WO2010087313A1 - Neurite elongation stimulator - Google Patents

Neurite elongation stimulator Download PDF

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Publication number
WO2010087313A1
WO2010087313A1 PCT/JP2010/050923 JP2010050923W WO2010087313A1 WO 2010087313 A1 WO2010087313 A1 WO 2010087313A1 JP 2010050923 W JP2010050923 W JP 2010050923W WO 2010087313 A1 WO2010087313 A1 WO 2010087313A1
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WIPO (PCT)
Prior art keywords
compound represented
general formula
test
group
neurite
Prior art date
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PCT/JP2010/050923
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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
Original Assignee
株式会社林原生物化学研究所
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Priority to JP2010548503A priority Critical patent/JPWO2010087313A1/en
Publication of WO2010087313A1 publication Critical patent/WO2010087313A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • 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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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
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    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
    • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
    • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
    • C07D421/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings
    • C07D421/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the present invention relates to a neurite outgrowth promoter comprising a compound represented by 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.
  • Neurodegenerative diseases are pathological conditions caused by systematic neuronal degeneration and disruption of the neural network based on loss, and many intractable diseases such as Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, cerebrovascular dementia, frontal side Known as lobar dementia, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, spinocerebellar degeneration, and the like.
  • 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.
  • neurodegenerative diseases vary depending on the disease, but they vary from minor to severe. Typical examples include tremor, rigidity, agitation, peristalsis, and movement. Slowness, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness or paralysis, etc. Both are major obstacles in daily life.
  • Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are serious diseases that cause degeneration of nerve cells, and various compounds are used as active ingredients to improve these diseases and their associated pathological conditions and neurological dysfunction.
  • International Publication No. WO97 / 030703, JP-A-11-228417, JP-A-2006-143708, and JP-A-2006-321737 See, for example, International Publication No. WO97 / 030703, JP-A-11-228417, JP-A-2006-143708, and JP-A-2006-321737.
  • an accelerator or the like has been proposed (see, for example, JP-A-2002-234841), an effective disease treatment method has not yet been found.
  • systemic administration such as subcutaneous or intravascular, has a low physical and mental burden on the patient, acts on nerve cells of the central nervous system, activates nerve cell bodies, suppresses neurite atrophy, or Therefore, development of a novel neurite outgrowth promoter capable of promoting neurite outgrowth and ameliorating neurological degeneration or dysfunction associated with neurodegeneration is eagerly desired.
  • An object of the present invention is to provide a novel neurite extension promoter.
  • the present invention mainly comprises a neurite outgrowth promoter containing 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.
  • the neurite outgrowth promoting agent of the present invention promotes neurite outgrowth and activates nerve function in various administration routes of oral administration and parenteral administration, and various symptoms associated with nerve dysfunction (for example, , Tremor, stiffness, immobility, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, swallowing Disorder, breathing problems, numbness and paralysis).
  • nerve dysfunction for example, Tremor, stiffness, immobility, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, swallowing Disorder, breathing problems, 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 reduction, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
  • the neurite extension promoter of the present invention contains 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.
  • 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.
  • R 7 to R 9 represent the same or different aliphatic hydrocarbon groups.
  • X 3 - represents an appropriate counter anion.
  • R 10 to R 12 represent the same or different aliphatic hydrocarbon groups.
  • X 4 - represents an appropriate counter anion.
  • 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 neurite outgrowth promoting action.
  • 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,
  • examples of the compound represented by the general formula 2 include 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-9694”).
  • NK-4 a compound having 4 carbon atoms in the side chain alkyl group (R4 to R6) of the general formula 2 (hereinafter, sometimes referred to as “NK-26”) and the like.
  • Examples thereof include compounds in which the alkyl group (R4 to R6) in the side chain of Formula 2 has 3 carbon atoms (hereinafter sometimes referred to as “NK-234”).
  • 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).
  • 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.
  • R 7 to R 9 represent the same alkyl group having 7 to 9 carbon atoms, and mX 3 — represents I 2 — or Cl 2 — .
  • 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.
  • NK-234 and NK-26 represented by any one of chemical formulas 1 to 9 are all cytotoxic factors such as starvation, radicals, and amyloid ⁇ peptide in addition to the nerve activation action and neurite outgrowth promoting action From the point of strength of its action and effect, it is more desirable as an active ingredient of the neurite outgrowth promoting agent of the present invention.
  • NK-9694 (a compound represented by Chemical Formula 1)
  • NK-150 a compound represented by Chemical Formula 3
  • NK-4, NK-234 and NK-26 is desirable and NK-4 is particularly desirable.
  • the compound represented by the general formula 1 used as an active ingredient of the neurite outgrowth promoter of the present invention is not limited in its origin or production method.
  • the neurite outgrowth promoter 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 represented by general formula 5. It contains one or more styryl dyes.
  • the neurite outgrowth promoting agent of the present invention if necessary, in addition to the compound represented by the general formula 1 which is an active ingredient, is pharmaceutically acceptable in the food field, cosmetic field, pharmaceutical field, quasi-drug It is provided in the form of a preparation containing one or more components used in the product 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.
  • a neurite outgrowth promoting agent having an active ingredient other than the compound represented by the general formula 1 and a therapeutic agent for neurodegenerative diseases, pathological conditions and neurological dysfunctions resulting therefrom can be advantageously implemented.
  • cerebrovascular disorders eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, arachnoid membrane) Etc.), brain tumors (eg, astrocytoma, brain abscess, etc.), blood volume reducing shock, traumatic shock, head injury and / or cerebrospinal trauma (eg, brain contusion, penetration, shear) ⁇
  • therapeutic agents for neurological dysfunction associated with compression / laceration, labor trauma, infant whiplash, etc. neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease
  • therapeutic agents for neurodegenerative diseases used in combination with the anti-neurodegenerative disease agent of the present invention and the pathological conditions and neurological dysfunctions resulting therefrom preferably, for example, cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombus) , Cerebral embolism, etc.), transient cerebral ischemic attack, cerebral hemorrhage (eg hypertensive intracerebral hemorrhage, subarachnoid hemorrhage, etc.), brain tumor treatment agent, cerebrospinal trauma (eg cerebral contusion, etc.)
  • Therapeutic agents for neurological dysfunction associated with neurodegenerative diseases eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration), motor neuropathy (eg muscle atrophy)
  • Therapeutic agents for lateral sclerosis, etc. therapeutic agents for demyelinating diseases (eg, multiple sclerosis), cerebrospinal diseases associated with infections (eg, men
  • Therapeutic agents for diseases eg, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, etc.
  • therapeutic agents for amyotrophic lateral sclerosis therapeutic agents for multiple sclerosis, Neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.
  • epilepsy and / or dystonia diabetes, diabetes Complication treatment and / or hyperlipidemia treatment agent, dopamine receptor agonist, dopamine release promoter, dopamine uptake inhibitor, dopamine agonist, central anticholinergic agent, aromatic L-amino acid decarboxylase Inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholinesterase inhibitor, NMDA (N-methyl-D-asparagine) Acid) receptor antagonist, ⁇ -secretase inhibitor, ⁇
  • the neurite extension promoter of the present invention is usually provided in the form of a parenteral injection preparation or the like. Moreover, when parenteral administration is difficult, it may be used in the form of an oral preparation.
  • the compound represented by the general formula 1, which is an active ingredient, is a process from the raw material stage to the completion of the product in consideration of the composition of the target injection preparation and oral preparation and the purpose of use. 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 methods such as loading are appropriately selected.
  • the neurite outgrowth promoting agent of the present invention in the case of a preparation for oral administration, depending on the target disease or symptom, for example, solution, syrup, mass kit, paste, powder, solid, Any shape such as granules, tablets, etc. may be used as it is, or as necessary, mixed with a filler, excipient, binder, etc. to form granules, spheres, short bars, plates, cubes It is also optional to use it in various shapes such as tablets and capsules.
  • 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.
  • solubility in a solvent or solubility in an aqueous medium when there is a problem with solubility in a solvent or solubility in an aqueous medium, or when preparing a sustained-release preparation, it is effective to use an amphiphilic solvent, an oily base material, an emulsifier, etc. 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 used, it may become unstable due to dissolved oxygen or the like. In that case, for example, if the dissolved oxygen concentration of the compound solution is reduced, Good.
  • 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.
  • 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.
  • the compound solution is prepared and stored under reduced pressure, or the oxygen dissolved in the compound solution is separated from another gas. Or a method of bringing the compound solution into contact with an oxygen scavenger is preferred.
  • 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 neurite outgrowth promoting agent of the present invention can be used in the form of an oral preparation, a haptic agent, a 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 neurite outgrowth promoting agent of the present invention produced in this way is a safe preparation without serious side effects even when used for a long time.
  • the daily dose of the neurite outgrowth promoting agent of the present invention is not particularly limited as long as the desired action and effect can be obtained.
  • it is represented by the general formula 1.
  • 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.
  • the compound represented by general formula 1, which is an active ingredient of the neurite outgrowth promoting agent of the present invention is generally administered subcutaneously in view of its low absorption rate from the digestive tract. It is necessary to set a higher dose than the case.
  • the neurite outgrowth promoting agent of the present invention in addition to the neurite outgrowth promoting action, protects the brain and nerve cells from damaging factors and suppresses degeneration, activates the nerve cells, inhibits neurite atrophy, Since neuronal survival and degeneration can be suppressed, neurodegenerative diseases, particularly diseases caused by degeneration of the central nervous system can be treated.
  • a neurodegenerative disease is a nerve cell (central nervous system (for example, cranial nerve, spinal nerve), and / or peripheral nerve (for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system) )
  • central nervous system for example, cranial nerve, spinal nerve
  • peripheral nerve for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system)
  • any disease that is generally regarded as a neurodegenerative disease may be used.
  • Parkinson's disease Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea-ataxia, progressive Supranuclear paralysis, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobar dementia, familial dementia, spinocerebellar degeneration (eg olive Bridge cerebellar atrophy, late cerebellar cortical atrophy, familial spinocerebellar ataxia (eg, Maccard Joseph disease, etc.), dentate nucleus erythrocytic Ryukyu atrophy, familial spastic paraplegia, Friedreich Disease), motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple sclerosis, multiple sclerosis, acute disseminated) Encephalomyelitis, acute encephalitis, transverse myelitis,
  • neurodegenerative diseases that are preferable as the neurite extension promoter of the present invention include, for example, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration, amyotrophic lateral sclerosis, Demyelinating diseases (for example, multiple sclerosis), cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombosis, cerebral embolism, etc.), transient cerebral ischemic attacks, cerebral hemorrhage (for example, hypertensive intracerebral hemorrhage) ), Brain tumors, traumatic shock, head injury and / or cerebrospinal trauma (eg, cerebral contusion), cerebrospinal disease (eg, meninges) Inflammation, influenza encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy, etc.), diseases derived from neurodegeneration of the central nervous system such as epilepsy, more preferably For
  • the neurite outgrowth promoting agent of the present invention can treat nerve dysfunction by activating nerve cells, extending neurites, and promoting synapse formation.
  • the target neurological dysfunction may be any neurological dysfunction, such as cognitive dysfunction, consciousness disorder, bilateral quadriplegia, contralateral hemiplegia, alternating hemiplegia. , Sensory disorders, transient blindness (eg, transient cataract), homonymous half-blindness, dizziness, nystagmus, double vision, aphasia, tinnitus, coma and the like. Particularly preferred are those neurological dysfunctions associated with the neurodegenerative diseases.
  • the neurological dysfunction associated with the above-mentioned neurodegenerative diseases for example, neurological dysfunction associated with cerebral infarction varies depending on the site of vascular occlusion, and the symptoms vary depending on the level to be impaired. It can be seen.
  • the presence or absence of neurological dysfunction in cerebral infarction may be determined by various diagnostic tests known in the art for detecting neurological dysfunction. Specific examples of the diagnostic test include, for example, a cognitive function score (Alzheimer's Dissease Assessment-cognitive part; ADAS-cog) used for evaluation of memory and cognitive impairment due to Alzheimer's disease, clinical symptoms, and the like.
  • a cognitive function score Alzheimer's Dissease Assessment-cognitive part
  • the neurite outgrowth promoting agent of the present invention is advantageous as a nerve cell protective agent, nerve cell activator, Purkinje cell degeneration / dropout inhibitor, therapeutic agent for neurodegenerative diseases and associated pathologies, therapeutic agent for neurological dysfunction, etc.
  • Can be used for The treatment of neurodegenerative diseases and neurological dysfunctions as used in the present invention refers to the pathology and dysfunction caused by neurodegeneration in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses progression and stops progression of the disease, Also includes the prevention of the onset of the disease itself.
  • NGF Neuro Growth Factor
  • PC-12HS cells NGF (Neural Growth Factor) hypersensitive strain of PC-12 cells derived from rat adrenal pheochromocytoma
  • PC-12HS cells which is a suitable model for studying human neurite outgrowth promoting action (Obtained from Human Science Research Resource Bank) was diluted with a 10 volume% FBS-added D-MEM medium to a collagen-coated 96-well microplate at 5 ⁇ 10 3 cells / well and seeded at 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. 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 are observed under a microscope to evaluate the presence or absence of neurite outgrowth.
  • the ratio of cells with neurite outgrowth is 30% or more, it is determined that there is a strong neurite outgrowth promoting action. 1 was marked with a circle.
  • the percentage of cells in which neurite outgrowth was observed was determined by observing cells under a microscope at a magnification containing about 100 cells in one field of view, and showing neurites at least twice the major axis of the cell body. The number of cells was counted, divided by the total number of cells in the same field, and multiplied by 100.
  • NGF NGF
  • ⁇ Experiment 2 Effect of pigment compound concentration on neurite outgrowth> Among the compounds determined to have a strong neurite outgrowth promoting effect in Experiment 1, the concentrations of NK-19, NK-53, NK-100, and NK-557 are related to neurite outgrowth of PC12-HS cells. The effect was examined using the same method as in Experiment 1. That is, the culture is continued by adding any one of NK-4, NK-19, NK-53, NK-100, and NK-557 so that the final concentration of the compound in the medium becomes the concentration shown in Table 2. The cells were fixed with glutaraldehyde. Table 2 shows the results of microscopic observation at a magnification including about 100 cells in one field, and determining the percentage (%) of cells in which neurite outgrowth was observed with respect to the total number of cells.
  • NK-19 which was confirmed to have a neurite outgrowth promoting action, was tested to confirm that its analogs had the same action. That is, a compound in which the alkyl group (R) in the side chain of the compound represented by the following general formula 3 has 1 to 12 carbon atoms (synthesized by Hayashibara Biochemical Laboratories Co., Ltd.) was synthesized. The strength of the neurite outgrowth promoting action on PC-12HS cells was examined using the proportion of cells in which neurite outgrowth was observed as an index.
  • NK-19 Twelve types of analogs of NK-19 including NK-19 shown in Table 3 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, and the concentration of the compound was diluted to 2 ⁇ g / ml to prepare a test sample solution. 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 D-MEM medium supplemented with 10% by volume FBS, and the test sample solutions were prepared by diluting the compound concentration to 2 ⁇ g / ml each.
  • 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 seeded at 100 ⁇ l / well. did. After 24 hours, 50 ⁇ l / well of each test sample solution adjusted to a compound concentration of 2 ⁇ g / ml and 50 ⁇ l / 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.
  • PC12-HS cells were cultured for 3 days only in 10% by volume FBS-added D-MEM medium, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the cells were counted by the same method as in Experiment 1 to determine the percentage (%) of cells in which neurite extension was observed. The results are also shown in Table 3. In addition, the test was implemented twice with each triplet for each test sample solution, and the average was obtained. In addition, when only NGF was added to this experimental system (5 ng / ml), the proportion of cells in which neurite outgrowth was observed was about 5%.
  • R 7 to R 9 represent the same alkyl group having 7 to 9 carbon atoms, and mX 3 — represents I 2 — or Cl 2 — .
  • NK-19 analog a compound having 3 to 12 carbon atoms in the side chain alkyl group has a neurite outgrowth promoting action, and the action is strong at 3 to 10; 10 having the structure represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., wherein the alkyl group in the side chain has 3 to 10 carbon atoms.
  • the compound is useful as a neurite outgrowth promoting agent, indicating that it can be used as a therapeutic agent for neurodegenerative diseases including Alzheimer's disease and cerebellar ataxia.
  • the neurite outgrowth promoting action of the NK-19 analog was not different depending on the type of counter anion.
  • NK-4 seven kinds of compounds shown in Table 4, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, each in 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 4 or 5, 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 compound concentration becomes the concentration shown in Table 4 or 5.
  • Table 4 shows the results of the cell growth promoting action
  • Table 5 shows the results of the neurite extension promoting action.
  • R 4 to R 6 represent the same alkyl group having 1 to 12 carbon atoms
  • X 2 represents I ⁇
  • m represents 2.
  • the NK-4 analog represented by the general formula 2 is a cell having 3 to 8 carbon atoms in the side chain alkyl group, which is the same or higher than NK-4. Growth and neurite outgrowth promoting effects were observed. When compared at a 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.
  • NK-19 analog compound was found to show strong cell proliferation and neurite outgrowth promoting activity.
  • NK-4 and its analogs can be used as neurite outgrowth promoters and therapeutic agents for neurodegenerative diseases, so human neurodegenerative diseases (such as spinocerebellar degeneration) Of cerebellar degenerative ataxia (hereinafter referred to as “cerebellar ataxia”) hamster (hereinafter referred to as “cerebellar ataxia hamster”) suitable as a model animal of NK-4, and its effects on the behavior and brain tissue The influence of was investigated.
  • pulmonary Purkinje cells were shed after 3 weeks of age, followed by spontaneous gene mutations known to spontaneously develop ataxia and cerebellar ataxia after 7 weeks of age ( Nna1 inhibition) 25 hamsters (see Akita K. et al., “J. Neurogenetics”, Volume 21, pages 1 to 11 (2007), reared at Hayashibara Biochemical Research Institute, Inc.) Test groups 1-5 were randomly assigned 5 animals in each group. Test group 1 hamsters were administered with 10 ml / kg / day of PBS before the onset of cerebellar ataxia (3 weeks of age).
  • the hamsters in test groups 2 to 4 were administered NK-4 at 20 ⁇ g / kg, 100 ⁇ g / kg, or 500 ⁇ g / kg / day before the onset of cerebellar ataxia (3 weeks of age).
  • the hamster of Test Group 5 is abbreviated as “IGF-1”, an insulin-like trophic factor-1 known as a neuronal trophic factor (sold by Assaypro, trade name “IGF-1, human”, human origin). ) was administered at 25 ⁇ g / kg / day. These administration components were administered intraperitoneally once daily until 10 weeks of age.
  • the degree of cerebellar ataxia and the effect of NK-4 on improving the symptoms were evaluated by using the rotarod test and slope endurance test described later once a week, with the improvement of hamster motor coordination as an index. Furthermore, at the age of 10 weeks, after measuring the number of hamster falls, the brain was removed and histologically evaluated, and the glutamic acid concentration in blood and cerebrospinal fluid (CSF) was also measured. As test group 6, 5 normal hamsters of the same age as the cerebellar ataxia hamster used in test groups 1 to 5 were treated with PBS at 10 ml / kg / day before the onset of cerebellar ataxia (3 weeks old). The same test as that of the cerebellar ataxia hamster was carried out by intraperitoneal administration once a day until 10 weeks of age.
  • ⁇ Rotarod test> The hamster walked with the rotation of the rotarod and used the duration of the exercise to stay on the rotarod as an indicator of motor coordination. Specifically, a hamster was placed on a rotarod apparatus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., rotarod diameter 60 mm) rotating at a constant speed (6 rpm), and the time until it dropped from the rotarod was measured (Fernandez et al., “Proc. Natl. Acad. Sci. USA, 95, 1253-1258 (1998)). The test is conducted 6 times for one hamster, and the first 5 times is a preliminary motion test for acclimatization to the rotational motion.
