WO2010087315A1 - Anti-alzheimer’s disease agent - Google Patents

Anti-alzheimer’s disease agent Download PDF

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Publication number
WO2010087315A1
WO2010087315A1 PCT/JP2010/050927 JP2010050927W WO2010087315A1 WO 2010087315 A1 WO2010087315 A1 WO 2010087315A1 JP 2010050927 W JP2010050927 W JP 2010050927W WO 2010087315 A1 WO2010087315 A1 WO 2010087315A1
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Prior art keywords
alzheimer
compound represented
general formula
disease
amyloid
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PCT/JP2010/050927
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French (fr)
Japanese (ja)
Inventor
人水 太田
研志 穐田
恒孝 太田
敏雄 河田
恵温 福田
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株式会社林原生物化学研究所
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Priority to JP2010548505A priority Critical patent/JPWO2010087315A1/en
Publication of WO2010087315A1 publication Critical patent/WO2010087315A1/en

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    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
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    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/415—1,2-Diazoles
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/32—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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Definitions

  • the present invention relates to an anti-Alzheimer's disease agent comprising a compound represented by the following general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • Alzheimer's disease is a pathological condition caused by systematic neuronal degeneration or loss caused by amyloid ⁇ peptide or the like, and the breakdown of the neural network based on omission, and is one of the major causes of dementia that has increased rapidly in recent years.
  • Alzheimer's disease atrophy of neurites (axons and dendrites) and a decrease in synapses are triggers that impair nerve function, and conversely, even after the trigger is triggered, It is said that if the nerve cells that survive the degeneration are activated, the neurites are extended, and the synapse is restored, the nerve function can be restored.
  • damaged axons in the peripheral nervous system regenerate, in the central nervous system, it is said that regeneration does not occur unless treatment such as transplantation of peripheral nerves is performed.
  • NGF nerve growth factor
  • systemic administration such as subcutaneously or intravascularly cannot be expected to have a therapeutic effect on neurodegenerative diseases caused by central nerve degeneration because it is difficult to cross the blood-brain barrier due to the polymer.
  • Surgical treatment is necessary to administer it into the brain with the expectation of an effect, and the patient is accompanied by a large physical and mental burden.
  • Alzheimer's disease vary from mild to severe, and typical examples include dementia. If the symptoms progress, as with other neurodegenerative diseases, for example, Tremor, rigidity, ataxia, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, breathing Obstacles, numbness, paralysis, etc. may appear, and these are major obstacles in conducting daily life.
  • neurodegenerative diseases for example, Tremor, rigidity, ataxia, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, breathing Obstacles, numbness, paralysis, etc. may appear, and these are major obstacles in conducting daily life.
  • Alzheimer's disease is a serious disease that causes degeneration of nerve cells, and therapeutic agents containing various compounds as active ingredients have been proposed in order to improve these diseases and the pathological conditions and neurological dysfunctions associated therewith (for example, International Publication WO 97/030703 pamphlet, Japanese Patent Application Laid-Open No. 11-228417, Japanese Patent Application Laid-Open No. 2006-143708, Japanese Patent Application Laid-Open No. 2006-321737, neurite extension promoters, etc.
  • an effective method for treating a disease has not yet been found.
  • systemic administration such as subcutaneous or intravascular, which has less physical and mental burden for patients, acts on nerve cells of the central nervous system, activates nerve cells, suppresses neurite atrophy, or
  • a novel anti-Alzheimer's disease agent that promotes neurite outgrowth to suppress neurodegeneration and treats the pathological conditions and clinical symptoms associated therewith is eagerly desired.
  • An object of the present invention is to provide a novel anti-Alzheimer's disease agent.
  • the present inventors have conducted extensive research and search.
  • the compound represented by the following general formula 1 has an excellent nerve cell activation action and a neurite extension promoting action. I found out.
  • these compounds have an inhibitory effect on neuronal cell death caused by amyloid ⁇ peptide, which activates central nervous system neuron even by systemic administration, suppresses neurodegeneration and is caused by cytotoxicity caused by amyloid ⁇ peptide.
  • the present invention was completed by finding that the onset of symptoms and pathological conditions to be delayed or improved. That is, the present invention mainly comprises an anti-Alzheimer's disease agent comprising a compound having a cation moiety and an anion moiety in the molecule represented by the general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • the anti-Alzheimer's disease agent of the present invention protects cells from cytotoxic factors including amyloid ⁇ peptide by administering parenterally, and promotes activation of nerve cells and extension of neurites, Suppresses neurodegeneration caused by these injury factors, and various pathologies and symptoms including Alzheimer's disease and associated dementia (for example, tremor, rigidity, immobility, peristalsis, slow motion, posture reflex disorder , Autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis) .
  • security of the compound represented by General formula 1 which is an active ingredient is very high.
  • the neurite means an axon and dendrite extending from a neuronal cell body.
  • the neurite outgrowth promoting action refers to the action of activating nerve cells to extend axons and / or dendrites, the action of suppressing neurite atrophy and decrease, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
  • Neurodegeneration as used in the present invention refers to a decrease in function, death, or decrease (dropout) of nerve cells, particularly neurons in the central nervous system. Atrophy and decrease of neurites, decrease of synapses, decrease of function of glial cells Including death and reduction.
  • the anti-Alzheimer's disease agent of the present invention contains a compound represented by the above general formula 1 as an active ingredient.
  • R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent
  • Z 1 represents a heterocyclic ring
  • Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1.
  • These heterocyclic rings and aromatic rings may have a substituent.
  • o represents an integer that is either 0, 1, or 2
  • p represents an integer that is either 0 or 1
  • p is 1 when o is 0 or 2
  • o is 1
  • P is 0.
  • X l - represents a suitable counter anion
  • q is an integer of either 1 or 2.
  • X l in the general formula 1 - represents an appropriate counter anion, usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodic acid ion, hexafluorophosphate ion, Rokudoruka Inorganic acid anions such as antimonate ions, hexafluorostannate ions, phosphate ions, borofluoride ions, tetrafluoroborate ions, thiocyanate ions, benzenesulfonate ions, naphthalenesulfonate ions, naphthalene disulfonate ions Organic acid anions such as p-toluenesulfonate ion, alkylsulfonate ion, benzenecarboxylate ion, alkylcarboxylate ion, trihaloalkylcarboxylate ion,
  • the compound represented by the general formula 1 include dye compounds such as a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and a dimethine styryl dye represented by the general formula 5 (hereinafter referred to as “a dye compound”). And may be simply referred to as “compound”).
  • R 4 to R 6 represent the same or different aliphatic hydrocarbon groups.
  • X 2 ⁇ represents an appropriate counter anion
  • m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
  • R 7 to R 9 represent the same or different aliphatic hydrocarbon groups.
  • X 3 - represents an appropriate counter anion
  • m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
  • R 10 to R 12 represent the same or different aliphatic hydrocarbon groups.
  • X 4 ⁇ represents an appropriate counter anion
  • m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
  • Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent.
  • Z 4 represents an aromatic ring or a heteroaromatic ring, and the heteroaromatic ring and the aromatic ring may have a substituent.
  • R 13 represents an aliphatic hydrocarbon group, and the aliphatic hydrocarbon group may have a substituent.
  • the R 14 is hydrogen atom or an appropriate substituent and, X 5 - represents a suitable counter anion.
  • the aliphatic hydrocarbon group represented by R 4 to R 13 in the general formulas 2 to 5 those having 1 to 12 carbon atoms are usually selected, those having 2 to 10 are preferable, and those having 2 to 9 are preferable. Those are more preferred.
  • the R4 to R6 aliphatic hydrocarbon group of the compound represented by the general formula 2 has 2 to 12 carbon atoms, or the R7 to R9 aliphatic hydrocarbon group of the compound represented by the general formula 3
  • a compound having 4 to 10 carbon atoms is particularly desirable because it has a strong neurodegeneration inhibitory effect.
  • Examples of the individual aliphatic hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a tert-pentyl group.
  • suitable counter anions represented by X 2 ⁇ to X 5 ⁇ in the general formulas 2 to 5 are usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodate ion , Inorganic acid anions such as hexafluorophosphate ion, hexafluoroantimonate ion, hexafluorostannate ion, phosphate ion, borofluoride ion, tetrafluoroborate ion, thiocyanate ion, benzenesulfonate ion , Naphthalene sulfonate ion, naphthalene disulfonate ion, p-toluene sulfonate ion, alkyl sulfonate ion, benzene carboxylate ion, alkyl carboxylate ion,
  • the compound represented by the general formula 2 includes a compound represented by the chemical formula 1 (hereinafter sometimes referred to as “NK-9694”) and a compound represented by the chemical formula 2 (hereinafter referred to as “ NK-4 ”) and compounds having 3 carbon atoms in the side chain alkyl groups (R4 to R6) of formula 2 (hereinafter also referred to as“ NK-234 ”). Can do.
  • NK-9694 a compound represented by the chemical formula 1
  • NK-4 a compound represented by the chemical formula 2
  • R4 to R6 side chain alkyl groups
  • NK-150 a compound represented by the chemical formula 3
  • NK-150 a compound represented by the chemical formula 4
  • NK-19 may be mentioned.
  • the counter anion of the NK-19 (I -) of Cl - in the compound represented by Chemical Formula 5 for changing (. Which hereinafter may be referred to as "NK-53"), similar to the NK-19 advantageously Available.
  • NK-100 a compound represented by the chemical formula 6
  • Examples of the compound represented by the general formula 5 include compounds represented by the chemical formulas 7 to 9 (hereinafter sometimes referred to as “NK-528”, “NK-557”, and “NK-1516”, respectively). can do.
  • any of the compounds represented by any one of Chemical Formulas 1 to 9 and NK-234 can be used in addition to neuronal activation and neurite outgrowth promoting effects from cytotoxic factors such as starvation, radicals, and amyloid ⁇ peptide. Since it also has an action of protecting cells and suppressing cell death and neurite atrophy, it is more desirable as an active ingredient of the anti-neurodegenerative disease agent of the present invention.
  • -26 compound represented by chemical formula 1
  • NK-4 compound represented by chemical formula 2
  • NK-234 compound having 3 carbon atoms in the side chain alkyl group of general formula 2)
  • NK-150 Compound represented by Chemical Formula 3
  • NK-4 and NK-234 are desirable, and NK-4 is particularly desirable, taking into account the strength of acetylcholinesterase (AchE) activity inhibitory activity, migration into the brain, ease of formulation, and the like.
  • AchE acetylcholinesterase
  • the compound represented by the general formula 1 used as an active ingredient of the anti-Alzheimer's disease agent of the present invention is not limited in its origin or production method.
  • the anti-Alzheimer's disease agent of the present invention is a compound represented by general formula 1, preferably a pentamethine cyanine dye represented by any one of general formulas 2 to 4 and / or a dimethine styryl represented by general formula 5. It contains one or more dyes.
  • the anti-Alzheimer's disease agent of the present invention contains, as needed, a pharmaceutically acceptable food field, cosmetic field, pharmaceutical field, quasi-drug in addition to the compound represented by the general formula 1 which is an active ingredient. It is provided in the form of a preparation containing one or more components used in the field.
  • Examples of pharmaceutically acceptable ingredients include additives, excipients, disintegrants, lubricants, stabilizers, surfactants, preservatives (antibacterial agents), fragrances, thickeners, and antioxidants.
  • blend combining suitably 1 type (s) or 2 or more types of these components, and to manufacture by a conventional method according to the target dosage form.
  • the anti-Alzheimer's disease agent of the present invention is a neurite outgrowth promoting agent that is said to have a therapeutic effect on Alzheimer's disease, its pathology and clinical symptoms,
  • the combined use with the therapeutic agent of the pathological condition and nerve dysfunction which originates can also be implemented advantageously.
  • Cerebrovascular disorder therapeutic agent neurodegenerative disease therapeutic agent, amyotrophic lateral sclerosis therapeutic agent, multiple sclerosis therapeutic agent, dopamine receptor agonist, dopamine release promoter, dopamine uptake inhibitor, dopamine Agonist, central anticholinergic, aromatic L-amino acid decarboxylase inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline)
  • DCI aromatic L-amino acid decarboxylase inhibitor
  • MAO-B monoamine oxidase
  • COMP catechol-O-methyltransferase
  • norepinephrine norepinephrine
  • NGF is desirable because the neurite extension promoting action of the compound represented by the general formula 1 used in the present invention is effectively enhanced.
  • the anti-Alzheimer's disease agent of the present invention is usually provided in the form of a parenteral injection preparation or the like.
  • the compound represented by the general formula 1, which is an inactive ingredient takes into account the composition of the parenteral preparation such as the injectable preparation and the purpose of use thereof, from the raw material stage until the product is completed. What is necessary is just to mix
  • the methods include, for example, mixing, kneading, dissolving, melting, dispersing, suspending, emulsifying, reverse micellization, infiltration, crystallization, spraying, application, adhesion, spraying, coating (coating), pouring, dipping, solidifying, One or more known methods such as loading are appropriately selected.
  • parenteral preparations such as injection preparations
  • parenteral preparations such as injection preparations
  • it is usually dissolved in an aqueous medium that does not contain pyrogen, depending on the target disease or symptom, and then intradermally, subcutaneously, intramuscularly, intracorporeally (intrathoracic) , Intraperitoneal, etc.), intravascular or intracerebral (including spinal cord)
  • the preparation may be a dry preparation or a liquid preparation.
  • a dry preparation it may be used by dissolving it in an aqueous medium such as purified water for injection, physiological saline, and glucose solution at the time of use.
  • a solution it may be administered as it is, or it may be added to an infusion solution or a perfusion solution.
  • an amphiphilic solvent when there is a problem with solubility in a solvent or solubility in an aqueous medium, or when preparing a sustained-release preparation, an amphiphilic solvent, an oily base material, an emulsifier, etc. are used. Increasing the solubility of the components in the solvent is optional.
  • the aqueous medium referred to in the present invention has water as an essential element, and if necessary, for example, alcohols such as ethanol, propanol and isopropanol, ketones such as acetone, ethers such as diethyl ether, dimethyl
  • aqueous medium comprising one or more hydrophilic organic solvents including sulfur-containing compounds such as sulfoxide (hereinafter sometimes abbreviated as “DMSO”).
  • DMSO hydrophilic organic solvents including sulfur-containing compounds such as sulfoxide
  • purified water for injection, physiological saline, Ringer's solution, etc. are used alone, or purified water for injection and physiological such as ethanol, propanol, isopropanol, diethyl ether, DMSO, etc. It is desirable to use a mixed solution with a hydrophilic organic solvent which is acceptable.
  • Such a liquid agent depending on the compound represented by the general formula 1, it may become unstable due to dissolved oxygen or the like. In this case, for example, the dissolved oxygen concentration of the compound solution may be reduced.
  • a liquid composition is usually prepared by a method through a step of dissolving the compound in an aqueous medium and a step of lowering the oxygen concentration in the atmospheric environment at normal temperature and pressure using the aqueous medium. be able to.
  • a predetermined amount of a compound is added to an appropriate amount of an aqueous medium, dissolved as necessary with heating and stirring, and then, if necessary, An aqueous medium may be added until the concentration reaches a predetermined level.
  • a solution of the compound represented by the general formula 1 is prepared under reduced pressure and stored, or the compound solution is added to the compound solution.
  • a method of replacing dissolved oxygen with another gas or contacting the compound solution with an oxygen scavenger is preferable.
  • a relatively inert gas such as nitrogen or a rare gas such as neon, argon, krypton, or xenon is used. Just bubbling.
  • the liquid composition is prepared by, for example, adding L-ascorbic acid, L-ascorbic acid stearate, sodium sulfite, sodium hydrogen sulfite, alphathioglycerin, sodium edetate, cysteine hydrochloride Citric acid, soybean lecithin, sodium thioglycolate, sodium thiomalate, sodium pyrosulfite, butylhydroxyanisole and the like may be added in appropriate amounts. These methods may be applied to the compound solution or to an aqueous medium before the compound is dissolved. In this case, the concentration of oxygen dissolved in the aqueous medium is usually 0.4 ppm or less, preferably 0.1 ppm or less.
  • the thus obtained solution of the compound represented by the general formula 1 may be stored in a state where it is sealed in an appropriate container that can block oxygen.
  • the material of the container is not particularly limited as long as it can hold the liquid composition in principle and can substantially block oxygen, but it is light-shielded like a brown bottle or brown ampoule. Sex containers are desirable.
  • the liquid composition is usually dispensed into containers such as glass ampoules and vials, sealed, and then sterilized by high-pressure sterilization or the like.
  • the anti-Alzheimer's disease agent of the present invention can be used in the form of a haptic agent, a transpulmonary sucking and spraying agent, etc. in addition to the injection, and in the form of a sustained-release preparation embedded in the body such as subcutaneously. It can also be used. It is also optional to treat animals other than humans, including pets that have developed neurodegenerative diseases, and to use as preventives or therapeutic agents for pathological conditions and neurological dysfunction associated with neurodegenerative diseases.
  • the anti-Alzheimer's disease agent of the present invention thus produced is a safe preparation without serious side effects even when used for a long time.
  • the daily dose of the anti-Alzheimer's disease agent of the present invention is not particularly limited as long as the desired action and effect can be obtained, and is usually represented by the general formula 1 in the case of subcutaneous to intraperitoneal administration.
  • 0.01 mg / kg ⁇ body weight / day or more is desirable, 0.1 to 20 mg / kg ⁇ body weight / day is more desirable, and 0.5 to 5 mg / kg ⁇ body weight / day is particularly desirable. Even when administered at 50 mg / kg ⁇ body weight / day or more, there is a case where the enhancement of the effect corresponding to the dose is not observed.
  • the anti-Alzheimer's disease agent of the present invention protects the brain and nerve cells from cytotoxic factors such as amyloid ⁇ peptide, suppresses degeneration, activates nerve cells, promotes neurite extension, suppresses atrophy, and nerve cells Can suppress the prolongation of survival and degeneration, and can treat or prevent neurological dysfunction associated with Alzheimer's disease.
  • the treatment of Alzheimer's disease referred to in the present invention refers to the pathological condition and functional disorder associated with Alzheimer's disease in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses deterioration and stops progression of disease, and further the onset of disease Including the prevention of itself.
  • NK-4 a compound represented by Chemical Formula 2 (manufactured by Hayashibara Biochemical Laboratories, Inc., “NK-4”) was used as a test sample. Since NK-4 is hardly soluble in water, it is dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then membrane filtered (Millipore, product name “Millex-LG SLLG025SS”, DMSO resistant membrane was used), and further diluted with Dulbecco's MEM medium (sold by Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as “D-MEM medium”), and subjected to the test.
  • D-MEM medium Dulbecco's MEM medium
  • PC-12HS cells ⁇ Influence of NK-4 on cell damage caused by nutrient starvation> NGF (nerve growth factor) hypersensitive strain of PC-12 cells derived from rat adrenal pheochromocytoma (hereinafter referred to as “PC-12HS cells”), which is suitable as a model for studying human neuronal degeneration. Obtained). Cultivation was performed using a serum-removed medium as a nutrient starvation environment. PC-12HS cells were thawed from cryopreserved cells, cultured in D-MEM medium supplemented with 10% by volume fetal bovine serum (FBS), and subjected to the test.
  • FBS fetal bovine serum
  • Cells used for the test were detached by a conventional method using a 0.25% by mass trypsin solution, diluted with 10% by volume FBS-added D-MEM medium, and coated with a collagen-coated 96-well plate (trade name, sold by Falcon). “Micro test plate for cell culture, flat bottom”) was seeded at 5 ⁇ 10 3 cells / 100 ⁇ l / well. After 24 hours, the culture supernatant was removed, diluted with D-MEM medium containing no FBS, and 100 ⁇ l / well of NK-4 adjusted to twice the final concentration shown in Table 1 was added for 3 days. Cultured.
  • PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, seeded at 2 ⁇ 10 4 cells / 100 ⁇ l / well in a collagen-coated 96-well plate, and cultured for 24 hours. did. After that, dilute 800 ⁇ M hydrogen peroxide solution (sales by Wako Pure Chemical Industries, Ltd.) with 50 ⁇ l / well (final concentration 200 ⁇ M) and 10% FBS-added D-MEM medium to a concentration 4 times the final concentration shown in Table 1.
  • NK-4 was added at the same time to each 50 ⁇ l / well (NK-4 final concentration 5 ng to 50 ⁇ g / ml), cultured in an incubator for 2 hours, and then 25 vol% glutaraldehyde (Wako Pure Chemical Industries, Ltd.) The cells were fixed by adding 20 ⁇ l / well (final concentration 20% by volume). 0.05% by mass of methylene blue (available from Wako) was added at 100 ⁇ l / well, and the absorbance of each well was measured by a die-up take method according to a conventional method. As a control, culture was carried out in the same manner except that hydrogen peroxide and NK-4 were not added, and methylene blue was added to measure the absorbance. The relative value when the number of cells of the control (absorbance) is 100 (%) is determined and shown in Table 1 as the cell viability (%) of each well.
  • a peptide fragment having an amino acid sequence corresponding to the 23rd to 35th amino acids from the amino terminus of amyloid ⁇ peptide (human origin), which is considered to be one of the main causes of neuronal cell death in Alzheimer's disease (sold by AnaSpec, hereinafter “Amyloid ⁇ Fragment ”) (a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing) was diluted with phosphate buffered saline (PBS) to a concentration of 2 mM, and 6 hours at 37 ° C before use. Aged and aggregated fragments were used to increase cytotoxicity.
  • PBS phosphate buffered saline
  • PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, and seeded in a collagen-coated 96-well plate at 5 ⁇ 10 3 cells / 100 ⁇ l / well. After culturing for 24 hours, the supernatant was removed, and amyloid ⁇ fragment solution diluted with 10% by volume FBS-added D-MEM medium was 50 ⁇ l / well (final concentration of amyloid ⁇ fragment was 50 ⁇ M), and NK-4 solution was 50 ⁇ l / well. (Final concentration 40 to 5000 ng / ml) was added and cultured for 3 days.
  • NK-4 has a difference in the effective concentration for each cytotoxic factor against nutrient starvation, hydrogen peroxide damage, and amyloid ⁇ fragment damage to PC12-HS cells. However, it has been found to have a protective effect on nerve cells. Comparing the concentrations of NK-4 that exert the protective action against the three types of cytotoxic factors shown in Table 1, the protective action against the damage of the amyloid ⁇ fragment is exerted from 40 ng / ml, whereas the nutrient starvation It was found that a concentration of 500 ng / ml was required for injury and a concentration of 5,000 ng / ml was required for hydrogen peroxide injury.
  • NK-4 aqueous solution having a concentration of 50,000 ng / ml or less does not have the ability to erase hydrogen peroxide, so NK-4 is free of peroxy radicals, hydroxy radicals and the like.
  • the cytoprotective action against hydrogen peroxide cytotoxicity in this test system is not directly due to the action of scavenging hydrogen peroxide, but acts on the cell side to prevent cell death. Judged to be suppressed.
  • the occupancy ratio of the cells having undergone apoptosis calculated from Hoechst staining image was 72% when amyloid ⁇ fragment was added (NK-4 concentration 0 ng / ml), whereas apoptosis was promoted. -4 (200 ng / ml) was added, the occupancy was 13%, which was close to the occupancy (5%) of apoptotic cells in the control, and NK-4 was reduced by amyloid ⁇ fragment.
  • NK-4 can be used as a therapeutic agent for Alzheimer's disease caused by cytotoxic factors such as amyloid ⁇ peptide because it has a neuroprotective action against neuronal cytotoxic factors including amyloid ⁇ fragment. Tells the story.
  • ⁇ Experiment 2 Protective action against a cytotoxic factor of a dye compound other than NK-4>
  • a dye compound other than NK-4 (hereinafter sometimes simply referred to as “compound”) was also used. It was examined whether there was a similar effect.
  • a test sample was prepared by diluting 200-fold or more with 10% by volume FBS-added D-MEM medium (Nissui Pharmaceutical) and subjected to the test (the structure of the compound corresponding to the NK number in the table is “Photosensitive Dye Table ”, published by Photosensitive Dye Research Institute (1969)). These compounds were all synthesized by Hayashibara Biochemical Laboratories.
  • ⁇ Evaluation method A Evaluation method of nerve cell proliferation promoting action>
  • PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a pre-collagen-coated 96-well microplate at 5 ⁇ 10 3 cells / well, and 100 ⁇ l / well. Sowing. After 24 hours, each test sample diluted with D-MEM (10% by volume FBS) and adjusted to 100 ng / ml was added at 100 ⁇ l / well and cultured in a 37 ° C., 5% by volume CO 2 incubator for 3 days.
  • ⁇ Evaluation method B Evaluation method of neurite extension action>
  • PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a 96-well microplate previously coated with collagen at 5 ⁇ 10 3 cells / well. And 100 ⁇ l / well.
  • each test sample was diluted with D-MEM (10% by volume FBS) and adjusted to 400 ng / ml, 50 ⁇ l / well, and D containing 20 ng / ml NGF (available from Chemicon, mouse, final concentration 5 ng / ml).
  • -MEM medium (10% by volume FBS) 50 ⁇ l / well was added and cultured for 3 days.
  • the cells were fixed with 10% by volume glutaraldehyde for 20 minutes at room temperature.
  • PC12-HS cells cultured for 3 days only in 10% by volume FBS-added D-MEM medium were fixed with glutaraldehyde.
  • the fixed cells were observed under a microscope to evaluate the presence or absence of neurite outgrowth, and when the neurite outgrowth rate was 30% or more, it was determined to be equal to or better than NK-4 ( ⁇ ).
  • the neurite outgrowth rate (%) is the same by observing cells under a microscope at a magnification including about 100 cells in one field, counting the number of cells having neurites more than twice the cell body, Dividing by the total number of cells in the field of view and multiplying by 100 was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%. The results are also shown in Table 2.
