WO2016080516A1 - INHIBITEUR DE POLYMÉRISATION DE LA Drp1 - Google Patents

INHIBITEUR DE POLYMÉRISATION DE LA Drp1 Download PDF

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WO2016080516A1
WO2016080516A1 PCT/JP2015/082688 JP2015082688W WO2016080516A1 WO 2016080516 A1 WO2016080516 A1 WO 2016080516A1 JP 2015082688 W JP2015082688 W JP 2015082688W WO 2016080516 A1 WO2016080516 A1 WO 2016080516A1
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cells
drp1
cilnidipine
group
mice
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PCT/JP2015/082688
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Japanese (ja)
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基宏 西田
石川　達也
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味の素株式会社
大学共同利用機関法人自然科学研究機構
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Priority to JP2016560298A priority Critical patent/JP6688503B2/ja
Publication of WO2016080516A1 publication Critical patent/WO2016080516A1/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
    • A61K31/33—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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine

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  • the present invention relates to a Drp1 polymerization inhibitor containing cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient, and a novel pharmaceutical composition using the Drp1 polymerization inhibitor.
  • This application claims the priority based on Japanese Patent Application No. 2014-236941 for which it applied to Japan on November 21, 2014, and uses the content here.
  • Mitochondria are organelles found in almost all eukaryotic cells, and are mainly responsible for ATP production (ADP phosphorylation) by oxidative phosphorylation by the electron transport system. It is known that mitochondria repeat fusion and division and are involved in the onset of cancer, cardiovascular diseases, neurodegenerative diseases and the like due to their abnormalities. For example, it has been suggested that in a non-infarcted lesion after myocardial infarction, mitochondrial morphological function abnormality (remodeling) causes an abnormal energy metabolism to cause chronic heart failure.
  • Mitochondrial fission is caused by activation of GTP-binding protein Drp1 (dynamin-related protein 1).
  • the activated Drp1 polymerizes to form a ring-shaped multimer around the mitochondria. This Drp1 multimer cleaves mitochondria to cause division.
  • cilnidipine is a kind of dihydropyridine calcium antagonist, and has both L-type and N-type calcium antagonistic actions and is known to have a blood pressure lowering action.
  • the antihypertensive effect of cilnidipine is characterized by a longer duration than other antihypertensive drugs. Therefore, cilnidipine is useful as a therapeutic agent for cardiovascular diseases such as heart failure and arrhythmia (see, for example, Patent Documents 1 to 3).
  • cilnidipine is useful for the treatment or prevention of cerebral infarction and cerebral hemorrhage in addition to the commonly used antihypertensive treatment by utilizing calcium antagonism (see, for example, Patent Document 4), and renal dysfunction. It is reported to be useful for the treatment or prevention of (see, for example, Patent Document 5).
  • cilnidipine has also been reported to have a side effect reducing action of an anticancer drug (see Patent Document 6).
  • An object of the present invention is to provide a Drp1 polymerization inhibitor useful for the treatment or prevention of diseases caused by induction of mitochondrial fission, and a pharmaceutical composition comprising the Drp1 polymerization inhibitor as an active ingredient.
  • the present invention provides the following Drp1 polymerization inhibitors, pharmaceutical compositions, and cell aging inhibitors of [1] to [7].
  • a Drp1 polymerization inhibitor characterized by containing cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient and inhibiting polymerization of Drp1 (dynamin-related protein 1).
  • a pharmaceutical composition comprising the Drp1 polymerization inhibitor of [1] as an active ingredient.
  • the pharmaceutical composition according to the above [2] which is used for prevention or treatment of chronic heart failure after myocardial infarction.
  • the pharmaceutical composition according to the above [2] which is used for reducing cardiomyocyte toxicity due to organic mercury.
  • the Drp1 polymerization inhibitor according to the present invention can inhibit mitochondrial division.
  • the said Drp1 polymerization inhibitor and the pharmaceutical composition which uses this as an active ingredient can be utilized as a preventive or therapeutic agent of various diseases caused by excessive division of mitochondria.
  • the Drp1 polymerization inhibitor can be used relatively safely in animals including humans. it can.
  • FIG. 1 is a diagram showing the results of analysis of the mitochondrial morphology of each cell in Reference Example 1.
  • FIG. 2 is a graph showing the measurement results of the percentage (%) of senescent cells with positive SA- ⁇ -Gal activity in each cell in Reference Example 1.
  • FIG. 3 is a diagram showing the calculation result of the relative value (%) of the amount of active oxygen in mitochondria of each cell in Reference Example 1.
  • FIG. 4 is a diagram showing the results of analysis of the mitochondrial morphology of each cell in Reference Example 1.
  • FIG. 5 shows the results of analysis of the mitochondrial morphology of cells hypoxically stimulated and reoxygenated in the presence or absence of cilnidipine in Example 1.
  • FIG. 6 is a graph showing the measurement results of the percentage (%) of senescent cells with positive SA- ⁇ -Gal activity in hypoxia-stimulated and reoxygenated cells in the presence or absence of cilnidipine in Example 1. It is.
  • FIG. 7 is a photomicrograph showing the localization of Drp1 and mitochondria in cells stimulated with hypoxia in the presence or absence of cilnidipine in Example 1.
  • FIG. 8 is a graph showing changes over time in the survival rate (%) of mice in each group in Example 2.
  • FIG. 9 is a graph showing measurement results of heart weight (mg / g) per body weight of each group of mice 4 weeks after myocardial infarction (after LAD ligation surgery) in Example 2.
  • FIG. 10 is a graph showing the measurement results of the left ventricular cardiomyocyte area ( ⁇ m 2 ) of each group of mice in Example 2.
