WO2023120407A1 - COMPOSITION FOR SUPPRESSING PRODUCTION AND/OR ACCUMULATION OF AMYLOID β - Google Patents

COMPOSITION FOR SUPPRESSING PRODUCTION AND/OR ACCUMULATION OF AMYLOID β Download PDF

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WO2023120407A1
WO2023120407A1 PCT/JP2022/046366 JP2022046366W WO2023120407A1 WO 2023120407 A1 WO2023120407 A1 WO 2023120407A1 JP 2022046366 W JP2022046366 W JP 2022046366W WO 2023120407 A1 WO2023120407 A1 WO 2023120407A1
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phe
amyloid
composition
cyclo
accumulation
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PCT/JP2022/046366
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French (fr)
Japanese (ja)
Inventor
嘉宏 中尾
シャンメイ ヨン
シャージャン リン
グレース カー ヤン チャン
翔太 野中
義 古元
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サントリーホールディングス株式会社
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Publication of WO2023120407A1 publication Critical patent/WO2023120407A1/en

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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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a composition for suppressing production and/or accumulation of amyloid ⁇ , and the like.
  • Amyloid ⁇ (A ⁇ ) is a type of protein produced in the brain. When amyloid ⁇ aggregates to oligomerize or fibrillate, it exhibits neuronal toxicity. In addition, aggregated amyloid ⁇ accumulates without being discharged from the brain, and is thought to trigger the onset of Alzheimer's disease. Therefore, suppressing the production or accumulation of amyloid ⁇ is considered effective, for example, in preventing Alzheimer's disease or in suppressing progression of the disease.
  • Acetylcholinesterase inhibitors are known as drugs that improve symptoms of Alzheimer's disease. Inhibition of acetylcholinesterase increases the amount of acetylcholine in the brain and is effective in suppressing the progression of cognitive decline. However, acetylcholinesterase inhibition cannot suppress the production of amyloid ⁇ or remove accumulated amyloid ⁇ . There is a demand for a substance that can be ingested on a daily basis as food or drink, etc., and that can suppress the production or accumulation of amyloid ⁇ .
  • Patent Document 1 describes an agent for suppressing and/or improving cognitive function decline containing Cyclo (Gly-Pro).
  • An object of the present invention is to provide a composition for suppressing the production and/or accumulation of amyloid ⁇ .
  • the present inventors have conducted intensive studies in view of the above problems, and have found that a cyclic dipeptide, cyclophenylalanylphenylalanine (Cyclo(Phe-Phe)), is effective in suppressing the production and/or accumulation of amyloid ⁇ . Found it.
  • the present invention relates to, but is not limited to, the following compositions for suppressing production and/or accumulation of amyloid ⁇ .
  • a composition for suppressing production and/or accumulation of amyloid ⁇ comprising Cyclo(Phe-Phe) or a salt thereof as an active ingredient.
  • the composition of [1] above which suppresses the production and/or accumulation of amyloid ⁇ by inhibiting ⁇ -secretase.
  • composition according to any one of [1] to [4] above, which is a food or drink or a pharmaceutical.
  • the composition "enhances cognitive function”, “suppresses deterioration of cognitive function”, “maintains good cognitive function”, “improves memory”, “suppresses deterioration of memory”, “improves memory “maintaining good memory”, “improving memory accuracy”, “preventing memory impairment”, “improving memory impairment”, “maintaining memory as a part of cognitive function”, and “preventing memory loss” functions that are suitable for people to become,””improve memory accuracy and judgment accuracy, which are part of cognitive function", “improve memory retention or consolidation”, “maintain enhanced cognitive function", “improves executive function”, “promotes attention and concentration”, “improves learning ability”, “maintains and improves orientation", “delays age-related cognitive decline", Labeling of one or more functions selected from the group consisting of "strengthening short-term and long-term memory,””promoting verbal and
  • compositions for suppressing the production and/or accumulation of amyloid ⁇ can be provided.
  • the composition of the present invention can be used as foods, beverages, pharmaceuticals, etc. for suppressing the production and/or accumulation of amyloid ⁇ .
  • FIG. 1 is a graph showing the ⁇ -secretase (BACE) inhibitory activity of Cyclo(Phe-Phe) and Cyclo(Leu-Ala).
  • FIG. 2 shows cells of SIM-A9 microglia (CFF) pretreated with Cyclo(Phe-Phe) for 24 hours and SIM-A9 microglia (vehicle control (VC)) not treated with Cyclo(Phe-Phe).
  • CFF SIM-A9 microglia
  • VC vehicle control
  • FIG. 3 shows the amount of internal amyloid- ⁇ monomer (*: p ⁇ 0.05 vs. vehicle control).
  • FIG. 5 is a graph showing the concentration of amyloid ⁇ 1-38 in the culture supernatant of H4 glioma cells (H4-hAPP cells) overexpressing the amyloid precursor protein wild type with the Swedish double mutation K595N/M596L. (***: p ⁇ 0.001 vs. vehicle control).
  • FIG. 6 is a graph showing the concentration of amyloid ⁇ 1-40 in the culture supernatant of H4-hAPP cells (***: p ⁇ 0.001 vs. vehicle control).
  • FIG. 7 is a graph showing the concentration of A ⁇ 1-42 in the culture supernatant of H4-hAPP cells (***: p ⁇ 0.001 vs. vehicle control).
  • the composition for suppressing production and/or accumulation of amyloid ⁇ of the present invention contains Cyclo(Phe-Phe) or a salt thereof.
  • the composition for suppressing production and/or accumulation of amyloid ⁇ of the present invention contains Cyclo(Phe-Phe) or a salt thereof as an active ingredient.
  • the composition for suppressing the production and/or accumulation of amyloid ⁇ of the present invention is sometimes referred to as the composition of the present invention.
  • the composition of the present invention is used to suppress the production of amyloid ⁇ and/or the accumulation of amyloid ⁇ .
  • Cyclo(Phe-Phe) (cyclophenylalanylphenylalanine) used in the present invention is a cyclic dipeptide having a structure in which two phenylalanine molecules are condensed.
  • Cyclo(Phe-Phe) is sometimes referred to as CFF.
  • the term "cyclic dipeptide" is characterized by having amino acids as constituent units, and is a diketopiperazine structure produced by dehydration condensation between the amino group of the N-terminal side amino acid and the carboxyl group of the C-terminal side amino acid.
  • the salt of Cyclo(Phe-Phe) is not particularly limited as long as it is a pharmacologically acceptable salt or a salt acceptable for food and drink, and may be either an acid salt or a basic salt.
  • Acid salts include, for example, inorganic salts such as hydrochlorides, sulfates, nitrates, phosphates; Examples include organic acid salts such as acid salts and propionate salts.
  • basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; Salts of cyclic dipeptides can be readily prepared by those skilled in the art by any method known in the art.
  • Cyclo(Phe-Phe) or a salt thereof are not particularly limited. Cyclo(Phe-Phe) or a salt thereof can be produced according to known methods. Cyclo(Phe-Phe) or a salt thereof may be derived from a natural product, may be artificially synthesized, may be produced by an enzymatic method or a microbial fermentation method, and may be a linear It may be synthesized by dehydrating and cyclizing the dipeptide. For example, protein hydrolysates, such as collagen hydrolysates, can be heated to yield heat-treated peptides rich in cyclic dipeptides, such as Cyclo(Phe-Phe).
  • Cyclo(Phe-Phe) or a salt thereof may be incorporated into the composition using a protein hydrolyzate containing it or a heat-treated product thereof, a concentrate of the protein hydrolyzate or a heat-treated product thereof, a dry powder Or you may mix
  • the composition of the present invention contains, for example, a protein hydrolyzate or heat treated product thereof, and Cyclo(Phe-Phe) or a salt thereof may be part of the protein hydrolyzate or heat treated product thereof. Cyclo(Phe-Phe) or a salt thereof can also be used as a commercially available product.
  • Amyloid- ⁇ is a peptide sometimes referred to as amyloid- ⁇ protein, amyloid- ⁇ peptide or ⁇ -amyloid.
  • Amyloid ⁇ is usually a peptide consisting of around 40 amino acids, and includes amyloid ⁇ 1-42 , amyloid ⁇ 1-40 , amyloid ⁇ 1-38 and the like. Unassociated amyloid- ⁇ is sometimes referred to as amyloid- ⁇ monomer.
  • Two or more amyloid ⁇ s may form an aggregate.
  • Amyloid ⁇ may form, for example, a soluble aggregate (amyloid ⁇ oligomer) in which two or more (eg, 2 to 50) amyloid ⁇ aggregates.
  • Amyloid ⁇ oligomers include amyloid ⁇ 1-42 oligomers, amyloid ⁇ 1-40 oligomers, amyloid ⁇ 1-38 oligomers, and the like.
  • Amyloid ⁇ in the present invention includes non-aggregated amyloid ⁇ and amyloid ⁇ forming aggregates such as oligomers.
  • Amyloid- ⁇ is produced in the form of amyloid- ⁇ monomers by the two-step cleavage of amyloid- ⁇ precursor protein (APP) by ⁇ -secretase and ⁇ -secretase.
  • APP amyloid- ⁇ precursor protein
  • ⁇ -Secretase is the rate-limiting enzyme for amyloid ⁇ production, and its cleavage regulates the total amyloid ⁇ production. Therefore, by inhibiting ⁇ -secretase, the production of amyloid ⁇ can be suppressed, and the accumulation of amyloid ⁇ can be suppressed.
  • Cyclo(Phe-Phe) or a salt thereof has a ⁇ -secretase inhibitory action.
  • the composition of the present invention can be used to suppress the production and/or accumulation of amyloid ⁇ by inhibiting ⁇ -secretase.
  • Cyclo(Phe-Phe) or salts thereof are useful as active ingredients for ⁇ -secretase inhibition.
  • Compositions for inhibiting ⁇ -secretase comprising Cyclo(Phe-Phe) or salts thereof are also encompassed by the present invention.
  • Cyclo(Phe-Phe) or its salts promote phagocytosis of microglial cells.
  • Microglial cells are central nervous system cells present in the brain. Microglial cells play an important role in maintaining the environment in the brain, and one of their actions is the phagocytosis of extracellular proteins such as amyloid ⁇ and brain waste products such as damaged cells. . Promoting phagocytosis of microglial cells promotes the removal of amyloid- ⁇ . Therefore, promotion of phagocytosis of microglial cells is effective in suppressing the accumulation of amyloid ⁇ . Cyclo(Phe-Phe) or a salt thereof is useful as an active ingredient for promoting phagocytosis of microglial cells.
  • the composition of the present invention can be used to suppress the accumulation of amyloid ⁇ by promoting phagocytosis (phagocytosis) of microglial cells.
  • phagocytosis phagocytosis
  • a composition for promoting phagocytosis of microglial cells containing Cyclo(Phe-Phe) or a salt thereof is also included in the present invention.
  • the composition of the present invention can be used to suppress the production and/or accumulation of amyloid ⁇ in the brain.
  • the composition of the present invention is used to suppress the production and/or accumulation of amyloid ⁇ by inhibiting ⁇ -secretase and/or promoting phagocytosis of microglial cells. be able to.
  • the composition of the present invention can be used to suppress production and/or accumulation of one or more types of amyloid ⁇ .
  • the composition of the present invention can be used for the prevention or amelioration of a condition or disease associated with amyloid ⁇ accumulation, preferably a condition or disease associated with amyloid ⁇ accumulation in the brain.
  • conditions or diseases include conditions or diseases associated with or caused by amyloid- ⁇ accumulation.
  • Conditions or diseases associated with or resulting from accumulation of amyloid ⁇ in the brain include, for example, Alzheimer's disease (including Alzheimer's dementia), mild cognitive impairment, and the like.
  • the composition of the present invention can be used to prevent or ameliorate the above conditions or diseases, and can be preferably used for the prevention of the above conditions or diseases.
  • prevention of a condition or disease includes preventing onset, delaying onset, reducing the rate of onset, reducing risk of onset, and the like.
  • Ameliorating a condition or disease includes ameliorating the subject from the condition or disease, alleviating symptoms of the condition or disease, ameliorating symptoms of the condition or disease, slowing progression of the condition or disease, preventing the condition or disease. etc.
  • Cognitive function is known to decline in Alzheimer's disease and mild cognitive impairment.
  • Symptoms of cognitive decline in Alzheimer's disease and mild cognitive impairment include, for example, memory loss, memory impairment (forgetfulness), aphasia (difficult to name things), apraxia, agnosia (walking in familiar places). (e.g., getting lost), decreased orientation (the ability to correctly recognize one's own situation, such as place, time, name of person, etc.), decreased verbal and non-verbal learning ability, decreased auditory and visual processing, executive function executive dysfunction (inability to plan and do things), decreased ability to concentrate, decreased attention, decreased judgment, decreased spatial awareness, decreased cognitive flexibility, decreased information processing speed, etc.
  • a preventive effect on cognitive function decline or an improving effect on cognitive function for example, an effect on preventing or improving the above symptoms can be obtained.
  • compositions of the present invention can be applied for either therapeutic use (medical use) or non-therapeutic use (non-medical use).
  • Non-therapeutic is a concept that does not involve medical intervention, i.e. human surgery, treatment or diagnosis.
  • the composition for suppressing production and/or accumulation of amyloid ⁇ of the present invention can be provided in the form of an agent as an example, but is not limited to this form.
  • the agent itself can be provided as a composition, or a composition containing the agent can be provided.
  • the composition for suppressing the production and/or accumulation of amyloid ⁇ of the present invention can also be said to be an agent for suppressing the production and/or accumulation of amyloid ⁇ .
  • a ⁇ -secretase inhibitory composition can also be referred to as a ⁇ -secretase inhibitor.
  • the composition for promoting phagocytosis of microglial cells can also be called an agent for promoting phagocytosis of microglial cells.
  • compositions of the invention may be either oral or parenteral.
  • the compositions of the invention are preferably oral compositions.
  • the composition of the present invention can be in the form of, for example, foods, beverages, pharmaceuticals, quasi-drugs, feeds, etc. Foods, beverages, or pharmaceuticals are preferred.
  • the composition of the present invention can also be used by adding it to foods and beverages, pharmaceuticals, quasi-drugs, feeds, and the like.
  • the form of the composition of the present invention is not particularly limited, and may be solid (for example, powder, granule, tablet, etc.), liquid, paste, or the like.
  • composition of the present invention contains Cyclo (Phe-Phe) or a salt thereof, and further contains various diluents, acidulants, antioxidants, stabilizers, preservatives, etc. that are acceptable as additives for food, beverages, pharmaceuticals, etc.
  • Ingredients, flavors, emulsifiers, pigments, seasonings, pH adjusters, nutritional enhancers, and the like may be added.
  • composition of the present invention when used as a food or drink, Cyclo (Phe-Phe) or a salt thereof may be added to components that can be used for food or drink (e.g., food materials, food additives used as necessary, etc.) can be blended to make various food and drink products.
  • Food and drink are not particularly limited, and examples thereof include general food and drink, health food, food with function claims, food for specified health use, health supplement, and food for the sick.
  • Health foods, foods with function claims, foods for specified health uses, health supplements, etc. are various formulations such as liquids, fine granules, tablets, granules, powders, capsules, chewable formulations, dry syrups, and liquid diets. Can be used as a form.
  • composition of the present invention When the composition of the present invention is used as a drug or quasi-drug, Cyclo (Phe-Phe) or a salt thereof is blended with a pharmacologically acceptable carrier, an optional additive, etc. , various dosage forms of pharmaceuticals or quasi-drugs.
  • Such carriers, additives, etc. may be those that can be used for pharmaceuticals or quasi-drugs and are pharmacologically acceptable.
