Use of amphiregulin (AREG) in methods for treating vascular hyperpermeability

Abstract

Disruption of endothelial barrier integrity, resulting in increased vascular permeability, contributes to many pathological processes. In a first cohort of 11 patients with cardiac arrest accompanied by vascular leakage, we confirmed that AREG expression was 56-fold higher than in controls. During a second prospective clinical study, we confirmed a strong association between AREG plasma levels and fluid balance in 77 independent patients hospitalized with cardiogenic shock. Finally, we demonstrated the important role of this protein in Areg- / - mice, showing that i) none of the KO mice achieved return of spontaneous circulation (ROSC), compared with 40% of wild-type mice (n = 6 per group, p < 0.05), and ii) intravenous infusion of 10 μg of recombinant AREG during resuscitation demonstrated a beneficial effect, with 80% of the infused mice achieving ROSC compared with 40% of controls (n = 6, p < 0.05). Finally, we used markers of extravasation to demonstrate that AREG administration reduced the level of vascular leakage. These results indicate that administering AREG to patients suffering from vascular permeability may be beneficial. Accordingly, the present invention relates to the use of amphiregulin (AREG) in methods for treating vascular permeability.

Description

[Technical Field]

[0001] Field of the invention: The present invention relates to the field of medicine, particularly vascular disease. [Background technology]

[0002] Background of the invention: The endothelial barrier plays an essential role in blood circulation and blood-tissue exchange. Its permeability, which enables these exchanges, is primarily regulated by adhesion molecules such as tight junctions. Studies have demonstrated that dysregulation of these cellular junctions, whether associated with structural changes in endothelial barrier cells or not, contributes to increased vascular permeability (Wautier JL et al., 2022, Int J Mol Sci). Disruption of the endothelial barrier integrity, leading to increased vascular permeability, contributes to many pathological processes, some of which develop rapidly within minutes or hours. For example, a condition in which acute vascular permeability is adverse is circulatory shock, which accounts for one-third of intensive care unit (ICU) admissions (20,000 per year in France) with a 40% mortality rate and is therefore a major public health problem. Vascular hyperpermeability (capillary leakage) is actually a key feature of circulatory failure. Inflammation caused by tissue lesions (SIRS: systemic inflammatory response syndrome) also induces massive vascular leakage, which affects both the macrocirculation and microcirculation. Fluid balance (the difference between fluid input and output) is independently correlated with mortality during sepsis and cardiogenic shock (Besnier, E., et al. (2020). Shock 53, 426-433. 2 Micek, ST, et al. (2013). Crit Care 17, R246. 3; Liu, L., et al. (2018). Basic Res Cardiol 113, 12.), and controlling capillary leak would be highly beneficial. Many studies have shown that diseases such as chronic inflammation, cancer, septic shock, diabetes, and acute hemorrhagic shock induce increased vascular permeability (Nagy JA et al., 2008, Angiogenesis; Pickkers P et al., 2005, Shock; Yuan SY et al., 2007, Microcirculation & Xia ZL et al., 1995, Respiration).It is known in the prior art that vascular permeability is essential for normal tissue health and is also a key feature of many disease states in which it is significantly increased (Nagy JA et al., 2008, Angiogenis). Increased vascular permeability induces biological dysfunction such as peripheral edema, tissue damage, or fluid retention in the lungs (Agostoni A et al., 1992, Int J Clin Lab; Paul R et al., 2001, Nat Med; Weis S et al., 2004, J Clin Invest & Groeneveld ABJ et al., 2002, Vascu Pharmacol).

[0003] Therefore, the deleterious effects associated with vascular permeability necessitate the development of treatments and therapeutic agents that can effectively and timely prevent, reduce, or counteract vascular permeability and protect tissues from ischemia / reperfusion injury.

