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WO2006064686A1 - Medicament et procede visant a prevenir ou a reduire la mort differee des cellules nerveuses - Google Patents

Medicament et procede visant a prevenir ou a reduire la mort differee des cellules nerveuses Download PDF

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Publication number
WO2006064686A1
WO2006064686A1 PCT/JP2005/022315 JP2005022315W WO2006064686A1 WO 2006064686 A1 WO2006064686 A1 WO 2006064686A1 JP 2005022315 W JP2005022315 W JP 2005022315W WO 2006064686 A1 WO2006064686 A1 WO 2006064686A1
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WIPO (PCT)
Prior art keywords
cell death
ischemia
neuronal cell
reperfusion
cerebral
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Ceased
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PCT/JP2005/022315
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English (en)
Japanese (ja)
Inventor
Seiji Shioda
Hirokazu Ohtaki
Hana Inoue
Yasunobu Okada
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Japan Science and Technology Agency
Showa University
National Institute of Natural Sciences
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Japan Science and Technology Agency
Showa University
National Institute of Natural Sciences
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Publication of WO2006064686A1 publication Critical patent/WO2006064686A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for protecting or relieving delayed cell death of nerve cells caused by ischemia due to blood flow disorder and reperfusion after ischemia, and protecting or relieving the delayed cell death. Therefore, the present invention relates to a drug for treating cerebrovascular diseases caused by the cell death. Furthermore, the present invention relates to a method for evaluating the above-mentioned cell death and a method for screening an agent that protects or rescues delayed neuronal cell death.
  • Cerebrovascular disease is one of the top causes of death in Japan. It is well known that cerebral blood flow blockage (cerebral ischemia) due to many cerebral infarctions and myocardial infarctions causes neuronal cell death (for example, see Non-Patent Document 1 and Non-Patent Document 2). Cerebral ischemic damage and ischemia ⁇ reperfusion injury (ie, damage caused by post-ischemic reperfusion) or hypoxic damage and hypoxia, reoxygenation damage (ie, reoxygenation after hypoxia) Neuronal cell death caused by the resulting disability significantly affects the patient's prognosis.
  • Apoptotic cell volume induced by non-ischemic stimulation induced by mitochondria-mediated stimulant (Staurosporin: STS), death receptor-mediated stimulant (Tumor Necrosis Factor—a: TNF ⁇ ), or Fas ligand in cultured cells
  • STS mitochondria-mediated stimulant
  • TNF ⁇ death receptor-mediated stimulant
  • Fas ligand Fas ligand in cultured cells
  • Non-Patent Document 10 protein tyrosine kinase (PTK) is involved in the function of volume-sensitive C1-channel (for example, see Non-Patent Document 10).
  • PTK protein tyrosine kinase
  • cerebral circulation is improved by inhibiting hemorrhoids, and as a result, neuronal cell death is suppressed (see Non-Patent Document 11).
  • Non-Patent Document 12 It is also known to be deceived (see Non-Patent Document 12).
  • hippocampal neurons have volume-sensitive C1-channels, and that acupuncture acts to suppress these hippocampal volume-sensitive C1-channels.
  • delayed neuronal cell death is cell death that occurs several days after ischemia / reperfusion. Drugs that are effective in preventing or remedying such delayed neuronal cell death have been completely displayed! Power against apoptosis-like neuronal death involving late neuronal death Drugs aimed at inhibiting apoptosis based on the inhibition of the spurase pathway may be under development, but due to the complex and diverse pathways of cell death, clinical applications are still in progress! ,. On the other hand, PTK inhibitors aimed at inhibiting metastasis of malignant tumors (ie cancer) have been developed, but no drugs for cerebral ischemic neuronal cell death have been developed.
  • the present invention pays attention to the activity control of the C ⁇ channel activated by the cell death signal, and a PTK inhibitor considered to be able to control the C ⁇ channel blocker and the C1-channel.
  • the drug according to the present invention is characterized in that it contains a C-blocker or a PTK inhibitor in order to protect or rescue nerve cell death under reperfusion conditions after cerebral ischemia.
  • the neuronal cell death is preferably delayed neuronal cell death in an individual.