  • antioxidants, coloring agents and the like can be mentioned.
  • administration (ingestion) forms of pharmaceuticals or quasi-drugs include oral or parenteral (transdermal, transmucosal, enteral, injection, etc.) forms of administration.
  • composition of the present invention When used as a drug or quasi-drug, it is preferably an oral drug or oral quasi-drug.
  • Dosage forms for oral administration include, for example, liquids, tablets, powders, fine granules, granules, dragees, capsules, suspensions, emulsions, chewables and the like.
  • Dosage forms for parenteral administration include, for example, injections, drops, ointments, lotions, patches, suppositories, nasal preparations, pulmonary preparations (inhalants) and the like.
  • the pharmaceutical may be a non-human veterinary pharmaceutical.
  • Cyclo (Phe-Phe) or a salt thereof may be added to the feed.
  • Feed also includes feed additives. Examples of feeds include livestock feeds for cows, pigs, chickens, sheep, horses, etc.; small animal feeds for rabbits, rats, mice, etc.; pet foods for dogs, cats, small birds, etc.;
  • the composition of the present invention when used as a food or drink, a drug, a quasi-drug, a feed, etc., the production method is not particularly limited, and Cyclo (Phe-Phe) or a salt thereof may be used to prepare a general It can be manufactured by a method.
  • the composition of the present invention is preferably a liquid composition, more preferably a beverage.
  • the beverage may be, for example, a functional beverage.
  • the form of the beverage is not particularly limited, and may be a packaged beverage.
  • Containers for packaged beverages are not particularly limited, and containers of any shape and material may be used. For example, metal containers such as aluminum cans and steel cans; resin containers such as PET bottles; Containers; glass containers such as glass bottles; and wooden containers such as barrels. Any of commonly used containers can be used.
  • a packaged beverage is obtained by filling and sealing such a container with a beverage.
  • the content of Cyclo(Phe-Phe) or a salt thereof contained in the composition of the present invention is not particularly limited, and can be set according to its form and the like.
  • the content of Cyclo(Phe-Phe) or a salt thereof in the composition of the present invention may be, for example, 1 ⁇ 10 ⁇ 6 wt % or more, preferably 1 ⁇ 10 ⁇ 5 wt % or more, Further, it may be 90% by weight or less, 1 ⁇ 10 ⁇ 1 % by weight or less, or 1 ⁇ 10 ⁇ 2 % by weight or less.
  • the content of Cyclo(Phe-Phe) or a salt thereof may be, for example, 1 ⁇ 10 ⁇ 6 to 90% by weight in the composition of the invention.
  • the content of Cyclo(Phe-Phe) or a salt thereof in the composition of the present invention may be from 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 1 wt %, and from 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 2 wt % is preferred, and 1 ⁇ 10 ⁇ 5 to 1 ⁇ 10 ⁇ 2 wt % is more preferred.
  • the content of Cyclo(Phe-Phe) or a salt thereof is 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 1 % by weight is preferred, 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 2 wt % is more preferred, and 1 ⁇ 10 ⁇ 5 to 1 ⁇ 10 ⁇ 2 wt % is even more preferred.
  • Cyclo(Phe-Phe) or a salt thereof can be quantified by a known method, for example, by liquid chromatography-mass spectrometry (LC/MS).
  • the composition of the present invention is preferably taken orally (orally administered). There is no particular limitation on the dose (which can also be referred to as intake) of the composition of the present invention.
  • the dosage of the composition of the present invention may be an amount that provides an effect of inhibiting the production and/or accumulation of amyloid ⁇ , and may be appropriately set according to the dosage form, administration method, body weight of the subject, and the like. .
  • the dosage of the composition of the present invention is preferably such that the ⁇ -secretase inhibitory effect and/or the microglial phagocytosis promoting effect can be obtained.
  • the dosage when the composition of the present invention is ingested or administered to a human (adult) subject, the dosage is preferably 1 ⁇ g or more per day as a dose of Cyclo(Phe-Phe), and more It is preferably 10 ⁇ g or more, more preferably 150 ⁇ g or more, and preferably 1000 ⁇ g or less, more preferably 500 ⁇ g or less, and still more preferably 300 ⁇ g or less.
  • the dose of Cyclo(Phe-Phe) when the composition of the present invention is ingested or administered to humans (adults), the dose of Cyclo(Phe-Phe) is preferably 1 to 1000 ⁇ g, more preferably 10 to 500 ⁇ g per day, More preferably 150 to 300 ⁇ g.
  • Cyclo(Phe-Phe) or a salt thereof in the above amount is preferably taken or administered orally.
  • the composition of the present invention may be an oral composition for ingesting or administering to humans Cyclo(Phe-Phe) or a salt thereof in the above amount per 60 kg body weight per day.
  • composition of the present invention is preferably taken or administered continuously. Continuous ingestion or administration of Cyclo(Phe-Phe) or a salt thereof is expected to provide greater effects.
  • the composition of the present invention is preferably taken or administered continuously for one week or longer, more preferably for four weeks or longer, and even more preferably for eight weeks or longer. Cyclo(Phe-Phe) or a salt thereof can be ingested as a food or drink, and from the viewpoint of safety, long-term ingestion is considered to cause few problems.
  • Subjects to whom the composition of the present invention is ingested or administered are not particularly limited. Humans or non-human mammals are preferred, and humans are more preferred.
  • the subject to which the composition of the present invention is administered is a subject who needs or desires suppression of amyloid ⁇ production and/or accumulation, and a subject who needs prevention or improvement of a condition or disease associated with amyloid ⁇ accumulation.
  • administration subjects in the present invention include middle-aged and elderly people.
  • Middle-aged and elderly people include elderly people.
  • a middle-aged person may be, for example, a person over the age of 40.
  • elderly people are preferred as subjects.
  • a senior citizen may be, for example, a human over the age of 60 or over the age of 65.
  • the subject of administration of the composition of the present invention may be a healthy subject.
  • it can be used in healthy individuals for the purpose of suppressing the production and/or accumulation of amyloid ⁇ in the brain, preventing the accumulation of amyloid ⁇ in the brain, preventing Alzheimer's disease or mild cognitive impairment, and the like.
  • composition of the present invention may be labeled with a function exerted by suppressing the production and/or accumulation of amyloid ⁇ .
  • indication is also called functionality indication.
  • the display is not particularly limited. Such indications include, for example, “increases cognitive function”, “suppresses deterioration of cognitive function”, “maintains good cognitive function”, “improves memory”, “suppresses deterioration of memory”, and “improves memory”.
  • the composition of the present invention is preferably a food or drink labeled with one or more of the above labels.
  • the above indication may be an indication that the above composition is used to obtain the above functions.
  • the label may be attached to the composition itself, or may be attached to the container or packaging of the composition.
  • the invention also includes the following methods and uses.
  • a method for suppressing production and/or accumulation of amyloid ⁇ comprising administering Cyclo(Phe-Phe) or a salt thereof.
  • a method of inhibiting ⁇ -secretase comprising administering Cyclo(Phe-Phe) or a salt thereof.
  • a method for promoting phagocytosis of microglial cells comprising administering Cyclo(Phe-Phe) or a salt thereof.
  • Use of Cyclo(Phe-Phe) or a salt thereof for suppressing production and/or accumulation of amyloid ⁇ Use of Cyclo(Phe-Phe) or salts thereof to inhibit ⁇ -secretase.
  • Cyclo(Phe-Phe) or salts thereof for promoting phagocytosis of microglial cells.
  • the method may be a therapeutic method or a non-therapeutic method.
  • the use may be therapeutic use or non-therapeutic use.
  • Cyclo(Phe-Phe) or a salt thereof can be used to suppress production and/or accumulation of one or more types of amyloid ⁇ .
  • Cyclo(Phe-Phe) or a salt thereof can be used to suppress the production and/or accumulation of amyloid ⁇ through ⁇ -secretase inhibition. In one aspect, Cyclo(Phe-Phe) or salts thereof can be used to inhibit amyloid- ⁇ accumulation by promoting phagocytosis of microglial cells.
  • Cyclo(Phe-Phe) or a salt thereof may be ingested or administered to the subject once or more times a day, for example, once to several times (for example, 2 to 3 times) a day. preferable.
  • the above uses are preferably in humans or non-human mammals, more preferably in humans.
  • Cyclo(Phe-Phe) or a salt thereof is used to prevent or ameliorate conditions or diseases associated with accumulation of amyloid ⁇ by inhibiting ⁇ -secretase and/or promoting phagocytosis of microglial cells. can be done.
  • the present invention also includes a method for preventing or ameliorating a condition or disease associated with amyloid ⁇ accumulation, comprising administering Cyclo(Phe-Phe) or a salt thereof.
  • Cyclo(Phe-Phe) or a salt thereof may be used in an amount (which can also be referred to as an effective amount) capable of suppressing the production and/or accumulation of amyloid ⁇ .
  • the above-mentioned effective amount is preferably an amount that provides ⁇ -secretase inhibitory effect and/or microglial cell phagocytosis promoting effect.
  • Cyclo(Phe-Phe) or a salt thereof is the same as the composition of the present invention described above with respect to the preferred dose, administration method, administration subject, and the like. Cyclo(Phe-Phe) or a salt thereof may be ingested or administered as is, or may be ingested or administered as a composition containing it. For example, a composition of the invention may be ingested or administered.
  • Cyclo(Phe-Phe) or a salt thereof can be used for the production of foods and drinks, pharmaceuticals, quasi-drugs, feeds, etc. that are used to suppress the production and/or accumulation of amyloid ⁇ .
  • the present invention also includes the use of Cyclo(Phe-Phe) or a salt thereof in the production of a composition for suppressing the production and/or accumulation of amyloid ⁇ .
  • the present invention also includes the use of Cyclo(Phe-Phe) or a salt thereof in the manufacture of a composition for inhibiting ⁇ -secretase or a composition for promoting phagocytosis of microglial cells. Cyclo(Phe-Phe) or a salt thereof can be used for the manufacture of a composition for preventing or improving conditions or diseases associated with amyloid ⁇ accumulation.
  • a range represented by “1-2” means from 1 to 2 and includes 1 and 2.
  • the upper limit and the lower limit may be any combination of ranges.
  • Example 1 ⁇ Examples 1 to 4 and Comparative Example 1> (Screening for compounds having ⁇ -secretase inhibitory activity) The test compounds were examined for ⁇ -secretase inhibitory activity. Cyclophenylalanylphenylalanine (Cyclo(Phe-Phe)) (Examples 1 to 4) and cycloleucylalanine (Cyclo(Leu-Ala)) (Comparative Example 1), which are cyclic dipeptides, were used as test compounds. (both from Bachem). A commercially available kit, Beta Secretase (BACE1) Activity Assay Kit (abcam), was used to evaluate the ⁇ -secretase inhibitory activity.
  • BACE1 Beta Secretase
  • abcam Activity Assay Kit
  • the ⁇ fluorescence intensity of the reaction solution ((fluorescence intensity at the end of the reaction (60 minutes)) ⁇ (fluorescence intensity before the start of the reaction (0 minutes)) was used to evaluate the enzyme activity.
  • a group to which no test compound was added was used as a control.
  • the inhibitor attached to the kit was used as a positive control.
  • the BACE activity shown in FIG. 1 is a relative value of the BACE activity of the test compound ( ⁇ fluorescence intensity of the reaction solution to which the test compound was added) when the BACE activity of the control ( ⁇ fluorescence intensity of the reaction solution of Control) is set to 1. is.
  • a decrease in ⁇ -secretase enzymatic activity was confirmed for Cyclo(Phe-Phe). Cyclo(Phe-Phe), a cyclic dipeptide, was found to have ⁇ -secretase inhibitory activity.
  • ⁇ -Amyloid 1-42
  • HiLyte TM Fluor 488 A ⁇ 1-42
  • 1% NH 4 OH solution purchased from Anaspec.
  • Triton® X-100 and paraformaldehyde (PFA) were obtained from Sigma Aldrich.
  • Cyclo-phenylalanine-phenylalanine was purchased from Bachem.
  • a soluble version of the A ⁇ 1-42 peptide (A ⁇ 1-42 monomer) was prepared according to the A ⁇ 1-42 preparation manufacturer's recommendations (Anaspec). Lyophilized A ⁇ 1-42 peptide was dissolved in 1% NH 4 OH solution at a concentration of 2 mg/mL. The peptide solution was further diluted with DPBS to a final concentration of 1 mg/mL stock solution. Reconstituted peptides were aliquoted into several freezer vials and stored at -20°C. Rhodamine B-labeled synthetic A ⁇ 1-42 peptide (GL Biochem, Shanghai, China) was dissolved in cold hexafluoroisopropanal (HFIP) at a concentration of 1 mM.
  • HFIP cold hexafluoroisopropanal
  • DMSO dimethylsulfoxide
  • MEM minimal essential medium
  • SIM-A9 mouse microglial cells (ATCC CRL-3265) were maintained in DMEM/F12 supplemented with 10% FBS, 5% HS and 1% P/S.
  • SIM-A9 cells were removed from culture flasks, pelleted, and resuspended in serum-free DMEM/F12.
  • Cells were seeded on PDL-coated 24-well plates at a density of 200,000 cells/well and pretreated with Cyclo(Phe-Phe) for 24 hours at 37° C. in a humidified gas chamber containing 5% CO 2 .
  • the final treatment concentration of Cyclo(Phe-Phe) was 2.2 ⁇ g/mL.
  • BV-2 mouse microglial cells were maintained in MEM supplemented with 10% FBS, 1% sodium pyruvate and 1% P/S. Cells were co-treated with 11.05 ⁇ g/mL Cyclo(Phe-Phe) and 2 ⁇ M A ⁇ 1-42 oligomers for 16 h at 37° C. in a humidified gas chamber containing 5% CO 2 . Cyclo(Phe-Phe) was dissolved in hydrochloric acid (HCl) and added to MEM.
  • HCl hydrochloric acid
  • Phagocytosis Assay Adherent SIM-A9 microglia were pretreated with Cyclo(Phe-Phe) (2.2 ⁇ g/mL) for 24 h, rinsed once with serum-free DMEM/F12, and then serum-free DMEM/F12. A ⁇ 1-42 (monomer) was added at a concentration of 4.51 ⁇ g/mL in medium and incubated at 37° C. for 6 hours. At the end of the incubation period, the A ⁇ 1-42 containing medium was removed and the cells were gently rinsed three times with HBSS and twice with DPBS to remove extracellular A ⁇ 1-42 .
  • BV-2 cells co-treated with Cyclo(Phe-Phe) and A ⁇ 1-42 oligomers were fixed with 4% PFA and micrographs were taken.
  • flow cytometric analysis cells were washed twice with 2% FBS, centrifuged at 1000 rpm for 5 minutes and blocked with Fc-Block (BD Bioscience) for 30 minutes. Fluorescence data were analyzed using FlowJo software (Tree Star, Ashland, OR).
  • FIGS. 3 and 4 depict the effect results of Cyclo(Phe-Phe) in enhancing phagocytosis of amyloid ⁇ 1-42 oligomers are shown in FIGS. 3 and 4.
  • FIG. 3 depicts BV-2 microglia treated with A ⁇ 1-42 oligomers (vehicle control (VC)) and BV-2 microglia co-treated with Cyclo(Phe-Phe) and A ⁇ 1-42 oligomers (CFF).
  • 3A, 3B and 3C vehicle control (VC)
  • 3D, 3E and 3F Cyclo(Phe-Phe) treatment).
  • CFF Cyclo(Phe-Phe) treated group
  • CFF Cyclo(Phe-Phe) treated group
  • BV-2 microglia were treated with CFF and A ⁇ 1-42 oligomers for 16 hours.
