WO2012172887A1 - Therapeutic agent for heart diseases and cell sheet for treating heart diseases - Google Patents

Abstract

It was discovered that a peptide having an amino acid sequence represented by formula (I), (II), (III), or (IV) has a cardiac function-improving effect. The peptide or a pharmaceutically acceptable salt thereof is useful as an active ingredient of a therapeutic agent for heart diseases. Further, when a cell sheet for treating heart diseases, which secretes the peptide, is transplanted into the heart suffering from a heart disease, the peptide can be allowed to act on the target site for a long time. X₁-X₂-Val-Tyr-X₅-X₆ (I) X₂-Val-Tyr-X₅-X₆-X₇ (II) Ser-X₂-X₃-(Tyr/Phe/Trp)-X₅-X₆ (III) X₂-X₃-(Tyr/Phe/Trp)-X₅-X₆-Arg (IV) In formulae (I), (II), (III), and (IV), X₁, X₂, X₃, X₅, X₆, and X₇ are the same or different and represent an arbitrary amino acid residue.

Description

Heart disease therapeutic agent and heart disease cell sheet

The present invention relates to a heart disease therapeutic drug and a cell sheet for heart disease treatment.

Cardiovascular disease is one of the top causes of death in the world, and the number of patients is expected to increase in the future. Heart failure, a condition in which the heart's pumping function is compromised and cannot pump enough blood to the body, is caused by various heart conditions. And the incidence and prevalence are increasing year by year with aging. Despite advances in drug treatment and surgical treatment, the prognosis for heart failure patients remains severe, and the development of new therapies is awaited. Myocardial infarction, which is a type of ischemic heart disease, is a disease in which the blood flow in the coronary artery that nourishes the myocardium is reduced or interrupted for a certain period of time and the myocardium in the perfusion region falls into necrosis. The necrotic part is eventually replaced with scar tissue, but the scar part has no contractile force and gradually deteriorates cardiac function. In recent years, research on new therapeutic methods using cell transplantation, gene therapy, cytokines, and angiogenic factors has been promoted for such pathological conditions.

However, growth factors such as HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) conventionally used for research and treatment are extracted proteins and recombinant proteins consisting of several hundred amino acids. There are problems that cannot be imagined in terms of side effects. On the other hand, peptides having about 10 amino acids linked are less susceptible to side effects in terms of antigenicity compared to various growth factors, are highly safe, are easily metabolized, and have a simple and highly efficient peptide design. There is an advantage that a synthesis method and a test method are established. Therefore, discovery and development of peptides having the ability to improve cardiac function are expected.

The present inventors have clarified that a peptide consisting of 7 amino acids (SVVYGLR) present in osteopontin (OPN), which is a kind of extracellular matrix, has an angiogenic action. It has been found that it is as high as VEGF, which plays a central role in angiogenic factors (see Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2). Further, the present inventors have found that the peptide has a mesenchymal cell proliferation promoting action (see Patent Document 2). However, it is not known that the peptide has an ability to improve cardiac function.

International Publication No. WO2003 / 030925 International Publication No. WO2008 / 026634

An object of the present invention is to find a peptide having an ability to improve cardiac function, provide a therapeutic agent for heart disease containing this as an active ingredient, and a cell sheet for treating cardiac disease that secretes the peptide.

The present invention includes the following inventions in order to solve the above problems.
[1] A peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having a cardiac function improving action, or a pharmaceutically acceptable salt thereof is effective. A therapeutic agent for heart diseases contained as an ingredient.
X ₁ -X ₂ -Val-Tyr-X ₅ -X ₆ (I)
(Wherein X ₁ , X ₂ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -Val-Tyr-X ₅ -X ₆ -X ₇ (II)
(Wherein X ₂ , X ₅ , X ₆ and X ₇ are the same or different and represent any amino acid residue.)
Ser-X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ (III)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ -Arg (IV)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
[2] The amino acid sequence represented by the formula (I), (II), (III) or (IV) is the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, Heart disease remedy.
[3] The above [1] or [2], wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 7, or a peptide having the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 ] The therapeutic agent for heart diseases according to the above.
[4] The therapeutic agent for heart disease according to any one of [1] to [3], wherein the total number of amino acid residues of the peptide is 50 or less.
[5] The therapeutic agent for heart disease according to any one of the above [1] to [4], which comprises cells that secrete the peptide.
[6] The therapeutic agent for heart diseases according to any one of [1] to [4], which comprises a cell sheet that secretes the peptide.
[7] The therapeutic agent for heart disease according to any one of [1] to [6], wherein the heart disease is ischemic heart disease or cardiomyopathy.
[8] For the treatment of heart disease, characterized by secreting a peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having an effect of improving cardiac function Cell sheet.
X ₁ -X ₂ -Val-Tyr-X ₅ -X ₆ (I)
(Wherein X ₁ , X ₂ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -Val-Tyr-X ₅ -X ₆ -X ₇ (II)
(Wherein X ₂ , X ₅ , X ₆ and X ₇ are the same or different and represent any amino acid residue.)
Ser-X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ (III)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ -Arg (IV)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
[9] The amino acid sequence represented by the formula (I), (II), (III) or (IV) is the amino acid sequence represented by any one of SEQ ID NOs: 1 to 6, Cell sheet.
[10] The cell sheet according to [8] or [9], wherein the peptide includes an amino acid sequence of a secretory signal peptide.
[11] The cell sheet according to [10], wherein the peptide is a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and the amino acid sequence of a secretory signal peptide.
[12] The cell sheet according to any one of [8] to [11], wherein the cells are myoblasts, smooth muscle cells, mesenchymal cells, or adipocytes.
[13] The cell sheet according to any one of [8] to [12], wherein the heart disease is ischemic heart disease or cardiomyopathy.

According to the present invention, it is possible to provide a therapeutic agent for heart disease containing a peptide having an ability to improve cardiac function as an active ingredient and a cell sheet for treating heart disease that secretes the peptide. Since the active ingredient is a peptide, the therapeutic agent for heart disease of the present invention has the advantages that side effects hardly occur from the viewpoint of antigenicity and safety is high. When the cell sheet for treating heart disease of the present invention is transplanted to a heart that has developed heart disease, the peptide that is an active ingredient of the therapeutic agent for heart disease of the present invention can be allowed to act on the target site for a long period of time.

It is a figure which shows the result of having evaluated the left ventricular diameter shortening rate (% FS), ejection fraction (EF), and left ventricular cavity area change rate (FAC) of the 3rd, 6th, and 9th week after operation. It is a figure which shows the hematoxylin eosin dyeing | staining image and sirius red dyeing | staining image of the left ventricle of the heart extract | collected 3 weeks after the operation. It is a figure which shows the result of having evaluated the myocardial fibrosis rate of the myocardial infarction peripheral part of the 3rd, 6th, and 9th week after an operation, (A) is a Sirius red dyeing | staining image of a myocardium, (B) is a myocardial fibrosis rate. It is a figure which shows the analysis result. It is a figure which shows the result of having measured the myocardial cell lateral diameter of the peripheral part of myocardial infarction of 3, 6, and 9 weeks after an operation, (A) is a PAS dyeing | staining image, (B) is a measurement result of myocardial cell lateral diameter FIG. It is a figure which shows the result of having measured the capillary density of the myocardial infarction peripheral part of the 3rd, 6th, and 9th week after an operation, (A) is an immunohistochemical dyeing | staining image using the antibody with respect to Von Willbrand Factor, (B ) Is a diagram showing the measurement results of capillary density. It is the chart which tested the synthesized peptide with the high performance liquid chromatograph mass spectrometer. It is the photograph of the isolated myoblast, The left is a phase-contrast microscope image, The right is a fluorescence microscope image. It is a figure which shows the result of having confirmed the mRNA expression of the target peptide in the myoblast which infected the lentiviral vector for peptide expression. It is a figure which shows the result of having confirmed the expression and secretion of the target peptide in the myoblast which infected the lentiviral vector for peptide expression, (A) is the result of the dot blotting which serially diluted the peptide of known concentration, ( B) is the result of dot blotting of the cell culture supernatant infected with the lentiviral vector for peptide expression, and (C) is a table showing the density of (B) in numerical form. It is a figure which shows the left ventricular ejection fraction (EF) of 2, 4, 6 and 8 weeks after cell sheet transplantation. It is a figure which shows the left ventricular diameter shortening rate (% FS) of 2, 4, 6 and 8 weeks after cell sheet transplantation. It is a figure which shows the left ventricular end-diastolic volume (EDV) 8 weeks after a cell sheet transplant. It is a figure which shows the left chamber diameter shortening rate (% FS) of the 8th week after cell sheet transplantation. It is a figure which shows the heart weight ratio (HW / BW) of the 8th week after cell sheet transplantation. It is a figure which shows the result of having evaluated the left ventricle wall thickness of the infarcted part of the left ventricular cavity at 8 weeks after cell sheet transplantation, (A) is a Masson trichrome stained image of the heart, and (B) is the left ventricular cavity. The figure which shows the evaluation result of the diameter of this, (C) is a figure which shows the evaluation result of the left ventricle wall thickness of an infarction part. It is a figure which shows the result of having evaluated the myocardial fibrosis rate of the myocardial infarction periphery 8 weeks after cell sheet transplantation, (A) is a Sirius red dyeing | staining image of a myocardium, (B) is an analysis of myocardial fibrosis rate It is a figure which shows a result. It is a figure which shows the result of having measured the myocardial cell horizontal diameter of the myocardial infarction periphery 8 weeks after a cell sheet transplant, (A) is a PAS dyeing | staining image, (B) shows the measurement result of myocardial cell horizontal diameter. FIG. It is a figure which shows the result of having counted the number of capillaries around myocardial infarction 8 weeks after cell sheet transplantation, (A) is an immunohistochemical staining image using an antibody against Von WillbrandｂFactor, (B) It is a figure which shows the measurement result of capillary blood vessel density. It is a figure which shows the result of having examined the distribution of the smooth muscle actin (SMA) positive cell in the ventricle 8 weeks after cell sheet transplantation.

