WO2009116546A1 - Excitatory chemical mediator and method for screening thereof - Google Patents

Excitatory chemical mediator and method for screening thereof Download PDF

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WO2009116546A1
WO2009116546A1 PCT/JP2009/055208 JP2009055208W WO2009116546A1 WO 2009116546 A1 WO2009116546 A1 WO 2009116546A1 JP 2009055208 W JP2009055208 W JP 2009055208W WO 2009116546 A1 WO2009116546 A1 WO 2009116546A1
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group
pharmaceutical composition
transporter
compound
neurotransmitter
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French (fr)
Japanese (ja)
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芳則 森山
弘志 表
成信 樹下
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国立大学法人 岡山大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to the field of excitatory chemical transmission regulators and screening methods thereof.
  • the excitatory chemical transmission modulators of the present invention also relate to the field of treatment of disorders caused by excessive neuronal firing such as sputum, and the field of treatment by suppressed neuronal activation.
  • Neurotransmitters are specific chemicals that are released from presynaptic cells (presynaptic cells) and stimulate or inhibit postsynaptic cells (postsynaptic cells) across the synapse. Neurotransmitters are produced in neurons, released from the presynaptic terminals, and act directly on the postsynaptic membrane. The response response evoked in the target cell by this action produces electrical excitement or suppression. Neurotransmitters that produce electrical excitement are called excitatory transmitters, and neurotransmitters that produce electrical inhibition are called inhibitory transmitters.
  • Neurotransmitters produced in neurons are accumulated in synaptic vesicles by neurotransmitter transporters present in synaptic vesicles of pre-neuronal cells, released into synaptic clefts by exocytosis, and cell surface of post-synaptic cells It binds to a specific receptor present in the cell and is recovered by a neurotransmitter transporter present in the cell membrane of presynaptic terminals and glial cells.
  • This series of neurotransmitter dynamics includes the neurotransmitter transporter (called vesicular neurotransmitter transporter or vesicular transporter) present in synaptic vesicles and the cell membrane of presynaptic terminal glial cells.
  • vesicular neurotransmitter transporter vesicular transporter
  • vesicular transporter present in synaptic vesicles
  • cell membrane of presynaptic terminal glial cells.
  • Existing neurotransmitter transporters plasma membrane transporters
  • these two neurotransmitter transporters are molecules that differ in structure and function.
  • Epilepsy is a chronic disease characterized by paroxysmal brain dysfunction caused by excessive neuronal discharge, and is usually a disease accompanied by modulation of consciousness. Seizures may be limited to elemental or complex disorders of behavior or may develop into generalized convulsions. The clinical findings of seizures range from complex behavioral abnormalities, including general and local convulsions, to instantaneous seizures of consciousness disturbances. There are various classifications of these clinical findings. Seizure type terms are descriptive and standardized, but not completely unified.
  • sputum is characterized by paroxysmal brain dysfunction due to excessive neuronal discharge, so the anti-epileptic effect is to reduce excitatory chemical transmission while inhibiting inhibitory chemical transmission. It is possible to increase it.
  • Valproic acid a known antidepressant
  • GABA GABA
  • Valproic acid is thought to be effective in almost all types of sputum, but has dose-dependent fire extinguisher and liver side effects and is teratogenic.
  • phenytoin is known as a drug that inhibits nerve excitability by Na channel suppression, nystagmus, dysarthria, ataxia, dizziness, diplopia, somnolence, abnormal behavior, cognitive impairment, hypersensitivity, rash, hirsutism, gingival proliferation, Side effects such as osteomalacia and macrocytic anemia are known.
  • TDM tumor necrosis factor
  • the target is a neurotransmitter transporter activity regulator as a development of antidepressant drugs through the reduction of excitatory chemical transmission, it is specific to excitatory neurotransmitters present in the plasma membrane of presynaptic terminals and glial cells Neurotransmitter transporter (cell membrane-type neurotransmitter transporter) activator or neurotransmitter transporter specific to excitatory neurotransmitter present in synaptic vesicles (vesicle-type neurotransmitter trans Porter) inhibitors.
  • the cell membrane type neurotransmitter transporter is activated, a decrease in excitatory chemical transmission is reasonably predicted by promoting clearance of the excitatory neurotransmitter released into the synaptic cleft.
  • inhibiting the vesicular neurotransmitter transporter can reduce the amount of excitatory neurotransmitter that accumulates in synaptic vesicles, resulting in a rational decrease in excitatory chemical transmission. This is because it is predicted.
  • glial cells are known to be associated with diseases such as epilepsy, and glial cells are known to secrete and inhibit excitatory neurotransmitters such as ATP and glutamate Therefore, it is considered that a therapeutic effect for these diseases can be expected (Non-Patent Documents 5 to 13). Therefore, compounds that inhibit the action of multiple types of excitatory neurotransmitters are required as candidate compounds for antidepressants.
  • neurotransmitter transporters are specific for various excitatory neurotransmitters regardless of whether they are cell membrane type or vesicle type. When developing antiepileptic drugs, a wide variety of neurotransmitter transporters should be targeted.
  • Non-patent Document 1 Inhibitors for vesicular glutamate transporters have been studied (Non-patent Document 1), but all of them are compounds having a structure similar to glutamate transporters or vesicular neurotransmitter transporter proteins. It is a compound centering on pigments that bind irreversibly. It is considered that a compound having a structure similar to the vesicular glutamate transporter cannot inhibit a plurality of types of vesicular neurotransmitter transporters. In addition, compounds centered on dyes that bind irreversibly to the vesicular neurotransmitter transporter protein irreversibly deactivate the vesicular neurotransmitter transporter, resulting in side effects that cannot be overlooked. Is predicted.
  • the vesicular glutamate transporter is known to be chloride ion-requiring (Non-Patent Document 2), and 2-keto-3-methylvaleric acid changes the chloride ion requirement of the vesicular glutamate transporter. It is also known (Non-Patent Documents 3 and 4).
  • 2-keto-3-methylvaleric acid has a very small inhibitory effect on the vesicular glutamate transporter, and thus is not recognized as a practical inhibitor of the vesicular glutamate transporter.
  • 2-keto-3-methylvaleric acid is a substance that accumulates in the body due to genetic enzyme deficiency, and neurotoxicity has been reported (Non-patent Documents 3 and 4). Therefore, 2-keto-3-methylvaleric acid is considered to act as a neurotoxin rather than being useful as a therapeutic agent for diseases such as hemorrhoids.
  • anti-epileptic drugs that reversibly bind to neurotransmitter transporters, molecules directly related to excitatory neurotransmitters, are expected to have fewer side effects. Therefore, there is a need for the development of activity modulators (eg, excitatory chemical transmission inhibitors) targeting a wide range of neurotransmitter transporters. There is also a need to develop drugs that activate inhibited neurons.
  • activity modulators eg, excitatory chemical transmission inhibitors
  • excitatory chemical transmission inhibitors can treat, prevent, treat / prevent diseases / conditions such as inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis. And / or useful prognosis.
  • the present invention provides an excitatory chemical transmission inhibitor and a screening method thereof.
  • the present invention provides an excitatory chemical transmission inhibitor that inhibits at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological chloride concentrations and a screening method thereof. Let it be an issue.
  • drugs useful for the treatment, prevention, and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis It is an object of the present invention to provide a screening method thereof.
  • the above challenge is to develop a novel technique for screening for inhibitors of vesicular neurotransmitter transporters that transport excitatory neurotransmitters, and to transport excitatory neurotransmitters by the screening method
  • identifying a group of compounds that identify compounds that have inhibitory effects on at least two of the vesicular neurotransmitter transporters the inventors have completed the present invention.
  • R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
  • R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O At least one of R 1 to R 4 contains an oxygen atom;
  • R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition according to item 1 are each independently selected from the group consisting of H and OH, and together O.
  • R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
  • the pharmaceutical composition according to item 2 Wherein R 1 and R 2 are each independently selected from the group consisting of H and together O. Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
  • the pharmaceutical composition according to item 4, Wherein R 3 and R 4 taken together are O.
  • R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
  • R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O At least one of R 1 to R 4 contains an oxygen atom;
  • R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition according to item 9 Wherein R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O.
  • R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
  • the pharmaceutical composition according to item 10 wherein R 1 and R 2 are each independently selected from the group consisting of H and together O. Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
  • the pharmaceutical composition according to item 9 wherein at least one of R 3 and R 4 contains an oxygen atom.
  • the pharmaceutical composition according to item 12, Wherein R 3 and R 4 taken together are O.
  • a method for screening candidate compounds for treating, preventing, and / or improving prognosis for diseases and / or conditions associated with excessive neural excitement comprising: : (A) preparing a vesicle comprising a vesicular neurotransmitter transporter; (B) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, and a vesicle of the excitatory neurotransmitter in the presence of chloride under physiological conditions Detecting the transport to (C) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, a physiological condition chloride, and a test compound; And (d) comparing the transport in the step (b) with the transport in the step (c), and using the
  • the vesicle comprising the vesicular neurotransmitter transporter is prepared by reconstituting an isolated vesicular neurotransmitter transporter into a liposome.
  • the ionophore is valinomycin.
  • the vesicular neurotransmitter transporter is selected from the group consisting of a glutamate transporter, an aspartate transporter, and a nucleotide transporter.
  • a method for screening a compound that cancels or activates inhibition of the transport activity of a vesicular neurotransmitter transporter comprising: (1) measuring the transport activity of the vesicular neurotransmitter transporter in the presence of an inhibitor of the vesicular neurotransmitter transporter; (2) a step of measuring the transport activity of the vesicular neurotransmitter transporter in the presence of the candidate compound and the inhibitor of (1) above; and (3) the activity of (2) above (1)
  • the candidate compound is identified as a compound having the ability to cancel or activate inhibition; Including the method. 23.
  • an excitatory chemical transmission inhibitor and a screening method thereof are provided.
  • an excitatory chemical transmission inhibitor that inhibits at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration and a screening method thereof are provided.
  • the treatment, prevention and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis.
  • Agents that are useful and methods of screening are provided, and specific active ingredients are provided.
  • an agent that activates a suppressed neuron is also provided.
  • FIG. 1 (A) is a graph showing glutamate transport activity in proteoliposomes reconstituted with F-ATPase and glutamate transporter. “+ ATP” indicates the transport activity in the presence of ATP, and “ ⁇ ATP” indicates the transport activity in the absence of ATP.
  • FIG. 1 (B) is a graph showing glutamate transport activity in proteoliposomes in which only the glutamate transporter is reconstituted without using F-ATPase. “+ Valinomycin” indicates transport activity in the presence of valinomycin, and “ ⁇ valinomycin” indicates transport activity in the absence of valinomycin.
  • FIG. 2 shows the results of testing the glutamate transport inhibitory activity of various candidate compounds in the presence of 5 mM chloride (5 mM Cl ⁇ ) and in the presence of 20 mM chloride (20 mM Cl ⁇ ).
  • FIG. 3 shows the results showing the concentration dependence of acetoacetate in the inhibition of glutamate transport activity by VGLUT.
  • FIG. 1 (B) is a graph showing glutamate transport activity in proteoliposomes in which only the glutamate transporter is reconstituted without using F-ATPase. “+ Valinomycin” indicates transport activity in the presence of valinomycin, and “ ⁇ valinomycin” indicates transport
  • FIG. 4 shows the results showing that inhibition of VGLUT activity by acetoacetate is eliminated by increasing the chloride ion concentration.
  • the black circle “ ⁇ ” is the result of not adding acetoacetate (control).
  • the black triangle “ ⁇ ” is the result of adding 0.2 mM acetoacetate.
  • the white circle “ ⁇ ” is the result of adding 1 mM acetoacetate.
  • the white triangle “ ⁇ ” is the result of adding 5 mM acetoacetate.
  • FIG. 5 is a diagram showing the structure of a candidate compound showing high inhibitory activity and the concentration required for 50% inhibition (IC 50 ).
  • FIG. 6A (VMAT) shows the results showing the inhibitory activity of 1 mM acetoacetate on the monoamine transporter.
  • FIG. 6B shows the results showing the inhibitory activity of 1 mM acetoacetate on the GAB transporter. All experiments were performed in the presence of 10 mM chloride ions.
  • FIG. 7A shows the results showing the inhibitory activity of acetoacetate on the aspartate transport activity by aspartate transporter (sialin).
  • FIG. 7B shows the results showing the inhibitory activity of acetoacetate on the ATP transport activity by the nucleotide transporter (VNUT).
  • FIG. 8 shows the results of examining the inhibitory effect of acetoacetate on glutamate exocytosis induced by KCl from hippocampal neurons.
  • FIG. 9A is a diagram schematically showing a method of administering 4AP and acetoacetate to a rat brain and a method of collecting a circulating fluid.
  • the upper diagram of FIG. 9B is a diagram schematically showing the timing of 4AP administration (downward arrow) and the timing of sampling (downward arrow).
  • the lower diagram of FIG. 9B shows the measurement results of circulating glutamic acid ( ⁇ in the upper graph) and dopamine ( ⁇ in the lower graph) sampled at each timing.
  • FIG. 9C shows the amount of glutamate ( ⁇ ) and dopamine ( ⁇ ) secreted at various acetoacetate concentrations. Glutamic acid secretion was recovered by washing after administration of 10 mM acetoacetate (arrow of “washing”).
  • SEQ ID NO: 1 is the nucleic acid sequence of glutamate transporter (VGLUT1).
  • SEQ ID NO: 2 is the amino acid sequence of glutamic acid transporter (VGLUT1).
  • SEQ ID NO: 3 is the nucleic acid sequence of aspartate transporter (sialin).
  • SEQ ID NO: 4 is the amino acid sequence of aspartic acid transporter (sialin).
  • SEQ ID NO: 5 is the nucleotide transporter (VNUT) nucleic acid sequence.
  • SEQ ID NO: 6 is the amino acid sequence of the nucleotide transporter (VNUT).
  • SEQ ID NO: 7 is the nucleic acid sequence of glutamate transporter (VGLUT2).
  • SEQ ID NO: 8 is the amino acid sequence of glutamic acid transporter (VGLUT2).
  • SEQ ID NO: 9 is the nucleic acid sequence of glutamate transporter (VGLUT3).
  • SEQ ID NO: 10 is the amino acid sequence of glutamic acid transporter (VGLUT3).
  • SEQ ID NO: 11 is a sense primer for PCR cloning of glutamate transporter (VGLUT2).
  • SEQ ID NO: 12 is an antisense primer for PCR cloning of glutamate transporter (VGLUT2).
  • SEQ ID NO: 13 is the sequence of the forward primer used for human sialin gene cloning.
  • SEQ ID NO: 14 is the reverse primer sequence used for human sialin gene cloning.
  • SEQ ID NO: 15 is the sequence of the forward primer used for human sialin gene cloning.
  • SEQ ID NO: 16 is the reverse primer sequence used for human sialin gene cloning.
  • SEQ ID NO: 17 is the sequence of mouse H183R 1 st PCR sense primer.
  • SEQ ID NO: 18 is the sequence of mouse H183R 1 st PCR antisense primer.
  • SEQ ID NO: 19 is the sequence of mouse H183R 2 nd PCR sense primer.
  • SEQ ID NO: 20 is the sequence of the sense primer used for cloning of the human VNUT gene.
  • SEQ ID NO: 21 is the sequence of the antisense primer used for cloning of the human VNUT gene.
  • excitatory neurotransmitter refers to a neurotransmitter that exhibits an excitatory action when released into the synaptic cleft.
  • excitatory neurotransmitters include, but are not limited to, glutamic acid, aspartic acid, and ATP.
  • vesicle-type neurotransmitter transporter refers to a transporter that is present in a synaptic vesicle of a neuron and actively transports an excitatory neurotransmitter into the synaptic vesicle.
  • physiological chloride concentration refers to the chloride concentration in the cytoplasm of mammalian cells (eg, neurons).
  • the chloride concentration under physiological conditions is 5-20 mM, for example 10 mM.
  • aryl is used interchangeably with “Ar” and refers to a compound that is a monocyclic or bicyclic aromatic residue, optionally containing one or more heteroatoms.
  • Ar refers to a compound that is a monocyclic or bicyclic aromatic residue, optionally containing one or more heteroatoms.
  • Representative examples of Ar include, but are not limited to, phenyl, naphthyl, quinolyl, pyridyl, pyrimidyl, benzothiazoyl, benzimidazolyl, and the like.
  • alkylaryl is used interchangeably with “(CH 2 ) m —Ar”, where m is 1 to 10 and Ar is as defined above. It is.
  • disease and / or condition associated with excessive neural excitement refers to a disease and / or abnormal condition resulting from excessive excitement of the nerve.
  • Diseases and / or conditions associated with excessive neural excitement include, for example, epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis However, it is not limited to this.
  • the term “disease and / or condition resulting from inhibition of a nerve” includes, for example, blurring, sensory impairment (eg, but not limited to taste disorders). However, it is not limited to these.
  • Glutamate transporter refers to a transporter that is a type of vesicular neurotransmitter transporter and transports glutamate into the vesicle or a variant thereof.
  • Glutamate transporters are typically: (A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, 7, or 9 and that encodes a polypeptide having glutamate transport activity; (B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1, 7, or 9 and encoding a polypeptide having glutamate transport activity; (C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 8, or 10; and (d) one or several mutations in the amino acid sequence set forth in SEQ ID NO: 2, 8, or 10.
  • the term “aspartic acid transporter” refers to a transporter that transports aspartic acid into a vesicle or a variant thereof, which is a type of vesicular neurotransmitter transporter.
  • Aspartate transporters are typically: (A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a polypeptide having aspartate transport activity; (B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and encoding a polypeptide having aspartate transport activity; (C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; and (d) comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 4 A nucleic
  • nucleotide transporter is a type of vesicular neurotransmitter transporter that transports nucleotides (eg, ATP, ADP and GTP) into vesicles or modifications thereof. Refers to the body.
  • nucleotides eg, ATP, ADP and GTP
  • Nucleotide transporters are typically: (A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a polypeptide having ATP transport activity; (B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having ATP transport activity; (C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6; and (d) comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 6 A nucleic acid encoding a polypeptide having an amino acid sequence and having ATP transport activity; A protein encoded by a nucleic acid selected from the group consisting of:
  • the term “transporter” refers to a substance that transports a substance (for example, glutamic acid, aspartic acid, and nucleotide) that cannot permeate the lipid bilayer across the lipid bilayer.
  • the transporter is a membrane protein present in the lipid bilayer.
  • a transporter that is a protein is used herein interchangeably with “transport protein”.
  • artificial membrane used in the present specification is a membrane artificially prepared from lipid as a raw material, and is preferably a lipid bilayer membrane, but is not limited thereto.
  • examples of the “artificial membrane” include, but are not limited to, liposomes.
  • transporter activity regulator refers to a substance that affects the transport activity of a transporter.
  • the “transporter activity regulator” may be a substance that promotes or inhibits transport activity.
  • excitatory neurotransmission inhibitor refers to a substance that inhibits excitatory neurotransmission by an excitatory neurotransmitter. This inhibitory action is typically brought about by, but not limited to, inhibition of the vesicular neurotransmitter transporter.
  • transport activity refers to an activity of transporting a substance that cannot permeate the lipid bilayer (for example, an excitatory neurotransmitter) across the lipid bilayer.
  • ionophore refers to a substance that increases the permeability of specific ions to a lipid bilayer.
  • the ionophore is preferably valinomycin.
  • kit refers to a product comprising a plurality of containers and manufacturer's instructions, and each container comprising a nucleic acid and / or protein of the present invention.
  • the kit of the present invention comprises (a) an artificial membrane such as a liposome, or a lipid for preparing an artificial membrane, (b) an excitatory neurotransmitter, and (c) an ionophore.
  • a “polynucleotide”, “nucleic acid” or “nucleic acid molecule” is a ribonucleotide (adenosine, guanosine, uridine, or cytidine in the form of a phosphate ester polymer that is in single-stranded, double-stranded, or other form; “RNA molecule”) or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine (DNA molecule)), or any phosphoester analog thereof (eg, phosphorothioate and thioester).
  • nucleic acid sequence or “nucleotide sequence” is a series of nucleotide bases (also referred to as “nucleosides”) in a nucleic acid (eg, DNA or RNA) and any chain of two or more nucleotides. Or its complementary strand.
  • nucleic acids of the present invention include the nucleic acid set forth in SEQ ID NO: 1 and its complementary strands, variants and fragments.
  • “Complementary strand” means a strand of nucleotides that can form base pairs with a nucleic acid sequence.
  • each strand of double-stranded DNA has a complementary base sequence, and the other strand is a complementary strand when viewed from one strand.
  • a “coding sequence” or a sequence that “encodes” an expression product is a nucleotide sequence that, when expressed, results in the production of that product.
  • Protein includes a continuous string of two or more amino acids.
  • Preferred peptides of the present invention include the peptide shown in SEQ ID NO: 2, as well as variants and fragments thereof.
  • Protein sequence refers to a series of two or more amino acids in a protein, peptide, or polypeptide.
  • homology of genes refers to the degree of identity of two or more gene sequences with each other.
  • identity of sequences refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions.
  • the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.
  • “similarity” of genes refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
  • the length of the fragment can be appropriately changed according to the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits.
  • examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
  • Non-integer lengths may also be appropriate as a lower limit.
  • the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above. However, the above numbers are not absolute and are limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include a number above and below that number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
  • the length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.
  • polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art.
  • Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained.
  • a polynucleotide that can be identified by washing the filter under conditions of 65 ° C using an SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate).
  • SSC saline-sodium citrate
  • concentration of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate.
  • Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Technical Approach, Second Edition, Oxford, etc.
  • the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions.
  • hybridizable polynucleotide refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions.
  • the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.
  • highly stringent conditions are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches.
  • Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide.
  • Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al.
  • Such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO 4 or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used.
  • concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions.
  • Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).
  • T m (° C.) 81.5 + 16.6 (log [Na + ]) + 0.41 (% G + C) ⁇ 600 / N ⁇ 0.72 (% formamide)
  • N is the length of the duplex formed
  • [Na + ] is the molar concentration of sodium ions in the hybridization or wash solution
  • % G + C is the (guanine + in the hybrid Cytosine) base percentage.
  • the melting temperature decreases by about 1 ° C. for each 1% mismatch.
  • the percentage of “identity”, “homology” and “similarity” of sequences is determined by comparing two sequences that are optimally aligned in a comparison window.
  • a portion of the polynucleotide or polypeptide sequence within the comparison window is a reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequence contains an addition, Reference sequences herein shall contain no additions or deletions), and may include additions or deletions (ie gaps).
  • Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window.
  • BLAST Basic Local Alignment Search Tool
  • J. Mol. Biol. 215: 403-410 Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402
  • BLAST Basic Local Alignment Search Tool
  • the BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified.
  • High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art.
  • a scoring matrix well known in the art.
  • a BLOSUM62 matrix Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61 is used as the scoring matrix.
  • PAM or PAM250 matrix can also be used (for example, Schwartz and Dayhoff, eds., 1978, Matrix for Detection Distance Relationships: Atlas of Protein Sequential Sequencial Sequenc thing).
  • the BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferable to evaluate the statistical significance of high-score segment pairs using Karlin's formula for calculating statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).
  • substitution, addition or deletion of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
  • the number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, the number can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.
  • the compounds of the present invention are capable of modulating (inhibiting or deblocking or activating) at least two vesicular neurotransmitter transporters.
  • the mechanism of inhibition by the compounds of the present invention is as follows: (a) vesicular neurotransmitter transporters, especially transporters that transport excitatory neurotransmitters (B) vesicular neurotransmitter transporter is active when chloride concentrations at physiological conditions are present (usually 5-20 mM, typically 10 mM).
  • the pharmaceutical compositions of the present invention can be utilized for the treatment, prevention and / or prognosis of diseases and / or conditions (eg, epilepsy) associated with excessive neural excitation.
  • the pharmaceutical composition of the present invention can be used for the treatment, prevention and / or prognosis of diseases and / or conditions resulting from nerve suppression.
  • the present invention also provides a method of screening for a substance having such activity.
  • the compounds of the present invention having inhibitory activity against multiple vesicular neurotransmitter transporters are useful for the treatment, prevention, and improvement of prognosis of diseases and / or conditions associated with excessive neural excitation. It is.
  • Diseases and / or conditions associated with excessive neural excitement include, for example, epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis However, it is not limited to this.
  • hemorrhoids is a chronic disease characterized by paroxysmal brain dysfunction due to excessive neuronal discharge, usually accompanied by modulation of consciousness. There are cases where it is limited to physical disorder and cases where it develops into generalized convulsions.
  • the clinical findings of seizures range from complex behavioral abnormalities, including generalized and local convulsions, to instantaneous seizures of consciousness disturbances. These clinical findings have various classifications, and terms that describe the type of seizure are described. Is standardized and standardized, but not completely unified.
  • the compound of the present invention having an inhibitory activity against a plurality of vesicular neurotransmitter transporters suppresses / inhibits excessive neural excitement, which is one of the causes of epilepsy. It is considered to be effective for drought.
  • R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
  • R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O At least one of R 1 to R 4 contains an oxygen atom;
  • R 5 is a compound selected from the group consisting of H, linear alkyl, branched alkyl, substituted alkyl, aryl, and alkylaryl, or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O, wherein R 3 and R 4 are each independently And H and OH and taken together are selected from the group consisting of O.
  • R 1 and R 2 are each independently selected from the group consisting of H and together O, wherein R 3 and R 4 are each independently H , And together are selected from the group consisting of O.
  • At least one of R 3 and R 4 contains an oxygen atom, and optionally R 3 and R 4 together are O.
  • Examples of the compound of the present invention include compounds selected from the group consisting of acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, ⁇ -keto- ⁇ -methyl-valeric acid, and lactic acid. However, it is not limited to this. Preferred compounds are those selected from the group consisting of acetoacetate and pyruvic acid.
  • pharmaceutically acceptable carrier refers to a substance that is used when producing an agrochemical such as a pharmaceutical or veterinary drug, and that does not adversely affect active ingredients.
  • Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or pharmaceutical adjuvants.
  • the type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like.
  • the frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art.
  • the frequency of monitoring the disease state includes, for example, daily-once every several months (eg, once a week-once a month). It is preferable to perform monitoring once a week to once a month while monitoring the progress.
  • two or more kinds of drugs may be used in the treatment of the present invention.
  • substances with similar properties or origins may be used, or drugs with different properties or origins may be used.
  • Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.
  • the present invention also provides a method of screening for a compound that cancels or activates inhibition of the transport activity of a vesicular neurotransmitter transporter.
  • a screening method is performed by selecting a compound having an effect of restoring transport activity from candidate compounds in the presence of the inhibitor of excitatory neurotransmitter transport of the present invention.
  • screening methods are for example: (1) measuring the transport activity of excitatory neurotransmitter transport protein in the presence of an inhibitor of excitatory neurotransmitter transport; (2) a step of measuring transport activity when a candidate compound is further added; and (3) a step of identifying a candidate compound as a compound having an ability of releasing inhibition or activating when the activity of (2) is higher than the activity of (1); Is included.
  • a preferred excitatory neurotransmitter transport protein is VGLUT2, but is not limited thereto.
  • the inhibitor of excitatory neurotransmitter transport is preferably acetoacetate, pyruvate, phenylpyruvate, 3-hydroxybutyrate, ⁇ -keto- ⁇ -methyl-valerate, or lactic acid, more preferably Acetoacetate, but is not limited thereto.
  • the compound thus identified can be used as an active ingredient of a drug used for the treatment, prevention and / or prognosis of diseases and / or conditions caused by nerve suppression.
  • the “effective amount” of a drug refers to an amount that allows the drug to exhibit the intended drug effect.
  • the minimum concentration may be referred to as the minimum effective amount.
  • Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art or can be determined as appropriate by those skilled in the art.
  • an animal model or the like can be used in addition to actual administration. The present invention is also useful in determining such effective amounts.
  • the type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like.
  • the frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency of monitoring the disease state include monitoring every day to once every several months (for example, once a week to once a month). It is preferable to perform monitoring once a week to once a month while monitoring the progress.
  • the present invention also provides for a disease or disorder (e.g., epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and the like by administration of an effective amount of a therapeutic agent to a subject.
  • a disease or disorder e.g., epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and the like.
  • a therapeutic agent e.g., epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and the like.
  • a therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).
  • the therapeutic agent takes into account the clinical condition of the individual patient (especially the side effects of therapeutic agent alone treatment), the site of delivery, the method of administration, the dosage regimen and other factors known to those skilled in the art, eg, “Therapeutics Manual 2006 Supervised by Medical Shoin: Takahisa Fumi / Yazaki Yoshio, edited by Seki Akira / Kitahara Mitsuo / Ueno Fumiaki / Echizen Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.
  • JNeurosci 27,3618-3625 As noted above, the dosage is left to therapeutic discretion. Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.
  • the therapeutic agent can be administered orally, rectally, parenterally, in the tank, vaginally, intraperitoneally, buccally, orally or as a nasal spray.
  • parenteral refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.
  • the therapeutic agent of the present invention is also appropriately administered by a sustained release system.
  • sustained release therapeutic agents can be administered orally, rectally, parenterally, in the tank, vaginally, intraperitoneally, buccally, or orally or as a nasal spray.
  • the therapeutic agent of the present invention is also appropriately administered by a sustained release system.
  • sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, in acceptable quality oils). Or an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).
  • Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and ⁇ -ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl Acetate (Langer et al., Ibid) or poly-D-(-)-3-hydroxybutyric acid (EP133,988).
  • polylactide US Pat. No. 3,773,919, EP 58,481
  • a copolymer of L-glutamic acid and ⁇ -ethyl-L-glutamate Sidman et al., Biopolymers 22: 547
  • Sustained release therapeutic agents also include the therapeutic agents of the present invention entrapped in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapeutic Diseases and Cancers, Cancer -See Berstein and Fiddler (eds.), Liss, New York, pages 317-327 and 353-365 (1989)).
  • Liposomes containing therapeutic agents can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.
  • liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for optimal therapeutic agents.
  • the therapeutic agents of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgary 88: 507 ( 1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)).
  • the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation.
  • a unit dosage injectable form solution, suspension or emulsion
  • the formulation preferably does not include oxidation and other compounds known to be harmful to therapeutic agents.
  • a formulation is prepared by contacting the therapeutic agent uniformly and intimately with a liquid carrier or a finely divided solid carrier or both.
  • the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.
  • the carrier suitably contains trace amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts.
  • Buffering agents such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.
  • polypeptides eg, polyarginine or tripeptides
  • proteins such as serum albumin, gelatin or immunoglobulins
  • polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, gluta
  • Any drug to be used for therapeutic administration may be free of organisms / viruses, that is, sterile. Aseptic conditions are easily achieved by filtration through a sterile filtration membrane (eg, 0.2 micron membrane).
  • the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper puncturable with a hypodermic needle.
  • Treatment agents are usually stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized formulations for reconstitution.
  • a lyophilized formulation a 10 ml vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous therapeutic agent and the resulting mixture is lyophilized.
  • the lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.
  • the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention.
  • a notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration. Represents institutional approval.
  • the therapeutic agent may be used in combination with other therapeutic compounds.
  • the therapeutic agent of the present invention can be administered alone or in combination with other therapeutic agents.
  • the combinations can be administered, for example, either simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual . Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.
  • the compounds selected from the group consisting of acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, and lactic acid which are representative compounds of the present invention, are all physiological substances, and are transported and metabolized in the body. • It is an excreted compound. Therefore, there are transporters that transport these in vivo (Muller et al., "Transportof ketone bodies and lactate in the sheepruminal epithelium by monocarboxylatetransporter 1", Am J Physiol GastrointestLiver Physiol.
  • Example 1 Cloning and expression of glutamate transporter VGLUT2, preparation of proteoliposome, and activity measurement
  • Rat VGLUT2 cDNA was amplified by PCR using the following primers (sense primer: 5'-CACCATGGAGTCGGTAAAACAAAGGATT-3 ': SEQ ID NO: 11, antisense primer: 5'-TTCGTTATGAATAATCATCTCGGTCCT-3': SEQ ID NO: 12), and TOPO cloning Using the system (Invitrogen), it was incorporated into the pENTER / D-TOPO vector.
  • This plasmid was incorporated into a destination vector, pDEST10, by LR recombination (pDEST10VGLUT2). This was transformed into E. coli DH10bac strain containing bacmid DNA to complete a VGLUT bacmid with 6 ⁇ His in the N-terminal side. VGLUT2 virus was obtained by transducing Sf9 cells by lipofection with a bacmid isolated from DH10bac strain.
  • MOI multiplicity of infection
  • buffer C (20 mM POP-tris pH 7.0, 1% octyl glucoside, 20% glycerol, 5 mM imidazole, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin) to obtain the same solution with 60 mM imidazole. And eluted. Stored at ⁇ 80 ° C. until use.
