JPWO2016080516A1 - Drp1 polymerization inhibitor - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to a Drp1 polymerization inhibitor, which contains cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient and inhibits the polymerization of Drp1 (dynamin-related protein 1), the Drp1 polymerization described above The pharmaceutical composition according to the above, which is used for a pharmaceutical composition comprising an inhibitor as an active ingredient, prevention or treatment of chronic heart failure after myocardial infarction, reduction of cardiomyocyte toxicity by organic mercury, or reduction of insulin-dependent hyperglycemia Composition.

Description

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 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 the 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.

  On the other hand, cilnidipine is a kind of dihydropyridine calcium antagonist and has both L-type and N-type calcium antagonistic activity and is known to have a blood pressure lowering effect. The antihypertensive effect of cilnidipine is characterized by a longer duration than other antihypertensive drugs. For this reason, cilnidipine is useful as a therapeutic agent for cardiovascular diseases such as heart failure and arrhythmia (see, for example, Patent Documents 1 to 3). Further, 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). In addition, cilnidipine has also been reported to have a side effect reducing action of an anticancer drug (see Patent Document 6).

Japanese Patent No. 4613396 JP 2008-048771 A International Publication No. 2013/147137 JP 2004-359675 A International Publication No. 2008/016171 JP 2013-014549 A

  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. And

  As a result of intensive studies to solve the above problems, the present inventors have found that cilnidipine can inhibit the polymerization of Drp1, and have completed the present invention.

That is, the present invention provides the following Drp1 polymerization inhibitors, pharmaceutical compositions, and cell aging inhibitors of [1] to [7].
[1] 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).
[2] A pharmaceutical composition comprising the Drp1 polymerization inhibitor of [1] as an active ingredient.
[3] The pharmaceutical composition according to the above [2], which is used for prevention or treatment of chronic heart failure after myocardial infarction.
[4] The pharmaceutical composition according to the above [2], which is used for reducing cardiomyocyte toxicity due to organic mercury.
[5] The pharmaceutical composition according to the above [2], which is used for reducing insulin-dependent hyperglycemia.
[6] The pharmaceutical composition according to the above [2], which is used for prevention or treatment of amyotrophic lateral sclerosis or Alzheimer's disease.
[7] A cell aging inhibitor comprising the Drp1 polymerization inhibitor of [1] as an active ingredient.

  The Drp1 polymerization inhibitor according to the present invention can inhibit mitochondrial division. For this reason, 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. In particular, since cilnidipine, which is an active ingredient of the Drp1 polymerization inhibitor, has already been clinically applied as an antihypertensive agent, 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 diagram showing the measurement results of the ratio (%) of senescent cells in which SA-β-Gal activity of each cell is positive 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-β-Gal activity in hypoxic 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 (μm 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 aged tissue having positive SA-β-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-β-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 μM 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. In the figure, “STZ” indicates the results of STZ mice not administered with cilnidipine, and “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 β 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. As a result of inhibition of Drp1 polymerization by the Drp1 polymerization inhibitor, 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:

(±) -2-methoxyethyl 3-phenyl-2- (E) propenyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) -3,5-pyridinedicarboxy Rate. 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 to a pharmaceutically acceptable salt, hydrate or solvate thereof as required. 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.). Hereinafter, 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 disrupted 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.

  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. As shown in Examples below, when cardiomyocytes are stimulated with hypoxia, 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. For this reason, 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. As shown in Examples below, 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. For this reason, 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.

  In addition, as shown in Examples below, 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. For example, 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 medicament.

  The Drp1 polymerization inhibitor according to the present invention or a pharmaceutical composition comprising this as an active ingredient (hereinafter sometimes simply referred to as “the pharmaceutical composition according to the present invention”) is administered to animals including humans. In this case, the administration form may be either oral administration or parenteral administration. As 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.

  From the viewpoint of reducing the burden on the patient, the pharmaceutical composition according to the present invention is preferably orally administered to the subject. A tablet is preferable as the dosage form for oral administration. On the other hand, for a subject who is difficult to administer orally, the pharmaceutical composition according to the present invention can be intravenously or arterally administered as an infusion. In addition, 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.