[Cardiac disease treatment]
The therapeutic agent for heart disease according to the present invention contains a peptide having a cardiac function improving action or a pharmaceutically acceptable salt thereof as an active ingredient. Examples of such a peptide include peptides having an amino acid sequence represented by the following formula (I), (II), (III) or (IV).
X ₁ -X ₂ -Val-Tyr-X ₅ -X ₆ (I)
(Wherein X ₁ , X ₂ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -Val-Tyr-X ₅ -X ₆ -X ₇ (II)
(Wherein X ₂ , X ₅ , X ₆ and X ₇ are the same or different and represent any amino acid residue.)
Ser-X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ (III)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ -Arg (IV)
(Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
In the formulas (III) and (IV), (Tyr / Phe / Trp) means Tyr, Phe or Trp.

In formulas (I) to (IV), X ₁ is not particularly limited, but for example, serine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable. X ₂ is not particularly limited, and for example, valine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable. Although X ₃ is not particularly limited, for example, valine, alanine, arginine, lysine, histidine, tryptophan, phenylalanine is preferred. X ₅ is not particularly limited, and for example, glycine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable. X ₆ is not particularly limited, and for example, leucine, alanine, arginine, lysine, histidine, tryptophan, and phenylalanine are preferable. X ₇ is not particularly limited, and for example, arginine, alanine, lysine, histidine, tryptophan, and phenylalanine are preferable.

As the amino acid sequences represented by the formulas (I) to (IV), the following sequences of SEQ ID NOs: 1 to 6 are preferable.
Ser-Val-Val-Tyr-Gly-Leu (SEQ ID NO: 1)
Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 2)
Ser-Val-Val-Phe-Gly-Leu (SEQ ID NO: 3)
Val-Val-Phe-Gly-Leu-Arg (SEQ ID NO: 4)
Ser-Val-Val-Trp-Gly-Leu (SEQ ID NO: 5)
Val-Val-Trp-Gly-Leu-Arg (SEQ ID NO: 6)

The peptide as an active ingredient of the therapeutic agent for heart disease of the present invention may be any peptide having any one of the amino acid sequences represented by the above formulas (I) to (IV), and has an amino acid sequence other than these. May be. The size of the peptide serving as the active ingredient of the therapeutic agent for heart disease of the present invention is not particularly limited, but the total number of amino acid residues is about 50 or less from the viewpoint of side effects such as ease of handling, production efficiency, and antigenicity. Is preferred. More preferably, it is about 30 residues or less, More preferably, it is about 20 residues or less, Most preferably, it is about 10 residues or less. The lower limit is 6 residues, preferably 7 residues or more.

The peptide serving as the active ingredient of the therapeutic agent for heart disease of the present invention has the amino acid sequence represented by SEQ ID NO: 1-6 or the amino acid sequence represented by SEQ ID NO: 7-9 below, and the total number of amino acid residues. Less than 20 peptides are preferred.
Ser-Val-Val-Tyr-Gly-Leu-Arg (SEQ ID NO: 7)
Ser-Val-Val-Phe-Gly-Leu-Arg (SEQ ID NO: 8)
Ser-Val-Val-Trp-Gly-Leu-Arg (SEQ ID NO: 9)
Among these, a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and having a total number of amino acid residues of 20 or less is more preferable, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 is further preferable.

The peptide of the present invention can be produced by a solid phase synthesis method (Fmoc method, Boc method) or a liquid phase synthesis method according to a known general peptide synthesis protocol. Moreover, it can manufacture using the transformant which introduce | transduced the expression vector containing DNA which codes the peptide of this invention.

The fact that the obtained peptide has a cardiac function improving action can be confirmed, for example, by evaluating using a heart disease model rat as shown in the Examples. Specifically, it can be confirmed by administering a peptide to a heart disease model rat, recording an echocardiogram after a certain period, and evaluating cardiac function. The cardiac function to be evaluated is not particularly limited. For example, the left ventricular diameter shortening rate (% fractional shortening;% FS), left ventricular ejection fraction (EF), left ventricular end-diastolic volume (LV end-diastolic volume; EDV), left ventricular end systolic volume (LV ESV), and the like. The evaluation time after administration is not particularly limited, but is preferably after about 3 weeks, more preferably after about 4 weeks, and even more preferably after about 5 weeks. If the cardiac function of the individual administered with the peptide is improved compared to the cardiac function of the individual not administered with the peptide, it can be determined that the peptide has a cardiac function improving action. In addition, for example, cardiomyocytes are isolated from newborn rats or adult rats, peptides are added to the isolated cardiomyocytes, and changes in cell morphology, protein expression level such as hypertrophy markers, cardiomyocyte viability, etc. are determined in vitro. This can be confirmed by evaluating with

The amino acid constituting the peptide of the present invention may be one in which the side chain is modified with an arbitrary substituent. Although a substituent is not specifically limited, For example, a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl group, a cycloalkyl group, an alkoxy group, an amino group etc. are mentioned. Preferably, the benzene ring of tryptophan or phenylalanine is modified with a substituent, and more preferably, the benzene ring of tryptophan or phenylalanine in the amino acid sequences represented by SEQ ID NOs: 1 to 9 is modified with a substituent. It is that.

In the peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl, α - _{C 6-12} aryl group such as naphthyl, for example, benzyl, _{C 7 - 14} aralkyl such as α- naphthyl _{-C 1-2} alkyl group such as a phenyl _{-C 1-2} alkyl or α- naphthylmethyl such phenethyl In addition to the group, a pivaloyloxymethyl group, which is widely used as an oral ester, can be mentioned. When the peptide of the present invention has a carboxyl group or a carboxylate other than the C-terminus, those in which these groups are amidated or esterified are also included in the peptide of the present invention.

Further, in the peptide of the present invention, the amino group of the N-terminal methionine residue is protected with a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl). N-terminal side cleaved in vivo, glutamyl group produced by pyroglutamine oxidation, substituent on amino acid side chain in molecule (for example, —OH, —SH, amino group, imidazole group, indole group) And a guanidino group) are protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl).

The peptide of the present invention may form a pharmaceutically acceptable salt. Examples of the salt include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, Salts with acids such as citric acid, oleic acid and palmitic acid; salts with alkali metals or alkaline earth metals such as sodium, potassium and calcium; or salts with aluminum hydroxides or carbonates; triethylamine, benzylamine , Salts with diethanolamine, t-butylamine, dicyclohexylamine, arginine and the like.

The heart disease to be treated is not particularly limited as long as it is a heart disease that exhibits a therapeutic effect by improving cardiac function. Specifically, ischemic heart disease (myocardial infarction, angina pectoris, etc.), cardiomyopathy (hypertrophic cardiomyopathy, secondary myocardial hypertrophy, dilated cardiomyopathy, restricted cardiomyopathy, etc.), myocarditis, heart failure, Endocarditis (such as bacterial endocarditis), valvular heart disease (such as mitral regurgitation), pericarditis (such as acute pericarditis, chronic constrictive pericarditis), congenital heart disease (atrium) Septal defect, ventricular septal defect, etc.), cardiac asthma, pulmonary heart and the like. Preferred are ischemic heart disease, cardiomyopathy and heart failure, more preferred are cardiomyopathy and heart failure.

The therapeutic agent for heart disease of the present invention can be formulated by using the above-mentioned peptide having a cardiac function-improving action as an active ingredient and appropriately blending a pharmaceutically acceptable carrier or additive. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions; parenterals such as injections, infusions, suppositories, ointments, patches, etc. can do. What is necessary is just to set suitably about the mixture ratio of a carrier or an additive based on the range normally employ | adopted in the pharmaceutical field | area. Carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorption Various additives such as an accelerator, a lubricant, a colorant, a corrigent, and a fragrance are included.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous liquid for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) are used. For example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage You may mix | blend with an agent (for example, benzyl alcohol, phenol, etc.), antioxidant, etc.

