Method for preserving and protecting cardiac muscle cells and reducing cardiac fibrosis after cardiac injury

Abstract

A method of treating a patient who has experienced myocardial infarction, also known as acute myocardial infarction, treats or mitigates the effects of said infarction. This treatment may comprise administering a composition containing a protein substantially similar or identical to pyruvate kinase M2 (PKM2) during the acute or early stage of the myocardial infarction.

Description

Technical Field

[0001] This application generally relates to methods for treating cardiovascular conditions. More specifically, this application relates to compositions, systems, and methods for improving recovery and tissue repair after cardiac injury by protecting cardiomyocytes and reducing cardiomyocyte cell death caused by myocardial injury. This application also relates to a pharmaceutical composition for treating, preventing, or alleviating cardiac fibrosis after myocardial injury, and more specifically, to a pharmaceutical composition for treating or preventing myocardial injury and the accompanying cardiac fibrosis. Background Art

[0002] The extensive loss of cardiomyocytes during heart attacks and other cardiovascular diseases is a major cause of morbidity and mortality. Preserving cardiomyocytes during cardiac injury could provide an effective strategy for managing these conditions. Acute myocardial infarction (MI) caused by coronary artery disease often leads to maladaptive myocardial remodeling, ultimately resulting in heart failure (HF).

[0003] Cardiomyocytes are the primary cell type in the adult heart and are responsible for maintaining cardiac function. Cardiomyocyte loss following myocardial infarction (MI) is a significant contributor to heart disease-related morbidity and mortality. Given the limited regenerative capacity of adult cardiomyocytes, acute cardiomyocyte loss following MI is irreversible. Preserving cardiomyocytes is crucial for ensuring survival after a heart attack. Notably, cardiomyocyte death rapidly activates local tissue repair mechanisms, spurring the involvement of cardiac fibroblasts. Once activated, cardiac fibroblasts produce an extracellular matrix (ECM), primarily composed of collagen, to form scar tissue to prevent post-MI myocardial rupture. However, persistent accumulation of ECM, particularly collagen, interferes with normal myocardial function, often leading to heart failure. It is generally believed that ECM originates from activated cardiac fibroblasts and replaces or fills the damaged myocardial tissue caused by cardiomyocyte death. Dying cardiomyocytes and cardiac fibroblasts maintain a close communication relationship, which influences fibroblast activation during MI. This communication primarily occurs through paracrine signaling and is facilitated by the release of cytokines, hormones, and growth factors. There is compelling evidence supporting the view that dying cardiomyocytes primarily activate cardiac fibroblasts, ultimately leading to cardiac fibrosis.

[0004] After fibrotic scar tissue replaces myocardium damaged by hypertension, the heart's elasticity decreases, affecting its function. Similarly, pulmonary fibrosis stiffens the lungs, impairing lung function. Fibrotic growth can spread and erode healthy adjacent tissue, persisting even after the initial injury has healed. In general, fibrosis is a reactive process influenced by various factors. These factors include an early inflammatory response, a local surge in fibroblast populations, changes in fibroblast synthesis function, and altered kinetics of collagen biosynthesis and degradation. Other contributing factors include inflammation of nearby tissues and a generalized inflammatory state characterized by an increase in circulating mediators. Unfortunately, there are currently no effective therapies to combat cardiac fibrosis or protect cardiomyocytes.

[0005] Therefore, there is a need for systems and methods to protect or preserve cardiomyocytes and reduce associated cardiac fibrosis. The present disclosure is made in response to this need and others. Summary of the Invention

[0006] The present application can treat acute cardiac injury, such as that caused by a heart attack, and promote cardiomyocyte proliferation. Specifically, the present application discloses that administration of a recombinant PKM2 mutant (e.g., G415R; as described in Yan et al., "SAICAR activates PKM2 in its dimeric form," published in Biochemistry, Aug. 23, 2016; 55(33):4731-4736), or recombinant PKM2, or a protein identical or similar to pyruvate kinase M2 (PKM2) (preferentially in a dimer form), helps protect cardiomyocytes, stimulate cardiomyocyte proliferation, and reduce cardiac fibrosis. This is particularly evident during and after myocardial infarction and is associated with fibroblast recruitment. A dimer form of PKM2 or a mutant thereof can be administered (e.g., acutely) as a treatment for a heart attack. The dimer form can be administered together with other forms (e.g., tetramers). A representative example of a recombinant PKM2 mutant suitable for use in the method is G415R, which primarily takes the dimer form. Administration of recombinant PKM2 mutants can prevent cardiomyocyte apoptosis and promote its proliferation under conditions such as hypoxia and oxidative stress. This applies to myocardial infarction, myocardial ischemia-reperfusion injury (IR), various cardiomyopathies (including hypertrophic cardiomyopathy, dilated cardiomyopathy and toxic cardiomyopathy), cardiotoxicity, congestive heart failure, and cardiac damage caused by viral or bacterial infection. Although the inventors do not wish to be limited to a specific mechanism, they have determined that the dimeric form of PKM2 and its variants and mutants can bind to integrin α on cardiomyocytes. v This interaction activates the FAK-PI3K signaling axis, which subsequently inhibits downstream phosphatase and tensin homolog (PTEN) expression, thereby enhancing survival and proliferation.

[0007] One aspect of the present application includes a method for treating acute cardiac injury in a patient or subject due to acute myocardial cell loss. The method comprises administering to the subject a composition comprising pyruvate kinase M2 (PKM2) or a composition comprising a PKM2 mutant. More specifically, the composition comprising PKM2 or a mutant thereof that preferentially dimerizes at equilibrium can be administered to the subject within 1 to 10 hours after cardiac injury.

[0008] Another aspect includes administering a recombinant PKM2 mutant (e.g., G415R) or recombinant PKM2 (rPKM2) or a protein similar or identical to pyruvate kinase M2 (PKM2) to protect cardiomyocytes during myocardial infarction, suggesting that rPKM2 (dimer mutant) is a therapeutic agent for heart attack and other cardiovascular diseases. Administration of rPKM2 or its mutants protects cardiomyocytes from death and promotes cardiomyocyte proliferation. In mice undergoing myocardial infarction and reperfusion injury, administration of recombinant PKM2 (e.g., at a dose that results in rPKM2 concentrations in the patient's blood of less than 1 micromolar to 5 micromolar) or a preferentially dimerizing mutant reduces activation of cardiac fibroblasts, thereby inhibiting fibrosis.

[0009] Another aspect provides an ischemia / reperfusion protection composition. The ischemia / reperfusion protection composition disclosed herein comprises a recombinant PKM2 mutant (eg, G415R) or recombinant PKM2 or a protein similar or identical to pyruvate kinase M2 (PKM2), which preferably adopts a dimer form.

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

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

[0012] Another aspect includes a method wherein administering occurs within 6 hours after the cardiac injury, or within 3 hours after the cardiac injury, or within 1 hour after the cardiac injury.

[0013] Another aspect includes a method wherein the subject experiences a heart attack and the cardiac damage results from the heart attack.

[0014] Another aspect includes a method wherein the proportion of PKM2 present in tetrameric form is less than 50%.

