WO2007080721A1 - Therapeutic agent for lewy body disease and prophylactic agent for lewy body disease - Google Patents

Therapeutic agent for lewy body disease and prophylactic agent for lewy body disease Download PDF

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Publication number
WO2007080721A1
WO2007080721A1 PCT/JP2006/324162 JP2006324162W WO2007080721A1 WO 2007080721 A1 WO2007080721 A1 WO 2007080721A1 JP 2006324162 W JP2006324162 W JP 2006324162W WO 2007080721 A1 WO2007080721 A1 WO 2007080721A1
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lewy body
body disease
disease
acid
synuclein
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PCT/JP2006/324162
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French (fr)
Japanese (ja)
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Masahito Yamada
Kenjiro Ono
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National University Corporation Kanazawa University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01L—SEMICONDUCTOR DEVICES NOT COVERED BY CLASS H10
    • H01L2924/00—Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
    • H01L2924/01—Chemical elements
    • H01L2924/01028—Nickel [Ni]

Definitions

  • the present invention relates to a therapeutic and prophylactic agent for Lewy body diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy.
  • Parkinson's disease is a typical neurodegenerative disease along with Arno-Ima disease, and it is known that Lewy bodies appear in the substantia nigra in the patient's brain. Lewy bodies are a protein aggregate of 140 amino acid residues called a-synuclein. In addition to Parkinson's disease, Lewy body diseases such as dementia with Lewy bodies, multiple system atrophy, etc. It is also known to appear in the brains of patients with the so-called diseases.
  • Patent Document 1 proposes a drug for a disease characterized by ⁇ -synuclein fibril formation such as Parkinson's disease, and discloses a drug containing any of tannic acid, myricetin, catechin, and the like. .
  • Patent Document 2 proposes an agent for promoting brain metabolism containing a substance having antioxidative ability and a therapeutic agent for improving brain function, which can treat or improve brain neurological diseases such as Parkinson's disease without side effects.
  • Patent Document 3 describes the use of L-cartine and the like together with an antioxidant for the prevention and treatment of Parkinson's disease and the like.
  • Patent Document 4 describes that a bis-dihydroxylyl compound having a specific structure is used for the treatment of ⁇ -synucleinopathies such as Parkinson's disease.
  • Patent Document 1 Special Table 2003-532634
  • Patent Document 2 JP-A-6-199690
  • Patent Document 3 Japanese Patent Laid-Open No. 10-87483
  • Patent Document 4 Japanese Translation of Special Publication 2005-531596 Disclosure of the invention
  • the present invention has been proposed in view of such a conventional situation, and aims to provide a therapeutic agent for Lewy body diseases capable of treating Lewy body diseases such as Parkinson's disease.
  • a therapeutic agent for Lewy body diseases capable of treating Lewy body diseases such as Parkinson's disease.
  • Target Another object of the present invention is to provide a preventive drug for Lewy body disease capable of suppressing the onset of Lewy body disease.
  • the present inventors have repeatedly studied for a long time. As a result, the inventors have obtained knowledge that a specific compound is extremely effective in suppressing or destabilizing the formation of ⁇ -synuclein fibers, and have completed the present invention.
  • the therapeutic agent for Lewy body disease according to the present invention is characterized by containing nordihydroguaiaretic acid.
  • the therapeutic agent for Lewy body disease according to the present invention is characterized by containing curcumin.
  • the therapeutic agent for Lewy body disease according to the present invention is characterized by containing rosemary acid.
  • the therapeutic agent for Lewy body disease according to the present invention is characterized by containing vitamin cocoon.
  • the therapeutic agent for Lewy body disease according to the present invention is characterized by containing ⁇ -carotene.
  • the Lewy body disease preventive agent according to the present invention is characterized by containing nordihydroguaiaretic acid.
  • the Lewy body disease-preventing drug according to the present invention is characterized by containing curcumin.
  • the Lewy body disease-preventing agent according to the present invention is characterized by containing rosemary acid.
  • the Lewy body disease preventive agent according to the present invention is characterized by containing vitamin cocoon.
  • the preventive drug for Lewy bodies according to the present invention is characterized by containing ⁇ -carotene.
  • a synuclein fiber which is the etiology of Lewy body disease
  • the therapeutic agent of the present invention makes it possible to control the pathology of Lewy body disease, such as delaying or reversing the progression of the disease.
  • a therapeutic agent for Lewy body disease containing vitamin A or beta-carotene which is a vitamin A precursor.
  • a Lewy body disease-preventing drug containing nordihydroguaiaretinic acid, curcumin or rosemary acid to a person without Lewy body disease, a-synuclein fibril formation is suppressed, It can suppress the accumulation of a-synuclein fibers in the brain and suppress the development of Lewy body disease.
  • a Lewy body prophylactic agent containing vitamin A or / 3 carotene which is a vitamin A precursor.
  • each of the above-mentioned compounds has an a-synuclein fibril formation inhibitory effect and an a-synuclein fiber instability effect is not clear, but for nordihydroguaiaretic acid, curcumin and rosemary acid, Since these compounds are reasonably small and have a symmetrical structure with aromatic rings at both ends, they are easy to bind to ex-synuclein and are suitable for suppressing synuclein fiber formation and destabilizing OC-synuclein fibers. It is guessed.
  • Patent Document 1 it is described that an antioxidant such as tannic acid, myricetin, catechin is used for the treatment of a disease characterized by a-synuclein fibril formation.
  • an antioxidant such as tannic acid, myricetin, catechin
  • the compounds classified as wine-related polyphenols such as these have a completely different structure from the compounds contained in the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention as active ingredients. is there.
  • nordihydroguaiaretic acid, curcumin and rosemary acid are similar in appearance to the compound group disclosed in Patent Document 4, but actually have different structures.
  • nordihydroguaiaretic acid, cucumin, and oral malic acid are superior to the compound group disclosed in Patent Document 4 in terms of a-synuclein fibril formation inhibitory effect and a synuclein fibril formation.
  • the effect of the invention showing the stabilizing effect
  • ⁇ synuclein fibers that have already accumulated can be destabilized and Lewy body disease can be controlled.
  • administration of the Lewy body disease preventive agent of the present invention to an unaffected person with Lewy body disease suppresses the formation of a-synuclein fibers and suppresses the accumulation of a-synuclein aggregates. The onset of the disease can be suppressed in advance.
  • FIG. 1 Fig. 1 (a) to (d) show the reaction rate of a-synuclein fibril formation from fresh ex-synuclein, nordihydroguaiaretic acid (a), rosemary acid (b), curcumin (c ) And tetracycline (d).
  • Figure 1 (e) is a characteristic diagram showing dose-dependent inhibition of OC-synuclein fibril formation from fresh ⁇ -synuclein.
  • FIG. 2 shows a reaction mixture containing 140 ⁇ ⁇ -synuclein, ⁇ 7.5, 20 mM Tris buffer, 100 mM NaCl, and 0 (a) or 50 ⁇ curcumin (b).
  • FIG. 3 Figures 3 (a) to 3 (d) show nordihydroguaiaretic acid (a), rosemary acid (b), curcumin (c), and tetracycline (d) as a function of the instability rate of a-synuclein fibrils.
  • FIG. Figure 3 (e) is a characteristic diagram showing the dose-dependent destabilization of ⁇ -synuclein fibrils.
  • FIG. 4 shows a reaction mixture containing 70 ⁇ ⁇ -synuclein fibrils, ⁇ 7.5, 20 mM Tris buffer, lOOmM NaCl, and 50 ⁇ curcumin at 37 ° C for 0 hour ( It is an electron micrograph after incubating a) or 6 hours (b). The horizontal bar indicates a length of 250 nm.
  • Fig. 5 show retinol (a), retinal (b), retinoic acid (c), CoQ (reaction rate) for the reaction rate of a-synuclein fibril formation from fresh ⁇ -synuclein. d)
  • Figure 10 is a characteristic diagram showing the influence.
  • Figure 5 (e) shows the dose-dependent inhibition of ⁇ -synuclein fibril formation from fresh ⁇ -synuclein.
  • Figure 6 shows that 140 ⁇ ⁇ ⁇ -synuclein, ⁇ 7.5 20 mM Tris buffer, 100 mM NaCl, and 0 (a, d), M retinol (b, e) or CoQ (c ⁇ f) for 6 hours at 37 ° C
  • FIG. 2 is an electron micrograph ((a) to (c)) and an atomic force microscope image ((d) to (f)) after incubation in FIG.
  • the horizontal bar indicates a length of 250 nm.
  • FIG. 7 Fig. 7 (a) to (d) show retinol versus instability of OL-synuclein fibrils.
  • FIG. 5 is a characteristic diagram showing the influence of (a), retinal (b), retinoic acid (c), and CoQ (d).
  • Fig 7
  • FIG. 8 shows 70 ⁇ ( ⁇ a-synuclein fibrils, ⁇ 7.5, 20 mM koji buffer, 1 OO mM NaCl, and 50 M retinol (ac) or CoQ (d). 0 hours (a), 1 o'clock
  • the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention are therapeutic agents and preventive agents for various diseases belonging to Lewy body disease.
  • Examples of the disease belonging to Lewy body disease include Parkinson's disease, Lewy body dementia, multiple system atrophy.
  • the therapeutic agent for Lewy body disease and the preventive agent for Lewy body of the present invention include nordihydroguaiaretic acid, curcumin, oral ⁇ smari ⁇ acid (rosmarinic aci d) Contains either vitamin A or j8 carotene. Vitamin A includes retinal, retinal and retinoic acid.
  • the dosage of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention may be determined as appropriate according to the intended use. However, since these active ingredients are highly safe, the dosage is large. It is considered that there is little worry about side effects even if it is administered continuously.
  • the dosage form of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention is not particularly limited.
  • it is an oral agent such as a tablet, granule or capsule, or an injection. be able to.
  • these can be manufactured in accordance with a conventionally well-known method.
  • the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention are conventional active agents such as nordihydroguaretic acid, curcumin, rosemary acid, vitamin A, or j8 carotene. It may contain known additives!
  • the administration method of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention is not particularly limited to oral administration, parenteral administration, etc., and conventionally known administration methods can be appropriately selected.
  • nordihydroguaiaretinic acid, curcumin, rosemary acid, vitamin A, or ⁇ -carotene is intended to promote health simply by being used as a therapeutic agent for Lewy body disease and a prophylactic agent for Lewy body disease. It can be used as a dietary supplement. In addition, it can also be used by adding to taste products such as food and drinks.
  • Fresh non-aggregated ⁇ -synuclein fibrils (f a S) were obtained by polymerizing fresh ex S just prior to the destabilization reaction.
  • the reaction mixture is 2000 ⁇ L and contains 140 ⁇ a S, 20 mM Tris-nofer, pH 7.5, and lOO mM NaCl.
  • Incubation was carried out at 37 ° C for 6 days under stirring.
  • the formation reaction reached equilibrium.
  • the progress of the formation reaction was measured by the fluorescence of thioflavin S.
  • Subsequent experiments estimate that the concentration of f «S in the final reaction mixture is 140 ⁇ .
  • ⁇ Fluorescence analysis> The fluorescence of a S was measured with a fluorescence spectrophotometer (Hitachi F-2500). The excitation wavelength and the fluorescence wavelength were 440 nm and 521 nm, respectively.
  • the reaction mixture contains 5 M thioflavine S, 50 mM pH 8.5 glycine-NaOH buffer.
  • ⁇ Electron Microscope> The reaction mixture was negatively colored with 1% phosphotungstic acid having a pH of 7.0 and spread on a carbon-coated grid. Then, observation was carried out under an electron microscope (JEM-1210) at an acceleration voltage of 75 kV.
  • ⁇ Polymerization Atsey> 140 ⁇ a S, 0-50 ⁇ NDGA, Cur, RA, FA, TA, Myr, Kmp, Cat, epi-Cat, RIF or TC, 1% dimethyl sulfoxide (DMSO ), 20 mM Tris buffer, pH 7.5, and lOO mM NaCl, a reaction mixture for polymerization assembly was prepared.
