Therapeutic agent for myocardial ischemia-reperfusion injury

Abstract

To provide a novel target for myocardial ischemia-reperfusion injury and a therapeutic agent therefor.SOLUTION: A therapeutic agent for myocardial ischemia-reperfusion injury comprising deferasirox is provided. The deferasirox may inhibit ferroptosis. The therapeutic agent for myocardial ischemia-reperfusion injury may further comprise cyclosporin A. The therapeutic agent for myocardial ischemia-reperfusion injury may be applied so as to be administered after the onset of myocardial infarction and prior to reperfusion after ischemia.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for myocardial ischemia-reperfusion injury. [Background technology]

[0002] Myocardial infarction is a fatal heart disease in which the myocardium becomes ischemic due to occlusion or stenosis of the coronary artery, leading to irreversible myocardial necrosis. Early revascularization by percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) has been established as a treatment for myocardial infarction. However, despite early revascularization, the size of the myocardial infarction is not sufficiently reduced, and an increasing number of patients develop post-infarction heart failure (ischemic cardiomyopathy) due to low cardiac function that occurs in the distant period after myocardial infarction.

[0003] One of the causes of post-infarction heart failure is myocardial ischemia-reperfusion injury. Myocardial ischemia-reperfusion injury is an injury caused by the production of various toxic substances in tissues when blood reperfusion to the tissue occurs due to early revascularization. In myocardial ischemia-reperfusion injury, MPT necrosis (mitochondrial permeability transition-driven necrosis) is one of the cell deaths that contributes most to infarction formation. Non-Patent Document 1 discloses that cyclosporine A, an inhibitor of the opening of the mitochondrial permeability transition pore, suppresses MPT necrosis caused by ischemia-reperfusion injury by suppressing the release of cytochrome c. However, Non-Patent Document 2 did not show the effectiveness of cyclosporine A in a phase III clinical trial with end points of remote cardiac function and heart failure. Therefore, there is a need to develop a therapeutic target as an injury mechanism different from MPT necrosis and a therapeutic drug for it. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] DJ Hausenloy et al., Cardiovascular Research 2002; 55: 534-543 [Non-Patent Document 2] Cung et al, N Engl J Med 2015; 373: 1021-31 [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2000-503625 [Patent Document 2] Special Publication No. 2023-518248 Summary of the Invention [Problem to be solved by the invention]

[0006] The purpose of the present invention is to provide a new therapeutic target for myocardial ischemia-reperfusion injury and a therapeutic agent therefor. [Means for solving the problem]

[0007] In one embodiment of the present invention, there is provided a therapeutic agent for myocardial ischemia-reperfusion injury, which comprises deferasirox.

[0008] Deferasirox may inhibit ferroptosis.

[0009] The therapeutic agent for myocardial ischemia-reperfusion injury may further contain cyclosporin A.

[0010] The therapeutic agent for myocardial ischemia-reperfusion injury contains deferasirox, and may be administered after the onset of myocardial infarction and before reperfusion after ischemia. Effect of the Invention