  Examples of excipients include sugars such as lactose, glucose and D-mannitol, organic excipients such as starches and celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin. As a lubricant, pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. Are stearic acid, fatty acid salts such as stearate, talc, silicates, etc., as solvent, purified water, physiological saline, etc., as disintegrant, low substituted hydroxypropylcellulose, chemically modified Cellulose and de Pungs, etc. include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, etc. as 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, saccharides, glycerin, urea, etc., as stabilizer, polyethylene glycol, sodium dextran sulfate, other amino acids, etc., as soothing agent, 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, and examples of the coloring agent 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. Can be set as appropriate. For example, the daily dose per adult is 0.0001 to 1000 mg, preferably 0.001 to 100 mg, particularly preferably 0.01 to 50 mg of the active ingredient is orally administered in one or several divided doses, or adult 1 As a daily dose per person, 0.0000001 to 100 mg, preferably 0.000001 to 50 mg, particularly preferably 0.0001 to 20 mg can be parenterally administered once a day to several times, or 1 hour per day To 24 hours and can be administered intravenously.

  EXAMPLES Next, although an Example etc. are shown and this invention is demonstrated further in detail, this invention is not limited to these.

[mouse]
In subsequent experiments, C57BL / 6J (SLC) mice purchased from Kudo Co., Ltd. were used.

[Preparation of mouse myocardial infarction model]
A 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). Whether or not myocardial infarction due to ligation was successful was confirmed by observing an increase in ST (ventricular excitement) on the electrocardiogram. Similarly, mice that had been anesthetized and thoracotomy but did not ligate LAD were assigned to the sham group.

[cell]
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. for 15 minutes, passed through a 70 μm nylon cell strainer (BD-Falcon Biosciences), and then centrifuged (1000 rpm, 2 minutes) to remove collagenase. The obtained cells were suspended in DMEM containing FBS (fetal bovine serum) (DMEM containing 10% by volume FBS, 100 unit / mL penicillin, and 100 μg / mL streptomycin), and then seeded in a non-coat dish. The culture was performed at 37 ° C. for 1 hour in a humidified atmosphere with vol% carbon dioxide (95 vol% air). Cells that did not adhere to the dish were then collected as NRCM cells and seeded on gelatin-coated dishes or plates. 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 μg / mL streptomycin). The culture medium was changed to DMEM) and cultured for 48 hours and used for each experiment.

  Hela cells were prepared in DMEM containing FBS (DMEM containing 10% by volume FBS, 100 unit / mL penicillin, and 100 μg / mL streptomycin) at 5 ° C. 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 DBS (DMEM containing 100 units / mL penicillin and 100 μg / mL streptomycin) the day before. And 3 μg of plasmid per 35 mm dish was introduced for 12 hours according to the attached instructions.

[Preparation of plasmids for expression of Drp1-EGFP and Drp1-Flag]
An expression plasmid for Drp1 (Drp1-EGFP) fused with EGFP on the C-terminal side and an expression plasmid for Drp1 (Drp1-Flag) fused with Flag on the C-terminal side were prepared as follows.
First, RNA prepared from a rat heart using RNeasy fibrous tissue mini kit (Qiagen) was used as a template, and Prime Script RT (Takara Bio) was used. Using the obtained cDNA as a template, PCR was performed using the Fw primer for GFP tag and the Rv primer for GFP tag, and the obtained PCR product was digested with BamHI and XhoI and then ligated to the pEGFP-C1 vector to obtain Drp1 -A plasmid for expression of EGFP was obtained. Similarly, 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. Thus, a plasmid for expression of Drp1-Flag was obtained.
The ligation product was transformed into Escherichia coli DH5α 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.

  In addition, the expression plasmid for the protein (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) is an expression of Drp1-EGFP. PCR was carried out using the C624S Fw primer, C624S Rv primer and PCR reagent (product name: KOD Fx, manufactured by Toyobo Co., Ltd.) after digesting the resulting PCR product with BamHI and XhoI. After ligation to the -C1 vector, it was prepared in the same manner as the expression plasmid for Drp1-EGFP.