  • NDGA, Cur, RA, FA, wine-related polyphenols (TA, Myr, Kmp, Cat ⁇ epi— Cat) RIF and TC are dissolved in DMSO at concentrations of 1 M, 10 M, 100 ⁇ M, ImM and 5 mM.
  • reaction mixture was added to final concentrations of 0.01 M, 0.1 M, 1 M, 10 M and 50 M, respectively.
  • DHA and EPA were dissolved in DMSO at concentrations of 5 mM and 10 mM, and then added to the reaction mixture so that the final concentrations were 50 M and 100 M, respectively.
  • FIG. Figures l (a)-(d) show 140 M a S, 20 mM Tris buffer at pH 7.5, lOO mM NaCl, and each compound at 0 M ( ⁇ ), 10 M ( ⁇ ), or 50 ⁇ This is a result of incubating a reaction mixture containing a concentration of ( ⁇ ) at 37 ° C for a predetermined time.
  • Figure 1 (e) shows 140 ⁇ ⁇ ⁇ 8, ⁇ 7.5, 20 mM Tris buffer, lOOmM NaCl, RA ((), NDGA ( ⁇ ), Cur (country), and TC (mouth).
  • a reaction mixture for measuring lability was prepared containing% DMSO, 20 mM Tris buffer at pH 7.5, lOOmM NaCl, and 1% (wtZvol) polybutyl alcohol.
  • Polyvinyl alcohol is
  • NDGA, Cur, RA, FA, wine-related polyphenols, RIF, and TC are dissolved in DMSO at concentrations of 1 M, 10 ⁇ , 100 ⁇ , lmM, 5 mM, followed by a final concentration of 0.01 / ⁇ ⁇ , The reaction mixture was charged to 0.1 / ⁇ , 1 / ⁇ , 10 ⁇ and 50 ⁇ . DHA and soot were dissolved in DMSO at concentrations of 5 mM and 10 mM, and then added to the reaction mixture to a final concentration of 50 M and 100 M.
  • FIG. Figure 3 (a)-(d) shows 70 ⁇ fa S, 20 mM Tris buffer pH 7.5, lOO mM NaCl, and each compound as M (fist), ⁇ ⁇ ( ⁇ ) or 50 ⁇ (mouth). This is a result of incubation of a reaction mixture containing a concentration of) at 37 ° C for a predetermined time. Each figure shows a typical pattern of three independent experiments.
  • Fig. 3 (e) shows 70 ⁇ ⁇ (20 mM Tris buffer with ⁇ a S, pH 7.5, lOOmM NaCl and RA (fist), NDGA ( ⁇ ), Cur (country), and TC (mouth). Results of incubation of reaction mixtures containing 0, 0.01, 0.1, 1, 10 or 50 M for 6 hours at 37 ° C. Each point represents the average of three independent experiments. The standard error at the point was within the symbol, and the mean without compound was taken as 100%.
  • the destabilized EC was an order of magnitude higher than the EC that inhibited the formation of f a S.
  • Samples were imaged immediately using a multimode scanning probe microscope equipped with a channel and a Nanoscope Ilia controller (Digital Instruments). All measurements were performed in a tapping mode using a single-beam silicon cantilever probe under ambient conditions. The size of the AFM image was usually 512 x 512 pixels. At least four locations on the mica substrate were measured to confirm that similar substances were present in the sample. The compartmental analysis was performed by drawing a line for each species and measuring the distance from the mica substrate surface to the top of the structure. This was repeated 10 times, and the average was obtained only when the fiber structure was confirmed. The results are shown in Figs.
  • ⁇ Polymerization Atssey> 140 ⁇ a S, 0-: LOO ⁇ NDGA, Myr, RIF ⁇ tetracytalin (TC), vitamins, ⁇ -carotene, CoQ or ⁇ -lipoic acid (LA), 1 % Dimethylsulfo
  • a reaction mixture for a polymerization assay was prepared containing xoxide (DMSO), pH 7.5, 20 mM Tris buffer, lOO mM NaCl.
  • NDGA, Myr, RIF, TC, Vitamin A (retinol, retinal, retinoic acid), ⁇ -carotene, CoQ (Sigma) are 1 M, 10 M,
  • vitamin B 2 riboflavin
  • vitamin B6 pyridoxine
  • vitamin C vitamin E
  • FIG. Figures 5 (a) to (d) show 140 ⁇ a S, 20 mM Tris buffer, pH 7.5, lOO mM NaCl, and each compound at 0 M ( ⁇ ), 10 M ( ⁇ ), or 50 This is the result of incubation of a reaction mixture containing a concentration of ⁇ (mouth) at 37 ° C for a predetermined time. Each figure shows a typical pattern of three independent experiments.
  • Figure 5 (e) shows 140 ⁇ a S, pH 7.5, 20 mM Trisnofer, lOO mM NaCl, NDGA ( ⁇ ), retinol ( ⁇ ), retinoic acid (garden), CoQ (mouth) ) 0, 0.01, 0.1,
  • NDGA Myr, RIF, TC, Vitamin A, ⁇ -Carotene, CoQ, 1 M, 10 M, 100
  • FIG. Figure 7 (a)-(d) shows 70 ⁇ fa S, 20 mM Tris buffer, pH 7.5, lOO mM NaCl, and ⁇ ⁇ (fist), ⁇ ⁇ ( ⁇ ), or 50 ⁇ (mouth) Reaction mixture containing retinol (a), retinal (b), retinoic acid (c), CoQ (d)
  • aS was defined as the concentration that caused instability to 50% of the control value.
  • the final equilibrium level decreased in a dose-dependent manner. Although data are not shown, 10 / zM and 50 M vitamin B2, vitamin B6, vitamin C, vitamin E, a lipoic acid did not inhibit f a S formation.
  • NDGA, Myr, RIF, TC, retinol, retinal, retinoic acid, ⁇ -carotene, and CoQ inhibit the formation and elongation of faS in a dose-dependent manner.
  • the formed faS was unstable in a dose-dependent manner.
  • the effective concentrations (EC) of NDGA, Myr, RIF, TC, retinal, retinal, retinoic acid, j8-carotene, and CoQ required to suppress fa S formation to 50% of the control value, and the control value
  • the concentration that causes f a S to become unstable in 50% was calculated by applying it to the sigmoid curve of the data as shown in Figs. 5 (e) and 7 (e). The results are shown in Table 2.
  • the effective concentration (EC) that suppresses f a S formation of NDGA, Myr, CoQ, RIF, and TC is f a S
  • the concentration (EC) of retinol, retinal, retinoic acid, and 13 carotene that destabilizes f a S suppresses the formation of f a S.
  • retinol, retinal, retinoic acid, and j8 carotene are useful as therapeutic agents for Lewy body diseases and preventive agents for Lewy body diseases.

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Abstract

[PROBLEMS] To provide: a therapeutic agent for a Lewy body disease which can be used for the treatment of a Lewy body disease such as Perkinson's disease, dementia with Lewy bodies and multiple system atrophy; and a prophylactic agent for a Lewy body disease which can prevent the development of a Lewy body disease. [MEANS FOR SOLVING PROBLEMS] A therapeutic agent for a Lewy body disease comprising any substance selected from nordihydroguaiaretic acid, curcumin, rosmarinic acid, vitamin A and β-carotene; and a prophylactic agent for a Lewy body disease comprising any substance selected from nordihydroguaiaretic acid, curcumin, rosmarinic acid, vitamin A and β-carotene.

Description

明 細 書  Specification
レビー小体病治療薬及びレビー小体病予防薬  Lewy body disease treatment and Lewy body disease prevention drug
技術分野  Technical field
[0001] 本発明は、パーキンソン病、レビー小体型認知症、多系統萎縮症等のレビー小体病 の治療薬及び予防薬に関する。  [0001] The present invention relates to a therapeutic and prophylactic agent for Lewy body diseases such as Parkinson's disease, Lewy body dementia, and multiple system atrophy.
背景技術  Background art
[0002] パーキンソン病はアルッノヽイマ一病と並ぶ代表的な神経変性疾患であり、患者の脳 には黒質にレビー小体 (Lewy body)が出現することが知られている。レビー小体と は、 a—シヌクレインと呼ばれる 140アミノ酸残基カゝらなるタンパク質の凝集体であり、 パーキンソン病の他にレビー小体型認知症、多系統萎縮症等のようなレビー小体病 と称される疾患の患者の脳にも出現することが知られている。  [0002] Parkinson's disease is a typical neurodegenerative disease along with Arno-Ima disease, and it is known that Lewy bodies appear in the substantia nigra in the patient's brain. Lewy bodies are a protein aggregate of 140 amino acid residues called a-synuclein. In addition to Parkinson's disease, Lewy body diseases such as dementia with Lewy bodies, multiple system atrophy, etc. It is also known to appear in the brains of patients with the so-called diseases.
[0003] ところで、レビー小体病の進行には、 α—シヌクレインの凝集、すなわち α—シヌタレ イン線維形成が重大な役割を果たしていると考えられている。そこで近年、 ひ一シヌ クレイン線維形成を抑制等する物質について各方面で活発に研究が進められている 。例えば特許文献 1においては、パーキンソン病等の α—シヌクレイン線維形成を特 徴とする疾患の医薬品が提案されており、タンニン酸、ミリセチン、カテキン等のいず れかを含む医薬品が開示されている。特許文献 2においては、抗酸化能を有する物 質を含有する脳代謝促進'脳機能改善治療剤が提案され、パーキンソン病等の脳神 経疾患を副作用なく治療又は改善することができるとされる。特許文献 3にお 、ては 、パーキンソン病等の予防及び治療のために、 L—カル-チン等を抗酸化剤とともに 使用することが記載されている。また、特許文献 4には、特定構造を有するビス—ジヒ ドロキシァリール化合物等をパーキンソン病のような αーシヌクレイノパチ一の処置に 使用することが記載されて 、る。 [0003] By the way, it is considered that α-synuclein aggregation, that is, α-synuclein fibril formation plays an important role in the progression of Lewy body disease. Therefore, in recent years, active research has been conducted in various fields on substances that inhibit the formation of synuclein fibers. For example, Patent Document 1 proposes a drug for a disease characterized by α-synuclein fibril formation such as Parkinson's disease, and discloses a drug containing any of tannic acid, myricetin, catechin, and the like. . Patent Document 2 proposes an agent for promoting brain metabolism containing a substance having antioxidative ability and a therapeutic agent for improving brain function, which can treat or improve brain neurological diseases such as Parkinson's disease without side effects. . Patent Document 3 describes the use of L-cartine and the like together with an antioxidant for the prevention and treatment of Parkinson's disease and the like. Patent Document 4 describes that a bis-dihydroxylyl compound having a specific structure is used for the treatment of α -synucleinopathies such as Parkinson's disease.
特許文献 1：特表 2003 - 532634号公報  Patent Document 1: Special Table 2003-532634
特許文献 2：特開平 6— 199690号公報  Patent Document 2: JP-A-6-199690
特許文献 3：特開平 10— 87483号公報  Patent Document 3: Japanese Patent Laid-Open No. 10-87483
特許文献 4 :特表 2005— 531596号公報 発明の開示 Patent Document 4: Japanese Translation of Special Publication 2005-531596 Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] 前述のように、パーキンソン病等のレビー小体病を治療又は予防することを目的とし て抗酸化剤のような多数の化合物が検討されている。しかし、レビー小体病に対する 理解を深め、レビー小体病を効果的に治療又は予防するためには、既存の化合物 では 、まだ不十分であり、新規な有効成分につ!、て検討する必要がある。  [0004] As described above, many compounds such as antioxidants have been studied for the purpose of treating or preventing Lewy body diseases such as Parkinson's disease. However, in order to deepen understanding of Lewy body disease and effectively treat or prevent Lewy body disease, existing compounds are still insufficient, and it is necessary to consider new active ingredients! There is.
