THERAPEUTIC AGENT FOR CARDIOMYOPATHY, OLD MYOCARDIAL INFARCTION AND CHRONIC HEART FAILURE

Description

Technical Field

[0001] The present invention provides a pharmaceutical composition for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the pharmaceutical composition comprises the substance described in any of (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

[0002] The present further provides a substance for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the substance is selected from (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.Background Art

[0003] Cardiomyopathy is defined as a "disease of myocardium associated with cardiac dysfunction" often accompanied by structural abnormalities of the heart, such as cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis; and presents symptoms of chronic heart failure as it progresses. Secondary cardiomyopathy may be ameliorated by treatment of the primary disease, but there is still no definitive treatment for idiopathic cardiomyopathy.

[0004] Myocardial infarction, which causes myocardial necrosis due to coronary artery occlusion, is a major underlying disorder of heart disease which is the leading cause of death in developed countries (first in the United States and second in Japan). While the acute-phase treatment outcome has been improved by the advancement of diagnostic technologies, catheter treatment, and coronary artery bypass grafting, cardiomegaly and cardiac hypertrophy progress and chronic heart failure often occur in cases of extensive infarction, severe reperfusion failure, and cases that have missed the opportunity for treatment.

[0005] When chronic heart failure caused by cardiomyopathy or old myocardial infarction becomes severe, existing drugs for chronic heart failure (ACE inhibitors, β blockers, etc.) cannot achieve sufficient improving effects in some cases, and heart transplantation may be required. However, the transplantation waiting period in Japan is as long as about 3 years (Non-Patent Document 1), and complications such as cerebral infarction and device infection often occur during the waiting period. Further, even if a heart transplant is received, there are problems such as increased risk of infectious diseases due to side effects of immunosuppressants which require to be continuously administered subsequently and possible complications such as coronary artery lesions; and the 10-year survival rate after the heart transplantation is about 50% (Non-Patent Document 2). Under such circumstances, development of new therapeutic agents for cardiomyopathy and old myocardial infarction as well as chronic heart failure caused thereby is desired.

[0006] Recently, regenerative medicine using mesenchymal stem cells has attracted attention, and regarding chronic heart failure after myocardial infarction, it has been reported that a cardiac function improving effect could be obtained by directly transplanting a cell sheet made of mesenchymal stem cells derived from the egg membrane, bone marrow, or adipose tissue into the heart in an animal model of chronic myocardial infarction (Non-Patent Document 3 and Patent Document 1). However, transplantation of the cell sheet is very burdensome to the patient as it requires a thoracotomy operation, and it cannot be applied to patients whose operation is difficult due to factors such as advanced age.

[0007] Also, the mechanism in which an injured tissue releases bone-marrow pluripotent stem cell recruiting factors into the blood to induce regeneration of the injured tissue has been confirmed. In previous studies, the present inventors found that fragment peptides of the High mobility group box 1 (HMGB 1) protein mobilize bone marrow mesenchymal stem cells from the bone marrow into the peripheral blood, and that administration of the fragment peptides in the acute phase of myocardial infarction leads to accumulation of bone marrow-derived mesenchymal stem cells at and near the site of infarction, resulting in an improved cardiac function (Patent Documents 2 and 3).

[0008] However, in the acute phase of myocardial infarction, a large quantity of cardiomyocytes are rapidly necrotized by ischemia and a strong inflammatory reaction is generated, whereas cardiomyopathy does not generally cause such phenomena and is characterized in that structural abnormalities such as cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis progress slowly. The pathologies of these two are greatly different. Necrosis and inflammation of cardiomyocytes are also settled in old myocardial infarction, and the progression of cardiomegaly and cardiac hypertrophy is characteristic for the disease. Therefore, it was unclear whether fragment peptides of the HMGB1 protein exert therapeutic effects on cardiomyopathy and old myocardial infarction.Citation List[Non-Patent Documents]

[0009] Non-Patent Document 1: Fukushima et al., "Registry Report on Heart Transplantation in Japan (June 2016),"Circulation Journal (Advance Publication, January 6, 2017, Article ID: CJ-16-0976) Non-Patent Document 2: Lund et al., "The Registry of the International Society for Heart and Lung Transplantation: Thirty-third Adult Heart Transplantation Report-2016; Focus Theme: Primary Diagnostic Indications for Transplant," J Heart Lung Transplant. 2016 Oct;35(10):1158-1169 Non-Patent Document 3: Ishikane Shin, "Development of multi-growth factor secreted fetal membrane-derived mesenchymal stem cell sheets" June 11, 2014, Grants-in-Aid for Scientific Research, Research report [Patent Documents]

[0010] Patent Document 1: WO2006 / 080434 Patent Document 2: WO2012 / 147470 Patent Document 3: WO2014 / 065347

[0011] NARUMI, T. et al., High-Mobility Group Box 1 Attenuates Mitochondrial Dysfunction and Apoptosis via Heat Shock Protein Beta 1 Induction in Doxorubicin-Induced Cardiomyopathy, 2015, vol. 33, no. 2, pp. 126-127 (Bulletin of Yamagata University (Medical Science)) suggest that full-length HMGB1 may be a new target for the treatment of doxorubicin induced cardiomyopathy. The reference is silent at least about fragments of HMGB1, let alone any therapeutic application thereof.

[0012] Takahashi et al. (Circulation. 2008;118; S106-S114) hypothesize that full-length HMGB1 injection could preferentially modulate local myocardial inflammation, attenuate ventricular remodeling, and subsequently improve cardiac performance of postinfarction chronic heart failure. The study conclude that HMGB1 injection modulated the local inflammation in the postinfarction chronically failing myocardium, particularly via reducing the accumulation of dendritic cells. This modulated inflammation resulted in attenuated fibrosis and cardiomyocyte hypertrophy, which thereby improved global cardiac function. Takahashi et al. suggest that HMGB1 may be valuable for the chronic heart failure treatment. The reference is silent at least about fragments of HMGB1, let alone any therapeutic application thereof.

[0013] Takahashi et al. (2011) vol. 27, supplement, p. S189, I-E-19Acta Cardiologica Paediatrica Japonica study the effect of full-length HMGB1 on chronic heart failure (3 weeks post infarction) and suggest that HMGB1 may improve cardiac function. The reference is silent at least about fragments of HMGB1, let alone any therapeutic application thereof.Summary of the Invention

[0014] As a result of studying the effects of HMGB1 fragment peptides in cardiomyopathy, the present inventors found that HMGB1 fragment peptides having particular amino acid sequences exhibit effects of improving cardiac functions, inhibiting structural abnormalities of the heart (cardiomyocyte hypertrophy and myocardial fibrosis), and promoting angiogenesis in an animal model of dilated cardiomyopathy. The present inventors also discovered that in an animal model of ischemic cardiomyopathy resulting from old myocardial infarction, the specific HMGB1 fragment peptides exhibit effects of improving cardiac function, inhibiting structural abnormalities of the heart (cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis), and promoting angiogenesis. Furthermore, the present inventors found that the specific HMGB1 fragment peptides exhibit an inhibitory effect on structural abnormalities of the heart (cardiomyocyte hypertrophy and myocardial fibrosis) in a hypertensive cardiomyopathy animal model.