  • VGLUT2 (6. Reconstitution of VGLUT into liposomes) 10 ⁇ g of purified VGLUT2 was mixed with 500 ⁇ g of liposomes and allowed to stand at ⁇ 80 ° C. for 15 minutes. After freezing, this was taken out and thawed quickly, and then immediately with buffer F (20 mMOPS-Tris pH 7.0, 150 mM sodium acetate, 5 mM magnesium acetate, 0.5 mM DTT, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin). The pellet was suspended in 200 ⁇ L of buffer F after doubling and centrifuging at 200,000 ⁇ g. This was used as a proteoliposome (reconstituted liposome) in subsequent experiments.
  • buffer F (20 mMOPS-Tris pH 7.0, 150 mM sodium acetate, 5 mM magnesium acetate, 0.5 mM DTT, 1 ⁇ g / ml pepstatin A, 1
  • E. coli DK8 containing the large F 0 F 1 expression plasmid pBWU13 was added to Tanaka medium (34 mM monopotassium phosphate, 64 mM dipotassium phosphate, 20 mM ammonium sulfate, 0.3 mM magnesium chloride, 1 ⁇ M) containing 0.5% glycerol. After culturing with iron sulfate, 1 ⁇ M calcium chloride, 1 ⁇ M zinc chloride, 100 ⁇ g / ml isoleucine, 100 ⁇ g / ml valine, 2 ⁇ g / ml thiamine), the cells were collected. All subsequent preparations were performed at 4 ° C.
  • the crushed liquid was centrifuged at 17,000 ⁇ g for 10 minutes, and the obtained supernatant was further centrifuged at 210,000 ⁇ g for 1 hour and 20 minutes.
  • the obtained membrane vesicle precipitate was suspended in F 0 F 1 preparation buffer (20 mM MOPS / NaOH (pH 7.0), 1 mM magnesium sulfate, 1 mM DTT, 1 mM PMSF, 0.8% octyl glucoside). Centrifuge again. 60 mg of membrane vesicle prepared as a precipitate was suspended in 3 ml of F 0 F 1 preparation buffer containing 2% octyl glucoside to solubilize F 0 F 1 .
  • the solubilized solution was centrifuged at 260,000 ⁇ g for 30 minutes, and F 0 F 1 was recovered from the supernatant fraction.
  • the recovered F 0 F 1 was purified by 10% (w / v) to 30% (w / v) glycerol density gradient centrifugation (330,000 ⁇ g for 5 hours).
  • a glycerol density gradient was made with F 0 F 1 preparation buffer containing 1% octyl glucoside. After density gradient centrifugation, the fraction was separated into 10 fractions from the bottom of the centrifuge tube, and the first 4 fractions were collected as F 0 F 1 and stored at ⁇ 80 ° C.
  • the F o F 1 -ATPase 90 ⁇ g and VGLUT2 10 [mu] g purified by the above method was mixed with the liposome 500 [mu] g, and allowed to stand for 15 minutes at -80 ° C., and frozen. This was immediately taken out, thawed quickly, diluted 20 times with F buffer (20 mM MOPS-Tris pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate), and centrifuged at 160,000 ⁇ g for 60 minutes. To the precipitate, 400 ⁇ l of F buffer was added and homogenized to obtain reconstituted proteoliposomes.
  • Example 2 Measurement of transport activity of reconstituted VGLUT2
  • liposomes reconstituted with F-ATPase and VGLUT2 glutamate transport activity was measured at various chloride ion concentrations in the presence (+ ATP) and absence (-ATP) of ATP.
  • reconstituted proteoliposomes (5 ⁇ g) were suspended in 20 mM MOPS-Tris pH 7.0, 5 mM magnesium acetate, 104 mM potassium acetate, and incubated at 27 ° C. for 3 minutes. A part of potassium acetate in the reaction solution was replaced with potassium chloride so as to contain a necessary concentration of chloride ions.
  • Example 3 Screening of inhibitors
  • Inhibitors of glutamate uptake were screened using an improved screening system for drugs that affect chloride ion sensitivity constructed in Example 2.
  • Candidate compound 5 mM was used, and glutamate transport activity was measured in the presence of 5 mM chloride ions and in the presence of 20 mM chloride ions.
  • the results are shown in FIG.
  • Acetoacetate, pyruvate, phenylpyruvate, 3-hydroxybutyrate, and ⁇ -keto- ⁇ -methylvalerate (KMV) showed high inhibitory activity under physiological conditions of chloride ion concentration (20 mM).
  • the graph of FIG. 4 shows the glutamate transport activity of VGLUT2 when various concentrations of chloride and acetoacetate were used. This result shows that inhibition of VGLUT2 activity by acetoacetate is eliminated by increasing the chloride ion concentration.
  • IC 50 of candidate compounds showing high inhibitory activity ie, acetoacetate, pyruvate, acetol, phenylpyruvate, 3-hydroxybutyrate, and ⁇ -keto- ⁇ -methylvalerate (KMV) are shown in FIG. Show.
  • IC 50 of lactate was> 20 mM.
  • Acetone, methylglyoxal, 1,2-propanediol, acetyl CoA, succinate, isoleucine, valine, phenylacetate and sialic acid did not show inhibitory activity at 10 mM (FIG. 5).
  • the inhibitory effect of acetoacetate was not a competitive inhibition on glutamic acid.
  • the Hill coefficient for chloride-mediated activation was about 3.3. This indicates a strong positive cooperativity with glutamate transport.
  • Acetoacetate did not affect the cooperativity between chloride and glutamic acid.
  • the effect of acetoacetate was completely reversible, and glutamate transport activity was completely restored by washing acetoacetate.
  • acetoacetate showed an inhibitory effect on VGLUT1 and VGLUT3.
  • VGLUT1 and VGLUT3 were inhibited by acetoacetate, glutamate transport activity was completely restored by washing.
  • inhibitors are considered to bind to the transporter protein reversibly from the structure and exhibit inhibitory activity.
  • Dyes that bind irreversibly to the vesicular neurotransmitter transporter protein are predicted to irreversibly deactivate the vesicular neurotransmitter transporter, resulting in side effects that cannot be overlooked.
  • the compounds exhibiting the inhibitory activity of the present invention are reversible in their mode of inhibition, so that they are particularly useful as inhibitors of vesicular excitatory neurotransmitter transporters, particularly diseases associated with excessive neural excitability and It is believed that / or the condition is useful for treating, preventing and / or improving prognosis.
  • Example 4 Inhibitory activity against inhibitory transmitter transporter and monoamine transporter by substance having VGLUT2 inhibitory activity
  • Example 3 a substance having an inhibitory activity against the vesicular glutamate transporter was identified. It was tested whether these inhibitors have inhibitory activity against inhibitory neurotransmitter transporters and / or monoamine transporters. Synaptic vesicles were used to measure the inhibitory activity on transporter transport activity.
  • the inhibitor of the present invention has no inhibitory activity against monoamine transporters and inhibitory neurotransmitter transporters.
  • Example 5 Inhibitory action on excitatory neurotransmitter transporters other than vesicular glutamate transporters
  • excitatory neurotransmitter transceptors other than vesicular glutamate transporters are tested.
  • mice sialin sense primer 5'-caccatgaggcccctgcttcggg-3 '(SEQ ID NO: 13) mouse sialin sense primer 5'-ccacggacacagaaactga-3' (SEQ ID NO: 14) mouse sialin sense primer 5'-caccatgaggtctccggttcgag-3 '(SEQ ID NO: 15) human sialin sense primer 5'-tcagtgtctgtgtccatggt-3 '(SEQ ID NO: 16) Human sialin antisense primer
  • the PCR reaction was performed at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 45 seconds, 56 ° C for 45 seconds, and 72 ° C for 2 minutes.
  • the PCR product was incorporated into the entry vector using the pENTR / D-TOPO cloning kit (No. K2400-20, Invitrogen). The method was performed according to the attached protocol.
  • Primers with the following sequences were used as primers: 5'-agctatgcgggccatgtgg-3 ', (SEQ ID NO: 17) mouse H183R1 st PCR sense primer 5'-accacagaggatcatgcataacc-3 '(SEQ ID NO: 18) mouse H183R1 st PCR antisense primer 5′-gtaaaacgacggccagtc-3 ′ (SEQ ID NO: 19) mouse H183R2 nd PCR sense primer.
  • 2nd PCR reaction 2 using a nd PCR sense primer and 1st PCR reaction products as primers, after heating 94 ° C. 2 min, 94 ° C. 45 seconds, 50 ° C. 1 min 30 sec, repeating 35 cycles 72 ° C. 3 min, And it kept at 72 ° C. for 5 minutes.
  • the PCR fragment was incorporated into an entry vector (pENTR, Invitrogen) using a TOPO cloning kit (Invitrogen).
  • the reaction solution (6 ⁇ l) is a solution containing 1 ⁇ l of salt solution (Invitrogen), 10 fmol of vector (Invitrogen), and 20 fmol of PCR product.
  • the reaction was carried out at room temperature for 10 minutes, and sialin was incorporated into the entry vector. This was used as a TOPO reaction solution.
  • sialin cDNA was cloned into the pDEST10 vector using LR clonase.
  • LR clonase To 150 mg of the plasmid prepared above, 300 ng of pDEST10 plasmid and 4 ⁇ l of LR clonase were added, incubated at 25 ° C. for 1 hour, and then 2 ⁇ l of proteinase K was added and incubated at 37 ° C. for 30 minutes.
  • the reaction solution was used to transform DH5 ⁇ competent E. coli. From the transformed DH5 ⁇ cells, the plasmid was recovered using QIAprepSpin Miniprep Kit (Qiagen) to obtain pDEST10 / SLC17A5.
  • pDEST10 / SLC17A5 20 pg of pDEST10 / SLC17A5 was added to 25 ⁇ l of DH10Bac competent cells (Invitrogen), left on ice for 30 minutes, and then 225 ⁇ l of SOC medium was added at 42 ° C. for 30 seconds. Incubated for 4 hours at 37 ° C., seeded with LB plates containing 50 ⁇ g / ml kanamycin, 7 ⁇ g / ml gentamicin, 10 ⁇ g / ml tetracycline and incubated overnight at 37 ° C. Well, the bacmid was recovered by the miniprep method.
  • the miniprep method used for the production of the recombinant bacmid was performed according to the following procedure. First, DH10Bac having a recombinant bacmid was inoculated into 3 ml of LB medium supplemented with 50 ⁇ g / ml kanamycin, 7 ⁇ g / ml gentamicin, and 10 ⁇ g / ml tetracycline, and cultured at 37 ° C. The cultured E.
  • coli is suspended in 200 ⁇ l of solution 1 (50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0), and then 200 ⁇ l of solution 2 (0.2 M NaOH, 1% SDS) is added, Mixed. After standing at room temperature for 5 minutes, 200 ⁇ l of solution 3 (3M KOAc, 11.5% (v / v) acetic acid) was added and mixed over the counter. After leaving at 4 ° C. for 10 minutes, the mixture was centrifuged (13,000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed. The precipitate was further washed twice with 70% ethanol. TE buffer (10 mM Tris / HCl pH 8.0, 1 mM EDTA) was aseptically added thereto, and stored at 4 ° C.
  • solution 1 50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0
  • the virus used in this example was prepared by the following procedure. First, 9 ⁇ 10 5 Sf9 cells were seeded in a 35 mm Petri dish. After exchanging the medium with Grace's Insect Medium (GIBCO) supplemented with 0.35 mg / ml sodium hydrogen carbonate, infection with Sf9 was performed by lipofection using 1 ⁇ g of bacmid containing sialin and 6 ⁇ l of cellfectin (Invitrogen). I let you. After incubating at 27 ° C. for 5 hours, the medium was replaced with 2 ml of completeTMN-FH, cultured until signs of infection were observed, and the medium was collected. This was designated as P1 virus.
  • GEBCO Grace's Insect Medium
  • washing buffer (20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin), and eluted buffer (20 mM MOPS- Purified sialin was eluted using Tris pH 7.0, 1% octyl glucoside, 20% glycerol, 60 mM imidazole, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin).
  • Reconstituted liposomes can be reacted with 20 mM MOPS-Tris (pH 7.0), 0.15 M potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride, or 20 mMOPS-Tris (pH 7.0), 54 mM potassium acetate, 5 2 ⁇ M valinomycin and 0.1 mM L- [2,3- 3 H] aspartic acid (0.5 MBq / ⁇ mol) were added to a reaction solution composed of mM magnesium acetate and 100 mM potassium chloride, and incubated at 27 ° C. for 2 minutes. The addition of reconstituted liposomes was the start of activity measurement.
  • reaction solution was centrifuged at 760 ⁇ g for 2 minutes at 4 ° C. using a Sephadex G-50 (fine) spin column.
  • the radioactivity contained in the reaction solution that passed through the column was measured with a liquid scintillation counter, and the transport activity of aspartic acid was measured. 200 ⁇ M acetoacetic acid was added before the reconstituted liposome was added.
  • PCR Cloning and expression of nucleotide transporter (VNUT), preparation of proteoliposome, and activity measurement
  • PCR was performed by adding 0.2 mM dNTP mixed solution, 1 pmol primer, and 1.5 U Ex Taq (TaKaRa) in Ex TaqBuffer (TaKaRa) to 50 ⁇ l.
  • the primers used were the forward primer (SEQ ID NO: 20: 5′-CACCCATGACCCTGACAAGCAGGCGCCAGGA-3 ′) and the reverse primer (SEQ ID NO: 21 ′ 5′-CTAGAGGTCCCTCATGGTAGAGCTC-3 ′).
  • As PCR conditions after heating at 94 ° C. for 3 minutes, 30 cycles of 94 ° C. for 3 minutes, 56 ° C. for 30 seconds, 72 ° C. for 2 minutes were performed, and then heating at 72 ° C. for 5 minutes was used.
  • the PCR fragment was incorporated into an entry vector (pENTR, Invitrogen) using a TOPO cloning kit (Invitrogen).
  • the reaction solution (6 ⁇ l) is a solution containing 1 ⁇ l of salt solution (Invitrogen), 10 fmol of vector (Invitrogen), and 20 fmol of PCR product.
  • the reaction was allowed to proceed at room temperature for 10 minutes, and SLC17A9 was incorporated into the entry vector. This was used as a TOPO reaction solution.
  • a vector (pENTR / SLC17A9) containing SLC17A9 was obtained from the cultured Escherichia coli using QIAprepSpinMiniprep Kit (Qiagen). SLC17A9 cDNA was sequenced using this vector.
  • the nucleic acid sequence of human SLC17A9 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
  • pDEST10 / SLC17A9 20 ng of pDEST10 / SLC17A9 was added to 25 ⁇ l of DH10Bac competent cells (Invitrogen), left on ice for 30 minutes, and then 225 ⁇ l of SOC medium was added at 42 ° C. for 30 seconds. Incubated for 4 hours at 37 ° C., seeded with LB plates containing 50 ⁇ g / ml kanamycin, 7 ⁇ g / ml gentamicin, 10 ⁇ g / ml tetracycline and incubated overnight at 37 ° C. Well, the bacmid was recovered by the miniprep method.
  • the miniprep method used for the production of the recombinant bacmid was performed according to the following procedure. First, DH10Bac having a recombinant bacmid was inoculated into 3 ml of LB medium supplemented with 50 ⁇ g / ml kanamycin, 7 ⁇ g / ml gentamicin, and 10 ⁇ g / ml tetracycline, and cultured at 37 ° C. The cultured E.
  • coli is suspended in 200 ⁇ l of solution 1 (50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0), and then 200 ⁇ l of solution 2 (0.2 M NaOH, 1% SDS) is added, Mixed. After standing at room temperature for 5 minutes, 200 ⁇ l of solution 3 (3M KOAc, 11.5% (v / v) acetic acid) was added and mixed by inversion. After leaving at 4 ° C. for 10 minutes, the mixture was centrifuged (13,000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed. The precipitate was further washed twice with 70% ethanol. TE buffer (10 mM Tris / HCl pH 8.0, 1 mM EDTA) was aseptically added thereto, and stored at 4 ° C.
  • solution 1 50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0
  • solution 2 0.2
  • the virus used in this example was prepared by the following procedure. First, 9 ⁇ 10 5 Sf9 cells were seeded in a 35 mm Petri dish. After exchanging the medium with Grace's Insect Medium (GIBCO) supplemented with 0.35 mg / ml sodium bicarbonate, infection with Sf9 was performed by lipofection using 1 ⁇ g of bacmid containing SLC17A9 and 6 ⁇ l of cellfectin (Invitrogen). I let you. After incubating at 27 ° C. for 5 hours, the medium was replaced with 2 ml of completeTMN-FH, cultured until signs of infection were observed, and the medium was collected. This was designated as P1 virus.
  • GEBCO Grace's Insect Medium
  • washing buffer (20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin), and eluted buffer (20 mM MOPS- Purified SLC17A9 was eluted using Tris pH 7.0, 1% octyl glucoside, 20% glycerol, 60 mM imidazole, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin).
  • Reconstituted liposomes can be reacted with 20 mM MOPS-Tris (pH 7.0), 0.15 M potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride, or 20 mMOPS-Tris (pH 7.0), 54 mM potassium acetate, 5 2 ⁇ M valinomycin and ATP (0.1 mM [ ⁇ 32 P] ATP 3.7 MBq / ⁇ mol) were added to a reaction solution composed of mM magnesium acetate and 100 mM potassium chloride, and incubated at 27 ° C. for 2 minutes. The addition of reconstituted liposomes was the start of activity measurement.
  • reaction solution was centrifuged at 760 ⁇ g for 2 minutes at 4 ° C. using a Sephadex G-50 (fine) spin column.
  • the radioactivity contained in the reaction solution that passed through the column was measured with a liquid scintillation counter, and the transport activity of aspartic acid was measured. 200 ⁇ M acetoacetic acid was added before the reconstituted liposome was added.
  • the experimental conditions for each lane are as follows: Lane 1 4 mM Cl - presence, valinomycin not added, Lane 2 4 mM Cl - presence, 2 [mu] M valinomycin added, Lane 3 in the presence of 4 mM Cl ⁇ , 2 ⁇ M valinomycin added, 200 ⁇ M acetoacetate added, Lane 4 100 mM Cl - presence, valinomycin not added, Lane 5 2 ⁇ M valinomycin added in the presence of 100 mM Cl ⁇ Lane 6 100 mM Cl - presence, 2 [mu] M valinomycin added, 200 [mu] M acetoacetate added.
  • acetoacetate inhibited the vesicular aspartate transporter (sialin) and vesicular nucleotide transporter (VNUT), but the inhibition was reduced by increasing the chloride ion concentration to 100 mM. / Disappeared.
  • Example 6 Inhibitory effect on glutamate release from vesicles It was investigated whether the compounds of the present invention inhibit glutamate exocytosis from glutamatergic cultured cells.
  • Example 7 Acupuncture treatment effect by glutamate transport inhibitor
  • FIG. 9A glutamate and dopamine secretion
  • FIG. 9D seizure
  • Seizures were assessed as follows: Each 20-minute behavior change was divided into the following 6 stages and scored. (0) No change in behavior. No epileptiform symptoms; (1) Move your mouth and face. Dress up. I smell it. Scratch your body. Shake your head. Excessive behavior; (2) Shake your head. Shake your whole body. Whole body stiffness; (3) The forefoot cramps. The hind legs extend; (4) Go back. Drool. Tonicity-clonic seizures; and (5) Epilepsy fainting. The scoring method was scored in all time courses according to MeursA et al. (Epilepsy Res. 2008, 78, 50-59). After adding 4AP, the action amount Totalseizure severity score (TSSS) for one hour was taken as the measured value. did.
  • TSSS Totalseizure severity score
  • the glutamate transport inhibitor of the present invention is useful as a therapeutic agent for epilepsy.
  • this result is not only for epilepsy but also for diseases and / or conditions associated with excessive neural excitation (eg, in addition to epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease)
  • diseases and / or conditions associated with excessive neural excitation eg, in addition to epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease
  • the glutamate secretion inhibitor of the present invention is useful as a therapeutic agent for diseases and / or conditions selected from the group consisting of marbleosis and osteoporosis.
  • Example 8 Screening of inhibitor or activator of glutamate transport inhibition
  • Acetoacetate is added to the reconstituted liposome prepared in Example 2 at a final concentration of 1 mM.
  • buffer A (20 mM MOPS-Tris pH 7.0, 5 mM magnesium acetate, 104 mM potassium acetate) is used.
  • a negative control or candidate drug (0.01 mM to 1 mM in buffer A) is added to the reconstituted liposomes to which acetoacetate has been added and left at room temperature for 5 minutes.
  • glutamate transport activity is measured at a chloride ion concentration of 5 mM in the presence of valinomycin (+ valinomycin) and in the absence ( ⁇ valinomycin).
  • buffer G (20 mM MOPS-Tris pH 7.0, 150 mM potassium acetate, 5 mM magnesium acetate, 10 mM KCl), 2 ⁇ M valinomycin, [2,3- 3 H] L-glutamic acid ( 0.5 MBq / pmol) and inhibitors were added and incubated in a 27 ° C. water bath for 3 minutes. The reaction is started by adding 0.5 ⁇ g of the prepared proteoliposome.
  • the present invention provides an excitatory chemical transmission inhibitor and a screening method thereof.
  • the present invention provides an excitatory chemical transmission inhibitor that inhibits at least two vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration and a screening method thereof.
  • drugs useful for the treatment, prevention, and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis And screening methods thereof are provided by the present invention.

Abstract

Disclosed is an inhibitor which can inhibit at least two vesicular neurotransmitter transporters for excitatory neurotransmitters under a chloride concentration included in physiological conditions. Also disclosed is a novel method for the screening of the inhibitor. The inhibitor is useful for the treatment of epilepsy, inflammations, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, osteopetrosis, osteoporosis or the like. Further disclosed is a method for the screening of a substance capable of activating a suppressed vesicular neurotransmitter transporter.

Description

興奮性化学伝達調節剤およびそのスクリーニング法Excitatory chemical transmission regulator and screening method thereof
　本発明は、興奮性化学伝達調節剤およびそのスクリーニング法の分野に関する。また、本発明の興奮性化学伝達調節剤は、癲癇のような過剰なニューロンの発火に起因する障害の治療薬の分野、および、抑制されたニューロンの活性化による治療の分野に関する。 The present invention relates to the field of excitatory chemical transmission regulators and screening methods thereof. The excitatory chemical transmission modulators of the present invention also relate to the field of treatment of disorders caused by excessive neuronal firing such as sputum, and the field of treatment by suppressed neuronal activation.
　神経伝達物質は、シナプス前の細胞（前シナプス細胞）から放出され，シナプスを横切ってシナプス後の細胞（後シナプス細胞）を刺激または阻害する特異的化学物質である。神経伝達物質は、ニューロンで生産されシナプス前終末から放出されて、シナプス後膜に直接作用する。この作用によって標的細胞で引き起こされる応答反応は、電気的興奮または抑制を生み出す。電気的興奮を生み出す神経伝達物質を興奮性伝達物質といい、電気的抑制を生み出す神経伝達物質を抑制性伝達物質という。ニューロンで生産された神経伝達物質は、前ニューロン細胞のシナプス小胞に存在する神経伝達物質トランスポーターによりシナプス小胞内に蓄積され、エキソサイトーシスによってシナプス間隙に放出され、後シナプス細胞の細胞表面に存在する特異的受容体に結合する一方で、シナプス前終末やグリア細胞の細胞膜に存在する神経伝達物質トランスポーターによって回収される。 Neurotransmitters are specific chemicals that are released from presynaptic cells (presynaptic cells) and stimulate or inhibit postsynaptic cells (postsynaptic cells) across the synapse. Neurotransmitters are produced in neurons, released from the presynaptic terminals, and act directly on the postsynaptic membrane. The response response evoked in the target cell by this action produces electrical excitement or suppression. Neurotransmitters that produce electrical excitement are called excitatory transmitters, and neurotransmitters that produce electrical inhibition are called inhibitory transmitters. Neurotransmitters produced in neurons are accumulated in synaptic vesicles by neurotransmitter transporters present in synaptic vesicles of pre-neuronal cells, released into synaptic clefts by exocytosis, and cell surface of post-synaptic cells It binds to a specific receptor present in the cell and is recovered by a neurotransmitter transporter present in the cell membrane of presynaptic terminals and glial cells.
　この一連の神経伝達物質の動態には、シナプス小胞に存在する神経伝達物質トランスポーター（小胞型神経伝達物質トランスポーターまたは小胞型トランスポーターと呼ばれる）およびシナプス前終末・グリア細胞の細胞膜に存在する神経伝達物質トランスポーター（原形質膜型トランスポーター）が関与するが、これら２種の神経伝達物質トランスポーターは構造および機能が異なる分子である。 This series of neurotransmitter dynamics includes the neurotransmitter transporter (called vesicular neurotransmitter transporter or vesicular transporter) present in synaptic vesicles and the cell membrane of presynaptic terminal glial cells. Existing neurotransmitter transporters (plasma membrane transporters) are involved, but these two neurotransmitter transporters are molecules that differ in structure and function.
　てんかん（癲癇）とは、過度のニューロンの放電による発作的な脳の機能障害を特徴とする慢性疾患で、通常、意識の変調を伴う疾患である。発作は行動の要素的または複合的障害に限られる場合と、全身的痙攣に発展する場合とがある。発作の臨床所見は、全身痙攣や局所痙攣を含む行動の複雑異常から、意識障害の瞬間的発作まで様々である。これらの臨床所見には種々の分類がある。発作の型を示す用語は記述的であり標準化されているが、完全に統一されたものものではない。 Epilepsy is a chronic disease characterized by paroxysmal brain dysfunction caused by excessive neuronal discharge, and is usually a disease accompanied by modulation of consciousness. Seizures may be limited to elemental or complex disorders of behavior or may develop into generalized convulsions. The clinical findings of seizures range from complex behavioral abnormalities, including general and local convulsions, to instantaneous seizures of consciousness disturbances. There are various classifications of these clinical findings. Seizure type terms are descriptive and standardized, but not completely unified.
　上記のように、癲癇は過度のニューロンの放電による発作的な脳の機能障害を特徴とすることから、抗癲癇薬の作用としては、興奮性化学伝達を低下させる一方で、抑制性化学伝達を亢進させることが考えられる。 As mentioned above, sputum is characterized by paroxysmal brain dysfunction due to excessive neuronal discharge, so the anti-epileptic effect is to reduce excitatory chemical transmission while inhibiting inhibitory chemical transmission. It is possible to increase it.
　例えば、公知の抗癲癇薬であるバルプロ酸の作用は、脳内における抑制性伝達物質であるＧＡＢＡ濃度を増加させることであると考えられている。バルプロ酸はほとんど全ての癲癇型に有効であると考えられるが、用量依存的な消火器および肝臓に対する副作用があり、また、催奇形性である。Ｎａチャンネル抑制による神経興奮性を阻害する薬剤としてフェニトインが公知であるが、眼振、構音障害、失調、めまい、複視、傾眠、異常行動、認知障害、過敏症、発疹、多毛、歯肉増殖、骨軟化症、および、大球性貧血などの副作用が知られている。既存の抗癲癇薬には例えば上記の副作用の問題があり、併用療法をしても副作用を増すのみであることから、通常は、単剤でＴＤＭ（治療効果や副作用に関する様々な因子をモニタリングしながらそれぞれの患者に個別化した薬物投与を行うこと）を利用して至適量まで使用するのが通常である。 For example, it is believed that the action of valproic acid, a known antidepressant, increases the concentration of GABA, an inhibitory transmitter in the brain. Valproic acid is thought to be effective in almost all types of sputum, but has dose-dependent fire extinguisher and liver side effects and is teratogenic. Although phenytoin is known as a drug that inhibits nerve excitability by Na channel suppression, nystagmus, dysarthria, ataxia, dizziness, diplopia, somnolence, abnormal behavior, cognitive impairment, hypersensitivity, rash, hirsutism, gingival proliferation, Side effects such as osteomalacia and macrocytic anemia are known. Existing antiepileptic drugs, for example, have the above-mentioned problems of side effects, and even if they are given in combination, they only increase the side effects. Therefore, TDM (various factors related to therapeutic effects and side effects are usually monitored as a single agent. However, it is usual to use the individual doses for each patient).
　興奮性化学伝達の低下を介する抗癲癇薬の開発として、神経伝達物質トランスポーターの活性調節剤を標的とするならば、シナプス前終末・グリア細胞の細胞膜に存在する興奮性神経伝達物質に特異的な神経伝達物質トランスポーター（細胞膜型神経伝達物質トランスポーター）の活性化剤、または、シナプス小胞に存在する興奮性神経伝達物質に特異的な神経伝達物質トランスポーター（小胞型神経伝達物質トランスポーター）の阻害剤が想定できる。なぜならば、細胞膜型神経伝達物質トランスポーターを活性化すると、シナプス間隙に放出された興奮性神経伝達物質のクリアランスを促進することで興奮性化学伝達の低下が合理的に予測されるからである。また、小胞型神経伝達物質トランスポーターを阻害することで、シナプス小胞に蓄積される興奮性神経伝達物質の量の低下が可能であり、その結果、興奮性化学伝達の低下が合理的に予測されるからである。 If the target is a neurotransmitter transporter activity regulator as a development of antidepressant drugs through the reduction of excitatory chemical transmission, it is specific to excitatory neurotransmitters present in the plasma membrane of presynaptic terminals and glial cells Neurotransmitter transporter (cell membrane-type neurotransmitter transporter) activator or neurotransmitter transporter specific to excitatory neurotransmitter present in synaptic vesicles (vesicle-type neurotransmitter trans Porter) inhibitors. This is because, when the cell membrane type neurotransmitter transporter is activated, a decrease in excitatory chemical transmission is reasonably predicted by promoting clearance of the excitatory neurotransmitter released into the synaptic cleft. In addition, inhibiting the vesicular neurotransmitter transporter can reduce the amount of excitatory neurotransmitter that accumulates in synaptic vesicles, resulting in a rational decrease in excitatory chemical transmission. This is because it is predicted.
　しかしながら、興奮性神経伝達物質は複数存在することから、癲癇においては、複数の興奮性神経伝達物質が関与すると考えられる。例えば、グリア細胞は癲癇などの疾患と関連していることが知られており、そして、グリア細胞は、ＡＴＰやグルタミン酸などの興奮性神経伝達物質を分泌することが知られ、その分泌を阻害することによって、これら疾患に対する治療効果が期待できると考えられている（非特許文献５～１３）。そのため、抗癲癇薬の候補化合物としては、複数種類の興奮性神経伝達物質の作用を阻害する化合物が求められる。 However, since there are a plurality of excitatory neurotransmitters, it is considered that a plurality of excitatory neurotransmitters are involved in acupuncture. For example, glial cells are known to be associated with diseases such as epilepsy, and glial cells are known to secrete and inhibit excitatory neurotransmitters such as ATP and glutamate Therefore, it is considered that a therapeutic effect for these diseases can be expected (Non-Patent Documents 5 to 13). Therefore, compounds that inhibit the action of multiple types of excitatory neurotransmitters are required as candidate compounds for antidepressants.
　その一方で、神経伝達物質トランスポーターは、細胞膜型であるか小胞型であるかに拘らず、各種の興奮性神経伝達物質に対して特異的であるため、神経伝達物質トランスポーターを標的として抗癲癇薬を開発する場合には、広範な種類の神経伝達物質トランスポーターを対象としなければならないと考えられる。 On the other hand, neurotransmitter transporters are specific for various excitatory neurotransmitters regardless of whether they are cell membrane type or vesicle type. When developing antiepileptic drugs, a wide variety of neurotransmitter transporters should be targeted.
　小胞型グルタミン酸トランスポーターに対する阻害剤については研究されているものの（非特許文献１）、そのいずれもが、グルタミン酸トランスポーターと類似の構造を有する化合物または、小胞型神経伝達物質トランスポータータンパク質に不可逆的に結合する色素類を中心とした化合物である。小胞型グルタミン酸トランスポーターと類似の構造を有する化合物は、複数種類の小胞型神経伝達物質トランスポーターを阻害することはできないと考えられる。また、小胞型神経伝達物質トランスポータータンパク質に不可逆的に結合する色素を中心とした化合物は、小胞型神経伝達物質トランスポーターを不可逆的に失活させ、その結果、看過できない副作用を生じることが予測される。 Inhibitors for vesicular glutamate transporters have been studied (Non-patent Document 1), but all of them are compounds having a structure similar to glutamate transporters or vesicular neurotransmitter transporter proteins. It is a compound centering on pigments that bind irreversibly. It is considered that a compound having a structure similar to the vesicular glutamate transporter cannot inhibit a plurality of types of vesicular neurotransmitter transporters. In addition, compounds centered on dyes that bind irreversibly to the vesicular neurotransmitter transporter protein irreversibly deactivate the vesicular neurotransmitter transporter, resulting in side effects that cannot be overlooked. Is predicted.