  • FIG. 11 is a diagram showing measurement results of collagen positive tissue region (CVF) (%) of mice in each group in Example 2.
  • FIG. 12 is a graph showing the measurement results of the ratio (area ratio) (%) of aging tissues with positive SA- ⁇ -Gal activity in non-infarcted myocardium after myocardial infarction in mice of each group in Example 2. It is.
  • FIG. 13 is a diagram showing the results of detecting Drp1 by Western blotting of the area around the myocardial infarction in the left ventricle of each group of mice in Example 2.
  • FIG. 14 is a diagram showing measurement results of FS (left ventricular diameter shortening rate) of mice in each group in Example 2.
  • FIG. 15 is a graph showing the measurement results of the percentage (%) of cells positive for SA- ⁇ -Gal activity of each cell in Example 3.
  • FIG. 16 is a graph showing changes over time in survival rate (%) from the time point of TAC treatment for mice in each group in Reference Example 2.
  • FIG. 17 is a graph showing the results of measuring the relative survival rate (%) of cells cultured in a medium supplemented with 0 or 0.5 ⁇ M methylmercury and various existing drugs in Example 4.
  • FIG. 18 is a graph showing the measurement results of the adventitious blood glucose level (left diagram) and fasting blood glucose level (right diagram) of STZ mice on Day 14 of cilnidipine administration in Example 5.
  • STZ indicates the results of STZ mice not administered with cilnidipine
  • STZ + cil indicates the results of STZ mice administered with cilnidipine.
  • FIG. 19 is a graph showing the results of measuring the cell viability (%) when amyloid ⁇ loading was applied in the presence and absence of cilnidipine in Example 6.
  • the Drp1 polymerization inhibitor according to the present invention comprises cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient.
  • mitochondrial fission is inhibited. That is, the Drp1 polymerization inhibitor according to the present invention functions as a mitochondrial division inhibitor.
  • Silnidipine is a compound known as an L / N-type calcium antagonist that inhibits both L-type and N-type calcium channels. Specifically, the following structural formula:
  • the cilnidipine in the present invention includes optical isomers based on the above structural formula, and any of them can be produced using a known production method (Japanese Patent Publication No. 3-14307, Japanese Patent Publication No. 6-43397, etc.) See). It is also commercially available.
  • Silnidipine can be converted into a pharmaceutically acceptable salt, hydrate or solvate thereof as necessary.
  • the pharmaceutically acceptable salt is not particularly limited, but for example, a salt with an inorganic acid (hydrochloride, hydrobromide, phosphate, nitrate, sulfate, etc.) or a salt with an organic acid (acetic acid) Salt, succinate, maleate, fumarate, malate, tartrate, lactate, citrate, etc.).
  • cilnidipine or a pharmaceutically acceptable salt thereof may be simply referred to as cilnidipine.
  • ⁇ Drp1 is activated and polymerized by stimulation of hyperglycemia, hypoxia, environmental pollutants, nitric oxide, etc., and mitochondria are split by tightening with the formed Drp1 multimer. Subsequent reoxygenation fuses split mitochondria and produces excessive amounts of ATP and active oxygen, leading to diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and the like. Silnidipine suppresses mitochondrial division by inhibiting Drp1 polymerization. For this reason, cilnidipine is effective in the treatment and prevention of diseases induced by mitochondrial division such as diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and the like.
  • hypoxia-inducing factors In non-infarcted lesions after myocardial infarction, the expression of hypoxia-inducing factors is increased, leading to premature cardiomyocyte aging, which causes chronic heart failure and sudden death.
  • mitochondrial division is induced transiently, and subsequent reoxygenation induces cell damage and cellular senescence.
  • Silnidipine suppresses mitochondrial division of cardiomyocytes due to hypoxic stimulation, and thus suppresses cell damage and aging after reoxygenation.
  • cilnidipine can be used as an active ingredient for suppressing cell aging, and is particularly preferably used for prevention or treatment of chronic heart failure after myocardial infarction and prevention of sudden death.
  • organic mercury such as methylmercury is known to be an environmental pollutant that increases cardiovascular risk.
  • exposure of cardiomyocytes to organic mercury at a low concentration that does not exhibit cytotoxicity induces mitochondrial division, induces cell death, and increases stretch stress sensitivity.
  • Silnidipine suppresses mitochondrial division of cardiomyocytes by organic mercury, and thus cell death.
  • cilnidipine is suitably used for reducing myocardial cell toxicity due to organic mercury, and particularly suitably used for suppressing the deterioration of cardiac function due to organic mercury in heart failure.
  • cilnidipine does not particularly affect blood glucose levels in a healthy state, but has an action of lowering blood glucose levels in an insulin-dependent hyperglycemia state. For this reason, cilnidipine is suitably used for the reduction of an insulin-dependent hyperglycemia state, and is suitable as an insulin-dependent diabetes drug.
  • the Drp1 polymerization inhibitor according to the present invention is preferably administered to animals, more preferably administered to mammals, humans, mice, rats, rabbits, guinea pigs, hamsters, monkeys. It is more preferable to administer to livestock and experimental animals such as sheep, horses, cows, pigs, donkeys, dogs and cats. Further, the Drp1 polymerization inhibitor according to the present invention may be administered alone to an animal, or may be administered in combination with a pharmaceutical composition containing a substance other than cilnidipine as an active ingredient.
  • the Drp1 polymerization inhibitor according to the present invention can also be used for the purpose of reducing side effects mainly caused by mitochondrial fission induced by highly electrophilic drugs such as anticancer agents.
  • a pharmaceutical composition having an effect of treating or preventing diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and the like may be administered in combination with the Drp1 polymerization inhibitor according to the present invention, These may be used as a kit.
  • the Drp1 polymerization inhibitor according to the present invention can be administered as a medicine.
