WO2014125663A1 - Médicament pour le traitement ou la prévention d'une maladie ischémique et son utilisation - Google Patents

Médicament pour le traitement ou la prévention d'une maladie ischémique et son utilisation Download PDF

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WO2014125663A1
WO2014125663A1 PCT/JP2013/068545 JP2013068545W WO2014125663A1 WO 2014125663 A1 WO2014125663 A1 WO 2014125663A1 JP 2013068545 W JP2013068545 W JP 2013068545W WO 2014125663 A1 WO2014125663 A1 WO 2014125663A1
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ischemic
cells
drug
treated
glucose
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PCT/JP2013/068545
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English (en)
Japanese (ja)
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匠徳 佐藤
幹昭 村越
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国立大学法人奈良先端科学技術大学院大学
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Priority to JP2015500086A priority Critical patent/JP6108569B2/ja
Publication of WO2014125663A1 publication Critical patent/WO2014125663A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a drug for treating or preventing ischemic disease and use thereof.
  • ischemic diseases When blood flow deteriorates due to occlusion or stenosis of a blood vessel and the blood flow is completely blocked or significantly reduced in a part of the body, oxygen deficiency, decreased substrate supply, and accumulation of metabolites proceed. Eventually leading to cell death and necrosis. Such diseases are collectively referred to as ischemic diseases.
  • the degree of ischemia is related to the rate of vascular occlusion, the duration, or the sensitivity of the tissue, and the degree of development of the accessory circulation. For example, the tissue leads to atrophy, degeneration, and necrosis.
  • ischemic diseases include ischemic heart diseases such as angina pectoris and myocardial infarction, ischemic brain diseases such as cerebral infarction (ischemic cerebrovascular disorder), and ischemic bowel diseases such as ischemic colitis. Etc. exist. In particular, the former two are the top causes of death among Japanese people and are on the rise.
  • myocardial infarction is a disease in which the myocardium becomes ischemic and necrotic due to disruption of blood flow in the coronary arteries.
  • the myocardium dies over time, and the cardiac function decreases.
  • collagen accumulates to fill the traces of necrotic cardiomyocytes, resulting in cardiac fibrosis.
  • Patent Document 1 administers a scar formation promoter containing at least one selected from SFRP2, SFRP4, midkine, pleiotrophin and thymosin ⁇ 10 as an effective element, without causing fibrosis and
  • a method of treating ischemic heart disease is disclosed that improves cardiac function by promoting scar formation while maintaining elasticity.
  • the present inventors have conducted intensive studies. As a result, the present invention does not affect cell survival in a normal environment, but causes mitochondrial dysfunction and promotes cell survival and proliferation in an ischemic environment. Drugs that can be suppressed were found. As a result of analyzing the mechanism of action of such a drug, the present invention is completed by finding new facts such as that the drug inhibits cellular energy metabolism, for example, part of mitochondrial aerobic respiration. It came to. That is, the present invention includes the following inventions.
  • a drug for the treatment or prevention of ischemic disease which contains as an active ingredient a substance having an activity that gives abnormalities to mitochondria and inhibits cell growth in an ischemic environment.
  • the substance is any one of (1) to (3), which is (i) a substance that inhibits a conversion reaction from pyruvic acid to acetyl-CoA and / or (ii) a substance that inhibits an electron transport system.
  • the drug according to Crab is any one of (1) to (3), which is (i) a substance that inhibits a conversion reaction from pyruvic acid to acetyl-CoA and / or (ii) a substance that inhibits an electron transport system.
  • the above ischemic diseases include transient cerebral ischemia, cerebral infarction, myocardial infarction, unstable angina, graft occlusion after coronary artery bypass surgery, coronary artery occlusion after percutaneous coronary angioplasty, and revascularization Later vascular occlusion, obstructive arteriosclerosis, obstructive thromboangiitis, glomerulonephritis, nephrotic syndrome, essential thrombocythemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, antiphospholipid antibody syndrome,
  • the drug according to any one of (1) to (6), which is any disease selected from Kawasaki disease, hepatitis, and renal fibrosis disease.
  • a method for screening an agent for treating or preventing ischemic disease which comprises the step of measuring an activity of inhibiting cell growth in an ischemic environment.
  • FIG. 1 It is a figure which shows the effect etc. which PP inhibits mitochondrial energy metabolism. It is a figure which shows the result etc. which investigated the persistence in the non-drug
  • FIG. 6 is a graph showing evaluation of recovery of survival of PP-treated cardiac fibroblasts in an ischemic environment by supplementing glucose. It is a figure which shows the path
  • FIG. 1 is a schematic diagram of a pathological process of myocardial infarction.
  • FIG. 6 is an evaluation of improved angiogenesis in a scarred heart treated with PP.
  • FIG. 2 is an evaluation of the role of classical Wnt and UPR in specific cytotoxicity during PP ischemia.
  • a to B representing a numerical range is “A or more (including A and greater than A) and B or less (including B and less than B)”, “%”. Means “% by mass”, and “part” means “part by mass”.
  • the present inventors have conducted extensive studies, and as a result, a certain drug causes an environment-independent abnormality in the mitochondria, and such a mitochondrial abnormality does not affect cell growth under a normal environment. However, it was revealed that cell growth is inhibited under ischemic environment. As a result of detailed analysis of the mechanism of action of the drug, it was found that the drug inhibits a part of cellular energy metabolism, in particular, inhibits mitochondrial aerobic respiration. Specifically, the drug inhibits metabolic flow from the glycolytic system to the citric acid cycle, and more specifically, (i) inhibits the conversion reaction from pyruvic acid to acetyl CoA. And / or (ii) that it inhibits the electron transport system.
  • the above-mentioned drug suppresses the survival and proliferation of fibroblasts in an ischemic environment (particularly undernutrition, such as a low glucose state), and in addition, cells resulting from the ischemic environment
  • the fibrosis can be suppressed, and further, by suppressing the fibrosis, the reduction of cardiac function after ischemic disease can be alleviated and thus the recovery of cardiac function can be improved, and the ischemic disease can be treated or prevented.
  • the present invention has been completed.
  • the inventors have also revealed that once the drug is administered, the pharmacological action continues to some extent (about 24 to 72 h) even in the absence of the drug thereafter. From this, it becomes possible to prevent the onset or worsening of an ischemic disease by administering the drug in advance.
