Amyloid-β aggregation inhibitor, pharmaceutical composition for amyloid-β aggregation diseases, and use application of same

Abstract

Provided is a new medical agent that inhibits aggregation of amyloid-β which causes Alzheimer's disease and the like.　This amyloid-β aggregation inhibitor is characterized by including a peptide (A1) or (A2). (A1) A peptide consisting of an amino acid sequence having five or more continuous amino acid residues in the amino acid sequence of SEQ ID NO: 1 YKNMRETLVYLTHLDYDDTE (SEQ ID NO: 1) (A2) A peptide consisting of an amino acid sequence in which one or two amino acids are deleted, substituted, or added in the amino acid sequence of (A1).

Description

Amyloid beta aggregation inhibitor, pharmaceutical composition for amyloid beta aggregation diseases, and use thereof

[0001] The present invention relates to an inhibitor of amyloid β aggregation, a pharmaceutical composition for amyloid β aggregation diseases, and uses thereof.

[0002] As the proportion of elderly people in the population increases with the rise in average life expectancy, Alzheimer's disease, which is largely diagnosed in old age, has become a serious problem. Alzheimer's disease is a progressive central neurodegenerative disorder that causes cognitive impairment and memory loss. It is believed that the cause is fibrous aggregates (amyloid fibrils) formed in the brain by the aggregation of amyloid beta through intermolecular association. However, no effective drugs have been developed in clinical practice, and further research into candidate drugs is needed. This problem is not limited to Alzheimer's disease, but is applicable to all diseases caused by amyloid fibrils.

[0003] Therefore, an object of the present invention is to provide a new drug that inhibits the aggregation of amyloid β, which causes Alzheimer's disease and the like.

[0004] The amyloid β aggregation inhibitor of the present invention is characterized by containing the following peptide (A1) or (A2): (A1) A peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues in the amino acid sequence of SEQ ID NO: 1: YKNMRETLVYLTHLDYDDTE (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).

[0005] The pharmaceutical composition for amyloid β aggregation diseases of the present invention is characterized by containing the peptide (A1) or (A2) above.

[0006] The method for inhibiting amyloid β aggregation of the present invention is characterized by adding the amyloid β aggregation inhibitor of the present invention to a test subject.

[0007] The method for treating amyloid β aggregation diseases of the present invention is characterized by administering the amyloid β aggregation inhibitor of the present invention to a subject.

[0008] The amyloid β aggregation inhibitor of the present invention can inhibit aggregation due to intermolecular association of amyloid β, thereby enabling the treatment, for example, prevention, inhibition of progression, or amelioration, of amyloid aggregation diseases caused by the aggregation of amyloid β, such as Alzheimer's disease.

[0009] FIG. 1 is a graph showing the fluorescence intensity of a reaction solution in which peptide YS was added to Aβ25-35 before the aggregation reaction. FIG. 2 is a graph showing the IC50 inhibitory ability of peptide YS against Aβ25-35 aggregation. FIG. 3 is an electron microscope photograph showing the degree of aggregation in a reaction solution in which peptide YS was added to Aβ25-35 before the aggregation reaction. FIG. 4 is a graph showing the fluorescence intensity of a reaction solution in which peptide YS was added to Aβ25-35 aggregates. FIG. 5 is a graph showing the fluorescence intensity of a reaction solution in which peptide YS was added to Aβ25-35 aggregates, where (A) shows the result for YS-11 and (B) shows the result for YS-16. FIG. 6 is an electron microscope photograph showing the degree of aggregation in a reaction solution in which peptide YS was added to Aβ25-35 aggregates. Figure 7 is a graph showing the alternation behavior rate of the mouse group, where Figure 7(A) shows the results of simultaneous administration of Aβ25-35 and peptide YS, and Figure 7(B) shows the results of administration of Aβ25-35 followed by administration of peptide YS.

[0010] <Amyloid β aggregation inhibitor> As described above, the amyloid β aggregation inhibitor of the present invention is characterized by containing the following peptide (A1) or (A2). The peptides (A1) and (A2) are collectively referred to as peptide (A), peptide YS, or aggregation-inhibitory peptide YS. The aggregation inhibitor of the present invention is characterized by containing peptide YS, and other configurations and conditions are not particularly limited.

[0011] (A1) A peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues in the amino acid sequence of SEQ ID NO: 1 YKNMRETLVYLTHLDYDDTE (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).

[0012] The length of the peptide YS in (A1) may be 5 or more amino acid residues, for example, 5 to 16 amino acid residues.