  • the Drp1 polymerization inhibitor according to the present invention or a pharmaceutical composition comprising this as an active ingredient is administered to animals including humans.
  • the administration form may be either oral administration or parenteral administration.
  • the dosage form if it is an oral administration agent, for example, a solid agent such as powder, granule, capsule, tablet, chewable agent; solution And liquids such as syrups and syrups, and in the case of parenteral agents, for example, injections, infusions, nasal and pulmonary sprays, and the like.
  • the pharmaceutical composition according to the present invention can be formulated into pharmaceuticals of these dosage forms by an ordinary method.
  • the pharmaceutical composition according to the present invention is preferably administered orally to the subject.
  • a tablet is preferable as the dosage form for oral administration.
  • the pharmaceutical composition according to the present invention can be intravenously or arterally administered as an infusion solution.
  • the pharmaceutical composition according to the present invention comprises an appropriate pharmaceutically acceptable carrier, such as an excipient, a binder, a lubricant, a solvent, a disintegrant, a solubilizing aid, as necessary in the formulation.
  • An agent, a suspending agent, an emulsifier, an isotonic agent, a stabilizer, a soothing agent, an antiseptic, an antioxidant, a corrigent, a coloring agent, and the like can be formulated.
  • excipients examples include sugars such as lactose, glucose, D-mannitol, organic excipients such as starches and celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin.
  • a lubricant pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc.
  • stearic acid fatty acid salts such as stearate, talc, silicates, etc.
  • solvent purified water, physiological saline, etc.
  • disintegrant low substituted hydroxypropylcellulose
  • chemically modified Cellulose for example, polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, etc.
  • suspending agents or emulsifiers Sodium sulfate, gum arabic, gelatin, lecithin, glyceryl monostearate, polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose sodium and other celluloses, polysorbates, polyoxyethylene hydrogenated castor oil, etc.
  • Potassium chloride, sugars, glycerin, urea, etc., stabilizers are polyethylene glycol, sodium dextran sulfate, other amino acids, etc.
  • soothing agents are glucose , Calcium gluconate, procaine hydrochloride, etc., as preservatives, paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc., as antioxidants, sulfite, ascorbic acid, etc.
  • examples of the flavoring agent include sweeteners and fragrances commonly used in the pharmaceutical and food fields
  • examples of the colorant include colorants commonly used in the pharmaceutical and food fields.
  • the dosage of the pharmaceutical composition according to the present invention varies depending on the type of anticancer agent, the age, weight, disease state, administration method, etc. of the subject, and is usually a clinical dose of the active ingredient cilnidipine or a pharmaceutically acceptable salt thereof.
  • 0.0001 to 1000 mg, preferably 0.001 to 100 mg, particularly preferably 0.01 to 50 mg of the active ingredient per day as an adult per day is orally administered in one or several divided doses
  • 0.0000001 to 100 mg, preferably 0.000001 to 50 mg, particularly preferably 0.0001 to 20 mg can be parenterally administered once to several times a day, or 1 hour per day To 24 hours and can be administered intravenously.
  • mouse myocardial infarction model was prepared with reference to the method of Nishida et al. (Nature Chemical Biology, vol. 8, p. 714-724 (2012)). Specifically, an 8-week-old mouse (C57BL / 6J (SLC)) was intraperitoneally administered with a triple anesthetic (dmitol: 0.75 mg / kg, midazolam: 4 mg / kg, betorfal: 5 mg / kg). After anesthesia, the thorax was opened and the left anterior descending coronary artery (LAD) was ligated using silk suture (No. 6-0) (MI group).
  • mice that had been anesthetized and thoracotomy but did not ligate LAD were assigned to the sham group.
  • NRCM Preparation of neonatal rat cardiac myocytes
  • NRCM Preparation of neonatal rat cardiac myocytes cells were obtained as follows. First, after the cold anesthesia of the SD rat fetus on the first day of feeding, the heart was taken out and incubated with 0.05% trypsin-EDTA at 4 ° C. for 16 hours. Then, trypsin was removed and incubated at 37 ° C. for 15 minutes in a collagenase II solution diluted with PBS (phosphate physiological saline) to 1 mg / mL. The residue was further incubated with collagenase II at 37 ° C.
  • PBS phosphate physiological saline
  • DMEM fetal bovine serum
  • penicillin 100 unit / mL penicillin
  • streptomycin 100 ⁇ g / mL streptomycin
  • the seeded NRCM cells were cultured in 5% by volume carbon dioxide (95% by volume air) in a humidified atmosphere at 37 ° C. for 24 hours, and then taurine-containing DMEM (containing 5 mM taurine, 100 unit / mL penicillin, and 100 ⁇ g / mL streptomycin). The culture medium was changed to DMEM) and cultured for 48 hours and used for each experiment.
  • taurine-containing DMEM containing 5 mM taurine, 100 unit / mL penicillin, and 100 ⁇ g / mL streptomycin.
  • Hela cells were FBS-containing DMEM (DMEM containing 10% by volume FBS, 100 unit / mL penicillin, and 100 ⁇ g / mL streptomycin) at 5% carbon dioxide (95% by volume air) in a humidified atmosphere at 37 ° C. Cultured. Transfection was performed on X-tremeGENE9 DNA transfection reagent (Roche) on Hela cells cultured overnight after changing the medium to DMEM without FBS (DMEM containing 100 unit / mL penicillin and 100 ⁇ g / mL streptomycin) the day before. And 3 ⁇ g of plasmid per 35 mm dish was introduced for 12 hours according to the attached instructions.