[0015] The present invention is set out in the appended set of claims.

[0016] The present invention provides a pharmaceutical composition for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the pharmaceutical composition comprises the substance described in any of (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

[0017] In one embodiment, said pharmaceutical composition for use according to the invention is for use in the treatment of cardiomyopathy.In one embodiment, the cardiomyopathy is idiopathic cardiomyopathy. In a further embodiment, the idiopathic cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy.

[0018] In one embodiment, the cardiomyopathy is a secondary cardiomyopathy. In a further embodiment, the secondary cardiomyopathy is selected from the group consisting of ischemic cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, drug-induced cardiomyopathy, alcoholic cardiomyopathy, mitochondrial cardiomyopathy, cardiomyopathy caused by cardiac sarcoidosis, cardiomyopathy caused by cardiac amyloidosis, cardiomyopathy caused by myocarditis, cardiomyopathy caused by muscular dystrophy, cardiomyopathy caused by cardiac Fabry's disease, and peripartum cardiomyopathy.

[0019] In one embodiment, the pharmaceutical composition for use according to the invention is for preventing and / or treating a chronic heart failure caused by cardiomyopathy.

[0020] In one embodiment, the pharmaceutical composition for use according to the invention is for use in inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis in a patient with cardiomyopathy.

[0021] In one embodiment, the pharmaceutical composition for use according to the invention is for use in treating a cardiomyopathy selected from the group consisting of dilated cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy.

[0022] In one embodiment, the chronic heart failure caused by cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy. In another embodiment, the chronic heart failure is caused by dilated cardiomyopathy or ischemic cardiomyopathy. In one embodiment, the chronic heart failure caused by dilated cardiomyopathy or ischemic cardiomyopathy is HFrEF.

[0023] In one embodiment, the chronic heart failure is caused by hypertensive cardiomyopathy.

[0024] In another embodiment, the chronic heart failure caused by hypertensive cardiomyopathy is HFpEF.

[0025] The present invention also provide a substance for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the substance is selected from (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

[0026] In one embodiment, said substance is for use in the treatment of cardiomyopathy. In another embodiment, the cardiomyopathy is idiopathic cardiomyopathy. In a further embodiment, the idiopathic cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy. In one embodiment, the cardiomyopathy is a secondary cardiomyopathy. In a further embodiment, the secondary cardiomyopathy is selected from the group consisting of ischemic cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, drug-induced cardiomyopathy, alcoholic cardiomyopathy, mitochondrial cardiomyopathy, cardiomyopathy caused by cardiac sarcoidosis, cardiomyopathy caused by cardiac amyloidosis, cardiomyopathy caused by myocarditis, cardiomyopathy caused by muscular dystrophy, cardiomyopathy caused by cardiac Fabry's disease, and peripartum cardiomyopathy.

[0027] In one embodiment, said substance is for use in treatment of a chronic heart failure caused by cardiomyopathy.

[0028] In one embodiment, said substance is for use in inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis in a patient with cardiomyopathy.

[0029] In one embodiment, said substance is for use in the treatment of a cardiomyopathy selected from the group consisting of dilated cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy.

[0030] In one embodiment, said substance is for use in treating a chronic heart failure caused by cardiomyopathy selected from the group consisting of dilated cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy. In further embodiment, the chronic heart failure is caused by dilated cardiomyopathy or ischemic cardiomyopathy. In one embodiment, the chronic heart failure caused by dilated cardiomyopathy or ischemic cardiomyopathy is HFrEF. In one embodiment, the chronic heart failure is caused by hypertensive cardiomyopathy. In a further embodiment, the chronic heart failure caused by hypertensive cardiomyopathy is HFpEF.Brief Description of the Drawings

[0031] FIG. 1 presents graphs showing the results of measurement of left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVDd), and left ventricular end-systolic diameter (LVDs) in the HMGB1 peptide (1-44) and PBS groups before, 4 weeks after, and 6 weeks after administration. FIG. 2 is a photograph showing the result of Sirius red staining of myocardial tissue sections and a graph showing the percentage of positively stained area. FIG. 3 is a photograph showing the result of immunostaining (anti-CD31 antibody) of myocardial tissue sections and a graph showing capillary density. FIG. 4 is a photograph showing the result of PAS staining of myocardial tissue sections and a graph showing the short diameter of cardiomyocytes. FIG. 5 is a photograph showing the result of immunostaining using antibodies against PDGFRα and CD29. PDGFRα, green; CD29, red; DAPI, blue. FIG. 6 is a graphical representation of the number of PDGFRα-positive and CD29-positive cells in the HMGB 1 peptide (1-44) and PBS groups. FIG. 7 is a graphical representation of the expression levels of VEGF and TSG-6 analyzed by RT-PCR. RQ, relative quantity. FIG. 8 is a photograph showing an observation result of cardiomyocytes by electron microscope. FIG. 9 presents graphs showing the results of the left ventricular ejection fraction (LVEF) measurement from pre-dose to 14 weeks post-dose in the HMGB1 peptide (1-44) group and PBS group. FIG. 10 is a graphical representation of the viability of the HMGB1 peptide (1-44) group and the PBS group. FIG. 11 presents a graph showing the results of the left ventricular ejection fraction (LVEF) measurement in the HMGB1 peptide (1-44) group and the PBS group before and 4 weeks after administration. FIG. 12 presents graphs showing the results of measurement of the left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) in the HMGB 1 peptide (1-44) group and the PBS group before and 4 weeks after administration. FIG. 13 presents a graph showing the results of the left ventricular ejection fraction (LVEF) measurement from pre-dose to 4 weeks post-dose in the HMGB1 peptide (1-44) and PBS groups. FIG. 14 is a photograph showing the result of Sirius red staining of myocardial tissue sections and a graph showing the percentage of positively stained area. FIG. 15 presents a graph showing capillary density at the infarct border and a photograph showing the result of von-Willebrand factor staining at the infarct border. FIG. 16 presents a graph showing the short diameter of cardiomyocytes at the infarct border. FIG. 17 is a fluorescent immunostaining image of the infarct border in the HMGB1 peptide (1-44) group. PDGFRα, green; CD90, red; DAPI, blue. The arrows in (a) indicate the PDGFRα- and CD90-double positive cells, and the broken line indicates the infarct border. FIG. 18 is a fluorescent immunostaining image of the infarct border in the HMGB1 peptide (1-44) group. PDGFRα, green; CD105, red; DAPI, blue. The arrows indicate the PDGFRα- and CD105-double positive cells, and the broken line indicates the infarct border. FIG. 19 is a fluorescent immunostaining image of the infarct border in the HMGB1 peptide (1-44) group and the PBS group. PDGFRα, green; CD90, red; DAPI, blue. The arrows indicate the PDGFRα- and CD90-double positive cells, and the broken line indicates the infarct border. FIG. 20 presents a graph showing heart weight at 15 weeks of age. FIG. 21 presents a graph showing the transition of LVAWd from 6 weeks of age to 15 weeks of age (however, no measurements were taken at 9 and 14 weeks of age). FIG. 22 is a photograph showing the results of Sirius red staining of myocardial tissue sections. FIG. 23 presents graphs showing the percentage of area positively stained with Sirius red in myocardial tissue sections. All p values between groups are 0.1 or more, except those shown in the figure. Detailed description of the invention

[0032] The present disclosure provides a pharmaceutical composition for use in treating (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, which comprises the substance described in any of (a) to (b) below: (a) an HMGB1 fragment peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

[0033] In one embodiment, the pharmaceutical composition is for use in treating a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis in a patient with cardiomyopathy.

