JP2008056565A - Agents and methods for protecting or remedying delayed neuronal cell death - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective drug or method for protecting or relieving delayed nerve cell death due to ischemia / reperfusion of nerve cells.
An agent comprising a Cl ^- channel blocker or a protein tyrosine kinase (PTK) inhibitor is used.
[Selection figure] None

Description

  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 the cell by protecting or relieving the delayed cell death. The present invention relates to a drug for treating cerebrovascular disease caused by death. Furthermore, the present invention relates to a method for evaluating the 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 results in neuronal cell death (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Ischemic damage and ischemia / reperfusion injury (ie, damage caused by post-ischemic reperfusion) or hypoxic and hypoxia / reoxygenation disorders (ie, reoxygenation after hypoxia) The neuronal cell death caused by the resulting disorder greatly affects the patient's prognosis. However, at present, most of the methods applied to suppress neuronal cell death caused by ischemia / reperfusion that cause cerebral infarction, etc. are treated in the acute phase (for example, hyperbaric oxygen therapy, thrombolytic therapy with urokinase) And a radical scavenging method using edaravone, etc., and a therapeutic method and / or a therapeutic agent for rapid treatment at the time of cerebral ischemia is required (see, for example, Non-Patent Document 3 and Non-Patent Document 4) thing). Until now, many efforts have been made to develop drugs for protecting and / or suppressing neuronal cell death caused by this ischemia / reperfusion, but they have not yet become definitive.

It is known that cell death that does not occur rapidly after ischemia / reperfusion of the brain but occurs several days later, that is, so-called delayed neuronal cell death (see Non-Patent Document 5 and Non-Patent Document 6) ). The present inventors have found that a fish larva extract mainly composed of nucleic acids and protamine has an inhibitory effect on delayed neuronal cell death caused by ischemia (see Patent Document 1). thing). This delayed neuronal cell death is considered to be a rescueable cell death because of its slow development. This late cell death is known to be involved in apoptosis (see Non-Patent Document 7). Apoptotic Volume Decrease (AVD) induced by mitochondrial-mediated stimulators (Staurosporin: STS), death receptor-mediated stimulators (Tumor Necrosis Factor-α: TNFα), or Fas ligand It has been clarified that KCl efflux due to the action of Cl ⁻ channel and K ⁺ channel is involved (see Non-Patent Document 8). The present inventors have developed a method for preventing apoptosis by administering an agent that blocks the function of a volume-sensitive Cl ⁻ channel (see, for example, Patent Document 2). Furthermore, it has been reported that Cl ^- channel blockers (administered before and after cardiac excision) also act suppressively on neuronal apoptotic cell death that occurs during ectopic transplantation of rat hearts (non-patent literature). 9). However, the development of a method for protecting or relieving delayed ischemic cell death caused by cerebral ischemia / reperfusion targeting the Cl ⁻ channel has not yet been developed.

It is well known that protein tyrosine kinase (PTK) is involved in the function of the volume sensitive Cl ⁻ channel (see, for example, Non-Patent Document 10). It is known that cerebral circulation is improved by inhibiting PTK, and as a result, neuronal cell death is suppressed (see Non-Patent Document 11), and PTK is also activated during cerebral ischemia. It is known (see Non-Patent Document 12). However, development of a method for protecting or relieving ischemia / reperfusion late neuronal cell death targeting PTK has not yet been performed.
JP 2004-196701 A (published July 15, 2004) JP 2002-3402 (released on January 9, 2002) Mizushima H, Zhou CJ, Dohi K, Horai R, Asano M, Iwakura Y, Hirabayashi T, Arata S, Nakajo S, Takaki A, Ohtaki H, Shioda S. (2002) "Reduced postischemic apoptosis in the hippocampus of mice deficient in interleukin-1. "J. Comp. Neurol. 448: 203-216. De Keyser J, Sulter G, Luiten PG (1999) "Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing?" Trends Neurosci. 22: 535-540. Schaller B, Graf R. (2004) "Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy." J. Cereb. Blood Flow Metab. 24: 351-371. Beresford IJ, Parsons AA, Hunter AJ. (2003) "Treatments for stroke." Expert. Opin. Emerg. Drugs. 8: 103-122. Kirino T. (1982) "Delayed neuronal death in the gerbil hippocampus following ischemia." Brain Res. 239: 57-69. Kirino T. (2000) "Delayed neuronal death." Neuropathology. 20 (Suppl): S95-S97. Zheng Z, Zhao H, Steinberg GK, Yenari MA (2003) "Cellular and molecular events underlying ischemia Induced neuronal apoptosis" Drug News Perspect. 16: 497-503. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. (2000) "Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis." Proc. Natl. Acad. Sci. USA 97: 9487-9492. Mizoguchi K, Maeta H, Yamamoto A, Oe M, Kosaka H. (2002) "Amelioration of myocardial global ischemia / reperfusion injury with volume-regulatory chloride channel inhibitors in vivo." Transplantation. 73: 1185-93. Abdullaev IF, Sabirov RZ, Okada Y. (2003) "Upregulation of swelling-Lctivated Cl- channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells." J. Physiol. 549: 749-758. Paul R, Zhang ZG, Eliceri BP, Jiang Q, Boccia AD, Zhang RL, Chopp M, Cheresh DA. (2001) "Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke." Nat. Med. 7: 222-227. Hon XY, Zhang GY, Yah JZ, Lin Y. (2003) "Increased tyrosine phosphorylation of alpha (1C) subunits of L-type voltage-gated calcium channels and interactions among Src / Fyn, PSD-95 and alpha (1C) in rat hippocampus after transient brain ischemia. "Brain Res. 979: 43-50.

