Method for Brain Injury After Stroke TECHNICAL FIELD [0001] This application relates to the systems and methods for improving recovery and tissue repair following brain stroke and reperfusion and hemorrhagic stroke by prevent neuron death and promote regeneration. BACKGROUND [0002] Ischemia and stroke are interrelated medical conditions characterized by restricted blood flow to the brain, leading to potential brain damage and neurological deficits. Ischemia refers to the inadequate blood supply to a tissue or organ, resulting in a reduced supply of oxygen and nutrients. This condition can occur in various parts of the body, but when it affects the brain, it is known as cerebral ischemia. Cerebral ischemia can result from a blockage or narrowing of the blood vessels supplying the brain, impeding the necessary flow of blood. [0003] Furthermore, a stroke is a medical emergency that happens when there is a sudden interruption of blood flow to a part of the brain, causing brain cell damage and impairment of neurological function. Stroke is the fifth leading cause of death in the United States and a significant cause of long-term disability. [0004] Ischemic stroke and ischemia-reperfusion injury are related concepts involving the restriction and subsequent restoration of blood flow to a tissue or organ, especially the brain during a stroke. Ischemia-reperfusion injury refers to the damage that occurs when blood flow is restored to a tissue or organ after a period of ischemia. While restoring blood flow is essential to prevent further damage, it can paradoxically exacerbate the initial injury due to a series of complex biochemical processes. This injury can worsen the outcome of a stroke, leading to increased brain damage and neurological deficits. Treating ischemic stroke involves rapidly restoring blood flow to the affected brain region to salvage as much viable tissue as possible. [0005] Accordingly there is an ongoing need for improved methods to treat or mitigate ischemia, stroke, and ischemia-reperfusion injury. Such new treatment strategies and interventions to address these conditions is impact on patients' health and quality of life. This application is directed to these needs among others.
SUMMARY [0006] This patent discloses aspects related to treating brain injury using a PKM2 protein variant. One aspect includes a method of treating ischemia-reperfusion brain injury (IRI) in a subject by administering a therapeutically effective amount of a PKM2 protein variant, wherein the PKM2 protein variant is delivered systemically and is capable of reducing tissue damage following reperfusion of ischemic brain tissue. In one aspect, the PKM2 protein variant in the method is a G415R mutant. In another aspect, the method is specifically for treating brain injury caused by a hemorrhagic stroke or by an ischemic stroke. In a further aspect, the PKM2 protein variant is administered intravenously and within 1 hour of reperfusion. [0007] Another aspect includes a method of improving recovery and tissue repair after ischemic or hemorrhagic stroke by administering a PKM2 protein variant with a G415R mutation. In one aspect, the PKM2 protein variant in the method is not a constitutive PKM2. In another aspect, the ischemic stroke being treated is an acute ischemic stroke. [0008] One aspect includes a method for treating brain injury caused by a hemorrhagic stroke using a systemically delivered PKM2 protein variant. In one aspect, the method preserves neural tissue. In another aspect, the PKM2 is derived from a human or non-human animal. [0009] Another aspect includes a method in which PKM2 (G415R) provides survival advantages. For example, G415R significantly reduced mortality rates in both MCAO and ICH models compared to other groups (FIGs.2 and 7). Additionally, in the ICH model, G415R decreased brain water content, indicating a reduction in edema (FIG.3). Furthermore, G415R significantly reduced the infarct size in the MCAO model (FIG.5). [0010] Additionally, one aspect includes the therapeutic agent being disposed within a pharmaceutically acceptable carrier and being systemically administered. In one aspect, the composition reduces neural cell death caused by ischemic injury or reduces fibrosis of the injured tissue. In another aspect, the composition improves brain function recovery after a stroke and the pyruvate kinase M2 is a dimer.
BRIEF DESCRIPTION OF THE FIGURES [0011] FIG.1 illustrates the significant improvement in the modified Neurological Severity Score (mNSS) test results for mice subjected to induced intracerebral hemorrhage (ICH) after G415R treatment. [0010] FIG. 2 illustrates increased survival of the intracerebral hemorrhage (ICH) mice after G415R treatment. [0011] FIG.3 illustrates the significant improvement in brain water content measurements in mice subjected to induced intracerebral hemorrhage (ICH) after G415R treatment. [0012] FIG. 4A and FIG. 4B demonstrate the significant improvement in neurological function Corner tests in mice treated with G415R. [0013] FIG.5 illustrates the reduction in infarction size in mice subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion after 90 minutes of occlusion and G415R treatment. [0014] FIG.6 illustrates the modified Neurological Severity Score (mNSS) test results for mice subjected to induced MCAO and G415R treatment. [0015] FIG.7 illustrates the survival rates of mice subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion after 90 minutes of occlusion and G415R treatment. DEFINITIONS [0012] The following definitions are provided to facilitate understanding of certain terms used throughout this disclosure. [0013] In the context of stroke, "brain injury" refers to the damage to brain tissue resulting from either an ischemic or hemorrhagic event. This injury can manifest in various forms, including cell death due to lack of oxygen and nutrients, inflammation and swelling in the brain tissue, accumulation of fluid leading to increased intracranial pressure (edema), and disruption of the blood-brain barrier. Additionally, brain injury encompasses reperfusion injury, where restored blood supply to brain tissue causes further damage due to oxidative stress and inflammation, and hemorrhage-related damage, where bleeding within or around the brain directly destroys brain cells and structures. These injuries result in immediate and long-term neurological deficits, such as impaired motor skills, cognitive functions, and speech, significantly affecting a patient's daily activities and quality of life.
