JP6688503B2 - Pharmaceutical composition - Google Patents

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.
The present application claims priority based on Japanese Patent Application No. 2014-236941 filed in Japan on November 21, 2014, the content of which is incorporated herein.

  Mitochondria are organelles in almost all eukaryotic cells, and are mainly responsible for the production of ATP by oxidative phosphorylation by the electron transport system (phosphorylation of ADP). It is known that mitochondria repeats fusion and division, and its abnormality is involved in the onset of cancer, cardiovascular disease, neurodegenerative disease and the like. For example, it has been suggested that, in a non-infarct lesion after myocardial infarction, abnormal energy metabolism due to abnormal morphological function (remodeling) of mitochondria causes chronic heart failure.

  Mitochondrial division is caused by activation of the GTP-binding protein Drp1 (dynamin-related protein 1). Activated Drp1 polymerizes to form ring-shaped multimers around mitochondria. Division occurs when the Drp1 multimer cleaves mitochondria.

  On the other hand, cilnidipine is a kind of dihydropyridine-based calcium antagonist, and is known to have both L-type and N-type calcium antagonism and have a blood pressure lowering action. The antihypertensive effect of cilnidipine is characterized by a longer duration than other antihypertensive agents. 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). Further, by utilizing calcium antagonism, cilnidipine is useful for treating or preventing cerebral infarction and cerebral hemorrhage in addition to the commonly used antihypertensive treatment (see, for example, Patent Document 4) and renal dysfunction. It has been reported that it is useful for the treatment or prophylaxis (for example, see Patent Document 5). In addition, it has been reported that cilnidipine has a side effect reducing action of anticancer agents (see Patent Document 6).

Japanese Patent No. 4613496 Japanese Patent Laid-Open No. 2008-044871 International Publication No. 2013/147137 JP, 2004-359675, A International Publication No. 2008/016171 JP, 2013-014549, A

  The present invention aims to provide a Drp1 polymerization inhibitor useful for treating or preventing a disease caused by induction of mitochondrial division, and a pharmaceutical composition containing 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 a pharmaceutical composition having the following [1].
[1] A pharmaceutical composition containing cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient and used for reducing cardiomyocyte toxicity due to organic mercury .

  The Drp1 polymerization inhibitor according to the present invention can inhibit mitochondrial division. Therefore, the Drp1 polymerization inhibitor and a pharmaceutical composition containing the same as an active ingredient can be used as prophylactic or therapeutic agents for 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 analysis results of mitochondrial morphology of each cell in Reference Example 1. FIG. 2 is a diagram showing the measurement results of the ratio (%) of senescent cells with positive SA-β-Gal activity of each cell in Reference Example 1. FIG. 3 is a diagram showing the calculation results of the relative value (%) of the amount of mitochondrial active oxygen in 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 is a diagram showing the results of analysis of the mitochondrial morphology of cells hypoxia-stimulated and reoxygenated in the presence or absence of cilnidipine in Example 1. FIG. 6 is a diagram showing the measurement results of the ratio (%) of senescent cells having a positive SA-β-Gal activity in cells hypoxia-stimulated and reoxygenated in the presence or absence of cilnidipine in Example 1. Is. FIG. 7 is a micrograph showing localization of Drp1 and mitochondria in cells hypoxia-stimulated in the presence or absence of cilnidipine in Example 1. FIG. 8 is a graph showing changes in the survival rate (%) of the mice in each group over time in Example 2. FIG. 9 is a diagram showing the measurement results of heart weight per body weight (mg / g) of each group of mice 4 weeks after myocardial infarction (after ligation surgery of LAD) in Example 2. FIG. 10 is a diagram 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 the measurement results of the collagen positive tissue region (CVF) (%) of the mice of each group in Example 2. FIG. 12 is a diagram showing the measurement results of the proportion (area ratio) (%) of senescent tissues having positive SA-β-Gal activity in the non-infarct region myocardium after myocardial infarction in mice of each group in Example 2. Is. FIG. 13 is a diagram showing the results of detecting Drp1 by Western blotting in the peripheral region of the myocardial infarction in the left ventricle of each group of mice in Example 2. FIG. 14 is a diagram showing the measurement results of FS (left ventricular diameter reduction rate) of each group of mice in Example 2. FIG. 15 is a diagram showing the measurement results of the ratio (%) of cells having a positive SA-β-Gal activity in each cell in Example 3. FIG. 16 is a diagram showing changes with time in survival rate (%) from the time of TAC treatment of mice in each group in Reference Example 2. FIG. 17 is a diagram showing the results of measuring the relative survival rate (%) of cells cultured in a medium containing 0 or 0.5 μM of methylmercury and various existing drugs in Example 4. FIG. 18 is a diagram showing the measurement results of the occasional blood glucose level (left figure) and the fasting blood glucose level (right figure) of STZ mice on day 14 after the administration of cilnidipine in Example 5. In the figure, “STZ” indicates the result of STZ mice not administered with cilnidipine, and “STZ + cil” indicates the result of STZ mice administered with cilnidipine. FIG. 19 is a diagram showing the results of measuring the cell viability (%) when amyloid β was loaded in the presence and absence of cilnidipine in Example 6.

  The Drp1 polymerization inhibitor according to the present invention is characterized by containing cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient. The Drp1 polymerization inhibitor inhibits Drp1 polymerization, resulting in inhibition of mitochondrial fission. That is, the Drp1 polymerization inhibitor according to the present invention functions as a mitochondrial mitosis inhibitor.

