α-synuclein aggregation inhibitor, pharmaceutical composition for α-synuclein disease, and use thereof

Abstract

The present invention provides a new medical agent that inhibits aggregation of α-synuclein, which is a main factor in Parkinson's disease, etc.　The α-synuclein aggregation inhibitor according to the present invention is characterized by including a peptide of (A1), (A2), (B1), or (B2). (A1) A peptide composed of an amino acid sequence having 6 or more contiguous residues in the amino acid sequence of SEQ ID NO: 1. RETLVYLTHLDYDDTE (SEQ ID NO: 1). (A2) A peptide composed of an amino acid sequence in which 1-2 amino acids have been deleted, substituted, or added in the amino acid sequence of (A1) above. (B1) A peptide composed of an amino acid sequence of SEQ ID NO: 4. YSNFNTDY (SEQ ID NO: 4). (B2) A peptide composed of an amino acid sequence in which 1-2 amino acids have been deleted, substituted, or added in the amino acid sequence of (B1) above.

Description

α-synuclein aggregation inhibitor, pharmaceutical composition for α-synuclein aggregation disease, and use thereof

[0001] The present invention relates to an α-synuclein aggregation inhibitor, a pharmaceutical composition for α-synuclein aggregation diseases, and uses thereof.

[0002] Parkinson's disease is a brain disorder that causes impaired movement. As there is no fundamental treatment for the disease, there is a need to elucidate its causes and develop drugs that can provide a fundamental treatment.

[0003] In recent years, it has been reported that aggregation of α-synuclein in the brain is a cause of Parkinson's disease, and therefore, if we can inhibit α-synuclein aggregation, it may become an effective drug.

[0004] Therefore, an object of the present invention is to provide a new drug that inhibits the aggregation of α-synuclein, which causes Parkinson's disease and the like.

[0005] The α-synuclein aggregation inhibitor of the present invention is characterized by comprising a peptide of the following (A1), (A2), (B1), or (B2): (A1) a peptide consisting of an amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1: RETLVYLTHLDYDDTE (SEQ ID NO: 1) (A2) a peptide consisting of an amino acid sequence in the amino acid sequence of (A1) in which 1 to 2 amino acids have been deleted, substituted, or added (B1) a peptide consisting of the amino acid sequence of SEQ ID NO: 4: YSNFNTDY (SEQ ID NO: 4) (B2) a peptide consisting of an amino acid sequence in the amino acid sequence of (B1) in which 1 to 2 amino acids have been deleted, substituted, or added

[0006] The pharmaceutical composition of the present invention for use in α-synuclein aggregation diseases is characterized by comprising the peptide (A1), (A2), (B1) or (B2).

[0007] The method for inhibiting α-synuclein aggregation of the present invention is characterized by adding the α-synuclein aggregation inhibitor of the present invention to a test sample.

[0008] The method for treating an α-synuclein aggregation disease of the present invention is characterized by administering the α-synuclein aggregation inhibitor of the present invention to a subject.

[0009] The α-synuclein (hereinafter also referred to as Syn) aggregation inhibitor of the present invention can inhibit aggregation due to intermolecular association of Syn, and therefore enables the treatment of Syn aggregation diseases caused by Syn aggregation, such as Parkinson's disease, including, for example, prevention, inhibition of progression, and improvement.

[0010] FIG. 1 is a graph showing the fluorescence intensity of the reaction solution to which the aggregating peptide was added in Example A1. FIG. 2 is a graph showing the alternation behavior rate of mice administered with the aggregating peptide in Example A1. FIG. 3 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibitory peptide was added in Example B1. FIG. 4 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibitory peptide was added in Example B2. FIG. 5 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibitory peptide was added in Example B3. FIG. 6 is a graph showing the alternation behavior rate of mice to which the aggregation-inhibitory peptide was added in Example B4. FIG. 7 is a graph showing the results of Rota-Rod analysis of mice to which the aggregation-inhibitory peptide was added in Example B4. FIG. 8 is a graph showing the alternation behavior rate of mice to which the aggregation-inhibitory peptide and the aggregating peptide were simultaneously added in Example B5. FIG. 9 is a graph showing the results of Rota-Rod analysis of mice to which the aggregation-inhibitory peptide and the aggregating peptide were simultaneously added in Example B5. Fig. 10 is a graph showing the cell viability when a reaction solution containing an aggregation-inhibiting peptide and an aggregating peptide was incubated and then added to the cells 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.

[0011] 1. Inhibition of α-synuclein aggregation The present invention relating to inhibition of α-synuclein aggregation is described below.

[0012] (1-1) α-Synuclein Aggregation Inhibitor The Syn aggregation inhibitor of the present invention (hereinafter also referred to as Syn aggregation inhibitor) is characterized in that it contains the following peptide (A1), (A2), (B1), or (B2), as described above. The peptide (A1), (A2), (B1), or (B2) will hereinafter be referred to as aggregation-inhibitory peptide, the peptide (A1) or (A2) will collectively be referred to as peptide (A) or hereinafter also referred to as YS, and the peptide (B1) or (B2) will collectively be referred to as peptide (B) or hereinafter also referred to as LYZA-3.

[0013] (A1) A peptide consisting of an amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1: RETLVYLTHLDYDDTE (SEQ ID NO: 1) (A2) 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 (A1) (B1) 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) YSNFNTDY (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)

[0014] The peptide YS of (A1) may be any amino acid sequence of 6 or more consecutive residues in the amino acid sequence of SEQ ID NO: 1, and its length may be any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In addition, the first amino acid residue of the peptide YS may be any of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, and 11th amino acid residues in the amino acid sequence of SEQ ID NO: 1.

[0015] Examples of the peptide YS in (A1) include 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), and a peptide consisting of the amino acid sequence of SEQ ID NO: 3 (hereinafter also referred to as YS-6), with YS-6 being preferred. YS-16 RETLVYLTHLDYDDTE (SEQ ID NO: 1) YS-11 RETLVYLTHLD (SEQ ID NO: 2) YS-6 DYDDTE (SEQ ID NO: 3)

[0016] As described above, the peptide (A2) has an amino acid sequence in which one or two amino acids have been deleted, substituted, or added relative to the amino acid sequence of the peptide (A1). When the peptide (A1) is the peptide YS-16, YS-11, or YS-6, the peptide (A2) has an amino acid sequence in which one or two amino acids have been deleted, substituted, or added relative to the amino acid sequence of the peptide YS-16, YS-11, or YS-6, for example.

[0017] Furthermore, the peptide (B2) has an amino acid sequence in which one or two amino acids are deleted, substituted or added relative to the amino acid sequence of the peptide (B1), as described above.

[0018] The Syn aggregation inhibitor of the present invention may contain, for example, only the YS, only the LYZA-3, or both as an active ingredient. Furthermore, the Syn aggregation inhibitor of the present invention may contain, for example, only the YS-11, only the YS-16, or only the YS-6, or may contain any two or more types thereof, or may contain three or more types thereof.

[0019] The peptide YS and the peptide LYZA-3 may contain, for example, only D-isomers or only L-isomers of amino acid residues as constituent units, or may contain both.

[0020] The peptide YS and the peptide LYZA-3 may be, for example, chemically modified peptide isosteres. The chemical modification may be, for example, of all or some of the amino acid residues. The type of chemical modification is not particularly limited, and examples include substitution of hydrogen atoms with halogen atoms, substitution of corresponding natural amino acids with non-natural amino acids, introduction of methyl groups, modification of amino acid side chains, cyclization of peptides, modification of carboxyl groups and / or amino groups, and amidation of terminal carboxyl groups.

[0021] The peptide YS and the peptide LYZA-3 can inhibit the aggregation of Syn or its fragment peptides (hereinafter also referred to as Syn fragments). In other words, they can inhibit the aggregation of Syn or Syn fragments due to intermolecular association, which can be said to inhibit the formation of aggregates, for example. The origin of Syn, the aggregation of which is inhibited by the peptide YS and the peptide LYZA-3, is not particularly limited and may be, for example, human or a non-human animal as described below, with human beings being preferred. The full-length amino acid sequence of human Syn is registered in the database (NCBI Reference Sequence) under accession number NP_001139527, for example, and is represented by SEQ ID NO: 5.