  • ⁇ Evaluation Method C Inhibitory Action on Cell Damage by Amyloid ⁇ Fragment>
  • the test sample in which the cell growth promoting action was recognized by the evaluation method A was examined by the same method as in Experiment 1 to determine whether it had a cytoprotective effect against cell damage caused by amyloid ⁇ fragment. Compared to the case where the test sample was not added, the case where the cell damage due to amyloid ⁇ fragment was significantly suppressed was determined to have an inhibitory effect ( ⁇ ). The results are also shown in Table 2.
  • NK-19 compound represented by chemical formula 4
  • NK-53 compound represented by chemical formula 5
  • NK-100 compound represented by chemical formula 6
  • NK-528 expressed by chemical formula 7
  • Compound Compound
  • NK-557 compound represented by chemical formula 8
  • NK-1516 compound represented by chemical formula 9
  • the inhibitory action with respect to the cytotoxicity by the amyloid (beta) fragment was recognized by all the compounds evaluated as having the inhibitory action with respect to the cytotoxicity by the amyloid (beta) fragment (evaluation method C) about the compound which has cell growth promotion activity. From the above results and the results of Experiment 1 and Experiment 2, all of the compounds having the cell growth promoting action and the neurite outgrowth promoting action shown in Table 6 activate neuronal cells. It tells us that it can be used as a disease agent. Among them, NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-100 (compound represented by chemical formula 6), NK-528 (expressed by chemical formula 7).
  • NK-557 compound represented by Chemical Formula 8
  • NK-1516 compound represented by Chemical Formula 9
  • these compounds particularly NK-4, NK-19, NK-53, NK-100, NK-528, NK-557, and NK-1516, are used as therapeutic agents for pathological conditions and neurological dysfunction associated with Alzheimer's disease. Also tells you that you can use it.
  • NK-19, NK-53, NK-100, NK-528, NK-557 are selected from the compounds that have been confirmed to have the effect of inhibiting cell damage caused by amyloid ⁇ fragment in the same manner as NK-4.
  • NK-1516 the effect of the concentration on cytotoxicity caused by amyloid ⁇ fragment was examined.
  • NK-19, NK-53, and NK-100 showed lower inhibitory activity against cell damage caused by amyloid ⁇ fragment at a lower concentration than NK-4.
  • NK-53 showed the highest inhibitory activity at the lowest concentration, and almost completely inhibited the cytotoxicity caused by amyloid ⁇ fragment at a concentration of 12.5 ng / ml (cell survival rate 115 ⁇ 16%).
  • the injury inhibition rate was highest at a concentration of 50 ng / ml, which was 102 ⁇ 27%, 88 ⁇ 12%, and 114 ⁇ 9%, respectively.
  • NK-53 was found to have a high inhibitory activity of 111 ⁇ 9% even at a concentration of 50 ng / ml.
  • NK-4 had the highest injury inhibition rate of 122 ⁇ 32% at a concentration of 200 ng / ml, whereas NK-19, NK-53, NK-100, and NK-557 had a cytoprotective effect.
  • NK-19, NK-53, NK-100, and NK-557 are NK- It was found to be lower than 4.
  • NK-528 and NK-1516 did not show a higher cytotoxic effect than NK-4. From the above results, it was suggested that NK-19, NK-53, NK-100, and NK-557 may exhibit superior effects at lower concentrations than NK-4 as anti-Alzheimer's disease agents.
  • NK-19 and NK-100 which showed a high cytotoxicity-inhibiting activity at a lower concentration than NK-4, had a higher molecular weight than the others, and NK19 and NK-53 bound to nitrogen in the thiazole ring. Since the number of carbon atoms in the side chain alkyl group is as large as 7 (other carbon number is 2), it was judged that strong activity was expressed because of high lipid solubility and high cell membrane permeability.
  • NK-4, NK-19, NK-53, NK-100, and NK-4 which were confirmed to have a neurite outgrowth-promoting action among the compounds in which the protective action against the cytotoxicity of amyloid ⁇ fragment was recognized.
  • a test for examining the effect of NK-557 on the aggregation of amyloid ⁇ peptide was conducted as follows. Specifically, each test sample was dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, and then membrane-filtered with Millex-LG (Millipore, product number “LLG025SS”, DMSO resistant). Using Tris-HCl buffer, a test sample solution was prepared to a concentration of 200 nM.
  • test sample When the test sample inhibits amyloid ⁇ peptide aggregation, the thioflavin-T fluorescence decreases.
  • the effect of the test sample on amyloid ⁇ peptide aggregation was examined by this method. That is, a human amyloid ⁇ peptide having an amino acid sequence consisting of 40 amino acids represented by SEQ ID NO: 2 in the Sequence Listing (sold by Ana Spec) was used after dissolving in sterile distilled water at a concentration of 400 ⁇ M. The test sample solution was diluted with Tris-HCl buffer.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test suppressed aggregation of amyloid ⁇ peptide
  • NK-19, NK- 53, NK-100 and NK-557 all showed stronger inhibitory activity than NK-4.
  • NK-53, NK-100 and NK-557 suppressed amyloid ⁇ peptide aggregation by 95% or more
  • NK-100 almost completely suppressed aggregation by 99%.
  • a compound having an action of suppressing cell damage caused by amyloid ⁇ fragment such as NK-4, NK-19, NK-53, NK-100 and NK-557 has an action of suppressing aggregation of amyloid ⁇ peptide. Therefore, it shows that it is useful not only as a therapeutic agent for Alzheimer's disease but also as a preventive agent.
  • AchE inhibitors such as donepezil have been clinically applied to Alzheimer-type dementia. It has been reported that AchE inhibitors activate the central cholinergic nervous system and improve cognitive function even in ischemic dementia. Therefore, in Experiment 7, NK-4, NK-19, NK-53, NK-100, and NK-557 were confirmed to have an effect of improving cerebral infarction and associated neurological dysfunction in rats with cerebral ischemia. A test was conducted to confirm whether there was an AchE inhibitory effect.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO were each diluted with a phosphate buffer, and a compound having a concentration 10 times that shown in Table 13 was added.
  • a solution was prepared to provide a test sample solution.
  • PC12-HS cells cultured in the same manner as in Experiment 1 were collected, and 5 volumes (volume) of 10 mM Tris-HCl buffer solution (1M NaCl, 50 mM MgCl 2 , 1% Triton X-100, pH 7.2) was added. After homogenizing uniformly by a conventional method, the mixture was centrifuged (10,000 g) at 4 ° C.
  • acetylcholinesterase AchE
  • a solution containing acetylcholinesterase AchE
  • 50 mM phosphate buffer pH 8.0
  • 10 ⁇ l of test sample solution 10 ⁇ l
  • AchE-containing solution 50 ⁇ l
  • 50 ⁇ l of a phosphate buffer containing 0.5 mM acetylthiocholine iodide available from Wako Pure Chemical Industries, Ltd.
  • 1 mM 2-nitrobenzoic acid available from Wako
  • the absorbance (A R ) at 405 nm was measured with a plate reader.
  • 10 ⁇ l of phosphate buffer was used instead of the AchE-containing solution, and the absorbance (A U ) was measured by performing an enzyme reaction for 30 minutes in a 37 ° C. incubator in the same manner as described above.
  • 10 ⁇ l of a phosphate buffer was used for an enzyme reaction for 30 minutes in an incubator at 37 ° C., and the absorbance (B R ) was measured.
  • the reaction was performed using 10 ⁇ l of a phosphate buffer instead of the AchE-containing solution of the control reaction solution, and the absorbance (B U ) was measured.
  • NK-100 showed the highest AchE inhibition in the low concentration range, and showed a significant AchE inhibitory effect at NK-100 concentrations of 0.78 ⁇ g / ml or more.
  • NK-4 having a structure similar to that of NK-100 was significantly inhibited at a concentration of 3.13 ⁇ g / ml or more.
  • the IC 50 of these compounds was 3.3 ⁇ g / ml for NK-4 and 11.8 ⁇ g / ml for NK-100.
  • NK-19 and NK-53 had weak AchE inhibitory activity, and NK-19 and NK-53 showed a significant decrease in activity only at a concentration of 25 ⁇ g / ml.
  • NK-557 showed almost no AchE inhibitory activity at concentrations up to 25 ⁇ g / ml. This result shows that the four compounds NK-4, NK-19, NK-53, and NK-100 can also activate the cholinergic nervous system and improve Alzheimer-type dementia even by the inhibitory action of AchE. Sex was suggested.
  • galantamine which is clinically used as a therapeutic agent for Alzheimer's disease, shows the same level of AchE inhibitory effect at a lower concentration than NK-4 (the IC 50 at that time is 442 ⁇ g / ml).
  • these compounds have also been reported to suppress cell death via the PI3K-Akt cascade, similar to NK-4, whereas NK-4 is effective at 3.3 g / ml, whereas Galantami Then, a concentration of about 100 times is required to obtain the same effect.
  • NK-4 (NK-19, NK-53, NK-100) NK-557) can be used as an anti-Alzheimer's disease agent having a different mechanism of action from existing Alzheimer's disease therapeutic agents.
  • NK-19 Neurotrophic factor-like activity of NK-19 analog
  • a test was conducted to confirm that NK-19, which has been confirmed to have a strong neurodegeneration inhibitory effect, has similar effects on its analogs. That is, a compound in which the alkyl group (R) in the side chain of the compound represented by the general formula 3 has 1 to 12 carbon atoms was synthesized (Synthesis by Hayashibara Biochemical Laboratories Co., Ltd.) The strength of cell growth promoting action and neurite extension promoting action on -12HS cells was examined. Twelve types of analogs of NK-19 including NK-19 shown in Table 6 were synthesized and dissolved in DMSO to a concentration of 5 mg / ml.
  • This solution was diluted with D-MEM medium supplemented with 10% by volume FBS to prepare a test sample solution so that the concentration of the compound was 100 ng / ml or 2 ⁇ g / ml, respectively.
  • the NK-24 and NK-19 is the counter anion I - Cl from - compounds instead (NK-56 and NK-53) was also dissolved at a 5 mg / ml in DMSO.
  • These solutions were diluted with 10% by volume FBS-added D-MEM medium to prepare test sample solutions by diluting the compound concentration to 100 ng / ml or 2 ⁇ g / ml, respectively.
  • the cells were cultured for 3 days only in D-MEM medium supplemented with 10% by volume FBS, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the presence or absence of neurite extension was evaluated by the same method as in Experiment 3. The results are also shown in Table 6. The test was performed twice with each triplet for each test sample solution except NK-56 and NK-53, and the average was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%.
  • R 7 to R 9 are the same carbon atoms, an alkyl group having 1 to 12 carbon atoms, X 3 - is I - or Cl - and, m represents 2.
  • NK-4 analog Neurotrophic factor-like activity of NK-4 analog>
  • the side chain alkyl group (R 4 to R 6 ) Seven types of compounds having 2 to 8 carbon atoms and a counter anion of I ⁇ were synthesized, and as in Experiment 3, the strength of cell proliferation promoting action and neurite extension promoting action on PC12-HS cells was increased. Examined.
  • NK-4 seven kinds of compounds shown in Table 7, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, were each added to DMSO at 5 mg / ml. It dissolved so that it might become. This solution was diluted with 10% by volume FBS-added D-MEM so that the final concentrations of the compounds were as shown in Table 7 or 8, respectively, to prepare test sample solutions.
  • NK-19 analogs NK-13, NK-392, NK-19 and NK-150 are dissolved in DMSO to a concentration of 5 mg / ml, so that the concentration of the compound becomes the concentration shown in Table 7 or 8.
  • Table 7 shows the results of the cell growth promoting action
  • Table 8 shows the results of the neurite extension promoting action.
  • R 4 to R 6 represent the same alkyl group having 6 to 8 carbon atoms
  • X 2 represents I ⁇
  • m represents 2.
  • the NK-4 analog represented by the general formula 2 has a compound having 3 to 8 carbon atoms in the side chain alkyl group, and has a cell growth equivalent to or higher than that of NK-4.
  • the cell growth promoting action was particularly strong for compounds having 4 to 6 carbon atoms in the side chain alkyl group.
  • the neurite outgrowth promoting action a strong activity was observed in the compounds having 4 and 5 carbon atoms in the side chain alkyl group.
  • a compound represented by the general formula 3 wherein the side chain alkyl group has 2 to 8 carbon atoms particularly an NK-19 analogue represented by the general formula 3 wherein the side chain alkyl group has 6 to 8 carbon atoms. It was revealed that somatic compounds show strong cell proliferation and neurite outgrowth promoting activity.
  • amyloid ⁇ fragment disorder and hydrogen peroxide disorder using primary neurons (neurons, astrocytes and microglia cells) prepared from rat fetal cerebral cortex instead of PC-12HS cells.
  • NK-4, NK-26, NK-234, NK-19 and NK-150 were examined for their effects on these cells, and these compounds have activity to inhibit amyloid ⁇ fragment damage and hydrogen peroxide damage to primary neurons It has been found. In addition, these compounds were found to suppress the production of NO produced by microglia cells in the presence of LPS.
  • AchE activity inhibitor is used as a therapeutic agent for Alzheimer-type dementia.
  • the strength of the Ache activity inhibitory action of the NK-4 analog was compared. That is, using the four types of NK-4 analogs having 2 to 5 carbon atoms in the side chain alkyl group of the compound represented by general formula 2 used in Experiment 11, the remaining AchE activity rate was the same as in Experiment 8. (%) was measured. In addition, the remaining percentage of AchE activity (%) was measured using NK-19 analog NK-150. Table 9 also shows the IC50 value (compound concentration that inhibits the Ache activity used in the experiment by 50%) calculated based on the results and the residual activity rate.
  • NK-4 analog showed a tendency that the residual ratio of AchE activity increased as the carbon number of the side chain alkyl group increased.
  • NK-4 and NK-234 are useful as a therapeutic agent for Alzheimer-type dementia.
  • AchE activity remaining rate (%) of donepezil hydrochloride (trade name “Aricept”) marketed for the treatment of Alzheimer-type dementia as an Ache activity inhibitor was measured in the same experimental system, the IC 50 was 0.9 ⁇ g. / Ml, NK-4 has almost the same activity of inhibiting AchE activity as donepezil hydrochloride.
  • NK-4 analog and NK-19 analog on model mice of human Alzheimer type dementia The above experiments suggested that NK-4 can be used as a therapeutic agent for Alzheimer's dementia.
  • NK-4 analogs and NK-19 analogs were modeled on human Alzheimer's model mice. The effect was verified.
  • NK-4, NK-234, NK-26, NK-19 and NK-150 were used as test samples.
  • physiological saline 200 ⁇ l / animal
  • Donepezil hydrochloride was used as control 2.
  • Each test sample was dissolved in DMSO to a concentration of 5 mg / ml, and diluted with physiological saline for administration.
  • mice manufactured by Charles River Japan, male, 5 weeks old, body weight 25 to 30 g
  • Chloral hydrate SIGMA, 350 mg / kg, body weight, intraperitoneal administration
  • Anesthetized mice were fixed on the back, and a midline incision was made on the head.
  • Evans blue solution (0.3 ⁇ g / 0.3 ⁇ l) is administered in advance for the insertion site instead of the amyloid ⁇ fragment solution, and the lateral ventricle, dorsal third ventricle, ventral third brain of the left and right forehead cross section It was confirmed that coloring was observed in the room. After the administration, the scalp was sutured, and one of the compounds was intraperitoneally administered once a day for 13 days from the next day, and behavioral evaluation was performed by the following method. The results and group composition are shown in Table 10.
  • ⁇ Evaluation method> ⁇ New object recognition test> The novel object recognition test uses the characteristics of the mouse when it likes the novelty, and unlike many other learning evaluation systems, it does not use artificial reinforcement factors.
  • the test consisted of three departments: acclimatization, training trials, and retention trials, which were performed 6-8 days after amyloid ⁇ fragment was administered into the ventricle.
  • An open field experimental device (40 cm long, 30 cm wide, 30 cm high) with wood chips on the floor was installed in a noise-free place under about 1,000 lux lighting. First, on the 6th day, the mouse was placed in the center of the experimental apparatus without the search object and allowed to search freely for 10 minutes (acclimation).
  • the object identification index is the ratio of the time allotted to the search for new objects to the total search time, and if the animal stores the object once searched, the value of the object identification index becomes large, If not stored, the value becomes smaller.
  • ⁇ Passive avoidance test> A step-through type using the property that the mouse prefers the dark room is adopted as an index of memory, which is the avoidance behavior shown to the aversive stimulation (electrical stimulation) once experienced by the animal.
  • the movement time to the dark room side when the mouse was put in the light room side of the device where the light room and dark room were connected by a door was used as an index of memory.
  • the passive avoidance test was performed 9 to 12 days after amyloid ⁇ fragment was administered into the ventricle. On the ninth day, the light room (1,000 lux, length 30 cm, width 30 cm, height 15 cm) was placed in a dark room (length 30 cm, width 30 cm, height 15 cm) for 2 minutes to acclimatize.
  • the acclimatization was similarly performed on the 10th day.
  • the mouse was first placed in the center of the light room, and at the same time the mouse moved into the dark room, the door between the light room and the dark room was closed, and electrical stimulation was applied (0.8 mA, 1 Seconds). After 24 hours (day 12), the mouse was placed in the center of the light room again as in the previous day, and the moving time (seconds) to the dark room was measured as a passive avoidance response. If the aversive stimulus by energization is memorized, the passive avoidance reaction becomes longer.
  • NK-4 is 500 ⁇ g / kg ⁇ body weight
  • NK-234 is 500 ⁇ g / kg ⁇ body weight
  • NK-26 is 50 ⁇ g / kg ⁇ body weight
  • NK-19 is 500 ⁇ g / kg ⁇ body weight
  • NK ⁇ In mice administered with 150 at 500 ⁇ g / kg ⁇ body weight, a significant improvement was observed in the novel object recognition test compared to mice administered with amyloid ⁇ fragment alone.
  • NK-4 is 50 ⁇ g / kg ⁇ body weight
  • NK-4 is 500 ⁇ g / kg ⁇ body weight
  • NK-234 is 500 ⁇ g / kg ⁇ body weight
  • NK-26 is 50 ⁇ g / kg ⁇ body weight
  • NK-26 is 500 ⁇ g / kg ⁇ body weight.
  • Significant improvement was observed in the passive avoidance test in mice administered with body weight and NK-19 at 500 ⁇ g / kg / body weight, compared with mice administered with amyloid ⁇ fragment alone.
  • NK-4, NK-234, NK-26, NK-19, and NK-150 all have an action to improve cognitive impairment caused by amyloid ⁇ peptide.
  • NK-4 has the strongest cognitive impairment improving effect.
  • ⁇ Experiment 10 Effect of NK-4 on APP transgenic mice> The effect of NK-4, which had the strongest effect on improving cognitive impairment in mice administered amyloid ⁇ fragment in Experiment 9, on commercially available APP transgenic mice (APP Tg mice) into which a causative gene mutation of Swedish Alzheimer's disease was introduced. Examined. Specifically, 45 APP Tg mice (taconic, female, 10 weeks old, body weight 15 to 23 g) were preliminarily reared for 10 days, then divided into 4 groups so that the body weights were equalized, and were reared alone, and NK- 4 was administered intraperitoneally 5 times a week for 12 weeks.
  • mice wild type, female, 10 weeks old, body weight 15 to 23 g
  • physiological saline was intraperitoneally injected 5 times a week.
  • physiological saline 200 ⁇ l / mouse
  • APP Tg mice were administered donepezil hydrochloride 5 times a week for 12 weeks.
  • ⁇ Water maze test method> A circular pool with a diameter of 130 cm was filled with water colored with white ink to a depth of 20 cm, and the water temperature was maintained at 23 ⁇ 1 ° C. with a water tank heater.
  • the pool was divided into four parts, and an evacuation platform was installed at a position 10 cm from the side of the pool so as to be 2 cm below the surface of the water. The platform position was fixed until the end of the test. From the day after the end of the passive avoidance test, the mouse was released on the surface of the pool toward the side of the pool, and the time taken to reach the platform hidden under the surface of the water was measured.
  • the starting position was 10 cm away from the central part of one of the fractions of the pool divided into 4 centimeters, and was randomly changed for each trial.
  • the mouse After searching the platform freely for 2 minutes, if the mouse could not reach the platform within 2 minutes, it was guided to the platform, allowed to stay on the platform for 30 seconds and then transferred to a cage with paper towels.
  • the second test started 1 minute after the end of the first test. This test was conducted for 4 consecutive days, and the average value of the two trials was taken as the value of the day.
  • mice administered NK-4 markedly improved the cognitive impairment of APP Tg mice in any of the object identification index, passive avoidance reaction, and water maze test. Moreover, the improvement effect was significantly stronger than that of commercially available Alzheimer-type dementia therapeutic agent donepezil hydrochloride. This result strongly suggests that NK-4 can be used as a therapeutic agent for Alzheimer-type cognitive impairment. During the study, no side effects believed to be caused by administration of NK-4 were observed.
  • the general formula 3 such as NK-19, NK-53, NK-150, etc., wherein the side chain alkyl group has 3 to 10 carbon atoms
  • the general formula The compound having 2 to 8 carbon atoms of the alkyl group in the side chain has a physiological function such as a neurotrophic factor-like action and a neurodegeneration inhibitory action, and also has an activity of suppressing cell damage caused by amyloid ⁇ fragment. Therefore, all of these compounds are useful as anti-Alzheimer's disease therapeutic agents.
  • NK-4, NK-234, NK-26, particularly NK-4 which are compounds represented by the general formula 2 and in which the side chain alkyl group has 2 to 4 carbon atoms, are particularly preferred. It shows that it has an effect of improving strong Alzheimer type cognitive impairment.
  • the anti-Alzheimer's disease agent of the present invention will be described with reference to examples, but the present invention is not limited to these examples.
  • ⁇ Liquid preparation for injection> A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, NK-4 (compound represented by Chemical Formula 2), NK-9694 as an active ingredient in 170 g of purified water for injection (Compound represented by chemical formula 1), NK-28 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 7), NK-147 (expressed by general formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 8 carbon atoms), NK-19 (a compound represented by Chemical Formula 4), NK-53 (a compound represented by Chemical Formula 5), NK- 150 (compound represented by chemical formula 3), NK-393 (compound in which the side chain alkyl group (R) of the compound represented by general formula 3 has 8 carbon atoms), NK-100 (compound represented by chemical
  • mice treated with amyloid ⁇ fragment > 65 ICR mice (available from Charles River Japan Co., Ltd.) were randomly divided into 13 groups, each of 5 mice.
  • the amyloid ⁇ fragment having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing used in Experiment 2 was aged at 37 ° C. for 4 days, and 5 ⁇ g / mouse was administered into the ventricles of all 65 mice.
  • Table 12 from the first day after administration of the amyloid ⁇ fragment, each of the 12 groups, 5 animals (test groups 1 to 12) contains any of the 12 compounds prepared in Example 1 as active ingredients.
  • any one of the formulations was intraperitoneally administered once a day until the ninth day, daily at 0.3 ml / animal (test groups 1 to 12).
  • the remaining 5 animals in 1 group were intraperitoneally administered with a 10% aqueous solution of sterilized maltose (pyrogen-free) once a day until the 9th day (control group).
  • the behavior of mice was observed from day 6 to day 10 after administration of amyloid ⁇ fragment, and the ratio (%) of the number of individuals in which some behavioral abnormality was observed during that period is also shown in Table 12.
  • mice were dissected and brains were collected, brain tissue samples were prepared by conventional methods, and congo red staining and thioflavin T were used to confirm the presence or absence of amyloid ⁇ fragment deposition (aggregation). Staining was performed. The stained specimen is observed with a fluorescence microscope, and the fluorescence intensity of the tissue is clearly weaker (2), the fluorescence intensity is slightly weaker (1), and there is no difference (0), compared with the case where the control is administered. Is scored by rating in 5 stages, with a slightly strong (-1) and a clearly strong fluorescence intensity (-2), and an average of 5 mice in each test group was obtained and expressed as the degree of amyloid ⁇ fragment accumulation. 12 is also shown.
  • pentamethine cyanine dye compounds (NK-4, NK-9694, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K -100, K-528, K-557, and NK-1516) (Experimental groups 1 to 9) improved behavioral abnormalities caused by amyloid ⁇ fragment administration and accumulation of fragments in the brain The effect was recognized.
  • ⁇ Liquid preparation for injection> A solution in which 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) is dissolved in 370 g of purified water for injection, and 168 g of purified lecithin for injection, 2 g of lecithin and an active ingredient, NK-4 (compound represented by chemical formula 2), NK-234 (compound in which the alkyl group (R) in the side chain of the compound represented by general formula 2 has 3 carbon atoms), NK-26 (compound represented by chemical formula 1), NK-28 (general formula 2), NK-147 (the number of carbon atoms in the side chain alkyl group (R) of the compound represented by the general formula 2 is 7).
  • NK-19 compound represented by chemical formula 4
  • NK-53 compound represented by chemical formula 5
  • NK-150 compound represented by chemical formula 3
  • NK-393 General The side chain alkyl group of the compound represented by Formula 3 ( )
  • NK-100 compound represented by chemical formula 6
  • NK-528 compound represented by chemical formula 7
  • NK-557 compound represented by chemical formula 8
  • NK-1516 compound represented by Chemical Formula 9) (both manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were mixed with a solution in which 120 mg each was dissolved, and the dissolved oxygen concentration was about 0. Nitrogen gas was bubbled to 0.05 ppm, and 1 ml was dispensed into brown ampules. After the ampules were sealed under a nitrogen stream, they were sterilized by filtration or autoclaved. All of these products are pyrogen-free and can be used as anti-Alzheimer's agents.
  • Each of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 was administered to 10 ddy mice (average body weight 25.6 g) in a single dose of 0.3 ml / body, and daily body weight for 1 week after administration.
