WO2024081960A2 - Procédés de conservation et de protection de cardiomyocytes et de réduction de la fibrose cardiaque à la suite d'une lésion cardiaque - Google Patents

Procédés de conservation et de protection de cardiomyocytes et de réduction de la fibrose cardiaque à la suite d'une lésion cardiaque Download PDF

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WO2024081960A2
WO2024081960A2 PCT/US2023/077001 US2023077001W WO2024081960A2 WO 2024081960 A2 WO2024081960 A2 WO 2024081960A2 US 2023077001 W US2023077001 W US 2023077001W WO 2024081960 A2 WO2024081960 A2 WO 2024081960A2
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pkm2
composition
mutant
pyruvate kinase
cardiac
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PCT/US2023/077001
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English (en)
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Zhi-Ren Liu
Huang YANG
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Georgia State University Research Foundation, Inc.
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  • This application relates in general to therapeutic methods for treating cardiovascular disorders. More particularly, this application relates to compositions, sy stems and methods for improving recovery and tissue repair following a cardiac injury by protecting cardiomyocytes and reducing cardiomyocyte death induced by cardiomyocyte injury' following myocardium damage. This application also relates to a pharmaceutical composition for treating or preventing or reducing cardiac fibrosis following myocardium damage, and more particularly, to a pharmaceutical composition for treating or preventing myocardium damage and the accompanying cardiac fibrosis.
  • MI Acute myocardial infarction
  • HF heart failure
  • Cardiomyocytes are the primary cell type in an adult heart, responsible for maintaining its function.
  • the loss of cardiomyocytes due to myocardial infarction stands as a significant factor for morbidity and mortality associated with heart diseases.
  • the acute loss of cardiomyocytes caused by MI cannot be replaced, given the limited regenerative capability of adult cardiomyocytes. It is crucial to preserve cardiomyocytes to ensure patient survival after a heart attack.
  • the death of cardiomyocytes swiftly activates a local tissue repair mechanism, prompting the engagement of cardiac fibroblasts. Once activated, cardiac fibroblasts produce an extracellular matrix (ECM), predominantly composed of collagen, forming scar tissue to avert myocardium rupture post-infarction.
  • ECM extracellular matrix
  • fibrosis is a reactionary process influenced by various factors. These factors include early inflammatory responses, a local surge in fibroblast cell populations, changes in the synthetic function of fibroblasts, and the altered dynamics of collagen biosynthesis and degradation. Other influential aspects comprise nearby tissue inflammation and a broad inflammatory state characterized by an uptick in circulating mediators. Unfortunately, no effective therapies exist to counteract cardiac fibrosis or to protect cardiomyocytes.
  • This application provides a treatment for acute cardiac injuries, such as those resulting from heart attacks, and promotes cardiomyocyte proliferation.
  • this application reveals that the 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 a recombinant PKM2, or a protein identical or similar to pyruvate kinase M2 (PKM2) - which preferentially assumes the dimeric form - aids in preserving cardiomyocytes, spurs cardiomyocyte proliferation, and reduces cardiac fibrosis.
  • 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
  • PKM2 pyruvate kina
  • the dimer form of PKM2 or its mutants can be administered (for instance, acutely) as a treatment for heart attacks.
  • the dimeric form can be administered with other forms (e.g., the tetramer).
  • a representative example of the recombinant PKM2 mutant suitable for the outlined methods is G415R, which chiefly adopts the dimeric form. Administering the recombinant PKM2 mutant can thwart cardiomyocytes from undergoing apoptosis and promote their proliferation under conditions such as hypoxia and oxidative stress.
  • One aspect of the application encompasses a method of treating acute cardiac injury caused by acute cardiomyocyte loss in a patient or a subject.
  • This method involves administering to the subject a composition containing pyruvate kinase M2 (PKM2) or a composition containing a mutant of PKM2. More specifically, a composition of PKM2 or a mutant of the same preferentially dimerizes at equilibrium can be administered to a subject within 1 to 10 hours following the cardiac injury.
  • PKM2 pyruvate kinase M2
  • Another aspect includes the administration of a recombinant PKM2 mutant (e.g., G415R) or a recombinant PKM2 (rPKM2) or a protein similar or identical to pyruvate kinase M2 (PKM2) to preserve cardiomyocytes during myocardial infarction, which shows that rPKM2 (the dimer mutant) is a therapeutic for heart attacks and other cardiovascular diseases.
  • a recombinant PKM2 mutant e.g., G415R
  • rPKM2 recombinant PKM2
  • PKM2 pyruvate kinase M2
  • the administration of rPKM2 or mutant thereof protects cardiomyocytes from death and promotes cardiomyocyte proliferation.
  • recombinant PKM2 for example at a dosage that results in a concentration of rPKM2 of less than 1 micromolar to 5 micromolar in the patient’s blood stream
  • a mutant that preferentially dimerizes decreases activation of cardiac fibroblasts, and therefore inhibits fibrosis, in the mice experiencing myocardial infarction and reperfusion injury .
  • An ischemia/reperfusion protection composition as disclosed herein comprises a recombinant PKM2 mutant (e.g., G415R) or a recombinant PKM2 or a protein similar or identical to pyruvate kinase M2 (PKM2) that preferentially adopts the dimeric form.
  • PKM2 mutant e.g., G415R
  • PKM2 pyruvate kinase M2
  • Another aspect includes a method of treating cardiac injury' in a subject that includes 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.