In addition, the therapeutic agent for heart disease of the present invention may be in a form in which a peptide having an effect of improving cardiac function as an active ingredient is bound to a carrier. The carrier is not particularly limited, and examples thereof include resins used for artificial organs, biopolymers such as proteins, and the like. Furthermore, the therapeutic agent for heart disease of the present invention may be in a form containing cells that secrete peptides having an effect of improving cardiac function of active ingredients. Preferably, it is a form including a cell sheet that secretes a peptide having an effect of improving cardiac function of an active ingredient. The cells and the cell sheet will be described later.

Since the preparation thus obtained is safe and has low toxicity, for example, it is administered to humans and other mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) can do.
Although the dose varies depending on the administration subject, target disease, administration route, etc., for example, when the therapeutic agent for heart disease of the present invention is orally administered for the purpose of treating cardiomyopathy, generally in an adult (with a body weight of 60 kg). Administers about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg of active ingredient per day. When administered parenterally, the single dose of the active ingredient varies depending on the administration subject, the target disease, etc. For example, for the purpose of treating cardiomyopathy, the therapeutic agent for heart disease of the present invention is usually in the form of injection. When administered to adults (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the active ingredient is administered by intravenous injection per day. Is preferred.

The present invention further includes the following inventions.
(A) a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) and having a cardiac function-improving action for a mammal, or a pharmaceutically A method of treating heart disease, comprising administering an effective amount of an acceptable salt.
(B) a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) for producing a therapeutic agent for heart disease, and having a cardiac function improving action, or Use of a pharmaceutically acceptable salt thereof.
(C) a peptide having an amino acid sequence represented by the above formula (I), (II), (III) or (IV) and having a cardiac function-improving action, for use in the treatment of heart disease, or Its pharmaceutically acceptable salt.

It has been demonstrated that the therapeutic agent for heart disease of the present invention maintains improvement in cardiac function even in the 9th week after single administration around the infarcted myocardium in myocardial infarction model rats (see Example 1). . On the other hand, angiogenesis therapy for inducing new blood vessels by introducing VEGF, HGF and bFGF (basic fibroblast growth factor), which are known angiogenesis-promoting factors for ischemic heart disease, has been constructed (Rissanen TT , Et al., Adv Genet. 117-167, 2004, Miyagawa S, et al., Transplantation. 81: 902-907, 2006, Rissanen TT, et al., FASEB. 10, 2002) Although the improvement of this is recognized, it has not yet reached the point where it can sufficiently improve the cardiac function in the long term. Therefore, the therapeutic agent for heart diseases of the present invention is a therapeutic agent for cardiac diseases superior to known angiogenesis-promoting factors such as VEGF, HGF, bFGF, etc. in that the effect of improving cardiac function is maintained for a long time.

[Cell sheet for heart disease treatment]
The cell sheet for treating heart disease of the present invention is characterized by secreting a peptide having a cardiac function improving action, which is an active ingredient of the above-mentioned therapeutic agent for heart disease of the present invention. Peptides having a cardiac function improving action are as described above, and include peptides having an amino acid sequence represented by the above formula (I), (II), (III) or (IV). Preferred examples of the amino acid sequence represented by (IV) include the sequences of SEQ ID NOs: 1 to 6.

The peptide having a cardiac function improving action secreted from the cell sheet for treating heart disease of the present invention preferably contains the amino acid sequence of a secretory signal peptide. The secretory signal peptide means a peptide region that plays a role of allowing a protein or peptide bound to the secretory signal peptide to permeate through the cell membrane. Therefore, by including the amino acid sequence of the secretory signal peptide, it is possible to secrete a peptide having an effect of improving cardiac function out of the cell. The amino acid sequences of such secretory signal peptides and the nucleic acid sequences encoding them are well known and reported in the art (eg, von Heijine G (1988) Biochim. Biohys. Acra 947: 307-333, von Heijine G (1990) J. Membr. Biol. 115: 195-201). The amino acid sequence of the secretory signal peptide and the nucleic acid sequence encoding it can be obtained from a known database (DDBJ / GenBank / EMBL, etc.). The secretory signal peptide suitable for the present invention is a secretory signal peptide that can function in mammalian cells, particularly human cells. For example, an Igκ chain leader sequence, a honey bee melittin signal sequence, an α factor secretion signal, and the like. The amino acid arrangement of the secretory signal peptide is preferably arranged on the N-terminal side of the peptide having a cardiac function improving action.

The peptide secreted from the cell sheet for treating heart disease of the present invention is preferably a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and the amino acid sequence of the secretory signal peptide. The total number of amino acid residues of the secreted peptide is preferably about 100 or less. More preferably, it is about 70 residues or less, More preferably, it is about 50 residues or less, Most preferably, it is about 40 residues or less. The lower limit is not particularly limited, but the lower limit is the number obtained by adding the number of amino acid residues of the secretory signal peptide to the 6 residues that are the lower limit of the peptide having a cardiac function improving action.

The cell sheet for treating heart disease of the present invention can be prepared by culturing cells that secrete peptides having a cardiac function improving action. A cell that secretes a peptide having a cardiac function improving action introduces a recombinant expression vector into which a polynucleotide encoding a peptide having a cardiac function improving action (including the amino acid sequence of a secretory signal peptide) is inserted into an appropriate cell. Can be produced. Hereinafter, a method for producing a cell secreting a peptide having a cardiac function improving action and a cell sheet for treating heart disease of the present invention will be described.

The cells used for the production of cells that secrete peptides having a cardiac function improving effect (hereinafter referred to as “peptide secreting cells”) are not particularly limited, but cells that have already been used for transplanting cell sheets into the heart are preferred. , Myoblasts (Ghostine S, Carrion C, Souza LC, Richard P, Bruneval P, Vilquin JT, Pouzet B, Schwartz K, Menasche P, Hagege AA ： Long-term efficacy of myoblast alplantfunction card functionplant Circulation. 106: I-131-I-136, 2002.), smooth muscle cells (Yoo KY, LiRK, Weisel RD, Mickle DAG, Li GM, Kim EJ, Tomita S, Yau TM: Autologous smooth muscle cell transplantation improved heart function in dilated cardiomyopathy. Ann Thorac Surg 70: 859-865, 2000., etc., mesenchymal cells (Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers JM, Pittenger BJ, Pittenger BJ, stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 73: 1919-25,2002), adipocytes (Valina C, Pinkernell K, Song YH, Bai X, Seaut J, TH, Alt E. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. Eur Heart J. 28: 2667-77, 2007.

Recombinant expression vectors can be prepared using known gene recombination techniques. As the expression vector, an appropriate expression vector containing a promoter that can function in a host cell may be selected. For example, retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA-11, pxT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. ) And the like. By inserting a polynucleotide encoding a peptide having a cardiac function improving action into such an expression vector, a target recombinant expression vector can be prepared. The polynucleotide may be DNA or RNA, or may be a DNA / RNA chimera. The polynucleotide may be double-stranded or single-stranded. Preferred is double-stranded DNA, particularly cDNA. The nucleotide sequence of a polynucleotide encoding a peptide having a cardiac function improving action (including the amino acid sequence of a secretory signal peptide) can be appropriately designed based on the amino acid sequence of the peptide. The polynucleotide can be prepared by chemical synthesis. The method for introducing the recombinant expression vector into the cell is not particularly limited, and a known method can be appropriately selected and used. For example, electroporation method, calcium phosphate method, lipofection method, DEAE dextran method and the like can be mentioned. In the case of a viral vector, the cells may be infected with the virus. Peptide-secreting cells are selected from the cells into which the recombinant expression vector has been introduced. The peptide-secreting cell is preferably a cell that stably expresses the target peptide.

After culturing the obtained peptide-secreting cells confluently using a suitable culture dish, the cell sheet for treating heart disease of the present invention can be produced by peeling the cells while maintaining the sheet state. Preferably, the culture is confluent using a culture dish coated with poly-N-isopropylacrylamide, which is a temperature-responsive polymer, and is peeled after low-temperature treatment (Medical History, 195, 203-204 (2000). )reference). A cell sheet having a three-dimensional structure composed of cells and extracellular matrix can be produced by a method using a culture dish coated with the temperature-responsive polymer.

The amount of the peptide having a cardiac function improving action secreted by the cell sheet for treating heart disease of the present invention is not particularly limited. The cell sheet for treating heart disease of the present invention can be laminated in a plurality of layers so that the secretion amount of the peptide having an effect of improving cardiac function becomes the target secretion amount. The transplanted cell sheet is thought to gradually fall out of the cell transplant site in about 2 weeks. In addition, it has been found that the SVVYGLR peptide improves the cardiac function in vivo in an amount of 20 to 100 ng. Therefore, a cell sheet capable of secreting 20-100 ng or more of a peptide within 2 weeks after transplantation is preferred.