  • a 10% maltose solution containing 0.2% lecithin was administered to 10 ddy mice (average body weight 26.3 g) as a control, and the course was observed.
  • no significant change in body weight was observed, and no other change in appearance was observed.
  • the LD 50 of the compound formulated as an active ingredient of the 13 types of anti-Alzheimer's disease agents prepared in Example 2 is 8.6 mg / kg ⁇ body weight or more, so these preparations were administered to humans. It shows that it is a safe formulation.
  • ⁇ Powder for injection> A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, 170 g of purified water for injection, 3 g of polysorbate 80 (sold by NOF Corporation), Compound represented by chemical formula 2), NK-234 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 3), NK-26 (represented by chemical formula 1) Compound), NK-28 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 7 carbon atoms), NK-147 (a compound in the side chain of the compound represented by Formula 2) A compound in which the alkyl group (R) has 8 carbon atoms), NK-19 (a compound represented by Formula 4), NK-53 (a compound represented by Formula 5), NK-150 (a compound represented by Formula 3) Compound), NK-393
  • All of these products are pyrogen-free. At the time of use, 2 to 10 ml of purified water for injection or physiological saline is added to the ampoule and dissolved, and then used by methods such as intravenous infusion, subcutaneous administration, and intraperitoneal administration. All of these products are pyrogen-free and can be used as anti-Alzheimer's agents.
  • the anti-Alzheimer's disease agent of the present invention is useful for preventing, treating and / or suppressing the progression of Alzheimer's disease or protecting nerve cells from neuronal cytotoxic factors that cause Alzheimer's disease such as amyloid ⁇ peptide.
  • cognitive impairment associated with Alzheimer's disease and various pathological and neurological dysfunctions eg tremor, rigidity, agitation, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory Disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis).
  • the anti-Alzheimer's disease agent of the present invention has no side effects even when administered for a long period of time, so it is highly safe and can be used with peace of mind.
  • the present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

Abstract

Disclosed is a novel anti-Alzheimer’s disease agent.  The anti-Alzheimer’s disease agent comprises a compound represented by general formula (1) as an active ingredient. In general formula (1), R1 to R3 independently represent a hydrogen atom or a proper substituent; Z1 represents a heterocyclic ring, and Z2 represents a heterocyclic ring which is the same as or different from the heterocyclic ring represented by Z1, or an aromatic ring, wherein each of the heterocyclic ring and the aromatic ring may have a substituent; o represents an integer of 0, 1 or 2, and p represents an integer of 0 or 1, wherein p is 1 when o is 0 or 2, p is 0 when o is 1, R1 and R2 do not exist when o is 0, and R3 does not exist and a carbon atom to which R2 is bound and Z2 are bound to each other through a single bond when p is 0; X1 - represents a proper counter anion; and q represents an integer of 1 or 2.

Description

抗アルツハイマー病剤Anti-Alzheimer's disease agent
　本発明は、下記一般式１で表される化合物を有効成分とする抗アルツハイマー病剤に関するものである。 The present invention relates to an anti-Alzheimer's disease agent comprising a compound represented by the following general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　アルツハイマー病は、アミロイドβペプチドなどにより引き起こされる系統的な神経細胞の変性、脱落に基づく神経回路網の破綻により引き起こされる病態であり、近年、急増する認知症の主因の一つとされている。 Alzheimer's disease is a pathological condition caused by systematic neuronal degeneration or loss caused by amyloid β peptide or the like, and the breakdown of the neural network based on omission, and is one of the major causes of dementia that has increased rapidly in recent years.
　アルツハイマー病をはじめとする神経変性疾患の原因である神経変性死のメカニズムには多くの分子群が複雑に関与し、それらの発現、機能異常が生じているものと予想される。しかしながら、その分子病態については解明されたものはほとんどなく、有効な神経変性の抑制方法も確立されていない。病因を取り除く治療に加えて重要なのが、神経回路網を再構築させることである。アルツハイマー病では、神経突起（軸索及び樹状突起）の萎縮とシナプスの減少が、神経機能が損なわれる引き金であり、逆に、その引き金が引かれた後でも、変性しきっていない神経細胞や変性を免れて生き残っている神経細胞を活性化して、神経突起を伸展させ、シナプスを回復できれば、神経機能を回復できるといわれている。しかしながら、損傷をうけた末梢神経系の軸索は再生するものの、中枢神経系では軸索は末梢神経を移植するなどの処置をしないと、再生は起こらないといわれている。 It is expected that many molecular groups are involved in the mechanism of neurodegenerative death, which is the cause of neurodegenerative diseases such as Alzheimer's disease, and that their expression and dysfunction are occurring. However, little has been elucidated about the molecular pathology, and no effective method for suppressing neurodegeneration has been established. In addition to the treatment to remove the etiology, it is important to rebuild the neural network. In Alzheimer's disease, atrophy of neurites (axons and dendrites) and a decrease in synapses are triggers that impair nerve function, and conversely, even after the trigger is triggered, It is said that if the nerve cells that survive the degeneration are activated, the neurites are extended, and the synapse is restored, the nerve function can be restored. However, although damaged axons in the peripheral nervous system regenerate, in the central nervous system, it is said that regeneration does not occur unless treatment such as transplantation of peripheral nerves is performed.
　神経成長因子（ｎｅｒｖｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ、以下、「ＮＧＦ」と略記する場合がある。）などの神経栄養因子群に属する蛋白は、神経細胞の分化や生存、シナプスの制御等に関与することが知られているものの、高分子ゆえに血液脳関門を通過し難いなどの点からも、皮下や血管内などの全身投与では、中枢神経の変性に起因する神経変性疾患に対する治療効果があまり期待できない。効果を期待して脳内へ投与するためには、外科的処置が必要となり、患者には、大きな肉体的・精神的負担が伴う。 Proteins belonging to the group of neurotrophic factors such as nerve growth factor (hereinafter sometimes abbreviated as “NGF”) are known to be involved in the differentiation and survival of neurons and the control of synapses. However, systemic administration such as subcutaneously or intravascularly cannot be expected to have a therapeutic effect on neurodegenerative diseases caused by central nerve degeneration because it is difficult to cross the blood-brain barrier due to the polymer. Surgical treatment is necessary to administer it into the brain with the expectation of an effect, and the patient is accompanied by a large physical and mental burden.
　アルツハイマー病の臨床症状とは、軽微なものから重篤なものまで様々であり、代表的なものとしては、認知症があり、症状が進行すれば、他の神経変性症と同様に、例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ又は麻痺等が現れることもあり、これらはいずれも日常生活を営む上で、大きな障害となっている。 The clinical symptoms of Alzheimer's disease vary from mild to severe, and typical examples include dementia.If the symptoms progress, as with other neurodegenerative diseases, for example, Tremor, rigidity, ataxia, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, breathing Obstacles, numbness, paralysis, etc. may appear, and these are major obstacles in conducting daily life.
　アルツハイマー病は、神経細胞に変性を来たす重大な疾患であり、これらの疾患やそれに伴う病態や神経機能障害を改善するために、種々の化合物を有効成分とする治療剤が提案されており（例えば、国際公開ＷＯ９７／０３０７０３号パンフレット、特開平１１－２２８４１７号公報、特開２００６－１４３７０８号公報、特開２００６－３２１７３７号公報を参照）、神経突起伸展促進剤なども提案されている（例えば特開２００２－２３４８４１号公報を参照）ものの、効果的な疾患の治療法はまだ見出されていない。 Alzheimer's disease is a serious disease that causes degeneration of nerve cells, and therapeutic agents containing various compounds as active ingredients have been proposed in order to improve these diseases and the pathological conditions and neurological dysfunctions associated therewith (for example, International Publication WO 97/030703 pamphlet, Japanese Patent Application Laid-Open No. 11-228417, Japanese Patent Application Laid-Open No. 2006-143708, Japanese Patent Application Laid-Open No. 2006-321737, neurite extension promoters, etc. However, an effective method for treating a disease has not yet been found.
　医療現場では、患者にとって肉体的・精神的負担の少ない、皮下や血管内などの全身投与により、中枢神経系の神経細胞に作用し、神経細胞を活性化し、神経突起の萎縮を抑制、乃至、神経突起の伸展を促進させて神経変性を抑制し、それに伴う病態や臨床症状を治療することができる新規抗アルツハイマー病剤の開発が切望されている。 In the medical field, systemic administration such as subcutaneous or intravascular, which has less physical and mental burden for patients, acts on nerve cells of the central nervous system, activates nerve cells, suppresses neurite atrophy, or The development of a novel anti-Alzheimer's disease agent that promotes neurite outgrowth to suppress neurodegeneration and treats the pathological conditions and clinical symptoms associated therewith is eagerly desired.
　本発明は、新規な抗アルツハイマー病剤を提供することを課題とする。 An object of the present invention is to provide a novel anti-Alzheimer's disease agent.
　本発明者等は、上記課題を解決するために、鋭意研究して検索した結果、下記一般式１で表される化合物が優れた神経細胞活性化作用を有し、神経突起伸展促進作用を有することを見出した。さらに、これらの化合物が、アミロイドβペプチドによる神経細胞死の抑制作用を有し、全身投与でも中枢神経系の神経細胞を活性化して、神経変性を抑制すると共に、アミロイドβペプチドによる細胞傷害に起因する症状や病態の発症を遅延乃至改善することを見出して、本発明を完成した。すなわち、本発明は、一般式１で表される分子内にカチオン部とアニオン部とをもつ化合物を有効成分とする抗アルツハイマー病剤を主な構成とする。 In order to solve the above-mentioned problems, the present inventors have conducted extensive research and search. As a result, the compound represented by the following general formula 1 has an excellent nerve cell activation action and a neurite extension promoting action. I found out. Furthermore, these compounds have an inhibitory effect on neuronal cell death caused by amyloid β peptide, which activates central nervous system neuron even by systemic administration, suppresses neurodegeneration and is caused by cytotoxicity caused by amyloid β peptide. The present invention was completed by finding that the onset of symptoms and pathological conditions to be delayed or improved. That is, the present invention mainly comprises an anti-Alzheimer's disease agent comprising a compound having a cation moiety and an anion moiety in the molecule represented by the general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　本発明の抗アルツハイマー病剤は、非経口的に投与することにより、アミロイドβペプチドをはじめとする細胞傷害因子から細胞を保護し、神経細胞の活性化や神経突起の伸展を促進することで、これらの傷害因子により引き起こされる神経変性を抑制して、アルツハイマー病やそれに伴う認知症をはじめとする種々の病態や症状（例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上・下肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ及び麻痺等を含む）を改善することができる。しかも、有効成分である一般式１で表される化合物の安全性は極めて高い。 The anti-Alzheimer's disease agent of the present invention protects cells from cytotoxic factors including amyloid β peptide by administering parenterally, and promotes activation of nerve cells and extension of neurites, Suppresses neurodegeneration caused by these injury factors, and various pathologies and symptoms including Alzheimer's disease and associated dementia (for example, tremor, rigidity, immobility, peristalsis, slow motion, posture reflex disorder , Autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis) . And the safety | security of the compound represented by General formula 1 which is an active ingredient is very high.
　本発明でいう神経突起とは、神経の細胞体から伸びる軸索及び樹状突起をいう。また、神経突起伸展促進作用とは、神経細胞を活性化して軸索及び／又は樹状突起を伸展させる作用をいい、神経突起の萎縮や減少の抑制作用、神経細胞間のシナプス形成の促進作用やシナプスの減少を抑制する作用を含む。 In the present invention, the neurite means an axon and dendrite extending from a neuronal cell body. The neurite outgrowth promoting action refers to the action of activating nerve cells to extend axons and / or dendrites, the action of suppressing neurite atrophy and decrease, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
　本発明でいう神経変性とは、神経細胞、とりわけ、中枢神経系の神経細胞の機能低下、死滅、や減少（脱落）をいい、神経突起の萎縮や減少、シナプスの減少、グリア細胞の機能低下、死滅や減少を含む。 Neurodegeneration as used in the present invention refers to a decrease in function, death, or decrease (dropout) of nerve cells, particularly neurons in the central nervous system. Atrophy and decrease of neurites, decrease of synapses, decrease of function of glial cells Including death and reduction.
　本発明の抗アルツハイマー病剤は、上記一般式１で表される化合物を有効成分とする。 The anti-Alzheimer's disease agent of the present invention contains a compound represented by the above general formula 1 as an active ingredient.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000010
　一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。 R 1 to R 3 in General Formula 1 each independently represent a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. These heterocyclic rings and aromatic rings may have a substituent. o represents an integer that is either 0, 1, or 2, p represents an integer that is either 0 or 1, p is 1 when o is 0 or 2, and o is 1 , P is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 does not exist, and the carbon to which R 2 is bonded and Z 2 form a single bond. X l - represents a suitable counter anion, q is an integer of either 1 or 2.
　一般式１におけるＸｌ －は適宜の対アニオンを表し、通常、例えば、弗素イオン、塩素イオン、臭素イオン、沃素イオン、過塩素酸イオン、過沃素酸イオン、六弗化燐酸イオン、六弗化アンチモン酸イオン、六弗化錫酸イオン、燐酸イオン、硼弗化水素イオン、四弗硼素酸イオンなどの無機酸アニオンや、チオシアン酸イオン、ベンゼンスルホン酸イオン、ナフタレンスルホン酸イオン、ナフタレンジスルホン酸イオン、ｐ－トルエンスルホン酸イオン、アルキルスルホン酸イオン、ベンゼンカルボン酸イオン、アルキルカルボン酸イオン、トリハロアルキルカルボン酸イオン、アルキル硫酸イオン、トリハロアルキル硫酸イオン、ニコチン酸イオン、アスパラギン酸イオンなどの有機酸アニオンから選択される。 X l in the general formula 1 - represents an appropriate counter anion, usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodic acid ion, hexafluorophosphate ion, Rokudoruka Inorganic acid anions such as antimonate ions, hexafluorostannate ions, phosphate ions, borofluoride ions, tetrafluoroborate ions, thiocyanate ions, benzenesulfonate ions, naphthalenesulfonate ions, naphthalene disulfonate ions Organic acid anions such as p-toluenesulfonate ion, alkylsulfonate ion, benzenecarboxylate ion, alkylcarboxylate ion, trihaloalkylcarboxylate ion, alkylsulfate ion, trihaloalkylsulfate ion, nicotinate ion, aspartate ion Selected from.
　一般式１で表される化合物としては、具体的には、一般式２乃至４のいずれかで表されるペンタメチン系シアニン色素及び一般式５で表されるジメチン系スチリル色素などの色素化合物（以下、単に「化合物」という場合がある。）を例示することができる。 Specific examples of the compound represented by the general formula 1 include dye compounds such as a pentamethine cyanine dye represented by any one of the general formulas 2 to 4 and a dimethine styryl dye represented by the general formula 5 (hereinafter referred to as “a dye compound”). And may be simply referred to as “compound”).
Figure JPOXMLDOC01-appb-C000011
 一般式２において、Ｒ４乃至Ｒ６は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ２ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000011
 In General Formula 2, R 4 to R 6 represent the same or different aliphatic hydrocarbon groups. X 2 − represents an appropriate counter anion, and m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
Figure JPOXMLDOC01-appb-C000012
 　一般式３において、Ｒ７乃至Ｒ９は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ３ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000012
 In the general formula 3, R 7 to R 9 represent the same or different aliphatic hydrocarbon groups. X 3 - represents an appropriate counter anion, m represents an integer of either charge become 1 or 2 to balance the charge of the cation.
Figure JPOXMLDOC01-appb-C000013
 　一般式４において、Ｒ１０乃至Ｒ１２は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ４ －は適宜の対アニオンを表し、ｍはカチオン部の電荷とバランスする電荷となる１又は２のいずれかである整数を表す。
Figure JPOXMLDOC01-appb-C000013
 In general formula 4, R 10 to R 12 represent the same or different aliphatic hydrocarbon groups. X 4 − represents an appropriate counter anion, and m represents an integer that is either 1 or 2 that has a charge balanced with the charge of the cation moiety.
Figure JPOXMLDOC01-appb-C000014
 　一般式５において、Ｚ３は複素芳香環を表し、その複素芳香環は置換基を有していてもよい。Ｚ４は芳香環又は複素芳香環を表し、それらの複素芳香環及び芳香環は置換基を有していてもよい。Ｒ１３は脂肪族炭化水素基を表し、その脂肪族炭化水素基は置換基を有していてもよい。Ｒ１４は水素原子又は適宜の置換基を、また、Ｘ５ －は適宜の対アニオンを表す。
Figure JPOXMLDOC01-appb-C000014
 In General Formula 5, Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent. Z 4 represents an aromatic ring or a heteroaromatic ring, and the heteroaromatic ring and the aromatic ring may have a substituent. R 13 represents an aliphatic hydrocarbon group, and the aliphatic hydrocarbon group may have a substituent. The R 14 is hydrogen atom or an appropriate substituent and, X 5 - represents a suitable counter anion.
　一般式２乃至５におけるＲ４乃至Ｒ１３で表される脂肪族炭化水素基としては、通常、炭素数が１乃至１２であるものが選択され、２乃至１０のものが好ましく、２乃至９のものがより好ましい。なかでも、一般式２で表される化合物のＲ４乃至Ｒ６の脂肪族炭化水素の基の炭素数が２乃至１２、又は、一般式３で表される化合物のＲ７乃至Ｒ９の脂肪族炭化水素の基の炭素数が４乃至１０の化合物は、神経変性抑制作用が強いので、特に望ましい。個々の脂肪族炭化水素基としては、例えば、メチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ペンチル基、イソペンチル基、ｔｅｒｔ－ペンチル基、１－メチルペンチル基、２－メチルペンチル基、ヘキシル基、イソヘキシル基、５－メチルヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基などが挙げられる。また、一般式２乃至５においてＸ２ －乃至Ｘ５ －で表される適宜の対アニオンは、通常、例えば、弗素イオン、塩素イオン、臭素イオン、沃素イオン、過塩素酸イオン、過沃素酸イオン、六弗化燐酸イオン、六弗化アンチモン酸イオン、六弗化錫酸イオン、燐酸イオン、硼弗化水素イオン、四弗硼素酸イオンなどの無機酸アニオンや、チアシアン酸イオン、ベンゼンスルホン酸イオン、ナフタレンスルホン酸イオン、ナフタレンジスルホン酸イオン、ｐ－トルエンスルホン酸イオン、アルキルスルホン酸イオン、ベンゼンカルボン酸イオン、アルキルカルボン酸イオン、トリハロアルキルカルボン酸イオン、アルキル硫酸イオン、トリハロアルキル硫酸イオン、ニコチン酸イオン、アスパラギン酸イオンなどの有機酸アニオンから選択される。 As the aliphatic hydrocarbon group represented by R 4 to R 13 in the general formulas 2 to 5, those having 1 to 12 carbon atoms are usually selected, those having 2 to 10 are preferable, and those having 2 to 9 are preferable. Those are more preferred. Among them, the R4 to R6 aliphatic hydrocarbon group of the compound represented by the general formula 2 has 2 to 12 carbon atoms, or the R7 to R9 aliphatic hydrocarbon group of the compound represented by the general formula 3 A compound having 4 to 10 carbon atoms is particularly desirable because it has a strong neurodegeneration inhibitory effect. Examples of the individual aliphatic hydrocarbon group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, and a tert-pentyl group. 1-methylpentyl group, 2-methylpentyl group, hexyl group, isohexyl group, 5-methylhexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group and the like. In addition, suitable counter anions represented by X 2 − to X 5 − in the general formulas 2 to 5 are usually, for example, fluorine ion, chlorine ion, bromine ion, iodine ion, perchlorate ion, periodate ion , Inorganic acid anions such as hexafluorophosphate ion, hexafluoroantimonate ion, hexafluorostannate ion, phosphate ion, borofluoride ion, tetrafluoroborate ion, thiocyanate ion, benzenesulfonate ion , Naphthalene sulfonate ion, naphthalene disulfonate ion, p-toluene sulfonate ion, alkyl sulfonate ion, benzene carboxylate ion, alkyl carboxylate ion, trihaloalkyl carboxylate ion, alkyl sulfate ion, trihaloalkyl sulfate ion, nicotinic acid Selected from organic acid anions such as ions and aspartate ions The
　一般式２で表される化合物としては、より具体的には、化学式１で表される化合物（以下、「ＮＫ－９６９４」という場合がある。）、化学式２で表される化合物（以下、「ＮＫ－４」という場合がある。）や一般式２の側鎖のアルキル基（Ｒ４乃至Ｒ６）の炭素数が３の化合物（以下、「ＮＫ－２３４」という場合がある。）を例示することができる。なお、本明細書中のＮＫ番号に対応する化合物の構造は、例えば、『感光色素表』、感光色素研究所発行（１９６９年）にも記載されている。 More specifically, the compound represented by the general formula 2 includes a compound represented by the chemical formula 1 (hereinafter sometimes referred to as “NK-9694”) and a compound represented by the chemical formula 2 (hereinafter referred to as “ NK-4 ”) and compounds having 3 carbon atoms in the side chain alkyl groups (R4 to R6) of formula 2 (hereinafter also referred to as“ NK-234 ”). Can do. The structure of the compound corresponding to the NK number in this specification is also described in, for example, “Photosensitive Dye Table”, published by Photosensitive Dye Research Institute (1969).
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　一般式３で表される化合物としては、具体的には、例えば、化学式３で表される化合物（以下、「ＮＫ－１５０」という場合がある。）や化学式４で表される化合物（以下、「ＮＫ－１９」という場合がある。）を例示することができる。さらには、ＮＫ－１９の対アニオン（Ｉ－）をＣｌ－に替えた化学式５で表される化合物（以下、「ＮＫ－５３」という場合がある。）も、ＮＫ－１９と同様に有利に利用できる。 Specific examples of the compound represented by the general formula 3 include, for example, a compound represented by the chemical formula 3 (hereinafter sometimes referred to as “NK-150”) and a compound represented by the chemical formula 4 (hereinafter, referred to as “NK-150”). "NK-19" may be mentioned). Furthermore, the counter anion of the NK-19 (I -) of Cl - in the compound represented by Chemical Formula 5 for changing (. Which hereinafter may be referred to as "NK-53"), similar to the NK-19 advantageously Available.
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
　一般式４で表される化合物としては、具体的には、例えば、化学式６で表される化合物（以下、「ＮＫ－１００」という場合がある。）を例示することができる。 Specific examples of the compound represented by the general formula 4 include a compound represented by the chemical formula 6 (hereinafter sometimes referred to as “NK-100”).
Figure JPOXMLDOC01-appb-C000020
Figure JPOXMLDOC01-appb-C000020
　一般式５で表される化合物としては、化学式７乃至９で表される化合物（以下、それぞれ、「ＮＫ－５２８」、「ＮＫ－５５７」、「ＮＫ－１５１６」という場合がある。）を例示することができる。
Figure JPOXMLDOC01-appb-C000021
 Examples of the compound represented by the general formula 5 include compounds represented by the chemical formulas 7 to 9 (hereinafter sometimes referred to as “NK-528”, “NK-557”, and “NK-1516”, respectively). can do.
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
　化学式１乃至９のいずれかで表される化合物やＮＫ－２３４は、いずれも、神経細胞活性化作用、神経突起伸展促進作用に加えて、飢餓、ラジカル、アミロイドβペプチドなどの細胞傷害因子から神経細胞を保護して細胞死や神経突起の萎縮を抑制する作用も有しているので、本発明の抗神経変性疾患剤の有効成分としてより望ましく、その作用効果の強さの点からは、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２の側鎖のアルキル基の炭素数が３の化合物）、ＮＫ－１５０（化学式３で表される化合物）が特に望ましい。アセチルコリンエステラーゼ（ＡｃｈＥ）活性抑制作用の強さ、脳内への移行性、製剤化の容易性などを加味すると、ＮＫ－４やＮＫ－２３４が望ましく、ＮＫ－４が特に望ましい。 Any of the compounds represented by any one of Chemical Formulas 1 to 9 and NK-234 can be used in addition to neuronal activation and neurite outgrowth promoting effects from cytotoxic factors such as starvation, radicals, and amyloid β peptide. Since it also has an action of protecting cells and suppressing cell death and neurite atrophy, it is more desirable as an active ingredient of the anti-neurodegenerative disease agent of the present invention. -26 (compound represented by chemical formula 1), NK-4 (compound represented by chemical formula 2), NK-234 (compound having 3 carbon atoms in the side chain alkyl group of general formula 2), NK-150 (Compound represented by Chemical Formula 3) is particularly desirable. NK-4 and NK-234 are desirable, and NK-4 is particularly desirable, taking into account the strength of acetylcholinesterase (AchE) activity inhibitory activity, migration into the brain, ease of formulation, and the like.
　本発明の抗アルツハイマー病剤の有効成分として使用する一般式１で表される化合物は、その由来や製法に制限はない。 The compound represented by the general formula 1 used as an active ingredient of the anti-Alzheimer's disease agent of the present invention is not limited in its origin or production method.
　本発明の抗アルツハイマー病剤は、一般式１で表される化合物、望ましくは、一般式２乃至４のいずれかで表されるペンタメチン系シアニン色素及び／又は一般式５で表されるジメチン系スチリル色素を、１種又は２種以上含有してなる。 The anti-Alzheimer's disease agent of the present invention is a compound represented by general formula 1, preferably a pentamethine cyanine dye represented by any one of general formulas 2 to 4 and / or a dimethine styryl represented by general formula 5. It contains one or more dyes.
　本発明の抗アルツハイマー病剤は、必要に応じて、有効成分である一般式１で表される化合物に加えて、製剤学的に許容される食品分野、化粧品分野、医薬品分野、医薬部外品分野で使用される成分の１種又は２種以上を配合した製剤の形態で提供される。 The anti-Alzheimer's disease agent of the present invention contains, as needed, a pharmaceutically acceptable food field, cosmetic field, pharmaceutical field, quasi-drug in addition to the compound represented by the general formula 1 which is an active ingredient. It is provided in the form of a preparation containing one or more components used in the field.