  Cirnidipine is a compound known as an L / N-type calcium antagonist that inhibits both L-type calcium channel and N-type calcium channel, and specifically, has the following structural formula:

Represented by (±) -2-methoxyethyl 3-phenyl-2- (E) propenyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) -3,5-pyridinedicarboxy The rate. The cilnidipine in the present invention also includes optical isomers based on the above structural formula, and both can be produced using known production methods (Japanese Patent Publication No. 3-14307, Japanese Patent Publication No. 6-43397, etc.). See). It is also commercially available.

  Cirnidipine can be made into its pharmaceutically acceptable salt, hydrate or solvate, if 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.) and the like. Hereinafter, cilnidipine or a pharmaceutically acceptable salt thereof may be simply referred to as cilnidipine.

  Drp1 is activated and polymerized by stimulation with hyperglycemia, hypoxia, environmental pollutants, nitric oxide and the like, and mitochondria are divided by tightening by the formed Drp1 multimer. Subsequent reoxygenation fuses the divided mitochondria and overproduces ATP and active oxygen, resulting in the induction of diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and the like. Cirnidipine suppresses mitochondrial fission by inhibiting the polymerization of Drp1. Therefore, cilnidipine is effective in treating and preventing diseases induced by mitochondrial division such as diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

  In non-infarct lesions after myocardial infarction, the expression of hypoxia-inducible factor increases, which induces premature aging of cardiomyocytes, which causes chronic heart failure and sudden death. As shown in Examples below, hypoxic stimulation of cardiomyocytes transiently induces mitochondrial division, and subsequent reoxygenation induces cell damage and cell senescence. Cirnidipine suppresses mitochondrial division of cardiomyocytes induced by hypoxia, and thus suppresses cell damage and cell senescence after reoxygenation. Therefore, 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.

  In addition, organic mercury such as methylmercury is known to be an environmental pollutant that increases cardiovascular risk. As shown in Examples described later, when cardiomyocytes are exposed to a low concentration of organic mercury that does not show cytotoxicity, mitochondrial division is induced, cell death is induced, and stretch stress sensitivity is increased. Cirnidipine suppresses mitochondrial division of cardiomyocytes by organic mercury, which in turn suppresses cell death. Therefore, cilnidipine is suitably used for reducing cardiomyocyte toxicity due to organic mercury, and is particularly preferably used for suppressing deterioration of cardiac function deterioration due to organic mercury in heart failure.

  Further, as shown in Examples below, cilnidipine has no particular effect on the blood glucose level in a healthy state, but has an action of lowering the blood glucose level in an insulin-dependent hyperglycemic state. Therefore, cilnidipine is suitably used for reducing the insulin-dependent hyperglycemic 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, and is preferably human, mouse, rat, rabbit, guinea pig, hamster, monkey. More preferably, it is administered to domestic animals such as sheep, horses, cows, pigs, donkeys, dogs and cats and experimental animals. 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 mitosis induced by highly electrophilic drugs such as anticancer drugs. For example, a pharmaceutical composition having an effect of treating or preventing diabetes, heart failure, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, etc. 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 containing the same as an active ingredient (hereinafter, may be simply referred to as “pharmaceutical composition according to the present invention”) is administered to animals including human. In this case, the dosage form may be either oral administration or parenteral administration, and the dosage form is an oral administration solid formulation such as powders, granules, capsules, tablets, chewable preparations; solutions. And liquids such as syrups, and examples of parenteral administrations include injections, infusions, nasal and transpulmonary sprays, and the like. The pharmaceutical composition according to the present invention can be formulated into a drug of these dosage forms by a conventional method.

  From the viewpoint of reducing the burden on the patient, the pharmaceutical composition according to the present invention is preferably orally administered to a subject. A tablet is preferable as a 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 solution. In addition, the pharmaceutical composition according to the present invention may be a suitable pharmaceutically acceptable carrier, for example, an excipient, a binder, a lubricant, a solvent, a disintegrating agent, or a solubilizing aid, if necessary for formulation. Formulations can be made by adding agents, suspending agents, emulsifiers, isotonic agents, stabilizers, soothing agents, preservatives, antioxidants, flavoring agents, coloring agents and the like.

  Examples of excipients include sugars such as lactose, glucose and D-mannitol, organic excipients such as starches and celluloses such as crystalline cellulose, inorganic excipients such as calcium carbonate and kaolin, and the like. Examples of the lubricant include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like. Are stearic acid, fatty acid salts such as stearates, talc, silicates, etc., solvents are purified water, physiological saline, etc., disintegrants are low-substituted hydroxypropyl cellulose, chemically modified Cellulose and de Puns and the like, as a solubilizing agent, polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like, and as a suspending agent or emulsifier, lauryl. Sodium sulfate, gum arabic, gelatin, lecithin, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethyl cellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc. , Potassium chloride, sugars, glycerin, urea and the like, stabilizers such as polyethylene glycol, dextran sodium sulfate and other amino acids, and soothing agents such as glucose, Calcium gluconate, procaine hydrochloride, etc., as preservatives, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc., antioxidants, sulfites, ascorbic acid, etc. Examples of the flavoring agent include sweeteners and flavors commonly used in the fields of medicine and food, and examples of the colorant include colorants commonly used in the fields of medicine and food.

  The dose of the pharmaceutical composition according to the present invention varies depending on the type of anticancer agent, the age, weight, condition of the subject, administration method, etc., and is usually a clinical dose of cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient. Can be appropriately set in consideration of the above. For example, the daily dose per adult is 0.0001 to 1000 mg, preferably 0.001 to 100 mg, and particularly preferably 0.01 to 50 mg is orally administered once or in several divided doses, or to adult 1 As a daily dose per person, 0.0000001 to 100 mg, preferably 0.000001 to 50 mg, and particularly preferably 0.0001 to 20 mg is parenterally administered once a day or divided into several times, or 1 hour a day. It can be continuously administered intravenously in the range of 1 to 24 hours.