[0014] 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 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 α-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. [0015] The term “conservatively modified variants” applies to both natural and non-natural amino acid sequences and natural and non-natural nucleic acid sequences, and combinations thereof. With respect to particular nucleic acid sequences, “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. By way of example, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or non-natural polypeptide also describes every possible silent variation of the natural or non-natural nucleic acid. One of skill will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) 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.
[0016] The term “ischemia-reperfusion injury” (IRI) refers to the damage that occurs when blood flow is restored to an area of tissue or an organ that previously experienced deficient blood flow due to an ischemic event. [0017] The term “individual” refers to a human or animal subject. [0018] The term “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 brain is referred to as “reperfusion.” [0019] The term “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. [0020] 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 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. [0021] The term “pharmaceutically acceptable”, as used herein, 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. [0022] The term “prophylactically effective amount,” as used herein, 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. [0023] The phrase “substantially similar,” in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 75%, preferably at least 85%,
more preferably at least 90%, 95%, 95% 98%, 99% or higher or any integral value therebetween nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as those described below for example, or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 10, preferably about 20, more preferable about 40-60 residues in length or any integral value therebetween, preferably over a longer region than 60-80 residues, more preferably at least about 90-100 residues, and most preferably the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example. A substantially similar polypeptide or nucleic acid may be that of a mutant or other protein that preferentially adopts a dimer form. In specific examples, the more effective proteins preferentially dimerized and were soluble. [0024] The term “synergistic”, as used herein, 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 lower 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. In some cases, 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. [0025] 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. By way of example only, 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, especially upon administration to a subject or upon administration in a cell culture assay. [0026] The term “subject” or “patient” as used herein includes mammals and humans.
[0027] The term “dosage” as used herein 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 bodyweight, mg/kg, mg/kg/day, M, nM, µM, or any other unit otherwise referred to in this disclosure or commonly used in the art. [0028] The term “therapeutic agent” or “therapeutic” 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. [0029] 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 brain injury and/or may be therapeutic in terms of ameliorating, improving, or eliminating one or more symptoms of brain damage. [0030] “Treatment,” as used herein, covers any treatment of brain injury in a mammal, particularly in a human, and includes: (a) preventing the brain injury from occurring in a subject; (b) relieving the brain injury; and (c) restoring the individual to a pre-brain injury state. “Treatment” may not indicate, or require, complete eradication or cure of the brain injury, or associated symptoms thereof.. [0031] PKM2, as used throughout this specification, refers to pyruvate kinase isoform M2. DETAILED DESCRIPTION [0032] The application described herein addresses a need for treating ischemia and ischemia- reperfusion injury, including brain ischemia and ischemia-reperfusion injury, by administering PKM2 (Pyruvate kinase M2) protein. A method of ameliorating and/or reducing remote ischemia- reperfusion injury (IRI), comprising administering an effective dose of a PKM2 (Pyruvate kinase M2) protein to an individual in need thereof.
[0033] One embodiment is a method of reducing an adverse consequence of ischemic stroke (middle cerebral artery occlusion, MCAO) and hemorrhagic stroke (intracerebral hemorrhage, ICH) in a patient comprising administering a composition containing a recombinant PKM2 mutant (e.g., G415R) or a recombinant PKM2, or a protein similar or identical to pyruvate kinase M2 (PKM2), which preferentially adopts a dimer form, to the patient during the acute stage of the brain injury. In some embodiments, the therapeutic PKM2 is not a constitutive PKM2. Administration of the protein substantially similar or identical to pyruvate kinase M2 (PKM2) may be commenced within 200 hours of the onset of a stroke. Further, the protein substantially similar or identical to pyruvate kinase M2 (PKM2) can be administered 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 within about 1 hour of the onset of a stroke. The patient can be a human or a non- human mammal. [0034] In one example, PKM2 (G415R) improves Behavior Test Scores: G415R treatment significantly improved mNSS and corner turn test scores compared to rPKM1 and vehicle groups in both MCAO and ICH models (FIGs. 1, 5, and 6). PKM2 (G415R) Provides Survival Advantages: G415R treatment significantly reduced mortality rates in both MCAO and ICH models. (FIGs.2 and 7). [0035] One embodiment is a method of reducing an adverse consequence of ischemia stroke (MCAO) and hemorrhage stroke (ICH) 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 pyruvate kinase M2 (PKM2), which that preferentially adopts a dimer form to the patient during the acute stage of the brain injury. In some embodiments the therapeutic PKM2 is not a constitutive PKM2. Physiologically, 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 brain stroke. The brain stroke can be an acute stroke both ischemia stroke and hemorrhage stroke. Administration of the protein substantially similar or identical to pyruvate kinase M2 (PKM2) may be commenced within 200 hours of onset of a brain stroke. Further, the protein substantially similar or identical to pyruvate kinase M2 (PKM2) can be commenced within about 100 hours, about 72 hours, within about 48 hours, within about 24 hours, or within about 12 hours, within about 6 hours, within about 4 hours, within about 3 hours, within about 2 hours or within about 1 hour. of onset of a brain stroke. The patient can be a human or a non-human mammal.