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

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

[0017] Another aspect includes a method wherein the cardiac injury is caused by acute cardiomyocyte loss.

[0018] Another aspect includes a method wherein myocardial preservation is achieved by administering the composition.

[0019] Another aspect includes a method wherein the composition is delivered extracellularly.

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

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

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

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

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

[0025] Another aspect includes a method of delivering a composition by intracardiac administration.

[0026] Another aspect includes a method of systemically delivering a composition.

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

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

[0029] Another aspect includes a method wherein the pyruvate kinase M2 exists predominantly as a dimer as compared to a tetramer.

[0030] Another aspect includes a method wherein PKM2 has a G415R mutation.

[0031] Another aspect includes a method wherein PKM2 or a PKM2 mutant is located in the extracellular space. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A It was demonstrated that recombinant G415R protected H9C2 cells from apoptosis under hypoxic conditions.

[0034] Figure 1B It was demonstrated that recombinant G415R promoted cell proliferation under hypoxic conditions.

[0035] Figure 1C We demonstrated that recombinant G415R has similar effects on primary human cardiomyocytes and protects them from apoptosis under conditions of hypoxia and oxidative stress.

[0036] Figures 1D and 1E show that recombinant G415R promotes primary cardiomyocyte proliferation under conditions of hypoxia and oxidative stress.

[0037] Figure 2A It is highlighted that administration of recombinant G415R significantly reduced mortality in MI mice at all observed time points.

[0038] Figure 2B It was shown that administration of recombinant G415R reduced heart weight (relative to body weight) in MI mice 30 days after infarction.

[0039] Figure 3A and 3B Depicted are histological analyses of infarcted hearts showing smaller infarct scar areas in mice treated with recombinant G415R compared to rPKM1 and vehicle-treated groups in both MI and IR models.

[0040] Figure 3C 、 3G and 3H show that recombinant G415R can protect cardiomyocytes from apoptosis, as confirmed by TUNEL staining of cardiac tissue 6, 24, and 168 hours after infarction, respectively.

[0041] Figure 3D The results showed that there was little apoptosis of myocardial cells observed by TUNEL staining of myocardial tissue 168 hours after infarction.

[0042] Figure 3E It was shown that in G415R-treated mice, cardiomyocyte proliferation was evident in the myocardial tissues of both MI and IR models at 4 and 7 days after infarction.

[0043] Figure 3F It was shown that G415R treatment significantly reduced cTnI levels in the blood of mice 7 days after infarction.

[0044] Figure 4A Wheat germ agglutinin (WGA) staining of myocardial tissue is shown, indicating that G415R-treated mice had less infarct scar in the infarcted area of the myocardium in both MI and IR models compared to rPKM1 and vehicle-treated groups.

[0045] Figures 4B and 4C illustrate analysis of cardiomyocytes (cross-sectional area) by co-staining with WGA and cTnI, demonstrating that recombinant G415R treatment reduced the cross-sectional area of cardiomyocytes in MI mice to a level similar to that of the sham-operated group.

[0046] Figures 4D and 4E show analysis of cardiomyocytes (cross-sectional area) by co-staining with WGA and cTnI, demonstrating that G415R treatment reduced the cross-sectional area of cardiomyocytes in IR mice to a level similar to that of the sham-operated group, indicating a significant reduction in cardiomyocyte hypertrophy.

[0047] Figures 4F and 4G depict that G415R reduced the activation of cardiac myofibroblasts in the infarcted myocardium of MI and IR mice (observed at 30 days), as indicated by IHC staining for α-SMA.

[0048] Figures 5A-5D It was shown that G415R reduced the expression of PTEN in cultured cardiomyocytes (demonstrated by immunoblotting) and in myocardial tissues of infarcted mice (IHC staining).

[0049] definition

[0050] The following definitions are provided to aid understanding of certain terms used throughout this disclosure.

[0051] The term "administer" refers to providing or delivering a therapeutic agent (e.g., an agent described herein) to a subject by any effective route. In some embodiments, the composition is administered to the same subject by multiple routes of administration. In some embodiments, multiple routes of administration include intravenous administration, intraarterial administration, intrathecal administration, intranasal administration, intraperitoneal administration, and / or periocular administration. In some instances, the composition having PKM2 or a mutant thereof can be administered intravenously to the subject's circulatory system. In some instances, the composition having PKM2 or a mutant thereof can be infused into a suitable liquid and administered to the subject's vein.

[0052] The terms "acute myocardial infarction" and "heart attack" refer to a condition in which localized myocardial ischemia leads to tissue death in a specific area. The most common cause of acute myocardial infarction is the rupture of an atherosclerotic lesion in a coronary artery. This rupture causes a blood clot to form, blocking the artery and preventing blood flow to the area of the heart it serves.

[0053] The term "amino acid" refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids include 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 pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, by way of example only, an α-carbon bound to a hydrogen, carboxyl, amino, and R groups. Such analogs may have modified R groups (e.g., norleucine) or may have a modified peptide backbone while still retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium.

[0054] The term "conservatively modified variant" applies to natural and non-natural amino acid sequences, natural and non-natural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variant" refers to natural and non-natural nucleic acids that encode identical or substantially identical natural and non-natural amino acid sequences, or, when the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to substantially identical sequences. For example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. This type of nucleic acid variation is a "silent variation," which is a type of conservatively modified variation. Thus, for example, every natural or non-natural nucleic acid sequence encoding a natural or non-natural polypeptide herein also describes every possible silent variation of the natural or non-natural nucleic acid. One skilled in the art will recognize that every codon in a natural or non-natural nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variation of natural and non-natural nucleic acids that encode natural and non-natural polypeptides is implicit in each described sequence.

[0055] The term "myocardial infarction (MI)" refers to the death of heart tissue caused by ischemia. "Ischemia" refers to a lack of blood supply to a region, usually due to constriction of blood vessels or poor blood flow to that region. Restoring blood flow to a previously ischemic tissue or organ (such as the heart) is called "reperfusion."

[0056] The term "ischemia-reperfusion" refers to the damage to tissue that occurs when blood supply is restored to the tissue after a period of ischemia. The lack of oxygen and nutrients in the blood results in inflammation and oxidative or peroxidative damage as circulation is restored.

[0057] As used herein, the term "nucleic acid sequence" refers to the order and identity of nucleotides that make up a nucleic acid.

[0058] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing backbone residues or bonds of known nucleotide analogs or modifications, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0059] 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 sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0060] A particular nucleic acid sequence also implicitly encompasses a "splice variant". Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of the nucleic acid. "Splice variants", as the name suggests, are products of alternative gene splicing. After transcription, the initial nucleic acid transcript can be spliced so that different (alternative) nucleic acid splicing products encode different polypeptides. The mechanisms for producing splice variants vary, but include alternative splicing of exons. This definition also encompasses alternative polypeptides derived from the same nucleic acid by read-through transcription. Any product of the splicing reaction, including recombinant forms of the splicing product, is included in this definition.

[0061] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0062] 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 the compound and is relatively non-toxic, i.e., the substance can be administered to a subject without causing adverse biological effects or interacting in a deleterious manner with any of the ingredients of the composition in which it is contained.

