ) of the evaluation criteria, the test substance is an aggregate of the flocculant, i.e., the It can be said that it has the ability to dissociate aggregates of aggregating peptide (C), and it can be said to have the ability to dissociate aggregates of full-length Syn. In addition, it can be said that the smaller the amount (Q 1 ) of the aggregates compared to the amount (Q 0 ) of the aggregates used as the evaluation criteria, the higher the dissociation ability.
  • the coexistence method of the aggregates of the aggregating agent and the test substance is not particularly limited, and may be, for example, in vitro or in vivo.
  • the coexistence When the coexistence is in vitro, it may be coexisted in a cell-free reaction system, or may be coexisted in a reaction system containing cells or tissues, for example, using containers such as wells and tubes.
  • the types of the cells and tissues are not particularly limited, and the origin thereof may be, for example, human or non-human animal.
  • the aggregating peptide (C) is dissolved in an aqueous solvent in the absence of the test substance. Aggregates of the aggregating peptide (C) are produced by incubating the lysate (liquid aggregating agent).
  • the concentration of the aggregating peptide (C) in the solution is, for example, 0.02 to 1 mmol/L (eg, 1 mmol/L).
  • the incubation conditions are not particularly limited, and the temperature is, for example, 30 to 38° C. (eg, 37° C.), and the time is, for example, 1 to 24 hours.
  • the final concentration of the aggregate in the reaction system is, for example, 0.02 to 1 mmol/L (e.g., 0.1 mmol/L) in terms of the aggregating peptide (C).
  • the final concentration is, for example, 0.1 ⁇ mol/L to 100 ⁇ mol/L.
  • the agglutinating agent that is, the aggregating peptide (C) is administered to an animal and allowed to stand for 1 to 2 weeks to generate the aggregate in vivo. Then, by further administering the test substance, both can coexist.
  • the aggregating agent is administered, for example, by dissolving the aggregating peptide (C) in an aqueous solvent and administering this solution (liquid aggregating agent).
  • the concentration of the aggregating peptide (C) in the solution is, for example, 0.1-1 mmol/L, 0.5-1 mmol/L.
  • Administration conditions are not particularly limited.
  • 1 to 7 ⁇ L (eg, 4 ⁇ L) of the solution is added at a rate of 1 to 2 ⁇ L/minute (eg, 1 ⁇ L/minute) per administration.
  • the dosage per administration is, for example, 2 to 4 nmol, and the number of administrations of the flocculant is not particularly limited, and is once.
  • the administration site of the aggregating agent is, for example, the brain, and the administration method may be, for example, direct administration to the brain by injection or the like, or indirect nasal administration or the like.
  • the animal is not particularly limited, and examples thereof include non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cows and camels.
  • the aggregation inhibitor screening method of the present invention comprises coexisting the aggregating agent of the present invention or an aggregate of the aggregating agent with a test substance, and aggregating the aggregating agent. and a selection step of selecting the test substance as an aggregation inhibitor when aggregation of the aggregating agent is suppressed.
  • the description in the evaluation method (2-2) above can be used unless otherwise specified.
  • the screening method of the present invention uses the aggregating agent of the present invention or an aggregate of the aggregating agent, as described in the evaluation method (2-2) above, to evaluate the anti-agglutination ability of a test substance. Therefore, the test substance can be screened for an aggregation inhibitor that suppresses aggregation of the aggregating agent.
  • the flocculant is preferably used as a liquid flocculant dissolved in an aqueous solvent, for example, as described above.
  • a test substance that suppresses the formation of aggregates also referred to as an aggregate formation inhibitory substance
  • an aggregate formation inhibitory substance a test substance that suppresses the formation of aggregates
  • dissociated test substances also called agglutination-dissociated substances
  • the screening method of the present invention allows the coexistence of the agglutinating agent and the test substance in the detection step, and the generation of aggregates from the aggregating agent can be detected. Then, when the formation of the aggregate is suppressed in the presence of the test substance, in the selection step, the test substance is used for the formation of the aggregate, that is, the aggregation of the agglutinating peptide (C). It can be selected as an aggregation inhibitor that suppresses the formation of aggregates (aggregate formation inhibitor).