　小胞型グルタミン酸トランスポーターは、クロライドイオン要求性であることが知られており（非特許文献２）、２－ケト－３－メチルバレリン酸が小胞型グルタミン酸トランスポーターのクロライドイオン要求性を変化させることも知られている（非特許文献３および４）。しかしながら、２－ケト－３－メチルバレリン酸は、小胞型グルタミン酸トランスポーターに対する阻害効果が非常に小さく、そのため、小胞型グルタミン酸トランスポーターの阻害剤として実用的であるとは認識されていない。しかも２－ケト－３－メチルバレリン酸は遺伝子性酵素欠損により体内蓄積する物質であり、神経毒性が報告されている（非特許文献３および４）。またそのため、２－ケト－３－メチルバレリン酸は、癲癇などの疾患の治療薬として有用であるどころか、神経毒として作用すると考えられる。 The vesicular glutamate transporter is known to be chloride ion-requiring (Non-Patent Document 2), and 2-keto-3-methylvaleric acid changes the chloride ion requirement of the vesicular glutamate transporter. It is also known (Non-Patent Documents 3 and 4). However, 2-keto-3-methylvaleric acid has a very small inhibitory effect on the vesicular glutamate transporter, and thus is not recognized as a practical inhibitor of the vesicular glutamate transporter. Moreover, 2-keto-3-methylvaleric acid is a substance that accumulates in the body due to genetic enzyme deficiency, and neurotoxicity has been reported (Non-patent Documents 3 and 4). Therefore, 2-keto-3-methylvaleric acid is considered to act as a neurotoxin rather than being useful as a therapeutic agent for diseases such as hemorrhoids.
　現時点では、神経伝達物質トランスポーター（特に、小胞型神経伝達物質トランスポーター）の構造および機能、ならびに阻害剤については、充分な知見がない。そのため、広範な神経伝達物質トランスポーターを対象とする活性調節剤の開発は多大な困難が予想される。 At present, there is insufficient knowledge about the structure and function of neurotransmitter transporters (particularly, vesicular neurotransmitter transporters) and inhibitors. Therefore, the development of activity modulators targeting a wide range of neurotransmitter transporters is expected to be very difficult.
　そのような困難性が予測されるにも拘らず、興奮性神経伝達物質に直接関わる分子である神経伝達物質トランスポーターを標的として可逆的に結合する抗癲癇薬は、副作用が少ないことが予測されるなどの利点から魅力的であるため、広範な神経伝達物質トランスポーターを対象とする活性調節剤（例えば、興奮性化学伝達阻害剤）の開発が求められている。また、抑制されたニューロンを活性化する薬剤の開発も求められている。 Despite such difficulties, anti-epileptic drugs that reversibly bind to neurotransmitter transporters, molecules directly related to excitatory neurotransmitters, are expected to have fewer side effects. Therefore, there is a need for the development of activity modulators (eg, excitatory chemical transmission inhibitors) targeting a wide range of neurotransmitter transporters. There is also a need to develop drugs that activate inhibited neurons.
　興奮性化学伝達阻害剤は、癲癇以外にも、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症などの疾患／状態の治療、予防、および／または予後に有用であると考えられる。 In addition to epilepsy, excitatory chemical transmission inhibitors can treat, prevent, treat / prevent diseases / conditions such as inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis. And / or useful prognosis.
　この出願の発明に関連する先行技術文献情報としては、次のものがある。
Thompson, C. M.ら、"Inhibitorof the glutamate vesiculartransporter (VGLUT)", Curr. Med. Chem. 12,2041-2056 (2005) Juge N, YoshidaY, Yatsushiro S, Omote H, Moriyama Y.、"Vesicularglutamate transportercontains two independent transport machineries."、JBiol Chem. 2006 Dec22;281(51):39499-506. Tavares RG ら、"Inhibitionof glutamate uptake into synapticvesicles of rat brain by the metabolitesaccumulating in maple syrup urinedisease."、J Neurol Sci. 2000 Dec1;181(1-2):44-9 Marcelo Reis,Mariana Farage, Herman Wolosker、"Chloride-dependentinhibition ofvesicular glutamate uptake by alpha-keto acids accumulated inmaple syrup urine Bekar L et al.,(2008) Adenosine is crucial for deep brainstimulation-mediated attenuation oftremor. Nature Med. 14, 75-80. Tian G-F etal., An astrocytic basis of epilepsy. Nature Med. 2005,973-981. noue K et al.,(2007) The role of nucleotides in the neuron-gliacommunication responsible forthe brain functions. J. Neurochem. 102, 1447-1458. INedergaard Mand Dirnagl U. (2005) Role of glical cells in cerebralischemia. Glia 50,281-286. Fields RD andBurnstock G. (2006) Purinergic signaling in neuron-gliainteractions. NatureNeuroscience 7, 423-436. Tian G-F etal., An astrocytic basis of epilepsy. Nature Med. 2005,973-981. Walts E (2007)Epilepsy controlled by low-carb diets effect on brainchannels. Nature Med. 13,516-517. Freeman J etal., (2006) The ketogenic diet: from molecularmechanisms to clinical effects.Epilepsy Res. 68, 145-180. Hartman AL etal., (2007) The neuropharmacology of the ketogenicdiet. Pediatr Neurol 36,281-292. Prior art document information relating to the invention of this application includes the following.
Thompson, C. M. et al., "Inhibitorof the glutamate vesiculartransporter (VGLUT)", Curr. Med. Chem. 12,2041-2056 (2005) Juge N, YoshidaY, Yatsushiro S, Omote H, Moriyama Y., "Vesicularglutamate transportercontains two independent transport machineries.", JBiol Chem. 2006 Dec22; 281 (51): 39499-506. Tavares RG et al., "Inhibitionof glutamate uptake into synapticvesicles of rat brain by the metabolitesaccumulating in maple syrup urinedisease.", J Neurol Sci. 2000 Dec1; 181 (1-2): 44-9 Marcelo Reis, Mariana Farage, Herman Wolosker, "Chloride-dependentinhibition ofvesicular glutamate uptake by alpha-keto acids accumulated inmaple syrup urine Bekar L et al., (2008) Adenosine is crucial for deep brainstimulation-mediated attenuation oftremor.Nature Med. 14, 75-80. Tian GF etal., An astrocytic basis of epilepsy.Nature Med. 2005,973-981. noue K et al., (2007) The role of nucleotides in the neuron-gliacommunication responsible for the brain functions. J. Neurochem. 102, 1447-1458. INedergaard Mand Dirnagl U. (2005) Role of glical cells in cerebralischemia. Glia 50,281-286. Fields RD and Burnstock G. (2006) Purinergic signaling in neuron-gliainteractions.NatureNeuroscience 7, 423-436. Tian GF etal., An astrocytic basis of epilepsy.Nature Med. 2005,973-981. Walts E (2007) Epilepsy controlled by low-carb diets effect on brainchannels.Nature Med. 13,516-517. Freeman J etal., (2006) The ketogenic diet: from molecularmechanisms to clinical effects.Epilepsy Res.68, 145-180. Hartman AL etal., (2007) The neuropharmacology of the ketogenicdiet. Pediatr Neurol 36,281-292.
　興奮性化学伝達阻害剤およびそのスクリーニング法を提供することを、本発明の課題とする。特に、興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害する興奮性化学伝達阻害剤およびそのスクリーニング法を提供することを、本発明の課題とする。さらに、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症などの疾患／状態の治療、予防、および／または予後に有用である薬剤およびそのスクリーニング法を提供することを、本発明の課題とする。 It is an object of the present invention to provide an excitatory chemical transmission inhibitor and a screening method thereof. In particular, the present invention provides an excitatory chemical transmission inhibitor that inhibits at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological chloride concentrations and a screening method thereof. Let it be an issue. In addition, drugs useful for the treatment, prevention, and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis It is an object of the present invention to provide a screening method thereof.
　上記課題は、興奮性神経伝達物質を輸送する小胞型神経伝達物質トランスポーターの阻害剤をスクリーニングするための新規の手法を開発し、そして、そのスクリーニング法によって、興奮性神経伝達物質を輸送する小胞型神経伝達物質トランスポーターの少なくとも２種類に対して阻害効果を有する化合物を同定する一群の化合物を同定することによって、本発明者らは、本発明を完成した。 The above challenge is to develop a novel technique for screening for inhibitors of vesicular neurotransmitter transporters that transport excitatory neurotransmitters, and to transport excitatory neurotransmitters by the screening method By identifying a group of compounds that identify compounds that have inhibitory effects on at least two of the vesicular neurotransmitter transporters, the inventors have completed the present invention.
　従って、本発明は、以下を提供する。
（項目１）　興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害するための、以下の式（Ｉ）の化合物： Accordingly, the present invention provides the following.
(Item 1) A compound of the following formula (I) for inhibiting at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration:
Figure JPOXMLDOC01-appb-C000003
 　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　Ｒ１～Ｒ４の少なくとも１つは酸素原子を含み、
　Ｒ５は、Ｈ、直鎖アルキル、分岐アルキル、置換アルキル、アリール、アルキルアリールからなる群から選択される化合物またはその医薬として許容される塩を含有する、薬学的組成物。
（項目２）　項目１に記載の薬学的組成物であって、
　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択される、薬学的組成物。
（項目３）　項目２に記載の薬学的組成物であって、
　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択される、薬学的組成物。
（項目４）　項目１に記載の薬学的組成物であって、Ｒ３およびＲ４の少なくとも１つは酸素原子を含む、薬学的組成物。
（項目５）　項目４に記載の薬学的組成物であって、
　ここで、Ｒ３およびＲ４は、一緒になって、Ｏである、薬学的組成物。
（項目６）　項目１に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、薬学的組成物。
（項目７）　項目６に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、および、ピルビン酸からなる群から選択される、薬学的組成物。
（項目８）　項目１に記載の薬学的組成物であって、ここで、前記興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種が、グルタミン酸トランスポーター、アスパラギン酸トランスポーター、および、ヌクレオチドトランスポーターからなる群から選択される、薬学的組成物。
（項目９）　過剰な神経の興奮に関連する疾患および／または状態を、治療するため、予防するため、および／または、予後を改善するための薬学的組成物であって、以下の式（Ｉ）の化合物：
Figure JPOXMLDOC01-appb-C000003
 Wherein R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O
At least one of R 1 to R 4 contains an oxygen atom;
R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
(Item 2) The pharmaceutical composition according to item 1,
Wherein R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
(Item 3) The pharmaceutical composition according to item 2,
Wherein R 1 and R 2 are each independently selected from the group consisting of H and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
(Item 4) The pharmaceutical composition according to item 1, wherein at least one of R 3 and R 4 contains an oxygen atom.
(Item 5) The pharmaceutical composition according to item 4,
Wherein R 3 and R 4 taken together are O.
(Item 6) The pharmaceutical composition according to item 1, wherein the compound is acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid And a pharmaceutical composition selected from the group consisting of lactic acid.
(Item 7) The pharmaceutical composition according to item 6, wherein the compound is selected from the group consisting of acetoacetate and pyruvic acid.
(Item 8) The pharmaceutical composition according to item 1, wherein at least two of the vesicular neurotransmitter transporters for the excitatory neurotransmitter are glutamate transporter, aspartate transporter, And a pharmaceutical composition selected from the group consisting of nucleotide transporters.
(Item 9) A pharmaceutical composition for treating, preventing and / or improving the prognosis of a disease and / or condition associated with excessive nerve excitation, comprising the following formula (I ) Compound:
Figure JPOXMLDOC01-appb-C000004
 　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　Ｒ１～Ｒ４の少なくとも１つは酸素原子を含み、
　Ｒ５は、Ｈ、直鎖アルキル、分岐アルキル、置換アルキル、アリール、アルキルアリールからなる群から選択される化合物またはその医薬として許容される塩を含有する、薬学的組成物。
（項目１０）　項目９に記載の薬学的組成物であって、
　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択される、薬学的組成物。
（項目１１）　項目１０に記載の薬学的組成物であって、
　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択される、薬学的組成物。
（項目１２）　項目９に記載の薬学的組成物であって、Ｒ３およびＲ４の少なくとも１つは酸素原子を含む、薬学的組成物。
（項目１３）　項目１２に記載の薬学的組成物であって、
　ここで、Ｒ３およびＲ４は、一緒になって、Ｏである、薬学的組成物。
（項目１４）　項目９に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、薬学的組成物。
（項目１５）　項目１４に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、および、ピルビン酸からなる群から選択される、薬学的組成物。
（項目１６）　過剰な神経の興奮に関連する疾患および／または状態が、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症からなる群から選択される、項目９に記載の薬学的組成物。
（項目１７）　過剰な神経の興奮に関連する疾患および／または状態が、項目９に記載の薬学的組成物。
（項目１８）　過剰な神経の興奮に関連する疾患および／または状態を、治療のため、予防のため、および／または、予後を改善するための候補化合物をスクリーニングするための方法であって、以下：
（ａ）小胞型神経伝達物質トランスポーターを含む小胞を調製する工程；
（ｂ）該小胞型神経伝達物質トランスポーターが輸送する興奮性神経伝達物質、該再構成した小胞、イオノフォア、および、生理条件のクロライド存在下で、該興奮性神経伝達物質の小胞内への輸送を検出する工程；
（ｃ）該小胞型神経伝達物質トランスポーターが輸送する興奮性神経伝達物質、該再構成した小胞、イオノフォア、生理条件のクロライド、および、試験化合物の存在下で、該興奮性神経伝達物質の輸送を検出する工程；ならびに
（ｄ）上記工程（ｂ）における輸送と、上記工程（ｃ）における輸送を比較して、該試験化合物が、輸送を阻害するか否かを指標として、該試験化合物が、過剰な神経の興奮に関連する疾患および／または状態を、治療のため、予防のため、および／または、予後を改善するための候補化合物であるか否かを決定する工程、
を包含する、方法。
（項目１９）　前記小胞型神経伝達物質トランスポーターを含む小胞が、リポソームに、単離された小胞型神経伝達物質トランスポーターを再構成することによって調製される、項目１８に記載の方法。
（項目２０）　前記イオノフォアがバリノマイシンである、項目１８に記載の方法。
（項目２１）　前記小胞型神経伝達物質トランスポーターが、グルタミン酸トランスポーター、アスパラギン酸トランスポーター、ヌクレオチドトランスポーターからなる群から選択される、項目１８に記載の方法。
（項目２２）　小胞型神経伝達物質トランスポーターの輸送活性について、阻害の解除もしくは活性化をする化合物をスクリーニングする方法であって、該方法は、以下：
（１）小胞型神経伝達物質トランスポーターの阻害剤存在下において、該小胞型神経伝達物質トランスポーターの輸送活性を測定する工程；
（２）候補化合物、および、上記（１）の阻害剤存在下において、該小胞型神経伝達物質トランスポーターの輸送活性を測定する工程；ならびに
（３）上記（２）の活性が上記（１）の活性より高い場合に、該候補化合物を、阻害の解除能もしくは活性化能を有する化合物として同定する工程；
を包含する、方法。
（項目２３）　項目２２に記載の方法であって、ここで、前記阻害剤が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、方法。
Figure JPOXMLDOC01-appb-C000004
 Wherein R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O
At least one of R 1 to R 4 contains an oxygen atom;
R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
(Item 10) The pharmaceutical composition according to item 9,
Wherein R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
(Item 11) The pharmaceutical composition according to item 10,
Wherein R 1 and R 2 are each independently selected from the group consisting of H and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
(Item 12) The pharmaceutical composition according to item 9, wherein at least one of R 3 and R 4 contains an oxygen atom.
(Item 13) The pharmaceutical composition according to item 12,
Wherein R 3 and R 4 taken together are O.
(Item 14) The pharmaceutical composition according to item 9, wherein the compound is acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid And a pharmaceutical composition selected from the group consisting of lactic acid.
(Item 15) The pharmaceutical composition according to item 14, wherein the compound is selected from the group consisting of acetoacetate and pyruvic acid.
(Item 16) Diseases and / or conditions associated with excessive neural excitement consist of epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis 10. The pharmaceutical composition according to item 9, selected from the group.
(Item 17) The pharmaceutical composition according to item 9, wherein the disease and / or condition associated with excessive nerve excitement is present.
18. A method for screening candidate compounds for treating, preventing, and / or improving prognosis for diseases and / or conditions associated with excessive neural excitement comprising: :
(A) preparing a vesicle comprising a vesicular neurotransmitter transporter;
(B) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, and a vesicle of the excitatory neurotransmitter in the presence of chloride under physiological conditions Detecting the transport to
(C) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, a physiological condition chloride, and a test compound; And (d) comparing the transport in the step (b) with the transport in the step (c), and using the test compound as an index to determine whether the test inhibits the transport. Determining whether the compound is a candidate compound for treating, preventing and / or improving prognosis of diseases and / or conditions associated with excessive neural excitability;
Including the method.
19. The method of claim 18, wherein the vesicle comprising the vesicular neurotransmitter transporter is prepared by reconstituting an isolated vesicular neurotransmitter transporter into a liposome. .
(Item 20) The method according to item 18, wherein the ionophore is valinomycin.
(Item 21) The method according to item 18, wherein the vesicular neurotransmitter transporter is selected from the group consisting of a glutamate transporter, an aspartate transporter, and a nucleotide transporter.
(Item 22) A method for screening a compound that cancels or activates inhibition of the transport activity of a vesicular neurotransmitter transporter, the method comprising:
(1) measuring the transport activity of the vesicular neurotransmitter transporter in the presence of an inhibitor of the vesicular neurotransmitter transporter;
(2) a step of measuring the transport activity of the vesicular neurotransmitter transporter in the presence of the candidate compound and the inhibitor of (1) above; and (3) the activity of (2) above (1) The candidate compound is identified as a compound having the ability to cancel or activate inhibition;
Including the method.
23. The method of claim 22, wherein the inhibitor is acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid, and A method selected from the group consisting of lactic acid.
　本発明に従って、興奮性化学伝達阻害剤およびそのスクリーニング法が提供される。特に、本発明に従って、興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害する興奮性化学伝達阻害剤およびそのスクリーニング法が提供される。さらに、本発明に従って、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症などの疾患／状態の治療、予防、および／または予後に有用である薬剤およびそのスクリーニング法が提供され、具体的な有効成分が提供される。また、本発明に従って、抑制されたニューロンを活性化する薬剤も提供される。 According to the present invention, an excitatory chemical transmission inhibitor and a screening method thereof are provided. In particular, according to the present invention, an excitatory chemical transmission inhibitor that inhibits at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration and a screening method thereof are provided. Furthermore, according to the present invention, the treatment, prevention and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis. Agents that are useful and methods of screening are provided, and specific active ingredients are provided. Also provided according to the invention is an agent that activates a suppressed neuron.
図１（Ａ）は、Ｆ－ＡＴＰａｓｅとグルタミン酸トランスポーターとを再構成したプロテオリポソームにおけるグルタミン酸輸送活性を示すグラフである。「＋ＡＴＰ」はＡＴＰ存在下での輸送活性を、「－ＡＴＰ」はＡＴＰ非存在下での輸送活性を示す。FIG. 1 (A) is a graph showing glutamate transport activity in proteoliposomes reconstituted with F-ATPase and glutamate transporter. “+ ATP” indicates the transport activity in the presence of ATP, and “−ATP” indicates the transport activity in the absence of ATP.
　　図１（Ｂ）は、Ｆ－ＡＴＰａｓｅを用いることなく、グルタミン酸トランスポーターのみを再構成したプロテオリポソームにおけるグルタミン酸輸送活性を示すグラフである。「＋バリノマイシン」はバリノマイシン存在下での輸送活性を、「－バリノマイシン」はバリノマイシン非存在下での輸送活性を示す。
図２は、５ｍＭクロライド存在下（５ｍＭ　Ｃｌ－）、および、２０ｍＭクロライド存在下（２０ｍＭ　Ｃｌ－）における、種々の候補化合物の、グルタミン酸輸送阻害活性を試験した結果である。 図３は、VGLUTによるグルタミン酸輸送の活性阻害における、アセトアセテートの濃度依存性を示す結果である。 図４は、アセトアセテートによるVGLUT活性阻害がクロライドイオン濃度を増やす事により解消することを示した結果である。黒丸「●」は、アセトアセテートを添加しなかった結果（コントロール）である。黒三角「▲」は、０．２ｍＭアセトアセテートを添加した結果である。白丸「○」は、１ｍＭアセトアセテートを添加した結果である。白三角「△」は、５ｍＭアセトアセテートを添加した結果である。 図５は、高い阻害活性を示した候補化合物の構造と、50%阻害に必要な濃度(IC50)を示す図である。 図６Ａ（ＶＭＡＴ）は、モノアミントランスポーターに対する１ｍＭ　アセトアセテートの阻害活性を示す結果である。図６Ｂ（ＶＩＡＡＴ）は、ＧＡＢトランスポーターに対する１ｍＭ　アセトアセテートの阻害活性を示す結果である。いずれの実験も１０ｍＭのクロライドイオン存在下で行った。 図７Ａは、アスパラギン酸トランスポーター（シアリン）によるアスパラギン酸輸送活性に対する、アセトアセテートの阻害活性を示す結果である。図７Ｂは、ヌクレオチドトランスポーター（ＶＮＵＴ）によるＡＴＰ輸送活性に対する、アセトアセテートの阻害活性を示す結果である。 図８は、海馬ニューロンからのＫＣｌによって誘起されるグルタミン酸のエキソサイトーシスに対するアセトアセテートの阻害効果を試験した結果である。 図９Ａは、ラット脳への４ＡＰおよびアセトアセテートの投与法および循環液採取法を模式的に示した図である。 図９Ｂの上図は、４ＡＰ投与のタイミング（下向きの矢印）、および、サンプリングのタイミング（下向きの矢印）を模式的に示した図である。図９Ｂの下図は、各タイミングでサンプリングされた循環中のグルタミン酸（上部グラフの▼）およびドーパミン（下部グラフの▼）の測定結果である。 図９Ｃは、種々のアセトアセテート濃度での、グルタミン酸（●）およびドーパミン（○）の分泌量を示す。１０ｍＭのアセトアセテート投与後の洗浄により、グルタミン酸分泌が回復した（「洗浄」の矢印）。 図９Ｄは、４ＡＰによって誘起された発作に対する、アセトアセテートの影響を示した結果である（ｎ＝４、*p<0.1、**p<0.01、***p<0.001）。 FIG. 1 (B) is a graph showing glutamate transport activity in proteoliposomes in which only the glutamate transporter is reconstituted without using F-ATPase. “+ Valinomycin” indicates transport activity in the presence of valinomycin, and “−valinomycin” indicates transport activity in the absence of valinomycin.
FIG. 2 shows the results of testing the glutamate transport inhibitory activity of various candidate compounds in the presence of 5 mM chloride (5 mM Cl − ) and in the presence of 20 mM chloride (20 mM Cl − ). FIG. 3 shows the results showing the concentration dependence of acetoacetate in the inhibition of glutamate transport activity by VGLUT. FIG. 4 shows the results showing that inhibition of VGLUT activity by acetoacetate is eliminated by increasing the chloride ion concentration. The black circle “●” is the result of not adding acetoacetate (control). The black triangle “▲” is the result of adding 0.2 mM acetoacetate. The white circle “◯” is the result of adding 1 mM acetoacetate. The white triangle “Δ” is the result of adding 5 mM acetoacetate. FIG. 5 is a diagram showing the structure of a candidate compound showing high inhibitory activity and the concentration required for 50% inhibition (IC 50 ). FIG. 6A (VMAT) shows the results showing the inhibitory activity of 1 mM acetoacetate on the monoamine transporter. FIG. 6B (VIAAT) shows the results showing the inhibitory activity of 1 mM acetoacetate on the GAB transporter. All experiments were performed in the presence of 10 mM chloride ions. FIG. 7A shows the results showing the inhibitory activity of acetoacetate on the aspartate transport activity by aspartate transporter (sialin). FIG. 7B shows the results showing the inhibitory activity of acetoacetate on the ATP transport activity by the nucleotide transporter (VNUT). FIG. 8 shows the results of examining the inhibitory effect of acetoacetate on glutamate exocytosis induced by KCl from hippocampal neurons. FIG. 9A is a diagram schematically showing a method of administering 4AP and acetoacetate to a rat brain and a method of collecting a circulating fluid. The upper diagram of FIG. 9B is a diagram schematically showing the timing of 4AP administration (downward arrow) and the timing of sampling (downward arrow). The lower diagram of FIG. 9B shows the measurement results of circulating glutamic acid (▼ in the upper graph) and dopamine (▼ in the lower graph) sampled at each timing. FIG. 9C shows the amount of glutamate (●) and dopamine (◯) secreted at various acetoacetate concentrations. Glutamic acid secretion was recovered by washing after administration of 10 mM acetoacetate (arrow of “washing”). FIG. 9D shows the effect of acetoacetate on the seizures induced by 4AP (n = 4, * p <0.1, ** p <0.01, *** p <0.001).
　配列番号１は、グルタミン酸トランスポーター（ＶＧＬＵＴ１）の核酸配列である。 SEQ ID NO: 1 is the nucleic acid sequence of glutamate transporter (VGLUT1).
　配列番号２は、グルタミン酸トランスポーター（ＶＧＬＵＴ１）のアミノ酸配列である。 SEQ ID NO: 2 is the amino acid sequence of glutamic acid transporter (VGLUT1).
　配列番号３は、アスパラギン酸トランスポーター（シアリン）の核酸配列である。 SEQ ID NO: 3 is the nucleic acid sequence of aspartate transporter (sialin).
　配列番号４は、アスパラギン酸トランスポーター（シアリン）のアミノ酸配列である。 SEQ ID NO: 4 is the amino acid sequence of aspartic acid transporter (sialin).
　配列番号５は、ヌクレオチドトランスポーター（ＶＮＵＴ）の核酸配列である。 SEQ ID NO: 5 is the nucleotide transporter (VNUT) nucleic acid sequence.
　配列番号６は、ヌクレオチドトランスポーター（ＶＮＵＴ）のアミノ酸配列である。 SEQ ID NO: 6 is the amino acid sequence of the nucleotide transporter (VNUT).
　配列番号７は、グルタミン酸トランスポーター（ＶＧＬＵＴ２）の核酸配列である。 SEQ ID NO: 7 is the nucleic acid sequence of glutamate transporter (VGLUT2).
　配列番号８は、グルタミン酸トランスポーター（ＶＧＬＵＴ２）のアミノ酸配列である。 SEQ ID NO: 8 is the amino acid sequence of glutamic acid transporter (VGLUT2).
　配列番号９は、グルタミン酸トランスポーター（ＶＧＬＵＴ３）の核酸配列である。 SEQ ID NO: 9 is the nucleic acid sequence of glutamate transporter (VGLUT3).
　配列番号１０は、グルタミン酸トランスポーター（ＶＧＬＵＴ３）のアミノ酸配列である。 SEQ ID NO: 10 is the amino acid sequence of glutamic acid transporter (VGLUT3).
　配列番号１１は、グルタミン酸トランスポーター（ＶＧＬＵＴ２）をＰＣＲクローニングするためのセンスプライマーである。 SEQ ID NO: 11 is a sense primer for PCR cloning of glutamate transporter (VGLUT2).
　配列番号１２は、グルタミン酸トランスポーター（ＶＧＬＵＴ２）をＰＣＲクローニングするためのアンチセンスプライマーである。 SEQ ID NO: 12 is an antisense primer for PCR cloning of glutamate transporter (VGLUT2).
　配列番号１３は、ヒトシアリン遺伝子クローニングに使用した正方向プライマーの配列である。 SEQ ID NO: 13 is the sequence of the forward primer used for human sialin gene cloning.
　配列番号１４は、ヒトシアリン遺伝子クローニングに使用した逆方向プライマーの配列である。 SEQ ID NO: 14 is the reverse primer sequence used for human sialin gene cloning.
　配列番号１５は、ヒトシアリン遺伝子クローニングに使用した正方向プライマーの配列である。 SEQ ID NO: 15 is the sequence of the forward primer used for human sialin gene cloning.
　配列番号１６は、ヒトシアリン遺伝子クローニングに使用した逆方向プライマーの配列である。 SEQ ID NO: 16 is the reverse primer sequence used for human sialin gene cloning.
　配列番号１７は、マウスH183R 1st PCRセンスプライマーの配列である。 SEQ ID NO: 17 is the sequence of mouse H183R 1 st PCR sense primer.
　配列番号１８は、マウスH183R 1st PCRアンチセンスプライマーの配列である。 SEQ ID NO: 18 is the sequence of mouse H183R 1 st PCR antisense primer.
　配列番号１９は、マウスH183R 2nd PCRセンスプライマーの配列である。 SEQ ID NO: 19 is the sequence of mouse H183R 2 nd PCR sense primer.
　配列番号２０は、ヒトＶＮＵＴ遺伝子のクローニングに使用したセンスプライマーの配列である。 SEQ ID NO: 20 is the sequence of the sense primer used for cloning of the human VNUT gene.
　配列番号２１は、ヒトＶＮＵＴ遺伝子のクローニングに使用したアンチセンスプライマーの配列である。 SEQ ID NO: 21 is the sequence of the antisense primer used for cloning of the human VNUT gene.
　以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。 Hereinafter, the present invention will be described. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.
　以下に本明細書において特に使用される用語の定義を列挙する。 The following lists the definitions of terms that are particularly used in this specification.
　本明細書において使用する場合、用語「興奮性神経伝達物質」とは、シナプス間隙に放出された場合に興奮性作用を示す神経伝達物質をいう。興奮性神経伝達物質としては、例えば、グルタミン酸、アスパラギン酸、ＡＴＰが挙げられるが、これに限定されない。 As used herein, the term “excitatory neurotransmitter” refers to a neurotransmitter that exhibits an excitatory action when released into the synaptic cleft. Examples of excitatory neurotransmitters include, but are not limited to, glutamic acid, aspartic acid, and ATP.
　本明細書において使用する場合、用語「小胞型神経伝達物質トランスポーター」とは、ニューロンのシナプス小胞に存在し、シナプス小胞内に興奮性神経伝達物質を能動輸送するトランスポーターをいう。 As used herein, the term “vesicle-type neurotransmitter transporter” refers to a transporter that is present in a synaptic vesicle of a neuron and actively transports an excitatory neurotransmitter into the synaptic vesicle.
　本明細書において使用する場合、用語「生理条件のクロライド濃度」とは、哺乳動物細胞（例えば、ニューロン）の細胞質中のクロライド濃度をいう。生理条件のクロライド濃度は、５～２０ｍＭ、例えば、１０ｍＭである。 As used herein, the term “physiological chloride concentration” refers to the chloride concentration in the cytoplasm of mammalian cells (eg, neurons). The chloride concentration under physiological conditions is 5-20 mM, for example 10 mM.
　本明細書において使用する場合、用語「アリール」とは、「Ａｒ」と互換可能に用いられ、単環状または双環状芳香族残基あり、１種またはそれ以上のヘテロ原子を任意に含む化合物をいう。Ａｒとしては、代表的には、フェニル、ナフチル、キノリル、ピリジル、ピリミジル、ベンゾチアゾイル、ベンズイミダゾイルなどが挙げられるが、これに限定されない。 As used herein, the term “aryl” is used interchangeably with “Ar” and refers to a compound that is a monocyclic or bicyclic aromatic residue, optionally containing one or more heteroatoms. Say. Representative examples of Ar include, but are not limited to, phenyl, naphthyl, quinolyl, pyridyl, pyrimidyl, benzothiazoyl, benzimidazolyl, and the like.
　本明細書において使用する場合、用語「アルキルアリール」とは、「（ＣＨ２）ｍ－Ａｒ」と互換可能に用いられ、ここでｍは１～１０であり、そしてＡｒ上記に定義されるとおりである。 As used herein, the term “alkylaryl” is used interchangeably with “(CH 2 ) m —Ar”, where m is 1 to 10 and Ar is as defined above. It is.
　本明細書において使用する場合、用語「過剰な神経の興奮に関連する疾患および／または状態」とは、神経が過剰に興奮することに起因する疾患および／または異常な状態をいう。過剰な神経の興奮に関連する疾患および／または状態としては、例えば、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症が挙げられるが、これに限定されない。 As used herein, the term “disease and / or condition associated with excessive neural excitement” refers to a disease and / or abnormal condition resulting from excessive excitement of the nerve. Diseases and / or conditions associated with excessive neural excitement include, for example, epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis However, it is not limited to this.
　本明細書において使用する場合、用語「神経が抑制されることに起因する疾患および／または状態」としては、例えば、ボケ、知覚障害（例えば味覚障害が挙げられるが、これに限定されない）が挙げられるが、これらに限定されない。 As used herein, the term “disease and / or condition resulting from inhibition of a nerve” includes, for example, blurring, sensory impairment (eg, but not limited to taste disorders). However, it is not limited to these.