  • PKM2 or PKM2 mutant may be a dimer.
  • Another aspect includes a method in which the PKM2 or PKM2 mutant dimerizes in the subject.
  • Another aspect includes a method in which the administration occurs within 6 hours post cardiac injury or within 3 hours post cardiac injury or within 1 hour post cardiac injury.
  • Another aspect includes a method in which the subject experienced a heart attack and the cardiac injury' is from the heart attack.
  • Another aspect includes a method in which the PKM2 exists less than 50% in its tetrameric form.
  • Another aspect includes a method in which the PKM2 predominantly forms a dimer at neutral pH.
  • composition includes PKM2 or a mutant that of PKM2 that preferentially adopts a dimeric state.
  • Another aspect includes a method in which the cardiac injury is caused by acute cardiomyocyte loss.
  • Another aspect includes a method in which the myocardium preservation is achieved by administration of the composition.
  • Another aspect includes a method in which the composition is delivered extracellularly.
  • Another aspect includes a method in which the myocardial infarction is characterized as acute.
  • composition comprises a PKM2 mutant or a protein highly similar to wild-type pyruvate kinase M2.
  • Another aspect includes a method in which the PKM2 is from either a human or another animal.
  • Another aspect includes a method in which the PKM2 has mutations differing from the wild-type sequence.
  • composition is contained within a pharmaceutically acceptable carrier.
  • compositions are delivered via intracardiac administration.
  • Another aspect includes a method in which the composition is systemically delivered.
  • Another aspect includes a method in which the composition mitigates cardiomyocyte death resulting from myocardial infarction.
  • Another aspect includes a method in which the composition decreases cardiac fibrosis in the infarcted myocardium.
  • Another aspect includes a method in which the pyruvate kinase M2 predominantly exists as a dimer compared to its tetrameric form.
  • Another aspect includes a method in which the PKM2 features a G415R mutation.
  • Another aspect includes a method in which the PKM2 or the PKM2 mutant is in the extracellular space.
  • FIG. 1 A illustrates that recombinant G415R protected H9C2 cells from apoptosis under hypoxic conditions.
  • FIG. IB demonstrates that recombinant G415R promoted cell proliferation under hypoxic conditions.
  • FIG. 1C indicates that recombinant G415R had comparable effects on primary human cardiomyocytes and protected them from apoptosis under both hypoxia and oxidative stress conditions.
  • FIGs ID and IE reveal that recombinant G415R promoted the proliferation of primary cardiomyocytes under hypoxic and oxidative stress conditions.
  • FIG. 2A highlights that the administration of recombinant G415R substantially reduced mortality in MI mice at all observed time points.
  • FIG. 2B presents that the administration of recombinant G415R lowered the heart weight (relative to body weight) in MI mice 30 days post-infarction.
  • FIGs. 3 A and 3B depict histological analyses of infarcted hearts, showing that mice treated with recombinant G415R had smaller infarction scar sizes compared to the rPKMl and vehicle-treated groups in both MI and IR models.
  • FIGs. 3C, 3G, and 3H show that recombinant G415R protects cardiomyocytes from apoptosis, as evidenced by TUNEL staining of heart tissues at 6-, 24-, and 168-hours postinfarction, respectively.
  • FIG. 3D indicates that cardiomyocyte apoptosis was minimally observed via TUNEL staining of myocardial tissues 168 hours post-infarction.
  • FIG. 3E demonstrates that in G415R-treated mice, cardiomyocyte proliferation was evident in myocardial tissue of both MI and IR models at days 4 and 7 post-infarction.
  • FIG. 3F shows that G415R treatments significantly reduced cTnl levels in the bloodstream of infarcted mice 7 days post-infarction.
  • FIG. 4A shows wheat germ agglutinin (WGA) staining of myocardial tissue, indicating that G415R-treated mice exhibited fewer infarction scars in the myocardium's infarcted regions in both MI and IR models compared to rPKMl and vehicle-treated groups.
  • FIGs. 4B and 4C illustrate analyses of cardiomyocyte (cross-sectional area) by WGA and cTnl co-staining, suggesting that recombinant G415R treatment reduced the cardiomyocyte cross-sectional area to levels similar to the sham groups in MI mice.
  • FIGs. 4D and 4E show analyses of cardiomyocyte (cross-sectional area) by WGA and cTnl co-staining, revealing that G415R treatment decreased cardiomyocyte cross-sectional area to levels similar to the sham groups in IR mice, signifying significantly reduced cardiomyocyte hypertrophy.
  • FIGs. 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 of a-SMA.
  • FIGs. 5A-5D show that G415R decreased PTEN expression in both cultured cardiomyocyte cells (evidenced by immunoblotting) and myocardial tissue (IHC staining) of infarcted mice.
  • administration refers to providing or delivering a therapeutic agent (e.g., an agent as described herein) to a subject by any effective route.
  • the composition is administered into the same subject by multiple routes of administration.
  • the multiple routes of administration comprise intravenous administration, intraarterial administration, intrathecal administration, intranasal administration, intraperitoneal administration, and/or periocular administration.
  • the composition having PKM2 or a mutant of the same can be administered intravenously to the circulatory’ system of the subject.
  • the composition having PKM2 or a mutant of the same can be infused in suitable liquid and administered into a vein of the subject.
  • acute myocardial infarction and "heart attack” refer to a condition where localized myocardial ischemia leads to the development of a defined region of tissue death.
