Myocardial regeneration-inducing agent

Abstract

An object of the present invention is to provide a novel cardiomyocyte growth agent and cardiac muscle regeneration inducer based on a new action mechanism.SOLUTION: The myocardial cell proliferating agent and the myocardial regeneration inducing agent contain a xanthine oxidase inhibiting compound or its salt as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for proliferating cardiomyocytes and an agent for inducing myocardial regeneration, which comprises a compound or a salt thereof that inhibits xanthine oxidase. The present invention also relates to a method for proliferating cardiomyocytes and a method for inducing myocardial regeneration, which use the compound or a salt thereof that inhibits xanthine oxidase. [Background technology]

[0002] Once damaged, the hearts of mammals, including humans, do not naturally regenerate. This is thought to be due to the fact that most of the cardiomyocytes that make up the majority of the heart permanently cease proliferation after birth (Non-Patent Documents 1 to 4). Therefore, for example, when myocardial tissue becomes necrotic due to myocardial infarction or other causes, the human heart has an extremely limited ability to repair itself through the regeneration of cardiomyocytes (myocardial regeneration), making treatment difficult.

[0003] The cardiac disease treatment drugs currently used in clinical practice are primarily intended to slow the progression of damage (Non-Patent Document 5), and no drugs have yet been developed that work by inducing regenerative capabilities, i.e., returning the body to the state before damage occurred. Additionally, clinical trials are currently underway for a treatment using human iPS-derived cardiomyocyte sheets as regenerative medicine (Non-Patent Document 6), but this is not a simple treatment method because it requires surgical procedures and involves the transplantation of human-derived cells. On the other hand, xanthine oxidase is a type of xanthine oxidoreductase, a type of enzyme that generates reactive oxygen species. It catalyzes the oxidation of hypoxanthine to xanthine, and further catalyzes the oxidation of xanthine to uric acid. It is known to play an important role in purine metabolism in several species, including humans (Non-Patent Document 7). Its inhibitors have been reported to be related to hyperuricemia, gout, heart disease, etc., and allopurinol, in particular, is used as a therapeutic agent for hyperuricemia and gout (Non-Patent Document 8). [Prior art documents] [Non-patent literature]

[0004]

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[0005] The present invention has been made in view of the above-mentioned problems of the prior art, and aims to provide a novel cardiomyocyte proliferation agent and a novel myocardial regeneration inducer based on a new mechanism of action. [Means for solving the problem]

[0006] The present inventors conducted extensive research to solve the above-mentioned problems. By comparing the metabolome and transcriptome of postnatal mouse and opossum hearts, they discovered that the purine nucleotide degradation pathway (xanthine metabolic pathway) is a novel pathway that induces cell cycle arrest in postnatal cardiomyocytes. Furthermore, through further detailed studies, the present inventors discovered for the first time that inhibiting the activity of xanthine oxidase, which is involved in the purine nucleotide degradation pathway, in the myocardium promotes cardiomyocyte proliferation and induces myocardial regeneration. Furthermore, they discovered that inhibiting xanthine oxidase activity in the myocardium maintains cardiomyocyte proliferation, suppresses oxidative DNA damage, inhibits cardiac hypertrophy and fibrosis, and restores cardiac function, leading to the completion of the present invention.

[0007] That is, the present invention relates to the following. [1] A cardiomyocyte proliferation agent comprising, as an active ingredient, a compound or a salt thereof that inhibits xanthine oxidase. [2] A myocardial regeneration inducer comprising, as an active ingredient, a compound or a salt thereof that inhibits xanthine oxidase. [3] The agent according to [1] or [2], wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton. [4] The agent according to [3], wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol. [5] A method for proliferating cardiomyocytes, which comprises contacting cardiomyocytes with a compound or a salt thereof that inhibits xanthine oxidase. [6] A method for inducing myocardial regeneration, which comprises contacting myocardium with a compound or a salt thereof that inhibits xanthine oxidase. [7] The method according to [5] or [6], wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton. [8] The method according to [7], wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a novel cardiomyocyte proliferation agent and a novel cardiomyocyte regeneration inducer having a mechanism of action different from that of the prior art. [Brief explanation of the drawings]

[0009] [Figure 1-1] FIG. 1 shows the results of metabolomic and transcriptomic analyses of postnatal mouse and opossum hearts. [Figure 1-2] FIG. 1 shows the results of metabolomic and transcriptomic analyses of postnatal mouse and opossum hearts. [Figure 1-3] FIG. 1 shows the results of metabolomic and transcriptomic analyses of postnatal mouse and opossum hearts. [Figure 2-1] FIG. 2 shows the results of metabolomic analysis of the hearts of newborn mice and opossums during cell cycle arrest. [Figure 2-2] FIG. 2 shows the results of metabolomic analysis of the hearts of newborn mice and opossums during cell cycle arrest. [Figure 3-1] FIG. 3 shows postnatal changes in the pentose phosphate pathway in mouse hearts. [Figure 3-2] FIG. 3 shows postnatal changes in the pentose phosphate pathway in mouse hearts. [Figure 4] FIG. 4 shows the reduction of PPP in postnatal mouse hearts by NAC administration. [Figure 5-1]FIG. 5 shows redox-dependent xanthine synthesis in the postnatal heart. [Figure 5-2] FIG. 5 shows redox-dependent xanthine synthesis in the postnatal heart. [Figure 6] FIG. 6 shows that inhibition of postnatal fatty acid metabolism expanded the cell cycle window of postnatal cardiomyocytes. [Figure 7-1] FIG. 7 shows that inhibition of postnatal fatty acid oxidation did not alter xanthine metabolism. [Figure 7-2] FIG. 7 shows that inhibition of postnatal fatty acid oxidation did not alter xanthine metabolism. [Figure 8] FIG. 8 shows postnatal changes in adenine-derived DNA building blocks. [Figure 9] FIG. 9 shows changes in pyrimidine metabolism in postnatal mouse hearts. [Figure 10] FIG. 10 shows the effect of allopurinol treatment on neonatal cardiomyocyte proliferation. [Figure 11-1] FIG. 11 shows the extension of the postnatal cardiomyocyte proliferation window by xanthine oxidase inhibition. [Figure 11-2] FIG. 11 shows the extension of the postnatal cardiomyocyte proliferation window by xanthine oxidase inhibition. [Figure 12] FIG. 12 shows the effect of allopurinol treatment on the postnatal mouse heart. [Figure 13-1] FIG. 13 shows that xanthine oxidase inhibition expanded the window of postnatal cardiac regenerative capacity. [Figure 13-2] FIG. 13 shows that xanthine oxidase inhibition expanded the window of postnatal cardiac regenerative capacity. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited to this.

