WO2023080687A1 - Procédé de criblage en masse à haute vitesse pour un médicament inhibiteur de multimère de bêta-amyloïde et composition comprenant de la doxorubicine ou un dérivé de celle-ci pour inhiber l'oligomérisation ou la fibrillation de bêta-amyloïde - Google Patents

Procédé de criblage en masse à haute vitesse pour un médicament inhibiteur de multimère de bêta-amyloïde et composition comprenant de la doxorubicine ou un dérivé de celle-ci pour inhiber l'oligomérisation ou la fibrillation de bêta-amyloïde Download PDF

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WO2023080687A1
WO2023080687A1 PCT/KR2022/017168 KR2022017168W WO2023080687A1 WO 2023080687 A1 WO2023080687 A1 WO 2023080687A1 KR 2022017168 W KR2022017168 W KR 2022017168W WO 2023080687 A1 WO2023080687 A1 WO 2023080687A1
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amyloid beta
present
doxorubicin
amyloid
antibody
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PCT/KR2022/017168
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Korean (ko)
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안성수
심규환
김단영
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가천대학교 산학협력단
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Priority claimed from KR1020210150098A external-priority patent/KR20230064470A/ko
Priority claimed from KR1020210150067A external-priority patent/KR20230064459A/ko
Application filed by 가천대학교 산학협력단 filed Critical 가천대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a kit for screening amyloid beta multimer inhibitory drug, a method for high-throughput screening of a drug for preventing or treating Alzheimer's disease using the kit, and the amyloid beta inhibitory effect of doxorubicin or a derivative thereof.
  • the present invention was made by the task identification number 1711115820 and detailed task number 2020R1A2B5B01002463 under the support of the Ministry of Science and ICT of the Republic of Korea. Multi variate analysis of biomarkers and etiologic mechanisms of Korean Alzheimer's disease-related mutations and development of Korean cell and animal models for new drug development”, Hosted by Gachon University, research period 2020.03.01 - 2023.02.28.
  • the present invention was made by the task identification number 1345334570 and detailed task number 2021R1A6A1A03038996 under the support of the Ministry of Education of the Republic of Korea.
  • the project name is "Bio-Nano Application Research Center", the host institution is Gachon University, and the research period is 2021.06.01 - 2030.05.31.
  • AD Alzheimer's disease
  • the number of Alzheimer's disease patients is expected to reach 70.7 million in 2030, and the number of Alzheimer's disease patients in Korea is currently about 800,000, which is expected to increase to 1.37 million in 2030 and 3.03 million in 2050.
  • Amyloid beta oligomer is known to cause Alzheimer's disease by causing cytotoxicity, and Alzheimer's disease patients show higher levels of amyloid beta oligomer than normal people.
  • amyloid beta (A ⁇ ) is entangled with each other to form an oligomer and continues to aggregate, further fibrillization proceeds to form a fibril form.
  • Aducanumab (jointly developed by Biogen of the US and Eisai of Japan; product name: AduhelmTM), which received conditional approval as a treatment for Alzheimer's disease from the US Food and Drug Administration (FDA) on June 7, 2021, is amyloid As a monoclonal antibody to beta, it has the effect of removing insoluble amyloid beta protein, and unlike existing treatments that showed symptomatic effects of alleviating the symptoms of Alzheimer's disease, it is a direct anti-inflammatory antibody that can fundamentally block or treat the disease. It is being evaluated as a treatment.
  • Eli Eli's Donanemab and Biogen and Eisai's Lecanemab were approved by the FDA in June 2021, and Hoffman-La Roche's Gantenerumab in October 2021. received breakthrough therapy designation.
  • Donanemab is an antibody also known as N3pG that targets abnormal amyloid beta produced in the bone marrow. It is a monoclonal antibody that binds to amyloid beta fibers. The three candidates mentioned above, which received breakthrough therapy designation, are currently undergoing clinical trials for FDA approval.
  • the present inventors have intensively researched to develop a composition, kit, or method capable of demonstrating a significant therapeutic effect from the discovery of a substance for treating Alzheimer's disease, in order to discover and preoccupy a lead substance for preventing or treating Alzheimer's disease.
  • the present invention was completed by constructing a composition and antibody concentration suitable for mass and rapid screening of drugs having an amyloid beta multimer inhibitory effect from candidate substances.
  • the present inventors have intensively researched to find a compound for inhibiting or detecting amyloid beta that is inexpensive compared to a conventional method of detecting amyloid beta using an antibody and can specifically bind to amyloid beta.
  • the present invention was completed by identifying that doxorubicin or a derivative thereof is a compound that inhibits oligomerization or fibrillation of amyloid beta and exhibits a color change and optical density difference in a specific wavelength band by binding to amyloid beta.
  • an object of the present invention is to provide a kit for screening amyloid beta multimer inhibitory drug.
  • Another object of the present invention is to provide a method for high-throughput screening of drugs for preventing or treating Alzheimer's disease using the above kit.
  • Another object of the present invention is to provide a composition for inhibiting oligomerization or fibrillation of amyloid beta, comprising doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Another object of the present invention is to provide a composition for detecting amyloid beta.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Alzheimer's disease.
  • Another object of the present invention is to provide a method for providing information for diagnosis of Alzheimer's disease.
  • the present inventors have intensively researched to develop a composition, kit, or method capable of demonstrating a significant therapeutic effect from the stage of discovering and preoccupying a lead substance for preventing or treating Alzheimer's disease.
  • the composition and antibody concentration of a composition suitable for rapid screening of drugs with amyloid beta multimer inhibitory effect in large quantities from candidate substances were established.
  • the present invention relates to a kit for screening an amyloid beta multimer inhibitor drug and a method for high-speed mass screening of a drug for preventing or treating Alzheimer's disease using the same.
  • the present invention provides a kit for screening amyloid beta multimer inhibitory drug comprising:
  • amyloid beta protein a) amyloid beta protein
  • the epitopes of amyloid beta to which the capture antibody and the detection antibody specifically bind overlap overlap.
  • the kit confirms whether or not multimerization of amyloid beta is inhibited by contacting a candidate drug for inhibiting amyloid beta multimerization with the amyloid beta protein.
  • a ⁇ amyloid beta
  • APP amyloid precursor protein
  • a ⁇ 40 which is composed of 40 amino acids, is the most abundant at about 80% to 90%, followed by A ⁇ 42, which is composed of 42 amino acids, at about 5% to 10%.
  • a ⁇ 42 which is more hydrophobic than other amyloid betas, is known to be a major component of amyloid plaques.
  • another amyloid beta species, the 43 amino acid A ⁇ 43 is known to contribute to the early onset of Alzheimer's disease.
  • amyloid beta 'multimer' refers to a polypeptide in which at least two amyloid beta monomers (amyloid beta monomers of a single polypeptide) are integrated with each other.
  • the amyloid beta multimer of the present invention has at least two amyloid beta N-terminals.
  • the amyloid beta multimer of the present invention exposes at least two amyloid beta N-terminals.
  • the amyloid beta multimer of the present invention may be an amyloid beta oligomer.
  • 'oligomer' of amyloid beta refers to a soluble or non-soluble form in which amyloid beta monomers are aggregated with each other.
  • 'oligomerization' of amyloid beta refers to a process in which soluble or insoluble oligomers in which A ⁇ monomers themselves are integrated with each other are formed.
  • kits for screening amyloid beta multimer inhibitors of the present invention in a multimer detection system (MDS) capable of discriminating amyloid beta multimers from amyloid beta monomers, among a plurality of candidate drugs, amyloid beta multimers High-throughput screening (HTS), which can quickly and efficiently search for drugs with inhibitory effects, is possible, so that drugs for preventing or treating Alzheimer's disease can be discovered and preoccupied.
  • MDS multimer detection system
  • HTS High-throughput screening
  • MDS multimer detection system
  • the term 'high throughput screening (HTS)' refers to a high-efficiency substance search method that simultaneously analyzes the pharmacological effects of a plurality of candidate drugs at high speed.
  • the high-speed, high-throughput screening of the present invention is a method capable of simultaneously searching for a large number of candidate drugs having an amyloid beta multimer inhibitory effect at high speed and with high efficiency.
  • MDS-HTS' refers to a method (HTS) for quickly and efficiently searching for and selecting drugs having an amyloid beta multimer inhibitory effect from a plurality of candidate substances using the multimer detection system (MDS). it means.
  • AD Alzheimer's disease
  • cognitive impairment symptoms such as memory decline, decline in language ability, decline in visuospatial ability, decline in judgment and daily life performance, gait disorder, and behavior disorder.
  • the term 'prevention' refers to prevention or protective treatment of a disease or disease state.
  • the term 'treatment' refers to reduction, suppression, sedation or eradication of a disease state.
  • the composition for forming amyloid beta multimers includes amyloid beta such that the concentration ratio of the amyloid beta multimer inhibitory candidate drug and amyloid beta is 0.5:1 to 50:1.
  • the concentration represents mass to volume (w/v).
  • the ratio is 2:1 to 10:1. In one specific embodiment of the present invention, the ratio is 3:1 to 6:1.
  • the concentration ratio of the amyloid beta multimer inhibitory candidate drug and amyloid beta in the composition for forming amyloid beta multimer is less than 0.5: 1, the concentration of the candidate drug is not sufficient to confirm the amyloid beta multimer inhibitory effect, In fact, it is difficult to distinguish whether the candidate drug has no effect on amyloid beta multimer formation or whether the concentration of the candidate drug is insufficient to exert an effect on amyloid beta multimer formation inhibitory effect.
  • the concentration ratio of the candidate drug for inhibiting amyloid beta multimers and the concentration ratio of amyloid beta in the composition for forming amyloid beta multimers is greater than 50:1, the concentration of amyloid beta multimer formation is difficult to confirm the effect of the candidate drug on formation of amyloid beta multimers. low, it is difficult to distinguish whether the induction of amyloid beta multimer formation is not well achieved or whether the candidate drug has an effect of actually inhibiting amyloid beta.
  • the present inventors investigated whether the candidate drug inhibits the formation of amyloid beta multimers when the concentration ratio of the amyloid beta multimer inhibiting candidate drug and amyloid beta in the composition for forming amyloid beta multimers is 0.5:1 to 50:1. It was suitable for judgment, and it was confirmed that it is useful for screening amyloid beta multimer inhibitory drug.
  • amyloid beta multimers Since the formation of amyloid beta multimers is appropriately induced within the above concentration ratio range, it can be determined whether or not the candidate drug has a substantially multimer inhibitory effect.
  • amyloid beta multimers is appropriately induced when the candidate drug does not have an amyloid multimer inhibitory effect, and the formation of amyloid beta multimers is inhibited when the candidate drug has an amyloid multimer inhibitory effect. do.
  • the kit for screening amyloid beta multimer inhibitory drug further includes a negative control.
  • the negative control is any small molecule, extract, nucleic acid molecule, antibody, antigen-binding fragment known to have no inhibitory effect on amyloid beta multimer formation, It may be an antibody-based drug, an antibody-drug conjugate (ADC), a targeted antibody, a peptide, an aptamer, a compound, a solvent, and the like, but is not necessarily limited thereto.
  • ADC antibody-drug conjugate
  • the negative control group may be one or more solvents selected from the group consisting of phosphate buffered saline (PBS), tris buffered saline (TBS), dimethyl sulfoxide (DMSO), and physiological saline.
  • PBS phosphate buffered saline
  • TBS tris buffered saline
  • DMSO dimethyl sulfoxide
  • physiological saline physiological saline.
  • the negative control can be a mixture of the solvents described above.
  • a negative control is included in the composition for forming amyloid beta multimer so that the concentration ratio of the negative control and amyloid beta is 0.5:1 to 50:1.
  • the amyloid beta multimer is an amyloid beta oligomer. In another embodiment of the present invention, the amyloid beta multimer is a nonfibrillar multimeric species or a fibrillar multimeric species of amyloid beta peptide.
  • the amyloid beta oligomer is a spherical oligomer, a protofibrillar oligomer, a fibrillar oligomer, a nonfibrillar oligomer, a disc- It may be an oligomer of disc-like morphology, globulomer, ⁇ -amyball, amylospheroid, or annular protofibril oligomer.
  • the amyloid beta oligomer may have a diameter of less than 4 nm. In yet another embodiment of the present invention, the amyloid beta oligomer may have a diameter of 4 nm to 10 nm.
  • the amyloid beta multimer is a dimer (2-mer), trimer (3-mer), tetramer (4-mer), pentamer ( pentamer (5-mer), hexamer (6-mer), heptamer (7-mer), octamer (8-mer), nonamer (9-mer), decamer ( decamer (10-mer), undecamer (11-mer), dodecamer (12-mer), tridecamer (13-mer), tetradecamer (14-mer), penta pentadecamer (15-mer), hexadecamer (16-mer), heptadecamer (17-mer), octadecamer (18-mer), nonadecamer (19 -mer), icosamer (20-mer), henicosamer (21-mer), docosamer (22-mer), tricosamer (23-mer), tetracosamer (tetracosamer, 24-mer), penta dimer (2-mer), trim
  • ADDL Alzheimer's disease
  • 'A ⁇ *56 (A ⁇ star 56)' refers to 56-kDa and 12-mer soluble amyloid beta-assembly accumulated extracellularly (Lesne S. et al. , A specific amyloid-beta protein assembly in the brain impairs memory.Nature.2006 Mar 16;440(7082):352-7.).