[0034] As used in the present disclosure, dysfunction of the heart (cardiac dysfunction) refers to impairment of the pump function by which the heart takes in and sends out blood, including impaired contractility and impaired diastolic capacity. Dysfunction of the heart may for example be impaired contractility or impaired diastolic capacity. Dysfunction of the heart may be impaired contractility or impaired diastolic capacity of the ventricle. Dysfunction of the heart may be impaired contractility or impaired diastolic capacity of the left ventricle.

[0035] In one embodiment, the pharmaceutical compositions for for use according to the invention comprises an HMGB1 fragment peptide consisting of the amino acid sequence described in SEQ ID NO: 1.

[0036] In the present disclosure, "cardiomyopathy" refers to a myocardial disease accompanied by cardiac dysfunction. Also, cardiomyopathy is often accompanied by structural abnormalities of the heart, such as cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis.

[0037] Among the cardiomyopathies, those having no obvious cause such as hypertension and coronary artery disease and having lesions predominantly in the myocardium are called idiopathic cardiomyopathy, and those having a clear relationship with the cause disease or systemic disease are called secondary cardiomyopathy.

[0038] Idiopathic cardiomyopathies include, but are not limited to, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and unclassified cardiomyopathy. "Dilated cardiomyopathy" refers to a disease characterized by left ventricular enlargement and impaired left ventricular contractility, without coincidence of a coronary artery disease or an abnormal load condition (hypertension or valvular disease) that can lead to diffuse contractile impairment.

[0039] Secondary cardiomyopathies include, but are not limited to, ischemic cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, drug-induced cardiomyopathy, alcoholic cardiomyopathy, mitochondrial cardiomyopathy, cardiomyopathy caused by cardiac sarcoidosis, cardiomyopathy caused by cardiac amyloidosis, cardiomyopathy caused by myocarditis, cardiomyopathy caused by muscular dystrophy, cardiomyopathy caused by cardiac Fabry's disease, and peripartum cardiomyopathy. In one aspect, the cardiomyopathy caused by muscular dystrophy is cardiomyopathy caused by Duchenne type-, Becker type-, or Emery-Dreifuss type-muscular dystrophy.

[0040] In the present disclosure, "ischemic cardiomyopathy" refers to a disease in which an ischemic heart disease (old myocardial infarction or angina pectoris) causes cardiomegaly and impairment of myocardial contractility. The HMGB1 fragment peptides can prevent and / or treat ischemic cardiomyopathy because the peptides have an inhibitory effect on cardiomegaly and an improving effect on myocardial contractility.

[0041] In the present disclosure, "hypertensive cardiomyopathy" refers to a disease in which hypertension causes cardiomyocyte hypertrophy and cardiac dysfunction (impaired diastolic capacity or impaired contractility). The HMGB1 fragment peptides can prevent and / or treat hypertensive cardiomyopathy because the peptides can suppress cardiomyocyte hypertrophy caused by hypertension.

[0042] In the present disclosure, the term "pharmaceutical composition" is used interchangeably with "pharmaceutical", "drug", and "pharmacological composition".

[0043] The term "old myocardial infarction" refers to a condition in which 30 days or more have elapsed after the onset of myocardial infarction in humans (14 days or more after the onset of myocardial infarction in rats), and which is accompanied by a structural abnormality or functional disorder of the heart. Old myocardial infarction with cardiomegaly and impaired cardiac contractility is classified as ischemic cardiomyopathy. The term "old myocardial infarction" is also used interchangeably with "chronic myocardial infarction".

[0044] The HMGB1 fragment peptides disclosed herein inhibit structural abnormalities (e.g., cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis) and dysfunction (e.g., impaired contractility) of the heart in old myocardial infarction. Also, the HMGB1 fragment peptides of the present disclosure exert an improving effect on cardiac function (e.g., contractility) in cardiomyopathy. Thus, the HMGB1 fragment peptides are believed to exert broad therapeutic effects on cardiac diseases associated with structural abnormality and / or dysfunction of the heart, such as old myocardial infarction and cardiomyopathy.

[0045] In one embodiment, the pharmaceutical composition is for the treatment of a chronic heart failure caused by cardiomyopathy, wherein the pharmaceutical composition comprises an HMGB1 fragment peptide consisting of the amino acid sequence described in SEQ ID NO: 1.

[0046] Cardiomyopathy leads to a condition of chronic heart failure when cardiac function deteriorates due to progression of the disease state. The HMGB1 fragment peptides can prevent and / or treat chronic heart failure in cardiomyopathy patients because they can suppress structural cardiac abnormalities such as cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis and improve cardiac function in cardiomyopathy.

[0047] In the present disclosure, "chronic heart failure" refers to a disease state in which the pump function of the heart deteriorates due to chronic myocardial impairment, and the absolute and relative volume of blood in proportion to the oxygen demand of the main peripheral organs cannot be pumped out, causing congestion of the lungs, the systemic venous system, or both systems, thereby impeding daily life. The types of chronic heart failure include heart failure with reduced left ventricular ejection fraction (heart failure with reduced ejection fraction: HFrEF) and heart failure with preserved left ventricular ejection fraction (heart failure with preserved ejection fraction: HFpEF). HFrEF is characterized by <50% left ventricular ejection fraction and has impaired contractility. HFpEF is characterized by a left ventricular ejection fraction of 50% or greater and has impaired diastolic capacity. In one aspect, the HFrEF is due to dilated cardiomyopathy or ischemic cardiomyopathy. In one aspect, the HFpEF is due to hypertensive cardiomyopathy.

[0048] In one embodiment, the pharmaceutical composition is for inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis in patients withcardiomyopathy, wherein pharmaceutical composition comprises an HMGB1 fragment peptide consisting of the amino acid sequence described in SEQ ID NO: 1. In one embodimeny, the pharmaceutical compositions for use in patients with cardiomyopathy to (i) inhibit a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, and myocardial fibrosis, (ii) promote angiogenesis in the heart, or (iii) improve the contractility or diastolic capacity of the heart. Contractility of the heart includes, but is not limited to, for example, ventricular contractility such as contractility of the left ventricle. Diastolic capacity of the heart includes, but is not limited to, for example, ventricular diastolic capacity such as diastolic capacity of the left ventricle.