As described above, late cell death is cell death that occurs several days after ischemia / reperfusion. No effective drug has been developed for preventing or remedying such late cell death. The present invention pays attention to the activity control of the Cl ⁻ channel activated by the cell death signal, and is used by ischemia / reperfusion by using a Cl ⁻ channel blocker and a PTK inhibitor considered to be able to control the Cl ⁻ channel. To provide a completely new method for protecting and / or relieving delayed cell death of nerve cells, and to prevent delayed neuronal cell death inhibitor and a disease caused by delayed cell death containing the cell death inhibitor It aims to provide a therapeutic drug.

Agents according to the present invention, in order to protect or rescue neuronal cell death in reperfusion conditions following ischemia, Cl ^- is characterized in that it comprises a channel blockers or PTK inhibitor.

  In the drug according to the present invention, the neuronal cell death is preferably delayed neuronal cell death.

  In the drug according to the present invention, the neuronal cell death is preferably accompanied by fragmentation of nuclear DNA.

  In the medicament according to the present invention, it is preferable that the neuronal cell death is accompanied by a significant 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.

In the drug according to the present invention, the Cl ^- channel blocker is preferably 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS).

  In the drug according to the present invention, the PTK inhibitor is preferably genistin.

  In the drug according to the present invention, it is preferable that the neuronal cell death occurs at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in cardiac transplantation or cerebral vascular anastomosis.

The method for protecting or rescue neuronal cell death according to the present invention, Cl ^- and a channel blockers or PTK inhibitor characterized by comprising the step of administering to neuronal cells in reperfusion conditions following ischemia.

  In the method according to the present invention, the neuronal cell death is preferably delayed neuronal cell death.

  In the method according to the present invention, the neuronal cell death is preferably accompanied by fragmentation of nuclear DNA.

  In the method according to the present invention, the neuronal cell death is preferably accompanied by a significant decrease in the activity of cytochrome c release from mitochondria causing cell death.

In the method according to the present invention, the Cl ^- channel blocker is preferably 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS).

  In the method according to the present invention, the PTK inhibitor is preferably genistin.

  In the method according to the present invention, the neuronal cell death is preferably caused during cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in cardiac transplantation or cerebral vascular anastomosis.

Evaluation of neuronal cell death in reperfusion conditions after ischemia according to the present invention, Cl ^- step of measuring cell death in the case of using the channel blocker or a protein tyrosine kinase inhibitor; measurements obtained by and the measurement Comparing the value to a reference value obtained by measuring cell death without using a Cl ^- channel blocker or a protein tyrosine kinase inhibitor.

  In the method for evaluating neuronal cell death according to the present invention, the neuronal cell death is preferably delayed neuronal cell death.

  The method for evaluating neuronal cell death according to the present invention comprises determining the survival rate of neurons at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in cardiac transplantation or cerebral vascular anastomosis. It is preferable to evaluate.

  In the method for evaluating neuronal cell death according to the present invention, the step of measuring cell death is preferably performed using the activity of mitochondrial dehydrogenase as an index.

  In the method for evaluating neuronal cell death according to the present invention, the step of measuring cell death is preferably performed using fragmentation of nuclear DNA as an index.

  The neuronal cell death evaluation method according to the present invention preferably further includes a step of measuring a change in cerebral blood flow.

  Preferably, the method for evaluating neuronal cell death according to the present invention further includes a step of temporarily reopening the both common carotid arteries of the mouse and then reopening them.

  By using the present invention, neuronal cell death, particularly delayed neuronal cell death caused by cerebral ischemia / reperfusion can be suppressed. When the administration route in the present invention is intravenous administration, application to clinical treatment becomes relatively easy. In addition, by applying the present invention at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in cardiac transplantation or cerebral vascular anastomosis, It is possible to reduce damage to certain brains, especially nerve cells.

No effective drugs have been developed to protect or rescue late neuronal cell death. The present inventors have intensively studied to solve the above problems. Specifically, the present inventors used mouse hippocampal primary cultured neurons,
(1) Is there a volume sensitive Cl ⁻ channel in mouse hippocampal primary cultured neurons?
(2) If present, it was examined whether the volume-sensitive Cl ⁻ channel of mouse hippocampal primary cultured neurons is suppressed by the above-described Cl ⁻ channel blocker or PTK inhibitor. Furthermore, using a transient mouse cerebral ischemia model,
(3) whether or not mouse cerebral ischemia / reperfusion delayed neuronal cell death can be suppressed by administering a Cl ^- channel blocker;
(4) Whether it can also be suppressed by administering a mouse cerebral ischemia / reperfusion delayed neuronal death PTK inhibitor,
(5) Whether or not apoptosis that occurs upon induction of mouse brain ischemia / reperfusion delayed neuronal death can be suppressed,
(6) We examined whether cerebral circulation blood flow during cerebral ischemia / reperfusion could be improved.