[0022] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 5)

[0023] 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). In the present invention, "treatment" includes, for example, prevention, inhibition of progression, and improvement (alleviation). "Prevention" also includes, for example, prevention of recurrence. The aggregation inhibitor of the present invention is useful for prevention and inhibition of progression, for example, because it can inhibit the formation of the aggregates themselves, and is also useful for inhibition of progression and improvement (alleviation), for example, because it can dissociate the aggregates. The Syn aggregation disease is not particularly limited, and is a disease that can be caused by the aggregation of Syn, and specific examples include Parkinson's disease, motor dysfunction, and memory impairment. In the present invention, "dissociation of aggregates" means, for example, that the aggregates are loosened and separated into Syns that constitute the aggregates, or separated into Syn fragments that constitute the aggregates. 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 dissociates the aggregates rather than decomposing Syn or Syn fragments.

[0024] The aggregation inhibitor of the present invention is also referred to as the aggregation-inhibiting 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 only the peptide YS, only the peptide LYZA-3, or both the peptide YS and the peptide LYZA-3. The aggregation inhibitor of the present invention may be, for example, a composition consisting of only the active ingredient, or a composition containing the active ingredient and other additive ingredients. The additive ingredients are not particularly limited, and examples include pharmacologically acceptable ingredients. With regard to the additive ingredients, for example, the explanation of the pharmaceutical composition described below can be used.

[0025] The aggregation inhibitor of the present invention can be used, for example, in an environment where Syn or a Syn fragment is present or is suspected to be present. The aggregation inhibitor of the present invention can be added, for example, to a test subject. The test subject may be, for example, a non-biological test subject not containing cells, or a biological test subject such as cells such as brain cells, tissues such as brain, or a living organism. In the case of the latter test subject, the addition can be performed, for example, in vivo or in vitro. The cells and tissues may be, for example, derived from humans or non-human animals, and the living organism may be, for example, humans or non-human animals. Examples of the non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels.

[0026] In the present invention, aggregation inhibition refers to, for example, inhibition of aggregation of Syn or Syn fragments, or inhibition by dissociation of already aggregated Syn aggregates or dissociation of Syn fragment aggregates. The peptide YS and peptide LYZA-3 in the present invention can, for example, dissociate Syn aggregates or Syn fragment aggregates, and can also inhibit aggregation itself. Dissociation of Syn aggregates means, for example, dissociating the aggregates into single Syns, and dissociation of Syn fragment aggregates means, for example, dissociating the aggregates into single Syn fragments. In this case, the aggregation inhibitor of the present invention can also be referred to as, for example, an aggregation dissociation agent. Hereinafter, Syn aggregates can be read as, for example, Syn fragment aggregates.

[0027] 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. The additional peptide may be, for example, bound to the peptide YS or the peptide LYZA-3. The additional peptide may have a DDS ability for a site where Syn, a Syn fragment, a Syn aggregate, or a Syn fragment aggregate is present. Furthermore, the additional peptide may be, for example, a signal peptide that is bound to the peptide YS or the peptide LYZA-3 when administered to a living body, and that is cleaved and removed from the peptide YS or the peptide LYZA-3 by an enzyme or the like in the living body after administration to the living body.

[0028] The aggregation inhibitor of the present invention can also be used, for example, as the pharmaceutical composition for Syn aggregation diseases of the present invention described below. Furthermore, the aggregation inhibitor of the present invention can be used, for example, in the method for inhibiting Syn aggregation of the present invention and the method for treating Syn aggregation diseases of the present invention described below. The explanations of the pharmaceutical composition for Syn aggregation diseases, the method for inhibiting Syn aggregation, and the method for treating Syn aggregation diseases of the present invention described below can be cited for the aggregation inhibitor of the present invention.

[0029] (1-2) Pharmaceutical Composition for Syn Aggregation Diseases As described above, the pharmaceutical composition for Syn aggregation diseases (hereinafter also referred to as the pharmaceutical composition) of the present invention is characterized by containing at least one of the peptide YS of (A) and the peptide LYZA-3 of (B).

[0030] The pharmaceutical composition of the present invention is characterized by containing at least one of the peptide YS and the peptide LYZA-3, which can inhibit aggregation of Syn or a Syn fragment, and other components are not particularly limited. The descriptions of the aggregation inhibitor of the present invention can be cited for the peptide YS and the peptide LYZA-3.

[0031] The pharmaceutical composition of the present invention contains at least one of peptide YS and peptide LYZA-3 as an active ingredient. The active ingredient contained in the pharmaceutical composition of the present invention may be peptide YS alone, peptide LYZA-3 alone, or both peptide YS and peptide LYZA-3. The pharmaceutical composition of the present invention may further contain another active ingredient for the Syn aggregation disorder. The other active ingredient may be, for example, an active ingredient that inhibits aggregate formation, similar to peptide YS and peptide LYZA-3, or an active ingredient that degrades formed aggregates. With regard to the other active ingredient, degradation of aggregates may be, for example, degradation by cleaving the aggregates using hydrolytic activity or the like, or degradation by dissociating the aggregates into their constituent molecules (Syn or Syn fragments).

[0032] The pharmaceutical composition of the present invention may be, for example, a composition consisting solely of the active ingredient, or a composition containing the active ingredient and other additive ingredients. The additive ingredients are not particularly limited and may be, for example, pharmacologically acceptable ingredients. The additive ingredients can be appropriately selected depending on, for example, the administration method, administration site, dosage form, etc. of the pharmaceutical composition of the present invention.

[0033] The method of administration of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include parenteral administration and oral administration.

[0034] Examples of the parenteral administration method include administration to an affected area, intravenous administration, subcutaneous administration, intradermal administration, nasal administration, transdermal administration, etc. Furthermore, for example, infusion, injection, etc. can be used for administration. In the case of parenteral administration, the 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, the site where the active ingredient of the pharmaceutical composition of the present invention can be delivered to the treatment site. Syn aggregation diseases are often caused by the formation of Syn aggregates in the brain, such as the aforementioned Parkinson's disease and memory disorders. Therefore, the treatment site is, for example, the brain, and the administration method is preferably, for example, direct administration to the brain by injection, nasal administration, etc.

[0035] The dosage form of the pharmaceutical composition of the present invention is not particularly limited and can be appropriately determined depending on the administration method. The dosage form of the pharmaceutical composition of the present invention at the time of administration is, for example, liquid, cream, gel, powder, etc. Furthermore, the dosage form of the pharmaceutical composition of the present invention before administration, specifically the dosage form during distribution, may be, for example, the same as or different from the dosage form at the time of administration. In the latter case, it may be a dosage form that can be adjusted to the dosage form at the time of administration by a pharmacist, nurse, doctor, etc. at the time of administration. Examples of the dosage form before administration include solid forms such as powder and granules, and concentrated liquid forms.

[0036] In the pharmaceutical composition of the present invention, the additive components can be appropriately selected depending on the administration method, dosage form, etc., as described above, and include, for example, solvents, diluents, excipients, carriers, etc. Examples of the solvent include aqueous solvents such as water, physiological saline, isotonic solutions, and buffer solutions, oily solvents such as soybean oil, and emulsifying solvents, which are mixtures of the aqueous and oily solvents. The peptide YS and peptide LYZA-3 of the present invention are soluble in, for example, the aqueous solvent. 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 in a form containing, for example, a carrier in which the active ingredient is encapsulated. Examples of the carrier include nanoparticles such as polymers. By encapsulating the active ingredient in this way, for example, the stability of the active ingredient can be maintained and the composition also serves as a DDS. In this case, for example, the pharmaceutical composition of the present invention is preferably used for administration by, for example, intravenous injection.

[0037] The subject (subject) to which the pharmaceutical composition of the present invention is administered can be, for example, a human or a non-human animal. The conditions for administering the pharmaceutical composition of the present invention are not particularly limited and can be appropriately determined depending on the species, age, body weight, sex, the presence or absence of a Syn aggregation disorder, the degree of progression, and the like. For example, when the subject is an adult male weighing 70 kg, the conditions for administering the pharmaceutical composition of the present invention are such that the total amount of peptide YS and / or peptide LYZA-3 per administration is 0.002 to 400 mg, the number of administrations per day is 1 to 3, and the intervals between administrations are every 1 to 10 days.

[0038] As used herein, the term "treatment" includes, for example, prevention, inhibition of progression, and improvement (alleviation), as described above. The pharmaceutical composition of the present invention may be used for, for example, any one of these purposes or for two or more purposes.

[0039] (1-3) Aggregation Inhibition Method The Syn aggregation inhibition method of the present invention is characterized by adding the Syn aggregation inhibitor of the present invention to a test sample, as described above. The inhibition method of the present invention is characterized by using the aggregation inhibitor of the present invention, and other steps and conditions are not limited in any way.