  • the neuronal cell death is preferably accompanied by fragmentation of nuclear DNA.
  • the neuronal cell death is preferably accompanied by a marked decrease in the release of cytochrome c from mitochondria.
  • the drug according to the present invention is preferably accompanied by an improvement in a decrease in cerebral blood flow that occurs during cerebral ischemia and reperfusion after ischemia.
  • the C1-channel blocker is preferably 4,4′-diisothiocy anostilbene-2,2,1 disulf onic acid (DIDS).
  • DIDS 4,4′-diisothiocy anostilbene-2,2,1 disulf onic acid
  • the drug according to the present invention is preferably genistein ⁇
  • the neuronal cell death is cerebral infarction or myocardial infarction, Or it is preferable to occur during cerebral ischemia or reperfusion after cerebral ischemia, which occurs during heart transplantation or cerebrovascular anastomosis.
  • the method for protecting or relieving neuronal cell death includes the step of administering a C1-channel blocker or a PTK inhibitor to neurons under reperfusion conditions after ischemia. It is characterized by
  • the neuronal cell death is preferably delayed neuronal cell death.
  • the neuronal cell death is preferably accompanied by fragmentation of nuclear DNA.
  • the neuronal cell death is preferably accompanied by a remarkable decrease in the activity of mitochondrial release of cytochrome c causing cell death.
  • the C1-channel blocker is preferably 4, 4′-diisothiocy anostilbene-2, 2, 1 disulf onic acid (DIDS).
  • the PTK inhibitor is genistein!
  • the neuronal cell death is caused by cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia that occurs during heart transplantation or cerebral vascular anastomosis. Preferred to occur ,.
  • the method for evaluating neuronal cell death under reperfusion conditions after ischemia using the individual according to the present invention measures cell death when a C1-channel blocker or a protein tyrosine kinase inhibitor is used. And comparing the measured value obtained by the above measurement with a reference value obtained by measuring cell death in the case where a C1-channel blocker or a protein tyrosine kinase inhibitor is not used. Yes.
  • the neuronal cell death is preferably cerebral ischemic delayed neuronal cell death.
  • the method for evaluating neuronal cell death according to the present invention is performed at the time of cerebral ischemia or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia that occurs during cardiac transplantation or cerebral vascular anastomosis. It is preferable to evaluate the survival rate of nerve cells.
  • the method for evaluating neuronal cell death according to the present invention is preferably performed using the above-described process capability of measuring cell death as an indicator of the activity of mitochondria dehydrogenase.
  • the step of measuring the cell death comprises a nuclear DN.
  • the method for evaluating neuronal cell death according to the present invention further includes a step of measuring a change in cerebral blood flow.
  • the method for evaluating neuronal cell death according to the present invention further includes a step of temporarily reopening the both common carotid arteries after the mice are temporarily blocked.
  • the screening method according to the present invention is for screening in vitro for a factor that protects or rescues delayed cell death of neurons.
  • the step of measuring cell death includes mitochondrial dehydrogenase activity, nuclear DNA fragment i, caspase activity, mitochondrial force, and release of cytochrome c into the cytoplasm.
  • FIG. 1 is a schematic diagram showing an experimental schedule using a transient mouse both common carotid artery occlusion forebrain ischemia model.
  • FIG. 2 is a graph showing volume-sensitive C1-current in mouse hippocampal primary cultured neurons.
  • A is a view showing a state in which a gradually activated current is observed when a low osmotic pressure is applied to a mouse hippocampal primary cultured neuron under a whole cell patch clamp.
  • B is a graph showing the current characteristics observed in A.
  • FIG. C is a graph showing that the current observed in A is outwardly rectifying.
  • D is a graph showing that the activity of hippocampal neurons is suppressed by volume-sensitive C channel inhibitor DIDS.
  • E is a graph showing that the activity of hippocampal neuronal cells is suppressed by the volume sensitive C1-channel inhibitor genistein.
  • FIG. 3 A is a photograph comparing the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion morphologically stained with toluidine blue staining.
  • B compares the hippocampal CA1 region of cerebral ischemia 'reperfusion day 4 with morphological staining with toluidine blue staining when DIDS (0.48 mg / kg) was administered 15 minutes before and after reperfusion It is a photograph. Furthermore, it is a photograph comparing the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion with morphological staining by toluidine blue staining when DIDS (12 mgZkg) was administered 15 minutes before ischemia and after reperfusion.