As a result, the present inventors revealed for the first time that volume-sensitive Cl ⁻ channels exist in neurons in the hippocampal region of mice. The present inventors have, Cl ^- or administering a channel blockers, or by the use of a PTK inhibitor, the volume-sensitive Cl ^- revealed that it is possible to inhibit the channel.

  Furthermore, based on these findings, the present inventors used a cerebral ischemia / reperfusion model in which both common carotid arteries of mice were transiently blocked and then reopened to prevent delayed neuronal cell death. Evaluation by morphological histological observation of living cells and / or degenerated neurons, increase of cytochrome c in the cytoplasm, or measurement of local cerebral blood flow.

  As a result, the present inventors have developed a therapeutic agent for delayed neuronal cell death that occurs at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis In addition to finding protective drugs and / or methods to determine the effects of the drugs.

With the present invention, the delayed neuronal death caused by cerebral ischemia-reperfusion, Cl intravenously ^- can be inhibited by or administered channel blocker, or administration of PTK inhibitor . This rescue mechanism is thought to be achieved by inhibiting the activation of volume-sensitive Cl ⁻ channels that lead to apoptotic cell volume reduction (AVD). Thus, the present invention provides a novel protection method against ischemia / reperfusion neuronal injury targeting volume-sensitive Cl ^- channel activation, which is an early reaction of late neuronal cell death, and its activation signal. I will provide a.

  As used herein, a “neuron cell” is a neuron in vivo or a commercially available cultured cell derived from a neuron, or a primary cultured neuron derived from a brain taken out from a living body. It may be.

  As used herein, the term “ischemia / reperfusion experimental system” refers to cultured cells or mice in an in vitro system using cultured cells or in an in vivo system using mice. ) Experimental systems that allow for reperfusion (or reoxygenation and glucose re-administration) treatment after treatment are contemplated. Further, as used herein, the term “ischemia / reperfusion” intends reperfusion performed after ischemia.

(1) Agent and method for protecting or relieving nerve cell death The present invention provides an agent for suppressing nerve cell death and a method for suppressing nerve cell death using the agent. 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 a means for suppressing cell death can be developed, an effective therapeutic agent or preventive agent for various diseases caused by the cell death can be obtained, which can be said to be very beneficial.

Agents according to the present invention, Cl ^- characterized in that it comprises a channel blockers or protein tyrosine kinase (PTK) inhibitors. In one embodiment, the Cl ^- channel blocker is preferably 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). In other embodiments, the PTK inhibitor is preferably genistein.

  In a preferred embodiment, the agent according to the present invention can be used to inhibit (ie, protect or rescue) neuronal cell death caused by ischemia or post-ischemic reperfusion, particularly delayed neuronal cell death. . Nerve cell death caused by ischemia or post-ischemic reperfusion includes cerebral infarction or myocardial infarction, or nerves that occur during cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis Examples include cell death.

  As described above, it is known that apoptosis is involved in the induction of delayed neuronal cell death. As used herein, the term “apoptosis” is used interchangeably with “cell death by apoptosis” or “apoptotic cell death”.

  The agent according to this embodiment is preferably used for suppressing apoptosis of nerve cells caused by ischemia or reperfusion after ischemia. More preferably, the agent according to the present 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.

  As used herein, the term “ischemia” is intended for local anemia. When ischemia occurs, the surrounding tissue changes variously (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. Produce. As used herein, the term “ischemic injury” intends an injury caused by an abnormality that occurs in tissue due to ischemia. Although ischemia usually causes various abnormalities in tissues, ischemic injury includes neuronal cell death and apoptotic cell volume reduction (AVD), and injury caused by post-ischemic reperfusion is also an ischemic injury. include. The disease (disorder) that is the treatment target of the drug according to the present invention may be one of the above ischemic disorders, a plurality of ischemic disorders, or a combination with another disease. Also good.

  When assessing apoptotic cell death, a marked decrease in nuclear DNA fragmentation or cytochrome c release from mitochondria can be used as an indicator. When such a phenomenon is observed, it can be determined that the cell death rate is high.

It should be noted that those skilled in the art who have read this specification are that Cl ^- channel blockers or protein tyrosine kinase (PTK) inhibitors are not only effective against cell death caused by ischemia or post-ischemic reperfusion, It is easily understood that it is effective for apoptotic cell death caused by other causes.

  The present invention further provides an agent 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, senile alopecia and the like. .

  The drug according to the present invention can be applied to mammals (human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.).

Agents according to the present invention, Cl ^- channel blockers or protein tyrosine kinase (PTK) inhibitor may be used as it is, it may include 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.

  In one embodiment, 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.