  • a ⁇ *56 is known to contribute to the cognitive impairment associated with Alzheimer's disease by impairing memory independently of plaque formation or neuronal loss.
  • composition for forming amyloid beta multimers of the present invention forms one or more of the amyloid beta multimers described above.
  • the amyloid beta multimers generated in the composition for forming amyloid beta multimers of the present invention may be a mixture of different types of amyloid beta multimers.
  • the concentration (w / v) ratio of the amyloid beta capture antibody and the amyloid beta detection antibody is 4: 1 to 12: 1.
  • the concentration of the amyloid beta-detecting antibody and the amyloid beta-capturing antibody is expressed in units of weight/volume, and can be specifically expressed in units of micrograms ( ⁇ g) per volume milliliters (ml). .
  • the ratio is 4:1 to 12:1, 5:1 to 12:1, 6:1 to 12:1, 7:1 to 12:1, 8:1 to 12 :1, 9:1 to 12:1, 10:1 to 12:1, 11:1 to 12:1, 4:1 to 11:1, 5:1 to 11:1, 6:1 to 11:1 , 7:1 to 11:1, 8:1 to 11:1, 9:1 to 11:1, 10:1 to 11:1, 4:1 to 10:1, 5:1 to 10:1, 6 :1 to 10:1, 7:1 to 10:1, 8:1 to 10:1, 9:1 to 10:1, 4:1 to 9:1, 5:1 to 9:1, 6:1 to 9:1, 7:1 to 9:1, 8:1 to 9:1, 4:1 to 8:1, 5:1 to 8:1, 6:1 to 8:1, 7:1 to 8 :1, 4:1 to 7:1, 5:1 to 7:1, 6:1 to 7:1, 4:1 to 6:1, 5:1 to 8:1, 6:1 to 8:1, 7:1 to 8 :1,
  • the ratio is 4:1 to 6:1. In one most specific embodiment of the present invention, the concentration ratio is 5:1.
  • the concentration ratio of the amyloid beta capture antibody and the amyloid beta detection antibody is 4:1 to 12:1 since the capture antibody is present at a higher concentration than the detection antibody, the capture antibody sufficiently binds to the amyloid beta N-terminus for capture.
  • Amyloid beta multimers can be easily detected by forming an antibody-amyloid beta complex, binding a detection antibody to the other amyloid beta N-terminus of the complex, and forming a capture antibody-amyloid beta-detection antibody complex.
  • the capture antibody and the detection antibody are in a competitive relationship for the same epitope or partially overlapping epitopes. Even if a small amount of capture antibody is used compared to the multimer detection technology, the capture antibody and the detection antibody cannot simultaneously bind to the amyloid beta monomer, but the capture antibody and the detection antibody can sufficiently simultaneously bind to the amyloid beta multimer.
  • this system of the present invention it is possible to determine whether a candidate substance has an inhibitory effect on amyloid beta multimer formation using a small amount of capture antibody and detection antibody, so it is accurate, high-speed, large-scale, and more economical.
  • a drug for preventing or treating Alzheimer's disease can be screened.
  • the concentration of the amyloid beta capture antibody is 0.08 ⁇ g/ml to 2.4 ⁇ g/ml.
  • the concentration is 0.08 ⁇ g/ml to 2.4 ⁇ g/ml, 0.08 ⁇ g/ml to 2.0 ⁇ g/ml, 0.08 ⁇ g/ml to 1.5 ⁇ g/ml, 0.08 ⁇ g/ml to 1.0 ⁇ g /ml, 0.08 ⁇ g/ml to 0.8 ⁇ g/ml, 0.08 ⁇ g/ml to 0.5 ⁇ g/ml, 0.08 ⁇ g/ml to 0.1 ⁇ g/ml, 0.1 ⁇ g/ml to 2.4 ⁇ g/ml, 0.1 ⁇ g/ml to 2.0 ⁇ g /ml, 0.1 ⁇ g/ml to 1.5 ⁇ g/ml, 0.1 ⁇ g/ml to 1.0 ⁇ g/ml, 0.1 ⁇ g/ml to 0.8 ⁇ g/ml, 0.1 ⁇ g/ml to 0.5 ⁇ g/ml, 0.5 ⁇ g/ml to 2.4 2.4 ⁇ g/ml
  • the concentration is 0.5 ⁇ g/ml.
  • the amyloid beta capture antibody is immobilized on a solid substrate.
  • solid substrates include hydrocarbon polymers such as polystyrene and polypropylene, glass, metals, and gels.
  • the solid substrate may include a dipstick, microtiter plate, particle (eg, bead), affinity column, and immunoblot membrane (eg, polyvinylidene fluoride membrane) form, but is not necessarily limited thereto (see : U.S. Patent Nos. 5,143,825, 5,374,530, 4,908,305 and 5,498,551).
  • the concentration of the amyloid beta detection antibody is 0.02 ⁇ g/ml to 0.2 ⁇ g/ml.
  • the concentration is 0.02 ⁇ g/ml to 0.2 ⁇ g/ml, 0.02 ⁇ g/ml to 0.15 ⁇ g/ml, 0.02 ⁇ g/ml to 0.1 ⁇ g/ml, 0.02 ⁇ g/ml to 0.05 ⁇ g /ml, 0.05 ⁇ g/ml to 0.2 ⁇ g/ml, 0.05 ⁇ g/ml to 0.15 ⁇ g/ml, 0.05 ⁇ g/ml to 0.1 ⁇ g/ml, 0.1 ⁇ g/ml to 0.2 ⁇ g/ml, 0.1 ⁇ g/ml to 0.15 ⁇ g /ml or 0.15 ⁇ g/ml to 0.2 ⁇ g/ml.
  • the concentration is 0.1 ⁇ g/ml.
  • the amyloid beta epitope recognized by the capture antibody and the detection antibody is the amyloid beta N-terminus.
  • the amyloid beta epitopes recognized by the amyloid beta capture antibody and the amyloid beta detection antibody overlap.
  • the term 'overlapping' refers to a state in which at least one amino acid residue is the same in an epitope recognized by the amyloid beta capture antibody and the detection antibody.
  • the overlap means that all of the amino acid residues constituting the epitope recognized by the capture antibody and the detection antibody are the same or only partially the same.
  • At least one amyloid beta epitope recognized by the amyloid beta capture antibody and the amyloid beta detection antibody overlaps.
  • the number of amyloid beta epitopes recognized by the amyloid beta capture antibody and the amyloid beta detection antibody is 1 to 8, 2 to 8, 2 to 6, 2 to 8, 4, 4-8, 4-6, or 6-8 overlaps.
  • two amyloid beta epitopes recognized by the amyloid beta capture antibody and the amyloid beta detection antibody overlap.
  • the amyloid beta epitope recognized by the amyloid beta capture antibody and the amyloid beta detection antibody is the amyloid beta N-terminus.
  • the epitope recognized by the amyloid beta capture antibody and the amyloid beta detection antibody is amino acid residues 1 to 8 of the amyloid beta N-terminus.
  • At least two of the amino acid residues 1 to 8 of the amyloid beta N-terminus of the epitope recognized by the amyloid beta capture antibody and the amyloid beta detection antibody are the same, that is, overlap.
  • the amyloid beta epitope of the amyloid beta capture antibody and the amyloid beta detection antibody is N-terminal 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 and 2, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 times, 2 times and 3 times, 3 times to 8 times, 3 times to 7 times, 3 times to 6 times, 3 times to 5 times, 3 times and 4 times, 5 times to 8 times, 5 times to 7 times At least two amino acid residues of positions 5 and 6, 6 to 8, or 7 and 8 are identical.
  • amyloid beta epitopes of the amyloid beta capture antibody and the amyloid beta detection antibody have the same N-terminal amino acid residues 3 and 4 of amyloid beta.
  • An aptamer having the same epitope as the amyloid beta capture antibody and the amyloid beta detection antibody, that is, specifically binding to the amyloid beta N-terminus, may be used instead of the antibody.
  • bases used for the aptamer are A, G, C, and U of these deoxy ( deoxy) form of bases.
  • the term 'aptamer' refers to an oligonucleotide molecule having high specificity and affinity for a predetermined target molecule, that is, amyloid beta.
  • the aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or cyclic.
  • Polyethylene glycol, idT (inverted deoxythymdine), LNA (Locked Nucliec Acid) between 5'-end, 3'-end, or 5'-end and 3'-end of aptamer to improve stability ), 2'-aminonucleoside, 2'F-nucleoside, amine linker, thiol linker, cholesterol, and the like can be modified.
  • the amyloid beta detection antibody has a labeling substance generating a detectable signal.
  • amyloid beta detection antibody is conjugated with a label that generates a specific wavelength.
  • the labeling material may be a compound label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescence label, or a FRET label, but is not necessarily limited thereto.
  • the labeling substance is peroxidase, horseradish peroxidase (HRP), glucose oxidase, luciferase, alkaline phosphatase, beta -Lactamase, beta-glucuronidase, beta-galactosidase, chloramphenicol acetyltransferase, iron oxide nanoparticle ) and at least one selected from the group consisting of hemin-containing complexes, but is not necessarily limited thereto.
  • HRP horseradish peroxidase
  • glucose oxidase glucose oxidase
  • luciferase alkaline phosphatase
  • beta -Lactamase beta-glucuronidase
  • beta-galactosidase beta-galactosidase
  • chloramphenicol acetyltransferase iron oxide nanoparticle
  • the radioactive label of the present invention may be I125 or C14.
  • FRET in the present invention means fluorescence resonance energy transfer.
  • the labeling material may be at least one fluorescent protein selected from the group consisting of, but is not necessarily limited thereto: IFP1, a near infrared fluorescent protein .4; Red fluorescent proteins RFP (red fluorescent protein), eRFP (enhanced red fluorescent protein), mRFP (modified red fluorescent protein), DsRed (Discosoma sp.
  • IFP1 a near infrared fluorescent protein .4
  • Red fluorescent proteins RFP red fluorescent protein
  • eRFP enhanced red fluorescent protein
  • mRFP modified red fluorescent protein
  • DsRed Discosoma sp.
  • red fluorescent protein HcR (HcRed, Heteractis crispa red fluorescent protein) , mCherry (monomeric cherry fluorescent protein), PAmcherry (photoactivatable mCherry fluorescent protein), Tag RFP, Tag RFP657, mRFP1, PaTagRFP, Turbo RFP, RFP693, tdRFP, mScarlet, mScarlet-i, mStrawberry (monomeric strawberry fluorescent protein), mRaspberry, mPlum, mApple, mGrape1, mGrape2, tdTomato, E2-Crimson, HcRed1, mKate, mKate2, mNeptune, mRubby, mRubby2, J-Red, mEOS4, katushka and mTangerine; orange fluorescent proteins mOrange (monomeric orange fluorescent protein), PSmOrange (photoconvertible orange fluorescent protein), mKO and mKO2; yellow fluorescent protein (YFP),
  • BFP blue fluorescent proteins
  • eBFP enhanced blue fluorescent protein
  • Azurite mKalamal
  • PS-CFP2 mTag BFP and mTag BFP2
  • cyan fluorescent protein (CFP) enhanced cyan fluorescent protein
  • eCFP cyan green fluorescent protein
  • CGFP cyan green fluorescent protein
  • TagCFP mTurquoise
  • mTurquosie2 CyPet, Cerulean, and monomeric teal fluorescent protein
  • mTFP green-red photoconvertible fluorescent protein
  • KikGR green-red fluorescent protein
  • the labeling substance is HRP or AP.
  • the coating buffer includes sodium carbonate-bicarbonate.
  • the coating buffer is pH 7.0 to pH 10.0.
  • the acidity of the coating buffer is pH 7.0 to pH 10.0.
  • the acidity of the coating buffer is pH 7.0 to pH 10.0, pH 7.0 to pH 9.5, pH 7.0 to pH 9.0, pH 7.0 to pH 8.5, pH 7.0 to pH 8.0, pH 7.0 to pH 7.5, pH 7.5 to pH 10.0, pH 7.5 to pH 9.5, pH 7.5 to pH 9.0, pH 7.5 to pH 8.5, pH 7.5 to pH 8.0, pH 8.0 to pH 10.0, pH 8.0 to pH 9.5, pH 8.0 to pH 9.0; pH 8.0 to pH 8.5, pH 8.5 to pH 10.0, pH 8.5 to pH 9.5, pH 8.5 to pH 9.0, pH 9.0 to pH 10.0, pH 9.0 to pH 9.5, or pH 9.5 to pH 10.0.
  • the acidity of the coating buffer is pH 7.0 to pH 7.5.
  • the acidity of the coating buffer is pH 7.0 to pH 7.5, pH 7.0 to pH 7.4, pH 7.0 to pH 7.3, pH 7.0 to pH 7.2, pH 7.0 to pH 7.1, pH 7.1 to pH 7.5, pH 7.1 to pH 7.4, pH 7.1 to pH 7.3, pH 7.1 to pH 7.2, pH 7.2 to pH 7.5, pH 7.2 to pH 7.4, pH 7.2 to pH 7.3, pH 7.3 to pH 7.5, pH 7.3 to pH 7.4 or pH 7.4 to pH 7.5 days can Specifically, the acidity of the coating buffer may be pH 7.2.
  • the acidity of the coating buffer is pH 9.1 to pH 9.7.