  • the evaluation criterion is, for example, the amount (P 0 ) of aggregates produced from the aggregating agent in the absence of the test substance. If the amount of aggregates produced from the flocculant in the presence of the test substance (P 1 ) is less than the production amount (P 0 ) of the evaluation criteria, the test substance is It can be selected as an aggregate formation-suppressing substance that suppresses aggregation, that is, suppresses the formation of aggregates from the aggregating peptide (C), or as an aggregate-suppressing substance that suppresses the formation of aggregates from full-length Syn. In addition, it can be said that the smaller the amount of production (P 1 ) compared to the amount of production (P 0 ) of the evaluation criteria, the higher the anti-aggregation ability of the test substance.
  • the method of coexistence of the agglutinating agent and the test substance is not particularly limited, and may be, for example, in vitro or in vivo. can be used.
  • the screening method of the present invention is characterized in that, in the detection step, the aggregate of the agglutinating agent (that is, the aggregate of the aggregating peptide (C)) and the test substance are allowed to coexist, and the Decrease can be detected. Then, when the aggregate is reduced in the presence of the test substance, in the selection step, the test substance dissociates the aggregate, that is, the aggregate of the agglutinating peptide (C) can be selected as an aggregation inhibitor (aggregate dissociation agent) that dissociates.
  • the aggregate of the agglutinating agent that is, the aggregate of the aggregating peptide (C)
  • the test substance dissociates the aggregate, that is, the aggregate of the agglutinating peptide (C) can be selected as an aggregation inhibitor (aggregate dissociation agent) that dissociates.
  • the degree of reduction of aggregates can be determined, for example, by comparison with evaluation criteria.
  • the evaluation criterion is, for example, the amount (Q 0 ) of the aggregate in the absence of the test substance. If the amount of aggregates (Q 1 ) in the presence of the test substance is less than the production amount (Q 0 ) of the evaluation criteria, the test substance is an aggregate of the flocculant, i.e., the An aggregate-dissociating substance that dissociates aggregates of the aggregating peptide (C) can be selected. In addition, it can be said that the smaller the amount (Q 1 ) of the aggregates compared to the amount (Q 0 ) of the aggregates used as the evaluation criteria, the higher the dissociation ability of the test substance.
  • the method of coexistence of the aggregates of the agglutinating agent and the test substance is not particularly limited, and may be, for example, in vitro or in vivo. The description of can be used.
  • aggregation inhibitors (aggregate formation inhibitors or aggregate dissociation agents) can be directly screened against the aggregating peptide (C), and indirectly, full-length Syn can be screened for aggregation inhibitors against Aggregation of full-length Syn causes, for example, Syn aggregation diseases such as Parkinson's disease as described above. Therefore, according to the screening method of the present invention, drugs for Syn aggregation diseases can be screened as a result. .
  • the present invention further includes a screening method for a drug for Syn aggregation disease, which comprises selecting an aggregation inhibitor selected by the method for screening an aggregation inhibitor of the present invention as a drug for Syn aggregation disease.
  • a screening method for a drug for Syn aggregation disease which comprises selecting an aggregation inhibitor selected by the method for screening an aggregation inhibitor of the present invention as a drug for Syn aggregation disease.
  • the method for producing a Syn aggregation disease model animal of the present invention is characterized by administering the agglutinating agent of the present invention into the brain of a non-human animal.
  • the aggregating peptide (C) in the aggregating agent of the present invention is soluble in an aqueous solvent. Therefore, a liquid agglutinating agent prepared by dissolving the aggregating peptide (C) in an aqueous solvent can be administered to animals to cause aggregation in the brain. Therefore, according to the present invention, a method for producing an aggregation model animal of the aggregating peptide (C) can be provided.