[0004] Amphiregulin (AREG) is a type II cytokine and a member of the epidermal growth factor family. The name AREG derives from its bifunctional role: it stimulates the proliferation of keratinocytes, normal fibroblasts, and tumor cells and inhibits the proliferation of several invasive cancer cell lines in vitro (Shoyab, Mohammed, et al. "Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7." Proceedings of the National Academy of Sciences 85.17 (1988): 6528-6532.). It is synthesized as a transmembrane propeptide and released as mature AREG after proteolytic cleavage, acting in a juxtacrine, autocrine, or paracrine manner. After associating with the EGF receptor (EGFR), AREG activates essential intracellular signaling cascades that govern cellular metabolism, inflammation, and the cell cycle. Elevated expression of AREG is associated with various inflammatory and pathological conditions. For example, AREG has been identified as a key regulatory factor secreted by both innate and adaptive immune cells, which not only promotes host resistance against pathogenic helminths but also assists in tissue repair and wound healing under various inflammatory conditions.Recombinant AREG has been shown to promote tissue repair processes in several infection-mediated injury models (Monticelli et al. “Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus”, Nature Immunology 12(11), 2011); Burzyn et al., “A Special Population of Regulatory T Cells Potentiates Muscle Repair”, Cell 155, 1282-1295, 2013; Jamieson et al. Role of tissue protection in lethal respiratory viral-bacterial coinfection Science 340(6137): 1230-12342013 2013; Jin, Richard M., Jordan Warunek, and Elizabeth A. Wohlfert. “Therapeutic administration of IL-10 and amphiregulin alleviates chronic skeletal muscle inflammation and damage induced by infection.” Immunohorizons 2.5 (2018): 142-154; Minutti et al. “A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation” Immunity 50, 645-654, 2019). These studies highlight the role of AREG in promoting tissue repair during the recovery phase by inducing cell proliferation and differentiation.AREG has been identified as a biomarker and therapeutic target, and prevention of AREG activity is being explored as a therapeutic approach in various cancers and chronic inflammatory and fibrotic conditions (Singh, Siddharth S., et al. "Amphiregulin in cellular physiology, health, and disease: Potential use as a biomarker and therapeutic target." Journal of Cellular Physiology 237.2 (2022): 1143-1156.). Similarly, in cardiovascular disease, AREG has been reported to promote the survival, differentiation, and proliferation of cardiac cells (International Publication No. WO2006081190). Pretreatment with AREG has also been reported to provide cardiac protection from ischemia and reperfusion injury in mice (Koeppen, Michael, et al. "Hypoxia-inducible factor 2-alpha-dependent induction of amphiregulin dampens myocardial ischemia-reperfusion injury." Nature Communications 9.1 (2018): 1-13).

[0005] However, the specific interest of inducing or administering AREG as an early measure to rapidly counter damage caused by vascular permeability has not previously been anticipated or considered. Summary of the Invention

[0006] Summary of the Invention: The invention is defined by the claims. In particular, the invention relates to the use of amphiregulin (AREG) in methods for treating vascular permeability.

[0007] Detailed description of the invention: Key definitions: As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with labeling moieties. Polypeptides, when discussed in the context of gene therapy, refer to the respective intact polypeptides or any fragments or genetically engineered derivatives thereof that retain the desired biochemical function of the intact protein.

[0008] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can comprise a polymer of synthetic subunits. The promoters of the present invention can be prepared by any method known to those of skill in the art, including chemical synthesis, recombination, and mutagenesis. In particular, the promoters of the present invention are DNA molecules typically synthesized by recombinant methods known to those of skill in the art.

[0009] As used herein, the term "derived from" refers to a process in which a first component (e.g., a first polypeptide or polynucleotide), or information from that first component, is used to isolate, derive, or create a different second component (e.g., a second polypeptide or polynucleotide that is different from the first one).

[0010] As used herein, the term "encode" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleic acid sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleic acid sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0011] As used herein, the term "amphiregulin" or "AREG" has its common meaning in the art and refers to a protein synthesized as a transmembrane glycoprotein by the AREG gene (Shoyab, Mohammed, et al. "Structure and function of human amphiregulin: a member of the epidermal growth factor family." Science 243.4894 (1989): 1074-1076.). The term is also known as AR; AREG; AREGB; colorectal cell-derived growth factor; CRDGF; MGC13647; schwannoma-derived growth factor; or SDGF. AREG is transcribed as a 1.4 kb mRNA containing six exons and encodes a 252 amino acid membrane-anchored precursor protein called pro-AREG. This precursor AREG protein contains many glycosylation motifs and cleavage sites that lead to different mature AREG proteins, affecting the biological activity of AREG in different cell types. Sequence analysis reveals the presence of an N-terminal domain with six spatially conserved cysteines and many other semi-conserved amino acid residues, which form disulfide bridges and create a three-loop structure called the EGF domain, which is involved in binding to EGFR. Subsequently, proteolytic cleavage of the precursor mediated by the metalloproteinase enzymes TACE or ADAM-17 releases mature soluble AREG containing the EGF motif, subsequently inducing autocrine or paracrine activation of EGFR, leading to a cascade of signaling events required for several cellular processes, including cell cycle, proliferation, and metabolism. An exemplary amino acid sequence of AREG is shown as SEQ ID NO: 1. The amino acid sequence of the EGF domain ranges from amino acid residue 142 to amino acid residue 182 of SEQ ID NO: 1. As used herein, the term "AREG polypeptide" refers to a polypeptide derived from AREG and containing the EGF domain of AREG or a functional variant thereof. [ka]

[0012] As used herein, the term "variant" refers to an amino acid sequence that differs from the original amino acid sequence but retains its essential properties. Generally, variants are overall very similar and, in many regions, identical to the original polypeptide. The sequence of a variant may differ by amino acid substitution, deletion, or insertion of one or more amino acid residues in the sequence, which does not impair the activity of the polypeptide. A variant may have the same length as the original sequence, or may be shorter or longer.