　製剤学的に許容される成分としては、例えば、添加剤、賦形剤、崩壊剤、滑沢剤、安定化剤、界面活性剤、防腐剤（抗菌剤）、香料、増粘剤、抗酸化剤、キレート剤、ビタミン類、アミノ酸類、水性媒体、糖質、水溶性高分子、ｐＨ調整剤、発泡剤、医薬品・医薬部外品・化粧品・食品用の添加剤、医薬用・医薬部外品用の有効成分など例示することができ、これらの成分の１種又は２種以上を適宜組み合わせて配合し、目的とする剤型に応じて、常法により製造すればよい。 Examples of pharmaceutically acceptable ingredients include additives, excipients, disintegrants, lubricants, stabilizers, surfactants, preservatives (antibacterial agents), fragrances, thickeners, and antioxidants. Agents, chelating agents, vitamins, amino acids, aqueous media, sugars, water-soluble polymers, pH adjusters, foaming agents, pharmaceuticals, quasi drugs, cosmetics, food additives, pharmaceuticals, quasi drugs The active ingredient for goods etc. can be illustrated, What is necessary is just to mix | blend combining suitably 1 type (s) or 2 or more types of these components, and to manufacture by a conventional method according to the target dosage form.
　また、本発明の抗アルツハイマー病剤は、一般式１で表される化合物以外の、アルツハイマー病、その病態や臨床症状に治療効果があるとされる神経突起伸展促進剤や、神経変性疾患やそれに起因する病態や神経機能障害の治療剤との併用も有利に実施できる。脳血管障害の治療薬、神経変性疾患の治療薬、筋萎縮性側索硬化症の治療薬、多発性硬化症の治療薬、ドーパミン受容体作動薬、ドーパミン遊離促進薬、ドーパミン取り込み阻害薬、ドーパミン作用薬、中枢性抗コリン薬、芳香族Ｌ－アミノ酸脱炭酸酵素阻害薬（ＤＣＩ）、モノアミン酸化酵素（ＭＡＯ－Ｂ）阻害薬、カテコール－Ｏ－メチルトランスフェラーゼ（ＣＯＭＴ）阻害薬、ノルエピネフリン（ノルアドレナリン）補充薬、アセチルコリンエステラーゼ阻害薬、神経栄養因子及びその他の脳機能賦活薬が挙げられる。中でも、ＮＧＦは、本発明で使用する一般式１で表される化合物の、神経突起伸展促進作用が効果的に増強されるので望ましい。 In addition, the anti-Alzheimer's disease agent of the present invention, other than the compound represented by the general formula 1, is a neurite outgrowth promoting agent that is said to have a therapeutic effect on Alzheimer's disease, its pathology and clinical symptoms, The combined use with the therapeutic agent of the pathological condition and nerve dysfunction which originates can also be implemented advantageously. Cerebrovascular disorder therapeutic agent, neurodegenerative disease therapeutic agent, amyotrophic lateral sclerosis therapeutic agent, multiple sclerosis therapeutic agent, dopamine receptor agonist, dopamine release promoter, dopamine uptake inhibitor, dopamine Agonist, central anticholinergic, aromatic L-amino acid decarboxylase inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) These include supplements, acetylcholinesterase inhibitors, neurotrophic factors and other brain function stimulants. Among them, NGF is desirable because the neurite extension promoting action of the compound represented by the general formula 1 used in the present invention is effectively enhanced.
　本発明の抗アルツハイマー病剤は、通常、非経口用の注射用の製剤などの形態で提供される。また、非有効成分である一般式１で表される化合物は、対象とする注射用の製剤などの非経口用製剤の組成やその使用目的を勘案して、原料の段階から製品が完成するまでの工程で配合すればよい。その方法としては、例えば、混和、混捏、溶解、融解、分散、懸濁、乳化、逆ミセル化、浸透、晶出、散布、塗布、付着、噴霧、被覆（コーティング）、注入、浸漬、固化、担持などの公知の１種又は２種以上の方法が適宜に選ばれる。 The anti-Alzheimer's disease agent of the present invention is usually provided in the form of a parenteral injection preparation or the like. In addition, the compound represented by the general formula 1, which is an inactive ingredient, takes into account the composition of the parenteral preparation such as the injectable preparation and the purpose of use thereof, from the raw material stage until the product is completed. What is necessary is just to mix | blend in the process of. The methods include, for example, mixing, kneading, dissolving, melting, dispersing, suspending, emulsifying, reverse micellization, infiltration, crystallization, spraying, application, adhesion, spraying, coating (coating), pouring, dipping, solidifying, One or more known methods such as loading are appropriately selected.
　注射用製剤などの非経口用の製剤の場合、対象となる疾患や症状などに応じて、通常、パイロジェンを含まない水性媒体に溶解して、皮内、皮下、筋肉内、体腔内（胸腔内、腹腔内など）、血管内又は脳内（脊髄内を含む）へ投与されるので、製剤の形態としては、乾燥製剤であってもよく、液剤であってもよい。乾燥製剤の場合は、使用時に、注射用の精製水、生理食塩水、ブドウ糖液などの水性媒体に溶解して使用すればよい。液剤の場合は、そのまま投与してもよく、輸液や灌流液などに添加して使用してもよい。また、溶媒への溶解性や水性媒体への溶解性に問題のある場合や、徐放性の製剤を調製する場合には、両親媒性溶媒、油性基材や乳化剤等を使用して、有効成分の溶媒への溶解性を高めることも随意である。 In the case of parenteral preparations such as injection preparations, it is usually dissolved in an aqueous medium that does not contain pyrogen, depending on the target disease or symptom, and then intradermally, subcutaneously, intramuscularly, intracorporeally (intrathoracic) , Intraperitoneal, etc.), intravascular or intracerebral (including spinal cord), the preparation may be a dry preparation or a liquid preparation. In the case of a dry preparation, it may be used by dissolving it in an aqueous medium such as purified water for injection, physiological saline, and glucose solution at the time of use. In the case of a solution, it may be administered as it is, or it may be added to an infusion solution or a perfusion solution. In addition, when there is a problem with solubility in a solvent or solubility in an aqueous medium, or when preparing a sustained-release preparation, an amphiphilic solvent, an oily base material, an emulsifier, etc. are used. Increasing the solubility of the components in the solvent is optional.
　本発明でいう水性媒体とは、水を必須の要素とし、必要に応じて、これに、例えば、エタノール、プロパノール、イソプロパノールなどのアルコール類、アセトンなどのケトン類、ジエチルエーテルなどのエーテル類、ジメチルスルホキシド（以下、「ＤＭＳＯ」と略記する場合がある。）などの含硫化合物をはじめとする親水性有機溶剤の１又は複数を配合してなる水性媒体一般を意味する。この発明による液剤における水性溶剤としては、注射用精製水、生理食塩、リンゲル液等、を単独で用いるか、あるいは、注射用精製水と、例えば、エタノール、プロパノール、イソプロパノール、ジエチルエーテル、ＤＭＳＯなどの生理学的に許容される親水性有機溶剤との混液を用いるのが望ましい。 The aqueous medium referred to in the present invention has water as an essential element, and if necessary, for example, alcohols such as ethanol, propanol and isopropanol, ketones such as acetone, ethers such as diethyl ether, dimethyl It means a general aqueous medium comprising one or more hydrophilic organic solvents including sulfur-containing compounds such as sulfoxide (hereinafter sometimes abbreviated as “DMSO”). As the aqueous solvent in the liquid preparation according to the present invention, purified water for injection, physiological saline, Ringer's solution, etc. are used alone, or purified water for injection and physiological such as ethanol, propanol, isopropanol, diethyl ether, DMSO, etc. It is desirable to use a mixed solution with a hydrophilic organic solvent which is acceptable.
　斯かる液剤の場合、一般式１で表される化合物によっては、溶存酸素などにより不安定になる場合があるので、その場合は、例えば、該化合物溶液の溶存酸素濃度を低減させればよい。このような液状組成物は、通常、該化合物を水性媒体に溶解する工程と、該水性媒体をしてその常温常圧の大気環境下における酸素濃度を下回らせる工程とを経由する方法により調製することができる。これらの化合物を水性媒体に溶解するには、例えば、所定の量の化合物を適量の水性媒体へ添加し、必要に応じて、加熱・攪拌しながら溶解させた後、必要に応じて、化合物の濃度が所定のレベルになるまで水性媒体を追加すればよい。 In the case of such a liquid agent, depending on the compound represented by the general formula 1, it may become unstable due to dissolved oxygen or the like. In this case, for example, the dissolved oxygen concentration of the compound solution may be reduced. Such a liquid composition is usually prepared by a method through a step of dissolving the compound in an aqueous medium and a step of lowering the oxygen concentration in the atmospheric environment at normal temperature and pressure using the aqueous medium. be able to. In order to dissolve these compounds in an aqueous medium, for example, a predetermined amount of a compound is added to an appropriate amount of an aqueous medium, dissolved as necessary with heating and stirring, and then, if necessary, An aqueous medium may be added until the concentration reaches a predetermined level.
　水性媒体の溶存酸素濃度を、常温常圧の大気環境下における濃度より低くするには、例えば、一般式１で表される化合物の溶液を減圧下で調製し、保存するか、該化合物溶液に溶存する酸素を別の気体で置換するか、あるいは、該化合物溶液を脱酸素剤へ接触させる方法が好適である。液状組成物に溶解する酸素を別の気体で置換するには、液状組成物中で、例えば、窒素などの比較的不活性な気体か、あるいは、ネオン、アルゴン、クリプトン、キセノンなどの希ガスをバブリングさせればよい。脱酸素剤を用いて酸素濃度を下げるには、液状組成物へ、例えば、Ｌ－アスコルビン酸、Ｌ－アスコルビン酸ステアリン酸エステル、亜硫酸ナトリウム、亜硫酸水素ナトリウム、アルファチオグリセリン、エデト酸ナトリウム、塩酸システイン、クエン酸、大豆レシチン、チオグリコール酸ナトウム、チオリンゴ酸ナトリウム、ピロ亜硫酸ナトリウム、ブチルヒドロキシアニソールなどを適量添加すればよい。これらの方法は、化合物溶液に適用しても、化合物を溶解する前の水性媒体へ適用してもよい。この場合の水性媒体に溶存する酸素の濃度は、通常、０．４ｐｐｍ以下、望ましくは、０．１ｐｐｍ以下とすればよい。 In order to make the dissolved oxygen concentration of the aqueous medium lower than the concentration in the atmospheric environment at normal temperature and pressure, for example, a solution of the compound represented by the general formula 1 is prepared under reduced pressure and stored, or the compound solution is added to the compound solution. A method of replacing dissolved oxygen with another gas or contacting the compound solution with an oxygen scavenger is preferable. In order to replace oxygen dissolved in the liquid composition with another gas, in the liquid composition, for example, a relatively inert gas such as nitrogen or a rare gas such as neon, argon, krypton, or xenon is used. Just bubbling. In order to reduce the oxygen concentration using an oxygen scavenger, the liquid composition is prepared by, for example, adding L-ascorbic acid, L-ascorbic acid stearate, sodium sulfite, sodium hydrogen sulfite, alphathioglycerin, sodium edetate, cysteine hydrochloride Citric acid, soybean lecithin, sodium thioglycolate, sodium thiomalate, sodium pyrosulfite, butylhydroxyanisole and the like may be added in appropriate amounts. These methods may be applied to the compound solution or to an aqueous medium before the compound is dissolved. In this case, the concentration of oxygen dissolved in the aqueous medium is usually 0.4 ppm or less, preferably 0.1 ppm or less.
　斯くして得られた一般式１で表される化合物の溶液は、酸素を遮断し得る、用途に応じた適宜の容器へ封入した状態で保存すればよい。容器の材質としては、原理上、液状組成物を保持することができ、かつ、酸素を実質的に遮断し得るものであるかぎり、特に制限がないが、褐色ビンや褐色のアンプルのような遮光性の容器が望ましい。用途にもよるけれども、通常、ガラスアンプル、バイアル瓶などの容器へ液状組成物を分注し、封止した後、高圧滅菌などにより滅菌する。 The thus obtained solution of the compound represented by the general formula 1 may be stored in a state where it is sealed in an appropriate container that can block oxygen. The material of the container is not particularly limited as long as it can hold the liquid composition in principle and can substantially block oxygen, but it is light-shielded like a brown bottle or brown ampoule. Sex containers are desirable. Although depending on the application, the liquid composition is usually dispensed into containers such as glass ampoules and vials, sealed, and then sterilized by high-pressure sterilization or the like.
　また、本発明の抗アルツハイマー病剤は、注射剤以外にも、ハップ剤や経肺用の吸飲噴霧剤等の形態で使用することもでき、皮下等の体内に埋め込む徐放製剤の形態で使用することもできる。また、神経変性疾患を発症したペットをはじめとするヒト以外の動物の治療や、神経変性症に伴う病態や神経機能障害の予防剤乃至治療剤として使用することも随意である。 Further, the anti-Alzheimer's disease agent of the present invention can be used in the form of a haptic agent, a transpulmonary sucking and spraying agent, etc. in addition to the injection, and in the form of a sustained-release preparation embedded in the body such as subcutaneously. It can also be used. It is also optional to treat animals other than humans, including pets that have developed neurodegenerative diseases, and to use as preventives or therapeutic agents for pathological conditions and neurological dysfunction associated with neurodegenerative diseases.
　このようにして製造される本発明の抗アルツハイマー病剤は、長期間連用しても、重篤な副作用もなく安全な製剤である。 The anti-Alzheimer's disease agent of the present invention thus produced is a safe preparation without serious side effects even when used for a long time.
　本発明の抗アルツハイマー病剤の、一日当たりの投与量は、所期の作用・効果が得られる量であれば特に制限はなく、通常、皮下乃至腹腔内投与の場合、一般式１で表される化合物を合計で、０．０１ｍｇ／ｋｇ・体重／日以上が望ましく、０．１乃至２０ｍｇ／ｋｇ・体重／日がより望ましく、０．５乃至５ｍｇ／ｋｇ・体重／日が特に望ましい。５０ｍｇ／ｋｇ・体重／日以上投与しても、その投与量に見合うほどの効果の増強は認められない場合がある。 The daily dose of the anti-Alzheimer's disease agent of the present invention is not particularly limited as long as the desired action and effect can be obtained, and is usually represented by the general formula 1 in the case of subcutaneous to intraperitoneal administration. In total, 0.01 mg / kg · body weight / day or more is desirable, 0.1 to 20 mg / kg · body weight / day is more desirable, and 0.5 to 5 mg / kg · body weight / day is particularly desirable. Even when administered at 50 mg / kg · body weight / day or more, there is a case where the enhancement of the effect corresponding to the dose is not observed.
　本発明の抗アルツハイマー病剤は、脳や神経細胞をアミロイドβペプチドなどの細胞傷害因子から保護して変性を抑制すると共に、神経細胞を活性化し、神経突起の伸展促進や萎縮の抑制、神経細胞の生存延長や変性を抑制し、アルツハイマー病に伴う神経機能障害を治療乃至予防することができる。なお、本発明でいうアルツハイマー病の治療とは、アルツハイマー病に伴う病態や機能障害を治癒の方向へ導く、いわゆる治療に加え、悪化を抑制し病態の進行をとどめる進展防止、さらには疾患の発症そのものの予防も含む。 The anti-Alzheimer's disease agent of the present invention protects the brain and nerve cells from cytotoxic factors such as amyloid β peptide, suppresses degeneration, activates nerve cells, promotes neurite extension, suppresses atrophy, and nerve cells Can suppress the prolongation of survival and degeneration, and can treat or prevent neurological dysfunction associated with Alzheimer's disease. The treatment of Alzheimer's disease referred to in the present invention refers to the pathological condition and functional disorder associated with Alzheimer's disease in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses deterioration and stops progression of disease, and further the onset of disease Including the prevention of itself.
　以下、実験により本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail by experiments.
＜実験１：神経細胞に対する傷害に及ぼすペンタメチン系シアニン色素の影響＞
　神経細胞は、栄養飢餓や活性酸素による傷害に対して非常に脆弱なことが知られている。この神経細胞の特性は、アルツハイマー病をはじめとする神経変性疾患で認められる神経細胞死の原因と考えられている。そこで、細胞傷害因子の神経細胞に対する傷害性に及ぼすペンタメチン系シアニン色素の影響を調べる試験を以下のように実施した。 <Experiment 1: Effect of Pentamethine Cyanine Dye on Injury to Neurons>
Neurons are known to be very vulnerable to nutrient starvation and active oxygen injury. This characteristic of nerve cells is thought to be the cause of nerve cell death observed in neurodegenerative diseases such as Alzheimer's disease. Then, the test which investigates the influence of the pentamethine type | system | group cyanine pigment | dye on the cytotoxicity with respect to the nerve cell of a cytotoxic factor was implemented as follows.
＜試験標品＞
　試験には、化学式２で表される化合物（株式会社林原生物化学研究所製造、「ＮＫ－４」）を試験標品として使用した。ＮＫ－４は水に難溶性のため、ＤＭＳＯ（ＳＩＧＭＡ社販売、商品番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、膜濾過（Ｍｉｌｌｉｐｏｒｅ社販売、商品名「Ｍｉｌｌｅｘ－ＬＧ　ＳＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性膜使用）し、ダルベッコのＭＥＭ培地（日水製薬株式会社販売、以下、「Ｄ－ＭＥＭ培地」と略記する。）でさらに希釈して、試験に供した。なお、ＤＭＳＯに溶解した試験標品を、Ｄ－ＭＥＭ培地で、試験に使用する濃度に希釈した場合、それに含まれる濃度のＤＭＳＯは、以下の各試験系に影響しないことを予め確認した。 <Test specimen>
In the test, a compound represented by Chemical Formula 2 (manufactured by Hayashibara Biochemical Laboratories, Inc., “NK-4”) was used as a test sample. Since NK-4 is hardly soluble in water, it is dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then membrane filtered (Millipore, product name “Millex-LG SLLG025SS”, DMSO resistant membrane was used), and further diluted with Dulbecco's MEM medium (sold by Nissui Pharmaceutical Co., Ltd., hereinafter abbreviated as “D-MEM medium”), and subjected to the test. When a test sample dissolved in DMSO was diluted with D-MEM medium to a concentration used for the test, it was confirmed in advance that the concentration of DMSO contained therein did not affect the following test systems.
＜細胞傷害抑制作用の測定方法＞
＜栄養飢餓による細胞傷害に及ぼすＮＫ－４の影響＞
　ヒトの神経細胞変性研究用のモデルとし好適な、ラット副腎褐色細胞腫由来のＰＣ－１２細胞のＮＧＦ（神経増殖因子）高感受性株（以下、「ＰＣ－１２ＨＳ細胞」という。ヒューマンサイエンス研究資源バンクより入手）を使用した。栄養飢餓環境として血清除去培地による培養をおこなった。ＰＣ－１２ＨＳ細胞は、凍結保存した細胞を解凍して、１０容積％ウシ胎児血清（ＦＢＳ）加Ｄ－ＭＥＭ培地を用いて培養して試験に供した。試験に用いる細胞は、常法により、０．２５質量％トリプシン溶液を用いて剥離し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレート（ファルコン社販売、商品名「マイクロテストプレート・細胞培養用、平底」）に、５´１０３個／１００μｌ／ウエルになるように播種した。２４時間後に、培養上清を除去後、ＦＢＳを含まないＤ－ＭＥＭ培地で希釈し、表１に示す終濃度の２倍に調整したＮＫ－４のいずれかを１００μｌ／ウエル添加し、３日間培養した。３日目に培養上清を除去し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で、１０質量／容積％に希釈したＡｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を、２００μｌ／ウエル添加し、６時間培養し、蛍光プレートリーダー（日本モレキュラーディバイス株式会社販売、商品名「ＳｐｅｃｔｒａＭａｘ　Ｇｅｍｉｎｉ　ＨＹ」）で５４４－５９０ｎｍの蛍光強度を測定した。対照として、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地（ＮＫ－４無添加）のみで細胞を培養し、同様に、Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を加えて培養後、各ウエルの蛍光強度を測定した。対照の蛍光強度を１００％とした時の相対値を求め、各ウエルの細胞の生存率（％）として表１に併せて示す。なお、本試験及び以下の試験では、ＰＣ－１２ＨＳ細胞は、３７℃、５容積％ＣＯ２の条件下で、インキュベーター内で培養した。 <Method for measuring cytotoxicity-inhibiting action>
<Influence of NK-4 on cell damage caused by nutrient starvation>
NGF (nerve growth factor) hypersensitive strain of PC-12 cells derived from rat adrenal pheochromocytoma (hereinafter referred to as “PC-12HS cells”), which is suitable as a model for studying human neuronal degeneration. Obtained). Cultivation was performed using a serum-removed medium as a nutrient starvation environment. PC-12HS cells were thawed from cryopreserved cells, cultured in D-MEM medium supplemented with 10% by volume fetal bovine serum (FBS), and subjected to the test. Cells used for the test were detached by a conventional method using a 0.25% by mass trypsin solution, diluted with 10% by volume FBS-added D-MEM medium, and coated with a collagen-coated 96-well plate (trade name, sold by Falcon). “Micro test plate for cell culture, flat bottom”) was seeded at 5 × 10 3 cells / 100 μl / well. After 24 hours, the culture supernatant was removed, diluted with D-MEM medium containing no FBS, and 100 μl / well of NK-4 adjusted to twice the final concentration shown in Table 1 was added for 3 days. Cultured. On day 3, the culture supernatant was removed, and Alamar blue (Trek Diagnostics) solution diluted to 10% by mass with 10% FBS-added D-MEM medium was added at 200 μl / well and cultured for 6 hours. Then, the fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader (trade name “SpectraMax Gemini HY”, sold by Nippon Molecular Devices Co., Ltd.). As a control, cells were cultured only in D-MEM medium supplemented with 10% by volume FBS (without NK-4). Similarly, after adding Alamar blue (Trek Diagnostics) solution, the fluorescence intensity of each well was increased. It was measured. The relative value when the fluorescence intensity of the control is taken as 100% is determined and shown in Table 1 as the cell viability (%) in each well. In this test and the following tests, PC-12HS cells were cultured in an incubator under conditions of 37 ° C. and 5% by volume CO 2 .
＜過酸化水素による細胞傷害に及ぼすＮＫ－４の影響＞
　上記と同様に培養したＰＣ１２－ＨＳ細胞を、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレートに、２´１０４個／１００μｌ／ウエルで播種して２４時間培養した。その後、８００μＭ過酸化水素水（和光純薬工業株式会社販売）を５０μｌ／ウエル（終濃度２００μＭ）と、表１に示す終濃度の４倍の濃度に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈したＮＫ－４とを、各々５０μｌ／ウエル（ＮＫ－４の終濃度５ｎｇ乃至５０μｇ／ｍｌ）を同時に添加して、インキュベーター内で２時間培養後、２５容積％グルタルアルデヒド（和光純薬工業株式会社販売）を２０μｌ／ウエル（終濃度２０容積％）添加して細胞を固定した。０．０５質量％メチレンブルー（Ｗａｋｏ社販売）を１００μｌ／ウエル加えて、ダイアップテイク法で、常法により、各ウエルの吸光度を測定した。対照として過酸化水素及びＮＫ－４を添加しなかった以外は同様に培養を行い、メチレンブルーを加えて吸光度を測定した。対照の細胞数（吸光度）を１００（％）とした時の相対値を求めて各ウエルの細胞の生存率（％）として表１に併せて示す。 <Influence of NK-4 on cell injury caused by hydrogen peroxide>
PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, seeded at 2 × 10 4 cells / 100 μl / well in a collagen-coated 96-well plate, and cultured for 24 hours. did. After that, dilute 800 μM hydrogen peroxide solution (sales by Wako Pure Chemical Industries, Ltd.) with 50 μl / well (final concentration 200 μM) and 10% FBS-added D-MEM medium to a concentration 4 times the final concentration shown in Table 1. NK-4 was added at the same time to each 50 μl / well (NK-4 final concentration 5 ng to 50 μg / ml), cultured in an incubator for 2 hours, and then 25 vol% glutaraldehyde (Wako Pure Chemical Industries, Ltd.) The cells were fixed by adding 20 μl / well (final concentration 20% by volume). 0.05% by mass of methylene blue (available from Wako) was added at 100 μl / well, and the absorbance of each well was measured by a die-up take method according to a conventional method. As a control, culture was carried out in the same manner except that hydrogen peroxide and NK-4 were not added, and methylene blue was added to measure the absorbance. The relative value when the number of cells of the control (absorbance) is 100 (%) is determined and shown in Table 1 as the cell viability (%) of each well.