  • ⁇ Slope durability test> Determine the angle at which the hamster's head is placed on a plate whose tilt angle can be changed and can rest for 5 seconds, and the slope endurance angle (Rivlin et al., “J. Neurosurg.”, Vol. 47, Vol. 577-581 (1997)). The tilt angle started from 25 degrees and increased by 5 degrees. If the vehicle falls in less than 5 seconds, subtract the angle at 1 degree intervals from the tilt angle, determine the angle at which it can stand for 5 seconds, measure the slope endurance tilt angle, The average was calculated. The results are shown in Table 8.
  • cerebellar ataxia hamster used in this test is known to have a reduced cell density in addition to Purkinje cells, which are inhibitory neurons, and granule cells, which are excitatory neurons. Therefore, cerebellar slices cut in the sagittal direction were stained with hematoxylin and eosin by a conventional method, and microscopically measured to determine the total number of Purkinje cells in the Purkinje cell layer (lobules I to X) and the number of granule cells per unit area. In addition, the number of individuals with demyelination in the cerebellar white matter was confirmed. The results are also shown in Table 9. In addition, since the cerebral volume did not recognize a significant difference between each test group, Table 9 shows only the calculation result of the cerebellum volume.
  • test group 4 (500 ⁇ g / kg ⁇ body weight), a significant effect of reducing the drop time was observed compared to test group 1. This inhibitory effect lasted until the end of the study at 10 weeks of age (7 weeks of administration). Even in Test Group 2 (20 ⁇ g / kg / body weight), after 2 weeks (5 weeks old) after the start of NK-4 administration, the effect of reducing the drop time was observed more than in Test Group 1, but Test Group 3 and Compared with test group 4, the effect was weak. In addition, when IGF-1 which is considered to be effective in the treatment of motor neurodegenerative diseases was administered (Test Group 5), the effect of suppressing the fall time was hardly observed.
  • NK-4 can be used as a therapeutic agent for human neurodegeneration and its pathological conditions and symptoms.
  • test group 6 normal hamster
  • test group 1 using cerebellar ataxia hamsters.
  • PBS administration group it was already significantly lower at 410.6 ⁇ 0.3 degrees at the start of the test, decreased with aging, further decreased after 8 weeks of age, and 35. It was 8 ⁇ 1.0 degrees.
  • test group 5 (IGF-1 administration)
  • 44.4 ⁇ 0.2 degrees at 4 weeks of age which is a significant slope durability inclination angle compared to 40.6 ⁇ 0.3 degrees in test group 1
  • the slope durability inclination angle decreased as in Test Group 1
  • no significant decrease suppressing effect was observed after 5 weeks of age.
  • test groups 2 to 4 (NK-4 administration)
  • no decrease in endurance tilt angle was observed throughout the test period at any dose used in the test, and a high slope endurance tilt angle decrease suppression effect was observed. It was.
  • the slope endurance slopes of the test groups 2 to 4 (NK-4 administration) at 10 weeks of age are 45.8 ⁇ 0.6, 51.6 ⁇ 0.6, and 51.8 ⁇ 0.4 degrees, respectively. Both were significantly higher than those in Test Group 1 (PBS administration) and Test Group 5 (IGF-1 administration).
  • test group 1 PBS administration
  • test group 6 IGF-1 administration
  • test group 5 IGF-1 administration
  • a significant cerebellar atrophy suppression effect was observed as compared to test group 1, which was 77.6 ⁇ 6.1 (mm 3 ).
  • a dose-dependent cerebellar atrophy suppression effect was also observed in the NK-4 administration group, and 76.0 ⁇ 8.2, 77.0 ⁇ 2.8, 80 in the 20, 100, and 500 ⁇ g / kg administration groups, respectively. 5 ⁇ 10.8 mm 3 (all significant at P ⁇ 0.05).
  • NK-4 improves motor coordination of cerebellar ataxia hamsters, effectively suppresses cerebellar atrophy, and its effect is superior to IGF-1. .
  • the granule cell density in the granule cell layer of the cerebellar cortex is 480 ⁇ 6 / 20,000 ⁇ m 2 in 10-week-old normal hamsters (test group 6).
  • test group 6 When PBS was administered to a cerebellar ataxia hamster (test group 1), the number was significantly reduced to 380 ⁇ 4/20000 ⁇ m 2 .
  • the granule cell density in test group 5 (IGF-1 administration) was 371 ⁇ 11 cells / 20,000 ⁇ m 2, which was not different from test group 1.
  • test groups 2 to 4 (NK-4 administration), the doses were 408 ⁇ 8, 419 ⁇ 6, 436 ⁇ 7 / 20,000 ⁇ m 2 , respectively, and an inhibitory effect on the decrease in granule cell density depending on the dose of NK-4 was observed. It was.
  • specific data are not shown, in microscopic observation of the cerebellar parenchyma, in the test groups 1 and 5, granule cell atrophy and degeneration were significant, whereas in the test groups 2 to 4, granule cells were observed. It was confirmed that atrophy and degeneration were suppressed.
  • NK-4 cerebellar ataxia hamster
  • NK-4 administration test groups 2 to 4 (NK-4 administration)
  • NK-4 Purkinje cells Suppression of atrophy of neurites (dendrites) in the body was also confirmed.
  • NK-4 is useful as a neurite extension promoter.
  • NK-4 also shows that it is useful as an inhibitor of cerebellar white matter demyelination, that is, as an inhibitor of Purkinje cell degeneration and shedding.
  • NK-4 administered intraperitoneally acted on cerebral neurons and suppressed cerebellar ataxia by suppressing neurite outgrowth (including suppression of atrophy) and suppressing neuronal function decline.
  • NK-4 is useful as a neurite outgrowth promoter as a therapeutic agent that can be systemically administered to human neurodegeneration and various pathological conditions and clinical symptoms associated therewith.
  • the body weight of the cerebellar ataxia hamster was measured once every week until the end of the test (10 weeks of age), and the average of each group was determined.
  • PBS, NK-4, IGF-1 administration group Since no significant difference was observed, NK-4 was judged to be a highly safe compound even when administered to a living body.
  • NK-4, NK-19, NK-53, NK-100, and NK-557 have been confirmed to have therapeutic effects on neurodegeneration.
  • atropine (fuso medicine, 0.3 mg / kg / body weight) was subcutaneously administered, and urethane (Sigma, 600 mg / kg / body weight) and ⁇ -chloralose (Sigma, 60 mg / kg / body weight) were intraperitoneally injected. Anesthesia was given, and it was fixed to a fixator with natural breathing. A midline cervical incision was made and the right carotid bifurcation was reached, paying attention to the preservation of the vagus nerve.
  • the common carotid artery and the external carotid artery were peeled off from the surrounding connective tissue, centering on the right carotid artery bifurcation, and ligated with 6-0 nylon yarn (Alfresser Pharma Co., Ltd., trade name “Nesscoacher”).
  • a 6-0 nylon thread was applied to the internal carotid artery to prepare for fixation after embolization.
  • the common carotid artery was incised, and an embolus (Doccol) made of 4-0 nylon thread coated with silicon at the tip was inserted into the internal carotid artery about 16 mm and fixed to the common carotid artery with a clip.
  • the tip portion of the embolus that is silicon-coated enters the anterior cerebral artery beyond the middle cerebral artery bifurcation and closes the middle cerebral artery entrance.
  • the cerebral artery is occluded for 2 hours on a thermal pad at 37 ° C., then the inserted embolus is removed, blood flow is resumed (reperfusion), and then bleeding from the common carotid artery incision is prevented. Therefore, the internal carotid artery was ligated near the carotid bifurcation. Since the right common carotid artery is ligated in this model, the blood flow is resumed from the left inner diameter artery, the vertebral artery, and the basilar artery via the anterior / posterior traffic artery.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test were each dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then the membrane. Filtration (sold by Millipore, trade name “Millex-LG SLLG025SS”, using DMSO resistant membrane) was performed. Each compound was dissolved in PBS to 25 ng / ml at the time of use, and any one of them was administered to 5 or 7 rats in 5 groups each 1 hour after middle cerebral artery occlusion and blood reperfusion.
  • DMSO product number “D8418”
  • test groups 1 to 5 Behavioral and histological evaluation was performed 24 hours after resumption of blood flow. Of the remaining 2 groups, 5 out of 5 animals, 1 group had 6 ml of PBS containing no compound as the control group 1 in the tail vein at 1 ml after middle cerebral artery occlusion and at the time of blood reperfusion. Administered.
  • control group 2 In addition, in the remaining 5 animals in 1 group, as control group 2, after ligation of the common neck, external carotid, and internal carotid artery, sham operation was resumed without inserting an embolus near the middle cerebral artery ( (Sham operation) was performed.
  • PBS containing no compound was also administered into the tail vein at 4 ml / kg ⁇ body weight, 1 hour after ligation of the common neck, external neck, and internal carotid artery, and at the time of blood reperfusion.
  • each rat was exsanguinated by severing the posterior vena cava while perfusing physiological saline from the left ventricle under ether anesthesia.
  • the brain is removed within 3 minutes after death, and sliced to a thickness of 2 mm in the coronal direction using a slicer (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.), and then the cerebral infarction site is specifically stained.
  • 2,3,5-triphenyltetrazole chloride (TTC) was incubated in PBS containing 2% by mass / volume at 37 ° C.
  • NK-4, NK-19, NK-53, NK-100 and NK-557 are effective in treating neurodegeneration including neurite atrophy caused by ischemia and associated neurological dysfunction. It tells you that it has an effect.
  • NK-4 analog or NK-19 analog Effect of NK-4 analog or NK-19 analog on rat model of cerebral infarction>
  • the effects of NK-4, NK-26, and NK-15, which were confirmed to have cell proliferation promoting action and neurite promoting action on PC-12HS cells, on cerebral infarction were examined using human cerebral infarction model rats. That is, according to Experiment 6, SD rats (manufactured by Charles River Japan, male, 8 weeks old, body weight 280 to 330 g) were embolized in the middle cerebral artery.
  • NK-150, NK-26, and NK-4 were each dissolved in DMSO at a concentration of 5 mg / ml, filtered through a membrane filter with a pore size of 0.45 ⁇ m, and further 2.5 to 0.05 mg / ml in DMSO. What was adjusted to the concentration was stored in the dark.
  • These compound solutions were diluted 250-fold with saline at the time of use and administered to the embolized rat through the tail vein twice after 1 hour of occlusion and at the time of reopening (fluid amount: 5 ml / kg / body weight).
  • NK-4 was prepared as a 10 mg / ml solution, diluted 250 to 167 times, and administered from the tail vein (fluid volume: 5 ml / kg / body weight).
  • physiological saline was administered as a negative control
  • an existing drug edaravone manufactured by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”
  • DMSO DMSO
  • a behavioral score was obtained by the same method as in Experiment 6 to evaluate neurological symptoms.
  • the actual volume value was obtained by dividing by the swelling rate.
  • the results and group composition are shown in Table 12.
  • 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.
  • the amyloid ⁇ fragment ( ⁇ -Amyloid 25-35 : Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met amino acid was determined by setting the insertion point 0.5 mm behind.
  • Peptide having sequence 9 nmol / 6 ⁇ l / animal of solution was administered into the ventricle (for the administration method, see “Brain Research”, 706, 181-193 (1996)).
  • a microsyringe equipped with a 28 gauge stainless needle (3 mm) was used for administration.
  • 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 13.
  • ⁇ 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 9th 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 are all against neurodegeneration including neurite atrophy caused by amyloid ⁇ peptide and neurological dysfunction associated therewith. It has a therapeutic effect.
  • 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. During the study, no side effects believed to be caused by administration of NK-4 were observed.
  • NK-4 Effects of NK-4 on cerebrovascular dementia model mice>
  • the above-mentioned experiments confirmed that NK-4 is effective in improving dyskinesia and Alzheimer-type cognitive impairment resulting from cerebral infarction.
  • mice 50 mg / kg were intraperitoneally administered and anesthetized, and a permanent ligation operation was performed on the right common carotid artery (refer to Japanese Patent Application Laid-Open No. 2008-193941 for the operation method). After the operation, all mice were reared alone, and were bred with free eating and drinking water. Of the 21 animals subjected to ligation surgery, 10 animals were reared as they were (ligation group), and the remaining 11 animals were administered NK-4 (administration group). In addition, 10 animals that had not undergone surgery were used as controls (no surgery group).
  • NK-4 has a therapeutic effect on neurodegeneration including neurite atrophy caused by ischemia and associated neurological dysfunction.
  • the above results are similar to those of the compounds having the structure represented by general formula 3, such as NK-19, NK-53, NK-150, etc., and having a side chain alkyl group having 3 to 10 carbon atoms.
  • the compound having the structure represented by 2 and having 2 to 8 carbon atoms in the side chain alkyl group is useful as a neurite extension promoter. These compounds all improve neurite atrophy in ischemia due to cerebral infarction and vascular injury, Alzheimer's disease, cerebellar ataxia, Parkinson's disease, etc., and cognitive impairment due to neurite atrophy or decrease And that it can improve various clinical symptoms.
  • the compound represented by the general formula 2 is desirable, and NK— in which the side chain alkyl group has 2 to 4 carbon atoms. 4, NK-234 and NK-26 were desirable, and NK-4 was particularly desirable.
  • ⁇ 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
  • All of these products are pyrogen-free and can be used as neurite outgrowth promoters.
  • this product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, or a therapeutic agent for pathological conditions or neurological dysfunction associated with neurodegeneration.
  • the remaining 10 animals in a group were intraperitoneally administered with a 10% aqueous solution of sterile maltose (pyrogen-free) from 3 weeks to 10 weeks of age, once daily for 56 days, daily at 0.5 ml / mouse. (Control group).
  • the day after the end of the administration period the weight of each hamster was measured in the same manner as in Experiment 5, and the rotarod test, the slope endurance test, and the number of falls were measured.
  • Table 16 shows the types of compounds that are active ingredients of the preparations administered to each group and the measurement results.
  • the average body weight at 3 weeks of hamster was 35.4 g
  • the average body weight at 10 weeks of age was 122.9 g
  • Table 16 shows only the rotarod test, the slope endurance test, and the measurement results of the number of falls.
  • a hamster cerebellar tissue specimen was prepared in the same manner as in Experiment 4, and it was determined by microscopic observation whether or not the cerebellar Purkinje cell atrophy was suppressed compared to the control group. The results are also shown in Table 16.
  • Example 1 As is clear from Table 16, the 12 preparations prepared in Example 1 all suppressed neurite (dendrites) atrophy of cerebellar Purkinje cells in a 10-week-old cerebellar ataxia hamster, The drop time from the rod, the slope endurance slope angle and the number of falls were significantly improved. Comparing the strength of the effect when 12 types of preparations were administered, in any test, preparations containing dimethine styryl dye compounds (NK-523, NK-557, and NK-1516) were administered.
  • NK-523, NK-557, and NK-1516 dimethine styryl dye compounds
  • the pentamethine cyanine dye compounds (NK-4, NK-9694, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K When -100, K-528, K-557, and NK-1516) were administered (Experimental groups 1 to 9), a stronger effect of improving motor coordination was observed. When the strength of the effect was improved among the pentamethine-based cyanine dye compounds, NK-4, NK-26, NK-9694, MK-150 and NK-393 were particularly effective in improving motor coordination. This result indicates that any of the prepared preparations is useful as a therapeutic agent for neurodegenerative diseases. Moreover, even if these preparations were administered for 56 days, the body weight of the hamster was not significantly different from that of the control group.
  • NK-19 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, and 2 g of lecithin and an active ingredient NK-4 (compound represented by Chemical Formula 2) in 170 g of purified water for injection, NK-234 (a compound in which the alkyl group (R) in the side chain of the compound represented by the general formula 2 has 3 carbon atoms), NK-26 (an alkyl group in the side chain of the compound represented by the general formula 2 ( R) is a compound having 4 carbon atoms), NK-9694 (a compound represented by Chemical Formula 1), NK-28 (a compound having a general formula 2 having a side chain alkyl group (R) having a carbon number of 7), NK-147 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 8 carbon atoms), NK-19 (a
  • Each of the 14 types of neurite outgrowth promoters prepared in Example 2 was administered to 10 ddy mice (average body weight 25.6 g) in a single dose of 0.3 ml / animal, and the body weight was daily for one 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 the active ingredient of the 12 types of neurite outgrowth promoting agents prepared in Example 2 is 8.6 mg / kg ⁇ body weight or more. It shows that it is safe to administer.
  • ⁇ 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, 3 g of polysorbate 80 (sold by Nippon Oil & Fats Co., Ltd.) in 170 g of purified water for injection, and NK-4 ( A compound represented by Chemical Formula 2), NK-234 (a 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 (a compound represented by General Formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 4 carbon atoms), NK-9694 (a compound represented by the chemical formula 1), NK-28 (a compound represented by the general formula 2) Compounds in which the alkyl group (R) has 7 carbon atoms), NK-147 (compounds in which the alkyl group (R) in the side chain of the compound
  • This product can be used as a neurite extension promoter.
  • the product can also be used as a neurode
  • ⁇ Capsule> Capsules containing the following components per tablet were prepared.
  • the mixture of the above component A was kneaded with an 8% aqueous solution of polyvinyl pyrrolidone K-90, dried at 60 ° C., pulverized, mixed with component B, filled into capsules at 150 mg per capsule, Capsule
  • the neurite outgrowth promoting agent of the present invention is used for the prevention, treatment and / or progression inhibition of Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, dementia, or stroke caused by neurodegeneration of nerve cells, or nerve from neurodegeneration.
  • Useful for cell protection for various pathological conditions and neurological dysfunctions associated with neurodegenerative diseases (for example, tremor, rigidity, ataxia, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, It is also useful for improving muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis.
  • the neurite outgrowth promoting 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 confidence.
  • the present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

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Abstract

Disclosed is a novel neurite elongation stimulator.  The neurite elongation stimulator 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

神経突起伸展促進剤Neurite outgrowth promoter
　本発明は、一般式１で表される化合物を有効成分とする神経突起伸展促進剤に関するものである。 The present invention relates to a neurite outgrowth promoter comprising a compound represented by 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.
　神経変性疾患は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、数多くの難病、例えば、アルツハイマー病、パーキンソン病、パーキンソン症候群、脳血管性認知症、前頭側頭葉型認知症、筋萎縮性側索硬化症、進行性核上性麻痺、ハンチントン病、脊髄小脳変性症などが知られている。 Neurodegenerative diseases are pathological conditions caused by systematic neuronal degeneration and disruption of the neural network based on loss, and many intractable diseases such as Alzheimer's disease, Parkinson's disease, Parkinson's syndrome, cerebrovascular dementia, frontal side Known as lobar dementia, amyotrophic lateral sclerosis, progressive supranuclear palsy, Huntington's disease, spinocerebellar degeneration, and the like.
　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。例えば、アミロイドβペプチドの細胞毒性がその一因と考えられてい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て、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, 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. For example, in Alzheimer's disease, which is thought to be due to the cytotoxicity of amyloid β peptide, atrophy of neurites (axons and dendrites) and a decrease in synapses are triggers that impair neuronal function. Even after the trigger is triggered, it is said that if the nerve cells that have not degenerated and those that survive the degeneration are activated, the neurites are extended, and the synapse is restored, the nerve function can be restored. ing. 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、シナプスの制御など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.
　神経変性疾患の臨床症状としては、それぞれの病気により異なるが、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、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、しびれ又は麻痺などが挙げられ、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 neurodegenerative diseases vary depending on the disease, but they vary from minor to severe. Typical examples include tremor, rigidity, agitation, peristalsis, and movement. Slowness, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness or paralysis, etc. Both are major obstacles in daily life.
　アルツハイマー病やパーキンソン病などに代表される神経変性疾患は、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。 Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are serious diseases that cause degeneration of nerve cells, and various compounds are used as active ingredients to improve these diseases and their associated pathological conditions and neurological dysfunction. (See, for example, International Publication No. WO97 / 030703, JP-A-11-228417, JP-A-2006-143708, and JP-A-2006-321737). Although an accelerator or the like has been proposed (see, for example, JP-A-2002-234841), an effective disease treatment method 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、神経細胞体を活性化し、神経突起の萎縮を抑制、乃至、神経突起の伸展を促進させ神経変性に伴う神経機能低下乃至機能障害を改善することが可能な新規神経突起伸展促進剤の開発が切望されている。 In the medical field, systemic administration, such as subcutaneous or intravascular, has a low physical and mental burden on the patient, acts on nerve cells of the central nervous system, activates nerve cell bodies, suppresses neurite atrophy, or Therefore, development of a novel neurite outgrowth promoter capable of promoting neurite outgrowth and ameliorating neurological degeneration or dysfunction associated with neurodegeneration is eagerly desired.