[0013] The peptide YS of (A1) is, for example, a peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues including at least one of the 5th amino acid residue R (underlined R in Table 1) and the 15th amino acid residue D (underlined R in Table 1) in SEQ ID NO: 1, and preferably a peptide consisting of an amino acid sequence of 11 or more consecutive amino acid residues including both the 5th amino acid residue R (underlined) and the 15th amino acid residue D (underlined) in SEQ ID NO: 1. More preferably, it is a peptide having the 5th amino acid residue R (underlined) in SEQ ID NO: 1 as its N-terminus, or the 15th amino acid residue D (underlined) as its C-terminus. Examples of such peptide YS include peptides consisting of the amino acid sequences of SEQ ID NOs: 2, 3, 4, and 5, as shown in Table 1.

[0014]

[0015] 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-5, YS-11, YS-15, or YS-16, 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-5, YS-11, YS-15, or YS-16, for example.

[0016] The peptide YS may contain, for example, only D-isomers or only L-isomers of amino acid residues as constituent units, or may contain both.

[0017] The peptide YS may be, for example, a chemically modified peptide isostere. 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 thereof 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 the peptide, modification of carboxyl groups and / or amino groups, and amidation of the terminal carboxyl group.

[0018] The aggregation inhibitor of the present invention may contain, as the peptide YS, for example, only one of YS-5, YS-11, YS-15, and YS-16, or may contain two or more of any of them, or may contain three or more of them.

[0019] In the present invention, aggregation of amyloid β (hereinafter also referred to as Aβ) refers to, for example, aggregation of amyloid β or its fragment peptides. Hereinafter, unless otherwise specified, Aβ aggregation also refers to aggregation of the fragment peptides.

[0020] Peptide YS can inhibit the aggregation of Aβ or its fragment peptides (hereinafter also referred to as Aβ fragments). In the present invention, aggregation inhibition refers, for example, to inhibition of aggregation of Aβ or Aβ fragments, or inhibition by dissociation of already aggregated Aβ aggregates or dissociation of Aβ fragment aggregates. Peptide YS in the present invention can, for example, dissociate Aβ aggregates or Aβ fragment aggregates, and can also inhibit aggregation itself. Dissociation of Aβ aggregates means, for example, dissociating the aggregates into single Aβ, and dissociation of Aβ fragment aggregates means, for example, dissociating the aggregates into single Aβ fragments. In this case, the aggregation inhibitor of the present invention can also be referred to, for example, as an aggregation dissociation agent. Hereinafter, Aβ aggregates can be read as, for example, Aβ fragment aggregates.

[0021] The origin of the Aβ or Aβ fragment whose aggregation is inhibited by the peptide YS is not particularly limited, and examples thereof include humans or non-human animals described below, preferably humans. The full-length amino acid sequence of human Aβ is, for example, 40-42 amino acid residues. The length of the full-length amino acid sequence of human amyloid β varies, for example, depending on the enzymatic cleavage site from the amyloid precursor (APP). Human amyloid β of 42 amino acid residues is registered in the database (PubChem) under accession number CID: 57339251 and is represented by SEQ ID NO: 6. Human amyloid β of 40 amino acid residues is the sequence from the first amino acid residue (D) at the N-terminus to the 40th amino acid residue (V) in SEQ ID NO: 6, with the 40th amino acid residue being the C-terminus.

[0022] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 6)

[0023] The aggregation inhibitor of the present invention can be used, for example, in the treatment of diseases caused by Aβ aggregation (hereinafter also referred to as Aβ 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), because it can dissociate the aggregates. The amyloid β aggregation disease is not particularly limited and is a disease that can be caused by the aggregation of amyloid β, and specific examples include, for example, memory impairment, cognitive dysfunction, Alzheimer's disease, cerebral amyloid angiopathy, etc.

[0024] The aggregation inhibitor of the present invention can, for example, inhibit neuronal cell death caused by Aβ aggregation. Neuronal cell death is known to be involved in the progression of pathological conditions such as Alzheimer's disease. Therefore, the aggregation inhibitor of the present invention can also exert a therapeutic effect on Aβ aggregation diseases such as Alzheimer's disease by inhibiting neuronal cell death through inhibition of Aβ aggregation.

[0025] 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 peptide YS as an active ingredient. The active ingredient contained in the aggregation inhibitor of the present invention may be peptide YS alone, or may further contain other substances that exhibit aggregation-inhibiting effects. 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 include, for example, pharmacologically acceptable ingredients. For the additive ingredients, the explanation for the pharmaceutical composition described below can be used, for example.