  • the agent for treatment or prevention of ischemic disease according to the present invention (hereinafter simply referred to as “the drug according to the present invention”) has an activity that gives abnormalities to mitochondria and inhibits cell growth in an ischemic environment. As long as it contains a substance having an active ingredient as an active ingredient, it is not limited to specific aspects such as other ingredients and dosage forms.
  • the substance does not affect the growth of cells in a normal environment.
  • “giving an abnormality to the mitochondria” refers to causing an abnormal state (abnormal state) of the function of the mitochondria, and particularly intended to cause an abnormality in the respiratory function of the mitochondria.
  • mitochondrial abnormality has occurred or not, measure mitochondrial energy metabolism, biochemical and physiological responses using, for example, an extracellular flux analyzer or other standard biochemical and physiological methods. Can be determined.
  • mitochondria derived from cells in a normal state for example, cells not administered with a drug
  • the fact that the shape of mitochondria has changed from a normal state by observation with an electron microscope can also be used as a criterion for occurrence of abnormality.
  • the occurrence of abnormalities in the mitochondria is preferably independent of the environment, that is, regardless of whether it is in a normal environment or ischemic environment.
  • the active ingredient is not particularly limited as long as it is a substance having the above function.
  • Substances include low molecular or high molecular compounds, polynucleotides (including DNA, RNA, and genes), oligopeptides, polypeptides (including proteins), antibodies, and the like. These substances may be used alone or in combination of two or more.
  • the substance inhibits cell energy metabolism.
  • the substance is preferably one that inhibits mitochondrial aerobic respiration, and more preferably one that inhibits the metabolic flow from the glycolytic system to the citric acid cycle.
  • the substance is particularly preferably (i) one that inhibits the conversion reaction of pyruvic acid to acetyl CoA and / or (ii) one that inhibits the electron transport system.
  • examples of “(i) a substance that inhibits the conversion reaction of pyruvate to acetyl CoA” include those that inhibit pyruvate reductase.
  • the non-administration group of the drug is used as a control (control), and compared with the control, the case where each of the above reactions is significantly reduced when the target drug is administered is regarded as being inhibited. Can be determined.
  • the said inhibition is recognized by the at least 1 analysis method shown in the Example mentioned later, it shall belong to the technical scope of this invention.
  • pyruvinium pamoate pyrivinium pamoate; hereinafter may be simply referred to as “PP”
  • PP pyruvinium pamoate
  • the present invention is not limited to this. It may also include new materials that are found or created.
  • a person skilled in the art can find various substances having functions similar to those of the above substances in the future by suitably utilizing the technology at the time of filing of the present application based on the disclosure of the present specification.
  • “Significant” as used in this specification refers to those based on statistical analysis in the t-test described in the examples. The significance level includes any of p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001.
  • the “ischemic environment” refers to an environment where the oxygen concentration is low and / or the substrate concentration is low.
  • the cause of the ischemic environment is not particularly limited, for example, the case where blood flow deteriorates due to blood vessel occlusion or stenosis, oxygen deficiency, decrease in substrate supply, accumulation of metabolites, progressing separately or simultaneously can be exemplified .
  • the substrate include conventionally known nutrients necessary for cell survival, such as saccharides, amino acids, nucleic acids, and vitamins, and are not particularly limited, but are in a state in which at least one selected from saccharides and amino acids is deficient. Is preferred.
  • the ischemic environment referred to in the present invention may include a state in which glucose and / or glutamine is deficient even if the oxygen concentration is normal.
  • the “normal environment” is paired with the ischemic environment and refers to a normal level that is not an ischemic state, such as an oxygen concentration, a substrate supply amount, etc., that is, a so-called normal state.
  • an oxygen concentration in a normal environment refers to an oxygen concentration necessary for a mammal or mammalian cells to survive, for example, 20 to 21%.
  • the case where it is lower than this range is referred to as a low oxygen state, for example, about 3%.
  • a saccharide concentration in a normal environment refers to a saccharide concentration necessary for the survival of a mammal or mammalian cells, for example, 25 mM for glucose. The case lower than this is referred to as a low glucose state, for example, about 300 ⁇ M.
  • An amino acid concentration in a normal environment refers to an amino acid concentration necessary for a mammal or a mammalian cell to survive. For example, 4 mM can be mentioned for glutamine. The case lower than this is referred to as a low glutamine state, for example, 100 ⁇ M or less.
  • the present invention is not limited to the above example, and the state in which oxygen, the substrate, and the like are in a normal environment may be any environment in which oxygen and the substrate are sufficiently supplied. In other words, the environment can be metabolized.
  • the environment can be metabolized.
  • the drug according to the present invention inhibits cell energy metabolism, but does not affect cells in a normal environment while suppressing cell growth in an ischemic environment. It is preferable.
  • suppressing cell growth is intended to suppress cell survival and / or proliferation. The presence or absence of the suppression can be determined to be suppressed when the survival and / or proliferation of the cells are significantly reduced with the non-administration group of the drug as a control.
  • the cell is not particularly limited, but is preferably a fibroblast.
  • fibroblasts survive and proliferate in an ischemic environment, but fibroblasts proliferated in an ischemic environment may produce a large amount of collagen outside the cell and cause fibrosis. is there.
  • the agent according to the present invention can also suppress fibrosis by suppressing the growth of fibroblasts in an ischemic environment.
  • the drug of the present invention for example, when the ischemic disease is an ischemic heart disease such as myocardial infarction, in addition to the above-described effects, it suppresses deterioration of the heart function after the ischemic heart disease. Can do. Furthermore, the functional recovery of the heart can be improved.
  • the drug according to the present invention is administered, the pharmacological action continues to some extent even in the absence of the drug thereafter.
  • 24h to 72h can be exemplified.
  • the disease targeted by the drug of the present invention is not particularly limited as long as it is an ischemic disease caused by blood flow deterioration due to occlusion or stenosis of blood vessels, oxygen deficiency, decreased substrate supply, etc.
  • examples include ischemic heart disease, ischemic brain disease (ischemic cerebrovascular disorder), ischemic colitis, and the like.
  • Vascular occlusion obstructive arteriosclerosis, obstructive thromboangiitis, glomerulonephritis, nephrotic syndrome, essential thrombocythemia, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, antiphospholipid antibody syndrome, and Examples include Kawasaki disease, hepatitis (general liver fibrosis), renal fibrosis in general.