[0049] In the present disclosure, an HMGB1 fragment peptide consisting of the amino acid sequence described in SEQ ID NO: 1 refers to a peptide consisting of a portion of an HMGB1 protein and consisting of the amino acid sequence described SEQ ID NO: 1. Such peptides can be obtained as genetic recombinants (recombinants) by incorporating DNA encoding the peptide into an appropriate expression system, or they can be synthesized artificially.

[0050] In the present disclosure, the HMGB1 protein includes, but is not limited to, proteins comprising the amino acid sequence described in SEQ ID NO:2 and proteins encoded by DNAs comprising the base sequence described in SEQ ID NO:3.

[0051] In the present disclosure, cells whose migration is stimulated by an HMGB1 fragment peptide include, but are not limited to, bone marrow cells or bone marrow-derived cells (e.g., bone marrow stem cells or bone marrow-derived stem cells).

[0052] In the present disclosure, "bone marrow cells" refers to cells present in the bone marrow, while "bone marrow-derived cells" refers to "bone marrow cells" mobilized from the bone marrow to the outside of the bone marrow. "Bone marrow cells" may also include undifferentiated cells such as stem cells and progenitor cells present in the bone marrow.

[0053] In the present disclosure, cells whose migration is stimulated by an HMGB1 fragment peptide also include, but are not limited to, mesenchymal stem cells. "Mesenchymal stem cells" are cells taken from the bone marrow or other tissues (e.g., blood such as umbilical cord blood, and skin, fat, dental pulp, etc.), which can be cultured and propagated as adherent cells on culture dishes (made of plastic or glass) and having the potential to differentiate into mesenchymal tissues such as bone, cartilage, fat, and muscle. The mesenchymal stem cells may also have an ability to differentiate into epithelial and nervous tissues. Mesenchymal stem cells may exist as a heterogeneous population of cells containing not only stem cells in a narrow sense but also progenitor cells, and they may include, under culture conditions, differentiated cells in addition to the narrow-sense stem cells and / or progenitor cells. The mesenchymal stem cells may be composed only of the narrow-sense stem cells, or they may be a cell population consisting of several types of progenitor cells.

[0054] Progenitor cells are defined herein as cells having a unidirectional differentiation potential into cells of a specific tissue other than the blood system, and include cells having a potential to differentiate into a mesenchymal tissue, an epithelial tissue, a nervous tissue, a parenchymal organ, or vascular endothelium.

[0055] In the present disclosure, cells whose migration is stimulated by an HMGB1 fragment peptide also include, but are not limited to, bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells. "Bone marrow mesenchymal stem cells" are cells present in the bone marrow, and are cells characterized in that they are collected from the bone marrow and can be cultured and proliferated as adherent cells on culture dishes (made of plastic or glass) and have the differentiation potential into mesenchymal tissues such as bone, cartilage, adipose, and muscle, as well as nervous and epithelial tissues. In the present disclosure, the term "bone marrow mesenchymal stem cells" is used interchangeably with "bone marrow mesenchymal stromal cells", "bone marrow pluripotent stem cells", and "bone marrow pluripotent stromal cells".

[0056] "Bone marrow-derived mesenchymal stem cells" refer to bone marrow mesenchymal stem cells mobilized from the bone marrow to the outside of the bone marrow, and they are cells that can be obtained by peripheral blood sampling and collection from mesenchymal tissues such as fat, epithelial tissues such as skin, and nerve tissues such as brain. In the present disclosure, the term "bone marrow-derived mesenchymal stem cell" is used interchangeably with "bone marrow-derived mesenchymal stromal cell", "bone marrow-derived pluripotent stem cell", and "bone marrow-derived pluripotent stromal cell".

[0057] Bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells are also characterized in that they have a potency to differentiate into epithelial tissues such as keratinocytes that constitute skin and tissues of the nervous system that constitute brain, when administered to a damaged portion of the living body immediately after collection or after once being adhered onto a culture dish.

[0058] Bone marrow mesenchymal stem cells and bone marrow-derived mesenchymal stem cells preferably have the potency to differentiate into osteoblasts (identifiable by observing calcification or such when differentiation is induced), chondrocytes (identifiable by positive Alcian blue staining, positive Safranin O staining or such), adipocytes (identifiable by positive Sudan III staining, etc.), as well as mesenchymal cells such as fibroblasts, smooth muscle cells, skeletal muscle cells, stromal cells, and tendon cells, nerve cells, pigment cells, epidermal cells, hair follicle cells (expressing the cytokeratin family, hair keratin family or such), epithelial cells (e.g., epidermal keratinocytes and intestinal epithelial cells express the cytokeratin family or such), endothelial cells, and moreover, cells of parenchymal organs such as liver, kidney, and pancreas. However, the cells after differentiation are not limited to the above-mentioned cells.

[0059] Examples of markers for human mesenchymal stem cells include, but are not limited to, all or some of PDGFRα positive, PDGFRβ positive, Lin negative, CD45 negative, CD44 positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive, CD73 positive, CD90 positive, CD71 positive, Stro-1 positive, CD106 positive, CD166 positive, CD31 negative, CD271 positive, and CD11b negative.

[0060] Examples of markers for murine mesenchymal stem cells include, but are not limited to, all or some of CD44 positive, PDGFRα positive, PDGFRβ positive, CD45 negative, Lin negative, Sca-1 positive, c-kit negative, CD90 positive, CD105 positive, CD29 positive, Flk-1 negative, CD271 positive, and CD11b negative.

[0061] Examples of markers for rat mesenchymal stem cells include, but are not limited to, all or some of PDGFRα positive, CD44 positive, CD54 positive, CD73 positive, CD90 positive, CD105 positive, CD29 positive, CD271 positive, CD31 negative, and CD45 negative.

[0062] In the present disclosure, cells whose migration is stimulated by an HMGB1 fragment peptide also include, but are not limited to, PDGFRα-positive cells. Examples of PDGFRα-positive cells whose migration is stimulated by an HMGB1 fragment peptide include, but are not limited to, PDGFRα-positive mesenchymal stem cells, PDGFRα-positive bone marrow-derived mesenchymal stem cells, and PDGFRα-positive bone marrow-derived cells which are obtained as adherent cells by means of cell culture of a monocyte fraction of blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection. Examples of PDGFRα-positive mesenchymal stem cells include cells that are positive for PDGFRα and CD44, cells that are positive for PDGFRα and CD90, cells that are positive for PDGFRα and CD105, cells that are positive for PDGFRα and CD29, and such. In one aspect, the PDGFRα-positive mesenchymal stem cells may be cells that are negative for CD44.

[0063] In one embodiment of the pharmaceutical composition for use according to the present invention, a peptide that consist an amino acid sequence with one or two amino acid residues modified (substitutions, deletions, insertions, or additions) in the amino acid sequence described in SEQ ID NO: 1 and have an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells can be used instead of or in conjunction with the HMGB1 fragment peptide comprising the amino acid sequence described in SEQ ID NO: 1..