[0011] It is possible to provide a new target for and a therapeutic agent against myocardial ischemia-reperfusion injury. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram illustrating the mechanism of action of a therapeutic agent for myocardial ischemia-reperfusion injury according to one embodiment of the present invention. [Figure 2A] 1 shows an experimental protocol for hypoxia in cultured cardiomyocytes according to an embodiment of the present invention. [Figure 2B] FIG. 1 is a diagram showing the amount of iron in the endoplasmic reticulum of isolated cultured cardiomyocytes under hypoxic stimulation according to an embodiment of the present invention. [Figure 3A] 1 shows an experimental protocol for hypoxia in cultured cardiomyocytes according to an embodiment of the present invention. [Figure 3B] 1 shows photographs of Western blotting of 4-hydroxynonenal modification of cardiac proteins, cyclooxygenase 2, and GAPDH in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 3C] FIG. 1 is a diagram showing the amount of 4-hydroxynonenal modification of cardiac proteins in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 3D] FIG. 2 is a graph showing the amount of cyclooxygenase 2 in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 4A] 1 shows an experimental protocol for hypoxia in cultured cardiomyocytes according to an embodiment of the present invention. [Figure 4B] 1 is a histogram of BODIPY-stained cells of isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 4C] FIG. 1 is a graph showing the number of BODIPY-positive cells in isolated and cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 5A] 1 shows an experimental protocol for hypoxia in cultured cardiomyocytes according to an embodiment of the present invention. [Figure 5B] FIG. 1 is a diagram showing the cell viability of isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation according to an embodiment of the present invention. [Figure 6A] 1 shows an experimental protocol for an ischemia-reperfusion injury model in mice according to an embodiment of the present invention. [Figure 6B] FIG. 2 is a diagram showing infarct size under myocardial ischemia-reperfusion injury in a mouse myocardial ischemia-reperfusion injury model according to an example of the present invention. [Figure 7A] 1 shows an M-mode image of a mouse myocardial ischemia-reperfusion injury model according to an embodiment of the present invention. [Figure 7B] FIG. 2 is a graph showing left ventricular ejection fraction (LVEF) in a mouse myocardial ischemia-reperfusion injury model according to an example of the present invention. [Figure 7C] FIG. 2 is a graph showing left ventricular end diastolic diameter (LVEDD) in a mouse myocardial ischemia-reperfusion injury model according to an embodiment of the present invention. [Figure 7D] FIG. 2 is a graph showing total heart weight per tibia length (TL) in a mouse myocardial ischemia-reperfusion injury model according to an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As a result of myocardial ischemia-reperfusion injury, the infarct size increases due to the death of cardiomyocytes and necrosis of myocardial tissue. MPT necrosis was thought to be the pathogenesis of myocardial ischemia-reperfusion injury. MPT necrosis is cell death caused by oxidative stress due to mitochondrial membrane permeability transition and Ca2+ overload in the cytosol. Although it has been suggested that cyclosporine A inhibits MPT necrosis, clinical trials have not shown sufficient effects on myocardial ischemia-reperfusion injury. In recent years, it has been suggested that ferroptosis, an iron-dependent cell death, is the causative cell death of myocardial necrosis due to myocardial ischemia-reperfusion injury, along with MPT necrosis (Miyamoto H et al. JACC Basic Transl Sci 2022; 7(8):800-819). Ferroptosis plays various physiological and pathological roles in living organisms. Accumulation of lipid bilayer damage (lipid peroxides) caused by iron-mediated hydroxyl radical generation (Fenton reaction) leads to breakdown of the lipid bilayer structure and induction of cell death (ferroptosis). It has been known that ferroptosis can be suppressed by iron chelators, lipophilic antioxidants, or endogenous lipid peroxide-specific antioxidant molecule GPX4. However, no ferroptosis inhibitors have been identified that are expected to be clinically applicable to myocardial ischemia-reperfusion injury. In clinical practice, myocardial infarction occurs suddenly, so the administration of inhibitors of myocardial ischemia-reperfusion injury is naturally limited to after the onset of myocardial infarction and before reperfusion. The present inventors have found that administration of deferasirox after the onset of myocardial infarction and before reperfusion suppresses myocardial ischemia-reperfusion injury, and have completed the present invention.

[0014] The therapeutic agent for myocardial ischemia-reperfusion injury according to one embodiment of the present invention will be described in detail below. However, the therapeutic agent for myocardial ischemia-reperfusion injury of the present invention should not be construed as being limited to the description of the following embodiments and examples.

[0015] The therapeutic agent for myocardial ischemia-reperfusion injury according to one embodiment of the present invention includes deferasirox. Deferasirox is 4-[3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid. Deferasirox is an iron (Fe 3+ Deferasirox is a chelating agent that has been shown to be effective against iron overload caused by blood transfusion. Deferasirox binds to trivalent iron in a 2:1 ratio, forming a chelate with excess iron present in the liver, heart, reticuloendothelial cells, etc., and excretes the iron mainly via bile into feces. Deferasirox is one of the iron chelating agents that has high cell membrane permeability and can penetrate and chelate iron in myocardial cells in a short time, so it can diffuse rapidly into the ischemic myocardium after reperfusion, making it particularly suitable as a therapeutic agent for myocardial ischemia-reperfusion injury.