  • FBS-containing DMEM DMEM containing 10% by volume FBS, 100 unit / mL penicillin, and 100 ⁇ g / mL streptomycin
  • PCR was performed using an Fw primer for GFP tag and an Rv primer for GFP tag, and the obtained PCR product was digested with BamHI and XhoI and then ligated to the pEGFP-C1 vector.
  • -A plasmid for expression of EGFP was obtained.
  • PCR is performed using the obtained cDNA as a template, Fw primer for GFP tag and Rv primer for GFP tag, and the obtained PCR product is digested with BamHI and XhoI and then ligated to pcDNA3.1 vector.
  • a plasmid for expression of Drp1-Flag was obtained.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ strain, a single colony was cultured, and then a plasmid was obtained by LaboPass (Hokkaido System Science Co., Ltd.).
  • the obtained plasmid was purified by Qiagen plasmid Maxi kit (manufactured by Qiagen) after confirming the sequence by sequencing, and used for each experiment.
  • Drp1 (C624S) -EGFP in which EGFP is fused to the C-terminal side of the C624S mutant of Drp1 (mutant in which the 624th cysteine is substituted with serine
  • Drp1-EGFP PCR was carried out using the C624S Fw primer, the C624S Rv primer, and a PCR reagent (product name: KOD ⁇ Fx, manufactured by Toyobo Co., Ltd.), and the resulting PCR product was digested with BamHI and XhoI and then pEGFP. After ligation to the -C1 vector, it was prepared in the same manner as the expression plasmid for Drp1-EGFP.
  • the MTT assay is an assay that examines mitochondrial NADH dehydrogenase activity. Specifically, MTT solution (MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-triphenyltetrazolium bromide)) was added to NRCM cells cultured in a 96-well plate at 5 mg / mL. The solution was dissolved so that the final concentration of MTT was 0.125 mg / mL, and cultured at 37 ° C. for 1 hour in a humidified atmosphere of 5% by volume carbon dioxide (95% by volume air).
  • the formazan produced was dissolved by removing the medium, adding 100 ⁇ L of DMSO, and gently shaking at room temperature for 30 minutes. Thereafter, the absorbance at 595 nm (Abs595) was measured using a plate reader (product name: SpectraMax i3, manufactured by Molecular devices).
  • Hypoxic stimulation of NRCM cells was performed by culturing NRCM cells at 37 ° C. for 16 hours in a humidified atmosphere with 1 vol% oxygen and 5 vol% carbon dioxide (94 vol% nitrogen). Reoxygenation was performed by culturing NRCM cells after hypoxic stimulation at 37 ° C. for 24 hours in a humidified atmosphere of 5% by volume carbon dioxide (95% by volume air).
  • Mitochondrial morphology was observed with a confocal laser scanning microscope (product name: FV10i, Olympus, magnification: x300) after staining the mitochondria of each cell with MitoTracker Green FM reagent (Life technologies). And analyzed.
  • the mitochondrial staining is performed by firstly inoculating a cell seeded in a 35 mm glass bottom dish with MitoTrakcer Green (0.5 ⁇ M), 5 vol% carbon dioxide (95 vol% air), and 37 ° C. in a humidified atmosphere. The reaction was incubated for a minute. After the reaction, the plate was washed twice with PBS to remove MitotTacker and then replaced with phenol red-free DMEM.
  • the form of mitochondria of each cell is divided into vesicles, tubules, and intermediate structures between vesicles and tubes, and the results of determining the respective abundances are shown in FIG.
  • N indicates the result of unstimulated cells
  • H indicates the results of cells after hypoxic stimulation
  • H / R indicates the results of cells reoxygenated after hypoxic stimulation. Show. Vesicular mitochondria increased with hypoxic stimulation but decreased after reoxygenation.
  • SA- ⁇ -gal activity measurement Acidic ⁇ -galactosidase (SA- ⁇ -Gal) activity related to senescence was measured and the proportion of senescent cells was measured. SA- ⁇ -Gal activity was measured using a senescence ⁇ -Galactosidase Staining Kit (manufactured by Cell signaling technology). Specifically, first, NRCM cells seeded in a 35 mm dish at 60% confluence were reoxygenated after hypoxic stimulation, and then washed once with PBS. As a control, unstimulated NRCM cells were also washed once with PBS.
  • Mdivi-1 (CAS No: 338967-87-6, manufactured by Sigma), which is a selective inhibitor for Drp1 and is a mitochondrial division inhibitor, is dissolved in DMSO so that the final concentration is 10 ⁇ M.
  • Unstimulated NRCM cells and NRCM cells reoxygenated after hypoxic stimulation were prepared in the same manner except that the added culture medium was used. Next, each cell was fixed by treatment with Fixative Solution included in the kit for 15 minutes at room temperature. The fixed cells were washed once with PBS, added with ⁇ -galactosidase staining solution included in the kit, and incubated overnight at 37 ° C.
  • the cells after the incubation were observed with an upright microscope (product name: Eclipse 80i, manufactured by Nikon) equipped with a color CCD camera (Nikon digital camera DXM1200F), and cells positive for SA- ⁇ -Gal activity (stained cells) ) Percentage (%).
  • FIG. 2 shows the measurement results of the percentage (%) of cells positive for SA- ⁇ -Gal activity.
  • “( ⁇ )” in the “H / R” column indicates the result of unstimulated NRCM cells
  • “(+)” indicates the result of NRCM cells reoxygenated after hypoxic stimulation.
  • “( ⁇ )” shows the result of NRCM cells without Midi-1
  • “(+)” shows the result of NRCM cells with Midi-1 added.
  • the proportion of cells positive for SA- ⁇ -Gal activity increased after hypoxic stimulation compared to unstimulated cells. It was confirmed that cell senescence was induced by oxygenation.