  As used herein, pharmacologically acceptable carriers include various organic or inorganic carrier substances that can be used as pharmaceutical materials, and include excipients, lubricants, and binders in solid formulations. Or a disintegrant, or a solvent, a solubilizing agent, a suspending agent, an isotonic agent, a buffering agent, or a soothing agent in a liquid preparation.

  Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica. Etc.

  Examples of the binder include pregelatinized starch, methylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

  Examples of the disintegrant include starch, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, and the like.

  Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, tricaprylin and the like.

  Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  As the suspending agent, for example, a surfactant such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, or polyvinyl alcohol, polyvinylpyrrolidone, Examples include hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like.

  Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.

  Examples of soothing agents include benzyl alcohol.

  Examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Examples of the antioxidant include sulfite and ascorbic acid.

The pharmaceutical composition which concerns on this embodiment can be manufactured in accordance with a method conventionally used in the formulation technical field. The content of the Cl ^- channel blocker or protein tyrosine kinase (PTK) inhibitor in the pharmaceutical composition according to the present embodiment is within the dosage range described below using the pharmaceutical composition in consideration of the administration form, administration method and the like. There is no particular limitation as long as it is an amount that allows administration of a Cl ^- channel blocker or a protein tyrosine kinase (PTK) inhibitor.

  Examples of the dosage form of the pharmaceutical composition according to this embodiment include tablets, capsules (including soft capsules and microcapsules), powders, granules, syrups and other oral preparations, or injections, suppositories, and pellets. And parenteral agents such as drops. The term “parenteral” as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular 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 appropriately set the optimum conditions according to the administration subject, administration route, symptom, etc. be able to.

  The pharmaceutical composition according to the present 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 according to the formulation form, administration method, purpose of use, and age, weight, and symptoms of the patient to whom the pharmaceutical is administered, and is not constant. In general, the dose of the active ingredient contained in the preparation is preferably 5 to 400 μg / kg per day for an adult. Of course, since the dosage varies depending on various conditions, an amount smaller than the above dosage may be sufficient or may be necessary beyond the range. Administration may be carried out in a single dose or divided into several doses within a desired dose range. Moreover, the pharmaceutical composition according to the present embodiment can be orally administered as it is, or can be added to any food or drink and taken daily.

Thus, agents according to the present invention, at least, Cl ^- said to it, including channel blockers or protein tyrosine kinase (PTK) inhibitors. That is, it should be noted that an agent containing a plurality of Cl ^- channel blockers or protein tyrosine kinase (PTK) inhibitors is also included in the technical scope of the present invention.

  By administering the drug according to the present invention, apoptotic cell death can be suppressed. In addition, by administering the drug according to the present invention, it is possible to protect nerve cells during cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebrovascular anastomosis. Furthermore, diseases caused by apoptosis can be treated by administering the drug according to the present invention.

(2) Method for evaluating cell death caused by ischemia and reperfusion after ischemia The present invention provides a method for evaluating neuronal cell death under reperfusion conditions after ischemia. Evaluation of neuronal cell death in reperfusion conditions after ischemia according to the present invention, Cl ^- step of measuring cell death in the case of using the channel blocker or a protein tyrosine kinase inhibitor; measurements obtained by and the measurement Comparing the value to a reference value obtained by measuring cell death without using a Cl ^- channel blocker or a protein tyrosine kinase inhibitor. In one embodiment, the method for evaluating neuronal cell death according to the present invention is preferably such that the neuronal cell death is delayed neuronal cell death. Preferably, the neuronal cell death evaluation method according to the present embodiment is a neuronal cell during cerebral infarction or myocardial infarction, or during reperfusion after cerebral ischemia or cerebral vascular anastomosis Assess the survival rate.

  In the method for evaluating neuronal cell death according to the present invention, the step of measuring cell death is preferably performed using the activity of mitochondrial dehydrogenase as an index, but may be performed using fragmentation of nuclear DNA as an index. The neuronal cell death evaluation method according to the present invention preferably further includes a step of measuring a change in cerebral blood flow.

  In one aspect, the present invention provides a method for assessing cell death caused by ischemia and post-ischemic reperfusion in a system using cultured cells in vitro.

  To induce ischemia / reperfusion injury (ie, damage caused by reperfusion after ischemia), induce hypoxia / reoxygenation injury (ie, damage caused by reoxygenation after hypoxia) May be. In order to provide hypoxic conditions or anoxic conditions using cultured cells, the cultured cells may be cultured in a buffer solution (medium) not containing glucose under hypoxic or anoxic conditions. The buffer used in this step is not particularly limited as long as it does not contain glucose. In order to apply the reoxygenation condition, cells subjected to the hypoxic treatment (anoxic treatment) may be cultured under aerobic conditions in a medium containing glucose.

  Also, for cell cultures under hypoxic or anoxic conditions, as used herein, the term “anoxic conditions” is intended to be in an atmosphere with 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 is a cell culture performed under carbon dioxide, nitrogen, or argon gas. For example, 95% argon gas + 5% carbon dioxide gas It is easily understood that the conditions are also anoxic conditions. It should be apparent to those skilled in the art that the instrument and / or culture method used for cell culture may be appropriately selected from those known in the art.

  By using the cell death evaluation method according to the present invention, 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.