  • An 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 it from supplying blood to the region of the heart it serves.
  • 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 the naturally occurring 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) and pyrolysine and selenocysteine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example only, an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones, while still retaining the same basic chemical structure as a naturally occurring amino acid.
  • Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • “conservatively modified variants” applies to both natural and non-natural amino acid sequences and natural and non-natural nucleic acid sequences, and combinations thereof.
  • “conservatively modified variants” refers to those natural and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations.
  • every' natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural poly peptide also describes every possible silent variation of the natural or non- natural nucleic acid.
  • each codon in a natural or non-natural nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a natural and non-natural nucleic acid which encodes a natural and non-natural polypeptide is implicit in each described sequence.
  • MI myocardial infarction
  • Ischemia refers to local deficiency of blood supply, generally produced by vasoconstriction or local obstacles to blood flow. Restoration of blood flow to a previously ischemic tissue or organ, such as the heart is referred to as “reperfusion.”
  • ischemia reperfusion refers to the damage caused to tissue when blood supply returns to the tissue after a period of ischemia.
  • the absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative or peroxidative damage.
  • nucleic acid sequence refers to the order and identity of the nucleotides comprising a nucleic acid.
  • 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, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates. chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptidenucleic acids (PNAs).
  • 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. Specifically, degenerate codon substitutions may 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.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • a particular nucleic acid sequence also implicitly encompasses “splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate 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.
  • 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 residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • pharmaceutically acceptable refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • prophylactically effective amount refers that amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.
  • the substantial identity' exists over a region of the sequences that is at least about 10, preferably about 20.
  • a substantially similar polypeptide or nucleic acid may be that of a mutant or other protein that preferentially adopts the dimeric form.
  • the more effective proteins preferentially dimerized and w ere soluble.
  • synergistic refers to a combination of prophylactic or therapeutic effective agents which is more effective than the additive effects of any two or more single agents.
  • a synergistic effect of a combination of prophylactic or therapeutic agents may permit the use of low er dosages of one or more of the agents and/or less frequent administration of the agents to a subject with a specific disease or condition.
  • a 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 any single therapy.
  • the term “therapeutically effective amount,'’ refers to the amount of a composition or biologic containing the protein administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least partially arrest, or relieve to some extent one or more of the signs, symptoms or causes of the disease, disorder or condition being treated.
  • the effectiveness of such compositions depends on the conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.
  • therapeutically effective amounts 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 variant thereof, that is sufficient to bring about a desired therapeutic result.
  • Therapeutically effective amount in the context of using a composition to treat heart cells after a heart attack, refers to the quantity of the composition that, when administered to an individual, produces the desired therapeutic effect to alleviate, mitigate, or reverse the damage to the heart cells 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 overall health, age, weight, and other medical considerations. In a typical subject of typical body weight, the therapeutically effective amount can range from 0. 1 to 10 mg/ml intravenously, or from 0.5 to 7 mg/ml intravenously, from 0. 1 to 6 mg/ml intravenously, or from 4 to 6 mg/ml intravenously 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 at for 5 minutes to 5 hours or more).
  • subj ect or “patient” as used herein includes mammals and humans.
  • the patient is having a heart attack.
  • dosage refers to the amount of a composition or biologic, such as pyruvate kinase M2 or variant thereof, administered to an animal or human or utilized in a cell culture assays.
  • 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 referred to in this disclosure or commonly used in the art. In one example, 5 mg/ml is administered.
  • therapeutic agent encompasses proteins, peptides, nucleic acids, vectors, pharmacological agents, or other macromolecules or compositions that are known in the art.
  • the therapeutic agent may be delivered to the recipient via inhalation, oral administration, subcutaneous injection; intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, or any other method of agent delivery used in the art.
  • Agents may be delivered as a single bolus or other one-time administration mechanisms; alternatively, agents may be administered via a sustained (continuous or intermittent) delivery.
  • the terms ’‘treat,” “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect, including without limitation achieving amelioration, improvement, or elimination of symptoms of ischemia.
  • the effect may be prophylactic in terms of completely or partially preventing cardiac fibrosis and/or may be therapeutic in terms of ameliorating, improving, or eliminating one or more symptoms of cardiomyocyte damage.
  • Treatment covers any treatment of acute myocardial infarction in a mammal, particularly in a human, and includes: (a) preventing the cardiac fibrosis from occurring in a subject; (b) relieving the cardiac fibrosis; and (c) restoring the individual to a pre-cardiac event state. “Treatment” may not indicate, or require, complete eradication or cure of the acute myocardial infarction, or associated symptoms thereof.
  • PKM2 refers to pyruvate kinase isoform M2.
  • PKM2 mutant as used throughout this specification, variant form of the pyruvate kinase M2 (PKM2) protein, in which one or more amino acid residues have been altered, added, or removed in comparison to the native or wild- type PKM2 sequence.
  • PKM2 pyruvate kinase M2
  • These alterations can be the result of genetic mutations, or they can be artificially introduced through methods such as genetic engineering. Mutations can lead to changes in the protein's function, stability, interaction partners, or other properties and
  • the application provides methods for treating and/or preventing a cardiac disease in a subj ect in need thereof.
  • the present inventors have made intensive research efforts to develop a method for protecting cardiomyocytes from death (e.g., apoptosis) and preventing or reducing cardiac fibrosis or heart disease accompanying cardiac fibrosis.