[0026] The aggregation inhibitor of the present invention can be used, for example, in an environment where Aβ or Aβ fragments are present or are 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 that does not contain cells, or a biological test subject, such as cells such as brain cells, tissues such as brains, or living organisms. 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.

[0027] The aggregation inhibitor of the present invention may have, for example, an additional peptide in addition to the peptide YS. The additional peptide may be, for example, bound to the peptide YS. The additional peptide may have DDS ability to target a site where Aβ, Aβ fragments, Aβ aggregates, or Aβ fragment aggregates are present. Furthermore, the additional peptide may be, for example, a signal peptide that is bound to the peptide YS when administered to a living body and is cleaved and removed from the peptide YS 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 neuronal cell death inhibitor of the present invention or the pharmaceutical composition for Aβ aggregation diseases described below. Furthermore, the aggregation inhibitor of the present invention can be used, for example, in the method for inhibiting Aβ aggregation, the method for inhibiting neuronal cell death, the method for treating Aβ aggregation diseases, etc. described below. The aggregation inhibitor of the present invention can be used in the same manner as the neuronal cell death inhibitor, the pharmaceutical composition for Aβ aggregation diseases, the method for inhibiting Aβ aggregation, the method for inhibiting neuronal cell death, and the method for treating amyloid β aggregation diseases described below.

[0029] <Neuronal Cell Death Inhibitor> As described above, the neuron death inhibitor of the present invention is characterized by containing the peptide (A) (the peptide YS), i.e., the peptide (A1) or (A2). The neuron death inhibitor of the present invention is characterized by containing the peptide YS, and other configurations and conditions are not particularly limited, and the descriptions of the Aβ aggregation inhibitor of the present invention can be cited.

[0030] The peptide YS of the present invention can suppress neuronal cell death. Specifically, for example, it can suppress neuronal cell death caused by Aβ aggregation. As mentioned above, neuronal cell death is known to be involved in the progression of pathologies such as Alzheimer's disease. Therefore, the neuronal cell death inhibitor of the present invention can suppress, for example, neuronal cell death induced by Aβ aggregation by suppressing Aβ aggregation. Furthermore, the suppression of neuronal cell death in this manner can also have a therapeutic effect on Aβ aggregation diseases (amyloid β aggregation diseases) such as Alzheimer's disease.

[0031] <Pharmaceutical Composition for Amyloid β Aggregation Disease> As described above, the pharmaceutical composition for amyloid β aggregation disease of the present invention (hereinafter also referred to as pharmaceutical composition) is characterized by containing the peptide (A) (the peptide YS), i.e., the peptide (A1) or (A2). The pharmaceutical composition of the present invention is characterized by containing the peptide YS, and other configurations and conditions are not particularly limited, and the descriptions of the Aβ aggregation inhibitor and neuronal cell death inhibitor of the present invention can be cited.

[0032] The pharmaceutical composition of the present invention contains peptide YS as an active ingredient. The active ingredient contained in the pharmaceutical composition of the present invention may be peptide YS alone, or may further contain another active ingredient for the Aβ aggregation disease. The other active ingredient may be, for example, an active ingredient that inhibits the formation of the aggregates, an active ingredient that dissociates the formed aggregates, or an active ingredient that degrades the aggregates. Degradation of the aggregates may be, for example, degradation by cleaving the aggregates using hydrolytic activity or the like.

[0033] 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.

[0034] 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.

[0035] Examples of the parenteral administration method include administration to the 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, it is a site where the active ingredient of the pharmaceutical composition of the present invention can be delivered to the treatment site. Aβ aggregation diseases are often caused by the generation of Aβ aggregates in the brain, such as the aforementioned memory impairment, cognitive dysfunction, Alzheimer's disease, etc. 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.

[0036] 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.

[0037] 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 emulsified solvents, which are mixtures of the aqueous and oily solvents. The pharmaceutical composition of the present invention may also contain, for example, alcohols, polyalcohols, surfactants, etc. as additive components. 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 also 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 it 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.

[0038] The subject (subject) to which the pharmaceutical composition of the present invention is administered can be, for example, a human or the 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 an Aβ 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 amount of peptide YS per administration is 0.002 to 400 mg, the number of administrations per day is 1 to 3, and the interval between administrations is every 1 to 10 days.

[0039] 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.

[0040] <Method for inhibiting amyloid-β aggregation> As described above, the method for inhibiting amyloid-β aggregation of the present invention is characterized by adding the Aβ aggregation inhibitor of the present invention (i.e., peptide YS as an active ingredient) to a test subject. The inhibition method of the present invention is characterized by using the aggregation inhibitor of the present invention, and other steps and conditions, etc. are not limited in any way.