  • agglutinating peptide (C) causes full-length Syn-like aggregation
  • a Syn-aggregation model animal can be produced according to the production method of the present invention.
  • the aggregating peptide (C) aggregates in the brain of the model animal, symptoms of Syn aggregation diseases such as Parkinson's disease caused by aggregation of the full-length Syn (e.g., deterioration of short-term cognitive function) appear. Therefore, according to the production method of the present invention, it can be said that Syn aggregation disease model animals can be produced.
  • the administration method of the flocculant of the present invention to the non-human animal is not particularly limited, and the in vivo administration method in the evaluation method (2-2) can be used.
  • non-human animals examples include mammals such as mice, rats, rabbits, horses, sheep, cows and camels.
  • the flocculant of the present invention is preferably administered to the non-human animal as a solution (liquid flocculant) dissolved in a water-soluble solvent.
  • the concentration of the aggregating peptide (C) is preferably, for example, 0.1 to 1 mmol/L, and as a specific example, in the case of Syn61-84, for example, 0.5 to 1 mmol/L. is preferred, and in the case of Syn70-95, for example, 0.5 to 1 mmol/L is preferred.
  • the administration site is, for example, the brain.
  • the agglutinating agent of the present invention may be directly administered to the brain, for example, using a syringe or the like, or may be administered indirectly through the nose.
  • the dose of the agglutinating agent of the present invention is not particularly limited, and can be appropriately determined according to the type, age, etc. of the non-human animal to be administered.
  • the non-human animal is a 30-35 g mouse, for example, 1-7 ⁇ L (eg, 4 ⁇ L) of the lysate is administered at a rate of 1-2 ⁇ L/minute (eg, 1 ⁇ L/minute) per dose.
  • the dosage per administration is, for example, 2 to 4 nmol, and the number of administrations of the flocculant is not particularly limited, and is once.
  • a test substance may be administered at the same time as the dissolution solution.
  • the test substance may be administered after leaving for a certain period of time (eg, 1 to 2 weeks) after the addition of the dissolution solution.
  • Example A1 Aggregation of Syn is known to be one of the causes of Parkinson's disease. Therefore, it was confirmed that the aggregating peptide (C) similarly aggregates as a substitute for Syn, and that administration of it to mice causes symptoms of Parkinson's disease.
  • Syn FPs Syn fragment peptides
  • the Syn61-84 (SEQ ID NO: 7) and the Syn70-95 (SEQ ID NO: 8) are the agglutinating peptide (C) of the example, and the peptide (SEQ ID NO: 9, Syn70-84) and the peptide of SEQ ID NO: 10 (Syn61-69) consisting of the region other than the underlined portion of Syn61-84 was used as a comparative peptide.
  • ThT Thioflavin T
  • a reaction solution with the following composition was prepared and dispensed into wells so that 100 ⁇ L of the above reaction solution per well.
  • FIG. 1 is a graph showing the fluorescence intensity of the reaction solution to which the peptides of the Examples (Syn61-84, Syn70-95) or the peptides of the Comparative Examples were added.
  • the vertical axis is the fluorescence intensity, and the unit is Fluorescence Intensity (FI).
  • FI Fluorescence Intensity
  • Syn61-84 and Syn70-95 were used as the aggregating peptide (C) in the above example, and Syn70-84 was used as the peptide in the comparative example.
  • Each peptide was dissolved in physiological saline to prepare a 1 mmol/L sample solution. Even when physiological saline was used as the aqueous solvent, precipitation of each peptide was not visually confirmed at room temperature.
  • the Y-maze test was performed by a general method under the following conditions. That is, the mouse was placed so that the nose tip was directed to the end wall of one specific arm among the three arms. Then, they were allowed to act freely for 10 minutes, their behavior was photographed on the monitor of an analysis device (trade name Time YM1, manufactured by Ohara Medical Sangyo), and the alternation rate (%) was calculated.