[0016] The myocardial ischemia-reperfusion injury therapeutic agent according to one embodiment of the present invention may further include an MPT necrosis inhibitor. As the MPT necrosis inhibitor, cyclosporin A is preferably used. Cyclosporin A is cyclo[L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-(3R,4R,6E)-6,7-didehydro-3-hydroxy-N,4-dimethyl-L-2-aminooctanoyl-L-2-aminobutanoyl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl]. Cyclosporin A is a cyclic polypeptide antibiotic consisting of 11 amino acids, and is recognized to have an effect as an immunosuppressant. Cyclosporin A is also an inhibitor of the opening of the mitochondrial permeability transition pore (mPTP), and inhibits the release of cytochrome c to inhibit necrosis caused by ischemia-reperfusion injury. Cyclosporine A, when used in combination with deferasirox, can inhibit both MPT necrosis and ferroptosis, making it particularly preferable as a therapeutic agent for myocardial ischemia-reperfusion injury.

[0017] It is believed that ischemia-reperfusion injury is caused by ferroptosis and MPT necrosis of cardiomyocytes. In ferroptosis, heme oxygenase-1 (HO-1), whose expression increases in response to stress due to ischemia and reperfusion, increases, and iron accumulates in the endoplasmic reticulum through enhanced degradation of heme, resulting in enhanced lipid peroxide production via iron, leading to ferroptosis. In MPT necrosis, the mitochondrial membrane potential is lost due to the continuous opening of the mitochondrial membrane transition pore accompanied by enhanced generation of reactive oxygen triggered by the resumption of blood and oxygen supply, leading to cell death. In isolated cultured cardiomyocytes, iron overload is induced by hypoxia-reoxygenation (H / R) stimulation. Deferasirox contained in the therapeutic agent for myocardial ischemia-reperfusion injury according to this embodiment can suppress the accumulation of iron in the endoplasmic reticulum induced by hypoxic stimulation of isolated cultured cardiomyocytes.

[0018] Hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes increases 4-hydroxynonenal (4-HNE) modification of cardiac proteins, which is one of the lipid peroxides that cause ferroptosis, and increases the expression of cyclooxygenase 2 (COX2), which is used as a marker of ferroptosis. Deferasirox can suppress the increase in 4-HNE modification caused by hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes. As a result, deferasirox contained in the myocardial ischemia-reperfusion injury treatment agent according to this embodiment can suppress the expression of COX2.

[0019] Lipid peroxides increase due to hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes. Lipid peroxides are specifically stained, and the number of stained cells is taken as the number of BODIPY (boron-dipyrromethene) positive cells. Deferasirox can reduce the number of BODIPY positive cells caused by hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes. That is, deferasirox contained in the therapeutic agent for myocardial ischemia-reperfusion injury according to this embodiment can suppress the accumulation of lipid peroxides caused by hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes.

[0020] Hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes induces cell death (ferroptosis). Deferasirox contained in the myocardial ischemia-reperfusion injury therapeutic agent according to this embodiment can suppress cell death (ferroptosis) caused by hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes. Cell death (ferroptosis) caused by hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes can be almost completely suppressed in the presence of, for example, 10 μM deferasirox.

[0021] Myocardial infarction occurs in a mouse myocardial ischemia-reperfusion injury model (anterior descending artery ligated, ligation released after 30 minutes of ischemia) that simulates a clinical scenario of myocardial infarction. Deferasirox can reduce infarct size and improve ischemia-reperfusion injury by administering it to mice before reperfusion after ischemia. Deferasirox can improve ischemia-reperfusion injury by orally administering 200 mg / kg (equivalent to 16 mg / kg in humans in terms of HED) deferasirox 10 minutes before reperfusion after ischemia. The effect of deferasirox in reducing infarct size may be almost the same as the effect of cyclosporine A in reducing infarct size. Furthermore, by administering both cyclosporine and deferasirox to mice before reperfusion after ischemia, the infarct size can be additively reduced and ischemia-reperfusion injury can be further improved.