  Next, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited thereto.

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

[Preparation of mouse myocardial infarction model]
The 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 (domitol: 0.75 mg / kg, midazolam: 4 mg / kg, betorfal: 5 mg / kg). After anesthesia, the chest was opened and the left anterior descending coronary artery (LAD) was ligated with a silk suture (No. 6-0) (MI group). Whether or not the myocardial infarction by ligation was successful was confirmed by observing the ST (extreme ventricular excitation period) elevation on the electrocardiogram. Mice were similarly anesthetized and thoracotomy was performed, but mice in which LAD was not ligated were set as a sham group.

[cell]
NRCM (Preparation of neonatal rat cardiac myocytes) cells were obtained as follows.
First, a fetus of an SD rat on the first day of feeding was anesthetized with cold, and the heart was taken out and incubated with 0.05% trypsin-EDTA at 4 ° C for 16 hours. After that, trypsin was removed, and the mixture was incubated at 37 ° C. for 15 minutes in a collagenase II solution diluted with PBS (phosphate saline) so that the concentration was 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 DBS containing FBS (fetal bovine serum) (DMEM containing 10% by volume FBS, 100 units / mL penicillin, and 100 μg / mL streptomycin), and seeded in a non-coat dish, and The cells were cultured at 37 ° C. for 1 hour in a humidified atmosphere of volume% carbon dioxide (95 volume% air). Next, cells that did not adhere to the dish were collected as NRCM cells and seeded on a gelatin-coated dish or plate. The seeded NRCM cells were cultured for 24 hours at 37 ° C. in a humidified atmosphere with 5 vol% carbon dioxide (95 vol% air), and then taurine-containing DMEM (containing 5 mM taurine, 100 unit / mL penicillin, and 100 μg / mL streptomycin was contained. The medium was exchanged with DMEM) and cultured for 48 hours, and used for each experiment.

  Hela cells were cultured in FBS-containing DMEM (DMEM containing 10% by volume FBS, 100 unit / mL penicillin, and 100 μg / mL streptomycin) at 5% by volume carbon dioxide (95% by volume air) at 37 ° C. in a humidified atmosphere. Cultured. The transfection was carried out by using the X-tremeGENE9 DNA transfection reagent (Roche, Inc.) for Hela cells that had been cultured overnight after medium exchange with FBS-free DMEM (DMEM containing 100 unit / mL penicillin and 100 μg / mL streptomycin). (Manufactured by Mitsui Chemicals Co., Ltd.) according to the attached instruction manual, and gene transfection was carried out for 12 hours with 3 μg of plasmid per 35 mm dish.

[Preparation of plasmid for expression of Drp1-EGFP and Drp1-Flag]
An expression plasmid of Drp1 (Drp1-EGFP) in which EGFP was fused to the C-terminal side and a plasmid for expression of Drp1 (Drp1-Flag) in which Flag was fused to the C-terminal side were prepared as follows.
First, RNA was prepared from rat heart using RNeasy fibrous tissue mini kit (manufactured by Qiagen), and Prime Script RT (manufactured by Takara Bio Inc.) was used as a template. PCR is performed using the obtained cDNA as a template and an Fw primer for GFP tag and an Rv primer for GFP tag, and the obtained PCR product is digested with BamHI and XhoI and then ligated to pEGFP-C1 vector to obtain Drp1. -A plasmid for expression of EGFP was obtained. Similarly, using the obtained cDNA as a template, PCR is carried out using the Fw primer for GFP tag and the Rv primer for GFP tag, the obtained PCR product is digested with BamHI and XhoI, and then ligated to pcDNA3.1 vector. Thus, a Drp1-Flag expression plasmid was obtained.
The ligation product was transformed into Escherichia coli DH5α strain to culture a single colony, and then a plasmid was obtained by LaboPass (manufactured by Hokkaido System Science Co., Ltd.). After confirming the sequence by sequencing, the obtained plasmid was purified with Qiagen plasmid Maxi kit (manufactured by Qiagen) and used in each experiment.

  Moreover, the expression plasmid of the protein (Drp1 (C624S) -EGFP) in which EGFP was fused to the C-terminal side of the C624S mutant of Drp1 (mutant in which the cysteine at the 624th position was replaced by serine) was the expression of Drp1-EGFP. PCR is performed using the plasmid for plasmid as a template, the Fw primer for C624S, the Rv primer for C624S, and the PCR reagent (product name: KOD Fx, manufactured by Toyobo Co., Ltd.), and the obtained PCR product is digested with BamHI and XhoI and then pEGFP After ligation into the -C1 vector, it was prepared in the same manner as the Drp1-EGFP expression plasmid.

[MTT assay]
The MTT assay is an assay for examining 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 in PBS at 5 mg / mL. Was added so that the final concentration of MTT was 0.125 mg / mL, and the mixture was cultured at 37 ° C. for 1 hour in a humidified atmosphere of 5 vol% carbon dioxide (95 vol% air). After the culture, the medium was removed, 100 μL of DMSO was added, and the produced formazan was dissolved by gently shaking at room temperature for 30 minutes. Then, 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 induces mitochondrial division and reoxygenation induces cellular senescence. Specifically, for unstimulated NRCM cells, NRCM cells after hypoxia stimulation, and NRCM cells reoxygenated after hypoxia stimulation, mitochondrial morphology and percentage of aged cells, and intracellular activity The oxygen amount (relative value) and the intracellular ATP concentration (μM / well) were examined.