  • D is a photograph comparing morphological staining of the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion with toluidine blue staining when DIDS (60 mg / kg) was administered 15 minutes before and after reperfusion. is there.
  • E shows cerebral ischemia 'reperfusion day 4 hippocampal CA1 region morphologically stained with toluidine blue staining when Genistein (0.24 mgZkg) was administered 15 minutes before and after reperfusion. It is a photograph.
  • F shows cerebral ischemia 'reperfusion day 4 hippocampal CA1 region morphologically stained with toluidine blue staining when Genistein (6.0 mg / kg) was administered 15 minutes before and after reperfusion It is a photograph.
  • G shows cerebral ischemia 'reperfusion day 4 hippocampal CA1 region morphologically stained with toluidine blue staining when Genistein (30 mg / kg) was administered 15 min before and after reperfusion It is a photograph.
  • H shows a comparison of morphological staining of the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion when Daidzein (6. OmgZkg) was administered 15 minutes before ischemia and after reperfusion. It is.
  • FIG. 4 is a graph comparing the number of remaining normal neurons in the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion shown in FIG.
  • FIG. 5A is a photograph of the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion stained with Fluoro-JadeB (FJB) staining.
  • B Staining the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion with Fluoro-JadeB (F JB) staining when DIDS (0.48 mg / kg) was administered 15 minutes before and after reperfusion It is a photograph.
  • C is a photograph of the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion stained with Fluoro-JadeB (FJB) when DIDS (12 mg / kg) was administered 15 minutes before and after reperfusion. is there.
  • D is a photograph of the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion stained with Fluoro-JadeB (FJB) when DIDS (60 mg / kg) was administered 15 minutes before and after reperfusion. It is.
  • E is a photograph of the hippocampal CA1 region stained with Fluoro-JadeB (FJB) staining on day 4 of cerebral ischemia / reperfusion when Genistein (0.24 mgZkg) was administered 15 minutes before and after reperfusion. is there.
  • F is a photograph of the hippocampal CA1 region stained with Fluoro-JadeB (FJB) stained on day 4 of cerebral ischemia 'reperfusion when Genistein (6.
  • G is a photograph of the hippocampal CA1 region on day 4 of cerebral ischemia 'reperfusion stained with Fluoro-JadeB (FJB) when Genistein (30 mgZkg) was administered 15 minutes before and after reperfusion.
  • H shows the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion when Daidzein (6. Omg / kg) is administered 15 minutes before and after reperfusion is stained with Fluoro-JadeB (FJB) staining It is a photograph.
  • FIG. 6 is a graph comparing the number of FJB positive cells in the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion shown in FIG.
  • FIG. 7 is a photograph showing the result of cytochrome c imnob fitting in the cytoplasmic fraction of the hippocampal region after cerebral ischemia / reperfusion.
  • FIG. 8 is a graph comparing local cerebral blood flow in the cerebral cortex in the middle cerebral artery region during cerebral ischemia / reperfusion.
  • volume-sensitive C1-channel exists in neurons in the hippocampal region of mice. Furthermore, the present inventors have shown that the volume-sensitive C1-channel can be inhibited by administering a C ⁇ channel blocking force or using a PTK inhibitor.
  • the present inventors used a cerebral ischemia / reperfusion model when both the common carotid arteries of a mouse were temporarily blocked and then reopened. Delayed neuronal cell death, morphological histological observation of living cells and Z or degenerated neurons, increased cytochrome c in the cytoplasm! ] Or by measuring local cerebral blood flow.
  • the present inventors have found that delayed neuronal cell death that occurs during cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis A therapeutic drug and Z or protective drug for this drug was found, and a method for determining the effect of the drug was established.
  • delayed neuronal cell death caused by cerebral ischemia / reperfusion is suppressed by repeatedly administering a C1-channel blocker or a PTK inhibitor into the vein. can do.