[0063] As used herein, the term "prophylactically effective amount" refers to an amount of a composition containing 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 and that will alleviate, to some extent, one or more symptoms of the disease, disorder, or condition being treated. In such prophylactic applications, such an amount may depend on the patient's health status, weight, etc. Those skilled in the art are fully capable of determining such a prophylactically effective amount through routine experimentation, including but not limited to dose escalation clinical trials.

[0064] In the context of two nucleic acids or polypeptides, the phrase "substantially similar" means that two or more sequences or subsequences have at least 75%, preferably at least 85%, more preferably at least 90%, 95%, 95%, 98%, 99% or more, or any integer value therebetween, nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as 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 over a region that is longer than 60-80 residues, more preferably over a region that is at least about 90-100 residues, and most preferably, the sequences are substantially identical over the entire length of the compared sequences, e.g., the coding region of a nucleotide sequence. Substantially similar polypeptides or nucleic acids can be mutants or polypeptides or nucleic acids of other proteins that preferentially adopt a dimer form. In specific examples, more effective proteins preferentially dimerize and are soluble.

[0065] As used herein, the term "synergistic" refers to a combination of prophylactic 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 can allow the use of lower doses of one or more agents and / or less frequent administration of agents to a subject suffering from a particular disease or condition. In some cases, the synergistic effect of a combination of prophylactic or therapeutic agents can be used to avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0066] The term "therapeutically effective amount" refers to the amount of a composition or biological agent containing the protein administered to a patient already suffering from a certain disease, disorder or condition, which is sufficient to cure or at least partially inhibit, or alleviate to some extent, one or more signs, symptoms or causes of the disease, disorder or condition being treated. The effectiveness of such a composition depends on a variety of conditions, including but not limited to the severity and course of the disease, disorder or condition, previous treatment, the patient's health status and response to drugs, and the judgment of the attending physician. By way of example only, a therapeutically effective amount can be determined by routine experimentation, including but not limited to a dose escalation clinical trial. The term "effective amount" is intended to include any amount of a composition or biological agent (e.g., pyruvate kinase M2 or a variant thereof) sufficient to produce the desired therapeutic result.

[0067] In the context of using a composition to treat heart cells following a heart attack, a "therapeutically effective amount" refers to the amount of the composition that, when administered to an individual, produces the desired therapeutic effect to alleviate, mitigate, or reverse the damage to heart cells caused by a heart attack. This amount may vary depending on factors such as the specific ingredients, the severity of the heart attack, the patient's overall health, age, weight, and other medical considerations. For a typical subject of a typical weight, a therapeutically effective amount can range from 0.1 to 10 mg / ml intravenously, or 0.5 to 7 mg / ml intravenously, 0.1 to 6 mg / ml intravenously, or 4 to 6 mg / ml or about 5 mg / ml intravenously. The dose can be administered as a single acute treatment, or delivered gradually over a specified duration (e.g., for 5 minutes to 5 hours or more).

[0068] As used herein, the term "subject" or "patient" includes mammals and humans. In some embodiments, the patient is having a heart attack.

[0069] As used herein, the term "dose" refers to the amount of a composition or biological preparation (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, pM, or any other unit otherwise mentioned in this disclosure or commonly used in the art. In one embodiment, 5 mg / ml is administered.

[0070] The term "therapeutic agent" or "therapeutic" encompasses proteins, peptides, nucleic acids, vectors, pharmaceutical agents, or other macromolecules or compositions known in the art. Therapeutic agents can be delivered to a recipient by inhalation, oral administration, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, or any other agent delivery method used in the art. The agent can be delivered as a single bolus or other one-time administration mechanism; alternatively, the drug can be administered by sustained (continuous or intermittent) delivery.

[0071] The terms "treat," "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect, including but not limited to achieving amelioration, improvement, or elimination of ischemic symptoms. The effect can be preventative in terms of completely or partially preventing cardiac fibrosis, and / or therapeutic in terms of ameliorating, improving, or eliminating one or more symptoms of myocardial cell damage.

[0072] As used herein, "treatment" encompasses any treatment of acute myocardial infarction in mammals, particularly humans, and includes: (a) preventing the development of cardiac fibrosis in a subject; (b) alleviating cardiac fibrosis; and (c) restoring the subject 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.

[0073] PKM2 used in this specification refers to pyruvate kinase isoform M2.

[0074] As used herein, a PKM2 mutant is 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 a genetic mutation or may be artificially introduced through methods such as genetic engineering. Mutations may result in changes in the function, stability, interaction partners, or other properties of the protein, and DETAILED DESCRIPTION

[0075] In general, the present application provides methods for treating and / or preventing heart disease in a subject in need thereof. The inventors have conducted extensive research to develop a method for protecting myocardial cells from death (e.g., apoptosis) and preventing or reducing cardiac fibrosis or heart disease associated with cardiac fibrosis. Specific embodiments relate to protecting myocardial cells and promoting the growth of myocardial cells. Treatable heart diseases include cardiomyopathies, such as hypertrophic cardiomyopathy, dilated cardiomyopathy, and toxic cardiomyopathy; cardiotoxicity; congestive heart failure; and heart damage caused by some infectious agents (e.g., viruses or bacteria).

[0076] One embodiment is a method of reducing adverse consequences of myocardial infarction in a patient, comprising administering to the patient during the acute phase of myocardial infarction a composition comprising a recombinant PKM2 mutant (e.g., G415R), or recombinant PKM2 or a protein similar or identical to PKM2 that preferentially exists in dimeric form or has at least some PKM2 dimeric form. In some embodiments, the therapeutic PKM2 is not PKM2 that exists primarily in tetrameric form. In some embodiments, the therapeutic PKM2 is not expressed intracellularly, for example, due to transfection of cardiomyocytes with a nucleic acid encoding PKM2. In specific embodiments, the PKM2 or PKM2 mutant in the composition exists in dimeric form or exists at least partially in dimeric form.

[0077] Physiologically, a period of time after cardiac injury or trauma is crucial and is sometimes referred to as the "golden hour" or the first hour after a myocardial infarction. The myocardial infarction may be an acute myocardial infarction. Administration of the ischemia / reperfusion protection composition may be initiated within 200 hours after a heart attack. In addition, a protein substantially similar or identical to pyruvate kinase M2 (PKM2) may be initiated within about 100 hours, about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour after a heart attack. The patient may be a human or non-human mammal.

[0078] Another embodiment provides an ischemia / reperfusion protection composition. The ischemia / reperfusion protection composition disclosed herein comprises a protein similar to or identical to PKM2, at a dosage resulting in a concentration of <1 μM to 5 μM in the patient's bloodstream under physiological conditions, or a mutant PKM2 that exists primarily in dimer form. In specific embodiments, PKM2 administered at a concentration of <1 μM under physiological conditions in a patient will be present in the form of approximately 70-85% dimers. In further specific embodiments, PKM2 administered at a concentration of >10 μM under physiological conditions in a subject will be present in the form of <25% dimers. In further specific embodiments, when the pharmaceutical formulation is administered to a patient at a concentration of 5 mg / ml, its dosage form is approximately 100 μM, but once administered and enters the patient's blood circulation, it will be diluted to <5 μM. In specific embodiments, the PKM2 mutant G415R is present in the form of >85% dimers at concentrations >50 μM. The ischemia / reperfusion protection compositions described herein can be administered to an individual to significantly reduce or prevent cardiac injury / reperfusion injury to all heart-related tissues.