　本発明は、新規な神経突起伸展促進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 neurite extension promoter.
　本発明者らは、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突起伸展促進作用を有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、下記一般式１で表される化合物を有効成分とする神経突起伸展促進剤を主な構成とする。 In order to solve the above problems, the present inventors have intensively researched and searched, and found that the compound represented by the following general formula 1 has an excellent neurite outgrowth promoting action, thereby completing the present invention. . That is, the present invention mainly comprises a neurite outgrowth promoter containing a compound represented by the following 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.
　本発明の神経突起伸展促進剤は、経口投与及び非経口的投与の何れの投与経路でも、神経突起の伸展を促進して、神経機能を活性化し、神経の機能障害に伴う種々の症状（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、しびれ及び麻痺など）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 neurite outgrowth promoting agent of the present invention promotes neurite outgrowth and activates nerve function in various administration routes of oral administration and parenteral administration, and various symptoms associated with nerve dysfunction (for example, , Tremor, stiffness, immobility, peristalsis, slow movement, postural reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, muscle atrophy, muscle weakness, upper / lower limb dysfunction, articulation disorder, swallowing Disorder, breathing problems, 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、神経細胞を賦活して軸索及び／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 reduction, and the action of promoting synapse formation between nerve cells. And the action of suppressing the decrease in synapses.
　本発明の神経突起伸展促進剤は、下記一般式１で表される化合物を有効成分とする。 The neurite extension promoter of the present invention contains a compound represented by the following 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.
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.
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.
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の脂肪族炭化水素基の炭素数が４乃至１０の化合物は、神経突起伸展促進作用が強いので、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 neurite outgrowth promoting action. 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、例えば、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。）、一般式２の側鎖のアルキル基（Ｒ４乃至Ｒ６）の炭素数が４の化合物（以下、「ＮＫ－２６」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, examples of the compound represented by the general formula 2 include 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-9694”). , “NK-4”), a compound having 4 carbon atoms in the side chain alkyl group (R4 to R6) of the general formula 2 (hereinafter, sometimes referred to as “NK-26”) and the like. Examples thereof include compounds in which the alkyl group (R4 to R6) in the side chain of Formula 2 has 3 carbon atoms (hereinafter sometimes referred to as “NK-234”). 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
 一般式３においてＲ７乃至Ｒ９は炭素数が同一で、炭素数が７乃至９のアルキル基を表し、ｍＸ３ －はＩ－又はＣｌ－を表す。
Figure JPOXMLDOC01-appb-C000017
 In the general formula 3, R 7 to R 9 represent the same alkyl group having 7 to 9 carbon atoms, and mX 3 — represents I 2 — or Cl 2 — .
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000019
Figure JPOXMLDOC01-appb-C000019
　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、ＮＫ－２３４、ＮＫ－２６は、いずれも神経活性化作用、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、本発明の神経突起伸展促進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－９６９４（化学式１で表される化合物）、ＮＫ－１５０（化学式３で表される化合物）が望ましい。また、脳梗塞、アルツハイマー病、血管性痴呆症糖などのモデル動物に対する認知障害や運動障害の改善効果の強さ、製剤化の容易さ、安全性などを考慮するとＮＫ－４、ＮＫ－２３４及びＮＫ－２６が望ましく、ＮＫ－４が特に望ましい。 Compounds NK-234 and NK-26 represented by any one of chemical formulas 1 to 9 are all cytotoxic factors such as starvation, radicals, and amyloid β peptide in addition to the nerve activation action and neurite outgrowth promoting action From the point of strength of its action and effect, it is more desirable as an active ingredient of the neurite outgrowth promoting agent of the present invention. NK-9694 (a compound represented by Chemical Formula 1) and NK-150 (a compound represented by Chemical Formula 3) are desirable. In addition, considering the strength of improvement in cognitive and motor disorders for model animals such as cerebral infarction, Alzheimer's disease, and vascular dementia sugar, ease of formulation, safety, etc., NK-4, NK-234 and NK-26 is desirable and NK-4 is particularly desirable.
　本発明の神経突起伸展促進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 neurite outgrowth promoter of the present invention is not limited in its origin or production method.
　本発明の神経突起伸展促進剤は、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 neurite outgrowth promoter 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 represented by general formula 5. It contains one or more styryl dyes.
　本発明の神経突起伸展促進剤は、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 neurite outgrowth promoting agent of the present invention, if necessary, in addition to the compound represented by the general formula 1 which is an active ingredient, is pharmaceutically acceptable in the food field, cosmetic field, pharmaceutical field, quasi-drug It is provided in the form of a preparation containing one or more components used in the product 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、一般式１で表される化合物以外を有効成分とす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。具体的には、例えば、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、再灌流障害、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍（例えば、星状膠細胞腫、脳膿瘍など）・血液量減少性ショック・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷・貫入・せん断・圧迫・裂傷、分娩時外傷、乳児むち打ち揺さぶり症候群など）に伴う神経機能障害の治療剤、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、線条体黒質変性症、ハンチントン病、舞踏病－無定位運動症、進行性核上麻痺、びまん性レビー小体病、大脳皮質基底核変性症、アルツハイマー病、老年性認知症、ピック病、前頭側頭葉型認知症、家族性認知症、脊髄小脳変性症（例えば、オリーブ橋小脳萎縮症、晩発性小脳皮質萎縮症、家族性脊髄小脳失調症（例えば、マッカードジョセフ病など）、歯状核赤核淡蒼球ルイ体萎縮症、家族性痙性対麻痺、フリードライヒ病など）など）の治療剤、運動神経病（例えば、筋萎縮性側索硬化症、家族性筋萎縮性側索硬化症など）の治療剤、脱髄性疾患（例えば、多発性硬化症、汎発硬化症、急性散在性脳脊髄炎、急性小脳炎、横断性脊髄炎、ギラン・バレー症候群など）の治療剤、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知など）の治療剤、毒物（ヒ素、カドミウム、有機水銀、サリン、ソマン、タブン、ＶＸガスなど）・放射線などによる神経機能障害の治療剤、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療剤、てんかんの治療剤、メージ症候群の治療剤、ジストニアの治療剤、ダウン症の治療剤及び／又は睡眠障害（例えば、過眠、ナルコレプシー、睡眠時無呼吸症候群など）の治療剤、糖尿病の治療剤、糖尿病合併症の治療剤及び／又は高脂血症の治療剤、ドーパミン受容体作動薬（ドーパミン受容体刺激薬）、ドーパミン遊離促進薬（ドーパミン分泌促進薬あるいはドーパミン放出促進薬）、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、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、ＡＭＰＡ（２－アミノ－３－（メチル－３－ヒドロキシイソオキサゾール－４－イル）プロパン酸）／カイニン酸受容体拮抗薬、ＧＡＢＡ受容体調節薬、アデノシンＡ２Ａ受容体遮断薬、ニコチン受容体調節薬、神経型一酸化窒素合成酵素（ｎ－ＮＯＳ）阻害薬、βアミロイドタンパクの産生、分泌、蓄積、凝集及び／又は沈着抑制薬（例えば、βセクレターゼ阻害薬、γセクレターゼ阻害作用薬、βアミロイドタンパク凝集抑制薬、βアミロイドタンパク分解酵素、βアミロイド用ワクチンなど）、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、ＮＧＦなどの増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、Ｒｈｏ－キナーゼ阻害薬、利尿薬（例えば、チアジド系利尿薬、ループ利尿薬、カリウム保持性利尿薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、ナトリウムチャネル遮断薬、カリウムチャネル開口薬、抗血小板薬、抗凝固薬、血栓溶解薬、トロンボキサンＡ２合成酵素阻害薬、マトリックスメタロプロテイナーゼ（ＭＭＰ）阻害剤、シクロオキシゲナーゼ（ＣＯＸ）－２阻害薬、非ステロイド性抗炎症薬、ステロイド薬、抗酸化薬、ビタミン類、疾患修飾性抗リウマチ薬、サイトカイン、抗サイトカイン薬（例えば、ＴＮＦ阻害薬、ＭＡＰキナーゼ阻害薬など）、性ホルモン又はその誘導体（例えば、プロゲステロン、エストラジオール、安息香酸エストラジオールなど）、副甲状腺ホルモン（例えば、ＰＴＨなど）及びカルシウム受容体拮抗薬などを例示することができる。これらの薬剤は、本発明の有効成分と混合剤の形態で投与してもよいし、個々の製剤を別々に投与することもできる。 In addition, a neurite outgrowth promoting agent having an active ingredient other than the compound represented by the general formula 1 and a therapeutic agent for neurodegenerative diseases, pathological conditions and neurological dysfunctions resulting therefrom can be advantageously implemented. Specifically, for example, cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, arachnoid membrane) Etc.), brain tumors (eg, astrocytoma, brain abscess, etc.), blood volume reducing shock, traumatic shock, head injury and / or cerebrospinal trauma (eg, brain contusion, penetration, shear)・ Therapeutic agents for neurological dysfunction associated with compression / laceration, labor trauma, infant whiplash, etc., neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea) Ataxia, progressive supranuclear palsy, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobe dementia, familial dementia, spinal cord Cerebellar degeneration For example, Olive Bridge cerebellar atrophy, late-onset cerebellar cortical atrophy, familial spinocerebellar ataxia (for example, Maccard Joseph disease, etc.), dentate nucleus red nucleus pallidal Louis atrophy, familial spastic paraplegia ), Therapeutic agents for motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple myelopathy) Therapeutic agents for sclerosis, general sclerosis, acute disseminated encephalomyelitis, acute cerebellar inflammation, transverse myelitis, Guillain-Barre syndrome, etc., cerebrospinal diseases associated with infections (eg meningitis, influenza encephalopathy) , Creutzfeldt-Jakob disease, cognition due to AIDS encephalopathy, etc., toxic agents (arsenic, cadmium, organic mercury, sarin, soman, soman, tabun, VX gas, etc.), therapeutic agents for neurological dysfunction due to radiation, mental illness ( For example, neurosis Therapeutic agents for psychosomatic disorders, anxiety, schizophrenia, manic depression etc., therapeutic agents for epilepsy, therapeutic agents for mage syndrome, therapeutic agents for dystonia, therapeutic agents for Down syndrome and / or sleep disorders (eg hypersomnia, narcolepsy) , Sleep apnea syndrome, etc.), diabetes treatment, diabetes complications and / or hyperlipidemia treatment, dopamine receptor agonist (dopamine receptor stimulant), dopamine release promotion Drugs (dopamine secretion promoters or dopamine release promoters), dopamine uptake inhibitors, dopamine agonists, central anticholinergic agents, aromatic L-amino acid decarboxylase inhibitors (DCI), monoamine oxidase (MAO-B) Inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholine Sterase inhibitor, NMDA (N-methyl-D-aspartic acid) receptor antagonist, AMPA (2-amino-3- (methyl-3-hydroxyisoxazol-4-yl) propanoic acid) / kainic acid receptor antagonist Drugs, GABA receptor modulators, adenosine A2A receptor blockers, nicotine receptor modulators, neuronal nitric oxide synthase (n-NOS) inhibitors, β amyloid protein production, secretion, accumulation, aggregation and / or Deposition inhibitor (eg, β-secretase inhibitor, γ-secretase inhibitor, β-amyloid protein aggregation inhibitor, β-amyloid proteinase, β-amyloid vaccine), apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophic Factors (eg, neurotrophin, TGF-β superfamily, neurokine family, NGF Which growth factors), other brain function activators (eg cerebral metabolism activators, cerebral circulation improvers, etc.), Rho-kinase inhibitors, diuretics (eg thiazide diuretics, loop diuretics, potassium retention) Diuretics, etc.), beta receptor blockers, calcium channel blockers (calcium antagonists), angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, sodium channel blockers, potassium channel openers, antiplatelet drugs Anticoagulants, thrombolytic agents, thromboxane A 2 synthase inhibitors, matrix metalloproteinase (MMP) inhibitors, cyclooxygenase (COX) -2 inhibitors, nonsteroidal anti-inflammatory drugs, steroid drugs, antioxidants, Vitamins, disease-modifying anti-rheumatic drugs, cytokines, anti-cytokine drugs (eg, TNF inhibition) Drugs, MAP kinase inhibitors, etc.), sex hormones or derivatives thereof (eg, progesterone, estradiol, estradiol benzoate, etc.), parathyroid hormones (eg, PTH, etc.) and calcium receptor antagonists. These agents may be administered in the form of a mixture with the active ingredient of the present invention, or individual preparations can be administered separately.
　本発明の抗神経変性疾患剤と併用される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の中で、好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）の治療剤、脳腫瘍の治療剤、脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害の治療剤、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症、脊髄小脳変性症など）の治療剤、運動神経病（例えば、筋萎縮性側索硬化症など）の治療剤、脱髄性疾患（例えば、多発性硬化症など）の治療剤、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）の治療剤、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療剤、てんかんの治療剤、ジストニアの治療剤、糖尿病の治療剤、糖尿病合併症の治療剤及び／又は高脂血症の治療剤、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、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、ＡＭＰＡ（２－アミノ－３－（メチル－３－ヒドロキシイソオキサゾール－４－イル）プロパン酸）／カイニン酸受容体拮抗薬、ＧＡＢＡＡ受容体調節薬（例えば、ＧＡＢＡＡ受容体作動薬など）、ＧＡＢＡＢ受容体調節薬、アデノシンＡ２Ａ受容体遮断薬、βセクレターゼ阻害薬、βアミロイドタンパク凝集抑制薬、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、Ｒｈｏ－キナーゼ阻害薬、利尿薬（例えば、チアジド系利尿薬、ループ利尿薬、カリウム保持性利尿薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、ナトリウムチャネル遮断薬、カリウムチャネル開口薬、抗血小板薬、抗凝固薬、血栓溶解薬、シクロオキシゲナーゼ（ＣＯＸ）－２阻害薬、非ステロイド性抗炎症薬、ステロイド薬、抗酸化薬及びビタミン類が挙げられ、より好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞など）の治療剤、脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害の治療剤、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症など）の治療剤、筋萎縮性側索硬化症の治療剤、多発性硬化症の治療剤、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）の治療剤、てんかんの治療剤及び／又はジストニアの治療剤、糖尿病の治療剤、糖尿病合併症の治療剤及び／又は高脂血症の治療剤、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、ＮＭＤＡ（Ｎ－メチル－Ｄ－アスパラギン酸）受容体拮抗薬、βセクレターゼ阻害薬、βアミロイドタンパク凝集抑制薬、アポトーシス阻害薬、神経分化・再生促進薬、神経栄養因子（例えば、ＮＧＦなどのニューロトロフィン、ＴＧＦ－βスーパーファミリー、ニューロカインファミリー、増殖因子など）、その他の脳機能賦活薬（例えば、脳代謝賦活薬、脳循環改善薬など）、β受容体遮断薬、カルシウムチャネル遮断薬（カルシウム拮抗薬）、アンジオテンシン変換酵素（ＡＣＥ）阻害薬、アンジオテンシンＩＩ受容体拮抗薬、抗血小板薬、抗凝固薬及び血栓溶解薬が挙げられ、とりわけ好ましくは、例えば、脳血管障害（例えば、脳卒中、脳梗塞など）の治療剤、神経変性疾患（例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病など）の治療剤、筋萎縮性側索硬化症の治療剤、多発性硬化症の治療剤、てんかんの治療剤及び／又は糖尿病合併症の治療剤、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。 Among the therapeutic agents for neurodegenerative diseases used in combination with the anti-neurodegenerative disease agent of the present invention and the pathological conditions and neurological dysfunctions resulting therefrom, preferably, for example, cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombus) , Cerebral embolism, etc.), transient cerebral ischemic attack, cerebral hemorrhage (eg hypertensive intracerebral hemorrhage, subarachnoid hemorrhage, etc.), brain tumor treatment agent, cerebrospinal trauma (eg cerebral contusion, etc.) Therapeutic agents for neurological dysfunction associated with neurodegenerative diseases (eg Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration), motor neuropathy (eg muscle atrophy) Therapeutic agents for lateral sclerosis, etc., therapeutic agents for demyelinating diseases (eg, multiple sclerosis), cerebrospinal diseases associated with infections (eg, meningitis, influenza encephalopathy, Creutzfeldt-Jako) Disease, dementia due to AIDS encephalopathy), treatment for mental disorders (eg, neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.), treatment for epilepsy, treatment for dystonia, diabetes , Diabetic complications and / or hyperlipidemia, 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) supplement, acetylcholinesterase inhibitor, NMDA (N- Methyl-D-aspartate receptor antagonist, AMPA (2-amino-3- (methyl-3-hydroxyisothio) Sasol-4-yl) propanoic acid) / kainate receptor antagonists, GABA A receptor modulators (e.g., GABA A receptor agonist), GABA B receptor modulators, adenosine A2A receptor blockers, beta Secretase inhibitor, β-amyloid protein aggregation inhibitor, apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophic factor (eg, neurotrophin, TGF-β superfamily, neurokine family, growth factor), other brains Functional activators (eg, cerebral metabolic activators, cerebral circulation improvers, etc.), Rho-kinase inhibitors, diuretics (eg, thiazide diuretics, loop diuretics, potassium-sparing diuretics), β receptor blockade Drugs, calcium channel blockers (calcium antagonists), angiotensin converting enzyme (ACE) inhibitors, angiotensi II receptor antagonist, sodium channel blocker, potassium channel opener, antiplatelet agent, anticoagulant, thrombolytic agent, cyclooxygenase (COX) -2 inhibitor, nonsteroidal anti-inflammatory agent, steroid drug, antioxidant And more preferably, for example, a therapeutic agent for cerebrovascular disorders (eg, stroke, cerebral infarction, etc.), a therapeutic agent for neurological dysfunction associated with cerebrospinal trauma (eg, cerebral contusion, etc.), neurodegeneration, etc. Therapeutic agents for diseases (eg, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, etc.), therapeutic agents for amyotrophic lateral sclerosis, therapeutic agents for multiple sclerosis, Neurosis, psychosomatic disorders, anxiety, schizophrenia, manic depression, etc.), epilepsy and / or dystonia, diabetes, diabetes Complication treatment and / or hyperlipidemia treatment agent, dopamine receptor agonist, dopamine release promoter, dopamine uptake inhibitor, dopamine agonist, central anticholinergic agent, aromatic L-amino acid decarboxylase Inhibitor (DCI), monoamine oxidase (MAO-B) inhibitor, catechol-O-methyltransferase (COMT) inhibitor, norepinephrine (noradrenaline) supplement, acetylcholinesterase inhibitor, NMDA (N-methyl-D-asparagine) Acid) receptor antagonist, β-secretase inhibitor, β-amyloid protein aggregation inhibitor, apoptosis inhibitor, neuronal differentiation / regeneration promoter, neurotrophin such as NGF, TGF-β superfamily, neuro Cain family, growth factors, etc.) and other brain function activation Drugs (for example, cerebral metabolism activator, cerebral circulation improving drug, etc.), β receptor blocker, calcium channel blocker (calcium antagonist), angiotensin converting enzyme (ACE) inhibitor, angiotensin II receptor antagonist, antiplatelet Drugs, anticoagulants and thrombolytic agents, and particularly preferably, for example, therapeutic agents for cerebrovascular disorders (eg, stroke, cerebral infarction, etc.), neurodegenerative diseases (eg, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease), amyotrophic lateral sclerosis, multiple sclerosis, epilepsy and / or diabetic complications, dopamine receptor agonist, dopamine release promotion Drugs, dopamine uptake inhibitors, dopamine agonists, central anticholinergics, aromatic L-amino acid decarboxylase inhibitors (DCI), Noamin oxidase (MAO-B) inhibitor, catechol -O- methyltransferase (COMT) inhibitors, norepinephrine (noradrenaline) replenishers, acetylcholinesterase inhibitors, neurotrophic factors and other brain function activation agents. 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.