  In the method for evaluating cell death in vitro according to the present invention, the cells to be evaluated are not particularly limited, and nerve cells, brain neurons, and brain glial cells can be suitably used. By using nerve cells, cell death caused by ischemia or reperfusion after ischemia caused by cerebral infarction or myocardial infarction can be evaluated. It can be said that cell death caused by ischemia or reperfusion after ischemia caused by cerebral infarction or the like can be evaluated by using cranial nerve cells or brain glial cells. Moreover, a commercially available cultured cell may be used for said cell, and a primary cultured cell may be used.

  In the method for evaluating cell death according to the present invention, the step of detecting cell death is not particularly limited. For example, a method of performing mitochondrial dehydrogenase activity as an index (MTT method or MTS method), A method of performing fragmentation as an indicator (DNA ladder test, etc.), a method using the activity of intracellular proteolytic enzyme (caspase) causing cell death as an indicator, a method using release of cytochrome c from the mitochondria to the cytoplasm as an indicator, and Examples thereof include a method using a change in the shape of the nucleus (for example, concentration or fragmentation of the nucleus) as an index.

  Since the activity of the mitochondrial dehydrogenase decreases when the cell dies, if the enzyme activity decreases with the activity of the mitochondrial dehydrogenase as an index, it can be determined that the cell death rate is high.

  When cells are killed by necrosis, the cells cause membrane damage and are stained with propidium iodide (PI). At this time, if the nuclei of all cells are stained using Hoechst 33342, the proportion of cells that have undergone necrotic cell death can be calculated.

  In addition, when cells are killed by apoptosis, the nuclear DNA is fragmented, so if the state of the nuclear DNA on the ladder is observed by electrophoresis etc., it can be easily determined whether the cell death is apoptotic. Can be determined.

  Furthermore, since it is known that caspase is activated (the activity is increased) when cells are killed by apoptosis, if the enzyme activity is increased using caspase activity as an index, the apoptotic cell death rate is high. It can be judged.

  In addition, when cells are killed by mitochondrial stimulation, cytochrome c is released from the mitochondria into the cytoplasm. Whether or not apoptotic cell death is present can be determined.

  In addition, when cells die due to apoptosis, nuclear morphological changes such as nuclear concentration and fragmentation occur, so it is easy to observe the nuclear morphological changes with an electron microscope or the like using the nuclear morphological changes as an index. Whether or not the cell death is apoptotic can be determined.

  In another aspect, the present invention provides a method for assessing cell death caused by ischemia and post-ischemic reperfusion in an in vivo animal system.

  As ischemia using a mouse and reperfusion after ischemia, it is preferable to use a cerebral ischemia / reperfusion model in which both common carotid arteries of a mouse are temporarily blocked and then restarted.

  As a method for evaluating delayed neuronal cell death, it is preferable to use morphological histological observation of living cells and / or degenerated neurons, increase of cytochrome c in the cytoplasm, or measurement of local cerebral blood flow.

  The method for evaluating cell death according to the present invention comprises the steps of restarting the mouse after temporarily blocking both common carotid arteries in order to successfully perform the above-described mouse ischemia and reperfusion after ischemia according to the present invention. It can be said that a brain ischemia / reperfusion model is used.

That is, the method for evaluating neuronal cell death under reperfusion conditions after ischemia according to the present invention measures cell death when using a Cl ^- channel blocker or a protein tyrosine kinase inhibitor, and uses the measured value as a reference. It can be said that it may be compared with the value.

  The invention is illustrated in more detail by the following examples, but should not be limited thereto.

[1: Procedure]
Confirmation of the expression of volume-sensitive Cl ⁻ channel (α-channel) in hippocampal neurons was performed by the following method.

Hippocampal neurons were isolated from fetuses collected from mice on day 16 of gestation. The obtained primary cultured hippocampal neurons were cultured at 37 ° C. in a 5% CO ₂ incubator in Dulbecco's essential medium supplemented with 10% fetal calf serum, streptomycin, and penicillin. Current measurement was performed by applying the whole cell patch clamp method to hippocampal neurons on the 5th to 7th days of culture. In the patch clamp experiment, a solution having the following composition was used. The extracellular fluid includes 124 mM N-methyl-D-glucamine (NMDG), 124 mM HCl, 10 mM NaCl, 4 mM MgCl ₂ , 0.33 mM NaH ₂ PO ₄ , 0.5 mM EGTA, 10 mM 2- [4- (2- Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2 mM 4-aminopiperidine (4-AP), 5.5 mM glucose was used to adjust the osmotic pressure by adjusting the mannitol concentration (normal osmotic pressure solution : 315 mOsm, low osmotic pressure solution: 280 mOsm). As the intracellular solution, 124 mM NMDG, 24 mM HCl, 100 mM aspartate, 2 mM MgCl ₂ , 5 mM Na ₂ ATP, 0.3 mM Na ₃ GTP, 1 mM EGTA, 10 mM HEPES were used, and the osmotic pressure was adjusted to 300 mOsm. The pH (both extracellular and internal fluids) was adjusted to 7.4 using NMDG. After the formation of the whole cell patch clamp mode, a current response was recorded by applying a test potential ± 60 mV from a holding potential of −40 mV in order to observe the passage of channel activation time. In addition, in order to examine the nature of the current after drug administration after normal osmotic pressure, low osmotic pressure stimulation, step responses from -100 mV to +100 mV were given as needed and current responses were recorded.