[0022] In the remote phase after ischemia-reperfusion in mice, the left ventricular ejection fraction (LVEF), an index of cardiac contractility, is reduced, and left ventricular dilation, as measured by the left ventricular end-diastolic diameter (LVEDD), occurs. When both cyclosporine A and deferasirox were administered to mice before ischemia-reperfusion, the left ventricular ejection fraction (LVEF) was maintained, and left ventricular dilation, as measured by the left ventricular end-diastolic diameter (LVEDD), was suppressed. Furthermore, when both cyclosporine A and deferasirox were administered to mice before ischemia-reperfusion, cardiac hypertrophy, as measured by the heart weight / tibia length, was also suppressed. Thus, cyclosporine A and deferasirox can improve cardiac function in the remote phase after ischemia-reperfusion in mice.

[0023] As shown in Fig. 1, the therapeutic agent for myocardial ischemia-reperfusion injury according to one embodiment of the present invention can suppress accumulation of lipid peroxides by deferasirox chelating iron in the endoplasmic reticulum, and can improve myocardial ischemia-reperfusion injury by suppressing ferroptosis. The therapeutic agent for myocardial ischemia-reperfusion injury according to one embodiment of the present invention can further improve myocardial ischemia-reperfusion injury by suppressing MPT necrosis by inhibiting the sustained opening of the mitochondrial permeability transition pore with cyclosporine A. EXAMPLES

[0024] Example 1: Deferasirox inhibits iron accumulation in the endoplasmic reticulum induced by hypoxic stimulation of isolated cultured cardiomyocytes. [Primary culture of isolated cardiomyocytes] Neonatal rat ventricular myocytes were prepared by euthanizing Sprague Dawley rats with an overdose of 5% isoflurane, followed by rapid excision of the hearts. After digestion of the myocardial tissue with trypsin (25300-062, Thermo Fisher Scientific) and type 2 collagenase (LS004176, Worthington Biochemical), the isolated cardiomyocytes were suspended in Dulbecco's modified Eagle's medium (DMEM, D5796, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, SH30910.03, HyClone Laboratories) and 1% penicillin / streptomycin (26253-84, Nacalai Tesque, Inc.). To reduce the number of non-myocytes, cells were seeded twice on 100 mm culture dishes every 70 min. Non-adherent cells were plated on culture dishes (Primaria, Corning) at approximately 2.5 × 10 5 The cells were seeded at a density of 100 / mL and cultured at 37°C in humidified air containing 5% CO2.

[0025] [Hypoxia experiments using isolated cultured cardiomyocytes] Figure 2A shows the experimental protocol for hypoxia in cultured cardiomyocytes according to this example. As shown in Figure 2A, hypoxia (Hx) was induced in cultured cardiomyocytes by replacing the standard medium with conditioned medium kept overnight in a hypoxic chamber (APM-50D, Astec, (5% CO2, 94.5% N2, 0.5% O2, 37°C)) and culturing the cells in the hypoxic chamber for 4 hours. Deferasirox (DFX: 200 μmol / L) was added 1 hour before Hx treatment.

[0026] [Iron detection in isolated cultured cardiac myocytes] The iron concentration in the endoplasmic reticulum of cultured cardiomyocytes was measured using FFR (FerroFarRed, GC903-01, Goryo Chemical). Cultured cardiomyocytes were seeded on 35 mm glass-bottom dishes (AGC Techno Glass), Hx was induced using conditioned medium in the presence or absence of 200 μmol / L DFX, and the cells were cultured in a hypoxic chamber for 4 hours. The cells were then washed three times with PBS (phosphate-buffered saline) and cultured for 1 hour in serum-free medium containing 5 μmol / L FFR and 0.1 μmol / L ERseeing (FDV-0038, Funakoshi Co., Ltd.). Thereafter, the cells were washed three times with PBS and fixed with 4% paraformaldehyde at 4°C for 10 minutes. The cells were washed again three times with PBS, mounted on microscope slides using a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vectashield H1200, Vector Laboratories), and observed under a BZ-X800 fluorescent microscope (Keyence Corporation). Image analysis was performed using ImageJ software v1.44 to measure fluorescence intensity.