  • the acidity of the coating buffer is pH 9.1 to pH 9.7, pH 9.1 to pH 9.6, pH 9.1 to pH 9.5, pH 9.1 to pH 9.4, pH 9.1 to pH 9.3, pH 9.1 to pH 9.2, pH 9.2 to pH 9.7, pH 9.2 to pH 9.6, pH 9.2 to pH 9.5, pH 9.2 to pH 9.4, pH 9.2 to pH 9.3, pH 9.3 to pH 9.7, pH 9.3 to pH 9.6, pH 9.3 to pH 9.5, pH 9.3 to pH 9.4, pH 9.4 to pH 9.7, pH 9.4 to pH 9.6, pH 9.4 to pH 9.5, pH 9.5 to pH 9.7, pH 9.5 to pH 9.6 or pH 9.6 to pH 9.7.
  • the acidity of the coating buffer may be pH 9.4.
  • the detection buffer contains a surfactant.
  • the surfactants used in the present invention are Tween ® 20, Tween ® 80, Triton X-100, Triton X-114, Triton X-200, NP-40, Polysorbate 20, Octyl glucoside, Octyl thioglucoside, SDS (Sodium dodecyl sulfate) , CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and CHAPSO (3-(3-cholamidopropyldimethylammonio)-2-hydroxy-1-propanesulfonate), but one or more selected from the group consisting of It is not limited.
  • the surfactant is Tween ® 20.
  • the surfactant used in the present invention may be at a concentration of 0.01% (v/v) to 5.0% (v/v) in the composition. Specifically, the surfactant may be present in a concentration of 0.05% (v / v) in the composition.
  • the detection buffer may include PBST containing Tween ® 20 in phosphate-buffered saline (PBS).
  • the detection buffer may include TBST containing Tween ® 20 in Tris-buffered saline (TBS).
  • the kit for screening amyloid beta multimer inhibitory drug additionally includes a positive control.
  • the positive control is doxorubicin or curcumin.
  • the kit further comprises doxorubicin as a positive control.
  • a positive control is included in the composition for forming amyloid beta multimer so that the concentration ratio of the positive control and amyloid beta is 0.5:1 to 50:1.
  • the kit further comprises 1% (w / v) to 10% (w / v) of albumin, a surfactant and a blocking buffer containing PBS.
  • the blocking buffer may include 1% (w / v) to 10% (w / v) of albumin.
  • the blocking buffer is 1% (w / v) to 10% (w / v), 1% (w / v) to 8% (w / v), 1% (w /v) to 6% (w/v), 1% (w/v) to 5% (w/v), 1% (w/v) to 4% (w/v), 1% (w/v) ) to 2% (w/v), 2% (w/v) to 10% (w/v), 2% (w/v) to 8% (w/v), 2% (w/v) to 6% (w/v), 2% (w/v) to 5% (w/v), 2% (w/v) to 4% (w/v), 4% (w/v), 4% (w/v), 4% (w/v), 1% (w/v) to 10% (w/v), 2% (w/v) to 8% (w
  • the blocking buffer contains 2% (w / v) of albumin.
  • the albumin is bovine serum albumin (BSA).
  • the blocking buffer of the present invention includes 2% (w / v) BSA, 0.05% (v / v) Tween ® 20 and PBS.
  • the kit is Western blot, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, radioimmunoassay, radio-immunodiffusion , double immunodiffusion method (Ouchterlony), rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, flow cytometry (Fluorescence Activated Cell Sorter, FACS), protein chip, etc. It may be used, but is not necessarily limited thereto.
  • the kit of the present invention can be used in various ELISA methods such as an immunoassay kit, an ELISA kit, and a sandwich ELISA, and the kit may further include essential components required to implement it.
  • the ELISA kit may include the amyloid beta capture antibody and the amyloid beta detection antibody, or an aptamer corresponding thereto.
  • the essential component may include a solid substrate to which the amyloid beta capture antibody binds.
  • solid substrates include hydrocarbon polymers such as polystyrene, polypropylene, glass, metals and gels.
  • the solid substrate may be in the form of dipsticks, microplates, particles, beads, magnetic beads, affinity columns, and immunoblot membranes such as PVDF membranes.
  • the microplate may be a 96 well black plate, a 96 well plate or a 384 well plate.
  • the present invention provides a method for high-throughput screening of drugs for preventing or treating Alzheimer's disease, comprising the following steps:
  • amyloid beta protein contacting amyloid beta protein with a candidate drug for preventing or treating Alzheimer's disease
  • the candidate drug When the capture antibody-amyloid beta multimer-detection antibody complex is not detected, select the candidate drug as a drug for preventing or treating Alzheimer's disease.
  • amyloid beta protein and the candidate drug may be contacted at the same concentrations as those mentioned in the above kit.
  • the method for high-throughput screening of drugs for preventing or treating Alzheimer's disease further comprises the following steps:
  • Each step included in the method for high-throughput screening of a drug for preventing or treating Alzheimer's disease according to the present invention may be performed regardless of the order listed above.
  • amyloid beta-capturing antibody may first be brought into contact with the composition for forming amyloid-beta multimers and the candidate drug, and then the amyloid-beta detection antibody may be brought into contact.
  • amyloid beta detection antibody and the amyloid beta capture antibody may be brought into contact with the composition for forming amyloid beta multimers and the candidate drug at the same time.
  • the step of contacting the amyloid beta detection antibody and the step of detecting the amyloid beta capture antibody-amyloid beta multimer-amyloid beta detection antibody complex may be simultaneously performed.
  • Each step included in the method for high-throughput screening of a drug for preventing or treating Alzheimer's disease according to the present invention may be performed without being limited to the sequence exemplified above.
  • each step is given a number. Those skilled in the art will understand that the assigned numbers do not mean the sequential execution order of each step or limit the order of each step.
  • composition for forming amyloid beta multimer i) contacting the composition for forming amyloid beta multimer with a candidate drug
  • This step is a step of inducing formation of amyloid beta multimers by adding amyloid beta to the candidate drug.
  • a candidate drug is brought into contact with the composition for forming amyloid beta multimers of the present invention.
  • a step of contacting a negative control group with the composition for forming amyloid beta multimers of the present invention may be further included.
  • the negative control may be a solution containing only PBS or DMSO.
  • the candidate drug is dissolved in PBS or DMSO.
  • the candidate drugs may be small molecule compounds, extracts, nucleic acid molecules, antibodies, antigen-binding fragments, peptides, aptamers, and antibody-drug conjugates, but are not necessarily limited thereto. Store the sample at -20°C until the next step.
  • Amyloid beta is diluted with PBS and stored at -80°C until the next step.
  • the sample is diluted with PBS to a concentration of 0.05 mg/ml to 5.0 mg/ml.
  • the diluted sample is added to the plate, and the prepared amyloid beta is also added to the plate.
  • the sample and the amyloid beta are added and reacted so that the concentration ratio is 0.5:1 to 50:1.
  • the reaction mixture of the sample and amyloid beta is reacted at room temperature for 30 minutes to 8 hours.
  • Room temperature in the present invention may be 20 °C to 25 °C. Specifically, the room temperature may be 20 °C to 22 °C.
  • the plate is spun down, i.e., centrifuged at 500 rpm for 20 seconds at room temperature and then allowed to react at room temperature for 30 minutes to 8 hours. Specifically, it is reacted at room temperature for 1 hour, 2 hours, 3 hours and/or 4 hours.
  • whether and/or the degree of amyloid beta multimer formation is determined depending on whether the sample has an amyloid beta multimer inhibitory effect in the experimental group to which the sample is added.
  • amyloid beta multimers are formed in the negative control group.
  • step i) the reaction product of step i) is added to the plate coated with the amyloid beta capture antibody of the present invention, and the primary antigen-antibody reaction between the amyloid beta capture antibody and the amyloid beta monomer and/or multimer of each reactant is added. This is the step that takes place.
  • a coating buffer containing the amyloid beta capture antibody according to the present invention and sodium carbonate-bicarbonate was added to the plate and reacted at 4° C. for 8 to 16 hours to coat the bottom of the plate with the capture antibody do.
  • the acidity of the coating buffer may be pH 7.0 to pH 10.0, pH 7.0 to pH 7.5, or pH 9.1 to pH 9.7.
  • the reactants of step i) are added to the plate and allowed to react for 5 minutes to 2 hours at room temperature.
  • a primary antigen-antibody complex is formed by binding the amyloid beta-capturing antibody coated on the bottom of the plate with the amyloid beta monomer and/or multimer.
  • This step is a step in which a secondary antigen-antibody reaction occurs in which the amyloid beta-detection antibody of the present invention binds to the amyloid beta N-terminus exposed in the primary antigen-antibody complex of step ii).
  • amyloid beta detection antibody When there is no amyloid beta N-terminus exposed in the primary antigen-antibody complex in step ii), that is, when only the amyloid beta monomer is bound to the amyloid beta capture antibody, the amyloid beta detection antibody does not bind to the complex .
  • amyloid beta detection antibody is detected by the capture antibody. It can bind to another N-terminus of the unoccupied amyloid beta multimer, resulting in a secondary antigen-antibody reaction.
  • the detection buffer according to the present invention is added to the plate and reacted at room temperature for 5 minutes to 1 hour.
  • the detection buffer contains any surfactant known in the art in an appropriate concentration and includes a suitable buffering material such as phosphate or Tris.
  • the amyloid beta detection antibody and the amyloid beta capture antibody may be brought into contact with the composition for forming amyloid beta multimers and the candidate drug at the same time.
  • the amyloid beta monomer bound to the amyloid beta detection antibody cannot additionally bind to the amyloid beta capture antibody, and vice versa.
  • the amyloid beta multimer bound to the amyloid beta detection antibody can additionally bind to the amyloid beta capture antibody, and vice versa. Therefore, even when contacted simultaneously, only when amyloid beta multimers are formed, secondary antigen-antibody complexes of amyloid beta capture antibody, amyloid beta multimer, and detection antibody are formed and fixed to the bottom of the plate.
  • This step is to detect the secondary antigen-antibody complex of the amyloid beta capture antibody, amyloid beta multimer, and amyloid beta detection antibody formed in step iii), that is, the amyloid beta capture antibody-amyloid beta multimer-amyloid beta detection antibody complex. It is a step. This is a step of measuring a signal or a specific wavelength value generated by a labeling substance bound or conjugated to the amyloid beta detection antibody.
  • the labeling material may be a compound label, an enzyme label, a radioactive label, a fluorescent label, a luminescent label, a chemiluminescent label, or a FRET label.
  • the labeling substance is HRP.
  • HRP a solution of TMB (3,3',5,5'-tetramethylbenzidine) is added, incubated for 15 minutes, and 1M sulfuric acid (H2SO4) is added as a stop solution to 450 Shows the maximum absorbance at the nm wavelength.
  • the value of the specific wavelength band may be measured by a device known in the field to which the present invention belongs, such as a microplate reader and a spectrophotometer.
  • a device known in the field to which the present invention belongs such as a microplate reader and a spectrophotometer.
  • HRP is the labeling material
  • absorbance can be measured at 450 nm using a microplate reader.
  • this step it is determined whether to select the candidate drug as a drug for preventing or treating Alzheimer's disease by analyzing the signal of step iv) or the value of a specific wavelength band.
  • the candidate drug With respect to the candidate drug, if the signal measured in step iv) or the value of the specific wavelength band increases, the candidate drug has no effect of inhibiting amyloid beta multimer, so the candidate drug is selected as a drug for preventing or treating Alzheimer's disease I never do that.
  • the candidate drug or the value of a specific wavelength band decreases, the candidate drug has an effect of inhibiting amyloid beta multimer, and thus the candidate drug is selected as a drug for preventing or treating Alzheimer's disease.
  • this step it is determined whether to select the candidate drug as a drug for preventing or treating Alzheimer's disease by comparing the signal or value of the specific wavelength range of step iv) with the signal or value of the specific wavelength range of the negative control group.
  • the signal measured in step iv) or the value of a specific wavelength band is compared. If there is no significant difference in the signal or value of a specific wavelength range of the candidate drug compared to the negative control group, or if there is a significant increase, the candidate drug has no effect of inhibiting amyloid beta multimer, so the candidate drug is used for preventing or treating Alzheimer's disease. It is not screened as a drug.
  • the candidate drug When the signal of the candidate drug or the value of a specific wavelength band is significantly decreased compared to the negative control, the candidate drug has an effect of inhibiting amyloid beta multimer, and thus the candidate drug is selected as a drug for preventing or treating Alzheimer's disease.
  • the solution contained in the plate between each step of the high-speed mass screening method for drugs for preventing or treating Alzheimer's disease of the present invention is discarded, and PBS containing a surfactant, specifically PBS containing Tween ® 20, that is, PBST It may include washing each well of the plate 1 to 4 times in an add-and-discard manner.
  • a blocking buffer containing 1% (w/v) to 10% (w/v) of albumin, a surfactant, and PBS was added to the plate to prevent antigen-antibody non-specific bonding can be reduced.
  • the candidate drug is a small molecule compound, extract, nucleic acid molecule, antibody, antigen-binding fragment, peptide, aptamer, and antibody-drug conjugate (antibody-drug conjugate) It is selected from the group consisting of, but is not necessarily limited thereto.
  • the term 'small molecule compound' refers to any organic compound or inorganic compound having a small molecular weight.