[0011] (definition) As used herein, "cardiomyocyte proliferation" means the proliferation of cardiac muscle cells through division.

[0012] As used herein, "myocardial regeneration" refers to the process by which cardiomyocytes that form cardiac muscle tissue (myocardium) divide and proliferate to produce new cells. In the present invention, induction of myocardial regeneration occurs when the proliferation of cardiomyocytes that form the myocardium results in the induction of myocardial regeneration.

[0013] As used herein, the term "xanthine oxidase" refers to one of the xanthine oxidoreductases that generate reactive oxygen species, and is a rate-limiting enzyme that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid.

[0014] As used herein, the term "compound that inhibits xanthine oxidase" refers to a compound that has the effect of suppressing the function of xanthine oxidase as defined above.

[0015] As used herein, the term "salt of a compound that inhibits xanthine oxidase" refers to the salt form of a compound that inhibits xanthine oxidase as defined above.

[0016] As used herein, "treatment" refers to the act of producing a therapeutic effect (e.g., a desired pharmacological and / or physiological effect) in a subject in need of treatment. The therapeutic effect may be prophylactic, in the sense of completely or partially preventing a disease or its symptoms, or therapeutic, in the sense of partially or completely curing the disease and / or side effects caused by the disease. "Treatment" includes, for example, the following: (1) preventing the onset of a disease; (2) delaying the onset of a disease; (3) alleviating a disease; (4) preventing the progression of a disease; and (5) delaying the progression of a disease.

[0017] As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient sufficient to effectively treat a disease when administered to a subject. The "therapeutically effective amount" may vary depending on the type of active ingredient, the disease and its severity, and the age, weight, etc. of the subject to be treated.

[0018] As used herein, the term "subject" refers to a test specimen to which the compound or its salt that inhibits xanthine oxidase, the cardiomyocyte proliferation agent, or the cardiomyocyte regeneration inducer of the present invention is applied, and includes, but is not limited to, rodents (e.g., rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs), etc. In one embodiment of the present invention, the "subject" is preferably a human.

[0019] (Xanthine oxidase inhibitor compound or its salt) In one embodiment of the present invention, the compound that inhibits xanthine oxidase is preferably a compound having a pyrazolopyrimidine skeleton. The compound having a pyrazolopyrimidine skeleton may be a compound that competitively inhibits xanthine oxidase.

[0020] Compounds that inhibit xanthine oxidase are preferably compounds having a pyrazolopyrimidine skeleton, such as allopurinol, tisopurine, oxypurinol, etc. Among these, allopurinol is particularly advantageous for medical applications because it has a proven track record of clinical treatment for other diseases.

[0021] Allopurinol, chemically known as 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, is a structural isomer of hypoxanthine. It has been used for many years as a drug for treating gout and hyperuricemia because it inhibits the activity of xanthine oxidase, thereby suppressing the production of uric acid in the body and preventing gout attacks.

[0022] The cardiomyocyte proliferation agent and cardiomyocyte inducer of the present invention may contain one type of compound that inhibits xanthine oxidase, or may contain two or more types of compounds.

[0023] The compound inhibiting xanthine oxidase may form a salt, and examples of such salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Suitable examples of metal salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; and aluminum salts. Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc.

[0024] Among these, pharmaceutically acceptable salts are preferred, including inorganic salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, barium salt, etc.); ammonium salts; salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.; and salts with organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.

[0025] (Cardiomyocyte proliferation agent and myocardial regeneration inducer) The cardiomyocyte proliferation agent and myocardial regeneration inducer of the present invention (hereinafter sometimes referred to as "the agent of the present invention") contains, as an active ingredient, a compound that inhibits xanthine oxidase or a salt thereof.

[0026] As described above, a compound or a salt thereof that inhibits xanthine oxidase has a cardiomyocyte proliferation effect, and therefore, by containing the compound, it can be used as a cardiomyocyte proliferation agent.Furthermore, since the compound or a salt thereof that inhibits xanthine oxidase induces cardiomyocyte proliferation and thereby induces myocardial regeneration, it can be used as a myocardial regeneration inducer.

[0027] In one embodiment of the present invention, the myocardial regeneration inducer can also be referred to as a myocardial regeneration inducer based on cardiomyocyte proliferation.

[0028] The agent of the present invention may contain any other active ingredient in addition to the compound or its salt that inhibits xanthine oxidase as an active ingredient.