[0027] Figure 2B shows the amount of iron in the endoplasmic reticulum of isolated cultured cardiomyocytes under hypoxic stimulation. As shown in Figure 2B, hypoxic stimulation of isolated cultured cardiomyocytes increased the amount of iron in the endoplasmic reticulum (Hx CTL). On the other hand, in the presence of deferasirox, the accumulation of iron in the endoplasmic reticulum induced by hypoxic stimulation of isolated cultured cardiomyocytes was suppressed (Hx DFX).

[0028] Example 2: Deferasirox suppresses 4-HNE modification and COX2 increase induced by hypoxia-reoxygenation in isolated cultured cardiomyocytes. [Hypoxia experiments using isolated cultured cardiomyocytes] FIG. 3A shows the experimental protocol of hypoxia-reoxygenation in cultured cardiomyocytes according to this embodiment. As shown in FIG. 3A, hypoxia (H) was induced in cultured cardiomyocytes by replacing the standard medium with conditioned medium kept overnight in a hypoxic chamber (APM-50D, Astec, (5% CO2, 94.5% N2, 0.5% O2, 37°C)) and culturing for 24 hours in the hypoxic chamber. Thereafter, reoxygenation (R) was induced by replacing the hypoxic medium with standard medium and culturing for 6 hours at 37°C in humidified air containing 5% CO2. DFX (200 μmol / L) was added 1 hour before H / R treatment.

[0029] [Detection of 4-HNE and COX2 in isolated cultured cardiomyocytes] The cells were washed three times with PBS, lysed in RIPA buffer, and collected, and proteins were separated by electrophoresis. The separated proteins were transferred to a membrane, and 4-HNE and COX2 were detected by Western blotting analysis. For Western blotting, 4-hydroxy-2-nonenal (4-HNE; MHN-020P, Nikken Seiru Co., Ltd., Japan Institute for Aging Control) and cyclooxygenase 2 (COX2; #12282, Cell Signaling Technology) were used as primary antibodies. An antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, internal standard; sc-32233, Santa Cruz Biotechnology Inc.) was used as an internal control to normalize the signal intensity.

[0030] FIG. 3B is a photograph of Western blotting of 4-hydroxynonenal modification of cardiac proteins, cyclooxygenase 2, and GAPDH in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. FIG. 3C is a diagram showing the amount of 4-hydroxynonenal modification of cardiac proteins in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. FIG. 3D is a diagram showing the amount of cyclooxygenase 2 in isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. As shown in FIG. 3B to FIG. 3D, hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes increased 4-hydroxynonenal (4-HNE) modification of cardiac proteins and increased the expression of cyclooxygenase 2 (COX2), which is used as a marker of ferroptosis (H / R Veh). On the other hand, in the presence of deferasirox, the increase in 4-HNE and the increase in COX2 expression due to hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes were suppressed (H / R DFX).

[0031] Example 3: Deferasirox reduces the number of BODIPY-positive cells induced by hypoxia-reoxygenation in isolated cultured cardiomyocytes. [Hypoxia experiments using isolated cultured cardiomyocytes] FIG. 4A shows the experimental protocol of hypoxia-reoxygenation in cultured cardiomyocytes according to this embodiment. As shown in FIG. 4A, hypoxia (H) was induced in cultured cardiomyocytes by replacing the standard medium with conditioned medium kept overnight in a hypoxic chamber (APM-50D, Astec Corporation, (5% CO2, 94.5% N2, 0.5% O2, 37°C)) and culturing for 24 hours in the hypoxic chamber. Thereafter, reoxygenation (R) was induced by replacing the hypoxic medium with standard medium and culturing for 1 hour at 37°C in humidified air containing 5% CO2. DFX (200 μmol / L) was added 1 hour before H / R treatment.