To examine the effects of Cl ^- channel blockers or PTK inhibitors on delayed neuronal cell death due to ischemia / reperfusion, both common carotid arteries of mice were occluded with cerebrovascular clips and re-removed by removing the clips. A perfusion transient ischemia / reperfusion model was used.

  FIG. 1 shows the protocol. Specifically, C57 / BL6Crj (9 to 11 weeks old, male) was injected into the DIDS (0.48, 12, 60 mg / kg), genstein (0.24, 6.0) from the jugular vein 20 minutes before ischemia. 30 mg / kg) or daidzein (30 mg / kg) was administered at a flow rate of 0.8 μL / min / g body weight (240-300 μL / animal) for 15 minutes. The control group (vehicle) received a solution without compound (PBS with 10% DMSO and 10% polysorbate 80). Thereafter, cerebral ischemia / reperfusion was performed by occlusion of both common carotid arteries for 12 minutes. In order to prevent a decrease in body temperature, the animals were raised overnight in a thermostatic chamber at 35 ° C. and then raised at room temperature (24 ± 1 ° C.) for 4 days. On the first, second and third days, the same amount of compound as that for intravenous administration was administered intraperitoneally. On day 4, the animals were sacrificed and histological examination was performed.

  In these cerebral ischemia models, about 10% of the nerve cells die on the first day of ischemia / reperfusion, but on the fourth day, nearly 50% of the nerve cells die, and so-called delayed nerve cells. Death was observed.

In addition, a similar experimental model system was used to elucidate whether this delayed neuronal cell death involves apoptosis and to investigate the effects of Cl ^- channel blockers or PTK inhibitors on this mechanism. , The increase of cytochrome c in the cytoplasmic fraction in the hippocampal region was observed by biochemical technique (Western blotting). In addition, the effect of the PTK inhibitor on the cerebral blood flow was measured with a laser Doppler local blood flow meter. The local cerebral blood flow was measured by measuring 6 to 9 left and right cerebral cortex in the middle cerebral artery region with a 1.0 mm laser diameter, and the average was shown. The calculation was made with the pre-compound administration as 100%.

[2: Volume-receptive Cl ⁻ channel exists in mouse hippocampal neurons]
The presence of volume-sensitive Cl ^- channels in neurons has been reported so far in rat sympathetic neurons (Leaney JL, Marsh SJ, Brown DA. (1997) "A swelling-activated chloride current in rat sympathetic neurons. "J. Physiol. 501: 555-564.), Rat carotid body type 1 neurosecretory cells (Carpenter E, Peers C. (1997)" Swelling- and camp-activated Cl ^- currents in isolated rat carotid. body type I cells. "J. Physiol. 503: 497-511.) and mouse cerebellar granule cells (Patel AJ, Lauritzen I, Lazdunski M, Honore E. (1998)" Disruption of mitochondrial respiration inhibits volume-regulated anion channels and provokes neuronal cell swelling. "See J. Neurosci. 18: 3117-3123.). Therefore, it was examined whether or not volume-sensitive Cl ⁻ channels exist in hippocampal neurons (FIG. 2).

Since the molecular entity of the volume-sensitive Cl ⁻ channel has not been clarified, immunohistological techniques cannot be applied. Therefore, the expression was confirmed by current recording by the patch clamp method.

As shown in FIG. 2A, when a low osmotic load was applied to mouse hippocampal primary cultured neurons under whole cell patch clamp, a gradually activated current was observed. The reversal potential (= −36 mV) of this activated current approximates the equilibrium potential (= −40 mV) of Cl ⁻ , and is inactivated at a high positive potential (> +80 mV) with outward rectification (FIG. 2C). Shown (FIG. 2B). All of these results are consistent with the volume sensitive Cl ^- current characteristics. Thus, it was revealed that volume-sensitive Cl ⁻ channels exist in mouse hippocampal primary cultured neurons.

[3: Volume-sensitive Cl ^- channel of mouse hippocampal primary cultured neurons is suppressed by DIDS and enistin]
It was confirmed whether or not the volume-sensitive Cl ⁻ channel of mouse hippocampal primary cultured neurons was suppressed by DIDS or genistin known as inhibitors (FIG. 2). The inhibitory effect of 500 μM DIDS was only seen on the positive potential and was voltage dependent (FIG. 2D). Moreover, the suppression by 100 μM genistein was independent of the potential, and exhibited the suppression effect at all potentials except the vicinity of the reversal potential (FIG. 2E). From this, it was confirmed that DIDS and genistin surely suppress the volume-sensitive Cl ⁻ channel of hippocampal neurons.