<Hypoxia stimulation and reoxygenation>
Hypoxia stimulation of NRCM cells was performed by culturing NRCM cells in 1 volume% oxygen and 5 volume% carbon dioxide (94 volume% nitrogen) in a humidified atmosphere at 37 ° C. for 16 hours. Reoxygenation was performed by culturing the NRCM cells after hypoxia stimulation in 5% by volume carbon dioxide (95% by volume air) in a humidified atmosphere at 37 ° C. for 24 hours.

<Mitochondrial morphology>
The mitochondrial morphology was observed with a confocal laser scanning microscope (product name: FV10i, Olympus, magnification: × 300) after staining the mitochondria of each cell with MitoTracker Green FM reagent (Life technologies). Was analyzed. More specifically, the mitochondrial staining was carried out by first incubating cells seeded in a 35 mm glass bottom dish with MitoTrakcer Green (0.5 μM) in 5% by volume carbon dioxide (95% by volume air) in a humidified atmosphere at 37 ° C. for 10 days. Incubated for a minute to react. 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 was divided into vesicles, tubules, and intermediate structures between vesicles and tubules, and the abundance ratios for each were shown in FIG. 1. In FIG. 1, “N” represents the result of unstimulated cells, “H” represents the result of cells after hypoxia stimulation, and “H / R” represents the result of cells reoxygenated after hypoxia stimulation. Show. Follicular mitochondria increased after hypoxia stimulation but decreased after reoxygenation.

<SA-β-gal activity measurement>
Aging-related acidic β-galactosidase (SA-β-Gal) activity was measured to determine the percentage of aged cells. 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 hypoxia stimulation and then washed once with PBS. As a control, unstimulated NRCM cells were also washed once with PBS. In addition, Mdivi-1 (CAS No: 338967-87-6, manufactured by Sigma), which is a selective inhibitor of Drp1 and is a mitochondrial mitotic inhibitor, was dissolved in DMSO to give a final concentration of 10 μM. Non-stimulated NRCM cells and NRCM cells reoxygenated after hypoxia stimulation were prepared in the same manner except that the added culture medium was used.
Next, each cell was fixed by treating with Fixative Solution included in the kit at room temperature for 15 minutes. The fixed cells were washed once with PBS, the β-galactosidase staining solution contained in the kit was added, and the mixture was incubated overnight at 37 ° C. The cells after the incubation are observed with an upright microscope (product name: Eclipse 80i, manufactured by Nikon Corporation) equipped with a color CCD camera (Nikon digital camera DXM1200F), and cells positive for SA-β-Gal activity (stained cells ) Was investigated.

  The measurement results of the ratio (%) of cells having a positive SA-β-Gal activity are shown in FIG. In FIG. 2, “(−)” in the “H / R” column shows the result of unstimulated NRCM cells, and “(+)” shows the result of NRCM cells reoxygenated after hypoxia stimulation. Further, in FIG. 2, “(−)” in the “Midivi-1” column shows the result of NRCM cells without Midivi-1 addition, and “(+)” shows the result of NRCM cells with Midivi-1 addition. . When Midivi was not added, the proportion of cells positive for SA-β-Gal activity was higher in the reoxygenated cells after hypoxia stimulation than in the non-stimulation cells. It was confirmed that oxygenation induces cell senescence. On the other hand, in the cells to which the Midivi-1 was added, the proportion of cells having a positive SA-β-Gal activity in the reoxygenated cells was low after the hypoxia stimulation, and Midi-1 reoxygenated the cells after the hypoxia stimulation. It was found that the cell senescence induced by the treatment was 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 on a 35 mm glass bottom dish were hypoxia-stimulated or reoxygenated after hypoxia stimulation, and then washed once with PBS. As a control, unstimulated NRCM cells were also washed once with PBS. Further, in the same manner, Mdiv-1 (manufactured by Sigma) dissolved in DMSO was added to the culture medium so that the final concentration was 10 μM, in the same manner as the non-stimulated NRCM cells, the hypoxia-stimulated NRCM cells, and the low NRCM cells. NRCM cells that were reoxygenated after oxygen stimulation were also prepared.
Then, MitoSOX (5 μM) was added to each cell and incubated at 37 ° C. for 10 minutes in a 5% by volume carbon dioxide (95% by volume air), humidified atmosphere to react. After the reaction, the cells were washed twice with PBS to remove MitoSOX, and then replaced with phenol red-free DMEM. The stained cells are observed with a confocal laser scanning microscope (product name: FV10i, manufactured by Olympus Co., magnification: × 300), and the fluorescence intensity per cell is measured for each cell to obtain no stimulation and Mdiv- The relative value with 100% of the fluorescence intensity per cell of the cell without 1 addition 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 active oxygen in mitochondria is shown in FIG. In FIG. 3, “N”, “H”, and “H / R” have the same meanings as those in FIG. 2, and in FIG. 3, “(−)” in the “Midivi-1” column is NRCM without Midivi-1 added. "(+)" Indicates the result of cells, and "(+)" indicates the result of NRCM cells to which Midivi-1 was added. As shown in FIG. 3, the amount of mitochondrial active oxygen increased by hypoxia stimulation and subsequent reoxygenation, but Midiv-1 treatment increased mitochondrial active oxygen content after hypoxia stimulation and after reoxygenation. The increase was suppressed.