[0032] [Detection of lipid peroxidation using BODIPY in isolated cultured cardiac myocytes] Lipid peroxidation in cultured cardiomyocytes was measured using C11-BODIPY581 / 591 (Thermo Fisher Scientific) as a probe. Cells were harvested by trypsinization and resuspended in PBS. Cells were then incubated with 2 mmol / L C11-BODIPY581 / 591 in PBS at 37°C for 15 min. Cells were strained through a 40 mm cell strainer (pluriSelect43-10040®, Life Sciences) and analyzed using a BD FACSLyric flow cytometer (BD Biosciences) equipped with a 488 nm laser for excitation. At least 10,000 cells were analyzed in each condition. Histograms were generated using FlowJo software v10.

[0033] FIG. 4B is a histogram of BODIPY-stained cells of isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. FIG. 4C is a diagram showing the number of BODIPY-positive cells of isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. As shown in FIG. 4B and FIG. 4C, the number of BODIPY (boron-dipyrromethene)-positive cells increased under hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes (H / R CTRL). On the other hand, in the presence of deferasirox, the increase in the number of BODIPY-positive cells due to hypoxia-reoxygenation stimulation of isolated cultured cardiomyocytes was suppressed (H / R DFX).

[0034] Example 4: Deferasirox inhibits cell death (ferroptosis) caused by hypoxia-reoxygenation in isolated cultured cardiomyocytes. [Hypoxia experiments using isolated cultured cardiomyocytes] FIG. 5A shows the experimental protocol of hypoxia-reoxygenation in cultured cardiomyocytes according to this embodiment. As shown in FIG. 5A, hypoxia (H) was induced in cultured cardiomyocytes by replacing the standard medium with conditioned medium kept overnight in a hypoxic chamber (APM-50D, Astec Corporation, (5% CO2, 94.5% N2, 0.5% O2, 37°C)) and culturing for 24 hours in the hypoxic chamber. Thereafter, reoxygenation (R) was induced by replacing the hypoxic medium with standard medium and culturing for 24 hours at 37°C in humidified air containing 5% CO2. DFX (1 μM, 10 μM, 100 μM, 200 μM) or Fer-1 (50 μM) was added 1 hour before H / R treatment.

[0035] [Cell viability assay using Calcein AM] Cell viability was assessed using Cell Counting Kit F (CK-06, Dojindo Laboratories, Inc.). Cultured cardiomyocytes were washed with PBS and then incubated with Calcein AM diluted in PBS (Calcein AM to PBS ratio 1:500) at 37°C for 15 min. Cell viability was then measured by fluorometry (excitation wavelength 490 nm and emission wavelength 520 nm) using a Varioskan® LUX multimode microplate reader (Thermo Fisher Scientific).

[0036] Figure 5B shows the cell viability of isolated cultured cardiomyocytes under hypoxia-reoxygenation stimulation. As shown in Figure 5B, cell death (ferroptosis) increased under hypoxia-reoxygenation stimulation in isolated cultured cardiomyocytes (H / R Veh). On the other hand, in the presence of 10 μM or more of deferasirox, cell death (ferroptosis) induced by hypoxia-reoxygenation stimulation in isolated cultured cardiomyocytes was suppressed (H / R DFX).