  • a low-molecular compound When a low-molecular compound is used as a drug, most of it can be administered orally, and can pass through a cell membrane to reach an intracellular target. In addition, the low-molecular-weight compound can be distributed to organs and tissues by acting in intracellular and extracellular regions. Small molecule compounds can be replicated through chemical synthesis, making identical copies easy, and most of them are not immunogenic because they show predictable chemical processes in the body. Small molecule compounds can be easily assayed for quality, safety and efficacy after manufacture and can generally be shipped at room temperature in blister packs that are easy to use and store. Low-molecular compounds are decomposed into active metabolites and inactive metabolites in the body, their clearance is generally linear, and have relatively short half-lives. Disadvantages include that the drug itself or its metabolites may be toxic in the body and may interact with other drugs.
  • the term 'extract' refers to Archaea, Bacteria, Protozoa, Fungi, algae, plant cells, plant tissues, animal cells, and animal tissues. , means a solution extracted from animal organs, includes solutions extracted from other prokaryotes and eukaryotes not mentioned above, and also includes solutions extracted from natural extracts and genetically modified organisms.
  • nucleic acid molecule' includes DNA, RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA), and microRNA.
  • the term 'antibody' refers to a protein that specifically binds to amyloid beta oligomer. In addition to complete antibody forms, antigen-binding fragments of antibody molecules are also included.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bonds.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types, and subclasses include gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).
  • the antibody or antigen-binding fragment thereof herein refers to a full-length or intact polyclonal or monoclonal antibody, as well as antigen-binding fragments thereof (eg, Fab, Fab', F(ab' )2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single chains Antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, other modified configurations of immunoglobulin molecules containing antigen recognition sites of the required specificity, glycosylation variants of antibodies, amino acid sequence variants of antibodies and Includes antibodies that have been covalently modified.
  • antigen-binding fragments thereof eg, Fab, Fab', F(ab' )2, Fab3, Fv and variants thereof
  • fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies,
  • modified antibodies and antigen-binding fragments thereof include affibodies, chemobodies, optobodies, nanobodies, AlbudAbs, dual affinity re-targeting (DARTs), and bispecific T-cell (BiTEs). engager), TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX- bodies (peptide modified antibodies), duobodies and triomAbs.
  • engager TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX- bodies (peptide modified antibodies), duobodies and triom
  • the expression 'specifically binds' or its equivalent means that an antibody or antigen-binding fragment thereof, or other construct, such as an scFv, forms a complex with an antigen that is relatively stable under physiological conditions, namely amyloid beta. do.
  • Specific binding is at least about 1 x 10 -6 M or less (eg, 9 x 10 -7 M, 8 x 10 -7 M, 7 x 10 -7 M, 6 x 10 -7 M , 5 x 10 -7 M , 4 x 10 -7 M, 3 x 10 -7 M, 2 x 10 -7 M, or 1 x 10 -7 M), preferably 1 x 10 -7 M or less (eg 9 x 10 -8 M , 8 x 10 -8 M, 7 x 10 -8 M, 6 x 10 -8 M, 5 x 10 -8 M, 4 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M , or 1 x 10 -8 M), more preferably 1 x 10 -8 M or less (eg, 9 x 10 -9 M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M , 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9
  • the antibody may be an antibody-based drug, an antibody-drug conjugate (ADC), a targeted antibody, or a bispecific antibody.
  • ADC antibody-drug conjugate
  • the antibody-based drug mainly refers to a monoclonal antibody.
  • the monoclonal antibody may be in a naked form.
  • the antibody-drug conjugate may be in the form of a chemical bond between a drug and a monoclonal antibody.
  • a monoclonal antibody against amyloid beta may be in a chemically bound form with a drug having an amyloid beta oligomer inhibitory effect.
  • the bispecific antibody may be an antibody having two binding sites for amyloid beta.
  • the term 'antigen-binding fragment' refers to a fragment having a binding function to an antigen, that is, amyloid beta.
  • antigen-binding fragments include Fab, F(ab'), F(ab') 2 , chemically linked F(ab') 2 and Fv, and the like.
  • the term 'aptamer' refers to an oligonucleotide molecule having high specificity and affinity for a specific target molecule, that is, amyloid beta.
  • the aptamer can specifically bind to amyloid beta, it can be used without limitation, and the base used for the aptamer is the deoxy form of A, G, C, and U, unless otherwise specified. It may be selected from the group consisting of bases of.
  • the aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or cyclic.
  • Polyethylene glycol, idT (inverted deoxythymidine), LNA (Locked Nucleic Acid) between 5'-end, 3'-end, or 5'-end and 3'-end of aptamer to improve stability ), 2'-aminonucleoside, 2'F-nucleoside, amine linker, thiol linker, cholesterol, and the like can be modified.
  • 'peptide' refers to a compound in which two or more amino acids that specifically bind to amyloid beta are linked by peptide bonds.
  • the peptide may be a target binding polypeptide.
  • target binding polypeptide refers to a non-immunoglobulin polypeptide molecule having binding affinity for a target antigen or hapten like an antibody, but structurally unrelated to the antibody.
  • the target-binding polypeptide is also called an antibody-like molecule or antibody mimetics, and generally has a molecular weight of 3-20 kDa, unlike an antibody having a molecular weight of about 150 kDa.
  • the target-binding polypeptide includes Affibody derived from protein A Z-domain of Staphlyococcus aureus , Affilin derived from gamma-B crystallin or ubiquitin, and cystatin.
  • Affimer derived from cystatin Affitin derived from Sac7d of Sulfolobus acidocaldarius , Alphabody derived from triple helix coiled coil, Lipocalin Anticalin derived from lipocalin, Avimer derived from the cell membrane receptor domain, DARPin derived from ankyrin repeat motif, and Fynomer derived from the SH3 domain of Fyn.
  • a Kunits domain peptide derived from the Kunits domain of a protease inhibitor, a monobody derived from the 10th type III domain of fibronectin, a cloth Tridium perfringens ( Clostridium perfringens ) NagH of carbohydrate binding module 32-2 (carbohydrate binding module 32-2) derived from nanoclamp (nanoCLAMP) and the like, but is not limited thereto.
  • the target-binding polypeptide described above can be engineered to have binding affinity for any target antigen or hapten through various screening methods known in the art, such as phage display and ribosome display.
  • the term 'drug' refers to any substance that exhibits pharmacological effects in vivo.
  • the pharmacological effect includes an inhibitory effect on amyloid beta multimer formation.
  • the term 'candidate drug' refers to any substance expected to exhibit pharmacological effects in vivo.
  • MDS-HTS assay of the present invention it is possible to screen whether or not the candidate drug has a pharmacological effect in vivo, that is, the presence or absence of an inhibitory effect on amyloid beta multimer formation and/or the degree of inhibition.
  • the concentration of the candidate drug is 0.05 mg/ml to 5.0 mg/ml.
  • the concentration is 0.05 mg/ml to 5.0 mg/ml, 0.05 mg/ml to 4.0 mg/ml, 0.05 mg/ml to 3.0 mg/ml, 0.05 mg/ml to 2.0 mg /ml, 0.05 mg/ml to 1.0 mg/ml, 0.05 mg/ml to 0.5 mg/ml, 0.05 mg/ml to 0.1 mg/ml, 0.1 mg/ml to 5.0 mg/ml, 0.1 mg/ml to 4.0 mg /ml, 0.1 mg/ml to 3.0 mg/ml, 0.1 mg/ml to 2.0 mg/ml, 0.1 mg/ml to 1.0 mg/ml, 0.1 mg/ml to 0.5 mg/ml, 0.5 mg/ml to 5.0 mg /ml, 0.5 mg/ml to 4.0 mg/ml, 0.5 mg/ml to 3.0 mg/ml, 0.5 mg/ml to 2.0 mg/ml, 0.5 mg/ml, 0.1 mg/ml
  • the concentration is 0.1 mg/ml.
  • the concentration of the candidate drug is 0.05 mM to 5.0 mM.
  • the concentration of the candidate drug is 0.05 mM to 5.0 mM, 0.05 mM to 4.0 mM, 0.05 mM to 3.0 mM, 0.05 mM to 2.0 mM, 0.05 mM to 1.0 mM, 0.05 mM to 0.5 mM mM, 0.05 mM to 0.1 mM, 0.1 mM to 5.0 mM, 0.1 mM to 4.0 mM, 0.1 mM to 3.0 mM, 0.1 mM to 2.0 mM, 0.1 mM to 1.0 mM, 0.1 mM to 0.5 mM, 0.5 mM to 5.0 mM, 0.5 mM to 4.0 mM, 0.5 mM to 3.0 mM, 0.5 mM to 2.0 mM, 0.5 mM to 1.0 mM, 0.5 mM to 0.5 mM, 0.5 mM to 5.0 mM, 0.5 mM to
  • the concentration is 0.1 mM.
  • the negative control containing only the solvent is PBS or DMSO.
  • the composition for forming amyloid beta multimers reacts with a candidate drug and DMSO or PBS at room temperature for 30 minutes to 8 hours to induce formation of amyloid beta multimers.
  • the composition for forming amyloid beta multimers is prepared by mixing a candidate drug and DMSO or PBS at room temperature for 30 minutes to 8 hours, 30 minutes to 7 hours, 30 minutes to 6 hours, 30 minutes to 30 minutes. 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 1 hour to 8 hours, 1 hour to 7 hours, 1 hour to 6 hours, 1 hour to 5 hours , 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 8 hours, 2 hours to 7 hours, 2 hours to 6 hours, 2 hours to 5 hours, 2 hours to 4 hours, 2 3 hours to 3 hours, 3 hours to 8 hours, 3 hours to 7 hours, 3 hours to 6 hours, 3 hours to 5 hours, 3 hours to 4 hours, 4 hours to 8 hours, 4 hours to 7 hours, 4 hours to 4 hours 6 hours, 4 hours to 5 hours, 5 hours to 4 hours, 3 hours to 4 hours, 4 hours to 8 hours, 4 hours to 7 hours, 4 hours to 4 hours 6 hours, 4 hours to 5 hours
  • the composition for forming amyloid beta multimers is reacted with a candidate drug and DMSO or PBS at room temperature for 1 hour, 2 hours, 3 hours, and/or 4 hours to form amyloid beta multimers. induce the formation of
  • the composition for forming amyloid beta multimers reacts with a candidate drug and DMSO or PBS at room temperature for 4 hours to induce formation of amyloid beta multimers.
  • the composition for forming amyloid beta multimers reacts with a candidate drug and DMSO or PBS at room temperature for 2 hours or 4 hours to induce formation of amyloid beta multimers.
  • the step of detecting the amyloid beta capture antibody-amyloid beta multimer-amyloid beta detection antibody complex is Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay (Immunoprecipitation) Assay), radioimmunoassay, radio-immunodiffusion, double immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, flow cytometry ( Fluorescence Activated Cell Sorter (FACS), protein chip, surface plasmon resonance (SPR), etc. may be performed, but it is not necessarily limited thereto.
  • FACS Fluorescence Activated Cell Sorter
  • SPR surface plasmon resonance
  • the ELISA may be a sandwich-ELISA. In another embodiment of the present invention, the ELISA may be a competitive ELISA.
  • the method for high-throughput screening of a drug for preventing or treating Alzheimer's disease uses the kit for screening amyloid beta multimer inhibitory drug, which is another aspect of the present invention described above, overlapping to avoid excessive complexity described herein The content is used and its description is omitted.
  • doxorubicin or a derivative thereof is a compound that inhibits oligomerization or fibrillation of amyloid beta and exhibits a color change and optical density difference in a specific wavelength band by binding to amyloid beta.
  • the present invention relates to a composition for inhibiting oligomerization or fibrillation of amyloid beta containing doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, a composition for detecting amyloid beta containing the same, or preventing or treating Alzheimer's disease.
  • the present invention is an oligomerization (oligomerization) or fibril of amyloid beta (amyloid beta) containing doxorubicin (doxorubicin), a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient
  • oligomerization oligomerization
  • fibril of amyloid beta amyloid beta
  • doxorubicin doxorubicin
  • a composition for inhibiting fibrillization is provided.
  • doxorubicin a derivative thereof, or a pharmaceutically acceptable salt thereof exhibits an effect of inhibiting oligomerization or fibrillation of amyloid beta.
  • 'doxorubicin (DOX)' used herein has the IUPAC name (7 S ,9 S )-7-[(2 R ,4 S ,5 S ,6 S )-4-amino-5-hydroxy- 6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7 H -tetracene-5 ,12-dione, and means a compound having a chemical structure shown in Formula 1 below.
  • Doxorubicin is an anthracycline antibiotic first isolated in 1969 from Streptomyces peucetius var. caesius. (Arcamone, F. et al., 11:1101-1110, 1969).
  • doxorubicin is sold under the trade name Adriamycin and is used for cancer treatment such as breast cancer, Kaposi's sarcoma, lymphoma, and acute leukemia.
  • 'A ⁇ monomer' refers to amyloid beta of a single polypeptide.
  • 'oligomerization' of amyloid beta refers to a process and/or a result thereof in which soluble oligomers or multimers in which A ⁇ monomers themselves are integrated with each other are formed.
  • the term 'fibrillization' of amyloid beta refers to a process in which insoluble fibrils in which oligomers of amyloid beta are accumulated are formed and aggregates as a result.