[4: Murine cerebral ischemic delayed neuronal cell death is rescued by Cl ^- channel blocker]
Apoptosis induced by staurosporine (STS) in cultured rat neuronal cells has been reported to be rescued by DIDS, a blocker of Cl ^- channel (Tanabe S, Maeno E, Okada Y (2002) " Prevention of staurosporine-induced apoptotic cell death by DIDS and SITS in rat cardiomyocytes in primary culture. "Jpn. J. Physiol. 52 (Suppl): S34). Therefore, cerebral ischemic neuronal cell death of the mice Cl ^- was examined whether the inhibition by channel blockers.

As shown in FIG. 3, when neuronal cell death was induced by 12 minutes of ischemia, when observed morphologically by toluidine blue staining, neuronal cells in the hippocampal CA1 region became unclear and cone cells. (Fig. 3, arrow A). The number of remaining morphologically normal cells per fixed area decreased from about 60 (results not shown) before cerebral ischemia to 37.1 (FIG. 2, first bar graph). Cl after ischemia 15 min before and reperfusion ^- idea to administer DIDS (0.48,12,60mg / kg) is the channel blocker, in the administration of 0.48 mg / kg or 60 mg / kg, the effect Was not so prominent (FIGS. 3 and 5). However, the morphological change decreased in the 12 mg / kg administration group (FIG. 3, D), and the number of remaining morphologically normal cells rose to 52.5 (FIG. 4, third bar graph).

Furthermore, as shown in FIG. 3, when a neuron showing a neurodegeneration image (detected by Fluoro-Jade B staining) was observed, the denatured neuron clearly and strongly fluorescently stained in the hippocampal CA1 region of the vehicle-administered mouse. Was observed (FIG. 5, arrow A). The number of degenerated neurons per unit area was measured to be 28.4 (FIG. 6, first bar graph). When DIDS 12 mg / kg was administered, the number of cells stained decreased (FIG. 5, D). The number of degenerated neurons was clearly reduced to 12.0 (FIG. 6, third bar graph). These results, Cl ^- indicates that a channel blockers DIDS acts remedial with respect to delayed neuronal cell death after mouse brain ischemia transient.

[4: Murine cerebral ischemic delayed neuronal cell death is rescued by PTK inhibitors]
As shown in FIG. 3, when a vehicle was administered 15 minutes before ischemia to induce 12 minutes of ischemic neuronal cell death, the number of morphologically normal cells remaining per fixed area was 37.1. (FIG. 4, first bar graph). Neurons in the hippocampal CA1 region when the PTK inhibitor genistein (1.2, 6, 30 mg / kg) was administered 15 times before ischemia and 1, 2 and 3 days after reperfusion. (FIGS. 3, E, F, and G), and the number of remaining morphologically normal cells per unit area increased to 48.2, 50.8, and 55.7, respectively (FIG. 4). , 5th, 6th, 7th bar graph). When degenerated neurons were observed, clearly denatured neurons were observed in the hippocampal CA1 region of the vehicle administration group (FIG. 5, A). When Genistine was administered, the appearance of degenerative neurons decreased (Fig. 5, E, F, G). The number of degenerated neurons per unit area was clearly reduced to 14.0, 11.7 and 5.8 in a concentration-dependent manner (FIG. 6, fifth, sixth and seventh bar graphs). Furthermore, in the administration group of daidzein (30 mg / kg), which is an analog having no PTK activity of genistein, the decrease in morphological normal cells and the increase in degenerated neurons could not be suppressed (FIG. 3, H; FIG. 4). , Eighth bar graph; FIG. 5, H; FIG. 6, eighth bar graph). These results indicate that genistin, a PTK inhibitor, has a rescue action against delayed neuronal cell death after transient mouse cerebral ischemia.

[5: Apoptosis due to mouse cerebral ischemic delayed neuronal cell death is suppressed by Cl ^- channel blocker or PTK blocker]
In the process of apoptotic cell death, it is known that the release of cytochrome c from the mitochondria to the cytoplasm is the most important phenomenon for the appearance of cell death (see 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 staurosporine-induced apoptotic cell". death by DIDS and SITS in rat cardiomyocytes in primary culture. "Jpn. J. Physiol. 52 (Suppl): S34).

Increased cytochrome c (cyt-c) was observed in the hippocampal cytoplasm after transient cerebral ischemia induction (FIG. 7, vehicle administration group (1 to 4 lanes)). Cl in 15 minutes before inducing transient ischemic ^- channel blockers (Fig. 7, the fifth to eighth lane (DIDS (12mg / kg)) ) or PTK inhibitors (FIG. 7, the ninth to 12 lane In the brain hippocampus region of mice administered with (genistein (30 mg / kg)), an increase in cytochrome c was suppressed. These results, Cl ^- indicates that a DIDS or PTK inhibitor is a channel blockers genistein acts relief to respect apoptosis following mouse brain ischemia transient.