[0079] In various embodiments, the present invention discloses, in part, administering to a subject an effective amount of a therapeutic composition to protect cardiomyocytes or promote cardiomyocyte growth. Specific embodiments contemplate treating a subject with a therapeutic agent substantially similar or identical to PKM2. In more specific embodiments, wild-type PKM2 and a PKM2 mutant that preferentially dimerizes at equilibrium comprise at least 51% dimer, at least 60% dimer, at least 70% dimer, at least 80% dimer, or at least 90% dimer. The composition reduces cardiomyocyte death caused by cardiac injury.

[0080] In various embodiments, the present invention discloses a method for reducing or inhibiting cardiac fibrosis by administering to a patient a composition comprising recombinant wild-type PKM2 in an amount such that, following administration to the patient, the concentration of wtPKM2 in the bloodstream is <1 μM. In various embodiments, the present invention discloses a method for reducing or inhibiting cardiac fibrosis by administering to the patient a composition comprising a PKM2 mutant that preferentially dimerizes at equilibrium. Without intending to be bound by a particular mechanism, it is hypothesized that the reduction in fibrosis is a result of reduced cardiomyocyte death.

[0081] Although PKM2 exists in both dimer and tetramer states, the biologically active protein possesses a dimeric state, and more potent mutants exist at equilibrium with a higher percentage of dimers relative to tetramers.PKM2 mutants that can adopt a dimer form are described herein and shown in the following exemplary references incorporated herein by reference: Gao, X., Mol Cell 2012 Mar 9; 45(5): 598-609.; Zhou. Zhifen et al. "Oncogenic kinase-induced PKM2 tyrosine 105phosphorylation converts nononcogenic PKM2 to a tumor promoter and induce s cancer stem-like cells." Cancer research 78.9(2018): 2248-2261.; Li, iScience 23, 101684, Nov 20, 2020; Liu, Vivian M. et al. "Cancer-associated mutations in human pyruvate kinase M2 impair enz ymeactivity." FEBS letters 594.4(2020):646-664;Chen.Tsan-Jan et al. “Mutations in thePKM2 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 kinaseM2 isozym e 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 excessive 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 nucleotide or amino acid residue identity compared to wild-type PKM2. PKM2 mutants that preferentially utilize dimerization can be used in methods and systems for treating MI, IR, and other conditions associated with myocardial cell damage.

[0082] In one embodiment, the compositions and methods are directed to cardiac disorders involving damaged cardiac tissue, such as myocardial damage caused by an ischemic event, ischemia-reperfusion injury, left ventricular damage (such as that caused by congestive heart failure), and heart valve damage caused by diseases such as 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 will benefit the patient.

[0083] In another embodiment, the composition can be administered with another pharmaceutical agent. Such pharmaceutical agents may include lipid-lowering drugs, antiplatelet drugs, antihypertensive drugs, vasodilators, hypoglycemic drugs, anticoagulants, thrombolytic drugs, hepatoprotective drugs, antiarrhythmic drugs, cardiotonic drugs, diuretics, anti-infective drugs, antiviral drugs, immunomodulators, inflammation modulators, anti-tumor drugs, or hormonal drugs.

[0084] Pyruvate kinase

[0085] Pyruvate kinase isoform M2 (PKM2) is the pyruvate kinase isoform expressed in mammalian cells. Pyruvate kinase regulates the final, rate-limiting event of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate to ADP to produce pyruvate and ATP. Of the four pyruvate kinase isoforms, PKM1 and PKM2 are ubiquitously expressed in different cell and tissue types. PKM2 is highly expressed in proliferating cells, including cancer cells. Unlike other isoforms, PKM2 expression and activity are regulated at multiple levels, including gene expression, alternative splicing, post-translational modifications, and metabolic intermediates and growth signaling pathways. Therefore, PKM2 is a unique multifaceted regulator that improves the adaptability of cellular metabolic programs to meet the physiological demands of diverse environments.

[0086] In addition to regulating glycolysis, PKM2 also has non-metabolic functions, such as regulating transcription and cell cycle progression. In contrast to mitochondrial respiratory responses, energy regeneration by these pyruvate kinases is independent of oxygen supply and allows organ survival under hypoxic conditions. PKM2 also acts as a coactivator of hypoxia-inducible factor 1-α (HIF-1α), a master transcription factor that regulates multiple signaling pathways in response to hypoxic insult. Increased PKM2 levels and activity are associated with enhanced motility and metastasis in tumor cells; 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 malignancies but rather originate in normal biology and physiological development. During physiological proliferation of neural progenitor cells or under hypoxic conditions, PKM2 helps reprogram energy metabolism to support growth and adaptation. Therefore, metabolic transformation is a co-option of developmental events essential for physiological growth. Because these important metabolic and non-metabolic roles of PKM2 have been primarily identified in cancerous tumor cells, its potential functions in normal cells or in response to ischemic events such as stroke or myocardial infarction have remained largely unknown.

[0087] 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 native pyruvate kinase, or a recombinant or other synthetic protein. The amino acid sequence of human pyruvate kinase is provided, for example, by GenBank accession number MP0011193727 Pyruvate Kinase. For example, the amino acid sequence identity of pyruvate kinase is highly conserved across species, with 98% amino acid sequence identity between humans and mice, hamsters, and rats. The amino acid sequence of human pyruvate kinase is disclosed herein (Example 1). In a specific embodiment, the active pyruvate kinase protein is a dimer, also referred to as PKM2 isoform M2. A wide range of proteins and therapeutic agents substantially similar to pyruvate kinase M2 can be used in specific embodiments.

[0088] The animal from which the native pyruvate kinase protein is purified can be, for example, a member of a cattle, sheep, pig, horse, dog, cat, primate, rodent, or other mammalian family. In at least some forms, the pyruvate kinase protein will be a human pyruvate kinase protein purified from bacterial production. Recombinant pyruvate kinase protein can have the same amino acid sequence as the native pyruvate kinase, or have one or more amino acid differences compared to the native protein. Amino acid changes can include one or more amino acid additions, deletions, and / or substitutions. Also included are amino acid inversions and other mutations that cause the native pyruvate kinase protein sequence to change. In addition, the recombinant protein can contain one or more amino acids that are not encoded by the genetic code.

[0089] Amino acid substitutions can be conservative substitutions or non-conservative substitutions. The term conservative amino acid substitution should be understood in accordance with the generally accepted meaning, i.e., replacing an amino acid residue with another amino acid having similar properties, which will not produce substantial adverse effects on the angiogenesis and / or wound healing activity of the pyruvate kinase protein. For example, conservative amino acid substitutions can involve replacing a basic amino acid (e.g., arginine) with another basic amino acid (e.g., lysine). Similarly, for example, a cysteine residue can be replaced by a serine, or a non-polar amino acid can be replaced by another non-polar amino acid (e.g., alanine). The amino acid that is easy to replace or lack in the pyruvate kinase protein amino acid sequence can be compared with a closely related pyruvate kinase protein to identify non-conservative amino acids, and determined by routine tests and experiments within the scope of the recipient's skills. Modified recombinant pyruvate kinase protein can be provided by introducing nucleotide changes in the nucleic acid sequence encoding the native protein so that the desired amino acid changes are achieved when the nucleic acid is expressed in a host cell.