[0041] 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.

[0042] <Method for inhibiting neuronal cell death> As described above, the method for inhibiting neuronal cell death of the present invention is characterized by adding the neuronal cell death inhibitor of the present invention (i.e., the peptide YS as an active ingredient) to a subject. The method for inhibiting neuronal cell death of the present invention is characterized by using the neuronal cell death inhibitor of the present invention, and other steps and conditions are not limited in any way.

[0043] The addition of the neuronal cell death inhibitor of the present invention to the subject can be referenced to the descriptions of the aggregation inhibitor, pharmaceutical composition, and Aβ aggregation inhibition method of the present invention. The neuronal cell death inhibition method of the present invention preferably further comprises an incubation step after the addition step of the neuronal cell death inhibitor of the present invention to the subject. When the subject is a non-biological system, for example, the incubation temperature is room temperature to 37°C, the incubation time is 4 to 72 hours, and the pH is 6.5 to 8. When the subject is a cell or tissue, for example, the incubation temperature is room temperature to 37°C, the incubation time is 1 to 7 days, and the pH is 6.5 to 8.

[0044] <Method for treating amyloid-β aggregation diseases> As described above, the method for treating amyloid-β aggregation diseases of the present invention is characterized by administering to a subject the Aβ aggregation inhibitor of the present invention or the pharmaceutical composition of the present invention (i.e., peptide YS as an active ingredient). The treatment method of the present invention is characterized by using the Aβ aggregation inhibitor of the present invention or the pharmaceutical composition of the present invention (i.e., peptide YS as an active ingredient), and other steps and conditions, etc. are not limited in any way.

[0045] As described above, the subject (subject) to which the amyloid-β aggregation inhibitor of the present invention is administered may be a human or a non-human animal, and preferably a human. Regarding the addition of the aggregation inhibitor of the present invention to the subject, the descriptions regarding the aggregation inhibitor and pharmaceutical composition of the present invention can be cited.

[0046] <Use of Peptide YS> The peptide of the present invention is peptide YS for use in inhibiting Aβ aggregation or neuronal cell death, i.e., the peptide (A1) or (A2) described above. The peptide of the present invention is also peptide YS for use in treating Aβ aggregation disorders caused by Aβ aggregation, i.e., the peptide (A1) or (A2) described above.

[0047] The peptide of the present invention is peptide YS for use in the production of an Aβ aggregation inhibitor or a neuronal cell death inhibitor, i.e., the peptide (A1) or (A2) described above. Also, the peptide of the present invention is peptide YS for use in the production of a medicament for an Aβ aggregation disorder caused by Aβ aggregation, i.e., the peptide (A1) or (A2) described above.

[0048] Example 1 The ability of the aggregation-inhibitory peptide YS to inhibit Aβ aggregation was confirmed.

[0049] (1) Confirmation of Aβ Aggregation Inhibitory Ability The peptides YS-11, YS-15, and YS-16 were used in this example. Each of the peptides was dissolved in MilliQ water to prepare three 1 mg / mL YS solutions.

[0050] For Aβ aggregation, an Aβ-derived fragment peptide was used. The fragment peptide selected was Aβ25-35, which has high aggregability. The Aβ25-35 is a peptide (SEQ ID NO: 7: GSNKGAIIGLM) consisting of 11 amino acid residues from the 25th to 35th positions in the full-length sequence of human Aβ (SEQ ID NO: 6). Aβ25-35 was dissolved in MilliQ water to prepare a 1 mmol / L Aβ25-35 solution.

[0051] The fluorescent dye thioflavin T (ThT) binds to aggregates and emits strong fluorescence upon binding, so the increase or inhibition of aggregation can be determined by measuring the fluorescence intensity. Therefore, we used ThT to confirm the inhibition of Aβ25-35 aggregation by each peptide YS.

[0052] Specifically, a reaction solution with the following composition was prepared and dispensed into wells so that 200 μL of the reaction solution was added per well. After incubating the reaction solution at 37°C for a predetermined time (0 hour, 4 hours), 10 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 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. As a control, a reaction solution to which water was added instead of the peptide YS was also measured in the same manner.

[0053]

[0054] These results are shown in Figure 1. Figure 1 is a graph showing the fluorescence intensity of the reaction solution to which peptide YS was added; (A) shows the result after 0 hours of incubation, and (B) shows the result after 4 hours of incubation. In Figure 1, the vertical axis represents fluorescence intensity, and the unit is Fluorescence Intensity (FI). As shown in Figure 1(B), after 4 hours of incubation, peptides YS-11, YS-15, and YS-16 all exhibited significantly lower fluorescence intensities than the control (Ab25-35) to which peptide YS was not added. In particular, YS-11 and YS-16 exhibited significantly lower values. These results demonstrate that peptide YS can inhibit Aβ aggregation (the formation of Aβ aggregates).

