Methods for preserving and protecting cardiomyocytes and reducing myocardial fibrosis after cardiac injury

Abstract

A method for treating a patient who has experienced a myocardial infarction, also known as acute myocardial infarction, treats or reduces the effects of the infarction and can include administering a composition containing a protein substantially similar to or identical to pyruvate kinase M2 (PKM2) during the acute or early stages of the myocardial infarction.

Description

[Technical Field]

[0001] The present application generally relates to therapeutic methods for treating cardiovascular disorders. More specifically, the present application relates to compositions, systems, and methods for improving recovery and tissue repair after cardiac injury by protecting myocardial cells, and for reducing myocardial cell death induced by myocardial cell damage after myocardial injury. The present application also relates to pharmaceutical compositions for treating or preventing or reducing myocardial fibrosis after myocardial injury, more specifically, to pharmaceutical compositions for treating or preventing myocardial injury and associated myocardial fibrosis. [Background technology]

[0002] The significant loss of cardiomyocytes during heart attacks and the development of other cardiovascular diseases is associated with significant morbidity and mortality. Preservation of cardiomyocytes during cardiac injury may provide an effective strategy for managing these conditions. Acute myocardial infarction (MI) due to coronary artery disease often leads to maladaptive myocardial remodeling, leading to heart failure (HF).

[0003] Cardiomyocytes are the predominant cell type in the adult heart and are responsible for maintaining its function. Cardiomyocyte loss due to myocardial infarction is a significant factor in cardiac disease-related morbidity and mortality. The acute loss of cardiomyocytes caused by MI cannot be replaced, given the limited regenerative capacity of adult cardiomyocytes. Preserving cardiomyocytes is crucial to ensure patient survival after a heart attack. Cardiac cell death rapidly activates local tissue repair mechanisms, promoting the involvement of cardiac fibroblasts. Upon activation, cardiac fibroblasts produce extracellular matrix (ECM), primarily composed of collagen, to form scar tissue to prevent myocardial rupture after infarction. Nevertheless, the continued accumulation of ECM, especially collagen, interferes with normal myocardial function and frequently leads to heart failure. It is widely believed that ECM derived from activated cardiac fibroblasts replaces or fills the myocardial tissue damaged by dying cardiomyocytes. Dying cardiomyocytes and cardiac fibroblasts maintain a close communication relationship, which influences fibroblast activation during the MI process. This communication occurs primarily through paracrine signaling and is facilitated by the release of cytokines, hormones, and growth factors. Compelling evidence supports the idea that dying cardiomyocytes primarily activate cardiac fibroblasts, ultimately leading to myocardial fibrosis.

[0004] After fibrous scar tissue replaces myocardium damaged by hypertension, the heart's elasticity decreases, thereby impairing its function. Similarly, pulmonary fibrosis stiffens the lungs and impedes lung function. Fibrotic growth can spread to and impinge on healthy adjacent tissues and may persist even after the initial injury has healed. Fibrosis is typically a reactive process influenced by various factors. These factors include an early inflammatory response, a local surge in fibroblast populations, altered fibroblast synthetic function, and altered dynamics of collagen biosynthesis and degradation. Other influential aspects include inflammation of nearby tissues and a widespread inflammatory state characterized by increased circulating mediators. Unfortunately, no effective treatments exist to combat myocardial fibrosis or protect cardiomyocytes.

[0005] Thus, there is a need for systems and methods that protect or preserve cardiomyocytes and reduce associated myocardial fibrosis. It is to this need, among other things, that the present disclosure is directed. Summary of the Invention

[0006] The present application provides a treatment for acute cardiac injury, such as that resulting from a heart attack, and promotes cardiomyocyte proliferation. Specifically, the present application demonstrates that administration of a recombinant PKM2 mutant (e.g., G415R, as referenced in Yan et al. "SAICAR activates PKM2 in its dimeric form," published in Biochemistry, 2016 Aug 23;55(33):4731-4736), or recombinant PKM2 selectively assuming a dimeric form, or a protein identical or similar to pyruvate kinase M2 (PKM2), helps preserve cardiomyocytes, promotes cardiomyocyte proliferation, and reduces myocardial fibrosis. This is particularly evident during and after myocardial infarction, where it is associated with fibroblast recruitment. The dimeric form of PKM2 or a mutant thereof can be administered (e.g., acutely) as a treatment for a heart attack. The dimeric form can also be administered together with other forms (e.g., tetramers). A representative example of a recombinant PKM2 mutant suitable for the outlined method is G415R, which primarily adopts a dimeric form. Administration of this recombinant PKM2 mutant can prevent cardiomyocytes from undergoing apoptosis and promote their proliferation under conditions such as hypoxia and oxidative stress. This is applicable in situations such as myocardial infarction, myocardial ischemia-reperfusion injury (IR), various cardiomyopathies (including hypertrophic, dilated, and toxic cardiomyopathy), cardiotoxicity, congestive heart failure, and cardiac damage resulting from infections, whether viral or bacterial. While not wishing to be limited to a particular mechanism, the inventors believe that the dimeric form of PKM2 and its variants and mutants interact with integrin a in cardiomyocytes. vb3. This interaction activates the FAK-PI3K signaling axis, which subsequently inhibits downstream expression of phosphatase and tensin homolog (PTEN), thereby enhancing survival and proliferation.

[0007] One embodiment of the present application encompasses a method for treating acute cardiac injury caused by acute cardiomyocyte loss in a patient or subject. The method comprises administering to the subject a composition comprising pyruvate kinase M2 (PKM2) or a PKM2 mutant. More specifically, a composition of PKM2 or a PKM2 mutant that selectively dimerizes in an equilibrium state can be administered to the subject within 1 to 10 hours after cardiac injury.

[0008] Another embodiment involves the administration of recombinant PKM2 mutants (e.g., G415R) or recombinant PKM2 (rPKM2), or proteins similar or identical to pyruvate kinase M2 (PKM2), to preserve cardiomyocytes during myocardial infarction, indicating that rPKM2 (dimerized mutants) are therapeutic agents for treating heart attacks and other cardiovascular diseases. Administration of rPKM2 or its mutants protects cardiomyocytes from death and promotes cardiomyocyte proliferation. Administration of selectively dimerizing recombinant PKM2 (e.g., at doses that result in concentrations of rPKM2 in the patient's bloodstream of less than 1 micromolar to 5 micromolar) or mutants reduces cardiac fibroblast activation and thus inhibits fibrosis in mice with myocardial infarction and reperfusion injury.

[0009] Another embodiment provides an ischemia / reperfusion protection composition. The ischemia / reperfusion protection composition disclosed herein comprises a recombinant PKM2 mutant (e.g., G415R) or a recombinant PKM2 that selectively adopts a dimeric form or a protein similar or identical to pyruvate kinase M2 (PKM2).

[0010] Another embodiment includes a method of treating cardiac injury in a subject, the method comprising administering to the subject a therapeutically effective amount of either pyruvate kinase M2 (PKM2) or a PKM2 mutant within 10 hours of cardiac injury, wherein the PKM2 or PKM2 mutant can be a dimer.

[0011] Another embodiment includes a method wherein said PKM2 or PKM2 mutant dimerizes in a subject.

[0012] Further embodiments include methods wherein the administration occurs within 6 hours after cardiac injury, or within 3 hours after cardiac injury, or within 1 hour after cardiac injury.

[0013] Another embodiment includes a method wherein the subject has experienced a heart attack and the cardiac damage is due to the heart attack.

[0014] Another embodiment includes the method, wherein less than 50% of said PKM2 is in its tetrameric form.

[0015] Another embodiment includes a method wherein said PKM2 forms predominantly dimers at neutral pH.

[0016] Another embodiment includes a method wherein the composition comprises PKM2 or a mutant of PKM2 that preferentially adopts a dimeric state.

[0017] Another embodiment includes a method wherein the cardiac injury is caused by acute myocardial cell loss.

[0018] Another embodiment includes a method in which myocardial preservation is achieved by administration of the composition.

[0019] Another embodiment includes a method in which the composition is delivered extracellularly.

[0020] Another embodiment includes the method wherein the myocardial infarction is characterized as acute.

[0021] Another embodiment includes a method wherein the composition comprises a PKM2 mutant or a protein highly similar to wild-type pyruvate kinase M2.