[0013] As used herein, the term "functional variant of the EGF domain of AREG" refers to a variant of the amino acid sequence ranging from amino acid residue 142 to amino acid residue 182 in SEQ ID NO: 1, which is capable of binding to EGFR and subsequently inducing autocrine or paracrine activation of EGFR, leading to a cascade of signaling events required for several cellular processes, including the cell cycle, proliferation, and metabolism (Berasain, Carmen, and Matias A. Avila. "Amphiregulin." Seminars in cell & developmental biology. Vol. 28. Academic Press, 2014.). Assays for assessing such functionality are well known in the art and typically include those described in Macdonald-Obermann, Jennifer L., and Linda J. Pike. "Different epidermal growth factor (EGF) receptor ligands show distinct kinetics and biased or partial agonism for homodimer and heterodimer formation." Journal of Biological Chemistry 289.38 (2014): 26178-26188.

[0014] As used herein, the term "percent identity" between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be achieved using a mathematical algorithm, as described below. Percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53). Percent identity between two nucleotide or amino acid sequences can also be determined using an algorithm such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, a false "end gap penalty," an "end gap open penalty" of 10, and an "end gap extend penalty" of 0.5. Generally, "percent identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, the identity is 60%. Percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules with identical primary amino acid or polynucleic acid sequences are identical regardless of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 80% identity to a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

[0015] As used herein, the term "patient" or "patient in need thereof" refers to a human or non-human mammal. Typically, the patient is affected or likely to be affected by vascular permeability.

[0016] As used herein, the term "vascular permeability" has its general meaning in the art and refers to the ability of vascular endothelial cells to pass through blood vessels, which function to control blood flow into the extracellular matrix. More specifically, the term refers to the outflow of plasma from the blood circulation system through capillary walls into surrounding tissues, muscle compartments, organs, or body cavities. The terms "vascular permeability" and "vascular leakage" are used interchangeably herein. The terms encompass "excessive vascular permeability" and "hyperpermeability." In particular, vascular permeability can be characterized by alterations in endothelial cell adhesion molecule regulation and / or alterations in endothelial cell structure.

[0017] As used herein, the term "vascular permeability-associated disease or condition" refers to any disease or condition resulting from, causing, characterized by, or otherwise associated with vascular permeability (typically excessive vascular permeability or hyperpermeability). Thus, the association between a disease or condition and vascular permeability may be direct or indirect, and may be separated in time and / or space. In the context of this specification, the terms vascular permeability or excessive vascular permeability and vascular leakage may be used interchangeably. In particular, the term "vascular permeability-associated disease or condition" refers to a disease caused by a lack of normal vascular permeability control.

[0018] As used herein, the term "capillary leak syndrome" or "vascular leak syndrome" has its common meaning in the art and refers to a syndrome characterized by leakage of plasma from the circulatory system through capillary walls into surrounding tissues, muscle compartments, organs, or body cavities. This phenomenon is most commonly seen in sepsis and other forms of circulatory failure, and less frequently in autoimmune diseases, differentiation syndrome, engraftment syndrome, hemophagocytic lymphohistiocytosis, ovarian hyperstimulation syndrome, viral hemorrhagic fever, and snakebite and ricin poisoning. Medications, including the chemotherapy drugs gemcitabine and denileukin diftitox, as well as certain interleukins and monoclonal antibodies, can also cause capillary leak. These conditions and factors are responsible for secondary capillary leak syndrome.

[0019] As used herein, the term "systemic capillary leak syndrome," also known as "Clarkson's disease" or "primary capillary leak syndrome," is a rare, severe, and paroxysmal medical condition observed primarily in middle-aged, otherwise healthy individuals. It is characterized by self-reversing attacks, usually lasting 1 to 3 days, in which the endothelial cells lining the capillaries of the limbs separate, causing leakage of plasma primarily into the muscular compartments of the arms and legs. While the abdomen, central nervous system, and organs (including the lungs) are typically spared, extravasation in the limbs can be large enough to cause circulatory shock and compartment syndrome, accompanied by dangerous hypotension (low blood pressure), hemoconcentration (thickening of the blood), and hypoalbuminemia (a decrease in the main protein, albumin), even in the absence of other causes of such abnormalities.