[0036] Another embodiment provides an ischemia/reperfusion protection composition. An ischemia/reperfusion protection composition as disclosed herein comprises a protein similar or identical to PKM2. The ischemia/reperfusion protection compositions described herein can be administered to individuals to significantly reduce or prevent ischemic damage/reperfusion injury to tissues all associated with the brain stroke. [0037] In various embodiments, the invention discloses, in part, administering an effective amount of a therapeutic composition to a subject to protect neurons or promote growth of neurons. Specific embodiments contemplate treating subject with a therapeutic substantially similar or identical to pyruvate kinase M2 (PKM2). The composition reduces neuron death induced by stroke. [0038] While PKM2 exists in both a dimeric and tetrameric state, the biologically active protein has a dimeric state and more effective mutants have a higher percentage for the dimeric state at equilibrium. PKM2 mutants that can adopt dimeric states are shown in the following illustrative references, which are incorporated herein by reference: Gao, X., Mol Cell 2012 Mar 9;45(5):598- 609.; Zhou, Zhifen, et al. “Oncogenic kinase–induced PKM2 tyrosine 105 phosphorylation converts nononcogenic PKM2 to a tumor promoter and induces cancer stem–like cells.” Cancer research 78.9 (2018): 2248-2261.; Li, iScience 23, 101684, November 20, 2020; Liu, Vivian M., et al. “Cancer‐associated mutations in human pyruvate kinase M2 impair enzyme activity.” FEBS letters 594.4 (2020): 646-664; Chen, Tsan-Jan, et al. “Mutations in the PKM2 exon-10 region are associated with reduced allostery and increased nuclear translocation. “Communications biology 2.1 (2019): 1-11; Gupta, Vibhor, et al. “Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy.” Journal of Biological Chemistry 285.22 (2010): 16864-16873.; Lv, Lei, et al. “Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization.” Molecular cell 52.3 (2013): 340-352. Other PKM2 mutants can be developed without undue experimentation. In certain embodiment, 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 a dimeric form are useful in the methods and systems for treating MI, IR and other conditions related to cardiomyocyte injury. [0039] In one embodiment, the composition and methods are directed to conditions involving brain injury, such as those arising from ischemic or hemorrhagic stroke. This includes brain tissue
damage due to restricted blood flow or subsequent reperfusion injury. In all aspects, improving the function of the damaged brain tissue by administering PKM2 or a protein substantially similar to PKM2 would be beneficial to the patient. [0040] In another embodiment, the composition may be administered with another agent. Such 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, antiarrhythmic drugs, cardiotonic drugs, diuretic drugs, anti-infective drugs, antiviral drugs, immunomodulatory drugs, inflammatory regulating drugs, anti-tumor drugs, or hormone drugs. Pyruvate Kinase [0041] 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. Among the 4 isoforms of pyruvate kinase, PKM1 and PKM2 are ubiquitously expressed in different types of cells and tissues. PKM2 is highly expressed in proliferating cells including cancer cells. Different from other isoforms, the expression and activity of PKM2 is regulated at multiple levels, including gene expression, alternative splicing, post-translational modification and by metabolic intermediates and growth signaling pathways. Hence, PKM2 is a unique multifaceted regulator that can improve cells adaptation in their metabolic program to match physiological needs in different environments. [0042] 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 allows survival of the organs under hypoxic conditions. PKM2 may also act as a co-activator of hypoxia- induced factor 1-alpha (HIF-1α); the later 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. It should be emphasized that increased aerobic glycolysis and cell proliferation or migration are not unique to cancer and malignancy but rather originate in normal biology and physiological development. In
physiological proliferation of neural progenitors or under hypoxia, PKM2 helps to reprogram energy metabolism to support growth and adaptation. Thus, metabolic transformation is a co- opting of developmental episodes integral to physiological growth. Since these important metabolic and non-metabolic roles of PKM2 were mainly identified in cancerous tumor cells, their potential function in normal cells or in the response to ischemic episodes such as stroke has until now been largely unknown. [0043] 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. The 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). In one specific embodiment, angiogenicly active pyruvate kinase protein is a dimmer, that is, the PKM2 subtype M2. A broad range of proteins and therapeutics substantially similar to pyruvate kinase M2 are useful with specific embodiments. [0044] 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. In at least some forms, 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. Moreover, a recombinant protein can comprise an amino acid or amino acids not encoded by the genetic code. [0045] The substitution of an amino acid can be a conservative or non-conservative substitution. The