[0022] Another embodiment includes a method wherein the PKM2 is derived from either a human or another animal.

[0023] Another embodiment includes a method wherein said PKM2 has a mutation that differs from said wild-type sequence.

[0024] Another embodiment includes a method wherein the composition is contained within a pharmaceutically acceptable carrier.

[0025] Another embodiment includes a method wherein the composition is delivered by intracardiac administration.

[0026] Another embodiment includes a method in which the composition is delivered systemically.

[0027] Another embodiment includes a method wherein the composition reduces myocardial cell death resulting from myocardial infarction.

[0028] Another embodiment includes a method wherein the composition reduces myocardial fibrosis in infarcted myocardium.

[0029] Another embodiment includes the method, wherein said pyruvate kinase M2 exists predominantly as a dimer as compared to its tetrameric form.

[0030] Another embodiment includes the method, wherein the PKM2 is characterized by a G415R mutation.

[0031] Another embodiment includes a method wherein said PKM2 or said PKM2 mutant is present in the extracellular space. [Brief explanation of the drawings]

[0032] [Figure 1A] 1 shows that recombinant G415R protected H9C2 cells from apoptosis under hypoxic conditions. [Figure 1B] We demonstrate that recombinant G415R promoted cell proliferation under hypoxic conditions. [Figure 1C]We show that recombinant G415R had a comparable effect on primary human cardiomyocytes and protected primary human cardiomyocytes from apoptosis under both hypoxic and oxidative stress conditions. [Figure 1D] We demonstrate that recombinant G415R promoted the proliferation of primary cardiomyocytes under conditions of hypoxia and oxidative stress. [Figure 1E] We demonstrate that recombinant G415R promoted the proliferation of primary cardiomyocytes under conditions of hypoxia and oxidative stress. [Figure 2A] We highlight that administration of recombinant G415R substantially reduced mortality in MI mice at all observed time points. [Figure 2B] Figure 1 shows that administration of recombinant G415R reduced heart weight (relative to body weight) in MI mice 30 days after infarction. [Figure 3A] Histological analysis of infarcted hearts shows that mice treated with recombinant G415R had smaller infarct scar size compared to rPKM1 and vehicle-treated groups in both MI and IR models. [Figure 3B] Histological analysis of infarcted hearts shows that mice treated with recombinant G415R had smaller infarct scar size compared to rPKM1 and vehicle-treated groups in both MI and IR models. [Figure 3C] As evidenced by TUNEL staining of cardiac tissue 6 hours after infarction, recombinant G415R protects cardiomyocytes from apoptosis. [Figure 3D] TUNEL staining of myocardial tissue 168 hours after infarction shows that minimal cardiomyocyte apoptosis was observed. [Figure 3E] 1 shows that in G415R-treated mice, proliferation of cardiomyocytes was evident in the myocardial tissue of both MI and IR models at 4 and 7 days after infarction. [Figure 3F] G415R treatment significantly lowers cTnI levels in the bloodstream of infarcted mice 7 days after infarction. [Figure 3G]As evidenced by TUNEL staining of cardiac tissue 24 hours after infarction, recombinant G415R protects cardiomyocytes from apoptosis. [Figure 3H] As evidenced by TUNEL staining of cardiac tissue at 168 hours after infarction, recombinant G415R protects cardiomyocytes from apoptosis. [Figure 4A] Wheat germ agglutinin (WGA) staining of myocardial tissue shows that mice treated with G415R had less infarct scar in the infarcted area of ​​the myocardium compared to the rPKM1 and vehicle-treated groups in both MI and IR models. [Figure 4B] Analysis of cardiomyocyte (cross-sectional area) by WGA and cTnI co-staining is shown, suggesting that recombinant G415R treatment reduced the cross-sectional area of ​​cardiomyocytes to a level similar to that of the sham group in MI mice. [Figure 4C] Analysis of cardiomyocyte (cross-sectional area) by WGA and cTnI co-staining is shown, suggesting that recombinant G415R treatment reduced the cross-sectional area of ​​cardiomyocytes to a level similar to that of the sham group in MI mice. [Figure 4D] Analysis of cardiomyocyte (cross-sectional area) by WGA and cTnI co-staining was shown, revealing that G415R treatment reduced the cross-sectional area of ​​cardiomyocytes to a level similar to that of the sham group in IR mice, indicating a significant reduction in cardiomyocyte hypertrophy. [Figure 4E] Analysis of cardiomyocyte (cross-sectional area) by WGA and cTnI co-staining was shown, revealing that G415R treatment reduced the cross-sectional area of ​​cardiomyocytes to a level similar to that of the sham group in IR mice, indicating a significant reduction in cardiomyocyte hypertrophy. [Figure 4F] Figure 1 shows that G415R reduced cardiac fibroblast activation in the infarcted myocardium of MI and IR mice (observed at day 30), as shown by IHC staining of α-SMA. [Figure 4G]Figure 1 shows that G415R reduced cardiac fibroblast activation in the infarcted myocardium of MI and IR mice (observed at day 30), as shown by IHC staining of α-SMA. [Figure 5A] Figure 1 shows that G415R reduced PTEN expression in cultured cardiomyocytes (proven by immunoblotting) from infarcted mice. [Figure 5B] 1 shows that G415R reduced PTEN expression in myocardial tissue (IHC staining) of infarcted mice. [Figure 5C] 1 shows that G415R reduced PTEN expression in both cultured cardiomyocytes (proven by immunoblotting) and myocardial tissue (IHC staining) from infarcted mice. [Figure 5D] 1 shows that G415R reduced PTEN expression in both cultured cardiomyocytes (proven by immunoblotting) and myocardial tissue (IHC staining) from infarcted mice.

[0033] definition The following definitions are provided to facilitate understanding of certain terms used throughout this disclosure.

[0034] The term "administration" refers to providing or delivering a therapeutic agent (e.g., an agent described herein) to a subject by any effective route. In some embodiments, a composition is administered to the same subject by multiple routes of administration. In some embodiments, the multiple routes of administration include intravenous, intraarterial, intrathecal, intranasal, intraperitoneal, and / or periocular administration. In some examples, a composition comprising PKM2 or a variant thereof may be administered intravenously to the subject's circulatory system. In some examples, a composition comprising PKM2 or a variant thereof may be injected into a suitable fluid and administered intravenously to the subject.

[0035] The terms "acute myocardial infarction" and "heart attack" refer to a condition in which focal myocardial ischemia results in the death of tissue in a defined area. Acute myocardial infarction is most commonly caused by the rupture of an atherosclerotic lesion in a coronary artery. This rupture results in the formation of a thrombus that occludes the artery, preventing blood flow to the area of ​​the heart that the artery supplies.

[0036] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, i.e., by way of example only, an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, yet still maintain the same basic chemical structure as a natural amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0037] The term "conservatively modified variants" applies to both natural and non-natural amino acid sequences, as well as natural and non-natural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to natural and non-natural nucleic acids that encode identical or essentially identical natural and non-natural amino acid sequences, or, if the natural and non-natural nucleic acids do not encode natural and non-natural amino acid sequences, to essentially identical sequences. For example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described herein without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Thus, by way of example, any natural or non-natural nucleic acid sequence herein that encodes a natural or non-natural polypeptide also represents any possible silent variation of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) of a natural or non-natural nucleic acid can be altered to yield a functionally identical molecule. Accordingly, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

[0038] The term "myocardial infarction (MI)" refers to the death of cardiac tissue caused by ischemia. "Ischemia" generally refers to a localized lack of blood supply caused by vasoconstriction or localized impaired blood flow. The restoration of blood flow to an ischemic tissue or organ, e.g., the heart, is called "reperfusion."

[0039] The term "ischemia-reperfusion" refers to the damage that occurs to tissue when blood supply is returned to that tissue after a period of ischemia. The lack of oxygen and nutrients in the blood creates a condition in which restoration of circulation leads to inflammation and oxidative or peroxidative damage.

[0040] As used herein, the term "nucleic acid sequence" refers to the order and identity of the nucleotides that comprise a nucleic acid.

[0041] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0042] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0043] A particular nucleic acid sequence also implicitly encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any proteins encoded by splice variants of that nucleic acid. A "splice variant," as the name suggests, is a product of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced ​​such that different (alternative) nucleic acid splice products encode different polypeptides. Mechanisms for the generation of splice variants vary but include alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.