<Measurement of intracellular ATP amount>
The intracellular ATP amount was measured using the Luciferase assay (Wako Pure Chemical Industries, Ltd.).
Specifically, first, NRCM cells seeded in a 96-well plate were hypoxia-stimulated or reoxygenated after hypoxia-stimulation, and then washed once with PBS. As a control, unstimulated NRCM cells were also washed once with PBS. Further, in the same manner, Mdiv-1 (manufactured by Sigma) dissolved in DMSO was added to the culture medium so that the final concentration was 10 μM, in the same manner as the non-stimulated NRCM cells, the hypoxia-stimulated NRCM cells, and the low NRCM cells. NRCM cells that were reoxygenated after oxygen stimulation were also prepared.
Then, the medium was removed and replaced with fresh DMEM (100 μL), 20 μL of Luciferase reagent was immediately added, and the mixture was incubated at room temperature for 10 minutes. The luminescence of the cells after the incubation was measured by SpectraMax i3 (manufactured by Molecular devices), and the ATP concentration per well was determined from the luminescence intensity. To calculate the ATP concentration from the emission intensity, the emission 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 emission intensity was obtained. The calibration curve calculated from the relationship between the ATP concentration and the ATP concentration was used.
The amount of ATP consumed per well was determined by adding 10 μM of carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to the cells and incubating at room temperature for 5 minutes to inhibit ATP synthesis from mitochondria, followed by Luciferase assay. Then, the amount of ATP remaining 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 meanings as in FIG. 2, “control” is the result of NRCM cells without Midivi-1 added, and “Midivi-1” is Midivi-. The results of NRCM cells with 1 addition are shown respectively. The intracellular ATP amount increased by reoxygenation after hypoxia stimulation, but this increasing tendency was suppressed by Midivi-1 treatment.

[Example 1]
Drp1-EGFP-expressing NRCM cells were subjected to hypoxic stimulation and reoxygenation treatment in the presence of various calcium antagonists to give intracellular ATP amount, mitochondrial morphology, aged cell ratio (%), and Drp1. Was investigated.
Specifically, NRCM cells are transfected with a plasmid for expressing Drp1-EGFP, and then DMSO solution of cilnidipine (CIL), amlodipine (Aml), verapamil (Ver), diltiazem (Dil) or ω-conotoxin (ω). -Ctx) phosphate buffer (PBS) was added to the culture medium to a final concentration of 10 μM to perform hypoxic stimulation and reoxygenation, and the amount of intracellular ATP and mitochondrial morphology of the cells after each treatment. , The percentage of aged cells, and the localization of Drp1 were examined. As a control, NRCM cells cultured in a culture medium containing an equal amount of DMSO alone were also measured in the same manner. Hypoxia stimulation and reoxygenation treatment were performed in the same manner as in Reference Example 1, and intracellular ATP amount, mitochondrial morphology, and aged cell ratio (%) 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, cells hypoxia stimulated and reoxygenated in the presence of a calcium antagonist other than cilnidipine were treated with hypoxia stimulation and reoxygenation in the same manner as the control (cells of culture medium containing only DMSO). Although the amount of intracellular ATP was increased, the amount of intracellular ATP after reoxygenation was apparently lower in the cells subjected to hypoxia stimulation and reoxygenation in the presence of cilnidipine. From these results, it was suggested that cilnidipine suppresses the increase in intracellular ATP amount due to hypoxia stimulation and reoxygenation, but this suppressive effect is an effect unrelated to the calcium antagonistic effect.

  The morphological morphology of the control cells (cells in the culture medium added with DMSO only) and the cells subjected to hypoxia stimulation and reoxygenation in the presence of cilnidipine were the same as in Reference Example 1, vesicular, tubular, and small. The abundance ratio of each was determined by dividing the structure into an intermediate structure of alveolar and tubular structures. Results are shown in FIG. In FIG. 5, “N”, “H”, and “H / R” have the same meanings as in FIG. 1, and “control” represents the result of the control cell, and “CIL” represents the result of hypoxia stimulation in the presence of cilnidipine. The results of reoxygenated cells are shown respectively. It was shown that cilnidipine suppressed hypoxia-stimulated mitochondrial mitochondrion, and suppressed hypoxia-stimulated mitochondrial division.

  FIG. 6 shows the measurement results of the percentage (%) of cells positive for SA-β-Gal activity in cells hypoxia-stimulated and reoxygenated in the presence or absence of cilnidipine. In FIG. 6, “(−)” in the “H / R” column shows the result of unstimulated NRCM cells, and “(+)” shows the result of NRCM cells reoxygenated after hypoxia stimulation. In FIG. 6, “(−)” in the “CIL” column shows the result of the control NRCM cells (cells of the culture medium added with DMSO only), and “(+)” shows the result of the NRCM cells added with cilnidipine. Are shown respectively. In control cells, the percentage (%) of cells positive for SA-β-Gal activity was increased by hypoxia stimulation and reoxygenation, while in cells in the presence of cilnidipine, SA-by hypoxia stimulation and reoxygenation was used. The increase in cells positive for β-Gal activity was suppressed. That is, it was shown that cilnidipine suppresses cell senescence due to hypoxic stimulation and reoxygenation.

  FIG. 7 shows a micrograph showing the localization of Drp1 and mitochondria in cells hypoxia-stimulated in the presence or absence of cilnidipine. In FIG. 7, “N” is the result of unstimulated cells, “H (Control)” is the result of hypoxic stimulation of control cells, and “H (CIL)” is the hypoxia of cells to which cilnidipine was added. The results after stimulation are shown respectively. In the unstimulated cells, the intracellular localization of Drp1-EGFP and mitochondria in cells to which cilnidipine was added was not particularly different from that in control cells, and Drp1-EGFP was broadly present in the cytoplasm. In cells after hypoxia stimulation, Drp1-EGFP was localized in the vicinity of mitochondrial vesicles in the control cells, whereas in cells to which cilnidipine was added, Drp1-EGFP became vesicular. Few were localized, and no co-localization with mitochondria was observed.

[Example 2]
Sirnidipine was administered to a mouse myocardial infarction model (MI group) prepared from C57BL / 6J mice and a sham group as a control, and the effect of cilnidipine on the heart after myocardial infarction was examined.