Regarding the transplantation of autologous myoblast cell sheets, it has shown excellent therapeutic results in experiments using various animal models of heart disease (Miyagawa S, et al., Transplantation. 80: 1586-95, 2005, Memon IA, et al., J Thorac Cardiovasc Surg. 130: 1333, 2005, Kondoh H, et al., Cardiovasc Res. 69: 466, 2006, Hata H, et al., J Thorac Cardiovasc Surg. 132: 918, 2006). However, it has been a drawback of cell sheet transplantation that the effect gradually decreases when the postoperative cardiac function is followed over a long period of time.
The cell sheet for treating heart disease of the present invention has been demonstrated to overcome the drawbacks of conventional cell sheets and maintain the improvement effect of cardiac function even 8 weeks after cell sheet transplantation (Example 3). And 4). In addition, since the cell sheet for treating heart disease of the present invention has both a cardiac function improving effect and an angiogenic action, it can be expected that the cardiac function can be improved more continuously. Therefore, it is considered to be extremely useful for treating not only ischemic heart diseases such as myocardial infarction but also cardiomyopathy. Furthermore, by using a cell sheet, unlike the single administration by the injection method, the peptide is continuously secreted from the sheet transplantation site, and the effect can be maintained. In addition, the injection method is known to induce arrhythmia, but there has been no such report on cell sheet transplantation. Therefore, if the cell sheet for treatment of heart disease of the present invention is used, a peptide having a function of improving cardiac function can be used in combination with the treatment of heart disease using a known cell sheet, and a synergistic treatment effect can be expected.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[Example 1: Evaluation of peptide using rat myocardial infarction model]
(1) Experimental method (1-1) Synthesis of peptide A peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 was synthesized by Fmoc method using a multi-item solid phase method automatic peptide synthesizer (PSSM-8; Shimadzu Corporation). did. More specifically, the graft copolymerization range Tentagel (particle size 80 μm) of polyethylene glycol and polystyrene was used as a support and was synthesized by a high-efficiency solid phase method. The obtained synthetic peptide was tested with a high performance liquid chromatograph mass spectrometer (LCMS; Shimadzu Corporation), and confirmed to be a single component consistent with the theoretical mass value (see FIG. 6). Hereinafter, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 is referred to as “WiDa”.

(1-2) Preparation of rat myocardial infarction model Sprague-Dawley rats (8 weeks old, male, body weight about 300 g) were inhaled by anesthesia with isoflurane, intubated, and managed intraoperatively under artificial ventilation. After confirming the thoracotomy and the left anterior descending coronary artery from the intercostal space, ligation was performed at the level of the left atrial appendage using 6-0 non-absorbable thread (nylon thread). An ST elevation was confirmed in the electrocardiogram, and the infarct was determined. Thereafter, 0.1 ml of PBS (Control) or WiDa (100 ng / ml) was administered around the infarcted myocardium, and the surgical field was closed with continuous sutures. As a healing model, a short-term treatment model for 3 weeks after peptide administration and a long-term treatment model for 6 weeks and 9 weeks were set. In addition to the WiDa group and the Control group (PBS administration), a Sham group in which only thoracotomy and closing was performed was provided.

(1-3) Evaluation of cardiac function Left ventricular function was evaluated using echocardiography on the 21st day (3 weeks), 42 days (6 weeks), and 63 days (9 weeks) after surgery. After anesthetizing anesthesia with isoflurane, echocardiograms were recorded using an ultrasonic diagnostic apparatus SONOS5500 (Aglient Technologies, Palo Alto, Calif.), Left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs), left The end-diastolic lumen area (LVEDA) and the left ventricular end-systolic lumen area (LVEDS) were obtained. From these values, the left ventricular diameter shortening rate (% Fractional shortening;% FS), left ventricular ejection fraction (EF), and left ventricular area change rate (FAC) were calculated (Yanagisawa M , Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 411-5.).
LV% FS = [(LVDd−LVDs) / LVDd] × 100
^{LVEF (%) = [(LVDd} 3 -LVDs 3) / LVDd 3] × 100
LVFAC (%) = [(LVEDA−LVEDS) / LVEDA] × 100

(1-4) Histological evaluation The heart was removed on day 21 (3 weeks), 42 days (6 weeks), and 63 days (9 weeks) after surgery, and fixed with 10% buffered formalin for 48 hours. went. After removing both atria, they were embedded in paraffin and sliced to a thickness of 3 μm. The sliced sections were stained with hematoxylin and eosin, and the infarct wall thickness was compared.
In order to measure the myocardial fibrosis rate, Sirius red staining was performed to specifically dye collagen fibers in red. For measurement of myocardial fibrosis, the border of the infarct was observed (× 200), and images were captured with ACT-2U software (NIKON). Images were analyzed with WinRoof software (MITANI CORPORATION). The results were expressed as the fibrosis rate per visual field.
In addition, Periodic Acid / Schiff reaction (PAS) staining was performed for the purpose of measuring the cell size of cardiomyocytes. After staining, observe with an optical microscope (× 400), randomly select 100 cells with nuclei at the infarct boundary and the left ventricular posterior wall, and capture the image with ACT-2U software. Were analyzed by WinRoof software. As an analysis method, the minor axis crossing the nucleus of the cardiomyocytes was measured and used as the cell transverse diameter.

In order to count the number of capillaries having vascular endothelial cells, immunohistochemical staining was performed using an antibody against Von Willbrand Factor, a marker of vascular endothelial cells (Anti-Human Von Willebrand factor, DAKO). The deparaffinized sections were dehydrated with alcohol and heat-treated to activate the antigen. Thereafter, endogenous peroxidase was inactivated with 0.1% hydrogen peroxide solution. After blocking with 5% skim milk, it was reacted overnight at 4 ° C. with an anti-Von Willbrand Factor antibody.
The section was washed with PBS-T and reacted with a biotinylated labeled anti-Rabbit IgG antibody (Anti-Rabbit Ig, biotinyated species-specific whole antibody, DAKO) as a secondary antibody. Color was developed with DAB (Diamino benzidine) using the LSAB (Labeled Streptavidin Biotinyated Antibody) method with HRP-labeled streptavidin (Streptavidin-Horseradish peroxidase conjugate, GE Healthcare). After staining, 15 fields were randomly selected with an optical microscope (× 400), and the number of capillaries having Von Willbrand Factor-positive vascular endothelial cells was counted to obtain the capillary density.

(1-5) Statistical analysis All statistical values were expressed as mean ± standard deviation or mean ± standard error. In order to compare the results of the previous experiments with Control, the student-t test was used and p <0.05 was considered significant.

(2) Results (2-1) Evaluation of cardiac function In order to examine the effect of WiDa on the recovery of left ventricular function, the left ventricular diameter shortening rate (% FS), which is a left ventricular function index, is measured using echocardiography. The output rate (EF) and the left ventricular area change rate (FAC) were evaluated. The results are shown in FIG.
First, the short-term effect after WiDa administration was evaluated at the third week after the operation. The left ventricular diameter shortening rate (% FS, FIG. 1a), ejection fraction (EF, FIG. 1b), left ventricular cavity area change rate In all items of (FAC, FIG. 1c), the WiDa group was significantly higher than the Control group (% FS; p = 0.005, EF; p = 0.005, FAC; p = 0.002) . Subsequently, the long-term effect of WiDa on ischemic myocardium was evaluated at 6 and 9 weeks after surgery. First, when the left ventricular diameter shortening rate (% FS, FIG. 1 dg) and the ejection fraction (EF, FIG. 1 eh) were evaluated, both the 6-week and 9-week models were significantly higher in the WiDa group than in the Control group. When the left ventricular area change rate (FAC, FIG. 1fi) was evaluated, the WiDa group was significantly improved with respect to the Control group in the 9-week model. On the other hand, in the 6-week model, no significant difference was observed between the Control group and the WiDa group, but the WiDa group showed an improvement trend compared with the Control group (p = 0.145).

(2-2) Histological examination (a) Effect on infarct lesion Myocardial infarction is the disruption of blood flow in the coronary artery that feeds the myocardium, resulting in necrosis of the myocardium in the perfusion region. The necrotic part is replaced with collagen fibers, and as a result, the cardiac function is considered to decrease. Therefore, in order to examine how WiDa affects infarcts, hematoxylin and eosin staining (hereinafter referred to as “HE staining”) and Sirius red staining that specifically stains collagen fibers are performed on heart sections removed each week in each group. Went.
The results are shown in FIG. Of each pair of photographs in FIGS. 2a-g, the left is the HE-stained image and the right is the Sirius red-stained image. In the figure, the scale bar represents 1000 μm. FIG. 2a (Sham group) only showed results for 3 weeks. In the Sirius red-stained images of FIGS. 2b to 2g, in the 3-week, 6-week and 9-week models, in the Control group, the wall thickness from the left ventricular anterior wall to the posterior wall became thinner and many collagen fibers were observed. The left ventricular wall thickness of the WiDa group was kept thicker than that of the Control group.
Regarding the expansion of the left ventricular cavity, in the HE-stained images of FIGS. 2b to g, the left ventricular cavity was gradually expanded in the Control group in both the 3-week, 6-week and 9-week models, whereas in the WiDa group, the Control Dilation was suppressed compared to the group.