  • the term 'pharmaceutically acceptable salt thereof' refers to a salt of doxorubicin, or a derivative of doxorubicin, which is a compound having a desired pharmacological effect, that is, an effect of inhibiting oligomerization or fibrillation of amyloid beta. .
  • These salts include inorganic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide; Tylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptano It can be formed using organic acids such as ate, hexanoate, 2-hydroxyethane sulfate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate, and undecanoate.
  • inorganic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide
  • Tylate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
  • the pharmaceutically acceptable salt of doxorubicin is doxorubicin hydrochloride (hydrochloride, HCl).
  • Doxorubicin hydrochloride refers to a compound having a chemical structure shown in Formula 2 below.
  • the expression 'comprising as an active ingredient' in the present specification means including an amount sufficient to achieve pharmacological efficacy or activity of doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof of the present invention, and delivery of a drug. , It means that various components can be additionally added for stabilization and formulation.
  • the term 'derivative thereof' refers to a compound obtained by chemically changing a part of a compound through the introduction of a functional group, oxidation, reduction, or substitution of atoms using a compound as a parent compound, and the structure and properties of the parent compound are similar compounds that have been altered without significantly changing the Usually, it refers to a compound in which a hydrogen atom or a specific group of atoms in a compound is substituted with another atom or group of atoms.
  • the derivative thereof is a derivative of doxorubicin.
  • the doxorubicin derivative has 4-methoxy-8,10-dihydro- 7H -tetracene-5,12-dione as a parent core.
  • the derivative is doxorubicinone, doxorubicinolone, or 7-deoxydoxorubicin aglycone, but is not necessarily limited thereto.
  • the derivative of doxorubicin, the active ingredient of the present invention has the same 4-methoxy-8,10-dihydro- 7H -tetracene-5,12-dione as doxorubicin as its parent nucleus, but introduces a functional group, oxidizes, It is a compound obtained by chemically changing a part of a compound through reduction, substitution of atoms, etc., and is a similar compound that has been changed to the extent that the structure and properties of the parent compound are not significantly changed, and has an inhibitory effect on amyloid beta oligomerization or fibrillation. It is a compound with
  • the term 'Doxorubicinone' refers to adriamycinone, adriamycin aglycone, 4'-deoxydoxorubicin 7-deoxyaglycone and 4-DOX-DONE
  • the IUPAC names are ( 7S , 9S )-6,7,9,11-tetrahydroxy-9-(2-hydroxyacetyl)-4-methoxy-8, It is 10-dihydro-7H-tetracene-5,12-dione and refers to a compound having a chemical structure shown in Formula 3 below.
  • the term 'Doxorubicinolone' means that the IUPAC name is (7 R ,9 R )-9-(1,2-dihydroxyethyl)-6,7,9,11-tetrahydroxy-4 -Methoxy-8,10-dihydro- 7H -tetracene-5,12-dione, and means a compound having a chemical structure shown in Formula 4 below.
  • the term '7-Deoxydoxorubicin aglycone' means that the IUPAC name is (9 R ) -6,9,11-trihydroxy-9- (2-hydroxyacetyl) -4- It is methoxy-8,10-dihydro- 7H -tetracene-5,12-dione, and means a compound having a chemical structure shown in Formula 5 below.
  • the derivatives of doxorubicin, doxorubicinone and doxorubicinolone are derived from the amino sugar at carbon 7 of doxorubicin ([(2 R ,4 S ,5 S ,6 S )-4-amino-5-hydroxy-6-methyloxane -2-yl]oxy) is a structural analogue or derivative in which the substituent is replaced by a hydroxy substituent. Additionally, in doxorubicinolone, the 2-hydroxyacetyl at carbon 9 of doxorubicin is substituted with 1,2-dihydroxyethyl.
  • Another derivative of doxorubicin, 7-deoxydoxorubicin aglycone is a structural analogue or derivative in which the aminosugar substituent at carbon 7 of doxorubicin is replaced by hydrogen.
  • the amyloid beta is composed of tissue, cerebrospinal fluid (CSF), serum, plasma, whole blood and interstitial fluid. Included in one or more species selected from the group, but is not necessarily limited thereto.
  • the present invention provides a composition for detecting amyloid beta containing doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the derivative is doxorubicinone, doxorubicinolone, 7-deoxydoxorubicin aglycone, or a mixture thereof.
  • doxorubicin does not interact with other proteins related to neurodegenerative diseases, but has an effect of causing color change by binding to amyloid beta.
  • the composition for detecting amyloid beta of the present invention interacts with tau or alpha-synuclein, which are other proteins related to neurodegenerative diseases, the color changes to brown, so that different proteins related to neurodegenerative diseases are indistinguishable from each other.
  • various amyloid beta species such as A ⁇ 43, A ⁇ 42 and A ⁇ 40, it shows a purple color change.
  • amyloid beta species exhibit a transparent color in a buffer solution such as PBS or TBS (Tris-buffered saline) and change to purple when doxorubicin is added, the presence of amyloid beta in a sample can be distinguished when using the composition.
  • a buffer solution such as PBS or TBS (Tris-buffered saline)
  • TBS Tris-buffered saline
  • the term 'detection' refers to the presence or absence of amyloid beta, amyloid beta oligomers and/or amyloid beta fibrils in a sample, measurement of the amount present in a sample and/or distribution pattern in a sample, or means decision.
  • the composition for detecting amyloid beta detects amyloid beta species in a sample.
  • amyloid beta species is in the form of multimers, oligomers and/or fibrils of at least one peptide represented by the following general formula 1:
  • the X is an integer selected from 1 or more and 12 or less
  • the Y is an integer selected from 33 or more and 43 or less.
  • the 'A ⁇ XY ' refers to a peptide sequence consisting of the Xth to Yth amino acids of the amyloid beta peptide sequence.
  • the amyloid beta species of the present invention may be a peptide including the entire amino acid sequence of A ⁇ 1-43 or a peptide fragment including a partial amino acid sequence of the entire amino acid sequence of A ⁇ 1-43 .
  • the amyloid beta species of the present invention may also be a peptide comprising the entire amino acid sequence of A ⁇ 1-42 or a peptide fragment comprising a partial amino acid sequence of the entire amino acid sequence of A ⁇ 1-42 .
  • amyloid beta species of the present invention are A ⁇ 1-33 , A ⁇ 1-34 , A ⁇ 1-36 , A ⁇ 1-37 , A ⁇ 1-38 , A ⁇ 1-39 , A ⁇ 1- 40 , A ⁇ 1-42 , A ⁇ 1-43 , A ⁇ 2-40 , A ⁇ 2-42 , A ⁇ 2-43 , A ⁇ 11-40 , A ⁇ 11-42 , and A ⁇ 11-43 .
  • Amyloid beta species but are not necessarily limited thereto.
  • a ⁇ 40 as used herein refers to A ⁇ 1-40
  • a ⁇ 42 refers to A ⁇ 1-42
  • a ⁇ 43 refers to A ⁇ 1-43 .
  • the composition for detecting amyloid beta changes color to purple when oligomers or fibrils of amyloid beta are present.
  • the composition for detecting amyloid beta changes color to purple when oligomers or fibrils of amyloid beta exist in the sample upon contact with the sample.
  • the color change is visually observed.
  • the color change is observed under a microscope.
  • the microscope is an optical microscope.
  • the composition for detecting amyloid beta has an absolute value of the difference in optical density in the 400 nm to 600 nm wavelength range before and after the amyloid beta reaction in the presence of oligomers or fibrils of amyloid beta of 0.05 to 0.25. .
  • the optical density can be measured and calculated in a wavelength range of 400 nm to 600 nm before and after contact between the composition for detecting amyloid beta and the sample using, for example, a microplate reader, If the absolute value of the optical density difference before and after contact is 0.05 to 0.25, it is determined that amyloid beta oligomers or fibrils are present in the sample, and if the absolute value is less than 0.05, amyloid beta oligomers or fibrils are not present in the sample. judge it to be
  • the optical density is the optical density value in the 400 nm to 600 nm wavelength range of the negative control group (including only the solution) and the positive control group (solution containing amyloid beta and doxorubicin whose concentrations are known) and the composition for detecting amyloid beta.
  • the absolute value of the difference in optical density in the 400 nm to 600 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25, 0.05 to 0.20, 0.05 to 0.15, 0.05 to 0.10, 0.10 to 0.25, 0.10 to 0.20, 0.10 to 0.15, 0.15 to 0.25, 0.20 to 0.20, or 0.20 to 0.25.
  • the composition for detecting amyloid beta is 400 nm to 600 nm, 400 nm to 580 nm, 400 nm before and after reaction with amyloid beta when oligomers or fibrils of amyloid beta are present.
  • the absolute value of the difference in optical density in the 400 nm to 440 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.1.
  • the absolute value of the difference in optical density in the 440 nm to 480 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25.
  • the absolute value of the difference in optical density in the 440 nm to 480 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25, 0.1 to 0.25, 0.15 to 0.25 or 0.2 to 0.25.
  • the absolute value of the difference in optical density in the 480 nm to 520 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25.
  • the composition for detecting amyloid beta, when oligomers or fibrils of amyloid beta are present the absolute value of the difference in optical density in the 480 nm to 520 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25, 0.1 to 0.25, 0.15 to 0.25 or 0.2 to 0.25.
  • the absolute value of the optical density difference in the 580 nm to 600 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25.
  • the composition for detecting amyloid beta, when oligomers or fibrils of amyloid beta are present the absolute value of the optical density difference in the 580 nm to 600 nm wavelength range before and after reaction with amyloid beta is 0.05 to 0.25, 0.1 to 0.25, 0.15 to 0.25 or 0.2 to 0.25.
  • the composition for detecting amyloid beta when oligomers or fibrils of amyloid beta exist, the absolute value of the difference in optical density in the 405 nm wavelength band before and after reaction with amyloid beta is 0.05 to 0.25 am.
  • the absolute value of the difference in optical density in the 405 nm wavelength band before and after reaction with amyloid beta is 0.05 to 0.05. is 0.1.
  • the absolute value of the difference in optical density in the 450 nm wavelength band before and after reaction with amyloid beta is 0.05 to 0.25 am.
  • the absolute value of the difference in optical density in the 450 nm wavelength band before and after reaction with amyloid beta is 0.10 to 0.10. It is 0.20.
  • the composition for detecting amyloid beta when oligomers or fibrils of amyloid beta exist, the absolute value of the difference in optical density in the 490 nm wavelength band before and after reaction with amyloid beta is 0.05 to 0.25 am.
  • the absolute value of the difference in optical density in the 490 nm wavelength band before and after reaction with amyloid beta is 0.10 to 0.10. It is 0.20.
  • the composition for detecting amyloid beta has an absolute value of the difference in optical density in the 590 nm wavelength band before and after reaction with amyloid beta when oligomers or fibrils of amyloid beta are present in the range of 0.05 to 0.25. .
  • the absolute value of the difference in optical density in the 590 nm wavelength band before and after reaction with amyloid beta is 0.15 to 0.25 or 0.20 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has an absolute difference in optical density between 400 nm and 600 nm before and after binding to amyloid beta in the range of 0.05 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has an absolute value of the difference in optical density at 405 nm before and after binding to amyloid beta in the range of 0.05 to 0.1.
  • the difference between after binding to amyloid beta and before binding to 405 nm by subtracting the optical density is -0.05 to -0.1.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has an absolute value of the difference in optical density at 450 nm before and after binding to amyloid beta in the range of 0.15 to 0.2.
  • the difference between after binding to amyloid beta and before binding to 450 nm by subtracting the optical density is -0.15 to -0.2.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has an absolute value of the difference in optical density at 490 nm before and after binding to amyloid beta in the range of 0.15 to 0.2.
  • the difference between after binding to amyloid beta and before binding to 490 nm by subtracting the optical density is -0.15 to -0.2.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has an absolute value of the difference in optical density at 590 nm before and after binding to amyloid beta in the range of 0.15 to 0.25 or 0.20 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicin as an active ingredient has a difference of 0.15 to 0.25 or 0.20 to 0.2 after subtracting the optical density at 590 nm before binding to amyloid beta.
  • the composition for detecting amyloid beta containing doxorubicinone as an active ingredient has an absolute difference in optical density between 400 nm and 600 nm before and after binding to amyloid beta in the range of 0.05 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicinone as an active ingredient has an absolute value of the difference in optical density at 450 nm before and after binding to amyloid beta in the range of 0.05 to 0.10.
  • the composition for detecting amyloid beta containing doxorubicinone as an active ingredient has a difference of -0.05 to -0.10 after subtracting the optical density at 450 nm before and after binding to amyloid beta.
  • the composition for detecting amyloid beta containing doxorubicinone as an active ingredient has an absolute value of the optical density difference at 590 nm before and after binding to amyloid beta of 0.15 to 0.25 or 0.20 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicinone as an active ingredient has a difference of 0.15 to 0.25 or 0.20 to 0.2 after subtracting the optical density at 590 nm before binding to amyloid beta. .
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has an absolute difference in optical density between 400 nm and 600 nm before and after binding to amyloid beta in the range of 0.05 to 0.25.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has an absolute value of the difference in optical density at 405 nm before and after binding to amyloid beta in the range of 0.05 to 0.1.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has a difference of -0.05 to -0.1 after subtracting the optical density at 405 nm before and after binding to amyloid beta.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has an absolute value of the optical density difference at 450 nm before and after binding to amyloid beta of 0.10 to 0.20 or 0.15 to 0.20.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has a difference of -0.10 to -0.20 or -0.15 after subtracting the optical density at 450 nm before and after binding to amyloid beta. to -0.20.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has an absolute value of the difference in optical density at 490 nm before and after binding to amyloid beta in the range of 0.10 to 0.15.