term 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. For example, a conservative amino acid substitution can involve substitution of a basic amino acid such as arginine with another basic amino acid such as lysine. Likewise, for instance 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. Variants of PKM2 or mutants of the same that are more useful are those that preferentially adopt the dimer form and the soluble in water. An exemplary pyruvate kinase M2 amino acid sequence or SEQ ID NO: 1: PKM2 Accession No. NP 002645 is as follows: 1 mskphseagt afiqtqqlha amadtflehm crldidsppi tarntgiict igpasrsvet 61 lkemiksgmn varlnfshgt heyhaetikn vrtatesfas dpilyrpvav aldtkgpeir 121 tglikgsgta evelkkgatl kitldnayme kcdenilwld yknickvvev gskiyvddgl 181 islqvkqkga dflvteveng gslgskkgvn lpgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie 301 ipaekvflaq kmmigrcnra gkpvicatqm lesmikkprp traegsdvan avldgadcim 361 lsgetakgdy pleavrmqhl iareaeaaiy hlqlfeelrr lapitsdpte atavgaveas 421 fkccsgaiiv ltksgrsahq varyrprapi iavtrnpqta rqahlyrgif pvlckdpvqe 481 awaedvdlrv nfamnvgkar gffkkgdvvi vltgwrpgsg ftntmrvvpv p [0046] An exemplary pyruvate kinase M2 amino acid sequence SEQ ID NO: 2 (R399E) is as follows: 1 mskphseagt afiqtqqlha amadtfelhm crldidsppi tarntgiict igpasrsvet 61 lkemiksgmn varlnfshgt heyhaetikn vrtatesfas dpilerpvav aldtkgpeir 121 tglikgsgta evelkkgatl kitldnayme kcdenilwld yknickvvev gskiyvddgl 181 islqvkqkga dflvteveng gslgskkgvn lpgaavdlpa vsekdiqdlk fgveqdvdmv
241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie 301 ipaekvflaq kmmigrcnra gkpvicatqm lesmikkprp traegsdvan avldgadcim 361 lsgetakgdy pleavrmqhl iareaeaaiy hlqlfeelrr lapitsdpte atavgaveas 421 fkccsgaiiv ltksgrsahq varyrprapi iavtrnpqta rqahlyrgif pvlckdpvqe 481 awaedvdlrv nfamnvgkar gffkkgdvvi vltgwrpgsg ftntmrvvpv p [0047] Another exemplary pyruvate kinase M2 amino acid sequence having three mutations (R399E, K422A, and N523A) or SEQ ID NO: 3 is as follows; MSKPHSEAGT AFIQTQQLHA AMADTFLEHM CRLDIDSPPI TARNTGIICT IGPASRSVET LKEMIKSGMN VARLNFSHGT HEYHAETIKN VRTATESFAS DPILYRPVAV ALDTKGPEIR TGLIKGSGTA EVELKKGATL KITLDNAYME KCDENILWLD YKNICKVVEV GSKIYVDDGL ISLQVKQKGA DFLVTEVENG GSLGSKKGVN LPGAAVDLPA VSEKDIQDLK FGVEQDVDMV FASFIRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELER LAPITSDPTE ATAVGAVEAS FACCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTATMRVVPV P [0048] Another exemplary pyruvate kinase M2 amino acid sequence having one mutation (G415R) or SEQ ID NO: 4 is as follows 1 mskphseagt afiqtqqlha amadtflehm crldidsppi tarntgiict igpasrsvet 61 lkemiksgmn varlnfshgt heyhaetikn vrtatesfas dpilyrpvav aldtkgpeir 121 tglikgsgta evelkkgatl kitldnayme kcdenilwld yknickvvev gskiyvddgl 181 islqvkqkga dflvteveng gslgskkgvn lpgaavdlpa vsekdiqdlk fgveqdvdmv 241 fasfirkasd vhevrkvlge kgknikiisk ienhegvrrf deileasdgi mvargdlgie 301 ipaekvflaq kmmigrcnra gkpvicatqm lesmikkprp traegsdvan avldgadcim 361 lsgetakgdy pleavrmqhl iareaeaaiy hlqlfeelrr lapitsdpte atavRaveas 421 fkccsgaiiv ltksgrsahq varyrprapi iavtrnpqta rqahlyrgif pvlckdpvqe
481 awaedvdlrv nfamnvgkar gffkkgdvvi vltgwrpgsg ftntmrvvpv p [0049] Recombinant or other synthetic pyruvate kinase protein useful in a method embodied by the invention can have amino acid sequence identity with the native pyruvate kinase of about 60% or greater, and, more commonly, at least about 70%, 80%, 90%, 95%, 98% or greater, or 100%. All sequence homologies and ranges thereof within those enumerated above are expressly encompassed. Sequence identity between amino acid sequences is determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison. The sequences are considered identical at a position if the amino acids at that position are the same. A gap, that is a position in an alignment where an amino acid residue is present in one sequence but not the other, is regarded as a position with non-identical residues. Alignment of sequences may be performed using any suitable program or algorithm. Computer assisted sequence alignment can be conveniently performed using standard software programs. [0050] The pyruvate kinase protein can also be chemically synthesized. The provision and use of fusion proteins incorporating a pyruvate kinase protein as described herein is also expressly encompassed by the invention. Nucleic acid encoding a fusion protein can be provided by joining separate DNA fragments encoding the pyruvate kinase protein and, for example, a lipophilic amino acid sequence for enhancing the lipophilic characteristics of the protein by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini and ligation of cohesive ends as required. [0051] Host cells that can be transfected for expression of recombinant pyruvate kinase proteins and fusion proteins as described herein include bacteria such as E. coli, Bacillus strains (eg., B. subtilis), Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells. The host cells are cultured in a suitable culture medium under conditions for expression of the introduced nucleic acid (typically in an appropriate expression vector) prior to purification of the expressed product from the host cells, and/or supernatants as required using standard purification techniques. [0052] Pyruvate kinase proteins as described herein can also be modified by coupling one or more proteinaceous or non-proteinaceous moieties to the protein to improve solubility, lipophilic characteristics, stability, biological half-life, or for instance to act as a label for subsequent detection or the like. Modifications can also result from post-translational or post-synthesis