[0064] Examples of cells whose migration is stimulated by these peptides include, but are not limited to, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, PDGFRα-positive cells, PDGFRα-positive mesenchymal stem cells, PDGFRα-positive bone marrow-derived mesenchymal stem cells, and PDGFRα-positive bone marrow-derived cells which are obtained as adherent cells by means of cell culture of a monocyte fraction of blood obtained by bone marrow collection (bone marrow cell collection) or peripheral blood collection.

[0065] An effective amount of a peptide for use according to the invention or a pharmaceutical composition comprising the peptide for use according to the invention (hereafter referred to as a peptide or such) is for administration to a subject for the treatment of a disease or symptom described herein.

[0066] An effective amount as used herein refers to an amount sufficient for the treatment of a disease or symptom as described herein. Treatment in the present disclosure includes, but is not limited to, alleviation, delay, blockade, improvement, remission, cure, complete cure, and such.

[0067] Subjects in the present disclosure include, without limitation, mammals, birds, fish, and such. Mammals include, but are not limited to, humans and non-human animals, for example, humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, whales, and such. In the present disclosure, the term "subject" is used interchangeably with "patient", "individual", and "animal".

[0068] There is no restriction to the site of administration of the peptide or pharmaceutical composition for use according to the invention, and the peptides and pharmaceutical composition for use according to the invention can exert their effects when administered to any site, such as a site with structural or functional abnormality of a tissue or a site nearby, a site different from them (other than those sites), a site distant from a site with structural or functional abnormality of a tissue, a site distal from a site with structural or functional abnormality of a tissue, or a site distal and ectopic to a site with structural or functional abnormality of a tissue.

[0069] For example, by administration at or near a site with structural or functional abnormality of the heart, cells (e.g., mesenchymal stem cells) are recruited to the site of administration to induce or promote regeneration of the cardiac tissue or improvement of structural or functional abnormality of the heart. Also, for example, by administration to a site different from the site with structural or functional abnormality of the heart or its vicinity, bone marrow cells (e.g., bone marrow mesenchymal stem cells) are mobilized from the bone marrow to a site with structural or functional abnormality of the heart or its vicinity via peripheral circulation to induce or promote regeneration of the cardiac tissue or improvement of structural or functional abnormality of the heart. Here, "peripheral circulation" is also referred to as "blood circulation" and "peripheral circulatory blood flow".

[0070] The peptide or pharmaceutical composition for use according to the invention can also exert its effects when administered to any tissue, such as a tissue different from the cardiac tissue, a tissue distant from the cardiac tissue, a tissue distal from the cardiac tissue, or a tissue distal and ectopic to the cardiac tissue. Thus, the peptide or pharmaceutical composition for use according to the invention is effectively utilized to regenerate the cardiac tissue, which is difficult to be administered with a drug directly from outside the body, or to ameliorate structural or functional abnormalities of the heart.

[0071] Modes of administration include, but are not limited to, oral administration and parenteral administration including intravascular (intra-arterial, intravenous, and such), intramuscular, subcutaneous, intradermal, intraperitoneal, nasal, pulmonary, and transdermal administrations. Also, the peptide pharmaceutical composition for use according to the inventionmay be for systemic or local administration by injection (e.g., subcutaneously, intradermally, or to the skin surface, eyeball or palpebral conjunctiva, nasal mucosa, oral and gastrointestinal mucosa, vaginal and endometrial mucosa, or injured site), for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection.

[0072] In addition, in place of the peptide as such, cells secreting the peptides of the present disclosure, gene therapy vectors into which DNAs encoding the peptides have been inserted, and pharmaceutical compositions containing them can be used.

[0073] Further, modes of administration can be appropriately selected according to the age and symptoms of the patient. For administration of the peptide, for example, the dosage can be selected from the range of 0.0000001 mg to 1000 mg per kilogram of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.00001 to 100000 mg / body per patient. Also when a cell secreting the peptide for use according the present invention or a gene therapy vector into which a DNA encoding the peptide has been inserted, administration can be performed so that the amount of the peptide is within the above range. However, the pharmaceutical compositions for use according to the present invention is not limited to these doses.

[0074] Pharmaceutical compositions for use according to the invention can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.), and may contain pharmaceutically acceptable carriers and additives together. Examples include, but are not limited to, surfactants, excipients, coloring agents, perfumes, preservatives, stabilizers, buffers, suspending agents, isotonizing agents, binding agents, disintegrants, lubricants, fluidity-promoting agents, and flavoring agents, and other commonly used carriers can be used as appropriate. Specific examples include, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, cornstarch, inorganic salts.

[0075] The present invention is further illustrated by, but not limited to, the examples below.Examples[Example 1] Efficacy evaluation of an HMGB1 fragment peptide for dilated cardiomyopathy (1) Materials and methods

[0076] J2N-k hamsters (18-week-old, male, 20 animals in total), which are model animals of dilated cardiomyopathy, were obtained from Japan SLC, acclimated for 2 weeks, and then used for experiments. J2N-k hamsters spontaneously develop dilated cardiomyopathy due to deletion mutations in the δ-sarcoglycan gene (specifically, cardiomyocyte shedding and fibrosis begin around 5 weeks of age, cardiomegaly and cardiac dysfunction appear at approximately 20 weeks of age, and eventually they die of congestive heart failure at approximately 1 year. J Biochem. 2003 Aug;134(2):269-76). Also, a peptide consisting of amino acid residues 1-44 (SEQ ID NO: 1) of the human HMGB1 protein was chemically synthesized by a solid-phase method. Hereinafter, the peptide is referred to as the HMGB1 peptide (1-44), and is abbreviated as "1-44" in the drawing corresponding to the Examples.

[0077] J2N-k hamsters were divided into the HMGB1 peptide (1-44)-treated (n=10) and PBS-treated (control, n=10) groups, and treatment was begun at 20 weeks of age (body weight about 120 g). Administration of the test substance was carried out by injecting a solution of the HMGB1 peptide (1-44), which has been adjusted to a concentration of 1 mg / ml with PBS as the vehicle, into the external jugular vein at a dose of 3 ml / kg (3 mg / kg as the peptide dosage) once a day for four consecutive days. In the control group, PBS was injected into the external jugular vein at a dose of 3 ml / kg once a day for four consecutive days. At 6 weeks after administration, a thoracotomy was performed under deep anesthesia, and the heart was removed. The mid portion of the heart was divided for cryopreservation and for paraffin fixation, and a histopathological examination was performed. Molecular biological studies were also performed using apical myocardium to evaluate effects of administration of the HMGB1 peptide (1-44).(2) Items for evaluationi) Cardiac function

[0078] Echocardiography was performed prior to dosing, and at 4 and 6 weeks after dosing; and left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), and left ventricular ejection fraction (LVEF) were measured and calculated to perform a cardiac function assessment.ii) Myocardial fibrosis

[0079] Myocardial tissue sections were stained with Sirius red, and the proportion of stain-positive area in the entire myocardial area was calculated as the percentage of fibrosis (%).iii) Angiogenesis