[0029] The pharmaceutical preparations of the present invention may contain, as an active ingredient, a compound or salt thereof that inhibits xanthine oxidase, as well as any other substance commonly used in the art (e.g., a pharmaceutically acceptable carrier). Examples of such substances include, but are not limited to, excipients, binders, disintegrants, emulsifiers, solubilizers, dispersants, lubricants, coating agents, colorants, stabilizers, and isotonicity agents. Examples of excipients include sugars such as lactose, sucrose, and glucose; inorganic substances such as starch, calcium carbonate, and calcium sulfate; crystalline cellulose; distilled water; purified water; sesame oil, soybean oil, corn oil, olive oil, and cottonseed oil, which are commonly used in pharmaceutical preparations.

[0030] The disease to be treated in the present invention is not particularly limited as long as it can be treated by the agent of the present invention, but is preferably a degenerative heart disease. More specifically, examples include heart failure, familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and ischemic cardiomyopathy such as myocardial infarction. Further examples include diseases accompanied by necrosis of myocardial tissue, including myocardial injury due to cardiac trauma and myocarditis.

[0031] The route of administration of the agent of the present invention is not particularly limited as long as it is an administration method that allows localization in the heart, and may be oral or parenteral. Parenteral administration includes, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, and intrarectal administration, with intravenous administration being preferred.

[0032] The dosage form can be appropriately selected depending on the purpose and subject of use, and can be, for example, an injection (liquid, suspension, etc.), tablet, pill, powder, liquid, suspension, emulsion, granule, capsule, etc. These dosage forms can be produced by methods conventionally known in the field of pharmaceutical formulations.

[0033] The dosage of the agent of the present invention can be appropriately determined depending on various factors such as the subject's age, body weight, general health, sex, diet, time of administration, route of administration, duration of treatment, other active ingredients used in combination, other relevant factors well known in the medical field, etc. Those skilled in the art can determine, by routine experimentation, an effective and non-toxic dosage required to treat the applicable condition.

[0034] The dosage of the agent of the present invention can be, for example, 0.01 to 3000 mg / day, preferably 0.1 to 2000 mg / day, more preferably 1 to 1000 mg / day.

[0035] (Method for Proliferating Cardiomyocytes and Method for Inducing Cardiomyocyte Regeneration) One embodiment of the present invention provides a method for proliferating cardiomyocytes, which comprises contacting cardiomyocytes with a compound or a salt thereof that inhibits xanthine oxidase. Another embodiment of the present invention provides a method for inducing myocardial regeneration, which comprises contacting cardiac muscle with a compound or a salt thereof that inhibits xanthine oxidase.

[0036] The compound or its salt that inhibits xanthine oxidase may be contacted with cardiomyocytes or cardiac muscle in vivo, in vitro, or ex vivo.

[0037] When the contact is performed in vivo, the contacting method is, for example, by the administration route described above in the section (Cardiomyocyte proliferation agent and myocardial regeneration inducer). When the contact is performed in vivo, the dosage is, for example, the amount described above in the section (Cardiomyocyte proliferation agent and myocardial regeneration inducer).

[0038] When the contact is carried out in vitro or ex vivo, it can be carried out, for example, by adding a compound or its salt that inhibits xanthine oxidase to a culture medium in a culture vessel that the myocardium is cultured in. When the contact is carried out in vitro or ex vivo, the compound or its salt that inhibits xanthine oxidase is added in an amount of, for example, 0.001 μmol / L to 100 mmol / L, preferably 0.1 μmol / L to 10 mmol / L, as a solution containing the compound or its salt.

[0039] Furthermore, when the contact is performed in vitro, the cardiomyocytes may be those extracted from a living body, or those prepared from pluripotent stem cells, etc., when the contact is performed ex vivo.

[0040] Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem cells (ntES cells) obtained by nuclear transfer, germline stem cells (GS cells), embryonic germ cells (EG cells), and induced pluripotent stem (iPS) cells.

[0041] Cardiomyocytes proliferated in vitro by contact with a compound or its salt that inhibits xanthine oxidase, and myocardium in which regeneration has been induced by contact with a compound or its salt that inhibits xanthine oxidase ex vivo, can be used, for example, as materials for regenerative medicine.

[0042] (Drug for maintaining proliferation ability of cardiomyocytes and method thereof) In one embodiment of the present invention, there is provided a drug for maintaining the proliferation ability of cardiomyocytes, which comprises, as an active ingredient, a compound or a salt thereof that inhibits xanthine oxidase. In another embodiment of the present invention, there is provided a method for maintaining the proliferation ability of cardiomyocytes, which comprises contacting cardiomyocytes with a compound or a salt thereof that inhibits xanthine oxidase.

[0043] The proliferation ability of cardiomyocytes can be maintained by the action of a compound or a salt thereof that inhibits xanthine oxidase.

[0044] (kit) In one embodiment of the present invention, there is provided a kit for cardiomyocyte proliferation, comprising a compound or a salt thereof that inhibits xanthine oxidase. The kit of the present invention is used, for example, for in vitro testing and research purposes.

[0045] As the compound or salt thereof that inhibits xanthine oxidase, for example, those described above in the section (Compound or salt thereof that inhibits xanthine oxidase) can be used.

[0046] In one embodiment of the present invention, the kit may include cardiomyocytes. Such cardiomyocytes may be derived from myocardium extracted from a living body, or may be prepared from pluripotent stem cells or the like.

[0047] As the pluripotent stem cells, for example, those described in the above section (Method for Proliferating Cardiomyocytes and Method for Inducing Cardiomyocyte Regeneration) can be used.