[MTT assay]
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). After culturing, the formazan produced was dissolved by removing the medium, adding 100 μL 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).

[Reference Example 1]
In NRCM cells, it was confirmed that hypoxia stimulation induced mitochondrial division and reoxygenation induced cellular senescence. Specifically, for unstimulated NRCM cells, NRCM cells after hypoxic stimulation, and NRCM cells reoxygenated after hypoxic stimulation, mitochondrial morphology and percentage of aged cells and intracellular activity The amount of oxygen (relative value) and intracellular ATP concentration (μM / well) were examined.

<Hypoxic stimulation and reoxygenation>
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>
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. In more detail, the mitochondrial staining is performed by firstly inoculating a cell seeded in a 35 mm glass bottom dish with MitoTrakcer Green (0.5 μM), 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 mitochondrial morphology of each cell is divided into vesicles, tubules, and intermediate structures between vesicles and tubes, and the results of the respective abundances are shown in FIG. In FIG. 1, “N” indicates the result of unstimulated cells, “H” indicates the results of cells after hypoxic stimulation, and “H / R” indicates the results of cells reoxygenated after hypoxic stimulation. Show. Vesicular mitochondria increased with hypoxic stimulation but decreased after reoxygenation.

<SA-β-gal activity measurement>
Acidic β-galactosidase (SA-β-Gal) activity related to aging was measured, and the proportion of aging cells was measured. SA-β-Gal activity was measured using senescence β-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. Further, 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 μM. 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 β-galactosidase staining solution included in the kit, and incubated overnight at 37 ° C. The cells after 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-β-Gal activity (stained cells) ) Percentage (%).

  The measurement results of the percentage (%) of cells positive for SA-β-Gal activity are shown in FIG. In FIG. 2, “(−)” in the “H / R” column indicates the result of unstimulated NRCM cells, and “(+)” indicates the result of NRCM cells reoxygenated after hypoxic stimulation. In FIG. 2, in the “Midivi-1” column, “(−)” indicates the result of the NRCM cell without Midi-1, and “(+)” indicates the result of the NRCM cell with Midi-1 added. . In the case of no addition of midi-1, the proportion of cells positive for SA-β-Gal activity increased in the reoxygenated cells after hypoxic stimulation than in the unstimulated cells. It was confirmed that cell senescence was induced by oxygenation. On the other hand, in the cells to which Midi-1 was added, the proportion of cells with positive SA-β-Gal activity in the reoxygenated cells after hypoxic stimulation was low, and the reoxygenation after hypoxic stimulation by Midi-1 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 so that the final concentration was 10 μM, unstimulated NRCM cells, hypoxia-stimulated NRCM cells, and low NRCM cells reoxygenated after oxygen stimulation were also prepared.
Subsequently, MitoSOX (5 μM) 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.

  The calculation result of the relative value (%) of the amount of mitochondrial active oxygen is shown in FIG. In FIG. 3, “N”, “H”, and “H / R” have the same meaning as in FIG. 2. In FIG. 3, “(−)” in the “Midivi-1” column is NRCM without Midi-1 added. The result of the cell, “(+)” indicates the result of the NRCM cell added with Midiv-1. As shown in FIG. 3, the mitochondrial active oxygen amount was increased by hypoxic stimulation and subsequent reoxygenation, but the mitochondrial active oxygen amount after hypoxic stimulation and in the subsequent reoxygenation was reduced by the Midi-1 treatment. The increase was suppressed.

<Measurement of intracellular ATP amount>
The amount of intracellular ATP was measured using Luciferase assay (manufactured by Wako Pure Chemical Industries, Ltd.).
Specifically, first, NRCM cells seeded in a 96-well plate 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 so that the final concentration was 10 μM, unstimulated NRCM cells, hypoxia-stimulated NRCM cells, and low NRCM cells reoxygenated after oxygen stimulation were also prepared.
Next, after removing the medium and replacing with fresh DMEM (100 μL), 20 μL of Luciferase reagent was immediately added and incubated at room temperature for 10 minutes. The luminescence of the cells after incubation was measured with SpectraMax i3 (Molecular devices), and the ATP concentration per well was determined from the luminescence intensity. For calculating the ATP concentration from the luminescence intensity, the luminescence intensity of DMEM containing ATP at a final concentration of 0, 0.01, 0.1, 1, or 10 μM 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 μM 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.