  • the proportion of cells positive for SA- ⁇ -Gal activity in the reoxygenated cells after hypoxia stimulation was low. It was found that cell senescence induced by this is suppressed.
  • ⁇ Measurement of mitochondrial active oxygen content The amount of active oxygen produced from mitochondria was measured using MitoSOX Red (manufactured by Life technologies). Specifically, first, NRCM cells seeded in a 35 mm glass bottom dish were washed once with PBS after hypoxic stimulation or after reoxygenation after hypoxic stimulation. As a control, unstimulated NRCM cells were also washed once with PBS. Further, except that Mdivi-1 (manufactured by Sigma) dissolved in DMSO was added to the culture medium to a final concentration of 10 ⁇ M, unstimulated NRCM cells, hypoxia-stimulated NRCM cells, and low NRCM cells reoxygenated after oxygen stimulation were also prepared.
  • MitoSOX Red manufactured by Life technologies. Specifically, first, NRCM cells seeded in a 35 mm glass bottom dish were washed once with PBS after hypoxic stimulation or after reoxygenation after hypoxic stimulation. As a control, unstimul
  • MitoSOX (5 ⁇ M) was added to each cell and reacted by incubating at 37 ° C. for 10 minutes in a humidified atmosphere with 5% by volume carbon dioxide (95% by volume air). After the reaction, it was washed twice with PBS to remove MitoSOX, and then replaced with phenol red-free DMEM. The stained cells were observed with a confocal laser scanning microscope (product name: FV10i, Olympus, magnification: x300), and the fluorescence intensity per cell was measured for each cell. The relative value with the fluorescence intensity per cell of one additive-free cell as 100% was calculated as the relative value (%) of the amount of active oxygen in mitochondria.
  • Fig. 3 shows the calculation result of the relative value (%) of the amount of active oxygen in mitochondria.
  • N”, “H”, and “H / R” have the same meaning as in FIG. 2, and in FIG. 3, “( ⁇ )” in the “Midivi-1” column is NRCM without Midi-1 added.
  • the results of the cells and “(+)” show the results of the NRCM cells to which Midiv-1 was added, respectively.
  • the amount of mitochondrial active oxygen increased by hypoxic stimulation and subsequent reoxygenation.
  • the treatment of mitochondrial active oxygen after hypoxic stimulation and subsequent reoxygenation by Midi-1 treatment The increase was suppressed.
  • the luminescence intensity of DMEM containing ATP at a final concentration of 0, 0.01, 0.1, 1, or 10 ⁇ M was determined in the same manner, and the obtained luminescence intensity was obtained.
  • a calibration curve obtained from the relationship between ATP concentration and ATP concentration was used.
  • the amount of ATP consumed per well was determined by adding 10 ⁇ M carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to the cells and incubating for 5 minutes at room temperature to inhibit ATP synthesis from mitochondria, followed by Luciferase assay The amount of residual ATP in the cells was measured.
  • CCCP carbonyl cyanide m-chlorophenyl hydrazine
  • FIG. 4 shows the measurement results of the intracellular ATP amount (ATP concentration per well) of each cell.
  • N “H”, and “H / R” have the same meaning as in FIG. 2
  • control is the result of NRCM cells without Midi-1
  • Modivi-1 is Midi ⁇ .
  • the results of 1 added NRCM cells are shown respectively.
  • the amount of intracellular ATP increases due to reoxygenation after hypoxic stimulation, but this increasing tendency was suppressed by Midi-1 treatment.
  • NRCM cells expressing Drp1-EGFP are subjected to hypoxic stimulation and reoxygenation treatment in the presence of various calcium antagonists, intracellular ATP content, mitochondrial morphology, percentage of aging cells (%), and Drp1 The localization of was investigated.
  • NRCM cells were transfected with a plasmid for expression of Drp1-EGFP, and then a DMSO solution of cilnidipine (CIL), amlodipine (Aml), verapamil (Ver), diltiazem (Dil) or ⁇ -conotoxin ( ⁇ -Ctx) Phosphate buffer solution (PBS) was added to a final culture concentration of 10 ⁇ M for hypoxia stimulation and reoxygenation, and intracellular ATP content and mitochondrial morphology of cells after each treatment The percentage of senescent cells (%) and the localization of Drp1 were examined.
  • CIL cilnidipine
  • Aml amlodipine
  • Ver verapamil
  • Dil diltiazem
  • ⁇ -Ctx ⁇ -conotoxin
  • NRCM cells cultured in a culture medium to which only an equal amount of DMSO was added were measured in the same manner.
  • Hypoxic stimulation and reoxygenation treatment were performed in the same manner as in Reference Example 1, and the amount of intracellular ATP, mitochondrial morphology, and percentage of aged cells (%) were measured in the same manner as in Reference Example 1.
  • the localization of Drp1 was examined by observing the fluorescence of fused EGFP with a confocal laser scanning microscope.
  • hypoxic stimulation and reoxygenation in the presence of a calcium antagonist other than cilnidipine were performed in the same manner as in the control (culture medium cells added with DMSO alone).
  • the amount of intracellular ATP was increased, the amount of intracellular ATP after reoxygenation was clearly lower in the cells subjected to hypoxic stimulation and reoxygenation in the presence of cilnidipine compared to the control. From these results, it was suggested that cilnidipine suppresses an increase in intracellular ATP level due to hypoxic stimulation and reoxygenation, but this inhibitory effect is an action unrelated to calcium antagonism.