[0048] The cardiomyocytes may be, for example, cardiomyocytes generated from pluripotent stem cells derived from a patient with a genetic factor that causes abnormalities in cardiac morphology or function. By including such cardiomyocytes, the kit of the present invention is suitable for use in testing and research on the genetic factor.

[0049] In one embodiment of the present invention, the kit may also include instructions describing how to use the kit.

[0050] (others) In one embodiment of the present invention, there is provided: [Item 1a] A method for treating heart disease, which comprises administering to a subject a therapeutically effective amount of a compound or a salt thereof that inhibits xanthine oxidase. [Item 2a] A method for inducing myocardial regeneration, which comprises administering to a subject a therapeutically effective amount of a compound or a salt thereof that inhibits xanthine oxidase. [Item 3a] The method according to [Item 1a] or [Item 2a], wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton. [Item 4a] The method according to [Item 3a], wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol.

[0051] In this embodiment, the compound or its salt that inhibits xanthine oxidase may be administered to a subject in the form of a cardiomyocyte proliferation agent or cardiomyocyte regeneration inducer containing the compound.

[0052] In another embodiment of the present invention, the following is provided: [Item 1b] Use of a compound or a salt thereof that inhibits xanthine oxidase in the manufacture of a medicament for treating heart disease. [Item 2b] Use of a compound or a salt thereof that inhibits xanthine oxidase in the manufacture of a medicament for inducing myocardial regeneration. [Item 3b] The use according to [Item 1b] or [Item 2b], wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton. [Item 4b] The use according to [Item 3b], wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol.

[0053] Furthermore, in another embodiment of the present invention, the following is provided: [Item 1c] A compound or a salt thereof that inhibits xanthine oxidase for use in treating heart disease. [Item 2c] A compound or a salt thereof that inhibits xanthine oxidase for use in inducing myocardial regeneration. [Item 3c] The compound or salt thereof that inhibits xanthine oxidase according to [Item 1c] or [Item 2c], wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton. [Item 4c] The compound or salt thereof inhibiting xanthine oxidase according to [Item 3c], wherein the compound inhibiting xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol. [Example]

[0054] The present invention will be described in more detail below using examples, but these are not intended to limit the scope of the present invention. Note that all references cited throughout this specification are incorporated herein by reference in their entirety.

[0055] (1) Mouse All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the RIKEN Kobe Institute and were performed using age-matched CD1(ICR) mice (provided by the RIKEN Institute for Animal Resources and Genetic Engineering and Oriental Yeast Co., Ltd.) or opossums (provided by the RIKEN Institute for Animal Resources and Genetic Engineering).

[0056] (2) Medication N-acetyl-L-cysteine ​​(Sigma, A7250) was reconstituted in PBS. Allopurinol (Wako, 011-12501) and sodium dichloroacetate (DCA, Sigma, 347795) were reconstituted in dimethyl sulfoxide (DMSO) diluted 1:10 with PBS. NAC, allopurinol, and DCA were subcutaneously injected into CD1 mice at daily doses of 75 mg / kg, 50 mg / kg, and 100 mg / kg, respectively, from postnatal day 0 (P0) to P7 or P14. For myocardial infarction (MI) experiments, allopurinol was injected into CD1 mice from P0 to P14, and MI was surgically induced at P7. Allopurinol was injected into control mice at P7 after MI induction, and also at P8 and P9. Echocardiography was performed at P28, and the hearts were excised.

[0057] (3) Surgical induction of myocardial infarction in neonatal mice Neonates were anesthetized by placing them in a plastic bag with air holes and placing the bag on ice to induce hypothermia. After anesthesia, myocardial infarction was induced in P7 neonatal mice by making a lateral chest incision and permanently ligating the left anterior descending coronary artery with 6-0 polypropylene sutures (Ethicon, EP8707). The chest wall incision was sutured with 6-0 polypropylene, and the external incision was sealed with skin adhesive (3M Vetbond). Neonates were warmed on a heating plate (42°C) until resuscitation. Left ventricular systolic function was measured in unsedated mice by echocardiography using an Affiniti 50 (Phillips) equipped with an L15-7 iotransducer.

[0058] (4) Immunohistochemistry Tissues were fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4°C, followed by freeze-thawing. They were embedded in tissue freezing medium and sectioned at 8 μm thickness. After antigen retrieval using epitope retrieval solution (IHC World) in 1 mM EDTA / 0.05% Tween 20 in boiling water or using a steamer (IHC-Tek Epitope Retrieval Streamer Set), sections were blocked with 10% host animal serum and incubated with primary antibodies overnight at 4°C. Subsequently, sections were washed with PBS and incubated with the corresponding secondary antibodies at room temperature for 1 hour. For 8OHG 454 staining, sections were blocked with the MOM Immunodetection Kit, Basic (Vector Laboratories), incubated with primary antibodies for 2 days at 4°C, and then incubated with alpaca recombinant secondary antibodies overnight at 4°C. The primary antibodies used were: Ser10-phosphorylated histone H3 (Millipore, 06-570; 1:100 dilution), cardiac troponin T (BD Pharmingen, 564766; 1:250), anti-sarcomeric α-actinin (Abcam, ab68167; 1:100), and anti-8-hydroxyguanosine (Abcam, ab62623; 1:50). Immune complexes were stained with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies (Invitrogen), Alexa Fluor 488- or Alexa Fluor 568-conjugated alpaca recombinant secondary antibodies (Invitrogen), or Alexa Fluor 647-conjugated wheat germ agglutinin (ThermoFisher Scientific, W32466; 50 μg / mL). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Nacalai Tesque, 11034-56). Slides were mounted with PLUS antifade mounting medium (Vector Laboratories). Histological images were captured using an X53 (Evident) or APX100 (Evident) microscope, and confocal images were acquired using an LSM800 microscope (Zeiss).