[0044] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers.

[0045] As used herein, the term "pharmaceutically acceptable" refers to a substance, including but not limited to, a salt, carrier, or diluent, that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting adversely with any of the components of the composition in which it is contained.

[0046] As used herein, the term "prophylactically effective amount" refers to the amount of a composition comprising at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide that is administered prophylactically to a patient to thereby alleviate to some extent one or more symptoms of the disease, condition, or disorder being treated. In such prophylactic applications, such amount may depend on the patient's state of health, weight, etc. It is considered well within the skill of one in the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, dose escalation clinical trials.

[0047] The phrase "substantially similar" in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have at least 75%, preferably at least 85%, more preferably at least 90%, 95%, 95%, 98%, 99%, or more nucleotide or amino acid residue identity, or any integer value therebetween, as determined, for example, using a sequence comparison algorithm such as those described below or by visual inspection. Preferably, substantial identity exists over a region of the sequences that is at least about 10, preferably about 20, more preferably about 40-60 residues in length, or any integer value therebetween, preferably longer than 60-80 residues, more preferably at least about 90-100 residues; most preferably, the sequences are substantially identical over the entire length of the compared sequences, e.g., the coding regions of the nucleotide sequences. Substantially similar polypeptides or nucleic acids can be variants or other proteins that preferentially adopt a dimeric form. In a specific example, more effective proteins are those that preferentially dimerize and are soluble.

[0048] As used herein, the term "synergistic" refers to a combination of prophylactically or therapeutically effective agents that is more effective than the additive effects of any two or more single agents. The synergistic effect of a combination of prophylactic or therapeutic agents may allow for the use of lower dosages and / or less frequent administration of one or more of the agents in a subject with a particular disease or condition. In some cases, the synergistic effect of a combination of prophylactic or therapeutic agents may be used to avoid or reduce adverse or unwanted side effects associated with the use of either agent alone.

[0049] The term "therapeutically effective amount" refers to an amount of a protein-containing composition or biologic administered to a patient already suffering from a disease, condition, or disorder sufficient to treat or at least partially arrest or alleviate to some extent one or more of the signs, symptoms, or causes of the disease, disorder, or condition being treated. The effectiveness of such a composition depends on factors including, but not limited to, the severity and course of the disease, disorder, or condition, previous treatments, the patient's health status and response to drugs, and the judgment of the treating physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose-escalation clinical trial. The term "effective amount" is meant to include any amount of a composition or biologic, such as pyruvate kinase M2 or a variant thereof, sufficient to produce the desired therapeutic result.

[0050] A "therapeutically effective amount," in the context of using a composition to treat cardiac cells after a heart attack, refers to the amount of the composition that, when administered to an individual, produces the desired therapeutic effect of reducing, mitigating, or repairing cardiac cell damage caused by the heart attack. This amount can vary based on factors such as the specific composition, the severity of the heart attack, the patient's general health, age, weight, and other medical considerations. For a normal subject of normal weight, the therapeutically effective amount can range from 0.1 to 10 mg / ml intravenously, or 0.5 to 7 mg / ml intravenously, or 0.1 to 6 mg / ml intravenously, or 4 to 6 mg / ml intravenously, or about 5 mg / ml intravenously. The dose can be administered as a single acute treatment or can be delivered gradually over a specific period of time (e.g., 5 minutes to 5 hours or more).

[0051] As used herein, the term "subject" or "patient" includes mammals and humans. In some embodiments, the patient has suffered a heart attack.

[0052] As used herein, the term "dosage" refers to the amount of a composition or biologic, e.g., pyruvate kinase M2 or a variant thereof, administered to an animal or human or used in a cell culture assay. Suitable dosage units for use in the methods of the present invention include, but are not limited to, ng / kg body weight, mg / kg, mg / kg / day, M, nM, μM, or any other unit otherwise mentioned in this disclosure or commonly used in the art. In one example, 5 mg / ml is administered.

[0053] The term "therapeutic agent" or "therapeutic" encompasses proteins, peptides, nucleic acids, vectors, drugs, or other macromolecules or compositions known in the art. Therapeutic agents may be delivered to a recipient via inhalation, oral administration, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, or any other drug delivery method used in the art. The agent may be delivered as a single bolus or other one-time administration mechanism. Alternatively, the agent may be administered via sustained release (continuous or intermittent) delivery.

[0054] The terms "treat," "treatment," "treating," and the like refer to achieving a desired pharmacological and / or physiological effect, including, but not limited to, achieving an improvement, amelioration, or elimination of symptoms of ischemia. The effect may be preventative, in that it completely or partially prevents myocardial fibrosis, and / or therapeutic, in that it improves, ameliorate, or eliminates one or more symptoms of cardiomyocyte injury.

[0055] As used herein, "treatment" refers to any treatment of acute myocardial infarction in a mammal, particularly a human, and includes (a) preventing the development of myocardial fibrosis in the subject, (b) reducing myocardial fibrosis, and (c) restoring the individual to a state prior to the cardiac event. "Treatment" may not indicate or require complete eradication or cure of acute myocardial infarction or its associated symptoms.

[0056] As used throughout this specification, PKM2 refers to pyruvate kinase isoform M2.

[0057] As used throughout this specification, a PKM2 mutant refers to a variant form of the pyruvate kinase M2 (PKM2) protein in which one or more amino acid residues have been altered, added, or removed compared to the native or wild-type PKM2 sequence. These alterations may be the result of genetic mutation or may be artificially introduced by methods such as genetic engineering. Mutations may lead to changes in the function, stability, interaction partners, or other properties of the protein. DETAILED DESCRIPTION OF THE INVENTION

[0058] Generally, the present application provides methods for treating and / or preventing cardiac disease in a subject in need thereof. The inventors have conducted extensive research efforts to develop methods for protecting cardiomyocytes from death (e.g., apoptosis) and preventing or alleviating myocardial fibrosis or cardiac diseases associated with myocardial fibrosis. Certain embodiments are directed to protecting cardiomyocytes and promoting their growth. Cardiac diseases that may be treated include cardiomyopathies, such as hypertrophic cardiomyopathy, dilated cardiomyopathy, and toxic cardiomyopathy, cardiotoxicity, congestive heart failure, and some infectious, e.g., viral or bacterial, cardiac damage.

[0059] One embodiment is a method for reducing the adverse effects of myocardial infarction in a patient, the method comprising administering a recombinant PKM2 mutant (e.g., G415R), or a composition comprising recombinant PKM2 or a protein similar or identical to PKM2, which selectively adopts the dimeric form of PKM2 or has at least some dimeric form, to a patient during the acute phase of myocardial infarction. In some embodiments, the therapeutic PKM2 is not PKM2 that exists primarily as a tetramer. In some embodiments, the therapeutic PKM2 is not expressed intracellularly, for example, as a result of transfection of cardiomyocytes with a nucleic acid encoding PKM2. In certain embodiments, the PKM2 or PKM2 mutant contained in the composition has at least a portion of the dimeric form or exists as a dimer.

[0060] Physiologically, the period immediately following cardiac injury or trauma is critical and is sometimes referred to as the "golden hour" or the first hour after myocardial infarction. The myocardial infarction can be acute myocardial infarction. Administration of the ischemia / reperfusion protection composition can be initiated within 200 hours of the onset of the heart attack. Furthermore, administration of a protein substantially similar or identical to pyruvate kinase M2 (PKM2) can be initiated within about 100 hours, about 72 hours, about 48 hours, about 24 hours, or about 12 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of the onset of the heart attack. The patient can be a human or non-human mammal.

[0061] Another embodiment provides an ischemia / reperfusion protection composition. The ischemia / reperfusion protection composition disclosed herein comprises a protein similar or identical to PKM2 at an amount that results in a concentration of less than 1 μM to 5 μM in the patient's bloodstream under physiological conditions, or a mutant PKM2 that exists primarily in a dimeric form. In certain embodiments, PKM2 administered to a patient at a concentration of less than 1 μM under physiological conditions is present at about 70-85% as a dimer. In even more specific embodiments, PKM2 administered to a subject at a concentration of greater than 10 μM under physiological conditions is present at less than 25% as a dimer. In even more specific embodiments, a 5 mg / ml formulation for administration to a patient has a concentration of approximately 100 μM in the formulation, but after administration, it is diluted to less than 5 μM once in the patient's circulation. In certain embodiments, the PKM2 mutant G415R exists at a concentration of greater than 50 μM and is present at greater than 85% as a dimer. The ischemia / reperfusion protection compositions described herein can be administered to an individual to significantly reduce or prevent cardiac damage / reperfusion injury to all tissues associated with the heart.