  • This rescue mechanism is responsible for apoptotic cell volume reduction (AVD This is thought to be achieved by suppressing the activity of the volume-sensitive Cl_ channel that leads to
  • the present invention is directed against ischemia / reperfusion neuronal injury targeting volume sensitive C1-channel activity, which is an early reaction of late neuronal cell death, and its activity signal. Provide new defense methods.
  • a "neural cell” is a neuron in vivo or a commercially available cultured cell derived from a neuron, or a brain derived from a biological force. Even primary cultured neurons.
  • ischemic 'reperfusion experimental system refers to ischemic culture cells or mice that are in vitro systems using cultured cells or in vivo systems using mice.
  • ischemia / reperfusion intends reperfusion performed after ischemia.
  • the present invention provides a drug for suppressing nerve cell death and a method for suppressing nerve cell death using the drug.
  • Nerve cell death occurs in various parts of the brain and causes various diseases.In particular, late neuronal cell death is considered to be a cell death that can be rescued due to its slow development. If such a means for suppressing cell death can be developed, an effective therapeutic or preventive agent for various diseases caused by the cell death can be obtained, which can be said to be very beneficial.
  • the agent according to the present invention is characterized by comprising a C1-channel blocker or a protein tyrosine kinase (PTK) inhibitor.
  • C1 channel blocking force
  • 4, 4 diisothiocyanostilbene—2, 2′—disulfonic acid (DIDS) is preferred.
  • the PTK inhibitor is preferably genistein.
  • the agent according to the present invention suppresses neuronal cell death caused by ischemia or reperfusion after ischemia, particularly delayed neuronal cell death (ie, protection or Can be used to rescue.
  • Neuronal cell death caused by ischemia or post-ischemic reperfusion may include cerebral or myocardial infarction or heart transplantation or cerebral vascular anastomosis Neuronal cell death that occurs during cerebral ischemia or reperfusion after cerebral ischemia.
  • apoptosis is involved in the induction of delayed neuronal cell death.
  • the term “apoptosis” is used interchangeably with “cell death by apoptosis” or “apoptotic cell death”.
  • the agent according to the present embodiment is preferably used for suppressing apoptosis of nerve cells caused by ischemia or reperfusion after ischemia. More preferably, the agent according to this embodiment can be used to suppress apoptosis induced by abnormalities occurring during ischemia in nerve cells. Abnormalities that occur during ischemia include, but are not limited to, cell swelling, tissue edema, and tissue destruction.
  • ischemia intends local ischemia. When ischemia occurs, the surrounding tissue changes in various ways (eg, apoptosis or necrosis) depending on the duration of ischemia, the sensitivity of the tissue to ischemia, or the presence or absence of vascular anastomoses at the site. ) Is generated.
  • ischemic injury intends an injury caused by an abnormality that occurs in a tissue due to ischemia. Forces that cause various abnormalities in tissues due to ischemia Usually, ischemic injury includes neuronal cell death and apoptotic cell volume reduction (AVD), and damage caused by post-ischemic reperfusion is also Included in injury.
  • a disease (disorder) that is a treatment target of the drug according to the present invention may be one of the above ischemic disorders, a combination of a plurality of ischemic disorders, or a combination with another disease. Moyo.
  • a marked increase in nuclear DNA fragmentation or mitochondrial force cytochrome c release into the cytoplasm can be used as an indicator. If such a phenomenon is observed, it can be judged that the cell death rate is high.
  • C1-channel blockers or protein tyrosin kinase (PTK) inhibitors are effective only against cell death caused by ischemia or post-ischemic reperfusion. It is easy to understand that it is effective for apoptotic cell death caused by other causes.
  • the present invention further provides a drug for preventing a disease caused by apoptosis.
  • diseases caused by apoptosis include, but are not limited to, cerebral infarction, myocardial infarction, vascular dementia, neurodegenerative diseases such as Alzheimer's disease, autoimmune diseases such as rheumatoid arthritis, and senile alopecia. .
  • the drug according to the present invention is a mammal (human, mouse, rat, usagi, inu, cat, ushi, horse.
  • the agent according to the present invention includes C1-channel blocker or protein tyrosine kinase (
  • PTK inhibitors may be used as is, but may contain a pharmaceutically acceptable carrier.