  • the drug according to the present invention can be in an appropriate pharmaceutically acceptable formulation form and administration method as necessary.
  • it can be administered orally as a preparation such as a tablet, capsule, granule, powder, or syrup, or parenterally as a preparation such as an injection, suppository, or patch.
  • parenteral administration methods include intraperitoneal administration, intramuscular administration, transdermal administration, respiratory administration, eye drop administration, nasal administration, and subcutaneous administration.
  • the drug according to the present invention includes, as necessary, excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, flavoring agents, diluents, solvents for injections, oily bases, water-soluble It is produced by a known method using an additive such as a base.
  • excipients include organic excipients and inorganic excipients.
  • examples of the organic excipient include sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, ⁇ -starch, dextrin and carboxymethyl starch; crystalline cellulose, low Degree of substitution Hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, cellulose derivatives such as internally cross-linked sodium carboxymethylcellulose; gum arabic; dextran; or pullulan.
  • examples of the inorganic excipient include silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate and calcium silicate; phosphates such as calcium phosphate; or sulfates such as calcium sulfate.
  • binder examples include the above-mentioned excipients; gelatin; polyvinyl pyrrolidone; or polyethylene glycol.
  • disintegrant examples include the above-mentioned excipients; chemically modified starch or cellulose derivatives such as croscarmellose sodium and sodium carboxymethyl starch; or crosslinked polyvinylpyrrolidone.
  • lubricant examples include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as beeswax and geirow; boric acid; glycol; D, L-leucine; Carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates such as sodium benzoate; sulfates such as sodium sulfate; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic anhydride, silicic acid hydration Silicic acids such as products; or starch derivatives in the above excipients.
  • emulsifiers include colloidal clays such as bentonite and bee gum; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; or polyoxyethylene alkyl Nonionic surfactants such as ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters may be mentioned.
  • the stabilizer examples include parahydroxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; Thimerosal; dehydroacetic acid; or sorbic acid.
  • parahydroxybenzoates such as methyl paraben and propyl paraben
  • alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol
  • benzalkonium chloride examples include phenols such as phenol and cresol; Thimerosal; dehydroacetic acid; or sorbic acid.
  • flavoring agent examples include sweeteners, acidulants, and fragrances that are usually used.
  • diluent examples include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.
  • Examples of the solvent for injection include water, ethanol, and glycerin.
  • oleaginous base examples include cocoa butter, lauric fat, coconut oil, palm kernel oil, camellia oil, liquid paraffin, white petrolatum, refined lanolin, glyceryl monostearate, polyoxyethylene hydrogenated castor oil, sorbitan fatty acid ester, Sucrose fatty acid ester, stearyl alcohol, or cetanol is mentioned.
  • water-soluble base examples include glycerin, polyethylene glycol, ethanol, and purified water.
  • the dosage of the drug according to the present invention can be appropriately set depending on the preparation form of the drug, the administration method, the age, sex, body weight, symptoms, etc. of the administration target of the drug.
  • ischemic diseases can be treated or prevented.
  • it can suppress fibrosis caused by ischemic disease, can suppress a decrease in the function of a tissue or organ after the disease, and / or speed up the functional recovery of a tissue or the like that has been decreased due to the disease. Is also possible.
  • a method for screening a drug for treatment or prevention of ischemic disease according to the present invention (hereinafter simply referred to as “screening method according to the present invention”) (I) a step of applying a test substance to a non-human mammal under an ischemic environment or a cell of a mammal under an ischemic environment in vitro or ex vivo; and (ii) the test substance.
  • screening method a method for screening a drug for treatment or prevention of ischemic disease according to the present invention (hereinafter simply referred to as “screening method according to the present invention”)
  • (I) a step of applying a test substance to a non-human mammal under an ischemic environment or a cell of a mammal under an ischemic environment in vitro or ex vivo and (ii) the test substance.
  • it is sufficient to have a step of measuring an activity that gives abnormalities to mitochondria and inhibits cell growth in an ischemic environment and other steps, conditions, etc. are not particularly limited.
  • Non-human mammals under ischemic environment means, for example, artificially causing ischemia by artificially causing occlusion or stenosis of blood vessels, worsening blood flow, lack of oxygen, decreased substrate supply, etc.
  • model animals of so-called ischemic diseases can be mentioned.
  • the non-human mammal may be any mammal other than a human, but a mouse, a rat, a guinea pig, a dog, a rabbit, a monkey, a chimpanzee and the like that are particularly suitable as an experimental animal are preferable.
  • human cells can be used, which is preferable.
  • mammalian cells in an ischemic environment in in vitro or ex vivo can be used. Such cells can be easily prepared, for example, by artificially creating an ischemic environment by reducing the oxygen concentration and / or the substrate concentration under the cell culture conditions.
  • the mammalian cell is not particularly limited as long as it is a cell that grows in an ischemic environment, but is preferably a cell involved in an ischemic disease.
  • a heart collected from a mammal for example, a heart collected from a mammal, digestive system (stomach, small intestine, large intestine), brain tissue (including hypothalamus, pituitary gland, etc.), adrenal gland, kidney, liver, limbs, or a piece of tissue obtained by chopping blood vessels
  • a mammal for example, a heart collected from a mammal, digestive system (stomach, small intestine, large intestine), brain tissue (including hypothalamus, pituitary gland, etc.), adrenal gland, kidney, liver, limbs, or a piece of tissue obtained by chopping blood vessels
  • cell lines derived from these organs or cells in the organs may be used.
  • a particularly preferred example is fibroblast.
  • Confirmation as to whether or not a non-human mammal or cell under an ischemic environment can be prepared can utilize a conventionally known one, and is not particularly limited. For example, analyzing whether there is an abnormality in the energy metabolism of the mitochondrion, in particular, whether an abnormality is occurring in a part of the mitochondrial aerobic respiration.
  • the step (i) can be performed by applying a test substance to the non-human mammal or cell.
  • a method for applying the test substance to the non-human mammal or cell is not particularly limited, and a technique performed by a conventionally known screening method can be suitably used.
  • the test substance may be administered to the non-human mammal.
  • the administration method include oral administration, intraperitoneal administration, intramuscular administration, transdermal administration, respiratory administration, eye drop administration, nasal administration and subcutaneous administration.