(B) Effect of infarct boundary on normal myocardial tissue In the healing process after myocardial infarction, normal myocardium at the infarct boundary is loaded, and cell hypertrophy and myocardial fibrosis progress. Therefore, in order to examine the influence of WiDa on normal myocardial cells in the vicinity of myocardial infarction, myocardial fibrosis rate was evaluated by Sirius red staining and myocardial cell lateral diameter was measured by PAS staining.
The results of Sirius red stained image and myocardial fibrosis rate are shown in FIGS. 3 (A) and 3 (B). (A) is a Sirius red stained image of the myocardium, and the scale bar represents 100 μm. (B) is a graph showing the analysis result of the myocardial fibrosis rate. In the 3-week model, no significant difference was observed between the Control group and the WiDa group in the myocardial fibrosis rate (FIG. 3 (B) a). On the other hand, in the 6-week and 9-week models, it was significantly decreased in the WiDa group as compared with the Control group (6 weeks; FIG. 3 (B) b, p = 0.00041, 9 weeks; FIG. 3 (B) c. , P = 0.00054).

4A and 4B show the PAS-stained image and the measurement results of the myocardial cell lateral diameter. (A) is a PAS-stained image of the infarct boundary, and the scale bar represents 100 μm. (B) is a graph showing the measurement results of the myocardial cell lateral diameter, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary. In any of the 3, 6 and 9 week models at the infarct boundary, the myocardial cell lateral diameter was significantly higher in the Control group and the WiDa group than in the sham group (p <0.0001). In addition, in the comparison between the Control group and the WiDa group, no significant difference was observed in the 3-week model, but in the 6-week and 9-week models, the WiDa group was significantly lower (6 weeks; p = 0.0001, 9 weeks; p = 0.00017). On the other hand, when the same evaluation was performed on the left ventricular posterior wall, no significant difference was observed in the Control group and the WiDa group in any of the 3-week, 6-week, and 9-week models.

(C) Evaluation of angiogenic ability in ischemic myocardial tissue In order to examine whether angiogenesis by WiDa is induced in myocardial tissue, capillary density was measured. Immunohistochemical staining was performed using an antibody against Von Willbrand Factor, which is a vascular endothelial cell marker, and the number of capillaries was counted.
The results are shown in FIGS. 5 (A) and (B). (A) is an immunohistochemically stained image of the infarct boundary, and the scale bar represents 100 μm. (B) is a graph showing the measurement result of capillary blood vessel density, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary. A significant increase in capillary density was observed in the WiDa group compared to the Sham group and the Control group in both the 3-week, 6-week and 9-week models at the infarct boundary (p <0.0001). On the other hand, there was no significant difference in capillary density in the left ventricular posterior wall of the Sham group, Control group and WiDa group.

(3) Discussion Many treatments and researches for ischemic heart disease models have been conducted so far, and various effects have been reported. However, the most important thing in considering the therapeutic effect of myocardial infarction is whether or not recovery of cardiac function is recognized. Therefore, in this study, we focused on the improvement of left ventricular function by WiDa. As a result of evaluating by echocardiography whether WiDa improves the cardiac function lost due to infarction, significant improvement in cardiac function was observed in the WiDa group compared to the Control group in the 3-week model. Moreover, compared with the Sham group, the cardiac function was significantly low in the Control group and the WiDa group. From these results, it was considered that cardiac function significantly decreased temporarily after the preparation of myocardial infarction, but WiDa administered immediately after myocardial infarction exhibited some function, and the cardiac function was greatly improved. Furthermore, similar effects were observed in the 6-week model and the 9-week model. The results in the 6-week and 9-week treatment long-term models suggested that WiDa is continuously involved in suppressing cardiac decline. In addition, in the myocardial infarction model, the administered WiDa significantly suppressed infarct wall thickness thinning, myocardial fibrosis, and cardiomyocyte hypertrophy. These were also significantly suppressed in the 6-week and 9-week long-term models. In the healing process of myocardial infarction, the work of normal myocardium at the infarct boundary increases, and cell hypertrophy and myocardial fibrosis gradually progress around the infarct. From the results of this study, it is considered that WIDA reduced the load on normal cells around the infarction and suppressed myocardial fibrosis and cardiomyocyte hypertrophy, and WiDa has an inhibitory effect on left ventricular remodeling after infarction. It has been suggested.

Studies have already been conducted on a myocardial infarction model of HGF having an angiogenesis-promoting action, and it has been clarified that HGF has an action for improving angiogenesis in addition to an angiogenesis-promoting action ([1] Jin , H. et ai., Curr Pharm Des 2004; 10: 2525-33. [2] Chen XH, et ai., J Card Fail 2007; 13: 874-83. [3] Li Y, et ai., Circulation 2003; 107: 2499-506. [4] Ueda H, et ai., CardiovascvasRes 2001; 51: 41-50.). According to a report on the therapeutic effect of gene therapy using HGF for a myocardial infarction model, the left ventricular function was 4% after treatment, the left ventricular diameter shortening rate (% FS) 20%, ejection fraction (EF) 42% Although an improvement effect was observed, a downward trend was observed thereafter (Miyagawa S. et ai., Circulation 2002; 105: 2556-2561.). On the other hand, in this study, the left ventricular function of the 3-week model is the left ventricular diameter shortening rate (% FS) of 25, even though only one administration of WiDa (100 ng / ml) was performed immediately after the creation of the myocardial infarction model. % And ejection fraction (EF) of 50%, and it was revealed that an effect equivalent to or higher than that obtained by HGF gene therapy can be obtained. Further, this effect lasted from 6 weeks to 9 weeks. WiDa is a low molecular weight peptide composed of 7 amino acid residues, and its half-life is considered to be short. However, a single administration of WiDa to the ischemic peripheral myocardium dramatically improved left ventricular function and the effect persisted for a long time. From this, it was found that WiDa has an effect of remarkably improving the cardiac function lost due to ischemia.

There have been many studies on the treatment of acute myocardial infarction using known angiogenesis-promoting factors and cytokines in addition to HGF, all of which improve cardiac function and suppress remodeling at 3 weeks after administration. Indicates. However, when the effects are examined over time such as 6 weeks and 9 weeks after administration, the therapeutic effect is remarkably lowered, and the current situation is that the treatment cannot be performed sufficiently. In contrast, when this peptide was administered to an acute myocardial infarction model, it improved cardiac function at the third week of administration and suppressed remodeling, as well as improved cardiac function in evaluations at the sixth and ninth weeks of administration. And the remodeling inhibitory effect was maintained. Therefore, it is expected that WiDa can be used as a new peptide drug that shows a therapeutic effect for 6 months and 9 weeks for acute myocardial infarction.

Also, in the myocardial infarction model, histological examination was performed in order to examine whether or not new blood vessels were induced by the angiogenic action of WiDa. When the capillary density was measured, in all of the 3, 6 and 9 week models, the number of capillaries was significantly increased in the WiDa group in the infarct boundary portion compared to the Sham group and the Control group. In addition, the increase in the number of capillaries observed in the 6-week and 9-week long-term models was almost the same as that observed in the short-term model at 3 weeks after treatment, and thus was induced by WiDa. It was suggested that the new blood vessels stabilized structurally and functionally and remained as nutrient vessels to the myocardium for a long time. Currently, VEGF, bFGF (basic fibroblast growth factor), etc. are known as angiogenic factors used in therapeutic research for ischemic heart disease ([1] Hader HKh, et ai., Circ Res 2008; 103: 1300-1308. [2] Rissanen TT, et ai., Adv Genet 2004; 52: 117-167. [3] Rissanen TT, et ai., The FASEB Journal 2003; 17: 100-102. [4] Hao X, et ai., BiochemBiophys Res Commun 2004; 322: 292-6.). However, these factors are concerned about side effects such as edema and inflammatory reaction. In this myocardial infarction model, WiDa induced more structurally and functionally stabilized trophic blood vessels from ischemic myocardial tissue from the angiogenic action of previous studies. Therefore, it can be said that WiDa is a new factor capable of overcoming side effects such as early regression of new blood vessels and edema.
From the above research results, it was considered that WiDa exhibited not only the conventionally known angiogenic action but also some function that markedly improved cardiac function.