[0090] One embodiment includes recombinant or other synthetic PKM2 that preferentially dimerizes. Such recombinant or other synthetic PKM2 includes the PKM2 G415R mutant. The amino acid sequence of the G415R mutant is shown in Example 2 and SEQ ID NO: _. More useful PKM2 variants or mutants thereof are those that preferentially adopt a dimer form and are soluble in water.

[0091] An exemplary pyruvate kinase M2 amino acid sequence or SEQ ID NO: 1: PKM2 (Accession No. NP 002645) is as follows:

[0092]

[0093] An exemplary mutant pyruvate kinase M2 amino acid sequence is SEQ ID NO: 2 (R399E) as follows:

[0094]

[0095] Another exemplary mutant pyruvate kinase M2 amino acid sequence having three mutations (R399E, K422A, and N523A) or SEQ ID NO: 3 is as follows;

[0096]

[0097] The recombinant or synthetic pyruvate kinase protein suitable for the method of the present invention should show at least 60% amino acid sequence identity with native pyruvate kinase. More commonly, the identity can be at least 70%, 80%, 90%, 95%, 98% or even 100%. Under the patient's physiological conditions, some proteins must form dimers. All sequence homologies and ranges specified above are clearly included. The sequence identity between amino acid sequences is determined by comparing the amino acids at each position in the best aligned sequences. Only when they match, the amino acids at a given position are considered to be identical. In the comparison, a gap (an amino acid residue that appears in one sequence but does not appear in another sequence) is considered to have a position with non-identical residues. Any appropriate program or algorithm can be used to realize sequence alignment, wherein computer-assisted sequence alignment is usually performed using standard software.

[0098] Pyruvate kinase protein can also be synthesized chemically. The present invention also encompasses the production and use of fusion proteins incorporating pyruvate kinase protein as described herein. Nucleic acids encoding fusion proteins can be generated by ligating separate DNA fragments encoding pyruvate kinase protein with, for example, a lipophilic amino acid sequence to enhance the lipophilicity of the protein. This can be achieved using methods such as using blunt ends and oligonucleotide linkers, digestion to provide staggered ends, and ligation of sticky ends as needed.

[0099] Pyruvate kinase protein as described herein can also be modified by coupling one or more proteins or non-protein moieties to the protein. This can improve aspects such as solubility, lipophilicity, stability, biological half-life, or as a label for subsequent detection. Modification can come from post-translational or post-synthetic processes, such as the attachment of carbohydrate moieties or chemical reactions that cause structural modifications (e.g., alkylation or acetylation of amino acid residues). For example, the pyruvate kinase protein can undergo modification, such as methylation, phosphorylation, oxidation of tyrosine and / or tryptophan residues, glycosylation, or S-methylcysteine covalent attachment. The size of the pyruvate kinase protein can be different from the intact protein. However, the length of the pyruvate kinase should promote dimer formation.

[0100] The C-terminal and N-terminal extensions of the native pyruvate kinase protein play a role in stabilizing the quaternary structure and generating protein aggregates. Therefore, pyruvate kinases lacking such extensions are less efficient at forming aggregates. Pyruvate kinases tend to form large aggregates. Electrostatic interactions between these proteins can also affect aggregate formation, and ionization of histidine residues at pH below 7 can disrupt these aggregates. Typically, the pyruvate kinase protein utilized in the methods of the present invention will exist as a dimer. Both 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 the native pyruvate kinase protein. Suitable conditions for alkaline phosphatase activity include the presence of a buffer containing zinc, magnesium, or calcium.

[0101] Partially hydrolyzed forms of pyruvate kinase protein may be purified for use in embodiments of the present invention using any suitable purification technique, including filtration and chromatography protocols.

[0102] Pyruvate kinase administration

[0103] Pyruvate kinase protein can be administered alone to a subject in need of such treatment, or co-administered with one or more other therapeutic agents. For example, pyruvate kinase can be co-administered with conventional therapeutic combinations for promoting angiogenesis, cell proliferation, or wound healing. "Co-administered" means administered simultaneously with the same formulation or two different formulations by the same or different routes, or administered sequentially by the same or different routes, such that the pyruvate kinase protein and other therapeutic agents exhibit overlapping therapeutic windows. "Sequential" administration means administration one after another. Such further agents that can be co-administered with pyruvate kinase protein include platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), platelet-derived wound healing factor, insulin growth factor (IGF), keratinocyte growth factor (KGF), anti-inflammatory agents, and antimicrobial agents. Further examples of other therapeutic agents for promoting angiogenesis and / or wound healing that can be co-administered with pyruvate kinase protein include indoleamine 2,3-dioxygenase (IDO), tryptophan dioxygenase (TDO), sphingosine-1-phosphate (SIP), N-acylethanolamines, grapefruit extract and other phytochemicals, including resveratrol, green tea catechins, melatonin, arginine and other amino acids that support angiogenesis. Additional therapeutic agents suitable for co-administration will be apparent to those of ordinary skill in the art.

[0104] The pyruvate kinase protein will generally be formulated as a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.

[0105] The pharmaceutical compositions described herein may also incorporate one or more preservatives (e.g., parabens, chlorobutanol, and sorbic acid), binders (e.g., 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 formulations, and solutions. The use of such ingredients and media for pharmaceutically active substances is well known. Unless any conventional media or agents are incompatible with pyruvate kinase protein, their use is explicitly contemplated.

[0106] Pharmaceutical compositions embodied by the present invention include therapeutic compositions for human or veterinary use.

[0107] Pharmaceutical compositions embodied in the present invention will typically contain at least about 0.001% up to about 80% by weight of the composition of pyruvate kinase protein. 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 mutant PKM2 that preferentially dimerizes. Pharmaceutical compositions embodied in the present invention will typically contain at least about 20 μg / ml up to about 1000 μg / ml of pyruvate kinase protein. 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, 1000 μg / ml of pyruvate kinase protein, or a substantially similar therapeutic agent or a mutant PKM2 that preferentially dimerizes. The amount of protein in the composition will be such that a suitable effective dose is delivered to the subject, taking into account the proposed mode of administration.

[0108] The dosage of the pyruvate kinase protein administered according to one embodiment of the present invention will depend on many factors, including whether the protein is administered for prophylactic or therapeutic purposes, the disease or condition for which the protein is intended to be administered, the severity of the condition, the sex and age of the subject, and related factors including the subject's weight and overall health, and can be determined according to generally accepted medical principles. For example, a lower dose may be administered initially, and then the dose may be increased with each administration after assessing the subject's response. Similarly, the frequency of administration may be determined in the same manner, i.e., by continuously monitoring the subject's response between each administration and, if necessary, increasing or decreasing the frequency of administration. In one example, the dosage may be between 1 mg / kg and 20 mg / kg; in another example, the dosage may be between 2 mg / kg and 10 mg / kg; in another example, the dosage may be between 1 mg / kg and 6 mg / kg; and in yet another example, the dosage may be between 2 mg / kg and 5 mg / kg.