  The measurement results of the intracellular ATP amount (ATP concentration per well) of each cell are shown in FIG. In FIG. 4, “N”, “H”, and “H / R” have the same meaning as in FIG. 2, “control” indicates the result of NRCM cells without Midi-1, and “Midivi-1” indicates 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 increase tendency was suppressed by Midi-1 treatment.

[Example 1]
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 aged cells (%), and Drp1 The localization of was investigated.
Specifically, 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 ω-conotoxin (ω -Ctx) phosphate buffer solution (PBS) was added to the culture medium so that the final concentration was 10 μM, and hypoxia stimulation and reoxygenation were performed. The percentage of senescent cells (%) and the localization of Drp1 were examined. As a control, 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.

  As a result, in the cells subjected to hypoxic stimulation and reoxygenation in the presence of a calcium antagonist other than cilnidipine, the hypoxic stimulation and reoxygenation were performed in the same manner as in the control (culture medium cells added with DMSO alone). Although 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-β-Gal activity in hypoxic stimulated and reoxygenated cells in the presence or absence of cilnidipine. In FIG. 6, “(−)” in the “H / R” column indicates the result of unstimulated NRCM cells, and “(+)” indicates the result of NRCM cells reoxygenated after hypoxic stimulation. In FIG. 6, “(−)” 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. Are shown respectively. In the control cells, hypoxia stimulation and reoxygenation increase the percentage of cells positive for SA-β-Gal activity, whereas in the presence of cilnidipine, SA- by hypoxia stimulation and reoxygenation increases. The increase in cells positive for β-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 hypoxia stimulated in the presence or absence of cilnidipine. In FIG. 7, “N” indicates the result of unstimulated cells, “H (Control)” indicates the result after hypoxic stimulation of the control cells, and “H (CIL)” indicates hypoxia of the cells to which cilnidipine was added. The results after stimulation are shown respectively. In unstimulated cells, the intracellular localization of Drp1-EGFP and mitochondria in silnidipine-added cells was not particularly different from control cells, and Drp1-EGFP was broadly present in the cytoplasm. In the cells after hypoxic stimulation, in the control cells, 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.

[Example 2]
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.

<Administration of cilnidipine>
The administration of cilnidipine was performed after myocardial infarction (LAD ligation) using an osmotic pump (product name: model 2004, manufactured by Alzet) in a state where DMSO and PEG300 were dissolved in a mixed solvent mixed at a volume ratio of 3: 7. Administration was continued from 24 hours after surgery. Of the mice administered so that the dose of cilnidipine was 30 mg / kg / day, 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. Similarly, among mice administered so that the dose of cilnidipine is 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 changes over time in the survival rate (%) of the Vehicle-MI group, the CIL30-MI group, and the CIL100-MI group. As a result, the survival rates of the CIL30-MI group and the CIL100-MI group were clearly higher than those of the Vehicle-MI group, and the survival rate of the CIL100-MI group was higher than that of the CIL30-MI group. These results suggested that cilnidipine can suppress sudden death after myocardial infarction.

  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 FIG. 9, “(−)” in the “CIL” column indicates the result of the Vehicle group, “30” indicates the result of the CIL 30 group, and “100” indicates the result of the CIL 100 group. As a result, in the sham group, the presence or absence of the administration of cilnidipine and the influence of the dosage on the heart weight were not observed, but in the MI group, the increase in the heart weight was suppressed by the administration of cilnidipine. From these results, it was suggested that cilnidipine can suppress the increase in heart weight after myocardial infarction.