<Administration of cilnidipine>
Cirnidipine was administered after myocardial infarction (ligation of LAD by using an osmotic pump (product name: model2004, manufactured by Alzet) in a state of being dissolved in a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7. It was continuously administered 24 hours after the operation. Among the mice administered at a dose of cilnidipine of 30 mg / kg / day, those administered to mice in MI group were designated as CIL30-MI group, and those administered to mice in sham group were designated as CIL30-sham group. Similarly, among mice administered at a dose of cilnidipine of 100 mg / kg / day, those administered to mice in MI group were CIL100-MI group, those administered to mice in sham group were CIL100-sham group. And 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 set as the Vehicle-MI group and the Vehicle-sham group, respectively.

  FIG. 8 shows changes with time in survival rate (%) of the Vehicle-MI group, the CIL30-MI group, and the CIL100-MI group. As a result, the survival rate of the CIL30-MI group and the CIL100-MI group was clearly higher than that 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 could suppress sudden death after myocardial infarction.

  FIG. 9 shows the measurement results of the heart weight per body weight (mg / g) of each group of mice 4 weeks after myocardial infarction (after ligation surgery of LAD). 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 CIL30 group, and “100” indicates the result of the CIL100 group. As a result, in the sham group, the presence or absence of the administration of cilnidipine on the heart weight and the effect of the dose were not observed, but in the MI group, the increase of the heart weight was suppressed by the administration of cilnidipine. These results suggested that cilnidipine could suppress the increase in heart weight after myocardial infarction.

<Observation of fibrosis and cardiomyocyte area>
The heart of each mouse at 4 weeks after myocardial infarction (after ligation surgery of LAD) was stained with 1-6 picrosirius red and HE (hematoxylin-eosin) to measure myocardial fibrosis and cardiomyocyte area by the method of Nishida et al. It was investigated according to The EMBO Journal (2008) vol.27, p.3104-3115).
First, the heart of a mouse was fixed by formalin treatment, the central region of the ventricle was cut out, and a ventricular specimen embedded with paraffin was prepared. Next, this ventricular specimen was sliced into 3 μm thick pieces, and paraffin was removed with xylene and ethanol. After removing the paraffin, the ventricular specimen was immersed in 0.1% Direct Red 80 solution prepared with hematoxylin solution or picric acid saturated solution for 60 minutes, and then fractionated with 1% hydrochloric acid alcohol, ethanol, xylene. It was dehydrated and sealed. The encapsulated specimen, especially the non-infarcted area, was observed with a microscope (product name: BZ-9000, manufactured by Keyence). The ratio of collagen content was calculated as the area stained and divided by the total area across the heart expressed as a percentage.

From the results of 1-6 picrosirius red-stained image and HE-stained image, CSA (cross sectional area; cardiomyocyte area (μm ² )) measurement results are shown in FIG. 10, and CVF (Collagen volume fraction; collagen positive region) (%). The measurement results of are shown in FIG. 11, respectively. As a result, both CSA and CVF were increased by myocardial infarction, but this increase was suppressed by administration of cilnidipine. These results suggest that cilnidipine can suppress left ventricular remodeling after myocardial infarction in a concentration-dependent manner.

<SA-β-gal activity measurement in heart section>
The SA-β-Gal activity in the heart slice was measured according to the method of Shlush et al. (BMC Cell Biology, 2011, vol. 12, 16), and the ratio of aged cells was measured.
Specifically, first, the heart was extracted 4 weeks after myocardial infarction (after ligation operation of LAD). The excised heart was washed in PBS, fixed with PBS containing 4% PFA, sucrose substitution was performed, and frozen and embedded using OCT compound (Sakura). Heart sections sliced from a frozen-embedded specimen to 10 μm using LEICA CM1100 were modified in the protocol of Senescence β-Galactosidase Staining Kit (manufactured by Cell signaling technology), and were placed in a β-Galactosidase solution at pH 4.0. The reaction was carried out by incubating at 0 ° C overnight. The incubated cells were washed with PBS and then mounted with VECTASHIELD Mounting Medium. The enclosed sample is observed by an upright microscope (product name: Eclipse 80i, manufactured by Nikon Corporation) equipped with a color CCD camera (Nikon digital camera DXM1200F), and [SA-β-Gal activity positive area ( β-Galactosidase positive area, blue color)] / [total tissue section area] × 100 (%) for analysis and quantification.

  FIG. 12 shows the measurement results of the proportion (area ratio) (%) of SA-β-Gal activity-positive tissues in the non-infarct region myocardium after myocardial infarction in the mice of each group. 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, the tissue in which the SA-β-Gal activity was obviously positive was increased in the MI group as compared with the sham group, and it was confirmed that the number of aging cells was increased in the non-infarct region myocardium after myocardial infarction. It was On the other hand, in the CIL100-MI group, the tissues in which the SA-β-Gal activity was positive were not significantly increased. From these results, it was suggested that cilnidipine could suppress cell senescence in the non-infarct region myocardium after myocardial infarction.

<Measurement of amount of dimer of Drp1>
After the myocardial infarction (after the LAD ligation operation), the peripheral region of the left ventricle of the heart of each mouse at 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 proteinase inhibitor cocktail (manufactured by Nacalai Tesque, Inc.)) and disrupted with a Polytron homogenizer, followed by centrifugation. After the separation treatment (9000 × g, 10 minutes, 4 ° C.), the supernatant was collected. The protein concentration of each sample (collected supernatant) was measured by the 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 performing Western blotting. The PVDF membrane after blocking was incubated with an anti-Drp1 antibody or an anti-GAPDH antibody (both manufactured by Santa Cruz Biotechnology) solution as a primary antibody, and then with an anti-horseradish peroxidase (HRP) antibody as a secondary antibody. After that, the ECL system (manufactured by Nacalai Tesque, Inc.) was used to detect the Drp1 and GAPDH bands.