  • Figures 3A and 3D show the cell densities observed by brightfield microscopy
  • Figures 3B and 3E show the presence of A ⁇ 1-42 peptide stained with rhodamine B
  • Figures 3C and 3F show the cells Merged images of Rhodamine B-stained A ⁇ 1-42 peptides and cells incorporated into .
  • CFF administration can significantly enhance the phagocytic activity of BV-2 cells incubated with A ⁇ 1-42 peptide oligomers. This was characterized by increased frequency of fluorescent cells and enhanced microglial mean fluorescence intensity (MFI) levels compared to untreated control cells (VC) (FIGS. 3 and 4).
  • MFI microglial mean fluorescence intensity
  • Cyclo(Phe-Phe) enhanced phagocytosis of amyloid ⁇ 1-42 monomers by SIM-A9 microglial cells and phagocytosis of amyloid ⁇ 1-42 oligomers by BV-2 microglial cells.
  • Amyloid precursor protein (APP) overexpression cell assay An in vitro assay was performed to investigate the effect of Cyclo(Phe-Phe) (CFF) on amyloid ⁇ accumulation.
  • the assay used H4 glioma cells overexpressing amyloid precursor protein wild-type with the Swedish double mutation K595N/M596L (H4-hAPP cells).
  • H4-hAPP cells H4 glioma cells overexpressing amyloid precursor protein wild-type with the Swedish double mutation K595N/M596L
  • H4-hAPP cells were obtained by stable transfection of human H4 glioma cell line with pAG3 vector containing human amyloid precursor protein 695 (APP695) with Swedish double mutation K595N/M596L.
  • the pAG3 vector is a modified pcDNA3 plasmid containing a hygromycin B resistance cassette with the transgene under the control of the fused CMV and chicken ⁇ -actin promoters.
  • H4-hAPP cells were maintained in Opti-MEM supplemented with 10% FCS, 1% P/S, 200 ⁇ g/mL hygromycin B and 2.5 ⁇ g/mL blasticidin S hydrochloride. . Cells were seeded in 96-well plates at a density of 25,000 cells/well and incubated overnight at 37°C in a humidified gas chamber containing 5% CO2 . The next day, H4-hAPP cells were treated with 22 mg/mL CFF. In the CFF treatment, CFF was added to the medium of H4-hAPP cells to 22 ⁇ g/mL, and the cells were incubated.
  • H4-hAPP cells were treated with 400 nM DAPT instead of CFF. After 24 hours of treatment, cell culture supernatants were collected for amyloid- ⁇ (A ⁇ ) analysis. H4-hAPP cells without CFF and DAPT treatment served as vehicle control and cell supernatants were collected.
  • a ⁇ amyloid- ⁇
  • Amyloid- ⁇ ELISA Assay Collected cell culture supernatants were diluted 1:10 and tested using the 6E10 Abeta Triplex Assay kit for human amyloid- ⁇ 1-38 (A ⁇ 1-38 ), human amyloid- ⁇ 1-40 (A ⁇ 1-40) . ), and human amyloid ⁇ 1-42 (A ⁇ 1-42 ) were analyzed. Multiplex assays were performed according to the manufacturer's instructions and plates were read on a Sector Imager 2400 (Meso Scale Discovery). Amyloid ⁇ concentrations were calculated with reference to a standard curve. The sensitivity of the multiplex kit was ⁇ 5 pg/mL.
  • FIG. 5 is a graph showing the concentration of amyloid ⁇ 1-38 in the culture supernatant of H4-hAPP cells.
  • FIG. 6 is a graph showing the concentration of amyloid ⁇ 1-40 in the culture supernatant of H4-hAPP cells.
  • FIG. 7 is a graph showing the concentration of amyloid ⁇ 1-42 in the culture supernatant of H4-hAPP cells.
  • CFF CFF-treated cells.
  • CFF suppressed the accumulation of A ⁇ 1-38 , A ⁇ 1-40 and A ⁇ 1-42 in H4-hAPP cells.
  • CFF has the effect of suppressing the accumulation of amyloid ⁇ 1-38 , amyloid ⁇ 1-40 and amyloid ⁇ 1-42 in H4-hAPP cells.

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Abstract

The purpose of the present invention is to provide a composition for suppressing production and/or accumulation of amyloid β. The present invention relates to the composition for suppressing production and/or accumulation of amyloid β, the composition containing Cyclo (Phe-Phe) or a salt thereof as an active component.

Description

アミロイドβの産生抑制及び／又は蓄積抑制用組成物Composition for suppressing production and/or accumulation of amyloid β
本発明は、アミロイドβの産生抑制及び／又は蓄積抑制用組成物等に関する。 The present invention relates to a composition for suppressing production and/or accumulation of amyloid β, and the like.
アミロイドβ（Ａβ）は、脳内で産生されるタンパク質の一種である。アミロイドβが凝集してオリゴマー化又は線維化すると、神経細胞毒性を示す。また、凝集したアミロイドβは脳から排出されずに蓄積し、アルツハイマー病発症の引き金になると考えられている。従って、アミロイドβの産生又は蓄積を抑制することは、例えば、アルツハイマー病の予防又は病気の進行の抑制に有効であると考えられる。 Amyloid β (Aβ) is a type of protein produced in the brain. When amyloid β aggregates to oligomerize or fibrillate, it exhibits neuronal toxicity. In addition, aggregated amyloid β accumulates without being discharged from the brain, and is thought to trigger the onset of Alzheimer's disease. Therefore, suppressing the production or accumulation of amyloid β is considered effective, for example, in preventing Alzheimer's disease or in suppressing progression of the disease.
アルツハイマー病の症状を改善する医薬品として、アセチルコリンエステラーゼ阻害剤が知られている。アセチルコリンエステラーゼの阻害は、脳内におけるアセチルコリン量を増加させ、認知機能低下の進行の抑制に有効である。しかしながら、アセチルコリンエステラーゼ阻害によって、アミロイドβの産生を抑制したり、蓄積したアミロイドβを除去したりすることはできない。飲食品等として日常的に摂取することができ、アミロイドβの産生又は蓄積を抑制することができる物質が求められている。 Acetylcholinesterase inhibitors are known as drugs that improve symptoms of Alzheimer's disease. Inhibition of acetylcholinesterase increases the amount of acetylcholine in the brain and is effective in suppressing the progression of cognitive decline. However, acetylcholinesterase inhibition cannot suppress the production of amyloid β or remove accumulated amyloid β. There is a demand for a substance that can be ingested on a daily basis as food or drink, etc., and that can suppress the production or accumulation of amyloid β.
一方、環状ジペプチドが脳機能に影響を与えることが報告されている。特許文献１には、Ｃｙｃｌｏ（Ｇｌｙ－Ｐｒｏ）を含有する、認知機能の低下抑制及び／又は改善剤が記載されている。 On the other hand, cyclic dipeptides have been reported to affect brain function. Patent Document 1 describes an agent for suppressing and/or improving cognitive function decline containing Cyclo (Gly-Pro).
特開２０２０－１９６６８６号公報JP 2020-196686 A
本発明は、アミロイドβの産生抑制及び／又は蓄積抑制用組成物を提供することを目的とする。 An object of the present invention is to provide a composition for suppressing the production and/or accumulation of amyloid β.
本発明者らは、上記課題に鑑み鋭意研究した結果、環状ジペプチドであるシクロフェニルアラニルフェニルアラニン（Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ））が、アミロイドβの産生抑制及び／又は蓄積抑制に有効であることを見出した。 The present inventors have conducted intensive studies in view of the above problems, and have found that a cyclic dipeptide, cyclophenylalanylphenylalanine (Cyclo(Phe-Phe)), is effective in suppressing the production and/or accumulation of amyloid β. Found it.
すなわち、本発明は、これに限定されるものではないが、以下のアミロイドβの産生抑制及び／又は蓄積抑制用組成物等に関する。
〔１〕Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を有効成分として含む、アミロイドβの産生抑制及び／又は蓄積抑制用組成物。
〔２〕β－セクレターゼを阻害することによりアミロイドβの産生及び／又は蓄積を抑制する、上記〔１〕に記載の組成物。
〔３〕ミクログリア細胞の貪食作用を促進することによりアミロイドβの蓄積を抑制する、上記〔１〕又は〔２〕に記載の組成物。
〔４〕上記組成物が、経口用である上記〔１〕～〔３〕のいずれかに記載の組成物。
〔５〕上記組成物が、飲食品又は医薬品である上記〔１〕～〔４〕のいずれかに記載の組成物。
〔６〕上記組成物が、「認知機能を高める」、「認知機能の低下を抑える」、「認知機能を良好に保つ」、「記憶力を高める」、「記憶力の低下を抑える」、「記憶力を良好に保つ」、「記憶の精度を高める」、「記憶障害を予防する」、「記憶障害を改善する」、「認知機能の一部である記憶力を維持する」、「記憶力の低下が気になる方に適した機能」、「認知機能の一部である記憶力の精度や判断の正確さを向上させる」、「記憶の保持又は統合を改善する」、「認知機能の強化を維持する」、「遂行機能を改善する」、「注意力と集中力を促進する」、「学習能力を改善する」、「見当識を維持・改善する」、「加齢に伴う認知機能低下を遅延する」、「短期及び長期記憶を強化する」、「言語的及び視空間記憶を促進する」及び「論理的思考力を維持・改善する」からなる群より選択される１又は２以上の機能の表示を付されたものである上記〔１〕～〔５〕のいずれかに記載の組成物。
〔７〕アミロイドβの産生抑制及び／又は蓄積抑制用組成物の製造における、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。
〔８〕アミロイドβの産生抑制及び／又は蓄積抑制のための、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。 That is, the present invention relates to, but is not limited to, the following compositions for suppressing production and/or accumulation of amyloid β.
[1] A composition for suppressing production and/or accumulation of amyloid β, comprising Cyclo(Phe-Phe) or a salt thereof as an active ingredient.
[2] The composition of [1] above, which suppresses the production and/or accumulation of amyloid β by inhibiting β-secretase.
[3] The composition of [1] or [2] above, which suppresses accumulation of amyloid β by promoting phagocytosis of microglial cells.
[4] The composition according to any one of [1] to [3] above, which is for oral use.
[5] The composition according to any one of [1] to [4] above, which is a food or drink or a pharmaceutical.
[6] The composition "enhances cognitive function", "suppresses deterioration of cognitive function", "maintains good cognitive function", "improves memory", "suppresses deterioration of memory", "improves memory "maintaining good memory", "improving memory accuracy", "preventing memory impairment", "improving memory impairment", "maintaining memory as a part of cognitive function", and "preventing memory loss" functions that are suitable for people to become,""improve memory accuracy and judgment accuracy, which are part of cognitive function", "improve memory retention or consolidation", "maintain enhanced cognitive function", "improves executive function", "promotes attention and concentration", "improves learning ability", "maintains and improves orientation", "delays age-related cognitive decline", Labeling of one or more functions selected from the group consisting of "strengthening short-term and long-term memory,""promoting verbal and visuospatial memory," and "maintaining/improving logical thinking ability." The composition according to any one of the above [1] to [5], which is obtained by
[7] Use of Cyclo(Phe-Phe) or a salt thereof in the production of a composition for inhibiting production and/or accumulation of amyloid β.
[8] Use of Cyclo(Phe-Phe) or a salt thereof for suppressing production and/or accumulation of amyloid β.
本発明によれば、アミロイドβの産生抑制及び／又は蓄積抑制用組成物を提供することができる。本発明の組成物は、アミロイドβの産生抑制及び／又は蓄積抑制のための飲食品、医薬品等として使用することができる。 According to the present invention, a composition for suppressing the production and/or accumulation of amyloid β can be provided. The composition of the present invention can be used as foods, beverages, pharmaceuticals, etc. for suppressing the production and/or accumulation of amyloid β.
図１は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）及びＣｙｃｌｏ（Ｌｅｕ－Ａｌａ）のβ－セクレターゼ（ＢＡＣＥ）阻害活性を示すグラフである。FIG. 1 is a graph showing the β-secretase (BACE) inhibitory activity of Cyclo(Phe-Phe) and Cyclo(Leu-Ala). 図２は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で２４時間前処理したＳＩＭ－Ａ９ミクログリア（ＣＦＦ）、及び、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理しなかったＳＩＭ－Ａ９ミクログリア（ビヒクルコントロール（ＶＣ））の細胞内アミロイドβモノマーの量を示すグラフである（＊：ｐ＜０．０５、ビヒクルコントロールに対して）。FIG. 2 shows cells of SIM-A9 microglia (CFF) pretreated with Cyclo(Phe-Phe) for 24 hours and SIM-A9 microglia (vehicle control (VC)) not treated with Cyclo(Phe-Phe). Graph showing the amount of internal amyloid-β monomer (*: p<0.05 vs. vehicle control). 図３は、Ａβ１－４２オリゴマーで処理したＢＶ－２ミクログリア（ビヒクルコントロール（ＶＣ））、並びに、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）及びＡβ１－４２オリゴマーで処理したＢＶ－２ミクログリア（ＣＦＦ）の顕微鏡写真である（図３Ａ、図３Ｂ及び図３Ｃ：ビヒクルコントロール、図３Ｄ、図３Ｅ及び図３Ｆ：Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）処理）。FIG. 3. Microscopic view of BV-2 microglia treated with Aβ 1-42 oligomers (vehicle control (VC)) and BV-2 microglia treated with Cyclo(Phe-Phe) and Aβ 1-42 oligomers (CFF). 3A, 3B and 3C: vehicle control; 3D, 3E and 3F: Cyclo(Phe-Phe) treatment). 図４は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理したＢＶ－２ミクログリア（ＣＦＦ）、及び、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理しなかったＢＶ－２ミクログリア（ビヒクルコントロール（ＶＣ））のアミロイドβオリゴマー貪食作用を調べた結果を示すグラフである（＊：ｐ＜０．０５、ビヒクルコントロールに対して）。Figure 4. Cyclo(Phe-Phe) treated BV-2 microglia (CFF) and BV-2 microglia not treated with Cyclo(Phe-Phe) (vehicle control (VC)) amyloid β oligomer phagocytosis. Graph showing the results of examining effects (*: p<0.05 vs. vehicle control). 図５は、スウェーデン二重変異Ｋ５９５Ｎ／Ｍ５９６Ｌを有するアミロイド前駆体タンパク質野生型を過剰発現するＨ４神経膠腫細胞（Ｈ４－ｈＡＰＰ細胞）の培養上清中のアミロイドβ１－３８の濃度を示すグラフである（＊＊＊：ｐ＜０．００１、ビヒクルコントロールに対して）。FIG. 5 is a graph showing the concentration of amyloid β 1-38 in the culture supernatant of H4 glioma cells (H4-hAPP cells) overexpressing the amyloid precursor protein wild type with the Swedish double mutation K595N/M596L. (***: p<0.001 vs. vehicle control). 図６は、Ｈ４－ｈＡＰＰ細胞の培養上清中のアミロイドβ１－４０の濃度を示すグラフである（＊＊＊：ｐ＜０．００１、ビヒクルコントロールに対して）。FIG. 6 is a graph showing the concentration of amyloid β 1-40 in the culture supernatant of H4-hAPP cells (***: p<0.001 vs. vehicle control). 図７は、Ｈ４－ｈＡＰＰ細胞の培養上清中のアミロイドβ１－４２の濃度を示すグラフである（＊＊＊：ｐ＜０．００１、ビヒクルコントロールに対して）。FIG. 7 is a graph showing the concentration of Aβ 1-42 in the culture supernatant of H4-hAPP cells (***: p<0.001 vs. vehicle control).