[0080] Immunostaining of the myocardial tissue sections was performed using an anti-CD31 antibody to determine the number of blood vessels (number of vascular endothelial cells). This measurement was performed in five different fields and the mean value was calculated.iv) Hypertrophy of cardiomyocytes

[0081] Periodic Acid Schiff (PAS) staining of the myocardial tissue sections was performed to measure the short diameter of cardiomyocytes retaining a nuclear architecture, and the average of the measurements was obtained. This measurement was performed in five different fields and the mean value was calculated.v) Recruitment of mesenchymal stem cells

[0082] Immunostaining of the myocardial tissue sections was performed using antibodies against PDGFRα and CD29, which are surface markers of mesenchymal stem cells, to assess whether accumulation of mesenchymal stem cells was observed. Staining of the nuclei was performed using DAPI.vi) RT-PCR

[0083] The intramyocardial expression of VEGF, an angiogenic factor secreted by mesenchymal myeloid stem cells, and that of TSG-6, an anti-inflammatory cytokine, were assessed by RT-PCR using apical myocardium.vii) Mitochondrial structure

[0084] The structure of cardiomyocytes was observed by electron microscopy. Specifically, the full-thickness myocardium of the mid-portion was sampled, and subjected to pre-fixation with 1 / 2 Karnofsky, post-fixation with 2% osmium tetroxide, block-staining with 0.5% uranium acetate aqueous solution and dehydration with ethanol, and then embedded and polymerized by infiltrating epoxy resin (Quetol812). Ultrathin sections (70-110 nm) were made, and electron staining with uranium acetate and a lead stain solution was performed, and then observed by electron microscopy (H-7500, Hitachi High Technologies).(3) Resultsi) Cardiac function

[0085] Cardiac function assessments at 4 and 6 weeks after dosing showed significantly higher levels of LVEF in the HMGB1 peptide (1-44) group compared with the PBS group, and reduction in the left ventricular contractility was inhibited (FIG. 1). These results indicate the improving effect of HMGB1 peptide (1-44) on the cardiac function. There were no significant differences in LVDd and LVDs at any time point after 4 and 6 weeks of dosing.ii) Myocardial fibrosis

[0086] Analysis showed that the percentage of Sirius red stain-positive area was significantly smaller in the HMGB1 peptide (1-44) group than in the PBS group, indicating that myocardial fibrosis was inhibited (FIG. 2).iii) Angiogenesis

[0087] Immunostaining results showed that in the mid layer, the HMGB1 peptide (1-44) group had a significantly higher number of blood vessels and enhanced angiogenesis than the PBS group (FIG. 3). In the subendocardial layer also, the number of blood vessels tended to be higher in the HMGB1 peptide (1-44) group than in the PBS group.iv) Hypertrophy of cardiomyocytes

[0088] PAS-staining results showed that the short diameter of cardiomyocytes was significantly smaller in the HMGB1 peptide (1-44) group than in the PBS group in both the subendocardial layer and mid layer, indicating that hypertrophy of the cardiomyocytes was inhibited (FIG. 4).v) Recruitment of mesenchymal stem cells

[0089] Immunostaining revealed that cells positive for both PDGFRα and CD29 were recruited to the myocardial tissue of the HMGB1 peptide (1-44) group (FIG. 5). Also, significantly more PDGFRα-positive and CD29-positive cells were present in the myocardial tissue of the HMGB 1 peptide (1-44) group than in the PBS group (FIG. 6).vi) RT-PCR

[0090] Analysis showed that both VEGF and TSG-6 had significantly higher expression levels in the HMGB1 peptide (1-44) group than in the PBS group (FIG. 7).vii) Mitochondrial structure

[0091] Electron microscopic examination of cardiomyocytes revealed loss of the mitochondrial cristae structure in the PBS group, whereas the cristae structure was maintained in the HMGB 1 peptide (1-44) group (FIG. 8). These results support the effect of the HMGB1 peptide (1-44) administration in maintaining the cardiac function.[Example 2] Efficacy evaluation of the HMGB1 fragment peptide for dilated cardiomyopathy (long-term observations)(1) Materials and methods

[0092] As in Example 1, twenty J2N-k hamsters were prepared and divided into the HMGB1 peptide (1-44)-treated group (n=11) and the PBS-treated group (control, n=9), and treatment was begun at 20 weeks of age. Administration of the test substance was carried out by injecting a solution of the HMGB1 peptide (1-44), which has been adjusted to a concentration of 1 mg / ml with PBS as the vehicle, into the external jugular vein at a dose of 3 ml / kg (3 mg / kg as the peptide dosage) once daily for four consecutive days. In the control group, PBS was injected into the external jugular vein at a dose of 3 ml / kg once a day for four consecutive days. Evaluation of the cardiac function and survival rate was continued thereafter while rearing the hamsters under normal conditions.(2) Items for evaluationi) Cardiac function

[0093] Echocardiography was performed prior to dosing, at 4 weeks after dosing, and every 2 weeks thereafter to measure LVEF.ii) Survival rate

[0094] After the last dose, the animals were kept under normal conditions and the survival rate was evaluated.(3) Resultsi) Cardiac function

[0095] The LVEF of the HMGB1 peptide (1-44) group remained significantly higher than that of the PBS group until 6 weeks after administration (FIG. 9).ii) Survival rate

[0096] Long-term observations showed a tendency toward higher viability in the HMGB1 peptide (1-44) group (FIG. 10).[Example 3] Comparative example evaluating the efficacy of the HMGB1 fragment peptide on improving the cardiac function after old myocardial infarction(1) Materials and methods

[0097] SD rats (7-week-old, male, body weight of approximately 250g) were anesthetized with the inhalation anesthetic sevofrane (or isoflurane) and intubated endotracheally after adequate suppression was achieved, and deep anesthesia was maintained with the inhalation anesthetic. A thoracotomy was performed in the supine position at the left fourth intercostal space, and the proximal portion of the left anterior descending coronary artery was ligated with 6-0 prolene sutures to create a broad-area myocardial infarction model. The cardiac function was evaluated by echocardiography 2 weeks after the infarction was made, and cases in which extensive infarction was obtained (LVEF<50%) were used as old myocardial infarction model rats (17 animals in total). Similar to the above examples, the HMGB1 peptide (1-44) consisting of SEQ ID NO: 1 was used.