[0005] そこで本発明はこのような従来の実情に鑑みて提案されたものであり、例えばパーキ ンソン病等のレビー小体病の治療が可能なレビー小体病治療薬を提供することを目 的とする。また、本発明は、レビー小体病の発症を抑えることが可能なレビー小体病 予防薬を提供することを目的とする。  [0005] Therefore, the present invention has been proposed in view of such a conventional situation, and aims to provide a therapeutic agent for Lewy body diseases capable of treating Lewy body diseases such as Parkinson's disease. Target. Another object of the present invention is to provide a preventive drug for Lewy body disease capable of suppressing the onset of Lewy body disease.
課題を解決するための手段  Means for solving the problem
[0006] 前述の目的を達成するために、本発明者らは長期に亘り検討を重ねてきた。その結 果、ある特定の化合物が α—シヌクレイン線維の形成抑制又は不安定化に極めて有 効であるとの知見を得、本発明を完成するに至った。  [0006] In order to achieve the above-mentioned object, the present inventors have repeatedly studied for a long time. As a result, the inventors have obtained knowledge that a specific compound is extremely effective in suppressing or destabilizing the formation of α-synuclein fibers, and have completed the present invention.
[0007] すなわち、本発明に係るレビー小体病治療薬は、ノルジヒドログアイァレチン酸を含 むことを特徴とする。本発明に係るレビー小体病治療薬は、クルクミンを含むことを特 徴とする。本発明に係るレビー小体病治療薬は、ローズマリー酸を含むことを特徴と する。本発明に係るレビー小体病治療薬は、ビタミン Αを含むことを特徴とする。本発 明に係るレビー小体病治療薬は、 βカロチンを含むことを特徴とする。  [0007] That is, the therapeutic agent for Lewy body disease according to the present invention is characterized by containing nordihydroguaiaretic acid. The therapeutic agent for Lewy body disease according to the present invention is characterized by containing curcumin. The therapeutic agent for Lewy body disease according to the present invention is characterized by containing rosemary acid. The therapeutic agent for Lewy body disease according to the present invention is characterized by containing vitamin cocoon. The therapeutic agent for Lewy body disease according to the present invention is characterized by containing β-carotene.
[0008] また、本発明に係るレビー小体病予防薬は、ノルジヒドログアイァレチン酸を含むこ とを特徴とする。本発明に係るレビー小体病予防薬は、クルクミンを含むことを特徴と する。本発明に係るレビー小体病予防薬は、ローズマリー酸を含むことを特徴とする 。本発明に係るレビー小体病予防薬は、ビタミン Αを含むことを特徴とする。本発明に 係るレビー小体病予防薬は、 βカロチンを含むことを特徴とする。  [0008] The Lewy body disease preventive agent according to the present invention is characterized by containing nordihydroguaiaretic acid. The Lewy body disease-preventing drug according to the present invention is characterized by containing curcumin. The Lewy body disease-preventing agent according to the present invention is characterized by containing rosemary acid. The Lewy body disease preventive agent according to the present invention is characterized by containing vitamin cocoon. The preventive drug for Lewy bodies according to the present invention is characterized by containing β-carotene.
[0009] ノルジヒドログアイァレチン酸、クルクミン又はローズマリー酸を含むレビー小体病治 療薬をレビー小体病患者に投与することで、レビー小体病の病因とされる a シヌク レイン線維を不安定ィ匕するため、 aーシヌクレイン線維のさらなる蓄積を抑制するとと もに、レビー小体病患者の脳組織の α シヌクレイン線維を分解することができる。し たがって、本発明の治療薬により、病気の進行を遅らせる、或いは逆戻りさせる等、レ ビー小体病の病状のコントロールが可能となる。ビタミン A、又はビタミン A前駆体で ある βカロチンを含むレビー小体病治療薬についても、同様の効果を得ることができ る。 [0009] By administering a therapeutic drug for Lewy body disease containing nordihydroguaiaretinic acid, curcumin or rosemary acid to a patient with Lewy body disease, a synuclein fiber, which is the etiology of Lewy body disease, is not treated. In order to stabilize, it can suppress further accumulation of a-synuclein fibers and degrade α-synuclein fibers in brain tissue of Lewy body disease patients. Shi Therefore, the therapeutic agent of the present invention makes it possible to control the pathology of Lewy body disease, such as delaying or reversing the progression of the disease. A similar effect can be obtained with a therapeutic agent for Lewy body disease containing vitamin A or beta-carotene, which is a vitamin A precursor.
[0010] また、ノルジヒドログアイァレチン酸、クルクミン又はローズマリー酸を含むレビー小体 病予防薬をレビー小体病未発症者に投与することで、 aーシヌクレインの線維形成 が抑制されるため、脳内への a—シヌクレイン線維蓄積を抑制し、レビー小体病の発 症を抑えることができる。ビタミン A、又はビタミン A前駆体である /3カロチンを含むレ ビー小体病予防薬についても、同様の効果を得ることができる。  [0010] Further, by administering a Lewy body disease-preventing drug containing nordihydroguaiaretinic acid, curcumin or rosemary acid to a person without Lewy body disease, a-synuclein fibril formation is suppressed, It can suppress the accumulation of a-synuclein fibers in the brain and suppress the development of Lewy body disease. A similar effect can be obtained with a Lewy body prophylactic agent containing vitamin A or / 3 carotene, which is a vitamin A precursor.
[0011] 前述した各化合物が aーシヌクレインの線維形成抑制効果及び aーシヌクレイン線 維の不安定ィ匕効果を有する理由は明確ではな 、が、ノルジヒドログアイァレチン酸、 クルクミン及びローズマリー酸に関しては、これら化合物は適度に小さく且つ両端に 芳香環を持つ対称的な構造を持っため、 exーシヌクレインに結合し易ぐ a シヌク レインの線維形成の抑制及び OCーシヌクレイン線維の不安定化に適しているものと 推測される。  [0011] The reason why each of the above-mentioned compounds has an a-synuclein fibril formation inhibitory effect and an a-synuclein fiber instability effect is not clear, but for nordihydroguaiaretic acid, curcumin and rosemary acid, Since these compounds are reasonably small and have a symmetrical structure with aromatic rings at both ends, they are easy to bind to ex-synuclein and are suitable for suppressing synuclein fiber formation and destabilizing OC-synuclein fibers. It is guessed.
[0012] 前述の特許文献 1にお 、ては、タンニン酸、ミリセチン、カテキン等の抗酸化剤を a —シヌクレインの線維形成を特徴とする疾患の処置に用いることが記載されている。 しかし、これらのようなワイン関連ポリフエノールに分類される化合物は、本発明のレビ 一小体病治療薬及びレビー小体病予防薬が有効成分として含む化合物とは全く異 なる構造を持つものである。また、ノルジヒドログアイァレチン酸、クルクミン及びロー ズマリー酸は、特許文献 4に開示されたィ匕合物群と一見類似しているものの、実際に は異なる構造を有している。この構造上の差により、ノルジヒドログアイァレチン酸、ク ルクミン及び口ーズマリ一酸は、特許文献 4に開示された化合物群に比較して優れた a—シヌクレイン線維形成抑制効果及び a シヌクレイン線維不安定化効果を示す 発明の効果  [0012] In the aforementioned Patent Document 1, it is described that an antioxidant such as tannic acid, myricetin, catechin is used for the treatment of a disease characterized by a-synuclein fibril formation. However, the compounds classified as wine-related polyphenols such as these have a completely different structure from the compounds contained in the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention as active ingredients. is there. In addition, nordihydroguaiaretic acid, curcumin and rosemary acid are similar in appearance to the compound group disclosed in Patent Document 4, but actually have different structures. Due to this structural difference, nordihydroguaiaretic acid, cucumin, and oral malic acid are superior to the compound group disclosed in Patent Document 4 in terms of a-synuclein fibril formation inhibitory effect and a synuclein fibril formation. The effect of the invention showing the stabilizing effect
[0013] 本発明のレビー小体病治療薬をレビー小体病患者に投与することにより、既に蓄積 した α シヌクレイン線維を不安定化し、レビー小体病をコントロールすることができ る。また、本発明のレビー小体病予防薬をレビー小体病の未発症者に投与すること により、 a—シヌクレイン線維形成を抑制し、 a—シヌクレイン凝集体の蓄積を抑制 するため、レビー小体病の発症を未然に抑えることができる。 [0013] By administering the therapeutic agent for Lewy body disease of the present invention to a Lewy body disease patient, α synuclein fibers that have already accumulated can be destabilized and Lewy body disease can be controlled. The In addition, administration of the Lewy body disease preventive agent of the present invention to an unaffected person with Lewy body disease suppresses the formation of a-synuclein fibers and suppresses the accumulation of a-synuclein aggregates. The onset of the disease can be suppressed in advance.
図面の簡単な説明 Brief Description of Drawings
[図 1]図 1 (a)〜（d)は、新鮮な exーシヌクレインからの aーシヌクレイン原線維形成の 反応速度に対するノルジヒドログアイァレチン酸 (a)、ローズマリー酸 (b)、クルクミン（ c)、及びテトラサイクリン (d)の影響を示す特性図である。図 1 (e)は、新鮮な α—シヌ クレインからの OC—シヌクレイン原線維形成の用量依存的抑制を示す特性図である。 [Fig. 1] Fig. 1 (a) to (d) show the reaction rate of a-synuclein fibril formation from fresh ex-synuclein, nordihydroguaiaretic acid (a), rosemary acid (b), curcumin (c ) And tetracycline (d). Figure 1 (e) is a characteristic diagram showing dose-dependent inhibition of OC-synuclein fibril formation from fresh α-synuclein.
[図 2]図 2は、 140 μ Μの α—シヌクレイン、 ρΗ7. 5、 20mMのトリスバッファー、 100 mMの NaCl、及び 0 (a)又は 50 μ Μのクルクミン（b)を含む反応混合物を、 37°Cで 6 時間インキュベートした後の電子顕微鏡写真である。横棒は長さ 250nmを示す。 [FIG. 2] FIG. 2 shows a reaction mixture containing 140 μΜ α-synuclein, ρΗ7.5, 20 mM Tris buffer, 100 mM NaCl, and 0 (a) or 50 μΜ curcumin (b). An electron micrograph after incubation at 37 ° C for 6 hours. The horizontal bar indicates a length of 250 nm.
[図 3]図 3 (a)〜（d)は、 aーシヌクレイン原線維の不安定ィ匕速度に対するノルジヒドロ グアイァレチン酸 (a)、ローズマリー酸 (b)、クルクミン (c)、及びテトラサイクリン (d)の 影響を示す特性図である。図 3 (e)は、 α—シヌクレイン原線維の用量依存的不安定 化を示す特性図である。 [Fig. 3] Figures 3 (a) to 3 (d) show nordihydroguaiaretic acid (a), rosemary acid (b), curcumin (c), and tetracycline (d) as a function of the instability rate of a-synuclein fibrils. FIG. Figure 3 (e) is a characteristic diagram showing the dose-dependent destabilization of α-synuclein fibrils.
[図 4]図 4は、 70 μ Μの α—シヌクレイン原線維、 ρΗ7. 5、 20mMのトリスバッファー 、 lOOmMの NaCl、及び 50 μ Μのクルクミンを含む反応混合物を、 37°Cで 0時間（a )又は 6時間（b)インキュベートした後の電子顕微鏡写真である。横棒は長さ 250nm を示す。  [FIG. 4] FIG. 4 shows a reaction mixture containing 70 μΜ α-synuclein fibrils, ρΗ7.5, 20 mM Tris buffer, lOOmM NaCl, and 50 μΜ curcumin at 37 ° C for 0 hour ( It is an electron micrograph after incubating a) or 6 hours (b). The horizontal bar indicates a length of 250 nm.
[図 5]図 5 (a)〜（d)は、新鮮な α—シヌクレインからの a—シヌクレイン原線維形成の 反応速度に対するレチノール (a)、レチナール (b)、レチノイン酸 (c)、 CoQ (d)の  [Fig. 5] Fig. 5 (a) to (d) show retinol (a), retinal (b), retinoic acid (c), CoQ (reaction rate) for the reaction rate of a-synuclein fibril formation from fresh α-synuclein. d)
10 影響を示す特性図である。図 5 (e)は、新鮮な α—シヌクレインからの α—シヌクレイ ン原線維形成の用量依存的抑制を示す図である。  10 is a characteristic diagram showing the influence. Figure 5 (e) shows the dose-dependent inhibition of α-synuclein fibril formation from fresh α-synuclein.