[0059] (5) Quantification of cardiomyocyte size Heart sections were stained with Alexa Fluor 488-conjugated wheat germ agglutinin (ThermoFisher Scientific, W11261; 50 μg / mL) to visualize cell edges and imaged with BX53 or APX100 across the left ventricle, right ventricle, basal, apical, and mid-septum. Cardiomyocyte size was quantified using ImageJ FIJI software53 (National Institutes of Health) by analyzing 500–1000 cells per group.

[0060] (6) Masson's trichrome staining Masson's trichrome staining was performed on 4% PFA-fixed frozen sections according to the manufacturer's protocol (ScyTek Laboratories, TRM-1). Histological images were taken using an APX100 (Evident) microscope. Quantification of the fibrotic area ratio in trichrome-stained sections was performed using ImageJ FIJI software. For each mouse, the average fibrotic area ratio was quantified for three sections at 600 μm intervals from the ligation site to the vertex.

[0061] (7) Metabolomic analysis For postnatal metabolomic comparisons between mouse and opossum hearts, hearts were harvested and snap-frozen. These frozen samples were analyzed by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS, Basic Scan, Human Metabolome Technologies Japan). Metabolite concentrations were calculated by absolute quantification. For P14 mouse hearts treated with NAC or DCA or postnatal mouse hearts, cardiac samples were immediately freeze-clamped using a pre-freezing clamp (Natsume Seisakusho, KN-838-S) (Reference 1). Briefly, metabolites were detected using an Orbitrap MS instrument (Q-Exactive focus, Thermo Fisher Scientific) connected to a high-performance IC system (ICS-5000+, Thermo Fisher Scientific). The obtained metabolomic data were analyzed using metaboanalyst 5.0 software. Heat maps were generated using Heatmapper.

[0062] (8) Enzyme activity measurement and uric acid measurement Enzyme activities were assessed using freeze-clamped mouse heart samples. Xanthine oxidase activity was measured using the Amplex™ Red Xanthine / Xanthine Oxidase Assay Kit (ThermoFisher Scientific, A22182), G6PD activity was measured using the Glucose-6-Phosphate Dehydrogenase Activity Assay Kit (Sigma, MAK015), and uric acid levels in cardiac lysates were measured using the Uric Acid Assay Kit (Sigma, MAK077) according to the respective manufacturer's protocols. Fluorescence and absorbance were measured using a multimode microplate reader (TACAN, Infinite 200).

[0063] (9) Cardiomyocyte culture Neonatal mouse ventricular cardiomyocytes were isolated from P1 mice using a Neonatal Heart Dissociation Kit for mice and rats (Miltenyi Biotec, 130-098-373) (10-15 hearts per isolation). Briefly, fibroblasts were transiently seeded twice onto uncoated dishes and incubated for 1.5 hours to remove fibroblasts. Isolated cardiomyocytes were then seeded onto fibronectin (Fibronectin Solution human, PromoCell, D13121)-coated 96-well plates and cultured for 2 days with allopurinol or control solvent (0.1%). Juvenile mouse cardiomyocytes were isolated from 514 P28 mice (one heart per isolation) according to a previously published protocol (Reference 2). Isolated cardiomyocytes were cultured for 2 days with allopurinol or control solvent (0.1% DMSO / PBS).

[0064] (10) EdU incorporation assay and immunocytochemistry For the primary cardiomyocyte proliferation assay based on EdU incorporation, we used the Click-it EdU Imaging Kit (ThermoFisher Scientific, C10337) according to the manufacturer's protocol. Briefly, cultured neonatal cardiomyocytes were incubated in culture medium containing 10 μM EdU solution for 2 days, after which the cardiomyocytes were fixed with 4% PFA for 10 minutes. After fixation, the cells were blocked with 5% serum from the host animal of the secondary antibody and incubated overnight at 4°C with anti-pericentriolar material 1 (Sigma, HPA023370; 1:2000 dilution) primary antibody. Subsequently, the cells were washed with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody (Invitrogen) for 1 hour at room temperature. After immunocytochemistry, EdU labeling was performed according to the manufacturer's protocol, and nuclei were stained with DAPI. Cell images were captured using an APX100 (Evident) microscope. The number of cardiomyocyte nuclei surrounded by PCM1 was quantified using ImageJ FIJI software (National Institutes of Health, USA).The number of EdU+ cardiomyocyte nuclei within the defined area was then counted to calculate the frequency of EdU+ cardiomyocytes.

[0065] (11) Cytoplasmic ROS assay Cytosolic ROS was assessed using CellROX Deep Red reagent (ThermoFisher Scientific, C10422) according to the manufacturer's protocol. Briefly, cultured immature cardiomyocytes were incubated in culture medium containing 5 μM CellROX Deep Red reagent for 30 minutes. After CellROX staining, the medium was replaced with FluoroBrite™ DMEM (Gibco, A1896701), and images were captured using an APX100 (Evident). The mean signal intensity of CellROX Deep Red was measured using ImageJ FIJI software53 (National Institutes of Health). The fluorescent signal of rod-shaped cardiomyocytes was quantified across multiple wells in each group.