modification such as by the attachment of carbohydrate moieties, or chemical reaction(s) resulting in structural modification(s) (e.g., the alkylation or acetylation of one or more amino acid residues or other changes involving the formation of chemical bonds). By way of a non-limiting example, the pyruvate kinase protein can have one or more modifications selected from the group consisting of methylation, phosphorylation, oxidation of tyrosine and/or tryptophan residues, glycosylation, and S-methylcysteine covalent attachment. [0053] The pyruvate kinase protein can vary in size from the complete protein. However, the pyruvate kinase should be of a length that enables a dimmer formation. [0054] C-terminal and N-terminal extensions of native pyruvate kinase proteins are involved in stabilization of quaternary structure and the generation of aggregates of the protein. Thus, pyruvate kinases lacking such C-terminal and N-terminal extensions forms aggregates poorly. Pyruvate kinases form large aggregates. Electrostatic interactions between pyruvate kinase proteins are also involved in pyruvate kinase aggregate formation, and ionization of histidine residues below a pH of 7 can disrupt the aggregates. Typically, the pyruvate kinase protein used in a method embodied by the invention will be in dimeric form. The intact and truncated forms of a pyruvate kinase protein useful in embodiments of the invention may be subjected to post translational modifications not limited to acetylation, methylation, ethylation, phosphorylation, oxidation and glycosylation modifications in the native pyruvate kinase protein. Suitable conditions for alkaline phosphatase activity include suitable zinc, magnesium or calcium containing buffers. [0055] Partially hydrolyzed forms of pyruvate kinase proteins can be purified for use in embodiments of the invention by any suitable purification technique including, e.g., filtration and chromatography protocols. [0056] Possible applications in accordance with this invention include preventing remote IRI by administering a PKM2 protein, in conjunction with surgical repair of the thoracic or suprarenal or abdominal aorta due to aneurysmal disease, but also in conjunction with those surgical procedures that induce or require transient occlusion or bypass of the visceral blood supply during and/or following major organ transplant, including liver, kidney, small intestine, extremities and pancreas. Also included is the prevention of remote IRI in conjunction with surgical procedures that result in the transient reduction or prevention of blood flow including hepatic and biliary surgical resections, total or partial pancreatectomy, total and partial gastrectomy, esophagectomy, colorectal surgery, vascular surgery for mesenteric vascular disease, or abdominal insufflation
during laparoscopic surgical procedures. Additional applications include blunt or penetrating trauma that results in interruption of blood flow to the visceral organs including those arising from stab wounds or from penetrating wounds or blunt abdominal trauma secondary to motor vehicle accident. Further applications include crush injuries, for example following a natural disaster. Additional applications include insertion of a device for delivery of pharmacologically active substances such as thrombolytic agents or vasodilators and/or for mechanical removal of complete or partial obstructions, and injection of pharmacologically active substances such as thrombolytic agents or vasodilators following onset of an initial thrombotic or thromboembolic or another ischemia-inducing disorder including but not limited to stroke, myocardial infarction, deep vein thrombosis, atherosclerosis or thrombotic events at foreign surfaces. [0057] Preferably, the surgical intervention is selected from the group consisting of orthopedic surgery, vascular surgery, cardiac surgery, catheter-directed procedures, cancer surgery and traumatic surgery. Orthopedic surgery is preferably selected from the group consisting of knee surgery, hand surgery, shoulder surgery, long bones in trauma, hip replacement, and back surgery. [0058] Vascular surgery may be due to repair and/or accidents, for example aortic aneurysms, etc. [0059] In another embodiment, the remote ischemia-reperfusion injury (IRI) affects the lung, the kidney, the brain, the liver, the heart, the intestine, the pancreas or other organs and extremities. [0060] In one embodiment, the surgery is traumatic surgery, for example due to car accidents, crash injuries and crush injuries in general, including major trauma with hypovolemia. [0061] In a specific embodiment the surgical intervention is transplantation, preferably of an organ. [0062] In one embodiment, the remote IRI is due to reperfusion of ischemic tissue(s) and/or organs after a surgical intervention. The surgical intervention includes any surgical procedure. [0063] Other applications include diseases or procedures that result in systemic hypotension that either disrupts or decreases the flow of blood to the visceral organs, including hemorrhagic shock due to blood loss, cardiogenic shock due to myocardial infarction or cardiac failure, neurogenic shock, nephrogenic shock, or anaphylaxis. [0064] PKM2 proteins are known. The PKM2 protein, also known as pyruvate kinase isozyme M2, is a well-known protein that plays a crucial role in cellular metabolism, particularly in glycolysis. [0065] The method can be performed in a number of different ways in accordance with the invention. Due to the nature of the active protein, however, administration is often performed