　本発明の神経突起伸展促進剤は、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、非経口投与が困難な場合には、経口用の製剤の形態で使用する場合もある。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や経口投与用の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１種又は２種以上の方法が適宜に選ばれる。 The neurite extension promoter of the present invention is usually provided in the form of a parenteral injection preparation or the like. Moreover, when parenteral administration is difficult, it may be used in the form of an oral preparation. The compound represented by the general formula 1, which is an active ingredient, is a process from the raw material stage to the completion of the product in consideration of the composition of the target injection preparation and oral preparation and the purpose of use. What is necessary is just to mix | blend. 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 methods such as loading are appropriately selected.
　本発明の神経突起伸展促進剤は、その形状を問わず、経口投与用の製剤の場合、対象となる疾患や症状などに応じて、例えば、溶液、シラップ、マスキット、ペースト、粉末、固状、顆粒、錠剤などの何れの形状であってもよく、そのままで、又は、必要に応じて、増量剤、賦形剤、結合剤などと混合して、顆粒、球状、短棒状、板状、立方体、錠剤、カプセル剤など各種形状に成型して使用することも随意である。 Regardless of its shape, the neurite outgrowth promoting agent of the present invention, in the case of a preparation for oral administration, depending on the target disease or symptom, for example, solution, syrup, mass kit, paste, powder, solid, Any shape such as granules, tablets, etc. may be used as it is, or as necessary, mixed with a filler, excipient, binder, etc. to form granules, spheres, short bars, plates, cubes It is also optional to use it in various shapes such as tablets and capsules.
　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、油性基材や乳化剤などを使用して、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, it is effective to use an amphiphilic solvent, an oily base material, an emulsifier, etc. 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、リンゲル液など、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、使用する一般式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 used, it may become unstable due to dissolved oxygen or the like. In that case, for example, if the dissolved oxygen concentration of the compound solution is reduced, Good. 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、該化合物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, the compound solution is prepared and stored under reduced pressure, or the oxygen dissolved in the compound solution is separated from another gas. Or a method of bringing the compound solution into contact with an oxygen scavenger is preferred. 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、本発明の神経突起伸展促進剤は、注射剤以外にも、経口剤、ハップ剤や吸飲噴霧剤などの形態で使用することもでき、皮下など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 neurite outgrowth promoting agent of the present invention can be used in the form of an oral preparation, a haptic agent, a 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神経突起伸展促進剤は、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 neurite outgrowth promoting agent of the present invention produced in this way is a safe preparation without serious side effects even when used for a long time.
　本発明の神経突起伸展促進剤の、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。なお、経口投与の場合には、本発明の神経突起伸展促進剤の有効成分である一般式１で表される化合物は、一般に消化管からの吸収率が低いことを勘案して、皮下投与の場合よりも高い投与量を設定する必要がある。 The daily dose of the neurite outgrowth promoting agent of the present invention is not particularly limited as long as the desired action and effect can be obtained. Usually, in the case of subcutaneous or intraperitoneal administration, it is represented by the general formula 1. 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. In the case of oral administration, the compound represented by general formula 1, which is an active ingredient of the neurite outgrowth promoting agent of the present invention, is generally administered subcutaneously in view of its low absorption rate from the digestive tract. It is necessary to set a higher dose than the case.
　本発明の神経突起伸展促進剤は、神経突起の伸展促進作用に加えて、脳や神経細胞を障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、神経突起の萎縮の抑制、神経細胞の生存延長や変性を抑制することもできるので、神経変性疾患、とりわけ、中枢神経の変性に起因する疾患を治療することができる。神経変性疾患とは、神経細胞（中枢神経（例えば、脳神経、脊髄神経など）及び／又は末梢神経（例えば、自律神経系（例えば、交感神経、副交感神経など）、運動神経系、知覚神経系））の変性を伴う疾患全てを包含し、その病因によって限定されるものではない。具体的には、一般的に神経変性疾患とされている疾患であればよく、例えば、パーキンソン病、パーキンソン症候群、線条体黒質変性症、ハンチントン病、舞踏病－無定位運動症、進行性核上麻痺、びまん性レビー小体病、大脳皮質基底核変性症、アルツハイマー病、老年性認知症、ピック病、前頭側頭葉型認知症、家族性認知症、脊髄小脳変性症（例えば、オリーブ橋小脳萎縮症、晩発性小脳皮質萎縮症、家族性脊髄小脳失調症（例えば、マッカードジョセフ病など）、歯状核赤核淡蒼球ルイ体萎縮症、家族性痙性対麻痺、フリードライヒ病など）、運動神経病（例えば、筋萎縮性側索硬化症、家族性筋萎縮性側索硬化症など）、脱髄性疾患（例えば、多発性硬化症、汎発硬化症、急性散在性脳脊髄炎、急性小脳炎、横断性脊髄炎、ギラン・バレー症候群など）、代謝性脳疾患、先天性及び遺伝性疾患（神経系リソソーム蓄積症など）などだけでなく、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、再灌流障害、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍（例えば、星状膠細胞腫、脳膿瘍など）・血液量減少性ショック・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷・貫入・せん断・圧迫・裂傷、分娩時外傷、乳児むち打ち揺さぶり症候群など）に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、毒物（ヒ素、カドミウム、有機水銀、サリン、ソマン、タブン、ＶＸガスなど）・放射線などによる神経機能障害、精神疾患（例えば、神経症、心身症、不安、統合失調症、躁うつ病など）、てんかん、メージ症候群、ジストニア、ダウン症、睡眠障害（例えば、過眠、ナルコレプシー、睡眠時無呼吸症候群など）なども含まれる。 The neurite outgrowth promoting agent of the present invention, in addition to the neurite outgrowth promoting action, protects the brain and nerve cells from damaging factors and suppresses degeneration, activates the nerve cells, inhibits neurite atrophy, Since neuronal survival and degeneration can be suppressed, neurodegenerative diseases, particularly diseases caused by degeneration of the central nervous system can be treated. A neurodegenerative disease is a nerve cell (central nervous system (for example, cranial nerve, spinal nerve), and / or peripheral nerve (for example, autonomic nervous system (for example, sympathetic nerve, parasympathetic nerve)), motor nervous system, sensory nervous system) ) Are included, and are not limited by the etiology. Specifically, any disease that is generally regarded as a neurodegenerative disease may be used. For example, Parkinson's disease, Parkinson's syndrome, striatal nigra degeneration, Huntington's disease, chorea-ataxia, progressive Supranuclear paralysis, diffuse Lewy body disease, basal ganglia degeneration, Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobar dementia, familial dementia, spinocerebellar degeneration (eg olive Bridge cerebellar atrophy, late cerebellar cortical atrophy, familial spinocerebellar ataxia (eg, Maccard Joseph disease, etc.), dentate nucleus erythrocytic Ryukyu atrophy, familial spastic paraplegia, Friedreich Disease), motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), demyelinating diseases (eg, multiple sclerosis, multiple sclerosis, acute disseminated) Encephalomyelitis, acute encephalitis, transverse myelitis, Guillain Valley syndrome, etc.), metabolic brain diseases, congenital and genetic diseases (neuropathic lysosomal storage diseases, etc.), as well as cerebrovascular disorders (eg, stroke, cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), Transient cerebral ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, subarachnoid hemorrhage, etc.), brain tumor (eg, astrocytoma, brain abscess, etc.), blood volume reducing shock, Neurological dysfunction associated with traumatic shock, head injury and / or cerebrospinal trauma (eg brain contusion, penetration, shear, compression, laceration, labor trauma, infant whiplash syndrome), cerebrospinal disease associated with infection (For example, meningitis, influenza encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy, etc.), toxic substances (arsenic, cadmium, organic mercury, sarin, soman, tabun, VX gas, etc.) Neurological dysfunction due to radiation, mental illness (eg, neurosis, psychosomatic disorder, anxiety, schizophrenia, manic depression, etc.), epilepsy, mage syndrome, dystonia, Down syndrome, sleep disorder (eg, hypersomnia, narcolepsy, sleep) Including apnea syndrome).
　本発明の神経突起伸展促進剤の対象として好ましい神経変性疾患としては、例えば、パーキンソン病、パーキンソン症候群、ハンチントン病、アルツハイマー病、老年性認知症、脊髄小脳変性症、筋萎縮性側索硬化症、脱髄性疾患（例えば、多発性硬化症など）、脳血管障害（例えば、脳卒中、脳梗塞（例えば、脳血栓、脳塞栓など）、一過性脳虚血発作、脳出血（例えば、高血圧性脳内出血、クモ膜下出血など）など）・脳腫瘍・外傷性ショック・頭部損傷及び／又は脳脊髄外傷（例えば、脳挫傷など）に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、てんかんなどの中枢神経系の神経変性に由来する疾患であり、より好ましくは、例えば、パーキンソン病、パーキンソン症候群、アルツハイマー病、筋萎縮性側索硬化症に伴う神経機能障害、感染症に伴う脳脊髄疾患（例えば、髄膜炎、インフルエンザ脳症、クロイツフェルト－ヤコブ病、エイズ脳症による認知症など）、てんかんなどであり、特に好ましくは、例えば、パーキンソン病、アルツハイマー病に伴う神経機能障害などである。 Examples of neurodegenerative diseases that are preferable as the neurite extension promoter of the present invention include, for example, Parkinson's disease, Parkinson's syndrome, Huntington's disease, Alzheimer's disease, senile dementia, spinocerebellar degeneration, amyotrophic lateral sclerosis, Demyelinating diseases (for example, multiple sclerosis), cerebrovascular disorders (for example, stroke, cerebral infarction (for example, cerebral thrombosis, cerebral embolism, etc.), transient cerebral ischemic attacks, cerebral hemorrhage (for example, hypertensive intracerebral hemorrhage) ), Brain tumors, traumatic shock, head injury and / or cerebrospinal trauma (eg, cerebral contusion), cerebrospinal disease (eg, meninges) Inflammation, influenza encephalopathy, Creutzfeldt-Jakob disease, dementia due to AIDS encephalopathy, etc.), diseases derived from neurodegeneration of the central nervous system such as epilepsy, more preferably For example, Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, neurological dysfunction associated with amyotrophic lateral sclerosis, cerebrospinal disease associated with infection (eg, meningitis, influenza encephalopathy, Creutzfeldt-Jakob disease, AIDS encephalopathy Dementia and the like), epilepsy and the like, particularly preferably, for example, neurological dysfunction associated with Parkinson's disease and Alzheimer's disease.
　本発明の神経突起伸展促進剤は、神経細胞を活性化し、神経突起を伸展させ、シナプスの形成を促進するなどにより、神経機能障害をも治療することができる。対象となる神経機能障害とは、神経機能の障害であればどのようなものであってもよいが、例えば、認知機能障害、意識障害、両側性四肢麻痺、反対側片麻痺、交代性片麻痺、感覚障害、一過性失明（例えば、一過性黒内障など）、同名性半盲、めまい、眼振、複視、失語、耳鳴、昏睡などが挙げられる。特に好ましくは、前記神経変性疾患に伴うこれらの神経機能障害などが対象となる。前記の神経変性疾患に伴う神経機能障害、例えば、脳梗塞に伴う神経機能障害は、血管閉塞部位により様々であり、また障害されるレベルによっても症状が異なるが、主に上記の神経機能障害が見られる。また、脳梗塞における神経機能障害は、神経機能障害を検出する当技術分野で公知の様々な診断試験によってその有無を判断してもよい。該診断試験の具体的な例としては、例えば、アルツハイマー病などによる記憶や認知機能障害の評価に使用されている認知機能スコア（Ａｌｚｈｅｉｍｅｒ’ｓ　Ｄｉｓｅａｓｅ　Ａｓｓｅｓｓｍｅｎｔ　Ｓｃａｌｅ－ｃｏｇｎｉｔｉｖｅ　ｐａｒｔ；ＡＤＡＳ－ｃｏｇ）、臨床症状改善スコア（Ａｌｚｈｅｉｍｅｒ’ｓ　Ｄｉｓｅａｓｅ　Ｃｏｏｐｅｒａｔｉｖｅ　Ｓｔｕｄｙ－Ｃｌｉｎｉｃａｌ　Ｇｌｏｂａｌ　Ｉｍｐｒｅｓｓｉｏｎ　ｏｆ　Ｃｈａｎｇｅ；ＡＤＣＳ－ＣＧＩＣ）、ミニ精神状態試験（Ｍｉｎｉ－Ｍｅｎｔａｌ　Ｓｔａｔｅ　Ｅｘａｍｉｎａｔｉｏｎ；ＭＭＳＥ）、長谷川式評価法などをあげることができ、さらには、グラスゴーアウトカムスケール（Ｇｌａｓｇｏｗ　Ｏｕｔｃｏｍｅ　Ｓｃａｌｅ：ＧＯＳ）、グラスゴーコーマスケール（Ｇｌａｓｇｏｗ　Ｃｏｍａ　Ｓｃａｌｅ：ＧＣＳ）、ランキンスケール（Ｒａｎｋｉｎ　Ｓｃａｌｅ：ＲＳ）、改変ランキンスケール（ｍｏｄｉｆｉｅｄ　Ｒａｎｋｉｎ　Ｓｃａｌｅ：ｍＲＳ）、能力障害関連スケール（Ｄｉｓａｂｉｌｉｔｙ　Ｒａｔｉｎｇ　Ｓｃａｌｅ：ＤＲＳ）、及び、ＮＩＨ卒中スケール（ＮＩＨ　Ｓｔｒｏｋｅ　Ｓｃａｌｅ：ＮＩＨＳＳ）などの公知の方法を用いて行うことができる。これらの神経機能障害を検出する診断試験は、物理的な脳の異常を検出する試験方法、例えば、ＣＴスキャンや頭蓋内圧の測定などと適宜組み合わせておこなってもよい。 The neurite outgrowth promoting agent of the present invention can treat nerve dysfunction by activating nerve cells, extending neurites, and promoting synapse formation. The target neurological dysfunction may be any neurological dysfunction, such as cognitive dysfunction, consciousness disorder, bilateral quadriplegia, contralateral hemiplegia, alternating hemiplegia. , Sensory disorders, transient blindness (eg, transient cataract), homonymous half-blindness, dizziness, nystagmus, double vision, aphasia, tinnitus, coma and the like. Particularly preferred are those neurological dysfunctions associated with the neurodegenerative diseases. The neurological dysfunction associated with the above-mentioned neurodegenerative diseases, for example, neurological dysfunction associated with cerebral infarction varies depending on the site of vascular occlusion, and the symptoms vary depending on the level to be impaired. It can be seen. In addition, the presence or absence of neurological dysfunction in cerebral infarction may be determined by various diagnostic tests known in the art for detecting neurological dysfunction. Specific examples of the diagnostic test include, for example, a cognitive function score (Alzheimer's Dissease Assessment-cognitive part; ADAS-cog) used for evaluation of memory and cognitive impairment due to Alzheimer's disease, clinical symptoms, and the like. Improvement score (Alzheimer's Dissease Cooperative Perspective-Clinical Global Impression of Change; ADCS-CGIC), Mini Mental State Exam (Mini-Mental State Examination; MMSE) Outcome scale (Glasgow Outcome Scale: GOS), glass -Comascale (Glasgow Coma Scale: GCS), Rankin Scale (Rakin Scale: RS), Modified Rankin Scale (Modified Rankin Scale: mRS), Disability-related Scale (Disability Rating Scale: DRS), and NS : NIHSS) or the like. These diagnostic tests for detecting neurological dysfunction may be performed in appropriate combination with a test method for detecting physical brain abnormalities, for example, a CT scan or intracranial pressure measurement.
　したがって、本発明の神経突起伸展促進剤は、神経細胞保護剤、神経細胞活性化剤、プルキンエ細胞変性・脱落抑制剤、神経変性疾患やそれに伴う病態の治療剤、神経機能障害治療剤などとして有利に使用することができる。本発明でいう神経変性疾患や神経機能障害の治療とは、神経変性に起因する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。 Therefore, the neurite outgrowth promoting agent of the present invention is advantageous as a nerve cell protective agent, nerve cell activator, Purkinje cell degeneration / dropout inhibitor, therapeutic agent for neurodegenerative diseases and associated pathologies, therapeutic agent for neurological dysfunction, etc. Can be used for The treatment of neurodegenerative diseases and neurological dysfunctions as used in the present invention refers to the pathology and dysfunction caused by neurodegeneration in the direction of healing, in addition to so-called treatment, prevention of progression that suppresses progression and stops progression of the disease, Also includes the prevention of the onset of the disease 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：神経突起に及ぼすペンタメチン系シアニン色素及びジメチン系スチリル色素の影響＞
　ペンタメチン系シアニン色素及びジメチン系スチリル色素を用いて、これらの色素に神経突起伸展作用があるかどうかを検討した。すなわち、表１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で表される２３２種類（合計２３９種類）の化合物について、以下に示す評価方法により、神経突起伸展促進作用の有無を調べた結果を表１に示す。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１では空欄とした。 <Experiment 1: Effect of pentamethine cyanine dye and dimethine styryl dye on neurite>
Using pentamethine cyanine dyes and dimethine styryl dyes, we examined whether these dyes have a neurite extension effect. That is, in addition to the compounds represented by Chemical Formula 2 and Chemical Formulas 4 to 9 shown in Table 1, 232 types (total 239 types) of compounds represented by the following Chemical Formulas 10 to 241 were evaluated according to the evaluation method described below. Table 1 shows the results of examining the presence or absence of the protrusion extension promoting action. In the following tests, when only an effect lower than each criterion was recognized, it was left blank in Table 1.
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000024
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-C000042
Figure JPOXMLDOC01-appb-C000042
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
＜試験試料＞
　表１に示す２３９種類の化合物は、水に難溶性のものが多いため、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 1 are hardly soluble in water, after dissolving in DMSO (SIGMA, catalog number “D8418”) at a concentration of 5 mg / ml, 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.
＜神経突起伸展促進作用の評価法＞
　ヒトの神経突起伸展促進作用を検討するために好適なモデルとされている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、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、神経突起伸展の見られた細胞の割合が３０％以上の場合を強い神経突起伸展促進作用ありと判定して、表１に○を付した。なお、神経突起伸展の見られた細胞の割合（％）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、細胞体の長径の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の見られた細胞の割合は５％程度であった。 <Evaluation method of neurite outgrowth promoting action>
NGF (Neural Growth Factor) hypersensitive strain of PC-12 cells derived from rat adrenal pheochromocytoma (hereinafter referred to as “PC-12HS cells”), which is a suitable model for studying human neurite outgrowth promoting action (Obtained from Human Science Research Resource Bank) was diluted with a 10 volume% FBS-added D-MEM medium to a collagen-coated 96-well microplate at 5 × 10 3 cells / well and seeded at 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 are observed under a microscope to evaluate the presence or absence of neurite outgrowth. When the ratio of cells with neurite outgrowth is 30% or more, it is determined that there is a strong neurite outgrowth promoting action. 1 was marked with a circle. The percentage of cells in which neurite outgrowth was observed was determined by observing cells under a microscope at a magnification containing about 100 cells in one field of view, and showing neurites at least twice the major axis of the cell body. The number of cells was counted, divided by the total number of cells in the same field, and multiplied by 100. In addition, when only NGF was added to this experimental system (5 ng / ml), the proportion of cells in which neurite outgrowth was observed was about 5%.
Figure JPOXMLDOC01-appb-T000047
Figure JPOXMLDOC01-appb-T000047
　表１の結果から明らかなように、試験した２３９種の化合物のうち、５６種類の化合物で、神経突起伸展の見られた細胞の割合が３０％以上となったので、これらの化合物には強い神経突起伸展促進作用があると判断した。以上の結果は、表１に示す強い神経突起伸展促進作用を有する化合物は、いずれも、神経突起伸展促進剤として有用であり、ヒトの神経変性疾患及びそれに伴う病態や神経機能障害の治療剤として利用できることを物語っている。 As is clear from the results of Table 1, among the 239 compounds tested, 56 compounds showed a neurite outgrowth ratio of 30% or more, which is strong against these compounds. Judged to have neurite outgrowth promoting action. The above results show that any of the compounds having a strong neurite outgrowth promoting action shown in Table 1 is useful as a neurite outgrowth promoting agent, and is used as a therapeutic agent for human neurodegenerative diseases and accompanying pathological conditions or neurological dysfunction. It tells you that you can use it.