  • the mitochondrial morphology of the control cells (culture medium cells to which only DMSO was added) and the hypoxic-stimulated and reoxygenated cells in the presence of cilnidipine were vesicular, tubular, small as in Reference Example 1. It was divided into an intermediate structure between the vesicular and tubular, and the abundance of each was determined. The results are shown in FIG. In FIG. 5, “N”, “H”, and “H / R” have the same meaning as in FIG. 1, “control” is the result of the control cell, “CIL” is hypoxic stimulation in the presence of cilnidipine and The results of the cells subjected to reoxygenation are shown respectively. Silnidipine showed that vesicular mitochondria by hypoxia stimulation hardly increased, and mitochondria division by hypoxia stimulation was suppressed.
  • FIG. 6 shows the measurement results of the percentage (%) of cells with positive SA- ⁇ -Gal activity in hypoxic-stimulated and reoxygenated cells in the presence or absence of cilnidipine.
  • “( ⁇ )” indicates the result of unstimulated NRCM cells
  • “(+)” indicates the result of NRCM cells reoxygenated after hypoxic stimulation.
  • “( ⁇ )” in the “CIL” column indicates the result of the control NRCM cells (culture medium cells added with DMSO alone), and “(+)” indicates the results of the NRCM cells added with cilnidipine.
  • CIL control NRCM cells
  • hypoxia stimulation and reoxygenation increase the percentage of cells positive for SA- ⁇ -Gal activity, whereas in the presence of cilnidipine, SA ⁇ due to hypoxia stimulation and reoxygenation increases.
  • the increase of cells with positive ⁇ -Gal activity was suppressed. That is, it was shown that cell senescence due to hypoxic stimulation and reoxygenation is suppressed by cilnidipine.
  • FIG. 7 shows a photomicrograph showing the localization of Drp1 and mitochondria in cells stimulated with hypoxia in the presence or absence of cilnidipine.
  • N indicates the result of unstimulated cells
  • H (Control) indicates the result after hypoxic stimulation of the control cells
  • H (CIL) indicates hypoxia of the cells to which cilnidipine was added. The results after stimulation are shown respectively.
  • unstimulated cells the intracellular localization of Drp1-EGFP and mitochondria in cilnidipine-added cells was not particularly different from control cells, and Drp1-EGFP was broadly present in the cytoplasm.
  • Drp1-EGFP was localized in the vicinity of mitochondrial vesicles, whereas Drp1-EGFP in the cells to which cilnidipine was added was in vesicles. There were few localities, and no co-localization with mitochondria was observed.
  • Cirnidipine was administered to a mouse myocardial infarction model (MI group) prepared from C57BL / 6J mice and a control sham group, and the influence of cilnidipine on the heart after myocardial infarction was examined.
  • cilnidipine administered to mice in the MI group.
  • CIL30-MI group those administered to mice in the MI group were designated as CIL30-MI group, and those administered to mice in the sham group were designated as CIL30-sham group.
  • mice administered at a dose of cilnidipine of 100 mg / kg / day those administered to mice in the MI group were administered to mice in the CIL100-MI group, and mice administered to the mice in the sham group were treated with the CIL100-sham group. It was. Further, as a control, mice in which only a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7 were administered to the MI group and the sham group were designated as the Vehicle-MI group and the Vehicle-sham group, respectively.
  • FIG. 8 shows time-dependent changes in the survival rate (%) of the Vehicle-MI group, the CIL30-MI group, and the CIL100-MI group.
  • FIG. 9 shows the measurement results of heart weight (mg / g) per body weight of each group of mice 4 weeks after myocardial infarction (after LAD ligation surgery).
  • the heart weight was measured by performing a cardiac ultrasonography according to a conventional method.
  • “( ⁇ )” in the “CIL” column indicates the result of the Vehicle group
  • “30” indicates the result of the CIL 30 group
  • “100” indicates the result of the CIL 100 group.
  • the ventricular specimen was immersed in a 0.1% Direct Red 80 solution prepared using a hematoxylin solution or a saturated picric acid solution, treated for 60 minutes, and further fractionated with 1% hydrochloric alcohol to obtain ethanol, xylene. Was dehydrated and sealed.
  • the encapsulated specimen, particularly the non-infarct region, was observed with a microscope (product name: BZ-9000, manufactured by Keyence). The ratio of the collagen content was calculated as a percentage obtained by dividing the dyed area by the total cross-cardiac area.
  • SA- ⁇ -Gal activity in heart sections was measured according to the method of Shlush et al. (BMC Cell Biology, 2011, vol. 12, 16), and the proportion of aged cells was measured. Specifically, first, the heart was removed 4 weeks after myocardial infarction (after LAD ligation surgery). The excised heart was washed in PBS, fixed with PBS containing 4% PFA, replaced with sucrose, and frozen and embedded using OCT compound (Sakura).
  • Heart sections sliced to 10 ⁇ m from a frozen-embedded specimen using LEICA CM1100 were modified from the Senescence ⁇ -Galactosidase Staining Kit (manufactured by Cell signaling technology) in a ⁇ -Galactosidase solution at pH 4.0. The reaction was performed by incubating overnight at 0 ° C. The cells after incubation were washed with PBS and then encapsulated with VECTASHIELD Mounting Medium.
  • the encapsulated specimen was observed with an upright microscope (product name: Eclipse 80i, manufactured by Nikon Corporation) equipped with a color CCD camera (Nikon digital camera DXM1200F), and the area of the area positive for SA- ⁇ -Gal activity ( ⁇ -Galactosidase positive area, blue color development)] / [total tissue section area] ⁇ 100 (%).
  • FIG. 12 shows the measurement results of the ratio (area ratio) (%) of the tissue with positive SA- ⁇ -Gal activity in the non-infarcted myocardium after myocardial infarction in mice of each group.
  • “( ⁇ )” in the “CIL” column indicates the result of the Vehicle group
  • “(+)” indicates the result of the CIL100 group.
  • “( ⁇ )” indicates the result of the sham group
  • “(+)” indicates the result of the MI group.