parenterally, e.g., by intraarterial, intravenous, subcutaneous, or intramuscular injection. The parenteral injection can be administered continuously or intermittently in discreet injections. As a general rule, the drugs can be administered in the form of a sterile aqueous solution buffered to a physiologically acceptable pH. The solution can be prepared well in advance of the administration or, depending upon the actual compounds employed, in direct conjunction with or just before administration. [0066] The term “ischemia-reperfusion injury” (IRI) refers to an injury resulting from the restoration of blood flow to an area of a tissue or organ that had previously experienced deficient blood flow due to an ischemic event. [0067] The term “individual” refers to a human or animal subject. [0068] The invention described herein addresses a critical need for treating ischemia and ischemia- reperfusion injury, including brain neuron ischemia and ischemia-reperfusion injury, through the administration of PKM2 (Pyruvate kinase M2) protein. This method involves administering an effective dose of PKM2 protein to an individual in need, aiming to ameliorate and/or reduce remote ischemia-reperfusion injury (IRI). The PKM2 protein, known for its role in glycolysis and cellular metabolism, has shown promise in protecting tissues from ischemic damage and the subsequent reperfusion injury that often exacerbates the initial harm. [0069] Possible applications of this invention are broad and encompass various medical scenarios where ischemia and reperfusion injury are concerns. One significant application is the prevention of remote IRI during surgical procedures involving the thoracic, suprarenal, or abdominal aorta, especially in cases of aneurysmal disease. The PKM2 protein can be administered in conjunction with surgeries that involve transient occlusion or bypass of visceral blood supply, such as major organ transplants (including liver, kidney, small intestine, extremities, and pancreas). Additionally, this method is applicable in surgeries resulting in transient reduction or cessation of blood flow, including hepatic and biliary resections, pancreatectomy, gastrectomy, esophagectomy, colorectal surgery, and vascular procedures for mesenteric vascular disease. It is also relevant during abdominal insufflation in laparoscopic surgeries. [0070] Preferably, the surgical interventions benefiting from this invention include orthopedic surgery, vascular surgery, cardiac surgery, catheter-directed procedures, cancer surgery, and trauma surgery. In orthopedic surgery, specific applications include knee surgery, hand surgery, shoulder surgery, long bone trauma, hip replacement, and back surgery.
[0071] Vascular surgeries that may utilize this method include repairs following accidents, such as aortic aneurysm repairs, where the risk of ischemia-reperfusion injury is significant. [0072] Another embodiment of the invention targets remote ischemia-reperfusion injury affecting various organs and extremities, including the lungs, kidneys, brain, liver, heart, intestines, pancreas, and others. In cases of traumatic surgery, such as those resulting from car accidents, crush injuries, or other major traumas with associated hypovolemia, this method offers potential benefits in mitigating remote IRI. [0073] A specific embodiment of this invention applies to transplantation surgeries, preferably involving organ transplants, where ischemia-reperfusion injury is a significant concern. [0074] In one embodiment, remote IRI is due to the reperfusion of ischemic tissues and/or organs following a surgical intervention. This encompasses any surgical procedure that may result in ischemia-reperfusion injury. [0075] Other applications include conditions or procedures leading to systemic hypotension, which disrupts or decreases blood flow to visceral organs. Examples include hemorrhagic shock due to blood loss, cardiogenic shock from myocardial infarction or cardiac failure, neurogenic shock, nephrogenic shock, and anaphylaxis. [0076] PKM2 proteins are well-known for their crucial role in cellular metabolism, particularly in glycolysis. The PKM2 protein, also known as pyruvate kinase isozyme M2, has been extensively studied and is recognized for its potential therapeutic benefits in protecting tissues from ischemic damage. [0077] The administration of PKM2 protein can be performed in various ways according to the invention. Given the nature of the active protein, administration is typically done parenterally, such as through intra-arterial, intravenous, subcutaneous, or intramuscular injections, and i.v. infusion. These injections can be administered continuously or intermittently in discrete doses. Generally, the drugs are delivered in the form of a sterile aqueous solution buffered to a physiologically acceptable pH. The solution can be prepared in advance or just prior to administration, depending on the specific compounds used.