＜実験２：色素化合物の濃度の神経突起伸展に及ぼす影響＞
　実験１で、強い神経突起伸展促進作用があると判定した化合物のうちＮＫ－１９、ＮＫ－５３、ＮＫ－１００、及び、ＮＫ－５５７について、その濃度が、ＰＣ１２－ＨＳ細胞の神経突起伸展に及ぼす影響を、実験１と同じ方法を用いて調べた。すなわち、培地中の化合物の終濃度が、表２に示す濃度となるように、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、ＮＫ－５５７のいずれかを加えて培養を継続し、細胞をグルタルアルデヒドで固定した。一視野に約１００個の細胞を含む倍率で顕微鏡観察し、全細胞数に対する神経突起伸展の見られた細胞の割合（％）を求めた結果を表２に示す。 <Experiment 2: Effect of pigment compound concentration on neurite outgrowth>
Among the compounds determined to have a strong neurite outgrowth promoting effect in Experiment 1, the concentrations of NK-19, NK-53, NK-100, and NK-557 are related to neurite outgrowth of PC12-HS cells. The effect was examined using the same method as in Experiment 1. That is, the culture is continued by adding any one of NK-4, NK-19, NK-53, NK-100, and NK-557 so that the final concentration of the compound in the medium becomes the concentration shown in Table 2. The cells were fixed with glutaraldehyde. Table 2 shows the results of microscopic observation at a magnification including about 100 cells in one field, and determining the percentage (%) of cells in which neurite outgrowth was observed with respect to the total number of cells.
Figure JPOXMLDOC01-appb-T000048
Figure JPOXMLDOC01-appb-T000048
　表２の結果から明らかなように、各化合物の濃度依存的に神経突起の伸展の見られる細胞の割合が増加した。この結果は、これらの化合物に、濃度依存的な神経突起伸展促進作用があること
を物語っている。また、試験に使用した５種類の化合物の中で、ＮＫ－１００を使用した場合は、神経突起の伸展の認められた細胞の割合が、濃度が２００ｎｇ／ｍｌのとき３６±２％、１６００ｎｇ／ｍｌのとき８９±６％となって、他の４種類の化合物を使用した場合よりも、低濃度から高濃度まで、強い神経突起伸展促進作用を示した。 As is clear from the results in Table 2, the proportion of cells in which neurite outgrowth was increased depending on the concentration of each compound. This result indicates that these compounds have a concentration-dependent neurite outgrowth promoting action. Of the five compounds used in the test, when NK-100 was used, the proportion of cells in which neurite outgrowth was observed was 36 ± 2% when the concentration was 200 ng / ml, and 1600 ng / ml. In the case of ml, it was 89 ± 6%, showing a stronger neurite outgrowth promoting action from a low concentration to a high concentration than when the other four kinds of compounds were used.
＜実験３：ＮＫ－１９類縁体の神経突起伸展促進作用＞
　上記実験で、神経突起伸展促進作用が確認された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、下記一般式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１と同様に、ＰＣ－１２ＨＳ細胞に対する神経突起伸展促進作用の強さを、伸展突起伸展の認められる細胞の割合を指標にして調べた。表３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、２μｇ／ｍｌに希釈して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２μｇ／ｍｌに希釈して試験試料溶液を調製した。 <Experiment 3: neurite outgrowth promoting action of NK-19 analog>
In the above experiment, NK-19, which was confirmed to have a neurite outgrowth promoting action, was tested to confirm that its analogs had the same action. That is, a compound in which the alkyl group (R) in the side chain of the compound represented by the following general formula 3 has 1 to 12 carbon atoms (synthesized by Hayashibara Biochemical Laboratories Co., Ltd.) was synthesized. The strength of the neurite outgrowth promoting action on PC-12HS cells was examined using the proportion of cells in which neurite outgrowth was observed as an index. Twelve types of analogs of NK-19 including NK-19 shown in Table 3 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, and the concentration of the compound was diluted to 2 μg / ml to prepare a test sample solution. 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 D-MEM medium supplemented with 10% by volume FBS, and the test sample solutions were prepared by diluting the compound concentration to 2 μg / ml each.
＜神経突起伸展促進作用の評価＞
　実験１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、ＰＣ１２－ＨＳ細胞を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１の場合と同じ方法で細胞をカウントして、神経突起伸展の見られた細胞の割合（％）を求めた。結果を表３に併せて示す。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。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の見られた細胞の割合は５％程度であった。 <Evaluation of neurite outgrowth promoting action>
As in Experiment 1, 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 seeded at 100 μl / well. did. 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, PC12-HS cells were cultured for 3 days only in 10% by volume FBS-added D-MEM medium, and the cells were fixed with glutaraldehyde. The fixed cells were observed under a microscope, and the cells were counted by the same method as in Experiment 1 to determine the percentage (%) of cells in which neurite extension was observed. The results are also shown in Table 3. In addition, the test was implemented twice with each triplet for each test sample solution, and the average was obtained. In addition, when only NGF was added to this experimental system (5 ng / ml), the proportion of cells in which neurite outgrowth was observed was about 5%.
Figure JPOXMLDOC01-appb-C000049
 　一般式３においてＲ７乃至Ｒ９は炭素数が同一で、炭素数が７乃至９のアルキル基を表し、ｍＸ３ －はＩ－又はＣｌ－を表す。
Figure JPOXMLDOC01-appb-C000049
 In the general formula 3, R 7 to R 9 represent the same alkyl group having 7 to 9 carbon atoms, and mX 3 — represents I 2 — or Cl 2 — .
Figure JPOXMLDOC01-appb-T000050
Figure JPOXMLDOC01-appb-T000050
　表３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を添加した場合には、ＮＧＦを単独で添加した場合に比して、神経突起伸展の見られた細胞の割合が増加し、３乃至１０のものではさらに増加し、５乃至１０のもので、最も増加した。また、ＮＫ－２４とＮＫ－５６、或いは、ＮＫ－１９とＮＫ－５３とを比較すると、何れの場合にも、神経突起伸展の見られた細胞の割合は、ほぼ同じなのであった。 As can be seen from Table 3, when 3 to 12 carbon atoms of the side chain alkyl group were added, the proportion of cells with neurite outgrowth was higher than when NGF was added alone. It increased, further increased with 3-10, and most increased with 5-10. In addition, when NK-24 and NK-56, or NK-19 and NK-53 were compared, the proportion of cells in which neurite outgrowth was observed was almost the same.
　以上の実験結果は、ＮＫ－１９類縁体では、その側鎖のアルキル基の炭素数が３乃至１２の化合物が神経突起伸展促進作用を有し、３乃至１０でその作用は強くなり、５乃至１０のもので、最も強くなることから、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表される構造を有し、側鎖のアルキル基の炭素数が３乃至１０個の化合物は、神経突起伸展促進剤として有用であり、アルツハイマー病や小脳失調症をはじめとする神経変性疾患治療剤として利用できることを物語っている。また、ＮＫ－１９類縁体の持つ神経突起伸展促進作用は、対アニオンの種類による差はないと判断した。 The above experimental results show that, in the NK-19 analog, a compound having 3 to 12 carbon atoms in the side chain alkyl group has a neurite outgrowth promoting action, and the action is strong at 3 to 10; 10 having the structure represented by the general formula 3 such as NK-19, NK-53, NK-150, etc., wherein the alkyl group in the side chain has 3 to 10 carbon atoms. The compound is useful as a neurite outgrowth promoting agent, indicating that it can be used as a therapeutic agent for neurodegenerative diseases including Alzheimer's disease and cerebellar ataxia. In addition, it was judged that the neurite outgrowth promoting action of the NK-19 analog was not different depending on the type of counter anion.
　＜実験４：ＮＫ－４類縁体の神経突起伸展促進作用＞
　実験３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４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４又は５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４又は５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４に、神経突起伸展促進作用の結果を表５にそれぞれ併せて示す。 <Experiment 4: neurite outgrowth promoting action of NK-4 analog>
As in Experiment 3, 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 4, NK-234, NK-26, NK-9815, NK-9694, NK-28 and NK-147, each in 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 4 or 5, 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 compound concentration becomes the concentration shown in Table 4 or 5. 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 4 shows the results of the cell growth promoting action, and Table 5 shows the results of the neurite extension promoting action.
Figure JPOXMLDOC01-appb-C000051
 　一般式２においてＲ４乃至Ｒ６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、Ｘ２はＩ－で、ｍは２を表す。
Figure JPOXMLDOC01-appb-C000051
 In General Formula 2, R 4 to R 6 represent the same alkyl group having 1 to 12 carbon atoms, X 2 represents I − , and m represents 2.
Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000053
Figure JPOXMLDOC01-appb-T000053
　表４及び５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と同など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で表される構造を有し、側鎖のアルキル基の炭素数が２乃至８のである化合物、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 4 and 5, the NK-4 analog represented by the general formula 2 is a cell having 3 to 8 carbon atoms in the side chain alkyl group, which is the same or higher than NK-4. Growth and neurite outgrowth promoting effects were observed. When compared at a 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. In addition, a compound having a structure represented by the general formula 3 and having a side chain alkyl group having 2 to 8 carbon atoms, particularly a side chain alkyl group represented by the general formula 3 having 6 to 8 carbon atoms. NK-19 analog compound was found to show strong cell proliferation and neurite outgrowth promoting activity.
　実験１乃至４の結果は、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表される構造を有し、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、ＮＫ－２６、ＮＫ－９８１５、ＮＫ－９６９４をはじめとする一般式２で表される構造を有し、側鎖のアルキル基の炭素数が２乃至８個の化合物は、神経突起伸展促進剤として有用であり、これらは、アルツハイマー病、小脳失調症、脳梗塞はじめとする神経変性症の治療剤として有用であることを物語っている。 The results of Experiments 1 to 4 are the same as those of the compounds having the structure represented by the general formula 3 such as NK-19, NK-53, NK-150, and the side chain alkyl group having 3 to 10 carbon atoms. , NK-26, NK-9815, NK-9694, and other compounds having a structure represented by general formula 2 and having 2 to 8 carbon atoms in the side chain alkyl group are neurite outgrowth promoters These are useful as therapeutic agents for neurodegenerative diseases such as Alzheimer's disease, cerebellar ataxia and cerebral infarction.
＜実験５：小脳変性運動失調症ハムスターの行動及び脳組織に及ぼすＮＫ－４投与の影響＞
　実験１乃至４で、ＮＫ－４やその類縁体が、神経突起伸展促進剤として利用でき、神経変性疾患治療剤として利用できることが確認されたので、ヒトの神経変性疾患（脊髄小脳変性症など）のモデル動物として好適な小脳変性運動失調症（以下、「小脳失調症」という。）ハムスター（以下、「小脳失調症ハムスター」という）を使用して、その行動及び脳組織に及ぼすＮＫ－４投与の影響を調べた。すなわち、生後３週齢以降に小脳のプルキンエ細胞の脱落が認められ、それに引き続いて、７週齢以降に運動失調と、小脳失調症を自然発症することが知られている自然発症遺伝子突然変異（Ｎｎａ１抑制）ハムスター（Ａｋｉｔａ　Ｋ．など、『Ｊ．Ｎｅｕｒｏｇｅｎｅｔｉｃｓ』、第２１巻、第１乃至１１頁（２００７年）参照）、株式会社林原生物化学研究所で飼育）２５匹を、表６に示す試験群１乃至５に、各群５匹を無作為に割り付けた。試験群１のハムスターには、小脳失調症発症前（３週齢）から、ＰＢＳを１０ｍｌ／ｋｇ／日投与した。試験群２乃至４のハムスターには、小脳失調症発症前（３週齢）から、ＮＫ－４を、２０μｇ／ｋｇ、１００μｇ／ｋｇ又は５００μｇ／ｋｇ／日投与した。試験群５のハムスターには、神経細胞栄養因子として知られるインスリン様栄養因子－１（Ａｓｓａｙｐｒｏ社販売、商品名「ＩＧＦ－１、ｈｕｍａｎ」、ヒト由来、以下、「ＩＧＦ－１」と略記する。）を２５μｇ／ｋｇ／日投与した。これらの投与成分は、１日１回１０週齢まで、毎日腹腔内に投与した。小脳失調症の症状の程度と、ＮＫ－４によるその症状の改善効果を、後述するロタロッド試験、斜面耐久試験を週１回行って、ハムスターの運動協調性の改善を指標に評価した。さらに、１０週齢で、ハムスターの転倒する回数を測定後、脳を摘出し、組織学的評価を行い、併せて、血中及び脳脊髄液（ＣＳＦ）中のグルタミン酸濃度を測定した。試験群６として、試験群１乃至５で使用した小脳失調症ハムスターと同一週齢の正常ハムスター５匹に、小脳失調症発症前（３週齢）から、ＰＢＳを１０ｍｌ／ｋｇ／日を、１日１回１０週齢まで、毎日腹腔内投与して、小脳失調症ハムスターと同じ試験を行った。 <Experiment 5: Effects of NK-4 administration on behavior and brain tissue of cerebellar degenerative ataxia hamster>
Experiments 1 to 4 confirmed that NK-4 and its analogs can be used as neurite outgrowth promoters and therapeutic agents for neurodegenerative diseases, so human neurodegenerative diseases (such as spinocerebellar degeneration) Of cerebellar degenerative ataxia (hereinafter referred to as “cerebellar ataxia”) hamster (hereinafter referred to as “cerebellar ataxia hamster”) suitable as a model animal of NK-4, and its effects on the behavior and brain tissue The influence of was investigated. In other words, pulmonary Purkinje cells were shed after 3 weeks of age, followed by spontaneous gene mutations known to spontaneously develop ataxia and cerebellar ataxia after 7 weeks of age ( Nna1 inhibition) 25 hamsters (see Akita K. et al., “J. Neurogenetics”, Volume 21, pages 1 to 11 (2007), reared at Hayashibara Biochemical Research Institute, Inc.) Test groups 1-5 were randomly assigned 5 animals in each group. Test group 1 hamsters were administered with 10 ml / kg / day of PBS before the onset of cerebellar ataxia (3 weeks of age). The hamsters in test groups 2 to 4 were administered NK-4 at 20 μg / kg, 100 μg / kg, or 500 μg / kg / day before the onset of cerebellar ataxia (3 weeks of age). The hamster of Test Group 5 is abbreviated as “IGF-1”, an insulin-like trophic factor-1 known as a neuronal trophic factor (sold by Assaypro, trade name “IGF-1, human”, human origin). ) Was administered at 25 μg / kg / day. These administration components were administered intraperitoneally once daily until 10 weeks of age. The degree of cerebellar ataxia and the effect of NK-4 on improving the symptoms were evaluated by using the rotarod test and slope endurance test described later once a week, with the improvement of hamster motor coordination as an index. Furthermore, at the age of 10 weeks, after measuring the number of hamster falls, the brain was removed and histologically evaluated, and the glutamic acid concentration in blood and cerebrospinal fluid (CSF) was also measured. As test group 6, 5 normal hamsters of the same age as the cerebellar ataxia hamster used in test groups 1 to 5 were treated with PBS at 10 ml / kg / day before the onset of cerebellar ataxia (3 weeks old). The same test as that of the cerebellar ataxia hamster was carried out by intraperitoneal administration once a day until 10 weeks of age.
＜ロタロッド試験＞
　ハムスターが、ロタロッドの回転にあわせて歩行運動し、ロタロッド上に留まるために行う運動の継続時間を運動協調性の指標とし使用した。すなわち、ハムスターを一定速度（６ｒｐｍ）で回転するロタロッド装置（株式会社林原生物化学研究所製造、ロタロッドの直径６０ｍｍ）に乗せ、ロタロッドから落下するまでの時間を測定した（Ｆｅｒｎａｎｄｅｚなど、『Ｐｒｏｃ．　Ｎａｔｌ．　Ａｃａｄ．　Ｓｃｉ．ＵＳＡ』、第９５号、第１２５３乃至１２５８頁（１９９８年）参照）。試験は、１匹のハムスターについて６回行い、最初から５回は、回転運動に馴化させるための予備運動試験とし、６回目の試験で、ロタロッドから落下するまでの時間（以下、「落下時間」という）を計測して、各群５匹の平均を求めた。結果を表６に示す。なお、落下時間は１８０秒まで計測した。さらに、この結果に基づき、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）の週齢と落下時間の関係を示すグラフを作成し、小脳失調症ハムスターにＮＫ－４（試験群２乃至４）又はＩＧＦ－１（試験群５）を投与した時の、５週齢以降（ＮＫ－４又はＩＧＦ－１投与２週間以降）について、各試験群の各週齢のハムスターの落下時間が、試験群１のハムスターの何週齢の落下時間と同じになるかをグラフから求め、試験群２乃至５のハムスターの小脳失調症の進行が遅延された日数を求めた（＝試験群２乃至５の週齢×７（日）－計算から求めた試験群２乃至５の週齢と同じ落下時間を示す試験群１の週齢×７（日））。結果を表７に示す。なお、表７に示すように、正常ハムスターでは、試験した何れの週齢においても、１８０秒間では、ロッドから落下したハムスターは認められなかったので、１８０秒以内に落下した場合には、ハムスターが小脳失調症を発症して運動協調性が低下したと判断した。 <Rotarod test>
The hamster walked with the rotation of the rotarod and used the duration of the exercise to stay on the rotarod as an indicator of motor coordination. Specifically, a hamster was placed on a rotarod apparatus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd., rotarod diameter 60 mm) rotating at a constant speed (6 rpm), and the time until it dropped from the rotarod was measured (Fernandez et al., “Proc. Natl. Acad. Sci. USA, 95, 1253-1258 (1998)). The test is conducted 6 times for one hamster, and the first 5 times is a preliminary motion test for acclimatization to the rotational motion. In the sixth test, the time until it falls from the rotarod (hereinafter referred to as “fall time”) And the average of 5 animals in each group was obtained. The results are shown in Table 6. The falling time was measured up to 180 seconds. Further, based on this result, a graph showing the relationship between the age of the cerebellar ataxia hamster when PBS was administered (test group 1) and the fall time was prepared. 4) or after 5 weeks of age (after 2 weeks of NK-4 or IGF-1 administration) when IGF-1 (test group 5) was administered, the fall time of each weekly hamster in each test group was tested From the graph, the number of weeks of fall of the hamsters in group 1 was the same as the fall time, and the number of days in which progression of cerebellar ataxia in hamsters in test groups 2 to 5 was delayed was calculated (= in test groups 2 to 5). Week age x 7 (days)-Test group 1 week age x 7 (days) showing the same drop time as that of test groups 2-5 as determined from the calculation. The results are shown in Table 7. As shown in Table 7, in any normal hamster, no hamster dropped from the rod in 180 seconds at any age tested, so if it falls within 180 seconds, It was judged that cerebellar ataxia developed and motor coordination decreased.
＜斜面耐久試験＞
　ハムスターの頭部を上にして、傾斜角度の変えられる板の上に乗せ、５秒間静止できる角度を判定して、斜面耐久傾斜角度（Ｒｉｖｌｉｎなど、『Ｊ．Ｎｅｕｒｏｓｕｒｇ．』、第４７巻、第５７７－５８１頁（１９９７年）参照）とした。傾斜角度は２５度から開始し、５度ずつ上昇させた。静止が５秒未満で落下した場合には、その傾斜角度から、１度間隔で角度を減じて、５秒間静止できる角度を判定して、斜面耐久傾斜角度を測定して、各群５匹の平均を求めた。結果を表８に示す。 <Slope durability test>
Determine the angle at which the hamster's head is placed on a plate whose tilt angle can be changed and can rest for 5 seconds, and the slope endurance angle (Rivlin et al., “J. Neurosurg.”, Vol. 47, Vol. 577-581 (1997)). The tilt angle started from 25 degrees and increased by 5 degrees. If the vehicle falls in less than 5 seconds, subtract the angle at 1 degree intervals from the tilt angle, determine the angle at which it can stand for 5 seconds, measure the slope endurance tilt angle, The average was calculated. The results are shown in Table 8.