[0055] (2) IC50 of YS peptide against Aβ25-35. Reaction solutions with different compositions (A, B, C, or D) were prepared and dispensed into wells so that 200 μL of the reaction solution was obtained per well. The concentrations of YS peptide (YS-11 or YS-16) in the YS solution added to the reaction solution (X mmol / L) were 0.01, 0.05, 0.1, 0.5, or 1 mmol / L. The reaction solutions were incubated at 37°C for 2 hours, after which 10 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 the measurement device (n=3). The measurement wavelengths were an excitation wavelength (ex) of 444 nm and an emission wavelength (em) of 480 nm.

[0056]

[0057] The fluorescence intensity FI of the reaction solution A to which the peptide YS was added and the Aβ25-35 was added Ａ From the above, the fluorescence intensity FI of the reaction solution B to which the peptide YS was added but the Aβ25-35 was not added Ｂ The fluorescence intensity FI of the reaction solution C to which the peptide YS was not added and the Aβ25-35 was added was calculated. Ｃ From the above, the fluorescence intensity FI of the reaction solution D to which the peptide YS was not added / the Aβ25-35 was not added Ｄ A blank correction was performed by subtracting the value of the YS-16 peptide from the YS-11 peptide. The results are shown in FIG. 2. In FIG. 2, the vertical axis represents fluorescence intensity (unit: FI), and the horizontal axis represents the concentration of the YS-16 peptide (unit: μmol / L). In FIG. 2, (A) represents the result for YS-11, and (B) represents the result for YS-16. As shown in FIG. 2, the IC50 of YS-11 was 3.6 μmol / L, and the IC50 of YS-16 was 0.6 μmol / L.

[0058] (3) Confirmation of Aggregation Inhibition by Peptide YS Using an Electron Microscope A reaction solution with the following composition was prepared in a 1.5 mL tube (Eppendorf). The reaction solution was incubated at 37°C for 4 hours, then centrifuged (12,000 rpm, 10 minutes) to collect the precipitate. 200 μL of MilliQ water was added to the collected precipitate, and the mixture was washed again by centrifugation (12,000 rpm, 10 minutes). This washing process was repeated three times. 100 μL of 1% phosphotungstic acid solution was then added to the collected precipitate, and the mixture was left at room temperature for 5 minutes. The precipitate was then collected by centrifugation (12,000 rpm, 10 minutes), washed with 200 μL of MilliQ water, and then centrifuged (12,000 rpm, 10 minutes). The precipitate was then collected. 100 μL of MilliQ water was added to the precipitate, and the mixture was suspended using a Vortex mixer. The suspension was applied to a glass slide, dried, and then observed under a scanning electron microscope.

[0059]

[0060] These results are shown in Figure 3. In Figure 3, the top panel shows the results without the addition of peptide YS, the middle panel shows the results with the addition of peptide YS-11, and the bottom panel shows the results with the addition of peptide YS-16 (800x magnification). In the photographs in Figure 3, the lined boxes indicate the areas where fibers were observed, and the thickness of the lines corresponds to the amount of fibers within that area; the more fibers there are, the thicker the lines. As shown in the top panel, when Ab25-35 was used alone without the addition of peptide YS, a large number of aggregated fibers were observed throughout the entire field of view. On the other hand, as shown in the middle panel, the coexistence of Ab25-35 and YS-11 significantly reduced the amount of fibrous material, demonstrating the suppression of aggregate formation compared to the case without the addition of peptide YS. Furthermore, as shown in the bottom panel, when Ab25-35 and YS-16 were used, almost no fibrous material was observed, demonstrating the significant suppression of aggregate formation.

[0061] Example 2 For peptide YS, dissociation of aggregates was examined as an indicator of the aggregation-inhibiting ability.

[0062] (1) Confirmation of Dissociation Ability for Aβ Aggregates First, a reaction solution with the following composition was prepared and incubated at 37°C for 18 hours to aggregate Aβ25-35 in the reaction solution. To this reaction solution, 100 μL of 1 mmol / L peptide YS (YS-5 or YS-16) was further added, and the mixture was incubated at 37°C for 5 days. After incubation, the reaction solution was dispensed into wells so that 200 μL was obtained per well. 10 μL of 2 mmol / L ThT was added per well, and the fluorescence intensity was measured using the measurement device (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 peptide YS was also similarly measured.