Examples [0078] PKM2 (G415R) demonstrates significant improvements in neurological function, survival rates, brain water content reduction, and infarction size reduction in both MCAO and ICH models, highlighting it as an effective treatment for ischemic and hemorrhagic stroke. Example 1 -- Ischemic Stroke and Ischemia-Reperfusion [0079] Intraluminal Suture Middle Cerebral Artery Occlusion (MCAO) was induced in male C57/6J mice (7-9 weeks old) under anesthesia. The middle cerebral artery (MCA) was surgically exposed and occluded using an intraluminal suture for 90 minutes to simulate ischemic stroke. Following occlusion, the suture was removed to allow reperfusion, and mice were returned to their cages for recovery. An initial intravenous (i.v.) dose of treatment was administered prior to recovery.’ [0080] Mice were randomly assigned to one of four groups: three treatment groups and one sham- operated control group. Treatment began within 30 minutes of reperfusion and consisted of intraperitoneal (i.p.) injections every other day for six days, with the first dose day 0 administered intravenously. On days 2, 4, and 6 post-MCAO, neurological function was assessed using the modified Neurological Severity Score (mNSS) and the corner turn test. After behavioral testing on day 7, brains were collected for analysis of water content (n=4) and FFPE tissue preparation (n=5) in each group. Post-surgery survival was also recorded. Example 2 -- Hemorrhagic stroke [0081] (Intracerebral Hemorrhage, ICH), C57/6J mice (7 - 9w, males), were anesthetized. The animals are placed in a stereotaxic frame under anesthesia. A 1-cm-long midline incision was made in the scalp. A hamilton syringe is used to inject 0.4 µL of collagenase into the CA1 region of the right hippocampus. The incision is sealed. Animals are returned to their home cage for recovery. The first dose of treatment (below) is given via i.v. injection before placing the mouse in its recovery cage. [0082] Randomly assigned to 4 groups (three treated groups and one sham group), are subjected for ICH modeling or sham operation (one group). Dosing starts immediately after procedure (within 30 minutes). Administration by i.p. injection (the first dose i.v. injection) for 6 days, one dose every other day. At the third day after the ICH surgery and at the end of treatment (7th day), the mice were
subjected to mNSS (Modified Neurological Severity Score) score and corner test to measure treatment effect. At the end of behavioral test, four mouse brains were taken to measure brain water contents, while five to prepare FFPE tissue in each group. Death of the experimental animals are recorded as post-surgery survival. Treatments: [0083] Recombinant PKM2 mutant (G415R), rPKM1, and vehicle are administered via i.p. injection (the first via i.v. right after MCAO or ICH induction day 0), day 2, day 4, and day 6 total 4 doses. Animals were accessed by mNSS and corner tests 3 and 7 days after the surgery. Animals are sacrificed after the tests for further analyses 7 days (1 day after last treatment dose). PKM2 (G415R) improves behavior test scores [0084] Neuropsychological tests (modified neurological severity score (mNSS)) and corner turn test) were performed with the mice that underwent MCAO and ICH procedures and treated with G415R, rPKM1 and vehicle (no treatment for animals in sham group) 3 and 7 days after surgery and 2 and 4 doses after the treatments. Animals in G415R group demonstrated improved test scores in both neuropsychological tests compared to rPKM1 and vehicle groups. [0085] FIG. 1 illustrates the results of modified Neurological Severity Score (mNSS) tests performed on mice subjected to induced intracerebral hemorrhage (ICH). The mice were treated immediately (day 0) after ICH induction with the indicated agents: G415R, recombinant PKM1 (rPKM1) at a dose of 4 mg/kg, and a vehicle control administered intraperitoneally (i.p.) once every other day for a total of four doses. The sham mice underwent a surgical procedure without ICH induction or subsequent treatments, serving as a baseline control group. The mNSS scores, representing the cumulative results of the neurological function tests, were recorded and analyzed to assess the therapeutic effects of the treatments on ICH-induced neurological deficits. The standard mNSS behavior tests were conducted on the third (3 d) and seventh (7 d) days following the ICH procedure. All animals were euthanized on day 7 post-ICH. Notably, the results demonstrated that G415R treatment significantly improved neurological outcomes compared to rPKM1 and the vehicle control.
[0086] FIG.5 demonstrates the significant improvement in neurological function in mice treated with G415R, highlighting its potential as an effective treatment for mitigating the adverse effects of MCAO-induced brain injury and promoting neurological recovery. The results indicate that G415R provides improved outcomes compared to the vehicle control and rPKM1 treated group. The results of the modified Neurological Severity Score (mNSS) behavior tests performed on mice subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion after 90 minutes of occlusion. The mice were treated immediately (day 0) after reperfusion with the indicated agents: G415R, recombinant PKM1 (rPKM1) at a dose of 4 mg/kg, and a vehicle control, administered intraperitoneally (i.p.) once every other day for a total of four doses. The standard mNSS behavior tests were conducted on the third (3 days) and seventh (7 days) days following the MCAO procedure to assess neurological function. All animals were euthanized on day 7 post-MCAO. [0087] FIG. 6 illustrates the results of modified Neurological Severity Score (mNSS) tests performed on mice subjected to induced MCAO, and reperfusion after 90 minutes ligation, and immediately (day 0) treated with indicated agents (G415R and rPKM14 mg/kg, and vehicle i.p. once every other day for three doses). The standard mNSS behavior tests of the MCAO mice were th performed at the third (3 d) and 7 (7 d) day after MCAO procedure. All animals were euthanized at day 7 after MCAO procedure. Sham mice were undergone surgery without MCAO and treatments. mNSS scores were the total test scores. Example 4 --PKM2 (G415R) Improves Neurological Function in Ischemic and Hemorrhagic Stroke Models [0088] Neurological function was assessed using the modified Neurological Severity Score (mNSS) and the corner turn test in mice subjected to middle cerebral artery occlusion (MCAO) or intracerebral hemorrhage (ICH). Mice were treated with G415R, recombinant PKM1 (rPKM1), or a vehicle control (sham group received no treatment) on days 0, 2, 4, and 6 after stroke induction. Behavioral tests were conducted on days 3 and 7 post-stroke. G415R treatment consistently improved neurological outcomes compared to rPKM1 and vehicle groups in both stroke models. [0089] FIG.1 illustrates the mNSS scores in mice subjected to ICH. G415R treatment significantly improved neurological deficits compared to rPKM1 and vehicle control on both day 3 and day 7 post-ICH.