＜ハムスターの転倒する回数＞
　１０週齢のハムスターを１匹ずつ飼育ケージに入れ、１分間に転倒する回数を肉眼観察により計測して、各群５匹の平均を求めた。結果を表９に示す。なお、この試験で使用した小脳失調症ハムスターは、９週齢以降に転倒頻度が増加することが知られている。 <Number of hamster falls>
One 10-week-old hamster was put in a breeding cage one by one, and the number of falls per minute was measured by visual observation, and the average of 5 animals in each group was determined. The results are shown in Table 9. In addition, it is known that the cerebellar ataxia hamster used in this test increases the frequency of falls after 9 weeks of age.
＜組織学的評価＞
　転倒回数を確認などの運動協調性に関する試験終了後の１０週齢のハムスターにペントバルビタールを５０ｍｇ／ｋｇ腹腔内投与し、麻酔下で、後大静脈より放血、致死せしめ、大脳及び小脳を摘出し、１０％ホルマリン溶液で固定後、上部より大脳及び小脳を、デジタルカメラで、一定の高さから撮影し、それぞれの矢状方向及び水平方向の直径を計測した。矢状方向長２×水平方向長×０．５を計算して、それぞれ大脳体積及び小脳体積として、各群５匹の平均を求めた。結果を表９に示す。さらに、この試験で使用した小脳失調症ハムスターは、抑制性神経細胞のプルキンエ細胞に加えて、興奮性神経細胞である顆粒細胞も細胞密度が減少することが知られている。そこで、矢状方向に切り出した小脳切片を、常法によりヘマトキシリン・エオシン染色し、検鏡してプルキンエ細胞層（小葉Ｉ乃至Ｘ）にある全プルキンエ細胞数及び単位面積当たりの顆粒細胞数を計測すると共に、小脳白質に脱髄が認められる個体数を確認した。結果を表９に併せて示す。なお、大脳体積は、各試験群間で有意の差が認められなかったので、表９には小脳体積の計算結果のみを示す。
Figure JPOXMLDOC01-appb-T000054
 <Histological evaluation>
50 mg / kg of pentobarbital was intraperitoneally administered to a 10-week-old hamster after completion of a test on motor coordination such as confirming the number of falls. Under anesthesia, blood was discharged from the posterior vena cava and lethal, and the cerebrum and cerebellum were removed. After fixing with a 10% formalin solution, the cerebrum and cerebellum were photographed from above at a certain height with a digital camera, and the diameters in the sagittal and horizontal directions were measured. Sagittal length 2 × horizontal length × 0.5 was calculated, and the average of 5 mice in each group was determined as cerebral volume and cerebellar volume, respectively. The results are shown in Table 9. Furthermore, the cerebellar ataxia hamster used in this test is known to have a reduced cell density in addition to Purkinje cells, which are inhibitory neurons, and granule cells, which are excitatory neurons. Therefore, cerebellar slices cut in the sagittal direction were stained with hematoxylin and eosin by a conventional method, and microscopically measured to determine the total number of Purkinje cells in the Purkinje cell layer (lobules I to X) and the number of granule cells per unit area. In addition, the number of individuals with demyelination in the cerebellar white matter was confirmed. The results are also shown in Table 9. In addition, since the cerebral volume did not recognize a significant difference between each test group, Table 9 shows only the calculation result of the cerebellum volume.
Figure JPOXMLDOC01-appb-T000054
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000055
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-T000056
Figure JPOXMLDOC01-appb-T000057
Figure JPOXMLDOC01-appb-T000057
　表６から明らかなよう、４週齢の正常ハムスター（試験群６）の落下時間は１８０秒以上だったのに対して、同じ４週齢の小脳失調症ハムスターを使用した試験群１（ＰＢＳ投与群）の落下時間は１０８±１０秒で、すでに、正常ハムスターに比較して有意な落下時間の短縮が認められ、以後、加齢と共に、さらに短縮した。１０週齢では、ロタロッドに留まることはできず、すぐに落下（０秒）した。これに対して、ＮＫ－４を投与したハムスターでは、投与開始後１週目（４週齢）から、用量依存的に、落下時間短縮の抑制が認められ、試験群３（１００μｇ／ｋｇ・体重）、及び、試験群４（５００μｇ／ｋｇ・体重）では、試験群１に比較して有意な落下時間短縮の抑制効果が認められた。この抑制効果は、試験終了の１０週齢（投与７週間）まで持続した。試験群２（２０μｇ／ｋｇ・体重）でもＮＫ－４投与開始後２週目（５週齢）以降に、試験群１よりも落下時間の短縮の抑制効果は認められたが、試験群３及び試験群４に比較してその効果は弱かった。また、運動神経変性疾患の治療に効果があるとされているＩＧＦ－１を投与した場合（試験群５）には、落下時間短縮の抑制効果はほとんど認められなかった。同様に、落下時間短縮の遅延日数を示す表７からも明らかなように、１０週齢（ＮＫ－４投与期間４９日）時点でも、試験群２乃至４では、各々、３２、３６、及び、３５日の、落下時間短縮の遅延が認められた。この試験期間を通して、試験群３及び４で最も高い落下時間短縮の抑制効果が認められ、小脳失調症の発症遅延効果が認められた。しかし、ＩＧＦ－１を投与した試験群５ではほとんど症状発症遅延効果は認められず、１０週齢時点では、落下時間に試験群１との差は認められなかった。この結果は、ＮＫ－４が、ヒトの神経変性症及びその病態や症状の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 6, the fall time of the 4-week-old normal hamster (Test Group 6) was 180 seconds or longer, whereas Test Group 1 (PBS administration) using the same 4-week-old cerebellar ataxia hamster The fall time of the group) was 108 ± 10 seconds, and a significant shortening of the fall time was already observed as compared with normal hamsters. At the age of 10 weeks, it was not possible to stay on the rotarod and it immediately dropped (0 seconds). In contrast, in the hamsters administered with NK-4, the reduction of the fall time was observed in a dose-dependent manner from the first week (4 weeks of age) after the start of administration, and in test group 3 (100 μg / kg / body weight). ) And test group 4 (500 μg / kg · body weight), a significant effect of reducing the drop time was observed compared to test group 1. This inhibitory effect lasted until the end of the study at 10 weeks of age (7 weeks of administration). Even in Test Group 2 (20 μg / kg / body weight), after 2 weeks (5 weeks old) after the start of NK-4 administration, the effect of reducing the drop time was observed more than in Test Group 1, but Test Group 3 and Compared with test group 4, the effect was weak. In addition, when IGF-1 which is considered to be effective in the treatment of motor neurodegenerative diseases was administered (Test Group 5), the effect of suppressing the fall time was hardly observed. Similarly, as is apparent from Table 7 showing the days of delay in reducing the fall time, at the time of 10 weeks of age (NK-4 administration period 49 days), in test groups 2 to 4, 32, 36, and A delay in shortening the fall time was observed on the 35th. Throughout this test period, the highest effect of reducing the fall time was observed in Test Groups 3 and 4, and the onset delay effect of cerebellar ataxia was observed. However, almost no symptom onset delay effect was observed in Test Group 5 administered with IGF-1, and no difference in drop time from Test Group 1 was observed at 10 weeks of age. This result demonstrates that NK-4 can be used as a therapeutic agent for human neurodegeneration and its pathological conditions and symptoms.
　表８の結果から明らかなように、試験群６（正常ハムスター）の斜面耐久傾斜角度は、試験期間を通して４６度から５２度の範囲内であったが、小脳失調症ハムスターを使用した試験群１（ＰＢＳ投与群）では、試験開始時には、すでに、４１０．６±０．３度と有意に低く、加齢に伴い、減少して、８週齢以降はさらに低下し、１０週齢では３５．８±１．０度となった。試験群５（ＩＧＦ－１投与）では、４週齢で４４．４±０．２度となって、試験群１の４０．６±０．３度に比較して有意な斜面耐久傾斜角度の低下抑制効果が認められたものの、それ以降は試験群１と同様に斜面耐久傾斜角度が低下し、５週齢以降は有意な低下抑制効果は認められなかった。これに対して、試験群２乃至４（ＮＫ－４投与）では、試験に使用した何れの投与量でも、試験期間を通して耐久傾斜角度低下は認められず、高い斜面耐久傾斜角度低下抑制効果が認められた。１０週齢での試験群２乃至４（ＮＫ－４投与）の斜面耐久傾斜角度は、各々、４５．８±０．６、５１．６±０．６、５１．８±０．４度となり、いずれも試験群１（ＰＢＳ投与）や試験群５（ＩＧＦ－１投与）に比較して有意に高かった。 As is clear from the results in Table 8, the slope endurance slope angle of test group 6 (normal hamster) was in the range of 46 to 52 degrees throughout the test period, but test group 1 using cerebellar ataxia hamsters. In the PBS administration group, it was already significantly lower at 410.6 ± 0.3 degrees at the start of the test, decreased with aging, further decreased after 8 weeks of age, and 35. It was 8 ± 1.0 degrees. In test group 5 (IGF-1 administration), 44.4 ± 0.2 degrees at 4 weeks of age, which is a significant slope durability inclination angle compared to 40.6 ± 0.3 degrees in test group 1 Although a decrease suppressing effect was observed, thereafter, the slope durability inclination angle decreased as in Test Group 1, and no significant decrease suppressing effect was observed after 5 weeks of age. In contrast, in test groups 2 to 4 (NK-4 administration), no decrease in endurance tilt angle was observed throughout the test period at any dose used in the test, and a high slope endurance tilt angle decrease suppression effect was observed. It was. The slope endurance slopes of the test groups 2 to 4 (NK-4 administration) at 10 weeks of age are 45.8 ± 0.6, 51.6 ± 0.6, and 51.8 ± 0.4 degrees, respectively. Both were significantly higher than those in Test Group 1 (PBS administration) and Test Group 5 (IGF-1 administration).
　表９の結果から明らかなように、１０週齢の正常ハムスター（試験群６）では、転倒は認められないのに対して、試験群１（ＰＢＳ投与）では、１２．８±０．５回／分の転倒が認められた。これに対して、試験群５（ＩＧＦ－１投与）では、１１．４±０．４回／分と試験群１と比較して、やや減少傾向にあったが、有意な転倒減少効果は認められなかった。これに対して、試験群２乃至４（ＮＫ－４投与）では、各々、４．０±１．０、１．６±０．９、１．２±０．８回／分と、いずれの群でも有意な転倒回数の減少が認められた。また、試験群１（ＰＢＳ投与）の小脳体積は、１０週齢で６４．６±９．４ｍｍ３と、同一週齢の正常ハムスター（試験群６）の９４．７±１１．４ｍｍ３に比較して有意な萎縮が認められた。これに対し、試験群５（ＩＧＦ－１投与）では、７７．６±６．１（ｍｍ３）と、試験群１に比して有意な小脳の萎縮抑制効果が認められた。また、ＮＫ－４投与群でも用量依存的な小脳萎縮抑制効果が認められ、２０、１００、５００μｇ／ｋｇ投与群で、それぞれ、７６．０±８．２、７７．０±２．８、８０．５±１０．８ｍｍ３となった（全てＰ＜０．０５で有意）。一方、何れの群においても大脳体積に有意の差は認められなかった（データ未掲載）。以上の結果から、ＮＫ－４は、小脳失調症ハムスターの運動協調性を改善するとともに、小脳萎縮を効果的に抑制すること、及び、その効果はＩＧＦ－１よりも優れていることが判明した。 As is apparent from the results of Table 9, no fall was observed in the 10-week-old normal hamster (Test Group 6), whereas in Test Group 1 (PBS administration), 12.8 ± 0.5 times A fall of / min was observed. In contrast, in test group 5 (IGF-1 administration), 11.4 ± 0.4 times / min was slightly decreased compared to test group 1, but a significant fall-reducing effect was observed. I couldn't. In contrast, in test groups 2 to 4 (NK-4 administration), 4.0 ± 1.0, 1.6 ± 0.9, and 1.2 ± 0.8 times / min, respectively, There was also a significant decrease in the number of falls in the group. In addition, the cerebellar volume of test group 1 (PBS administration) was 64.6 ± 9.4 mm 3 at 10 weeks of age and 94.7 ± 11.4 mm 3 of normal hamsters (test group 6) of the same age. As a result, significant atrophy was observed. In contrast, in test group 5 (IGF-1 administration), a significant cerebellar atrophy suppression effect was observed as compared to test group 1, which was 77.6 ± 6.1 (mm 3 ). In addition, a dose-dependent cerebellar atrophy suppression effect was also observed in the NK-4 administration group, and 76.0 ± 8.2, 77.0 ± 2.8, 80 in the 20, 100, and 500 μg / kg administration groups, respectively. 5 ± 10.8 mm 3 (all significant at P <0.05). On the other hand, there was no significant difference in cerebral volume in any group (data not shown). From the above results, it was found that NK-4 improves motor coordination of cerebellar ataxia hamsters, effectively suppresses cerebellar atrophy, and its effect is superior to IGF-1. .
　また、表９の結果から明らかなように、小脳皮質の顆粒細胞層にある顆粒細胞密度は、１０週齢の正常ハムスター（試験群６）が、４８０±６個／２００００μｍ２であるのに対し、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）は３８０±４個／２００００μｍ２にまで、有意に減少した。試験群５（ＩＧＦ－１投与）の顆粒細胞密度は、３７１±１１個／２００００μｍ２と、試験群１と差は認められなかった。試験群２乃至４（ＮＫ－４投与）では、各々、４０８±８、４１９±６、４３６±７個／２００００μｍ２となり、ＮＫ－４の投与量に依存した顆粒細胞密度減少抑制効果が認められた。また、具体的なデータは示さないが、小脳実質組織の顕微鏡観察では、試験群１や５では顆粒細胞の萎縮や変性が顕著であったのに対して、試験群２乃至４では、顆粒細胞の萎縮や変性が抑制されていることが確認された。さらに、小脳失調症ハムスターにＰＢＳを投与した場合（試験群１）では、小脳白質の脱髄が全個体（５匹中５匹）に認められたが、ＮＫ－４投与群では試験群２乃至４（ＮＫ－４投与）では、各々、５匹中４匹、５匹中１匹、５匹中０匹に脱髄が認められたのみであり、これらのＮＫ－４投与群では、プルキンエ細胞の持つ神経突起（樹状突起）の萎縮の抑制も確認された。この結果は、ＮＫ－４を神経突起伸展促進剤として有用であることを物語っている。また、ＮＫ－４は、小脳白質の脱髄の抑制、則ち、プルキンエ細胞の変性や脱落の抑制剤としても有用であることを物語っている。 As is clear from the results in Table 9, the granule cell density in the granule cell layer of the cerebellar cortex is 480 ± 6 / 20,000 μm 2 in 10-week-old normal hamsters (test group 6). When PBS was administered to a cerebellar ataxia hamster (test group 1), the number was significantly reduced to 380 ± 4/20000 μm 2 . The granule cell density in test group 5 (IGF-1 administration) was 371 ± 11 cells / 20,000 μm 2, which was not different from test group 1. In test groups 2 to 4 (NK-4 administration), the doses were 408 ± 8, 419 ± 6, 436 ± 7 / 20,000 μm 2 , respectively, and an inhibitory effect on the decrease in granule cell density depending on the dose of NK-4 was observed. It was. In addition, although specific data are not shown, in microscopic observation of the cerebellar parenchyma, in the test groups 1 and 5, granule cell atrophy and degeneration were significant, whereas in the test groups 2 to 4, granule cells were observed. It was confirmed that atrophy and degeneration were suppressed. Furthermore, when PBS was administered to a cerebellar ataxia hamster (test group 1), demyelination of cerebellar white matter was observed in all individuals (5 of 5 animals), whereas in the NK-4 administration group, test groups 2 to 4 (NK-4 administration), only 4 out of 5 animals, 1 out of 5 animals, and 0 out of 5 animals were demyelinated. In these NK-4 administration groups, Purkinje cells Suppression of atrophy of neurites (dendrites) in the body was also confirmed. This result shows that NK-4 is useful as a neurite extension promoter. NK-4 also shows that it is useful as an inhibitor of cerebellar white matter demyelination, that is, as an inhibitor of Purkinje cell degeneration and shedding.
　以上のように、ＮＫ－４を投与した小脳失調症ハムスターでは、ロタロッド試験、斜面耐久試験、転倒回数の全ての試験項目で症状改善が認められ、その効果は、ＮＫ－４の投与量に依存し、投与量が１００及び５００μｇ／ｋｇ・体重／日で、特に高い効果が得られた。また、小脳失調症ハムスターの症状発症の原因となる小脳プルキンエ細胞では、神経突起の萎縮の抑制が確認された。この結果は、腹腔内に投与したＮＫ－４は、脳神経細胞に作用して、その神経突起伸展作用（萎縮抑制を含む）により、神経機能低下を抑制し小脳失調症を抑制したことを示している。これらの結果は、ＮＫ－４が、神経突起伸展促進剤として、ヒトの神経変性症やそれに伴う種々の病態や臨床症状に対する全身投与可能な治療剤として有用であることを物語っている。なお、小脳失調ハムスターの体重を試験終了（１０週齢）まで毎週１回計測して、各群の平均を求めたところ、何れの週齢においても、ＰＢＳ、ＮＫ－４、ＩＧＦ－１投与群間に有意な差は認められなかったことから、ＮＫ－４は、生体へ投与しても安全性は高い化合物と判断した。なお、的に具体的なデータは示さないが、ＮＫ－４を経口摂取させ、本実験と同じ系でその効果を調べたところ、ＮＫ－４を腹腔内投与した場合と同じ効果を得るためには、腹腔内投と比較して約１００倍量のＮＫ－４の摂取が必要であった。 As described above, in cerebellar ataxia hamsters administered with NK-4, symptom improvement was observed in all test items of rotarod test, slope endurance test, and number of falls, and the effect depends on the dose of NK-4 However, particularly high effects were obtained at doses of 100 and 500 μg / kg · body weight / day. Moreover, suppression of neurite atrophy was confirmed in cerebellar Purkinje cells that cause cerebellar ataxia hamsters. This result indicates that NK-4 administered intraperitoneally acted on cerebral neurons and suppressed cerebellar ataxia by suppressing neurite outgrowth (including suppression of atrophy) and suppressing neuronal function decline. Yes. These results indicate that NK-4 is useful as a neurite outgrowth promoter as a therapeutic agent that can be systemically administered to human neurodegeneration and various pathological conditions and clinical symptoms associated therewith. The body weight of the cerebellar ataxia hamster was measured once every week until the end of the test (10 weeks of age), and the average of each group was determined. At any age, PBS, NK-4, IGF-1 administration group Since no significant difference was observed, NK-4 was judged to be a highly safe compound even when administered to a living body. Although specific data is not specifically shown, when NK-4 was orally ingested and its effect was examined in the same system as this experiment, in order to obtain the same effect as when NK-4 was administered intraperitoneally Required about 100 times the intake of NK-4 compared to intraperitoneal injection.
＜実験６：各色素化合物の脳虚血モデルラットに及ぼす影響＞
　実験１乃至５で、ＮＫ－４や、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００、及び、ＮＫ－５５７に神経変性症に対する治療効果が確認されたので、これらの化合物の投与が、ヒトの脳梗塞の好適なモデルとされている脳虚血モデルラットに及ぼす影響を、行動学的指標及び脳梗塞部位の体積を指標として調べた。 <Experiment 6: Effect of each dye compound on cerebral ischemia model rats>
In Experiments 1 to 5, NK-4, NK-19, NK-53, NK-100, and NK-557 have been confirmed to have therapeutic effects on neurodegeneration. The effect on the cerebral ischemia model rat, which is a suitable model of cerebral infarction, was examined using the behavioral index and the volume of the cerebral infarction site as an index.