　本明細書において使用する場合、用語「グルタミン酸トランスポーター」とは、小胞型神経伝達物質トランスポーターの一種でグルタミン酸を小胞内に輸送するトランスポーターまたはその改変体をいう。グルタミン酸トランスポーターは、代表的には：
（ａ）配列番号１、７または９に記載の核酸配列を含む核酸の相補鎖とストリンジェントな条件下でハイブリダイズする核酸であって、グルタミン酸輸送活性を有するポリペプチドをコードする核酸；
（ｂ）配列番号１、７または９に記載の核酸配列と少なくとも８０％相同な配列を含む核酸であって、グルタミン酸輸送活性を有するポリペプチドをコードする核酸；
（ｃ）配列番号２、８または１０に記載されるアミノ酸配列を有するポリペプチドをコードする核酸；および
（ｄ）配列番号２、８または１０に記載されるアミノ酸配列において１または数個の変異、置換、挿入または欠失を含むアミノ酸配列を有し、かつグルタミン酸輸送活性を有するポリペプチドをコードする核酸；
からなる群から選択される、核酸によってコードされるタンパク質である。 As used herein, the term “glutamate transporter” refers to a transporter that is a type of vesicular neurotransmitter transporter and transports glutamate into the vesicle or a variant thereof. Glutamate transporters are typically:
(A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 1, 7, or 9 and that encodes a polypeptide having glutamate transport activity;
(B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 1, 7, or 9 and encoding a polypeptide having glutamate transport activity;
(C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, 8, or 10; and (d) one or several mutations in the amino acid sequence set forth in SEQ ID NO: 2, 8, or 10. A nucleic acid encoding a polypeptide having an amino acid sequence containing a substitution, insertion or deletion and having glutamate transport activity;
A protein encoded by a nucleic acid selected from the group consisting of:
　本明細書において使用する場合、用語「アスパラギン酸トランスポーター」とは、小胞型神経伝達物質トランスポーターの一種でアスパラギン酸を小胞内に輸送するトランスポーターまたはその改変体をいう。アスパラギン酸トランスポーターは、代表的には：
（ａ）配列番号３に記載の核酸配列を含む核酸の相補鎖とストリンジェントな条件下でハイブリダイズする核酸であって、アスパラギン酸輸送活性を有するポリペプチドをコードする核酸；
（ｂ）配列番号３に記載の核酸配列と少なくとも８０％相同な配列を含む核酸であって、アスパラギン酸輸送活性を有するポリペプチドをコードする核酸；
（ｃ）配列番号４に記載されるアミノ酸配列を有するポリペプチドをコードする核酸；および
（ｄ）配列番号４に記載されるアミノ酸配列において１または数個の変異、置換、挿入または欠失を含むアミノ酸配列を有し、かつアスパラギン酸輸送活性を有するポリペプチドをコードする核酸；
からなる群から選択される、核酸によってコードされるタンパク質である。 As used herein, the term “aspartic acid transporter” refers to a transporter that transports aspartic acid into a vesicle or a variant thereof, which is a type of vesicular neurotransmitter transporter. Aspartate transporters are typically:
(A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 3, and that encodes a polypeptide having aspartate transport activity;
(B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 3 and encoding a polypeptide having aspartate transport activity;
(C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4; and (d) comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 4 A nucleic acid encoding a polypeptide having an amino acid sequence and having aspartic acid transport activity;
A protein encoded by a nucleic acid selected from the group consisting of:
　本明細書において使用する場合、用語「ヌクレオチドトランスポーター」とは、小胞型神経伝達物質トランスポーターの一種でヌクレオチド（例えば、ＡＴＰ、ＡＤＰおよびＧＴＰ）を小胞内に輸送するトランスポーターまたはその改変体をいう。ヌクレオチドトランスポーターは、代表的には：
（ａ）配列番号５に記載の核酸配列を含む核酸の相補鎖とストリンジェントな条件下でハイブリダイズする核酸であって、ＡＴＰ輸送活性を有するポリペプチドをコードする核酸；
（ｂ）配列番号５に記載の核酸配列と少なくとも８０％相同な配列を含む核酸であって、ＡＴＰ輸送活性を有するポリペプチドをコードする核酸；
（ｃ）配列番号６に記載されるアミノ酸配列を有するポリペプチドをコードする核酸；および
（ｄ）配列番号６に記載されるアミノ酸配列において１または数個の変異、置換、挿入または欠失を含むアミノ酸配列を有し、かつＡＴＰ輸送活性を有するポリペプチドをコードする核酸；
からなる群から選択される、核酸によってコードされるタンパク質である。 As used herein, the term “nucleotide transporter” is a type of vesicular neurotransmitter transporter that transports nucleotides (eg, ATP, ADP and GTP) into vesicles or modifications thereof. Refers to the body. Nucleotide transporters are typically:
(A) a nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO: 5, and that encodes a polypeptide having ATP transport activity;
(B) a nucleic acid comprising a sequence at least 80% homologous to the nucleic acid sequence set forth in SEQ ID NO: 5 and encoding a polypeptide having ATP transport activity;
(C) a nucleic acid encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6; and (d) comprising one or several mutations, substitutions, insertions or deletions in the amino acid sequence set forth in SEQ ID NO: 6 A nucleic acid encoding a polypeptide having an amino acid sequence and having ATP transport activity;
A protein encoded by a nucleic acid selected from the group consisting of:
　本明細書において使用される用語「トランスポーター」とは、脂質二重膜を透過できない物質（例えば、グルタミン酸、アスパラギン酸、および、ヌクレオチド）を、脂質二重膜を越えて輸送する物質をいう。典型的には、トランスポーターは、脂質二重膜中に存在する膜タンパク質である。タンパク質であるトランスポーターは、本明細書において「輸送タンパク質」と互換可能に使用される。 As used herein, the term “transporter” refers to a substance that transports a substance (for example, glutamic acid, aspartic acid, and nucleotide) that cannot permeate the lipid bilayer across the lipid bilayer. Typically, the transporter is a membrane protein present in the lipid bilayer. A transporter that is a protein is used herein interchangeably with “transport protein”.
　本明細書において使用される用語「人工膜」とは、脂質を原料として人工的に調製された膜であり、好ましくは、脂質二重膜であるがこれに限定されない。「人工膜」としては、例えば、リポソームが挙げられるが、これに限定されない。 The term “artificial membrane” used in the present specification is a membrane artificially prepared from lipid as a raw material, and is preferably a lipid bilayer membrane, but is not limited thereto. Examples of the “artificial membrane” include, but are not limited to, liposomes.
　本明細書において使用される用語「トランスポーターの活性調節剤」とは、トランスポーターの輸送活性に影響を与える物質をいう。「トランスポーターの活性調節剤」は、輸送活性を促進する物質であっても、阻害する物質であってもよい。 As used herein, the term “transporter activity regulator” refers to a substance that affects the transport activity of a transporter. The “transporter activity regulator” may be a substance that promotes or inhibits transport activity.
　本明細書において使用される用語「興奮性神経伝達阻害剤」とは、興奮性神経伝達物質による興奮性神経伝達を阻害する物質をいう。この阻害作用は、限定されることはないが、代表的には、小胞型神経伝達物質トランスポーターの阻害によってもたらされる。 As used herein, the term “excitatory neurotransmission inhibitor” refers to a substance that inhibits excitatory neurotransmission by an excitatory neurotransmitter. This inhibitory action is typically brought about by, but not limited to, inhibition of the vesicular neurotransmitter transporter.
　本明細書において使用される用語「輸送活性」とは、脂質二重膜を透過できない物質（例えば、興奮性神経伝達物質）を、脂質二重膜を越えて輸送する活性をいう。 As used herein, the term “transport activity” refers to an activity of transporting a substance that cannot permeate the lipid bilayer (for example, an excitatory neurotransmitter) across the lipid bilayer.
　本明細書において使用する場合、用語「イオノフォア」とは、脂質二重膜に対する特定のイオンの透過性を増加させる物質をいう。イオノフォアは、好ましくは、バリノマイシンである。 As used herein, the term “ionophore” refers to a substance that increases the permeability of specific ions to a lipid bilayer. The ionophore is preferably valinomycin.
　本明細書において使用される場合、「キット」とは、複数の容器、および製造業者の指示書を含み、そして各々の容器が、本発明の核酸および／またはタンパク質を含む製品をいう。必要に応じて、本発明のキットは、（ａ）リポソームのような人工膜、あるいは、人工膜を調製するための脂質、（ｂ）興奮性神経伝達物質、および、（ｃ）イオノフォアを含む。 As used herein, “kit” refers to a product comprising a plurality of containers and manufacturer's instructions, and each container comprising a nucleic acid and / or protein of the present invention. Optionally, the kit of the present invention comprises (a) an artificial membrane such as a liposome, or a lipid for preparing an artificial membrane, (b) an excitatory neurotransmitter, and (c) an ionophore.
　「ポリヌクレオチド」、「核酸」または「核酸分子」は、一本鎖形態、二本鎖形態、または他の形態である、リン酸エステルポリマー形態のリボヌクレオチド（アデノシン、グアノシン、ウリジン、もしくはシチジン；「ＲＮＡ分子」）またはデオキシリボヌクレオチド（デオキシアデノシン、デオキシグアノシン、デオキシチミジン、もしくはデオキシシチジン（ＤＮＡ分子」）、またはそれらの任意のホスホエステルアナログ（例えば、ホスホロチオエートおよびチオエステル）を指し得る。 A “polynucleotide”, “nucleic acid” or “nucleic acid molecule” is a ribonucleotide (adenosine, guanosine, uridine, or cytidine in the form of a phosphate ester polymer that is in single-stranded, double-stranded, or other form; “RNA molecule”) or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine (DNA molecule)), or any phosphoester analog thereof (eg, phosphorothioate and thioester).
　「ポリヌクレオチド配列」、「核酸配列」または「ヌクレオチド配列」は、核酸（例えば、ＤＮＡまたはＲＮＡ）中の一連のヌクレオチド塩基（「ヌクレオシド」とも呼ばれる）であり、２つの以上のヌクレオチドの任意の鎖またはその相補鎖を意味する。本発明の好ましい核酸は、配列番号１に示される核酸、ならびにその相補鎖、改変体およびフラグメントを包含する。 A “polynucleotide sequence”, “nucleic acid sequence” or “nucleotide sequence” is a series of nucleotide bases (also referred to as “nucleosides”) in a nucleic acid (eg, DNA or RNA) and any chain of two or more nucleotides. Or its complementary strand. Preferred nucleic acids of the present invention include the nucleic acid set forth in SEQ ID NO: 1 and its complementary strands, variants and fragments.
　「相補鎖」とは、ある核酸配列に対して塩基対を形成し得るようなヌクレオチドの鎖を意味する。例えば、二本鎖ＤＮＡの各々の鎖は互いに相補的な塩基配列を有し、一方の鎖から見て他方の鎖は相補鎖である。 “Complementary strand” means a strand of nucleotides that can form base pairs with a nucleic acid sequence. For example, each strand of double-stranded DNA has a complementary base sequence, and the other strand is a complementary strand when viewed from one strand.
　「コード配列」または発現生成物（例えば、ＲＮＡ、ポリペプチド、タンパク質、もしくは酵素）を「コードする」配列は、発現された場合にその生成物の生成をもたらすヌクレオチド配列である。 A “coding sequence” or a sequence that “encodes” an expression product (eg, RNA, polypeptide, protein, or enzyme) is a nucleotide sequence that, when expressed, results in the production of that product.
　「タンパク質」、「ペプチド」または「ポリペプチド」は、２つ以上のアミノ酸の連続列を含む。本発明の好ましいペプチドは、配列番号２に示されるペプチド、ならびにその改変体およびフラグメントを包含する。 “Protein”, “peptide” or “polypeptide” includes a continuous string of two or more amino acids. Preferred peptides of the present invention include the peptide shown in SEQ ID NO: 2, as well as variants and fragments thereof.
　「タンパク質配列」、「ペプチド配列」、または「ポリペプチド配列」または「アミノ酸配列」は、タンパク質、ペプチド、またはポリペプチド中にある一連の２つ以上のアミノ酸を指す。 “Protein sequence”, “peptide sequence”, or “polypeptide sequence” or “amino acid sequence” refers to a series of two or more amino acids in a protein, peptide, or polypeptide.
　本明細書において遺伝子（例えば、核酸配列、アミノ酸配列など）の「相同性」とは、２以上の遺伝子配列の、互いに対する同一性の程度をいう。また、本明細書において配列（核酸配列、アミノ酸配列など）の同一性とは、２以上の対比可能な配列の、互いに対する同一の配列（個々の核酸、アミノ酸など）の程度をいう。従って、ある２つの遺伝子の相同性が高いほど、それらの配列の同一性または類似性は高い。２種類の遺伝子が相同性を有するか否かは、配列の直接の比較、または核酸の場合ストリンジェントな条件下でのハイブリダイゼーション法によって調べられ得る。２つの遺伝子配列を直接比較する場合、その遺伝子配列間でＤＮＡ配列が、代表的には少なくとも５０％同一である場合、好ましくは少なくとも７０％同一である場合、より好ましくは少なくとも８０％、９０％、９５％、９６％、９７％、９８％または９９％同一である場合、それらの遺伝子は相同性を有する。本明細書において、遺伝子（例えば、核酸配列、アミノ酸配列など）の「類似性」とは、上記相同性において、保存的置換をポジティブ（同一）とみなした場合の、２以上の遺伝子配列の、互いに対する同一性の程度をいう。従って、保存的置換がある場合は、その保存的置換の存在に応じて相同性と類似性とは異なる。また、保存的置換がない場合は、相同性と類似性とは同じ数値を示す。 As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In this specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.
　本明細書では、アミノ酸配列および塩基配列の類似性、同一性および相同性の比較は、配列分析用ツールであるＦＡＳＴＡを用い、デフォルトパラメータを用いて算出される。 In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using FASTA, which is a sequence analysis tool, and using default parameters.
　本明細書において「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド（長さがｎ）に対して、１～ｎ－１までの配列長さを有するポリペプチドまたはポリヌクレオチドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例えば、その長さの下限としては、ポリペプチドの場合、３、４、５、６、７、８、９、１０、１５，２０、２５、３０、４０、５０およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙していない整数で表される長さ（例えば、１１など）もまた、下限として適切であり得る。また、ポリヌクレオチドの場合、５、６、７、８、９、１０、１５，２０、２５、３０、４０、５０、７５、１００およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙していない整数で表される長さ（例えば、１１など）もまた、下限として適切であり得る。本明細書において、ポリペプチドおよびポリヌクレオチドの長さは、上述のようにそれぞれアミノ酸または核酸の個数で表すことができるが、上述の個数は絶対的なものではなく、同じ機能を有する限り、上限または下限としての上述の個数は、その個数の上下数個（または例えば上下１０％）のものも含むことが意図される。そのような意図を表現するために、本明細書では、個数の前に「約」を付けて表現することがある。しかし、本明細書では、「約」のあるなしはその数値の解釈に影響を与えないことが理解されるべきである。本明細書において有用なフラグメントの長さは、そのフラグメントの基準となる全長タンパク質の機能のうち少なくとも１つの機能が保持されているかどうかによって決定され得る。 As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above. However, the above numbers are not absolute and are limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include a number above and below that number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.
　本明細書において、「ストリンジェントな条件でハイブリダイズするポリヌクレオチド」とは、当該分野で慣用される周知の条件をいう。本発明のポリヌクレオチド中から選択されたポリヌクレオチドをプローブとして、コロニー・ハイブリダイゼーション法、プラーク・ハイブリダイゼーション法あるいはサザンブロットハイブリダイゼーション法等を用いることにより、そのようなポリヌクレオチドを得ることができる。具体的には、コロニーあるいはプラーク由来のＤＮＡを固定化したフィルターを用いて、０．７～１．０ＭのＮａＣｌ存在下、６５℃でハイブリダイゼーションを行った後、０．１～２倍濃度のＳＳＣ（ｓａｌｉｎｅ－ｓｏｄｉｕｍ　ｃｉｔｒａｔｅ）溶液（１倍濃度のＳＳＣ溶液の組成は、１５０ｍＭ　塩化ナトリウム、１５ｍＭ　クエン酸ナトリウムである）を用い、６５℃条件下でフィルターを洗浄することにより同定できるポリヌクレオチドを意味する。ハイブリダイゼーションは、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　２ｎｄ　ｅｄ．，Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｓｕｐｐｌｅｍｅｎｔ　１～３８、ＤＮＡ　Ｃｌｏｎｉｎｇ　１：Ｃｏｒｅ　Ｔｅｃｈｎｉｑｕｅｓ，Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，Ｓｅｃｏｎｄ　Ｅｄｉｔｉｏｎ，Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ（１９９５）等の実験書に記載されている方法に準じて行うことができる。ここで、ストリンジェントな条件下でハイブリダイズする配列からは、好ましくは、Ａ配列のみまたはＴ配列のみを含む配列が除外される。「ハイブリダイズ可能なポリヌクレオチド」とは、上記ハイブリダイズ条件下で別のポリヌクレオチドにハイブリダイズすることができるポリヌクレオチドをいう。ハイブリダイズ可能なポリヌクレオチドとして具体的には、本発明で具体的に示されるアミノ酸配列を有するポリペプチドをコードするＤＮＡの塩基配列と少なくとも６０％以上の相同性を有するポリヌクレオチド、好ましくは８０％以上の相同性を有するポリヌクレオチド、さらに好ましくは９５％以上の相同性を有するポリヌクレオチドを挙げることができる。 As used herein, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C using an SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Technical Approach, Second Edition, Oxford, etc. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.
　本明細書において「高度にストリンジェントな条件」は、核酸配列において高度の相補性を有するＤＮＡ鎖のハイブリダイゼーションを可能にし、そしてミスマッチを有意に有するＤＮＡのハイブリダイゼーションを除外するように設計された条件をいう。ハイブリダイゼーションのストリンジェンシーは、主に、温度、イオン強度、およびホルムアミドのような変性剤の条件によって決定される。このようなハイブリダイゼーションおよび洗浄に関する「高度にストリンジェントな条件」の例は、０．００１５Ｍ塩化ナトリウム、０．００１５Ｍ　クエン酸ナトリウム、６５～６８℃、または０．０１５Ｍ　塩化ナトリウム、０．００１５Ｍクエン酸ナトリウム、および５０％　ホルムアミド、４２℃である。このような高度にストリンジェントな条件については、Ｓａｍｂｒｏｏｋｅｔ　ａｌ．，Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ、第２版、Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（ＣｏｌｄＳｐｒｉｎｇ　Ｈａｒｂｏｒ，Ｎ，Ｙ．１９８９）；およびＡｎｄｅｒｓｏｎ　ｅｔ　ａｌ．、Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ：ａ　Ｐｒａｃｔｉｃａｌ　ａｐｐｒｏａｃｈ、ＩＶ、ＩＲＬ　Ｐｒｅｓｓ　Ｌｉｍｉｔｅｄ（Ｏｘｆｏｒｄ，Ｅｎｇｌａｎｄ）．Ｌｉｍｉｔｅｄ，Ｏｘｆｏｒｄ，Ｅｎｇｌａｎｄを参照のこと。必要により、よりストリンジェントな条件（例えば、より高い温度、より低いイオン強度、より高いホルムアミド、または他の変性剤）を、使用してもよい。他の薬剤が、非特異的なハイブリダイゼーションおよび／またはバックグラウンドのハイブリダイゼーションを減少する目的で、ハイブリダイゼーション緩衝液および洗浄緩衝液に含まれ得る。そのような他の薬剤の例としては、０．１％ウシ血清アルブミン、０．１％ポリビニルピロリドン、０．１％ピロリン酸ナトリウム、０．１％ドデシル硫酸ナトリウム（ＮａＤｏｄＳＯ４またはＳＤＳ）、Ｆｉｃｏｌｌ、Ｄｅｎｈａｒｄｔ溶液、超音波処理されたサケ精子ＤＮＡ（または別の非相補的ＤＮＡ）および硫酸デキストランであるが、他の適切な薬剤もまた、使用され得る。これらの添加物の濃度および型は、ハイブリダイゼーション条件のストリンジェンシーに実質的に影響を与えることなく変更され得る。ハイブリダイゼーション実験は、通常、ｐＨ６．８～７．４で実施されるが；代表的なイオン強度条件において、ハイブリダイゼーションの速度は、ほとんどｐＨ独立である。Ａｎｄｅｒｓｏｎ　ｅｔ　ａｌ．、ＮｕｃｌｅｉｃＡｃｉｄ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ：ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ、第４章、ＩＲＬ　Ｐｒｅｓｓ　Ｌｉｍｉｔｅｄ（Ｏｘｆｏｒｄ，Ｅｎｇｌａｎｄ）を参照のこと。 As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO 4 or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).
　ＤＮＡ二重鎖の安定性に影響を与える因子としては、塩基の組成、長さおよび塩基対不一致の程度が挙げられる。ハイブリダイゼーション条件は、当業者によって調整され得、これらの変数を適用させ、そして異なる配列関連性のＤＮＡがハイブリッドを形成するのを可能にする。完全に一致したＤＮＡ二重鎖の融解温度は、以下の式によって概算され得る。
Ｔｍ（℃）＝８１．５＋１６．６（ｌｏｇ［Ｎａ＋］）＋０．４１（％Ｇ＋Ｃ）－６００／Ｎ－０．７２（％ホルムアミド）
　ここで、Ｎは、形成される二重鎖の長さであり、［Ｎａ＋］は、ハイブリダイゼーション溶液または洗浄溶液中のナトリウムイオンのモル濃度であり、％Ｇ＋Ｃは、ハイブリッド中の（グアニン＋シトシン）塩基のパーセンテージである。不完全に一致したハイブリッドに関して、融解温度は、各１％不一致（ミスマッチ）に対して約１℃ずつ減少する。 Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
T m (° C.) = 81.5 + 16.6 (log [Na + ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na + ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + in the hybrid Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.
　本明細書において配列（アミノ酸または核酸など）の「同一性」、「相同性」および「類似性」のパーセンテージは、比較ウィンドウで最適な状態に整列された配列２つを比較することによって求められる。ここで、ポリヌクレオチド配列またはポリペプチド配列の比較ウィンドウ内の部分には、２つの配列の最適なアライメントについての基準配列（他の配列に付加が含まれていればギャップが生じることもあるが、ここでの基準配列は付加も欠失もないものとする）と比較したときに、付加または欠失（すなわちギャップ）が含まれる場合がある。同一の核酸塩基またはアミノ酸残基がどちらの配列にも認められる位置の数を求めることによって、マッチ位置の数を求め、マッチ位置の数を比較ウィンドウ内の総位置数で割り、得られた結果に１００を掛けて同一性のパーセンテージを算出する。検索において使用される場合、相同性については、従来技術において周知のさまざまな配列比較アルゴリズムおよびプログラムの中から、適当なものを用いて評価する。このようなアルゴリズムおよびプログラムとしては、ＴＢＬＡＳＴＮ、ＢＬＡＳＴＰ、ＦＡＳＴＡ、ＴＦＡＳＴＡおよびＣＬＵＳＴＡＬＷ（Ｐｅａｒｓｏｎ　ａｎｄ　Ｌｉｐｍａｎ，１９８８，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８５（８）：２４４４－２４４８、　Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９０，Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５（３）：４０３－４１０、Ｔｈｏｍｐｓｏｎ　ｅｔ　ａｌ．，１９９４，Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．２２（２）：４６７３－４６８０、Ｈｉｇｇｉｎｓ　ｅｔ　ａｌ．，１９９６，Ｍｅｔｈｏｄｓ　Ｅｎｚｙｍｏｌ．２６６：３８３－４０２、Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９０，Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５（３）：４０３－４１０、Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９３，Ｎａｔｕｒｅ　Ｇｅｎｅｔｉｃｓ　３：２６６－２７２）があげられるが、何らこれに限定されるものではない。特に好ましい実施形態では、従来技術において周知のＢａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ（ＢＬＡＳＴ）（たとえば、Ｋａｒｌｉｎ　ａｎｄ　Ａｌｔｓｃｈｕｌ，１９９０，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８７：２２６７－２２６８、Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９０，Ｊ．Ｍｏｌ．Ｂｉｏｌ．２１５：４０３－４１０、Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９３，Ｎａｔｕｒｅ　Ｇｅｎｅｔｉｃｓ　３：２６６－２７２、Ａｌｔｓｃｈｕｌ　ｅｔ　ａｌ．，１９９７，Ｎｕｃ．Ａｃｉｄｓ　Ｒｅｓ．２５：３３８９－３４０２を参照のこと）を用いてタンパク質および核酸配列の相同性を評価する。特に、５つの専用ＢＬＡＳＴプログラムを用いて以下の作業を実施することによって比較または検索が達成され得る。 As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, a portion of the polynucleotide or polypeptide sequence within the comparison window is a reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequence contains an addition, Reference sequences herein shall contain no additions or deletions), and may include additions or deletions (ie gaps). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res. 22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol. 266: 383-402. , Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410, Altschul. . T al, 1993, Nature Genetics 3: 266-272), but the like, is not intended to be limited to this. In a particularly preferred embodiment, Basic Local Alignment Search Tool (BLAST) (eg Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990, well known in the prior art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.
　（１）　ＢＬＡＳＴＰおよびＢＬＡＳＴ３でアミノ酸のクエリー配列をタンパク質配列データベースと比較；
　（２）　ＢＬＡＳＴＮでヌクレオチドのクエリー配列をヌクレオチド配列データベースと比較；
　（３）　ＢＬＡＳＴＸでヌクレオチドのクエリー配列（両方の鎖）を６つの読み枠で変換した概念的翻訳産物をタンパク質配列データベースと比較；
　（４）　ＴＢＬＡＳＴＮでタンパク質のクエリー配列を６つの読み枠（両方の鎖）すべてで変換したヌクレオチド配列データベースと比較；
　（５）　ＴＢＬＡＳＴＸでヌクレオチドのクエリ配列を６つの読み枠で変換したものを、６つの読み枠で変換したヌクレオチド配列データベースと比較。 (1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.
　ＢＬＡＳＴプログラムは、アミノ酸のクエリ配列または核酸のクエリ配列と、好ましくはタンパク質配列データベースまたは核酸配列データベースから得られた被検配列との間で、「ハイスコアセグメント対」と呼ばれる類似のセグメントを特定することによって相同配列を同定するものである。ハイスコアセグメント対は、多くのものが従来技術において周知のスコアリングマトリックスによって同定（すなわち整列化）されると好ましい。好ましくは、スコアリングマトリックスとしてＢＬＯＳＵＭ６２マトリックス（Ｇｏｎｎｅｔ　ｅｔ　ａｌ．，１９９２，Ｓｃｉｅｎｃｅ　２５６：１４４３－１４４５、Ｈｅｎｉｋｏｆｆ　ａｎｄ　Ｈｅｎｉｋｏｆｆ，１９９３，Ｐｒｏｔｅｉｎｓ　１７：４９－６１）を使用する。このマトリックスほど好ましいものではないが、ＰＡＭまたはＰＡＭ２５０マトリックスも使用できる（たとえば、Ｓｃｈｗａｒｔｚ　ａｎｄ　Ｄａｙｈｏｆｆ，ｅｄｓ．，１９７８，Ｍａｔｒｉｃｅｓ　ｆｏｒ　Ｄｅｔｅｃｔｉｎｇ　Ｄｉｓｔａｎｃｅ　Ｒｅｌａｔｉｏｎｓｈｉｐｓ：　Ａｔｌａｓ　ｏｆ　Ｐｒｏｔｅｉｎ　Ｓｅｑｕｅｎｃｅ　ａｎｄ　Ｓｔｒｕｃｔｕｒｅ，Ｗａｓｈｉｎｇｔｏｎ：　Ｎａｔｉｏｎａｌ　Ｂｉｏｍｅｄｉｃａｌ　Ｒｅｓｅａｒｃｈ　Ｆｏｕｎｄａｔｉｏｎを参照のこと）。ＢＬＡＳＴプログラムは、同定されたすべてのハイスコアセグメント対の統計的な有意性を評価し、好ましくはユーザー固有の相同率などのユーザーが独自に定める有意性の閾値レベルを満たすセグメントを選択する。統計的な有意性を求めるＫａｒｌｉｎの式を用いてハイスコアセグメント対の統計的な有意性を評価すると好ましい（Ｋａｒｌｉｎ　ａｎｄ　Ａｌｔｓｃｈｕｌ，１９９０，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８７：２２６７－２２６８参照のこと）。 The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, PAM or PAM250 matrix can also be used (for example, Schwartz and Dayhoff, eds., 1978, Matrix for Detection Distance Relationships: Atlas of Protein Sequential Sequencial Sequenc thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferable to evaluate the statistical significance of high-score segment pairs using Karlin's formula for calculating statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).
　本明細書において、ポリペプチドまたはポリヌクレオチドの「置換、付加または欠失」とは、もとのポリペプチドまたはポリヌクレオチドに対して、それぞれアミノ酸もしくはその代替物、またはヌクレオチドもしくはその代替物が、置き換わること、付け加わることまたは取り除かれることをいう。このような置換、付加または欠失の技術は、当該分野において周知であり、そのような技術の例としては、部位特異的変異誘発技術などが挙げられる。置換、付加または欠失は、１つ以上であれば任意の数でよく、そのような数は、その置換、付加または欠失を有する改変体において目的とする機能（例えば、ホルモン、サイトカインの情報伝達機能など）が保持される限り、多くすることができる。例えば、そのような数は、１または数個であり得、そして好ましくは、全体の長さの２０％以内、１０％以内、または１００個以下、５０個以下、２５個以下などであり得る。 As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such a number may be a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, the number can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.
　本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手法は、当該分野において周知であり慣用されるものであり、例えば、Ｓａｍｂｒｏｏｋ　Ｊ．ｅｔ　ａｌ．（１９８９）．Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒおよびその３ｒｄ　Ｅｄ．（２００１）；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９８７）．Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ　ａｎｄ　Ｗｉｌｅｙ－Ｉｎｔｅｒｓｃｉｅｎｃｅ；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９８９）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔ　ＥＳ　ａｎｄ　Ｗｉｌｅｙ－Ｉｎｔｅｒｓｃｉｅｎｃｅ；Ｉｎｎｉｓ，Ｍ．Ａ．（１９９０）．ＰＣＲ　Ｐｒｏｔｏｃｏｌｓ：Ａ　Ｇｕｉｄｅ　ｔｏ　Ｍｅｔｈｏｄｓ　ａｎｄ　Ａｐｐｌｉｃａｔｉｏｎｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９９２）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９９５）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ；Ｉｎｎｉｓ，Ｍ．Ａ．ｅｔ　ａｌ．（１９９５）．ＰＣＲ　Ｓｔｒａｔｅｇｉｅｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９９９）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｗｉｌｅｙ，ａｎｄ　ａｎｎｕａｌ　ｕｐｄａｔｅｓ；Ｓｎｉｎｓｋｙ，Ｊ．Ｊ．ｅｔ　ａｌ．（１９９９）．ＰＣＲ　Ａｐｐｌｉｃａｔｉｏｎｓ：Ｐｒｏｔｏｃｏｌｓ　ｆｏｒ　Ｆｕｎｃｔｉｏｎａｌ　Ｇｅｎｏｍｉｃｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ、別冊実験医学「遺伝子導入＆発現解析実験法」羊土社、１９９７などに記載されており、これらは本明細書において関連する部分（全部であり得る）が参考として援用される。 Molecular biological techniques, biochemical techniques, and microbiological techniques used in the present specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associate ES and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annular updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, separate volume of experimental medicine “Gene Transfer & Expression Analysis Experiment Method” Yodosha, 1997, etc., which are related parts (may be all) Is incorporated by reference.
　人工的に合成した遺伝子を作製するためのＤＮＡ合成技術および核酸化学については、例えば、Ｇａｉｔ，Ｍ．Ｊ．（１９８５）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｓｙｎｔｈｅｓｉｓ：Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，ＩＲＬＰｒｅｓｓ；Ｇａｉｔ，Ｍ．Ｊ．（１９９０）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｓｙｎｔｈｅｓｉｓ：Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，ＩＲＬ　Ｐｒｅｓｓ；Ｅｃｋｓｔｅｉｎ，Ｆ．（１９９１）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ　ａｎｄ　Ａｎａｌｏｇｕｅｓ：Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃ，ＩＲＬ　Ｐｒｅｓｓ；Ａｄａｍｓ，Ｒ．Ｌ．ｅｔ　ａｌ．（１９９２）．Ｔｈｅ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　ｏｆ　ｔｈｅ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，Ｃｈａｐｍａｎ＆Ｈａｌｌ；Ｓｈａｂａｒｏｖａ，Ｚ．ｅｔ　ａｌ．（１９９４）．Ａｄｖａｎｃｅｄ　Ｏｒｇａｎｉｃ　Ｃｈｅｍｉｓｔｒｙ　ｏｆ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，Ｗｅｉｎｈｅｉｍ；Ｂｌａｃｋｂｕｒｎ，Ｇ．Ｍ．ｅｔ　ａｌ．（１９９６）．Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　ｉｎ　Ｃｈｅｍｉｓｔｒｙ　ａｎｄ　Ｂｉｏｌｏｇｙ，Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ；Ｈｅｒｍａｎｓｏｎ，Ｇ．Ｔ．（Ｉ９９６）．Ｂｉｏｃｏｎｊｕｇａｔｅ　Ｔｅｃｈｎｉｑｕｅｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓなどに記載されており、これらは本明細書において関連する部分が参考として援用される。 For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. et al. J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Blackberry, G. Advanced Organic Chemistry of Nucleic Acids, Weinheim; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference for relevant portions.