  • the test substance may be brought into contact with the cells, and a known method such as addition to a medium can be used.
  • test substance can be the above-described drug according to the present invention, that is, (a) whether or not it has an activity to give an abnormality to mitochondria and inhibit cell growth in an ischemic environment, (b Whether to inhibit mitochondrial aerobic respiration, (c) whether to inhibit metabolic flow from the glycolytic pathway to the citrate cycle, or (d) (i) conversion of pyruvate to acetyl-CoA Any substance for confirming whether to inhibit the reaction and / or (ii) whether to inhibit the electron transport system, etc. may be used, and examples include compounds, polynucleotides, polypeptides, antibodies, etc. Can do. If necessary, it may be applied to cells and the like as a pharmacologically acceptable salt.
  • This step (ii) may be any step as long as the test substance is measured for its activity to give abnormalities to mitochondria and inhibit cell growth in an ischemic environment.
  • This step (ii) for example, there is a method of simply measuring whether or not cell growth in an ischemic environment is inhibited.
  • Other methods include (a) inhibiting mitochondrial aerobic respiration, (b) inhibiting metabolic flow from the glycolytic system to the citric acid cycle, or (c) (i) acetyl-CoA from pyruvic acid And / or (ii) a method for determining whether to inhibit the electron transport system, or the like.
  • the various methods described above are the same as those described in the column of the drug described above, and thus description thereof is omitted.
  • the method for determining the presence or absence of various inhibitors is not particularly limited as public methods can be used.For example, for non-human mammals, the non-administration group of the test substance is compared with the administration group, and it is caused artificially.
  • a method for evaluating or measuring whether or not the symptoms of the ischemic disease are alleviated or improved In the case of cells, a method for evaluating or measuring cytotoxicity in an ischemic environment by comparing the presence of a test substance with the absence of a test substance can be mentioned.
  • the inhibition of mitochondrial aerobic respiration is associated with inhibition of pyruvate reductase and / or inhibition of electron transport system.
  • a method of measuring the activity of the above-described pyruvate reductase or a factor involved in the electron transport system can also be used.
  • a conventionally well-known method can be utilized about activity measurement of this enzyme etc., It does not specifically limit.
  • a method in which a reference substance for example, PP is applied is set as a reference value, and the effect of the test substance is evaluated or measured Is also included.
  • the effect of the test substance may be quantified and measured more simply.
  • a candidate substance that can be a drug for treatment or prevention of ischemic disease can be screened easily and accurately.
  • Such a substance is useful for the development of a medicament as a candidate substance for a therapeutic / preventive drug for ischemic disease, like the drug according to the present invention described above.
  • Method for inhibiting cell growth under ischemic environment according to the present invention (hereinafter simply referred to as “method according to the present invention”) May be any non-human mammal or in vitro or ex vivo mammalian cell method that gives abnormalities to mitochondria and inhibits cell growth in an ischemic environment, and other specific steps, etc. Is not particularly limited.
  • the content demonstrated by the said (1), (2) column is also used suitably also in this method.
  • the method according to the present invention causes an environment-independent abnormality in mitochondria and does not affect the growth under normal conditions, but is preferably a method that inhibits cell growth only under an ischemic environment. .
  • a method for example, in a non-human mammal or a mammalian cell in in vitro or ex vivo, (a) inhibiting mitochondrial aerobic respiration, (b) from glycolysis to citrate cycle Examples thereof include a method of inhibiting metabolic flow, (c) (i) inhibiting a conversion reaction from pyruvate to acetyl CoA, and / or (ii) inhibiting an electron transport system.
  • the method according to the present invention may include a method of inhibiting cell growth in an ischemic environment by giving an abnormality to the mitochondria by genetic engineering techniques.
  • the method according to the present invention is useful for elucidating the mechanism of ischemic disease and for developing new therapeutic methods and therapeutic agents.
  • the present invention includes not only the above-described invention but also the following invention.
  • a method for treating or preventing an ischemic disease using the drug according to the present invention A method for producing a drug according to the present invention, wherein a substance that gives an abnormality to mitochondria and inhibits cell growth in an ischemic environment is used as an active ingredient.
  • Treatment and prevention of ischemic heart diseases such as myocardial infarction, which targets mitochondrial abnormalities and targets energy metabolism in an ischemic environment.
  • Use of pyribinium pamoate as a drug that causes mitochondrial dysfunction in cardiac fibroblasts under ischemic environment.
  • Use of pyribinium pamoate as a therapeutic and prophylactic agent for ischemic heart diseases such as myocardial infarction.
  • FIG. 1A is shown as panel A in FIG. 1
  • FIG. 1B is shown as panel B in FIG. 1
  • FIG. 2A is shown as panel A in FIG. The same applies to the other drawings.
  • PP is an anthelmintic drug that suppresses the activity of NADH fumarate reductase (NADH-FR) in parasite mitochondria, but it remains unclear whether it suppresses the same target in mammalian cells.
  • NADH-FR NADH fumarate reductase
  • the present inventors have identified two possible mechanisms. As can be inferred from the results shown in FIG. 7, the first possibility is that PP acts on a target present in cells in a normal environment rich in glucose, while glucose and / or glutamine are deficient. It is to exert cytotoxicity only under. A second possibility is that PP acts only on cells that are present only in cells that are deficient in glucose and / or glutamine, but not in cells in a normal environment that is rich in glucose and / or glutamine. That is.
  • FIG. 1B cardiac fibroblasts were cultured under conditions where glucose and glutamine were deficient (Glc low / Gln low ) after temporarily treating the cardiac fibroblasts with PP under normal culture conditions. In this case, it was found that cell survival was sufficiently suppressed (FIG. 1B). Since treatment with PP had no effect on cells in normal culture medium, this cell treatment inhibition effect by PP treatment is considered to be unique to the Glc low / Gln low conditions (FIG. 1B). The IC50 of PP in this temporary effect was 61.8 nM (FIG. 1C).
  • the inventors have shown that the PP-induced inhibitory effect on mitochondrial energy metabolism is not able to survive in any medium in which cardiac fibroblasts are deficient in glucose and / or glutamine, It was examined whether it was possible to survive in a medium rich in glucose and / or glutamine.