  • the difference between after binding to amyloid beta and before binding to 490 nm by subtracting the optical density is -0.10 to -0.15.
  • the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has an absolute value of the difference in optical density at 590 nm before and after binding to amyloid beta in the range of 0.15 to 0.25. In another specific embodiment of the present invention, the composition for detecting amyloid beta containing doxorubicinolone as an active ingredient has a difference of 0.15 to 0.25 after subtracting the optical density at 590 nm before and after binding to amyloid beta.
  • the change in color can determine the amount of beta oligomer in the sample by measuring the wavelength range of 400 nm to 600 nm. For example, by using a microplate reader, the wavelength range values of a negative control group (including only a solution) and a positive control group containing a known concentration of amyloid beta and a composition for detecting amyloid beta containing doxorubicin of the present invention are measured, , A composition for detecting amyloid beta is brought into contact with a sample whose concentration is to be known, the wavelength value thereof is measured, and then the amount of amyloid beta present in the sample, that is, the concentration, can be determined through extrapolation.
  • a negative control group including only a solution
  • a positive control group containing a known concentration of amyloid beta and a composition for detecting amyloid beta containing doxorubicin of the present invention are measured, , A composition for detecting amyloid beta is brought into contact with a sample whose concentration is to be known, the wavelength value thereof is
  • the change in color can determine the amount of oligomeric beta in a sample by measuring a wavelength range of 400 nm to 600 nm.
  • the sample for which the concentration is to be known includes one or more selected from the group consisting of tissue, cerebrospinal fluid, serum, plasma, whole blood, and interstitial fluid, but is not necessarily limited thereto.
  • the composition for detecting amyloid beta according to the present invention can be used to determine the distribution pattern of amyloid beta in tissues. For example, the presence or absence of amyloid beta in brain tissue by reacting the sliced brain tissue with the composition for detecting amyloid beta of the present invention under appropriate conditions and observing the resulting color change with the naked eye or a microscope, abundance, and/or distribution pattern can be determined.
  • the composition for detecting amyloid beta is postmortem brain tissue isolated from a species selected from the group consisting of mouse, rat, guinea pig, rabbit, pig, cow, non-human primate and human It can be used to determine the distribution pattern of amyloid beta in Such non-human primates include apes, old world monkeys, and new world monkeys.
  • the non-human primate is a rhesus monkey or a cynomolgus monkey.
  • composition for detecting amyloid beta according to the present invention includes the composition for inhibiting oligomerization or fibrillation of amyloid beta, which is another aspect of the present invention described above, duplicate contents are used to avoid excessive complexity described in the present specification. , the description is omitted.
  • the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • composition of the present invention has a fundamental therapeutic effect of inhibiting oligomerization and/or fibrillation of amyloid beta, it can be usefully used for preventing or treating Alzheimer's disease.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier of the present invention is commonly used in the art to which the present invention pertains and is pharmacologically compatible with doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof, which is an active ingredient of the present invention.
  • Pharmaceutically acceptable carriers of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose , water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.
  • the pharmaceutical composition of the present invention may further include excipients, stabilizers, diluents, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
  • excipients stabilizers, diluents, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like.
  • Suitable pharmaceutically acceptable carriers, vehicles, excipients, stabilizers or diluents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, percutaneous injection ), intracerebral injection, intraspinal injection, etc.
  • the suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
  • the dosage of the pharmaceutical composition of the present invention is preferably 0.0001-100 mg/kg (body weight) per day.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
  • doxorubicin is sold under the trade name Adriamycin and is used for cancer treatment such as breast cancer, Kaposi's sarcoma, lymphoma, and acute leukemia.
  • Doxorubicin was approved for medicinal use in the United States in 1974 and is designated as an essential drug by the WHO. Therefore, from the viewpoint of drug repurposing, the preventive or therapeutic effect on Alzheimer's disease, which is a new indication of doxorubicin newly identified in the present invention, has a fundamental therapeutic effect and can lower the overall development cost and shorten the development time. , it is judged to be a promising alternative for prevention or treatment of Alzheimer's disease.
  • doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof has an effect of inhibiting amyloid beta oligomerization and/or fibrillation, and thus can be used for preventing or treating Alzheimer's disease, while amyloid beta species are present in the sample.
  • the term 'theragnostic composition' refers to a composition capable of simultaneously diagnosing and treating a specific disease, disease or medical condition.
  • the present invention provides a theragnosis composition containing doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, and capable of simultaneously diagnosing and treating Alzheimer's disease.
  • the pharmaceutical composition for preventing or treating Alzheimer's disease or the theragnosis composition according to the present invention includes the composition for inhibiting oligomerization or fibrillation of amyloid beta, which is another aspect of the present invention, excessive complexity described herein In order to avoid, duplicate contents are used, and descriptions thereof are omitted.
  • the present invention provides a kit for diagnosing Alzheimer's disease comprising the composition for detecting amyloid beta according to one embodiment.
  • the kit for diagnosing Alzheimer's disease is not only the composition for detecting amyloid beta according to the present invention, but also other components well-known in the art suitable for measuring color change, such as compositions, solutions, buffers (buffers), or devices.
  • compositions, solutions, buffers (buffers), or devices can include
  • devices that may be included in the kit of the present invention include a microplate reader capable of measuring color change, a silane-coated slide glass for observing color change under a microscope, and a slide glass A slide glass cover, a microscope, etc., a constant temperature reactor for the formation of amyloid beta oligomers and/or fibrils, specifically a 37° C. constant temperature reactor, an incubator, a shaker, etc. may be included, but is not necessarily limited thereto. .
  • solutions that may be included in the kit of the present invention include a 10% (v/v) sucrose solution to prevent tissue and/or cell deformation by freezing, and tissue and/or cell damage during rapid tissue freezing.
  • Isopentane for blocking formalin for tissue fixation, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, etc., paraffin for tissue sectioning, OCT compound, etc.
  • Known mounting that can mount tissue sections on slides medium with/without DAPI may be included, but is not necessarily limited thereto.
  • the kit of the present invention may include a buffer solution (buffer) for diluting a sample to be analyzed, for example, PBS, DMSO, glycine-sodium hydroxide buffer, etc., but is not necessarily limited thereto.
  • buffer solution for diluting a sample to be analyzed
  • PBS PBS
  • DMSO DMSO
  • glycine-sodium hydroxide buffer etc., but is not necessarily limited thereto.
  • the buffer solution is Tween ® 20, Tween ® 80, Triton X-100, Triton X-114, NP-40, Polysorbate 20, Octyl glucoside, Octyl thioglucoside, SDS (sodium dodecyl sulfate), CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (3-(3-Cholamidopropyldimethylammonio)-2-hydroxy-1-propanesulfonate), and the like may be further included.
  • kit of the present invention examples include a negative control composition containing only the buffer solution, a positive control composition containing the buffer solution and amyloid beta oligomers and/or fibrils, amyloid beta oligomers and / or an amyloid beta monomer for fibril formation may be included, but is not necessarily limited thereto.
  • the present invention provides a method for providing information necessary for diagnosis of Alzheimer's disease comprising the following steps:
  • the step (b) is a step of observing the color change of the sample after contact with the color of the sample before contacting the composition with the sample.
  • diagnosis means confirming the presence or characteristics of a pathological condition.
  • diagnosis is to determine whether or not Alzheimer's disease has occurred, how far the disease has progressed, or whether it is possible to develop it.
  • the sample may include at least one selected from the group consisting of tissue, cerebrospinal fluid, serum, plasma, whole blood, and interstitial fluid.
  • step (b) when the color changes to purple in step (b), it is determined that amyloid beta is present.
  • the color of the sample before contacting the composition with the sample is a transparent color.
  • amyloid beta when the color of the sample after contacting the composition with the sample is brown, it is determined that amyloid beta is not present in the sample.
  • the amyloid beta may be oligomers and/or fibrils of amyloid beta, and any amyloid beta species may be included without limitation.
  • amyloid beta when the color of the sample after contacting the composition with the sample is purple, it is determined that amyloid beta is present in the sample.
  • the amyloid beta may be oligomers and/or fibrils of amyloid beta, and any amyloid beta species may be included without limitation.
  • the method further comprises the following step:
  • the negative control is PBS, TBS, or PBS or TBS containing a surfactant.
  • the positive control is PBS containing 3 to 5 different concentrations of amyloid beta, or TBS.
  • the positive control is PBS containing 3 to 5 different concentrations of amyloid beta and a surfactant, or TBS.
  • the positive control group is at least 3, at least 4, at least 5, 3 to 5, 3 to 8, 3 to 10, 3 to 15, 3 to 20, 5 to 8, 5 to 10, 5 to 15, 5 to 20, 8 to 10, 8 to 15, 8 to 20, 10 to 15, 10 to 20, or 15 to 20 different concentrations of amyloid beta, or PBS or TBS comprising different concentrations of amyloid beta described above and a surfactant.
  • the surfactant is Tween ® 20, Tween ® 80, Triton X-100, Triton X-114, NP-40, Polysorbate 20, Octyl glucoside, Octyl thioglucoside, SDS (Sodium dodecyl sulfate), CHAPS (3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate) and CHAPSO (3-(3-Cholamidopropyldimethylammonio)-2-hydroxy-1-propanesulfonate).
  • the method further comprises the following steps:
  • the negative control exhibits a color change, exhibiting a clear color before contact with the composition and exhibiting a brown color after contact with the composition.
  • the positive control shows a color change, showing a transparent color before contacting the composition and showing a purple color after contacting the composition.
  • the color change of the sample shows a color change from transparent to brown before and after contact with the composition, it is determined that the color change of the negative control is similar, and it is determined that amyloid beta does not exist in the sample.
  • the presence or absence of amyloid beta, amyloid beta oligomers, amyloid beta fibrils, and various amyloid beta species in the sample can be determined, thereby causing the onset of Alzheimer's disease. It is possible to provide information necessary for determining whether or not the disease progresses or whether there is a possibility of onset, that is, information necessary for diagnosing Alzheimer's disease.
  • the step (b) is a step of measuring the optical density of the 400 nm to 600 nm wavelength band of the sample before and after the step (a), and the sample before and after the step (a).
  • the absolute value of the optical density difference in the 400 nm to 600 nm wavelength range is 0.05 to 0.25, it is determined that amyloid beta is present.
  • determining that amyloid beta exists is to provide information necessary for diagnosis of Alzheimer's disease.
  • the method further comprises the following step:
  • the method further comprises the following step:
  • the positive control is PBS or TBS containing 3 to 5 different concentrations of amyloid beta, and the measured value of the 400 nm to 600 nm wavelength band of the sample is 400 nm to 600 nm of the positive control. If included between the measured values of the wavelength band of , it is determined that amyloid beta exists in the sample, and the concentration of amyloid beta in the sample is determined by extrapolation; or
  • the positive control is PBS or TBS containing 3 to 5 different concentrations of amyloid beta, and the measured value of the 400 nm to 600 nm wavelength band of the sample is 400 nm to 600 nm of the positive control. If it is not included between the measured values of the wavelength range of , the sample is further diluted and steps (a), (b), (d-1), (d-2) and (d-5) are repeated. .
  • the method of the present invention when diagnosing a patient as having Alzheimer's disease by the method, provides the diagnosed patient with an Alzheimer's disease drug, the above-mentioned doxorubicin, a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • a step of administering a salt or a candidate substance for the treatment of Alzheimer's disease may be additionally included.
  • the method for providing information for diagnosis of Alzheimer's disease uses the composition for detecting amyloid beta, which is another aspect of the present invention described above, redundant content is used to avoid excessive complexity described herein, omit that description.
  • the present invention provides a kit for screening amyloid beta multimer inhibitory drug.
  • the present invention provides a method for high-throughput screening of drugs for preventing or treating Alzheimer's disease.
  • the present invention provides a composition for inhibiting oligomerization or fibrillation of amyloid beta, comprising doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a composition for detecting amyloid beta comprising the composition.
  • the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising the composition and a pharmaceutically acceptable carrier.
  • the present invention provides a method for providing information necessary for diagnosis of Alzheimer's disease.
  • drugs having an amyloid beta multimer inhibitory effect can be rapidly screened in large quantities from candidate substances, thereby discovering and treating drugs for preventing or treating Alzheimer's disease. It can be useful for preemption.
  • composition for inhibiting oligomerization or fibrillation of amyloid beta containing doxorubicin, a derivative thereof, or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient is used, oligomerization or fibrillation of amyloid beta is prevented. Since it inhibits significantly, it can be usefully used for preventing, improving, or treating Alzheimer's disease, as well as providing information necessary for detecting amyloid beta and diagnosing Alzheimer's disease.
  • MDS-HTS multimer detection system-high throughput system
  • Figure 2 shows the results of screening for potential inhibitory effects of small molecule compounds on amyloid beta multimers using MDS-HTS according to an embodiment of the present invention.
  • Figure 3 shows the results of screening for potential inhibitory effects on amyloid beta multimers of extracts using MDS-HTS according to an embodiment of the present invention.
  • Figure 4a shows the MDS-HTS analysis results for curcumin known to have an amyloid beta oligomer inhibitory effect.