[0040] The addition of the aggregation inhibitor of the present invention to the test subject can be referred to in the description of the aggregation inhibitor and pharmaceutical composition of the present invention. Preferably, the aggregation inhibition method of the present invention further comprises an incubation step after the addition step of adding the aggregation inhibitor of the present invention to the test subject. When the test subject is a non-biological system, the incubation temperature is, for example, room temperature to 37°C, the incubation time is 4 to 72 hours, and the pH is 6.5 to 8. When the test subject is a cell or tissue, the incubation temperature is, for example, room temperature to 37°C, the incubation time is 1 to 7 days, and the pH is 6.5 to 8.

[0041] (1-4) Method for Treating Syn Aggregation Disorders As described above, the method for treating Syn aggregation disorders of the present invention is characterized by administering the Syn aggregation inhibitor of the present invention to a subject. The method for treating Syn aggregation disorders of the present invention is characterized by using the pharmaceutical composition of the present invention, and other steps and conditions are not limited in any way.

[0042] As described above, the subject (subject) to which the Syn aggregation inhibitor of the present invention is administered may be a human or a non-human animal, and preferably a human. The description of the aggregation inhibitor and pharmaceutical composition of the present invention can be applied to the addition of the aggregation inhibitor of the present invention to the subject.

[0043] (1-5) Use of Peptides The peptide of the present invention is the peptide (A1), (A2), (B1), or (B2) described above for use in inhibiting α-synuclein (Syn) aggregation. The peptide of the present invention is also the peptide (A1), (A2), (B1), or (B2) described above for use in treating Syn aggregation disorders caused by Syn aggregation.

[0044] The peptide of the present invention is the peptide (A1), (A2), (B1), or (B2) described above for use in the production of a Syn aggregation inhibitor. Also, the peptide of the present invention is the peptide (A1), (A2), (B1), or (B2) described above for use in the production of a medicament for a Syn aggregation disorder caused by Syn aggregation.

[0045] 2. α-Synuclein Fragment Peptide The present invention relating to an α-synuclein fragment peptide (also referred to as Syn fragment) will be described below.

[0046] Since Syn aggregation in the brain is known to be a cause of Parkinson's disease, it is believed that inhibiting Syn aggregation could be a potential treatment for Parkinson's disease. However, Syn is poorly soluble in aqueous solvents. For this reason, it has been difficult to establish, for example, an in vitro reaction system for confirming the inhibition of Syn aggregation by a test substance, or to establish an in vivo animal model of Parkinson's disease in which Syn aggregation occurs upon administration to an animal.

[0047] Therefore, an object of the present invention is to provide a soluble aggregating agent that can be used as an alternative to the poorly soluble full-length Syn.

[0048] The aggregating agent of the present invention is characterized by containing the following peptide (C1) or (C2): (C1) A peptide comprising an amino acid sequence of 16 to 26 consecutive amino acid residues including the 10th to 24th region in the amino acid sequence of SEQ ID NO: 6: EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 6) (C2) A peptide comprising an amino acid sequence in which 1 to 2 amino acids have been deleted, substituted or added in the amino acid sequence of (C1).

[0049] The peptides (C1) and (C2) are collectively referred to as peptide (C). As described above, the peptide (C2) has an amino acid sequence in which one or two amino acids are deleted, substituted, or added in the amino acid sequence of the peptide (C1).

[0050] The method for evaluating the aggregation-inhibiting ability of the present invention includes a detection step of coexisting the agglutinant of the present invention or an aggregate of the agglutinant with a test substance and detecting the aggregation of the agglutinant, and is characterized in that the aggregation-inhibiting ability of the test substance is evaluated based on the degree of aggregation of the agglutinant in the presence of the test substance.

[0051] The screening method for aggregation inhibitors of the present invention is characterized by comprising a detection step in which the agglutinant of the present invention or an aggregate of the agglutinant is coexisted with a test substance and aggregation of the agglutinant is detected, and a selection step in which, if aggregation of the agglutinant is inhibited, the test substance is selected as an agglutination inhibitor.

[0052] 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.

[0053] The peptide (C) in the aggregating agent of the present invention is soluble in aqueous solvents and undergoes aggregation similar to that of full-length Syn. Therefore, the aggregating agent of the present invention can be used, for example, as a substitute for the poorly soluble full-length Syn, to construct a reaction system for confirming inhibition of Syn aggregation, or to create an animal model of Syn aggregation by administering the aggregating agent to non-human animals. Therefore, the present invention can be said to be extremely useful, for example, in elucidating Syn aggregation diseases such as Parkinson's disease and developing drugs therefor.

[0054] As mentioned above, the peptide (C) can be used for aggregation as a substitute for full-length Syn, and therefore, hereinafter, it is also referred to as aggregating peptide (C).

[0055] (2-1) Flocculant The flocculant of the present invention is characterized by containing the peptide of (C) above, i.e., the peptide of (C1) or (C2) below: (C1) A peptide comprising an amino acid sequence of 16 to 26 consecutive amino acid residues including the 10th to 24th region in the amino acid sequence of SEQ ID NO: 6: EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 6) (C2) A peptide comprising an amino acid sequence in which 1 to 2 amino acids have been deleted, substituted or added in the amino acid sequence of (C1) above.

[0056] As mentioned above, full-length 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 Syn fragments, the aggregating peptide (C) of the present invention exhibits aggregating 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 aggregating reagent can be prepared. Furthermore, this liquid aggregating reagent can be added to an in vitro reaction system or administered to an animal in vivo, for example. The aggregating agent of the present invention is also referred to as a Syn-like aggregating agent or Syn-like aggregating reagent because it is a substitute for full-length Syn and produces aggregating properties similar to full-length Syn.

[0057] In the present invention, "solubility" can be determined as "poorly soluble" when a precipitate forms with the naked eye upon mixing with an aqueous solvent, and as "soluble or soluble" when no precipitate forms with the naked eye upon mixing with an aqueous solvent. As a specific example, when 1 mmol of full-length Syn is mixed with 1 L of an aqueous solvent (e.g., water) at room temperature (20 to 25°C) (1 mmol / L) and the mixture is allowed to stand at room temperature (20 to 25°C) for 5 to 10 minutes, a precipitate is visually observed, and therefore the full-length Syn can be determined to be poorly soluble. In contrast, when the aggregating peptide (C) is mixed with an aqueous solvent (e.g., water) (1 mmol / L) under the same conditions and allowed to stand under the same conditions, no precipitate is visually observed, and therefore the aggregating peptide can be determined to be soluble.

[0058] In the present invention, the state in which the peptide is dissolved in an aqueous solvent may be, for example, a true solution or a colloidal solution, with a true solution being preferred.

[0059] As described above, the aggregating peptide (C) exhibits aggregation properties similar to those of full-length Syn. The aggregating peptide (C) can, for example, mimic the aggregation behavior of full-length α-synuclein (Syn) in vivo. That is, the aggregating peptide (C) can be caused to aggregate by, for example, incubating it in an aqueous solvent under temperature conditions similar to those in vivo. The temperature conditions similar to body temperature are, for example, 30 to 38°C or 35 to 38°C, and in experimental systems such as in vitro, the temperature is preferably set to, for example, 37°C±1°C, preferably 37°C.

[0060] When the aggregating peptide (C) is aggregated, the concentration of the aggregating peptide (C) in the reaction system is not particularly limited. The lower limit of the concentration of the aggregating peptide (C) in the reaction system is, for example, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 0.01 mmol / L, 0.02 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, etc., and preferably 50 μmol / L, 0.1 mmol / L, 0.2 mmol / L. The upper limit is not particularly limited and is, for example, 0.1 mmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, and the range is, for example, 0.5 μmol / L to 5 mmol / L, 1 μmol / L to 5 mmol / L, 0.05 to 5 mmol / L, 0.05 to 2 mmol / L, 0.01 to 0.1 mmol / L, 0.02 to 1 mmol / L, 0.1 to 1 mmol / L, or 0.5 to 1 mmol / L.

[0061] The amino acid sequence of SEQ ID NO: 6 is a region of 35 residues from positions 61 to 95 in the amino acid sequence of full-length α-synuclein (Syn) (the aforementioned SEQ ID NO: 5). The aggregating peptide (C1) in the aggregating agent of the present invention is a fragment peptide thereof, and includes the underlined region from positions 10 to 24 in the amino acid sequence of SEQ ID NO: 6, with a length of 16 to 26 consecutive amino acid residues in SEQ ID NO: 6. 61 EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV 95 (SEQ ID NO: 6)

[0062] 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. In the amino acid sequence of SEQ ID NO: 7, the underlined portion corresponds to the region from positions 10 to 24 in the amino acid sequence of SEQ ID NO: 6. Since the amino acid sequence of SEQ ID NO: 7 corresponds to the region from positions 61 to 84 in the amino acid sequence of the full-length Syn, hereinafter it is also referred to as Syn61-84. EQVTNVGGAVVTGVTAVAQKTVEG (SEQ ID NO: 7)

[0063] An example of the aggregating peptide (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 8. In the amino acid sequence of SEQ ID NO: 8, the underlined portion corresponds to the region from 10 to 24 in the aforementioned SEQ ID NO: 6. Since the amino acid sequence of SEQ ID NO: 8 corresponds to the region from 70 to 95 in the aforementioned full-length Syn amino acid sequence, it is hereinafter also referred to as Syn70-95. VVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 8)

[0064] In the aggregating agent of the present invention, the aggregating peptide (C) may be, for example, one type or two or more types. Among them, the aggregating peptide (C) is preferably the peptide (C1), and the aggregating agent may contain, for example, one type or two or more types of the peptide (C1).