＜脳虚血ラット＞
　ＳＤラット（雄性、７乃至８週齢、体重２８０乃至３３０ｇ、日本チャールスリバー株式会社販売）を、無作為に７群各５乃至７匹を割り付けた。予め、アトロピン（扶桑薬品、０．３ｍｇ／ｋｇ・体重）を皮下に投与した、ウレタン（シグマ社販売６００ｍｇ／ｋｇ・体重）及びα－クロラロース（シグマ社販売、６０ｍｇ／ｋｇ・体重）を腹腔内に投与して、麻酔を施し、自然呼吸のまま、固定器に固定した。頚部正中切開を加え、迷走神経の保存に留意しつつ、右頚動脈分岐部に達した。右頚動脈分岐部を中心に、総頚動脈及び外頚動脈を周囲結合織より剥離し、それぞれ６－０ナイロン糸（アルフレッサーファーマ株式会社販売、商品名「ネスコスーチャー」）にて結紮した。次に、内頚動脈に６－０ナイロン糸をかけ、塞栓挿入後の固定に備えた。次いで、総頚動脈を切開し、同部より先端をシリコンコートした４－０ナイロン糸で作成した塞栓（Ｄｏｃｃｏｌ）を内頚動脈に向けて約１６ｍｍ挿入し、総頚動脈にクリップで固定した。（例えば、小泉ら、「脳卒中」、第８巻、第１号、第１乃至８頁（１９８６年）参照）。なお、この方法により、塞栓のシリコンコートした先端部分は、中大脳動脈分岐部を越えて、前大脳動脈内に２ｍｍ程度入り、中大脳動脈入口を閉塞する。この状態のまま３７℃の保温パッド上で２時間中大脳動脈を閉塞した後、挿入した塞栓を抜き去り、血流を再開通（再灌流）させ、その後、総頚動脈切開部からの出血を防ぐため、内頚動脈を頚動脈分岐部近傍で結紮した。このモデルでの血流再開は、右総頚動脈が結紮されているため、左内径動脈及び、椎骨動脈、脳底動脈より、前・後交通動脈を介して行われる。 <Brain ischemic rat>
SD rats (male, 7 to 8 weeks old, body weight 280 to 330 g, sold by Nippon Charles River Co., Ltd.) were randomly assigned to 5 to 7 animals in 7 groups. In advance, atropine (fuso medicine, 0.3 mg / kg / body weight) was subcutaneously administered, and urethane (Sigma, 600 mg / kg / body weight) and α-chloralose (Sigma, 60 mg / kg / body weight) were intraperitoneally injected. Anesthesia was given, and it was fixed to a fixator with natural breathing. A midline cervical incision was made and the right carotid bifurcation was reached, paying attention to the preservation of the vagus nerve. The common carotid artery and the external carotid artery were peeled off from the surrounding connective tissue, centering on the right carotid artery bifurcation, and ligated with 6-0 nylon yarn (Alfresser Pharma Co., Ltd., trade name “Nesscoacher”). Next, a 6-0 nylon thread was applied to the internal carotid artery to prepare for fixation after embolization. Next, the common carotid artery was incised, and an embolus (Doccol) made of 4-0 nylon thread coated with silicon at the tip was inserted into the internal carotid artery about 16 mm and fixed to the common carotid artery with a clip. (See, for example, Koizumi et al., “Stroke”, Vol. 8, No. 1, pp. 1-8 (1986)). By this method, the tip portion of the embolus that is silicon-coated enters the anterior cerebral artery beyond the middle cerebral artery bifurcation and closes the middle cerebral artery entrance. In this state, the cerebral artery is occluded for 2 hours on a thermal pad at 37 ° C., then the inserted embolus is removed, blood flow is resumed (reperfusion), and then bleeding from the common carotid artery incision is prevented. Therefore, the internal carotid artery was ligated near the carotid bifurcation. Since the right common carotid artery is ligated in this model, the blood flow is resumed from the left inner diameter artery, the vertebral artery, and the basilar artery via the anterior / posterior traffic artery.
＜色素化合物の投与＞
　試験に使用したＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、各々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）した。各化合物を、使用時に、各々ＰＢＳに２５ｎｇ／ｍｌとなるように溶解して、そのいずれかを、５群各５匹又は７匹のラットに、中大脳動脈閉塞１時間後及び血液の再灌流時に尾静脈内に投与した（４ｍｌ／ｋｇ・体重、化合物の投与量１００μｇ／ｋｇ・体重）（試験群１乃至５）。血流再開２４時間後に、行動学的及び組織学的評価を行った。残りの２群各５匹の内の１群６匹には、対照群１として、化合物を含まないＰＢＳを４ｍｌ／ｋｇ・体重、中大脳動脈閉塞１時間後及び血液の再灌流時に尾静脈内投与した。また、残りの１群５匹には、対照群２として、総頚、外頚、内頚動脈の結紮した後、塞栓を中大脳動脈付近に挿入せずに、血流を再開させた偽手術（Ｓｈａｍ手術）を行った。対照群２にも、化合物を含まないＰＢＳを４ｍｌ／ｋｇ・体重、総頚、外頚、内頚動脈の結紮１時間後及び血液の再灌流時に尾静脈内投与した。 <Dye compound administration>
NK-4, NK-19, NK-53, NK-100 and NK-557 used in the test were each dissolved in DMSO (SIGMA, product number “D8418”) at a concentration of 5 mg / ml, and then the membrane. Filtration (sold by Millipore, trade name “Millex-LG SLLG025SS”, using DMSO resistant membrane) was performed. Each compound was dissolved in PBS to 25 ng / ml at the time of use, and any one of them was administered to 5 or 7 rats in 5 groups each 1 hour after middle cerebral artery occlusion and blood reperfusion. Occasionally, it was administered into the tail vein (4 ml / kg · body weight, compound dose of 100 μg / kg · body weight) (test groups 1 to 5). Behavioral and histological evaluation was performed 24 hours after resumption of blood flow. Of the remaining 2 groups, 5 out of 5 animals, 1 group had 6 ml of PBS containing no compound as the control group 1 in the tail vein at 1 ml after middle cerebral artery occlusion and at the time of blood reperfusion. Administered. In addition, in the remaining 5 animals in 1 group, as control group 2, after ligation of the common neck, external carotid, and internal carotid artery, sham operation was resumed without inserting an embolus near the middle cerebral artery ( (Sham operation) was performed. In control group 2, PBS containing no compound was also administered into the tail vein at 4 ml / kg · body weight, 1 hour after ligation of the common neck, external neck, and internal carotid artery, and at the time of blood reperfusion.
＜効果の評価方法＞
＜行動学的及び組織学的評価＞
＜行動学的評価＞
　表１０に示す基準に基づいて、各評価項目の症状の程度をスコア化し、個体ごとに積算して、行動学的スコアを求めた（Ｐｅｔｕｌｌｏ　Ｄ．など、『Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ』、第６４巻、第１３号、第１０９９乃至１１０８頁（１９９９年）参照）（最大スコア６）。結果を表１１に示す。 <Evaluation method of effect>
<Behavioral and histological evaluation>
<Behavioral evaluation>
Based on the criteria shown in Table 10, the degree of symptom of each evaluation item was scored and accumulated for each individual to obtain a behavioral score (Petullo D. et al., “Life Sciences”, Vol. 64, Vol. 13, pp. 1099 to 1108 (1999)) (maximum score 6). The results are shown in Table 11.
＜組織学的評価＞
　試験終了後、各々のラットをエーテル麻酔下で、左心室からの生理食塩水を灌流させながら後大静脈を切断して脱血した。死後３分以内に脳を摘出し、スライサー（株式会社林原生物化学研究所製造）を使用して、冠状方向に、２ｍｍの厚さにスライスにした後、脳梗塞部位を、特異的に染色する２，３，５－ｔｒｉｐｈｅｎｙｌｔｅｔｒａｚｏｌｉｕｍ　ｃｈｌｏｒｉｄｅ（ＴＴＣ）を２質量／容積％含有するＰＢＳ中で３７℃、３０分間インキュベートし、１０％ホルマリン溶液中で１時間固定した（Ｂｅｎｄｅｒｓｏｎ　Ｊ．Ｂ．など，　『Ｓｔｒｏｋｅ』、第１７巻、第１３０４乃至１３０８頁（１９８６年）参照）。ＴＴＣで染色された脳梗塞部位の面積を、画像解析フリーソフト（Ｓｃｉｏｎ社販売、商品名「Ｓｃｉｏｎ　Ｉｍａｇｅ」）を用いて解析し、梗塞部位と脳全体の体積を算出した。梗塞部位の体積を、脳の全体積で除し、１００倍して、脳全体に占める梗塞部位の割合（％）を計算した。さらに、対照群１のラットの脳梗塞部位の脳全体に占める割合を１００％としたときの、実験群１乃至５のラットの脳梗塞部位の脳全体に占める割合の相対値を計算して、脳梗塞部位の抑制率（％）とした。結果を表１１に併せて示す。 <Histological evaluation>
After completion of the test, each rat was exsanguinated by severing the posterior vena cava while perfusing physiological saline from the left ventricle under ether anesthesia. The brain is removed within 3 minutes after death, and sliced to a thickness of 2 mm in the coronal direction using a slicer (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.), and then the cerebral infarction site is specifically stained. 2,3,5-triphenyltetrazole chloride (TTC) was incubated in PBS containing 2% by mass / volume at 37 ° C. for 30 minutes and fixed in 10% formalin solution for 1 hour (Benderson JB et al., “Stroke 』, Vol. 17, pp. 1304 to 1308 (1986)). The area of the cerebral infarction site stained with TTC was analyzed using image analysis free software (trade name “Scion Image”, sold by Scion), and the volume of the infarct site and the entire brain was calculated. The volume of the infarcted area was divided by the total volume of the brain, multiplied by 100, and the ratio (%) of the infarcted area to the entire brain was calculated. Furthermore, the relative value of the ratio of the cerebral infarction site of the rats of the experimental groups 1 to 5 to the entire brain when the ratio of the cerebral infarction site of the rats of the control group 1 to 100% is calculated, The inhibition rate (%) of the cerebral infarction site was used. The results are also shown in Table 11.
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000058
Figure JPOXMLDOC01-appb-T000059
Figure JPOXMLDOC01-appb-T000059
　表１１から明らかなように、偽手術を行ったラット（対照群２）の脳には梗塞部位は認められず、行動も全て正常であった。脳虚血を起こしたラット（対照群１）は、行動学的スコアが、５．８±０．１となって、評価した全ての項においてその運動能の殆どを喪失した。これに対して、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７のいずれかを投与したラット（試験群１乃至５）では、何れの場合にも、対照群１に比較して、運動能の喪失が有意に抑制された。脳梗塞の起こった部位の体積で見ても、脳虚血を起こしたラット（対照群１）と比較して、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７のいずれかを投与したラット（試験群１乃至５）では、何れの場合にも、脳梗塞を起こした部位が小さく、ＮＫ－４、ＮＫ－１９、ＮＫ－５３投与（試験群１乃至３）では、有意な脳梗塞部位の縮小が認められた。効果の強さを比較すると、ＮＫ－１９を投与した場合に、行動学的スコア及び脳梗塞部位の抑制率共に、最も強い改善が認められた。この結果は、ＮＫ－４、ＮＫ－１９、ＮＫ－５３、ＮＫ－１００及びＮＫ－５５７は、虚血が原因で引き起こされる神経突起の萎縮をはじめとする神経変性及びそれに伴う神経機能障害に対する治療効果を有することを物語っている。 As is clear from Table 11, the infarcted area was not observed in the brains of the sham-operated rats (control group 2), and the behavior was all normal. Rats with cerebral ischemia (control group 1) had a behavioral score of 5.8 ± 0.1 and lost most of their motility in all terms evaluated. On the other hand, in rats (test groups 1 to 5) administered with any of NK-4, NK-19, NK-53, NK-100 and NK-557, in any case, the control group 1 In comparison, loss of motor ability was significantly suppressed. In terms of the volume of the site where cerebral infarction occurred, NK-4, NK-19, NK-53, NK-100 and NK-557 were compared with those of rats (control group 1) that had cerebral ischemia. In either case (test groups 1 to 5), the site of cerebral infarction was small in any case, and in NK-4, NK-19, and NK-53 (test groups 1 to 3) A significant reduction in the cerebral infarction site was observed. When the strength of the effect was compared, the strongest improvement was observed in both the behavioral score and the inhibition rate of the cerebral infarction site when NK-19 was administered. As a result, NK-4, NK-19, NK-53, NK-100 and NK-557 are effective in treating neurodegeneration including neurite atrophy caused by ischemia and associated neurological dysfunction. It tells you that it has an effect.
＜実験７：ＮＫ－４類縁体又はＮＫ－１９類縁体の脳梗塞モデルラットに及ぼす影響＞
　ＰＣ－１２ＨＳ細胞に対する細胞増殖促進作用及び神経突起促進作用が確認されたＮＫ－４、ＮＫ－２６、ＮＫ－１５が脳梗塞に及ぼす影響をヒトの脳梗塞モデルラットを用いて調べた。すなわち、実験６に準じて、ＳＤラット（日本チャールスリバー社販売、オス、８週齢、体重２８０乃至３３０ｇ）に中大脳動脈に塞栓を行った。ＮＫ－１５０、ＮＫ－２６及びＮＫ－４を、それぞれＤＭＳＯに５ｍｇ／ｍｌの濃度で溶解後、ポワサイズ０．４５μｍの膜フィルターで膜濾過し、さらにＤＭＳＯで２．５乃至０．０５ｍｇ／ｍｌの濃度に調製したものを遮光し保存した。これらの化合物溶液を、使用時に生食で２５０倍希釈し、塞栓したラットに、閉塞１時間後および再開通時の２回、尾静脈より投与した（液量：５ｍｌ／ｋｇ・体重）。ＮＫ－４は１０ｍｇ／ｍｌの溶液を調製し、２５０乃至１６７倍希釈し、尾静脈より投与した（液量：５ｍｌ／ｋｇ・体重）。ＮＫ－４と同じ投与スケジュールで、陰性対照として生理食塩水を投与し、陽性対照として既存薬エダラボン（田辺三菱製薬社製、商品名「ラジカット」）をＤＭＳＯにより希釈し静脈内投与した。実験６と同じ方法により行動学的スコアを求め神経症状を評価した。また、脳梗塞部位の体積（ｍｍ３）は、梗塞に伴う脳の浮腫の影響を排除するため、予め腫脹率（＝虚血側大脳半球の体積÷健常側大脳半球の体積）を求め、梗塞体積の実測値を腫脹率で除して求めた。その結果と群構成を表１２に示した。 <Experiment 7: Effect of NK-4 analog or NK-19 analog on rat model of cerebral infarction>
The effects of NK-4, NK-26, and NK-15, which were confirmed to have cell proliferation promoting action and neurite promoting action on PC-12HS cells, on cerebral infarction were examined using human cerebral infarction model rats. That is, according to Experiment 6, SD rats (manufactured by Charles River Japan, male, 8 weeks old, body weight 280 to 330 g) were embolized in the middle cerebral artery. NK-150, NK-26, and NK-4 were each dissolved in DMSO at a concentration of 5 mg / ml, filtered through a membrane filter with a pore size of 0.45 μm, and further 2.5 to 0.05 mg / ml in DMSO. What was adjusted to the concentration was stored in the dark. These compound solutions were diluted 250-fold with saline at the time of use and administered to the embolized rat through the tail vein twice after 1 hour of occlusion and at the time of reopening (fluid amount: 5 ml / kg / body weight). NK-4 was prepared as a 10 mg / ml solution, diluted 250 to 167 times, and administered from the tail vein (fluid volume: 5 ml / kg / body weight). In the same administration schedule as NK-4, physiological saline was administered as a negative control, and an existing drug edaravone (manufactured by Mitsubishi Tanabe Pharma Corporation, trade name “Radicut”) was diluted with DMSO and administered intravenously as a positive control. A behavioral score was obtained by the same method as in Experiment 6 to evaluate neurological symptoms. Further, the volume of the cerebral infarction site (mm 3 ) is obtained in advance by determining the swelling rate (= volume of ischemic cerebral hemisphere ÷ volume of healthy cerebral hemisphere) in order to eliminate the influence of cerebral edema associated with infarction. The actual volume value was obtained by dividing by the swelling rate. The results and group composition are shown in Table 12.
Figure JPOXMLDOC01-appb-T000060
Figure JPOXMLDOC01-appb-T000060
　表１２から明らかなように、ＮＫ－４、ＮＫ－２６及びＮＫ－１５０を投与したラットでは、至適投与量に差はあるものの、いずれも脳梗塞に伴う行動学的スコアの有意な改善と、脳梗塞部位の体積増加の有意な抑制が認められた。エダラボンの投与では行動学的スコアの改善は認められたものの、脳梗塞部位の体積増加の有意な抑制が認められなかった。この結果は、ＮＫ－４、ＮＫ－２６、ＮＫ－１５０は、虚血が原因で引き起こされる神経突起の萎縮をはじめとする神経変性及びそれに伴う神経機能障害に対する治療効果を有することを物語っている。 As can be seen from Table 12, in rats administered with NK-4, NK-26 and NK-150, although there is a difference in the optimum dose, all of them showed a significant improvement in behavioral scores associated with cerebral infarction. In addition, a significant suppression of volume increase at the cerebral infarction site was observed. Administration of edaravone improved behavioral scores, but did not significantly suppress volume increase at the cerebral infarction site. This result indicates that NK-4, NK-26, and NK-150 have therapeutic effects on neurodegeneration including neurite atrophy caused by ischemia and associated neurological dysfunction. .
＜実験８：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 8: 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刺入しアミロイドβフラグメント（β－Ａｍｙｌｏｉｄ２５－３５：Ｇｌｙ－Ｓｅｒ－Ａｓｎ－Ｌｙｓ－Ｇｌｙ－Ａｌａ－Ｉｌｅ－Ｉｌｅ－Ｇｌｙ－Ｌｅｕ－Ｍｅｔのアミノ酸配列を有するペプチド）：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１３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 amyloid β fragment (β-Amyloid 25-35 : Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met amino acid was determined by setting the insertion point 0.5 mm behind. Peptide having sequence): 9 nmol / 6 μl / animal of solution was administered into the ventricle (for the administration method, see “Brain Research”, 706, 181-193 (1996)). 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 13.
Figure JPOXMLDOC01-appb-T000061
Figure JPOXMLDOC01-appb-T000061
＜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 9th 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１３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 13, 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神経突起の萎縮をはじめとする神経変性及びそれに伴う神経機能障害に対する治療効果を有することを物語っている。 As a result, NK-4, NK-234, NK-26, NK-19 and NK-150 are all against neurodegeneration including neurite atrophy caused by amyloid β peptide and neurological dysfunction associated therewith. It has a therapeutic effect.
＜実験９：ＮＫ－４のＡＰＰトランスジェニックマウスに及ぼす影響＞
　実験１４で、アミロイドβフラグメント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、体重が均などになるように、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１４に示す。 <Experiment 9: Effect of NK-4 on APP transgenic mice>
In Experiment 14, the effect of NK-4, which had the strongest effect on improving cognitive impairment in mice treated with amyloid β fragment, 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, 15 to 23 g body weight) were preliminarily raised for 10 days, then divided into 4 groups so that their body weights were equal, and were bred alone. -4 was intraperitoneally administered 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 14.
Figure JPOXMLDOC01-appb-T000062
Figure JPOXMLDOC01-appb-T000062
＜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１４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、水迷路試験の何れにおいても、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。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 clear from Table 14, 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. During the study, no side effects believed to be caused by administration of NK-4 were observed.