  • both vehicle-treated cardiac fibroblasts and PP-treated cardiac fibroblasts have a flux of both TCA cycle and / or cataprotic response.
  • a large amount of the intermediate metabolite of the TCA cycle was consumed, and energy was produced in an environment deficient in glucose (that is, in an environment in which no glycolysis exists) (FIG. 2B).
  • citric acid, cis-aconitic acid and isocitric acid can be barely detected in culture in Glc low / Gln low media up to 12 hours after PP treatment. It can be seen that it is consumed or completely consumed (FIG. 2B).
  • the present inventors also examined whether the decrease in OCR induced by PP is sustained even in the absence of PP (FIG. 2C).
  • OCR extracellular wall acidification rate
  • FIG. 2C After treatment of cardiac fibroblasts with PP in normal medium for 1 hour, thorough washing and removal of excess PP, both OCR and extracellular wall acidification rate (ECAR) (the latter The sugar system is displayed in real time.) was measured in either a normal medium or a Glc low / Gln low medium (FIG. 2C).
  • ECAR extracellular wall acidification rate
  • FIG. 3C A method is provided (FIG. 3C).
  • cardiac fibroblasts do not utilize mitochondrial energy metabolism, but utilize activated glycolysis and produce enough energy to survive.
  • glucose availability is limited, cardiac fibroblasts rely primarily on mitochondrial energy metabolism due to a dramatic reduction in energy production via the glycolysis.
  • the present inventors examined the effect of PP on cardiac fibroblasts in vivo in a myocardial infarction mouse model.
  • the left coronary artery is continuously occluded by surgical ligation.
  • cardiac fibroblasts begin to proliferate and migrate, and 7 days after ligation The number of fibroblasts becomes the highest (FIG. 19).
  • Secretion and deposition of collagen fibers by these activated cardiac fibroblasts leads to fibrosis (FIG. 19).
  • the number of cardiac myofibroblasts actively involved in fibrosis is specifically confirmed by staining the cells with anti- ⁇ -smooth muscle actin ( ⁇ SMA) both at the border and in the blocked area And quantified (FIG. 4C, FIG. 4D).
  • ⁇ SMA anti- ⁇ -smooth muscle actin
  • the inventors It was found that by 14 days after ligation, the degree of fibrosis in hearts treated with PP decreased (FIGS. 4E, 4F). This reduced fibrosis is due to a reduction in the area of obstruction (cardiomyocyte death / necrosis is reduced, as there is no difference in infarct size between hearts treated with vehicle or PP. (FIGS. 4E and 4F).
  • Fibrosis in the obstructive heart decreased by PP treatment was further confirmed by the level of decrease in the content of hydroxyproline (numerical target of grown collagen molecules) in the heart (FIG. 4G).
  • PP reduces the number of cardiac fibroblasts that proliferate in vivo in ischemic heart tissue caused by myocardial infarction, and thus reduces fibrosis in vivo. Yes.
  • 14 days after ligation infarcted hearts treated with PP showed improved left ventricular function, as shown by increased ejection fraction, compared to hearts treated with vehicle alone. (FIG. 4H). This result suggests that PP can reduce fibrosis and prevent obstructed heart function decline.
  • PP also effectively kills various cancer cells that are deficient in glucose, but the mode of action is controversial (refs. 15-19).
  • Such studies also provide important insights into putative metabolic properties shared by both cardiac fibroblasts and cancer cells that have the unique ability to survive in the absence of glucose .
  • the present inventors have clarified a metabolic mechanism in which cardiac fibroblasts adapt to an environment deficient in glucose (FIG. 3C). Furthermore, the inventors have discovered that temporary exposure of cardiac fibroblasts to anthelmintic drugs (PP) induces a sedative effect of permanent mitochondrial energy metabolism (FIG. 3C). This mode of drug action is unprecedented, with only one drug inducing a sedative “memory” of mitochondrial energy metabolism that persists beyond the period in which the drug is present. PP-induced mitochondrial metabolic memory in cardiac fibroblasts inhibits metabolic adaptation to glucose-deficient environments and is essentially unable to survive under metabolic stress in the absence of the drug (FIG. 3C).
  • PP anthelmintic drugs
  • the present inventors have provided that the fibroblasts of the heart treated with the drug have a condition of “recovering” by allowing the fibroblasts to reset the metabolic adaptation to an environment deficient in glucose, It has been shown that this memory can be erased (FIG. 18).
  • FIG. PP has the effect of inhibiting mitochondrial energy metabolism, and this effect inhibits the survival of cardiac fibroblasts, especially in microenvironments that are deficient in glucose and / or glutamine after the drug is excreted.
  • FIG. PP A schematic diagram of a hypothesis.
  • E Normal structure of mitochondria (arrow) in cells treated with vehicle (DMSO) (-PP) or cells treated with PP (+ PP) in normal medium for 1 hour. Scale bar: 400 nm.
  • 13 C uptake is derived from [ 13 C-Glc] in the TCA cycle metabolites.
  • the results after 0 minutes, 20 minutes, 120 minutes, and 720 minutes after adding [ 13 C-Glc] / DMSO (-PP) or [ 13 C-Glc] / PP (+ PP) to the normal medium are shown.
  • the absolute amount of metabolite that has incorporated 13 C (black) or the absolute amount of metabolite that has not incorporated 13 C (eg, 12 C) (white) is shown.
  • Data Mean ⁇ standard deviation. N. D. ; Undetectable. * P ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001.
  • n 3.
  • D Energy sufficiency in cardiac fibroblasts treated with PP.
  • PP-treated cells black in a normal medium for 1 hour, and then each of the above cells in a nutrient-deficient medium (-Glc / Gln) in the absence of PP.
  • the absolute values of AMP, ADP, ATP, and “energy sufficiency” of cells cultured over time are shown.
  • the energy sufficiency is calculated by ([ATP] +1/2 [ADP]) / ([ATP] + [ADP] + [AMP]), and all units of [ATP], [ADP], and [AMP] are pmol / 10 5 cells.
  • FIG. Glucose rather than pyruvate or glutamine, supports the survival of cardiac fibroblasts treated with PP, and the effects of PP on metabolism in cardiac fibroblasts adapted to glucose-deficient environments.
  • Diagram showing summary A: Cardiac fibroblasts treated with PP survive with glucose, not pyruvate.