[0062] In various embodiments, the present invention discloses, in part, administering an effective amount of a therapeutic composition to a subject to protect cardiomyocytes or promote cardiomyocyte growth. Certain embodiments contemplate treating a subject with a therapeutic agent substantially similar or identical to PKM2. In more specific embodiments, wild-type PKM2 and mutant PKM2 that selectively dimerize in equilibrium comprise at least 51% dimers, at least 60% dimers, at least 70% dimers, at least 80% dimers, or at least 90% dimers. The composition reduces cardiomyocyte death induced by cardiac injury.

[0063] In various embodiments, the present invention discloses methods for reducing or inhibiting myocardial fibrosis by administering a composition comprising recombinant wild-type PKM2 in an amount that results in a concentration of wt PKM2 in the bloodstream of less than 1 μM after administration to a patient. In various embodiments, the present invention discloses methods for reducing or inhibiting myocardial fibrosis by administering a composition comprising a mutant form of PKM2 that selectively dimerizes in an equilibrium state. While not intending to be bound by a particular mechanism, it is hypothesized that the reduction in fibrosis is the result of reduced myocardial cell death.

[0064] PKM2 exists in both dimeric and tetrameric states, but the biologically active protein has the dimeric state, and more effective mutants exist as a higher percentage of dimers relative to tetramers at equilibrium. PKM2 mutants that can adopt a dimeric form are described herein and are illustrated in the following exemplary references, which are incorporated herein by reference: Gao, X., Mol Cell 2012 Mar 9;45(5):598-609; Zhou, Zhifen, et al. "Oncogenic kinase-induced PKM2 tyrosine 105 phosphorylation converts nononcogenic PKM2 to a tumor promoter and induces cancer stem-like cells." Cancer research 78.9(2018):2248-2261; Li, iScience 23,101684, November 20, 2020; Liu, Vivian M., et al. "Cancer-associated mutations in human pyruvate kinase M2 impair enzyme activity." FEBS letters 594.4(2020):646-664; Chen, Tsan-Jan, et al. "Mutations in the PKM2 exon-10 region are associated with reduced allostery and increased nuclear translocation.“Communications biology 2.1(2019):1-11;Gupta,Vibhor,et al.“Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy.”Journal of Biological Chemistry 285.22(2010):16864-16873., Lv.Lei,et al."Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization." Molecular Cell 52.3 (2013): 340-352. Other PKM2 mutants can be developed without undue experimentation. In certain embodiments, the PKM2 mutant has at least 75%, preferably at least 85%, more preferably at least 90%, 95%, 98%, 99%, or more, or any integer value therebetween, nucleotide or amino acid residue identity compared to wild-type PKM2. PKM2 mutants that selectively adopt a dimeric form are useful in methods and systems for treating MI, IR, and other conditions associated with cardiomyocyte injury.

[0065] In one embodiment, the compositions and methods are directed to cardiac conditions including damaged cardiac tissue, e.g., damaged myocardium resulting from an ischemic event, ischemia-reperfusion injury, left ventricular damage, e.g., resulting from congestive heart failure, and heart valve damage resulting from disease, e.g., coronary artery disease. In all aspects, improving the function of damaged portions of cardiac tissue by administering PKM2 or a protein substantially similar to PKM2 is beneficial to the patient.

[0066] In another embodiment, the composition may be administered with another drug, which may include a lipid-lowering drug, an antiplatelet drug, an antihypertensive drug, a vasodilator, a hypoglycemic drug, an anticoagulant, a thrombolytic drug, a hepatoprotective drug, an antiarrhythmic drug, a cardiac inotropic drug, a diuretic, an anti-infective drug, an antiviral drug, an immunomodulatory drug, an inflammation-modulating drug, an anti-tumor drug, or a hormonal drug.

[0067] pyruvate kinase Pyruvate kinase isoform M2 (PKM2) is an isoform of pyruvate kinase expressed in mammalian cells. Pyruvate kinase regulates the final, rate-limiting event in glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate to ADP, generating pyruvate and ATP. Among the four isoforms of pyruvate kinase, PKM1 and PKM2 are ubiquitously expressed in different cell types and tissues. PKM2 is highly expressed in proliferating cells, including cancer cells. Unlike other isoforms, PKM2 expression and activity are regulated by metabolic intermediates and growth signaling pathways at multiple levels, including gene expression, alternative splicing, and post-translational modifications. Therefore, PKM2 is a unique pleiotropic regulator that can improve the adaptation of cells' metabolic programs to meet the physiological demands of different environments.

[0068] In addition to regulating glycolysis, PKM2 has non-metabolic functions such as regulating transcription and cell cycle progression. In contrast to mitochondrial respiration, energy regeneration by these pyruvate kinases is independent of oxygen supply, enabling organ survival under hypoxic conditions. PKM2 can also function as a coactivator of hypoxia-inducible factor 1-alpha (HIF-1a). The latter acts as a master transcription factor regulating multiple signaling pathways in response to hypoxia. Increased levels and activity of PKM2 are associated with enhanced tumor cell motility and metastasis. The molecular mechanisms underlying increased cell migration remain unclear. It should be emphasized that increased aerobic glycolysis and cell proliferation or migration are not unique to cancer and malignant tumors but rather stem from normal biology and physiological development. During physiological proliferation of neural progenitor cells or under hypoxic conditions, PKM2 helps reprogram energy metabolism, supporting growth and adaptation. Thus, metabolic transformation is a developmental event essential for physiological growth. Because these important metabolic and non-metabolic roles of PKM2 have been identified primarily in cancerous tumor cells, their potential functions in normal cells or in response to ischemic insults, such as stroke or myocardial infarction, have remained largely unknown until now.

[0069] The angiogenic activity and / or endothelial cell proliferation or migration potential of pyruvate kinase M2 or therapeutic agents substantially similar to pyruvate kinase can be assessed by assays and methods. The pyruvate kinase protein can be any vertebrate or mammalian pyruvate kinase, and can be a naturally occurring pyruvate kinase or a recombinant or other synthetic protein. The amino acid sequence of human pyruvate kinase is provided, for example, by GenBank Accession No. MP0011193727. For example, the amino acid sequence identity of pyruvate kinase is highly conserved across species, with humans sharing 98% amino acid sequence identity with mice, hamsters, and rats. The amino acid sequence of human pyruvate kinase is disclosed herein (Example 1). In one particular embodiment, the active pyruvate kinase protein is a dimer, also known as PKM2 subtype M2. A wide range of proteins and therapeutic agents substantially similar to pyruvate kinase M2 are useful in certain embodiments.

[0070] The animal from which the native pyruvate kinase protein is purified can be, for example, bovine, ovine, porcine, equine, canine, feline, primate, rodent, or other member of a mammalian family. In at least some forms, the pyruvate kinase protein is a human pyruvate kinase protein purified from bacterial production. The recombinant pyruvate kinase protein may have an amino acid sequence identical to that of a native pyruvate kinase or may have one or more amino acid differences compared to the native protein. The amino acid changes may include the addition, deletion, and / or substitution of one or more amino acids. Amino acid inversions and other mutational changes that result in modifications of the native pyruvate kinase protein sequence are also encompassed. Furthermore, the recombinant protein may contain an amino acid(s) not encoded by the genetic code.

[0071] The amino acid substitution may be conservative or non-conservative. The term "conservative amino acid substitution" should be interpreted in the commonly accepted sense of replacing an amino acid residue with another amino acid having similar properties without substantially adversely affecting the angiogenesis and / or wound healing activity of the pyruvate kinase protein. For example, a conservative amino acid substitution may include substituting a basic amino acid, such as arginine, with another basic amino acid, such as lysine. Similarly, a cysteine ​​residue may be replaced with serine, or a non-polar amino acid may be replaced with another non-polar amino acid, such as alanine. Amino acids suitable for substitution or deletion in the amino acid sequence of a pyruvate kinase protein can be determined by comparing the sequence with closely related pyruvate kinase proteins to identify non-conserved amino acids, as well as by routine testing and experimentation well within the skill of the applicant. Modified recombinant pyruvate kinase proteins can be provided by introducing nucleotide change(s) into a nucleic acid sequence encoding a native protein, such that the desired amino acid change is achieved upon expression of the nucleic acid in a host cell.