＜アミロイドβフラグメントによる細胞傷害に及ぼすＮＫ－４の影響＞
　アルツハイマー病における神経細胞死の主要原因の一つと考えられているアミロイドβペプチド（ヒト由来）のアミノ末端から２３乃至３５番目に相当するアミノ酸配列を有するペプチドフラグメント（ＡｎａＳｐｅｃ社販売、以下、「アミロイドβフラグメント」という。）（配列表における配列番号１のアミノ酸配列を有するペプチド）を、リン酸緩衝生理食塩水（ＰＢＳ）で希釈して２ｍＭの濃度に調整し、使用前に６時間、３７℃でエイジングさせ、フラグメントを凝集させて、細胞毒性を上昇させたものを使用した。上記と同様に培養したＰＣ１２－ＨＳ細胞を、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、コラーゲンコートした９６ウエルプレートに、５´１０３個／１００μｌ／ウエルになるように播種し、２４時間培養後に上清を除去して、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈したアミロイドβフラグメント溶液を５０μｌ／ウエル（アミロイドβフラグメントの終濃度５０μＭ）と、ＮＫ－４溶液を５０μｌ／ウエル（終濃度４０乃至５０００ｎｇ／ｍｌ）添加して３日間培養した。３日目に培養上清を除去し、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で、１０質量％濃度に希釈したＡｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）溶液を、２００μｌ／ウエル添加し、６時間培養し、蛍光プレートリーダーで５４４－５９０ｎｍの蛍光強度を測定した。対照として、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで細胞を培養（アミロイドβフラグメント及びＮＫ－４の両方とも無添加）し、同様にＡｌａｍａｒ　ｂｌｕｅ溶液を加えて、蛍光強度を測定した。対照の蛍光強度を１００％する相対値を求めて、各ウエルの細胞の生存率（％）として表１に併せて示す。また、別途、前記と同じ条件で培養した細胞の培養上清を除去後、ＰＢＳで希釈した１容積％グルタルアルデヒドを１００μｌ／ウエル添加して３０分間固定した後、１ｍＭヘキスト３３２５８色素（Ｓｉｇｍａ社販売）で５分間染色した後、位相差顕微鏡下及び蛍光顕微鏡下で、一視野に約１００個の細胞を含む倍率で細胞を観察し、細胞をカウントして、同一視野内の全細胞中に占めるアポトーシスを起こしている細胞の占有率（％）を計算した結果を、表１に併せて示す。なお、アポトーシスを起こしている細胞の判定は、細胞の核の断片化乃至核内のクロマチンの凝集を指標とした。 <Influence of NK-4 on cell injury by amyloid β fragment>
A peptide fragment having an amino acid sequence corresponding to the 23rd to 35th amino acids from the amino terminus of amyloid β peptide (human origin), which is considered to be one of the main causes of neuronal cell death in Alzheimer's disease (sold by AnaSpec, hereinafter “Amyloid β Fragment ") (a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing) was diluted with phosphate buffered saline (PBS) to a concentration of 2 mM, and 6 hours at 37 ° C before use. Aged and aggregated fragments were used to increase cytotoxicity. PC12-HS cells cultured in the same manner as above were diluted with 10% by volume FBS-added D-MEM medium, and seeded in a collagen-coated 96-well plate at 5 × 10 3 cells / 100 μl / well. After culturing for 24 hours, the supernatant was removed, and amyloid β fragment solution diluted with 10% by volume FBS-added D-MEM medium was 50 μl / well (final concentration of amyloid β fragment was 50 μM), and NK-4 solution was 50 μl / well. (Final concentration 40 to 5000 ng / ml) was added and cultured for 3 days. On the third day, the culture supernatant was removed, and Alamar blue (Trek Diagnostics) solution diluted to 10% by mass with 10% by volume FBS-added D-MEM medium was added at 200 μl / well and cultured for 6 hours. The fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader. As a control, cells were cultured only in D-MEM medium supplemented with 10% by volume FBS (no addition of both amyloid β fragment and NK-4), and Alamar blue solution was added in the same manner, and fluorescence intensity was measured. The relative value for 100% of the fluorescence intensity of the control was determined and is shown in Table 1 as the cell viability (%) of each well. Separately, after removing the culture supernatant of cells cultured under the same conditions as described above, 100 μl / well of 1% by volume glutaraldehyde diluted with PBS was added and fixed for 30 minutes, and then 1 mM Hoechst 33258 dye (sold by Sigma) ) For 5 minutes, and under a phase contrast microscope and a fluorescence microscope, the cells are observed at a magnification containing about 100 cells in one field, and the cells are counted and occupied in all cells in the same field The results of calculating the occupancy rate (%) of cells undergoing apoptosis are also shown in Table 1. The determination of cells undergoing apoptosis was based on cell nucleus fragmentation or aggregation of chromatin in the nucleus.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
　表１から明らかなように、ＮＫ－４は、ＰＣ１２－ＨＳ細胞に対する栄養飢餓、過酸化水素傷害、アミロイドβフラグメントによる傷害のいずれに対しても、各々の細胞傷害因子に対する有効濃度に差はあるものの、神経細胞に対する保護作用を有することが明らかとなった。表１に示す３種類の細胞傷害因子に対する保護作用が発揮されるＮＫ－４の濃度を比較すると、アミロイドβフラグメントの傷害に対する保護作用は４０ｎｇ／ｍｌから発揮されているのに対して、栄養飢餓傷害では５００ｎｇ／ｍｌ、過酸化水素傷害では５，０００ｎｇ／ｍｌの濃度が各々必要であることが判明した。傷害の発生する速度を見ると、過酸化水素が最も早く、アポトーシス誘導を伴うアミロイドβフラグメントが最も遅いと考えられるので、これらの細胞傷害因子に対するＮＫ－４による細胞保護作用は、傷害の発生する速度が速い程、高い濃度を必要とすることを物語っている。なお、具体的なデータは示さないが、濃度が５０，０００ｎｇ／ｍｌ以下のＮＫ－４水溶液は過酸化水素に対する消去能は有していないので、ＮＫ－４はペルオキシラジカルやヒドロキシラジカルなどのフリーラジカルの消去能も有しているものの、この試験系における過酸化水素による細胞傷害に対する細胞保護作用は、直接過酸化水素を消去する作用によるものではなく、細胞側に作用して、細胞死を抑制していると判断した。また、ヘキスト染色像より算出したアポトーシスを起こした細胞の占有率は、アミロイドβフラグメントを添加した場合（ＮＫ－４濃度０ｎｇ／ｍｌ）７２％となってアポトーシスが促進されたのに対して、ＮＫ－４（２００ｎｇ／ｍｌ）を添加した場合の占有率は１３％となって、対照のアポトーシスを起こした細胞の占有率（５％）にほぼ近い値となり、ＮＫ－４が、アミロイドβフラグメントにより誘導されるアポトーシスを抑制することが確認された。また、具体的なデータは示さないが、位相差顕微鏡観察でもアミロイドβフラグメントの添加により、細胞の凝集及び細胞死が認められたのに対して、ＮＫ－４の添加により細胞の凝集や細胞死が抑制されていることが確認された。さらに、ヘキスト染色像でも、アミロイドβフラグメントの添加により認められるアポトーシスによるクロマチンの凝集及び核の断片化が、ＮＫ－４を添加することにより、抑制されることが確認された。以上の結果は、ＮＫ－４は、アミロイドβフラグメントを初めとする神経細胞傷害因子に対する神経細胞保護作用があることからアミロイドβペプチドをはじめとする細胞傷害因子により引き起こされるアルツハイマー病治療剤として利用できることを物語っている。 As is clear from Table 1, NK-4 has a difference in the effective concentration for each cytotoxic factor against nutrient starvation, hydrogen peroxide damage, and amyloid β fragment damage to PC12-HS cells. However, it has been found to have a protective effect on nerve cells. Comparing the concentrations of NK-4 that exert the protective action against the three types of cytotoxic factors shown in Table 1, the protective action against the damage of the amyloid β fragment is exerted from 40 ng / ml, whereas the nutrient starvation It was found that a concentration of 500 ng / ml was required for injury and a concentration of 5,000 ng / ml was required for hydrogen peroxide injury. When looking at the rate at which injury occurs, hydrogen peroxide is the fastest and amyloid β fragment with apoptosis induction is considered to be the slowest, so the cytoprotective action by NK-4 against these cytotoxic factors causes injury. The higher the speed, the higher the concentration required. Although specific data is not shown, NK-4 aqueous solution having a concentration of 50,000 ng / ml or less does not have the ability to erase hydrogen peroxide, so NK-4 is free of peroxy radicals, hydroxy radicals and the like. Although it has radical scavenging ability, the cytoprotective action against hydrogen peroxide cytotoxicity in this test system is not directly due to the action of scavenging hydrogen peroxide, but acts on the cell side to prevent cell death. Judged to be suppressed. In addition, the occupancy ratio of the cells having undergone apoptosis calculated from Hoechst staining image was 72% when amyloid β fragment was added (NK-4 concentration 0 ng / ml), whereas apoptosis was promoted. -4 (200 ng / ml) was added, the occupancy was 13%, which was close to the occupancy (5%) of apoptotic cells in the control, and NK-4 was reduced by amyloid β fragment. It was confirmed to suppress the induced apoptosis. Although specific data are not shown, cell aggregation and cell death were observed by addition of amyloid β fragment in phase contrast microscopy, whereas cell aggregation and cell death were observed by addition of NK-4. Was confirmed to be suppressed. Furthermore, it was also confirmed in the Hoechst-stained image that the addition of NK-4 suppresses the aggregation of chromatin and the fragmentation of the nucleus due to apoptosis, which is recognized by the addition of amyloid β fragment. The above results indicate that NK-4 can be used as a therapeutic agent for Alzheimer's disease caused by cytotoxic factors such as amyloid β peptide because it has a neuroprotective action against neuronal cytotoxic factors including amyloid β fragment. Tells the story.
＜実験２：ＮＫ－４以外の色素化合物の細胞傷害性因子に対する保護作用＞
　実験１において、ＮＫ－４に、アミロイドβフラグメントなどの細胞傷害性因子に対する保護作用が確認されたので、ＮＫ－４以外の色素化合物（以下、単に「化合物」という場合がある。）についても、同様の作用があるかどうかを検討した。すなわち、表２に示す化学式２、化学式４乃至９で表される化合物に加えて、下記化学式１０乃至２４１で表わされる２３２種類（合計２３９種類）の化合物について、以下に示す細胞増殖促進活性（評価法Ａ）、神経突起伸展促進作用（評価法Ｂ）及びアミロイドβフラグメントによる細胞傷害に対する抑制作用（評価法Ｃ）により、ＰＣ１２－ＨＳ細胞に対する細胞増殖活性、神経突起伸展促進作用及びアミロイドβフラグメントによる細胞傷害に対する抑制作用の有無を調べた結果を表２に併せて示す。なお、下記試験において、各々の判定基準よりも低い効果しか認められなかった場合、表２は空欄とした。 <Experiment 2: Protective action against a cytotoxic factor of a dye compound other than NK-4>
In Experiment 1, since NK-4 was confirmed to have a protective action against cytotoxic factors such as amyloid β fragment, a dye compound other than NK-4 (hereinafter sometimes simply referred to as “compound”) was also used. It was examined whether there was a similar effect. That is, in addition to the compounds represented by Chemical Formula 2 and Chemical Formulas 4 to 9 shown in Table 2, 232 types (total 239 types) of compounds represented by the following Chemical Formulas 10 to 241, the following cell growth promoting activity (evaluation) Method A), neurite outgrowth promoting action (assessment method B) and inhibitory action against cell damage by amyloid β fragment (evaluation method C), cell proliferation activity against neurite outgrowth, neurite outgrowth promoting action and amyloid β fragment Table 2 also shows the results of examining the presence or absence of an inhibitory effect on cell injury. In the following tests, Table 2 was left blank when only an effect lower than each criterion was recognized.
Figure JPOXMLDOC01-appb-C000025
Figure JPOXMLDOC01-appb-C000025
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000029
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000047
＜試験試料＞
　表２に示す２３９種類の化合物は、水に難溶性のものが多いため、ＮＫ－４の場合と同様に、ＤＭＳＯ（ＳＩＧＭＡ社販売、カタログ番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、Ｍｉｌｌｅｘ－ＬＧ（Ｍｉｌｌｉｐｏｒｅ社販売、製品番号「ＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性）で膜ろ過し、遮光して２５℃で保存した。使用時には、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地（日水製薬）で２００倍以上に希釈して試験試料を調製し、試験に供した（表中のＮＫ番号に対応する化合物の構造は、『感光色素表』、感光色素研究所発行（１９６９年）参照）。これらの化合物は、いずれも株式会社林原生物化学研究所で合成したものを使用した。 <Test sample>
Since 239 kinds of compounds shown in Table 2 are hardly soluble in water, they were dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, as in the case of NK-4. Thereafter, the membrane was filtered with Millex-LG (sold by Millipore, product number “LLG025SS”, DMSO resistant), and stored at 25 ° C. protected from light. At the time of use, a test sample was prepared by diluting 200-fold or more with 10% by volume FBS-added D-MEM medium (Nissui Pharmaceutical) and subjected to the test (the structure of the compound corresponding to the NK number in the table is “ Photosensitive Dye Table ”, published by Photosensitive Dye Research Institute (1969)). These compounds were all synthesized by Hayashibara Biochemical Laboratories.
＜評価法Ａ：神経細胞増殖促進作用の評価法＞
　実験１と同じ方法で、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後にＤ－ＭＥＭ（１０容積％ＦＢＳ）で希釈し、１００ｎｇ／ｍｌに調整した各試験試料を１００μｌ／ウエル添加し、３７℃、５容積％ＣＯ２インキュベーター内で３日間培養した。３日目に培養上清を除去し、１０質量％Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ社販売）／１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を２００μｌ／ウエルずつ添加し、６時間、３７℃、５容積％ＣＯ２インキュベーター中で培養し、蛍光プレートリーダー（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ社販売）で５４４－５９０ｎｍの蛍光強度を測定した。各試験サンプルの細胞増殖促進作用は、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を１００μｌ／ウエル添加した対照の値を１００とした時の相対値が１４０乃至１９９の場合をＮＫ－４と同等（○）、２００以上をＮＫ－４より強い作用（◎）と判定した。結果を表２に併せて示す。 <Evaluation method A: Evaluation method of nerve cell proliferation promoting action>
In the same manner as in Experiment 1, PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a pre-collagen-coated 96-well microplate at 5 × 10 3 cells / well, and 100 μl / well. Sowing. After 24 hours, each test sample diluted with D-MEM (10% by volume FBS) and adjusted to 100 ng / ml was added at 100 μl / well and cultured in a 37 ° C., 5% by volume CO 2 incubator for 3 days. On the third day, the culture supernatant was removed, 10% by weight Alamar blue (sold by Trek Diagnostics) / 10 volume% FBS-added D-MEM medium was added in an amount of 200 μl / well for 6 hours at 37 ° C., 5 volume% CO. The cells were cultured in two incubators, and the fluorescence intensity at 544 to 590 nm was measured with a fluorescence plate reader (available from Molecular Devices). The cell growth promoting effect of each test sample is equivalent to that of NK-4 when the relative value is 140 to 199 when the control value obtained by adding 100 μl / well of 10% FBS-added D-MEM medium is 100 (○ ), 200 or more were judged to be stronger (◎) than NK-4. The results are also shown in Table 2.
＜評価法Ｂ：神経突起伸展作用の評価法＞
　細胞増殖促進活性の測定の場合と同様に、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５×１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に、Ｄ－ＭＥＭ（１０容積％ＦＢＳ）で希釈して４００ｎｇ／ｍｌに調整した各試験試料５０μｌ／ウエルと、２０ｎｇ／ｍｌＮＧＦ（Ｃｈｅｍｉｃｏｎ社販売、マウス由来、終濃度５ｎｇ／ｍｌ）含有Ｄ－ＭＥＭ培地（１０容積％ＦＢＳ）５０μｌ／ウエルとを加えて、３日間培養した。培養３日目に１０容積％グルタルアルデヒドで室温２０分間固定した。対照として１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで３日間培養したＰＣ１２－ＨＳ細胞をグルタルアルデヒドで固定した。固定した細胞を、顕微鏡下で観察し、神経突起伸展の有無を評価し、神経突起伸展率が３０％以上の場合をＮＫ－４と同等以上（○）と判定した。なお、神経突起伸展率（％）は、顕微鏡下で、一視野に約１００個の細胞を含む倍率で細胞を観察し、細胞体の２倍以上の神経突起を有する細胞数をカウントし、同一視野内にある全細胞数で除し、１００倍して求めた。また、この実験系にＮＧＦのみを添加（５ｎｇ／ｍｌ）したときの神経突起伸展率は５％程度であった。結果を表２に併せて示す。 <Evaluation method B: Evaluation method of neurite extension action>
As in the case of the measurement of cell growth promoting activity, PC12-HS cells were diluted with D-MEM medium supplemented with 10% by volume FBS to a 96-well microplate previously coated with collagen at 5 × 10 3 cells / well. And 100 μl / well. After 24 hours, each test sample was diluted with D-MEM (10% by volume FBS) and adjusted to 400 ng / ml, 50 μl / well, and D containing 20 ng / ml NGF (available from Chemicon, mouse, final concentration 5 ng / ml). -MEM medium (10% by volume FBS) 50 μl / well was added and cultured for 3 days. On the third day of culture, the cells were fixed with 10% by volume glutaraldehyde for 20 minutes at room temperature. As a control, PC12-HS cells cultured for 3 days only in 10% by volume FBS-added D-MEM medium were fixed with glutaraldehyde. The fixed cells were observed under a microscope to evaluate the presence or absence of neurite outgrowth, and when the neurite outgrowth rate was 30% or more, it was determined to be equal to or better than NK-4 (◯). The neurite outgrowth rate (%) is the same by observing cells under a microscope at a magnification including about 100 cells in one field, counting the number of cells having neurites more than twice the cell body, Dividing by the total number of cells in the field of view and multiplying by 100 was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%. The results are also shown in Table 2.
＜評価法Ｃ：アミロイドβフラグメントによる細胞傷害に対する抑制作用＞
　評価法Ａで細胞増殖促進作用の認められた試験試料について、実験１と同じ方法で、アミロイドβフラグメントによる細胞傷害に対する細胞保護効果があるかどうかを検討した。試験試料無添加の場合に比して、アミロイドβフラグメントによる細胞傷害を有意に抑制した場合を抑制効果あり（○）と判定した。結果を表２に併せて示す。 <Evaluation Method C: Inhibitory Action on Cell Damage by Amyloid β Fragment>
The test sample in which the cell growth promoting action was recognized by the evaluation method A was examined by the same method as in Experiment 1 to determine whether it had a cytoprotective effect against cell damage caused by amyloid β fragment. Compared to the case where the test sample was not added, the case where the cell damage due to amyloid β fragment was significantly suppressed was determined to have an inhibitory effect (◯). The results are also shown in Table 2.
Figure JPOXMLDOC01-appb-T000048
Figure JPOXMLDOC01-appb-T000048
　表２の結果に示すように、試験した２３９種の化合物は、細胞増殖促進作用（評価法Ａ）、神経突起伸展促進作用（評価法Ｂ）或いはアミロイドβ傷害抑制作用（評価法Ｃ）を有するものがあることが判明した。とりわけ、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１００（化学式６で表される化合物）、ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、ＮＫ－１５１６（化学式９で表される化合物）は、ＮＫ－４と同等以上の細胞増殖促進作用と、神経突起伸展促進作用を示した。また、細胞増殖促進活性をもつ化合物について、アミロイドβフラグメントによる細胞傷害に対する抑制作用（評価法Ｃ）があると評価した化合物は、いずれも、アミロイドβフラグメントによる細胞傷害に対する抑制作用が認められた。以上の結果、及び、実験１、実験２の結果から、表６に示す細胞増殖促進作用や神経突起伸展促進作用を有する化合物は、いずれも、神経細胞を活性化しているので、ヒトの抗アルツハイマー病剤として利用できることを物語っている。なかでも、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１００（化学式６で表される化合物）、ＮＫ－５２８（化学式７で表わされる化合物）、ＮＫ－５５７（化学式８で表される化合物）、ＮＫ－１５１６（化学式９で表される化合物）は、細胞増殖促進作用や神経突起伸展促進作用が強いので、特にヒトの抗アルツハイマー病剤として有用であることを物語っている。さらに、これらの化合物、とりわけ、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５２８、ＮＫ－５５７、ＮＫ－１５１６は、アルツハイマー病に伴う病態や神経機能障害の治療剤としても利用できることを物語っている。 As shown in the results of Table 2, the 239 compounds tested have a cell growth promoting action (Evaluation Method A), a neurite outgrowth promoting action (Evaluation Method B), or an amyloid β injury inhibiting action (Evaluation Method C). It turns out that there is something. In particular, NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-100 (compound represented by chemical formula 6), NK-528 (expressed by chemical formula 7) Compound), NK-557 (compound represented by chemical formula 8), and NK-1516 (compound represented by chemical formula 9) have cell growth promoting effects and neurite outgrowth promoting actions equivalent to or higher than those of NK-4. Indicated. Moreover, the inhibitory action with respect to the cytotoxicity by the amyloid (beta) fragment was recognized by all the compounds evaluated as having the inhibitory action with respect to the cytotoxicity by the amyloid (beta) fragment (evaluation method C) about the compound which has cell growth promotion activity. From the above results and the results of Experiment 1 and Experiment 2, all of the compounds having the cell growth promoting action and the neurite outgrowth promoting action shown in Table 6 activate neuronal cells. It tells us that it can be used as a disease agent. Among them, NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-100 (compound represented by chemical formula 6), NK-528 (expressed by chemical formula 7). Compound), NK-557 (compound represented by Chemical Formula 8), and NK-1516 (compound represented by Chemical Formula 9) have a strong cell growth promoting action and neurite outgrowth promoting action. It tells us that it is useful as a disease agent. Furthermore, these compounds, particularly NK-4, NK-19, NK-53, NK-100, NK-528, NK-557, and NK-1516, are used as therapeutic agents for pathological conditions and neurological dysfunction associated with Alzheimer's disease. Also tells you that you can use it.
＜実験３：各化合物の濃度がアミロイドβフラグメントによる細胞傷害に及ぼす影響＞
　実験２において、ＮＫ－４と同様にアミロイドβフラグメントによる細胞傷害を抑制する作用を持つことが確認された化合物の中からＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５２８、ＮＫ－５５７及びＮＫ－１５１６について、その濃度が、アミロイドβフラグメントによる細胞傷害に及ぼす影響を調べた。すなわち、前記６種の化合物及びＮＫ－４を試験標品として使用し、各化合物が表３に示す終濃度となるように、ＰＣ１２－ＨＳ細胞を播種したウエルに添加した以外は、実験３の評価法Ｃと同一の条件で、これらの化合物のアミロイドβフラグメントによる細胞傷害に対する抑制効果を評価した。結果を細胞生存率（％）として表３に示す。 <Experiment 3: Effect of Compound Concentration on Cell Damage Caused by Amyloid β Fragment>
In Experiment 2, NK-19, NK-53, NK-100, NK-528, NK-557 are selected from the compounds that have been confirmed to have the effect of inhibiting cell damage caused by amyloid β fragment in the same manner as NK-4. And NK-1516, the effect of the concentration on cytotoxicity caused by amyloid β fragment was examined. That is, except that the above six compounds and NK-4 were used as test preparations, and each compound was added to the wells seeded with PC12-HS cells so that the final concentrations shown in Table 3 were obtained, the experiment 3 Under the same conditions as in Evaluation Method C, the inhibitory effect of these compounds on cytotoxicity by amyloid β fragment was evaluated. The results are shown in Table 3 as cell viability (%).
Figure JPOXMLDOC01-appb-T000049
Figure JPOXMLDOC01-appb-T000049
　表３から明らかなように、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００はＮＫ－４よりも低濃度で、アミロイドβフラグメントによる細胞傷害に対して、高い抑制活性を示した。このうち、最も低濃度で高い抑制活性を示したのは、ＮＫ－５３で、１２．５ｎｇ／ｍｌの濃度でほぼ完全にアミロイドβフラグメントによる細胞傷害を抑制した（細胞生存率１１５±１６％）。ＮＫ－１９、ＮＫ－１００、ＮＫ－５５７では、５０ｎｇ／ｍｌの濃度で傷害抑制率が最高となり、それぞれ１０２±２７％、８８±１２％、１１４±９％となった。この効果は全て、５０μｇ／ｍｌのＮＫ－４を添加した場合に得られた細胞傷害抑制率（６９±７％）よりも高かった。ＮＫ－５３は、濃度が５０ｎｇ／ｍｌでも傷害抑制率が１１１±９％と高い抑制活性が認められた。一方、ＮＫ－４は２００ｎｇ／ｍｌの濃度で、傷害抑制率が１２２±３２％と最高になったのに対して、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７では細胞保護効果の低下が認められ、これらの化合物の細胞傷害抑制作用には至適濃度が存在することが確認され、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７の至適濃度は、ＮＫ－４より低いことが判明した。ＮＫ－５２８及びＮＫ－１５１６では、ＮＫ－４より高い細胞傷害抑制効果は認められなかった。以上の結果から、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７は、抗アルツハイマー病剤としてＮＫ－４より低濃度で、優れた効果を発揮する可能性が示唆された。また、ＮＫ－４よりも低濃度で、高い細胞傷害抑制活性の認められたＮＫ－１９及びＮＫ－１００は、他のものより分子量が大きく、ＮＫ１９やＮＫ－５３ではチアゾール環の窒素に結合した側鎖のアルキル基の炭素数が７（他は炭素数２）と多いことから、脂溶性が高く、細胞膜透過性が高いため、強い活性が発現されたと判断した。 As is clear from Table 3, NK-19, NK-53, and NK-100 showed lower inhibitory activity against cell damage caused by amyloid β fragment at a lower concentration than NK-4. Among these, NK-53 showed the highest inhibitory activity at the lowest concentration, and almost completely inhibited the cytotoxicity caused by amyloid β fragment at a concentration of 12.5 ng / ml (cell survival rate 115 ± 16%). . In NK-19, NK-100, and NK-557, the injury inhibition rate was highest at a concentration of 50 ng / ml, which was 102 ± 27%, 88 ± 12%, and 114 ± 9%, respectively. All of these effects were higher than the cytotoxicity inhibition rate (69 ± 7%) obtained when 50 μg / ml of NK-4 was added. NK-53 was found to have a high inhibitory activity of 111 ± 9% even at a concentration of 50 ng / ml. On the other hand, NK-4 had the highest injury inhibition rate of 122 ± 32% at a concentration of 200 ng / ml, whereas NK-19, NK-53, NK-100, and NK-557 had a cytoprotective effect. It was confirmed that there was an optimal concentration for the cytotoxic effect of these compounds, and the optimal concentrations of NK-19, NK-53, NK-100, and NK-557 are NK- It was found to be lower than 4. NK-528 and NK-1516 did not show a higher cytotoxic effect than NK-4. From the above results, it was suggested that NK-19, NK-53, NK-100, and NK-557 may exhibit superior effects at lower concentrations than NK-4 as anti-Alzheimer's disease agents. In addition, NK-19 and NK-100, which showed a high cytotoxicity-inhibiting activity at a lower concentration than NK-4, had a higher molecular weight than the others, and NK19 and NK-53 bound to nitrogen in the thiazole ring. Since the number of carbon atoms in the side chain alkyl group is as large as 7 (other carbon number is 2), it was judged that strong activity was expressed because of high lipid solubility and high cell membrane permeability.