[Example 2: Production of WiDa-secreting myoblast sheet]
(1) Experimental method (1-1) Myoblast isolation Anterior tibial muscle was collected from 3-week-old Lewis rats, washed with cold HBSS (Hanks balanced salt solution), and trypsin (Invitrogen life Technologies, Carlsbad, CA, USA) was added, and tendons, fibrous tissue, adipose tissue, etc. were carefully removed to prevent mixing of fibroblasts. Mince with trypsin and treated with 0.2% type II collagenase (Worthingto Biochemical corporation, Lakewood, NJ, USA) for 45 minutes. After centrifugation and filtration, the cells were seeded on a collagen type I (Nitta Gelatin Inc, Osaka, Japan) coat dish (10 μg / ml) in order to adhere fibroblasts. After incubating at 37 ° C. with 5% CO ₂ for 4 hours, the supernatant was seeded again on a collagen type I coat dish (10 μg / ml). After another 24 hours, the supernatant was matrigel (Becton Dickinson Bioscience, Flanklin Lakes, NJ, USA) Coated dishes (0.5 mg / ml) were reseeded, and the cells adhered here were used as myoblasts. As a medium, 20% FBS (fetal bovine serum, Biowest, Minami, FL, USA), 1% Antibiotic-Antimytic (Invitrogen life Technologies), and Orgadron (Nihonoruganon, Osaka, Japan) -containing DMEM (Dulbecco modified Eagle's medium) (Nihonseiyaku , Toky, Japan). In the experiment, cells having passage numbers from 1 to 4 were used.

(1-2) Immunofluorescence staining In order to confirm that the cells isolated from the anterior tibial muscle were myoblasts, the expression of Desmin present in the muscle cells was confirmed by immunofluorescence staining. The isolated cells were seeded on a Matrigel-coated 12 mm diameter cover glass (Matsunami Glass IND LTD, Tokyo, Japan), and after cell adhesion, fixed with a 4% PFA (paraformaldehyde) / PBS (phosphate buffered solution) solution. Treated with 5% Triton X-100 / PBS solution and blocked with 2% BSA / PBS solution. After blocking, anti-Desmin antibody (Sigma, St. Louis, MO, USA) was reacted as a primary antibody for 1 hour at room temperature, and FITC-labeled anti-rabbit IgG (DAKO, Glostrup, Denmark) was reacted as a secondary antibody for 30 minutes at room temperature. It was sealed with a water-soluble mounting medium (Invitrogen life Technologies) containing (4 ′, 6-diamidino-2-phenylindole). Then, it observed with the fluorescence microscope (ECLIPSE E600, Nikon).

(1-3) Gene Construction A DNA encoding WiDa with a secretion signal (Igκ chain leader sequence) on the N-terminal side and an HA tag on the C-terminal side was constructed. That is, the following four DNAs were synthesized with a DNA synthesizer and joined together to obtain a target DNA fragment.
foward1; 5'-GCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGT-3 '(SEQ ID NO: 10)
foward2; 5'-TCCACTGGTGACGCGGCCCAGCCGGCCAGTGTGGTTTATGGACTGAGGCTCGAGTACCCATACGATGTTCCAGATTACGCTTAAC-3 '(SEQ ID NO: 11)
reverse1; 5'-TCGAGTTAAGCGTAATCTGGAACATCGTATGGGTACTCGAGCCTCAGTCCATAAACCACACT-3 '(SEQ ID NO: 12)
reverse2; 5'-GGCCGGCTGGGCCGCGTCACCAGTGGAACCTGGAACCCAGAGCAGCAGTACCCATAGCAGGAGTGTGTCTGTCTCCATGGTGGCG-3 '(SEQ ID NO: 13)
The obtained DNA fragment was ligated to a lentiviral vector pCS-CG treated with restriction enzymes NheI and XhoI, and it was confirmed by sequencing that the DNA fragment of the correct base sequence was incorporated. The pCS-CG in which DNA encoding WiDa is incorporated is hereinafter referred to as “WiDa / pCS-CG”.

(1-4) Gene transfer (i) Production of lentivirus Human kidney epithelium-derived cells HEK293T cells were prepared in a state of 80% confluence in 10 cm dishes coated with collagen. Gene transfer was performed by the calcium phosphate method. After preparing WiDa / pCS-CG (20 μg) and three packaging vectors of pMD2.G, pRSV-Rev and pMDLg / pRRE (each 10 μg) in sterile water, add 50 μl of 2.5 M CaCl ₂ and mix did. While stirring this solution, 500 μl of 2 × HBS (pH 7) was added and incubated at room temperature for 10 minutes to prepare a complex of calcium phosphate and DNA. The medium of each dish was replaced with 10% FBS DMEM, and the DNA complex was added to the culture supernatant to introduce the gene. After 12 hours, the medium was replaced with 6 ml of 10% FBS DMEM, and the virus-containing supernatant was collected 48 hours and 72 hours after gene introduction. The collected virus-containing supernatant was passed through a 0.45 μm filter (Millipore, Billerica, MA, USA) to remove floating cells and the like, and Lenti-XTM Concentrator (Clontech Laboratories, Inc., Mountain View, CA, USA). Concentrated. A virus-containing supernatant (mock) into which a pCS-CG empty vector used as a negative control for expression studies was introduced was also collected.

(Ii) Lentiviral vector infection Isolated myoblasts are seeded in a 35 mm dish the day before infection so that the number of isolated myoblasts is 4 × 10 ⁴ cells / dish at the time of infection. At the time of infection, the medium was replaced with 500 μl of serum-free DMEM containing 8 μg / ml of polybrene (Sigma), and the virus-containing supernatant collected there was added dropwise, and 500 μl of 20% FBS DMEM was added after 4 hours. The cells were incubated for 48 hours at 37 ° C. with 5% CO ₂ and infected. The medium was changed to a medium suitable for each experiment 48 hours after the infection and used for the experiment 96 to 120 hours later.

(1-5) RT-PCR
The expression of WiDa mRNA was examined by RT-PCR. The medium was exchanged with 20% FBS DMEM 48 hours after infection, and 1 ml of Sepasol-RNA1 Super G (nacalai tesque, Kyoto, Japan) was added 120 hours after infection. After 5 minutes, the cells were detached with a scraper and transferred to a 1.5 ml tube, and then 200 μl of chloroform was added and mixed vigorously. It was centrifuged (12000 rpm, 15 minutes, 4 ° C.) and 400 μl of the supernatant was transferred to a new 1.5 μl tube. An equal amount of isopropanol was added thereto, mixed by inversion, incubated at room temperature for 10 minutes, and then centrifuged (12000 rpm, 10 minutes, 4 ° C.). After centrifugation, the supernatant was removed, and the extracted RNA pellet was washed with 75% ethanol and centrifuged (8500 rpm, 5 minutes, 4 ° C.). After centrifugation, the supernatant was removed and dried. The obtained total RNA was suspended in 20 μl of DEPC (diethypyrocarbonate) water and allowed to stand on ice for 15 minutes for dissolution. Thereafter, the absorbance at 260 nm was measured to determine the concentration.
5 × AMV buffer 5 μl, 10 mM dNTP 2 μl, 50 μM Oligo dT 1 μl, RNasion 1 μl, AMV reverse transcriptase (Sigma) 1 μl was added, and a reverse transcription reaction was performed under reaction conditions of 42 ° C. for 60 minutes, 65 ° C. for 10 minutes, and 4 ° C.
CDNA obtained by reverse transcription was used as a template. The primers used are shown below. GAPDH (Glycelaldehyde-3-phospate dehydrogenase) was used as an internal control.
SVVYGLR (forward); 5'-CTAGCGCCACCATGGAGACAGACA-3 '(SEQ ID NO: 14)
SVVYGLR (reverse); 5'-AGCGTAATCTGGAACATCGTATGG-3 '(SEQ ID NO: 15)
GAPDH (forward); 5'-ACTGGCGTCTTCACCACCAT-3 '(SEQ ID NO: 16)
GAPDH (reverse); 5'-AGTGAGCTTCCCGTTCAGCT-3 '(SEQ ID NO: 17)

(1-6) Confirmation of WiDa Production and Secretion Production / secretion of WiDa in infected myoblasts was examined by the dot blotting method. 48 hours after infection, the medium was replaced with serum-free DMEM, and after 72 hours, the culture supernatant was collected and centrifuged (12,000 rpm, 5 minutes, 4 ° C.). 100 μl each of serial dilutions (12.5 μg / ml to 0.2 ng / ml) of the collected culture supernatant and WiDa-HA peptide were immobilized on a microplate overnight at 4 ° C. and blocked with 5% skim milk. . A high HA polyclonal antibody was used as the primary antibody, an HRP-labeled anti-rabbit IgG antibody was used as the secondary antibody, and light was emitted with Super signal West Femto, followed by development and analysis. The image of the film was taken into Image J, and the density of Dot of each well was quantified and examined and evaluated.

(1-7) Preparation of cell sheet 72 hours after infection, 3 × 10 ⁶ cells / dish of infected cells were seeded in a 35 mm temperature-responsive culture dish (Cell Seed, Tokyo, Japan), 5% CO ² , 37 ° C. Incubated with. Sixteen hours after seeding the cells, the dish was allowed to stand at room temperature for 30 minutes, and the cells were peeled off into a sheet.