[図 6]図 6は、 140 μ Μの α—シヌクレイン、 ρΗ7. 5の 20mMのトリス緩衝液、 100m Mの NaCl、及び 0 (a、 d)、 Mレチノール（b、 e)又は CoQ (cゝ f)を 6時間 37°C  [Figure 6] Figure 6 shows that 140 μ μ α-synuclein, ρΗ7.5 20 mM Tris buffer, 100 mM NaCl, and 0 (a, d), M retinol (b, e) or CoQ (cゝ f) for 6 hours at 37 ° C
10  Ten
でインキュベーションした後の電子顕微鏡写真（ (a)〜 (c) )及び原子間力顕微鏡画 像（（d)〜 (f) )である。横棒は長さ 250nmを示す。 2 is an electron micrograph ((a) to (c)) and an atomic force microscope image ((d) to (f)) after incubation in FIG. The horizontal bar indicates a length of 250 nm.
[図 7]図 7 (a)〜（d)は、 OLーシヌクレイン原線維の不安定ィ匕速度に対するレチノール (a)、レチナール (b)、レチノイン酸 (c)、 CoQ (d)の影響を示す特性図である。図 7 [Fig. 7] Fig. 7 (a) to (d) show retinol versus instability of OL-synuclein fibrils. FIG. 5 is a characteristic diagram showing the influence of (a), retinal (b), retinoic acid (c), and CoQ (d). Fig 7
10  Ten
(e)は、新鮮なひ一シヌクレインからのひ一シヌクレイン原線維形成の用量依存的抑 制を示す図である。  (e) shows dose-dependent inhibition of synuclein fibril formation from fresh synuclein.
[図 8]図 8は、 70 μ Μ(Ό a—シヌクレイン原線維、 ρΗ7. 5、 20mMの卜ジス緩衝液、 1 OOmMの NaCl、及び 50 Mのレチノール（a〜c)又は CoQ (d)を 0時間（a)、 1時  [FIG. 8] FIG. 8 shows 70 μΜ (Ό a-synuclein fibrils, ρΗ7.5, 20 mM koji buffer, 1 OO mM NaCl, and 50 M retinol (ac) or CoQ (d). 0 hours (a), 1 o'clock
10  Ten
間（b)、 6時間（c、 d)、 37°Cでインキュベーションした後の電子顕微鏡写真（（a)〜（d ) )及び原子間力顕微鏡画像 ( (e)〜 (g) )である。横棒は長さ 250nmを示す。  (B), 6 hours (c, d), electron micrographs after incubation at 37 ° C ((a)-(d)) and atomic force microscope images ((e)-(g)) . The horizontal bar indicates a length of 250 nm.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 以下、本発明を適用したレビー小体病治療薬及びレビー小体病予防薬について、 詳細に説明する。 [0015] Hereinafter, the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease to which the present invention is applied will be described in detail.
[0016] 本発明のレビー小体病治療薬及びレビー小体病予防薬は、レビー小体病に属する 各種疾患に対する治療薬及び予防薬である。レビー小体病に属する疾患としては、 パーキンソン病、レビー小体型痴呆症、多系統萎縮症等が挙げられる。  [0016] The therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention are therapeutic agents and preventive agents for various diseases belonging to Lewy body disease. Examples of the disease belonging to Lewy body disease include Parkinson's disease, Lewy body dementia, multiple system atrophy.
[0017] 本発明のレビー小体病治療薬及びレビー小体病予防薬は、ノルジヒドログアイァレチ ン酸 (nordihydroguaiaretic acid)、クノレク^ン (curcumin)、口 ~~ズマリ ~~酸 (rosmarinic aci d)、ビタミン A、又は j8カロチンのいずれかを含むものである。ビタミン Aには、レチノ ール、レチナール及びレチノイン酸が含まれる。  [0017] The therapeutic agent for Lewy body disease and the preventive agent for Lewy body of the present invention include nordihydroguaiaretic acid, curcumin, oral ~~ smari ~~ acid (rosmarinic aci d) Contains either vitamin A or j8 carotene. Vitamin A includes retinal, retinal and retinoic acid.
[0018] 本発明のレビー小体病治療薬及びレビー小体病予防薬の投与量については、その 使用目的に応じて適宜決定すればよいが、これら有効成分の安全性は高いので、多 量を継続的に投与しても副作用の心配は少ないと考えられる。  [0018] The dosage of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention may be determined as appropriate according to the intended use. However, since these active ingredients are highly safe, the dosage is large. It is considered that there is little worry about side effects even if it is administered continuously.
[0019] 本発明のレビー小体病治療薬及びレビー小体病予防薬の剤型としては特に限定さ れないが、例えば錠剤、顆粒剤、カプセル剤等の経口剤や、注射剤等とすることがで きる。また、これらは、従来公知の方法に従って製造することができる。また、本発明 のレビー小体病治療薬及びレビー小体病予防薬は、有効成分であるノルジヒドログ アイァレチン酸、クルクミン、ローズマリー酸、ビタミン A、又は j8カロチンの他に、安 定剤等の従来公知の添加剤等を含有してもよ!ヽ。本発明のレビー小体病治療薬及 びレビー小体病予防薬の投与方法としては、経口投与、非経口投与等、特に限定さ れず、従来公知の投与方法を適宜選択することができる。 [0020] なお、ノルジヒドログアイァレチン酸、クルクミン、ローズマリー酸、ビタミン A、又は β カロチンは、レビー小体病の治療薬及びレビー小体病の予防薬として用いるだけで なぐ健康増進を目的とした栄養補助食品として用いることができる。また、飲食物や 酒類などの嗜好品に添加して用いることも可能である。 [0019] The dosage form of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention is not particularly limited. For example, it is an oral agent such as a tablet, granule or capsule, or an injection. be able to. Moreover, these can be manufactured in accordance with a conventionally well-known method. In addition, the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention are conventional active agents such as nordihydroguaretic acid, curcumin, rosemary acid, vitamin A, or j8 carotene. It may contain known additives! The administration method of the therapeutic agent for Lewy body disease and the preventive agent for Lewy body disease of the present invention is not particularly limited to oral administration, parenteral administration, etc., and conventionally known administration methods can be appropriately selected. [0020] In addition, nordihydroguaiaretinic acid, curcumin, rosemary acid, vitamin A, or β-carotene is intended to promote health simply by being used as a therapeutic agent for Lewy body disease and a prophylactic agent for Lewy body disease. It can be used as a dietary supplement. In addition, it can also be used by adding to taste products such as food and drinks.
実施例  Example
[0021] 以下、本発明について、実験結果を参照して詳細に説明する。（実験 1) 本実験で は、ノルジヒドログアイァレチン酸（NDGA)、クルクミン（Cur)、ローズマリー酸 (RA) 、フェルラ酸（FA, ferulic acid)又はワイン関連ポリフエノール（タン-ン酸（TA, tanni c acid) ,ミリセチン (Myr, myricetin)、カンフェローノレ (Kmp, kaempferol)、力テキン ( Cat, (+)-catechin)、ェピカテキン（epi— Cat, (-) -epicatechin) )、ドコサへキサェン 酸（DHA, cis-4, 7 , 10 , 13 , 16 , 19-docosahexaenoic acid)、エイコサペンタエン酸（EPA , cis-5,8, 11,14, 17-eicosapentaenoic acid)、リファンピシン（RIF, rifampicin)、及びテ トラサイクリン (TC, tetracycline)の、 α—シヌクレインの線維形成に与える影響につ いて調べた。また、本実験では、前記各化合物の OCーシヌクレイン原線維不安定ィ匕 効果についても調べた。本実験で用いた NDGA、 Cur、 RA、 FA、 TA、 Myr, Kmp 、 Cat, epi -Cat, RIF及び TCの構造を、下記に示す。  Hereinafter, the present invention will be described in detail with reference to experimental results. (Experiment 1) In this experiment, nordihydroguaiaretic acid (NDGA), curcumin (Cur), rosemary acid (RA), ferulic acid (FA, ferulic acid) or wine-related polyphenols (tan-acid ( TA, tanni c acid), myricetin (Myr, myricetin), camphoronore (Kmp, kaempferol), force techin (Cat, (+)-catechin), epicatechin (epi- Cat, (-) -epicatechin)), docosa Hexaenoic acid (DHA, cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid), eicosapentaenoic acid (EPA, cis-5,8, 11,14, 17-eicosapentaenoic acid), rifampicin (RIF, The effects of rifampicin) and tetracycline on α-synuclein fibril formation were investigated. In this experiment, the effect of each compound on OC-synuclein fibril instability was also examined. The structures of NDGA, Cur, RA, FA, TA, Myr, Kmp, Cat, epi-Cat, RIF and TC used in this experiment are shown below.
[0022] [化 1]  [0022] [Chemical 1]
Figure imgf000008_0001
Figure imgf000008_0001
NDGA NDGA
[0023] [化 2] [0023] [Chemical 2]
[S^ ] [9200] [S ^] [9200]
Figure imgf000009_0001
Figure imgf000009_0001
Ζ9Ϊ 900Zdf/ェ:) d L ΪΖ.080/.00Ζ OAV Ζ9Ϊ 900Zdf / e :) d L ΪΖ.080 / .00Ζ OAV
Figure imgf000010_0001
Figure imgf000010_0001
[0028] [ィ匕 7]
Figure imgf000011_0001
[0028] [Yi 7]
Figure imgf000011_0001
SS0O
Figure imgf000012_0001
SS0O
Figure imgf000012_0001
RIF  RIF
[0032] [化 11]  [0032] [Chemical 11]
Figure imgf000012_0002
Figure imgf000012_0002
TC  TC
[0033] < aーシヌクレイン及び aーシヌクレイン原線維の準備〉 aーシヌクレイン（ α S)は、  [0033] <Preparation of a-synuclein and a-synuclein fibrils> a-synuclein (α S) is
Recombinant Peptide Technologiesより購入した。新鮮な凝集していない α—シヌク レイン原線維 (f a S)は、不安定化反応の直前に新鮮な ex Sを重合反応させること〖こ よって得た。反応混合物は 2000 μ Lとし、 140 μ Μの a S、 pH7. 5の 20mMのトリス ノ ッファー、 lOOmMの NaClを含むものである。 37°Cで 6日間、撹拌環境下でインキ ュベーシヨンした。形成反応は平衡に到達した。形成反応の進行の度合いはチオフ ラビン Sの蛍光により測定した。その後の実験により、最終的な反応混合物中の f « S の濃度は 140 μ Μと推測される。  Purchased from Recombinant Peptide Technologies. Fresh non-aggregated α-synuclein fibrils (f a S) were obtained by polymerizing fresh ex S just prior to the destabilization reaction. The reaction mixture is 2000 μL and contains 140 μΜ a S, 20 mM Tris-nofer, pH 7.5, and lOO mM NaCl. Incubation was carried out at 37 ° C for 6 days under stirring. The formation reaction reached equilibrium. The progress of the formation reaction was measured by the fluorescence of thioflavin S. Subsequent experiments estimate that the concentration of f «S in the final reaction mixture is 140 μΜ.
[0034] 〈蛍光分析〉 a Sの蛍光を蛍光分光光度計 (Hitachi F-2500)で測定した。励起波長 及び蛍光波長は、それぞれ 440nm、 521nmとした。反応混合物は 5 Mのチオフラ ビン S、 50mMの pH8. 5グリシン一 NaOHバッファーを含むものである。 [0035] 〈電子顕微鏡〉 pH7. 0の 1%リンタングステン酸で反応混合物をネガティブに着色 し、カーボン被覆グリッド上に広げた。そして、電子顕微鏡 (JEM-1210)下で加速電 圧 75kVにて観察を行った。 <Fluorescence analysis> The fluorescence of a S was measured with a fluorescence spectrophotometer (Hitachi F-2500). The excitation wavelength and the fluorescence wavelength were 440 nm and 521 nm, respectively. The reaction mixture contains 5 M thioflavine S, 50 mM pH 8.5 glycine-NaOH buffer. <Electron Microscope> The reaction mixture was negatively colored with 1% phosphotungstic acid having a pH of 7.0 and spread on a carbon-coated grid. Then, observation was carried out under an electron microscope (JEM-1210) at an acceleration voltage of 75 kV.