  • the drug according to the present invention can be produced according to a known method as a method for producing a pharmaceutical composition.
  • the medicament according to the present invention may be a pharmaceutical composition.
  • the pharmaceutically acceptable carrier used in the pharmaceutical composition can be selected according to the dosage form and dosage form of the pharmaceutical composition.
  • pharmacologically acceptable carriers include various organic or inorganic carrier materials that can be used as pharmaceutical materials, and excipients in solid formulations, It is incorporated as a lubricant, binder, or disintegrant, or as a solvent, solubilizer, suspension, isotonic agent, buffer, or soothing agent in a liquid preparation.
  • excipient examples include lactose, sucrose, D-manntol, xylitol, sorbitol, erythritol, starch, crystalline cellulose and the like, and examples of the lubricant include magnesium stearate and calcium stearate. , Talc and colloidal silica.
  • binder examples include pregelatinized starch, methylcellulose, crystalline cellulose, sucrose, D-mannitol, trenorose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polybulurpyrrolidone, and the like.
  • disintegrant examples include starch, carboxymethylcellulose, low-substituted hydroxypropinoresenorelose, canoleoxymethinoresenorelose canolecium, croscanolemellose sodium, carboxymethyl starch sodium, and the like. It is done.
  • Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, tricaprylin and the like.
  • solubilizers include polyethylene glycol, propylene glycol, D-mann-tol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, citrate. Sodium etc. are mentioned.
  • suspending agent examples include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate.
  • surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate.
  • hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxy methenoresenorelose, hydroxyethinoresenorelose, and hydroxypropinoresenorelose can be used.
  • Examples of the isotonic agent include sodium chloride salt, glycerin, D-manntol and the like.
  • buffer solutions of phosphate, acetate, carbonate, citrate, and the like examples include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.
  • Examples of the soothing agent include benzyl alcohol.
  • Examples of the preservative include noroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidants examples include sulfite and ascorbic acid.
  • the pharmaceutical composition according to the present embodiment can be produced according to a method commonly used in the field of pharmaceutical technology.
  • the content of the C1-channel blocking force mono- or protein tyrosine kinase (PTK) inhibitor is determined using the pharmaceutical composition, which will be described later, in consideration of the administration form, administration method, etc.
  • the C1—channel blocker or protein tyrosine kinase (PTK) inhibitor can be administered in a range of amounts.
  • Examples of the dosage form of the pharmaceutical composition according to the present embodiment include tablets, capsules (including soft force capsules and microcapsules), powders, granules, syrups and other oral preparations, or Examples include parenteral preparations such as injections, suppositories, pellets, and drops.
  • parenteral as used herein is intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and refers to the mode of administration including intranodal injection and infusion.
  • the dosage of the pharmaceutical composition according to this embodiment varies depending on the administration subject, administration route, symptom, and the like, but those skilled in the art will determine the optimal dose depending on the administration subject, administration route, symptom, etc. Appropriate conditions can be set as appropriate.
  • the pharmaceutical composition according to this embodiment can be administered by an appropriate administration route according to the preparation form.
  • the administration method is not particularly limited, and can be applied by internal use, external use or injection.
  • the injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, or the like.
  • the dosage of the pharmaceutical composition according to the present embodiment is appropriately set depending on the preparation form, administration method, purpose of use, and age, weight, and symptoms of the patient to whom the pharmaceutical is administered, and is not constant.
  • the dose of the active ingredient contained in the preparation is preferably 5 to 400 / ⁇ 8 per day for an adult.
  • an amount smaller than the above dose may be sufficient or may be necessary beyond the range.
  • Administration may be carried out within a desired dose range, in a single day or in several divided portions.
  • the pharmaceutical composition according to the present embodiment can be administered orally as it is, or can be added daily to any food or drink.
  • the drug according to the present invention may contain at least a C1-channel blocker or a protein synthase ( ⁇ ) inhibitor. That is, it should be noted that an agent containing a plurality of C channel blockers or protein tyrosine kinase ( ⁇ ) inhibitors is also included in the technical scope of the present invention.
  • apoptotic cell death can be suppressed.
  • administration of the drug according to the present invention can protect nerve cells during cerebral ischemia or reperfusion after cerebral ischemia that occurs during heart transplantation or cerebral vascular anastomosis.