[0090] FIG. 5 further demonstrates the superior efficacy of G415R in improving neurological function after MCAO. G415R-treated mice showed significantly lower mNSS scores compared to the other groups on both day 3 and day 7 post-MCAO. [0091] FIG.6 corroborates the findings of FIG.5, again showing the significant improvement in mNSS scores in G415R-treated mice following MCAO. [0092] These results collectively demonstrate the therapeutic potential of G415R in promoting neurological recovery after ischemic and hemorrhagic stroke. Example 5 -- PKM2 (G415R) provides survival advantages for both MCAO and ICH mice. [0093] The mice upon MCAO and ICH procedure exhibited a high death rate compared to the sham mice. However, administration of PKM2 (G415R) dramatically reduced death rate compared to the rPKM1 and vehicle groups. [0094] FIG.2 illustrates the results from the intracerebral hemorrhage (ICH) survival test. Mice were induced with ICH and immediately (day 0) treated with the indicated agents: G415R, recombinant PKM1 (rPKM1) at a dose of 4 mg/kg, and a vehicle control, administered intraperitoneally (i.p.) once every other day for a total of four doses. The survival rates of the ICH mice under different treatments were plotted over the observation period. [0095] FIG.7 illustrates the results from the intracerebral hemorrhage (MCAO) survival test. Mice were induced with MCAO and followed by reperfusion after 90 minutes of occlusion. The mice were immediately (day 0) treated with the indicated agents: G415R, recombinant PKM1 (rPKM1) at a dose of 4 mg/kg, and a vehicle control, administered intraperitoneally (i.p.) once every other day for a total of four doses. The survival rates of the MCAO mice under different treatments were plotted over the observation period. G415R decreased brain water contents [0096] Brain swell (increase in water contents) is a severe and potentially fatal complication after stroke. Therefore, measurement of brain water contents after stroke is a measurement for stroke severity. Brain tissues samples from the mice that underwent ICH procedures and treated with G415R, rPKM1 and vehicle (no treatment for animals in sham group) 7 days after surgery and four doses after the treatments were collected. The water contents in these brain samples were
analyzed. Animals in G415R group exhibited similar water contents and less than that in the rPKM1 and vehicle treated groups. [0097] FIG.3 illustrates the results of the brain water content measurement in mice subjected to induced intracerebral hemorrhage (ICH). The mice were treated immediately (day 0) after ICH induction with the indicated agents: G415R, recombinant PKM1 (rPKM1) at a dose of 4 mg/kg, and a vehicle control, administered intraperitoneally (i.p.) once every other day for a total of four doses. The brain tissues of the ICH mice were collected on the seventh day (day 7) post-ICH procedure following the euthanasia of the test animals. The brain water content is presented as a percentage of the total cerebral water content. G415R Demonstrates Improved Neurological Function in Corner Turn Test [0098] FIG.4A and FIG.4B illustrate the results of the corner turn test, a behavioral assessment of neurological function, conducted on days 3 and 7 post-MCAO, respectively. Mice were subjected to MCAO followed by reperfusion and treated with G415R, rPKM1 (4 mg/kg), or a vehicle control. G415R-treated mice exhibited significantly lower corner turn scores compared to both the vehicle control and rPKM1 groups at both time points (FIG.4A, 4B). Lower corner turn scores indicate improved neurological function, suggesting a therapeutic benefit of G415R in mitigating MCAO-induced brain injury and promoting neurological recovery. G415R Reduces Infarct Size After MCAO [0099] Brain sections from mice subjected to MCAO and treated with G415R, rPKM1, or vehicle control were prepared 7 days after surgery and four doses of treatment. The sections were analyzed using TTC staining, which stains viable tissue red and infarcted (damaged) tissue pale. G415R- treated mice exhibited significantly smaller infarct sizes compared to both rPKM1 and vehicle- treated groups. [0100] FIG.5 illustrates the quantification of infarct size in mice subjected to MCAO followed by reperfusion. Mice were treated immediately after reperfusion with G415R, rPKM1 (4 mg/kg), or vehicle control (i.p., every other day for a total of four doses). Brain sections were analyzed on day 7 post-MCAO, and infarct size is presented as a percentage of total brain volume. The results demonstrate that G415R treatment significantly reduced infarct size, indicating a protective effect against ischemic brain injury.
G415R Improves Survival Rates after MCAO [0101] FIG.7 illustrates the survival rates of mice subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. Mice were treated immediately after reperfusion with G415R, rPKM1 (4 mg/kg), or a vehicle control (i.p., every other day for a total of four doses). G415R treatment significantly improved the survival rate of mice following MCAO compared to both the rPKM1 and vehicle control groups. This finding underscores the potential of G415R as a therapeutic agent for mitigating the mortality associated with ischemic stroke. [0102] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.