本発明のアミロイドβの産生抑制及び／又は蓄積抑制用組成物は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を含む。本発明のアミロイドβの産生抑制及び／又は蓄積抑制用組成物は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を有効成分として含むものである。本明細書中、本発明のアミロイドβの産生抑制及び／又は蓄積抑制用組成物を、本発明の組成物ということもある。本発明の組成物は、アミロイドβの産生を抑制するため、及び／又は、アミロイドβの蓄積を抑制するために使用されるものである。 The composition for suppressing production and/or accumulation of amyloid β of the present invention contains Cyclo(Phe-Phe) or a salt thereof. The composition for suppressing production and/or accumulation of amyloid β of the present invention contains Cyclo(Phe-Phe) or a salt thereof as an active ingredient. In the present specification, the composition for suppressing the production and/or accumulation of amyloid β of the present invention is sometimes referred to as the composition of the present invention. The composition of the present invention is used to suppress the production of amyloid β and/or the accumulation of amyloid β.
本発明において使用されるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）（シクロフェニルアラニルフェニルアラニン）は、フェニルアラニン２分子が縮合結合した構造を有する環状ジペプチドである。本明細書中、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）を、ＣＦＦと記載することもある。
本明細書において「環状ジペプチド」とは、アミノ酸を構成単位とすることを特徴とし、Ｎ末端側アミノ酸のアミノ基とＣ末端側アミノ酸のカルボキシル基とが脱水縮合することにより生成したジケトピペラジン構造を有する化合物をいう。 Cyclo(Phe-Phe) (cyclophenylalanylphenylalanine) used in the present invention is a cyclic dipeptide having a structure in which two phenylalanine molecules are condensed. In this specification, Cyclo(Phe-Phe) is sometimes referred to as CFF.
As used herein, the term "cyclic dipeptide" is characterized by having amino acids as constituent units, and is a diketopiperazine structure produced by dehydration condensation between the amino group of the N-terminal side amino acid and the carboxyl group of the C-terminal side amino acid. A compound having
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の塩としては、薬理学的に許容される塩又は飲食品に許容される塩であれば特に限定されず、酸性塩及び塩基性塩のいずれであってもよい。酸性塩として、例えば、塩酸塩、硫酸塩、硝酸塩、リン酸塩等の無機酸塩；酢酸塩、クエン酸塩、マレイン酸塩、リンゴ酸塩、シュウ酸塩、乳酸塩、コハク酸塩、フマル酸塩、プロピオン酸塩等の有機酸塩等が挙げられる。塩基性塩として、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩等が挙げられる。環状ジペプチドの塩は、当該分野で公知の任意の方法により、当業者によって容易に調製され得る。 The salt of Cyclo(Phe-Phe) is not particularly limited as long as it is a pharmacologically acceptable salt or a salt acceptable for food and drink, and may be either an acid salt or a basic salt. Acid salts include, for example, inorganic salts such as hydrochlorides, sulfates, nitrates, phosphates; Examples include organic acid salts such as acid salts and propionate salts. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; Salts of cyclic dipeptides can be readily prepared by those skilled in the art by any method known in the art.
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の由来及び製造方法は特に制限されない。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、公知の方法に従って製造することができる。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、天然物由来のものであってもよく、人工的に合成したものであってもよく、酵素法又は微生物発酵法により製造されてもよく、直鎖状ジペプチドを脱水及び環化させることによって合成されてもよい。例えば、コラーゲン加水分解物などのタンパク質加水分解物を加熱して、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）等の環状ジペプチドを豊富に含むペプチド熱処理物を得ることができる。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、これを含むタンパク質加水分解物又はその熱処理物を使用して組成物に配合してもよいし、タンパク質加水分解物若しくはその熱処理物の濃縮物、乾燥粉末又は精製度を高めたものを使用して組成物に配合してもよい。本発明の組成物は、例えば、タンパク質加水分解物又はその熱処理物を含有し、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、タンパク質加水分解物又はその熱処理物の一部であってもよい。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、市販品を使用することもできる。 The origin and production method of Cyclo(Phe-Phe) or a salt thereof are not particularly limited. Cyclo(Phe-Phe) or a salt thereof can be produced according to known methods. Cyclo(Phe-Phe) or a salt thereof may be derived from a natural product, may be artificially synthesized, may be produced by an enzymatic method or a microbial fermentation method, and may be a linear It may be synthesized by dehydrating and cyclizing the dipeptide. For example, protein hydrolysates, such as collagen hydrolysates, can be heated to yield heat-treated peptides rich in cyclic dipeptides, such as Cyclo(Phe-Phe).
Cyclo(Phe-Phe) or a salt thereof may be incorporated into the composition using a protein hydrolyzate containing it or a heat-treated product thereof, a concentrate of the protein hydrolyzate or a heat-treated product thereof, a dry powder Or you may mix|blend in a composition using what raised the refinement|purification. The composition of the present invention contains, for example, a protein hydrolyzate or heat treated product thereof, and Cyclo(Phe-Phe) or a salt thereof may be part of the protein hydrolyzate or heat treated product thereof. Cyclo(Phe-Phe) or a salt thereof can also be used as a commercially available product.
アミロイドβは、アミロイドβタンパク質、アミロイドβペプチド又はβアミロイドと呼ばれることもあるペプチドである。アミロイドβは、通常、４０個前後のアミノ酸からなるペプチドであり、アミロイドβ１－４２、アミロイドβ１－４０、アミロイドβ１－３８等が挙げられる。非会合状態のアミロイドβは、アミロイドβモノマーと呼ばれることもある。アミロイドβは、２個以上で凝集体を形成していてもよい。アミロイドβは、例えば、アミロイドβが２個以上（例えば２～５０個）凝集した可溶性凝集体（アミロイドβオリゴマー）を形成していてもよい。アミロイドβオリゴマーとして、アミロイドβ１－４２オリゴマー、アミロイドβ１－４０オリゴマー、アミロイドβ１－３８オリゴマー等が挙げられる。本発明におけるアミロイドβには、非会合状態のアミロイドβ、オリゴマー等の凝集体を形成しているアミロイドβが含まれる。 Amyloid-β is a peptide sometimes referred to as amyloid-β protein, amyloid-β peptide or β-amyloid. Amyloid β is usually a peptide consisting of around 40 amino acids, and includes amyloid β 1-42 , amyloid β 1-40 , amyloid β 1-38 and the like. Unassociated amyloid-β is sometimes referred to as amyloid-β monomer. Two or more amyloid βs may form an aggregate. Amyloid β may form, for example, a soluble aggregate (amyloid β oligomer) in which two or more (eg, 2 to 50) amyloid β aggregates. Amyloid β oligomers include amyloid β 1-42 oligomers, amyloid β 1-40 oligomers, amyloid β 1-38 oligomers, and the like. Amyloid β in the present invention includes non-aggregated amyloid β and amyloid β forming aggregates such as oligomers.
アミロイドβは、アミロイドβ前駆体タンパク質（ＡＰＰ）が、β－セクレターゼ及びγ－セクレターゼによって２段階で切断されてアミロイドβモノマーの形態で生成する。β－セクレターゼは、アミロイドβ産生の律速酵素であり、その切断が総アミロイドβ産生量を規定している。このため、β－セクレターゼを阻害することによって、アミロイドβの産生を抑制することができ、アミロイドβの蓄積を抑制することができる。 Amyloid-β is produced in the form of amyloid-β monomers by the two-step cleavage of amyloid-β precursor protein (APP) by β-secretase and γ-secretase. β-Secretase is the rate-limiting enzyme for amyloid β production, and its cleavage regulates the total amyloid β production. Therefore, by inhibiting β-secretase, the production of amyloid β can be suppressed, and the accumulation of amyloid β can be suppressed.
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、β－セクレターゼ阻害作用を有する。本発明の組成物は、β－セクレターゼを阻害することにより、アミロイドβの産生を抑制及び／又は蓄積を抑制するために使用することができる。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、β－セクレターゼ阻害のための有効成分として有用である。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を含む、β－セクレターゼ阻害用組成物も本発明に包含される。 Cyclo(Phe-Phe) or a salt thereof has a β-secretase inhibitory action. The composition of the present invention can be used to suppress the production and/or accumulation of amyloid β by inhibiting β-secretase. Cyclo(Phe-Phe) or salts thereof are useful as active ingredients for β-secretase inhibition. Compositions for inhibiting β-secretase comprising Cyclo(Phe-Phe) or salts thereof are also encompassed by the present invention.
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、ミクログリア細胞の貪食作用を促進する。
ミクログリア細胞は、脳内に存在する中枢神経系の細胞である。ミクログリア細胞は、脳内の環境の維持のために重要な役割を果たしており、その作用の一つに、アミロイドβ等の細胞外タンパク質、損傷を受けた細胞等の脳内老廃物の貪食がある。ミクログリア細胞の貪食作用を促進することで、アミロイドβの除去が促進される。このため、ミクログリア細胞の貪食作用の促進は、アミロイドβの蓄積抑制に有効である。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、ミクログリア細胞の貪食作用促進のための有効成分として有用である。 Cyclo(Phe-Phe) or its salts promote phagocytosis of microglial cells.
Microglial cells are central nervous system cells present in the brain. Microglial cells play an important role in maintaining the environment in the brain, and one of their actions is the phagocytosis of extracellular proteins such as amyloid β and brain waste products such as damaged cells. . Promoting phagocytosis of microglial cells promotes the removal of amyloid-β. Therefore, promotion of phagocytosis of microglial cells is effective in suppressing the accumulation of amyloid β. Cyclo(Phe-Phe) or a salt thereof is useful as an active ingredient for promoting phagocytosis of microglial cells.
一態様において、本発明の組成物は、ミクログリア細胞の貪食作用（貪食）を促進することにより、アミロイドβの蓄積を抑制するために使用することができる。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を含む、ミクログリア細胞の貪食作用促進用組成物も本発明に包含される。 In one aspect, the composition of the present invention can be used to suppress the accumulation of amyloid β by promoting phagocytosis (phagocytosis) of microglial cells. A composition for promoting phagocytosis of microglial cells containing Cyclo(Phe-Phe) or a salt thereof is also included in the present invention.
本発明の組成物は、脳内におけるアミロイドβの産生抑制及び／又は蓄積抑制のために使用することができる。一態様において、本発明の組成物は、β－セクレターゼを阻害することにより及び／又はミクログリア細胞の貪食作用を促進することにより、アミロイドβの産生を抑制及び／又は蓄積を抑制するために使用することができる。本発明の組成物は、１種又は２種以上のアミロイドβの産生抑制及び／又は蓄積抑制のために使用することができる。 The composition of the present invention can be used to suppress the production and/or accumulation of amyloid β in the brain. In one aspect, the composition of the present invention is used to suppress the production and/or accumulation of amyloid β by inhibiting β-secretase and/or promoting phagocytosis of microglial cells. be able to. The composition of the present invention can be used to suppress production and/or accumulation of one or more types of amyloid β.
一態様において、本発明の組成物は、アミロイドβの蓄積に関連する状態又は疾患、好ましくは、脳におけるアミロイドβの蓄積に関連する状態又は疾患の予防又は改善のために使用することができる。このような状態又は疾患として、アミロイドβの蓄積を伴う又はアミロイドβの蓄積に起因する状態又は疾患が挙げられる。脳におけるアミロイドβの蓄積を伴う又はアミロイドβの蓄積に起因する状態又は疾患として、例えば、アルツハイマー病（アルツハイマー型認知症を含む）、軽度認知障害等が挙げられる。
本発明の組成物は、上記の状態又は疾患を予防又は改善するために使用することができ、上記の状態又は疾患の予防に好適に使用することができる。
本明細書において、状態又は疾患の予防は、発症を防止すること、発症を遅延させること、発症率を低下させること、発症のリスクを軽減すること等を包含する。状態又は疾患の改善は、対象を状態又は疾患から回復させること、状態又は疾患の症状を軽減すること、状態又は疾患の症状を好転させること、状態又は疾患の進行を遅延させること、防止すること等を包含する。 In one aspect, the composition of the present invention can be used for the prevention or amelioration of a condition or disease associated with amyloid β accumulation, preferably a condition or disease associated with amyloid β accumulation in the brain. Such conditions or diseases include conditions or diseases associated with or caused by amyloid-β accumulation. Conditions or diseases associated with or resulting from accumulation of amyloid β in the brain include, for example, Alzheimer's disease (including Alzheimer's dementia), mild cognitive impairment, and the like.
The composition of the present invention can be used to prevent or ameliorate the above conditions or diseases, and can be preferably used for the prevention of the above conditions or diseases.
As used herein, prevention of a condition or disease includes preventing onset, delaying onset, reducing the rate of onset, reducing risk of onset, and the like. Ameliorating a condition or disease includes ameliorating the subject from the condition or disease, alleviating symptoms of the condition or disease, ameliorating symptoms of the condition or disease, slowing progression of the condition or disease, preventing the condition or disease. etc.
アルツハイマー病及び軽度認知障害では、認知機能が低下することが知られている。アルツハイマー病、軽度認知障害における認知機能低下の症状として、例えば、記憶力低下、記憶障害（物忘れ）、失語（ものの名前が出にくくなる）、失行、失認（よく知っているはずの場所で道に迷う等）、見当識（場所、時間、人の名前など、自分が置かれた状況を正しく認識する能力）の低下、言語及び非言語学習能力の低下、聴覚及び視覚処理の低下、遂行機能低下、遂行機能障害（計画を立てて物事を行うことができなくなる）、集中力低下、注意力低下、判断力低下、空間認識力低下、認知柔軟性低下、情報処理速度低下等が挙げられる。アミロイドβの産生又は蓄積を抑制することによって、認知機能低下の予防又は認知機能の改善効果、例えば、上記の症状の予防又は改善効果が得られることが期待できる。 Cognitive function is known to decline in Alzheimer's disease and mild cognitive impairment. Symptoms of cognitive decline in Alzheimer's disease and mild cognitive impairment include, for example, memory loss, memory impairment (forgetfulness), aphasia (difficult to name things), apraxia, agnosia (walking in familiar places). (e.g., getting lost), decreased orientation (the ability to correctly recognize one's own situation, such as place, time, name of person, etc.), decreased verbal and non-verbal learning ability, decreased auditory and visual processing, executive function executive dysfunction (inability to plan and do things), decreased ability to concentrate, decreased attention, decreased judgment, decreased spatial awareness, decreased cognitive flexibility, decreased information processing speed, etc. By suppressing the production or accumulation of amyloid β, it can be expected that a preventive effect on cognitive function decline or an improving effect on cognitive function, for example, an effect on preventing or improving the above symptoms can be obtained.
本発明の組成物は、治療的用途（医療用途）又は非治療的用途（非医療用途）のいずれにも適用することができる。非治療的とは、医療行為、すなわち人間の手術、治療又は診断を含まない概念である。
本発明のアミロイドβの産生抑制及び／又は蓄積抑制用組成物は、一例として、剤の形態で提供することができるが、本形態に限定されるものではない。当該剤をそのまま組成物として、又は、当該剤を含む組成物として提供することもできる。一態様において、本発明のアミロイドβの産生抑制及び／又は蓄積抑制用組成物は、アミロイドβの産生抑制及び／又は蓄積抑制剤ということもできる。β－セクレターゼ阻害用組成物は、β－セクレターゼ阻害剤ということもできる。ミクログリア細胞の貪食作用促進用組成物は、ミクログリア細胞の貪食作用促進剤ということもできる。 The compositions of the present invention can be applied for either therapeutic use (medical use) or non-therapeutic use (non-medical use). Non-therapeutic is a concept that does not involve medical intervention, i.e. human surgery, treatment or diagnosis.