  FIG. 13 shows the results of detection of Drp1 by Western blotting in 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 “Cilnidipine” indicates the result of the CIL100 group. No dimers of Drpl were detected in the sham group with or without administration of cilnidipine. On the other hand, although the Drp1 dimer was detected in the MI group, the amount of the Drp1 dimer was apparently lower in the CIL100 group than in the Vehicle group. From this result, it was found that cilnidipine suppressed the dimerization of Drp1.

<Measurement of FS (left chamber diameter reduction rate)>
From the time before the myocardial infarction treatment to the treatment for 4 weeks after the myocardial infarction, the mouse was subjected to an echocardiographic examination every week according to the usual method to measure the FS (%) (Nature Chemical Biology, vol. (2012)).

  FIG. 14 shows the FS measurement result of the mice in each group. In FIG. 14, “(−)” in the “CIL” column indicates the result of the Vehicle group, “30” indicates the result of the CIL30 group, and “100” indicates the result of the CIL100 group. As a result, it was found that FS reduced by myocardial infarction was recovered by administration of cilnidipine.

[Example 3]
NRCM cells were examined for cell senescence when cultured in the presence of cilnidipine or Mdivi-1.
Specifically, after culturing NRCM cells for 72 hours in a medium to which cilnidipine was added so that the final concentration was 1 μM and a medium to which Mdivi-1 was added so that the final concentration was 10 μM, respectively, Reference Example 1 SA-β-Gal activity was measured in the same manner as in. As a control, NRCM cells were similarly cultured in a medium to which neither cilnidipine nor Mdivi-1 was added, and the SA-β-Gal activity was measured.

  FIG. 15 shows the measurement results of the proportion (%) of cells having positive SA-β-Gal activity in each cell. As a result, when the cells were cultivated in the presence of Mdivi-1, the number of cells having a positive SA-β-Gal activity increased, but when cultivated in the presence of cilnidipine, SA-β-Gal activity was higher than that in the case of no addition. The proportion of cells positive for β-Gal activity was reduced, suggesting that cilnidipine, unlike Mdivi-1, may suppress cellular senescence.

[Reference example 2]
Transverse aortic stenosis (TAC) treatment was performed on a mouse that had been ingested with water containing 10 ppm of methylmercury, and weight change (%), survival rate (%), and heart weight per body weight (mg / g) were examined. .
First, administration of drinking water containing 10 ppm of methylmercury was started to 22 5-week-old mice (C57BL / 6J (SLC)) (MeHg group). Mice were allowed to freely drink water containing methylmercury. Similarly, 23 5-week-old mice (C57BL / 6J (SLC)) were allowed to freely take drinking water containing no methylmercury (Vehicle group).
Thirteen days after the start of methylmercury intake, thoracotomy was performed on 14 mice in the MeHg group and 15 in the Vehicle group (TAC group), and 8 mice in the MeHg group and 8 in the Vehicle group were TAC treated. It was sutured as it was without treatment (sham group).
Mice were bred for 4 weeks after TAC treatment, and changes in body weight and survival rate were examined. In addition, at 1st, 2nd, 3rd, and 4th weeks after the TAC treatment, echocardiography was performed according to a conventional method to measure the heart weight. The intake of drinking water containing methylmercury was continued until the 4th week.

  FIG. 16 shows the time-dependent changes 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 between the groups. On the other hand, as for the survival rate, all the mice in the MeHg-sham group survived to the end as in the Vehicle-sham group, but the final survival rate in the MeHg-TAC group was only about 60%. From these results, it was found that even ingestion of methylmercury at a low concentration that does not induce cytotoxicity in healthy individuals has an action of promoting deterioration of cardiac function due to TAC.

  In addition, when the heart weight per mouse body weight (mg / g) 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 MeHg-TAC group had a greater increase in heart weight than 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 were seeded in a 96-well plate, a culture medium containing 0.1 μM methylmercury, a culture medium containing 10 μM Midivi-1, 0.1 μM methylmercury and 10 μM Midivi-1. Was cultivated at 37 ° C. for 24 hours in a humidified atmosphere containing 5% by volume carbon dioxide (95% by volume air) in a culture medium containing the above or a culture medium containing neither methylmercury nor Midivi-1.

  For the cells after this culture, the ATP concentration (μM) per well was determined 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 Midivi-1) The relative value was calculated with the ATP concentration per 100% as 100%. The intracellular ATP amount of each cell was decreased by exposure to methylmercury, and it was found that this reduction of ATP production amount by methylmercury can be restored by Midivi-1.

  In addition, the cells after culturing in each culture medium were placed in a cell extension device to extend them by 20%, and then MTT assay was performed. From the absorbance at 595 nm (MTT value) of each cell, a relative value with Abs595 of control cells (cells cultured in a culture medium containing neither methylmercury nor Midivi-1) as 100 was calculated. As a result, in the cells exposed to methylmercury, the MTT value decreased and the number of dead cells increased after the extension stress, but in the cells exposed to methylmercury in the presence of Midivi-1, the MTT value of the cells increased even after the extension stress. No reduction was observed. From these results, it was suggested that although organic mercury increases sensitivity to extensional stress, Midivi suppresses the effect of organic mercury and enhances adaptability to extensional stress.