[0065] The flocculant of the present invention may contain only the aggregating peptide (C), or may further contain other additives. Examples of the additives include aqueous solvents, such as water, physiological saline, and buffer solutions. When the flocculant of the present invention is used in an in vitro or in vivo reaction system, as described above, it is preferably a liquid flocculant containing the aggregating peptide (C) and the aqueous solvent. Furthermore, the flocculant of the present invention may be, for example, a ready-to-use agent that is prepared into a liquid containing the aggregating peptide (C) and the aqueous solvent at the time of use.

[0066] When the aggregating agent of the present invention is a liquid aggregating agent in which the aggregating peptide (C) is the aggregating peptide (C), the concentration of the aggregating peptide (C) is not particularly limited, and the lower limit is, for example, 1 μmol / L, 5 μmol / L, 0.01 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 1 mmol / L, or 2 mmol / L, and the upper limit is, for example, 10 mmol / L or 5 mmol / L, and the range is, for example, 1 μmol / L to 10 mmol / L, 0.05 to 10 mmol / L, 0.05 to 5 mmol / L, 1 to 5 mmol / L, or 2 to 5 mmol / L.

[0067] The method for using the aggregating agent of the present invention is not particularly limited, and it can be used, for example, in the methods for evaluating aggregation-inhibiting ability, screening methods, and methods for producing model animals, which will be described later.

[0068] (2-2) Evaluation method for aggregation inhibition ability The evaluation method of the present invention is characterized in that it includes a detection step of causing the agglutinant of the present invention or an aggregate of the agglutinant to coexist with a test substance and detecting the aggregation of the agglutinant, and evaluating the aggregation inhibition ability of the test substance based on the degree of aggregation of the agglutinant in the presence of the test substance.

[0069] As described above, the aggregating peptide (C) in the aggregating agent of the present invention exhibits aggregating properties similar to full-length Syn, and, unlike full-length Syn, is soluble in aqueous solvents. Therefore, for example, the aggregating peptide (C) or its aggregates dissolved in an aqueous solvent can be coexisted with the test substance in vivo or in vitro. Then, 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. Furthermore, since the aggregating peptide (C) has aggregating properties similar to full-length Syn, according to the present invention, for example, the aggregation-inhibiting ability of the aggregating peptide (C) can be directly evaluated, and the aggregation-inhibiting ability of the test substance can be indirectly evaluated.

[0070] In the evaluation method of the present invention, the flocculant is preferably used as a liquid flocculant dissolved in an aqueous solvent, as described above.

[0071] According to the evaluation method of the present invention, the aggregation-inhibiting ability can be evaluated, for example, by the ability to inhibit the formation of aggregates, and also by the ability to dissociate already formed aggregates. The former is exemplified as Form 1, and the latter as Form 2, as follows.

[0072] In the case of Form 1, the evaluation method of the present invention allows the aggregating agent and the test substance to coexist and enables the detection of aggregate formation from the aggregating agent in the detection step. In this case, for example, the aggregation-inhibiting ability of the test substance, i.e., the ability to inhibit the formation of aggregates of the aggregating peptide (C), can be evaluated based on the degree of aggregate formation in the presence of the test substance.

[0073] The degree of aggregate formation can be determined, for example, by comparing with an evaluation standard. The evaluation standard is, for example, the amount of aggregates formed from the agglutinant in the absence of the test substance (P ０ The amount of aggregates produced by the agglutinant in the presence of the test substance (P １ ) is the amount of evaluation criteria generated (P ０ ), the test substance can be said to have the ability to inhibit aggregation of the aggregating agent, i.e., the aggregating peptide (C), and can also be said to have the ability to inhibit aggregation of full-length Syn. ０ ) compared to the amount of production (P １ ) is smaller, the aggregation-inhibiting ability is higher.

[0074] In the detection step, the method for causing the agglutinating agent and the test substance to coexist is not particularly limited, and may be, for example, in vitro or in vivo.

[0075] When the coexistence is in vitro, the coexistence may be in a cell-free reaction system in a container such as a well or a tube, or in a reaction system containing cells or tissues. The types of cells and tissues are not particularly limited, and they may be derived from, for example, humans or non-human animals. When the coexistence is in vitro, for example, the final concentration of the aggregating peptide (C) in the reaction system is set to 0.01 to 0.1 mmol / L (e.g., 0.1 mmol / L), and the final concentration of the test substance is set to 0.1 μmol / L to 100 μmol / L. It is preferable that the aggregating peptide (C) is added as an aggregating agent in the liquid dissolved in an aqueous solvent, and the test substance is added as a test liquid dissolved in a solvent such as an aqueous solvent.

[0076] When the coexistence is in vivo, for example, the coexistence may be achieved by administering the aggregating agent and the test substance to an animal. The animal is not particularly limited, and examples thereof include non-human animals. Examples of the non-human animal include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels. When the coexistence is in vivo, for example, the aggregating agent and the test substance are administered to the non-human animal. For example, the aggregating agent is administered as a solution (liquid aggregating agent) obtained by dissolving the aggregating peptide (C) in an aqueous solvent. The concentration of the aggregating peptide (C) in the solution is, for example, 0.1 to 1 mmol / L, or 0.5 to 1 mmol / L. The administration conditions for the aggregating agent are not particularly limited, and for example, in the case of a 30-35 g mouse, 1 to 7 μL (e.g., 4 μL) of the solution is administered at a rate of 1 to 2 μL / min (e.g., 1 μL / min) per administration. The amount administered per administration is, for example, 2 to 4 nmol, and the number of administrations of the aggregating agent is not particularly limited, and may be, for example, once. The test substance is preferably added as a test solution dissolved in a solvent such as an aqueous solvent, and the amount of the test substance administered is not particularly limited. Then, for example, the aggregating peptide and the test subject are administered, and after leaving for 1 to 2 weeks, it is possible to confirm whether the aggregating peptide has aggregated.

[0077] In the case of Form 2, the evaluation method of the present invention allows the coexistence of aggregates of the aggregating agent (i.e., aggregates of the aggregating peptide (C)) with the test substance in the detection step, and enables the detection of a decrease in the aggregates. In this case, for example, the aggregation-inhibiting ability of the test substance, i.e., the dissociation ability of the aggregating peptide (C) to dissociate aggregates, can be evaluated based on the degree of decrease in the aggregates in the presence of the test substance.

[0078] The degree of reduction of the aggregates can be determined, for example, by comparison with an evaluation standard. The evaluation standard can be, for example, the amount of the aggregates in the absence of the test substance (Q ０ The amount of the aggregate in the presence of the test substance (Q １ ) is the amount of evaluation criteria generated (Q ０), the test substance can be said to have the ability to dissociate from the aggregates of the aggregating agent, i.e., the aggregates of the aggregating peptide (C), and can be said to have the ability to dissociate from the aggregates of full-length Syn. ０ ) compared to the amount of aggregates (Q １ ) the lower the deviation ability.

[0079] In the detection step, the method for causing the aggregates of the agglutinating agent and the test substance to coexist is not particularly limited, and may be, for example, in vitro or in vivo.

[0080] When the coexistence is in vitro, the coexistence may be in a cell-free reaction system using a container such as a well or a tube, or in a reaction system containing cells or tissues. The types of cells and tissues are not particularly limited, and they may be derived from, for example, humans or non-human animals. A specific method for coexisting the aggregates of the aggregating agent and the test substance includes, for example, first dissolving the aggregating peptide (C) in an aqueous solvent in the absence of the test substance, and incubating this solution (liquid aggregating agent) to generate aggregates of the aggregating peptide (C). The concentration of the aggregating peptide (C) in the solution is, for example, 0.02 to 1 mmol / L (e.g., 1 mmol / L). The incubation conditions are not particularly limited, and the temperature is, for example, 30 to 38°C (e.g., 37°C), and the incubation time is, for example, 1 to 24 hours. The aggregates and the test substance are then added to a reaction system containing the cells or tissues to allow them to coexist. 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), and the final concentration of the test substance is, for example, 0.1 μmol / L to 100 μmol / L.