[0066] (12) Quantification and statistical analysis Statistical evaluation was generally performed using GraphPad Prism 9, with unpaired t-tests for comparisons between two groups and one-way analysis of variance (ANOVA) with Tukey's test for comparisons between multiple groups. For comparisons between different species, independent statistical analysis was performed on the intraspecies variation only. Error bars are expressed as mean ± SEM. No data or samples were excluded throughout the study.

[0067] Interspecies comparison of postnatal metabolome during cell cycle arrest To identify unknown metabolic mechanisms controlling postnatal cardiomyocyte cell cycle arrest, we attempted metabolomic profiling of postnatal mammalian hearts. Several studies have performed comprehensive metabolomic profiling of postnatal mammalian hearts (Refs. 3, 4). However, identifying metabolic pathways that directly control cardiomyocyte cell cycle progression has been challenging, given the dramatic and multifaceted nature of postnatal metabolic switching in the heart. Recently, we discovered that cardiomyocyte cell cycle arrest and loss of regenerative capacity occur 2–4 weeks after birth in the marsupial gray short-tailed opossum (Monodelphis domestica, hereafter simply referred to as "opossum"). Taking advantage of this exceptionally long proliferation period in the opossum heart, we aimed to distinguish metabolic pathways that directly control cardiomyocyte cell cycle arrest from broader postnatal metabolic changes. Our approach is based on the hypothesis that metabolic pathways controlling the cell cycle remain unchanged at birth but should change in the opossum heart simultaneously with cardiomyocyte cell cycle withdrawal.

[0068] We therefore compared the metabolomic profiles of mouse hearts at postnatal day 1 (P1) and postnatal day 14 (P14) with those of opossum hearts at postnatal day 28 (P28) (Figure 1A, Figure 2A-H). PCA analysis revealed that metabolites in opossum hearts changed similarly to those in mouse hearts during postnatal development (Figure 1B, PC1). Consistent with changes in cardiomyocyte proliferation rates (ref. 5), the postnatal developmental axis (PC1) of P14 opossum hearts resembled P1 mouse hearts, and that of postnatal day 56 (P56) opossum hearts resembled P14 mouse hearts (Figure 1B). Nearly half of the metabolites significantly increased in mouse hearts (Figure 1C) were also increased in opossum hearts (Figure 1D), and vice versa. Meanwhile, only a few decreased metabolites were common to both species (Figure 1C-D). Pathway analysis of commonly altered metabolites revealed significant changes in purine metabolism ( Fig. 1E ).

[0069] Because metabolic flux is not simply reflected by the abundance of individual metabolites, we performed an integrated omics analysis of the transcriptome and metabolome in postnatal mouse hearts. The top five pathways based on the pathway impact score of the integrated omics analysis included purine metabolism, as well as metabolic pathways related to cardiomyocyte cell cycle arrest, such as the TCA cycle, glycolysis, and pyruvate metabolism (Ref. 6) (Figure 1F). These results prompted us to investigate the dynamics of purine nucleotide metabolism in postnatal hearts. First, we compared metabolites (Figure 1G) and genes encoding enzymes involved in purine metabolism (Figure 1H; some genes lack their counterparts in opossums) in postnatal mouse and opossum hearts, revealing significant changes in purine metabolism in both species. Quantifying the expression of these metabolites and genes encoding purine metabolic pathway enzymes over time revealed that expression of the adenine nucleotide degradation pathway (adenine to uric acid) consistently increased after birth (Figure 1I-J). The only exception was the expression of Hprt, which encodes an enzyme controlling the salvage pathway that converts hypoxanthine to IMP (Figure 1J, L). On the other hand, adenosine levels increased in P14 mouse hearts compared with P1 mouse hearts, while adenine levels remained unchanged, suggesting no change in adenine synthesis from AMP (Figure 2I). Furthermore, expression of Ada, which encodes an enzyme controlling inosine synthesis from adenosine, remained unchanged (Figure 2J), suggesting that adenosine-mediated xanthine production, unlike IMP-mediated xanthine production, is not upregulated in mouse hearts from P1 to P14. Furthermore, a postnatal increase in XO enzyme activity was detected in mouse hearts from P1 to P14 (Figure 1K). These interspecies comparisons of metabolic changes identified a dramatic and evolutionarily conserved activation of hypoxanthine- and xanthine-mediated adenine nucleotide degradation during the cardiomyocyte cell cycle in postnatal mammalian hearts (Figure 1L).

[0070] Postnatal metabolic shift in PPP Our data indicate that during cardiomyocyte cell cycle arrest, adenine nucleotide catabolism is activated without significant changes in its salvage pathway. To comprehensively assess purine nucleotide homeostasis, we analyzed de novo purine biosynthesis, primarily mediated by the PPP, in postnatal hearts. Interestingly, the PPP was the second most significantly altered metabolic pathway in postnatal hearts based on the P value of cross-species metabolome analysis (Figure 1E), and it showed the highest Pathway Impact score in integrated analysis of the transcriptome and metabolome of mouse hearts (Figure 1F). Therefore, we performed a time-course quantification of PPP intermediate metabolites in postnatal mouse hearts (Figure 3). Most PPP metabolites, except for fructose-6-phosphate (F6P), were decreased at P14 compared to P1 (Figure 3A-B). This is consistent with the decreased demand for DNA synthesis in the heart as cell proliferation decreases from P1 to P14. F6P is an intermediate metabolite in the pathway back to glycolysis rather than de novo nucleotide synthesis, as shown in the schematic diagram in Figure 3G, suggesting that de novo nucleotide synthesis is reduced in the postnatal heart. The enzymatic activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme mediating the first step of PPP21, was reduced in P14 mouse hearts, further supporting a reduction in PPP flux (Figure 3C).