[0109] Route of administration includes but is not limited to local, respiratory, intravenous, oral, intraperitoneal, subcutaneous, intramuscular, rectal, local, directly enter the heart and implant.For intravenous approach, particularly suitable approach is by injection into the blood vessel (such as superior vena cava and inferior vena cava) that supplies blood to target tissue to be treated. Pyruvate kinase protein can also be delivered to cavity, such as pleural cavity or peritoneal cavity, cranial cavity, or is directly injected into tissue to be treated, such as left ventricle or right ventricle of heart. For oral administration, pyruvate kinase protein can be encapsulated or otherwise provided in enteric-coated tablets, so as to pass through the stomach and discharge in the small intestine. Any suitable enteric formulation or coating can be used. In addition, these systems and methods can use any medically acceptable mode of administration to put into practice, which means that any cell regeneration or tissue repair that can produce effective levels can not cause clinically unacceptable adverse reactions.

[0110] In addition, pyruvate kinase protein can also be coated onto the surface of a catheter (such as an angioplasty catheter) or a stent or balloon of other surgical instruments for application to the inner wall of a blood vessel during angioplasty or other surgical procedures. For example, pyruvate kinase can be applied to the blood vessel wall in this manner in the form of a gel or any other suitable formulation to promote wound healing and / or angiogenesis, epithelial cell migration, or cell regeneration at the treatment site.

[0111] Suitable pharmaceutically acceptable carriers and formulations that can be used in the compositions embodied by the present invention can be found, for example, in handbooks and texts.

[0112] Medicaments, kits and implementation plans

[0113] Medicaments include the following categories and specific examples. Categories are not intended to be limited to specific examples. Those of ordinary skill in the art will be able to easily identify medicaments useful inside or outside the central nervous system. Those of ordinary skill in the art will also recognize many other compounds that belong to the described categories and are useful according to the present invention.

[0114] One embodiment also includes a kit for improving recovery after a heart attack or injury.The combination of agents is provided so as to be administered in therapeutically effective amounts and frequencies to produce cell regeneration after a heart attack or injury.

[0115] In some embodiments, it may be necessary to increase the solubility and blood circulation time of PKM2 or a substantially similar therapeutic agent. In order to increase polypeptide solubility and blood circulation time, polyethylene glycol can be used to derivatize the polypeptide of the present invention, including, for example, poly (ethylene glycol) (PEG), poly (vinyl pyrrolidone), polyoxyethylene ether, polysorbate and poly (vinyl alcohol), of which PEG polymers are particularly preferred. PEG polymers are PEG polymers having a molecular weight of about 100 to about 40,000. In addition to those exemplified above, those skilled in the art will readily know other suitable hydrophilic polymers based on this disclosure. In general, the polymer used may include a polymer that can be attached to the polypeptide of the present invention by an alkylation or acylation reaction. In one example, PKM2 or a substantially similar therapeutic agent is PEGylated with a 20 kDa PEG chain.

[0116] When attaching a polyethylene glycol molecule (or other chemical moiety) to a polypeptide, the effect on the functional domain or antigenic domain of the polypeptide should be considered. A variety of attachment methods are available to those skilled in the art. For example, polyethylene glycol can be covalently bound via reactive groups (e.g., free amino groups or carboxyl groups) via amino acid residues. Reactive groups are groups that activate polyethylene glycol molecules to bind to them. Amino acid residues with free amino groups may include lysine residues and N-terminal amino acid residues; amino acid residues with free carboxyl groups may 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 to an amino group is preferred, for example, to an N-terminal or lysine group. People may particularly need polypeptides that are chemically modified at the N-terminus. Using polyethylene glycol as an example of the composition of the present invention, it can be selected from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to polypeptide (polypeptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method for obtaining the selected N-terminal pegylated polypeptide. Under appropriate reaction conditions, substantially selective derivatization of polypeptides at the N-terminus with carbonyl-containing polymers is achieved.

[0117] Certain specific 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 in a dimer form rather than a tetramer form, 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, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the carrier. Saline solutions, as well as aqueous dextrose solutions and aqueous 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, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories with conventional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of PKM2 or a substantially similar therapeutic agent and an appropriate amount of carrier to provide an appropriate administration form for the patient. The formulation should be suitable for the mode of administration.

[0118] The dosage of PKM2 or a substantially similar therapeutic agent that will effectively aid in the recovery of an ischemic attack can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to help determine the optimal dosage range. The precise dosage used in the formulation will also depend on the route of administration and the severity of the disease or condition and should be determined based on the physician's judgment and the circumstances of each patient. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0119] More specifically, prior to use in humans, a pharmaceutical agent or pharmaceutical composition can be tested in vitro and then tested in vivo for its desired therapeutic or preventive activity. For example, in vitro assays demonstrating the therapeutic or preventive efficacy of a compound or pharmaceutical composition include the effects of the compound on a cell line or patient tissue sample. Techniques known to those skilled in the art can be used to determine the effects of a compound or composition on a cell line and / or tissue sample, including but not limited to rosette formation assays and cell lysis assays. According to the present invention, in vitro assays that can be used to determine whether a particular compound needs to be administered include in vitro cell culture assays, in which patient tissue samples are grown in culture and exposed to the compound or administered in other ways, and the effects of such compounds on the tissue sample are observed.

[0120] It is expected that PKM2 or a substantially similar therapeutic agent can be formulated as a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic (e.g., lidocaine) to relieve pain at the injection site. Typically, the components are either provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate, packed in a sealed container (such as an ampoule or a pouch) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule containing sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0121] Various delivery systems are known and can be used to administer the compounds of the present invention, for example, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds, receptor-mediated endocytosis, constructing nucleic acids as part of retroviruses or other vectors, etc. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelium or mucosal skin lining (e.g., oral mucosa, rectum, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or topical. Additionally, the pharmaceutical compounds or compositions of the present invention can also be introduced into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection can be facilitated by intraventricular catheters.

[0122] In a specific embodiment, it may be necessary to administer the therapeutic composition (ischemia / reperfusion protective composition) locally to the area in need of treatment. This can be achieved by, for example, but not limited to, local injection (e.g., injection into the myocardium) or infusion during surgery, by injection, by catheter, by suppository, or by implant, the implant being made of porous, non-porous, or colloidal material, including membranes (e.g., silicone rubber membranes) or fibers. When administering a polypeptide, care should be taken to use a material that the polypeptide does not absorb.

[0123] It is expected that regulatory genes and sequences can be used for the expression and replication of PKM2 or substantially similar therapeutic agents. The nature of gene expression regulatory sequences may vary depending on the species or cell type, but generally should include 5' non-transcribed sequences and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, as needed, such as TATA boxes, capping sequences, CAAT sequences, etc. Promoters can be constitutive or inducible. Regulatory sequences may also include enhancer sequences or upstream activator sequences as needed.