[0081] When the coexistence occurs in vivo, for example, the aggregating agent, i.e., the aggregating peptide (C), is first administered to an animal and left for 1-2 weeks to generate the aggregates in vivo, and then the test substance is administered, thereby allowing the two to coexist. The aggregating agent is, for example, administered as a solution (liquid aggregating agent) by dissolving the aggregating peptide (C) in an aqueous solvent. The concentration of the aggregating peptide (C) in the solution is, for example, 0.1-1 mmol / L or 0.5-1 mmol / L. The administration conditions are not particularly limited. For example, in the case of a 30-35 g mouse, 1-7 μL (e.g., 4 μL) of the solution is administered at a rate of 1-2 μL / min (e.g., 1 μL / min) per administration. The dose per administration is, for example, 2-4 nmol, and the number of administrations of the aggregating agent is not particularly limited, and may be one. The administration site of the agglutinating 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 administration via nasal administration or the like. The animal is not particularly limited, and examples thereof include non-human animals. Examples of the non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels.

[0082] (2-3) Screening method for aggregation inhibitors The screening method for aggregation inhibitors of the present invention is characterized by comprising a detection step of detecting aggregation of the agglutinant in the presence of the agglutinant of the present invention or an aggregate of the agglutinant and a test substance, and a selection step of selecting the test substance as an aggregation inhibitor when aggregation of the agglutinant is inhibited. Unless otherwise specified, the description of the evaluation method in (2-2) above can be used for the screening method of the present invention.

[0083] The screening method of the present invention uses the flocculant of the present invention or an aggregate of the flocculant, and as described in the evaluation method (2-2) above, can evaluate the aggregation inhibitory ability of the test substance, and therefore can screen for an aggregation inhibitor that inhibits the aggregation of the flocculant from the test substance.

[0084] In the screening method of the present invention, the agglutinant is preferably used as a liquid agglutinant dissolved in an aqueous solvent, as described above.

[0085] According to the screening method of the present invention, it is possible to screen for, as the aggregation inhibitor, a test substance that inhibits the formation of aggregates (also referred to as an aggregate formation inhibitor), and it is also possible to screen for a test substance that dissociates already formed aggregates (also referred to as an agglutination dissociation substance). The former is exemplified as Form 1, and the latter as Form 2, as shown below.

[0086] In the case of Form 1, the screening method of the present invention allows the agglutinant and the test substance to coexist in the detection step, and can detect the formation of aggregates from the agglutinant. If the formation of aggregates is inhibited in the presence of the test substance, the test substance can be selected in the selection step as an aggregation inhibitor (aggregate formation inhibitor) that inhibits the formation of aggregates, i.e., the formation of aggregates of the aggregating peptide (C).

[0087] The inhibition of aggregate formation can be determined, for example, by comparing with an evaluation criterion. The evaluation criterion is, for example, the amount of aggregates formed from the agglutinant in the absence of the test substance (P ０ The amount of aggregates produced by the agglutinant in the presence of the test substance (P １ ) is the amount of evaluation criteria generated (P ０ ), the test substance can be selected as an aggregate formation inhibitor that inhibits the aggregation of the aggregating agent, i.e., an aggregate formation inhibitor that inhibits the formation of aggregates from the aggregating peptide (C), or an aggregate formation inhibitor that inhibits the formation of aggregates from full-length Syn. ０ ) compared to the amount of production (P １ ) is lower, the test substance has higher aggregation inhibitory ability.

[0088] In the detection step, the method for coexistence of the agglutinant and the test substance is not particularly limited, and may be, for example, in vitro or in vivo, and the description of form 1 in the evaluation method (2-2) above can be cited.

[0089] In the case of Form 2, the screening method of the present invention allows the coexistence of aggregates of the aggregating agent (i.e., aggregates of the aggregating peptide (C)) with the test substance in the detection step, and is able to detect a decrease in the aggregates. Then, if the aggregates decrease in the presence of the test substance, the test substance can be selected in the selection step as an aggregation inhibitor (aggregate dissociation agent) that dissociates the aggregates, i.e., dissociates aggregates of the aggregating peptide (C).

[0090] The degree of reduction of the aggregates can be determined, for example, by comparison with an evaluation standard. The evaluation standard can be, for example, the amount of the aggregates in the absence of the test substance (Q ０ The amount of the aggregate in the presence of the test substance (Q １ ) is the amount of evaluation criteria generated (Q ０ ), the test substance can be selected as an aggregate-dissociating substance that dissociates aggregates of the aggregating agent, i.e., aggregates of the aggregating peptide (C). ０ ) compared to the amount of aggregates (Q １ The smaller the difference, the higher the dissociation ability of the test substance.

[0091] In the detection step, the method for coexistence of the agglutinant aggregates and the test substance is not particularly limited, and may be, for example, in vitro or in vivo, and the description of form 2 in the evaluation method of (2-2) above can be used.

[0092] According to the screening method of the present invention, it is possible to directly screen for aggregation inhibitors (aggregate formation inhibitors or aggregate dissociation agents) for the aggregating peptide (C), and indirectly screen for aggregation inhibitors for full-length Syn. Since aggregation of full-length Syn causes Syn aggregation diseases such as the aforementioned Parkinson's disease, the screening method of the present invention ultimately makes it possible to screen for drugs for Syn aggregation diseases.

[0093] Therefore, the present invention further includes a method for screening drugs for Syn aggregation diseases, comprising selecting an aggregation inhibitor selected by the above-mentioned method for screening aggregation inhibitors of the present invention as a drug for Syn aggregation diseases. The same description as for the above-mentioned method for screening aggregating agents of the present invention can be applied to this method for screening drugs for Syn aggregation diseases of the present invention.

[0094] (2-4) Method for Producing Model Animals The method for producing a model animal for Syn aggregation disease of the present invention is characterized by administering the aggregating agent of the present invention into the brain of a non-human animal.

[0095] As described above, full-length Syn is poorly soluble in aqueous solvents, and therefore, it was not possible to produce a model animal in which Syn aggregates in the brain by administering it to the animal's brain. On the other hand, the aggregating peptide (C) in the aggregating agent of the present invention is soluble in aqueous solvents. Therefore, it is possible to cause aggregation in the brain by administering a liquid aggregating agent prepared by dissolving the aggregating peptide (C) in an aqueous solvent to an animal. Therefore, the present invention provides a method for producing an aggregation model animal of the aggregating peptide (C). Furthermore, since the aggregating peptide (C) acts as a substitute for full-length Syn and causes full-length Syn-like aggregation, it can be said that the production method of the present invention can produce a Syn aggregation model animal. Furthermore, aggregation of the aggregating peptide (C) in the brain of the model animal results in symptoms of Syn aggregation diseases such as Parkinson's disease (e.g., short-term cognitive decline), which are caused by the aggregation of full-length Syn. Therefore, it can be said that the production method of the present invention can produce a Syn aggregation disease model animal.

[0096] The method of administering the agglomerating agent of the present invention to the non-human animal is not particularly limited, and the in vivo administration method in the evaluation method in (2-2) above can be used.

[0097] Examples of the non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cattle, and camels.

[0098] As described above, the aggregating agent of the present invention is preferably administered to the non-human animal as a solution (liquid aggregating agent) dissolved in an aqueous solvent. In the liquid aggregating agent, the concentration of the aggregating peptide (C) is preferably, for example, 0.1 to 1 mmol / L. Specifically, in the case of Syn61-84, the concentration is preferably, for example, 0.5 to 1 mmol / L, and in the case of Syn70-95, the concentration is preferably, for example, 0.5 to 1 mmol / L.

[0099] The administration site is, for example, the brain. The aggregating agent of the present invention may be administered directly to the brain using, for example, a syringe, or indirectly via intranasal administration. The dose of the aggregating agent of the present invention is not particularly limited and can be appropriately determined depending on the type, age, and the like of the non-human animal to be administered. When the non-human animal is a mouse weighing 30-35 g, for example, 1 to 7 μL (e.g., 4 μL) of the solution is administered at a rate of 1 to 2 μL / min (e.g., 1 μL / min) per administration. The dose per administration is, for example, 2 to 4 nmol, and the number of administrations of the aggregating agent is not particularly limited and is one administration. When the model animal is used, for example, to confirm the inhibition of aggregate formation, for example, a test substance may be administered simultaneously with the solution. When the model animal is used, for example, to confirm the dissociation of the generated aggregates, for example, the test substance may be administered after leaving the animal for a certain period of time (e.g., 1 to 2 weeks) following the addition of the solution.