[6: Decrease in cerebral blood flow during mouse cerebral ischemia / reperfusion is suppressed by PTK inhibitors]
PP1, which is one of the inhibitors of PTK, has been reported to exhibit a vasodilatory effect during cerebral ischemia (see 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 decreases to 25% compared with that before cerebral ischemia (100%), but gradually recovers after 10 minutes of reperfusion, Return to the vicinity of the blood (in the figure, Genistine administration group (black circle), vehicle group (white circle) and control (vehicle non-administration group)). However, when administration of genistein (30 mg / kg) for 15 minutes before cerebral ischemia is performed, it is as high as 30 to 35% of cerebral blood flow during ischemia, and the local cerebral blood flow after reperfusion is about 2 Recover in minutes. In particular, it recovers 50% in 30 seconds immediately after reperfusion and recovers to 80% before ischemia. However, the vehicle group recovers only about 20% (recovered to 40% before ischemia). This result may be caused by the PTK inhibitor genistein, which causes circulatory improvement such as vasodilation during transient mouse cerebral ischemia / reperfusion, and consequently suppresses cerebral blood flow decline. It shows a remedy for late neuronal cell death.

  Until now, no effective drug or method has been developed for protecting or relieving delayed neuronal cell death due to ischemia / reperfusion of neurons, so that the present invention allows for delayed onset. It becomes possible to protect and / or rescue neuronal cell death, and greatly contributes to the development of techniques for effective treatment and / or prevention against various brain disorders caused by delayed cell death of neurons.

FIG. 1 is a schematic diagram showing an experimental schedule according to a transient mouse both common carotid artery occlusion forebrain ischemia model. FIG. 2 is a graph showing volume-sensitive Cl ⁻ current in mouse hippocampal primary cultured neurons. FIG. 3 is a photograph comparing the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion with morphological staining by toluidine blue staining. FIG. 4 is a graph showing a comparison of the number of remaining normal neurons in the hippocampal CA1 region on the 4th day of cerebral ischemia / reperfusion. FIG. 5 is a photograph comparing the degenerated neurons by staining the hippocampal CA1 region on the 4th day of cerebral ischemia / reperfusion with Fluoro-JadeB (FJB) staining. FIG. 6 is a graph comparing the number of FJB positive cells in the hippocampal CA1 region on day 4 of cerebral ischemia / reperfusion. FIG. 7 is a photograph showing the results of cytochrome c immunoblotting 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.

Claims (22)

An agent for protecting or relieving neuronal cell death after ischemia, comprising a Cl ^- channel blocker or a protein tyrosine kinase (PTK) inhibitor.   The drug according to claim 1, wherein the neuronal cell death is delayed neuronal cell death.   The drug according to claim 1 or 2, wherein the neuronal cell death is accompanied by fragmentation of DNA in the nucleus.   The drug according to any one of claims 1 to 3, wherein the neuronal cell death is accompanied by a significant decrease in the release of cytochrome c from mitochondria. The drug according to any one of claims 1 to 4, wherein the Cl ^- channel blocker is 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS).   The drug according to any one of claims 1 to 4, wherein the PTK inhibitor is genistein.   The neuron death 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. The drug according to claim 1. Cl ^- methods for neuronal cell death protection or remedy, characterized in that the channel blocker or PTK inhibitor comprising administering to neuronal cells in reperfusion conditions following ischemia.   The method according to claim 8, wherein the neuronal cell death is delayed neuronal cell death.   The method according to claim 8 or 9, wherein the neuronal cell death is accompanied by fragmentation of DNA in the nucleus.   11. The method according to any one of claims 8 to 10, wherein the neuronal cell death is accompanied by a significant decrease in the activity of cytochrome c release from mitochondria causing cell death.   The method according to any one of claims 8 to 11, which is accompanied by an improvement in a decrease in cerebral blood flow that occurs during cerebral ischemia and reperfusion after ischemia. The method according to any one of claims 8 to 12, wherein the Cl ^- channel blocker is 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS).   The method according to any one of claims 8 to 12, wherein the PTK inhibitor is genistein.   15. The neuron death occurs at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis. The method according to claim 1. A method for evaluating neuronal cell death under reperfusion conditions after ischemia,
A step of measuring cell death when using a Cl ^- channel blocker or a protein tyrosine kinase inhibitor; and a measurement value obtained by the above measurement is used to determine cell death without using a Cl ^- channel blocker or a protein tyrosine kinase inhibitor. A method comprising the step of comparing with a reference value obtained by measurement.   The method of evaluating neuronal cell death according to claim 16, wherein the neuronal cell death is delayed neuronal cell death.   18. The neuronal cell survival rate at the time of cerebral infarction or myocardial infarction, or at the time of cerebral ischemia or reperfusion after cerebral ischemia in heart transplantation or cerebral vascular anastomosis is evaluated. The evaluation method of neuronal cell death as described in 2.   The method for evaluating neuronal cell death according to any one of claims 16 to 18, wherein the step of measuring cell death is performed using the activity of mitochondrial dehydrogenase as an index.   The method for evaluating neuronal cell death according to any one of claims 16 to 18, wherein the step of measuring cell death is performed using fragmentation of nuclear DNA as an index.   The method for evaluating neuronal cell death according to any one of claims 16 to 20, further comprising a step of measuring a change in cerebral blood flow.   The method for evaluating neuronal cell death according to any one of claims 16 to 21, further comprising a step of temporarily reopening the both common carotid arteries of the mouse and then reopening them.

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