[0124] In one embodiment, a polynucleotide encoding PKM2 or a substantially similar therapeutic agent may be fused to a polynucleotide encoding a signal sequence that directs the localization of the polypeptide to a specific compartment of a prokaryotic or eukaryotic cell and / or directs the secretion of the polypeptide. For example, in E. coli, one may wish to direct the expression of a protein to the periplasmic space. Several vectors are commercially available for constructing fusion proteins that direct protein localization.

[0125] One specific embodiment provides a stent comprising a generally tubular structure, which may include, for example, a helical shape. As described above, the surface of this structure is coated with PKM2 or a substantially similar therapeutic agent. The stent is typically a generally cylindrical stent that can be inserted into a body passage (e.g., a bile duct, an artery, a vein) or a portion thereof. The passage may be narrowed, irregularly contoured, blocked, or occluded due to a disease process (e.g., tumor growth), preventing the passage from closing or reclosing.

[0126] A specific embodiment also provides for the use of PKM2 or substantially similar therapeutic agents in a variety of surgical procedures. For example, surgical mesh coated with PKM2 can be used in any procedure where surgical mesh may be used.

[0127] Example

[0128] Example 1

[0129] The following is an exemplary wild-type pyruvate kinase M2 amino acid sequence or SEQ ID NO:4.

[0130]

[0131] Example 2 (PKM2 G415R mutant sequence)

[0132] The following is the amino acid sequence of a modified pyruvate kinase protein, wherein at least one glycine is replaced by an arginine. This protein preferentially adopts a dimer form relative to wild-type EcPKM2 or SEQ ID NO. 5.

[0133]

[0134] Example 3 (Extracellular administration of PKM2 G415R mutant)

[0135] Studies have shown that integrin α vβ3 is expressed in cardiomyocytes under myocardial infarction conditions. The present inventors investigated the expression of integrins in H9C2 cells by immunofluorescence (IF) staining. Integrins are highly expressed in cells under hypoxic conditions, but not under normoxic conditions. In addition, the present inventors analyzed the expression of integrins in primary human cardiomyocytes under hypoxic and oxidative stress conditions. Integrins are expressed in cardiomyocytes under stress conditions, while integrins are not expressed under normal culture conditions. In order to confirm the clinical relevance of integrin expression, integrin expression in cardiac tissue of patients with myocardial infarction was analyzed. Integrins are highly expressed in the infarcted area, but are barely detected in the normal non-infarcted area. Under hypoxic and oxidative stress conditions, integrin a in cardiomyocytes v b3 is upregulated.

[0136] Extracellular PKM2 (EcPKM2) interacts with integrin αvβ3 on angiogenic 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 Escherichia coli. 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. Compared to recombinant PKM2, G415R exhibited enhanced solubility and prolonged stability.

[0137] Figure 1A Recombinant G415R was shown to protect H9C2 cells from death (eg, apoptosis) under hypoxic conditions. Figure 1B This indicates that the G415R mutant promoted cell growth under these conditions. Figure 1C Recombinant G415R was shown to have similar effects on primary human cardiomyocytes, specifically protecting them from cell death under conditions of hypoxia and oxidative stress. Similarly, Figures 1D and 1E depict the promotion of cell growth in primary cardiomyocytes by the G415R mutant under conditions of hypoxia and oxidative stress.

[0138] Example 4 (Recombinant G415R preserves cardiomyocytes in a mouse myocardial infarction model)

[0139] Protecting cardiomyocytes from death and promoting cardiomyocyte proliferation under conditions of hypoxia and oxidative stress can protect the heart during myocardial infarction, so the inventors used the commonly used mouse left anterior descending coronary artery ligation model to determine the effect of this PKM2 mutant on myocardial infarction. 24 hours before arterial ligation, animals were pretreated with a single dose (50 μg / ml) of G415R. The next day, myocardial infarction (MI) or ischemia-reperfusion (IR) was induced. Infarcted mice (both MI and IR) received the same dose of G415R twice a week for two weeks. Ultrasound and MR imaging showed that myocardial infarction and IR were successfully induced due to arterial ligation. Figure 2A It was shown that administration of G415R significantly reduced the mortality of MI mice at all time points. Figure 2B G415R administration also reduced heart weight and body weight in MI mice 30 days after infarction. MR imaging analysis of cardiac blood flow showed that G415R treatment improved blood flow in MI mice and restored blood flow in IR mice to almost the same level as in sham-operated mice.

[0140] Figure 3A and 3B Histological analysis of infarcted hearts was presented, showing that the infarct scar area was reduced in mice treated with G415R compared to rPKM1 and vehicle-treated groups in both MI and IR mice. To determine whether G415R could protect cardiomyocytes from apoptosis, TUNEL staining was performed on mouse heart tissues at 6, 24, and 168 hours after myocardial infarction. Figure 3C As depicted, G415R treatment significantly reduced cardiomyocyte apoptosis at 6 and 24 hours. At 168 hours after infarction, cardiomyocyte apoptosis was almost undetectable using TUNEL staining, and no significant differences were observed between the treatment groups. Figure 3D shown.

[0141] Studies have found that G415R can stimulate the proliferation of human primary cardiomyocytes under conditions of hypoxia and oxidative stress. Therefore, the inventors used Ki67 staining to investigate whether G415R also enhances cardiomyocyte proliferation. As expected, G415R treatment did not trigger cardiomyocyte proliferation within 6 hours after infarction. However, in mice treated with G415R, significant cardiomyocyte proliferation was observed in the myocardial tissue of both MI and IR mice on days 4 and 7 after infarction ( Figure 3E The effect of G415R on cardiomyocyte proliferation weakened from day 4 to day 7 and was almost absent by day 28 post-infarction in both MI and IR mice. This suggests that the optimal period for promoting cardiomyocyte proliferation / regeneration is 1 to 7 days post-infarction.

[0142] Cardiomyocyte death leads to the release of cardiac troponin I (cTnI) into the blood, a key molecular marker of cardiac damage. Plasma samples from infarcted mice were analyzed for cTnI levels. Almost no cTnI was detected in the plasma of sham-operated mice. cTnI concentrations peaked 24 hours after infarction. However, G415R treatment significantly reduced circulating cTnI levels in infarcted mice by day 7 after infarction.

[0143] In summary, our experiments show that administration of the recombinant PKM2 mutant G415R to mice with MI and IR effectively protects cardiomyocytes from apoptosis and promotes cardiomyocyte proliferation after infarction. Because G415R is known to act through the same mechanism as PKM2, these results demonstrate the effectiveness of PKM2 and proteins that are substantially similar to PKM2.

[0144] Example 4 (EcPKM2 alleviates cardiac fibrosis caused by infarction)

[0145] Infarction-induced cardiomyocyte loss triggers cardiac fibroblast activation, which releases ECM / collagen to repair the damaged myocardium. Continuous activation of cardiac fibroblasts leads to ECM accumulation in the myocardium and cardiac fibrosis. Figure 4A WGA staining of myocardial tissues was shown, demonstrating that G415R-treated mice had less infarct scar in the infarcted myocardial area compared to rPKM1 and vehicle-treated groups in both MI and IR models.

[0146] Figures 4B and 4C show Masson-Trichrome staining of myocardial tissue. Staining showed that compared with mice treated with rPKM1 and vehicle groups, mice treated with G415R had reduced collagen accumulation in the infarcted myocardial area of MI and IR models. Wheat germ agglutinin (WGA) staining of infarcted myocardial tissue further confirmed these findings.