  • the PVDF membrane after blocking was incubated with anti-Drp1 antibody or anti-GAPDH antibody (both manufactured by Santa Cruz Biotechnology) solution as a primary antibody, and then incubated with anti-horseradish peroxidase (HRP) antibody as a secondary antibody. Then, bands of Drp1 and GAPDH were detected using an ECL system (manufactured by Nacalai Tesque).
  • FIG. 13 shows the results of detecting Drp1 by Western blotting of the area around the myocardial infarction in the left ventricle of each group of mice.
  • sham indicates the result of the sham group
  • MI indicates the result of the MI group.
  • Vehicle indicates the result of the Vehicle group
  • Cilinipine indicates the result of the CIL100 group.
  • sham group no Drp1 dimer was detected regardless of whether or not cilnidipine was administered.
  • MI group a Drp1 dimer was detected, but the DIL dimer amount was clearly lower in the CIL100 group than in the Vehicle group. From this result, it was found that cilnidipine suppresses the dimerization of Drp1.
  • FIG. 14 shows the FS measurement results for each group of mice.
  • “( ⁇ )” in the “CIL” column indicates the result of the Vehicle group
  • “30” indicates the result of the CIL 30 group
  • “100” indicates the result of the CIL 100 group.
  • Example 3 Cell senescence when NRCM cells were cultured in the presence of cilnidipine or Mdiv-1 was examined. Specifically, after culturing NRCM cells for 72 hours in a medium to which cilnidipine was added to a final concentration of 1 ⁇ M and a medium to which Mdiv-1 was added to a final concentration of 10 ⁇ M, Reference Example 1 In the same manner, SA- ⁇ -Gal activity was measured. As a control, NRCM cells were similarly cultured in a medium to which neither cilnidipine nor Mdivi-1 was added, and SA- ⁇ -Gal activity was measured.
  • FIG. 15 shows the measurement results of the percentage (%) of cells positive for SA- ⁇ -Gal activity of each cell.
  • mice fed with water containing 10 ppm methylmercury were treated with transverse aortic stenosis (TAC), and body weight change (%), survival rate (%), and heart weight per body weight (mg / g) were examined.
  • TAC transverse aortic stenosis
  • body weight change %
  • survival rate %
  • heart weight per body weight mg / g
  • TAC TAC
  • cardiac ultrasonography was performed according to a conventional method at 1 week, 2 weeks, 3 weeks, and 4 weeks after TAC treatment, and the heart weight was measured. The intake of drinking water containing methylmercury continued until the 4th week.
  • FIG. 16 shows the change over time in the survival rate (%) of the mice in each group from the time of TAC treatment.
  • both the MeHg-TAC group and the Vehicle-TAC group had increased heart weight.
  • the heart weight increased more in the MeHg-TAC group than in the Vehicle-TAC group.
  • NRCM cells were exposed to low concentrations of methylmercury (0.1 ⁇ M) that did not induce cytotoxicity in the presence and absence of Midivi-1, and the effects on ATP production and stretch stress sensitivity were examined. Specifically, NRCM cells are seeded in a 96-well plate, a culture medium containing 0.1 ⁇ M methylmercury, a culture medium containing 10 ⁇ M Midi-1, 0.1 ⁇ M methylmercury and 10 ⁇ M Midi-1 In a culture medium containing 1 or 5% by volume carbon dioxide (95% by volume air) in a humidified atmosphere at 37 ° C. for 24 hours.
  • the ATP concentration ( ⁇ M) per well was obtained in the same manner as in Reference Example 1, and the wells of control cells (cells cultured in a culture medium containing neither methylmercury nor Midi-1) were obtained. The relative value was calculated with the per-ATP concentration as 100%. The amount of intracellular ATP in each cell was decreased by exposure to methylmercury, and it was found that this decrease in ATP production by methylmercury can be recovered by Midiv-1.
  • the cells after culturing in each culture medium were expanded by 20% using a cell extension device, and then MTT assay was performed. From the absorbance (MTT value) at 595 nm of each cell, a relative value was calculated by setting Abs595 of the control cell (cultured in a culture medium containing neither methylmercury nor Midi-1) as 100. As a result, in the cells exposed to methylmercury, the MTT value decreased after extension stress and the number of dead cells increased, but in the cells exposed to methylmercury in the presence of Midi-1, the MTT value was increased even after extension stress. No decrease was observed. From these results, it was suggested that organic mercury increases stretch stress sensitivity, but midi-1 suppresses the effects of organic mercury and increases adaptability to stretch stress.
  • Example 4 We searched for existing drugs that reduce myocardial cytotoxicity caused by methylmercury.
  • the drugs used were cilnidipine (1 ⁇ M), Mdivi-1 (10 ⁇ M), DIDS (4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid) (100 ⁇ M), ROX (1 ⁇ M), diazoxide (Diazoxide) (100 ⁇ M) Rottererin (5 ⁇ M), AICAR (5-amino-1-bD-ribofuranosyl-imidazole-4-carboxamide) (500 ⁇ M), Amlodipine (1 ⁇ M), and ET-1 (0.1 ⁇ M). .
  • the survival rate of each cell was calculated when the survival rate of cells cultured in a medium to which no drug was added and methylmercury was not added was defined as 100%.
  • the calculation results are shown in FIG. As a result, cilnidipine, Mdivi-1, DIDS, diazoxide, and AICAR reduced the toxicity of methylmercury.
  • Cirnidipine was administered to diabetic model mice, and the effect on blood glucose level was examined.
  • 48 C57BL / 6J (SLC) mice were divided into 4 groups of 12 mice each, and a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7 to 2 groups (24 mice) of these mice.