[0063]

[0064] These results are shown in Figure 4. Figure 4 is a graph showing the fluorescence intensity of the reaction solution to which peptide YS was added. In Figure 4, the vertical axis represents fluorescence intensity, and the units are fluorescence intensity (FI). As shown in Figure 4, the addition of peptide YS reduced the fluorescence intensity compared to the control (Aβ25-35) to which peptide YS was not added. In particular, when peptide YS-16 was used, a significant reduction in fluorescence intensity was observed, with the fluorescence intensity decreasing to 50% of the control (Aβ25-35). These results confirm that peptide YS can dissociate Aβ aggregates, i.e., inhibit Aβ aggregation, and it can be said that YS-16 in particular was able to dissociate 50% of the control aggregates.

[0065] (2) Relationship between peptide YS concentration and dissociation ability toward Aβ aggregates. First, a reaction solution with the following composition was prepared and dispensed into wells so that 200 μL of the reaction solution was distributed per well. The reaction solution was then incubated at 37°C for 18 hours to aggregate Aβ25-35. To this reaction solution, 100 μL of X mmol / L peptide YS solution (YS-11 or YS-16) was added, and the mixture was incubated at 37°C for 5 days. The peptide YS concentrations (X mmol / L) of the peptide YS solution were 0.01, 0.05, 0.1, 0.5, or 1 mmol / L. After the incubation, 10 μL of 2 mmol / L ThT was added per well, and the fluorescence intensity was measured using the measurement device (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 (Aβ25-35) to which water was added instead of the peptide YS was also measured in the same manner.

[0066]

[0067] These results are shown in Figure 5. Figure 5 is a graph showing the fluorescence intensity of the reaction solution to which the peptide YS was added. (A) shows the result for YS-11, and (B) shows the result for YS-16. In Figure 5, the vertical axis represents fluorescence intensity, and the units are Fluorescence Intensity (FI). As shown in Figure 5(A), the peptide YS-11 exhibited excellent dissociation ability when the final concentration in the reaction solution was in the range of 0.005 to 0.1 mmol / L. Furthermore, as shown in Figure 5(B), the peptide YS-16 exhibited excellent dissociation ability in a concentration-dependent manner when the final concentration in the reaction solution was in the range of 0.01 to 0.1 mmol / L. In particular, when the aggregates of the control (Aβ25-35) were taken as 100%, adding YS-16 to a final concentration of 0.1 mmol / L was able to dissociate 48% of the aggregates.

[0068] (3) Confirmation of Aggregate Dissociation by YS Peptide Using Electron Microscopy A reaction solution having the following composition was prepared in a 1.5 mL tube (manufactured by Eppendorf) and incubated at 37°C for 18 hours to aggregate the Aβ25-35. Then, 100 μL of a peptide YS solution at a predetermined concentration was added to the reaction solution, followed by further incubation at 37°C for 5 days. The peptide YS solutions used were 0.05 mmol / L YS-11 solution, 1 mmol / L YS-16 solution, and 1 mmol / L YS-6 solution. As a control, MilliQ water was used instead of the peptide YS solution. After incubation, the mixture was centrifuged (12,000 rpm, 10 minutes) to recover the precipitate. The precipitate was treated in the same manner as in Example 1(3) and observed using a scanning electron microscope.

[0069] These results are shown in Figure 6. In Figure 6, the top graph shows the results without the addition of peptide YS, the middle graph shows the results with the addition of peptide YS-11, and the bottom graph shows the results with peptide YS-16 (magnification 200x). In this example, Aβ25-35 was aggregated in advance, and the dissociation of the aggregates by peptide YS was confirmed. As shown in the top graph, when Ab25-35 alone was used without the addition of peptide YS, a large number of aggregated fibers were observed throughout the entire field of view. On the other hand, as shown in the bottom graph, in the coexistence of Ab25-35 and YS-16, a decrease in fibrous aggregates was confirmed, particularly in the area enclosed by the line. Furthermore, as shown in the middle graph, in the coexistence of Ab25-35 and YS-11, a further decrease in fibrous aggregates was confirmed, particularly in the area enclosed by the line.

[0070] Example 3 The aggregation-inhibitory peptide was further administered to mice to which the Aβ25-35 had been administered, and the effect was confirmed.