　（治療、予防および／または予後における用途）
　本発明の化合物は、少なくとも２種類の小胞型神経伝達物質トランスポーターを調節（阻害または阻害の解除もしくは活性化）することが可能である。原理にとらわれることを望まないが、本発明の化合物による阻害メカニズムは、以下のとおりである：（ａ）小胞型神経伝達物質トランスポーター、なかでも特に興奮性神経伝達物質を輸送するトランスポーターは、その輸送活性にクロライドを必要とする；（ｂ）生理条件のクロライド濃度が存在する場合（通常、５～２０ｍＭ、代表的には、１０ｍＭ）、小胞型神経伝達物質トランスポーターは活性である；（ｃ）本発明の化合物を添加すると、小胞型神経伝達物質トランスポーターのクロライド感受性が変化し、その結果、輸送活性を示すためには、より高濃度のクロライドを要求する一方で、生理条件のクロライド濃度では、充分な輸送活性を示さない；（ｄ）そのため、生理条件のクロライド濃度下では、本発明の化合物は、濃度依存的に、小胞型神経伝達物質トランスポーターの輸送活性を調節（阻害または阻害の解除もしくは活性化、好ましくは、阻害）する。 (Use in treatment, prevention and / or prognosis)
The compounds of the present invention are capable of modulating (inhibiting or deblocking or activating) at least two vesicular neurotransmitter transporters. Although not wishing to be bound by principle, the mechanism of inhibition by the compounds of the present invention is as follows: (a) vesicular neurotransmitter transporters, especially transporters that transport excitatory neurotransmitters (B) vesicular neurotransmitter transporter is active when chloride concentrations at physiological conditions are present (usually 5-20 mM, typically 10 mM). (C) adding the compound of the present invention changes the chloride sensitivity of the vesicular neurotransmitter transporter, resulting in a higher concentration of chloride in order to exhibit transport activity, The condition chloride concentration does not show sufficient transport activity; (d) Therefore, under physiological condition chloride concentration, the compounds of the present invention Exist manner, vesicular neurotransmitter regulating the transporter transport activity (inhibition or inhibition of release or activation, preferably, inhibit).
　また、理論に拘束されることは望まないが、小胞型神経伝達物質トランスポーターの輸送活性を阻害すると、シナプス小胞内への興奮性神経伝達物質の輸送が阻害され、その結果、興奮性神経伝達物質によって生み出される電気的興奮が阻害されることとなる。そのため、本発明の薬学的組成物は、過剰な神経の興奮に関連する疾患および／または状態（例えば、癲癇）の治療、予防および／または予後に利用することが可能である。 Although not wishing to be bound by theory, inhibiting the transport activity of the vesicular neurotransmitter transporter inhibits the transport of excitatory neurotransmitters into synaptic vesicles, resulting in excitability. The electrical excitement produced by the neurotransmitter will be inhibited. As such, the pharmaceutical compositions of the present invention can be utilized for the treatment, prevention and / or prognosis of diseases and / or conditions (eg, epilepsy) associated with excessive neural excitation.
　また、小胞型神経伝達物質トランスポーターの輸送活性について、阻害の解除もしくは活性化をすると、シナプス小胞内への興奮性神経伝達物質の輸送について、阻害の解除もしくは活性化され、その結果、興奮性神経伝達物質によって生み出される電気的興奮が増強されることとなる。そのため、本発明の薬学的組成物は、神経の抑制に起因する疾患および／または状態の治療、予防および／または予後に利用することが可能である。本発明はまた、そのような活性を有する物質をスクリーニングする方法を提供する。 In addition, when the inhibition or activation of the transport activity of the vesicular neurotransmitter transporter is canceled or activated, the inhibition or activation of the excitatory neurotransmitter transport into the synaptic vesicle is released. The electrical excitement produced by the excitatory neurotransmitter will be enhanced. Therefore, the pharmaceutical composition of the present invention can be used for the treatment, prevention and / or prognosis of diseases and / or conditions resulting from nerve suppression. The present invention also provides a method of screening for a substance having such activity.
　癲癇のような過剰な神経の興奮に関連する疾患および／または状態には複数の興奮性神経伝達物質が関与すると予測され、その一方で、複数種類の薬剤を併用する弊害が予測されることから、単独の薬剤によって、複数の小胞型神経伝達物質トランスポーターを阻害する化合物が、過剰な神経の興奮に関連する疾患および／または状態の処置のために望まれていた。その一方で、小胞型神経伝達物質トランスポーターの構造、機能、および阻害剤についての知見が少ないため、そのような複数の小胞型神経伝達物質トランスポーターに対して阻害活性を有する物質の探索は非常に困難であると予測されていたが、本発明は、予想外にも、複数の小胞型神経伝達物質トランスポーターに対して阻害活性を有する化合物を提供するものである。 Diseases and / or conditions associated with excessive neural excitement, such as hemorrhoids, are expected to involve multiple excitatory neurotransmitters, while the adverse effects of combining multiple drugs are predicted Compounds that inhibit multiple vesicular neurotransmitter transporters with a single agent have been desired for the treatment of diseases and / or conditions associated with excessive neural excitation. On the other hand, because there is little knowledge about the structure, function, and inhibitors of vesicular neurotransmitter transporters, the search for substances having inhibitory activity against such vesicular neurotransmitter transporters The present invention unexpectedly provides compounds having inhibitory activity against multiple vesicular neurotransmitter transporters.
　その結果、複数の小胞型神経伝達物質トランスポーターに対して阻害活性を有する本発明の化合物は、過剰な神経の興奮に関連する疾患および／または状態の、治療、予防、予後の改善に有用である。過剰な神経の興奮に関連する疾患および／または状態としては、例えば、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症が挙げられるが、これに限定されない。 As a result, the compounds of the present invention having inhibitory activity against multiple vesicular neurotransmitter transporters are useful for the treatment, prevention, and improvement of prognosis of diseases and / or conditions associated with excessive neural excitation. It is. Diseases and / or conditions associated with excessive neural excitement include, for example, epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis However, it is not limited to this.
　これら疾患の中で、癲癇とは、過度のニューロンの放電による発作的な脳の機能障害を特徴とする慢性疾患で、通常、意識の変調を伴う疾患であり、発作は行動の要素的または複合的障害に限られる場合と、全身的痙攣に発展する場合とがある。発作の臨床所見は、全身痙攣や局所痙攣を含む行動の複雑異常から、意識障害の瞬間的発作まで様々であり、これらの臨床所見には種々の分類があり、発作の型を示す用語は記述的であり標準化されているが、完全に統一されたものものではない。しかしながら、複数の小胞型神経伝達物質トランスポーターに対して阻害活性を有する本発明の化合物は、癲癇の原因の一つである過剰な神経の興奮を抑制／阻害することから、全てのタイプの癲癇に有効であると考えられる。 Among these diseases, hemorrhoids is a chronic disease characterized by paroxysmal brain dysfunction due to excessive neuronal discharge, usually accompanied by modulation of consciousness. There are cases where it is limited to physical disorder and cases where it develops into generalized convulsions. The clinical findings of seizures range from complex behavioral abnormalities, including generalized and local convulsions, to instantaneous seizures of consciousness disturbances. These clinical findings have various classifications, and terms that describe the type of seizure are described. Is standardized and standardized, but not completely unified. However, the compound of the present invention having an inhibitory activity against a plurality of vesicular neurotransmitter transporters suppresses / inhibits excessive neural excitement, which is one of the causes of epilepsy. It is considered to be effective for drought.
　（本発明の化合物）
　興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害する本発明の化合物は、以下の式（Ｉ）： (Compound of the present invention)
The compounds of the present invention that inhibit at least two vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration have the following formula (I):
Figure JPOXMLDOC01-appb-C000005
 　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
　Ｒ１～Ｒ４の少なくとも１つは酸素原子を含み、
　Ｒ５は、Ｈ、直鎖アルキル、分岐アルキル、置換アルキル、アリール、アルキルアリールからなる群から選択される化合物またはその医薬として許容される塩である。
Figure JPOXMLDOC01-appb-C000005
 Wherein R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
Wherein R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O
At least one of R 1 to R 4 contains an oxygen atom;
R 5 is a compound selected from the group consisting of H, linear alkyl, branched alkyl, substituted alkyl, aryl, and alkylaryl, or a pharmaceutically acceptable salt thereof.
　一つの局面において、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択され、ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択される。 In one aspect, R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O, wherein R 3 and R 4 are each independently And H and OH and taken together are selected from the group consisting of O.
　別の局面において、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択され、ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択される。 In another aspect, R 1 and R 2 are each independently selected from the group consisting of H and together O, wherein R 3 and R 4 are each independently H , And together are selected from the group consisting of O.
　さらなる局面において、Ｒ３およびＲ４の少なくとも１つは酸素原子を含み、必要に応じて、Ｒ３およびＲ４は、一緒になって、Ｏである。 In a further aspect, at least one of R 3 and R 4 contains an oxygen atom, and optionally R 3 and R 4 together are O.
　本発明の化合物としては、例えば、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される化合物が挙げられるが、これに限定されない。好ましい化合物は、アセトアセテート、および、ピルビン酸からなる群から選択される化合物である。 Examples of the compound of the present invention include compounds selected from the group consisting of acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid, and lactic acid. However, it is not limited to this. Preferred compounds are those selected from the group consisting of acetoacetate and pyruvic acid.
　本明細書において「薬学的に受容可能なキャリア」は、医薬または動物薬のような農薬を製造するときに使用される物質であり、有効成分に有害な影響を与えないものをいう。そのような薬学的に受容可能なキャリアとしては、例えば、以下が挙げられるがそれらに限定されない：抗酸化剤、保存剤、着色料、風味料、および希釈剤、乳化剤、懸濁化剤、溶媒、フィラー、増量剤、緩衝剤、送達ビヒクル、賦形剤および／または薬学的アジュバント。 As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing an agrochemical such as a pharmaceutical or veterinary drug, and that does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or pharmaceutical adjuvants.
　本発明の処置方法において使用される薬剤の種類および量は、本発明の方法によって得られた情報（例えば、疾患に関する情報）を元に、使用目的、対象疾患（種類、重篤度など）、患者の年齢、体重、性別、既往歴、投与される被検体の部位の形態または種類などを考慮して、当業者が容易に決定することができる。本発明のモニタリング方法を被検体（または患者）に対して施す頻度もまた、使用目的、対象疾患（種類、重篤度など）、患者の年齢、体重、性別、既往歴、および治療経過などを考慮して、当業者が容易に決定することができる。疾患状態をモニタリングする頻度としては、例えば、毎日－数ヶ月に１回（例えば、１週間に１回－１ヶ月に１回）のモニタリングが挙げられる。１週間－１ヶ月に１回のモニタリングを、経過を見ながら施すことが好ましい。 The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (eg, once a week-once a month). It is preferable to perform monitoring once a week to once a month while monitoring the progress.
　必要に応じて、本発明の治療では、２種類以上の薬剤が使用してもよい。２種類以上の薬剤を使用する場合、類似の性質または由来の物質を使用してもよく、異なる性質または由来の薬剤を使用してもよい。このような２種類以上の薬剤を投与する方法のための疾患レベルに関する情報も、本発明の方法によって入手することができる。 If necessary, two or more kinds of drugs may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.
　本発明はまた、小胞型神経伝達物質トランスポーターの輸送活性について、阻害の解除もしくは活性化をする化合物をスクリーニングする方法を提供する。そのようなスクリーニング法は、本発明の興奮性神経伝達物質輸送の阻害剤存在下において、候補化合物の中から輸送活性を回復させる効力を有する化合物を選択することによって行われる。そのようなスクリーニング法は、例えば：
（１）興奮性神経伝達物質輸送の阻害剤存在下において、興奮性神経伝達物質輸送タンパク質の輸送活性を測定する工程；
（２）さらに、候補化合物を添加した場合の、輸送活性を測定する工程；および、
（３）上記（２）の活性が上記（１）の活性より高い場合に、候補化合物を、阻害の解除能もしくは活性化能を有する化合物として同定する工程；
を包含する。この方法において、好ましい、興奮性神経伝達物質輸送タンパク質は、ＶＧＬＵＴ２であるが、これに限定されない。興奮性神経伝達物質輸送の阻害剤は、好ましくは、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、または、乳酸であり、より好ましくは、アセトアセテートであるが、これらに限定されない。 The present invention also provides a method of screening for a compound that cancels or activates inhibition of the transport activity of a vesicular neurotransmitter transporter. Such a screening method is performed by selecting a compound having an effect of restoring transport activity from candidate compounds in the presence of the inhibitor of excitatory neurotransmitter transport of the present invention. Such screening methods are for example:
(1) measuring the transport activity of excitatory neurotransmitter transport protein in the presence of an inhibitor of excitatory neurotransmitter transport;
(2) a step of measuring transport activity when a candidate compound is further added; and
(3) a step of identifying a candidate compound as a compound having an ability of releasing inhibition or activating when the activity of (2) is higher than the activity of (1);
Is included. In this method, a preferred excitatory neurotransmitter transport protein is VGLUT2, but is not limited thereto. The inhibitor of excitatory neurotransmitter transport is preferably acetoacetate, pyruvate, phenylpyruvate, 3-hydroxybutyrate, α-keto-β-methyl-valerate, or lactic acid, more preferably Acetoacetate, but is not limited thereto.
　このようにして同定された化合物は、神経の抑制に起因する疾患および／または状態の治療、予防および／または予後に利用する薬剤の有効成分として利用することが可能である。 The compound thus identified can be used as an active ingredient of a drug used for the treatment, prevention and / or prognosis of diseases and / or conditions caused by nerve suppression.
　（薬学的組成物）
　本明細書において薬剤の「有効量」とは、その薬剤が目的とする薬効を発現することができる量をいう。本明細書において、そのような有効量のうち、最小の濃度を最小有効量ということがある。そのような最小有効量は、当該分野において周知であり、通常、薬剤の最小有効量は当業者によって決定されているか、または当業者は適宜決定することができる。そのような有効量の決定には、実際の投与のほか、動物モデルなどを用いることも可能である。本発明はまた、このような有効量を決定する際に有用である。 (Pharmaceutical composition)
In the present specification, the “effective amount” of a drug refers to an amount that allows the drug to exhibit the intended drug effect. In the present specification, among such effective amounts, the minimum concentration may be referred to as the minimum effective amount. Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art or can be determined as appropriate by those skilled in the art. In order to determine such an effective amount, an animal model or the like can be used in addition to actual administration. The present invention is also useful in determining such effective amounts.
　本発明の処置方法において使用される薬剤の種類および量は、本発明の方法によって得られた情報（例えば、疾患に関する情報）を元に、使用目的、対象疾患（種類、重篤度など）、患者の年齢、体重、性別、既往歴、投与される被検体の部位の形態または種類などを考慮して、当業者が容易に決定することができる。本発明のモニタリング方法を被検体（または患者）に対して施す頻度もまた、使用目的、対象疾患（種類、重篤度など）、患者の年齢、体重、性別、既往歴、および治療経過などを考慮して、当業者が容易に決定することができる。疾患状態をモニタリングする頻度としては、例えば、毎日～数ヶ月に１回（例えば、１週間に１回～１ヶ月に１回）のモニタリングが挙げられる。１週間～１ヶ月に１回のモニタリングを、経過を見ながら施すことが好ましい。 The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency of monitoring the disease state include monitoring every day to once every several months (for example, once a week to once a month). It is preferable to perform monitoring once a week to once a month while monitoring the progress.
　本発明では、いったん類似の種類（例えば、ヒトに対するマウスなど）の生物、培養細胞、組織などに関し、ある特定の糖鎖構造の分析結果と、疾患レベルとが相関付けられた場合、対応する糖鎖構造の分析結果と、疾患レベルとが相関付けることができることは、当業者は容易に理解する。そのような事項は、例えば、動物培養細胞マニュアル、瀬野ら編著、共立出版、１９９３年などに記載され支持されており、本明細書においてこのすべての記載を援用する。 In the present invention, once an analysis result of a specific sugar chain structure and a disease level are correlated with organisms, cultured cells, tissues, etc. of similar types (for example, mice against humans), the corresponding sugar Those skilled in the art will readily understand that the analysis results of the chain structure can be correlated with the disease level. Such matters are described and supported by, for example, the Animal Culture Cell Manual, edited by Seno et al., Kyoritsu Shuppan, 1993, etc., all of which are incorporated herein by reference.
　（処方）
　本発明はまた、有効量の治療剤の被験体への投与による、疾患または障害（例えば、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症が挙げられるが、これに限定されない）の処置および／または予防の方法を提供する。治療剤は、薬学的に受容可能なキャリア型（例えば、滅菌キャリア）と組み合せた、本発明の組成物を意味する。 (Prescription)
The present invention also provides for a disease or disorder (e.g., epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and the like by administration of an effective amount of a therapeutic agent to a subject. Methods of treatment and / or prevention of osteoporosis (including but not limited to) are provided. By therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).
　治療剤を、個々の患者の臨床状態（特に、治療剤単独処置の副作用）、送達部位、投与方法、投与計画および当業者に公知の他の因子を考慮に入れ、例えば、「治療薬マニュアル２００６」医学書院　監修：高久　史麿／矢崎　義雄、編集：関　顕／北原　光夫／上野　文昭／越前　宏俊に従って処方および投薬する。従って、本明細書において目的とする「有効量」は、このような考慮を行って決定される。 The therapeutic agent takes into account the clinical condition of the individual patient (especially the side effects of therapeutic agent alone treatment), the site of delivery, the method of administration, the dosage regimen and other factors known to those skilled in the art, eg, “Therapeutics Manual 2006 Supervised by Medical Shoin: Takahisa Fumi / Yazaki Yoshio, edited by Seki Akira / Kitahara Mitsuo / Ueno Fumiaki / Echizen Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.
　ケト原食の場合には、１ｍＭ～２ｍＭ程度の血中濃度が達成されることから、１ｍＭ～２ｍＭ程度の血中濃度によって効果があると考えられる。なお、マウスの場合には、１０ｍｇ／ｋｇの血中濃度で毒性が観察されなかった。（Rhoet al., (2002) Acetoacetate, acetone, and dibenzylamine (acontaminant inL-(+)-beta-hydroxybutyrate) exhibit direct anticonvulsantactions in vivo.Epilepsia 43, 358-361.およびMa W and Yallen G. (2007) Ketogenicdiet metabolitesreduce firing in central neurons by operating KATP channels. JNeurosci 27,3618-3625.）
　なお、上記のように投与量は治療的裁量に委ねられる。静脈内用バッグ溶液もまた使用し得る。変化を観察するために必要な処置期間および応答が生じる処置後の間隔は、所望の効果に応じて変化するようである。 In the case of a keto diet, a blood concentration of about 1 mM to 2 mM is achieved, and it is considered that a blood concentration of about 1 mM to 2 mM is effective. In the case of mice, no toxicity was observed at a blood concentration of 10 mg / kg. (Rhoet al., (2002) Acetoacetate, acetone, and dibenzylamine (acontaminant inL-(+)-beta-hydroxybutyrate) exhibit direct anticonvulsantactions in vivo.Epilepsia 43, 358-361. And Ma W and Yallen G. (2007) Ketogenicdiet metabolitesreduce firing in central neurons by operating KATP channels. JNeurosci 27,3618-3625.)
As noted above, the dosage is left to therapeutic discretion. Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.
　治療剤を、経口的、直腸内、非経口的、槽内、膣内、腹腔内、口内あるいは経口または鼻腔スプレーとして投与し得る。本明細書で用いる用語「非経口的」とは、静脈内、筋肉内、腹腔内、胸骨内、皮下および関節内の注射および注入を含む投与の様式をいう。 The therapeutic agent can be administered orally, rectally, parenterally, in the tank, vaginally, intraperitoneally, buccally, orally or as a nasal spray. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.
　本発明の治療剤はまた、徐放性システムにより適切に投与される。徐放性治療剤の適切な例は、経口的、直腸内、非経口的、槽内、膣内、腹腔内、口内あるいは経口または鼻腔スプレーとして投与され得る。 The therapeutic agent of the present invention is also appropriately administered by a sustained release system. Suitable examples of sustained release therapeutic agents can be administered orally, rectally, parenterally, in the tank, vaginally, intraperitoneally, buccally, or orally or as a nasal spray.
　本発明の治療剤はまた、徐放性システムにより適切に投与される。徐放性治療剤の適切な例は、適切なポリマー物質（例えば、成形品（例えば、フィルムまたはマイクロカプセル）の形態の半透過性ポリマーマトリックス）、適切な疎水性物質（例えば、許容品質油中のエマルジョンとして）またはイオン交換樹脂、および貧可溶性誘導体（例えば、貧可溶性塩）を包含する。 The therapeutic agent of the present invention is also appropriately administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, in acceptable quality oils). Or an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).
　徐放性マトリックスとしては、ポリラクチド（米国特許第３，７７３，９１９号、ＥＰ５８，４８１）、Ｌ－グルタミン酸およびγ－エチル－Ｌ－グルタメートのコポリマー（Ｓｉｄｍａｎら、Ｂｉｏｐｏｌｙｍｅｒｓ　２２：５４７－５５６（１９８３））、ポリ（２－ヒドロキシエチルメタクリレート）（Ｌａｎｇｅｒら、Ｊ．Ｂｉｏｍｅｄ．Ｍａｔｅｒ．Ｒｅｓ．１５：　１６７－２７７（１９８１）、およびＬａｎｇｅｒ，Ｃｈｅｍ．Ｔｅｃｈ．１２：９８－１０５（１９８２））、エチレンビニルアセテート（Ｌａｎｇｅｒら、同書）またはポリ－Ｄ－（－）－３－ヒドロキシ酪酸（ＥＰ１３３，９８８）が挙げられる。 Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl Acetate (Langer et al., Ibid) or poly-D-(-)-3-hydroxybutyric acid (EP133,988).
　徐放性治療剤はまた、リポソームに包括された本発明の治療剤を包含する（一般に、Ｌａｎｇｅｒ，Ｓｃｉｅｎｃｅ　２４９：１５２７－１５３３（１９９０）；Ｔｒｅａｔら，Ｌｉｐｏｓｏｍｅｓ　ｉｎ　ｔｈｅ　Ｔｈｅｒａｐｙ　ｏｆ　Ｉｎｆｅｃｔｉｏｕｓ　Ｄｉｓｅａｓｅ　ａｎｄ　Ｃａｎｃｅｒ，Ｌｏｐｅｚ－Ｂｅｒｅｓｔｅｉｎ　ａｎｄ　Ｆｉｄｌｅｒ（編），Ｌｉｓｓ，Ｎｅｗ　Ｙｏｒｋ，３１７－３２７頁および３５３－３６５（１９８９）を参照のこと）。治療剤を含有するリポソームは、それ自体が公知である方法により調製され得る：ＤＥ３，２１８，１２１；Ｅｐｓｔｅｉｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８２：３６８８－３６９２（１９８５）；Ｈｗａｎｇら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　７７：４０３０－４０３４（１９８０）；ＥＰ５２，３２２；ＥＰ３６，６７６；ＥＰ８８，０４６；ＥＰ１４３，９４９；ＥＰ１４２，６４１；日本国特許出願第８３－１１８００８号；米国特許第４，４８５，０４５号および同第４，５４４，５４５号ならびにＥＰ第１０２，３２４号。通常、リポソームは、小さな（約２００～８００Å）ユニラメラ型であり、そこでは、脂質含有量は、約３０モル％コレステロールよりも多く、選択された割合が、最適治療剤のために調整される。 Sustained release therapeutic agents also include the therapeutic agents of the present invention entrapped in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapeutic Diseases and Cancers, Cancer -See Berstein and Fiddler (eds.), Liss, New York, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Patent No. 4,485,045 and No. 4,544,545 and EP No. 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for optimal therapeutic agents.
　なおさらなる実施態様において、本発明の治療剤は、ポンプにより送達される（Ｌａｎｇｅｒ、前出；Ｓｅｆｔｏｎ、ＣＲＣ　Ｃｒｉｔ．Ｒｅｆ．Ｂｉｏｍｅｄ．Ｅｎｇ．１４：２０１（１９８７）；Ｂｕｃｈｗａｌｄら、Ｓｕｒｇｅｒｙ　８８：５０７（１９８０）；Ｓａｕｄｅｋら、Ｎ．Ｅｎｇｌ．Ｊ．Ｍｅｄ．３２１：５７４（１９８９）を参照のこと）。 In still further embodiments, the therapeutic agents of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgary 88: 507 ( 1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)).
　他の制御放出系は、Ｌａｎｇｅｒ（Ｓｃｉｅｎｃｅ　２４９：１５２７－１５３３（１９９０））による総説において議論される。 Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).
　非経口投与のために、１つの実施態様において、一般に、治療剤は、それを所望の程度の純度で、薬学的に受容可能なキャリア、すなわち用いる投薬量および濃度でレシピエントに対して毒性がなく、かつ処方物の他の成分と適合するものと、単位投薬量の注射可能な形態（溶液、懸濁液または乳濁液）で混合することにより処方される。例えば、この処方物は、好ましくは、酸化、および治療剤に対して有害であることが知られている他の化合物を含まない。 For parenteral administration, in one embodiment, in general, the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidation and other compounds known to be harmful to therapeutic agents.
　一般に、治療剤を液体キャリアまたは微細分割固体キャリアあるいはその両方と均一および緊密に接触させて処方物を調製する。次に、必要であれば、生成物を所望の処方物に成形する。好ましくは、キャリアは、非経口的キャリア、より好ましくはレシピエントの血液と等張である溶液である。このようなキャリアビヒクルの例としては、水、生理食塩水、リンゲル溶液およびデキストロース溶液が挙げられる。不揮発性油およびオレイン酸エチルのような非水性ビヒクルもまた、リポソームと同様に本明細書において有用である。 Generally, a formulation is prepared by contacting the therapeutic agent uniformly and intimately with a liquid carrier or a finely divided solid carrier or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.
　キャリアは、等張性および化学安定性を高める物質のような微量の添加剤を適切に含有する。このような物質は、用いる投薬量および濃度でレシピエントに対して毒性がなく、このような物質としては、リン酸塩、クエン酸塩、コハク酸塩、酢酸および他の有機酸またはその塩類のような緩衝剤；アスコルビン酸のような抗酸化剤；低分子量（約１０残基より少ない）ポリペプチド（例えば、ポリアルギニンまたはトリペプチド）；血清アルブミン、ゼラチンまたは免疫グロブリンのようなタンパク質；ポリビニルピロリドンのような親水性ポリマー；グリシン、グルタミン酸、アスパラギン酸またはアルギニンのようなアミノ酸；セルロースまたはその誘導体、ブドウ糖、マンノースまたはデキストリンを含む、単糖類、二糖類、および他の炭水化物；ＥＤＴＡのようなキレート剤；マンニトールまたはソルビトールのような糖アルコール；ナトリウムのような対イオン；および／またはポリソルベート、ポロキサマーもしくはＰＥＧのような非イオン性界面活性剤が挙げられる。 The carrier suitably contains trace amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts. Buffering agents; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.
　治療的投与に用いられるべき任意の薬剤は、生物・ウイルスを含まない状態、すなわち、無菌状態であり得る。滅菌濾過膜（例えば０．２ミクロンメンブラン）で濾過することにより無菌状態は容易に達成される。一般に、治療剤は、滅菌アクセスポートを有する容器、例えば、皮下用注射針で穿刺可能なストッパー付の静脈内用溶液バッグまたはバイアルに配置される。 Any drug to be used for therapeutic administration may be free of organisms / viruses, that is, sterile. Aseptic conditions are easily achieved by filtration through a sterile filtration membrane (eg, 0.2 micron membrane). In general, the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper puncturable with a hypodermic needle.
　治療剤は、通常、単位用量または複数用量容器、例えば、密封アンプルまたはバイアルに、水溶液または再構成するための凍結乾燥処方物として貯蔵される。凍結乾燥処方物の例として、１０ｍｌのバイアルに、滅菌濾過した１％（ｗ／ｖ）治療剤水溶液５ｍｌを充填し、そして得られる混合物を凍結乾燥する。凍結乾燥した治療剤を、注射用静菌水を用いて再構成して注入溶液を調製する。 Treatment agents are usually stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or lyophilized formulations for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous therapeutic agent and the resulting mixture is lyophilized. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.
　本発明はまた、本発明の治療剤の１つ以上の成分を満たした一つ以上の容器を備える薬学的パックまたはキットを提供する。医薬品または生物学的製品の製造、使用または販売を規制する政府機関が定めた形式の通知が、このような容器に付属し得、この通知は、ヒトへの投与に対する製造、使用または販売に関する政府機関による承認を表す。さらに、治療剤を他の治療用化合物と組み合わせて使用し得る。 The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration. Represents institutional approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.
　本発明の治療剤は、単独または他の治療剤と組合わせて投与され得る。組合わせは、例えば、混合物として同時に；同時にまたは並行してだが別々に；あるいは経時的のいずれかで投与され得る。これは、組み合わされた薬剤が、治療用混合物として共に投与されるという提示、およびまた、組み合わされた薬剤が、別々にしかし同時に、例えば、同じ個体に別々の静脈ラインを通じて投与される手順を含む。「組み合わせて」の投与は、一番目、続いて二番目に与えられる化合物または薬剤のうち１つの別々の投与をさらに含む。 The therapeutic agent of the present invention can be administered alone or in combination with other therapeutic agents. The combinations can be administered, for example, either simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual . Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.
　本発明の代表的な化合物である、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、および、乳酸からなる群から選択される化合物はいずれも生理物質であり、体内で輸送・代謝・排泄される化合物である。従って、生体内には、これらを運搬するトランスポーターが存在する（Mullerら、"Transportof ketone bodies and lactate in the sheepruminal epithelium by monocarboxylatetransporter 1"、Am J Physiol GastrointestLiver Physiol. 2002Nov;283(5):G1139-46、および、Martin PMら、"Identity of SMCT1(SLC5A8) as aneuron-specific Na+-coupled transporter for active uptake ofL-lactate andketone bodies in the brain"、J Neurochem. 2006Jul;98(1):279-88）。生体内に存在する、このトランスポーターおよび類似のトランスポーターによって本発明の化合物を輸送すると考えられることから、本発明の化合物を経口的および非経口的に生体に投与した場合、標的部位に送達され、その結果、興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害することが理解できる。 The compounds selected from the group consisting of acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, and lactic acid, which are representative compounds of the present invention, are all physiological substances, and are transported and metabolized in the body. • It is an excreted compound. Therefore, there are transporters that transport these in vivo (Muller et al., "Transportof ketone bodies and lactate in the sheepruminal epithelium by monocarboxylatetransporter 1", Am J Physiol GastrointestLiver Physiol. 2002Nov; 283 (5): G1139-G 46, and Martin PM et al., “Identity of SMCT1 (SLC5A8) as aneuron-specific Na + -coupled transporter for active uptake ofL-lactate andketone bodies in the brain", J Neurochem. 2006Jul; 98 (1): 279-88) . Since this transporter and similar transporters existing in vivo are considered to transport the compound of the present invention, when the compound of the present invention is orally and parenterally administered to the living body, it is delivered to the target site. As a result, it can be understood that at least two types of vesicular neurotransmitter transporters for excitatory neurotransmitters are inhibited under physiological chloride concentrations.
　以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的のみに提供される。従って、本発明の範囲は、上記発明の詳細な説明にも下記実施例にも限定されるものではなく、請求の範囲によってのみ限定される。 Hereinafter, the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above detailed description of the invention nor the following examples, but is limited only by the scope of the claims.