＜実験４：アミロイドβペプチドの凝集に及ぼす影響＞
　実験２及び３において、アミロイドβフラグメントの細胞傷害性に対する保護作用が認められた化合物のなかから、神経突起伸展促進作用も確認されたＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を試験試料に使用してアミロイドβペプチドの凝集に及ぼす影響を調べる試験を以下のように実施した。すなわち、各試験試料をＤＭＳＯ（ＳＩＧＭＡ社販売、カタログ番号「Ｄ８４１８」）に５ｍｇ／ｍｌの濃度で溶解した後、Ｍｉｌｌｅｘ－ＬＧ（Ｍｉｌｌｉｐｏｒｅ社販売、製品番号「ＬＬＧ０２５ＳＳ」、ＤＭＳＯ耐性）で膜ろ過し、Ｔｒｉｓ－ＨＣｌ緩衝液を使用して、濃度の２００ｎＭの濃度に調製して試験試料溶液とした。 <Experiment 4: Effect on Aggregation of Amyloid β Peptide>
In Experiments 2 and 3, NK-4, NK-19, NK-53, NK-100, and NK-4, which were confirmed to have a neurite outgrowth-promoting action among the compounds in which the protective action against the cytotoxicity of amyloid β fragment was recognized. A test for examining the effect of NK-557 on the aggregation of amyloid β peptide was conducted as follows. Specifically, each test sample was dissolved in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, and then membrane-filtered with Millex-LG (Millipore, product number “LLG025SS”, DMSO resistant). Using Tris-HCl buffer, a test sample solution was prepared to a concentration of 200 nM.
＜アミロイドβペプチド凝集の測定法＞
　アミロイドβペプチドの凝集はチオフラビンＴを用いた方法で測定した（例えば、Ｈｉｌａｌ　Ａ．Ｌａｓｈｕｅｌら、『Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ』、第２７７巻、第４５号、第４２８８１－４２８９０頁（２００２年）参照）。チオフラビン－Ｔは、凝集したアミロイドβペプチドのβ－シート構造に結合し、蛍光を発する。この蛍光量を蛍光プレートリーダーで検出しアミロイドβペプチド凝集の指標とした。試験試料がアミロイドβペプチド凝集を抑制した場合、チオフラビン－Ｔの蛍光が減少する。この方法で試験試料のアミロイドβペプチド凝集に及ぼす影響を調べた。すなわち、配列表における配列番号２で表される４０個のアミノ酸からなるアミノ酸配列を有するヒトアミロイドβペプチドＡｎａ　Ｓｐｅｃ社販売）を、４００μＭの濃度に滅菌蒸留水に溶解して使用した。試験試料溶液は、Ｔｒｉｓ－ＨＣｌ緩衝液にて希釈した。反応用の容器に１００ｍＭ　アミロイドβペプチド１５μｌおよび試験試料溶液（２００ｎＭ）４５μｌを入れて、混合し、３７℃で６日間反応させた。反応後の溶液を５０μｌとり、の１０μＭチオフラビンＴ４５０μｌと混合し、３０分後に、蛍光度計で測定した（励起波長４５０ｎｍ、吸収波長４８２ｎｍ）。チオフラビンＴのみの蛍光度を０％、１００ｍＭ　アミロイドβペプチド１５μｌおよびＴｒｉｓ－ＨＣｌ緩衝液４５μｌを入れ、混合し、３７℃で６日間反応させたときの蛍光度を１００％とし、各試験試料溶液を加えて反応させたときの蛍光強度の相対値を求め１００％から減じてアミロイドβペプチド凝集抑制効果（％）として表４に示す。 <Measurement method of amyloid β peptide aggregation>
Aggregation of amyloid β peptide was measured by a method using thioflavin T (see, for example, Hilal A. Laschel et al., “Journal of Biological Chemistry”, Vol. 277, No. 45, pp. 42881-42890 (2002)). . Thioflavin-T binds to the β-sheet structure of aggregated amyloid β peptide and emits fluorescence. This amount of fluorescence was detected with a fluorescence plate reader and used as an indicator of amyloid β peptide aggregation. When the test sample inhibits amyloid β peptide aggregation, the thioflavin-T fluorescence decreases. The effect of the test sample on amyloid β peptide aggregation was examined by this method. That is, a human amyloid β peptide having an amino acid sequence consisting of 40 amino acids represented by SEQ ID NO: 2 in the Sequence Listing (sold by Ana Spec) was used after dissolving in sterile distilled water at a concentration of 400 μM. The test sample solution was diluted with Tris-HCl buffer. In a reaction container, 15 μl of 100 mM amyloid β peptide and 45 μl of a test sample solution (200 nM) were mixed, mixed, and reacted at 37 ° C. for 6 days. 50 μl of the solution after the reaction was taken and mixed with 450 μl of 10 μM thioflavin T, and measured with a fluorometer after 30 minutes (excitation wavelength 450 nm, absorption wavelength 482 nm). Fluorescence of only thioflavin T was 0%, 100 mM amyloid β peptide 15 μl and Tris-HCl buffer 45 μl were mixed, mixed, and reacted at 37 ° C. for 6 days. The fluorescence was 100%. In addition, the relative value of the fluorescence intensity when the reaction was carried out was determined and subtracted from 100% and shown in Table 4 as the amyloid β peptide aggregation inhibitory effect (%).
Figure JPOXMLDOC01-appb-T000050
Figure JPOXMLDOC01-appb-T000050
　表４から明らかなように、試験に使用したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７はいずれも、アミロイドβペプチドの凝集を抑制し、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７はいずれも、ＮＫ－４よりも強い抑制活性を示した。なかでも、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、アミロイドβペプチドの凝集を９５％以上抑制し、ＮＫ－１００では９９％とほぼ完全に凝集を抑制した。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７などの、アミロイドβフラグメントによる細胞傷害を抑制する作用を有する化合物は、アミロイドβペプチドの凝集を抑制する作用も有することから、アルツハイマー病の治療剤としてだけでなく、予防剤としても有用であることを物語っている。 As is apparent from Table 4, all of NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test suppressed aggregation of amyloid β peptide, and NK-19, NK- 53, NK-100 and NK-557 all showed stronger inhibitory activity than NK-4. Among them, NK-53, NK-100 and NK-557 suppressed amyloid β peptide aggregation by 95% or more, and NK-100 almost completely suppressed aggregation by 99%. As a result, a compound having an action of suppressing cell damage caused by amyloid β fragment such as NK-4, NK-19, NK-53, NK-100 and NK-557 has an action of suppressing aggregation of amyloid β peptide. Therefore, it shows that it is useful not only as a therapeutic agent for Alzheimer's disease but also as a preventive agent.
＜実験５：色素化合物のアセチルコリンエステラーゼ（ＡｃｈＥ）活性に及ぼす影響＞
　アルツハイマー型認知症にはドネぺジルをはじめとするＡｃｈＥ阻害薬が臨床応用されている。ＡｃｈＥ阻害薬は中枢コリン神経系を賦活化し、虚血性認知症においても認知機能を改善することが報告されている。そこで、実験７で脳虚血を起こしたラットの脳梗塞及びそれに伴う神経機能障害を改善する効果が確認された、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７に、ＡｃｈＥ阻害作用があるかどうかを確認する試験を行った。すなわち、ＤＭＳＯに溶解したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７を、各々リン酸緩衝液で希釈し、濃度が表１３に示す濃度の１０倍濃度の化合物を含む溶液を調製して、試験試料溶液とした。実験１と同様に培養したＰＣ１２－ＨＳ細胞を回収し、その５倍量（容積）の１０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（１Ｍ　ＮａＣｌ、５０ｍＭ　ＭｇＣｌ２、１％Ｔｒｉｔｏｎ　Ｘ－１００、ｐＨ７．２）を加えて、常法により均一にホモジナイズした後、４℃にて３０分間遠心分離（１０，０００ｇ）し、その上清を回収してアセチルコリンエステラーゼ（ＡｃｈＥ）含有溶液とした。９６ウエルプレート（住友ベークライト社販売、商品名「μテストプレート、細胞培養用、平底」）に、５０ｍＭリン酸緩衝液（ｐＨ８．０）３０μｌ、試験試料溶液１０μｌ、ＡｃｈＥ含有溶液１０μｌを入れ、さらに、反応基質液として０．５ｍＭヨウ化アセチルチオコリン（和光純薬工業株式会社販売）と１ｍＭ　２－ニトロ安息香酸（Ｗａｋｏ社販売）を含むリン酸緩衝液５０μｌを加えた。３７℃のインキュベーターで３０分間酵素反応を行った後、プレートリーダーで４０５ｎｍにおける吸光度（ＡＲ）を測定した。また、ＡｃｈＥ含有溶液の代わりにリン酸緩衝液１０μｌを用いて、上記と同様に、３７℃のインキュベーターで３０分間酵素反応を行って吸光度（ＡＵ）を測定した。対照におけるＡｃｈＥ含有液に代えてリン酸緩衝液１０μｌを用いて３７℃のインキュベーターで３０分間酵素反応を行い、吸光度（ＢＲ）を測定した。さらに、対照の対照として、対照の反応液のＡｃｈＥ含有液に代えてリン酸緩衝液１０μｌを用いて反応を行い、吸光度（ＢＵ）を測定した。ＡｃｈＥ活性残存率（％）（＝｛（ＡＲ－Ａｕ）÷（ＢＲ－ＢＵ）｝×１００）を求め、ＡｃｈＥ活性を５０％阻害するときの化合物の濃度（ＩＣ５０）を求めた。結果を表５に示す。 <Experiment 5: Effect of pigment compound on acetylcholinesterase (AchE) activity>
AchE inhibitors such as donepezil have been clinically applied to Alzheimer-type dementia. It has been reported that AchE inhibitors activate the central cholinergic nervous system and improve cognitive function even in ischemic dementia. Therefore, in Experiment 7, NK-4, NK-19, NK-53, NK-100, and NK-557 were confirmed to have an effect of improving cerebral infarction and associated neurological dysfunction in rats with cerebral ischemia. A test was conducted to confirm whether there was an AchE inhibitory effect. That is, NK-4, NK-19, NK-53, NK-100 and NK-557 dissolved in DMSO were each diluted with a phosphate buffer, and a compound having a concentration 10 times that shown in Table 13 was added. A solution was prepared to provide a test sample solution. PC12-HS cells cultured in the same manner as in Experiment 1 were collected, and 5 volumes (volume) of 10 mM Tris-HCl buffer solution (1M NaCl, 50 mM MgCl 2 , 1% Triton X-100, pH 7.2) was added. After homogenizing uniformly by a conventional method, the mixture was centrifuged (10,000 g) at 4 ° C. for 30 minutes, and the supernatant was recovered to obtain a solution containing acetylcholinesterase (AchE). In a 96-well plate (sold by Sumitomo Bakelite Co., Ltd., trade name “μ test plate, for cell culture, flat bottom”) is added 30 μl of 50 mM phosphate buffer (pH 8.0), 10 μl of test sample solution, and 10 μl of AchE-containing solution. As a reaction substrate solution, 50 μl of a phosphate buffer containing 0.5 mM acetylthiocholine iodide (available from Wako Pure Chemical Industries, Ltd.) and 1 mM 2-nitrobenzoic acid (available from Wako) was added. After an enzyme reaction for 30 minutes in a 37 ° C. incubator, the absorbance (A R ) at 405 nm was measured with a plate reader. In addition, 10 μl of phosphate buffer was used instead of the AchE-containing solution, and the absorbance (A U ) was measured by performing an enzyme reaction for 30 minutes in a 37 ° C. incubator in the same manner as described above. In place of the AchE-containing solution in the control, 10 μl of a phosphate buffer was used for an enzyme reaction for 30 minutes in an incubator at 37 ° C., and the absorbance (B R ) was measured. Further, as a control for the control, the reaction was performed using 10 μl of a phosphate buffer instead of the AchE-containing solution of the control reaction solution, and the absorbance (B U ) was measured. AchE activity remaining rate (%) (= {(A R −A u ) ÷ (B R −B U )} × 100) was determined, and the concentration of compound at which AchE activity was inhibited by 50% (IC 50 ) was determined. It was. The results are shown in Table 5.
Figure JPOXMLDOC01-appb-T000051
Figure JPOXMLDOC01-appb-T000051
　表５から明らかなように、低濃度域ではＮＫ－１００に最も高いＡｃｈＥ阻害が認められ、ＮＫ－１００の濃度が０．７８μｇ／ｍｌ以上で有意なＡｃｈＥ阻害作用を示した。また、ＮＫ－１００と構造の類似しているＮＫ－４でも３．１３μｇ／ｍｌ以上の濃度で有意な阻害が認められた。これらの化合物のＩＣ５０は、ＮＫ－４は３．３μｇ／ｍｌ、ＮＫ－１００は１１．８μｇ／ｍｌであった。これに対し、ＮＫ－１９及びＮＫ－５３のＡｃｈＥ阻害活性は弱く、ＮＫ－１９及びＮＫ－５３では２５μｇ／ｍｌの濃度でのみで有意な活性の低下が認められた。一方、ＮＫ－５５７では２５μｇ／ｍｌまでの濃度でほとんどＡｃｈＥ阻害活性を示さなかった。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００の４種の化合物では、ＡｃｈＥの阻害作用によっても、コリン作動性神経系を賦活化し、アルツハイマー型認知症を改善する可能性が示唆された。 As is apparent from Table 5, NK-100 showed the highest AchE inhibition in the low concentration range, and showed a significant AchE inhibitory effect at NK-100 concentrations of 0.78 μg / ml or more. In addition, NK-4 having a structure similar to that of NK-100 was significantly inhibited at a concentration of 3.13 μg / ml or more. The IC 50 of these compounds was 3.3 μg / ml for NK-4 and 11.8 μg / ml for NK-100. In contrast, NK-19 and NK-53 had weak AchE inhibitory activity, and NK-19 and NK-53 showed a significant decrease in activity only at a concentration of 25 μg / ml. On the other hand, NK-557 showed almost no AchE inhibitory activity at concentrations up to 25 μg / ml. This result shows that the four compounds NK-4, NK-19, NK-53, and NK-100 can also activate the cholinergic nervous system and improve Alzheimer-type dementia even by the inhibitory action of AchE. Sex was suggested.
　なお、アルツハイマー病の治療薬として臨床利用されているガランタミンは、ＮＫ－４に比較して低い濃度で同程度のＡｃｈＥ阻害効果を示す（その時のＩＣ５０は、４４２μｇ／ｍｌ）。最近、これらの化合物では、ＮＫ－４と同様のＰＩ３Ｋ－Ａｋｔカスケードを介した細胞死の抑制も報告されているが、ＮＫ－４では３．３ｇ／ｍｌで効果が得られるのに対し、ガランタミでは同等の効果を得るのに約１００倍濃度を要する。また、Ｉｎ　ｖｉｔｒｏのアミロイドβフラグメント傷害モデルでの細胞保護効果についても同様の濃度のオーダーが異なっているので、これらのことを考慮すると、ＮＫ－４（ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７を含む）は既存のアルツハイマー病治療薬とは異なる作用機作を有する抗アルツハイマー病剤として利用できることを物語っている。 Note that galantamine, which is clinically used as a therapeutic agent for Alzheimer's disease, shows the same level of AchE inhibitory effect at a lower concentration than NK-4 (the IC 50 at that time is 442 μg / ml). Recently, these compounds have also been reported to suppress cell death via the PI3K-Akt cascade, similar to NK-4, whereas NK-4 is effective at 3.3 g / ml, whereas Galantami Then, a concentration of about 100 times is required to obtain the same effect. Further, since the order of the same concentration is different for the cytoprotective effect in the in vitro amyloid β fragment injury model, considering these, NK-4 (NK-19, NK-53, NK-100) NK-557) can be used as an anti-Alzheimer's disease agent having a different mechanism of action from existing Alzheimer's disease therapeutic agents.
＜実験６：ＮＫ－１９類縁体の神経栄養因子様活性＞
　上記実験で、強い神経変性抑制作用が確認されたＮＫ－１９について、その類縁体にも同様の作用があることを確認する試験をおこなった。すなわち、一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が１乃至１２である化合物を合成（株式会社林原生物化学研究所合成）し、実験２と同様に、ＰＣ－１２ＨＳ細胞に対する細胞増殖促進作用及び神経突起伸展促進作用の強さを調べた。表６に示すＮＫ－１９を含む、ＮＫ－１９の類縁体１２種類の化合物を合成し、各々ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。この溶液を１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、化合物の濃度が、各々１００ｎｇ／ｍｌ又は２μｇ／ｍｌにとなるように試験試料溶液を調製した。また、ＮＫ－２４及びＮＫ－１９については、その対アニオンをＩ－からＣｌ－に替えた化合物（ＮＫ－５６及びＮＫ－５３）も、ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。これらの溶液を１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈して、化合物の濃度を、各々１００ｎｇ／ｍｌ又は２μｇ／ｍｌに希釈して試験試料溶液を調製した。 <Experiment 6: Neurotrophic factor-like activity of NK-19 analog>
In the above experiment, a test was conducted to confirm that NK-19, which has been confirmed to have a strong neurodegeneration inhibitory effect, has similar effects on its analogs. That is, a compound in which the alkyl group (R) in the side chain of the compound represented by the general formula 3 has 1 to 12 carbon atoms was synthesized (Synthesis by Hayashibara Biochemical Laboratories Co., Ltd.) The strength of cell growth promoting action and neurite extension promoting action on -12HS cells was examined. Twelve types of analogs of NK-19 including NK-19 shown in Table 6 were synthesized and dissolved in DMSO to a concentration of 5 mg / ml. This solution was diluted with D-MEM medium supplemented with 10% by volume FBS to prepare a test sample solution so that the concentration of the compound was 100 ng / ml or 2 μg / ml, respectively. Moreover, the NK-24 and NK-19 is the counter anion I - Cl from - compounds instead (NK-56 and NK-53) was also dissolved at a 5 mg / ml in DMSO. These solutions were diluted with 10% by volume FBS-added D-MEM medium to prepare test sample solutions by diluting the compound concentration to 100 ng / ml or 2 μg / ml, respectively.
＜細胞増殖促進作用の評価＞
　実験２の評価法Ａと同様に、コラーゲンコートした９６ウエルプレートに５Ｘ１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、化合物の濃度を１００ｎｇ／ｍｌに調整した各試験試料溶液を１００μｌ／ウエル添加し、３７℃、５容積％ＣＯ２インキュベーター内で３日間培養した。培養３日目に培養上清を除去し、１０質量％Ａｌａｍａｒ　ｂｌｕｅ（Ｔｒｅｋ　Ｄｉａｇｎｏｓｔｉｃ）／１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を２００μｌ／ウエルずつ添加し、６時間、３７℃、５容積％ＣＯ２インキュベーター中で培養し、蛍光プレートリーダー（Ｍｏｌｅｃｕｌａｒ　Ｄｅｖｉｃｅｓ）で５４４－５９０ｎｍの蛍光強度を測定した。各試験試料溶液を添加した場合の細胞増殖促進作用は、１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地を１００μｌ／ウエル添加した対照の値を１００とした時の相対値として求めた。結果を表６に示す。なお、試験は、各試験試料溶液につき、トリプレットで２回実施し、その平均を求めた。 <Evaluation of cell proliferation promoting action>
Similar to Evaluation Method A in Experiment 2, a 96-well plate coated with collagen was diluted with 10% by volume FBS-added D-MEM medium to 5 × 10 3 cells / well, and seeded at 100 μl / well. After 24 hours, each test sample solution diluted with 10% by volume FBS-added D-MEM medium and adjusted to a compound concentration of 100 ng / ml was added at 100 μl / well, and the mixture was kept at 37 ° C. in a 5% by volume CO 2 incubator for 3 days. Cultured. On the third day of culture, the culture supernatant was removed, and 10% by weight Alamar blue (Trek Diagnostics) / 10 volume% FBS-added D-MEM medium was added in an amount of 200 μl / well for 6 hours at 37 ° C., 5 volume% CO 2. After culturing in an incubator, the fluorescence intensity at 544-590 nm was measured with a fluorescence plate reader (Molecular Devices). The cell growth promoting effect when each test sample solution was added was determined as a relative value when the value of the control added with 100 μl / well of 10% by volume FBS-added D-MEM medium was taken as 100. The results are shown in Table 6. In addition, the test was implemented twice with each triplet for each test sample solution, and the average was obtained.
＜神経突起伸展促進作用の評価＞
　実験２の評価Ｂと同様に、ＰＣ１２－ＨＳ細胞を、予めコラーゲンコートした９６ウエルマイクロプレートに５´１０３個／ウエルになるように１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地で希釈し、１００μｌ／ウエルで播種した。２４時間後に、化合物の濃度を２μｇ／ｍｌに調整した各試験試料溶液５０μｌ／ウエルとＮＧＦ（終濃度５ｎｇ／ｍｌ）５０μｌ／ウエルとを添加し、培養３日目に１０容積％グルタルアルデヒドで室温２０分間固定した。対照として１０容積％ＦＢＳ加Ｄ－ＭＥＭ培地のみで３日間培養し、細胞をグルタルアルデヒドで固定した。固定した細胞を、顕微鏡下で観察し、実験３の場合と同じ方法で神経突起伸展の有無を評価した。結果を表６に併せて示す。なお、試験は、ＮＫ－５６及びＮＫ－５３を除く各試験試料溶液につき、トリプレットで２回実施し、その平均を求めた。また、この実験系にＮＧＦのみを添加（５ｎｇ／ｍｌ）したときの神経突起伸展率は５％程度であった。 <Evaluation of neurite outgrowth promoting action>
Similar to Evaluation B in Experiment 2, PC12-HS cells were diluted with 10 volume% FBS-added D-MEM medium to a 96-well microplate previously coated with collagen at 5 × 10 3 cells / well, and 100 μl / Seeded in wells. After 24 hours, 50 μl / well of each test sample solution adjusted to a compound concentration of 2 μg / ml and 50 μl / well of NGF (final concentration 5 ng / ml) were added, and 10% by volume glutaraldehyde was added at room temperature on the third day of culture. Fixed for 20 minutes. As a control, the cells were cultured for 3 days only in D-MEM medium supplemented with 10% by volume FBS, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the presence or absence of neurite extension was evaluated by the same method as in Experiment 3. The results are also shown in Table 6. The test was performed twice with each triplet for each test sample solution except NK-56 and NK-53, and the average was obtained. Further, when only NGF was added to this experimental system (5 ng / ml), the neurite extension rate was about 5%.
Figure JPOXMLDOC01-appb-C000052
 　一般式３においてＲ７乃至Ｒ９は炭素数が同一で、炭素数が１乃至１２のアルキル基を表し、Ｘ３ －はＩ－又はＣｌ－で、ｍは２を表す。
Figure JPOXMLDOC01-appb-C000052
 In the general formula 3 R 7 to R 9 are the same carbon atoms, an alkyl group having 1 to 12 carbon atoms, X 3 - is I - or Cl - and, m represents 2.
Figure JPOXMLDOC01-appb-T000053
Figure JPOXMLDOC01-appb-T000053
　表６から明らかなように、側鎖のアルキル基の炭素数が３乃至１２で、細胞増殖及び神経突起伸展の促進作用が認められ、３乃至１０でその作用は強くなり、細胞増殖促進作用は４乃至９のものが最も強い活性を示し、神経突起伸展作用は５乃至１０のものが最も強い活性を示した。また、ＮＫ－２４とＮＫ－５６、或いは、ＮＫ－１９とＮＫ－５３とを比較すると、いずれの場合にも、神経突起伸展促進作用の程度に差は認められないことから、ＮＫ－１９類縁体の持つ神経栄養因子様活性には、対アニオンの種類による差異はないと判断した。 As is apparent from Table 6, when the side chain alkyl group has 3 to 12 carbon atoms, the cell growth and neurite outgrowth promoting effects are recognized, and 3 to 10 the action becomes stronger, and the cell growth promoting action is Those with 4 to 9 showed the strongest activity, and those with neurite outgrowth of 5 to 10 showed the strongest activity. In addition, when comparing NK-24 and NK-56, or NK-19 and NK-53, there is no difference in the degree of neurite outgrowth promoting action in either case. The neurotrophic factor-like activity of the body was judged to have no difference depending on the type of counter anion.
　以上の実験結果は、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表される化合物は、神経栄養因子様作用、神経変性抑制作用などの生理機能を持っているので、これらの化合物は抗アルツハイマー病剤として有用であることを物語っている。 The above experimental results show that the compounds represented by general formula 3 such as NK-19, NK-53, and NK-150 have physiological functions such as neurotrophic factor-like action and neurodegeneration inhibitory action. This compound is useful as an anti-Alzheimer's disease agent.