(2) Results (2-1) Confirmation of myoblasts In order to confirm that cells isolated from the anterior tibial muscle are myoblasts, the expression of Desmin present in muscle cells was determined by immunofluorescence staining. investigated. The results are shown in FIG. The left is a photograph by a phase contrast microscope, and the right is a photograph by a fluorescence microscope. In the fluorescence micrograph, Desmin is dyed green and the nucleus is dyed blue. As a result of the observation, 90% or more of the cells were positive for Desmin, and it was confirmed that high-purity myoblasts could be isolated.

(2-2) Confirmation of WiDa mRNA expression The results are shown in FIG. In FIG. 8, WT represents wild-type (non-infected virus) myoblasts, mock represents empty vector-infected myoblasts, and WiDa / pCS-CG represents WiDa / pCS-CG-infected cells. As is apparent from FIG. 8, WiDa mRNA was detected only from WiDa / pCS-CG-infected cells.

(2-3) Confirmation of WiDa production and secretion The results are shown in FIG. (A) is the result of dot blotting in which a known concentration of WiDa-HA peptide is serially diluted, (B) is the result of dot blotting of WiDa / pCS-CG-infected cell culture supernatant, and (C) is (B ) Is a table showing the darkness of the numerical value. From the results shown in FIG. 9, it was confirmed that WiDa was produced and secreted in WiDa / pCS-CG-infected cell culture. Further, from the comparison of the difference in the lightness and the lightness of the serially diluted dots in FIG. 9A, it was calculated that about 3.125 to 6.25 ng / ml WiDa was secreted in 72 hours.

[Example 3: Evaluation of WiDa secreting myoblast sheet using rat heart failure model I]
(1) Experimental method (1-1) Preparation of rat heart failure model F344 / NJcl-rnu / rnu rats (8 weeks old, female) were inhaled and anesthetized with isoflurane. Thereafter, endotracheal intubation was performed and intraoperative management was performed under artificial ventilation. A thoracotomy was performed from the heartbeat area, and a visual field was secured using a retractor. After the lungs were protected with gauze, the left anterior descending coronary artery was ligated with 7-0 non-absorbable thread (nylon thread) at the height of the left atrial appendage to create a myocardial infarction in the left ventricular anterior wall. Echocardiography was taken 2 weeks after ligation, and left ventricular ejection fraction (EF) of 45-35% was used as a myocardial infarction model rat in the experiment. In addition, a myoblast sheet resuming 2 weeks after ligation and secreting WiDa (hereinafter referred to as “WiDa-rSkM”), a wild type myoblast sheet (hereinafter referred to as “WT-rSkM”) Transplanted to the infarct site. A cell sheet that was not transplanted was designated as Control. Each cell sheet was composed of three layers. This model starts treatment at the second week after ligation of the left anterior descending coronary artery. The motility of the left ventricular anterior wall at the second week is reduced, and in the untreated state as it is, the left ventricular wall motion further decreases with time, and the left ventricular wall function is significantly impaired. It can be said that the pressure load on the left ventricular wall also rises and the left ventricular cavity gradually expands, causing heart failure.

(1-2) Cardiac function evaluation Left ventricular function evaluation was performed using echocardiography at 2, 4, 6, and 8 weeks after cell sheet transplantation. After anesthetizing anesthesia with isoflurane, echocardiograms were recorded using an ultrasonic diagnostic apparatus SONOS5500 (Aglient Technologies, Palo Alto, Calif.), Left ventricular end diastolic diameter (LVDd), left ventricular end systolic diameter (LVDs), left The end-diastolic lumen area (LVEDA) and the left ventricular end-systolic lumen area (LVEDS) were obtained. From these values, left ventricular diameter shortening rate (% Fractional shortening;% FS), left ventricular ejection fraction (EF), left ventricular end-diastolic volume (EDV), left ventricular end systole The volume (LV end-systolic volume; ESV) was calculated.
LV% FS = [(LVDd−LVDs) / LVDd] × 100
^{LVEF (%) = [(LVDd} 3 -LVDs 3) / LVDd 3] × 100
EDV (ml) = LVIDd ³ × (0.98 × LVIDd + 5.9)
ESV (ml) = LVIDs ³ × (1.14 × LVIDs + 4.18)

(1-3) Heart weight ratio (HW / BW)
The body weight of the rat was measured 8 weeks after transplantation of the cell sheet, and the heart was removed. The weight of the extracted heart was measured, and the heart weight / body weight ratio was calculated.

(1-4) Histological Evaluation The heart was removed 8 weeks after the sheet transplantation, and fixed with 10% buffered formalin for 48 hours. After removing both atria, they were embedded in paraffin, sliced and HE stained.
Masson trichrome staining was performed to examine left ventricular cavity dilation and left ventricular wall thickness at the infarcted area. The size of the left ventricular cavity was evaluated by the average of the diameters passing through the cavity, and the left ventricular wall thickness was evaluated by the ratio of the left ventricular wall thickness of the infarcted part to the wall thickness of the normal part (left ventricular rear wall).
In order to evaluate the myocardial fibrosis rate, Sirius red staining was performed to specifically dye collagen fibers in red. For measurement of myocardial fibrosis, the infarct boundary was observed with an optical microscope at a magnification of 20x, and an image taken in ACT-2U software (NIKON) was analyzed by image analysis software Image J. Evaluation was shown by the fibrosis rate per visual field.
Periodic Acid / Schiff reaction (PAS) staining was performed to evaluate the lateral diameter of cardiomyocytes. After staining, observation was performed with an optical microscope at a magnification of 40x, and 100 cells with nuclei were randomly selected at the infarct boundary and the left ventricular posterior wall, and the minor axis crossing the nucleus was defined as the cell transverse diameter. .

In order to count the number of capillaries having vascular endothelial cells, immunohistochemical staining was performed using an antibody against Von Willbrand Factor, a marker of vascular endothelial cells (Anti-Human Von Willebrand factor, DAKO). The deparaffinized sections were dehydrated with alcohol and heat-treated to activate the antigen. Thereafter, endogenous peroxidase was inactivated with 0.1% hydrogen peroxide solution. After blocking with 5% skim milk, it was reacted overnight at 4 ° C. with an anti-Von Willbrand Factor antibody.
The section was washed with PBS-T and reacted with a biotinylated labeled anti-Rabbit IgG antibody (Anti-Rabbit Ig, biotinyated species-specific whole antibody, DAKO) as a secondary antibody. Color was developed with DAB (3,3-diaminobenzidine, Sigma, St. Louis, MO, USA) using LSAB (Labeled Streptavidin Biotinyated Antibody) method with HRP-labeled streptavidin (Streptavidin-Horseradish peroxidase conjugate, GE Healthcare). After staining, observe with an optical microscope at 40x objective, randomly select 24 journals in the infarct boundary and left ventricular posterior wall, and count the number of capillaries with Von Willbland Factor positive vascular endothelial cells did. Evaluation was shown by the number of blood vessels per field of view (high power field).

(1-5) Statistical analysis All statistical values were expressed as mean ± standard deviation or mean ± standard error.
Regarding cardiac function evaluation (EF,% FS), first, analysis by duplicate measurement-analysis of variance (repeated meature ANOVA), and then by one-factor analysis of variance (one-factor ANOVA) using Turkey-Kramer post hoc test Tests were conducted to examine significant differences at each time point. p <0.05 was considered significant.
For other statistical values, the student-t test was used and p <0.05 was considered significant.

(2) Results (2-1) Cardiac function evaluation using EF (left ventricular ejection fraction) and% FS (left ventricular diameter shortening rate) as indices EF at 2, 4, 6, and 8 weeks after cell sheet transplantation FIG. 10 shows% FS at 2, 4, 6, and 8 weeks after cell sheet transplantation, respectively. Before cell sheet transplantation (base line in the figure), there was no significant difference in EF and% FS between the groups. The WT-rSkM group and the WiDa-rSkM group showed significant improvement in cardiac function in both EF and% FS compared to the Control group at 2, 4, 6 and 8 weeks after transplantation of the cell sheet to the infarcted region. (P <0.05). Furthermore, at 2, 6, and 8 weeks after transplantation, the WiDa-rSkM group showed a significant improvement in cardiac function in both EF and% FS compared to the WT-rSkM group (p <0.05). .

(2-2) Evaluation of cardiac function using EDV (left ventricular end-diastolic volume) and ESV (left ventricular end-systolic volume) as indices. FIG. 12 shows EDV at 8 weeks after cell sheet transplantation, and 8 weeks after cell sheet transplantation. The ESVs are shown in FIG. In EDV, the WiDa-rSkM group showed a significant decrease compared to the Control group and the WT-rSkM group. Even in ESV, the WiDa-rSkM group showed a significant decrease compared to the Control group and the WT-rSkM group. For both EDV and ESV, no significant difference was observed between the Control group and the WT-rSkM group.