The composition for suppressing production and/or accumulation of amyloid β of the present invention can be provided in the form of an agent as an example, but is not limited to this form. The agent itself can be provided as a composition, or a composition containing the agent can be provided. In one aspect, the composition for suppressing the production and/or accumulation of amyloid β of the present invention can also be said to be an agent for suppressing the production and/or accumulation of amyloid β. A β-secretase inhibitory composition can also be referred to as a β-secretase inhibitor. The composition for promoting phagocytosis of microglial cells can also be called an agent for promoting phagocytosis of microglial cells.
本発明の組成物は、経口用又は非経口用のいずれであってもよい。本発明の組成物は、好ましくは経口用組成物である。本発明の組成物は、例えば、飲食品、医薬品、医薬部外品、飼料等の形態とすることができ、飲食品又は医薬品が好ましい。本発明の組成物は、飲食品、医薬品、医薬部外品、飼料等に添加して使用することもできる。本発明の組成物の形態は特に限定されず、固体状（例えば、粉末状、顆粒状、タブレット状等）、液状、ペースト状等のいずれであってもよい。 The compositions of the invention may be either oral or parenteral. The compositions of the invention are preferably oral compositions. The composition of the present invention can be in the form of, for example, foods, beverages, pharmaceuticals, quasi-drugs, feeds, etc. Foods, beverages, or pharmaceuticals are preferred. The composition of the present invention can also be used by adding it to foods and beverages, pharmaceuticals, quasi-drugs, feeds, and the like. The form of the composition of the present invention is not particularly limited, and may be solid (for example, powder, granule, tablet, etc.), liquid, paste, or the like.
本発明の組成物は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を含み、更に飲食品又は医薬品等への添加物として許容されている各種の希釈剤、酸味料、抗酸化剤、安定剤、保存料、香料、乳化剤、色素類、調味料、ｐＨ調整剤、栄養強化剤等が添加されていてもよい。 The composition of the present invention contains Cyclo (Phe-Phe) or a salt thereof, and further contains various diluents, acidulants, antioxidants, stabilizers, preservatives, etc. that are acceptable as additives for food, beverages, pharmaceuticals, etc. Ingredients, flavors, emulsifiers, pigments, seasonings, pH adjusters, nutritional enhancers, and the like may be added.
例えば、本発明の組成物を飲食品とする場合、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩に、飲食品に使用可能な成分（例えば、食品素材、必要に応じて使用される食品添加物等）を配合して、種々の飲食品とすることができる。飲食品は特に限定されず、例えば、一般的な飲食品、健康食品、機能性表示食品、特定保健用食品、健康補助食品、病者用食品等が挙げられる。上記健康食品、機能性表示食品、特定保健用食品、健康補助食品等は、例えば、液剤、細粒剤、錠剤、顆粒剤、散剤、カプセル剤、チュアブル剤、ドライシロップ剤、流動食等の各種製剤形態として使用することができる。 For example, when the composition of the present invention is used as a food or drink, Cyclo (Phe-Phe) or a salt thereof may be added to components that can be used for food or drink (e.g., food materials, food additives used as necessary, etc.) can be blended to make various food and drink products. Food and drink are not particularly limited, and examples thereof include general food and drink, health food, food with function claims, food for specified health use, health supplement, and food for the sick. Health foods, foods with function claims, foods for specified health uses, health supplements, etc. are various formulations such as liquids, fine granules, tablets, granules, powders, capsules, chewable formulations, dry syrups, and liquid diets. Can be used as a form.
本発明の組成物を医薬品又は医薬部外品とする場合、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩に、薬理学的に許容される担体、必要に応じて添加される添加剤等を配合して、各種剤形の医薬品又は医薬部外品とすることができる。そのような担体、添加剤等は、医薬品又は医薬部外品に使用可能な、薬理学的に許容されるものであればよく、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、抗酸化剤、着色剤等の１又は２以上が挙げられる。医薬品又は医薬部外品の投与（摂取）形態としては、経口又は非経口（経皮、経粘膜、経腸、注射等）投与の形態が挙げられる。本発明の組成物を医薬品又は医薬部外品とする場合、経口用医薬品又は経口用医薬部外品とすることが好ましい。経口投与のための剤形としては、例えば、液剤、錠剤、散剤、細粒剤、顆粒剤、糖衣錠、カプセル剤、懸濁液、乳剤、チュアブル剤等が挙げられる。非経口投与のための剤形としては、例えば、注射剤、点滴剤、軟膏剤、ローション剤、貼付剤、坐剤、経鼻剤、経肺剤（吸入剤）等が挙げられる。医薬品は、非ヒト動物用医薬であってもよい。 When the composition of the present invention is used as a drug or quasi-drug, Cyclo (Phe-Phe) or a salt thereof is blended with a pharmacologically acceptable carrier, an optional additive, etc. , various dosage forms of pharmaceuticals or quasi-drugs. Such carriers, additives, etc. may be those that can be used for pharmaceuticals or quasi-drugs and are pharmacologically acceptable. One or more of antioxidants, coloring agents and the like can be mentioned. Examples of administration (ingestion) forms of pharmaceuticals or quasi-drugs include oral or parenteral (transdermal, transmucosal, enteral, injection, etc.) forms of administration. When the composition of the present invention is used as a drug or quasi-drug, it is preferably an oral drug or oral quasi-drug. Dosage forms for oral administration include, for example, liquids, tablets, powders, fine granules, granules, dragees, capsules, suspensions, emulsions, chewables and the like. Dosage forms for parenteral administration include, for example, injections, drops, ointments, lotions, patches, suppositories, nasal preparations, pulmonary preparations (inhalants) and the like. The pharmaceutical may be a non-human veterinary pharmaceutical.
本発明の組成物を飼料とする場合には、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を飼料に配合すればよい。飼料には飼料添加剤も含まれる。飼料としては、例えば、牛、豚、鶏、羊、馬等に用いる家畜用飼料；ウサギ、ラット、マウス等に用いる小動物用飼料；犬、猫、小鳥等に用いるペットフードなどが挙げられる。 When the composition of the present invention is used as a feed, Cyclo (Phe-Phe) or a salt thereof may be added to the feed. Feed also includes feed additives. Examples of feeds include livestock feeds for cows, pigs, chickens, sheep, horses, etc.; small animal feeds for rabbits, rats, mice, etc.; pet foods for dogs, cats, small birds, etc.;
本発明の組成物を、例えば、飲食品、医薬品、医薬部外品、飼料等とする場合、その製造方法は特に限定されず、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を用いて、一般的な方法により製造することができる。 For example, when the composition of the present invention is used as a food or drink, a drug, a quasi-drug, a feed, etc., the production method is not particularly limited, and Cyclo (Phe-Phe) or a salt thereof may be used to prepare a general It can be manufactured by a method.
一態様において、本発明の組成物は、液状組成物であることが好ましく、飲料であることがより好ましい。飲料は、例えば、機能性飲料であってよい。飲料の形態は特に限定されず、容器詰飲料であってよい。容器詰飲料の容器は特に限定されず、いずれの形態及び材質の容器を用いてもよく、例えば、アルミ缶、スチール缶等の金属製容器；ペットボトル等の樹脂製容器；紙パック等の紙容器；ガラス瓶等のガラス製容器；樽等の木製容器等の通常用いられる容器のいずれも用いることができる。このような容器に飲料を充填及び密閉することにより、容器詰飲料が得られる。 In one aspect, the composition of the present invention is preferably a liquid composition, more preferably a beverage. The beverage may be, for example, a functional beverage. The form of the beverage is not particularly limited, and may be a packaged beverage. Containers for packaged beverages are not particularly limited, and containers of any shape and material may be used. For example, metal containers such as aluminum cans and steel cans; resin containers such as PET bottles; Containers; glass containers such as glass bottles; and wooden containers such as barrels. Any of commonly used containers can be used. A packaged beverage is obtained by filling and sealing such a container with a beverage.
本発明の組成物に含まれるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の含有量は特に限定されず、その形態等に応じて設定することができる。
一態様において、本発明の組成物におけるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の含有量は、例えば、１×１０－６重量％以上であってよく、１×１０－５重量％以上が好ましく、また、９０重量％以下であってよく、１×１０－１重量％以下であってよく、１×１０－２重量％以下であってよい。一態様において、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の含有量は、例えば、本発明の組成物中に１×１０－６～９０重量％であってよい。一態様において、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の含有量は、本発明の組成物中に、１×１０－６～１×１０－１重量％であってよく、１×１０－６～１×１０－２重量％が好ましく、１×１０－５～１×１０－２重量％がより好ましい。一態様において、本発明の組成物が飲料等の液体組成物の場合は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の含有量は、組成物中に、１×１０－６～１×１０－１重量％が好ましく、１×１０－６～１×１０－２重量％がより好ましく、１×１０－５～１×１０－２重量％が更に好ましい。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、例えば、液体クロマトグラフィー質量分析法（ＬＣ／ＭＳ）により公知の方法で定量することが可能である。 The content of Cyclo(Phe-Phe) or a salt thereof contained in the composition of the present invention is not particularly limited, and can be set according to its form and the like.
In one aspect, the content of Cyclo(Phe-Phe) or a salt thereof in the composition of the present invention may be, for example, 1×10 −6 wt % or more, preferably 1×10 −5 wt % or more, Further, it may be 90% by weight or less, 1×10 −1 % by weight or less, or 1×10 −2 % by weight or less. In one aspect, the content of Cyclo(Phe-Phe) or a salt thereof may be, for example, 1×10 −6 to 90% by weight in the composition of the invention. In one aspect, the content of Cyclo(Phe-Phe) or a salt thereof in the composition of the present invention may be from 1×10 −6 to 1×10 −1 wt %, and from 1×10 −6 to 1×10 −2 wt % is preferred, and 1×10 −5 to 1×10 −2 wt % is more preferred. In one aspect, when the composition of the present invention is a liquid composition such as a beverage, the content of Cyclo(Phe-Phe) or a salt thereof is 1×10 −6 to 1×10 −1 % by weight is preferred, 1×10 −6 to 1×10 −2 wt % is more preferred, and 1×10 −5 to 1×10 −2 wt % is even more preferred.
Cyclo(Phe-Phe) or a salt thereof can be quantified by a known method, for example, by liquid chromatography-mass spectrometry (LC/MS).
本発明の組成物は、経口で摂取（経口投与）されることが好ましい。本発明の組成物の投与量（摂取量ということもできる）は特に限定されない。本発明の組成物の投与量は、アミロイドβの産生抑制及び／又は蓄積抑制効果が得られるような量であればよく、投与形態、投与方法、対象の体重等に応じて適宜設定すればよい。本発明の組成物の投与量は、β－セクレターゼ阻害効果及び／又はミクログリアの貪食作用促進効果が得られるような量であることが好ましい。 The composition of the present invention is preferably taken orally (orally administered). There is no particular limitation on the dose (which can also be referred to as intake) of the composition of the present invention. The dosage of the composition of the present invention may be an amount that provides an effect of inhibiting the production and/or accumulation of amyloid β, and may be appropriately set according to the dosage form, administration method, body weight of the subject, and the like. . The dosage of the composition of the present invention is preferably such that the β-secretase inhibitory effect and/or the microglial phagocytosis promoting effect can be obtained.
一態様において、本発明の組成物をヒト（成人）を対象に摂取させる又は投与する場合、その投与量は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の投与量として、１日あたり、好ましくは１μｇ以上、より好ましくは１０μｇ以上、さらに好ましくは１５０μｇ以上、また、好ましくは１０００μｇ以下、より好ましくは５００μｇ以下、さらに好ましくは３００μｇ以下である。一態様において、本発明の組成物をヒト（成人）に摂取させる又は投与する場合、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の投与量として、１日あたり、好ましくは１～１０００μｇ、より好ましくは１０～５００μｇ、さらに好ましくは１５０～３００μｇである。
上記量を、１日１回以上、例えば、１日１回で又は数回（例えば２～３回）に分けて、摂取又は投与することが好ましい。一態様においては、上記量のＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を、経口で摂取させる又は投与することが好ましい。ヒト（成人）の場合、１日当たり体重６０ｋｇ当たり上記量のＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を摂取させる又は投与することが好ましい。一態様において、本発明の組成物は、ヒトに、体重６０ｋｇあたり、１日あたり上記量のＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を摂取させる又は投与するための経口用組成物であってよい。 In one aspect, when the composition of the present invention is ingested or administered to a human (adult) subject, the dosage is preferably 1 μg or more per day as a dose of Cyclo(Phe-Phe), and more It is preferably 10 µg or more, more preferably 150 µg or more, and preferably 1000 µg or less, more preferably 500 µg or less, and still more preferably 300 µg or less. In one aspect, when the composition of the present invention is ingested or administered to humans (adults), the dose of Cyclo(Phe-Phe) is preferably 1 to 1000 μg, more preferably 10 to 500 μg per day, More preferably 150 to 300 μg.
It is preferable to take or administer the above amount once or more a day, for example, once a day or divided into several times (eg, 2 to 3 times). In one aspect, Cyclo(Phe-Phe) or a salt thereof in the above amount is preferably taken or administered orally. For humans (adults), it is preferable to ingest or administer Cyclo(Phe-Phe) or a salt thereof in the above amount per 60 kg of body weight per day. In one aspect, the composition of the present invention may be an oral composition for ingesting or administering to humans Cyclo(Phe-Phe) or a salt thereof in the above amount per 60 kg body weight per day.
本発明の組成物は、継続して摂取又は投与されるものであることが好ましい。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を継続的に摂取させる又は投与することによって、より高い効果が得られることが期待される。一態様において、本発明の組成物は、好ましくは１週間以上、より好ましくは４週間以上、さらに好ましくは８週間以上継続して摂取又は投与されることが好ましい。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、飲食品などとして摂取可能であり、安全性の観点から、例えば長期摂取することにも問題が少ないと考えられる。 The composition of the present invention is preferably taken or administered continuously. Continuous ingestion or administration of Cyclo(Phe-Phe) or a salt thereof is expected to provide greater effects. In one aspect, the composition of the present invention is preferably taken or administered continuously for one week or longer, more preferably for four weeks or longer, and even more preferably for eight weeks or longer.
Cyclo(Phe-Phe) or a salt thereof can be ingested as a food or drink, and from the viewpoint of safety, long-term ingestion is considered to cause few problems.
本発明の組成物を摂取させる又は投与する対象（投与対象ということもできる）は、特に限定されない。好ましくはヒト又は非ヒト哺乳動物であり、より好ましくはヒトである。
一態様において、本発明の組成物の投与対象として、アミロイドβの産生抑制及び／又は蓄積抑制を必要とする又は希望する対象、アミロイドβの蓄積に関連する状態又は疾患の予防又は改善を必要とする又は希望する対象等が挙げられる。一態様において、本発明における投与対象として、中高年者が挙げられる。中高年者は、高齢者を含む。中高年者は、例えば、４０歳以上のヒトであってよい。一態様において中高年者の中でも、対象として高齢者が好ましい。高齢者は、例えば、６０歳以上又は６５歳以上のヒトであってよい。一態様において、本発明の組成物の投与対象は、健常者であってよい。例えば、脳内におけるアミロイドβの産生抑制及び／又は蓄積抑制、脳内におけるアミロイドβの蓄積予防、アルツハイマー病又は軽度認知障害の予防等を目的として、健常者に対して使用することもできる。 Subjects to whom the composition of the present invention is ingested or administered (can also be referred to as administration subjects) are not particularly limited. Humans or non-human mammals are preferred, and humans are more preferred.