  • Glycolysis and mitochondrial energy metabolism in cells treated with vehicle (DMSO) (black) and cells treated with PP (gray) are measured in real time, in particular ECAR (mpH / min) and OCR (pmol / min) It shows using.
  • Data: Mean ⁇ standard deviation. n 3.
  • FIG. 1 Schematic diagram showing the effect of PP on cardiac fibroblasts.
  • the impedance of pyruvate (glycolytic metabolite) entry to TCA cycle metabolism induced by PP is expressed in the “wide space” between the arrow indicating glycolysis and the ring indicating TCA cycle in the schematic diagram.
  • the inhibitory effect of ETC induced by PP is represented by “X” in the schematic diagram. Note that ETC means an electron transmission system.
  • FIG. 4 Effect of PP on cardiac fibrosis in myocardial infarction model mice
  • A It is the figure which investigated the influence of PP with respect to the cell proliferation in the infarcted heart.
  • Cells of Ki67 + (green when displayed in color) and DAPI + (dark blue when displayed in color) were identified by immunofluorescence.
  • Scale bar 50 ⁇ m.
  • Ki67 + proliferating cells of A The result of quantifying Ki67 + proliferating cells of A above.
  • the number of ⁇ SMA + myofibroblasts at the infarct zone and border was counted and expressed as% ( ⁇ SMA + / DAPI + ).
  • the area of ⁇ SMA + was also measured and expressed as% (area of ⁇ SMA + / area of all sites).
  • H It is a figure which shows the effect of PP with respect to the heart function after myocardial infarction. Cardiac function after myocardial infarction was measured by echocardiography and expressed as ejection fraction (EF:%). Echocardiography was performed on mice treated with vehicle (-PP) or PP (+ PP) 7 days and 14 days after surgery for control (Sham) or ligation.
  • Data Mean ⁇ standard deviation. * P ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001. N. S. : Not significant.
  • FIG. Metabolomic analysis of cardiac fibroblasts under ischemic environment A: Heat map showing the overall quantitative change of intracellular metabolites in cardiac fibroblasts cultured under O 2 low / Glc low / Gln low conditions (ischemic environment). Intracellular metabolites were quantitatively analyzed using CE-TOFMS. A scale bar representing the amount of each metabolite is shown on the left side (when displayed in color, it is displayed as a change from green to red. Note that the change from green to red corresponds to a low level ⁇ a high level. ). The results of hierarchical cluster analysis (HCA) are shown as the left side cluster tree.
  • HCA hierarchical cluster analysis
  • Normal culture conditions are 20-21% O 2 , DMEM (ie, 25 mM Glc, 4 mM Gln), 10% FBS.
  • the culture conditions of the ischemic environment (O 2 low / Glc low / Gln low ) (hereinafter sometimes simply referred to as “ischemia”) are 3% O 2 , DMDE (Glc ⁇ , Gln ⁇ ), 10 % FBS (ie, 300 ⁇ M Glc, ⁇ 100 ⁇ M Gln).
  • Cardiac fibroblasts were cultured and collected for 72 hours under each condition and processed for CE-TOFMS analysis. In each condition, three independent culture dishes (shown as 1, 2, 3) were processed for CE-TOFMS analysis.
  • FIG. Evaluation of PP cytotoxicity Cytotoxicity of PP against cardiac fibroblasts was examined under normal (black line) and ischemic (gray line) conditions.
  • the IC50 of PP is 9.5 nM (PP) in cardiac fibroblasts under O 2 low / Glc low / Gln low culture conditions (ischemia).
  • FIG. Evaluation of recovery of survival of cardiac fibroblasts treated with PP in an ischemic environment by supplementing glucose is a diagram of examining the specific impact of complementary oxygen to cytotoxic (+ O 2) and glutamine (+ Glu) of PP.
  • Results with cells in each culture condition for 24 hours in the presence or absence of PP are shown as a white bar graph or a black bar graph, respectively.
  • the number of viable cells under each culture condition at 0 hours is shown as 100%.
  • Data: Mean ⁇ standard deviation. * P ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001. N. S. : Not significant. n 3.
  • FIG. 13 C-Glu Path map for metabolomics analysis of flux
  • F Treated with vehicle (DMSO) for 1 hour and cultured under normal culture conditions for 12 hours.
  • G Treated with PP for 1 hour and cultured under normal culture conditions for 12 hours.
  • H After 12 hours without PP treatment, cultured under nutrient deficient (Glc low / Gln low ) culture conditions for 12 hours.
  • I Treated with vehicle (DMSO) for 1 hour and cultured under nutrient-deficient (Glc low / Gln low ) culture conditions for 12 hours.
  • J Treated with PP for 1 hour and cultured under nutrient deficient (Glc low / Gln low ) culture conditions for 12 hours.
  • the bar graph shows the results of measuring three different samples under each condition (AJ).
  • FIG. Restoration of metabolic adaptation and recovery of mitochondrial energy metabolism in PP-treated cardiac fibroblasts in culture conditions deficient in glucose
  • Heart fibroblasts treated with vehicle (DMSO) (for 1 hour in normal medium) or cardiac fibroblasts treated with PP (for 1 hour in normal medium) were treated with normal culture medium. Cultured over time (the cells were fed with fresh normal medium daily). After this “recovery” period, the cells were cultured in Glc low / Gln low medium for 72 hours, and the results of measuring the viability of the cells are shown in Panel A. 100% is the number of cells at the time when the culture is started in Glc low / Gln low medium.
  • FIG. 1 Schematic diagram of pathological process of myocardial infarction
  • FIG. Improved angiogenesis in scarred hearts treated with PP A: CD31 + capillaries at the infarcted area of the scarred heart and its border. Capillaries were identified using anti-CD31 antibodies in heart portions prepared from mice administered vehicle (DMSO) (-PP) or PP (+ PP). Scale bar: 50 ⁇ m.
  • FIG. Evaluation of the role of classical Wnt and UPR in specific cytotoxicity during PP ischemia A: Classical Wnt in cardiac fibroblasts cultured for 12 hours under normal and O 2 low / Glc low / Gln low (ischemic) conditions in the presence or absence of PP (87 ⁇ M) It is the figure which investigated the expression of two downstream genes (Axin2 and c-Myc) of a signal. The expression level is shown as fold-induction (the expression of each gene in cells cultured in the absence of PP and under normal conditions is shown as 1-fold-induction). Double or triple analysis was performed and the expression levels were quantified by qRT-PCR in 4 independent experiments.