(2-3) Heart-weight ratio (HW / BW)
The results are shown in FIG. The WiDa-rSkM group showed a significant decrease compared to the Control group and the WT-rSkM group. There was no significant difference between the Control group and the WT-rSkM group.

(2-4) Histological examination (a) Expansion of left ventricular cavity and histological evaluation of left ventricular wall of infarcted part are shown in FIGS. 15 (A), (B) and (C). (A) is a Masson trichrome stained image of the heart, and the scale bar represents 1000 μm. (B) is a graph which shows the evaluation result of the left ventricular wall thickness of an infarction part. (C) is a graph showing the evaluation result of the diameter of the left ventricular cavity.
As is clear from FIGS. 15A and 15B, the left ventricular wall at the infarcted site was thinned in the Control group and the WT-rSkM group, whereas the left ventricular wall was kept thick in the WiDa-rSkM group. It was leaning. When statistical analysis was performed, the left ventricular wall of the infarct was significantly thicker in the WiDa-rSkM group compared to the Control group and the WT-rSkM group (p <0.01). There was no significant difference between the Control group and the WT-rSkM group.
Further, as is clear from FIGS. 15A and 15C, the WiDa-rSkM group significantly suppressed the expansion of the left ventricular cavity as compared with the Control group and the WT-rSkM group (p <0). .05). There was no significant difference between the Control group and the WT-rSkM group.

(B) Histological evaluation of the effect on the myocardial tissue around the infarction In order to examine the effect on the normal myocardial tissue around the infarction, the evaluation of the myocardial fibrosis rate by Sirius Red staining and the cell lateral diameter by PAS staining Measurements were made.
The results of Sirius red stained image and myocardial fibrosis rate are shown in FIGS. 16 (A) and 16 (B). (A) is a Sirius red stained image of the myocardium, and the scale bar represents 100 μm. (B) is a graph showing the analysis result of the myocardial fibrosis rate. The myocardial fibrosis rate in the WiDa-rSkM group was significantly lower than that in the Control group and the WT-rSkM group (p <0.01). There was no significant difference between the Control group and the WT-rSkM group.

FIG. 17 (A) and (B) show the PAS-stained image and the measurement results of the cardiomyocyte lateral diameter. (A) is a PAS-stained image of the infarct boundary, and the scale bar represents 50 μm. (B) is a graph showing the measurement results of the myocardial cell lateral diameter, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary. The myocardial cell transverse diameter at the infarct boundary in the WiDa-rSkM group was significantly lower than that in the Control group and the WT-rSkM group (p <0.01). There was no significant difference between the Control group and the WT-rSkM group. There was no significant difference in the cardiomyocyte lateral diameter of the left ventricular posterior wall between any groups.

(C) Examination of angiogenesis promoting action Immunohistochemical staining was performed using an antibody against Von Willbrand Factor, and the results of counting the number of capillaries are shown in FIGS. 18 (A) and (B). (A) is an immunohistochemically stained image of the infarct boundary, and the scale bar represents 100 μm. (B) is a graph showing the measurement result of capillary blood vessel density, white is the result of the left ventricular rear wall, and black is the result of the infarct boundary. The number of capillaries at the infarct boundary in the WiDa-rSkM group was significantly increased compared to the Control group and the WT-rSkM group (p <0.01). There was no significant difference between the Control group and the WT-rSkM group. Capillary density in the left ventricular posterior wall was not significantly different between any groups.

(3) Discussion In Example 1 (peptide administration experiment by the injection method), the peptide was administered immediately after ligating the descending branch of the left ventricle, and the therapeutic effect on acute myocardial infarction was evaluated. In contrast, in Example 3 (WiDa secretion myoblast cell sheet transplantation), a cell sheet was transplanted two weeks after ligation of the left ventricular descending branch. The second week after ligation of the left ventricular anterior descending branch is a stage in which acute myocardial infarction has progressed and heart failure has occurred. That is, Example 3 evaluates the usefulness of WiDa in the treatment of heart failure. Similar to Example 3, in a study in which HGF, which is a known angiogenic factor, was introduced into a myoblast sheet and transplanted into a heart failure model rat, although a remodeling inhibitory effect was observed, sustained cardiac function was confirmed. No improvement was observed (Siltanen A, Kitabayashi K, Lakkisto P, Makela J, Patila T, Ono M, Tikkanen I, Sawa Y, Kankuri E, Harjula A. hHGF Overexpression in Myoblast Sheets Enhances Their Angiogenic Potential in Rat Chronic Heart Failure. PLoS One. 2011,26; 6: e19161.). In contrast, the transplantation of WiDa-secreting myoblasts showed an improvement in cardiac function and an effect of suppressing remodeling in heart failure model rats also at 4, 6, and 8 weeks after the second week after the transplantation. From this, it can be said that WiDa can be a useful therapeutic agent not only for acute myocardial infarction but also for heart failure.

[Example 4: Evaluation of WiDa secreting myoblast sheet using rat heart failure model II]
A rat heart failure model was prepared using F344 / NJcl-rnu / rnu rats (8 weeks old, female) in the same manner as in Example 1-1 (1-1). Eight weeks after the transplantation, the heart was removed and fixed with 10% buffered formalin for 48 hours. After removing both atria, they were embedded in paraffin and sliced. In order to examine the distribution of smooth muscle actin (SMA) positive cells, immunohistochemical staining was performed using an anti-smooth muscle actin antibody and observed with an optical microscope.

The results are shown in FIG. The upper part of FIG. 19 is a 40 × observation image, and the scale bar represents 500 μm. The lower row is a 200-fold observation image, and the scale bar represents 100 μm. As is clear from FIG. 19, accumulation of SMA positive cells was observed in the infarct region in the WiDa-rSkM group. On the other hand, almost no SMA positive cells were observed in the control group and WT-rSkM group. SMA positive cells such as smooth muscle cells and myofibroblasts have contractility. Therefore, the WiDA peptide secreted from the myoblast cell sheet significantly increases SMA positive cells in the infarct region, and the increased SMA positive cells constrict the infarct wall, thereby significantly improving cardiac function. It is thought that it was connected.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (13)

1. A peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having a cardiac function improving action, or a pharmaceutically acceptable salt thereof as an active ingredient To treat heart disease.
   X ₁ -X ₂ -Val-Tyr-X ₅ -X ₆ (I)
   (Wherein X ₁ , X ₂ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
   X ₂ -Val-Tyr-X ₅ -X ₆ -X ₇ (II)
   (Wherein X ₂ , X ₅ , X ₆ and X ₇ are the same or different and represent any amino acid residue.)
   Ser-X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ (III)
   (Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
   X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ -Arg (IV)
   (Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
2. The therapeutic agent for heart disease according to claim 1, wherein the amino acid sequence represented by the formula (I), (II), (III) or (IV) is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6. .
3. The heart disease according to claim 1 or 2, wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 2, or 7, or a peptide having the amino acid sequence represented by SEQ ID NO: 1, 2, or 7. Remedy.
4. 4. The therapeutic agent for heart disease according to claim 1, wherein the total number of amino acid residues of the peptide is 50 or less.
5. The heart disease therapeutic agent according to any one of claims 1 to 4, comprising a cell secreting the peptide.
6. The heart disease therapeutic agent according to any one of claims 1 to 4, comprising a cell sheet that secretes the peptide.
7. The heart disease therapeutic agent according to any one of claims 1 to 6, wherein the heart disease is ischemic heart disease or cardiomyopathy.
8. A cell sheet for treating heart disease, characterized by secreting a peptide having an amino acid sequence represented by the following formula (I), (II), (III) or (IV) and having an effect of improving cardiac function.
   X ₁ -X ₂ -Val-Tyr-X ₅ -X ₆ (I)
   (Wherein X ₁ , X ₂ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
   X ₂ -Val-Tyr-X ₅ -X ₆ -X ₇ (II)
   (Wherein X ₂ , X ₅ , X ₆ and X ₇ are the same or different and represent any amino acid residue.)
   Ser-X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ (III)
   (Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
   X ₂ -X _3- (Tyr / Phe / Trp) -X ₅ -X ₆ -Arg (IV)
   (Wherein X ₂ , X ₃ , X ₅ and X ₆ are the same or different and represent any amino acid residue.)
9. The cell sheet according to claim 8, wherein the amino acid sequence represented by the formula (I), (II), (III) or (IV) is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 6.
10. The cell sheet according to claim 8 or 9, wherein the peptide comprises an amino acid sequence of a secretory signal peptide.
11. The cell sheet according to claim 10, wherein the peptide is a peptide comprising the amino acid sequence represented by SEQ ID NO: 1, 2, or 7 and the amino acid sequence of a secretory signal peptide.
12. The cell sheet according to any one of claims 8 to 11, wherein the cells are myoblasts, smooth muscle cells, mesenchymal cells or adipocytes.
13. The cell sheet according to any one of claims 8 to 12, wherein the heart disease is ischemic heart disease or cardiomyopathy.

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