[0100] [Example A1] Syn aggregation is known to be one of the causes of Parkinson's disease. Therefore, it was confirmed that the aggregating peptide (C) aggregates in a similar manner as a Syn substitute, and that administration of the aggregating peptide (C) to mice induces symptoms of Parkinson's disease.

[0101] (1) Peptides The following four types of peptides were used as Syn FPs (fragment peptides of Syn). Syn61-84 (SEQ ID NO: 7) and Syn70-95 (SEQ ID NO: 8) are aggregating peptides (C) of the example, and the peptide consisting of the underlined region of Syn61-84 (SEQ ID NO: 9, Syn70-84) and the peptide of SEQ ID NO: 10 (Syn61-69) consisting of the region of Syn61-84 other than the underlined region were peptides of comparative examples.

[0102] Example peptide Syn61-84 EQVTNVGGAVVTGVTAVAQKTVEG (SEQ ID NO: 7) Syn70-95 VVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 8) Comparative example peptide Syn70-84 VVTGVTAVAQKTVEG (SEQ ID NO: 9) Syn61-69 EQVTNVGGA (SEQ ID NO: 10)

[0103] Then, for each of the peptides of the Examples (Syn FP) and the Comparative Examples, 1 mmol / L peptide solutions were prepared using MilliQ water as the aqueous solvent. Both the 1 mmol / L Syn FP solution obtained by dissolving the Syn FP of the Examples in water and the 1 mmol / L peptide solution obtained by dissolving the peptide of the Comparative Examples in water were transparent solutions at room temperature, with no visible precipitates. No precipitates formed even after prolonged storage at room temperature after preparation. On the other hand, when full-length Syn was mixed with MilliQ water at the same concentration (1 mmol / L), precipitation of Syn was immediately visible upon standing at room temperature after mixing. The same results were obtained when physiological saline, buffer solutions, etc. were used as the aqueous solvent in addition to water.

[0104] (2) Confirmation of aggregation in vitro The fluorescent dye thioflavin T (ThT) binds to aggregates and emits strong fluorescence upon binding, so the presence or absence of aggregation can be determined by measuring the fluorescence intensity. Therefore, aggregation of the peptides of the above examples and the above comparative examples was confirmed using ThT.

[0105] Specifically, a reaction solution with the following composition was prepared and dispensed into wells so that 100 μL of the reaction solution was distributed per well. For the comparative example, the comparative peptide solution was used, in which the comparative peptide was dissolved, instead of the 1 mmol / L Syn FP solution. At room temperature, all of the aggregating peptides were dissolved in the reaction solution, and no precipitates were observed. The reaction solution was then incubated at 37°C for 24 hours. 5 μL of 2 mmol / L ThT was added per well, and the fluorescence intensity was measured using a Cytation 5 (BioTek) measurement device (n=3). The measurement wavelengths were an excitation wavelength (ex) of 444 nm and an emission wavelength (em) of 480 nm.

[0106]

[0107] These results are shown in Figure 1. Figure 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. In Figure 1, the vertical axis represents fluorescence intensity, and the units are Fluorescence Intensity (FI). As shown in Figure 1, when the peptide Syn70-84 of the Comparative Examples, which has a sequence common to Syn61-84 and Syn70-95, or the peptide Syn61-69 of the Comparative Examples, which has a sequence partially common to Syn61-84, was added and incubated at 37°C, almost no aggregation was observed. In contrast, when the peptides Syn61-84 and Syn70-95 of the Examples were incubated at 37°C, the fluorescence intensity of each peptide increased significantly, confirming the formation of aggregates.

[0108] (3) Confirmation of short-term cognitive function in mice The aggregating peptide (C) was administered to the brain of a mouse, and it was confirmed that a decline in short-term cognitive function similar to that seen in Parkinson's disease, which is caused by aggregation of Syn, occurred.

[0109] The aggregating peptides (C) used in the examples were Syn61-84 and Syn70-95, and the peptide used in the comparative examples was Syn70-84. 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, no precipitation of each peptide was visually observed at room temperature.

[0110] For each peptide, an administration group (n=3) of ICR mice (7 weeks old) was prepared. 4 μL of the sample solution was directly administered into the cerebral ventricles of the mice using a microsyringe. Then, for each administration group, a Y-maze (short-term memory evaluation) test was performed 14 days after administration.

[0111] The Y-maze test was performed using a standard method under the following conditions. Specifically, a mouse was placed with its nose facing the wall at the end of one of the three arms. The mouse was then allowed to move freely for 10 minutes, and its behavior was recorded on the monitor of an analyzer (product name Time YM1, manufactured by Ohara Medical Industries), and the alternation rate (%) was calculated.

[0112] First, the results of the Y-maze test are shown in Figure 2. Figure 2 shows the results of the alternation rate (%). Compared to the mice administered with the comparative peptide Syn61-69, in which no aggregation was confirmed in Example A1, the mice administered with the peptides Syn61-84 and Syn70-95 of the examples all had a reduced alternation rate (%).

[0113] As described above, it was found that the aggregating peptides Syn61-84 and Syn70-95 of the Examples cause aggregation in a manner similar to that of full-length Syn, and that administration of these peptides to the brains of mice causes short-term cognitive decline, a symptom of Parkinson's disease. This demonstrates that the aggregating peptides can be used as Syn substitutes to construct a reaction system related to Syn aggregation, and that administration of these peptides to animals such as mice makes it possible to produce model animals for Syn aggregation.

[0114] Example A2 Syn61-84 (SEQ ID NO: 7) from Example A1 was dissolved in MilliQ water as an aqueous solvent at room temperature to prepare Syn FP solutions of various concentrations, as in Example A1. In vitro aggregation was confirmed in the same manner as in Example A1(2), except that the final concentrations of Syn61-84 in the reaction solutions were 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L (0.05 mmol / L), 100 μmol / L (0.1 mmol / L), and 200 μmol / L (0.2 mmol / L). A control (blank) was prepared using a reaction solution to which MilliQ water had been added instead of the Syn FP solution.

[0115] 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.

[0116] These results are shown in Figure 11. Figure 11 is a graph showing the fluorescence intensity of the reaction solution to which the peptide (Syn61-84) of the above example was added. In Figure 11, the vertical axis represents fluorescence intensity, and the unit is Fluorescence Intensity (FI). As shown in Figure 11, in all reaction solutions containing Syn61-84, incubation at 37°C confirmed a fluorescence intensity that showed a significant difference compared to the control. In particular, significantly higher fluorescence intensities compared to the control were confirmed in reaction solutions with final concentrations of 50 µmol / L (0.05 mmol / L) or higher.

[0117] [Example B1] The Syn aggregation-inhibitory ability of the aggregation-inhibitory peptides YS and LYZA-3 was confirmed.

[0118] As aggregation-inhibitory peptides in the examples, 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.

[0119] The fluorescent dye thioflavin T (ThT) binds to aggregates and emits strong fluorescence upon binding, allowing the increase or inhibition of aggregation to be determined by measuring the fluorescence intensity. Therefore, the inhibition of Syn aggregation by each peptide was confirmed using ThT. In confirming the inhibition of aggregation, the Syn61-84 (SEQ ID NO: 7), a fragment peptide of Syn, was used as a substitute for full-length Syn. It has been confirmed in Example A1 that this aggregating peptide Syn61-84 aggregates in the same way as full-length Syn.

[0120] Specifically, a reaction solution with the following composition was prepared and dispensed into wells so that 100 μL of the reaction solution was distributed per well. In the following composition, 1 mg / mL peptides (aggregation-inhibiting peptide solutions: YS-16, YS-11, YS-6, and LYZA-3) were prepared using MilliQ water, and 1 mmol / L Syn61-84 (aggregating peptide solution) was prepared using MilliQ water as the aqueous solvent. After incubating the reaction solution at 37°C for 24 hours, 5 μL of 2 mmol / L ThT (reagent name: Thioflavine T, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added per well, and the fluorescence intensity was measured using a Cytation 5 measuring device (manufactured by BioTek) (n=3). The measurement wavelengths were an excitation wavelength (ex) of 444 nm and an emission wavelength (em) of 480 nm. As a control, a reaction solution to which water was added instead of the aggregation-inhibiting peptides (YS-16, YS-11, YS-6, LYZA-3) was also subjected to similar measurements.