  • Cardiac fibroblasts were cultured for 24 hours under normal conditions or O 2 low / Glc low / Gln low (ischemic) conditions using vehicle ( ⁇ AEBSF) or AEBSF (300 ⁇ M) (+ AEBSF) The number of viable cells in each condition was counted.
  • Collagenase / trypsin treated samples were further separated by pipetting up to 50 times. Each suspension of tissue was transferred to a 50 ml tube containing 20 ml of DMEM (Nacalai) with 10% FBS, 1% penicillin, 1% streptomycin. The separated cells were centrifuged at 100 ⁇ g for 5 minutes at 4 ° C., suspended in DMEM supplemented with 20% FBS, 1% penicillin, 1% streptomycin, and in the same medium. The culture was continued for about 1 week until storage.
  • DMEM Nacalai
  • Heart fibroblasts were plated on collagen-coated dishes (IWAKI). After culturing in culture medium (high glucose DMEM, 10% FBS, 1% penicillin, 1% streptomycin) for 24 hours, the cells were subjected to the corresponding oxygen and Cultured under nutrient conditions. Low glucose and glutamine conditions were performed by using DMEM without glucose and glutamine. Trace amounts of glucose and glutamine were present as a culture medium containing 10% FBS. The final concentration was determined to be 300 ⁇ M glucose and ⁇ 100 ⁇ M glutamine. Hypoxic conditions were performed by using a Hypoxia chamber (Veritas) and a gas container to be mixed (3% O 2 , 10% CO 2 , 87% N 2 ). Viable cells were identified and counted by the exclusion of trypan blue dye (GIBCO).
  • GEBCO trypan blue dye
  • the extracted metabolite was concentrated using a centrifugal concentrator (Tomy) and stored at ⁇ 80 ° C. until analysis. Appropriate amount of MilliQ water was added to dissolve the concentrated metabolites just prior to sample injection into CE-TOFMS.
  • Methanol-water (50% v / v) containing 0.1 M hexakis (2,2-difluorothoxy) phosphazene was fed at 10 ⁇ l / min as a protective solution.
  • the ESI-TOFMS was set to positive ion mode and the capillary voltage was set to 4 kV. Heat drying was performed (heating temperature was 300 ° C.), and the flow rate of nitrogen gas was maintained at 10 psig.
  • the fragmentor voltage, skimmer voltage, and Oct RFV voltage were set to 75V, 50V, and 125V, respectively.
  • a sample solution (30 nL) was injected at 50 mbar for 30 seconds using a voltage of ⁇ 30 kV.
  • the temperature of the capillary was adjusted to 20 ° C. using a thermostat, and the sample pan was cooled to a temperature lower than 5 ° C.
  • CE interface at 10 ⁇ L / min with 5 mM ammonium acetate dissolved in 50% (v / v) methanol-water containing 0.1 M hexakis using an Agilent 1100 series pump with a 1: 100 splitter.
  • the solution was sent to (CE interface). It is used as a sheath fluid outside the CE capillary and provides a stable electrical connection between the capillary tip and a grounded electrospray needle.
  • ESI-TOFMS was performed by negative ionization (capillary voltage set to 3,500V).
  • fragmentor voltage, skimmer voltage, and Oct The RFV voltage was set to 100V, 50V and 200V, respectively.
  • the flow rate of dry nitrogen gas was maintained at 7 L / min.
  • the annotation table was performed by CE-ESI-TOFMS measurements of standard compounds and aligned with the data set according to similar m / z values and normalized MT values.
  • PCA main component analysis
  • JMP version 9.0.2 SAS Institute, Cary, NC
  • Mev TM4 software version 4.7.4. Dana-Farber Cancer Institute, Boston, MA
  • PB phosphate buffer
  • PFA paraformaldehyde
  • GA glutaraldehyde
  • OsO 4 osmium tetroxide
  • the sample was dehydrated through a series of graded ethanols (50%, 70%, 90%, 100%) (50% and 70% for 5 minutes at 4 ° C, 90% respectively) was carried out in 3 portions at room temperature for 5 minutes, and in the case of 100% at room temperature for 10 minutes each.) After dehydration, the sample was embedded in resin (Quetol-812; Nisshin EM Co., Tokyo, Japan) and then polymerized at 60 ° C. for 48 hours. The thinnest part (thickness 70 nm) was prepared using a diamond knife using an ultramicrotome (ULTRACUT UCT; Leica), and the part was placed on a copper grid.
  • resin Quetol-812; Nisshin EM Co., Tokyo, Japan
  • the part is colored with uranyl acetate for 15 minutes at room temperature, then secondary colored with lead coloring solution (Sigma-Aldrich Co.,) for 3 minutes at room temperature, followed by Washed with distilled water.
  • the copper lattice was observed using a transmission electron microscope (JEM-1200EX; JEOL Ltd.) with an acceleration voltage of 80 kV.
  • Digital images (2048 ⁇ 2048 pixels) were taken using a CCD camera (VELETA; Olympus Soft Imaging Solutions GmbH).
  • a myocardial infarction was generated by ligating a coronary artery to an ICR male mouse (8-12 weeks old) by the method already disclosed in the conventional literature (Reference 25). Mice were anesthetized by inhalation of vaporized 2-2.5% isoflurane (Abbott Japan) and intubated with a 20 gauge infusion container. Animals were ventilated using a respirator (Harvard Apparatus) with a respiration rate of 110 cycles per minute and a volume controlled at 200 ⁇ l per cycle. After thoracotomy, the left coronary artery was ligated using 8-0 nylon sutures 1-2 mm below the tip of the left atrial appendage.
  • Occlusion was confirmed by a change in the color (pale white) of the left ventricular anterior wall.
  • the chest cavity and skin were closed using 5-0 silk sutures.
  • Tubular organs were removed from the animals and allowed to recover from surgery on a 1 hour warm plate.
  • the same procedure was performed on sham-operated mice, but not sutured, but by passing under the coronary artery.
  • Suspend PP so that the final concentration is 400 ⁇ g / ml in 4% DMSO dissolved in saline and 0.5 ml suspension or vehicle (dissolved in saline) in the stomach.