[0036] 〈重合アツセィ〉 140 μ Μの a S、 0〜50 μ Μの NDGA、 Cur、 RA、 FA、 TA、 Myr 、 Kmp、 Cat, epi— Cat、 RIF又は TC、 1%ジメチルスルホキシド（DMSO)、 pH7. 5の 20mMトリスバッファー、及び lOOmMの NaClを含む重合アツセィ用反応混合 物を調製した。 NDGA、 Cur、 RA、 FA、ワイン関連ポリフエノール (TA、 Myr, Kmp 、 Catゝ epi— Cat) RIF及び TCは、 1 M、 10 M、 100 μ M、 ImM及び 5mMの 濃度で DMSOに溶解された後、最終濃度がそれぞれ 0. 01 M、 0. 1 M、 1 M 、 10 M及び 50 Mとなるように反応混合物に添カ卩した。 DHA及び EPAは、 5m M及び 10mMの濃度で DMSOに溶解された後、最終濃度がそれぞれ 50 M及び 100 Mとなるように反応混合物に添カ卩した。  [0036] <Polymerization Atsey> 140 μΜ a S, 0-50 μΜ NDGA, Cur, RA, FA, TA, Myr, Kmp, Cat, epi-Cat, RIF or TC, 1% dimethyl sulfoxide (DMSO ), 20 mM Tris buffer, pH 7.5, and lOO mM NaCl, a reaction mixture for polymerization assembly was prepared. NDGA, Cur, RA, FA, wine-related polyphenols (TA, Myr, Kmp, Cat ゝ epi— Cat) RIF and TC are dissolved in DMSO at concentrations of 1 M, 10 M, 100 μM, ImM and 5 mM. After that, the reaction mixture was added to final concentrations of 0.01 M, 0.1 M, 1 M, 10 M and 50 M, respectively. DHA and EPA were dissolved in DMSO at concentrations of 5 mM and 10 mM, and then added to the reaction mixture so that the final concentrations were 50 M and 100 M, respectively.
[0037] 前記混合物のうち 30 μ Lをオイルフリー PCRチューブに移した。反応チューブをイン キュベータ一に入れ、プレート温度を開始温度 4°Cから 37°Cまで最大速度で上昇さ せた。そして、後の図で示すように、反応チューブ内をマイクロビーズを用いて 0〜6 日間撹拌し、氷上に置くことで反応を停止さ  [0037] 30 μL of the mixture was transferred to an oil-free PCR tube. The reaction tube was placed in an incubator and the plate temperature was increased from the starting temperature of 4 ° C to 37 ° C at maximum speed. Then, as shown in the following figure, the reaction tube was stirred for 0-6 days using microbeads and placed on ice to stop the reaction.
せた。それぞれの反応チューブ力 5 L分量を 3つ分取し、蛍光分光計で調べ、そ れぞれ 3つの平均値を決定した。チオフラビン S溶液中では、前記化合物は反応混 合物濃度の 200分の 1まで希釈された。これら化合物がチオフラビン Sの蛍光を前記 希釈濃度で消光させないことは実験的に確かめられている。  Let Three 5 L aliquots of each reaction tube were collected and examined with a fluorescence spectrometer, and the average value of each was determined. In thioflavin S solution, the compound was diluted to 1/200 of the reaction mixture concentration. It has been experimentally confirmed that these compounds do not quench the fluorescence of thioflavin S at the diluted concentration.
[0038] 結果を図 1に示す。図 l (a)〜（d)は、 140 Mの a S、 pH7. 5の 20mMのトリスバッ ファー、 lOOmMの NaCl、及び各化合物を 0 M (參）、 10 M (〇）、又は 50 μ Μ ( □)の濃度で含む反応混合物を 37°Cで所定時間インキュベートした結果である。そ れぞれの図は、 3つの独立した実験の代表的なパターンを示している。また、図 1 (e) は、 140 ^ Μ α 8, ρΗ7. 5、 20mMのトリスバッファー、 lOOmMの NaCl、及び RA( 參）、 NDGA(〇）、 Cur (國）、及び TC (口）を 0、 0. 01、 0. 1、 1、 10又は 50 M含 む反応混合物を、 37°Cで 6時間インキュベートした結果である。それぞれのポイント は、 3つの独立した実験の平均を表している。全てのポイントで、標準誤差は記号の 内側であった。化合物なしの平均を 100%とみなした。 [0038] The results are shown in FIG. Figures l (a)-(d) show 140 M a S, 20 mM Tris buffer at pH 7.5, lOO mM NaCl, and each compound at 0 M (參), 10 M (◯), or 50 μΜ This is a result of incubating a reaction mixture containing a concentration of (□) at 37 ° C for a predetermined time. Each figure shows a typical pattern of three independent experiments. Figure 1 (e) shows 140 ^ Μ α8, ρΗ7.5, 20 mM Tris buffer, lOOmM NaCl, RA ((), NDGA (〇), Cur (country), and TC (mouth). This is the result of incubation of a reaction mixture containing 0, 0.01, 0.1, 1, 10 or 50 M at 37 ° C for 6 hours. Each point represents the average of three independent experiments. At all points, the standard error is the symbolic It was inside. The average without compound was taken as 100%.
[0039] 〈原線維不安定化活性の測定〉 70 μ Μの新鮮な f a S、 0〜50 μ Μの NDGA、 Cur 、 RA、 FA、ワイン関連ポリフエノール、 DHA、 EPA、 RIF又は TC、 l %DMSO、 20 mMの pH7. 5のトリスバッファー、 lOOmMの NaCl、及び 1 % (wtZvol)ポリビュル アルコールを含む不安定ィ匕測定用反応混合物を調製した。ポリビニルアルコールは[0039] <Measurement of fibril destabilizing activity> 70 μΜ fresh fa S, 0-50 μΜ NDGA, Cur, RA, FA, wine-related polyphenols, DHA, EPA, RIF or TC, l A reaction mixture for measuring lability was prepared containing% DMSO, 20 mM Tris buffer at pH 7.5, lOOmM NaCl, and 1% (wtZvol) polybutyl alcohol. Polyvinyl alcohol is
、 f a Sの凝集と反応中の反応チューブ内壁への f a Sの吸着とを回避する目的で用 いたものである。 NDGA、 Cur、 RA、 FA、ワイン関連ポリフエノール、 RIF、及び TC は、濃度 1 M、 10 Μ、 100 μ Μ、 lmM、 5mMとして DMSOに溶解された後、 最終濃度 0. 01 /ζ Μ、 0. 1 /ζ Μ、 1 /ζ Μ、 10 Μ及び 50 Μとなるように反応混合 物に添カ卩した。 DHA及び ΕΡΑは、濃度 5mM及び 10mMとして DMSOに溶解され た後、最終濃度 50 M及び 100 Mとなるように反応混合物に添加した。 This is used for the purpose of avoiding the aggregation of f a S and the adsorption of f a S to the inner wall of the reaction tube during the reaction. NDGA, Cur, RA, FA, wine-related polyphenols, RIF, and TC are dissolved in DMSO at concentrations of 1 M, 10 Μ, 100 μΜ, lmM, 5 mM, followed by a final concentration of 0.01 / ζ Μ, The reaction mixture was charged to 0.1 / ζΜ, 1 / ζΜ, 10Μ and 50Μ. DHA and soot were dissolved in DMSO at concentrations of 5 mM and 10 mM, and then added to the reaction mixture to a final concentration of 50 M and 100 M.
[0040] ピペッティング後、 30 μ Lを PCRチューブに移した。反応チューブをインキュベータ 一に入れ、プレート温度を開始温度 4°Cから 37°Cまで最大速度で上昇させた。そし て、後の図で示すように、反応チューブ内をマイクロビーズを用いて 0〜6日間撹拌し 、氷上に置くことで反応を停止させた。それぞれの反応チューブから 5 L分量を 3つ 分取し、蛍光分光計で調べ、それぞれ 3つの平均値を決定した。前記希釈濃度で、 4 °Cと 37°Cのどちらにおいても、前述の化合物は f a Sに対してチオフラビン Sと競合し なかった (データ示さず。）。  [0040] After pipetting, 30 μL was transferred to a PCR tube. The reaction tube was placed in an incubator and the plate temperature was increased at a maximum rate from a starting temperature of 4 ° C to 37 ° C. Then, as shown in the following figure, the reaction tube was stirred for 0-6 days using microbeads and placed on ice to stop the reaction. Three 5 L aliquots were taken from each reaction tube and examined with a fluorescence spectrometer to determine the average of each. At the dilution concentrations, the compounds did not compete with thioflavin S for f a S at either 4 ° C or 37 ° C (data not shown).
[0041] 結果を図 3に示す。図 3 (a)〜（d)は、 70 μ Μの f a S、 pH7. 5の 20mMトリスバッフ ァー、 lOOmMの NaCl、及び各化合物を M (拳）、 ΙΟ Μ (〇）又は 50 Μ (口） の濃度で含む反応混合物を、 37°Cで所定時間インキュベートした結果である。それ ぞれの図は、 3つの独立した実験の代表的なパターンを示している。また、図 3 (e)は 、 70 ^ Μ( ί a S, pH7. 5の 20mMトリスバッファー、 lOOmMの NaCl及び RA (拳） 、 NDGA(〇）、 Cur (國）、及び TC (口）を 0、 0. 01、 0. 1、 1、 10又は 50 M含む 反応混合物を 37°Cで 6時間インキュベートした結果である。それぞれのポイントは、 3 つの独立した実験の平均を表している。全てのポイントで、標準誤差は記号の内側 であった。化合物なしの平均を 100%とみなした。  [0041] The results are shown in FIG. Figure 3 (a)-(d) shows 70 μΜ fa S, 20 mM Tris buffer pH 7.5, lOO mM NaCl, and each compound as M (fist), ΙΟ Μ (〇) or 50 Μ (mouth). This is a result of incubation of a reaction mixture containing a concentration of) at 37 ° C for a predetermined time. Each figure shows a typical pattern of three independent experiments. Fig. 3 (e) shows 70 ^ Μ (20 mM Tris buffer with ί a S, pH 7.5, lOOmM NaCl and RA (fist), NDGA (〇), Cur (country), and TC (mouth). Results of incubation of reaction mixtures containing 0, 0.01, 0.1, 1, 10 or 50 M for 6 hours at 37 ° C. Each point represents the average of three independent experiments. The standard error at the point was within the symbol, and the mean without compound was taken as 100%.
[0042] 〈他の分析方法〉 遠心分離後の反応混合物の上清のタンパク質濃度は、タンパク質 アツセィキット（Bio- rad Laboratories社製）を用いたブラッドフォード法（1976)によつ て決定した。有効濃度 (EC )は、 f a Sの形成又は伸長を対照値の 50%に抑制する [0042] <Other analysis methods> The protein concentration in the supernatant of the reaction mixture after centrifugation is the protein concentration It was determined by the Bradford method (1976) using an Atsey kit (Bio-rad Laboratories). Effective concentration (EC) inhibits fa S formation or elongation to 50% of control value
50  50
NDGA、 Cur、 RA、 FA、ワイン関連ポリフエノール、 RIF又は TCの濃度、又は、 f a Sを対照値の 50%に不安定ィ匕させる濃度として定義した。 EC は、 Igor Pro ver.5を  NDGA, Cur, RA, FA, wine-related polyphenols, RIF or TC concentrations, or concentrations that make f a S unstable to 50% of control values. EC uses Igor Pro ver.5
50  50
用い、図 1 (e)及び図 3 (e)に示すようなデータのシグモイドカーブに当てはめて算出 した。  Calculations were made using the sigmoid curve of the data shown in Fig. 1 (e) and Fig. 3 (e).