  • Figure 4b is a result of showing the area under the curve (AUC, area under the curve) for the amyloid beta 42 oligomer ratio values over time in Figure 4a based on the AUC value of the negative control group, showing the effect of curcumin on reducing A ⁇ oligomers .
  • Figure 5 shows the absorbance change at a wavelength of 450 nm with time (h) as a result of MDS-HTS analysis of doxorubicin.
  • Figure 7 shows the change in fluorescence signal according to time (min) as a result of thioflavin thi (ThT) analysis of doxorubicin.
  • FIG. 9 shows color changes according to the reaction between doxorubicin and amyloid beta, tau and alpha-synuclein.
  • FIG. 9a shows a photograph before treatment of each neurodegenerative disease-related protein with doxorubicin
  • FIG. 9b shows a photograph after treatment of each neurodegenerative disease-related protein with doxorubicin.
  • MDS-HTS is a method for high throughput screening (HTS) of drugs having an amyloid beta multimer inhibitory effect from candidate substances using a multimer detection system (MDS) capable of detecting amyloid beta multimers.
  • HTS high throughput screening
  • MDS multimer detection system
  • the antibodies used in the MDS-HTS experiment include a capture antibody and a detection antibody that specifically bind to amyloid beta.
  • the capture antibody is coated on the bottom of a solid substrate such as a plate, and binds to amyloid beta to fix the amyloid beta to the bottom of the plate.
  • the detection antibody also binds to amyloid beta, and at the same time is labeled with a labeling substance and conjugation, for example, HRP, so that it is sequentially combined with TMB (3,3',5,5'-tetramethylbenzidine) and stop solution (H2SO4, 1 M). Reaction shows maximum absorbance at a wavelength of 450 nm.
  • Epitopes recognized by the capture antibody and the detection antibody overlap each other at the N-terminus of amyloid beta. More specifically, the capture antibody according to an embodiment of the present invention has amyloid beta N-terminal residues 3 to 8 as an epitope, and the detection antibody has amyloid beta N-terminal residues 1 to 4 as an epitope , the epitopes of the two antibodies overlap at residues 3 and 4 of the N-terminus of amyloid beta. In the present invention, this was referred to as an epitope-overlapping antibody. Those skilled in the art will understand that any antibody having an overlapping epitope at the N-terminus of amyloid beta, specifically, any antibody having at least one or all identical amino acid residues of the epitope may be used in the present invention without limitation.
  • the capture antibody and the detection antibody are epitope-overlapping antibodies, the amyloid beta monomer whose amyloid beta N-terminus is already occupied by the capture antibody cannot additionally bind to the detection antibody (vice versa) (see the bottom of FIG. 1), Since the amyloid beta multimer has at least two N-terminals, even if one of the amyloid beta N-terminals is already occupied by the capture antibody, the detection antibody can bind to another N-terminus (vice versa) (see top of FIG. 1). ).
  • the MDS of the present invention can detect only the multimer form of amyloid beta, distinguishing it from the amyloid beta monomer.
  • amyloid beta and a candidate drug that is, a sample
  • sample-A ⁇ reaction see FIG. 1
  • amyloid beta multimer formation is induced, and then the capture antibody and the detection antibody are sequentially, simultaneously, or oppositely
  • the capture antibody and the detection antibody are sequentially, simultaneously, or oppositely
  • This method of the present invention was referred to as MDS-HTS.
  • the specific method is as follows.
  • amyloid beta multimer inhibitory effect of small molecule compounds Samples 1 and 2, extracts Samples 3 and 4, negative control (solvent) and positive control (doxorubicin) was investigated using MDS-HTS.
  • Example 1 samples 1 and 2, which are small molecule compounds, and samples 3 and 4, which are extracts in Example 2 (see FIG. 3), investigate the amyloid beta multimer inhibitory effect.
  • the negative control used in Examples 1 and 2 is a control containing only PBS (phosphate buffered saline) or DMSO (dimethyl sulfoxide) solvent
  • the positive control is the amyloid beta oligomer
  • Doxorubicin a compound confirmed to have a significant inhibitory effect on
  • Curcumin a compound already known to have a beta oligomer inhibitory effect, was compared with a positive control (doxorubicin).
  • Lyophilized amyloid beta 42 peptide was purchased from rPeptide.
  • the A ⁇ 42 peptide was diluted with PBS, and the diluted A ⁇ 42 peptide solution was aliquoted into a polypropylene (PP) tube by 270 ⁇ l, and then stored at -80°C until use.
  • PP polypropylene
  • the A ⁇ 42 peptide stored at -80 ° C in Preparation Example 2 was diluted once more with PBS and added to the 384 black well plate by 32 ⁇ l per well.
  • the 384 black well plate was then centrifuged at 500 rpm for 20 seconds at room temperature, i.e. spun down, to allow the samples, negative control and positive control to react with amyloid beta, respectively. Then, it was reacted at room temperature for 0 hour, 1 hour, 2 hours, 3 hours, and 4 hours. At each reaction time, 5 ⁇ l of sample or control was taken and put into a 1.5 ml PP tube and diluted with PBST. As a result, the reactants were stored at -80 °C until the next experiment.
  • a coating buffer was prepared by diluting mouse monoclonal antibody 6E10 (BioLegend), an amyloid beta capture antibody, in sodium carbonate-bicarbonate of pH 9.4 to a concentration of 0.5 ⁇ g/ml. . 100 ⁇ l of the coating buffer was dispensed into a 96-well plate, incubated overnight in a 4° C. refrigerator, and the plate was coated with the capture antibody.
  • the detection buffer is PBST containing the detection antibody WO2-HRP antibody (Absolute Antibody) at a concentration of 0.1 ⁇ g/ml.
  • TMB (3,3',5,5'-tetramethylbenzidine) solution was dispensed into each well, followed by incubation for 15 minutes. 50 ⁇ l of stop solution (H2SO4, 1 M) was dispensed into each well. The signal was then measured using a microplate reader (PerkinElmer Victor-3®) at a wavelength of 450 nm.
  • the samples 1 to 4 have an inhibitory effect on amyloid beta multimers, since the detection antibody does not bind to amyloid beta multimers, no signal is generated at a wavelength of 450 nm (see FIG. 1). Conversely, if the sample does not have an inhibitory effect on the amyloid beta multimer, since the detection antibody binds to the amyloid beta multimer, a signal is generated at a wavelength of 450 nm (see FIG. 1).
  • amyloid beta oligomer ratio (A ⁇ 42 oligomer ratio) was calculated.
  • the amyloid beta oligomer ratio is a ratio obtained by dividing the result value measured at each time point in the 450 nm wavelength band of each sample by the average value of the values measured at 0 hour in the 450 nm wavelength band of the negative control group.
  • the results for the low-molecular-weight compound are shown in Figure 2, the results for the extract in Figure 3, and the results for curcumin in Figure 4.
  • Figure 2 shows the results of samples 1 and 2, which are low molecular weight compounds, positive control group (doxorubicin) and negative control group.
  • the negative control showed an increase in the ratio of A ⁇ 42 oligomers over time, indicating that amyloid beta multimers were formed in the MDS-HTS condition constructed in the present invention. Confirmed.
  • the positive control showed a significantly lower A ⁇ 42 oligomer ratio compared to the negative control. This indicates that the positive control group has a significant inhibitory effect on amyloid beta multimer formation.
  • sample 1 Compared to the ratios of the negative control group and the positive control group, sample 1 showed no significant difference in A ⁇ 42 oligomer ratio compared to the negative control group at all reaction times or was rather high, indicating no amyloid beta oligomer inhibitory effect. In addition, the A ⁇ 42 oligomer ratio of sample 2 was lower than that of the negative control group, and showed a pattern similar to that of the positive control group over time.
  • sample 2 has a significant inhibitory effect on amyloid beta oligomers, and suggests that it is a potential Alzheimer's preventive or therapeutic drug that can be usefully used for the prevention or treatment of Alzheimer's disease.
  • Figure 3 shows the results of extracts Sample 3 and Sample 4, positive control (doxorubicin) and negative control.
  • the negative control group formed multimers by increasing the ratio of A ⁇ 42 oligomers with the lapse of reaction time.
  • the positive control group showed a significantly lower A ⁇ 42 oligomer ratio compared to the negative control group. It was confirmed once again that the positive control group had an inhibitory effect on amyloid beta oligomer.
  • sample 3 Compared to the A ⁇ 42 oligomer ratios of the negative and positive controls, sample 3 showed a change in A ⁇ 42 oligomer ratio over time similar to that of the negative control at all reaction times, so sample 3 had no amyloid beta oligomer inhibitory effect. sound was confirmed. In contrast, in sample 4 (sample 4), there was no significant difference in the ratio of amyloid beta 42 oligomers at 2 and 3 hours of reaction compared to 0 hour of reaction.
  • sample 4 may have an effect of inhibiting the formation of amyloid beta oligomers, and suggest that it is a potential drug for preventing or treating Alzheimer's disease that can be usefully used for preventing or treating Alzheimer's disease.
  • Curcumin is already known to have an inhibitory effect on amyloid beta oligomers (see Yang F, et al. J Biol Chem. 2005 Feb 18;280(7):5892-901.), but the availability of curcumin in the brain Curcumin's bioavailability is low due to low bioavailability compared to other organs, low solubility, poor absorption, rapid metabolism and elimination, and curcumin's use in medicine and food is limited (Vareed SK, et al., Cancer Epidemiol Biomarkers Prev. 2008 Jun; 17(6):1411-7.).
  • the negative control group showed no significant change in the A ⁇ 42 oligomer ratio over time.
  • the ratio of the positive control group blue dotted line
  • doxorubicin a positive control of the present invention, has a significant inhibitory effect on amyloid beta oligomer formation.
  • curcumin (solid line) showed a similar aspect to the negative control group at all reaction times, but showed a decreased A ⁇ oligomer ratio compared to the negative control group. It seems that curcumin, which is known to have an amyloid beta oligomer inhibitory effect, has an inhibitory effect on A ⁇ oligomer formation compared to the negative control group.
  • FIG. 4B is a graph showing the area under the A ⁇ 42 oligomer ratio graph with time (AUC) of FIG. 4A.
  • the positive control doxorubicin showed a significant decrease in the area compared to the negative control (p ⁇ 0.05). This means that doxorubicin, which is a positive control of the present invention, has an inhibitory effect on the formation of amyloid beta multimers, as examined in the results of the above examples.
  • curcumin also showed a significantly reduced area ( p ⁇ 0.05), but positive control It did not show as great an amyloid beta multimer inhibitory effect as doxorubicin.
  • MDS-HTS analysis method of the present invention is specialized for analyzing the inhibitory effect of a candidate drug on amyloid beta oligomer formation compared to the conventional method for confirming the amyloid beta inhibitory effect.
  • the present inventors were able to prove that the MDS-HTS of the present invention is a method capable of more precisely discovering drugs having an inhibitory effect on amyloid beta multimers by rapidly screening a large amount of candidate substances compared to the conventional screening method.
  • Those skilled in the art will understand that any substance having a potential inhibitory effect on amyloid beta multimer formation can be applied to MDS-HTS, even if the candidate substance is in the form of other than a small molecule compound and an extract.
  • the composition of a composition suitable for rapid screening of drugs with amyloid beta multimer inhibitory effect in large quantities from various types of candidate substances and MDS-HTS with built-in antibody concentrations are provided.
  • doxorubicin which exhibits a significant amyloid beta multimer formation inhibitory effect as a result of MDS-HTS analysis, also exhibits an inhibitory effect on amyloid beta oligomerization and fibrillization, a derivative of doxorubicin. It was also investigated whether the same or similar effects were exhibited and whether doxorubicin could be used for the detection of amyloid beta.
  • Doxorubicin purchased from Sigma-Aldrich was diluted in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL (10 mM) to make a stock, and stored at -20°C until use. did
  • Lyophilized A ⁇ 42 peptide was purchased from rPeptide.
  • the A ⁇ 42 peptide was diluted with PBS, and the diluted A ⁇ 42 peptide solution was aliquoted into a polypropylene (PP) tube by 270 ⁇ l, and then stored at -80°C until use.
  • PP polypropylene
  • MDS-HTS Multimer Detection System-High Throughput Screening
  • the MDS-HTS experiment is an amyloid beta multimer disaster drug screening method developed by the present inventors, using a system (MDS) that can detect amyloid beta multimers (oligomers) by distinguishing them from amyloid beta monomers. It is a method for rapid and precise screening (HTS) of drugs with an amyloid beta multimer inhibitory effect from candidates for the treatment of Alzheimer's disease in large quantities.
  • MDS amyloid beta multimer disaster drug screening method developed by the present inventors, using a system (MDS) that can detect amyloid beta multimers (oligomers) by distinguishing them from amyloid beta monomers. It is a method for rapid and precise screening (HTS) of drugs with an amyloid beta multimer inhibitory effect from candidates for the treatment of Alzheimer's disease in large quantities.
  • HTS rapid and precise screening
  • doxorubicin has an effect of inhibiting the formation of amyloid beta multimers, that is, amyloid beta oligomers.
  • Doxorubicin at a concentration of 10 mg/mL (10 mM) stored at -20 ° C in the above preparation was dissolved at room temperature, diluted 20-fold with PBS, and doxorubicin at a final concentration of 0.5 mg / mL (0.5 mM) was used in the experiment. .