  • the mice were forced to eat so that 4% DMSO) entered.
  • Oral administration started 1 day after ligation and continued daily until 14 days after ligation.
  • Ki67 + signal and CD31 + signal were detected by Alexa 488 and Alexa 647, respectively, conjugated with secondary antibody (Invitrogen).
  • Cell nuclei were counterstained with DAPI using ProLong® Gold antifade reagent (Invitrogen) containing DAPI.
  • concentrations of Ki67 + cells and capillaries were calculated using Image J (NIH). Using BZ-9000 BZII software (Keyence Japan, Inc.), ⁇ SMA-positive cells and ⁇ SMA + area were calculated.
  • RNA isolation, cDNA synthesis and quantitative RT-PCR All RNA was isolated using TRIZOL reagent (Invitrogen), and cDNA was synthesized using SuperScript III first-strand synthesis system (Invitrogen). Real-time qRT-PCR was performed using QuantiTect SYBER Green PCR kit (Qiagen) according to the manufacturer's procedure using the primers described below. All qRT-PCR results were normalized to the level of B2m ( ⁇ -2-microglobulin) transcript.
  • B2m (F: 5′-GCTCGGTGACCCTGGTCTTT -3 ′ (SEQ ID NO: 1), R: 5′-AATGTGAGGCGGGTGGAACT -3 ′ (SEQ ID NO: 2))
  • Axin2 (F: 5′-GAGAGTGAGCGGCAGAGC-3 ′ (SEQ ID NO: 3), R: 5′-CGGCTGACTCGTTCTCCT-3 ′ (SEQ ID NO: 4))
  • c-Myc (F: 5′-CCTAGTGCTGCATGAGGAGA-3 ′ (SEQ ID NO: 5), R: 5′-TCCACAGACACCACATCAATTT -3 ′ (SEQ ID NO: 6))
  • Grp78 / BiP (F: 5′-CTGAGGCGTATTTGGGAAAG-3 ′ (SEQ ID NO: 7), R: 5′-TCATGACATTCAGTCCAGCAA-3 ′ (SEQ ID NO: 8))
  • Grp94 (F: 5′-AG
  • PP replaces quiescent mitochondria with fresh mitochondria that are not affected by mechanisms that mediate (or are associated with) autophagy.
  • PP suppresses the autophagy of cancer cells, particularly in the environment of glucose starvation (Reference 15). Therefore, PP can inhibit the replacement of dysfunctional mitochondria with normal mitochondria by suppressing the autophagy.
  • the event disclosed in the present specification is useful, and the extent of the inhibitory activity of PP's autophagy is estimated. To do is a future research subject.
  • FIG. 19 Inhibition of fibrosis after myocardial infarction by a combination of starting and daily administration of PP one or two days after myocyte death, but not simultaneously with coronary artery ligation, and heart PP validates the beneficial effect on improving function.
  • One or two days after ligating the coronary arteries it is important to treat the infarcted heart with PP to minimize adverse effects on muscle cells in the ischemic area.
  • the inventors do not want to kill the cardiac muscle cells by administering PP when the cardiac muscle cells are dying. For this reason, the present inventors started administration of PP on the next day after ligation (that is, one day after ligation) (FIG. 19).
  • Wnt or UPR signals are not involved in the ischemic-specific cytotoxic effects of PP in cardiac fibroblasts.
  • PP has been reported to inhibit two non-NADH-FR pathways (classical Wnt pathway and endoplasmic reticulum stress response (UPR) pathway) (References 19 to 21). Therefore, the inventors have determined whether these signals are activated by ischemia or whether inhibition of such signals mediates PP-induced ischemia-specific cytotoxicity of cardiac fibroblasts.
  • Figure 21 Activation of the classical Wnt signal is typically observed by increased expression of genes that are the subject of direct transcription of ⁇ -catenin (eg, Axin2 and c-Myc) (http: //www.stanford.
  • FIG. 21A Compared with cells cultured under normal conditions, the expression of Axin2 was slightly suppressed in cells under ischemic conditions (FIG. 21A). Suppression of Axin2 expression in cardiac fibroblasts under ischemic conditions was further suppressed by PP (FIG. 21A). In contrast, c-Myc expression was elevated in cardiac fibroblasts under ischemic conditions (FIG. 21A). However, the increase in c-Myc expression was not affected by PP (FIG. 21A).
  • FIG. 21C The potential for inhibition of UPR signaling to mediate PP-specific ischemic cytotoxic effects was evaluated (FIG. 21C).
  • ATF6, IRE1 and PERK There are three distinct pathways (ATF6, IRE1 and PERK) underlying the UPR signal (Reference 40).
  • the present inventors examined the expression of a chaperone (Grp78 / BiP, Grp94) downstream of the UPR signal and the mediator (Xbp1) of ATF6 and IRE1 signals, and cultured cardiac fibroblasts under ischemic conditions.
  • the present invention can be used for treatment or prevention of ischemic diseases. Moreover, it is useful for the development of a drug for treating or preventing a new ischemic disease.

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Abstract

L'invention concerne un médicament comprenant, comme principe actif, une substance qui a une activité destinée à rendre anormales des mitochondries et à inhiber la croissance cellulaire dans un environnement ischémique, une maladie ischémique pouvant être traitée ou prévenue.
PCT/JP2013/068545 2013-02-15 2013-07-05 Médicament pour le traitement ou la prévention d'une maladie ischémique et son utilisation WO2014125663A1 (fr)

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JP2009514787A (ja) * 2005-11-08 2009-04-09 財団法人先端医療振興財団 虚血性心疾患の治療方法

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JP2009514787A (ja) * 2005-11-08 2009-04-09 財団法人先端医療振興財団 虚血性心疾患の治療方法

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Title
CHIKAOMI YAMADA ET AL.: "Shinkin Kosoku ni Okeru Sen'ika no Seigyo Kiko", ANNUAL REVIEW JUNKANKI, vol. 2012, 2012, pages 54 - 60 *
GOHIL V.M. ET AL.: "Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis", NAT. BIOTECHNOL., vol. 28, 2010, pages 249 - 255 *
LAM A.P. ET AL.: "beta-catenin signaling: a novel mediator of fibrosis and potential therapeutic target", CURR. OPIN. RHEUMATOL., vol. 23, 2011, pages 562 - 567 *

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