[Example 4]
We searched for existing drugs that reduce the cardiomyocyte toxicity 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). , Lottelerin (5 μM), AICAR (5-amino-1-bD-ribofuranosyl-imidazole-4-carboxamide) (500 μM), Amordipine (1 μM), and ET-1 (0.1 μM). .
Specifically, NRC M cells were seeded in a 96-well plate, cultured for 24 hours, and then the medium was replaced with taurine-containing DMEM (DMEM containing 5 mM taurine, 100 unit / mL penicillin, and 100 μg / mL streptomycin), and further. It was cultured for 24 hours. After culturing, each drug was added to the medium to a predetermined concentration and cultivated for 1 hour. After culturing, the medium was replaced with a DMEM medium containing 0, 0.5, or 1 μM methylmercury, and after culturing for 24 hours, MTT assay was performed to measure Abs595 of each cell. Based on the Abs595 measurement value of each cell, the survival rate of each cell was calculated when the survival rate of the cells cultured in the medium to which neither drug was added nor methylmercury was added was 100%. The calculation result is shown in FIG. As a result, cilnidipine, Mdiv-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 was examined.
First, 48 C57BL / 6J (SLC) mice were divided into 4 groups with 12 mice each, and 2 groups (24 mice) out of them were mixed with DMSO and PEG300 at a volume ratio of 3: 7. Streptozotocin (STZ) in a dissolved state was intraperitoneally administered at 200 mg / kg to prepare an STZ mouse that is an insulin-dependent diabetic model mouse. To the remaining 2 groups (24 mice), as a control, only a mixed solvent in which DMSO and PEG300 were mixed at a volume ratio of 3: 7 was administered (control mouse).

  Eighteen days after the administration of STZ, an osmotic pump containing cilnidipine was implanted into the abdominal cavity to start continuous administration. A dose of cilnidipine of 5 mg / kg / day was administered to one of the two groups of STZ mice and one of the two groups of control mice, which were designated as STZ + cil group and Veh + cil group, respectively. . Further, as a control, only the mixed solvent of DMSO and PEG300 mixed at a volume ratio of 3: 7 was administered to the remaining 1 group of STZ mice and the remaining 1 group of control mice, respectively. In groups.

  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), the effect of administration of cilnidipine on blood glucose was not observed. On the other hand, in STZ mice, no difference was observed between the STZ + veh group and the STZ + cil group in the ad libitum blood glucose level and the fasting blood glucose level 18 days after the administration of STZ and before the administration of cilnidipine, but 10 From the day afterward, a tendency that the blood glucose level decreased in the STZ + cil group was observed. FIG. 18 shows the measurement results of the ad libitum blood glucose level and the fasting blood glucose level of the STZ group and the STZ + cil group on the 14th day after the administration of cilnidipine (32nd day after the STZ administration). These results suggested that cilnidipine could reduce insulin-dependent hyperglycemic state.

[Example 6]
In Alzheimer's disease patients, many deposits containing amyloid β protein as a tumor component have been observed, and accumulation of amyloid β protein is considered to be involved in the onset of Alzheimer's disease. Further, when a large amount of amyloid β protein is taken up into cells, endoplasmic reticulum stress is increased and apoptosis is induced. Therefore, using the cultured cell line PC-12 cells derived from rat pheochromocytoma, the effect of cilnidipine on the cytotoxicity due to amyloid β loading was examined. 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, Ltd.) is used with 5% FBS, 5% HS (horse serum) and 1% penicillin-. A medium containing streptomycin (manufactured by Nakarai Co., Ltd.) was used. In addition, as a medium when amyloid β is loaded on PC-12, a serum-free medium (D-MEM (High Glucose) (manufactured by Wako Pure Chemical Industries, Ltd.) containing 1% penicillin-streptomycin (manufactured by Nakarai)) Conditioned medium) was used.

First, a 24-well plate was injected with a final concentration of 0.001% Poly-L-lysine (PLL) solution, allowed to stand at 37 ° C. for 30 minutes, washed twice with PBS, and then air-dried to give wells. The inner wall was coated with PLL. Undifferentiated PC-12 cells were seeded on a PLL-coated 24-well plate at 1 × 10 ⁵ cells / well (500 μL / well) and cultured in the culture medium at 37 ° C. for 24 hours.
Then, the cells in each well were washed once with a serum-free medium, and then a serum-free medium containing 500 μL of cilnidipine (final concentration 1 μM) and Amyloid β _25-35 (final concentration 10 μM) per well. , Serum-free medium containing only cilnidipine (final concentration 1 μM), serum-free medium containing only Amyloid β _25-35 (final concentration 10 μM), or serum-free medium, at 37 ° C. Incubated for hours. Then, after removing the medium from each well, 12.5 μL of MTT solution (a solution of MTT dissolved in PBS so as to be 5 mg / mL) was added to each well at 12.5 μL and shaken gently, and then 37 ° C. And incubated for 1 hour. Then, after removing the MTT solution from each well, DMSO was added at 500 μL per well, and the produced formazan was dissolved by shaking firmly. Then, 100 μL of the solution in each well was dispensed into each well of a 96-well plate, the 96-well plate was placed on a plate reader (product name: SpectraMax i3, manufactured by Molecular devices), and the absorbance at 595 nm (Abs595) was set. 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 It was calculated as the survival rate (%). The calculation result is shown in FIG. As a result, the survival rate of cells loaded with amyloid β in the presence of cilnidipine (“Cil (+)” in the figure) is the survival rate of cells loaded with amyloid β in the absence of cilnidipine (“ Cil (−) ”), cilnidipine suppressed the induction of apoptosis by amyloid β loading. From these results, it is clear that cilnidipine reduces amyloid β load on cells, and can be expected to be effective in treating Alzheimer's disease.

Claims (1)

A pharmaceutical composition containing cilnidipine or a pharmaceutically acceptable salt thereof as an active ingredient and used for reducing cardiomyocyte toxicity due to organic mercury .