[0098] The old myocardial infarction model rats were divided into the HMGB1 peptide (1-44)-treated group (n=9) and the PBS-treated group (control, n=8), and treatment was begun 2 weeks after the infarction was made. Administration of the test substance was carried out by injecting a solution of the HMGB1 peptide (1-44), which has been adjusted to a concentration of 1 mg / ml with PBS as vehicle, into the femoral veins at a dose of 3 ml / kg (3 mg / kg as the peptide dosage) once daily for four consecutive days. In the control group, PBS was injected at a dose of 3 ml / kg once a day via the femoral vein for four consecutive days. At 4 weeks after dosing, the heart was removed by re-thoracotomy under deep anesthesia. At the time of removal, 5 ml or more blood was collected by puncture from the right atrium toward the inferior vena cava, and the infarction area of heart was cut into four short-axis slices of equal thickness and divided for cryopreservation and paraffin fixation. Both groups were comparatively studied using cardiac physiological, histopathological, and molecular biological techniques to evaluate the effects of administering the HMGB1 peptide (1-44). The condition after 2 weeks (14 days) or more have passed following the onset of myocardial infarction in model rats is equivalent to the condition after 30 days or more have passed following the onset of myocardial infarction in humans. In addition, the old myocardial infarction model rats prepared in this study can be regarded as a model of ischemic cardiomyopathy because the LVEF was decreased to 42% at the time point prior to dosing, and the progression of cardiomegaly was observed in the control group.(2) Items for evaluationi) Cardiac function

[0099] Echocardiography was performed at 1, 2, and 4 weeks after dosing, and the left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), and left ventricular ejection fraction (LVEF) were measured and calculated to perform a cardiac function assessment.ii) Myocardial fibrosis

[0100] Myocardial tissue sections were stained with Sirius red, and the proportion of stain-positive area in the total area of the left ventricular myocardium was calculated as the percentage of fibrosis (%).iii) Angiogenesis

[0101] Von-Willebrand factors were stained at the infarct border to determine the number of blood vessels (number of vascular endothelial cells). This measurement was performed in 10 different fields and the mean value was calculated.iv) Hypertrophy of cardiomyocytes

[0102] Periodic Acid Schiff (PAS) staining was performed at the infarct border, and the short diameter of cardiomyocytes retaining a nuclear architecture at the infarct border was measured, and the average of the measurements was obtained. This measurement was performed in 10 different fields and the mean value was calculated.v) Recruitment of mesenchymal stem cells at the infarct boundary

[0103] Immunostaining of the myocardial tissue was performed using antibodies against PDGFRα, CD90, and CD105, which are surface markers of mesenchymal stem cells, to evaluate whether accumulation of mesenchymal stem cells was observed at the infarct border. Staining of the nuclei was performed using DAPI.(3) Resultsi) Cardiac function

[0104] Cardiac function assessment at 4 weeks after dosing showed significantly higher levels of LVEF in the HMGB1 peptide (1-44) group compared to the PBS group (FIG. 11). In addition, LVDd and LVDs at 4 weeks after dosing showed smaller values in the HMGB1 peptide (1-44) group than in the PBS group, and inhibition of cardiomegaly was observed (FIG. 12).

[0105] In addition, with respect to the change in LVEF over time, the PBS group showed a decrease in LVEF over time after dosing, whereas the HMGB1 peptide (1-44) group showed an increase in LVEF over time up to 4 weeks after dosing, with an average improvement in LVEF of about 7% with respect to before the dosing (FIG. 13).ii) Myocardial fibrosis

[0106] Analysis showed that the percentage of Sirius red stain-positive area was significantly smaller in the HMGB1 peptide (1-44) group than in the PBS group, indicating that myocardial fibrosis was inhibited (FIG. 14).iii) Angiogenesis

[0107] Von-Willebrand factor-staining results at the infarct border showed that the HMGB1 peptide (1-44) group had significantly more vessels than the PBS group (P = 0.05) indicating that angiogenesis was enhanced (FIG. 15).iv) Hypertrophy of cardiomyocytes

[0108] The results of PAS staining at the infarct border showed that the short diameter of cardiomyocytes was significantly smaller in the HMGB1 peptide (1-44) group than in the PBS group (P = 0.05), indicating that the hypertrophy of cardiomyocytes was inhibited (FIG. 16).v) Recruitment of mesenchymal stem cells at the infarct border

[0109] Immunostaining of the myocardial tissue revealed that cells positive for both PDGFRα and CD90 and cells positive for both PDGFRα and CD105 were recruited near the infarct border of the HMGB1 peptide (1-44) group (FIGS. 17 and 18). There were also more PDGFRα-positive and CD90-positive cells in the vicinity of the infarct border in the HMGB1 peptide (1-44) group than in the PBS group (FIG. 19).[Example 4] Efficacy evaluation of the HMGB1 fragment peptide for hypertensive cardiomyopathy(1) Materials and methods

[0110] The Dahl salt-sensitive rats (hereafter also referred to as DIS / Eis rats) were used in the experiments. DIS / Eis rats are hypertensive when fed with a high-salt diet and are a model that develop heart failure at a high incidence (e.g., when fed with a diet supplemented with 8% NaCl from 6 weeks of age, blood pressure reaches 250 mm Hg at 9 weeks of age, death begin to appear from 12 weeks of age, and death is highly probable by 16 weeks of age).

[0111] Ten DIS / Eis rats (6-weeks-old, male, body weight about 200g, Japan SLC) were prepared and divided into two groups, a high-salinity diet group (n=7) and a low-salinity diet group (n=3), and feeding was continued until 15 weeks of age in which the former was fed with a diet supplemented with 8% NaCl and the latter was fed with a diet supplemented with 0.3% NaCl. Aside from the meals, nothing interfered with free movement and they were kept clean and rested without restriction on drinking water, feeding, and such in their cages.

[0112] At 11 weeks of age, the high-salinity diet group (n=7) was divided into the HMGB1 peptide (1-44)-treated group (n=3) and the control group (n=4), and treatment was initiated. Administration of the test substance was carried out by injecting a solution of the HMGB 1 peptide (1-44), which has been adjusted to a concentration of 1 mg / ml with PBS as the vehicle, into the tail vein at a dose of 3 ml / kg (3 mg / kg as the peptide dosage) once daily for four consecutive days. The control group was injected with PBS at a dose of 3 ml / kg once a day via the tail vein for four consecutive days. Similar to the control group, the low-salinity diet group (n = 3) also received PBS via the tail vein for four consecutive days. At 4 weeks after dosing (at 15 weeks of age), hearts were removed from the rats by thoracotomy under general anesthesia, and their weight was measured, and then histological sections of the left ventricular myocardial cross sections were prepared at the papillary muscle level and subjected to histological analysis. In the following description, the HMGB1 peptide (1-44) administration group fed with a high-salinity diet is abbreviated as the "High-1-44 group"; the control group fed with a high-salinity diet is abbreviated as the "High-PBS group" and the low-salinity diet group is abbreviated as the "Low-PBS group".(2) Items for evaluationi) Heart weight

[0113] At 15 weeks of age, hearts were removed from rats in each group and weighed.ii) Cardiac function and ventricular wall thickness

[0114] Echocardiography was performed prior to dosing and every week after initiation of dosing to determine the left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs), left ventricular ejection fraction (LVEF), end-diastolic left ventricular anterior wall thickness (LVAWd), and end-diastolic left ventricular posterior wall thickness (LVPWd).iii) Myocardial fibrosis

[0115] Myocardial tissue sections were stained with Sirius red, and the proportion of stain-positive area in the area of the whole heart or the left ventricle was calculated as the percentage of fibrosis (%).(3) Resultsi) Heart weight