[0071] The Aβ25-35 was dissolved in physiological saline to prepare a 3 mmol / L Aβ25-35 solution. The Aβ25-35 and the YS-11 were dissolved in physiological saline to prepare a mixed solution containing 3 mmol / L Aβ25-35 and 3 mmol / L YS-11. The YS-11 was dissolved in physiological saline to prepare a 10 mg / mL YS-11 solution. Physiological saline (Saline) was used as a control.

[0072] ICR mice (6 weeks old) were prepared in the Aβ25-35 administration group (n=15), the control (salaine) administration group (n=15), the mixed solution administration group (n=6), and the peptide YS administration group (n=7).

[0073] The administration schedule was intracerebroventricular administration on Day 1, and intranasal administration on Days 14, 17, 20, and 23. A microsyringe was used for intracerebroventricular administration, and a pipette was used for intranasal administration. Specifically, each administration group was administered as follows.

[0074] (1) Aβ25-35 administration group Day 1: 3 μL of Aβ25-35 solution (Aβ25-35 9 nmol) Day 14, Day 17, Day 20, Day 23: Unadministered (2) Control administration group (Saline) Day 1: 3 μL of saline Day 14: 3 μL of saline Day 17: 3 μL of saline Day 20: 3 μL of saline Day 23: 3 μL of saline (3) Mixture administration group Day 1: 3 μL of the above mixture (Aβ25-35 9 nmol, YS-11 9 nmol) Day 14: 3 μL of saline Day 17: 3 μL of saline Day 20: 3 μL saline Day 23: 3 μL saline (4) Peptide YS administration group Day 1: 3 μL Aβ25-35 solution (Aβ25-35 9 nmol) Day 14: 10 μL YS-11 solution (YS-11 74 nmol) Day 17: 10 μL YS-11 solution (YS-11 74 nmol) Day 20: 10 μL YS-11 solution (YS-11 74 nmol) Day 23: 10 μL YS-11 solution (YS-11 74 nmol)

[0075] On Day 14 and Day 28, short-term memory was assessed using the Y-maze test. The Y-maze test was performed using a standard method under the following conditions. Specifically, the mouse was placed with its nose facing the wall at the end of one of the three arms. The mouse was then allowed to behave 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. A relatively high alternation rate indicates better short-term memory, while a relatively low rate indicates worse short-term memory.

[0076] Figure 7 shows the results of the alternation rate (%) calculated by Y-maze. Figure 7(A) shows the results comparing the Aβ25-35 administration group (Aβ25-35), the control administration group (Control), and the mixed solution administration group (Mixed solution), while Figure 7(B) shows the results comparing the Aβ25-35 administration group (Aβ25-35), the control administration group (Control), and the peptide YS administration group (YS-11). In Figure 7, the vertical axis shows the alternation rate (%).

[0077] First, as shown in Figures 7(A) and (B), the control group (Control) not administered Aβ25-35 had an alternation behavior rate of approximately 70-75% on Day 14 and Day 28, and did not change over time. In contrast, the Aβ25-35 administration group (Aβ25-35) administered only with Aβ25-35 had a lower alternation behavior rate than the control group (Control) on both Day 14 and Day 28. In other words, it is believed that the alternation behavior rate decreased due to the aggregation of Aβ25-35.

[0078] 7(A), the mixed solution administration group (Mixed solution) in which Aβ25-35 and YS-11 were administered simultaneously showed a higher alternation behavior rate than the Aβ25-35 administration group (Aβ25-35) in which only Aβ25-35 was administered, and showed a similar alternation behavior rate to the control group not administered Aβ25-35, on Day 14. In the mixed solution administration group, Aβ25-35 and YS-11 were administered simultaneously, which is thought to have suppressed the formation of Aβ25-35 aggregates, thereby suppressing the decrease in alternation behavior rate caused by Aβ25-35 aggregation.

[0079] Furthermore, the peptide YS administration group (YS-11) received intracerebroventricular administration of Aβ25-35 on Day 1, and short-term memory was assessed by Y-maze on Day 14. The results were similar to those of the Aβ25-35 administration group (Aβ25-35) administered with only Aβ25-35 (Figure 7(A)). That is, on Day 14, the peptide YS administration group (YS-11) had a lower alternation behavior rate than the control group (Control) (not shown). Subsequently, peptide YS-11 was administered intranasally four times, on Day 14, Day 17, Day 20, and Day 23, and short-term memory was assessed again on Day 28 by Y-maze. The results are shown in Figure 7(B). The alternation behavior rate was lower than that of the control group on Day 14 due to intracerebroventricular administration of Aβ25-35, but as shown in Figure 7 (B), by administering peptide YS-11 four times from Day 14, on Day 28, the Aβ25-35 administration group (Aβ25-35) administered only Aβ25-35 showed a higher alternation behavior rate than the control group not administered Aβ25-35, and the alternation behavior rate was similar to that of the control group not administered Aβ25-35, and a recovery of the alternation behavior rate was observed. In the peptide YS administration group, YS-11 was administered after administration of Aβ25-35, so it is understood that the decrease in the alternation behavior rate caused by the aggregation of Aβ25-35 was suppressed by dissociating the aggregates formed from the previously administered Aβ25-35 with YS-11.