  • an analysis device trade name Time YM1, manufactured by Ohara Medical Sangyo
  • FIG. 2 shows the results of the Y-maze test.
  • FIG. 2 shows the results of Alteration Rate (%).
  • the mice administered with the peptide Syn61-69 of the comparative example in which aggregation was not confirmed in Example A1
  • the mice administered with the peptides Syn61-84 and Syn70-95 of the example had a shift behavior rate (%) decreased.
  • aggregation occurred in the same manner as full-length Syn, and administration to the brain of mice caused short-term cognitive function, which is a symptom of Parkinson's disease. It was found that a decrease in From this, it was confirmed that the agglutinating peptide can constitute a reaction system for Syn aggregation as a substitute for Syn, and that an animal model of Syn aggregation can be produced by administering it to an animal such as a mouse. .
  • Example A2 Syn61-84 (SEQ ID NO: 7) of Example A1 was dissolved in MilliQ water as an aqueous solvent at room temperature in the same manner as in Example A1 to prepare Syn FP solutions of various concentrations. Then, the final concentration of Syn61-84 in the reaction solution was 1 ⁇ mol/L, 5 ⁇ mol/L, 10 ⁇ mol/L, 50 ⁇ mol/L (0.05 mmol/L), 100 ⁇ mol/L (0.1 mmol/L), 200 ⁇ mol/L. Aggregation in vitro was confirmed by the same method as in Example A1(2), except that (0.2 mmol/L) was used. As a control (blank), a reaction solution to which MilliQ water was added instead of the Syn FP solution was used.
  • each reaction solution containing Syn61-84 at the above final concentration was a transparent solution, and no precipitate was generated at room temperature after preparation.
  • FIG. 11 is a graph showing the fluorescence intensity of the reaction solution to which the peptide (Syn61-84) of the above example was added.
  • the vertical axis is the fluorescence intensity, and the unit is Fluorescence Intensity (FI).
  • FI Fluorescence Intensity
  • Example B1 Anti-agglutination peptides YS and LYZA-3 were confirmed for Syn aggregation-inhibiting ability.
  • YS-16 (SEQ ID NO: 1), YS-11 (SEQ ID NO: 2), YS-6 (SEQ ID NO: 3), and LYZA-3 (SEQ ID NO: 4) were used as aggregation-inhibiting peptides in the examples.
  • the fluorescent dye Thioflavin T (ThT) binds to aggregates and emits strong fluorescence upon binding, so it is possible to determine whether aggregation is increased or suppressed by measuring fluorescence intensity. Therefore, suppression of Syn aggregation by each peptide was confirmed using ThT.
  • the Syn61-84 (SEQ ID NO: 7) fragment peptide of Syn was used as a substitute for full-length Syn. It has already been confirmed in Example A1 that this aggregating peptide Syn61-84 aggregates in the same manner as full-length Syn.
  • a reaction solution with the following composition was prepared and dispensed into wells so that 100 ⁇ L of the above reaction solution per well.
  • 1 mg/mL peptides aggregation-inhibiting peptide solutions: YS-16, YS-11, YS-6, LYZA-3
  • MilliQ water 1 mg/mL peptides
  • Syn61-84 aggregating Peptide solution
  • FIG. 3 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibiting peptide was added.
  • the vertical axis represents the relative value of fluorescence intensity (FI), specifically, the relative value with the fluorescence intensity of the control (Syn61-84) set to 1.
  • FI fluorescence intensity
  • each aggregation-inhibiting peptide significantly decreased the fluorescence intensity compared to the control (Syn61-84) to which the aggregation-inhibiting peptide was not added.
  • Example B2 Aggregation-inhibiting peptides YS-11 and LYZA-3 were confirmed to inhibit formation of aggregates as aggregation-inhibiting ability.
  • a reaction solution with the following composition was prepared and dispensed into wells so that the reaction solution was 200 ⁇ L per well.