[0116] Compared with the Low-PBS group, the High-1-44 and High-PBS groups had greater heart weights. Further, the High-1-44 group had smaller heart weights than the High-PBS group, indicating that administration of the HMGB1 peptide (1-44) inhibited cardiac hypertrophy (FIG. 20).ii) Cardiac function

[0117] During the observation period, LVEF remained at a value of 70% or more in all groups (no significant differences between the groups).iii) Ventricular wall thickness

[0118] The changes in LVAWd during the observation period are shown in FIG. 21. At 15 weeks of age, the LVAWd of the High-PBS and High-1-44 groups were greater than that of the Low-PBS group, but the LVAWd of the High-1-44 group was smaller than that of the High-PBS group, showing the tendency for the increase of the left ventricular anterior wall thickness to be inhibited by administration of the HMGB1 peptide (1-44).iv) Myocardial fibrosis

[0119] The results of Sirius red staining of the myocardial tissue sections are shown in FIG. 22. Compared with the Low-PBS group, the High-PBS group had increased fibrosis around the coronary arteries. The percentage of Sirius red-stained positive area (percentage of fibrosis) in the whole heart was smaller in the High-1-44 group than in the High-PBS group, showing the tendency for fibrosis of the myocardium to be inhibited by the administration of the HMGB1 peptide (1-44) (FIG. 23A). The percentage of fibrosis in the left ventricle was smaller in the High-1-44 group than in the High-PBS group, indicating that myocardial fibrosis was inhibited by the administration of the HMGB1 peptide (1-44) (FIG. 23B).(4) Discussion

[0120] DIS / Eis rats fed with a high-salinity diet develop hypertension. It was also confirmed that the rats actually developed cardiac hypertrophy and cardiomyocyte hypertrophy. Therefore, the high-salinity diet-fed DIS / Eis rats used in the examples can be evaluated as a model of hypertensive cardiomyopathy.

[0121] Cardiac hypertrophy / cardiomyocyte hypertrophy is a structural abnormality that results in a reduced diastolic capacity of the heart. The high-salinity diet-fed DIS / Eis rats used in the present examples can also be evaluated as an HFpEF model because they developed cardiac hypertrophy / cardiomyocyte hypertrophy while maintaining normal left ventricular contractility.

[0122] The HMGB1 peptide (1-44) of the present invention inhibits cardiac hypertrophy and cardiomyocyte hypertrophy in high-salinity diet-fed DIS / Eis rats and thus can be used for the prevention and / or treatment of hypertensive cardiomyopathy and HFpEF.Industrial Applicability

[0123] Pharmaceutical composition for use according to the invention and the substance for use according to the invention are expected to provide great benefits to patients, who are difficult to be operated due to factors such as advanced age, and who cannot receive a sufficient effect with existing drugs for chronic heart failure. In addition, the pharmaceutical compositions comprising the peptide exert effects such as improvement of the cardiac function and inhibition of cardiomyocyte hypertrophy in multiple cardiomyopathy models that are dilated cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy, and thus they can be expected to exhibit broad therapeutic effects on various types of cardiomyopathy including idiopathic and secondary cardiomyopathies.

Claims

1. A pharmaceutical composition for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the pharmaceutical composition comprises the substance described in any of (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

2. The pharmaceutical composition for use according to claim 1, which is for use in treating cardiomyopathy.

3. The pharmaceutical composition for use according to claim 1, which is for use in treating a chronic heart failure caused by cardiomyopathy.

4. The pharmaceutical composition for use according to claim 1, which is for use in inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy.

5. The pharmaceutical composition for use according to any one of claims 1-4, wherein the cardiomyopathy is a secondary cardiomyopathy.

6. The pharmaceutical composition for use according to claim 5, wherein the secondary cardiomyopathy is selected from the group consisting of ischemic cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, drug-induced cardiomyopathy, alcoholic cardiomyopathy, mitochondrial cardiomyopathy, cardiomyopathy caused by cardiac sarcoidosis, cardiomyopathy caused by cardiac amyloidosis, cardiomyopathy caused by myocarditis, cardiomyopathy caused by muscular dystrophy, cardiomyopathy caused by cardiac Fabry's disease, and peripartum cardiomyopathy.

7. The pharmaceutical composition for use according to any one of claims 1-4, wherein the cardiomyopathy is idiopathic cardiomyopathy.

8. The pharmaceutical composition for use according to claim 7, wherein the idiopathic cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy.

9. The pharmaceutical composition for use according to any one of claims 1-4, wherein the cardiomyopathy is dilated cardiomyopathy, ischemic cardiomyopathy, or hypertensive cardiomyopathy.

10. The pharmaceutical composition for use according to any one of claims 1-9, wherein the peptide is selected from (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one amino acid has been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

11. The pharmaceutical composition for use according to any one of claims 1-9, wherein the peptide is a peptide consisting of the amino acid sequence described in SEQ ID NO: 1.

12. A substance for use in (1) treating cardiomyopathy, (2) treating a chronic heart failure caused by cardiomyopathy, or (3) inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy, wherein the substance is selected from (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

13. The substance for use according to claim 12, which is for use in treating cardiomyopathy.

14. The substance for use according to claim 12, which is for use in treating a chronic heart failure caused by cardiomyopathy.

15. The substance for use according to claim 12, which is for use in inhibiting a structural abnormality of the heart selected from the group consisting of cardiomegaly, cardiomyocyte hypertrophy, or myocardial fibrosis in a patient with cardiomyopathy.

16. The substance for use according to any one of claims 12-15, wherein the cardiomyopathy is a secondary cardiomyopathy.

17. The substance for use according to claim 16, wherein the secondary cardiomyopathy is selected from the group consisting of ischemic cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, drug-induced cardiomyopathy, alcoholic cardiomyopathy, mitochondrial cardiomyopathy, cardiomyopathy caused by cardiac sarcoidosis, cardiomyopathy caused by cardiac amyloidosis, cardiomyopathy caused by myocarditis, cardiomyopathy caused by muscular dystrophy, cardiomyopathy caused by cardiac Fabry's disease, and peripartum cardiomyopathy.

18. The substance for use according to any one of claims 12-15, wherein the cardiomyopathy is idiopathic cardiomyopathy.

19. The substance for use according to claim 18, wherein the idiopathic cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy.

20. The substance for use according to any one of claims 12-15, wherein the cardiomyopathy is dilated cardiomyopathy, ischemic cardiomyopathy, or hypertensive cardiomyopathy.

21. The substance for use according to any one of claims 12-20, wherein the peptide is selected from (a) or (b) below: (a) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (b) a peptide consisting of an amino acid sequence in which one amino acid has been substituted, deleted, inserted, or added in the amino acid sequence described in SEQ ID NO: 1 and having an activity of stimulating migration of PDGFRα-positive and CD29-positive mesenchymal stem cells.

22. The substance for use according to any one of claims 12-20, wherein the peptide is a peptide consisting of the amino acid sequence described in SEQ ID NO: 1.