  • Specific embodiments are directed to protecting cardiomyocytes and promoting the growth of cardiomyocytes.
  • Cardiac disease that can be treated include cardiomyopathy, such as hypertrophic cardiomyopathy, dilated cardiomyopathy, and toxic cardiomyopathy; cardiotoxicity; congestive heart failure; and cardiac damages due to some infectious e g., virus or bacteria.
  • One embodiment is a method of reducing an adverse consequence of myocardial infarction in a patient comprising administering a composition having a recombinant PKM2 mutant (e.g., G415R), or a recombinant PKM2 or a protein similar or identical to PKM2 that preferentially adopts the dimeric form or has a least some dimeric form of PKM2, to the patient during the acute stage of the myocardial infarction.
  • the therapeutic PKM2 is not PKM2 that exists primarily as a tetramer.
  • the therapeutic PKM2 is not expressed intracellularly, e.g., as a result of transfection of cardiomyocytes with a nucleic acid encoding PKM2.
  • the PKM2 or PKM2 mutant in the composition has a dimer form or exists as a dimer at least is some part.
  • the period immediately after heart injury or heart trauma is critical and is sometimes referred to as the “golden hour” or the first hour following a myocardial infarction.
  • the myocardial infarction can be an acute myocardial infarction.
  • Administration of the ischemia/reperfusion protection composition may be commenced within 200 hours of onset of a heart attack.
  • the protein substantially similar or identical to pyruvate kinase M2 PLM2
  • PLM2 protein substantially similar or identical to pyruvate kinase M2
  • the patient can be a human or a non-human mammal.
  • An ischemia/reperfusion protection composition as disclosed herein comprises a protein similar or identical to PKM2 at a dosage that results in a concentration of ⁇ 1pm to 5 pm under physiological conditions in a patient’s blood stream or a mutant PKM2 that exists primarily in dimeric form.
  • PKM2 administered such that it is at a concentration of ⁇ 1pm under physiological conditions in a patient will exist as about 70-85% dimer.
  • PKM2 administered such that it is at a concentration of > 10 pm under physiological conditions in a subject will exists as ⁇ 25% dimer.
  • a 5 mg/ml concentration as the drug formulation for patient administration will be approximately 100 pm in dosage form but will be diluted to ⁇ 5 pm once it is administered and is in the patient’s blood circulation.
  • PKM2 mutant G415R exists as > 85% dimer at concentrations that are > 50 pm.
  • the invention discloses, in part, administering an effective amount of a therapeutic composition to a subject to protect cardiomyocytes or promote growth of cardiomyocytes.
  • a therapeutic composition comprising 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 induced by cardiac injury.
  • the invention discloses a method of reducing or inhibiting cardiac fibrosis by administering a composition comprising recombinant wild type PKM2 in an amount that results in a concentration of wt PKM2 of ⁇ 1pm in the bloodstream after administration to a patient.
  • the invention discloses a method of reducing or inhibiting cardiac fibrosis by administering r a composition comprising a mutant of PKM2 that preferentially dimerizes at equilibrium. Without intending to be bound to a particular mechanism, it is postulated that the reduction in fibrosis is a result of reduced cardiomyocyte death.
  • PKM2 mutants have a least 75%, preferably at least 85%, more preferably at least 90%, 95%, 98%, 99% or higher or any integral value therebetween nucleotide or amino acid residue identity when compared to wild-type PKM2.
  • PKM2 mutants that preferentially adopt the dimeric form are useful in the methods and systems for treating MI, IR and other conditions related to cardiomyocyte injury.
  • composition and methods are directed to cardiac conditions that involve damaged cardiac tissue such as damage cardiac muscles arising from ischemic events, ischemia reperfusion injury, damage to the left ventricle such as those arising from congestive heart failure, damage to the heart valves arising from diseases, such as coronary artery’ diseases.
  • damaged cardiac tissue such as damage cardiac muscles arising from ischemic events, ischemia reperfusion injury, damage to the left ventricle such as those arising from congestive heart failure, damage to the heart valves arising from diseases, such as coronary artery’ diseases.
  • PKM2 damage to the left ventricle
  • diseases such as coronary artery
  • the composition may be administered with another agent.
  • an agent can include a hypolipidemic drug, an antiplatelet drug, a blood pressure lowering drug, a dilated vascular drug, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a liver protection drug, anti arrhythmic drugs, cardiotonic drugs, diuretic drugs, anti -infective drugs, antiviral drugs, immunomodulatory’ drugs, inflammatory’ regulating drugs, anti-tumor drugs, or hormone drugs.
  • Pyruvate kinase isoform M2 (PKM2) is a 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.
  • PKM1 and PKM2 are ubiquitously expressed in different types of cells and tissues. PKM2 is highly expressed in proliferating cells including cancer cells.
  • PKM2 is a unique multifaceted regulator that can improve cells adaptation in their metabolic program to match physiological needs in different environments.
  • PKM2 In addition to regulating glycolysis, PKM2 has non-metabolic functions such as regulation of transcription and cell cycle progression. In contrast to the mitochondrial respiratory reaction, energy regeneration by these pyruvate kinases is independent from oxygen supply and allow s survival of the organs under hypoxic conditions. PKM2 may also act as a co-activator of hypoxia-induced factor 1 -alpha (HIF-la); the latter behaves as a master transcription factor to regulate multiple signaling pathways in response to hypoxic insults. Increased levels and activities of PKM2 are associated with enhanced motility and metastasis of tumor cells; the molecular mechanism of the increased cell migration is so far poorly understood.