  • diseases caused by apoptosis can be treated by administering the drug according to the present invention.
  • the present invention provides a method for evaluating neuronal cell death under reperfusion conditions after ischemia.
  • the method for evaluating neuronal cell death under post-ischemic reperfusion conditions according to the present invention comprises cell death when a C1-channel blocker or a protein tyrosine kinase inhibitor is used. And a step of comparing the measured value obtained by the above measurement with a reference value obtained by measuring cell death in the case where a C1-channel blocker or a protein tyrosine kinase inhibitor is not used. It is a feature.
  • the neuronal cell death is preferably delayed neuronal cell death.
  • the neuronal cell death evaluation method according to the present embodiment is a neuronal cell during cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis Assess the survival rate.
  • the method for evaluating neuronal cell death according to the present invention is preferably performed by using the process power of measuring cell death as described above, and the activity of mitochondria dehydrogenase as an index, but by performing fragmentation of nuclear DNA as an index. Also good.
  • the neuronal cell death evaluation method according to the present invention preferably further includes a step of measuring a change in cerebral blood flow.
  • the present invention provides a method for evaluating cell death caused by ischemia and post-ischemic reperfusion in a system using cultured cells in the in vitro port.
  • hypoxia 'reoxygenation injury ie, caused by reoxygenation after hypoxia
  • ischemia ⁇ reperfusion injury ie, damage caused by reperfusion after ischemia
  • the buffer solution used in this step is not particularly limited as long as it does not contain dalcose.
  • cells subjected to the hypoxic treatment may be cultured in an aerobic condition in a medium containing glucose.
  • anoxic conditions refers to an oxygen concentration of 1% or less. It is preferable that oxygen is hardly contained at all (0.01% or less). Accordingly, those skilled in the art will understand that the “anoxic conditions” described herein are cell cultures performed under carbon dioxide, nitrogen gas, and argon gas, for example, 95% argon gas + 5% carbon. It will be readily understood that the conditions of acid gas are also oxygen-free conditions.
  • the instrument and Z or culture method used for cell culture are appropriately selected from those known in the art. It will be apparent to those skilled in the art that the choice may be made.
  • the cell to be evaluated experiences reperfusion after ischemia. Therefore, by using the in vitro cell death evaluation method according to the present invention, cell death such as cell viability is evaluated before and after ischemia treatment or before and after reperfusion treatment after ischemia. By comparison, reperfusion cell death can be easily evaluated.
  • the cells to be evaluated are not particularly limited, and nerve cells, brain neurons, and brain glial cells are preferably used. Can do. By using nerve cells, cell death caused by ischemia or reperfusion after ischemia caused by cerebral infarction or myocardial infarction can be evaluated. By using cranial nerve cells or brain glial cells, it can be said that cell death caused by reperfusion after ischemia or ischemia caused by cerebral infarction or the like can be evaluated. In addition, as the above cells, commercially available cultured cells or primary cultured cells may be used.
  • the step of detecting cell death is not particularly limited.
  • a method of performing mitochondrial dehydrogenase activity as an index MTT method or MTS method
  • Methods that use nuclear DNA fragmentation as an indicator such as DNA ladder testing
  • methods that use the activity of intracellular proteolytic enzymes (caspases) that cause cell death and mitochondrial force that releases cytochrome c into the cytoplasm.
  • Examples include an index method, and a method using an index of nuclear morphological change (for example, nuclear concentration or fragmentation).
  • cytochrome c is released into the cytoplasm as well as mitochondrial force. Therefore, mitochondrial force is also measured as cytochrome c release into the cytoplasm Can be easily determined whether the force is apoptotic cell death.
  • nuclear morphological changes such as nuclear concentration and fragmentation occur. Therefore, the nuclear morphological changes can be observed with an electron microscope or the like using the nuclear morphological changes as an index. For example, it can be easily determined whether apoptotic cell death occurs.
  • the present invention provides a method for assessing cell death caused by ischemia and post-ischemic reperfusion in an in vivo animal system.