[0147] Figures 4D and 4E show analysis of cardiomyocyte cross-sectional area using co-staining with WGA and cTnI. The data indicate that, for both MI models, G415R treatment reduced cardiomyocyte cross-sectional area to an extent almost identical to that of the sham-operated group.

[0148] Figure 4E Figures 4 and 4F depict the results from the IR model, highlighting that G415R treatment of infarcted mice significantly inhibited cardiomyocyte hypertrophy. A potential explanation for the reduced ECM / collagen fiber accumulation in the myocardium of G415R-treated mice is that G415R treatment may lead to a reduction in activated cardiac fibroblasts in MI and IR mice due to the preservation of cardiomyocytes.

[0149] Finally, Figures 4F and 4G demonstrate the activation of cardiac fibroblasts in the infarcted myocardium of MI and IR mice, as confirmed by IHC staining of α-SMA (a molecular marker of myofibroblasts). Treatment with G415R resulted in a significant reduction in α-SMA staining in the myocardial tissue of MI and IR mice.

[0150] Example 5 (Systemic administration of G415R-preserved cardiomyocytes in infarcted hearts)

[0151] After 4 days, PKM2 was expressed in infarcted heart tissue, while no PKM2 staining was detected in normal healthy heart tissue. Extracellular PKM2 was visible in the staining of myocardial infarction tissue from patients. The present inventors also performed IHC staining of PKM2 in infarcted heart tissue from mice at different time points after infarction induction. No PKM2 staining was observed 6 hours, 24 hours, and 3 days after infarction induction. 4 days after infarction induction, PKM2 staining was detected in the infarcted area. EcPKM2 was visible in IHC staining. No PKM2 was detected in the plasma of sham-operated mice or mice 6 hours and 24 hours after myocardial infarction. 7 days after myocardial infarction, PKM2 was detected in mouse plasma (not shown). Our experiments and those of other laboratories have shown that PKM2 is expressed from, for example, dead cells and released into the extracellular space at late time points after myocardial infarction induction (approximately 4 days after infarction).

[0152] Example 6 (EcPKM2 and integrin α v β3 interacts and activates it)

[0153] Co-immunoprecipitation experiments were performed using G415R and extracts from primary human cardiomyocytes exposed to hypoxia. Figures 5A-5D As shown in Figure 3, G415R effectively reduced PTEN expression in cultured cardiomyocytes (demonstrated by immunoblotting) and myocardial tissues of infarcted mice (observed by IHC staining). Figures 5A-5D G415R was shown to have affinity for integrin β3, as evidenced by its coprecipitation, confirmed using antibodies specific for PKM2 or integrin β3.

[0154] To determine whether EcPKM2 initiates cardiomyocyte responses through integrin signaling, the present inventors first measured the level of integrin signaling activation. After G415R was introduced into the culture medium of H9C2 cells, FAK activation was significantly increased. However, Figure 5CAs indicated, this FAK activation was inhibited by the antibodies LM609 and IgGPK. Subsequent assessment of PI3K activation in H9C2 cells following G415R treatment by immunoblotting and specific PI3K activity assays confirmed its upregulation and activation in these cells. Furthermore, PI3K activation was abrogated by a FAK inhibitor. Therefore, if the cardioprotective and proliferative effects of EcPKM2 are primarily through PI3K activation, PI3K inhibitors would abrogate their effects. Supporting this notion, the effects of G415R on H9C2 proliferation were abrogated by commercially available FAK and PI3K inhibitors. EcPKM2 promotes a beneficial interaction with the integrin avβ3. This interaction, in turn, triggers integrin signaling in cardiomyocytes during hypoxia and oxidative stress, providing a barrier against apoptosis and promoting cell proliferation.

[0155] Given the well-established role of the PI3K-PTEN pathway in regulating cardiomyocyte behavior and the effects of reduced PTEN expression or its pharmacological inhibition on cardiomyocyte survival and proliferation, EcPKM2 may direct cardiomyocyte responses to hypoxia and oxidative stress, potentially through FAK-PI3K signaling, primarily by downregulating PTEN levels in cardiomyocytes.

[0156] Reducing PTEN expression in cardiomyocytes or pharmacologically inhibiting PTEN can prevent cardiomyocyte death and stimulate its proliferation. Therefore, the present inventors speculate that EcPKM2 may regulate cardiomyocyte apoptosis and proliferation under conditions of hypoxia and oxidative stress by reducing PTEN levels in cardiomyocytes through the same FAK-PI3K signaling pathway. Initially, the present inventors examined PTEN levels and activity in H9C2 cells treated with G415R under hypoxia and normoxia. Under normoxia, PTEN expression in cells is low. Introducing G415R into cell culture has minimal effect on PTEN expression. However, PTEN is significantly expressed under hypoxia. Figure 6H shows that adding G415R to the culture medium, but not rPKM1 or vehicle, reduces PTEN in cells. This suggests that EcPKM2 may contribute to the downregulation of PTEN in cardiomyocytes during myocardial infarction, thereby protecting cardiomyocytes from apoptosis and stimulating proliferation. To further verify the effect of EcPKM2 on PTEN in cardiomyocytes, the present inventors used IHC to assess PTEN levels in myocardial tissue from infarcted mouse hearts. Sham-operated mice showed only trace amounts of PTEN expression, whereas PTEN levels were elevated in both MI and IR mice. G415R treatment reduced PTEN levels threefold. This observation supports the conclusion that EcPKM2 activates the integrin avβ3-FAK-PI3K signaling pathway, thereby reducing PTEN levels in cardiomyocytes during MI.

[0157] The foregoing detailed description and accompanying drawings are for illustration and description purposes only. They are not intended to be exhaustive or to limit the scope of the invention. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application. Those skilled in the art will recognize that many variations may be made to the invention disclosed in this specification 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 pyruvate kinase M2 (PKM2) or a PKM2 mutant within 10 hours after the cardiac injury, wherein the PKM2 or PKM2 mutant is in a dimer form.

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

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

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

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

6. The method of claim 1, wherein the subject experienced a heart attack and the cardiac damage was caused by the heart attack. The method of claim 1 , wherein the proportion of PKM2 in its tetrameric form is less than 50%.

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

9. The method of claim 1, wherein the PKM2 or the PKM2 mutant preferentially 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 myocardial preservation is achieved by administering the 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. The method according to claim 1 , wherein the PKM2 is derived from humans or other animals.

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 cardiomyocyte death resulting from myocardial infarction.

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

22. The method of claim 1, wherein pyruvate kinase M2 exists predominantly as a dimer as compared to a tetramer.

23. The method of claim 1, wherein the PKM2 has a G415R mutation.

24. The method of claims 1 to 18, wherein the PKM2 or the PKM2 mutant is located in the extracellular space.

25. The method of claims 1 to 18, wherein the PKM2 or the PKM2 mutant in the composition is between 1 mg / kg and 6 kg.

26. The method of claims 1 to 18, wherein the PKM2 or the PKM2 mutant in the composition is between 2 mg / kg and 5 kg.

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