[0071] G6PD not only initiates the first step of de novo purine nucleotide biosynthesis but also synthesizes NADPH. NADPH is an essential molecule for maintaining the cellular redox state by providing most of the cellular reduction potential (Ref. 7). We hypothesized that a decline in G6PD activity after birth would shift the redox state of cardiomyocytes toward oxidation. To test this, we measured the redox state of glutathione, a major physiological antioxidant and an indicator of cytosolic redox state, since the majority of glutathione molecules are localized in the cytosol (Ref. 8). Oxidized glutathione (GSSG) levels significantly increased in the heart over the first 2 weeks after birth, while reduced glutathione (GSH) levels significantly decreased (Figure 3D–F), as shown in previous studies (Ref. 9). These results indicate that the cytosolic redox state in the postnatal heart shifts toward oxidation over time, likely due to a decline in PPP activity. This postnatal downregulation of the PPP is likely a result of reduced DNA demand as cardiomyocytes withdraw from the cell cycle ( Fig. 3G ).

[0072] Xanthine metabolism is regulated by cellular redox state in the postnatal heart Our data clearly demonstrate that although de novo purine nucleotide biosynthesis declines as cardiomyocytes exit the cell cycle shortly after birth, purine nucleotide degradation continuously increases in the heart throughout the first month of life. Therefore, we hypothesized that despite the decreased PPP in the postnatal heart, de novo synthesis of purine nucleotides continues as a by-product of G6PD-mediated NADPH synthesis in response to increased oxidative status in myocardial tissue. To assess whether the redox status further downregulates PPP in postnatal hearts, we injected neonatal mice with N-acetylcysteine ​​(NAC), a glutathione precursor reported to reduce oxidative status in postnatal mouse hearts (Figure 4A). We then analyzed the metabolomic profile of P14 hearts (Figure 4B). NAC treatment decreased PPP metabolites, including 6-phosphogluconate (6PG), ribulose-5-phosphate (Ribulose5P), xylulose-5-phosphate (Xylulose5P), and ribose-5-phosphate (Ribose5P) (Figures 4C-D). G6PD activity tended to decrease, but did not reach statistical significance, likely due to considerable variability across samples (Figure 4E). These results suggest that postnatal PPP activity declines in parallel with the decrease in DNA demand, but de novo nucleotide synthesis via the PPP persists because NADPH is required to maintain a cytoplasmic reducing state (Figures 3G and 4F).

[0073] Previous studies have shown that cytosolic oxidation is associated with the induction of xanthine metabolism in cryopreserved human red blood cells (Ref. 10). To investigate whether oxidation is a driver of xanthine metabolic activation in postnatal myocardium, we analyzed metabolites related to xanthine metabolism in NAC-treated P14 hearts. NAC treatment significantly altered purine metabolism (Figure 5A). Importantly, several intermediate metabolites of xanthine metabolism, such as IMP and hypoxanthine, were significantly decreased in NAC-treated hearts (Figure 5B). Detailed analysis revealed that most metabolites in the xanthine metabolic pathway were decreased in P14 NAC-treated hearts (Figure 5C-D). These results suggest that reducing the redox state of postnatal hearts by glutathione supplementation reverses the postnatal activation of purine nucleotide degradation (Figure 5E), thus highlighting the role of cellular oxidation as a key factor promoting xanthine metabolism in postnatal hearts.

[0074] Mitochondrial oxidation of fatty acids is a key trigger for cardiomyocyte cell cycle arrest in the postnatal heart through the induction of oxidative stress (Ref. 11). Therefore, we evaluated the relationship between mitochondrial fatty acid oxidation and xanthine metabolism in the postnatal heart. Pyruvate dehydrogenase kinase isoform 4 (PDK4) plays a central role in the cardiac switch from glucose oxidation to fatty acid oxidation by redirecting pyruvate from the cytosol to mitochondria (Refs. 12, 13). Previous studies have shown that the PDK4 inhibitor dichloroacetic acid (DCA) promotes glucose oxidation, suppresses mitochondrial fatty acid oxidation, reduces mitochondrial reactive oxygen species, and induces cell cycle reentry in adult mouse cardiomyocytes (Ref. 11). This prompted us to examine whether DCA treatment could extend the postnatal period of cardiomyocyte proliferation in neonatal mice (Figure 6A). DCA treatment increased the number of proliferative cardiomyocytes at P14 (Figures 6B-C), suggesting that mitochondrial fatty acid utilization regulates cardiomyocyte proliferation in the neonatal heart. Metabolomic profiling of DCA-treated hearts at P14 revealed that DCA primarily affected the glucose-alanine cycle (Ref. 14), reflecting the altered balance between glycolysis and fatty acid oxidation due to PDK4 inhibition (Figure 7A-D). However, neither the PPP nor xanthine metabolism was affected by DCA treatment (Figure 7E-F). Because NADPH flux, a key determinant of cellular reductive capacity, is independently regulated in the cytosol and mitochondria (Ref. 15), activation of xanthine metabolism in postnatal hearts may be induced by cytosolic ROS rather than mitochondrial ROS.

[0075] To gain further mechanistic insight into changes in DNA demand and purine metabolism in the postnatal heart, we examined deoxyadenosine triphosphate (dATP), a component of DNA synthesis (Ref. 16) (Figure 8). Concurrent with the decline in cardiac cell proliferation and DNA demand from P1 to P14, dATP levels in mouse hearts significantly decreased (Figure 8A). NAC treatment increased dATP levels in P14 hearts (Figure 8B), and DCA treatment also showed a trend toward increased dATP levels (Figure 8C), consistent with the extension of the cardiomyocyte proliferation window (Ref. 9) (Figure 6). These results suggest that DNA demand, unlike cellular oxidative status, may not determine xanthine metabolic flux (Figures 4, 7, and 8).