<Observation of fibrosis and cardiomyocyte area>
The heart of each mouse 4 weeks after myocardial infarction (after LAD ligation surgery) was stained with 1-6 picirius red and HE (hematoxylin and eosin), and myocardial fibrosis and myocardial cell area were determined by the method of Nishida et al. The EMBO Journal (2008) vol.27, p.3104-3115).
First, the heart of a mouse was fixed by formalin treatment, and then a central part of the ventricle was cut out to prepare a ventricular specimen embedded with paraffin. Subsequently, this ventricular specimen was sliced into a thickness of 3 μm, and paraffin was removed with xylene and ethanol. After removing the paraffin, 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.

1-6 From the results of the Picirius red stained image and the HE stained image, the measurement results of CSA (cross sectional area; myocardial cell area (μm ² )) are shown in FIG. 10, CVF (Collagen volume fraction; collagen positive area) (%) The measurement results are shown in FIG. As a result, both CSA and CVF increased due to myocardial infarction, but this increase was suppressed by administration of cilnidipine. From these results, it was suggested that cilnidipine can suppress left ventricular remodeling after myocardial infarction in a concentration-dependent manner.

<SA-β-gal activity measurement in heart section>
SA-β-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 aging 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 μm from a frozen-embedded specimen using LEICA CM1100 were modified from the Senescence β-Galactosidase Staining Kit (manufactured by Cell signaling technology), and 37 in a β-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 [area of a region positive for SA-β-Gal activity ( β-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-β-Gal activity in the non-infarcted myocardium after myocardial infarction in each group of mice. In FIG. 12, “(−)” in the “CIL” column indicates the result of the Vehicle group, and “(+)” indicates the result of the CIL100 group. In the “MI” column, “(−)” indicates the result of the sham group, and “(+)” indicates the result of the MI group. As a result, in the Vehicle group, it was confirmed that the tissue positive in SA-β-Gal activity was clearly increased in the MI group than in the sham group, and cells aging in the non-infarcted myocardium increased after myocardial infarction. It was. On the other hand, in the CIL100-MI group, tissues positive for SA-β-Gal activity did not increase so much. From these results, it was suggested that cilnidipine can suppress cell aging in non-infarcted myocardium after myocardial infarction.

<Measurement of the amount of the dimer of Drp1>
After myocardial infarction (after ligation surgery of LAD), the area around the myocardial infarction in the left ventricle of each mouse 1 week and 4 weeks was treated with 200 μL of GTP-binding buffer (50 mM HEPES (pH 7.5), 1 mM EGTA). , 1.5 mM MgCl ₂ , 150 mM NaCl, 10% (v / v) Glycerol, 1% (v / v) TritonX-100, and protease inhibitor cocktail (manufactured by Nacalai Tesque)) After separation (9000 × g, 10 minutes, 4 ° C.), the supernatant was collected. Each sample (collected supernatant) was measured for protein concentration by Bradford assay.
The proteins in each sample were separated by SDS-PAGE and then electrically transferred to a PVDF membrane at ² mA / cm ² . The PVDF membrane to which the protein was transferred was blocked with a 1% BSA solution for 1 hour before Western blotting. 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. In FIG. 13, “sham” indicates the result of the sham group, and “MI” indicates the result of the MI group. In addition, “Vehicle” indicates the result of the Vehicle group, and “Cilinipine” indicates the result of the CIL100 group. In the sham group, no Drp1 dimer was detected regardless of whether or not cilnidipine was administered. On the other hand, in the 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.

<Measurement of FS (Left chamber inner diameter reduction rate)>
From a pre-myocardial infarction treatment period to 4 weeks after the treatment, mice were subjected to cardiac ultrasonography every week according to a conventional method, and FS (%) was measured (Nature Chemical Biology, vol. 8, p. 714-724). (2012)).

  FIG. 14 shows the measurement results of FS of each group of mice. In FIG. 14, “(−)” in the “CIL” column indicates the result of the Vehicle group, “30” indicates the result of the CIL 30 group, and “100” indicates the result of the CIL 100 group. As a result, it was found that FS decreased by myocardial infarction was recovered by cilnidipine administration.

[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 added with cilnidipine to a final concentration of 1 μM and a medium added with Mdiv-1 to a final concentration of 10 μM, Reference Example 1 In the same manner, SA-β-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-β-Gal activity was measured.