  • Doxorubicin at a concentration of 0.5 mM was added to a 384 black well plate at 8 ⁇ L per well.
  • doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone were dissolved in DMSO and PBS, respectively, at a concentration of 0.5 mM, and 8 ⁇ L per well was added to a 384 black well plate.
  • the A ⁇ 42 peptide stored at -80 ° C of the above preparation was diluted so that the concentration ratio of doxorubicin (0.5 mg / mL) and A ⁇ 42 peptide was 0.5: 1 to 50: 1, and the diluted A ⁇ 42 peptide was diluted with 32 ⁇ L per well. were added to black well plates.
  • the 384 black well plate was then briefly centrifuged at 500 rpm for 20 seconds at room temperature, i.e. spun down, to allow doxorubicin and its derivatives to react with amyloid beta.
  • the reaction was carried out at room temperature for 0 hour, 2 hours and 4 hours.
  • 5 ⁇ L of the reaction was put into a 1.5 mL PP tube and diluted using PBST.
  • the reactants were stored at -80 °C until the next experiment.
  • mouse monoclonal antibody 6E10 (BioLegend, USA), an amyloid beta capturing antibody, was diluted in sodium carbonate-bicarbonate of pH 9.4 to a concentration of 0.5 ⁇ g/mL, and the coating buffer (coating buffer) buffer) was prepared. 100 ⁇ L of the coating buffer was dispensed into a 96-well plate, incubated overnight in a 4° C. refrigerator, and the plate was coated with the capture antibody.
  • a blocking buffer containing 2% BSA bovine serum albumin
  • the detection buffer is PBST containing a detecting antibody, WO2-HRP antibody (PeopleBio Inc., South Korea) at a concentration of 0.1 ⁇ g/ml.
  • TMB (3,3',5,5'-tetramethylbenzidine) solution was dispensed into each well and incubated for 15 minutes.
  • the signal was then measured using a microplate reader (PerkinElmer Victor- 3® ) at a wavelength of 450 nm. A signal was measured at a wavelength of 450 nm together with a negative control.
  • amyloid beta-capturing antibody and the amyloid beta-detection antibody used in MDS-HTS are epitope-overlapping antibodies in which at least one amino acid residue in the epitope of amyloid beta is the same. That is, the capture antibody and the detection antibody are in a competitive relationship with respect to the epitope of amyloid beta.
  • amyloid beta oligomers are not formed and exist as amyloid beta monomers, and since the epitope of these amyloid beta monomers is already occupied by the capture antibody, the detection antibody is Since it does not bind to oligomers, no signal is generated at a wavelength of 450 nm.
  • amyloid beta oligomers are formed if the sample does not have an inhibitory effect on amyloid beta oligomerization, even if the epitope of any part of the amyloid beta oligomer formed by the capture antibody is occupied, the detection antibody is amyloid beta oligomer. Since it can bind to oligomers, a signal is generated at a wavelength of 450 nm.
  • amyloid beta 42 oligomer ratio (A ⁇ 42 oligomer ratio) was calculated.
  • the amyloid beta oligomer ratio is a ratio obtained by dividing the result value measured at each time point in the 450 nm wavelength band of each sample by the average value of the values measured at 0 hour in the 450 nm wavelength band of the negative control group. The resulting graphs are shown in FIGS. 5 and 6 .
  • doxorubicin (red solid line) significantly decreased the A ⁇ 42 oligomer ratio according to the reaction time. It was once again confirmed that doxorubicin had a significant inhibitory effect on amyloid beta oligomerization.
  • doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone which are derivatives of doxorubicin, showed an increase in the A ⁇ 42 oligomer ratio over time similar to that of the negative control, so all of them did not show an inhibitory effect on amyloid beta oligomerization. I was able to confirm that it wasn't.
  • doxorubicin exhibits a significant inhibitory effect on amyloid beta oligomerization, which is characteristic of doxorubicin compared to its structural derivatives.
  • Thioflavin T assay (ThT assay) was performed to confirm whether doxorubicin exhibits an inhibitory effect on amyloid beta fibrillation as well as the inhibitory effect on amyloid beta oligomerization described above.
  • the doxorubicin of Preparation Example was reacted with the Thioflavin T solution, and then the fluorescence signal was measured by reacting with the amyloid beta of Preparation Example.
  • doxorubicin of the preparation example was diluted with PBS, and 10 ⁇ L of doxorubicin at a concentration of 0.5 mg/mL was added to each well.
  • Prepare the A ⁇ 42 peptide of the preparation so that the concentration ratio of doxorubicin (0.5 mg / mL) and A ⁇ 42 peptide is 0.5: 1 to 50: 1, add 30 ⁇ L of it to each well, and react at 37 ° C. for 24 hours .
  • As a negative control only the A ⁇ 42 peptide prepared above was reacted with the thioflavin T solution without the addition of doxorubicin, and the fluorescence signal was measured.
  • ThT analysis for doxorubicin, doxorubicinone, doxorubicinolone and 7-deoxydoxorubicin aglycone was also performed in the same manner as the above-described method, and the results are shown in FIG. 8 .
  • doxorubicin had a significant inhibitory effect on not only amyloid beta oligomerization but also amyloid beta fibrillation.
  • doxorubicin derivatives of doxorubicin, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone also showed significantly lower fluorescence signals depending on the reaction time. This means that the above three doxorubicin derivatives also exhibit significant inhibitory effects on amyloid beta fibrillation.
  • the doxorubicin derivative exhibits a significant inhibitory effect on amyloid beta fibrillation similarly to doxorubicin.
  • doxorubicin the active ingredient of the present invention, exhibits significant inhibitory effects on both amyloid beta oligomerization and fibrillation, and doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone, which are derivatives of doxorubicin, amyloid beta fibrils. It was confirmed that it showed a significant inhibitory effect only on fire.
  • the MDS-HTS method used in the present invention is a screening specialized for detecting an inhibitory effect on amyloid beta oligomers.
  • doxorubicin had an effect of significantly inhibiting oligomerization and fibrillation of amyloid beta.
  • Doxorubicin and amyloid beta were reacted and color change was confirmed.
  • the color changes of tau and alpha-synuclein, which are other proteins associated with neurodegenerative diseases were also investigated.
  • a solution containing only PBS was used as a negative control.
  • a ⁇ 43, A ⁇ 42, A ⁇ 40, tau, and alpha-synuclein were obtained from rPeptide, diluted with doxorubicin (0.5 mg/mL) and PBS so that their concentration ratios were 0.5:1 to 50:1, respectively.
  • a ⁇ 43, A ⁇ 42, A ⁇ 40, tau and alpha-synuclein were first mixed with 32 ⁇ L each of 1.5 mL of E.P. added to the tube.
  • the tube containing only PBS before addition of doxorubicin was transparent, but the tube containing only PBS immediately after addition of doxorubicin showed a brown color.
  • tubes containing tau and alpha-synuclein which are proteins associated with neurodegenerative diseases, known as another causative protein of dementia, showed a transparent color before doxorubicin addition and a brown color immediately after doxorubicin addition.
  • doxorubicin exhibits a brown color in PBS, but turns purple when it reacts with amyloid beta.
  • a ⁇ 43, A ⁇ 42, and A ⁇ 40 which are amyloid beta species having various C-terminals, all reacted with doxorubicin and turned purple, it is assumed that doxorubicin can bind to all amyloid beta species.
  • the tube color of A ⁇ 40 after reaction with doxorubicin shows a lighter purple color than other amyloid beta species, A ⁇ 43 and A ⁇ 42, it is thought that doxorubicin has binding specificity for the C-terminal part of amyloid beta.
  • doxorubicin specifically turns purple when reacting with amyloid beta species, which is distinct from other proteins related to neurodegenerative diseases, in PBS as well as TBS (Tris-Buffered Saline). It was confirmed that a characteristic color change was exhibited.
  • doxorubicin exhibited a characteristic color change by specifically binding only to amyloid beta species, which is distinct from other proteins associated with neurodegenerative diseases. This result indicates that doxorubicin can be usefully used as an amyloid beta-specific detection compound.
  • Example 4 Optical density difference analysis according to the binding of doxorubicin and its derivatives and amyloid beta
  • doxorubicin the active ingredient of the present invention, exhibits a characteristic color change that changes to purple upon reaction with amyloid beta, so doxorubicin is useful for confirming the presence or absence of amyloid beta in a sample.
  • the present inventors go beyond visually observing the color change of doxorubicin upon reaction with amyloid beta and measure the optical density (OD) before and after the reaction between doxorubicin and its derivatives and amyloid beta, thereby amyloid beta in the sample. It was intended to confirm the presence and/or amount of
  • Doxorubicin, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone were prepared at a concentration of 0.5 mM in DMSO and PBS, and then added to a 384 black well plate at 8 ⁇ L per well, using a microplate reader (PerkinElmer Victor-3 ® ) was used to measure the optical density at wavelengths of 340 nm, 405 nm, 450 nm, 490 nm, 590 nm and 635 nm.
  • the concentration ratio of doxorubicin (0.5 mM) and A ⁇ 42 peptide per well in the plate was diluted so that the concentration ratio of 0.5: 1 to 50: 1, and the A ⁇ 42 peptide was diluted, and added to a 384 black well plate by 32 ⁇ L per well.
  • doxorubicin, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicin aglycone were each reacted with A ⁇ 42 by spinning down. The reaction proceeded for 2 hours at room temperature.
  • the optical density was measured at 340 nm, 405 nm, 450 nm, 490 nm, 590 nm and 635 nm wavelengths using a microplate reader (PerkinElmer Victor-3 ® ).
  • Doxorubicinolone a derivative of doxorubicin, showed no significant difference in optical density after and before the reaction with A ⁇ 42 at wavelengths of 405 nm, 450 nm, and 490 nm, but showed a significant difference in optical density at a wavelength of 590 nm after and before the reaction with A ⁇ 42. showed up
  • Doxorubicin showed significant differences in optical densities before and after the reaction with A ⁇ 42 at 405 nm, 450 nm, 490 nm, and 590 nm.
  • doxorubicin interacts with A ⁇ 42, that is, when doxorubicin binds/binds to amyloid beta and inhibits oligomerization and/or fibrillation of amyloid beta
  • the 405 nm, 450 nm, 490 nm and 590 nm wavelength bands It indicates a change in optical density and indicates the presence of amyloid beta species in the sample.
  • the amyloid beta species may be oligomeric and/or fibril forms of amyloid beta.
  • doxorubicinone a derivative of doxorubicin
  • Doxorubicinone showed significant optical density differences at wavelengths of 405 nm, 450 nm, 490 nm and 590 nm after and before the reaction with A ⁇ 42. This is when doxorubicinone interacts with A ⁇ 42, i.e.
  • amyloid beta species may be oligomeric and/or fibril forms of amyloid beta.
  • the present inventors found that when doxorubicin and its derivatives, doxorubicinone and doxorubicinolone, were used, a significant difference in optical density after and before the reaction with A ⁇ 42 was observed in the wavelength range of 400 nm to 600 nm. It was confirmed that the presence and/or amount of amyloid beta in the sample could be measured by measuring the optical density in the wavelength range.
  • doxorubicin and doxorubicinone showed significant optical density differences before and after the reaction with A ⁇ 42
  • doxorubicin, doxorubicinone and doxorubicinolone showed significant differences in optical density between A ⁇ 42 and doxorubicinone. showed a significant difference in optical density before and after the reaction with
  • doxorubicin and/or derivatives thereof exhibited an effect of significantly inhibiting amyloid beta oligomerization and/or fibrillation, and thus doxorubicin and derivatives thereof could be usefully used for the improvement, prevention, or treatment of Alzheimer's disease. Confirmed.
  • doxorubicin and/or its derivatives specifically bind to amyloid beta, cause a characteristic color change, and show a significant optical density difference in the 400 nm to 600 nm wavelength range before and after reacting with amyloid beta, the present invention It was confirmed that doxorubicin or a derivative thereof according to the present invention can be usefully used for detecting amyloid beta.

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Abstract

La présente invention concerne un kit de criblage pour un médicament inhibiteur de multimère de bêta-amyloïde, un procédé de criblage en masse à haute vitesse d'un médicament pour la prévention ou le traitement de la maladie d'Alzheimer l'utilisant, et un effet inhibiteur de la doxorubicine et d'un dérivé de celle-ci sur le bêta-amyloïde. Plus spécifiquement, il peut cribler rapidement et avec précision des médicaments ayant un effet inhibiteur sur le multimère de bêta-amyloïde parmi des matériaux candidats, en grandes quantités, et peut ainsi être utilement employé pour découvrir et préempter un médicament pour prévenir ou traiter la maladie d'Alzheimer, et étant donné que la doxorubicine ou un dérivé de celle-ci inhibe de manière significative l'oligomérisation et la fibrillation de bêta-amyloïde, elle peut être utilement employée non seulement pour prévenir, améliorer ou traiter la maladie d'Alzheimer, mais également pour fournir des informations nécessaires à la détection de bêta-amyloïde et au diagnostic de la maladie d'Alzheimer.
PCT/KR2022/017168 2021-11-03 2022-11-03 Procédé de criblage en masse à haute vitesse pour un médicament inhibiteur de multimère de bêta-amyloïde et composition comprenant de la doxorubicine ou un dérivé de celle-ci pour inhiber l'oligomérisation ou la fibrillation de bêta-amyloïde WO2023080687A1 (fr)

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