[0076] We also examined pyrimidine metabolism in postnatal mouse hearts (Figure 9). Intermediate metabolites in the de novo pyrimidine biosynthetic pathway, such as carbamoyl-aspartate, orotate, UMP, and TMP, decreased from P1 to P14 (Figure 9A). Concomitantly, expression of Tk1, a gene encoding the cytosolic form of thymidine kinase that mediates the pyrimidine salvage pathway, recycling thymidine (17) to TMP, also decreased significantly (Figure 9B). Thymidine, a substrate of thymidine kinase, also decreased postnatally (Figure 9C). In contrast, uridine and uracil, intermediate metabolites in the pyrimidine catabolic pathway, increased, while dihydrouracil, a downstream metabolite, significantly decreased (Figure 9D). These findings suggest that pyrimidine metabolism in postnatal hearts exhibits decreased synthesis and increased degradation, consistent with the trends observed in purine metabolism.

[0077] Xanthine oxidase induces cell cycle arrest in cardiomyocytes Previous studies have highlighted the important role of mitochondrial ROS in cell cycle regulation in postnatal cardiomyocytes (Refs. 6, 9). Given that xanthine oxidase is an important source of ROS in the cytosol (Ref. 18), we hypothesized that cytosolic ROS generated by xanthine oxidase might induce cell cycle exit in postnatal cardiomyocytes. First, we evaluated the effect of xanthine oxidase inhibition with allopurinol (Ref. 19), a purine analog widely used in clinical practice, in primary cultures of neonatal mouse ventricular cardiomyocytes (NMVCs; Figure 10A). Allopurinol treatment dose-dependently increased EdU incorporation in cardiomyocytes (Figure 10B). Next, we investigated whether activation of xanthine metabolism contributes to cell cycle arrest in postnatal cardiomyocytes in vivo (Figure 11A). Uric acid levels were reduced by allopurinol treatment in P14 hearts (Figure 11B). This suggests that cardiac xanthine oxidase activity is suppressed. Allopurinol administration did not affect heart weight at P7 and P14 (Figures 11C and 12A) but significantly reduced cardiomyocyte size (Figure 11D). Importantly, inhibition of xanthine oxidase by allopurinol was sufficient to reduce oxidative stress in postnatal cardiomyocytes (Figure 11E). Furthermore, allopurinol treatment increased cardiomyocyte proliferation at both P7 and P14 (Figure 11F). These results suggest that postnatal activation of xanthine oxidase causes cell cycle arrest in cardiomyocytes via the induction of oxidative DNA damage.

[0078] Inhibition of xanthine oxidase can extend the window for cardiac regeneration Given the strong correlation between cardiac regeneration and cardiomyocyte proliferation (Refs. 20, 21-23), we investigated whether allopurinol treatment preserves postnatal cardiac regeneration. Neonatal mice were injected with allopurinol daily for 14 days, and myocardial infarction (MI) was induced at P7. This abolished regenerative capacity (Figure 13A). Xanthine oxidase has been reported as an important source of reactive oxygen species that induces myocardial injury after MI and ischemia-reperfusion injury (Refs. 24, 25). To distinguish between the acute protective effect of xanthine oxidase inhibition and a true regenerative response, control mice were also treated with allopurinol for 2 days after MI (Figure 13A). Cardiomyocyte proliferation at 7 days postinfarction (dpi) was significantly enhanced in the allopurinol-treated group compared with the control group (Figure 13B). Furthermore, at 21 dpi, allopurinol treatment significantly suppressed cardiomyocyte hypertrophy (Figure 13C) and myocardial fibrosis (Figure 13D). Consistent with these histological changes, echocardiographic analysis showed significant improvements in left ventricular systolic function and dilation in the allopurinol-treated group (Fig. 13E).

[0079] To investigate whether inhibition of xanthine oxidase reduces cytosolic ROS levels in immature cardiomyocytes, cardiomyocytes were isolated from P28 mice, cultured, and then treated with allopurinol. Allopurinol-treated immature cardiomyocytes showed a significant decrease in cytosolic ROS levels (Figure 13F). These results suggest that postnatal xanthine metabolism contributes to the loss of postnatal cardiac regenerative potential through the production of cytosolic ROS by xanthine oxidase. [Industrial Applicability]

[0080] The present invention is useful in fields where proliferation of cardiomyocytes is desired, particularly in the field of myocardial regeneration therapy.

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Claims

1. A cardiomyocyte proliferation agent comprising, as an active ingredient, a compound or a salt thereof that inhibits xanthine oxidase.

2. A myocardial regeneration inducer comprising, as an active ingredient, a compound or a salt thereof that inhibits xanthine oxidase.

3. The agent according to claim 1 or 2, wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton.

4. The agent according to claim 3, wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol.

5. A method for proliferating cardiomyocytes, which comprises contacting cardiomyocytes with a compound or a salt thereof that inhibits xanthine oxidase.

6. A method for inducing myocardial regeneration, which comprises contacting myocardium with a compound or a salt thereof that inhibits xanthine oxidase.

7. The method according to claim 5 or 6, wherein the compound that inhibits xanthine oxidase is a compound having a pyrazolopyrimidine skeleton.

8. 8. The method of claim 7, wherein the compound that inhibits xanthine oxidase is at least one selected from allopurinol, tisopurine, and oxypurinol.

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