[0043] 〈新鮮な a Sからの f a S形成反応速度に対する各化合物の影響〉 以下、実験 1の結 果について説明する。 図 1 (a)〜（d)に示すように、新鮮な a Sを 37°Cでインキュべ ーシヨンすることにより生成したチオフラビン Sの蛍光は、特徴的なシグモイドカーブを 示した。このカーブは、核依存性重合モデルに一致している。また、データは示さな Vヽが、 10 μ Μ及び 50 μ Μの DHA及び EPAは、 f a S形成を抑制しなかった。  <Effect of each compound on the reaction rate of f a S formation from fresh a S> Hereinafter, the results of Experiment 1 will be described. As shown in Fig. 1 (a) to (d), the fluorescence of thioflavin S produced by incubating fresh a S at 37 ° C showed a characteristic sigmoid curve. This curve is consistent with the nucleus dependent polymerization model. In addition, data are not shown. DHA and EPA with V 10 of 10 μΜ and 50 μΜ did not inhibit f a S formation.
[0044] なお、 Cur等を含まない反応混合物を 37°Cで 6時間インキュベーションした後、電子 顕微鏡により観察したところ、 f « Sの形成が明確に確認された（図 2 (a) )。一方、 Cur を含む反応混合物を同条件でインキュベーションし、電子顕微鏡により観察したとこ ろ、 f a S形成が抑制されて ヽることが確認された（図 2 (b) )。  [0044] The reaction mixture containing no Cur or the like was incubated at 37 ° C for 6 hours, and then observed with an electron microscope. As a result, the formation of f «S was clearly confirmed (Fig. 2 (a)). On the other hand, when the reaction mixture containing Cur was incubated under the same conditions and observed with an electron microscope, it was confirmed that the formation of fa S was suppressed (FIG. 2 (b)).
[0045] 〈原線維不安定ィ匕アツセィ〉 図 3 (a)〜（d)に示すように、化合物を添加しな 、状態 で新鮮な f a Sを 37°Cでインキュベーションしている間、チオフラビン Sの蛍光はほと んど変化しなカゝつた。他方、 DHA及び EPAの添加を除き、 Cur等を反応混合物に 添加した後、チオフラビン Sの蛍光は直ちに減少した。  <Fibrillar instability> As shown in Fig. 3 (a) to (d), thioflavine was incubated at 37 ° C with fresh fa S in the state without compound addition. The fluorescence of S was almost unchanged. On the other hand, the fluorescence of thioflavin S immediately decreased after adding Cur etc. to the reaction mixture, except for the addition of DHA and EPA.
[0046] なお、インキュベーション後の反応混合物を電子顕微鏡により観察した。インキュべ ーシヨン前の反応混合物にお 、ては、 f a Sが明瞭に観察された（図 4 (a) )のに対し、 50 μ Μの Curを含む反応混合物を 1時間インキュベーションした後には、短くせん断 された原線維が多数観察された。 6時間インキュベーションした後の前記反応混合物 においては、原線維の数は著しく減少し、小さな不定形の凝集体が時折観察された（ 図 4 (b) )。なお、 TA、 TC、 FA、 Myr、 Kmp、 Cat及び epi— Catにおいても形態学 的に不安定化所見が見られたが、 DHA及び EPAは f a Sを不安定ィ匕させなカゝつた。  [0046] The reaction mixture after the incubation was observed with an electron microscope. In the pre-incubation reaction mixture, fa S was clearly observed (Figure 4 (a)), whereas after incubation of the reaction mixture containing 50 μΜ Cur for 1 hour, A number of sheared fibrils were observed. In the reaction mixture after 6 hours of incubation, the number of fibrils was markedly reduced and small amorphous aggregates were occasionally observed (Figure 4 (b)). TA, TC, FA, Myr, Kmp, Cat, and epi-Cat also showed morphological destabilization, but DHA and EPA did not destabilize faS.
[0047] 1. 6 X 104gで 4°C、 2時間の遠心分離を行った後の上清を、ブラッドフォードアツセィ により分析した。その結果、上清力もタンパク質は検出されな力つた。これは、前述し た化合物は f a Sを電子顕微鏡的に観察できる凝集体にまで不安定化することができ るけれども、 a Sのモノマー又はオリゴマーに脱重合 (depolymerize)することはできな いということを意味している。 [0047] 1. After centrifugation at 6 x 10 4 g for 4 hours at 4 ° C, the supernatant was added to Bradford Atsey Was analyzed. As a result, the supernatant force was also strong without protein being detected. This is because the aforementioned compounds can destabilize fa S to aggregates that can be observed by electron microscopy, but cannot be depolymerized to a monomer or oligomer of a S. It means that.
[0048] 〈各化合物の活性の比較〉 図 1 (e)及び図 3 (e)に示すように、 NDGA、 Cur、 RA及 び TCは用量依存的に f a Sの形成及び伸長を抑制し、同様に用量依存的に予め形 成しておいた f a Sを不安定ィ匕した。ここで、図 1 (e)及び図 3 (e)に示すようなデータ のシグモイド曲線に当てはめて、 EC (f a Sの形成を対照値の 50%に抑制する ND <Comparison of the activity of each compound> As shown in Fig. 1 (e) and Fig. 3 (e), NDGA, Cur, RA and TC inhibited fa S formation and elongation in a dose-dependent manner, Similarly, the pre-formed fa S was unstable in a dose-dependent manner. Here, it is applied to the sigmoid curve of the data as shown in Fig. 1 (e) and Fig. 3 (e) to suppress EC (f a S formation to 50% of the control value.
50  50
GA、 Cur、 RA、 FA、ワイン関連ポリフエノール、 RIF又は TCの濃度、又は f a Sを対 照値の 50%に不安定ィ匕させる濃度）を算出した。結果を表 1に示す。  The concentration of GA, Cur, RA, FA, wine-related polyphenol, RIF or TC, or the concentration that makes f a S unstable to 50% of the reference value) was calculated. The results are shown in Table 1.
[0049] [表 1] [0049] [Table 1]
Figure imgf000016_0001
Figure imgf000016_0001
TA、 NDGA、 Cur、 RA、 Myr、 RIF及び TCの f a Sの形成を抑制する EC EC that suppresses the formation of f a S in TA, NDGA, Cur, RA, Myr, RIF and TC
50は、 f a 50 is f a
S不安定化の EC と類似していた。他方、 FA、 Kmp、 Cat及び epi— Catの f a Sを Similar to EC with S instability. On the other hand, FA, Kmp, Cat, and epi-Cat f a S
50  50
不安定化させる EC は、 f a Sの形成を抑制する EC より一桁高い値を示した。前記  The destabilized EC was an order of magnitude higher than the EC that inhibited the formation of f a S. Above
50 50  50 50
表 1のデータより、検討した化合物の f a Sに関する抗原線維形成及び原線維不安定 化活性の強さは、以下の順であることが明らかとなった。 TA=NDGA= Cur=RA = Myr>Kmp = FA > Cat = epi- Cat >RIF=TC From the data in Table 1, antigen fibril formation and fibril instability related to fa S of the studied compounds It became clear that the strength of the activating activity was in the following order. TA = NDGA = Cur = RA = Myr> Kmp = FA> Cat = epi- Cat> RIF = TC
[0051] 以上の実験より、 NDGA、 Cur及び RA力 インビトロで用量依存的に f a Sの形成を 抑制し、予め形成しておいた f a Sを不安定ィ匕するということが明ら力となった。また、 NDGA、 Cur及び RAは、 a Sの線維形成抑制効果を持つ化合物としてこれまでに 知られている TA及び Myrのようなワイン関連ポリフエノールと同等に、非常に強い a S線維形成抑制効果及び不安定ィ匕効果を持つことが明らかとなった。したがって、こ れら 3種の化合物は、 a Sの凝集が関与する各種レビー小体病のコントロールに有用 であることが確認された。また、これら 3種の化合物は、 f a Sを不安定ィ匕することが可 能であることから、 ex Sの凝集が関与する各種レビー小体病の予防に有用であること が確認された。 [0051] From the above experiments, it is clear that NDGA, Cur, and RA force inhibit fa S formation in a dose-dependent manner in vitro and destabilize pre-formed fa S. It was. In addition, NDGA, Cur and RA have a very strong a S-fibrogenesis inhibitory effect similar to wine-related polyphenols such as TA and Myr, which have been known as compounds that have a S-fibrosis-inhibiting effect. And it became clear that it has an unstable effect. Therefore, it was confirmed that these three compounds are useful for the control of various Lewy body diseases in which a S aggregation is involved. In addition, these three compounds were able to destabilize fa S, and thus were confirmed to be useful for the prevention of various Lewy body diseases involving ex S aggregation.
[0052] (実験 2) 本実験では、ビタミン A (全トランスレチノール、全トランスレチナール、全ト ランスレチノイン酸）、 βカロチン、補酵素 Q (η= 10) (CoQ )等の f a S形成に与え  [0052] (Experiment 2) In this experiment, it was given to fa S formation such as vitamin A (all-trans retinol, all-trans retinal, all-trans retinoic acid), β-carotene, coenzyme Q (η = 10) (CoQ).
10  Ten
る影響について調べた。また、 f a S不安定ィ匕効果についても調べた。本実験で用い た各化合物の構造を下記に示す。  We investigated the effects of this. We also investigated the f a S instability effect. The structure of each compound used in this experiment is shown below.
[0053] [化 12] [0053] [Chemical 12]
Figure imgf000017_0001
レ /— Jレ
Figure imgf000017_0001
Les / — J
[0054] [化 13]
Figure imgf000017_0002
レチナ ^—ル [0055] [化 14] [0054] [Chemical 13]
Figure imgf000017_0002
Retina ^ —Le [0055] [Chemical 14]
Figure imgf000018_0001
Figure imgf000018_0001
レチノィン  Retinoin
[0056] [化 15]
Figure imgf000018_0002
[0056] [Chemical 15]
Figure imgf000018_0002
力□チン  Power □ Chin
[0057] [化 16] [0057] [Chemical 16]
Figure imgf000018_0003
Figure imgf000018_0003
し OW10  OW10
[0058] く《S及び f a Sの準備〉 前述の実験 1と同様にして準備した。  <Preparation of S and f a S> Preparations were made in the same manner as in Experiment 1 described above.
[0059] 〈蛍光分析〉 前述の実験 1と同様にして行った。 [0060] 〈原子間力顕微鏡 (AFM)〉 f « S溶液をゆっくりと混合した後、 AFM分析用に一定 分量（20 μ 1)を取り出した。取り出した f a S溶液を新たに劈開した雲母基板に適用し 、常温で 10分放置した。 Millipore社のフィルターを通した水で雲母基板をすすぎ（ 3 X 50 1)、軽く結合しているタンパク質を取り除き、 COの流れで乾かした。 JVスキ <Fluorescence analysis> [0059] The analysis was performed in the same manner as in Experiment 1 described above. <Atomic Force Microscope (AFM)> f «S solution was slowly mixed, and then an aliquot (20 μ 1) was taken out for AFM analysis. The taken out fa S solution was applied to a newly cleaved mica substrate and left at room temperature for 10 minutes. The mica substrate was rinsed with water through a Millipore filter (3 X 50 1) to remove lightly bound proteins and dried with CO flow. JV Ski
2  2
ャナーを装備したマルチモード走査型プローブ顕微鏡と Nanoscope Ilia制御装置 (Digital Instruments社製）を用いてサンプルをただちにイメージ化した。全ての 測定は、周辺条件下でシングルビーム方式のシリコン片持ち梁探触子を使 、タツピ ングモードで行われた。 AFM画像の大きさは通常 512 X 512ピクセルとした。サンプ ル中に同様の物質が存在して 、ることを裏付けるために、雲母基板上の少なくとも 4 箇所を測定した。区分解析は、個々の種に線を引き、雲母基板表面から構造の上部 までの距離を測定して行った。これを 10回繰り返し、線維構造が確かめられたときの み平均を求めた。結果を図 6、図 8に示す。  Samples were imaged immediately using a multimode scanning probe microscope equipped with a channel and a Nanoscope Ilia controller (Digital Instruments). All measurements were performed in a tapping mode using a single-beam silicon cantilever probe under ambient conditions. The size of the AFM image was usually 512 x 512 pixels. At least four locations on the mica substrate were measured to confirm that similar substances were present in the sample. The compartmental analysis was performed by drawing a line for each species and measuring the distance from the mica substrate surface to the top of the structure. This was repeated 10 times, and the average was obtained only when the fiber structure was confirmed. The results are shown in Figs.