　（実施例１：グルタミン酸トランスポーターVGLUT2のクローニング、発現、プロテオリポソームの調製、および、活性測定）
　（１．クローニングおよび発現用プラスミドの構築）
　ラットVGLUT2　cDNAを以下のプライマー(センスプライマー:5’-CACCATGGAGTCGGTAAAACAAAGGATT-3’：配列番号１１、アンチセンスプライマー:5’-TTCGTTATGAATAATCATCTCGGTCCT-3’：配列番号１２)を用いてPCRにより増幅し、TOPOクローニンングシステム(インビトロジェン)を用いて、pENTER/D-TOPOベクターに組み込んだ。このプラスミドをLRリコンビネーションによりデスティネーションベクター、pDEST10に組み込んだ(pDEST10VGLUT2)。これをバクミドDNAの入った大腸菌DH10bac株に形質転換し、N末側に6×Hisのはいった、VGLUTのバクミドを完成させた。DH10bac株から単離したバクミドをリポフェクション法により、Sf9細胞に形質導入することにより、VGLUT2ウイルスを得た。 (Example 1: Cloning and expression of glutamate transporter VGLUT2, preparation of proteoliposome, and activity measurement)
(1. Construction of cloning and expression plasmids)
Rat VGLUT2 cDNA was amplified by PCR using the following primers (sense primer: 5'-CACCATGGAGTCGGTAAAACAAAGGATT-3 ': SEQ ID NO: 11, antisense primer: 5'-TTCGTTATGAATAATCATCTCGGTCCT-3': SEQ ID NO: 12), and TOPO cloning Using the system (Invitrogen), it was incorporated into the pENTER / D-TOPO vector. This plasmid was incorporated into a destination vector, pDEST10, by LR recombination (pDEST10VGLUT2). This was transformed into E. coli DH10bac strain containing bacmid DNA to complete a VGLUT bacmid with 6 × His in the N-terminal side. VGLUT2 virus was obtained by transducing Sf9 cells by lipofection with a bacmid isolated from DH10bac strain.
　（２．HighFive 細胞の培養とウイルスの感染）
　感染はHighFive 細胞を用いて行った。High Five 細胞を培地(GIBCO Express Five SFM、 0.2mML-Glutamine、 10μg/ml gentamaicine)をいれた蓋つきフラスコにおいて27℃で培養した。感染時、1×107細胞を１０cm2シャーレに蒔きMOI(multiplicity of infection)= 1で感染し、感染後のインキュベートは48時間行った。 (2. HighFive cell culture and virus infection)
Infection was performed using HighFive cells. High Five cells were cultured at 27 ° C. in a flask with a lid containing medium (GIBCO Express Five SFM, 0.2 mM L-Glutamine, 10 μg / ml gentamaicine). At the time of infection, 1 × 10 7 cells were seeded in a 10 cm 2 petri dish and infected with MOI (multiplicity of infection) = 1, and incubation after infection was performed for 48 hours.
　（３．細胞の回収と膜画分の可溶化）
　感染48 h後の細胞をセルスクレーバーにより回収し、700×g　10分遠心して上清を取り除いた。これを緩衝液A(20mM Tris-HCl pH 8.0、0.1 M 酢酸カリウム、10%グリセロール、5mM　DTT、1μg/ml　ペプスタチンＡ、1μg/ml　ロイペプチン)で懸濁し、もう一度700×g　10分遠心して上清を取り除いた。これを緩衝液Aで懸濁した。これをTOMYUD200tip sonifier により粉砕した後、700×g　10分遠心して上清を回収し、160,000×g　１時間で超遠心して、膜画分とした。このペレットを緩衝液B（20mMMOPS-tris pH 7.0、2%　オクチルグルコシド、10%グリセロール、１μg/ml　ペプスタチンA、１μg/ml　ロイペプチン)をいれホモジナイザーを用いて懸濁し、260,000×g　30分間の遠心操作を行い、その上清を膜の可溶化画分とした。 (3. Cell recovery and solubilization of membrane fraction)
Cells 48 hours after infection were collected with a cell scraper and centrifuged at 700 × g for 10 minutes to remove the supernatant. This was suspended in buffer A (20 mM Tris-HCl pH 8.0, 0.1 M potassium acetate, 10% glycerol, 5 mM DTT, 1 μg / ml pepstatin A, 1 μg / ml leupeptin), and centrifuged again at 700 × g for 10 minutes to obtain a supernatant. Removed. This was suspended in buffer A. This was pulverized with TOMYUD200tip sonifier and then centrifuged at 700 × g for 10 minutes to recover the supernatant, and ultracentrifuged at 160,000 × g for 1 hour to obtain a membrane fraction. Suspend this pellet in buffer B (20mMMOPS-tris pH 7.0, 2% octylglucoside, 10% glycerol, 1μg / ml pepstatin A, 1μg / ml leupeptin) using a homogenizer, and centrifuge at 260,000 xg for 30 minutes The supernatant was used as the solubilized fraction of the membrane.
　（４．アフィニティカラムを用いたVGLUT2 の精製）
　QIAGENNi-NTA スーパーフローレジンをエコノカラムに充填し（１ｍＬ；50%スラリー)、蒸留水にて洗浄した後 pH 8.0 の緩衝液Bで平衡化する。ここに先ほどの可溶化画分を入れ、4℃、4時間シーソー上で揺らしながら吸着させる。これを１０ｍＬの緩衝液C（20mMMOPS-tris pH 7.0、１％　オクチルグルコシド、20%　グリセロール、5 mM　イミダゾール、1μg/ml　ペプスタチンＡ、1μg/ml　ロイペプチン)で洗浄し、イミダゾールを60mMにした同液にて溶出した。使用するまで、－８０℃に保管した。 (4. Purification of VGLUT2 using affinity column)
Pack QIAGENNi-NTA Super Flow Resin on Econocolumn (1 mL; 50% slurry), wash with distilled water, and equilibrate with Buffer B at pH 8.0. The solubilized fraction is added to this and adsorbed while shaking on a seesaw at 4 ° C for 4 hours. This was washed with 10 mL of buffer C (20 mM POP-tris pH 7.0, 1% octyl glucoside, 20% glycerol, 5 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin) to obtain the same solution with 60 mM imidazole. And eluted. Stored at −80 ° C. until use.
　（５．リポソームの調製）
　ダイズレクチン20mgを20 mM MOPS-NaOH pH 7.0、1 mM DTT 2 mL に加え、バス式ソニケーターにて 110 V、10 min ソニケーションした。液が澄明で均一となったらソニケーションを終了した。使用時まで－８０℃にて保管した。 (5. Preparation of liposome)
20 mg of soybean lectin was added to 2 mM of 20 mM MOPS-NaOH pH 7.0 and 1 mM DTT, and sonicated at 110 V for 10 min with a bath sonicator. The sonication was terminated when the liquid became clear and uniform. Stored at −80 ° C. until use.
　（６．VGLUTのリポソームへの再構成）
　精製したVGLUT2　10μｇをリポソーム500μｇと混合し、－８０℃で15分間静置した。凍結後これを取り出し、迅速に解凍し、その後直ちに緩衝液Ｆ（20mMMOPS-Tris pH 7.0、150 mM　酢酸ナトリウム、5 mM　酢酸マグネシウム、0.5 mM DTT、1μg/ml　ペプスタチンＡ、1μg/ml　ロイペプチン）で60倍に希釈し、200,000×gで遠心しペレットを200μＬの緩衝液Ｆで懸濁した。これをプロテオリポソーム（再構成リポソーム）として以後の実験に用いた。 (6. Reconstitution of VGLUT into liposomes)
10 μg of purified VGLUT2 was mixed with 500 μg of liposomes and allowed to stand at −80 ° C. for 15 minutes. After freezing, this was taken out and thawed quickly, and then immediately with buffer F (20 mMOPS-Tris pH 7.0, 150 mM sodium acetate, 5 mM magnesium acetate, 0.5 mM DTT, 1 μg / ml pepstatin A, 1 μg / ml leupeptin). The pellet was suspended in 200 μL of buffer F after doubling and centrifuging at 200,000 × g. This was used as a proteoliposome (reconstituted liposome) in subsequent experiments.
　（７．Ｆ－ＡＴＰａｓｅとVGLUTのリポソームへの再構成）
　（ＦｏＦ１－ＡＴＰａｓｅの精製）
Ｍｏｒｉｙａｍａ　Ｙら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６６，２２１４１－２２１４６（１９９１）に記載の手順に従って、プロトンポンプであるＦ０Ｆ１タンパク質を調製した。 (7. Reconstitution of F-ATPase and VGLUT into liposomes)
(Purification of F o F 1 -ATPase)
Moriyama Y et al. Biol. Chem. 266, 22141-22146 (1991), the proton pump F 0 F 1 protein was prepared.
　Ｆ０Ｆ１の大量発現プラスミドｐＢＷＵ１３を含有している大腸菌ＤＫ８を、０．５％グリセロールを含むＴａｎａｋａ培地（３４ｍＭ一カリウムリン酸、６４ｍＭ二カリウムリン酸、２０ｍＭ硫酸アンモニウム、０．３ｍＭ塩化マグネシウム、１μＭ硫酸鉄、１μＭ塩化カルシウム、１μＭ塩化亜鉛、１００μｇ／ｍｌイソロイシン、１００μｇ／ｍｌバリン、２μｇ／ｍｌチアミン）で培養した後、菌体を回収した。以降の調製を全て４℃で行った。 E. coli DK8 containing the large F 0 F 1 expression plasmid pBWU13 was added to Tanaka medium (34 mM monopotassium phosphate, 64 mM dipotassium phosphate, 20 mM ammonium sulfate, 0.3 mM magnesium chloride, 1 μM) containing 0.5% glycerol. After culturing with iron sulfate, 1 μM calcium chloride, 1 μM zinc chloride, 100 μg / ml isoleucine, 100 μg / ml valine, 2 μg / ml thiamine), the cells were collected. All subsequent preparations were performed at 4 ° C.
　菌体（ＤＫ８／ｐＢＷＵ１３）約１０ｇを、４０ｍｌの膜調製緩衝液（４℃の５０ｍＭ
　Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）、２ｍＭ塩化マグネシウム、０，５ｍＭ　ＥＤＴＡ、１ｍＭ　ＰＭＳＦ、１μｇ／ｍｌロイペプチン、１μｇ／ｍｌペプスタチンＡ、１０％（ｖ／ｖ）グリセロール、１ｍＭ　ＤＴＴ）に懸濁し、フレンチプレス（１，５００ｋｇ／ｃｍ２）で細胞を破砕した。破砕液を１７，０００×ｇにて１０分間遠心分離し、得られた上清をさらに２１０，０００×ｇで１時間２０分間遠心分離した。得られた膜小胞の沈澱を、Ｆ０Ｆ１調製用緩衝液（２０ｍＭ　ＭＯＰＳ／ＮａＯＨ（ｐＨ７．０）、１ｍＭ硫酸マグネシウム、１ｍＭ　ＤＴＴ、１ｍＭ　ＰＭＳＦ、０．８％オクチルグルコシド）中に懸濁し、再度遠心分離した。沈澱物として調製した膜小胞６０ｍｇを、２％のオクチルグルコシドを含む３ｍｌのＦ０Ｆ１調製用緩衝液中に懸濁し、Ｆ０Ｆ１を可溶化した。可溶化溶液を、２６０，０００×ｇで３０分間遠心分離し、上清画分からＦ０Ｆ１を回収した。回収したＦ０Ｆ１を、１０％（ｗ／ｖ）～３０％（ｗ／ｖ）のグリセロール密度勾配遠心分離（３３０，０００×ｇで５時間）によって精製した。グリセロール密度勾配を、１％オクチルグルコシドを含むＦ０Ｆ１調製用緩衝液で作製した。密度勾配遠心後、遠心管の底から１０分画に分けて分離し、最初の４画分をＦ０Ｆ１として回収し、－８０℃で保存した。 About 10 g of microbial cells (DK8 / pBWU13) was added to 40 ml of membrane preparation buffer (50 mM at 4 ° C.
Suspended in Tris-HCl (pH 8.0), 2 mM magnesium chloride, 0.5 mM EDTA, 1 mM PMSF, 1 μg / ml leupeptin, 1 μg / ml pepstatin A, 10% (v / v) glycerol, 1 mM DTT), French press Cells were disrupted with (1,500 kg / cm 2 ). The crushed liquid was centrifuged at 17,000 × g for 10 minutes, and the obtained supernatant was further centrifuged at 210,000 × g for 1 hour and 20 minutes. The obtained membrane vesicle precipitate was suspended in F 0 F 1 preparation buffer (20 mM MOPS / NaOH (pH 7.0), 1 mM magnesium sulfate, 1 mM DTT, 1 mM PMSF, 0.8% octyl glucoside). Centrifuge again. 60 mg of membrane vesicle prepared as a precipitate was suspended in 3 ml of F 0 F 1 preparation buffer containing 2% octyl glucoside to solubilize F 0 F 1 . The solubilized solution was centrifuged at 260,000 × g for 30 minutes, and F 0 F 1 was recovered from the supernatant fraction. The recovered F 0 F 1 was purified by 10% (w / v) to 30% (w / v) glycerol density gradient centrifugation (330,000 × g for 5 hours). A glycerol density gradient was made with F 0 F 1 preparation buffer containing 1% octyl glucoside. After density gradient centrifugation, the fraction was separated into 10 fractions from the bottom of the centrifuge tube, and the first 4 fractions were collected as F 0 F 1 and stored at −80 ° C.
　（ＦｏＦ１－ＡＴＰａｓｅと精製ＶＧＬＵＴ２のリポソームへの再構成）
　２０ｍｇの大豆レシチン（Ｓｉｇｍａ　ｔｙｐｅＩＩＳ）を緩衝液（２０ｍＭ　ＭＯＰＳ／ＮａＯＨ　ｐＨ７．０、０．５ｍＭ　ＤＴＴ）に懸濁し、バスタイプ超音波装置で透明になるまで超音波処理した。調製したリポソームは分注し、－８０℃で保存した。 (Reconstitution of F o F 1 -ATPase and purified VGLUT2 into liposomes)
20 mg of soybean lecithin (Sigma type IIS) was suspended in a buffer (20 mM MOPS / NaOH pH 7.0, 0.5 mM DTT) and sonicated with a bath-type ultrasonic device until clear. The prepared liposomes were dispensed and stored at −80 ° C.
　次に、上記の手法で精製したＦｏＦ１－ＡＴＰａｓｅ　９０μｇとＶＧＬＵＴ２　１０μｇを上記リポソーム５００μｇに混合し、－８０℃で１５分間静置し、凍結した。ただちにこれを取り出し、迅速に解凍しＦ緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、１００ｍＭ　酢酸カリウム、５ｍＭ　酢酸マグネシウム）にて２０倍に希釈し、１６０，０００×ｇ　６０分遠心した。沈澱にＦ緩衝液　４００μｌを添加し、ホモジナイズし、再構成プロテオリポソームを得た。 Then, the F o F 1 -ATPase 90μg and VGLUT2 10 [mu] g purified by the above method was mixed with the liposome 500 [mu] g, and allowed to stand for 15 minutes at -80 ° C., and frozen. This was immediately taken out, thawed quickly, diluted 20 times with F buffer (20 mM MOPS-Tris pH 7.0, 100 mM potassium acetate, 5 mM magnesium acetate), and centrifuged at 160,000 × g for 60 minutes. To the precipitate, 400 μl of F buffer was added and homogenized to obtain reconstituted proteoliposomes.
　（実施例２：再構成ＶＧＬＵＴ２の輸送活性測定）
　Ｆ－ＡＴＰａｓｅとＶＧＬＵＴ２を再構成したリポソームを用いて、ＡＴＰ存在下（＋ＡＴＰ）、および非存在下（－ＡＴＰ）における、種々のクロライドイオン濃度でのグルタミン酸輸送活性測定を行った。具体的には、再構成プロテオリポソーム（５μｇ）を２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、５ｍＭ　酢酸マグネシウム、１０４ｍＭの酢酸カリウム中に懸濁し、２７℃で３分間インキュベートした。この反応液の酢酸カリウムの一部を塩化カリウムで置換することで、必要な濃度のクロライドイオンを含むようにした。その後、終濃度２ｍＭのＡＴＰを加え、さらに３分間インキュベートした。[2,3-3H]L-グルタミン酸(0.5 MBq/μmol)を加えて反応を開始し、各時間ごとに130μＬずつサンプル液を取り、セファデックスG-50ファインに通した。760×ｇで2分間遠心し、その溶出液をクリアゾル３ｍＬに溶かし、液体シンチレーションカウンターにより計測した。結果を図１（Ａ）に示す。 (Example 2: Measurement of transport activity of reconstituted VGLUT2)
Using liposomes reconstituted with F-ATPase and VGLUT2, glutamate transport activity was measured at various chloride ion concentrations in the presence (+ ATP) and absence (-ATP) of ATP. Specifically, reconstituted proteoliposomes (5 μg) were suspended in 20 mM MOPS-Tris pH 7.0, 5 mM magnesium acetate, 104 mM potassium acetate, and incubated at 27 ° C. for 3 minutes. A part of potassium acetate in the reaction solution was replaced with potassium chloride so as to contain a necessary concentration of chloride ions. Thereafter, ATP at a final concentration of 2 mM was added and further incubated for 3 minutes. [2,3-3H] L-glutamic acid (0.5 MBq / μmol) was added to initiate the reaction, and 130 μL of sample solution was taken at each time and passed through Sephadex G-50 Fine. Centrifugation was performed at 760 × g for 2 minutes, the eluate was dissolved in 3 mL of clear sol, and measured with a liquid scintillation counter. The results are shown in FIG.
　次に、ＶＧＬＵＴ２のみを再構成したリポソームをもちいて、バリノマイシン存在下（＋バリノマイシン）、および非存在下（－バリノマイシン）における、種々のクロライドイオン濃度でのグルタミン酸輸送活性測定を行った。具体的には、試験管内に緩衝液Ｇ（20mM MOPS-Tris pH 7.0、150 mM 酢酸カリウム、5 mM 酢酸マグネシウム、10 mM KCl）、2μＭ　バリノマイシン、[2,3-3H]L-グルタミン酸(0.5 MBq/pmol)、と阻害剤を加え、27℃水浴において3分間インキュベートした。ここに作製したプロテオリポソーム0.5μｇを加え反応を開始した。各時間ごとに130μＬずつサンプル液を取り、セファデックスG-50ファインに通した。760×ｇで2分間遠心し、その溶出液をクリアゾル３ｍＬに溶かし、液体シンチレーションカウンターにより計測した。結果を図１（Ｂ）に示す。 Next, glutamate transport activity was measured at various chloride ion concentrations in the presence of valinomycin (+ valinomycin) and in the absence (-valinomycin) using liposomes reconstituted only with VGLUT2. Specifically, buffer G (20 mM MOPS-Tris pH 7.0, 150 mM potassium acetate, 5 mM magnesium acetate, 10 mM KCl), 2 μM valinomycin, [2,3- 3 H] L-glutamic acid (0.5 MBq / pmol) and an inhibitor were added and incubated in a 27 ° C. water bath for 3 minutes. The prepared proteoliposome 0.5 μg was added to initiate the reaction. 130 μL of sample solution was taken at each time and passed through Sephadex G-50 Fine. Centrifugation was performed at 760 × g for 2 minutes, the eluate was dissolved in 3 mL of clear sol, and measured with a liquid scintillation counter. The results are shown in FIG.
　Ｆ－ＡＴＰａｓｅとトランスポーターをともに再構成する従来法（Ａ）では、クロライドイオン濃度が増えるに従って、輸送活性は一過性に上昇後、急速に低下する。いわゆるオーバーシュート現象を示す。これはおそらく形成された膜電位がＦ－ＡＴＰａｓｅ経由で漏れてしまうためであると推定された。従って、この従来法の測定システムでは高濃度の塩素イオンによるVGLUTへの影響を調べることは難しい。そこで、本発明者らは膜電位をバリノマイシンにより起こすことにより精製VGLUTのみを含むリポソームでグルタミン酸輸送を測定する系を構築し、グルタミン酸輸送活性の測定に成功した（Ｂ）。これにより高濃度のクロライドイオン存在下でも安定したグルタミン酸輸送活性を示すことが判明。この測定系を用いてクロライドイオン感受性に影響を与える薬物をスクリーニングすることが初めて可能になった。 In the conventional method (A) in which both F-ATPase and transporter are reconstituted, the transport activity increases transiently and then rapidly decreases as the chloride ion concentration increases. The so-called overshoot phenomenon is shown. This was presumably because the formed membrane potential leaked via F-ATPase. Therefore, it is difficult to investigate the influence of high concentration of chlorine ions on VGLUT with this conventional measurement system. Therefore, the present inventors constructed a system for measuring glutamate transport using liposomes containing only purified VGLUT by causing membrane potential with valinomycin, and succeeded in measuring glutamate transport activity (B). As a result, it was found that stable glutamate transport activity was exhibited even in the presence of a high concentration of chloride ions. For the first time, it has become possible to screen for drugs that affect chloride ion sensitivity using this measurement system.
　（実施例３：阻害剤のスクリーニング）
　実施例２において構築した、クロライドイオン感受性に影響を与える薬物の改善されたスクリーニング系を用いて、グルタミン酸取り込みの阻害剤をスクリーニングした。候補化合物５ｍＭを使用し、５ｍＭクロライドイオン存在下、および、２０ｍＭクロライドイオン存在下でグルタミン酸輸送活性を測定した。結果を、図２に示す。アセトアセテート、ピルベート、フェニルピルベート、３－ヒドロキシブチレート、および、α－ケト－β－メチルバレレート（ＫＭＶ）が、生理条件のクロライドイオン濃度（２０ｍＭ）下で、高い阻害活性を示した。 (Example 3: Screening of inhibitors)
Inhibitors of glutamate uptake were screened using an improved screening system for drugs that affect chloride ion sensitivity constructed in Example 2. Candidate compound 5 mM was used, and glutamate transport activity was measured in the presence of 5 mM chloride ions and in the presence of 20 mM chloride ions. The results are shown in FIG. Acetoacetate, pyruvate, phenylpyruvate, 3-hydroxybutyrate, and α-keto-β-methylvalerate (KMV) showed high inhibitory activity under physiological conditions of chloride ion concentration (20 mM).
　最も高い阻害活性を示したアセトアセテートについて、種々の濃度を用いた場合の、ＶＧＬＵＴ２のグルタミン酸輸送活性を図３のグラフに示した。この結果は、アセトアセテートによる阻害活性が濃度依存性であることを示す。 For the acetoacetate that showed the highest inhibitory activity, the glutamic acid transport activity of VGLUT2 when various concentrations were used is shown in the graph of FIG. This result shows that the inhibitory activity by acetoacetate is concentration-dependent.
　種々の濃度のクロライドおよびアセトアセテートを用いた場合における、ＶＧＬＵＴ２のグルタミン酸輸送活性を図４のグラフに示した。この結果は、アセトアセテートによるＶＧＬＵＴ２活性阻害がクロライドイオン濃度を増やす事により解消することを示す。 The graph of FIG. 4 shows the glutamate transport activity of VGLUT2 when various concentrations of chloride and acetoacetate were used. This result shows that inhibition of VGLUT2 activity by acetoacetate is eliminated by increasing the chloride ion concentration.
　高い阻害活性を示した、候補化合物、すなわち、アセトアセテート、ピルベート、アセトール、フェニルピルベート、３－ヒドロキシブチレート、および、α－ケト－β－メチルバレレート（ＫＭＶ）のＩＣ５０を図５に示す。なお、ラクテートのＩＣ５０は＞２０ｍＭであった。また、アセトン、メチルグリオキサール、1,2－プロパンジオール、アセチルCoA、サクシネート、イソロイシン、バリン、フェニルアセテート、および、シアル酸は、10mMで阻害活性を示さなかった（図５）。 The IC 50 of candidate compounds showing high inhibitory activity, ie, acetoacetate, pyruvate, acetol, phenylpyruvate, 3-hydroxybutyrate, and α-keto-β-methylvalerate (KMV) are shown in FIG. Show. Incidentally, IC 50 of lactate was> 20 mM. Acetone, methylglyoxal, 1,2-propanediol, acetyl CoA, succinate, isoleucine, valine, phenylacetate and sialic acid did not show inhibitory activity at 10 mM (FIG. 5).
　アセトアセテートによる阻害効果は、グルタミン酸に対する競合的阻害ではなかった。クロライド媒介性活性化のヒル係数は、約３．３であった。このことは、グルタミン酸輸送との、強力な正の協同性を示す。アセトアセテートは、クロライドとグルタミン酸との協同性に影響しなかった。アセトアセテートの効果は、完全に可逆的であり、アセトアセテートを洗浄することによって、グルタミン酸輸送活性は完全に復活した。なお、アセトアセテートは、ＶＧＬＵＴ１およびＶＧＬＵＴ３に対しても、阻害効果を示した。また、ＶＧＬＵＴ１およびＶＧＬＵＴ３をアセトアセテートによって阻害した場合も、洗浄によってグルタミン酸輸送活性は、完全に復活した。 The inhibitory effect of acetoacetate was not a competitive inhibition on glutamic acid. The Hill coefficient for chloride-mediated activation was about 3.3. This indicates a strong positive cooperativity with glutamate transport. Acetoacetate did not affect the cooperativity between chloride and glutamic acid. The effect of acetoacetate was completely reversible, and glutamate transport activity was completely restored by washing acetoacetate. In addition, acetoacetate showed an inhibitory effect on VGLUT1 and VGLUT3. In addition, when VGLUT1 and VGLUT3 were inhibited by acetoacetate, glutamate transport activity was completely restored by washing.
　これら阻害剤は、その構造から、可逆的にトランスポータータンパク質に結合し、阻害活性を示すと考えられる。小胞型神経伝達物質トランスポータータンパク質に不可逆的に結合する色素は、小胞型神経伝達物質トランスポーターを不可逆的に失活させ、その結果、看過できない副作用を生じることが予測される。これに対して、本発明の阻害活性を示す化合物は、阻害様式が可逆的なため、小胞型興奮性神経伝達物質トランスポーターの阻害剤として、特に、過剰な神経の興奮に関連する疾患および／または状態を、治療するため、予防するため、および／または、予後を改善するために有用であると考えられる。 These inhibitors are considered to bind to the transporter protein reversibly from the structure and exhibit inhibitory activity. Dyes that bind irreversibly to the vesicular neurotransmitter transporter protein are predicted to irreversibly deactivate the vesicular neurotransmitter transporter, resulting in side effects that cannot be overlooked. In contrast, the compounds exhibiting the inhibitory activity of the present invention are reversible in their mode of inhibition, so that they are particularly useful as inhibitors of vesicular excitatory neurotransmitter transporters, particularly diseases associated with excessive neural excitability and It is believed that / or the condition is useful for treating, preventing and / or improving prognosis.
　（実施例４：ＶＧＬＵＴ２阻害活性を有する物質による、抑制性伝達物質トランスポーターおよびモノアミントランスポーターに対する阻害活性）
　実施例３において、小胞型グルタミン酸トランスポーターに対する阻害活性を有する物質が同定された。これら阻害剤が、抑制性神経伝達物質のトランスポーターおよび／またはモノアミントランスポーターに対して阻害活性を有するか否かを試験した。トランスポーターの輸送活性に対する阻害活性を測定するために、シナプス小胞を用いた。 (Example 4: Inhibitory activity against inhibitory transmitter transporter and monoamine transporter by substance having VGLUT2 inhibitory activity)
In Example 3, a substance having an inhibitory activity against the vesicular glutamate transporter was identified. It was tested whether these inhibitors have inhibitory activity against inhibitory neurotransmitter transporters and / or monoamine transporters. Synaptic vesicles were used to measure the inhibitory activity on transporter transport activity.
　（１．シナプス小胞の調製）
　Wister 系ラット3週齢雄の全脳を採取し、これをSME緩衝液(0.3M スクロース、10 mM MOPS-Tris pH 7.0、5 mMEDTA-NaOH pH 7.0、1μg/ml　ペプスタチンＡ、1μg/ml　ロイペプチン）内で洗浄し、機械ホモジナイザー(Iuchi)900 rpmで脳をホモジナイズした。これを、900×g　10分遠心して上清を回収し、2,000×g15分遠心しペレットを回収する。ペレットをSME緩衝液に再懸濁し、2,000×g15分遠心しペレットを回収する。このペレットを2 mL SME緩衝液のはいった10mM MOPS-Tris pH 7.0 緩衝液20 mL に懸濁し、20分静置した。その後、11,000×g20分遠心して上清を回収後、160,000×g　1時間　超遠心して、ペレットを回収した。回収したペレットを5mg/mLになるようにSME緩衝液を加えて懸濁し、使用するまで－８０℃に保存した。 (1. Preparation of synaptic vesicles)
The whole brain of a Wister rat 3-week-old male was collected and used as an SME buffer (0.3 M sucrose, 10 mM MOPS-Tris pH 7.0, 5 mM EDTA-NaOH pH 7.0, 1 μg / ml pepstatin A, 1 μg / ml leupeptin). The brain was homogenized with a mechanical homogenizer (Iuchi) 900 rpm. The supernatant is collected by centrifugation at 900 × g for 10 minutes, and the pellet is collected by centrifugation at 2,000 × g for 15 minutes. Resuspend the pellet in SME buffer and centrifuge at 2,000 xg for 15 minutes to recover the pellet. This pellet was suspended in 20 mL of 10 mM MOPS-Tris pH 7.0 buffer containing 2 mL SME buffer and allowed to stand for 20 minutes. Thereafter, the supernatant was collected by centrifugation at 11,000 × g for 20 minutes, and then ultracentrifuged at 160,000 × g for 1 hour to collect the pellet. The recovered pellet was suspended by adding SME buffer to 5 mg / mL and stored at −80 ° C. until use.
　（２．RI標識した化合物を用いたシナプス小胞の活性測定）
　試験管内に緩衝液(10 mM MOPS-Tris pH 7.0、5 mM　酢酸マグネシウム、10 mM KCl、0.28 M　スクロース)、阻害剤（１ｍＭ　アセトアセテート）と50μｇのシナプス小胞を加え、27℃水浴において3分間インキュベートした。その後、2mMATPを加え27℃水浴において3分間インキュベートした。100μＭ　［２，３－３Ｈ］－ＧＡＢＡ（0.5MBq/μmol）、または10μＭ　５－ヒドロキシ［３－３Ｈ］トリプタミン（0.5MBq/μmol）を加え、反応を開始した。各時間に100μＬずつサンプル液を取り、0.45μｍフィルター（ミリポア社）に通し、このフィルターを緩衝液１０ｍｌで洗浄後、クリアゾル３ｍｌに溶かし、液体シンチレーションカウンターにより計測した。 (2. Activity measurement of synaptic vesicles using RI-labeled compounds)
Add buffer (10 mM MOPS-Tris pH 7.0, 5 mM magnesium acetate, 10 mM KCl, 0.28 M sucrose), inhibitor (1 mM acetoacetate) and 50 μg synaptic vesicles in a test tube, and then in a 27 ° C. water bath for 3 minutes. Incubated. Then, 2mMATP was added and it incubated for 3 minutes in a 27 degreeC water bath. 100 μM [2,3- 3 H] -GABA (0.5 MBq / μmol) or 10 μM 5-hydroxy [3- 3 H] tryptamine (0.5 MBq / μmol) was added to initiate the reaction. 100 μL of sample solution was taken at each time, passed through a 0.45 μm filter (Millipore), this filter was washed with 10 ml of buffer solution, dissolved in 3 ml of clear sol, and measured with a liquid scintillation counter.
　（３．結果）
　モノアミントランスポーターによる輸送活性の指標として、１ｍＭのアセトアセテート存在下（＋アセトアセテート）および非存在下（－アセトアセテート）での、シナプス小胞のセロトニン輸送活性を測定した。結果を図６Ａ（ＶＭＡＴ）に示す。抑制性神経伝達物質トランスポーターによる輸送活性の指標として、１ｍＭのアセトアセテート存在下（＋アセトアセテート）および非存在下（－アセトアセテート）での、シナプス小胞のＧＡＢＡ輸送活性を測定した。結果を図６Ａ（ＶＩＡＡＴ）に示す。 (3. Results)
As an indicator of the transport activity by the monoamine transporter, the serotonin transport activity of synaptic vesicles in the presence (+ acetoacetate) and absence (−acetoacetate) of 1 mM acetoacetate was measured. The results are shown in FIG. 6A (VMAT). As an indicator of transport activity by the inhibitory neurotransmitter transporter, GABA transport activity of synaptic vesicles in the presence (+ acetoacetate) and absence (-acetoacetate) of 1 mM acetoacetate was measured. The results are shown in FIG. 6A (VIAAT).
　これらの結果に示されるように、本発明の阻害剤は、モノアミントランスポーターや抑制性神経伝達物質トランスポーターの対する阻害活性を有さないことが実証された。 As shown in these results, it was demonstrated that the inhibitor of the present invention has no inhibitory activity against monoamine transporters and inhibitory neurotransmitter transporters.
　（実施例５：小胞型グルタミン酸トランスポーター以外の興奮性神経伝達物質トランスポーターに対する阻害作用）
　本発明の阻害剤が、小胞型グルタミン酸トランスポーター以外の興奮性神経伝達物質トランスポーターに対する阻害作用を有するか否かを試験するために、小胞型グルタミン酸トランスポーター以外の興奮性神経伝達物質トランスポーターである小胞型アスパラギン酸トランスポーター（シアリン）および小胞型ヌクレオチドトランスポーター（ＶＮＵＴ）に対する阻害活性を測定した。 (Example 5: Inhibitory action on excitatory neurotransmitter transporters other than vesicular glutamate transporters)
In order to test whether the inhibitor of the present invention has an inhibitory action on excitatory neurotransmitter transporters other than vesicular glutamate transporters, excitatory neurotransmitter transceptors other than vesicular glutamate transporters are tested. The inhibitory activity against the vesicle-type aspartate transporter (sialin) and vesicle-type nucleotide transporter (VNUT), which are porters, was measured.