  FIG. 15 shows the measurement results of the percentage (%) of cells positive for SA-β-Gal activity of each cell. As a result, when cultured in the presence of Mdiv-1, the number of cells positive for SA-β-Gal activity was increased. However, when cultured in the presence of cilnidipine, SA- The proportion of cells with positive β-Gal activity was reduced, and it was suggested that cilnidipine can suppress cell senescence unlike Mdiv-1.

[Reference Example 2]
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. .
First, 22 5-week-old mice (C57BL / 6J (SLC)) were started to receive drinking water containing 10 ppm of methylmercury (MeHg group). Drinking water containing methylmercury was freely given to mice. Similarly, 23 5-week-old mice (C57BL / 6J (SLC)) were allowed to freely take drinking water not containing methylmercury (Vehicle group).
Mice 10 days after the start of methylmercury intake, thoracotomy was performed, 14 in the MeHg group and 15 in the Vehicle group were treated with TAC (TAC group), 8 in the MeHg group and 8 in the Vehicle group were TAC. The suture was performed without any treatment (sham group).
Mice were bred for 4 weeks after TAC treatment, and changes in body weight and survival rate were examined. In addition, 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. As a result, there was no particular difference in body weight in each group. On the other hand, as for the survival rate, in the MeHg-sham group, as in the Vehicle-sham group, all mice survived to the end, but the MeHg-TAC group had a final survival rate of only about 60%. From these results, it was found that even ingestion of a low concentration of methylmercury that does not induce cytotoxicity in healthy individuals has an effect of enhancing cardiac function deterioration due to TAC.

  In addition, when the heart weight per body weight (mg / g) of the mice at 1 week and 4 weeks after the TAC treatment was examined, 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.

[Reference Example 3]
NRCM cells were exposed to low concentrations of methylmercury (0.1 μM) 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 μM methylmercury, a culture medium containing 10 μM Midi-1, 0.1 μM methylmercury and 10 μM Midi-1 Was cultured in a culture medium containing NO or methylmercury and Midi-1 at 37 ° C. for 24 hours in a humidified atmosphere with 5% by volume carbon dioxide (95% by volume air).

  About the cell after this culture | cultivation, ATP density | concentration (micromol) per well was calculated | required similarly to the reference example 1, and the well of the control cell (The cell cultured in the culture medium which does not contain any of methylmercury and Midi-1) The relative value was calculated with the per-ATP concentration as 100%. The amount of intracellular ATP in each cell was reduced by exposure to methylmercury, but it was found that this reduction in ATP production by methylmercury can be recovered by Midi-1.

  In addition, the cells after culturing in each culture medium were stretched by 20% using a cell stretcher, 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 (a 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 effect 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 μM), Mdivi-1 (10 μM), DIDS (4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid) (100 μM), ROX (1 μM), diazoxide (Diazoxide) (100 μM) Rottererin (5 μM), AICAR (5-amino-1-bD-ribofuranosyl-imidazole-4-carboxamide) (500 μM), Amlodipine (1 μM), and ET-1 (0.1 μM). .
Specifically, after seeding NRCM cells in a 96-well plate and culturing for 24 hours, the medium was changed to taurine-containing DMEM (DMEM containing 5 mM taurine, 100 units / mL penicillin, and 100 μg / mL streptomycin), and Cultured for 24 hours. After culturing, each drug was added to the medium to a predetermined concentration and cultured for 1 hour. After the culture, the medium was replaced with a DMEM medium containing 0, 0.5, or 1 μM methylmercury, and after culturing for 24 hours, an MTT assay was performed to measure Abs595 of each cell. Based on the measured value of Abs595 of each cell, 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.

[Example 5]
Cirnidipine was administered to diabetic model mice, and the effect on blood glucose level was examined.
First, 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. For the remaining two groups (24 mice), only a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7 was administered as a control (control mice).

  After 18 days from the administration of STZ, an osmotic pump containing cilnidipine was implanted into the abdominal cavity and continuous administration was started. 1 group of 2 groups of STZ mice and 1 group of 2 groups of control mice were administered so that the dose of cilnidipine was 5 mg / kg / day, which was used as STZ + cil group and Veh + cil group, respectively. . Further, as a control, the remaining one group of STZ mice and the remaining one group of control mice were administered only a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7, respectively. Grouped.