[0061] 〈重合アツセィ〉 140 μ Μの a S、 0〜： LOO μ Μの NDGA、 Myr、 RIFゝテトラサイタリ ン（TC)、ビタミン類、 βカロチン、 CoQ 又は α—リポ酸（LA)、 1 %ジメチルスルホ [0061] <Polymerization Atssey> 140 μΜ a S, 0-: LOO μΜ NDGA, Myr, RIF ゝ tetracytalin (TC), vitamins, β-carotene, CoQ or α-lipoic acid (LA), 1 % Dimethylsulfo
10  Ten
キシド（DMSO)、 pH7. 5、 20mMトリスバッファー、 lOOmMの NaClを含む重合ァ ッセィ用反応混合物を調製した。 NDGA、 Myr、 RIF、 TC、ビタミン A (レチノール、 レチナール、レチノイン酸）、 βカロチン、 CoQ (Sigma社製）は、 1 M、 10 M、  A reaction mixture for a polymerization assay was prepared containing xoxide (DMSO), pH 7.5, 20 mM Tris buffer, lOO mM NaCl. NDGA, Myr, RIF, TC, Vitamin A (retinol, retinal, retinoic acid), β-carotene, CoQ (Sigma) are 1 M, 10 M,
10  Ten
100 /z M、 lmM、 5mMの濃度で DMSOに溶解した後、最終濃度がそれぞれ 0. 0 1、 0. 1、 1、 10、 50 Mになるように反応混合物に添カ卩した。他のビタミン (ビタミン B 2 (リボフラビン）、ビタミン B6 (ピリドキシン）、ビタミン C、ビタミン E)は、 5mM及び 10 mMとなるよう DMSOに溶解し、最終濃度が 50 M及び 100 Mとなるように反応 混合物に添加した。  After dissolving in DMSO at concentrations of 100 / z M, lmM, and 5 mM, they were added to the reaction mixture so that the final concentrations were 0.01, 0.1, 1, 10, and 50 M, respectively. Other vitamins (vitamin B 2 (riboflavin), vitamin B6 (pyridoxine), vitamin C, vitamin E) are dissolved in DMSO to 5 mM and 10 mM, and reacted to a final concentration of 50 M and 100 M. Added to the mixture.
[0062] その後は、実験 1と同様にして測定した。すなわち、前記混合物のうち 30 Lをオイ ルフリー PCRチューブに移した。反応チューブをインキュベーターに入れ、プレート 温度を開始温度 4°Cから 37°Cまで最大速度で上昇させた。そして、後の図で示すよ うに、反応チューブ内をマイクロビーズを用いて 0〜6日間撹拌し、氷上に置くことで 反応を停止させた。それぞれの反応チューブ力 5 L分量を 3つ分取し、蛍光分光 計 で調べ、それぞれ 3つの平均値を決定した。チオフラビン S溶液中では、前記化合物 は反応混合物濃度の 200分の 1まで希釈された。これら化合物がチオフラビン Sの蛍 光を前記希釈濃度で消光させないことは実験的に確かめられている。 [0062] Thereafter, measurement was performed in the same manner as in Experiment 1. That is, 30 L of the mixture was transferred to an oil-free PCR tube. The reaction tube was placed in an incubator and the plate temperature was increased from the starting temperature of 4 ° C to 37 ° C at maximum speed. Then, as shown in the subsequent figure, the reaction tube was stirred for 0-6 days using microbeads and placed on ice to stop the reaction. Take three reaction tube forces (5 L) and use a fluorescence spectrometer. And determined three average values for each. In thioflavin S solution, the compound was diluted to 1/200 of the reaction mixture concentration. It has been experimentally confirmed that these compounds do not quench the thioflavin S fluorescence at the diluted concentration.
[0063] 結果を図 5に示す。図 5 (a)〜（d)は、 140 μ Μの a S、 pH7. 5の 20mMのトリス緩 衝液、 lOOmMの NaCl、及び各化合物を 0 M (參）、 10 M (〇）、又は 50 μ Μ ( 口）の濃度で含む反応混合物を 37°Cで所定時間インキュベーションした結果である 。それぞれの図は、 3つの独立した実験の代表的なパターンを示している。また、図 5 (e)は、 140 μ Μの a S、 pH7. 5、 20mMのトリスノ ッファー、 lOOmMの NaCl、及 び NDGA (參）、レチノール（〇）、レチノイン酸（園）、 CoQ (口）を 0、 0. 01、 0. 1、 [0063] The results are shown in FIG. Figures 5 (a) to (d) show 140 μΜ a S, 20 mM Tris buffer, pH 7.5, lOO mM NaCl, and each compound at 0 M (參), 10 M (◯), or 50 This is the result of incubation of a reaction mixture containing a concentration of μΜ (mouth) at 37 ° C for a predetermined time. Each figure shows a typical pattern of three independent experiments. Figure 5 (e) shows 140 μΜ a S, pH 7.5, 20 mM Trisnofer, lOO mM NaCl, NDGA (參), retinol (〇), retinoic acid (garden), CoQ (mouth) ) 0, 0.01, 0.1,
10  Ten
1、 10又は 50 /z M含む反応混合物を、 37°Cで 6時間インキュベーションした結果で ある。それぞれのポイントは、 3つの独立した実験の平均を表している。全てのポイン トで、標準誤差は記号の内側であった。化合物なしの平均を 100%とみなした。  This is the result of incubation of a reaction mixture containing 1, 10 or 50 / z M at 37 ° C for 6 hours. Each point represents the average of three independent experiments. At all points, the standard error was inside the symbol. The average without compound was taken as 100%.
[0064] 〈原線維不安定化活性の測定〉 原線維不安定化活性は、実験 1と同様に測定した。 <Measurement of fibril destabilizing activity> The fibril destabilizing activity was measured in the same manner as in Experiment 1.
NDGA、 Myr、 RIF、 TC、ビタミン A、 βカロチン、 CoQ は、 1 M、 10 M、 100  NDGA, Myr, RIF, TC, Vitamin A, β-Carotene, CoQ, 1 M, 10 M, 100
10  Ten
M、 lmM、 5mMの濃度で DMSOに溶解した後、最終濃度がそれぞれ 0. 01、 0 . 1、 1、 10、 50 Mになるように反応混合物に添加された。その他のビタミンや LA は、 5mMと 10mMの濃度で DMSOに溶解した後、最終濃度がそれぞれ 50、 100 μ Μになるように反応混合物に添加された。それぞれの反応チューブから 5 Lを取 り出し蛍光分光計で調べ、 30 Lを PCRチューブに入れた。前記希釈濃度では、 4 °Cと 37°Cのどちらにおいても、前述の化合物は f a Sに対してチオフラビン Sと競合し なかった。  After dissolving in DMSO at concentrations of M, lmM and 5 mM, they were added to the reaction mixture to final concentrations of 0.01, 0.1, 1, 10, and 50 M, respectively. Other vitamins and LA were dissolved in DMSO at concentrations of 5 mM and 10 mM, and then added to the reaction mixture so that the final concentrations were 50 and 100 μΜ, respectively. 5 L was taken from each reaction tube and examined with a fluorescence spectrometer, and 30 L was placed in a PCR tube. At the dilution concentration, the compound did not compete with thioflavin S for f a S at either 4 ° C or 37 ° C.
[0065] 結果を図 7に示す。図 7 (a)〜（d)は、 70 μ Μの f a S、 pH7. 5の 20mMのトリスバッ ファー、 lOOmMの NaCl、および Ο Μ (拳）、 ΙΟ Μ (〇）、または 50 Μ (口）の濃 度でレチノール (a)、レチナール (b)、レチノイン酸 (c)、 CoQ (d)を含む反応混合  The results are shown in FIG. Figure 7 (a)-(d) shows 70 μΜ fa S, 20 mM Tris buffer, pH 7.5, lOO mM NaCl, and Ο Μ (fist), ΙΟ Μ (〇), or 50 Μ (mouth) Reaction mixture containing retinol (a), retinal (b), retinoic acid (c), CoQ (d)
10  Ten
物を 37°Cで所定時間インキュベーションした結果である。それぞれのポイントは、 3つ の独立した実験の平均を表している。全てのポイントで、標準誤差は記号の内側であ つた。化合物なしの平均を 100%とみなした。また、図 7 (e)は、 70 Mf α S、 20m Mのトリスバッファー、 pH7. 5、 lOOmMの NaCl、及び NDGA (參）、レチノール（〇 )、レチノイン酸（園）、 CoQ (口）を 0、 0. 01、 0. 1、 1、 10、 50/zM含む反応混合 This is the result of incubation of the product at 37 ° C for a predetermined time. Each point represents the average of three independent experiments. At all points, the standard error was inside the symbol. The average without compound was taken as 100%. Figure 7 (e) shows 70 Mf α S, 20 mM Tris buffer, pH 7.5, lOO mM NaCl, NDGA (參), and retinol (〇). ), Retinoic acid (garden), CoQ (mouth) containing 0, 0.01, 0.1, 1, 10, 50 / zM
10  Ten
物を、 37°Cで 6時間インキュベーションした結果である。それぞれのポイントは、 3つ の独立した実験の平均を表している。全てのポイントで、標準誤差は記号の内側であ つた。化合物なしの平均を 100%とみなした。  This is the result of incubation of the product at 37 ° C for 6 hours. Each point represents the average of three independent experiments. At all points, the standard error was inside the symbol. The average without compound was taken as 100%.
[0066] 〈他の分析方法〉 遠心分離後の反応混合物の上清のタンパク質濃度は、実験 1と同 様にして決定した。有効濃度 (EC )は、 f a Sの形成又は伸長を対照値の 50%に抑 <Other Analysis Methods> The protein concentration of the supernatant of the reaction mixture after centrifugation was determined in the same manner as in Experiment 1. The effective concentration (EC) suppresses f a S formation or elongation to 50% of the control value.
50  50
制する NDGA, Mvr， RIF， TC，ビタミン A, j8カロチン又は CoQ の濃度、又は、 f  NDGA, Mvr, RIF, TC, vitamin A, j8 carotene or CoQ concentration or f
10  Ten
aSを対照値の 50%に不安定ィ匕させる濃度として定義した。 EC は実験 1と同様に  aS was defined as the concentration that caused instability to 50% of the control value. EC as in Experiment 1
50  50
算出した。  Calculated.
[0067] 〈新鮮な《Sからの fa S形成反応速度に対する各化合物の影響〉 以下、実験 2の結 果について説明する。 図 5から明らかなように、 10/ζΜ、 50 Μのレチノール、レチ ナール、レチノイン酸又は CoQ と a Sとを組み合わせてインキュベーションすると、  [0067] <Effect of each compound on the reaction rate of fa S formation from fresh S> Hereinafter, the results of Experiment 2 will be described. As is clear from FIG. 5, incubation with 10 / ζΜ, 50 レ retinol, retinal, retinoic acid or CoQ in combination with a S
10  Ten
最終平衡レベルは用量依存的に減少した。データは示さないが、 10/zM及び 50 Mのビタミン B2、ビタミン B6、ビタミン C、ビタミン E、 a リポ酸は、 f a S形成を抑制 しなかった。  The final equilibrium level decreased in a dose-dependent manner. Although data are not shown, 10 / zM and 50 M vitamin B2, vitamin B6, vitamin C, vitamin E, a lipoic acid did not inhibit f a S formation.
[0068] また、電子顕微鏡 (EM)及び原子間力顕微鏡 (AFM)を使った観察により、ビタミン A、 j8カロチン、 CoQ 力 a S形成を抑制することがわかった（図 6)。ビタミン A等を  [0068] Further, observation using an electron microscope (EM) and an atomic force microscope (AFM) showed that formation of vitamin A, j8 carotene, and CoQ force a S was suppressed (Fig. 6). Vitamin A, etc.