  • hypoxia-induced factor 1 -alpha HIF-la
  • the angiogenic activity and/or endothelial cell proliferative or migration potential of a pyruvate kinase M2, or a therapeutic substantially similar to pyruvate kinase can be assessed by assays and methodology.
  • the pyruvate kinase protein can be any vertebrate or mammalian pyruvate kinase, and may be a native pyruvate kinase, or a recombinant or other synthetic protein.
  • the amino acid sequence for human pyruvate kinase is for instance provided by GenBank Accession No. MP0011193727 pyruvate kinase.
  • amino acid sequence identity of pyruvate kinase for example is highly conserved between species with human having 98% amino acid sequence identity with mouse, hamster, and rat.
  • the amino acid sequence for human pyruvate kinase is disclosed herein (Example 1).
  • active pyruvate kinase protein is a dimer, also known as PKM2 subtype M2.
  • PKM2 subtype M2 A broad range of proteins and therapeutics substantially similar to pyruvate kinase M2 are useful with specific embodiments.
  • An animal from which native pyruvate kinase protein is purified can for instance be a member of the bovine, ovine, porcine, equine, canine, feline, primate, rodent or other mammalian family.
  • the pyruvate kinase protein will be a human pyruvate kinase protein purified from bacterial production.
  • a recombinant pyruvate kinase protein can have an identical amino acid sequence to the native pyruvate kinase or one or more amino acid differences compared to the native protein.
  • the amino acid changes can comprise the addition, deletion and/or substitution of one or more amino acids. Inversion of amino acids and other mutational changes that result in modification of the native pyruvate kinase protein sequence are also encompassed.
  • a recombinant protein can comprise an amino acid or amino acids not encoded by the genetic code.
  • the substitution of an amino acid can be a conservative or non-conservative substitution.
  • conservative amino acid substitution is to be taken in the normally accepted sense of replacing an amino acid residue with another amino acid having similar properties, which does not have a substantial and adverse effect the angiogenic and/or wound healing activity of the pyruvate kinase protein.
  • a conservative amino acid substitution can involve substitution of a basic amino acid such as arginine with another basic amino acid such as lysine.
  • a cysteine residue can be replaced with serine, or a non-polar amino acid may be substituted with another non-polar amino acid such as alanine.
  • Amino acids amenable to substitution or deletion in a pyruvate kinase protein amino acid sequence may be determined by comparison of the sequence with closely related pyruvate kinase proteins to identify non-conserved amino acids and by routine trial and experimentation well within the skill of the addressee.
  • a modified recombinant pyruvate kinase protein can be provided by introducing nucleotide change(s) in nucleic acid sequence encoding the native protein such that the desired amino acid changes are achieved upon expression of the nucleic acid in a host cell.
  • One embodiment includes an a 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 also SEQ ID NO: .
  • Variants of PKM2 or mutants of the same that are more useful are those that preferentially adopt the dimeric form and are soluble in water.
  • PKM2 (Accession No. NP 002645) is as follows:
  • 181 islqvkqkga dflvteveng gslgskkgvn Ipgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie
  • 181 islqvkqkga dflvteveng gslgskkgvn Ipgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie
  • Another exemplary mutant pyruvate kinase M2 amino acid sequence having three mutations (R399E, K422A, and N523A) or SEQ ID NO: 3 is as follows;
  • a recombinant or synthetic pyruvate kinase protein suitable for the methods of this invention should exhibit an amino acid sequence identity with native pyruvate kinase of at least 60%. More commonly, the identity may be at least 70%, 80%, 90%, 95%, 98%, or even 100%. Some of the protein must fonn a dimer under physiological conditions in a patient. All sequence homologies and ranges specified above are explicitly included. Sequence identity between amino acid sequences is determined by comparing amino acids at each position in optimally aligned sequences. Amino acids at a given position are considered identical only if they match.
  • a gap — where an amino acid residue appears in one sequence but not in another — is treated as a position with non-identical residues.
  • Aligning sequences can be achieved using any appropriate program or algorithm, with computer-assisted sequence alignments ty pically performed using standard software.
  • the pyruvate kinase protein can also be chemically synthesized.
  • the creation and use of fusion proteins incorporating a pyruvate kinase protein, as described herein, are also encompassed by the invention.
  • Nucleic acids encoding a fusion protein can be generated by joining separate DNA fragments encoding the pyruvate kinase protein and, for instance, a lipophilic amino acid sequence to enhance the lipophilic characteristics of the protein. This can be achieved using methods such as employing blunt-endedtemiini and oligonucleotide linkers, digestion to provide staggered termini, and ligation of cohesive ends as required.
  • Pyruvate kinase proteins described herein can also be modified by coupling one or more proteinaceous or non-proteinaceous moieties to the protein. This improves aspects like solubility, lipophilic characteristics, stability, biological half-life, or serves as a label for subsequent detection. Modifications can arise from post-translational or post-synthesis processes such as the attachment of carbohydrate moieties or chemical reactions leading to structural modifications (e.g., alky lation or acetylation of amino acid residues).
  • the pyruvate kinase protein may undergo modifications like methylation, phosphorylation, oxidation of tyrosine and/or tryptophan residues, glycosylation, or S-methylcysteine covalent attachment.
  • the pyruvate kinase protein can vary in size from the complete protein. However, the pyruvate kinase should be of a length that facilitates dimer formation.