  • the cell death evaluation method according to the present invention includes a method of transiently blocking both common carotid arteries of a mouse in order to successfully perform reperfusion after ischemia and ischemia of the mouse according to the present invention described above. After that, use the cerebral ischemia / reperfusion model.
  • the method for evaluating neuronal cell death under reperfusion conditions after ischemia measures cell death when using a C1-channel blocker or a protein tyrosine kinase inhibitor, and measures the measurement. Compare the value with the reference value.
  • the present invention provides a method for screening in vitro a factor that protects or rescues delayed cell death of neurons.
  • the screening method according to the present invention includes a step of preparing cells that can be cultured by preferably using a culture system. In order to lead the prepared cultured cells to delayed cell death, as described above, hypoxia's reoxygenation injury may be induced rather than induction of ischemia / reperfusion injury.
  • the screening method according to the present invention comprises a step of preparing a cell for culturing; the cell is subjected to hypoxic conditions or anoxic conditions in a glucose-free medium. Including a hypoxic treatment step of culturing in step 1; a reoxygenation step of culturing cells subjected to the ischemic treatment under aerobic conditions in a medium containing dalcose; and a step of measuring cell death, and Compare cell death measured at the time of the hypoxia treatment step or at the time of the reoxygenation treatment step, or before and after adding the candidate factor into the medium, to the cell death measured without adding the candidate factor. Process.
  • the step of measuring cell death includes the mitochondrial dehydrogenase activity, fragmentation of DNA in the nucleus, caspase activity, and mitochondrial force as described above.
  • the index may be the release of cytochrome c into the nucleus or the change in the shape of the nucleus.
  • volume sensitive C1-channel-channel in hippocampal neurons was confirmed by the following method.
  • Hippocampal neurons were isolated from the fetus from which the mouse force on the 16th day of pregnancy was also collected. The resulting primary cultured hippocampal neurons were added with 10% urchin fetal serum, streptomycin, and penicillin. In a Dulbecco essential medium, the cells were cultured at 37 ° C in a 5% CO incubator. Culture 5
  • HEPMS Hydroxyethyl 1 piperazyl] ethanesulfonic acid
  • 2-AP 2 mM 4 aminobiperidine
  • glucose 5.5 mM glucose
  • osmotic pressure was adjusted by adjusting the mannitol concentration (normal) Osmotic pressure solution: 315 mOsm, low osmotic pressure solution: 280 m Osm).
  • the osmotic pressure was adjusted to 300 mOsm.
  • the pH both extracellular and internal fluids
  • the pH was adjusted to 7.4 using NMDG.
  • the current response was recorded by applying a test potential of ⁇ 60 mV as the holding potential of 40 mV.
  • step responses from 100mV to + lOOmV were given as needed, and current responses were recorded.
  • Fig. 1 shows the protocol. Specifically, C57ZBL6Crj (9-: L 1 week old, male) and jugular vein force from DIAS (0.48, 12, 60 mg / kg), genistein (0.24, 6.0) from 20 minutes before ischemia 30 mgZkg) or daidzein (30 mgZkg) was administered at a flow rate of 0.8 LZ min Zg body weight (240-300 ⁇ LZ mice) for 15 min.
  • the control group vehicle
  • received a solution PBS containing 10% DMSO and 10% polysorvate 80
  • cerebral ischemia / reperfusion was performed by occlusion of both common carotid arteries for 12 minutes.
  • volume-sensitive C1-channel force in mouse hippocampal primary cultured neurons We confirmed whether it was suppressed by DIDS and genistein, which are known as inhibitors (Fig. 2).
  • the inhibitory effect of 500 M DIDS was only seen on the positive potential and was voltage-dependent (Fig. 2D).
  • suppression by 1OO ⁇ M genistein was independent of the potential, and it exhibited the suppression effect at all potentials except near the reversal potential (Fig. 2E). This force confirmed that DIDS and genistein reliably suppressed the volume-sensitive C1-channel of hippocampal neurons.
  • Apoptosis induced by staurosporine (STS) in cultured rat neurons is reported to be rescued by DIDS, a blocker of C1-channels (Ta nabe S, Maeno E, Okada Y (2002) Prevention of staurosporine- induced apoptotic cell death by DIDS and SITS in rat cardiomyocytes in primary culture.