[0080] As described above, administration of Aβ25-35 to the brain of mice causes aggregation of Aβ25-35, resulting in a decline in short-term cognitive function, a symptom of Alzheimer's disease. However, it was found that administration of peptide YS can suppress the decline in short-term cognitive function.

[0081] 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.

[0082] This application claims priority based on Japanese Patent Application No. 2021-80513, filed on May 11, 2021, the disclosure of which is incorporated herein in its entirety.

[0083] The amyloid β aggregation inhibitor of the present invention can inhibit aggregation due to intermolecular association of amyloid β, thereby enabling the treatment, for example, prevention, inhibition of progression, or amelioration, of amyloid aggregation diseases caused by the aggregation of amyloid β, such as Alzheimer's disease.

Claims

1. An agent for inhibiting amyloid β aggregation, comprising the following peptide (A1) or (A2): (A1) A peptide consisting of an amino acid sequence of 5 or more consecutive residues in the amino acid sequence of SEQ ID NO:

1. YKNMRETLVYLTHLDYDDTE (SEQ ID NO: 1) (A2) A peptide consisting of the amino acid sequence of (A1) above in which 1 to 2 amino acids have been deleted, substituted or added.

2. The aggregation inhibitor according to claim 1, wherein the peptide (A1) has 5 to 16 amino acid residues.

3. The aggregation inhibitor according to claim 1 or 2, wherein the peptide (A1) is a peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues including at least one of the 5th amino acid residue R and the 15th amino acid residue D in SEQ ID NO:

1.

4. The aggregation inhibitor according to claim 1 or 2, wherein the peptide (A1) is a peptide consisting of an amino acid sequence of 11 or more consecutive amino acid residues including both the 5th amino acid residue R and the 15th amino acid residue D in SEQ ID NO:

1.

5. The aggregation inhibitor according to claim 1 or 2, wherein the peptide (A1) is a peptide having the 5th amino acid residue R in SEQ ID NO: 1 as its N-terminus, or a peptide having the 15th amino acid residue D as its C-terminus.

6. The aggregation inhibitor according to claim 1 or 2, wherein the peptide (A1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5. YKNMR (SEQ ID NO:2) RETLVYLTHLD (SEQ ID NO: 3) YKNMRETLVYLTHLD (SEQ ID NO: 4) RETLVYLTHLDYDDTE (SEQ ID NO:5)

7. A pharmaceutical composition for an amyloid β aggregation disease caused by amyloid β aggregation, comprising the following peptide (A1) or (A2): (A1) A peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues in the amino acid sequence of SEQ ID NO:

1. YKNMRETLVYLTHLDYDDTE (SEQ ID NO: 1) (A2) A peptide consisting of the amino acid sequence of (A1) above in which 1 to 2 amino acids have been deleted, substituted or added.

8. The pharmaceutical composition according to claim 7, wherein the peptide (A1) has 5 to 16 amino acid residues.

9. The pharmaceutical composition according to claim 7 or 8, wherein the peptide (A1) is a peptide consisting of an amino acid sequence of 5 or more consecutive amino acid residues including at least one of the 5th amino acid residue R and the 15th amino acid residue D in SEQ ID NO:

1.

10. The pharmaceutical composition according to claim 7 or 8, wherein the peptide (A1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 3, 4, or 5. YKNMR (SEQ ID NO:2) RETLVYLTHLD (SEQ ID NO: 3) YKNMRETLVYLTHLD (SEQ ID NO: 4) RETLVYLTHLDYDDTE (SEQ ID NO:5)

11. The pharmaceutical composition according to claim 7 or 8, wherein the amyloid β aggregation disease is at least one selected from the group consisting of memory impairment, cognitive dysfunction, Alzheimer's disease, and cerebral amyloid angiopathy.

12. A method for inhibiting amyloid β aggregation, comprising adding the amyloid β aggregation inhibitor according to claim 1 or 2 to a subject in vitro.

13. A method for inhibiting amyloid beta aggregation, comprising adding an amyloid beta aggregation inhibitor described in claim 1 or 2 to a non-human animal.