  • Both intact and truncated forms of the protein might 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 zinc, magnesium, or calcium-containing buffers.
  • Partially hydrolyzed forms of pyruvate kinase proteins can be purified for use in the invention's embodiments using any appropriate purification technique, including filtration and chromatography protocols.
  • the pyruvate kinase protein can be administered to a subject in need of such treatment alone or be co-administered with one or more other therapeutic agents.
  • pyruvate kinase can be co-administered in combination with therapeutic agents conventionally used for promoting angiogenesis, cellular proliferation, or wound healing.
  • co- administered is meant 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 other therapeutic agent(s) exhibit overlapping therapeutic windows.
  • ‘'sequential” administration is meant one is administered after the other.
  • Such further agents that may be co-administered with the pyruvate kinase protein include platelet-derived growth factor (PDGF), transforming growth factor-.beta. (TGF-0), platelet- derived wound healing factor, insulin growth factor (IGF), keratinocyte gro th factor (KGF), anti-inflammatory agents and anti-microbial agents.
  • PDGF platelet-derived growth factor
  • TGF-0 transforming growth factor-.beta.
  • IGF insulin growth factor
  • KGF keratinocyte gro th factor
  • anti-inflammatory agents anti-microbial agents.
  • therapeutic agents used for promoting angiogeneisis and/or wound healing include indoleamine 2,3-dioxygenase (IDO), tryptophan dioxygenase (TDO), spingosine-1 -phosphate (SIP), N- acylethanolamines, grapefruit extract and other plant phytochemicals including ascein, green tea catechins, melatonin, arginine and other amino acids for support of vessel growth. Additional therapeutic agents appropriate for co-admini strati on will be obvious to those of ordinary skill in the art.
  • the pyruvate kinase protein will generally be formulated into a pharmaceutical composition comprising the protein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition as described herein can also incorporate one or more preservatives such as parabens, chlorobutanol, and sorbic acid, binders such as com starch or gelatin, thickening agents, emulsifiers, surfactants, gelling agents, and other components typically used in such compositions.
  • Pharmaceutically acceptable carriers include any suitable conventionally known physiologically acceptable solvents, dispersion media, isotonic preparations and solutions. Use of such ingredients and media for pharmaceutically active substances is w ell known. Except insofar as any conventional media or agent is incompatible with the pyruvate kinase protein, use thereof is expressly encompassed.
  • compositions embodied by the invention include therapeutic compositions for human or veterinary use.
  • a pharmaceutical composition embodied by the invention will generally contain at least about 0.001% by weight of the pyruvate kinase protein up to about 80% w/w of the composition.
  • the pharmaceutical composition can 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, substantially similar therapeutic or mutant PKM2 that preferentially dimerizes.
  • a pharmaceutical composition embodied by the invention will generally contain at least about 20pg/ml of the pyruvate kinase protein up to about 1000 pg/ml.
  • the pharmaceutical composition can contain about 50pg/ml, 60pg/ml, 70pg/ml, 80pg/ml, 90pg/ml, lOOpg/ml, 200pg/mL 300pg/ml, 400pg/mL 500pg/ml, 600pg/ml, 700pg/ml, 800pg/ml, 900
  • the amount of the protein in the composition will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed mode of administration.
  • the dosage of the pyruvate kinase protein administered in accordance with an embodiment of the invention will depend on a number of factors including whether the protein is to be administered for prophylactic or therapeutic use, 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 weight and general health of the subject, and can be determined in accordance with accepted medical principles. For instance, a low' dosage can initially be given which is subsequently increased at each administration following evaluation of the subject's response. Similarly, the frequency of administration can be determined in the same way that is, by continuously monitoring the subject's response betw een each dosage and if desirable, increasing the frequency of administration or alternatively, reducing the frequency of administration.
  • 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.
  • Routes of administration include but are not limited to topically, respiratorialy, intravenously, orally, intraperitoneally, subcutaneously, intramuscularly, rectally, topically, directly into the heart, and by implant.
  • intravenous routes particularly suitable routes are via injection into blood vessels (e.g., superior and inferior vena cava) which supply the target tissue to be treated.
  • the pyruvate kinase protein can also be delivered into cavities such as for example the pleural or peritoneal cavity, cranial or be injected directly into the tissues to be treated, e.g., the left or right ventricle of the heart.
  • the pyruvate kinase protein can be encapsulated or otherwise provided in an enteric for passage through the stomach and release in the small intestine. Any suitable such enteric formulation or coating can be utilized. Furthermore, these systems and methods may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of cellular regeneration or tissue repair without causing clinically unacceptable adverse effects.
  • a pyruvate kinase protein can also be coated onto the surface of a stent or balloon of a catheter such as an angioplasty catheter, or other surgical instrument for application to the interior wall of a blood vessel during angioplasty or other surgical procedure.
  • the pyruvate kinase can for instance be applied to the wall of the blood vessel in this manner in the form of a gel or any other appropriate formulation to promote wound healing and/or angiogenesis, epithelial cell migration, or cellular regeneration at the site of treatment.
  • Suitable pharmaceutically acceptable carriers and formulations useful in compositions embodied by the invention can for instance be found in handbooks and texts.
  • Pharmaceutical agents include the following categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will be able to identify readily those phannaceutical agents that have utility within or outside of the central nervous system. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the invention.
  • One embodiment also includes a kit for improving recovery following a cardiac episode or injury.