　（Ａ：アスパラギン酸トランスポーター（シアリン）のクローニング、発現、プロテオリポソームの調製、および、活性測定）
　（１．ＰＣＲによるマウスおよびヒトのシアリンcDNAのクローン化）
　以下のプライマーを用いたPCRによりマウスおよびヒトシアリンcDNAを得た。 (A: Cloning and expression of aspartate transporter (sialin), preparation of proteoliposome, and activity measurement)
(1. Cloning of mouse and human sialin cDNA by PCR)
Mouse and human sialin cDNA were obtained by PCR using the following primers.
　5’-caccatgaggcccctgcttcggg-3’（配列番号１３）マウスシアリンセンスプライマー
　5’-ccacggacacagaaactga-3’（配列番号１４）マウスシアリンセンスプライマー
　5’-caccatgaggtctccggttcgag-3’（配列番号１５）ヒトシアリンセンスプライマー
　5’-tcagtgtctgtgtccatggt-3’（配列番号１６）ヒトシアリンアンチセンスプライマー
　PCR反応は、94℃　2分の後、94℃　45秒、56℃　45秒、および、72℃　2分を３５サイクル行い、次に、72℃　5分インキュベートし、ExTaqBuffer (Takara)、0.25 mM dNTP mix、1.6 pmol/μl Primer、0.5 U Ex Taq (Takara)を加えてtotalvolume16 μlにした。 5'-caccatgaggcccctgcttcggg-3 '(SEQ ID NO: 13) mouse sialin sense primer 5'-ccacggacacagaaactga-3' (SEQ ID NO: 14) mouse sialin sense primer 5'-caccatgaggtctccggttcgag-3 '(SEQ ID NO: 15) human sialin sense primer 5'-tcagtgtctgtgtccatggt-3 '(SEQ ID NO: 16) Human sialin antisense primer The PCR reaction was performed at 94 ° C for 2 minutes, followed by 35 cycles of 94 ° C for 45 seconds, 56 ° C for 45 seconds, and 72 ° C for 2 minutes. The mixture was incubated at 72 ° C. for 5 minutes, and ExTaqBuffer (Takara), 0.25 mM dNTP mix, 1.6 pmol / μl Primer and 0.5 U Ex Taq (Takara) were added to make a total volume of 16 μl.
　pENTR/D-TOPOクローニングキット(No.K2400-20, Invitrogen)を用いてエントリーベクターにPCR産物を組み込んだ。方法は付属のプロトコールに準拠して行った。 The PCR product was incorporated into the entry vector using the pENTR / D-TOPO cloning kit (No. K2400-20, Invitrogen). The method was performed according to the attached protocol.
　（部位特異的変異体の作成）
　プライマーとして、以下の配列のプライマーを用いた：
5’-agctatgcgggccatgtgg-3’、（配列番号１７）マウスH183R1st PCRセンスプライマー
5’-accacagaggatcatgcataacc-3’（配列番号１８）マウスH183R1st PCR アンチセンスプライマー
5’-gtaaaacgacggccagtc-3’（配列番号１９）マウスH183R2nd PCRセンスプライマー。 (Creation of site-specific mutants)
Primers with the following sequences were used as primers:
5'-agctatgcgggccatgtgg-3 ', (SEQ ID NO: 17) mouse H183R1 st PCR sense primer
5'-accacagaggatcatgcataacc-3 '(SEQ ID NO: 18) mouse H183R1 st PCR antisense primer
5′-gtaaaacgacggccagtc-3 ′ (SEQ ID NO: 19) mouse H183R2 nd PCR sense primer.
　1st PCR反応は、94℃　2分の後、94℃　45秒、55℃　45秒、72℃　1分を３５サイクル繰り返し、そして、72℃　3分保温した。反応溶液は、ExTaqBuffer (Takara)、0.15 mM dNTP mix、1.6 pmol/μl Primer、0.5 U Ex Taq (Takara)を加えてtotalvolume16 μlにした。 1st PCR reaction was performed at 94 ° C. for 2 minutes, followed by 35 cycles of 94 ° C. for 45 seconds, 55 ° C. for 45 seconds, 72 ° C. for 1 minute, and incubated at 72 ° C. for 3 minutes. ExTaqBuffer (Takara), 0.15 mM dNTPs mix, 1.6 pmol / μl Primer, and 0.5 U Ex-Taq (Takara) were added to make a total volume of 16 μl.
　2nd PCR反応は、2ndPCRセンスプライマーと1st PCR反応産物とをプライマーとして用い、94℃　2分の加熱後、94℃　45秒、50℃　1分30秒、72℃　3分を３５サイクル繰り返し、そして、72℃　5分保温した。反応溶液は、1stPCR産物、ExTaq Buffer (Takara)、0.38 mM dNTP mix、1.3 pmol/μl Primer、0.5 U Ex Taq(Takara)を加えて総容量16μlにした。 2nd PCR reaction 2 using a nd PCR sense primer and 1st PCR reaction products as primers, after heating 94 ° C. 2 min, 94 ° C. 45 seconds, 50 ° C. 1 min 30 sec, repeating 35 cycles 72 ° C. 3 min, And it kept at 72 ° C. for 5 minutes. The reaction solution, 1 st PCR product, ExTaq Buffer (Takara), 0.38 mM dNTP mix, 1.3 pmol / μl Primer, and the total volume 16μl added to 0.5 U Ex Taq (Takara).
　（２．エントリーベクターへの連結）
　ＰＣＲフラグメントをＴＯＰＯクローニングキット（Invitrogen）を用いて、エントリーベクター（pENTR、Invitrogen）に組み込んだ。反応溶液（６μｌ）は、塩溶液　１μｌ（Invitrogen）、ベクター　10fmol（Invitrogen）、およびＰＣＲ産物　20fmolを含む溶液である。室温で１０分間反応させ、シアリンをエントリーベクターに組み込んだ。これをＴＯＰＯ反応液とした。 (2. Link to entry vector)
The PCR fragment was incorporated into an entry vector (pENTR, Invitrogen) using a TOPO cloning kit (Invitrogen). The reaction solution (6 μl) is a solution containing 1 μl of salt solution (Invitrogen), 10 fmol of vector (Invitrogen), and 20 fmol of PCR product. The reaction was carried out at room temperature for 10 minutes, and sialin was incorporated into the entry vector. This was used as a TOPO reaction solution.
　（３．形質転換）
　大腸菌Ｍａｃｈ－１コンピテントセル（Invitrogen）５０μｌに上記ＴＯＰＯ反応液２μｌを加えた。氷上で３０分間放置後、ＳＯＣ培地（Invitrogen）２５０μｌを添加し、３７℃で１時間反応させ、５０μｇ／ｍｌカナマイシンを含むＬＢプレートに全量を播種した。プレートを３７℃で一晩培養し、シングルコロニーをピックアップし、５０μｇ／ｍｌカナマイシンを含むＬＢ培地３ｍｌで一晩培養した。培養した大腸菌からQIAprepSpinMiniprep Kit(Qiagen)を用いて、シアリンを含むベクターを得た。 (3. Transformation)
2 μl of the above TOPO reaction solution was added to 50 μl of E. coli Mach-1 competent cell (Invitrogen). After leaving on ice for 30 minutes, 250 μl of SOC medium (Invitrogen) was added, reacted at 37 ° C. for 1 hour, and the whole amount was seeded on an LB plate containing 50 μg / ml kanamycin. The plate was cultured overnight at 37 ° C., a single colony was picked up, and cultured overnight in 3 ml of LB medium containing 50 μg / ml kanamycin. A vector containing sialin was obtained from the cultured Escherichia coli using QIAprepSpinMiniprep Kit (Qiagen).
　（４．ｐＤＥＳＴ１０への組換え）
　上記で調製したベクターから、ＬＲクロナーゼを用いてシアリンのｃＤＮＡをｐＤＥＳＴ１０ベクターへクローニングした。上記で調製したプラスミド１５０ｍｇにｐＤＥＳＴ１０プラスミド３００ｎｇとＬＲクロナーゼ４μｌを加え、２５℃で１時間インキュベートし、その後、プロテイナーゼＫを２μｌ加え、３７℃で３０分間インキュベートした。反応液を用いて、ＤＨ５αコンピテント大腸菌を形質転換した。形質転換したＤＨ５α細胞から、QIAprepSpin Miniprep Kit(Qiagen)を用いてプラスミドを回収し、ｐＤＥＳＴ１０／ＳＬＣ１７Ａ５とした。 (4. Recombination into pDEST10)
From the vector prepared above, sialin cDNA was cloned into the pDEST10 vector using LR clonase. To 150 mg of the plasmid prepared above, 300 ng of pDEST10 plasmid and 4 μl of LR clonase were added, incubated at 25 ° C. for 1 hour, and then 2 μl of proteinase K was added and incubated at 37 ° C. for 30 minutes. The reaction solution was used to transform DH5α competent E. coli. From the transformed DH5α cells, the plasmid was recovered using QIAprepSpin Miniprep Kit (Qiagen) to obtain pDEST10 / SLC17A5.
　（５．組換えバックミドの作製）
　BaculovirusExpression System with Gateway Technology（Invitrogen）を用いて、ｐＤＥＳＴ１０／ＳＬＣ１７Ａ５からシアリンのｃＤＮＡをバキュロウイルスゲノム（バックミド）に組み込んだ。 (5. Production of recombinant bacmid)
Using Baculovirus Expression System with Gateway Technology (Invitrogen), the sialin cDNA from pDEST10 / SLC17A5 was integrated into the baculovirus genome (bacmid).
　具体的には、ＤＨ１０Ｂａｃコンピテント細胞（Invitrogen）２５μｌにｐＤＥＳＴ１０／ＳＬＣ１７Ａ５　２０ｐｇを加え、氷上で３０分間放置後、４２℃、３０秒、ＳＯＣ培地２２５μｌ加えた。３７℃で４時間インキュベートし、５０μｇ／ｍｌカナマイシン、７μｇ／ｍｌゲンタマイシン、１０μｇ／ｍｌテトラサイクリンを含むＬＢプレートの播種し、３７℃で一晩インキュベートした。そしれ、ミニプレップ法にてバックミドを回収した。 Specifically, 20 pg of pDEST10 / SLC17A5 was added to 25 μl of DH10Bac competent cells (Invitrogen), left on ice for 30 minutes, and then 225 μl of SOC medium was added at 42 ° C. for 30 seconds. Incubated for 4 hours at 37 ° C., seeded with LB plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline and incubated overnight at 37 ° C. Well, the bacmid was recovered by the miniprep method.
　（６．ミニプレップ法；バックミド用）
　組換えバックミドの作製に用いたミニプレップ法は、以下の手順で行った。まず、５０μｇ／ｍｌカナマイシン、７μｇ／ｍｌゲンタマイシン、１０μｇ／ｍｌテトラサイクリンを添加したＬＢ培地３ｍｌに組換えバックミドを有するＤＨ１０Ｂａｃを植菌し、３７℃で培養した。培養した大腸菌を溶液１（５０ｍＭ　グルコース、２５ｍＭ　Ｔｒｉｓ／ＨＣｌ　ｐＨ８．０，１０ｍＭ　ＥＤＴＡ　ｐＨ８．０）２００μｌ中に懸濁し、次に、溶液２（０．２Ｍ　ＮａＯＨ、１％　ＳＤＳ）２００μｌを加え、転倒混和した。室温で５分間放置後、溶液３（３Ｍ　ＫＯＡｃ、１１．５％（ｖ／ｖ）酢酸）を２００μｌ加え、店頭混和した。そして、４℃で１０分間放置した後、遠心（１３，０００ｒｐｍ、１５分、４℃）し、上清を除いた。沈澱を、さらに、７０％エタノールで２回洗浄した。これにＴＥ緩衝液（１０ｍＭ　Ｔｒｉｓ／ＨＣｌ　ｐＨ８．０、１ｍＭ　ＥＤＴＡ）を無菌的に添加し、４℃に保存した。 (6. Miniprep method; for bacmid)
The miniprep method used for the production of the recombinant bacmid was performed according to the following procedure. First, DH10Bac having a recombinant bacmid was inoculated into 3 ml of LB medium supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline, and cultured at 37 ° C. The cultured E. coli is suspended in 200 μl of solution 1 (50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0), and then 200 μl of solution 2 (0.2 M NaOH, 1% SDS) is added, Mixed. After standing at room temperature for 5 minutes, 200 μl of solution 3 (3M KOAc, 11.5% (v / v) acetic acid) was added and mixed over the counter. After leaving at 4 ° C. for 10 minutes, the mixture was centrifuged (13,000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed. The precipitate was further washed twice with 70% ethanol. TE buffer (10 mM Tris / HCl pH 8.0, 1 mM EDTA) was aseptically added thereto, and stored at 4 ° C.
　（７．ウイルスの調製）
　本実施例に用いたウイルスは、以下の手順によって調製した。まず、３５ｍｍのペトリ皿に９×１０５個のＳｆ９細胞を播種した。培地を、０．３５ｍｇ／ｍｌの炭酸水素ナトリウムを添加したGrace'sInsect Medium（GIBCO）に交換した後、シアリンを含むバックミド１μｇと、cellfectin（Invitrogen）６μｌを用い、リポフェクション法にて、Ｓｆ９に感染させた。２７℃で５時間インキュベートした後、２ｍｌのcompleteTMN-FHに交換し、感染兆候が見られるまで培養し、培地を回収した。これをＰ１ウイルスとした。そして、１００ｍｍペトリ皿に６×１０６個のＳｆ９細胞を播種し（５０％コンフルエント）、１０倍段階希釈したウイルス液１ｍｌを添加し、室温で１時間振とうした。completeTMN-FH：４％Sea Plaque Agarose＝３：１となるように、混合したペトリ皿の培地を取り除いた後、これを１０ｍｌの重層アガロースを用いて２７℃で７～１０日密閉して培養し、形成したプラークをピックアップして、再度感染させ、７２時間後、この培地をＰ１ウイルスの場合と同様に回収し、Ｐ２ウイルスとした。 (7. Preparation of virus)
The virus used in this example was prepared by the following procedure. First, 9 × 10 5 Sf9 cells were seeded in a 35 mm Petri dish. After exchanging the medium with Grace's Insect Medium (GIBCO) supplemented with 0.35 mg / ml sodium hydrogen carbonate, infection with Sf9 was performed by lipofection using 1 μg of bacmid containing sialin and 6 μl of cellfectin (Invitrogen). I let you. After incubating at 27 ° C. for 5 hours, the medium was replaced with 2 ml of completeTMN-FH, cultured until signs of infection were observed, and the medium was collected. This was designated as P1 virus. Then, 6 × 10 6 Sf9 cells were seeded in a 100 mm Petri dish (50% confluent), 1 ml of a 10-fold diluted virus solution was added, and the mixture was shaken at room temperature for 1 hour. CompleteTMN-FH: 4% Sea Plaque Agarose = 3: 1 After removing the mixed Petri dish medium, this was sealed and cultured at 27 ° C for 7-10 days with 10 ml of layered agarose. The formed plaque was picked up and infected again, and after 72 hours, this medium was recovered in the same manner as in the case of the P1 virus, and designated as P2 virus.
　（８．細胞の回収と膜画分の可溶化）
　HighFive細胞にＰ２ウイルスをM.O.I.＝１で感染させ、２７℃で培養した。感染６０時間後の細胞をセルスクレーパーにより回収し、７００×ｇ　１０分間遠心して上清を取り除いた。これを破壊緩衝液（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０、１００ｍＭ　酢酸カリウム、１０％　グリセロール、５ｍＭ　ＤＴＴ、１μｇ／ｍｌ　ペプスタチンＡ（ペプチド研究所）、１μｇ／ｍｌ　ロイペプチン（ペプチド研究所））中に懸濁し、再度７００×ｇ　１０分間遠心して上清を取り除いた。これを破壊緩衝液で懸濁し、超音波処理（ＴＯＭＹultrasonicdisruptorにて、Output 4、３０秒×８回）後、７００×ｇ　１０分間遠心して上清を回収し、１００，０００×ｇ　１時間、４℃で超遠心して、得られた沈澱を膜画分とした。この分画を、可溶化緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、２％オクチルグルコシド（同仁化学）、１０％　グリセロール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）を添加し、ホモジナイザーを用いて懸濁し、１００，０００×ｇ　３０分の遠心操作を行い、その上清を可溶化画分とした。 (8. Cell recovery and solubilization of membrane fraction)
HighFive cells were infected with P2 virus at MOI = 1 and cultured at 27 ° C. Cells 60 hours after infection were collected with a cell scraper and centrifuged at 700 × g for 10 minutes to remove the supernatant. This was suspended in a disruption buffer (20 mM Tris-HCl pH 8.0, 100 mM potassium acetate, 10% glycerol, 5 mM DTT, 1 μg / ml pepstatin A (Peptide Institute), 1 μg / ml leupeptin (Peptide Institute)). The supernatant was removed again by centrifugation at 700 × g for 10 minutes. This was suspended in a disruption buffer, sonicated (TOMY Ultrasonic Disruptor, Output 4, 30 seconds × 8 times), centrifuged at 700 × g for 10 minutes, and the supernatant was collected. 100,000 × g for 1 hour, 4 hours Ultracentrifugation was performed at 0 ° C., and the resulting precipitate was used as a membrane fraction. To this fraction, solubilization buffer (20 mM MOPS-Tris pH 7.0, 2% octylglucoside (Dojindo), 10% glycerol, 1 μg / ml pepstatin A, 1 μg / ml leupeptin) was added, and a homogenizer was used. The suspension was subjected to centrifugation at 100,000 × g for 30 minutes, and the supernatant was used as a solubilized fraction.
　（９．アフィニティーカラムを用いたシアリンの精製）
　QIAGENNi-NTA super flowレジンをエコノカラムに充填し（１ｍＬ；５０％　スラリー）、蒸留水にて洗浄した後、ｐＨ８．０の可溶化緩衝液で平衡化した。ここに上記の可溶化画分を入れ、４℃、４時間攪拌しながら、吸着させた。これを１５ｍｌの洗浄緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、１％オクチルグルコシド、２０％　グリセロール、５ｍＭ　イミダゾール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）で洗浄し、溶出緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、１％オクチルグルコシド、２０％　グリセロール、６０ｍＭ　イミダゾール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）を用いて、精製シアリンを溶出した。 (9. Purification of sialin using affinity column)
The QIAGENNi-NTA super flow resin was packed in an Econo column (1 mL; 50% slurry), washed with distilled water, and equilibrated with a solubilization buffer having a pH of 8.0. The above-mentioned solubilized fraction was added thereto and adsorbed while stirring at 4 ° C. for 4 hours. This was washed with 15 ml of washing buffer (20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin), and eluted buffer (20 mM MOPS- Purified sialin was eluted using Tris pH 7.0, 1% octyl glucoside, 20% glycerol, 60 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin).
　（１０．精製したシアリンの再構成）
　精製したアスパラギン酸トランスポーター（シアリン）40μｇと大豆由来の脂質0.5 mgを混ぜ、－８０℃で10分以上インキュベーションした。これを手で素早く解かし、20mMMOPS-Tris(pH7.0)、0.15 M 酢酸ナトリウム、5 mM 酢酸マグネシウム、0.5 mM DTTを含む緩衝液で30倍希釈した。これを200,000×g、1時間、4℃で遠心し、上清を捨て、同じ緩衝液200μＬで再懸濁した。これを再構成リポソーム（プロテオリポソーム）として、以後の実験に用いた。 (10. Reconstitution of purified sialin)
40 μg of purified aspartic acid transporter (sialin) and 0.5 mg of soybean-derived lipid were mixed and incubated at −80 ° C. for 10 minutes or longer. This was quickly solved by hand, and diluted 30-fold with a buffer containing 20 mM POP-Tris (pH 7.0), 0.15 M sodium acetate, 5 mM magnesium acetate, and 0.5 mM DTT. This was centrifuged at 200,000 × g for 1 hour at 4 ° C., and the supernatant was discarded and resuspended in 200 μL of the same buffer. This was used as a reconstituted liposome (proteoliposome) in subsequent experiments.
　（１１．アスパラギン酸輸送活性の測定）
　再構成リポソームを20 mM MOPS-Tris(pH7.0)、0.15 M 酢酸カリウム、5 mM 酢酸マグネシウム、4 mM 塩化カリウムからなる反応液、または20mMMOPS-Tris(pH7.0)、54 mM 酢酸カリウム、5 mM 酢酸マグネシウム、100 mM 塩化カリウムからなる反応液に、2μＭ　バリノマイシン、0.1mML-[2,3-3H]アスパラギン酸(0.5 MBq/μmol)を加え、27℃で2分間インキュベーションした。再構成リポソームの添加を活性測定の開始とした。2分後に反応液120μＬをセファデックスG-50(fine)スピンカラムにて760×g、2分間、4℃で遠心した。カラムを通過した反応液に含まれる放射活性を液体シンチレーションカウンターにて測定し、アスパラギン酸の輸送活性を測定した。また、200μＭアセト酢酸は再構成リポソームを添加する前に加えた。 (11. Measurement of aspartate transport activity)
Reconstituted liposomes can be reacted with 20 mM MOPS-Tris (pH 7.0), 0.15 M potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride, or 20 mMOPS-Tris (pH 7.0), 54 mM potassium acetate, 5 2 μM valinomycin and 0.1 mM L- [2,3- 3 H] aspartic acid (0.5 MBq / μmol) were added to a reaction solution composed of mM magnesium acetate and 100 mM potassium chloride, and incubated at 27 ° C. for 2 minutes. The addition of reconstituted liposomes was the start of activity measurement. Two minutes later, 120 μL of the reaction solution was centrifuged at 760 × g for 2 minutes at 4 ° C. using a Sephadex G-50 (fine) spin column. The radioactivity contained in the reaction solution that passed through the column was measured with a liquid scintillation counter, and the transport activity of aspartic acid was measured. 200 μM acetoacetic acid was added before the reconstituted liposome was added.
　（Ｂ：ヌクレオチドトランスポーター（ＶＮＵＴ）のクローニング、発現、プロテオリポソームの調製、および、活性測定）
　（１．ＰＣＲによるＳＬＣ１７Ａ９遺伝子の単離）
　Ex TaqBuffer（TaKaRa）中に、0.2mM dNTP混合液、1pmol プライマー、1.5U Ex Taq (TaKaRa)を添加し５０μｌとしてＰＣＲを行った。使用したプライマーは、正方向プライマー（配列番号２０：５’－ＣＡＣＣＡＴＧＡＣＣＣＴＧＡＣＡＡＧＣＡＧＧＣＧＣＣＡＧＧＡ－３’）および逆方向プライマー（配列番号２１：５’－ＣＴＡＧＡＧＧＴＣＣＴＣＡＴＧＧＧＴＡＧＡＧＣＴＣ－３’）である。ＰＣＲ条件は、９４℃　３分で加熱した後、９４℃　３分、５６℃　３０秒、７２℃　２分のサイクルを３０サイクル行い、その後、７２℃で５分加熱を用いた。 (B: Cloning and expression of nucleotide transporter (VNUT), preparation of proteoliposome, and activity measurement)
(1. Isolation of SLC17A9 gene by PCR)
PCR was performed by adding 0.2 mM dNTP mixed solution, 1 pmol primer, and 1.5 U Ex Taq (TaKaRa) in Ex TaqBuffer (TaKaRa) to 50 μl. The primers used were the forward primer (SEQ ID NO: 20: 5′-CACCCATGACCCTGACAAGCAGGCGCCAGGA-3 ′) and the reverse primer (SEQ ID NO: 21 ′ 5′-CTAGAGGTCCCTCATGGTAGAGCTC-3 ′). As PCR conditions, after heating at 94 ° C. for 3 minutes, 30 cycles of 94 ° C. for 3 minutes, 56 ° C. for 30 seconds, 72 ° C. for 2 minutes were performed, and then heating at 72 ° C. for 5 minutes was used.
　（２．エントリーベクターへの連結）
　ＰＣＲフラグメントをＴＯＰＯクローニングキット（Invitrogen）を用いて、エントリーベクター（pENTR、Invitrogen）に組み込んだ。反応溶液（６μｌ）は、塩溶液　１μｌ（Invitrogen）、ベクター　10fmol（Invitrogen）、およびＰＣＲ産物　20fmolを含む溶液である。室温で１０分間反応させ、ＳＬＣ１７Ａ９をエントリーベクターに組み込んだ。これをＴＯＰＯ反応液とした。 (2. Link to entry vector)
The PCR fragment was incorporated into an entry vector (pENTR, Invitrogen) using a TOPO cloning kit (Invitrogen). The reaction solution (6 μl) is a solution containing 1 μl of salt solution (Invitrogen), 10 fmol of vector (Invitrogen), and 20 fmol of PCR product. The reaction was allowed to proceed at room temperature for 10 minutes, and SLC17A9 was incorporated into the entry vector. This was used as a TOPO reaction solution.
　（３．形質転換）
　大腸菌Ｍａｃｈ－１コンピテントセル（Invitrogen）５０μｌに上記ＴＯＰＯ反応液２μｌを加えた。氷上で３０分間放置後、ＳＯＣ培地（Invitrogen）２５０μｌを添加し、３７℃で１時間反応させ、５０μｇ／ｍｌカナマイシンを含むＬＢプレートに全量を播種した。プレートを３７℃で一晩培養し、シングルコロニーをピックアップし、５０μｇ／ｍｌカナマイシンを含むＬＢ培地３ｍｌで一晩培養した。培養した大腸菌からQIAprepSpinMiniprep Kit(Qiagen)を用いて、ＳＬＣ１７Ａ９を含むベクター（ｐＥＮＴＲ／ＳＬＣ１７Ａ９）を得た。このベクターを用いて、ＳＬＣ１７Ａ９のｃＤＮＡの配列決定を行った。ヒトＳＬＣ１７Ａ９の核酸配列を配列番号５に、アミノ酸配列を配列番号６に示す。 (3. Transformation)
2 μl of the above TOPO reaction solution was added to 50 μl of E. coli Mach-1 competent cell (Invitrogen). After leaving on ice for 30 minutes, 250 μl of SOC medium (Invitrogen) was added, reacted at 37 ° C. for 1 hour, and the whole amount was seeded on an LB plate containing 50 μg / ml kanamycin. The plate was cultured overnight at 37 ° C., a single colony was picked up, and cultured overnight in 3 ml of LB medium containing 50 μg / ml kanamycin. A vector (pENTR / SLC17A9) containing SLC17A9 was obtained from the cultured Escherichia coli using QIAprepSpinMiniprep Kit (Qiagen). SLC17A9 cDNA was sequenced using this vector. The nucleic acid sequence of human SLC17A9 is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
　（４．ｐＤＥＳＴ１０への組換え）
　上記で調製したｐＥＮＴＲ／ＳＬＣ１７Ａ９から、ＬＲクロナーゼを用いてＳＬＣ１７Ａ９のｃＤＮＡをｐＤＥＳＴ１０ベクターへクローニングした。ｐＥＮＴＲ／ＳＬＣ１７Ａ９プラスミド１５０ｎｇにｐＤＥＳＴ１０プラスミド３００ｎｇとＬＲクロナーゼ４μｌを加え、２５℃で１時間インキュベートし、その後、プロテイナーゼＫを２μｌ加え、３７℃で３０分間インキュベートした。反応液を用いて、ＤＨ５αコンピテント大腸菌を形質転換した。形質転換したＤＨ５α細胞から、QIAprepSpin Miniprep Kit(Qiagen)を用いてプラスミドを回収し、ｐＤＥＳＴ１０／ＳＬＣ１７Ａ９とした。 (4. Recombination into pDEST10)
From pENTR / SLC17A9 prepared above, SLC17A9 cDNA was cloned into pDEST10 vector using LR clonase. To 150 ng of pENTR / SLC17A9 plasmid, 300 ng of pDEST10 plasmid and 4 μl of LR clonase were added, incubated at 25 ° C. for 1 hour, and then 2 μl of proteinase K was added and incubated at 37 ° C. for 30 minutes. The reaction solution was used to transform DH5α competent E. coli. From the transformed DH5α cells, the plasmid was recovered using QIAprepSpin Miniprep Kit (Qiagen) to obtain pDEST10 / SLC17A9.
　（５．組換えバックミドの作製）
　BaculovirusExpression System with Gateway Technology（Invitrogen）を用いて、ｐＤＥＳＴ１０／ＳＬＣ１７Ａ９からＳＬＣ１７Ａ９のｃＤＮＡをバキュロウイルスゲノム（バックミド）に組み込んだ。 (5. Production of recombinant bacmid)
Using the Baculovirus Expression System with Gateway Technology (Invitrogen), the cDNA of pDEST10 / SLC17A9 to SLC17A9 was integrated into the baculovirus genome (bacmid).
　具体的には、ＤＨ１０Ｂａｃコンピテント細胞（Invitrogen）２５μｌにｐＤＥＳＴ１０／ＳＬＣ１７Ａ９　２０ｎｇを加え、氷上で３０分間放置後、４２℃、３０秒、ＳＯＣ培地２２５μｌ加えた。３７℃で４時間インキュベートし、５０μｇ／ｍｌカナマイシン、７μｇ／ｍｌゲンタマイシン、１０μｇ／ｍｌテトラサイクリンを含むＬＢプレートの播種し、３７℃で一晩インキュベートした。そしれ、ミニプレップ法にてバックミドを回収した。 Specifically, 20 ng of pDEST10 / SLC17A9 was added to 25 μl of DH10Bac competent cells (Invitrogen), left on ice for 30 minutes, and then 225 μl of SOC medium was added at 42 ° C. for 30 seconds. Incubated for 4 hours at 37 ° C., seeded with LB plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline and incubated overnight at 37 ° C. Well, the bacmid was recovered by the miniprep method.
　（６．ミニプレップ法；バックミド用）
　組換えバックミドの作製に用いたミニプレップ法は、以下の手順で行った。まず、５０μｇ／ｍｌカナマイシン、７μｇ／ｍｌゲンタマイシン、１０μｇ／ｍｌテトラサイクリンを添加したＬＢ培地３ｍｌに組換えバックミドを有するＤＨ１０Ｂａｃを植菌し、３７℃で培養した。培養した大腸菌を溶液１（５０ｍＭ　グルコース、２５ｍＭ　Ｔｒｉｓ／ＨＣｌ　ｐＨ８．０，１０ｍＭ　ＥＤＴＡ　ｐＨ８．０）２００μｌ中に懸濁し、次に、溶液２（０．２Ｍ　ＮａＯＨ、１％　ＳＤＳ）２００μｌを加え、転倒混和した。室温で５分間放置後、溶液３（３Ｍ　ＫＯＡｃ、１１．５％（ｖ／ｖ）酢酸）を２００μｌ加え、転倒混和した。そして、４℃で１０分間放置した後、遠心（１３，０００ｒｐｍ、１５分、４℃）し、上清を除いた。沈澱を、さらに、７０％エタノールで２回洗浄した。これにＴＥ緩衝液（１０ｍＭ　Ｔｒｉｓ／ＨＣｌ　ｐＨ８．０、１ｍＭ　ＥＤＴＡ）を無菌的に添加し、４℃に保存した。 (6. Miniprep method; for bacmid)
The miniprep method used for the production of the recombinant bacmid was performed according to the following procedure. First, DH10Bac having a recombinant bacmid was inoculated into 3 ml of LB medium supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline, and cultured at 37 ° C. The cultured E. coli is suspended in 200 μl of solution 1 (50 mM glucose, 25 mM Tris / HCl pH 8.0, 10 mM EDTA pH 8.0), and then 200 μl of solution 2 (0.2 M NaOH, 1% SDS) is added, Mixed. After standing at room temperature for 5 minutes, 200 μl of solution 3 (3M KOAc, 11.5% (v / v) acetic acid) was added and mixed by inversion. After leaving at 4 ° C. for 10 minutes, the mixture was centrifuged (13,000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed. The precipitate was further washed twice with 70% ethanol. TE buffer (10 mM Tris / HCl pH 8.0, 1 mM EDTA) was aseptically added thereto, and stored at 4 ° C.