[0121]

[0122] These results are shown in Figure 3. Figure 3 is a graph showing the fluorescence intensity of the reaction solution to which the aggregation-inhibitory peptide was added. In Figure 3, the vertical axis represents the relative fluorescence intensity (FI), specifically, the relative value when the fluorescence intensity of the control (Syn61-84) was set to 1. As shown in Figure 3, each aggregation-inhibitory peptide significantly reduced the fluorescence intensity compared to the control (Syn61-84) to which no aggregation-inhibitory peptide was added. These results demonstrate that peptide YS and peptide LYZA-3 can inhibit Syn aggregation.

[0123] [Example B2] The aggregation-inhibiting peptides YS-11 and LYZA-3 were examined for their aggregation-inhibiting ability, as evidenced by their inhibition of aggregate formation.

[0124] Specifically, a reaction solution with the following composition was prepared and dispensed into wells so that 200 μL of the reaction solution was dispensed per well. In the following composition, X mmol / L peptide (aggregation-inhibiting peptide solution: YS-11, LYZA-3) was prepared using MilliQ water, and the X mmol / L concentration in the reaction solution was set to 1 μmol / L, 10 μmol / L, or 100 μmol / L. In addition, in the following composition, 1 mmol / L Syn61-84 (aggregating peptide solution) was prepared using MilliQ water as the aqueous solvent. After incubating the reaction solution at 37°C for 24 hours, 10 μL of 2 mmol / L ThT was added per well, and the fluorescence intensity was measured using a Cytation 5 (trade name, manufactured by BioTek) (n=3). The measurement wavelengths were an excitation wavelength (ex) of 444 nm and an emission wavelength (em) of 480 nm. As a control, a reaction solution to which water was added instead of the aggregation-inhibiting peptides (YS-11 and LYZA-3) was also subjected to similar measurements.

[0125]

[0126] These results are shown in Figure 4. In Figure 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. In Figure 4, the vertical axis represents fluorescence intensity, and the units are fluorescence intensity (FI). As shown in Figures 4(A) and (B), when either peptide LYZA-3 or peptide YS-11 was used, the fluorescence intensity decreased in a concentration-dependent manner compared to the control (Syn61-84). These results confirmed that both peptide LYZA-3 and peptide YS-11 inhibit the formation of Syn aggregates, i.e., inhibit Syn aggregation. Furthermore, a comparison of Figures 4(A) and (B) confirmed that peptide LYZA-3 had a stronger aggregation-inhibiting ability than peptide YS-11 when used at the same concentration.

[0127] [Example B3] For the aggregation-inhibitory peptides YS-11 and LYZA-3, dissociation of aggregates was examined as an indicator of the aggregation-inhibitory ability.

[0128] Specifically, the aggregating peptide Syn61-84 was first dissolved in PBS buffer to a final concentration of 1 mmol / L. The solution was then incubated at 37°C for 24 hours to prepare a solution containing Syn61-84 aggregates formed by aggregation of the aggregating peptide (final concentration of Syn61-84 aggregates: 1 mmol / L). A reaction solution having the following composition was then prepared, and 200 μL was dispensed per well. After incubating the reaction solution at 37°C for 24 hours, 10 μL of 2 mmol / L ThT was added per well, and the fluorescence intensity was measured using a measuring device (product name: Cytation 5, manufactured by BioTek) (n=3). The measurement wavelengths were an excitation wavelength (ex) of 444 nm and an emission wavelength (em) of 480 nm. In the following composition, X mmol / L peptides (aggregation-inhibitory peptides: YS-11, LYZA-3) were prepared using MilliQ water, and the concentrations X mmol / L in the reaction solution were set to 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L. As a control, a reaction solution to which water was added instead of the peptide YS-11 and peptide LYZA-3 was also similarly measured.

[0129]

[0130] These results are shown in Figure 5. In Figure 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. In Figure 5, the vertical axis represents fluorescence intensity, and the units are fluorescence intensity (FI). As shown in Figures 5(A) and (B), when either peptide LYZA-3 or peptide YS-11 was used, the fluorescence intensity decreased compared to the control (Syn61-84). In particular, when peptide YS-11 was used, the fluorescence intensity decreased in a concentration-dependent manner, as shown in Figure 5(B). These results confirmed that both peptide LYZA-3 and peptide YS-11 dissociate Syn aggregates, i.e., inhibit Syn aggregation. Furthermore, by comparing Figures 5(A) and (B), it was confirmed that when used at the same concentration, peptide YS-11 had a stronger aggregation inhibitory ability than peptide YS-11.

[0131] [Example B4] Parkinson's disease model mice were generated using the aggregating peptide Syn61-84, and the aggregation-inhibiting peptides YS-11 or LYZA-3 were further administered to confirm the effects.

[0132] The aggregating peptide Syn61-84 was dissolved in physiological saline to prepare a 1 mmol / L solution. Physiological saline (saline) was used as a control. Then, 4 μL of the Syn61-84 solution or 4 μL of saline was administered intracerebroventricularly to ICR mice (7 weeks old) using a microsyringe (Day 0). The Syn61-84 administration group consisted of n=4, and the saline administration group consisted of n=3.

[0133] The aggregation-inhibitory peptide (YS-11 or LYZA-3) was dissolved in physiological saline to prepare a 1 mmol / L solution. Then, on day 56, 4 μL of 1 mmol / L YS-11 was administered to n=2 of the Syn61-84 administration group (n=4) in the same manner (YS-11 administration group), and 4 μL of 1 mmol / L LYZA-3 was administered to the remaining n=2 (LYZA-3 administration group). Note that the aggregation-inhibitory peptide was not administered to the saline administration group (n=3).

[0134] On Day 14, Day 56, and Day 74, short-term memory was assessed using the Y-maze, and on Day 21, Day 55, Day 63, and Day 70, motor function was assessed using the Rota-Rod. The Y-maze was performed in the same manner as in Example A1. The Rota-Rod was performed using a rotarod device for mice (product name MK-670, Muromachi Kikai Co., Ltd.), with the rotor speed set to 10 rpm or 20 rpm, and the time it took for the mouse to fall off the rotating rotor was measured.

[0135] FIG. 6 shows the results of the alternation rate (%) calculated using Y-maze. First, in the first Y-maze session (Y-maze-1) on Day 14 and the second Y-maze session (Y-maze-2) on Day 56, a decrease in the alternation rate was confirmed in the Syn61-84 administration group compared to the saline administration group. These results demonstrated that the administration of Syn61-84 resulted in a decline in cognitive function. Then, in the third Y-maze session (Y-maze-3) on Day 74, which was performed after the administration of the aggregation-inhibitory peptide on Day 56, the alternation rate increased in both the YS-11 administration group and the LYZA-3 administration group compared to the saline administration group. These results demonstrated that the administration of the aggregation-inhibitory peptide improved the decline in cognitive function.

[0136] Figure 7 shows the results of the Rota-Rod test. In Figure 7, 10 rpm and 20 rpm represent the rotor rotation speed, and the vertical axis represents the time until the mouse falls from the rotor. The longer the time until the mouse falls, the higher the motor ability can be evaluated, and the shorter the time, the lower the motor ability can be evaluated. As shown in Figure 7, first, in the first Rota-Rod test (RR-1) on Day 21 and the second Rota-Rod test (RR-2) on Day 55, the Syn61-84-administered group showed a significant decrease in the time spent on the rotor compared to the Saline-administered group. These results revealed that administration of Syn61-84 caused motor dysfunction. Then, on the third and fourth Rota-Rod tests (RR-3, RR-4) on Day 63, which were performed after administration of the aggregation-inhibitory peptide on Day 56, the saline-administered group showed a decrease in residence time, whereas both the YS-11-administered group and the LYZA-3-administered group showed an increase in residence time. In particular, in the 10 rpm test, the LYZA-3-administered group showed a significant increase, and in RR-4, the residence time recovered to a level exceeding that of the saline-administered group. These results demonstrated that administration of the aggregation-inhibitory peptide improved motor dysfunction.

[0137] [Example B5] The aggregating peptide Syn61-84 and the aggregation-inhibiting peptide YS-11 or LYZA-3 were simultaneously administered to mice, and the effects were confirmed.

[0138] The aggregating peptide Syn61-84 was dissolved in physiological saline to prepare a 0.5 mmol / L solution. The aggregating peptide Syn61-84 and the aggregation-inhibiting peptide (YS-11 or LYZA-3) were dissolved in physiological saline to prepare a mixed solution with each having a concentration of 0.5 mmol / L. Then, 4 μL of the Syn61-84 solution or 4 μL of the mixed solution was administered intracerebroventricularly to ICR mice (7 weeks old) using a microsyringe at a rate of 1 μL / min (Day 0). As a control, 4 μL of physiological saline was administered at the same rate as the aggregating peptide or the aggregation-inhibiting peptide (Day 0). The Syn61-84 administration group and the mixed solution administration group each had an n=4 group, and the saline administration group had an n=3 group.