10  Ten
添加しない場合、原線維形成が明確に確認された（図 6(a)、（d))。図 6(a)から、 fa Sは非分岐繊維構造をとると推測される。タッピングモード AFMでは EMよりも高 、解 像度で画像ィ匕できた。 AFMによると、この典型的な繊維状構造の高さは 6. 96±1. 08nm (平均士標準偏差、 n=10)だった（図 6(d))。 50 Mレチノールとともにイン キュベーシヨンした場合、 fa S形成は強く抑制された（図 6(b)、（e))。同様に、レチ ナール、レチノイン酸、 j8カロチンも fa Sの形成を抑制した（データ示さず)。 CoQ  When not added, fibril formation was clearly confirmed (FIGS. 6 (a) and (d)). From Fig. 6 (a), fa S is assumed to have an unbranched fiber structure. Tapping mode AFM was higher than EM, and the image was able to be displayed with high resolution. According to AFM, the height of this typical fibrous structure was 6.96 ± 1.08 nm (mean standard deviation, n = 10) (Figure 6 (d)). When incubated with 50 M retinol, fa S formation was strongly suppressed (Figs. 6 (b) and (e)). Similarly, retinal, retinoic acid, and j8 carotene also inhibited fa S formation (data not shown). CoQ
10 も、 f a Sの形成抑制が電子顕微鏡により観察された（図 6(c))。 AFMによると、 CoQ で抑制された短繊維構造の高さは 4. 84±0. 78nm (平均士標準偏差、 n=10) 10, the suppression of the formation of f a S was observed with an electron microscope (Fig. 6 (c)). According to AFM, the height of the short fiber structure suppressed by CoQ is 4.84 ± 0.78nm (average standard deviation, n = 10)
10 Ten
だった (図 6(f))。  (Fig. 6 (f)).
[0069] 〈原線維不安定ィ匕アツセィ〉 図 7から明らかなように、ビタミン A、 |8カロチン、 CoQ  [0069] <Fibrotic instability 匕 Atssey> As shown in Fig. 7, vitamin A, | 8 carotene, CoQ
10 はチオフラビン Sの蛍光を減少させた。新鮮な 70 μ Μの a Sと 50 μ Μのレチノールと を 1時間インキュベーションすると、短ぐせん断された線維が多数観察された（図 8 ( b)、（f) )。 AFMによると、アツセィ前の伸長していた線維は高さ 7. 44±0. 49nm、 1時間後の短くせん断された線維は 5. 42±0. 67nmだった（平均士標準偏差、 n= 10) (図 8 (e)、（f) )。 6時間後には線維の数は著しく減少し、小さな不定形の凝集体 が時折観察された（図 8 (c)、（g) )。レチナール、レチノイン酸、 |8カロチンも予め形 成してぉ 、た f a Sを不安定化させた（データ示さず）。 50 μ Μの CoQ の存在下で 6 10 decreased thioflavin S fluorescence. With 70 μΜ fresh a S and 50 μΜ retinol After incubation for 1 hour, many short sheared fibers were observed (Fig. 8 (b), (f)). According to AFM, the stretched fibers before Atsey were 7.44 ± 0.49 nm in height, and the short sheared fibers after 1 hour were 5.42 ± 0.67 nm (mean standard deviation, n = 10) (Fig. 8 (e), (f)). After 6 hours, the number of fibers decreased markedly, and small irregular aggregates were occasionally observed (Fig. 8 (c), (g)). Retinal, retinoic acid, and | 8 carotene were also pre-formed to destabilize fa S (data not shown). 6 in the presence of 50 μΜ CoQ
10  Ten
時間インキュベーションすると、短ぐせん断された線維が多数観察された (図 8 (D) ) 。これに対し、ビタミン B2、ビタミン B6、ビタミン C、ビタミン E、 a—リポ酸は、あらかじ め形成してぉ 、た f a Sを不安定ィ匕させなかった（データ示さず)。  Many short sheared fibers were observed after incubation for a period of time (Fig. 8 (D)). In contrast, vitamin B2, vitamin B6, vitamin C, vitamin E, and a-lipoic acid formed in advance did not destabilize f a S (data not shown).
[0070] 1. 6 X 104gで 4°C、 2時間の遠心分離を行った後の上清を、ブラッドフォードアツセィ により分析した。その結果、上清力もタンパク質は検出されな力つた。これは、前述し た化合物は f a Sを電子顕微鏡的に観察できる凝集体にまで不安定ィ匕することができ るけれども、 a Sのモノマー又はオリゴマーに脱重合 (depolymerize)することはできな いということを意味している。 [0070] 1. The supernatant after centrifugation at 6 × 10 4 g at 4 ° C. for 2 hours was analyzed by Bradford Atsey. As a result, the supernatant force was also strong without protein being detected. This is because the compounds described above can destabilize fa S to aggregates that can be observed by electron microscopy, but cannot depolymerize to a S monomer or oligomer. It means that.
[0071] 〈各化合物の活性の比較〉  <Comparison of the activity of each compound>
図 5 (e)及び図 7 (e)から、 NDGA、 Myr、 RIF、 TC、レチノール、レチナール、レチ ノイン酸、 βカロチン、 CoQ は用量依存的に f a Sの形成及び伸長を抑制し、予め  From Fig. 5 (e) and Fig. 7 (e), NDGA, Myr, RIF, TC, retinol, retinal, retinoic acid, β-carotene, and CoQ inhibit the formation and elongation of faS in a dose-dependent manner.
10  Ten
形成しておいた f a Sを用量依存的に不安定ィ匕させることが確認された。ここで、 f a S の形成を対照値の 50%に抑制するために必要な NDGA、 Myr、 RIF、 TC、レチノ ール、レチナール、レチノイン酸、 j8カロチン、 CoQ の有効濃度（EC )と、対照値  It was confirmed that the formed faS was unstable in a dose-dependent manner. Here, the effective concentrations (EC) of NDGA, Myr, RIF, TC, retinal, retinal, retinoic acid, j8-carotene, and CoQ required to suppress fa S formation to 50% of the control value, and the control value
10 50  10 50
の 50%に f a Sを不安定ィ匕させる濃度を、図 5 (e)及び 7 (e)に示すようなデータのシ グモイドカーブに当てはめて算出した。結果を表 2に示す。  The concentration that causes f a S to become unstable in 50% was calculated by applying it to the sigmoid curve of the data as shown in Figs. 5 (e) and 7 (e). The results are shown in Table 2.
[0072] [表 2] -シヌクレイン ひ-シヌクレイン [0072] [Table 2] -Synuclein Hi-Synuclein
原線維形成の 原線維不安定化の  Fibril formation fibril destabilization
EC50 ( / M) EC50 ( jU M) EC 50 (/ M) EC 50 (jU M)
レチノール 0.19 11  Retinol 0.19 11
レチナール 0.19 1.0  Retinal 0.19 1.0
レチノイン酸 3.0 24  Retinoic acid 3.0 24
カロチン 0.25 4.1  Carotene 0.25 4.1
CoQ10 10 48 CoQ 10 10 48
ノルジヒドログアイァレチン酸 0.22 0.37  Nordihydroguaiaretic acid 0.22 0.37
ミリセチン 0.22 0.25  Myricetin 0.22 0.25
リファンピシン 7.1 19  Rifampicin 7.1 19
テトラサイクリン 6.4 19  Tetracycline 6.4 19
NDGA、Myr、CoQ 、 RIF、 TCの f a S形成を抑制する有効濃度（EC )は、 f a S The effective concentration (EC) that suppresses f a S formation of NDGA, Myr, CoQ, RIF, and TC is f a S
10 50 を不安定ィ匕させる濃度と類似していた。一方で、レチノール、レチナール、レチノイン 酸、 13カロチンの f a Sを不安定化させる濃度 (EC )は、 f a Sの形成を抑制するそ  It was similar to the concentration that made 10 50 unstable. On the other hand, the concentration (EC) of retinol, retinal, retinoic acid, and 13 carotene that destabilizes f a S suppresses the formation of f a S.
50  50
れらの濃度よりも一桁高い値を示した。前記表 2のデータより、実験 2において検討し た化合物の f a Sに関する抗原線維形成及び原線維不安定化活性の強さは、以下の 順であることが明ら力となった。 NDGA= Myr=レチノール =レチナ一ル> j8カロチ ン〉レチノイン酸〉 CoQ =RIF =TC。そして、ビタミン Aであるレチノール、レチナ The value was an order of magnitude higher than these concentrations. From the data in Table 2 above, it became clear that the strength of antigen fibril formation and fibril destabilization activity related to f a S of the compounds examined in Experiment 2 was in the following order. NDGA = Myr = Retinol = Retinal> j8 Caroten> Retinoic acid> CoQ = RIF = TC. And vitamin A, retinol, retina
10  Ten
ール、レチノイン酸、及びビタミン A前駆体である |8カロチンは、 NDGA等と同様、強 いひーシヌクレイン線維形成抑制効果及び不安定ィ匕効果を持つことが明らかとなつ た。したがって、レチノール、レチナール、レチノイン酸、及び j8カロチンはレビー小 体病治療薬及びレビー小体病予防薬として有用であることがわ力 た。 , Retinoic acid, and vitamin A precursor | 8 carotene, as with NDGA, were found to have strong hyosinuclein fibril formation inhibition and instability effects. Therefore, it was proved that retinol, retinal, retinoic acid, and j8 carotene are useful as therapeutic agents for Lewy body diseases and preventive agents for Lewy body diseases.

Claims

請求の範囲 The scope of the claims
[I] ノルジヒドログアイァレチン酸を含むことを特徴とするレビー小体病治療薬。  [I] A therapeutic agent for Lewy body disease comprising nordihydroguaiaretic acid.
[2] クルクミンを含むことを特徴とするレビー小体病治療薬。 [2] A therapeutic agent for Lewy body disease comprising curcumin.
[3] ローズマリー酸を含むことを特徴とするレビー小体病治療薬。  [3] A therapeutic agent for Lewy body disease comprising rosemary acid.
[4] ビタミン Aを含むことを特徴とするレビー小体病治療薬。  [4] A therapeutic agent for Lewy body disease comprising vitamin A.
[5] βカロチンを含むことを特徴とするレビー小体病治療薬。 [5] A therapeutic agent for Lewy body disease comprising β-carotene.
[6] 前記レビー小体病がパーキンソン病、レビー小体型認知症、多系統萎縮症から選 ばれる少なくとも 1種であることを特徴とする請求項 1〜5のいずれか 1項記載のレビ 一小体病治療薬。  6. The Lewy body disease according to any one of claims 1 to 5, wherein the Lewy body disease is at least one selected from Parkinson's disease, Lewy body dementia, and multiple system atrophy. Somatic remedy.
[7] ノルジヒドログアイァレチン酸を含むことを特徴とするレビー小体病予防薬。  [7] A preventive drug for Lewy body disease comprising nordihydroguaiaretic acid.
[8] クルクミンを含むことを特徴とするレビー小体病予防薬。  [8] A Lewy body prophylactic agent comprising curcumin.
[9] ローズマリー酸を含むことを特徴とするレビー小体病予防薬。  [9] An agent for preventing Lewy body disease, comprising rosemary acid.
[10] ビタミン Αを含むことを特徴とするレビー小体病予防薬。  [10] A preventive drug for Lewy bodies characterized by containing vitamin Α.
[II] βカロチンを含むことを特徴とするレビー小体病予防薬。  [II] A Lewy body disease preventive drug characterized by containing β-carotene.
[12] 前記レビー小体病がパーキンソン病、レビー小体型認知症、多系統萎縮症から選 ばれる少なくとも 1種であることを特徴とする請求項 7〜 11の 、ずれ力 1項記載のレビ 一小体病予防薬。  12. The Lewy body according to claim 7, wherein the Lewy body disease is at least one selected from Parkinson's disease, Lewy body dementia, and multiple system atrophy. Anti-body disease drug.
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