  • X mmol/L peptide aggregation-inhibiting peptide solution: YS-11, LYZA-3
  • concentration X mmol/L was 1 ⁇ mol/L
  • 10 ⁇ mol/ L was set to 100 ⁇ mol/L.
  • 1 mmol/L Syn61-84 aggregating peptide solution was prepared using MilliQ water as an aqueous solvent. After incubating the reaction solution at 37° C.
  • FIG. 4 (A) is a graph showing the fluorescence intensity of the reaction solution to which peptide LYZA-3 was added, and (B) is a graph showing the fluorescence intensity of the reaction solution to which peptide YS-11 was added.
  • the vertical axis is the fluorescence intensity, and the unit is Fluorescence Intensity (FI).
  • FI Fluorescence Intensity
  • Example B3 Regarding the aggregation-inhibiting peptides YS-11 and LYZA-3, aggregate dissociation was confirmed as aggregation-inhibiting ability.
  • Measurement wavelengths were an excitation wavelength (ex) of 444 nm and a fluorescence wavelength (em) of 480 nm.
  • X mmol/L peptides (aggregation-inhibiting peptides: YS-11, LYZA-3) were prepared using MilliQ water, and the concentrations X mmol/L were 0.1 ⁇ mol/L and 1 ⁇ mol in the reaction solution. /L, 10 ⁇ mol/L, and 100 ⁇ mol/L.
  • a reaction solution to which water was added instead of peptide YS-11 and peptide LYZA-3 was similarly measured.
  • FIG. 5 (A) is a graph showing the fluorescence intensity of the reaction solution to which peptide LYZA-3 was added, and (B) is a graph showing the fluorescence intensity of the reaction solution to which peptide YS-11 was added.
  • the vertical axis is the fluorescence intensity, and the unit is Fluorescence Intensity (FI).
  • FI Fluorescence Intensity
  • Example B4 Parkinson's disease model mice were prepared using the aggregating peptide Syn61-84, and the aggregation-inhibiting peptide YS-11 or LYZA-3 was administered to confirm the effect.
  • Rota-Rod uses a ro-rod device for mice (product name: MK-670, Muromachi Kikai Co., Ltd.), sets the number of rotations of the rotor to 10 rpm or 20 rpm, and rotates the rotor until the mouse falls. time was measured.
  • Fig. 6 shows the results of the Alternation Rate (%) calculated by Y-maze.
  • the first Y-maze (Y-maze-1) on Day 14 and the second Y-maze (Y-maze-2) on Day 56 the Syn61-84 administration group compared with the Saline administration group A decrease in the shift behavior rate was confirmed. These results revealed that the administration of Syn61-84 reduced cognitive function.
  • the third Y-maze (Y-maze-3) on Day 74 which was performed after administration of the aggregation-inhibiting peptide on Day 56, compared with the Saline-administered group, the YS-11-administered group and the LYZA- All of the 3 administration groups increased the alternation behavior rate.
  • the administration of the aggregation-inhibiting peptide ameliorated the decline in cognitive function.
  • Fig. 7 shows the results of the Rota-Rod test.
  • 10 rpm and 20 rpm are the rotation speeds of the rotor, and the vertical axis is the time until the mouse on the rotor falls. The longer the time until falling, the higher the athletic ability can be evaluated, and the shorter the time, the lower the athletic ability can be evaluated.
  • the Syn61- In the 84-administered group it was confirmed that the staying time in the rotor was significantly reduced.
  • Example B5 The aggregation-promoting peptide Syn61-84 and the aggregation-inhibiting peptide YS-11 or LYZA-3 were co-administered to mice to confirm their effects.
  • the aggregating peptide Syn61-84 was dissolved in physiological saline to prepare a 0.5 mmol/L solution.
  • the aggregation peptide Syn61-84 and the aggregation-inhibiting peptide (YS-11 or LYZA-3) were dissolved in physiological saline to prepare a mixed solution with a concentration of 0.5 mmol/L for each.