  • the combination of agents is provided to allow administration in an amount and frequency therapeutically effective to produce cellular regeneration following a cardiac episode or injury.
  • polyethylene glycol may be used to derivatize polypeptides of the invention, include, for example, poly (ethylene glycol) (PEG), poly (vinylpyrrolidone), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred.
  • PEG polymers are PEG polymers having a molecular weight of from about 100 to about 40,000.
  • Other suitable hydrophilic polymers in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure.
  • the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.
  • the PKM2 or a substantially similar therapeutic was PEGylated with a PEG-chain of 20 kDa.
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C- terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to polypeptide (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated polypeptide. Under the appropriate reaction conditions, substantially selective derivatization of the polypeptide at the N-terminus with a carbonyl group containing polymer is achieved.
  • compositions comprise a therapeutically effective amount of active component (e.g., PKM2 dimer, PKM2 mutant that exists more as a dimer than as a tetramer, or a substantially similar therapeutic), and a pharmaceutically acceptable carrier.
  • active component e.g., PKM2 dimer, PKM2 mutant that exists more as a dimer than as a tetramer, or a substantially similar therapeutic
  • pharmaceutically acceptable carrier 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 and the like. Water is a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for inj ectable 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 and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository', with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Such compositions will contain a therapeutically effective amount of the PKM2 or a substantially similar therapeutic together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the amount of the PKM2 or a substantially similar therapeutic that will be effective in assisting with recovery from an ischemic episode can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the agent or pharmaceutical compositions can be tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
  • in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of a 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 utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
  • in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is gro ⁇ n in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage fonn, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • 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 linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the therapeutic composition ischemia/reperfusion protection composition
  • This may be achieved by. for example, and not by way of limitation, local injection (for example, injection into the myocardium) or infusion during surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • regulatory genes and sequence may be used with the expression and replication of the PKM2 or a substantially similar therapeutic.
  • the nature of the regulatory sequences for gene expression may vary between species or cell types, but shall in general include, as necessary. 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Promoters may be constitutive or inducible. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired.
  • polynucleotides encoding the PKM2 or a substantially similar therapeutic may be fused to polynucleotides encoding signal sequences which will direct the localization of a polypeptide to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a polypeptide.
  • signal sequences which will direct the localization of a polypeptide to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of a polypeptide.
  • E. coli one may wish to direct the expression of the protein to the periplasmic space.
  • vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein.
  • stents that comprise a generally tubular structure, which may include, for example, spiral shapes.
  • the surface of this structure is coated with PKM2 or a therapeutic that is substantially similar, as described above.
  • a stent is ty pically a scaffolding, usually cylindrical in shape, which can be inserted into a body passageway (e.g., bile ducts, arteries, veins) or a portion thereof.
  • This passageway may have been narrowed. irregularly contoured, obstructed, or occluded due to a disease process (e.g., tumor ingrow th) to prevent the closure or reclosure of the passageway.
  • One specific embodiment also provides use of PKM2 or a substantially similar therapeutic in a wide variety of surgical procedures.
  • surgical meshes which have been coated with PKM2 may be utilized in any procedure wherein a surgical mesh might be utilized.
  • the following is an exemplary wild-type pyruvate kinase M2 amino acid sequence or SEQ ID NO: 4 .
  • integrin a v p3 is expressed in cardiomyocytes under cardiac infarction condition.
  • the inventors probed the expression of the integrin in H9C2 cells by immunofluorescence (IF) staining. Integrin was expressed in high levels in the cells under hypoxia condition and not under normoxia condition. Further, the inventors analyzed the integrin expression in primary human cardiomyocytes under hypoxia and oxidative stress conditions. The integrin was expressed in cardiomyocytes under the stress conditions, while the integrin is not expressed under normal culture conditions. To confirm the clinical relevance of the integrin expression, the integrin expression was analyzed in heart tissues from infarction patients. Integrin was highly expressed in the infarction region and was almost undetectable in the normal non-infraction region. Integrin a v b3 was upregulated in cardiomyocytes under hypoxia and oxidative stress conditions.
  • Extracellular PKM2 (EcPKM2) interacts to integrin avP3 found on angiogenic endothelial cells and myofibroblasts, safeguarding them from apoptosis.
  • rPKMl recombinant PKM1
  • G415R shoed enhanced solubility- and prolonged stability compared to the recombinant PKM2.
  • FIG. 1A reveals that the recombinant G415R protected H9C2 cells from death (e.g., apoptosis) during hypoxic conditions.
  • FIG. IB demonstrates that the G415R mutant promoted cell growth in these conditions.
  • FIG. 1C shows that recombinant G415R had analogous effects on primary human cardiomyocytes, specifically protecting them from cell death in hypoxia and oxidative stress scenarios.
  • FIGs ID and IE depict the G415R mutant's promotion of cell growth in primary cardiomyocytes under hypoxia and oxidative stress conditions.
  • Example 4 The recombinant G415R preserved cardiomyocytes in infarction mouse model
  • FIG. 2A shows that administration of G415R dramatically decreased MI mice death at all time points.
  • FIG. 2B shows that administration of G415R also decreased heart weight vs body weight both in the MI mice at 30 days after infarction.
  • MR imaging analyses of blood flow in the heart showed that G415R treatment improved the blood flow of MI mice and restored blood flow of IR mice almost to the same level of the sham mice.