　（７．ウイルスの調製）
　本実施例に用いたウイルスは、以下の手順によって調製した。まず、３５ｍｍのペトリ皿に９×１０５個のＳｆ９細胞を播種した。培地を、０．３５ｍｇ／ｍｌの炭酸水素ナトリウムを添加したGrace'sInsect Medium（GIBCO）に交換した後、ＳＬＣ１７Ａ９を含むバックミド１μｇと、cellfectin（Invitrogen）６μｌを用い、リポフェクション法にて、Ｓｆ９に感染させた。２７℃で５時間インキュベートした後、２ｍｌのcompleteTMN-FHに交換し、感染兆候が見られるまで培養し、培地を回収した。これをＰ１ウイルスとした。そして、１００ｍｍペトリ皿に６×１０６個のＳｆ９細胞を播種し（５０％コンフルエント）、１０倍段階希釈したウイルス液１ｍｌを添加し、室温で１時間振とうした。completeTMN-FH：４％Sea Plaque Agarose＝３：１となるように、混合したペトリ皿の培地を取り除いた後、これを１０ｍｌの重層アガロースを用いて２７℃で７～１０日密閉して培養し、形成したプラークをピックアップして、再度感染させ、７２時間後、この培地をＰ１ウイルスの場合と同様に回収し、Ｐ２ウイルスとした。 (7. Preparation of virus)
The virus used in this example was prepared by the following procedure. First, 9 × 10 5 Sf9 cells were seeded in a 35 mm Petri dish. After exchanging the medium with Grace's Insect Medium (GIBCO) supplemented with 0.35 mg / ml sodium bicarbonate, infection with Sf9 was performed by lipofection using 1 μg of bacmid containing SLC17A9 and 6 μl of cellfectin (Invitrogen). I let you. After incubating at 27 ° C. for 5 hours, the medium was replaced with 2 ml of completeTMN-FH, cultured until signs of infection were observed, and the medium was collected. This was designated as P1 virus. Then, 6 × 10 6 Sf9 cells were seeded in a 100 mm Petri dish (50% confluent), 1 ml of a 10-fold diluted virus solution was added, and the mixture was shaken at room temperature for 1 hour. CompleteTMN-FH: 4% Sea Plaque Agarose = 3: 1 After removing the mixed Petri dish medium, this was sealed and cultured at 27 ° C for 7-10 days with 10 ml of layered agarose. The formed plaque was picked up and infected again, and after 72 hours, this medium was recovered in the same manner as in the case of the P1 virus, and designated as P2 virus.
　（８．細胞の回収と膜画分の可溶化）
　HighFive細胞にＰ２ウイルスをM.O.I.＝１で感染させ、２７℃で培養した。感染６０時間後の細胞をセルスクレーパーにより回収し、７００×ｇ　１０分間遠心して上清を取り除いた。これを破壊緩衝液（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０、１００ｍＭ　酢酸カリウム、１０％　グリセロール、５ｍＭ　ＤＴＴ、１μｇ／ｍｌ　ペプスタチンＡ（ペプチド研究所）、１μｇ／ｍｌ　ロイペプチン（ペプチド研究所））中に懸濁し、再度７００×ｇ　１０分間遠心して上清を取り除いた。これを破壊緩衝液で懸濁し、超音波処理（ＴＯＭＹultrasonicdisruptorにて、Output 4、３０秒×８回）後、７００×ｇ　１０分間遠心して上清を回収し、１００，０００×ｇ　１時間、４℃で超遠心して、得られた沈澱を膜画分とした。この分画を、可溶化緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、２％オクチルグルコシド（同仁化学）、１０％　グリセロール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）を添加し、ホモジナイザーを用いて懸濁し、１００，０００×ｇ　３０分の遠心操作を行い、その上清を可溶化画分とした。 (8. Cell recovery and solubilization of membrane fraction)
HighFive cells were infected with P2 virus at MOI = 1 and cultured at 27 ° C. Cells 60 hours after infection were collected with a cell scraper and centrifuged at 700 × g for 10 minutes to remove the supernatant. This was suspended in a disruption buffer (20 mM Tris-HCl pH 8.0, 100 mM potassium acetate, 10% glycerol, 5 mM DTT, 1 μg / ml pepstatin A (Peptide Institute), 1 μg / ml leupeptin (Peptide Institute)). The supernatant was removed again by centrifugation at 700 × g for 10 minutes. This was suspended in a disruption buffer, sonicated (TOMY Ultrasonic Disruptor, Output 4, 30 seconds × 8 times), centrifuged at 700 × g for 10 minutes, and the supernatant was collected. 100,000 × g for 1 hour, 4 hours Ultracentrifugation was performed at 0 ° C., and the resulting precipitate was used as a membrane fraction. To this fraction, solubilization buffer (20 mM MOPS-Tris pH 7.0, 2% octylglucoside (Dojindo), 10% glycerol, 1 μg / ml pepstatin A, 1 μg / ml leupeptin) was added, and a homogenizer was used. The suspension was subjected to centrifugation at 100,000 × g for 30 minutes, and the supernatant was used as a solubilized fraction.
　（９．アフィニティーカラムを用いたＳＬＣ１７Ａ９の精製）
　QIAGENNi-NTA super flowレジンをエコノカラムに充填し（１ｍＬ；５０％　スラリー）、蒸留水にて洗浄した後、ｐＨ８．０の可溶化緩衝液で平衡化した。ここに上記の可溶化画分を入れ、４℃、４時間攪拌しながら、吸着させた。これを１５ｍｌの洗浄緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、１％オクチルグルコシド、２０％　グリセロール、５ｍＭ　イミダゾール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）で洗浄し、溶出緩衝液（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、１％オクチルグルコシド、２０％　グリセロール、６０ｍＭ　イミダゾール、１μｇ／ｍｌ　ペプスタチンＡ、１μｇ／ｍｌ　ロイペプチン）を用いて、精製ＳＬＣ１７Ａ９を溶出した。 (9. Purification of SLC17A9 using affinity column)
The QIAGENNi-NTA super flow resin was packed in an Econo column (1 mL; 50% slurry), washed with distilled water, and equilibrated with a solubilization buffer having a pH of 8.0. The above-mentioned solubilized fraction was added thereto and adsorbed while stirring at 4 ° C. for 4 hours. This was washed with 15 ml of washing buffer (20 mM MOPS-Tris pH 7.0, 1% octylglucoside, 20% glycerol, 5 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin), and eluted buffer (20 mM MOPS- Purified SLC17A9 was eluted using Tris pH 7.0, 1% octyl glucoside, 20% glycerol, 60 mM imidazole, 1 μg / ml pepstatin A, 1 μg / ml leupeptin).
　（１０．精製したヌクレオチドトランスポーター（ＶＮＵＴ）の再構成）
　精製したヌクレオチドトランスポーター（ＶＮＵＴ）40μｇと大豆由来の脂質0.5 mgを混ぜ、－８０℃で10分以上インキュベーションした。これを手で素早く解かし、20mMMOPS-Tris(pH7.0)、0.15 M 酢酸ナトリウム、5 mM 酢酸マグネシウム、0.5 mM DTTを含む緩衝液で30倍希釈した。これを200,000×g、1時間、4℃で遠心し、上清を捨て、同じ緩衝液200μＬで再懸濁した。これを再構成リポソーム（プロテオリポソーム）として、以後の実験に用いた。 (10. Reconstitution of purified nucleotide transporter (VNUT))
40 μg of the purified nucleotide transporter (VNUT) and 0.5 mg of soybean-derived lipid were mixed and incubated at −80 ° C. for 10 minutes or longer. This was quickly solved by hand, and diluted 30-fold with a buffer containing 20 mM POP-Tris (pH 7.0), 0.15 M sodium acetate, 5 mM magnesium acetate, and 0.5 mM DTT. This was centrifuged at 200,000 × g for 1 hour at 4 ° C., and the supernatant was discarded and resuspended in 200 μL of the same buffer. This was used as a reconstituted liposome (proteoliposome) in subsequent experiments.
　（１１．ＡＴＰ輸送活性の測定）
　再構成リポソームを20 mM MOPS-Tris(pH7.0)、0.15 M 酢酸カリウム、5 mM 酢酸マグネシウム、4 mM 塩化カリウムからなる反応液、または20mMMOPS-Tris(pH7.0)、54 mM 酢酸カリウム、5 mM 酢酸マグネシウム、100 mM 塩化カリウムからなる反応液に、２μＭ　バリノマイシン、ＡＴＰ（０．１ｍＭ　［α３２Ｐ］ＡＴＰ　３．７ＭＢｑ／μｍｏｌ）を加え、27℃で2分間インキュベーションした。再構成リポソームの添加を活性測定の開始とした。2分後に反応液120μＬをセファデックスG-50(fine)スピンカラムにて760×g、2分間、4℃で遠心した。カラムを通過した反応液に含まれる放射活性を液体シンチレーションカウンターにて測定し、アスパラギン酸の輸送活性を測定した。また、200μＭアセト酢酸は再構成リポソームを添加する前に加えた。 (11. Measurement of ATP transport activity)
Reconstituted liposomes can be reacted with 20 mM MOPS-Tris (pH 7.0), 0.15 M potassium acetate, 5 mM magnesium acetate, 4 mM potassium chloride, or 20 mMOPS-Tris (pH 7.0), 54 mM potassium acetate, 5 2 μM valinomycin and ATP (0.1 mM [α 32 P] ATP 3.7 MBq / μmol) were added to a reaction solution composed of mM magnesium acetate and 100 mM potassium chloride, and incubated at 27 ° C. for 2 minutes. The addition of reconstituted liposomes was the start of activity measurement. Two minutes later, 120 μL of the reaction solution was centrifuged at 760 × g for 2 minutes at 4 ° C. using a Sephadex G-50 (fine) spin column. The radioactivity contained in the reaction solution that passed through the column was measured with a liquid scintillation counter, and the transport activity of aspartic acid was measured. 200 μM acetoacetic acid was added before the reconstituted liposome was added.
　（Ｃ：小胞型アスパラギン酸トランスポーター（シアリン）および小胞型ヌクレオチドトランスポーター（ＶＮＵＴ）に対する阻害活性の測定）
　アスパラギン酸輸送に対するアセトアセテートの阻害活性（図７Ａ）、および、ＡＴＰ輸送に対するアセトアセテートの阻害活性（図７Ｂ）を測定した。各レーンの実験条件は、以下のとおりである：
　レーン１　４ｍＭ　Ｃｌ－存在下、バリノマイシン非添加、
　レーン２　４ｍＭ　Ｃｌ－存在下、２μＭ　バリノマイシン添加、
　レーン３　４ｍＭ　Ｃｌ－存在下、２μＭ　バリノマイシン添加、２００μＭ　アセトアセテート添加、
　レーン４　１００ｍＭ　Ｃｌ－存在下、バリノマイシン非添加、
　レーン５　１００ｍＭ　Ｃｌ－存在下、２μＭ　バリノマイシン添加、
　レーン６　１００ｍＭ　Ｃｌ－存在下、２μＭ　バリノマイシン添加、２００μＭ　アセトアセテート添加。 (C: Measurement of inhibitory activity against vesicle-type aspartate transporter (sialin) and vesicle-type nucleotide transporter (VNUT))
The inhibitory activity of acetoacetate on aspartate transport (FIG. 7A) and the inhibitory activity of acetoacetate on ATP transport (FIG. 7B) were measured. The experimental conditions for each lane are as follows:
Lane 1 4 mM Cl - presence, valinomycin not added,
Lane 2 4 mM Cl - presence, 2 [mu] M valinomycin added,
Lane 3 in the presence of 4 mM Cl − , 2 μM valinomycin added, 200 μM acetoacetate added,
Lane 4 100 mM Cl - presence, valinomycin not added,
Lane 5 2 μM valinomycin added in the presence of 100 mM Cl −
Lane 6 100 mM Cl - presence, 2 [mu] M valinomycin added, 200 [mu] M acetoacetate added.
　クロライド濃度が低い場合、アセトアセテートは小胞型アスパラギン酸トランスポーター（シアリン）および小胞型ヌクレオチドトランスポーター（ＶＮＵＴ）を阻害したが、その阻害は、クロライドイオン濃度を１００ｍＭまで増加することによって、減少／消失した。 At low chloride concentrations, acetoacetate inhibited the vesicular aspartate transporter (sialin) and vesicular nucleotide transporter (VNUT), but the inhibition was reduced by increasing the chloride ion concentration to 100 mM. / Disappeared.
　（実施例６：小胞からのグルタミン酸放出に対する阻害作用）
　本発明の化合物がグルタミン酸作動性の培養細胞からのグルタミン酸のエキソサイトーシスを阻害するか否かを検討した。 (Example 6: Inhibitory effect on glutamate release from vesicles)
It was investigated whether the compounds of the present invention inhibit glutamate exocytosis from glutamatergic cultured cells.
　具体的には、培養中の海馬ニューロンからのＫＣｌによって誘起されるグルタミン酸のエキソサイトーシスに対するアセトアセテートの効果を試験した。その結果、０．５ｍＭ以上のアセトアセテートを培養培地中へ添加した場合、グルタミン酸のエキソサイトーシスが阻害された（図８）。この阻害作用は、培地からアセトアセテートを除去することによって、完全に回復した（図８）。グルタミン酸作動性の島α細胞であるαＴＣ６細胞を用いた場合においても、同様の結果が得られた。 Specifically, the effect of acetoacetate on glutamate exocytosis induced by KCl from hippocampal neurons in culture was examined. As a result, glutamate exocytosis was inhibited when 0.5 mM or more of acetoacetate was added to the culture medium (FIG. 8). This inhibitory effect was completely restored by removing acetoacetate from the medium (FIG. 8). Similar results were obtained when αTC6 cells, which are glutamatergic islet α cells, were used.
　（実施例７：グルタミン酸輸送阻害剤による癲癇治療効果）
　本発明の化合物が癲癇治療薬として有用であるか否かを決定するために、４－アミノピリジン（４ＡＰ）によってグルタミン酸分泌と発作が誘起されたラットに対する本発明のグルタミン酸輸送阻害剤の効果を検討した。 (Example 7: Acupuncture treatment effect by glutamate transport inhibitor)
In order to determine whether the compound of the present invention is useful as an acupuncture drug, the effect of the glutamate transport inhibitor of the present invention on rats in which glutamate secretion and seizures were induced by 4-aminopyridine (4AP) was examined. did.
　４ＡＰを、マイクロピペットを用いて、ラット脳に局所投与し、グルタミン酸およびドーパミンの分泌（図９Ａ）、ならびに、発作（図９Ｄ）を測定した。 4AP was locally administered to the rat brain using a micropipette, and glutamate and dopamine secretion (FIG. 9A) and seizure (FIG. 9D) were measured.
　発作の評価は、以下のように行った：
各２０分間の行動変化を次の6段階に分け、スコア化した。
(0) 行動変化なし。てんかん様症状なし；
(1) 口や顔を動かす。身繕いをする。臭いをかぐ。体をかきむしる。首を振る。過度な行動；
(2) 頭を大きく揺らす。全身をふるわせる。全身硬直；
(3) 前足が攣縮する。後ろ足が伸展する；
(4) 後ずさりをする。よだれをだす。強直性-間代性けいれん；および、
(5) てんかん性の失神。
スコア化の方法は、MeursAら（Epilepsy Res. 2008, 78, 50-59）に従い、全タイムコースにおいてスコア化し、4APを加えてから一時間分の行動量Totalseizure severity score (TSSS)を測定値とした。 Seizures were assessed as follows:
Each 20-minute behavior change was divided into the following 6 stages and scored.
(0) No change in behavior. No epileptiform symptoms;
(1) Move your mouth and face. Dress up. I smell it. Scratch your body. Shake your head. Excessive behavior;
(2) Shake your head. Shake your whole body. Whole body stiffness;
(3) The forefoot cramps. The hind legs extend;
(4) Go back. Drool. Tonicity-clonic seizures; and
(5) Epilepsy fainting.
The scoring method was scored in all time courses according to MeursA et al. (Epilepsy Res. 2008, 78, 50-59). After adding 4AP, the action amount Totalseizure severity score (TSSS) for one hour was taken as the measured value. did.
　４ＡＰ投与（図９Ｂの下向きの矢印）の前後、循環液４０μｌを採取して、グルタミン酸量を測定した（図９Ｂの上向きの矢印）。これに並行して、サンプル中のドーパミン量も測定した（図９Ｂの上向きの矢印）。４ＡＰの投与によって、脳におけるグルタミン酸分およびドーパミン分泌、ならびに、重篤な発作が誘起された。本発明のグルタミン酸輸送阻害剤のひとつであるアセトアセテート（１０ｍＭ）の投与により、４ＡＰによって誘起されたグルタミン酸分泌は抑制されたが、ドーパミン分泌は、影響を受けなかった（図９ＢおよびＣ）。アセトアセテートを除去すると、再度、脳におけるグルタミン酸分およびドーパミン分泌、ならびに、重篤な発作が誘起された（図９Ｄ）。 Before and after 4AP administration (downward arrow in FIG. 9B), 40 μl of circulating fluid was collected and the amount of glutamic acid was measured (upward arrow in FIG. 9B). In parallel with this, the amount of dopamine in the sample was also measured (upward arrow in FIG. 9B). Administration of 4AP induced glutamate and dopamine secretion in the brain and severe seizures. Administration of acetoacetate (10 mM), one of the glutamate transport inhibitors of the present invention, suppressed glutamate secretion induced by 4AP, but dopamine secretion was not affected (FIGS. 9B and C). Removal of acetoacetate again induced glutamate and dopamine secretion in the brain and severe seizures (FIG. 9D).
　上記の結果から、本発明のグルタミン酸輸送阻害剤は、癲癇の治療薬として有用であることが示された。さらに、この結果は、癲癇のみならず、過剰な神経の興奮に関連する疾患および／または状態（たとえば、癲癇に加えて、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症からなる群から選択される疾患および／または状態）の治療薬として、本発明のグルタミン酸分泌阻害剤が有用であることを示す。 From the above results, it was shown that the glutamate transport inhibitor of the present invention is useful as a therapeutic agent for epilepsy. In addition, this result is not only for epilepsy but also for diseases and / or conditions associated with excessive neural excitation (eg, in addition to epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease) This shows that the glutamate secretion inhibitor of the present invention is useful as a therapeutic agent for diseases and / or conditions selected from the group consisting of marbleosis and osteoporosis.
　（実施例８：グルタミン酸輸送の阻害解除剤または活性化剤のスクリーニング）
　本実施例の方法を用いることによって、グルタミン酸輸送の阻害解除剤または活性化剤のスクリーニングすることができる。 (Example 8: Screening of inhibitor or activator of glutamate transport inhibition)
By using the method of this example, it is possible to screen for an inhibitor or an activator of glutamate transport inhibition.
　実施例２において調製した再構成リポソームに、アセトアセテートを終濃度１ｍＭで添加する。ネガティブコントロールとして、緩衝液Ａ（２０ｍＭ　ＭＯＰＳ－Ｔｒｉｓ　ｐＨ７．０、５ｍＭ　酢酸マグネシウム、１０４ｍＭの酢酸カリウム）を用いる。ネガティブコントロール、または、候補薬物（緩衝液Ａ中で０．０１ｍＭ～１ｍＭ）を、アセトアセテートを添加した再構成リポソームに添加し、室温で５分間放置する。 Acetoacetate is added to the reconstituted liposome prepared in Example 2 at a final concentration of 1 mM. As a negative control, buffer A (20 mM MOPS-Tris pH 7.0, 5 mM magnesium acetate, 104 mM potassium acetate) is used. A negative control or candidate drug (0.01 mM to 1 mM in buffer A) is added to the reconstituted liposomes to which acetoacetate has been added and left at room temperature for 5 minutes.
　次に、実施例２に記載したように、バリノマイシン存在下（＋バリノマイシン）、および非存在下（－バリノマイシン）における、５ｍＭのクロライドイオン濃度でのグルタミン酸輸送活性測定を行う。具体的には、試験管内に緩衝液Ｇ（20 mM MOPS-Tris pH 7.0、150 mM 酢酸カリウム、5 mM 酢酸マグネシウム、10 mM KCl）、2μＭ　バリノマイシン、[2,3-3H]L-グルタミン酸(0.5 MBq/pmol)、と阻害剤を加え、27℃水浴において3分間インキュベートした。ここに作製したプロテオリポソーム0.5μｇを加え反応を開始する。130μＬのサンプル液を取り、セファデックスG-50ファインに通す。760×ｇで2分間遠心し、その溶出液をクリアゾル３ｍＬに溶かし、液体シンチレーションカウンターにより計測し、輸送活性を測定する。ネガティブコントロールの場合と比較して、候補化合物を添加することにより高い輸送活性が観察された場合、その候補化合物を、阻害解除剤または活性化剤として同定する。 Next, as described in Example 2, glutamate transport activity is measured at a chloride ion concentration of 5 mM in the presence of valinomycin (+ valinomycin) and in the absence (−valinomycin). Specifically, buffer G (20 mM MOPS-Tris pH 7.0, 150 mM potassium acetate, 5 mM magnesium acetate, 10 mM KCl), 2 μM valinomycin, [2,3- 3 H] L-glutamic acid ( 0.5 MBq / pmol) and inhibitors were added and incubated in a 27 ° C. water bath for 3 minutes. The reaction is started by adding 0.5 μg of the prepared proteoliposome. Take 130 μL of sample solution and pass through Sephadex G-50 Fine. Centrifuge at 760 × g for 2 minutes, dissolve the eluate in 3 mL of clear sol, measure with a liquid scintillation counter, and measure transport activity. When a high transport activity is observed by adding a candidate compound as compared to the negative control, the candidate compound is identified as an inhibitor release agent or activator.
　以上のように、本発明の好ましい実施形態を用いて本発明を例示してきたが、本発明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に基づいて等価な範囲を実施することができることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。 As described above, the present invention has been exemplified using the preferred embodiment of the present invention, but the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.
　本発明によって、興奮性化学伝達阻害剤およびそのスクリーニング法が提供される。特に、興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害する興奮性化学伝達阻害剤およびそのスクリーニング法が本発明によって提供される。さらに、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症などの疾患／状態の治療、予防、および／または予後に有用である薬剤およびそのスクリーニング法が本発明によって提供される。 The present invention provides an excitatory chemical transmission inhibitor and a screening method thereof. In particular, the present invention provides an excitatory chemical transmission inhibitor that inhibits at least two vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration and a screening method thereof. In addition, drugs useful for the treatment, prevention, and / or prognosis of diseases / conditions such as epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis And screening methods thereof are provided by the present invention.

Claims (23)

  1. 興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種を、生理条件のクロライド濃度下で阻害するための、以下の式（Ｉ）の化合物：
    Figure JPOXMLDOC01-appb-C000001
     　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
    　Ｒ１～Ｒ４の少なくとも１つは酸素原子を含み、
    　Ｒ５は、Ｈ、直鎖アルキル、分岐アルキル、置換アルキル、アリール、アルキルアリールからなる群から選択される化合物またはその医薬として許容される塩を含有する、薬学的組成物。     A compound of the following formula (I) for inhibiting at least two vesicular neurotransmitter transporters for excitatory neurotransmitters under physiological conditions of chloride concentration:
    Figure JPOXMLDOC01-appb-C000001
     Wherein R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O
    At least one of R 1 to R 4 contains an oxygen atom;
    R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
  2. 請求項１に記載の薬学的組成物であって、
    　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択される、薬学的組成物。     A pharmaceutical composition according to claim 1, comprising:
    Wherein R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
  3. 請求項２に記載の薬学的組成物であって、
    　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択される、薬学的組成物。     A pharmaceutical composition according to claim 2, comprising:
    Wherein R 1 and R 2 are each independently selected from the group consisting of H and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
  4. 請求項１に記載の薬学的組成物であって、Ｒ３およびＲ４の少なくとも１つは酸素原子を含む、薬学的組成物。 The pharmaceutical composition according to claim 1, wherein at least one of R 3 and R 4 comprises an oxygen atom.
  5. 請求項４に記載の薬学的組成物であって、
    　ここで、Ｒ３およびＲ４は、一緒になって、Ｏである、薬学的組成物。     A pharmaceutical composition according to claim 4, comprising:
    Wherein R 3 and R 4 taken together are O.
  6. 請求項１に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、薬学的組成物。 2. The pharmaceutical composition of claim 1, wherein the compound is acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid, and A pharmaceutical composition selected from the group consisting of lactic acid.
  7. 請求項６に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、および、ピルビン酸からなる群から選択される、薬学的組成物。 The pharmaceutical composition according to claim 6, wherein the compound is selected from the group consisting of acetoacetate and pyruvic acid.
  8. 請求項１に記載の薬学的組成物であって、ここで、前記興奮性神経伝達物質に対する小胞型神経伝達物質トランスポーターの少なくとも２種が、グルタミン酸トランスポーター、アスパラギン酸トランスポーター、および、ヌクレオチドトランスポーターからなる群から選択される、薬学的組成物。 2. The pharmaceutical composition according to claim 1, wherein at least two of the vesicular neurotransmitter transporters for the excitatory neurotransmitter are glutamate transporter, aspartate transporter, and nucleotide. A pharmaceutical composition selected from the group consisting of transporters.
  9. 過剰な神経の興奮に関連する疾患および／または状態を、治療するため、予防するため、および／または、予後を改善するための薬学的組成物であって、以下の式（Ｉ）の化合物：
    Figure JPOXMLDOC01-appb-C000002
     　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、ＯＨ、および、低級アルキル（１～４Ｃ）、ならびに、一緒になって、Ｏからなる群から選択され、
    　Ｒ１～Ｒ４の少なくとも１つは酸素原子を含み、
    　Ｒ５は、Ｈ、直鎖アルキル、分岐アルキル、置換アルキル、アリール、アルキルアリールからなる群から選択される化合物またはその医薬として許容される塩を含有する、薬学的組成物。     A pharmaceutical composition for treating, preventing and / or improving the prognosis of diseases and / or conditions associated with excessive neural excitement comprising a compound of formula (I):
    Figure JPOXMLDOC01-appb-C000002
     Wherein R 1 and R 2 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H, OH, and lower alkyl (1-4C), and together O
    At least one of R 1 to R 4 contains an oxygen atom;
    R 5 is a pharmaceutical composition comprising a compound selected from the group consisting of H, straight chain alkyl, branched alkyl, substituted alkyl, aryl, alkylaryl or a pharmaceutically acceptable salt thereof.
  10. 請求項９に記載の薬学的組成物であって、
    　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、ＯＨ、ならびに、一緒になって、Ｏからなる群から選択される、薬学的組成物。     A pharmaceutical composition according to claim 9, comprising:
    Wherein R 1 and R 2 are each independently selected from the group consisting of H and OH, and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H and OH, and together O.
  11. 請求項１０に記載の薬学的組成物であって、
    　ここで、Ｒ１およびＲ２は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択され、
    　ここで、Ｒ３およびＲ４は、それぞれ独立して、Ｈ、および、一緒になって、Ｏからなる群から選択される、薬学的組成物。     A pharmaceutical composition according to claim 10, comprising
    Wherein R 1 and R 2 are each independently selected from the group consisting of H and together O.
    Wherein R 3 and R 4 are each independently selected from the group consisting of H and taken together O.
  12. 請求項９に記載の薬学的組成物であって、Ｒ３およびＲ４の少なくとも１つは酸素原子を含む、薬学的組成物。 The pharmaceutical composition according to claim 9, wherein at least one of R 3 and R 4 comprises an oxygen atom.
  13. 請求項１２に記載の薬学的組成物であって、
    　ここで、Ｒ３およびＲ４は、一緒になって、Ｏである、薬学的組成物。     A pharmaceutical composition according to claim 12, comprising:
    Wherein R 3 and R 4 taken together are O.
  14. 請求項９に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、薬学的組成物。 10. The pharmaceutical composition of claim 9, wherein the compound is acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid, and A pharmaceutical composition selected from the group consisting of lactic acid.
  15. 請求項１４に記載の薬学的組成物であって、ここで、前記化合物が、アセトアセテート、および、ピルビン酸からなる群から選択される、薬学的組成物。 15. The pharmaceutical composition according to claim 14, wherein the compound is selected from the group consisting of acetoacetate and pyruvic acid.
  16. 過剰な神経の興奮に関連する疾患および／または状態が、癲癇、炎症、振せん、とう痛、高血圧、虚血、パーキンソン病、アルツハイマー病、大理石症、および、骨粗相症からなる群から選択される、請求項９に記載の薬学的組成物。 The disease and / or condition associated with excessive neural excitation is selected from the group consisting of epilepsy, inflammation, tremor, pain, hypertension, ischemia, Parkinson's disease, Alzheimer's disease, marbleosis, and osteoporosis The pharmaceutical composition according to claim 9.
  17. 過剰な神経の興奮に関連する疾患および／または状態が、請求項９に記載の薬学的組成物。 10. The pharmaceutical composition according to claim 9, wherein the disease and / or condition associated with excessive neural excitation.
  18. 過剰な神経の興奮に関連する疾患および／または状態を、治療のため、予防のため、および／または、予後を改善するための候補化合物をスクリーニングするための方法であって、以下：
    （ａ）小胞型神経伝達物質トランスポーターを含む小胞を調製する工程；
    （ｂ）該小胞型神経伝達物質トランスポーターが輸送する興奮性神経伝達物質、該再構成した小胞、イオノフォア、および、生理条件のクロライド存在下で、該興奮性神経伝達物質の小胞内への輸送を検出する工程；
    （ｃ）該小胞型神経伝達物質トランスポーターが輸送する興奮性神経伝達物質、該再構成した小胞、イオノフォア、生理条件のクロライド、および、試験化合物の存在下で、該興奮性神経伝達物質の輸送を検出する工程；ならびに
    （ｄ）上記工程（ｂ）における輸送と、上記工程（ｃ）における輸送を比較して、該試験化合物が、輸送を阻害するか否かを指標として、該試験化合物が、過剰な神経の興奮に関連する疾患および／または状態を、治療のため、予防のため、および／または、予後を改善するための候補化合物であるか否かを決定する工程、
    を包含する、方法。     A method for screening for candidate compounds for treating, preventing and / or improving prognosis of diseases and / or conditions associated with excessive neural excitation, comprising:
    (A) preparing a vesicle comprising a vesicular neurotransmitter transporter;
    (B) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, and a vesicle of the excitatory neurotransmitter in the presence of chloride under physiological conditions Detecting the transport to
    (C) an excitatory neurotransmitter transported by the vesicular neurotransmitter transporter, the reconstituted vesicle, an ionophore, a physiological condition chloride, and a test compound; And (d) comparing the transport in the step (b) with the transport in the step (c), and using the test compound as an index to determine whether the test inhibits the transport. Determining whether the compound is a candidate compound for treating, preventing and / or improving prognosis of diseases and / or conditions associated with excessive neural excitability;
    Including the method.
  19. 前記小胞型神経伝達物質トランスポーターを含む小胞が、リポソームに、単離された小胞型神経伝達物質トランスポーターを再構成することによって調製される、請求項１８に記載の方法。 19. The method of claim 18, wherein the vesicle comprising the vesicular neurotransmitter transporter is prepared by reconstituting an isolated vesicular neurotransmitter transporter into a liposome.
  20. 前記イオノフォアがバリノマイシンである、請求項１８に記載の方法。 The method of claim 18, wherein the ionophore is valinomycin.
  21. 前記小胞型神経伝達物質トランスポーターが、グルタミン酸トランスポーター、アスパラギン酸トランスポーター、ヌクレオチドトランスポーターからなる群から選択される、請求項１８に記載の方法。 19. The method of claim 18, wherein the vesicular neurotransmitter transporter is selected from the group consisting of a glutamate transporter, an aspartate transporter, and a nucleotide transporter.
  22. 小胞型神経伝達物質トランスポーターの輸送活性について、阻害の解除もしくは活性化をする化合物をスクリーニングする方法であって、該方法は、以下：
    （１）小胞型神経伝達物質トランスポーターの阻害剤存在下において、該小胞型神経伝達物質トランスポーターの輸送活性を測定する工程；
    （２）候補化合物、および、上記（１）の阻害剤存在下において、該小胞型神経伝達物質トランスポーターの輸送活性を測定する工程；ならびに
    （３）上記（２）の活性が上記（１）の活性より高い場合に、該候補化合物を、阻害の解除能もしくは活性化能を有する化合物として同定する工程；
    を包含する、方法。     A method for screening a compound that cancels or activates the transport activity of a vesicular neurotransmitter transporter, which comprises the following:
    (1) measuring the transport activity of the vesicular neurotransmitter transporter in the presence of an inhibitor of the vesicular neurotransmitter transporter;
    (2) a step of measuring the transport activity of the vesicular neurotransmitter transporter in the presence of the candidate compound and the inhibitor of (1) above; and (3) the activity of (2) above (1) The candidate compound is identified as a compound having the ability to cancel or activate inhibition;
    Including the method.
  23. 請求項２２に記載の方法であって、ここで、前記阻害剤が、アセトアセテート、ピルビン酸、フェニルピルビン酸、３－ヒドロキシブチレート、α－ケト－β－メチル－バレリン酸、および、乳酸からなる群から選択される、方法。 24. The method of claim 22, wherein the inhibitor is from acetoacetate, pyruvic acid, phenylpyruvic acid, 3-hydroxybutyrate, α-keto-β-methyl-valeric acid, and lactic acid. A method selected from the group consisting of:
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