[0072] One embodiment includes recombinant or other synthetic PKM2 that selectively dimerizes. Such recombinant or other synthetic PKM2 includes the G415R mutant of PKM2. The amino acid sequence of the G415R mutant is shown in Example 2 and also in SEQ ID NO: _. More useful PKM2 variants or mutants thereof are those that selectively adopt a dimeric form and are water soluble.

[0073] The amino acid sequence of an exemplary pyruvate kinase M2 or SEQ ID NO:1:PKM2 (Accession No. NP_002645) is as follows: 1 mskphseagt afiqtqqlha amadtflehm crldidsppi tarntgiict igpasrsvet 61 lkemiksgmn varlnfshgt heyhaetikn vrtatesfas dpilyrpvav aldtkgpeir 121 tglikgsgta evelkkgatl kitldnayme kcdenilwld yknickvvev gskiyvddgl 181 islqvkqkga dflvteveng gslgskkgvn lpgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie 301 ipaekvflaq kmmigrcnra gkpvicatqm lesmikkprp traegsdvan avldgadcim 361 lsgetakgdy pleavrmqhl iareaeaaiy hlqlfeelrr lapitsdpte atavgaveas 421 fkccsgaiiv ltksgrsahq varyrprapi iavtrnpqta rqahlyrgif pvlckdpvqe 481 awaedvdlrv nfamnvgkar gffkkgdvvi vltgwrpgsg ftntmrvvpv p

[0074] The amino acid sequence of an exemplary mutant pyruvate kinase M2, SEQ ID NO:2:PKM2(R399E), is as follows: 1 mskphseagt afiqtqqlha amadtfelhm crldidsppi tarntgiict igpasrsvet 61 lkemiksgmn varlnfshgt heyhaetikn vrtatesfas dpilerpvav aldtkgpeir 121 tglikgsgta evelkkgatl kitldnayme kcdenilwld yknickvvev gskiyvddgl 181 islqvkqkga dflvteveng gslgskkgvn lpgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie 301 ipaekvflaq kmmigrcnra gkpvicatqm lesmikkprp traegsdvan avldgadcim 361 lsgetakgdy pleavrmqhl iareaeaaiy hlqlfeelrr lapitsdpte atavgaveas 421 fkccsgaiiv ltksgrsahq varyrprapi iavtrnpqta rqahlyrgif pvlckdpvqe 481 awaedvdlrv nfamnvgkar gffkkgdvvi vltgwrpgsg ftntmrvvpv p

[0075] The amino acid sequence of another exemplary mutant pyruvate kinase M2 having three mutations (R399E, K422A, and N523A), or SEQ ID NO:3, is as follows: MSKPHSEAGT AFIQTQQLHA AMADTFLEHM CRLDIDSPPI TARNTGIICT IGPASRSVET LKEMIKSGMN VARLNFSHGT HEYHAETIKN VRTATESFAS DPILYRPVAV ALDTKGPEIR TGLIKGSGTA EVELKKGATL KITLDNAYME KCDENILWLD YKNICKVVEV GSKIYVDDGL ISLQVKQKGA DFLVTEVENG GSLGSKKGVN LPGAAVDLPA VSEKDIQDLK FGVEQDVDMV FASFIRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELER LAPITSDPTE ATAVGAVEAS FACCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTATMRVVPV P

[0076] Recombinant or synthetic pyruvate kinase proteins suitable for the methods of the present invention should exhibit at least 60% amino acid sequence identity with native pyruvate kinase. More generally, the identity can be at least 70%, 80%, 90%, 95%, 98%, or even 100%. A portion of the protein must form a dimer under physiological conditions in a patient. All sequence homologies and ranges specified above are expressly included. Sequence identity between amino acid sequences is determined by comparing the amino acids at each position of optimally aligned sequences. Amino acids at a given position are considered identical only if they match. In alignment, gaps, where an amino acid residue appears in one sequence but not in another, are treated as positions containing non-identical residues. Sequence alignment can be achieved using any suitable program or algorithm, usually by computer-assisted sequence alignment using standard software.

[0077] The pyruvate kinase protein can also be chemically synthesized. The creation and use of fusion proteins incorporating the pyruvate kinase proteins described herein are also encompassed by the present invention. Nucleic acids encoding fusion proteins can be generated by combining separate DNA fragments encoding the pyruvate kinase protein with, for example, a lipophilic amino acid sequence to enhance the lipophilicity of the protein. This can be accomplished using methods such as the use of blunt ends and oligonucleotide linkers, digestion to create sticky ends, and, if necessary, ligation of the sticky ends.

[0078] The pyruvate kinase proteins described herein can also be modified by attaching one or more proteinaceous or non-proteinaceous moieties to the protein. This can improve aspects such as solubility, lipophilicity, stability, biological half-life, or serve as a label for subsequent detection. Modifications can result from post-translational or post-synthetic processes, such as chemical reactions resulting in the attachment of carbohydrate moieties or structural modifications (e.g., alkylation or acetylation of amino acid residues). For example, pyruvate kinase proteins can undergo modifications such as methylation, phosphorylation, oxidation of tyrosine and / or tryptophan residues, glycosylation, or covalent attachment of S-methylcysteine. The pyruvate kinase protein can differ in size from the intact protein. However, the pyruvate kinase should be of a length that promotes dimer formation.

[0079] The C- and N-terminal extensions of native pyruvate kinase protein are involved in stabilizing the quaternary structure and generating protein aggregates. Therefore, pyruvate kinase lacking such extensions forms aggregates less efficiently. Pyruvate kinase tends to form large aggregates. Electrostatic interactions between these proteins also affect aggregate formation, and ionization of histidine residues at pH below 7 can disrupt these aggregates. Typically, pyruvate kinase proteins used in the methods of the present invention exist in a dimeric form. Both the intact and truncated forms of the protein useful in embodiments of the present invention may undergo post-translational modifications, including, but not limited to, acetylation, methylation, ethylation, phosphorylation, oxidation, and glycosylation, found in native pyruvate kinase proteins. Conditions suitable for alkaline phosphatase activity include the presence of zinc-, magnesium-, or calcium-containing buffers.

[0080] The partially hydrolyzed form of pyruvate kinase protein may be purified for use in embodiments of the present invention using any suitable purification technique, including filtration and chromatographic protocols.

[0081] Pyruvate kinase administration The pyruvate kinase protein may be administered alone or co-administered with one or more other therapeutic agents to a subject in need of such treatment. For example, pyruvate kinase may be co-administered in combination with a therapeutic agent conventionally used to promote angiogenesis, cell proliferation, or wound healing. "Co-administered" refers to simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes, whereby the pyruvate kinase protein and the other therapeutic agent(s) exhibit an overlapping therapeutic window. "Sequential" administration refers to administration of one agent after the other. Examples of such additional agents that may be co-administered with the pyruvate kinase protein include platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), platelet-derived wound healing factor, insulin growth factor (IGF), keratinocyte growth factor (KGF), anti-inflammatory agents, and antibacterial agents. Further examples of other therapeutic agents that can be used to promote angiogenesis and / or wound healing and that can be co-administered with the pyruvate kinase protein include indoleamine 2,3-dioxygenase (IDO), tryptophan dioxygenase (TDO), sphingosine-1-phosphate (SIP), N-acylethanolamines, grapefruit extract, and other phytochemicals, including ascein, green tea catechins, melatonin, arginine, and other amino acids to support blood vessel growth. Additional therapeutic agents suitable for co-administration will be apparent to those skilled in the art.

[0082] The pyruvate kinase protein is generally formulated into a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.

[0083] The pharmaceutical compositions described herein can also contain one or more preservatives, such as parabens, chlorobutanol, and sorbic acid, binders, such as corn starch or gelatin, thickeners, emulsifiers, surfactants, gelling agents, and other ingredients commonly used in such compositions.Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations, and solutions.The use of such ingredients and media for pharmaceutically active substances is well known.Unless any conventional media or agent is incompatible with the pyruvate kinase protein, its use is expressly included.

[0084] Pharmaceutical compositions embodied by the present invention include therapeutic compositions for human or veterinary use.