[0139] Then, on Day 14 and Day 74, short-term memory was evaluated using the Y-maze, and motor function was evaluated using the Rota-Rod on Day 21, Day 55, Day 63, and Day 70. The Y-maze was performed using the same method as in Example A1. The Rota-Rod was performed using the same method as in Example B4.

[0140] FIG. 8 shows the results of the alternation rate (%) calculated by Y-maze. First, in the first Y-maze (Y-maze-1) on Day 14, a decrease in the alternation rate was confirmed in the Syn61-84 administration group compared to the saline administration group. These results demonstrated that administration of the aggregating peptide Syn61-84 decreased cognitive function. In contrast, the alternation rates of the groups administered a mixed solution in which the aggregating peptide was added simultaneously with the aggregating peptide (Syn61-84 / LYZA-3 and Syn61-84 / YS-11) were higher than those of the Syn61-84 administration group and were similar to or higher than those of the saline administration group. Furthermore, during the third Y-maze (Y-maze-3) on Day 74, the Syn61-84 administration group showed a lower alternation rate compared to the saline administration group. However, the alternation rates of the mixed solution administration groups (Syn61-84 / LYZA-3 and Syn61-84 / YS-11) were higher than those of the Syn61-84 administration group and were similar to or higher than those of the saline administration group. In particular, the Syn61-84 / LYZA-3 administration group showed a higher value than the Syn61-84 administration group and the saline administration group. These results demonstrated that the simultaneous administration of the aggregation-inhibitory peptide with Syn61-84 prevented cognitive decline.

[0141] Figure 9 shows the results of the Rota-Rod test. In Figure 9, 10 rpm and 20 rpm represent the rotor rotation speed, and the vertical axis represents the time it took for the mouse to fall off the rotor. A longer time to fall indicates a higher motor ability, while a shorter time indicates a lower motor ability. As shown in Figure 9, in the first Rota-Rod test (RR-1) on Day 21, the Syn61-84-administered group showed a significant decrease in the time spent on the rotor compared to the saline-administered group. This result revealed that administration of Syn61-84 caused motor dysfunction. Thereafter, the Syn61-84-administered group showed a slight increase in the time spent on the rotor, but did not recover to the time spent on the rotor of the saline-administered group. In contrast, the mixed solution-administered groups (Syn61-84 / LYZA-3 and Syn61-84 / YS-11) both showed lower residence times than the saline-administered group in RR-1, but showed significantly longer residence times than the Syn61-84-administered group, and subsequently showed an increase rather than a decrease in residence time. In RR-4, they showed a longer residence time than the saline-administered group. In particular, the Syn61-84 / LYZA-3-administered group showed a significantly longer residence time than the saline-administered group. These results demonstrate that the simultaneous administration of the aggregation-inhibitory peptide with Syn61-84 prevented the deterioration of motor dysfunction.

[0142] [Example B6] The effect of aggregating peptides on cells and the inhibition of cell death by the aggregation-inhibiting peptide YS-11 were confirmed.

[0143] The aggregating peptide Syn61-84 was dissolved in PBS buffer to a concentration of 1 mmol / L. The aggregation-inhibitory peptide YS-11 was dissolved in PBS to a predetermined concentration (0.001 to 1 mmol / L). These were then used to prepare a reaction solution with the following composition. This reaction solution was incubated at 37°C for 24 hours. As a control, PBS incubated in the same manner was used instead of the reaction solution.

[0144]

[0145] On the other hand, 0.15 mL of medium was placed in each well of a 96-well plate, and mouse neuroblastoma (Neuro2a) cells were added at a rate of 2.66 × 10 cells per well. ４ After culturing at 37°C for 24 hours, 50 μL of the reaction solution after incubation was added per well, and the cells were further cultured at 37°C for 24 hours. The cell viability (%) of each well was then measured by the WST-8 method.

[0146] These results are shown in Figure 10. In Figure 10, the vertical axis represents viability (%), and the values ​​for YS-11 and Syn61-84 in the figure represent the final concentrations (µmol / L) in the wells. As a result, when the viability of the control, to which neither the aggregating peptide nor the aggregation-inhibitory peptide was added, was taken as 100%, the viability of the system to which the reaction solution containing only the aggregating peptide Syn61-84 was added after incubation (YS-11 0 µmol / L) was significantly reduced. In contrast, a concentration-dependent increase in viability was confirmed in the system to which the reaction solution containing the aggregating peptide Syn61-84 and the aggregation-inhibitory peptide YS-11 was added after incubation. The reaction solution containing no aggregation-inhibitory peptide caused cell death due to the formation of aggregates upon incubation, but the reaction solution containing the aggregation-inhibitory peptide further suppressed aggregate formation even upon incubation. Consequently, it is believed that cell death was suppressed even when added to the cell culture medium. Note that the reaction solution containing only the aggregation-inhibitory peptide did not affect cell viability.

[0147] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0148] This application claims priority based on Japanese Patent Application No. 2021-80512, filed on May 11, 2021, the disclosure of which is incorporated herein in its entirety.

[0149] The Syn aggregation inhibitor of the present invention can inhibit aggregation due to intermolecular association of Syn, thereby making it possible to treat, for example, prevent, inhibit the progression, or improve Syn aggregation diseases caused by Syn aggregation, such as Parkinson's disease.

Claims

1. 1. An agglutinating agent comprising a peptide represented by (C1) or (C2) below: (C1) A peptide comprising an amino acid sequence of 16 to 26 consecutive amino acid residues including the 10th to 24th regions in the amino acid sequence of SEQ ID NO:

6. EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 6) (C2) A peptide consisting of the amino acid sequence of (C1) above in which 1 to 2 amino acids have been deleted, substituted or added.

2. The aggregating agent according to claim 1 , wherein the peptide (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO:

7. EQVTNVGGAVVTGVTAVAQKTVEG (SEQ ID NO: 7)

3. The aggregating agent according to claim 1 , wherein the peptide (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO:

8. VVTGVTAVAQKTVEGAGSIAAATGFV (SEQ ID NO: 8)

4. The method includes a detection step of detecting the agglutination of the agglutinant according to any one of claims 1 to 3, in which the agglutinant or an aggregate of the agglutinant is allowed to coexist with a test substance, and the agglutination of the agglutinant is detected, A method for evaluating aggregation-inhibiting ability, comprising evaluating the aggregation-inhibiting ability of the test substance based on the degree of aggregation of the agglutinant in the presence of the test substance.

5. In the detection step, the agglutinant and the test substance are allowed to coexist, and the generation of an aggregate from the agglutinant is detected; The evaluation method according to claim 4 , wherein the aggregation-inhibiting ability of the test substance is evaluated based on the degree of formation of the aggregate in the presence of the test substance.

6. In the detection step, the aggregates of the agglutinant are allowed to coexist with the test substance, and a reduction in the aggregates is detected; The evaluation method according to claim 4 , wherein the aggregation-inhibiting ability of the test substance is evaluated based on the degree of reduction of the aggregates in the presence of the test substance.

7. The evaluation method according to claim 4 , wherein the agglutinant or the aggregate of the agglutinant and the test substance are allowed to coexist in a non-human animal in vivo or in vitro.

8. A detection step of allowing the flocculant according to any one of claims 1 to 3 or an aggregate of the flocculant and a test substance to coexist and detecting the agglutination of the flocculant; and a selection step of selecting the test substance as an aggregation inhibitor when aggregation by the agglutinant is inhibited.

9. In the detection step, the agglutinant and the test substance are allowed to coexist, and the generation of an aggregate from the agglutinant is detected; The screening method according to claim 8 , wherein, in the selection step, if formation of the aggregates is inhibited, the test substance is selected as an aggregation inhibitor that inhibits formation of the aggregates.

10. In the detection step, the aggregates of the agglutinant are allowed to coexist with the test substance, and a reduction in the aggregates is detected; The screening method according to claim 8 , wherein, in the selection step, when the number of aggregates is decreased, the test substance is selected as an aggregation inhibitor that dissociates the aggregates.

11. The screening method according to claim 8 , wherein the agglutinating agent or the aggregate of the agglutinating agent and the test substance are allowed to coexist in a non-human animal in vivo or in vitro.

12. A method for screening a drug for an α-synuclein aggregation disease, comprising selecting an aggregation inhibitor selected by the method for screening an aggregation inhibitor according to claim 8 as a drug for an α-synuclein aggregation disease.

13. A method for producing an animal model for an α-synuclein aggregation disease, comprising administering the aggregating agent according to any one of claims 1 to 3 into the brain of a non-human animal.