  • DIDS a blocker of C1-channels
  • FIG. 3 when neuronal death was induced by 12 minutes of ischemia, the nucleolus of the hippocampal CA1 region neurons became unclear when morphologically observed by toluidine blue staining. Reduction of pyramidal cells was observed (Fig. 3, arrow A). The number of remaining morphologically normal cells per fixed area decreased from about 60 before cerebral ischemia (results not shown) to 37.1 ( Figure 2, first bar graph). If DIDS (0.48, 12, 60 mgZkg), a C1-channel blocker, was administered 15 minutes before ischemia and after reperfusion, 0.48 mgZkg or When administered at 60 mgZkg, the effect was less pronounced (Figs. 3 and 5). However, the morphological change decreased in the 12 mg Zkg group (Fig. 3, D), and the number of remaining morphologically normal cells increased to 52.5 (Fig. 3, third bar graph).
  • Non-Patent Document 7 DIDS has also been reported to rescue apoptosis induced by staurosporine (STS) in cultured rat neurons (Tanabe S, Maeno E, Okada Y (2002) Prevention of staurosporme- induced apoptotic cell death by DI DS and SITS in rat cardiomyocytes in primary culture. "Jpn. J. Physiol. 52 (Suppl): S 34).
  • Non-Patent Document 11 Local cerebral blood flow was measured using a laser Doppler blood flow meter. As shown in Fig. 8, the local blood flow in the cerebral cortex during ischemia is reduced to 25% compared to before cerebral ischemia (100%). Return to the vicinity (in the figure, Genistein administration group (black circle), vehicle group (white circle) and control mouth (vehicle non-administration group)). However, when genistein (30 mgZkg) was administered for 15 minutes before cerebral ischemia, it increased to 30-35% of cerebral blood flow during ischemia, and the local cerebral blood flow after reperfusion was approximately 2%. Recover in minutes.
  • the present invention reveals the presence of volume-sensitive C1-channel in hippocampal primary cultured neurons, and suppresses the volume-sensitive C1-channel force ⁇ channel blocker or PTK inhibitor that controls cr channel. It was completed on the basis of finding that Furthermore, based on the knowledge found in the primary neuronal cultured cells, the inventor's original “Evaluation system for cerebral ischemia in individuals” is used to circulate by dissolving the drug in a solvent containing a solubilizing agent. It would have been impossible to develop a new method to improve the pharmacokinetics of the drug and to repeatedly administer the drug over several days as well as before cerebral ischemia.
  • the present invention By using the present invention, it is possible to suppress neuronal cell death in an individual, particularly delayed neuronal cell death caused by cerebral ischemia / reperfusion.
  • the administration route in the present invention is intravenous administration, application to clinical treatment becomes relatively easy.
  • the method of the repeated administration method used in the present invention is useful for the development of a useful technique for more efficiently applying the present invention.
  • this invention by applying this invention during cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebrovascular anastomosis, It is possible to reduce damage to the organ brain, especially nerve cells.

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Abstract

La présente invention a trait à la prévention ou à la réduction de la mort différée des cellules nerveuses touchant les cellules nerveuses suite à une ischémie résultant d’un trouble du flux sanguin et à une reperfusion post-ischémique; l’invention décrit un médicament destiné à prévenir ou à réduire la mort différée des cellules nerveuses telle que décrite ci-dessus, qui permet donc de traiter les accidents vasculaires cérébraux induits par la mort cellulaire ; un procédé permettant d’évaluer la mort cellulaire décrite ci-dessus; ainsi qu’un procédé de criblage d’un facteur de prévention ou de réduction de la mort différée des cellules nerveuses.
PCT/JP2005/022315 2004-12-13 2005-12-05 Medicament et procede visant a prevenir ou a reduire la mort differee des cellules nerveuses Ceased WO2006064686A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2949324A1 (fr) * 2014-05-27 2015-12-02 Consorci Institut Catala de Ciencies Cardiovasculars Prévention et/ou traitement d'une blessure de reperfusion / ischémie
WO2015181216A1 (fr) * 2014-05-27 2015-12-03 Consorci Institut Català De Ciències Cardiovasculars Prévention et/ou traitement de lésions par ischémie/reperfusion

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