＜実験７：ＮＫ－４類縁体の神経栄養因子様活性＞
　実験６と同様に、ＮＫ－４の類縁体に同様の作用効果があることを確認するために、下記一般式２で表される化合物において、側鎖のアルキル基（Ｒ４乃至Ｒ６）の炭素数が２乃至８で、対アニオンがＩ－である７種類の化合物を合成し、実験３と同様に、ＰＣ１２－ＨＳ細胞に対する細胞増殖促進作用、及び、神経突起伸展促進作用の強さを調べた。すなわち、ＮＫ－４に加えて、表７に示すＮＫ－２３４、ＮＫ－２６、ＮＫ－９８１５、ＮＫ－９６９４、ＮＫ－２８及びＮＫ－１４７の７種類の化合物を、各々ＤＭＳＯに５ｍｇ／ｍｌとなるように溶解した。この溶液をそれぞれ化合物の終濃度が表７又は８に示す濃度となるように、１０容積％ＦＢＳ加Ｄ－ＭＥＭで希釈し試験試料溶液を調製した。また、ＮＫ－１９の類縁体ＮＫ－１３、ＮＫ－３９２、ＮＫ－１９及びＮＫ－１５０をＤＭＳＯに５ｍｇ／ｍｌとなるように溶解し、化合物の濃度が表７又は８に示す濃度となるように１０容積％ＦＢＳ加Ｄ－ＭＥＭで希釈し、試験試料溶液を調製した。なお、試験は、いずれも、各試験試料溶液につきトリプレットで２回実施し、その平均を求めた。細胞増殖促進作用の結果を表７に、神経突起伸展促進作用の結果を表８にそれぞれ併せて示す。 <Experiment 7: Neurotrophic factor-like activity of NK-4 analog>
As in Experiment 6, in order to confirm that the analog of NK-4 has the same action and effect, in the compound represented by the following general formula 2, the side chain alkyl group (R 4 to R 6 ) Seven types of compounds having 2 to 8 carbon atoms and a counter anion of I − were synthesized, and as in Experiment 3, the strength of cell proliferation promoting action and neurite extension promoting action on PC12-HS cells was increased. Examined. That is, in addition to NK-4, seven kinds of compounds shown in Table 7, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, were each added to DMSO at 5 mg / ml. It dissolved so that it might become. This solution was diluted with 10% by volume FBS-added D-MEM so that the final concentrations of the compounds were as shown in Table 7 or 8, respectively, to prepare test sample solutions. In addition, NK-19 analogs NK-13, NK-392, NK-19 and NK-150 are dissolved in DMSO to a concentration of 5 mg / ml, so that the concentration of the compound becomes the concentration shown in Table 7 or 8. Was diluted with 10% by volume FBS-added D-MEM to prepare a test sample solution. Each test was carried out twice with each triplet for each test sample solution, and the average was obtained. Table 7 shows the results of the cell growth promoting action, and Table 8 shows the results of the neurite extension promoting action.
Figure JPOXMLDOC01-appb-C000054
 　一般式２においてＲ４乃至Ｒ６は炭素数が同一で、炭素数が６乃至８のアルキル基を表し、Ｘ２はＩ－で、ｍは２を表す。
Figure JPOXMLDOC01-appb-C000054
 In General Formula 2, R 4 to R 6 represent the same alkyl group having 6 to 8 carbon atoms, X 2 represents I − , and m represents 2.
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-T000056
　表７及び８の結果から明らかなように、一般式２で表されるＮＫ－４類縁体は、側鎖のアルキル基の炭素数が３乃至８化合物で、ＮＫ－４と同等乃至高い細胞増殖及び神経突起伸展の促進作用が認められ、化合物の濃度８０ｎｇ／ｍｌで比較すると、細胞増殖促進作用は側鎖のアルキル基の炭素数が４乃至６の化合物で特に強い作用が認められた。神経突起進展促進作用は側鎖のアルキル基の炭素数が４及び５の化合物で強い活性の発現が認められた。また、一般式３で表され側鎖のアルキル基の炭素数が２乃至８である化合物、とりわけ、一般式３で表され側鎖のアルキル基の炭素数が６乃至８であるＮＫ－１９類縁体化合物は強い細胞増殖及び神経突起伸展の促進活性を示すことが明らかになった。 As is clear from the results in Tables 7 and 8, the NK-4 analog represented by the general formula 2 has a compound having 3 to 8 carbon atoms in the side chain alkyl group, and has a cell growth equivalent to or higher than that of NK-4. In comparison with the compound concentration of 80 ng / ml, the cell growth promoting action was particularly strong for compounds having 4 to 6 carbon atoms in the side chain alkyl group. As for the neurite outgrowth promoting action, a strong activity was observed in the compounds having 4 and 5 carbon atoms in the side chain alkyl group. Further, a compound represented by the general formula 3 wherein the side chain alkyl group has 2 to 8 carbon atoms, particularly an NK-19 analogue represented by the general formula 3 wherein the side chain alkyl group has 6 to 8 carbon atoms. It was revealed that somatic compounds show strong cell proliferation and neurite outgrowth promoting activity.
　なお、具体的なデータは示さないが、ＰＣ－１２ＨＳ細胞に替えて、ラット胎児大脳皮質から調製した初代神経細胞（ニューロン、アストロサイト及びミクログリア細胞）を用い、アミロイドβフラグメント障害及び過酸化水素障害に対するＮＫ－４、ＮＫ－２６、ＮＫ－２３４、ＮＫ－１９及びＮＫ－１５０の影響を調べたところ、これらの化合物は初代神経細胞に対するアミロイドβフラグメント障害及び過酸化水素障害を抑制する活性を有することが判明した。また、これらの化合物は、ＬＰＳ存在下においてミクログリア細胞が産生するＮＯの産生を抑制することが判明した。
＜実験８：ＮＫ－４類縁体のＡｃｈＥ活性阻害作用の比較＞
　既述のごとく、ＡｃｈＥ活性抑制剤はアルツハイマー型認知症治療剤として用いられている。そこで、ＮＫ－４類縁体をアルツハイマー症に適用することを想定しＮＫ－４類縁体のＡｃｈｅ活性阻害作用の強さを比較した。すなわち、実験１１で用いた一般式２で表される化合物の側鎖のアルキル基の炭素数が２乃至５の４種のＮＫ－４類縁体を用い、実験８と同じ方法でＡｃｈＥ活性残存率（％）を測定した。併せて、ＮＫ－１９類縁体のＮＫ－１５０を用いてＡｃｈＥ活性残存率（％）を測定した。その結果及斯かる活性残存率に基づき計算したＩＣ５０値（実験で用いたＡｃｈｅ活性を５０％抑制する化合物濃度）を表９に併せて示す。 Although specific data are not shown, amyloid β fragment disorder and hydrogen peroxide disorder using primary neurons (neurons, astrocytes and microglia cells) prepared from rat fetal cerebral cortex instead of PC-12HS cells. NK-4, NK-26, NK-234, NK-19 and NK-150 were examined for their effects on these cells, and these compounds have activity to inhibit amyloid β fragment damage and hydrogen peroxide damage to primary neurons It has been found. In addition, these compounds were found to suppress the production of NO produced by microglia cells in the presence of LPS.
<Experiment 8: Comparison of AchE activity inhibitory action of NK-4 analog>
As described above, the AchE activity inhibitor is used as a therapeutic agent for Alzheimer-type dementia. Therefore, assuming that the NK-4 analog is applied to Alzheimer's disease, the strength of the Ache activity inhibitory action of the NK-4 analog was compared. That is, using the four types of NK-4 analogs having 2 to 5 carbon atoms in the side chain alkyl group of the compound represented by general formula 2 used in Experiment 11, the remaining AchE activity rate was the same as in Experiment 8. (%) Was measured. In addition, the remaining percentage of AchE activity (%) was measured using NK-19 analog NK-150. Table 9 also shows the IC50 value (compound concentration that inhibits the Ache activity used in the experiment by 50%) calculated based on the results and the residual activity rate.
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000057
　表９から明らかなように、ＮＫ－４類縁体は側鎖のアルキル基の炭素数が増えるにつれてＡｃｈＥ活性の残存率が高くなる傾向を示した。ＮＫ－４及びＮＫ－２３４、とりわけＮＫ－４はＡｃｈＥ活性を強く抑制した。また、ＮＫ－１９類縁体のＮＫ－１５０は、ＮＫ－４類縁体と比較しＡｃｈｅ抑制作用が低いことが判明した。 As is clear from Table 9, the NK-4 analog showed a tendency that the residual ratio of AchE activity increased as the carbon number of the side chain alkyl group increased. NK-4 and NK-234, especially NK-4, strongly suppressed AchE activity. It was also found that NK-19 analog NK-150 has a lower Ache inhibitory effect than NK-4 analog.
　この結果は、ＮＫ－４及びＮＫ－２３４、とりわけＮＫ－４はアルツハイマー型認知症治療剤として有用であることを物語っている。また、Ａｃｈｅ活性抑制剤としてアルツハイマー型認知症治療用に市販されているドネペジル塩酸塩（商品名『アリセプト』）のＡｃｈＥ活性残存率（％）を同じ実験系で測定したところＩＣ５０は０．９μｇ／ｍｌとなったことから、ＮＫ－４はドネペジル塩酸塩とほぼ同等のＡｃｈＥ活性抑制活性を有することを物語っている。 This result indicates that NK-4 and NK-234, especially NK-4, is useful as a therapeutic agent for Alzheimer-type dementia. Further, when the AchE activity remaining rate (%) of donepezil hydrochloride (trade name “Aricept”) marketed for the treatment of Alzheimer-type dementia as an Ache activity inhibitor was measured in the same experimental system, the IC 50 was 0.9 μg. / Ml, NK-4 has almost the same activity of inhibiting AchE activity as donepezil hydrochloride.
＜実験９：ＮＫ－４類縁体及びＮＫ－１９類縁体のヒトアルツハイマー型認知症のモデルマウスに及ぼす影響＞
　既述の実験からＮＫ－４がアルツハイマー型認知症治療剤として利用できる可能性が示唆されたので、本実験ではＮＫ－４類縁体及びＮＫ－１９類縁体のヒトアルツハイマー型認知症のモデルマウスに及ぼす影響を検証した。 <Experiment 9: Effects of NK-4 analog and NK-19 analog on model mice of human Alzheimer type dementia>
The above experiments suggested that NK-4 can be used as a therapeutic agent for Alzheimer's dementia. In this experiment, NK-4 analogs and NK-19 analogs were modeled on human Alzheimer's model mice. The effect was verified.
＜被験試料＞
　被験試料としてＮＫ－４、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９及びＮＫ－１５０を用いた。対照１として生理食塩水（２００μｌ／匹）を投与した。対照２としてドネペジル塩酸塩を用いた。各被験試料はＤＭＳＯにより５ｍｇ／ｍｌの濃度に溶解し、生理食塩水に希釈し投与した。 <Test sample>
NK-4, NK-234, NK-26, NK-19 and NK-150 were used as test samples. As a control 1, physiological saline (200 μl / animal) was administered. Donepezil hydrochloride was used as control 2. Each test sample was dissolved in DMSO to a concentration of 5 mg / ml, and diluted with physiological saline for administration.
＜実験方法＞
　ＩＣＲマウス（日本チャールスリバー社販売、オス、５週齢、体重２５乃至３０ｇ）１００匹を無作為に１０群各１０匹に群分けし、試験終了まで単独飼育した。抱水クロラール（シグマ社販売、３５０ｍｇ／ｋｇ・体重、腹腔内投与）麻酔を施したマウスを、背面固定し、頭部正中切開を加え、骨縫合を確認した後、ブレグマの左側方１．０ｍｍ、後方０．５ｍｍに刺入点を定め、３ｍｍ刺入し、配列表における配列番号１で示すアミノ酸配列を有するアミロイドβフラグメント（β－Ａｍｙｌｏｉｄ２５－３５）溶液９ｎｍｏｌ／６μｌ／匹を脳室内に投与した（投与法は、『Ｂｒａｉｎ　Ｒｅｓｅａｒｃｈ』、第７０６巻、１８１－１９３頁（１９９６年）参照。）。投与には２８ゲージステンレス針（３ｍｍ）を装着したマイクロシリンジを用いた。刺入部位は、予めアミロイドβフラグメント溶液の代わりにエバンスブルー溶液（０．３μｇ／０．３μｌ）を投与し、左右前額断面の側脳室、背側第三脳室、腹側第三脳室などに着色が認められることを確認し決定した。投与後、頭皮を縫合し、翌日より化合物のいずれかを腹腔内に１日１回１３日間投与し、以下に示す方法により行動学的評価をおこなった。その結果と群構成を表１０に示した。 <Experiment method>
100 ICR mice (manufactured by Charles River Japan, male, 5 weeks old, body weight 25 to 30 g) were randomly divided into 10 groups of 10 mice and reared alone until the end of the test. Chloral hydrate (SIGMA, 350 mg / kg, body weight, intraperitoneal administration) Anesthetized mice were fixed on the back, and a midline incision was made on the head. After confirming bone suture, 1.0 mm on the left side of Bregma The insertion point was determined 0.5 mm backward, 3 mm insertion was performed, and 9 nmol / 6 μl / animal solution of amyloid β fragment (β-Amyloid 25-35 ) having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing was placed in the ventricle (Refer to “Brain Research”, 706, 181-193 (1996) for the administration method.) For administration, a microsyringe equipped with a 28 gauge stainless needle (3 mm) was used. Evans blue solution (0.3 μg / 0.3 μl) is administered in advance for the insertion site instead of the amyloid β fragment solution, and the lateral ventricle, dorsal third ventricle, ventral third brain of the left and right forehead cross section It was confirmed that coloring was observed in the room. After the administration, the scalp was sutured, and one of the compounds was intraperitoneally administered once a day for 13 days from the next day, and behavioral evaluation was performed by the following method. The results and group composition are shown in Table 10.
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000058
＜評価法＞
＜新奇物体認識試験＞
　新奇物体認識試験は、マウスの新奇性を好むとい特性を利用したもので、他の多くの学習評価系と異なり人為的な強化因子を用いない。試験は、順化、訓練試行、保持試行の３部門で構成され、アミロイドβフラグメントを脳室内に投与して後６乃至８日目に実施した。床にウッドチップを敷き詰めたオープンフィールドの実験装置（縦４０ｃｍ、横３０ｃｍ、高さ３０ｃｍ）を、約１，０００ルックス（ｌｕｘ）の照明下、雑音のない場所に設置した。まず６日目に、マウスを探索物体の入っていない実験装置中央に入れ、１０分間自由に探索させた（順化）。その２４時間後（７日目）、実験装置内に２種類の物体（ＡとＢ）をそれぞれ側面から１０ｃｍの位置に設置し、マウスを実験装置中央に入れ、１０分間自由に探索させた（訓練試行）。さらにその２４時間後（８日目）、実験装置内に前日探索させた物体Ａを前日の物体Ａ（一度記憶した対象物）と同じ位置に、前日の物体Ｂと異なる物体Ｃ（新しい対象物）を物体Ｂと同じ位置に設置し、マウスを装置の中央に入れ１０分間自由に探索させた（保持試行）。この時、マウスが鼻先を物体に向け、鼻先から物体までの距離が２ｃｍ以内にある時、又はマウスの鼻先が物体に接触している時を物体探索中とみなし、その時間をストップウオッチで計測した。物体識別指数（＝（新しい対象物の探索に費やした時間－一度記憶した対象物の探索に費やした時間）／（新しい対象物の探索に費やした時間＋一度記憶した対象物の探索に費やした時間））を求めた。この場合、物体識別指数は、新規対象物の探索により多く割かれた時間の全探索時間に対する割合であり、一度探索した対象物を動物が記憶していれば物体識別指数の値が大きくなり、記憶していなければ値が小さくなる。 <Evaluation method>
<New object recognition test>
The novel object recognition test uses the characteristics of the mouse when it likes the novelty, and unlike many other learning evaluation systems, it does not use artificial reinforcement factors. The test consisted of three departments: acclimatization, training trials, and retention trials, which were performed 6-8 days after amyloid β fragment was administered into the ventricle. An open field experimental device (40 cm long, 30 cm wide, 30 cm high) with wood chips on the floor was installed in a noise-free place under about 1,000 lux lighting. First, on the 6th day, the mouse was placed in the center of the experimental apparatus without the search object and allowed to search freely for 10 minutes (acclimation). Twenty-four hours later (7th day), two types of objects (A and B) were placed in the experimental apparatus at positions 10 cm from the side, and the mouse was placed in the center of the experimental apparatus and allowed to search freely for 10 minutes ( Training trial). Furthermore, 24 hours later (8th day), the object A searched for in the experiment device the previous day is located at the same position as the object A (the object stored once) on the previous day, and the object C (new object) different from the object B on the previous day. ) Was placed at the same position as the object B, and the mouse was placed in the center of the apparatus and allowed to search freely for 10 minutes (holding trial). At this time, when the mouse pointed at the tip of the nose and the distance from the tip of the nose to the object was within 2 cm, or when the nose of the mouse was in contact with the object, the object search was considered and the time was measured with a stopwatch. . Object identification index (= (time spent searching for a new object−time spent searching for an object once stored) / (time spent searching for a new object + searching for an object stored once) Time)). In this case, the object identification index is the ratio of the time allotted to the search for new objects to the total search time, and if the animal stores the object once searched, the value of the object identification index becomes large, If not stored, the value becomes smaller.
＜受動的回避試験＞
　動物が一度経験した嫌悪刺激（電気刺激）に対して示す回避行動を記憶の指標とするもので、マウスが暗室を好む性質を利用したステップスルー型を採用した。明室と暗室が扉でつながった装置の明室側にマウスを入れた時の暗室側への移動時間を記憶の指標とした。受動的回避試験はアミロイドβフラグメントを脳室内に投与して後９乃至１２日目に実施した。９日目に明室（１，０００ルックス、縦３０ｃｍ、横３０ｃｍ、高さ１５ｃｍ）に１分間、暗室（縦３０ｃｍ、横３０ｃｍ、高さ１５ｃｍ）に２分間入れ順化させた。１０日目も同様に順化を行った。１１日目の訓練試行で、まず明室の中央にマウスを入れ、マウスが暗室内に移動すると同時に明室と暗室のとの間の扉を閉め、電気刺激を与えた（０．８ｍＡ、１秒）。２４時間後（１２日目）、前日と同様に再び明室の中央にマウスを入れ、暗室への移動時間（秒）を受動的回避的反応として測定した。通電による嫌悪刺激を記憶していれば受動的回避的反応が長くなる。 <Passive avoidance test>
A step-through type using the property that the mouse prefers the dark room is adopted as an index of memory, which is the avoidance behavior shown to the aversive stimulation (electrical stimulation) once experienced by the animal. The movement time to the dark room side when the mouse was put in the light room side of the device where the light room and dark room were connected by a door was used as an index of memory. The passive avoidance test was performed 9 to 12 days after amyloid β fragment was administered into the ventricle. On the ninth day, the light room (1,000 lux, length 30 cm, width 30 cm, height 15 cm) was placed in a dark room (length 30 cm, width 30 cm, height 15 cm) for 2 minutes to acclimatize. The acclimatization was similarly performed on the 10th day. In the training trial on the 11th day, the mouse was first placed in the center of the light room, and at the same time the mouse moved into the dark room, the door between the light room and the dark room was closed, and electrical stimulation was applied (0.8 mA, 1 Seconds). After 24 hours (day 12), the mouse was placed in the center of the light room again as in the previous day, and the moving time (seconds) to the dark room was measured as a passive avoidance response. If the aversive stimulus by energization is memorized, the passive avoidance reaction becomes longer.
　表１０から明らかなように、ＮＫ－４を５００μｇ／ｋｇ・体重、ＮＫ－２３４を５００μｇ／ｋｇ・体重、ＮＫ－２６を５０μｇ／ｋｇ・体重、ＮＫ－１９を５００μｇ／ｋｇ・体重及びＮＫ－１５０を５００μｇ／ｋｇ・体重投与マウスでは、アミロイドβフラグメントのみ投与マウスに比べて、新奇物体認識試験で有意の改善が認められた。また、ＮＫ－４を５０μｇ／ｋｇ・体重、ＮＫ－４を５００μｇ／ｋｇ・体重、ＮＫ－２３４を５００μｇ／ｋｇ・体重、ＮＫ－２６を５０μｇ／ｋｇ・体重、ＮＫ－２６を５００μｇ／ｋｇ・体重、及び、ＮＫ－１９を５００μｇ／ｋｇ・体重投与マウスでは、アミロイドβフラグメントのみを投与マウスに比べて、受動的回避試験で有意の改善が認められた。なかでも、ＮＫ－４を５００μｇ／ｋｇ・体重投与した群では、新奇物体認識試験及び受動的回避試験の両方で、ドネペジル塩酸塩１，０００μｇ／ｋｇ・体重投与した群（対照２）よりも著明な改善が認められた。なお、試験中ＮＫ－４、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９或いはＮＫ－１５０の投与に起因すると思われる副作用は認められなかった。 As is apparent from Table 10, NK-4 is 500 μg / kg · body weight, NK-234 is 500 μg / kg · body weight, NK-26 is 50 μg / kg · body weight, NK-19 is 500 μg / kg · body weight and NK− In mice administered with 150 at 500 μg / kg · body weight, a significant improvement was observed in the novel object recognition test compared to mice administered with amyloid β fragment alone. Also, NK-4 is 50 μg / kg · body weight, NK-4 is 500 μg / kg · body weight, NK-234 is 500 μg / kg · body weight, NK-26 is 50 μg / kg · body weight, NK-26 is 500 μg / kg · body weight. Significant improvement was observed in the passive avoidance test in mice administered with body weight and NK-19 at 500 μg / kg / body weight, compared with mice administered with amyloid β fragment alone. In particular, in the group administered NK-4 at 500 μg / kg / body weight, both the novel object recognition test and the passive avoidance test were more marked than the group treated with donepezil hydrochloride at 1,000 μg / kg / body weight (control 2). A clear improvement was observed. During the study, no side effects believed to be caused by administration of NK-4, NK-234, NK-26, NK-19 or NK-150 were observed.
　この結果は、ＮＫ－４、ＮＫ－２３４、ＮＫ－２６、ＮＫ－１９及びＮＫ－１５０は、いずれもアミロイドβペプチドに起因する認知障害を改善する作用を有し、この実験条件において、試験に用いた４種類の化合物の中ではＮＫ－４が最も強い認知障害改善作用を有していることを物語っている。 As a result, NK-4, NK-234, NK-26, NK-19, and NK-150 all have an action to improve cognitive impairment caused by amyloid β peptide. Of the four compounds used, NK-4 has the strongest cognitive impairment improving effect.
＜実験１０：ＮＫ－４のＡＰＰトランスジェニックマウスに及ぼす影響＞
　実験９でアミロイドβフラグメントを投与したマウスの認知障害改善作用の最も強かったＮＫ－４につき、スウェーデン型アルツハイマー病の原因遺伝子変異を導入した市販のＡＰＰトランスジェニックマウス（ＡＰＰ　Ｔｇマウス）に及ぼす影響を調べた。すなわち、ＡＰＰ　Ｔｇマウス（Ｔａｃｏｎｉｃ社販売、メス、１０週齢、体重１５乃至２３ｇ）４５匹を１０日間予備飼育した後、体重が均等になるように、４群に分け、単独飼育とし、ＮＫ－４を腹腔内に週５回、１２週間投与した。対照１として遺伝子変異を導入していないマウス（野生型、メス、１０週齢、体重１５乃至２３ｇ）１０匹を１０日間予備飼育後、単独飼育とし、生理食塩水を腹腔内に週５回、１２週間投与した。対照２としてＡＰＰ　Ｔｇマウスに生理食塩水（２００μｌ／匹）を週５回、１２週間投与した。対照３としてＡＰＰ　Ｔｇマウスにドネペジル塩酸塩を週５回、１２週間投与した。ＮＫ－４、生理食塩水或いはドネペジル塩酸塩投与１２週目に、実験１５と同じ方法で、最初に新奇物体認識試験、次に受動的回避試験を行い、引き続き下記方法による水迷路試験を行った。その結果と群構成を表１１に示す。 <Experiment 10: Effect of NK-4 on APP transgenic mice>
The effect of NK-4, which had the strongest effect on improving cognitive impairment in mice administered amyloid β fragment in Experiment 9, on commercially available APP transgenic mice (APP Tg mice) into which a causative gene mutation of Swedish Alzheimer's disease was introduced. Examined. Specifically, 45 APP Tg mice (taconic, female, 10 weeks old, body weight 15 to 23 g) were preliminarily reared for 10 days, then divided into 4 groups so that the body weights were equalized, and were reared alone, and NK- 4 was administered intraperitoneally 5 times a week for 12 weeks. As a control 1, 10 mice (wild type, female, 10 weeks old, body weight 15 to 23 g) that were not introduced with a genetic mutation were preliminarily raised for 10 days, and then alone, and physiological saline was intraperitoneally injected 5 times a week. Administered for 12 weeks. As control 2, physiological saline (200 μl / mouse) was administered to APP Tg mice 5 times a week for 12 weeks. As a control 3, APP Tg mice were administered donepezil hydrochloride 5 times a week for 12 weeks. At the 12th week after administration of NK-4, physiological saline or donepezil hydrochloride, a novel object recognition test and then a passive avoidance test were first conducted in the same manner as in Experiment 15, followed by a water maze test by the following method. . The results and group composition are shown in Table 11.