＜実験１０：脳血管性認知症モデルマウスに及ぼすＮＫ－４の影響＞
　既述の実験により、ＮＫ－４が脳梗塞に由来する運動障害やアルツハイマー型認知障害の改善に有効であることが確認できたので、本実験では脳血管性認知障害に対するＮＫ－４の影響を調べた。すなわち、Ｃ５７ＢＬ／６Ｊマウス（日本クレア社販売、雄、１２週齢）３１匹を１週間予備飼育後、２１匹にアトロピン（０．３ｍｇ／ｋｇ、皮下投与）を前投与した後、ペントバルビタールナトリウム（５０ｍｇ／ｋｇ）を腹腔内投与し麻酔し右総頸動脈の永久結紮手術を行った（手術法については特開２００８－１９３９４１号公報参照）。手術して後全てのマウスを単独飼育とし、自由飲食、飲水で飼育した。結紮手術を施した２１匹のうち１０匹はそのまま飼育（結紮群）し、残りの１１匹はＮＫ－４を投与した（投与群）。また手術を行っていない１０匹を対照（無手術群）とした。手術後２日目より、無手術群および結紮群には生理食塩水を、ＮＫ－４投与群にはＮＫ－４（１００μｇ／ｋｇ）を連日（週５日間）腹腔内投与した。手術３週目、４週目に、実験１５と同じ方法により新奇物体認識試験を実施した。その結果を表１５に示す。 <Experiment 10: Effects of NK-4 on cerebrovascular dementia model mice>
The above-mentioned experiments confirmed that NK-4 is effective in improving dyskinesia and Alzheimer-type cognitive impairment resulting from cerebral infarction. In this experiment, we examined the effects of NK-4 on cerebrovascular cognitive impairment. Examined. Specifically, 31 C57BL / 6J mice (manufactured by Claire Japan, male, 12 weeks old) were preliminarily raised for 1 week, and then atropine (0.3 mg / kg, subcutaneous administration) was pre-administered to 21 mice, followed by pentobarbital sodium. (50 mg / kg) was intraperitoneally administered and anesthetized, and a permanent ligation operation was performed on the right common carotid artery (refer to Japanese Patent Application Laid-Open No. 2008-193941 for the operation method). After the operation, all mice were reared alone, and were bred with free eating and drinking water. Of the 21 animals subjected to ligation surgery, 10 animals were reared as they were (ligation group), and the remaining 11 animals were administered NK-4 (administration group). In addition, 10 animals that had not undergone surgery were used as controls (no surgery group). From the second day after the operation, physiological saline was administered intraperitoneally to the non-operative group and ligation group, and NK-4 (100 μg / kg) was intraperitoneally administered daily (5 days a week) to the NK-4 administration group. A novel object recognition test was performed in the same manner as in Experiment 15 on the third and fourth weeks of the operation. The results are shown in Table 15.
Figure JPOXMLDOC01-appb-T000063
Figure JPOXMLDOC01-appb-T000063
　表１５から明らかなように、ＮＫ－４投与群の物体識別指数は、無手術群と差が無く、右総頸動脈結紮による脳血管性認知障害をほぼ完全に回復させた。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。この結果は、ＮＫ－４は、虚血が原因で引き起こされる神経突起の萎縮をはじめとする神経変性及びそれに伴う神経機能障害に対する治療効果を有することを物語っている。 As is apparent from Table 15, the object identification index of the NK-4 administration group was not different from the non-operative group, and cerebrovascular cognitive impairment due to right common carotid artery ligation was almost completely recovered. During the study, no side effects believed to be caused by administration of NK-4 were observed. This result indicates that NK-4 has a therapeutic effect on neurodegeneration including neurite atrophy caused by ischemia and associated neurological dysfunction.
　以上の結果は、ＮＫ－１９、ＮＫ－５３、ＮＫ－１５０などの一般式３で表される構造を有し、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で表される構造を有し、側鎖のアルキル基の炭素数が２乃至８である化合物は神経突起伸展促進剤として有用であることを物語っている。また、これらの化合物はいずれも、脳梗塞や血管障害による虚血、アルツハイマー症、小脳失調症、パーキンソン氏病などにみられる神経突起の萎縮を改善し、神経突起の萎縮や減少による、認知障害や各種臨床症状を改善できることを物語っている。なかでも、モデル動物での実験結果、製剤化の容易さ、安全性なども考慮すると、一般式２で表される化合物が望ましく、側鎖のアルキル基の炭素数が２乃至４であるＮＫ－４、ＮＫ－２３４、ＮＫ－２６が望ましく、ＮＫ－４が特に望ましいことが示唆された。 The above results are similar to those of the compounds having the structure represented by general formula 3, such as NK-19, NK-53, NK-150, etc., and having a side chain alkyl group having 3 to 10 carbon atoms. The compound having the structure represented by 2 and having 2 to 8 carbon atoms in the side chain alkyl group is useful as a neurite extension promoter. These compounds all improve neurite atrophy in ischemia due to cerebral infarction and vascular injury, Alzheimer's disease, cerebellar ataxia, Parkinson's disease, etc., and cognitive impairment due to neurite atrophy or decrease And that it can improve various clinical symptoms. Among these, considering the experimental results in model animals, ease of formulation, safety, etc., the compound represented by the general formula 2 is desirable, and NK— in which the side chain alkyl group has 2 to 4 carbon atoms. 4, NK-234 and NK-26 were desirable, and NK-4 was particularly desirable.
　以下、本発明の神経突起伸展促進剤について、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 neurite extension promoter 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、神経突起伸展促進剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤や、神経変性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。 <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 neurite outgrowth promoters. In addition, this product can be used as a neurodegeneration inhibitor, a nerve cell protective agent, or a therapeutic agent for pathological conditions or neurological dysfunction associated with neurodegeneration.
　これらの製剤を使用して、小脳失調症ハムスターの運動協調性低下に対する治療効果を確認した。
＜小脳失調症ハムスターの運動協調性低下に対する効果＞
　実験５と同様に、同一週に生まれた３週齢の小脳失調症ハムスター１３０匹を、無作為に、１３群各１０匹に分けた。そのうちの１２群各１０匹には、表１６に示すように、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、３週齢から１０週齢まで、５６日間、１日１回、毎日、０．５ｍｌ／匹で腹腔内投与した（試験群１乃至１２）。残りの１群１０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、３週齢から１０週齢まで、５６日間、１日１回、毎日、０．５ｍｌ／匹で腹腔内投与した（対照群）。投与期間終了の翌日、実験５と同様に、各ハムスターの体重を測定し、ロタロッド試験、斜面耐久試験及び転倒回数の測定を行った。各群に投与した製剤の有効成分である化合物の種類と、測定結果とを表１６に示す。なお、対照群のハムスターの３週齢の平均体重は３５．４ｇ、１０週齢の平均体重は１２２．９ｇで、実施例１で調製した製剤を投与した試験群１乃至１２の何れの群においても、その平均体重に対照群と有意の差は認められなかったので、表１６には、ロタロッド試験、斜面耐久試験及び転倒回数の測定結果のみを示す。また、試験終了後、実験４と同様の方法でハムスターの小脳組織標本を作製し、顕微鏡観察により、対照群と比較して小脳プルキンエ細胞の樹状突起の萎縮が抑制されているかどうかを判定して、その結果を表１６に併せて示す。 These preparations were used to confirm the therapeutic effect on reduced motor coordination in cerebellar ataxia hamsters.
<Effects of cerebellar ataxia hamsters on motor coordination decline>
As in Experiment 5, 130 cerebellar ataxia hamsters born in the same week were randomly divided into 10 animals in 13 groups. As shown in Table 16, in each of 10 groups of 12 groups, any one of the preparations containing any of the 12 compounds prepared in Example 1 as an active ingredient was administered from 3 weeks of age to 10 weeks. Until the age, it was intraperitoneally administered at 0.5 ml / mouse once a day for 56 days (test groups 1 to 12). The remaining 10 animals in a group were intraperitoneally administered with a 10% aqueous solution of sterile maltose (pyrogen-free) from 3 weeks to 10 weeks of age, once daily for 56 days, daily at 0.5 ml / mouse. (Control group). The day after the end of the administration period, the weight of each hamster was measured in the same manner as in Experiment 5, and the rotarod test, the slope endurance test, and the number of falls were measured. Table 16 shows the types of compounds that are active ingredients of the preparations administered to each group and the measurement results. In the control group, the average body weight at 3 weeks of hamster was 35.4 g, the average body weight at 10 weeks of age was 122.9 g, and in any of the test groups 1 to 12 to which the preparation prepared in Example 1 was administered. However, since there was no significant difference in the average body weight from the control group, Table 16 shows only the rotarod test, the slope endurance test, and the measurement results of the number of falls. In addition, after the test was completed, a hamster cerebellar tissue specimen was prepared in the same manner as in Experiment 4, and it was determined by microscopic observation whether or not the cerebellar Purkinje cell atrophy was suppressed compared to the control group. The results are also shown in Table 16.
Figure JPOXMLDOC01-appb-T000064
Figure JPOXMLDOC01-appb-T000064
　表１６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、１０週齢の小脳失調症ハムスターの、小脳プルキンエ細胞の神経突起（樹状突起）の萎縮を抑制し、ロッドからの落下時間低下、斜面耐久傾斜角度の低下及び転倒回数の増加を、顕著に改善した。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、何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（試験群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（試験験群１乃至９）の方が、強い運動協調性の改善効果が認められた。ペンタメチン系シアニン色素化合物間で、効果の強さを改善すると、ＮＫ－４、ＮＫ－２６、ＮＫ－９６９４、ＭＫ－１５０及びＮＫ－３９３で特に強い運動協調性の改善効果が認められた。この結果は、製造した製剤は、いずれも、神経変性疾患の治療剤としても有用であることを物語っている。また、これらの製剤は、５６日間投与しても、ハムスターの体重は、対照群と有意な差が認められなかったので、いずれも、安全性は高いと判断した。 As is clear from Table 16, the 12 preparations prepared in Example 1 all suppressed neurite (dendrites) atrophy of cerebellar Purkinje cells in a 10-week-old cerebellar ataxia hamster, The drop time from the rod, the slope endurance slope angle and the number of falls were significantly improved. Comparing the strength of the effect when 12 types 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 cases (test groups 10 to 12), the pentamethine cyanine dye compounds (NK-4, NK-9694, NK-28, NK-147, NK-19, NK-53, NK-150, NK-393, K When -100, K-528, K-557, and NK-1516) were administered (Experimental groups 1 to 9), a stronger effect of improving motor coordination was observed. When the strength of the effect was improved among the pentamethine-based cyanine dye compounds, NK-4, NK-26, NK-9694, MK-150 and NK-393 were particularly effective in improving motor coordination. This result indicates that any of the prepared preparations is useful as a therapeutic agent for neurodegenerative diseases. Moreover, even if these preparations were administered for 56 days, the body weight of the hamster was not significantly different from that of the control group.
＜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、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（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４である化合物）、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、神経突起伸展促進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, and 2 g of lecithin and an active ingredient NK-4 (compound represented by Chemical Formula 2) in 170 g of purified water for injection, NK-234 (a compound in which the alkyl group (R) in the side chain of the compound represented by the general formula 2 has 3 carbon atoms), NK-26 (an alkyl group in the side chain of the compound represented by the general formula 2 ( R) is a compound having 4 carbon atoms), NK-9694 (a compound represented by Chemical Formula 1), NK-28 (a compound having a general formula 2 having a side chain alkyl group (R) having a carbon number of 7), NK-147 (a compound in which the alkyl group (R) in the side chain of the compound represented by Formula 2 has 8 carbon atoms), NK-19 (a compound represented by Formula 4), NK-53 (compound represented by Chemical Formula 5), NK-1 0 (compound represented by chemical formula 3), NK-393 (compound in which the alkyl group (R) in the side chain of the compound represented by general formula 3 has 8 carbon atoms), 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) (all of which are Hayashibara Co., Ltd.) 120 mg each of any one of the Biochemical Research Laboratories) was mixed with a solution, and nitrogen gas was bubbled until the dissolved oxygen concentration was about 0.05 ppm, and 1 ml was dispensed into a brown ampule. The ampoule was sealed under a nitrogen stream, and then sterilized by filtration or autoclaved. All of these products are pyrogen-free and can be used as neurite outgrowth promoters.
　実施例２で調製した１４種類の神経突起伸展促進剤を、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１２種類の神経突起伸展促進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 14 types of neurite outgrowth promoters prepared in Example 2 was administered to 10 ddy mice (average body weight 25.6 g) in a single dose of 0.3 ml / animal, and the body weight was daily for one 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 the active ingredient of the 12 types of neurite outgrowth promoting agents prepared in Example 2 is 8.6 mg / kg · body weight or more. It shows that it is safe to administer.
＜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（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４である化合物）、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（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、神経突起伸展促進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, 3 g of polysorbate 80 (sold by Nippon Oil & Fats Co., Ltd.) in 170 g of purified water for injection, and NK-4 ( A compound represented by Chemical Formula 2), NK-234 (a 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 (a compound represented by General Formula 2) A compound in which the alkyl group (R) in the side chain of the compound has 4 carbon atoms), NK-9694 (a compound represented by the chemical formula 1), NK-28 (a compound represented by the general formula 2) Compounds in which the alkyl group (R) has 7 carbon atoms), NK-147 (compounds in which the alkyl group (R) in the side chain of the compound represented by the general formula 2 has 8 carbon atoms), NK-19 ( Compound represented by Chemical Formula 4), NK-53 ( Compound represented by Formula 5), NK-150 (compound represented by Formula 3), NK-393 (the alkyl group (R) in the side chain of the compound represented by Formula 3 has 8 carbon atoms) Certain compounds), 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 (chemical formula 9). A compound obtained by dissolving 60 mg of any one of the compounds (all manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) After mixing and sterilizing by filtration, 10 ml each was dispensed into brown ampules, freeze-dried by a conventional method, and then sealed under 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. This product can be used as a neurite extension promoter.
＜カプセル剤＞
　１錠あたり、下記量の成分を含有するカプセル剤を調製した。
　＜成分Ａ＞
　　実施例１で使用した１２種の色素化合物の
　　　いずれか１種　　　　　　　　　　　　　　　　　３０ｍｇ
　乳糖　　　　　　　　　　　　　　　　　　　　　　３５ｍｇ
　とうもろこし澱粉　　　　　　　　　　　　　　　　５３ｍｇ
　不溶性ポリビニルピロリドン（ＢＡＳＦ
　　　ジャパン株式会社販売、商品名「コ
　リドンＣＬ」）　　　　　　　　　　　　　　　　２ｍｇ
　＜成分Ｂ＞
　ポリビニルピロリドンＫ－９０　　　　　　　　　　　５ｍｇ
　結晶セルロース（旭化成株式会社販売、
　　　商品名「アビセルＰＨ３０２」）　　　　　　　１８ｍｇ
　ステアリン酸マグネシウム　　　　　　　　　　　　　２ｍｇ
上記成分Ａの混合物をポリビニルピロリドンＫ－９０の８％水溶液で練合し、６０℃で乾燥後、粉砕し、成分Ｂと混合して、１カプセル当たり１５０ｍｇをカプセルに充填し、常法により、カプセル剤を得た。本品は、神経突起伸展促進剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤、神経変性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。 <Capsule>
Capsules containing the following components per tablet were prepared.
<Component A>
30 mg of any one of the 12 dye compounds used in Example 1
Lactose 35mg
Corn starch 53mg
Insoluble polyvinylpyrrolidone (BASF
Japan Co., Ltd., trade name “Collidon CL”) 2mg
<Component B>
Polyvinylpyrrolidone K-90 5mg
Crystalline cellulose (sales from Asahi Kasei Corporation,
Product name "Avicel PH302") 18mg
Magnesium stearate 2mg
The mixture of the above component A was kneaded with an 8% aqueous solution of polyvinyl pyrrolidone K-90, dried at 60 ° C., pulverized, mixed with component B, filled into capsules at 150 mg per capsule, Capsules were obtained. This product can be used as a neurite extension promoter. The product can also be used as a neurodegeneration inhibitor, a nerve cell protective agent, or a therapeutic agent for pathological conditions or neurological dysfunction associated with neurodegeneration.
＜カプセル剤＞
　１錠あたり、下記量の成分を含有するカプセル剤を調製した。
　＜成分Ａ＞
　ＮＫ－１５０（株式会社林原生物化学研究所
　　　製造）　　　　　　　　　　　　　　　　　　　１０ｍｇ
　実施例１で使用した、ＮＫ－１５０を除く
　　　１１種の化合物のいずれか１種　　　　　　　　　２０ｍｇ
　乳糖　　　　　　　　　　　　　　　　　　　　　　３５ｍｇ
　とうもろこし澱粉　　　　　　　　　　　　　　　　５３ｍｇ
　不溶性ポリビニルピロリドン（ＢＡＳＦ
　　　ジャパン株式会社販売、商品名「コ
　リドンＣＬ」）　　　　　　　　　　　　　　　　２ｍｇ
　＜成分Ｂ＞
　ポリビニルピロリドンＫ－９０　　　　　　　　　　　５ｍｇ
　結晶セルロース（旭化成株式会社販売、
　　　商品名「アビセルＰＨ３０２」）　　　　　　　１８ｍｇ
　ステアリン酸マグネシウム　　　　　　　　　　　　　２ｍｇ
上記成分Ａの混合物をポリビニルピロリドンＫ－９０の８％水溶液で練合し、６０℃で乾燥後、粉砕し、成分Ｂと混合して、１カプセル当たり１５０ｍｇをカプセルに充填し、常法により、カプセル剤を得た。本品は、神経突起伸展促進剤として利用できる。また、本品は、神経変性抑制剤、神経細胞保護剤、神経変性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。 <Capsule>
Capsules containing the following components per tablet were prepared.
<Component A>
NK-150 (Manufactured by Hayashibara Biochemical Laboratories, Inc.) 10mg
20 mg of any one of 11 compounds used in Example 1, excluding NK-150
Lactose 35mg
Corn starch 53mg
Insoluble polyvinylpyrrolidone (BASF
Japan Co., Ltd., trade name “Collidon CL”) 2mg
<Component B>
Polyvinylpyrrolidone K-90 5mg
Crystalline cellulose (sales from Asahi Kasei Corporation,
Product name "Avicel PH302") 18mg
Magnesium stearate 2mg
The mixture of the above component A was kneaded with an 8% aqueous solution of polyvinyl pyrrolidone K-90, dried at 60 ° C., pulverized, mixed with component B, filled into capsules at 150 mg per capsule, Capsules were obtained. This product can be used as a neurite extension promoter. The product can also be used as a neurodegeneration inhibitor, a nerve cell protective agent, or a therapeutic agent for pathological conditions or neurological dysfunction associated with neurodegeneration.
　本発明の神経突起伸展促進剤は、神経細胞の神経変性に起因するパーキンソン病、パーキンソン症候群、アルツハイマー病、認知症、又は、脳卒中などの予防、治療及び／又は進展抑制、又は神経変性からの神経細胞保護に有用である。また、神経変性疾患に伴う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、しびれ及び麻痺など）の改善にも有用である。しかも、本発明の神経突起伸展促進剤は、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 neurite outgrowth promoting agent of the present invention is used for the prevention, treatment and / or progression inhibition of Parkinson's disease, Parkinson's syndrome, Alzheimer's disease, dementia, or stroke caused by neurodegeneration of nerve cells, or nerve from neurodegeneration. Useful for cell protection. In addition, various pathological conditions and neurological dysfunctions associated with neurodegenerative diseases (for example, tremor, rigidity, ataxia, peristalsis, slow movement, posture reflex disorder, autonomic disorder, lunging phenomenon, gait disorder, depression, memory disorder, It is also useful for improving muscle atrophy, muscle weakness, upper limb dysfunction, articulation disorder, dysphagia, respiratory disorder, numbness and paralysis. Moreover, the neurite outgrowth promoting 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 confidence. 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神経突起伸展促進剤。
    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。）     A neurite extension promoter containing a compound represented by 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項記載の神経突起伸展促進剤。
    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 neurite extension promoter 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項記載の神経突起伸展促進剤。 The neurite extension promoter 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１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項記載の神経突起伸展促進剤。
    Figure JPOXMLDOC01-appb-C000006
    Figure JPOXMLDOC01-appb-C000007
         The neurite extension promoter according to claim 2, wherein the compound represented by the general formula 2 is a compound represented by the chemical formula 1, 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神経突起伸展促進剤。 The neurite outgrowth promoting 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項記載の神経突起伸展促進剤。 6. The neurite extension promoter 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神経突起伸展促進剤。 The neurite extension promoter 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神経突起伸展促進剤。 The neurite outgrowth promoting 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. 　神経突起伸展促進剤が、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神経突起伸展促進剤。 The neurite outgrowth promoting agent is a neuronal degeneration inhibitor, neurite outgrowth promoting agent, nerve cell protecting agent, or ataxia ameliorating agent associated with neuronal cell degeneration, any one of claims 1 to 8 A neurite outgrowth promoting agent according to claim 1.
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