In one aspect, the subject to which the composition of the present invention is administered is a subject who needs or desires suppression of amyloid β production and/or accumulation, and a subject who needs prevention or improvement of a condition or disease associated with amyloid β accumulation. The subject etc. which do or desire are mentioned. In one aspect, administration subjects in the present invention include middle-aged and elderly people. Middle-aged and elderly people include elderly people. A middle-aged person may be, for example, a person over the age of 40. In one aspect, among middle-aged and elderly people, elderly people are preferred as subjects. A senior citizen may be, for example, a human over the age of 60 or over the age of 65. In one aspect, the subject of administration of the composition of the present invention may be a healthy subject. For example, it can be used in healthy individuals for the purpose of suppressing the production and/or accumulation of amyloid β in the brain, preventing the accumulation of amyloid β in the brain, preventing Alzheimer's disease or mild cognitive impairment, and the like.
本発明の組成物には、アミロイドβの産生抑制及び／又は蓄積抑制により発揮される機能の表示が付されていてもよい。このような表示は機能性表示ともいう。上記表示は、特に限定されない。このような表示として、例えば、「認知機能を高める」、「認知機能の低下を抑える」、「認知機能を良好に保つ」、「記憶力を高める」、「記憶力の低下を抑える」、「記憶力を良好に保つ」、「記憶の精度を高める」、「記憶障害を予防する」、「記憶障害を改善する」、「認知機能の一部である記憶力を維持する」、「記憶力の低下が気になる方に適した機能」、「認知機能の一部である記憶力の精度や判断の正確さを向上させる」、「記憶の保持又は統合を改善する」、「認知機能の強化を維持する」、「遂行機能を改善する」、「注意力と集中力を促進する」、「学習能力を改善する」、「見当識を維持・改善する」、「加齢に伴う認知機能低下を遅延する」、「短期及び長期記憶を強化する」、「言語的及び視空間記憶を促進する」、「論理的思考力を維持・改善する」、及び、これらと同視できる表示又は機能性表示が挙げられる。
本発明の一態様において、本発明の組成物は、上記の表示が１又は２以上付された飲食品であることが好ましい。また上記の表示は、上記の機能を得るために上記組成物を用いる旨の表示であってもよい。当該表示は、組成物自体に付されてもよいし、組成物の容器又は包装に付されていてもよい。 The composition of the present invention may be labeled with a function exerted by suppressing the production and/or accumulation of amyloid β. Such indication is also called functionality indication. The display is not particularly limited. Such indications include, for example, "increases cognitive function", "suppresses deterioration of cognitive function", "maintains good cognitive function", "improves memory", "suppresses deterioration of memory", and "improves memory". "maintaining good memory", "improving memory accuracy", "preventing memory impairment", "improving memory impairment", "maintaining memory as a part of cognitive function", and "preventing memory loss" functions that are suitable for people to become,""improve memory accuracy and judgment accuracy, which are part of cognitive function", "improve memory retention or consolidation", "maintain enhanced cognitive function", "improves executive function", "promotes attention and concentration", "improves learning ability", "maintains and improves orientation", "delays age-related cognitive decline", Examples include "strengthening short-term and long-term memory", "promoting verbal and visuospatial memory", "maintaining/improving logical thinking ability", and indications or functional indications equated with these.
In one aspect of the present invention, the composition of the present invention is preferably a food or drink labeled with one or more of the above labels. Moreover, the above indication may be an indication that the above composition is used to obtain the above functions. The label may be attached to the composition itself, or may be attached to the container or packaging of the composition.
本発明は、以下の方法及び使用も包含する。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を投与する、アミロイドβの産生抑制及び／又は蓄積抑制方法。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を投与する、β－セクレターゼ阻害方法。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を投与する、ミクログリア細胞の貪食作用促進方法。
アミロイドβの産生抑制及び／又は蓄積抑制のための、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。
β－セクレターゼを阻害するための、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。
ミクログリア細胞の貪食作用促進のための、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。
上記方法は、治療的な方法であってもよく、非治療的な方法であってもよい。上記使用は、治療的な使用であってもよく、非治療的な使用であってもよい。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、１種又は２種以上のアミロイドβの産生抑制及び／又は蓄積抑制のために使用することができる。 The invention also includes the following methods and uses.
A method for suppressing production and/or accumulation of amyloid β, comprising administering Cyclo(Phe-Phe) or a salt thereof.
A method of inhibiting β-secretase, comprising administering Cyclo(Phe-Phe) or a salt thereof.
A method for promoting phagocytosis of microglial cells, comprising administering Cyclo(Phe-Phe) or a salt thereof.
Use of Cyclo(Phe-Phe) or a salt thereof for suppressing production and/or accumulation of amyloid β.
Use of Cyclo(Phe-Phe) or salts thereof to inhibit β-secretase.
Use of Cyclo(Phe-Phe) or salts thereof for promoting phagocytosis of microglial cells.
The method may be a therapeutic method or a non-therapeutic method. The use may be therapeutic use or non-therapeutic use.
Cyclo(Phe-Phe) or a salt thereof can be used to suppress production and/or accumulation of one or more types of amyloid β.
一態様において、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、β－セクレターゼ阻害によって、アミロイドβの産生を抑制及び／又は蓄積を抑制するために使用することができる。一態様において、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、ミクログリア細胞の貪食作用を促進することによって、アミロイドβの蓄積を抑制するために使用することができる。 In one aspect, Cyclo(Phe-Phe) or a salt thereof can be used to suppress the production and/or accumulation of amyloid β through β-secretase inhibition. In one aspect, Cyclo(Phe-Phe) or salts thereof can be used to inhibit amyloid-β accumulation by promoting phagocytosis of microglial cells.
上記方法及び使用においては、１日に１回以上、例えば、１日１回～数回（例えば２～３回）、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を対象に摂取させる又は投与することが好ましい。上記の使用は、好ましくはヒト又は非ヒト哺乳動物、より好ましくはヒトにおける使用である。一態様において、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、β－セクレターゼ阻害及び／又はミクログリア細胞の貪食作用促進によって、アミロイドβの蓄積に関連する状態又は疾患を予防又は改善するために使用することができる。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を投与する、アミロイドβの蓄積に関連する状態又は疾患の予防又は改善方法も本発明に包含される。 In the above methods and uses, Cyclo(Phe-Phe) or a salt thereof may be ingested or administered to the subject once or more times a day, for example, once to several times (for example, 2 to 3 times) a day. preferable. The above uses are preferably in humans or non-human mammals, more preferably in humans. In one aspect, Cyclo(Phe-Phe) or a salt thereof is used to prevent or ameliorate conditions or diseases associated with accumulation of amyloid β by inhibiting β-secretase and/or promoting phagocytosis of microglial cells. can be done. The present invention also includes a method for preventing or ameliorating a condition or disease associated with amyloid β accumulation, comprising administering Cyclo(Phe-Phe) or a salt thereof.
上記方法及び使用においては、アミロイドβの産生抑制及び／又は蓄積抑制効果が得られる量（有効量ということもできる）のＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を使用すればよい。上記有効量は、β－セクレターゼ阻害効果及び／又はミクログリア細胞の貪食作用促進効果が得られるような量であることが好ましい。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の好ましい投与量、投与方法、投与対象等は上述した本発明の組成物と同じである。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、そのまま摂取又は投与してもよく、これを含む組成物として摂取又は投与してもよい。例えば、本発明の組成物を摂取又は投与してもよい。 In the method and use described above, Cyclo(Phe-Phe) or a salt thereof may be used in an amount (which can also be referred to as an effective amount) capable of suppressing the production and/or accumulation of amyloid β. The above-mentioned effective amount is preferably an amount that provides β-secretase inhibitory effect and/or microglial cell phagocytosis promoting effect. Cyclo(Phe-Phe) or a salt thereof is the same as the composition of the present invention described above with respect to the preferred dose, administration method, administration subject, and the like. Cyclo(Phe-Phe) or a salt thereof may be ingested or administered as is, or may be ingested or administered as a composition containing it. For example, a composition of the invention may be ingested or administered.
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、アミロイドβの産生抑制及び／又は蓄積抑制のために使用される飲食品、医薬品、医薬部外品、飼料等の製造のために使用することができる。一態様において、本発明は、アミロイドβの産生抑制及び／又は蓄積抑制用組成物の製造における、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用、も包含する。本発明は、β－セクレターゼ阻害用組成物又はミクログリア細胞の貪食作用促進用組成物の製造における、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用、も包含する。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩は、アミロイドβの蓄積に関連する状態又は疾患の予防又は改善用組成物の製造のために使用することができる。 Cyclo(Phe-Phe) or a salt thereof can be used for the production of foods and drinks, pharmaceuticals, quasi-drugs, feeds, etc. that are used to suppress the production and/or accumulation of amyloid β. In one aspect, the present invention also includes the use of Cyclo(Phe-Phe) or a salt thereof in the production of a composition for suppressing the production and/or accumulation of amyloid β. The present invention also includes the use of Cyclo(Phe-Phe) or a salt thereof in the manufacture of a composition for inhibiting β-secretase or a composition for promoting phagocytosis of microglial cells.
Cyclo(Phe-Phe) or a salt thereof can be used for the manufacture of a composition for preventing or improving conditions or diseases associated with amyloid β accumulation.
本明細書において下限値と上限値によって表されている数値範囲、即ち「下限値～上限値」は、それら下限値及び上限値を含む。例えば、「１～２」により表される範囲は、１以上２以下を意味し、１及び２を含む。本明細書において、上限及び下限は、いずれの組み合わせによる範囲としてもよい。 Numerical ranges represented by lower and upper limits herein, ie, "lower and upper limits", include these lower and upper limits. For example, a range represented by "1-2" means from 1 to 2 and includes 1 and 2. In this specification, the upper limit and the lower limit may be any combination of ranges.
以下、本発明を実施例によりさらに詳しく説明するが、これにより本発明の範囲を限定するものではない。 EXAMPLES The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited by these examples.
＜実施例１～４及び比較例１＞
（β－セクレターゼ阻害活性を有する化合物のスクリーニング）
被験化合物について、β－セクレターゼ阻害活性を調べた。被験化合物には、環状ジペプチドであるシクロフェニルアラニルフェニルアラニン（Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ））（実施例１～４）及びシクロロイシルアラニン（Ｃｙｃｌｏ（Ｌｅｕ－Ａｌａ））（比較例１）を使用した（いずれもＢａｃｈｅｍ社）。
β－セクレターゼ阻害活性の評価には、市販のキットＢｅｔａ　Ｓｅｃｒｅｔａｓｅ　（ＢＡＣＥ１）　Ａｃｔｉｖｉｔｙ　Ａｓｓａｙ　Ｋｉｔ（ａｂｃａｍ）を用いた。キットに記載の方法に従って、β－セクレターゼ酵素活性の阻害効果を評価した。
９６　Ｗｅｌｌ　Ｐｌａｔｅ（Ｃｏｒｎｉｎｇ）上でサンプル液（被験化合物溶液）と酵素液を混合した後、３７℃で１０分間プレインキュベートした。その後、基質液を添加し、３７℃で６０分間反応させた。反応開始後０分から６０分まで５分ごとに、マイクロプレートリーダー　ＦｌｅｘＳｔａｔｉｏｎ　３（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ）で、反応液の蛍光強度（ＲＦＵ）（Ｅｘ／Ｅｍ＝３３５ｎｍ／４９５ｎｍ）を測定した。ｎ＝２で反応を行った。評価における各被験化合物の濃度（最終濃度）は、表１の通りとして実施した。 <Examples 1 to 4 and Comparative Example 1>
(Screening for compounds having β-secretase inhibitory activity)
The test compounds were examined for β-secretase inhibitory activity. Cyclophenylalanylphenylalanine (Cyclo(Phe-Phe)) (Examples 1 to 4) and cycloleucylalanine (Cyclo(Leu-Ala)) (Comparative Example 1), which are cyclic dipeptides, were used as test compounds. (both from Bachem).
A commercially available kit, Beta Secretase (BACE1) Activity Assay Kit (abcam), was used to evaluate the β-secretase inhibitory activity. The inhibitory effect on β-secretase enzymatic activity was evaluated according to the method described in the kit.
After mixing the sample solution (test compound solution) and the enzyme solution on a 96 Well Plate (Corning), the mixture was preincubated at 37°C for 10 minutes. After that, a substrate solution was added and reacted at 37° C. for 60 minutes. The fluorescence intensity (RFU) (Ex/Em=335 nm/495 nm) of the reaction solution was measured with a microplate reader FlexStation 3 (Molecular Devices) every 5 minutes from 0 to 60 minutes after the start of the reaction. Reactions were performed with n=2. The concentration (final concentration) of each test compound in the evaluation was carried out as shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
酵素活性の評価には反応液のΔ蛍光強度（（反応終了時の蛍光強度（６０分））－（反応開始前蛍光強度（０分）））を使用した。被験化合物非添加群をＣｏｎｔｒｏｌとした。また、陽性対照にはキットに付属の阻害剤を使用した。 The Δ fluorescence intensity of the reaction solution ((fluorescence intensity at the end of the reaction (60 minutes))−(fluorescence intensity before the start of the reaction (0 minutes))) was used to evaluate the enzyme activity. A group to which no test compound was added was used as a control. In addition, the inhibitor attached to the kit was used as a positive control.
図１は、被験化合物のβ－セクレターゼ（ＢＡＣＥ）阻害活性を示すグラフである。結果は、平均±標準偏差（ｎ＝２）で示した。図１に示すＢＡＣＥ活性は、ＣｏｎｔｒｏｌのＢＡＣＥ活性（Ｃｏｎｔｒｏｌの反応液のΔ蛍光強度）を１とした場合の、被験化合物のＢＡＣＥ活性（被験化合物を添加した反応液のΔ蛍光強度）の相対値である。
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）について、β－セクレターゼの酵素活性の低下が確認された。環状ジペプチドであるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）がβ－セクレターゼ阻害活性を有していることが明らかになった。 FIG. 1 is a graph showing the β-secretase (BACE) inhibitory activity of test compounds. Results are shown as mean±standard deviation (n=2). The BACE activity shown in FIG. 1 is a relative value of the BACE activity of the test compound (Δ fluorescence intensity of the reaction solution to which the test compound was added) when the BACE activity of the control (Δ fluorescence intensity of the reaction solution of Control) is set to 1. is.
A decrease in β-secretase enzymatic activity was confirmed for Cyclo(Phe-Phe). Cyclo(Phe-Phe), a cyclic dipeptide, was found to have β-secretase inhibitory activity.
＜実施例５＞
（貪食作用の測定）
目的
ｉｎ　ｖｉｔｒｏアッセイを使用して脳ミクログリアによるアミロイドβ（Ａβ）１－４２（モノマー及びオリゴマー）貪食作用（ファゴサイトシス）の増強におけるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）（ＣＦＦ）の効果を調べた。 <Example 5>
(Measurement of phagocytosis)
Objective The effect of Cyclo(Phe-Phe) (CFF) in enhancing amyloid β (Aβ) 1-42 (monomeric and oligomeric) phagocytosis by brain microglia was investigated using an in vitro assay.