  • FIGs, 3 A and 3B display histological analyses of the infarcted heart, revealing that mice treated with G415R had reduced infarction scar sizes compared to the rPKMl and vehicle- treated groups in both MI and IR mice.
  • TUNEL staining was conducted on tissues from mice hearts at 6, 24, and 168 hours post-infarction. G415R treatments notably decreased cardiomyocyte apoptosis at 6 and 24 hours, as depicted in FIG. 3C. By 168 hours post-infarction, cardiomyocyte apoptosis was barely detected using TUNEL staining, with no significant variations observed among the various treatment groups, as shown in FIG. 3D.
  • G415R was found to stimulate human primary cardiomyocyte proliferation under hypoxia and oxidative stress conditions. Thus, the inventors investigated if G415R also enhanced cardiomyocyte proliferation using Ki67 staining. As anticipated, G415R treatment did not trigger cardiomyocyte proliferation 6 hours post-infarction. Nevertheless, in the G415R-treated mice, evident cardiomyocyte proliferation was observed in myocardium tissues of both MI and IR mice on days 4 and 7 post-infarction (Fig. 3E). The effects of G415R on cardiomyocyte proliferation diminished from day 4 to day 7 and were virtually absent by day 28 post-infarction in both MI and IR mice. This suggests that the optimal period for promoting cardiomyocyte proliferation/regeneration lies between 1 to 7 days post-infarction.
  • cTnl cardiac troponin I
  • FIG. 4A shows WGA staining of myocardium tissue revealing that G415R treated mice resulted in less infarction scars in infarction myocardium region in both MI and IR models compared to rPKMl and ventical treated group.
  • FIGs. 4B and 4C display Masson-Tri chrome staining of myocardium tissue.
  • the staining reveals that mice treated with G415R exhibited reduced collagen accumulation in the infarcted myocardium region for both MI and IR models compared to those treated with rPKMl and the vehicle group.
  • WGA wheat germ agglutinin
  • FIG. 4D and 4E present analyses of the cardiomyocyte cross-section area, using WGA and cTnl co-staining.
  • the data indicate that G415R treatment diminished the cardiomyocyte cross-section area nearly to the same extent as seen in the sham groups for both MI models.
  • FIGs. 4E and 4F depict results for IR models, highlighting that G415R treatment in infarcted mice significantly curtailed cardiomyocyte hypertrophy.
  • a potential explanation for the decreased accumulation of ECM/collagen fibers in the myocardium of G415R-treated mice is that the G415R treatment may have caused fewer cardiac fibroblasts to activate in the MI and IR mice due to the preservation of cardiomyocytes
  • FIGs. 4F and 4G showcase the activation of cardiac fibroblasts in the infarcted myocardium of MI and IR mice, as evidenced by IHC staining of a-SMA — a molecular marker for myofibroblasts.
  • a-SMA a molecular marker for myofibroblasts.
  • PKM2 was expressed in the infarcted heart tissues after 4 days, while no PKM2 staining was detected in normal healthy cardiac tissue. Extracellular PKM2 was seen in the staining of the patient myocardial infarction tissues, the inventors also performed IHC staining of PKM2 in the tissue of mouse infarction heart after different time points of induction of infarction. No PKM2 staining was observed at 6 hours, 24 hours, and 3 days after infarction induction. PKM2 staining was detected in the infarction region 4 days after infarction induction. EcPKM2 was visible in the IHC staining. PKM2 was not detected in plasma of sham mice and the mice at 6 and 24 hours post myocardial infarction.
  • PKM2 was detected in plasma of mice 7d after myocardial infarction (not shown).
  • the experiments from our and other laboratories suggest that PKM2 is expressed and released into the extracellular space at a late time point of myocardial infarction induction ( ⁇ 4 days post infarction) from, e.g., dead cells.
  • FIGs 5A-5D Co-immunoprecipitation experiments were performed using G415R and extracts from primary human cardiomyocyte cells exposed to hypoxic conditions. As shown in FIGs 5A-5D, G415R effectively reduced PTEN expression in cultured cardiomyocy te cells (evidenced by immunoblotting) as well as in myocardium tissue (observed through IHC staining) from mice with infarctions. Interestingly, FIGs 5A-5D illustrate that G415R has an affinity with integrin P3, as evidenced by its co-precipitation, confirmed using antibodies specific for either PKM2 or integrin [33.
  • EcPKM2 should the cardioprotective and proliferative effects of EcPKM2 be primarily through PI3K activation, they'd be rendered ineffective by a PI3K inhibitor. Supporting this notion, G415R's influence on H9C2 proliferation was nullified by commercially available FAK and PI3K inhibitors. EcPKM2 fosters a beneficial interaction with integrin avP3. This interaction, in turn, triggers integrin signaling in cardiomyocytes during periods of hypoxic and oxidative stress, providing a shield against apoptosis and promoting cellular proliferation.

Abstract

Procédés de traitement de patients qui ont subi un infarctus du myocarde, également connus sous le nom d'infarctus aigu du myocarde, et de traitement ou d'atténuation des effets de l'infarctus. Ce traitement peut comprendre l'administration d'une composition contenant une protéine qui est sensiblement similaire ou identique à la pyruvate kinase M2 (PKM2) pendant la phase aiguë ou initiale de l'infarctus du myocarde.
PCT/US2023/077001 2022-10-14 2023-10-16 Procédés de conservation et de protection de cardiomyocytes et de réduction de la fibrose cardiaque à la suite d'une lésion cardiaque WO2024081960A2 (fr)

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