[0085] Pharmaceutical compositions embodied by the present invention generally contain at least about 0.001% by weight of the pyruvate kinase protein and up to about 80% w / w of the composition. For example, the pharmaceutical composition may contain about 0.05%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% by weight of the pyruvate kinase protein, a substantially similar therapeutic agent, or a selectively dimerizing mutant PKM2. Pharmaceutical compositions embodied by the present invention generally contain at least about 20 μg / ml of the pyruvate kinase protein and up to about 1000 μg / ml. For example, the pharmaceutical composition may contain about 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1000 μg / ml of the pyruvate kinase protein, or substantially similar therapeutic agent, or selectively dimerizing mutant PKM2. The amount of protein in the composition is such that an appropriate effective dosage is delivered to the subject, taking into account the proposed method of administration.

[0086] The dosage of pyruvate kinase protein administered according to embodiments of the present invention depends on several factors, including whether the protein is administered prophylactically or therapeutically, the disease or condition for which the protein is to be administered, the severity of the condition, the subject's sex and age, and related factors including the subject's weight and overall health, and can be determined according to generally accepted medical principles. For example, a low dosage can be administered initially, which is then increased with each administration after evaluating the subject's response. Similarly, the frequency of administration can be determined in the same manner by continuously observing the subject's response between each administration and increasing or decreasing the frequency of administration as desired. In one example, the dosage can be 1 mg / kg to 20 mg / kg; in another example, the dosage can be 2 mg / kg to 10 mg / kg; in another example, the dosage can be 1 mg / kg to 6 mg / kg; and in yet another example, the dosage can be 2 mg / kg to 5 mg / kg.

[0087] Routes of administration include, but are not limited to, topical, respiratory, intravenous, oral, intraperitoneal, subcutaneous, intramuscular, rectal, local, direct to the heart, and via implantation. With regard to the intravenous route, a particularly suitable route is via injection into a blood vessel (e.g., the superior vena cava or inferior vena cava) supplying the target tissue to be treated. The pyruvate kinase protein may also be delivered to cavities such as the thoracic or peritoneal cavity, the skull, or directly injected into the tissue to be treated, e.g., the left or right ventricle of the heart. For oral administration, the pyruvate kinase protein may be encapsulated or enterically provided for passage through the stomach and release in the small intestine. Any suitable such enteric formulation or coating may be used. Furthermore, these systems and methods may be implemented using any medically acceptable administration method, i.e., any method that results in effective levels of cell regeneration or tissue repair without causing clinically unacceptable side effects.

[0088] Furthermore, pyruvate kinase proteins can be coated onto the surface of catheters, such as angioplasty catheters, stents or balloons, or other surgical devices for application to the interior wall of a blood vessel during angioplasty or other surgical procedures. The pyruvate kinase can be applied to the vessel wall in this manner in the form of a gel or any other suitable formulation, for example, to promote wound healing and / or angiogenesis, epithelial cell migration, or cell regeneration at the treatment site.

[0089] Suitable pharmaceutically acceptable carriers and formulations useful in compositions embodied by the present invention can be found, for example, in handbooks and textbooks.

[0090] Pharmaceuticals, kits, and embodiments Pharmaceuticals include the following categories and specific examples. The categories are not intended to be limited by the specific examples. One of ordinary skill in the art will be able to readily identify pharmaceuticals that have utility within or outside the central nervous system. One of ordinary skill in the art will also recognize many other compounds that fall within the categories and are useful according to the present invention.

[0091] One embodiment also includes a kit for improving recovery after a heart attack or injury, where a combination of agents is provided that allows for administration in a therapeutically effective amount and frequency to effect cellular regeneration after a heart attack or injury.

[0092] In some embodiments, it may be desirable to increase the solubility and blood circulation time of PKM2 or a substantially similar therapeutic agent. To increase the solubility and blood circulation time of the polypeptide, the polypeptide of the present invention may be derivatized with polyethylene glycol, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidone), polyoxomers, polysorbates, and poly(vinyl alcohol), with PEG polymers being particularly preferred. The PEG polymer has a molecular weight of about 100 to about 40,000. In addition to the examples exemplified above, other suitable hydrophilic polymers will be readily apparent to those skilled in the art based on this disclosure. Generally, the polymer used may include polymers that can be conjugated to the polypeptide of the present invention via alkylation or acylation reactions. In one example, the PKM2 or a substantially similar therapeutic agent is pegylated with a 20 kDa PEG chain.

[0093] When attaching a polyethylene glycol molecule (or other chemical moiety) to a polypeptide, consideration should be given to the impact on the functional or antigenic domain of the polypeptide. Several attachment methods are available to those skilled in the art. For example, polyethylene glycol can be covalently attached through an amino acid residue via a reactive group, such as a free amino or carboxyl group. The reactive group is one to which an activated polyethylene glycol molecule can be attached. Examples of amino acid residues with a free amino group include lysine residues and N-terminal amino acid residues, while examples of amino acid residues with a free carboxyl group include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Sulfhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. For therapeutic purposes, attachment via an amino group, e.g., via the N-terminus or lysine group, is preferred. Polypeptides chemically modified at the N-terminus may be particularly desirable. When using polyethylene glycol as an example of the present composition, various polyethylene glycol molecules (based on molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to polypeptide molecules in the reaction mixture, the type of PEGylation reaction performed, and the method for obtaining the selected N-terminally PEGylated polypeptide may be selected. Under appropriate reaction conditions, substantially selective derivatization at the N-terminus of the polypeptide with a carbonyl group-containing polymer is achieved.

[0094] Certain embodiments also provide pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an active ingredient (e.g., a PKM2 dimer, a PKM2 mutant that exists more as a dimer than as a tetramer, or a substantially similar therapeutic agent) and a pharmaceutically acceptable carrier. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is the carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions contain a therapeutically effective amount of PKM2 or a substantially similar therapeutic agent, together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should be suited to the method of administration.

[0095] The amount of PKM2 or a substantially similar therapeutic agent that is effective in assisting recovery from ischemic stroke can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0096] More specifically, the agent or pharmaceutical composition can be tested in vitro and then in vivo for the desired therapeutic or prophylactic activity before use in humans. For example, in vitro assays for demonstrating the therapeutic or prophylactic usefulness of a compound or pharmaceutical composition include the effect of the compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and / or tissue sample can be determined using techniques known in the art, including, but not limited to, rosette formation assays and cytolytic assays. According to the present invention, in vitro assays that can be used to determine whether administration of a particular compound is desirable include in vitro cell culture assays, in which a patient tissue sample is grown in culture and exposed to or otherwise administered a compound, and the effect of the compound on the tissue sample is observed.

[0097] It is contemplated that the PKM2 or substantially similar therapeutic agent can be formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate, in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0098] Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, and construction of nucleic acids as part of retroviral or other vectors, are known and can be used to administer the compounds of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral, rectal, and intestinal mucosa), or may be administered together with other bioactive agents. Administration may be systemic or local. Furthermore, it may be desirable to introduce the pharmaceutical agents or pharmaceutical compositions of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter.

[0099] In certain embodiments, it may be desirable to administer the therapeutic composition (ischemia / reperfusion protection composition) locally to the area that needs treatment.This may be achieved, for example, but not limited to, by local injection (e.g., injection into the myocardium) or infusion during surgery, by injection, by catheter, by suppository, or by implant, which may be made of porous, non-porous, or gel-like material, including membranes, for example, sialastic membranes, or fibers.When administering polypeptides, care must be taken to use materials that the polypeptides do not absorb.

[0100] It is contemplated that regulatory genes and sequences can be used to express and replicate the PKM2 or substantially similar therapeutic agent. The nature of the regulatory sequences for gene expression may vary between species or cell types, but will generally include, as necessary, 5' untranscribed and 5' untranslated sequences involved in initiation of transcription and translation, respectively, e.g., a TATA box, capping sequence, CAAT sequence, etc. Promoters can be constitutive or inducible. Regulatory sequences can also include enhancer sequences or upstream activator sequences, as desired.

[0101] In one embodiment, the polynucleotide encoding the PKM2 or substantially similar therapeutic agent may be fused to a polynucleotide encoding a signal sequence that directs localization of the polypeptide to a specific compartment of a prokaryotic or eukaryotic cell and / or directs secretion of the polypeptide. For example, in E. coli, it may be advantageous to direct expression of the protein to the periplasmic space. Several vectors are commercially available for constructing fusion proteins that direct protein localization.