材料と方法
Ａ．試薬
Ｇｉｂｃｏ　Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ　Ｍｉｘｔｕｒｅ　Ｆ－１２　Ｈａｍ　１：１（ＤＭＥＭ／Ｆ１２）、ピルビン酸ナトリウム、ウシ胎児血清（ＦＢＳ）、ウマ血清（ＨＳ）、ペニシリン／ストレプトマイシン（Ｐ／Ｓ）、ポリ－Ｄ－リジン（ＰＤＬ）コート２４－ｗｅｌｌプレート、Ｈａｎｋｓ’　Ｂａｌａｎｃｅｄ　Ｓａｌｔ　Ｓｏｌｕｔｉｏｎ（ＨＢＳＳ）、Ｄｕｌｂｅｃｃｏ’ｓ　ｐｈｏｓｐｈａｔｅ－ｂｕｆｆｅｒｅｄ　ｓａｌｉｎｅ（ＤＰＢＳ）、ＢｌｏｃｋＡｉｄＴＭ　Ｂｌｏｃｋｉｎｇ　Ｓｏｌｕｔｉｏｎ、ＩＢＡ１ポリクローナル抗体（ＩＢＡ１）及びＨｏｅｃｈｓｔ　３３３４２，Ｔｒｉｈｙｄｒｏｃｈｌｏｒｉｄｅ，Ｔｒｉｈｙｄｒａｔｅは、ＴｈｅｒｍｏＦｉｓｃｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃから入手した。
β－アミロイド（１－４２）、ＨｉＬｙｔｅＴＭ　Ｆｌｕｏｒ　４８８（Ａβ１－４２）及び１％　ＮＨ４ＯＨ　ｓｏｌｕｔｉｏｎはＡｎａｓｐｅｃから購入した。Ｔｒｉｔｏｎ（登録商標）Ｘ－１００及びパラホルムアルデヒド（ＰＦＡ）は、Ｓｉｇｍａ　Ａｌｄｒｉｃｈから入手した。シクロ－フェニルアラニン－フェニルアラニン（Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）：ＣＦＦ）はＢａｃｈｅｍ社から購入した。 Materials and MethodsA. Reagents Gibco Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 Ham 1:1 (DMEM/F12), sodium pyruvate, fetal bovine serum (FBS), horse serum (HS), penicillin/streptomycin (P/S ), poly-D-lysine (PDL) coated 24-well plate, Hanks' Balanced Salt Solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), BlockAid ™ Blocking Solution, IBA1 polyclonal antibody (IBA1) and Hoechst 33342 , Trihydrochloride, Trihydrate were obtained from ThermoFischer Scientific.
β-Amyloid (1-42), HiLyte ™ Fluor 488 (Aβ 1-42 ) and 1% NH 4 OH solution were purchased from Anaspec. Triton® X-100 and paraformaldehyde (PFA) were obtained from Sigma Aldrich. Cyclo-phenylalanine-phenylalanine (Cyclo(Phe-Phe): CFF) was purchased from Bachem.
Ｂ．Ａβ１－４２の調製
メーカー（Ａｎａｓｐｅｃ）の推奨に従って、Ａβ１－４２ペプチド（Ａβ１－４２モノマー）の可溶性種を調製した。凍結乾燥したＡβ１－４２ペプチドを１％ＮＨ４ＯＨ溶液に２ｍｇ／ｍＬの濃度で溶解した。ペプチド溶液をＤＰＢＳでさらに希釈し、最終濃度１ｍｇ／ｍＬのストック溶液にした。再構成されたペプチドをいくつかのフリーザーバイアルに分注し、－２０℃で保存した。
ローダミンＢ標識合成Ａβ１－４２ペプチド（ＧＬ　Ｂｉｏｃｈｅｍ社、上海、中国）を冷たいヘキサフルオロイソプロパナール（ＨＦＩＰ）に１ｍＭの濃度で溶解した。溶液を室温で１時間インキュベートし、ｔｅｒｍｏｖａｐサンプル濃縮器で乾燥させた。残留物をジメチルスルホキシド（ＤＭＳＯ）に溶解して５ｍＭの濃度にし、さらに最小必須培地（ＭＥＭ）で最終濃度２００μＭに希釈し、ストック溶液とした。希釈したＡβ１－４２を４℃で２４時間インキュベートして、オリゴマーのＡβ１－４２(Ａβ１－４２オリゴマー)を得た。 B. A soluble version of the Aβ 1-42 peptide (Aβ 1-42 monomer) was prepared according to the Aβ 1-42 preparation manufacturer's recommendations (Anaspec). Lyophilized Aβ 1-42 peptide was dissolved in 1% NH 4 OH solution at a concentration of 2 mg/mL. The peptide solution was further diluted with DPBS to a final concentration of 1 mg/mL stock solution. Reconstituted peptides were aliquoted into several freezer vials and stored at -20°C.
Rhodamine B-labeled synthetic Aβ 1-42 peptide (GL Biochem, Shanghai, China) was dissolved in cold hexafluoroisopropanal (HFIP) at a concentration of 1 mM. The solution was incubated at room temperature for 1 hour and dried with a termovap sample concentrator. Residues were dissolved in dimethylsulfoxide (DMSO) to a concentration of 5 mM and further diluted with minimal essential medium (MEM) to a final concentration of 200 μM to serve as stock solutions. Diluted Aβ 1-42 was incubated at 4° C. for 24 hours to obtain oligomeric Aβ 1-42 (Aβ 1-42 oligomers).
Ｃ．細胞培養及び処理
ＳＩＭ－Ａ９マウスミクログリア細胞（ＡＴＣＣ　ＣＲＬ－３２６５）を、１０％ＦＢＳ、５％ＨＳ及び１％Ｐ／Ｓを添加したＤＭＥＭ／Ｆ１２中で保持した。貪食作用アッセイでは、ＳＩＭ－Ａ９細胞を培養フラスコから取り出し、ペレット化し、無血清ＤＭＥＭ／Ｆ１２に再懸濁した。細胞を２０００００細胞／ウェルの密度でＰＤＬコート２４ウェルプレートに播種し、５％ＣＯ２を含む加湿ガスチャンバー内で、３７℃で２４時間、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で前処理した。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の最終処理濃度は２．２μｇ／ｍＬであった。 C. Cell Culture and Treatment SIM-A9 mouse microglial cells (ATCC CRL-3265) were maintained in DMEM/F12 supplemented with 10% FBS, 5% HS and 1% P/S. For phagocytosis assays, SIM-A9 cells were removed from culture flasks, pelleted, and resuspended in serum-free DMEM/F12. Cells were seeded on PDL-coated 24-well plates at a density of 200,000 cells/well and pretreated with Cyclo(Phe-Phe) for 24 hours at 37° C. in a humidified gas chamber containing 5% CO 2 . The final treatment concentration of Cyclo(Phe-Phe) was 2.2 μg/mL.
ＢＶ－２マウスミクログリア細胞を、１０％ＦＢＳ、１％ピルビン酸ナトリウム及び１％Ｐ／Ｓを添加したＭＥＭ中で保持した。細胞を、１１．０５μｇ／ｍＬのＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）及び２μＭ　Ａβ１－４２オリゴマーで、５％ＣＯ２を含む加湿ガスチャンバー内で３７℃で１６時間共処理した。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）は、塩酸（ＨＣｌ）に溶解させてＭＥＭに添加した。 BV-2 mouse microglial cells were maintained in MEM supplemented with 10% FBS, 1% sodium pyruvate and 1% P/S. Cells were co-treated with 11.05 μg/mL Cyclo(Phe-Phe) and 2 μM Aβ 1-42 oligomers for 16 h at 37° C. in a humidified gas chamber containing 5% CO 2 . Cyclo(Phe-Phe) was dissolved in hydrochloric acid (HCl) and added to MEM.
Ｄ．貪食作用アッセイ
接着性のＳＩＭ－Ａ９ミクログリアをＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）（２．２μｇ／ｍＬ）で２４時間前処理した後、無血清ＤＭＥＭ／Ｆ１２で１回リンスし、次いで、無血清ＤＭＥＭ／Ｆ１２中で４．５１μｇ／ｍＬの濃度でＡβ１－４２（モノマー）を添加し、３７℃で６時間インキュベートした。インキュベーション期間の終わりに、Ａβ１－４２含有培地を除去し、細胞をＨＢＳＳで３回、ＤＰＢＳで２回穏やかにリンスして、細胞外Ａβ１－４２を除去した。次に、細胞を４％ＰＦＡで固定し、０．２％Ｔｒｉｔｏｎ（登録商標）　Ｘ－１００で透過処理し、ＢｌｏｃｋＡｉｄＴＭ　Ｂｌｏｃｋｉｎｇ　Ｓｏｌｕｔｉｏｎで、室温で１時間ブロックした。ＩＢＡ１抗体を使用して細胞の形状を視覚化し、Ｈｏｅｃｈｓｔ３３３４２を使用して核を染色した。分析のためにウェルごとに５セットの画像を取得し、細胞内Ａβ１－４２を含む細胞をＡβ１－４２／ＩＢＡ１／核シグナルの共局在化によって決定した。細胞内Ａβ１－４２を含む細胞の数は、ビヒクルグループ（Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理していない細胞、ビヒクルコントロール）に対して正規化された貪食細胞の平均数として表す。 D. Phagocytosis Assay Adherent SIM-A9 microglia were pretreated with Cyclo(Phe-Phe) (2.2 μg/mL) for 24 h, rinsed once with serum-free DMEM/F12, and then serum-free DMEM/F12. Aβ 1-42 (monomer) was added at a concentration of 4.51 μg/mL in medium and incubated at 37° C. for 6 hours. At the end of the incubation period, the Aβ 1-42 containing medium was removed and the cells were gently rinsed three times with HBSS and twice with DPBS to remove extracellular Aβ 1-42 . Cells were then fixed with 4% PFA, permeabilized with 0.2% Triton® X-100, and blocked with BlockAid ™ Blocking Solution for 1 hour at room temperature. The IBA1 antibody was used to visualize cell shape and Hoechst 33342 was used to stain the nuclei. Five sets of images were acquired per well for analysis and cells containing intracellular Aβ 1-42 were determined by co-localization of the Aβ 1-42 /IBA1/nuclear signal. The number of cells containing intracellular Aβ 1-42 is expressed as the mean number of phagocytic cells normalized to the vehicle group (cells not treated with Cyclo(Phe-Phe), vehicle control).
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）とＡβ１－４２オリゴマーで共処理したＢＶ－２細胞を４％ＰＦＡで固定し、顕微鏡写真を撮影した。フローサイトメトリー分析では、細胞を２％ＦＢＳで２回洗浄し、１０００ｒｐｍで５分間遠心分離し、Ｆｃ－Ｂｌｏｃｋ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅ）で３０分間ブロックした。ＦｌｏｗＪｏソフトウェア（Ｔｒｅｅ　Ｓｔａｒ、Ａｓｈｌａｎｄ、ＯＲ）を使用して蛍光データを分析した。 BV-2 cells co-treated with Cyclo(Phe-Phe) and Aβ 1-42 oligomers were fixed with 4% PFA and micrographs were taken. For flow cytometric analysis, cells were washed twice with 2% FBS, centrifuged at 1000 rpm for 5 minutes and blocked with Fc-Block (BD Bioscience) for 30 minutes. Fluorescence data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Ｅ．統計分析
統計分析は、ＧｒａｐｈＰａｄ　Ｐｒｉｓｍ　ｖｅｒｓｉｏｎ　５．０（Ｓａｎ　Ｄｉｅｇｏ，　Ｃａｌｉｆｏｒｎｉａ，　ＵＳＡ）を使用して行った。結果は全て、平均±標準偏差として示す。統計分析は、Ｓｔｕｄｅｎｔ’ｓ　ｔ－ｔｅｓｔによって実施した。Ｐ値がｐ＜０．０５の場合、データを有意であるとみなした。 E. Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.0 (San Diego, California, USA). All results are presented as mean ± standard deviation. Statistical analysis was performed by Student's t-test. Data were considered significant if the P-value was p<0.05.
（結果）
脳ミクログリア細胞によるアミロイドβ１－４２（モノマー及びオリゴマー）貪食作用の増強におけるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の効果を評価するために、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理していない細胞（ビヒクルコントロール）とＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）処理細胞とを比較した。 (result)
To assess the effect of Cyclo(Phe-Phe) in enhancing amyloid β 1-42 (monomeric and oligomeric) phagocytosis by brain microglial cells, cells not treated with Cyclo(Phe-Phe) (vehicle control) and Cyclo(Phe-Phe) treated cells were compared.
Ａ．アミロイドβ１－４２モノマー貪食作用の増強におけるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の効果
図２は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で２４時間前処理したＳＩＭ－Ａ９ミクログリア（ＣＦＦ）、及び、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理しなかったＳＩＭ－Ａ９ミクログリア（ビヒクルコントロール（ＶＣ））の細胞内アミロイドβモノマーの量を示すグラフである。＊は、ビヒクルコントロール（ＶＣ）に対して有意差（ｐ＜０．０５）があったことを示す。ミクログリア内のアミロイドβ量は、ビヒクルコントロールのアミロイドβ量を１００とした相対値（％ＶＣ）で示した（平均±ＳＤ、ｎ＝３）。
図２に示すように、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）は、未処理の細胞（ビヒクルコントロール）と比較して、有意にアミロイドβ１－４２モノマー貪食作用を増加させた。 A. Effect of Cyclo(Phe-Phe) in enhancing amyloid β 1-42 monomer phagocytosis. ) is a graph showing the amount of intracellular amyloid-β monomer in SIM-A9 microglia (vehicle control (VC)) not treated with ). * indicates that there was a significant difference (p<0.05) from the vehicle control (VC). The amount of amyloid β in microglia was shown as a relative value (%VC) with the amount of amyloid β in the vehicle control set to 100 (mean±SD, n=3).
As shown in Figure 2, Cyclo(Phe-Phe) significantly increased amyloid β 1-42 monomer phagocytosis compared to untreated cells (vehicle control).
Ｂ．アミロイドβ１－４２オリゴマー貪食作用の増強におけるＣｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）の効果
結果を図３及び図４に示す。
図３は、Ａβ１－４２オリゴマーで処理したＢＶ－２ミクログリア（ビヒクルコントロール（ＶＣ））、並びに、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）及びＡβ１－４２オリゴマーで共処理したＢＶ－２ミクログリア（ＣＦＦ）の顕微鏡写真である（図３Ａ、図３Ｂ及び図３Ｃ：ビヒクルコントロール（ＶＣ）、図３Ｄ、図３Ｅ及び図３Ｆ：Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）処理）。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）処理群（ＣＦＦ）では、ＢＶ－２ミクログリアを１６時間ＣＦＦ及びＡβ１－４２オリゴマーで処理した。図３Ａ及び図３Ｄは、明視野顕微鏡で観察された細胞密度を示し、図３Ｂ及び図３Ｅは、ローダミンＢで染色されたＡβ１－４２ペプチドの存在を示し、図３Ｃ及び図３Ｆは、細胞に取り込まれたローダミンＢで染色されたＡβ１－４２ペプチドと細胞のマージ画像（Ｍｅｒｇｅｄ）である。 B. The effect results of Cyclo(Phe-Phe) in enhancing phagocytosis of amyloid β 1-42 oligomers are shown in FIGS. 3 and 4. FIG.
FIG. 3 depicts BV-2 microglia treated with Aβ 1-42 oligomers (vehicle control (VC)) and BV-2 microglia co-treated with Cyclo(Phe-Phe) and Aβ 1-42 oligomers (CFF). 3A, 3B and 3C: vehicle control (VC), 3D, 3E and 3F: Cyclo(Phe-Phe) treatment). In the Cyclo(Phe-Phe) treated group (CFF), BV-2 microglia were treated with CFF and Aβ 1-42 oligomers for 16 hours. Figures 3A and 3D show the cell densities observed by brightfield microscopy, Figures 3B and 3E show the presence of Aβ 1-42 peptide stained with rhodamine B, and Figures 3C and 3F show the cells Merged images of Rhodamine B-stained Aβ 1-42 peptides and cells incorporated into .
図４は、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理したＢＶ－２ミクログリア（ＣＦＦ）と、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）で処理しなかったＢＶ－２ミクログリア（ビヒクルコントロール（ＶＣ））のアミロイドβオリゴマー貪食作用を調べた結果を示すグラフである。縦軸は、ミクログリア当たりのアミロイドβ１－４２密度を示す（平均±ＳＤ、ｎ＝３）。＊は、ビヒクルコントロール（ＶＣ）に対して有意差（ｐ＜０．０５）があったことを示す。 FIG. 4. Phagocytosis of BV-2 microglia (CFF) treated with Cyclo(Phe-Phe) and BV-2 microglia not treated with Cyclo(Phe-Phe) (vehicle control (VC)). It is a graph which shows the result of having investigated. The vertical axis indicates the amyloid β 1-42 density per microglia (mean ± SD, n=3). * indicates that there was a significant difference (p<0.05) from the vehicle control (VC).