  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. In control mice (Veh group and Veh + cil group), no effect of cilnidipine administration on blood glucose level was observed. On the other hand, in 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. FIG. 18 shows the results of measurement of the blood glucose level and the fasting blood glucose level of the STZ group and STZ + cil group on the 14th day (32 days after the STZ administration) on the start of cilnidipine administration. These results suggest that cilnidipine can reduce insulin-dependent hyperglycemia.

[Example 6]
In Alzheimer's disease patients, many deposits containing amyloid β protein as a tumor component are observed, and accumulation of amyloid β protein is considered to be involved in the onset of Alzheimer's disease. In addition, when a large amount of amyloid β protein is taken into cells, endoplasmic reticulum stress becomes strong and apoptosis is induced. Therefore, the influence of cilnidipine on cell damage caused by amyloid β loading was examined using a cultured cell line PC-12 cells derived from rat pheochromocytoma. As the amyloid β protein, Amyloid β _25-35 (manufactured by Sigma-Aldrich, catalog number: A4559) was used.

  As a culture medium for culturing undifferentiated PC-12 cells, D-MEM (High Glucose) (manufactured by Wako Pure Chemical Industries), 5% FBS, 5% HS (horse serum) and 1% penicillin- A medium containing streptomycin (Nacalai) was used. In addition, as a medium when PC-12 is loaded with amyloid β, a serum-free medium (D-MEM (High Glucose) (manufactured by Wako Pure Chemical Industries)) contains 1% penicillin-streptomycin (manufactured by Nacalai). Medium) was used.

First, a poly-L-lysine (PLL) solution having 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 μL / well) in a PLL-coated 24-well plate and cultured at 37 ° C. for 24 hours in the culture medium.
Next, the cells in each well were washed once with a serum-free medium, and then serum-free medium containing 500 μL of cilnidipine (final concentration 1 μM) and Amyloid β _25-35 (final concentration 10 μM) per well. Then, a serum-free medium containing only cilnidipine (final concentration 1 μM), a serum-free medium containing only Amyloid β _25-35 (final concentration 10 μM), or a serum-free medium was added at 37 ° C. for 24 hours. Incubate for hours. Thereafter, after removing the medium from each well, 12.5 μL of MTT solution (a solution in which MTT was dissolved in PBS so as to be 5 mg / mL) was added at 12.5 μL per well and lightly shaken. And incubated for 1 hour. Thereafter, the MTT solution was removed from each well, and then DMSO was added at 500 μL per well and shaken firmly to dissolve the produced formazan. Thereafter, 100 μL 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.

For Amyloid β _25-35 Abs595 of cells cultured in serum-free medium containing no, Amyloid beta ratio of Abs595 of cells cultured in serum-free medium which contains _25-35 (percent), the cell The survival rate (%) was calculated. The calculation results are shown in FIG. As a result, the survival rate of cells subjected to amyloid β loading in the presence of cilnidipine (“Cil (+)” in the figure) is the survival rate of cells subjected to amyloid β loading in the absence of cilnidipine (in the figure, “ Cil (−) ”), and cilnidipine suppressed the induction of apoptosis due to amyloid β loading. From these results, it is clear that cilnidipine reduces the amyloid β load on cells, and can be expected to be effective in the treatment of Alzheimer's disease.

Claims (7)

  A Drp1 polymerization inhibitor comprising 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 according to claim 1 as an active ingredient.   The pharmaceutical composition according to claim 2, which is used for prevention or treatment of chronic heart failure after myocardial infarction.   The pharmaceutical composition according to claim 2, which is used for reducing cardiomyocyte toxicity caused by organic mercury.   The pharmaceutical composition according to claim 2, which is used for reducing insulin-dependent hyperglycemia.   The pharmaceutical composition according to claim 2, which is used for prevention or treatment of amyotrophic lateral sclerosis or Alzheimer's disease.   A cell aging inhibitor comprising the Drp1 polymerization inhibitor according to claim 1 as an active ingredient.