[0102] One particular embodiment provides a stent comprising a generally tubular structure, which may comprise, for example, a helical shape. The surface of this structure is coated with PKM2 or a substantially similar therapeutic agent, as described above. The stent is typically a scaffold, typically cylindrical, and can be inserted into a body passageway (e.g., bile duct, artery, vein) or a portion thereof. This passageway may be narrowed, irregularly shaped, blocked, or obstructed due to a disease process (e.g., tumor ingrowth), and the stent prevents the passageway from closing or reclosing.

[0103] One particular embodiment also provides for the use of PKM2 or a substantially similar therapeutic agent in a wide variety of surgical procedures. For example, a surgical mesh coated with PKM2 can be used in any procedure in which a surgical mesh can be used. [Example]

[0104] Example 1 Below is the amino acid sequence of an exemplary wild-type pyruvate kinase M2, or SEQ ID NO:4. MSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPITARNTGIICTIGPASRSVETLKEM IKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSG TAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKG ADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASD VHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEIPAEKVFLAQKMMI GRCNRAGKPVICATQMLESMIKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAV RMQHLIAREAEAAMFHRKLFEELVRASSHSTDLMEAMGSVEASYKCLAAALIVLTESGR SAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVG KARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP

[0105] Example 2 (PKM2 G415R mutant sequence) Below is the amino acid sequence of a modified pyruvate kinase protein in which at least glycine has been replaced with arginine, which protein more preferentially adopts a dimeric form compared to wild-type EcPKM2 or SEQ ID NO:5. MSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPITARNTGIICTIGPASRSVETLKEM IKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSG TAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKG ADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASD VHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEIPAEKVFLAQKMMI GRCNRAGKPVICATQMLESMIKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAV RMQHLIAREAEAAMFHRKLFEELVRASSHSTDLMEAMAM R SVEASYKCLAAALIVLTESGR SAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVG KARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP

[0106] Example 3 (Extracellular administration of PKM2 G415R mutant) Research has shown that integrin α v It has been demonstrated that β3 is expressed in cardiomyocytes under myocardial infarction conditions. We probed the expression of integrins in H9C2 cells by immunofluorescence (IF) staining. Integrins were expressed at high levels in cells under hypoxic conditions but not under normoxia. Furthermore, we analyzed integrin expression in primary human cardiomyocytes under hypoxic and oxidative stress conditions. Integrins were expressed in cardiomyocytes under stress conditions, whereas integrins were not expressed under normal culture conditions. To confirm the clinical relevance of integrin expression, integrin expression was analyzed in cardiac tissue from infarcted patients. Integrins were highly expressed in the infarcted area and were barely detectable in the normal, non-infarcted area. Integrin av b3 was upregulated in cardiomyocytes under hypoxic and oxidative stress conditions.

[0107] Extracellular PKM2 (EcPKM2) interacts with the integrin αvβ3 found in vascular-derived endothelial cells and myofibroblasts, protecting them from apoptosis. An exemplary recombinant PKM2 mutant, G415R, which exists primarily as a dimer, was expressed and purified from E. coli bacteria. This mutant, designated G415R or recombinant G415R, was used at a concentration of 50 μg / ml in place of EcPKM2. As a control, recombinant PKM1 (rPKM1), also expressed and purified from E. coli, was used. G415R exhibited improved solubility and long-term stability compared to recombinant PKM2.

[0108] Figure 1A reveals that recombinant G415R protected H9C2 cells from death (e.g., apoptosis) under hypoxic conditions. Figure 1B demonstrates that the G415R mutant promoted cell proliferation under these conditions. Figure 1C shows that recombinant G415R has a similar effect on primary human cardiomyocytes, specifically protecting them from cell death under conditions of hypoxia and oxidative stress. Similarly, Figures 1D and 1E show the promotion of cell proliferation of the G415R mutant in primary cardiomyocytes under conditions of hypoxia and oxidative stress.

[0109] Example 4 (Recombinant G415R preserved cardiomyocytes in an infarcted mouse model) Because protection from cardiomyocyte death under conditions of hypoxia and oxidative stress and promotion of cardiomyocyte proliferation protect the heart during myocardial infarction, we used a commonly used mouse model of left anterior descending coronary artery ligation to identify the effect of this PKM2 mutant on myocardial infarction. Animals were pretreated with a single dose of G415R (50 μg / ml) 24 hours before arterial ligation. The following day, myocardial infarction (MI) or ischemia-reperfusion (IR) was induced. Infarcted mice (both MI and IR) were treated with the same dose of G415R twice weekly for 2 weeks. Ultrasound and MR imaging demonstrated the successful creation of MI and IR by arterial ligation. Figure 2A shows that administration of G415R dramatically reduced death in MI mice at all time points. Figure 2B also shows that administration of G415R reduced heart weight relative to body weight in both MI mice 30 days after infarction. MR imaging analysis of intracardiac blood flow showed that G415R treatment improved blood flow in MI mice and restored blood flow in IR mice to levels similar to those in sham mice.

[0110] Figures 3A and 3B show histological analysis of infarcted hearts, revealing that mice treated with G415R had reduced infarct scar size compared with rPKM1 and vehicle-treated mice in both MI and IR mice. To determine whether G415R protects cardiomyocytes from apoptosis, TUNEL staining was performed on tissues from mouse hearts at 6, 24, and 168 hours after infarction. As shown in Figure 3C, G415R treatment significantly reduced cardiomyocyte apoptosis at 6 and 24 hours. As shown in Figure 3D, by 168 hours after infarction, cardiomyocyte apoptosis was barely detectable using TUNEL staining, and no significant changes were observed among the various treatment groups.

[0111] G415R was found to stimulate the proliferation of human primary cardiomyocytes under conditions of hypoxia and oxidative stress. Therefore, we investigated whether G415R similarly promoted cardiomyocyte proliferation using Ki67 staining. As expected, G415R treatment did not induce cardiomyocyte proliferation at 6 h after infarction. Nevertheless, clear cardiomyocyte proliferation was observed in the myocardial tissue of both MI and IR mice treated with G415R on days 4 and 7 after infarction (Figure 3E). The effect of G415R on cardiomyocyte proliferation decreased from days 4 to 7 in both MI and IR mice and was almost nonexistent by day 28 after infarction. This suggests that the optimal period for promoting cardiomyocyte proliferation / regeneration is days 1 to 7 after infarction.

[0112] Cardiomyocyte death results in the release of cardiac troponin I (cTnI), a crucial molecular marker of cardiac injury, into the bloodstream. cTnI levels were analyzed in plasma samples from infarcted mice. Virtually no cTnI was detected in the plasma of sham mice. cTnI concentrations peaked 24 hours after infarction. Furthermore, G415R treatment significantly reduced cTnI levels in the circulation of infarcted mice by day 7 after infarction.

[0113] In summary, these experiments demonstrate that administration of the recombinant PKM2 mutant G415R to MI and IR mice effectively protects cardiomyocytes from apoptosis and promotes cardiomyocyte proliferation after infarction. Because G415R is known to act via the same mechanism as PKM2, the results of these experiments demonstrate the efficacy of PKM2 and proteins substantially similar to PKM2.

[0114] Example 4 (EcPKM2 reduces myocardial fibrosis resulting from infarction) Loss of cardiomyocytes due to infarction triggers the activation of cardiac fibroblasts, which release ECM / collagen to repair the damaged myocardium. Sustained activation of cardiac fibroblasts leads to the accumulation of ECM in the myocardium and myocardial fibrosis. Figure 4A shows WGA staining of myocardial tissue, revealing that mice treated with G415R had less infarct scar in the infarcted myocardial area compared with rPKM1 and vehicle-treated groups in both MI and IR models.

[0115] Figures 4B and 4C show Masson's trichrome staining of myocardial tissue, revealing that G415R-treated mice exhibited reduced collagen accumulation in the infarcted myocardial region in both MI and IR models compared with rPKM1- and vehicle-treated mice. These findings were further supported by wheat germ agglutinin (WGA) staining of infarcted myocardial tissue.

[0116] Figures 4D and 4E show analysis of cardiomyocyte cross-sectional area using co-staining for WGA and cTnI. These data show that G415R treatment reduced cardiomyocyte cross-sectional area to a similar extent as that seen in the sham group in both MI models.