  • 4 ⁇ L of the Syn61-84 solution or 4 ⁇ L of the mixed solution was administered into the ventricles of ICR mice (7 weeks old) at 1 ⁇ L/min (Day 0).
  • physiological saline was used as a system in which neither the aggregating peptide nor the aggregation-inhibiting peptide was administered, and 4 ⁇ L was administered at the same rate (Day 0).
  • Fig. 8 shows the results of the alternation rate (%) calculated by Y-maze.
  • the Syn61-84 administration group had a lower alternation behavior rate than the Saline administration group, but the mixed solution administration group
  • the alternation behavior rate of (Syn61-84/LYZA-3 and Syn61-84/YS-11) was higher than that of the Syn61-84 administration group and similar to or higher than that of the Saline administration group.
  • the Syn61-84/LYZA-3 administration group showed higher values than the Syn61-84 administration group and the Saline administration group.
  • Fig. 9 shows the results of the Rota-Rod test.
  • 10 rpm and 20 rpm are the rotation speeds of the rotor, and the vertical axis is the time until the mouse on the rotor falls. The longer the time until falling, the higher the athletic ability can be evaluated, and the shorter the time, the lower the athletic ability can be evaluated.
  • the Syn61-84-administered group showed a significant decrease in the time spent in the rotor. was confirmed.
  • the Syn61-84-administered group showed a slight increase in the stay time, but no recovery to the stay time of the Saline-administered group was observed.
  • the mixed solution administration group (Syn61-84/LYZA-3 and Syn61-84/YS-11) showed a value lower than the residence time of the Saline administration group in the RR-1.
  • the residence time was significantly longer than that of the Syn61-84 administration group, and even after that, the residence time increased rather than decreased, and RR-4 showed a longer residence time than the Saline administration group.
  • the Syn61-84/LYZA-3-administered group exhibited significantly longer residence time than the Saline-administered group. From these results, it was clarified that simultaneous administration of the aggregation-inhibiting peptide and Syn61-84 prevented the deterioration of motor dysfunction.
  • Example B6 The effect of the aggregating peptide on cells and the inhibition of cell death by the aggregation-inhibiting peptide YS-11 were confirmed.
  • the aggregating peptide Syn61-84 was dissolved in PBS buffer to 1 mmol/L.
  • the aggregation-inhibiting peptide YS-11 was dissolved in PBS to a predetermined concentration (0.001 to 1 mmol/L). Then, using these, a reaction solution having the following composition was prepared. The reaction was incubated at 37°C for 24 hours. As a control, PBS incubated in the same manner was used instead of the reaction solution.
  • mice neuroblastoma Neuroblastoma (Neuro2a) cells were seeded at 2.66 ⁇ 10 4 cells per well. After culturing at 37° C. for 24 hours, 50 ⁇ L of the above-described reaction solution after incubation was added per well, and the cells were further cultured at 37° C. for 24 hours. Then, the survival rate (%) of cells was measured for each well by the WST-8 method.
  • the vertical axis indicates the survival rate (%)
  • the values of YS-11 and Syn61-84 in the figure indicate the final concentration in the well ( ⁇ mol/L).
  • reaction solution containing no aggregation-inhibiting peptide In the reaction solution containing no aggregation-inhibiting peptide, aggregation was generated by incubation, which caused cell death. In the reaction solution containing the aggregation-inhibiting peptide, the aggregation was inhibited even by incubation. As a result, it is thought that cell death was suppressed even when added to the cell culture medium. The reaction solution containing only the aggregation-inhibiting peptide did not affect the viability of the cells.
  • Syn aggregation inhibitor of the present invention since aggregation due to intermolecular association of Syn can be suppressed, Syn aggregation diseases such as Parkinson's disease caused by Syn aggregation can be treated, for example, prevented, inhibited from progressing, improved, etc. be possible.

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