ＣＦＦ投与は、Ａβ１－４２ペプチドオリゴマーとインキュベートしたＢＶ－２細胞の貪食活性を有意に促進できることが分かった。これは、未処理のコントロール細胞（ＶＣ）との比較で、蛍光細胞の頻度の増加とミクログリアの平均蛍光強度（ＭＦＩ）レベルの向上で特徴づけられた（図３及び図４）。 It was found that CFF administration can significantly enhance the phagocytic activity of BV-2 cells incubated with Aβ 1-42 peptide oligomers. This was characterized by increased frequency of fluorescent cells and enhanced microglial mean fluorescence intensity (MFI) levels compared to untreated control cells (VC) (FIGS. 3 and 4).
以上のように、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）は、ＳＩＭ－Ａ９ミクログリア細胞によるアミロイドβ１－４２モノマーの貪食及びＢＶ－２ミクログリア細胞によるアミロイドβ１－４２オリゴマーの貪食作用を増強した。 As described above, Cyclo(Phe-Phe) enhanced phagocytosis of amyloid β 1-42 monomers by SIM-A9 microglial cells and phagocytosis of amyloid β 1-42 oligomers by BV-2 microglial cells.
＜実施例６＞
アミロイド前駆体タンパク質（ＡＰＰ）過剰発現細胞アッセイ 
Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）（ＣＦＦ）のアミロイドβ蓄積抑制効果を調べるため、ｉｎ　ｖｉｔｒｏアッセイを実施した。アッセイには、スウェーデン二重変異Ｋ５９５Ｎ／Ｍ５９６Ｌを有するアミロイド前駆体タンパク質野生型を過剰発現するＨ４神経膠腫細胞（Ｈ４－ｈＡＰＰ細胞）を使用した。
Ｈ４－ｈＡＰＰ細胞によるアミロイドβの蓄積に対するＣＦＦの効果を評価するために、ＣＦＦで処理していない細胞（ビヒクルコントロール）とＣＦＦ処理細胞とを比較した。 <Example 6>
Amyloid precursor protein (APP) overexpression cell assay
An in vitro assay was performed to investigate the effect of Cyclo(Phe-Phe) (CFF) on amyloid β accumulation. The assay used H4 glioma cells overexpressing amyloid precursor protein wild-type with the Swedish double mutation K595N/M596L (H4-hAPP cells).
To assess the effect of CFF on amyloid-β accumulation by H4-hAPP cells, cells not treated with CFF (vehicle control) were compared with CFF-treated cells.
材料及び方法
Ａ．試薬及び細胞
試薬及び細胞は、下記のものを使用した。
Ｇｉｂｃｏ　Ｏｐｔｉ－ＭＥＭＴＭ　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍ　（Ｏｐｔｉ－ＭＥＭ）（細胞培養培地）、ウシ胎児血清（ＦＣＳ）、ペニシリン／ストレプトマイシン（Ｐ／Ｓ）、ハイグロマイシンＢ及びブラストサイジンＳ塩酸塩は、ＴｈｅｒｍｏＦｉｓｃｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製を用いた。
ＭＳＤ（登録商標）　９６－ｗｅｌｌ　ＭＵＬＴＩＳＰＯＴ（登録商標）　６Ｅ１０　Ａｂｅｔａ　Ｔｒｉｐｌｅｘ　Ａｓｓａｙ　ｋｉｔ（アミロイドβ検出キット）はＭｅｓｏ　Ｓｃａｌｅ　Ｄｉｓｃｏｖｅｒｙ社製を用いた。
Ｎ－［Ｎ－（３，５－ジフルオロフェナセチル－Ｌ－アラニル）］－Ｓ－フェニルグリシンｔ－ブチルエステル（ＤＡＰＴ、γ－セクレターゼ阻害剤）は、Ｃａｌｂｉｏｃｈｅｍ社製を用いた。Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）（ＣＦＦ）は、Ｂａｃｈｅｍ社製を用いた。
Ｈ４－ｈＡＰＰ細胞は、ヒトＨ４神経膠腫細胞株に、スウェーデン二重変異Ｋ５９５Ｎ／Ｍ５９６Ｌを有するヒトアミロイド前駆体タンパク質６９５(ＡＰＰ６９５)を含むｐＡＧ３ベクターを安定導入（ｓｔａｂｌｅ　ｔｒａｎｓｆｅｃｔｉｏｎ）して得た。ｐＡＧ３ベクターは、融合されたＣＭＶとニワトリβ－アクチンプロモーターの制御下に導入遺伝子を有するハイグロマイシンＢ耐性カセットを含む改変ｐｃＤＮＡ３プラスミドである。 Materials and MethodsA. Reagents and Cells The following reagents and cells were used.
Gibco Opti-MEM ™ I Reduced Serum Medium (Opti-MEM) (cell culture medium), fetal calf serum (FCS), penicillin/streptomycin (P/S), hygromycin B and blasticidin S hydrochloride were obtained from ThermoFischer Scientific I used the company's product.
MSD (registered trademark) 96-well MULTISPOT (registered trademark) 6E10 Abeta Triplex Assay kit (amyloid β detection kit) manufactured by Meso Scale Discovery was used.
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) was manufactured by Calbiochem. Cyclo(Phe-Phe) (CFF) manufactured by Bachem was used.
H4-hAPP cells were obtained by stable transfection of human H4 glioma cell line with pAG3 vector containing human amyloid precursor protein 695 (APP695) with Swedish double mutation K595N/M596L. The pAG3 vector is a modified pcDNA3 plasmid containing a hygromycin B resistance cassette with the transgene under the control of the fused CMV and chicken β-actin promoters.
Ｂ．細胞培養及び処理
Ｈ４－ｈＡＰＰ細胞は、１０％ＦＣＳ、１％Ｐ／Ｓ、２００μｇ／ｍＬのハイグロマイシンＢ及び２．５μｇ／ｍＬのブラストサイジンＳ塩酸塩を添加したＯｐｔｉ－ＭＥＭ中で維持した。細胞を、２５，０００細胞／ウェルの密度で９６ウェルプレートに播種し、５％ＣＯ２を含む加湿ガスチャンバー内で、３７℃で一晩インキュベートした。翌日、２２ｍｇ／ｍＬのＣＦＦでＨ４－ｈＡＰＰ細胞を処理した。ＣＦＦ処理においては、Ｈ４－ｈＡＰＰ細胞の培地にＣＦＦを２２μｇ／ｍＬとなるように添加し、細胞をインキュベートした。陽性対照として、ＣＦＦの代わりに、４００ｎＭのＤＡＰＴでＨ４－ｈＡＰＰ細胞を処理した。２４時間処理した後、アミロイドβ（Ａβ）分析のために細胞培養上清を収集した。ＣＦＦ処理及びＤＡＰＴ処理を行わないＨ４－ｈＡＰＰ細胞を、ビヒクルコントロールとし、細胞上清を収集した。 B. Cell Culture and Treatment H4-hAPP cells were maintained in Opti-MEM supplemented with 10% FCS, 1% P/S, 200 μg/mL hygromycin B and 2.5 μg/mL blasticidin S hydrochloride. . Cells were seeded in 96-well plates at a density of 25,000 cells/well and incubated overnight at 37°C in a humidified gas chamber containing 5% CO2 . The next day, H4-hAPP cells were treated with 22 mg/mL CFF. In the CFF treatment, CFF was added to the medium of H4-hAPP cells to 22 μg/mL, and the cells were incubated. As a positive control, H4-hAPP cells were treated with 400 nM DAPT instead of CFF. After 24 hours of treatment, cell culture supernatants were collected for amyloid-β (Aβ) analysis. H4-hAPP cells without CFF and DAPT treatment served as vehicle control and cell supernatants were collected.
Ｃ．アミロイドβ　ＥＬＩＳＡアッセイ
収集した細胞培養上清を１：１０に希釈し、６Ｅ１０　Ａｂｅｔａ　Ｔｒｉｐｌｅｘ　Ａｓｓａｙ　ｋｉｔを用いてヒトアミロイドβ１－３８（Ａβ１－３８）、ヒトアミロイドβ１－４０（Ａβ１－４０）、及びヒトアミロイドβ１－４２（Ａβ１－４２）を分析した。マルチプレックスアッセイは製造元の指示に従って実行され、プレートをＳｅｃｔｏｒ　Ｉｍａｇｅｒ　２４００（Ｍｅｓｏ　Ｓｃａｌｅ　Ｄｉｓｃｏｖｅｒｙ社製）で読み取った。アミロイドβの濃度は、標準曲線を参照して計算した。マルチプレックスキットの感度は＜５ｐｇ／ｍＬであった。 C. Amyloid-β ELISA Assay Collected cell culture supernatants were diluted 1:10 and tested using the 6E10 Abeta Triplex Assay kit for human amyloid-β 1-38 (Aβ 1-38 ), human amyloid-β 1-40 (Aβ 1-40) . ), and human amyloid β 1-42 (Aβ 1-42 ) were analyzed. Multiplex assays were performed according to the manufacturer's instructions and plates were read on a Sector Imager 2400 (Meso Scale Discovery). Amyloid β concentrations were calculated with reference to a standard curve. The sensitivity of the multiplex kit was <5 pg/mL.
Ｄ．統計分析
Ｐｒｉｓｍバージョン５．０（ＧｒａｐｈＰａｄ社製）を使用して統計分析を実施した。すべての結果は、平均±標準偏差として表した。統計分析は、分散分析（ＡＮＯＶＡ）と、それに続く事後Ｔｕｋｅｙの多重比較検定により行った。ｐ値がｐ＜０．０５の場合、データは有意であると見なした。 D. Statistical analysis Statistical analysis was performed using Prism version 5.0 (GraphPad). All results were expressed as mean ± standard deviation. Statistical analysis was performed by analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparison test. Data were considered significant if the p-value was p<0.05.
結果
図５、図６及び図７に、結果を示す。図５は、Ｈ４－ｈＡＰＰ細胞の培養上清中のアミロイドβ１－３８の濃度を示すグラフである。図６は、Ｈ４－ｈＡＰＰ細胞の培養上清中のアミロイドβ１－４０の濃度を示すグラフである。図７は、Ｈ４－ｈＡＰＰ細胞の培養上清中のアミロイドβ１－４２の濃度を示すグラフである。
図５、図６及び図７中、ＣＦＦは、ＣＦＦ処理した細胞である。Ｃｏｎｔｒｏｌはビヒクルコントロールであり、ＤＡＰＴは陽性対照である。値は平均値±標準偏差（ｎ＝６）を表す。＊＊＊は、ビヒクルコントロールに対してｐ＜０．００１の有意差を示す。 Results Figures 5, 6 and 7 show the results. FIG. 5 is a graph showing the concentration of amyloid β 1-38 in the culture supernatant of H4-hAPP cells. FIG. 6 is a graph showing the concentration of amyloid β 1-40 in the culture supernatant of H4-hAPP cells. FIG. 7 is a graph showing the concentration of amyloid β 1-42 in the culture supernatant of H4-hAPP cells.
In Figures 5, 6 and 7, CFF is CFF-treated cells. Control is vehicle control and DAPT is positive control. Values represent mean±standard deviation (n=6). *** indicates significant difference with p<0.001 from vehicle control.
ＣＦＦは、Ｈ４－ｈＡＰＰ細胞におけるアミロイドβ１－３８、アミロイドβ１－４０及びアミロイドβ１－４２の蓄積を抑制した。ＣＦＦは、Ｈ４－ｈＡＰＰ細胞におけるアミロイドβ１－３８、アミロイドβ１－４０及びアミロイドβ１－４２の蓄積を抑制する効果を有する。 CFF suppressed the accumulation of Aβ 1-38 , Aβ 1-40 and Aβ 1-42 in H4-hAPP cells. CFF has the effect of suppressing the accumulation of amyloid β 1-38 , amyloid β 1-40 and amyloid β 1-42 in H4-hAPP cells.

Claims (8)

  1. Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩を有効成分として含む、アミロイドβの産生抑制及び／又は蓄積抑制用組成物。 A composition for suppressing the production and/or accumulation of amyloid β, comprising Cyclo(Phe-Phe) or a salt thereof as an active ingredient.
  2. β－セクレターゼを阻害することによりアミロイドβの産生及び／又は蓄積を抑制する、請求項１に記載の組成物。 The composition according to claim 1, which suppresses the production and/or accumulation of amyloid β by inhibiting β-secretase.
  3. ミクログリア細胞の貪食作用を促進することによりアミロイドβの蓄積を抑制する、請求項１又は２に記載の組成物。 3. The composition according to claim 1, which suppresses accumulation of amyloid β by promoting phagocytosis of microglial cells.
  4. 前記組成物が、経口用である請求項１又は２に記載の組成物。 3. The composition according to claim 1 or 2, wherein said composition is for oral use.
  5. 前記組成物が、飲食品又は医薬品である請求項１又は２に記載の組成物。 3. The composition according to claim 1 or 2, wherein the composition is a food or drink or a pharmaceutical.
  6. 前記組成物が、「認知機能を高める」、「認知機能の低下を抑える」、「認知機能を良好に保つ」、「記憶力を高める」、「記憶力の低下を抑える」、「記憶力を良好に保つ」、「記憶の精度を高める」、「記憶障害を予防する」、「記憶障害を改善する」、「認知機能の一部である記憶力を維持する」、「記憶力の低下が気になる方に適した機能」、「認知機能の一部である記憶力の精度や判断の正確さを向上させる」、「記憶の保持又は統合を改善する」、「認知機能の強化を維持する」、「遂行機能を改善する」、「注意力と集中力を促進する」、「学習能力を改善する」、「見当識を維持・改善する」、「加齢に伴う認知機能低下を遅延する」、「短期及び長期記憶を強化する」、「言語的及び視空間記憶を促進する」及び「論理的思考力を維持・改善する」からなる群より選択される１又は２以上の機能の表示を付されたものである請求項１又は２に記載の組成物。 The composition "enhances cognitive function", "suppresses decline in cognitive function", "maintains good cognitive function", "enhances memory", "suppresses decline in memory", "maintains good memory" , "Improve memory accuracy", "Prevent memory impairment", "Improve memory impairment", "Maintain memory which is a part of cognitive function", "For those who are concerned about memory decline" "improve memory accuracy and judgment accuracy", "improve memory retention or consolidation", "maintain enhanced cognitive function", "executive function" "improve attention and concentration", "improve learning ability", "maintain and improve orientation", "delay age-related cognitive decline", "short-term and Labeled with one or more functions selected from the group consisting of "strengthening long-term memory", "promoting verbal and visuospatial memory" and "maintaining/improving logical thinking ability" The composition according to claim 1 or 2, wherein
  7. アミロイドβの産生抑制及び／又は蓄積抑制用組成物の製造における、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。 Use of Cyclo(Phe-Phe) or a salt thereof in the production of a composition for suppressing production and/or accumulation of amyloid β.
  8. アミロイドβの産生抑制及び／又は蓄積抑制のための、Ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ）又はその塩の使用。 Use of Cyclo(Phe-Phe) or a salt thereof for suppressing production and/or accumulation of amyloid β.
PCT/JP2022/046366 2021-12-23 2022-12-16 COMPOSITION FOR SUPPRESSING PRODUCTION AND/OR ACCUMULATION OF AMYLOID β WO2023120407A1 (en)

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