  • STZ mice which are insulin-dependent diabetes model mice, were prepared by intraperitoneally administering streptozotocin (STZ) in a dissolved state to 200 mg / kg.
  • STZ mice streptozotocin
  • the blood glucose level and fasting blood glucose level of each mouse were measured every 3 to 4 days from the start of STZ administration.
  • control mice Veh group and Veh + cil group
  • no effect of cilnidipine administration on blood glucose level was observed.
  • STZ mice no difference was observed between the STZ + veh group and the STZ + cil group in the occasional blood glucose level and the fasting blood glucose level at the time before 18 days after administration of STZ but before cilnidipine administration. After the day, a tendency for the blood glucose level to decrease in the STZ + cil group was observed.
  • Example 6 In Alzheimer's disease patients, many deposits containing amyloid ⁇ protein as a tumor component are observed, and accumulation of amyloid ⁇ protein is considered to be involved in the onset of Alzheimer's disease. In addition, when a large amount of amyloid ⁇ protein is taken into cells, endoplasmic reticulum stress becomes strong and apoptosis is induced. Thus, the influence of cilnidipine on cell damage caused by amyloid ⁇ loading was examined using a cultured cell line PC-12 cells derived from rat pheochromocytoma. As the amyloid ⁇ protein, Amyloid ⁇ 25-35 (manufactured by Sigma-Aldrich, catalog number: A4559) was used.
  • D-MEM High Glucose
  • FBS fetal bovine serum
  • HS fetal bovine serum
  • penicillin- A medium containing streptomycin Nacalai
  • D-MEM High Glucose
  • penicillin-streptomycin manufactured by Nacalai
  • a poly-L-lysine (PLL) solution with a final concentration of 0.001% was poured into a 24-well plate, allowed to stand at 37 ° C. for 30 minutes, washed twice with PBS, and air-dried. The inner wall was coated with PLL. Undifferentiated PC-12 cells were seeded at 1 ⁇ 10 5 cells / well (500 ⁇ L / well) in a PLL-coated 24-well plate and cultured at 37 ° C. for 24 hours in the culture medium.
  • serum-free medium containing 500 ⁇ L of cilnidipine (final concentration 1 ⁇ M) and Amyloid ⁇ 25-35 (final concentration 10 ⁇ M) per well.
  • a serum-free medium containing only cilnidipine (final concentration 1 ⁇ M), a serum-free medium containing only Amyloid ⁇ 25-35 (final concentration 10 ⁇ M), or a serum-free medium was added at 37 ° C. for 24 hours. Incubate for hours.
  • MTT solution a solution in which MTT was dissolved in PBS so as to be 5 mg / mL
  • MTT solution a solution in which MTT was dissolved in PBS so as to be 5 mg / mL
  • DMSO was added at 500 ⁇ L per well and shaken firmly to dissolve the produced formazan.
  • 100 ⁇ L of the solution in each well was dispensed into each well of a 96-well plate, and this 96-well plate was placed in a plate reader (product name: SpectraMax i3, manufactured by Molecular devices), and absorbance at 595 nm (Abs595) was measured. It was measured.

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Abstract

La présente invention concerne une composition pharmaceutique contenant de la cilnidipine ou un sel pharmaceutiquement acceptable de celle-ci en tant que principe actif. La présente invention concerne : un inhibiteur de polymérisation de la protéine apparentée à la dynamine 1 (Drp1 : dynamin-related protein 1) caractérisé par le fait qu'il contient de la cilnidipine ou un sel pharmaceutiquement acceptable de celle-ci en tant que principe actif et également caractérisé en par le fait qu'il est capable d'inhiber la polymérisation de la Drp1; une composition pharmaceutique contenant l'inhibiteur de polymérisation de la Drp1 en tant que principe actif; et une composition pharmaceutique telle que décrite ci-dessus, qui peut être utilisée pour prévenir ou traiter une insuffisance cardiaque chronique après un infarctus du myocarde ou pour réduire la toxicité cardiomyocytaire provoquée par du mercure organique ou encore pour atténuer un état hyperglycémique insulino-dépendant.
PCT/JP2015/082688 2014-11-21 2015-11-20 INHIBITEUR DE POLYMÉRISATION DE LA Drp1 WO2016080516A1 (fr)

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WO2022098642A1 (fr) * 2020-11-03 2022-05-12 Rdiscovery, LLC Thérapies pour le traitement du cancer et de maladies associées à une déficience en phagocytose
WO2022114187A1 (fr) * 2020-11-30 2022-06-02 国立大学法人九州大学 Composé ayant une action inhibitrice contre des divisions mitochondriales excessives

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Cited By (7)

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CN114206441A (zh) * 2019-05-31 2022-03-18 R发现有限责任公司 Drp1-细丝蛋白复合物形成抑制剂
JP2022525697A (ja) * 2019-05-31 2022-05-18 アールディスカバリー エルエルシー Ｄｒｐ１－フィラミン複合体形成阻害剤
EP3976177A4 (fr) * 2019-05-31 2023-05-31 RDiscovery, LLC Inhibiteurs de formation de complexe drp1-filamine
JP7333105B2 (ja) 2019-05-31 2023-08-24 アールディスカバリー エルエルシー Ｄｒｐ１－フィラミン複合体形成阻害剤
CN114206441B (zh) * 2019-05-31 2024-07-02 R发现有限责任公司 Drp1-细丝蛋白复合物形成抑制剂
WO2022098642A1 (fr) * 2020-11-03 2022-05-12 Rdiscovery, LLC Thérapies pour le traitement du cancer et de maladies associées à une déficience en phagocytose
WO2022114187A1 (fr) * 2020-11-30 2022-06-02 国立大学法人九州大学 Composé ayant une action inhibitrice contre des divisions mitochondriales excessives

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