[0117] Figures 4E and 4F show the results of the IR model and highlight that G415R treatment significantly suppressed cardiomyocyte hypertrophy in infarcted mice. A possible explanation for the reduced accumulation of ECM / collagen fibers in the myocardium of G415R-treated mice is that G415R treatment may have preserved cardiomyocytes, resulting in less cardiac fibroblast activation in MI and IR mice.

[0118] Finally, Figures 4F and 4G show the activation of cardiac fibroblasts in the infarcted myocardium of MI and IR mice, as evidenced by IHC staining for α-SMA, a molecular marker of myofibroblasts. Treatment with G415R significantly reduced α-SMA staining in the myocardial tissue of both MI and IR mice.

[0119] Example 5 (Systemic administration of G415R preserved cardiomyocytes in infarcted hearts) PKM2 was expressed in infarcted cardiac tissue after 4 days, but PKM2 staining was not detected in normal, healthy cardiac tissue. Extracellular PKM2 was observed in the staining of myocardial infarcted tissue from patients. We also performed IHC staining of PKM2 in infarcted cardiac tissue from mice after different infarction induction time points. PKM2 staining was not observed at 6 hours, 24 hours, and 3 days after infarction induction. PKM2 staining was detected in the infarcted area 4 days after infarction induction. EcPKM2 was revealed by IHC staining. PKM2 was not detected in the plasma of sham mice or mice 6 hours and 24 hours after myocardial infarction. PKM2 was detected in the plasma of mice 7 days after myocardial infarction (not shown). Experiments by the present inventors and other laboratories suggest that PKM2 is expressed late after myocardial infarction induction (approximately 4 days after infarction) and is released into the extracellular space from dead cells, for example.

[0120] Example 6 (EcPKM2 is an integrin α v interacts with and activates β3) Co-immunoprecipitation experiments were performed using G415R and extracts from primary human cardiomyocytes exposed to hypoxic conditions. As shown in Figures 5A-5D, G415R effectively reduced PTEN expression in cultured cardiomyocytes (proven by immunoblotting) and myocardial tissue (observed by IHC staining) from infarcted mice. Interestingly, Figures 5A-5D show that G415R has affinity for integrin β3, as evidenced by co-precipitation confirmed using antibodies specific for either PKM2 or integrin β3.

[0121] To determine whether EcPKM2 initiates cardiomyocyte responses through integrin signaling, we first measured the activation level of integrin signaling. After introducing G415R into the H9C2 cell culture medium, a significant increase in FAK activation was evident. However, as shown in Figure 5C, this FAK activation was suppressed by the antibody LM609 and IgGPK. Subsequent evaluation of PI3K activation in H9C2 cells after G415R treatment by both immunoblotting and specific PI3K activity assays confirmed its upregulation and activation in these cells. Furthermore, PI3K activation was attenuated by a FAK inhibitor. Thus, if the cardioprotective and proliferative effects of EcPKM2 are primarily due to PI3K activation, they are abrogated by PI3K inhibitors. Supporting this idea, the effects of G415R on H9C2 proliferation were abrogated by commercially available FAK and PI3K inhibitors. EcPKM2 promotes beneficial interactions with the integrin avβ3. This interaction in turn triggers integrin signaling in cardiomyocytes during periods of hypoxia and oxidative stress, providing protection against apoptosis and promoting cell proliferation.

[0122] Considering the well-documented role of the PI3K-PTEN pathway in regulating cardiomyocyte behavior and the impact of reducing PTEN expression or its pharmacological inhibition on cardiomyocyte survival and proliferation, it is likely that EcPKM2 can induce cardiomyocyte responses in hypoxic and oxidative stress environments. This may occur primarily through the FAK-PI3K signaling channel by downregulating PTEN levels in cardiomyocytes.

[0123] Reduction or pharmacological inhibition of PTEN expression in cardiomyocytes protects them from death and stimulates proliferation. Therefore, we hypothesized that EcPKM2 might regulate cardiomyocyte apoptosis and proliferation under hypoxic and oxidative stress conditions via the same FAK-PI3K signaling pathway by reducing PTEN levels in cardiomyocytes. First, we examined PTEN levels and activity in H9C2 cells treated with G415R under both hypoxic and normoxic conditions. Under normoxic conditions, PTEN was not highly expressed in the cells. Introduction of G415R into cell culture had only a slight effect on PTEN expression. However, PTEN was substantially expressed under hypoxic conditions. Figure 6H shows that addition of G415R, but not rPKM1 or vehicle, to the culture medium reduced intracellular PTEN. This suggests that EcPKM2 may contribute to the downregulation of PTEN in cardiomyocytes during myocardial infarction, subsequently protecting cardiomyocytes from apoptosis and stimulating proliferation. To further verify the effect of EcPKM2 on PTEN in cardiomyocytes, we evaluated PTEN levels in myocardial tissue derived from infarcted mouse hearts using IHC. PTEN expression was minimal in sham mice, whereas PTEN levels were elevated in both MI and IR mice. G415R treatment reduced PTEN levels by one-third. This observation supports the conclusion that EcPKM2 activates the integrin avβ3-FAK-PI3K signaling pathway, which then reduces PTEN levels in infarcted cardiomyocytes.

[0124] The foregoing detailed description and accompanying drawings have been presented for purposes of illustration and description only. They are not intended to be exhaustive or to limit the scope of the invention. The embodiments have been chosen and described in order to best explain the principles of the invention and its practical application. Those skilled in the art will understand that many modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method for treating cardiac injury in a subject, comprising administering to the subject a therapeutically effective amount of either pyruvate kinase M2 (PKM2) or a PKM2 mutant within 10 hours of the cardiac injury, wherein the PKM2 or PKM2 mutant is in the form of a dimer.

2. The method of claim 1 , wherein the PKM2 or PKM2 mutant dimerizes in the subject.

3. 10. The method of claim 1, wherein the administration occurs within 6 hours after cardiac injury.

4. 10. The method of claim 1, wherein the administration occurs within 3 hours after cardiac injury.

5. 10. The method of claim 1, wherein the administration occurs within 1 hour after cardiac injury.

6. 10. The method of claim 1, wherein the subject has experienced a heart attack and the cardiac damage is due to the heart attack.

7. 2. The method of claim 1, wherein PKM2 is present less than 50% in its tetrameric form.

8. The method of claim 1 , wherein PKM2 forms predominantly dimers at neutral pH.

9. The method of claim 1 , wherein the PKM2 or the PKM2 mutant selectively adopts a dimeric state.

10. The method of claim 1 , wherein the cardiac injury is caused by acute myocardial cell loss.

11. The method of claim 1 , wherein the preservation of myocardium is achieved by administration of a composition.

12. The method of claim 1 , wherein the composition is delivered extracellularly.

13. The method of claim 1 , wherein the myocardial infarction is characterized as acute.

14. The method of claim 1, wherein the composition comprises a PKM2 mutant or a protein highly similar to wild-type pyruvate kinase M2.

15. 2. The method of claim 1, wherein the PKM2 is derived from either a human or another animal.

16. The method of claim 1 , wherein the PKM2 has a mutation that differs from the wild-type sequence.

17. The method of claim 12, wherein the composition is contained in a pharmaceutically acceptable carrier.

18. The method of claim 1 , wherein the composition is delivered by intracardiac administration.

19. The method of claim 1 , wherein the composition is delivered systemically.

20. The method of claim 1 , wherein the composition reduces myocardial cell death resulting from myocardial infarction.

21. 10. The method of claim 1, wherein the composition reduces myocardial fibrosis in infarcted myocardium.

22. 2. The method of claim 1, wherein the pyruvate kinase M2 exists predominantly as a dimer compared to its tetrameric form.

23. 2. The method of claim 1, wherein the PKM2 is characterized by a G415R mutation.

24. The method according to any one of claims 1 to 18, wherein the PKM2 or the PKM2 mutant is present in the extracellular space.

25. The method according to any one of claims 1 to 18, wherein the PKM2 or PKM2 variant contained in the composition is 1 mg / kg to 6 kg.

26. The method according to any one of claims 1 to 18, wherein the amount of PKM2 or the PKM2 variant contained in the composition is 2 mg / kg to 5 kg.

Images