[0037] Example 5: Deferasirox reduces infarct size and ameliorates ischemia-reperfusion injury when administered before reperfusion after ischemia in mice. [A mouse model of myocardial ischemia-reperfusion injury simulating a myocardial infarction scenario] The myocardial ischemia-reperfusion injury model was induced in C57BL / 6J mice. Figure 6A shows the experimental protocol of the ischemia-reperfusion injury model in mice according to this example. Male mice aged 9 to 12 weeks were anesthetized by inhalation of 1 to 2% isoflurane. Then, the intercostal space was opened under mechanical ventilation, and myocardial ischemia was induced by ligating the left anterior descending artery (LAD) for 30 minutes, and then reperfusion was performed by releasing the ligation. Deferasirox (diluted to 40 mg / mL DFX with polyethylene glycol (28214-05, Nacalai Tesque, Inc.); D5905, Tokyo Chemical Industry Co., Ltd.) was orally administered to the mice at 200 mg / kg 10 minutes before reperfusion. Cyclosporine A (diluted in saline to 7.5 mg / mL CsA; 3999406A1032, Novartis International) was injected at 2.5 mg / kg via the femoral vein into mice 10 min before reperfusion. Mouse hearts were excised 24 h after reperfusion for histological and biochemical analysis.

[0038] [Measurement of infarct size] To identify the area at risk (AAR) 24 h after reperfusion, the LAD was reoccluded and 0.3 mL of 2% Evans blue dye (E2129, Sigma) was injected into the right jugular vein. Once the distal limbs turned blue, the heart was rapidly excised, rinsed with saline, and the left ventricle (LV) was frozen in liquid nitrogen. The LV was then cut into five 1 mm thick slices and incubated with 1% 2,3,5-TTC (T0520, Tokyo Chemical Industry) at 37 °C for 15 min. The infarcted area (IFA; white area) and the area exposed to ischemia (area at risk, AAR (red and white areas)) of each slice were measured using ImageJ software v1.44 (NIH). The AAR / LV, IFA / AAR, and IFA / LV ratios were then calculated.

[0039] Figure 6B shows the infarct size after myocardial ischemia-reperfusion injury in a mouse myocardial ischemia-reperfusion injury model. As shown in Figure 6B, myocardial infarction was generated by myocardial ischemia-reperfusion injury (IFA / AAR I / R Veh). On the other hand, in the presence of deferasirox or cyclosporine A, the increase in infarct size due to myocardial ischemia-reperfusion injury was suppressed to the same extent (IFA / AAR I / R DFX or IFA / AAR I / R CsA). In the presence of deferasirox and cyclosporine A, the infarct size due to myocardial ischemia-reperfusion injury was additively suppressed (IFA / AAR I / R DFX+CsA).

[0040] Example 6: Deferasirox and cyclosporine A improve long-term cardiac function when administered before reperfusion after ischemia in mice. A mouse myocardial ischemia-reperfusion injury model simulating a myocardial infarction scenario was created in the same manner as in Example 5. 14 days after reperfusion, the mouse was anesthetized with 1-2% isoflurane, and M-mode images were obtained from short-axis images at the papillary muscle level using a Vevo (registered trademark) 1100 ultrasound examination system (FUJIFILM VisualSonics). Figure 7A is an M-mode image in a mouse myocardial ischemia-reperfusion injury model. Figure 7B is a diagram showing the left ventricular ejection fraction (LVEF) in a mouse myocardial ischemia-reperfusion injury model. Figure 7C is a diagram showing the left ventricular end-diastolic diameter (LVEDD) in a mouse myocardial ischemia-reperfusion injury model. Figure 7D is a diagram showing the total heart weight (TL) per tibia length in a mouse myocardial ischemia-reperfusion injury model. As shown in Figures 7B to 7D, the combined treatment significantly improved the decrease in left ventricular ejection fraction (LVEF) and suppressed the left ventricular end-diastolic diameter (LVEDD). Furthermore, combined treatment also reduced cardiac hypertrophy, expressed as heart weight / tibia length.

Claims

1. A therapeutic agent for myocardial ischemia-reperfusion injury, comprising deferasirox.

2. The method of claim 1, wherein the deferasirox inhibits ferroptosis.

3. The therapeutic agent for myocardial ischemia-reperfusion injury according to claim 2, further comprising cyclosporin A.

4. The therapeutic agent for myocardial ischemia-reperfusion injury according to any one of claims 1 to 3, which is administered after the onset of myocardial infarction and before reperfusion after ischemia.

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