Figure JPOXMLDOC01-appb-T000059
Figure JPOXMLDOC01-appb-T000059
　＜水迷路試験法＞
　直径１３０ｃｍの円形プールに、白色インクで着色した水を深さ２０ｃｍまで満たし、水槽用ヒーターで水温を２３±１℃に維持した。プールを４分割し一画の中央に、プールの側面から１０ｃｍの位置に避難用のプラットホームを水面下２ｃｍになるように設置した。このプラットホームの位置は、試験終了まで一定の場所とした。受動的回避試験終了の翌日よりマウスをプールの側面に向けて水面上に放ち、水面下に隠れたプラットホームに到着するまでの時間を計測した。スタート位置は、プールを４分割したいずれかの画分の中央部、壁面より１０ｃｍ離れた場所とし、試行毎にランダムに変更した。２分間自由にプラットホームを探索させた後、マウスが２分以内にプラットホームに到着できなかった場合は、プラットホームへ誘導し、３０秒間プラットホームに留まらせた後、ペーパータオルを敷いたケージに移した。２回目の試験は、１回目の試験終了１分後に開始した。この試験を４日間連続で行ない、２回の試行の平均値を１日の値とした。 <Water maze test method>
A circular pool with a diameter of 130 cm was filled with water colored with white ink to a depth of 20 cm, and the water temperature was maintained at 23 ± 1 ° C. with a water tank heater. The pool was divided into four parts, and an evacuation platform was installed at a position 10 cm from the side of the pool so as to be 2 cm below the surface of the water. The platform position was fixed until the end of the test. From the day after the end of the passive avoidance test, the mouse was released on the surface of the pool toward the side of the pool, and the time taken to reach the platform hidden under the surface of the water was measured. The starting position was 10 cm away from the central part of one of the fractions of the pool divided into 4 centimeters, and was randomly changed for each trial. After searching the platform freely for 2 minutes, if the mouse could not reach the platform within 2 minutes, it was guided to the platform, allowed to stay on the platform for 30 seconds and then transferred to a cage with paper towels. The second test started 1 minute after the end of the first test. This test was conducted for 4 consecutive days, and the average value of the two trials was taken as the value of the day.
　表１１から明らかなように、ＮＫ－４を投与したマウスは、物体識別指数、受動的回避反応、水迷路試験のいずれにおいても、ＡＰＰ　Ｔｇマウスの認知障害を顕著に改善した。また、その改善作用は、市販のアルツハイマー型認知症治療剤ドネペジル塩酸塩より有意に強かった。この結果は、ＮＫ－４がアルツハイマー型認知障害の治療剤として利用できることを強く示唆している。なお、試験中ＮＫ－４の投与に起因すると思われる副作用は認められなかった。 As is apparent from Table 11, the mice administered NK-4 markedly improved the cognitive impairment of APP Tg mice in any of the object identification index, passive avoidance reaction, and water maze test. Moreover, the improvement effect was significantly stronger than that of commercially available Alzheimer-type dementia therapeutic agent donepezil hydrochloride. This result strongly suggests that NK-4 can be used as a therapeutic agent for Alzheimer-type cognitive impairment. During the study, no side effects believed to be caused by administration of NK-4 were observed.
　以上の実験１乃至１１の結果から、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表され、側鎖のアルキル基の炭素数が３乃至１０の化合物と同様に、一般式２で表され、側鎖のアルキル基の炭素数が２乃至８個の化合物は、神経栄養因子様作用、神経変性抑制作用などの生理機能を持ち、アミロイドβフラグメントによる細胞傷害を抑制する活性も有しているので、これらの化合物はいずれも抗アルツハイマー病治療剤として有用であることを物語っている。また、これらの化合物の中では、一般式２で表され側鎖のアルキル基の炭素数が２乃至４である化合物であるＮＫ－４、ＮＫ－２３４、ＮＫ－２６、とりわけＮＫ－４が特に強いアルツハイマー型認知障害の改善効果を有していることを物語っている。 From the results of the above experiments 1 to 11, similar to the compounds represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., wherein the side chain alkyl group has 3 to 10 carbon atoms, the general formula The compound having 2 to 8 carbon atoms of the alkyl group in the side chain has a physiological function such as a neurotrophic factor-like action and a neurodegeneration inhibitory action, and also has an activity of suppressing cell damage caused by amyloid β fragment. Therefore, all of these compounds are useful as anti-Alzheimer's disease therapeutic agents. Among these compounds, NK-4, NK-234, NK-26, particularly NK-4, which are compounds represented by the general formula 2 and in which the side chain alkyl group has 2 to 4 carbon atoms, are particularly preferred. It shows that it has an effect of improving strong Alzheimer type cognitive impairment.
　以下、本発明の抗アルツハイマー病剤について、実施例により説明するが、本発明はこれら実施例に何ら限定されるものではない。 Hereinafter, the anti-Alzheimer's disease agent of the present invention will be described with reference to examples, but the present invention is not limited to these examples.
＜注射用の液剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１７０ｇに、有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－９６９４（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々１２ｍｇ溶解した溶液とを混合し、溶存する酸素の濃度が約０．１ｐｐｍになるまで窒素ガスをバブリングして、褐色アンプルに１ｍｌずつ分注し、窒素気流下でアンプルを封止した後、濾過滅菌又は高圧滅菌した。本品は、いずれもパイロジェンフリーであり、抗アルツハイマー病剤として利用できる。 <Liquid preparation for injection>
A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, NK-4 (compound represented by Chemical Formula 2), NK-9694 as an active ingredient in 170 g of purified water for injection (Compound represented by chemical formula 1), NK-28 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 7), NK-147 (expressed by general formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 8 carbon atoms), NK-19 (a compound represented by Chemical Formula 4), NK-53 (a compound represented by Chemical Formula 5), NK- 150 (compound represented by chemical formula 3), NK-393 (compound in which the side chain alkyl group (R) of the compound represented by general formula 3 has 8 carbon atoms), NK-100 (compound represented by chemical formula 6) Compound) NK-528 (expressed by chemical formula 7) Compound), NK-557 (compound represented by chemical formula 8), and NK-1516 (compound represented by chemical formula 9) (all manufactured by Hayashibara Biochemical Laboratories, Inc.) After mixing 12 mg each of the dissolved solutions, bubbling nitrogen gas until the dissolved oxygen concentration was about 0.1 ppm, dispensing 1 ml each into a brown ampule, and sealing the ampule under a nitrogen stream, Filter sterilized or autoclaved. All of these products are pyrogen-free and can be used as anti-Alzheimer's agents.
　これらの製剤を使用して、アミロイドβフラグメント投与マウス（アルツハイマー病モデル）に対する治療効果を確認した。 These therapeutic agents were used to confirm the therapeutic effects on mice administered with amyloid β fragment (Alzheimer's disease model).
＜アミロイドβフラグメント投与マウス（アルツハイマー病モデル）に対する効果＞
　ＩＣＲマウス（日本チャールスリバー社販売）６５匹を無作為に５匹ずつ、１３群に分けた。実験２で使用した配列表における配列番号１で表されるアミノ酸配列を有するアミロイドβフラグメントを、３７℃で４日間エイジングさせて、６５匹のマウス全ての脳室内に５μｇ／匹投与した。アミロイドβフラグメント投与後１日目から表１２に示すように、１２群各５匹（試験群１乃至１２）には、実施例１で調製した１２種類の化合物のいずれかを有効成分として含有する製剤のいずれか１種を、１日１回９日目まで毎日、０．３ｍｌ／匹、腹腔内投与した（試験群１乃至１２）。残りの１群５匹には、滅菌したマルトースの１０％水溶液（パイロジェンフリー）を、１日１回９日目まで毎日、０．３ｍｌ／匹、腹腔内投与した（対照群）。アミロイドβフラグメント投与６日目から１０日目まで、マウスの行動を観察して、その期間中に何らかの行動異常が認められた個体数の割合（％）を表１２に併せて示す。また、投与１０日目に、マウスを解剖して脳を採取し、常法により脳の組織標本を作製し、アミロイドβフラグメントの沈着（凝集）の有無を確認するためにコンゴーレッド染色及びチオフラビンＴ染色を行った。染色した標本を蛍光顕微鏡で観察し、対照を投与した場合に比して、組織の蛍光強度が明らかに弱い（２）、蛍光強度が少し弱い（１）、差がない（０）、蛍光強度が少し強い（－１）、蛍光強度が明らかに強い（－２）の５段階で評価してスコア化し、各試験群５匹のマウスの平均を求めて、アミロイドβフラグメントの蓄積の程度として表１２に併せて示す。 <Effects on mice treated with amyloid β fragment (Alzheimer's disease model)>
65 ICR mice (available from Charles River Japan Co., Ltd.) were randomly divided into 13 groups, each of 5 mice. The amyloid β fragment having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing used in Experiment 2 was aged at 37 ° C. for 4 days, and 5 μg / mouse was administered into the ventricles of all 65 mice. As shown in Table 12 from the first day after administration of the amyloid β fragment, each of the 12 groups, 5 animals (test groups 1 to 12) contains any of the 12 compounds prepared in Example 1 as active ingredients. Any one of the formulations was intraperitoneally administered once a day until the ninth day, daily at 0.3 ml / animal (test groups 1 to 12). The remaining 5 animals in 1 group were intraperitoneally administered with a 10% aqueous solution of sterilized maltose (pyrogen-free) once a day until the 9th day (control group). The behavior of mice was observed from day 6 to day 10 after administration of amyloid β fragment, and the ratio (%) of the number of individuals in which some behavioral abnormality was observed during that period is also shown in Table 12. On day 10 of administration, mice were dissected and brains were collected, brain tissue samples were prepared by conventional methods, and congo red staining and thioflavin T were used to confirm the presence or absence of amyloid β fragment deposition (aggregation). Staining was performed. The stained specimen is observed with a fluorescence microscope, and the fluorescence intensity of the tissue is clearly weaker (2), the fluorescence intensity is slightly weaker (1), and there is no difference (0), compared with the case where the control is administered. Is scored by rating in 5 stages, with a slightly strong (-1) and a clearly strong fluorescence intensity (-2), and an average of 5 mice in each test group was obtained and expressed as the degree of amyloid β fragment accumulation. 12 is also shown.
Figure JPOXMLDOC01-appb-T000060
Figure JPOXMLDOC01-appb-T000060
　表１２から明らかなように、実施例１で調製した１２種類の製剤は、いずれも、アミロイドβフラグメント投与マウスの異常行動の発現を抑制すると共に、脳内へのアミロイドβフラグメントの蓄積を抑制した。１２種類の製剤を投与した場合の効果の強さを比較すると、いずれの試験においても、ジメチン系スチリル色素化合物（ＮＫ－５２３、ＮＫ－５５７、及び、ＮＫ－１５１６）を含有する製剤を投与した場合（実験群１０乃至１２）よりも、ペンタメチン系シアニン色素化合物（ＮＫ－４、ＮＫ－９６９４、ＮＫ－２８、ＮＫ－１４７、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０、ＮＫ－３９３、Ｋ－１００、Ｋ－５２８、Ｋ－５５７、及び、ＮＫ－１５１６）を投与した場合（実験群１乃至９）の方が、アミロイドβフラグメント投与による行動異常やフラグメントの脳内蓄積に対して強い改善効果が認められた。 As is clear from Table 12, all of the 12 preparations prepared in Example 1 suppressed the expression of abnormal behavior in amyloid β fragment-administered mice and also suppressed the accumulation of amyloid β fragments in the brain. . Comparing the strength of the effect when 12 kinds of preparations were administered, in any test, preparations containing dimethine styryl dye compounds (NK-523, NK-557, and NK-1516) were administered. More than the case (experimental groups 10 to 12), pentamethine cyanine dye compounds (NK-4, NK-9694, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K -100, K-528, K-557, and NK-1516) (Experimental groups 1 to 9) improved behavioral abnormalities caused by amyloid β fragment administration and accumulation of fragments in the brain The effect was recognized.
＜注射用の液剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１６８ｇに、レシチン２ｇと有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が３である化合物）、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々１２０ｍｇ溶解した溶液とを混合し、溶存する酸素の濃度が約０．０５ｐｐｍになるまで窒素ガスをバブリングして、褐色アンプルに１ｍｌずつ分注し、窒素気流下でアンプルを封止した後、濾過滅菌又は高圧滅菌した。本品は、いずれもパイロジェンフリーであり、抗アルツハイマー病剤として利用できる。 <Liquid preparation for injection>
A solution in which 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) is dissolved in 370 g of purified water for injection, and 168 g of purified lecithin for injection, 2 g of lecithin and an active ingredient, NK-4 (compound represented by chemical formula 2), NK-234 (compound in which the alkyl group (R) in the side chain of the compound represented by general formula 2 has 3 carbon atoms), NK-26 (compound represented by chemical formula 1), NK-28 (general formula 2), NK-147 (the number of carbon atoms in the side chain alkyl group (R) of the compound represented by the general formula 2 is 7). 8), NK-19 (compound represented by chemical formula 4), NK-53 (compound represented by chemical formula 5), NK-150 (compound represented by chemical formula 3), NK-393 (general The side chain alkyl group of the compound represented by Formula 3 ( )), NK-100 (compound represented by chemical formula 6), NK-528 (compound represented by chemical formula 7), NK-557 (compound represented by chemical formula 8), and , NK-1516 (compound represented by Chemical Formula 9) (both manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were mixed with a solution in which 120 mg each was dissolved, and the dissolved oxygen concentration was about 0. Nitrogen gas was bubbled to 0.05 ppm, and 1 ml was dispensed into brown ampules. After the ampules were sealed under a nitrogen stream, they were sterilized by filtration or autoclaved. All of these products are pyrogen-free and can be used as anti-Alzheimer's agents.
　実施例２で調製した１３種類の抗神経変性疾患剤を、各々、１０匹のｄｄｙマウス（平均体重２５．６ｇ）に、０．３ｍｌ／匹単回投与して、投与後１週間、毎日体重を測定しながら、経過を観察したところ、対照として１０匹のｄｄｙマウス（平均体重２６．３ｇ）に、０．２％のレシチンを含む１０％マルトース溶液を投与して、経過を観察した場合に比して、体重に優位な変化は認められず、他に外観的な変化も認められなかった。この結果は、実施例２で調製した１３種類の抗アルツハイマー病剤の有効成分として配合した化合物のＬＤ５０は、いずれも８．６ｍｇ／ｋｇ・体重以上となるので、これらの製剤がヒトに投与しても安全な製剤であることを物語っている。 Each of the 13 types of anti-neurodegenerative disease agents prepared in Example 2 was administered to 10 ddy mice (average body weight 25.6 g) in a single dose of 0.3 ml / body, and daily body weight for 1 week after administration. As a control, a 10% maltose solution containing 0.2% lecithin was administered to 10 ddy mice (average body weight 26.3 g) as a control, and the course was observed. In comparison, no significant change in body weight was observed, and no other change in appearance was observed. As a result, the LD 50 of the compound formulated as an active ingredient of the 13 types of anti-Alzheimer's disease agents prepared in Example 2 is 8.6 mg / kg · body weight or more, so these preparations were administered to humans. It shows that it is a safe formulation.
＜注射用の粉末剤＞
　注射用精製水３７０ｇに注射用精製マルトース（株式会社林原製造）６０ｇを溶解した溶液と、注射用精製水１７０ｇに、ポリソルベイト８０（日本油脂株式会社販売）３ｇと、有効成分として、ＮＫ－４（化学式２で表される化合物）、ＮＫ－２３４（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が３である化合物）、ＮＫ－２６（化学式１で表される化合物）、ＮＫ－２８（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が７である化合物）、ＮＫ－１４７（一般式２で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１９（化学式４で表される化合物）、ＮＫ－５３（化学式５で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）、ＮＫ－３９３（一般式３で表される化合物の側鎖のアルキル基（Ｒ）の炭素数が８である化合物）、ＮＫ－１００（化学式６で表される化合物）ＮＫ－５２８（化学式７で表される化合物）、ＮＫ－５５７（化学式８で表される化合物）、及び、ＮＫ－１５１６（化学式９で表される化合物）（いずれも株式会社林原生物化学研究所製造）のいずれか１種を、各々６０ｍｇ溶解した溶液とを混合して濾過滅菌後、褐色アンプルに１０ｍｌずつ分注し、常法により凍結乾燥後、窒素気流下でアンプルを封止した。本品は、いずれもパイロジェンフリーであり、用時に、アンプルに注射用精製水乃至生理食塩水２乃至１０ｍｌを加えて溶解し、点滴静注、皮下投与、腹腔内投与などの方法で使用する。本品は、いずれもパイロジェンフリーであり、抗アルツハイマー病剤として利用できる。 <Powder for injection>
A solution prepared by dissolving 60 g of purified maltose for injection (manufactured by Hayashibara Co., Ltd.) in 370 g of purified water for injection, 170 g of purified water for injection, 3 g of polysorbate 80 (sold by NOF Corporation), Compound represented by chemical formula 2), NK-234 (compound in which the carbon number of the alkyl group (R) in the side chain of the compound represented by general formula 2 is 3), NK-26 (represented by chemical formula 1) Compound), NK-28 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 7 carbon atoms), NK-147 (a compound in the side chain of the compound represented by Formula 2) A compound in which the alkyl group (R) has 8 carbon atoms), NK-19 (a compound represented by Formula 4), NK-53 (a compound represented by Formula 5), NK-150 (a compound represented by Formula 3) Compound), NK-393 (general formula 3) A compound in which the carbon number of the alkyl group (R) in the side chain of the compound is 8), NK-100 (a compound represented by Chemical Formula 6), NK-528 (a compound represented by Chemical Formula 7), NK— A solution in which 60 mg of each of 557 (compound represented by chemical formula 8) and NK-1516 (compound represented by chemical formula 9) (both manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) were dissolved, After sterilizing by filtration, 10 ml each was dispensed into brown ampules, freeze-dried by a conventional method, and sealed in a nitrogen stream. All of these products are pyrogen-free. At the time of use, 2 to 10 ml of purified water for injection or physiological saline is added to the ampoule and dissolved, and then used by methods such as intravenous infusion, subcutaneous administration, and intraperitoneal administration. All of these products are pyrogen-free and can be used as anti-Alzheimer's agents.
　本発明の抗アルツハイマー病剤は、アルツハイマー病の予防、治療及び／又は進展抑制、又は、アミロイドβペプチドなどのアルツハイマー病の原因となる神経細胞傷害因子からの神経細胞保護に有用である。また、アルツハイマー病に伴う認知障害や種々の病態や神経機能障害（例えば、振戦、固縮、無動、寡動、動作緩慢、姿勢反射障害、自律神経障害、突進現象、歩行障害、うつ、記憶障害、筋萎縮、筋力低下、上肢機能障害、構音障害、嚥下障害、呼吸障害、しびれ及び麻痺等）の改善にも有用である。しかも、本発明の抗アルツハイマー病剤は、長期間投与しても、副作用がないので、安全性が高く、安心して利用することができる。本発明は、斯くも顕著な作用効果を奏する発明であり、斯界に多大の貢献をする、誠に意義のある発明である。 The anti-Alzheimer's disease agent of the present invention is useful for preventing, treating and / or suppressing the progression of Alzheimer's disease or protecting nerve cells from neuronal cytotoxic factors that cause Alzheimer's disease such as amyloid β peptide. Also, cognitive impairment associated with Alzheimer's disease and various pathological and neurological dysfunctions (eg tremor, rigidity, agitation, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory Disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis). In addition, the anti-Alzheimer's disease agent of the present invention has no side effects even when administered for a long period of time, so it is highly safe and can be used with peace of mind. The present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

Claims (9)

  1. 　一般式１で表される化合物を有効成分として含有する抗アルツハイマー病剤。
    Figure JPOXMLDOC01-appb-C000001
     （一般式１におけるＲ１乃至Ｒ３は、それぞれ独立に、水素原子又は適宜の置換基を表し、Ｚ１は複素環を、また、Ｚ２はＺ１と同じか異なる複素環又は芳香環を表し、それらの複素環及び芳香環は置換基を有していてもよい。ｏは０又は１、２のいずれかである整数を表し、ｐは、０又は１のいずれかである整数を表し、ｏが０又は２のとき、ｐは１であり、ｏが１のとき、ｐは０である。ｏが０の場合、Ｒ１、Ｒ２は存在せず、ｐが０の場合、Ｒ３は存在せず、Ｒ２が結合する炭素とＺ２とは一重結合となる。Ｘｌ －は適宜の対アニオンを表し、ｑは１又は２のいずれかである整数を表す。）     An anti-Alzheimer's disease agent comprising a compound represented by the general formula 1 as an active ingredient.
    Figure JPOXMLDOC01-appb-C000001
     (R 1 to R 3 in General Formula 1 each independently represents a hydrogen atom or an appropriate substituent, Z 1 represents a heterocyclic ring, and Z 2 represents a heterocyclic ring or an aromatic ring that is the same as or different from Z 1. And the heterocyclic ring and the aromatic ring may have a substituent, o represents an integer of 0, 1 or 2, and p represents an integer of 0 or 1. , O is 0 or 2, p is 1, and when o is 1, p is 0. When o is 0, R 1 and R 2 do not exist, and when p is 0, R 3 is not present, the single bond between carbon and Z 2 wherein R 2 is attached .X l - represents a suitable counter anion, q is an integer of either 1 or 2).
  2. 　一般式１で表される化合物が、一般式２乃至５のいずれかで表される化合物である請求の範囲第１項記載の抗アルツハイマー病剤。
    Figure JPOXMLDOC01-appb-C000002
     （一般式２において、Ｒ４乃至Ｒ６は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ２ －は適宜の対アニオンを表す。）
    Figure JPOXMLDOC01-appb-C000003
     （一般式３において、Ｒ７乃至Ｒ９は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ３ －は適宜の対アニオンを表す。）
    Figure JPOXMLDOC01-appb-C000004
     （一般式４において、Ｒ１０乃至Ｒ１２は互いに同じか異なる脂肪族炭化水素基を表す。Ｘ４ －は適宜の対アニオンを表す。）
    Figure JPOXMLDOC01-appb-C000005
     （一般式５において、Ｚ３は複素芳香環を表し、その複素芳香環は置換基を有していてもよい。Ｚ４は芳香環又は複素芳香環を表し、それらの複素芳香環及び芳香環は置換基を有していてもよい。Ｒ１３は脂肪族炭化水素基を表し、その脂肪族炭化水素基は置換基を有していてもよい。Ｒ１４は水素原子又は適宜の置換基を、また、Ｘ５ －は適宜の対アニオンを表す。）     The anti-Alzheimer's disease agent according to claim 1, wherein the compound represented by the general formula 1 is a compound represented by any one of the general formulas 2 to 5.
    Figure JPOXMLDOC01-appb-C000002
     (In General Formula 2, R 4 to R 6 represent the same or different aliphatic hydrocarbon groups. X 2 — represents an appropriate counter anion.)
    Figure JPOXMLDOC01-appb-C000003
     (In General Formula 3, R 7 to R 9 represent the same or different aliphatic hydrocarbon groups. X 3 — represents an appropriate counter anion.)
    Figure JPOXMLDOC01-appb-C000004
     (In General Formula 4, R 10 to R 12 represent the same or different aliphatic hydrocarbon groups. X 4 — represents an appropriate counter anion.)
    Figure JPOXMLDOC01-appb-C000005
     (In General Formula 5, Z 3 represents a heteroaromatic ring, and the heteroaromatic ring may have a substituent. Z 4 represents an aromatic ring or a heteroaromatic ring, and these heteroaromatic ring and aromatic ring R 13 represents an aliphatic hydrocarbon group, the aliphatic hydrocarbon group may have a substituent, and R 14 represents a hydrogen atom or an appropriate substituent. in addition, X 5 - represents a suitable counter anion).
  3. 　一般式１乃至５のいずれかで表される化合物の対アニオンが、沃素イオン又は塩素イオンである請求の範囲第１項又は第２項記載の抗アルツハイマー病剤。 The anti-Alzheimer's disease agent according to claim 1 or 2, wherein the counter anion of the compound represented by any one of the general formulas 1 to 5 is an iodine ion or a chlorine ion.
  4. 　一般式２で表される化合物が化学式２で表される化合物であり、一般式３で表される化合物が化学式３で表される化合物である請求の範囲第２項記載の抗アルツハイマー病剤。
    Figure JPOXMLDOC01-appb-C000006
    Figure JPOXMLDOC01-appb-C000007
         The anti-Alzheimer's disease agent according to claim 2, wherein the compound represented by the general formula 2 is a compound represented by the chemical formula 2, and the compound represented by the general formula 3 is a compound represented by the chemical formula 3.
    Figure JPOXMLDOC01-appb-C000006
    Figure JPOXMLDOC01-appb-C000007
  5. 　製剤学的に許容される１種又は２種以上の成分を含んでなる請求の範囲第１項乃至第４項のいずれかに記載の抗アルツハイマー病剤。 The anti-Alzheimer's disease agent according to any one of claims 1 to 4, comprising one or more pharmaceutically acceptable components.
  6. 　製剤学的に許容される成分が、水性媒体である請求の範囲第５項記載の抗アルツハイマー病剤。 6. The anti-Alzheimer's disease agent according to claim 5, wherein the pharmaceutically acceptable ingredient is an aqueous medium.
  7. 　神経が小脳プルキンエ細胞である請求の範囲第１項乃至第６項のいずれかに記載の抗アルツハイマー病剤。 The anti-Alzheimer's disease agent according to any one of claims 1 to 6, wherein the nerve is a cerebellar Purkinje cell.
  8. 　神経変性疾患が、パーキンソン病、アルツハイマー病、認知症、脊髄小脳変性症、脳梗塞又は運動失調症である請求の範囲第１項乃至第７項のいずれかに記載の抗アルツハイマー病剤。 The anti-Alzheimer's disease agent according to any one of claims 1 to 7, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, dementia, spinocerebellar degeneration, cerebral infarction or ataxia.
  9. 　抗アルツハイマー病剤が、神経細胞変性抑制剤、神経突起伸展促進剤、神経細胞保護剤、又は、神経細胞変性に伴う運動失調症改善剤である請求の範囲第１項乃至第８項のいずれかに記載の抗アルツハイマー病剤。
          9. The any one of claims 1 to 8, wherein the anti-Alzheimer's disease agent is a neuronal degeneration inhibitor, a neurite outgrowth promoting agent, a neuronal cell protective agent, or an ataxia ameliorating agent associated with neuronal degeneration. Anti-Alzheimer's disease agent as described in.
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