[0020] As used herein, the term "vascular endothelial cell barrier" refers to a layer of cells lining the luminal surface of blood vessels, which acts as a selective barrier between the vascular lumen and surrounding tissue by controlling the passage of fluids, substances, and cells, such as bone marrow cells and leukocytes, into and out of the bloodstream. Excessive or persistent increases in the permeability of the vascular endothelial cell barrier result in tissue edema / swelling. Thus, the phrase "preserving the integrity of the vascular endothelial cell barrier" refers to maintaining the vascular endothelial cell barrier by avoiding or limiting the permeability of the barrier.

[0021] As used herein, the term "ischemic condition" has its general meaning in the art and refers to any condition resulting from a restriction of blood supply to at least one organ or tissue. Ischemic conditions typically result from vascular obstruction. For example, ischemic conditions include, but are not limited to, renal ischemia, retinal ischemia, cerebral ischemia, and myocardial ischemia. More specifically, the term includes, but is not limited to, coronary artery bypass graft surgery, global cerebral ischemia due to cardiac arrest, focal cerebral infarction, cerebral hemorrhage, hemorrhagic infarction, hypertensive hemorrhage, bleeding due to rupture of intracranial vascular abnormalities, subarachnoid hemorrhage due to rupture of intracranial aneurysms, hypertensive encephalopathy, carotid artery stenosis or occlusion leading to cerebral ischemia, cardiogenic thromboembolism, cerebral stroke, spinal cord stroke and spinal cord injury, and cerebrovascular diseases such as atherosclerosis, vasculitis, macular degeneration, myocardial infarction, cardiac ischemia, and supraventricular tachyarrhythmia. Alternatively, ischemia in all these organs can be caused by circulatory failure without vascular occlusion. These conditions include cardiogenic shock, sepsis and septic shock, hemorrhagic and anaphylactic shock, and post-resuscitation (or post-cardiac arrest syndrome) (Mehta S., Granton J., Gordon AC, Cook DJ, Lapinsky S., Newton G., et al. “Cardiac ischemia in patients with septic shock randomized to vasopressin or norepinephrine”. Crit Care. 2013 Jun 20;17(3):R117; Geri G., Grimaldi G., Seguin T., Lamhaut L., Marin N., Chiche JD, et al. “Hemodynamic efficiency of hemodialysis treatment with a high cut-off membrane during the early period of post-resuscitation shock: The Hyperdia trial”. Resuscitation. 2019 Jul;140:170-177; Haertel F., Reisberg D., Peters M., Nuding S., Schroeder J., Werdan K., and Ebelt H. “Prognostoc value of tissue oxygen saturation using a vascular occlusion test in patients in the early phase of multiple organ dysfunction syndrome”. Shock. 2019 Jun;51(6):706-712; Cour M., Klouche K., Souweine B., Quenot JP, Schwebel C., Perinel S., et al. “Remote ischemic conditioning in septic shock: The RECO-sepsis randomized clinical trial”. Intensive Care Med. 2022 Nov; 48(11):1563-1572). .

[0022] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and therapeutic or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients suffering from or diagnosed with a disease or medical condition, and includes the suppression of clinical recurrence. Treatment can be administered to a patient with a medical disorder or a patient who is likely to ultimately acquire a disorder in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or to extend the patient's survival beyond that expected in the absence of such treatment.

[0023] As used herein, the term "reduce" or other forms such as the word "reduce" or "reduction" refer to a decrease in an event or characteristic (e.g., vascular permeability). This is typically relative to some baseline or expected value, in other words, relative, although it is understood that a baseline or relative value need not always be stated. As used herein, the terms "inhibit" and "inhibition" also refer to the reduction or prevention of vascular leakage or inappropriate vascular permeability. Vascular permeability is considered "reduced" if it is reduced by at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, including complete inhibition or 100%, in a given permeability assay. Methods for assessing vascular permeability are well known in the art (see, for example, Wollborn, Jakob, et al. "Diagnosing capillary leak in critically ill patients: development of an innovative scoring instrument for non-invasive detection." Annals of Intensive Care 11.1 (2021): 1-13.).

[0024] As used herein, the term "prevent" or other forms, such as the words "preventing" or "prevention," means to stop a particular event or characteristic (e.g., vascular permeability), stabilize or delay the occurrence or progression of a particular event or characteristic (e.g., vascular permeability), or minimize the likelihood that a particular event or characteristic (e.g., vascular permeability) will occur. Prevention is typically more absolute than, for example, reduction and therefore does not require a comparison to a control. As used herein, something can be reduced but not prevented, but reducing something can also be prevented. Similarly, something can be prevented but not reduced, but preventing something can also be reduced. When reduction or prevention is used, it is understood that the use of the other term is expressly disclosed unless specifically stated otherwise.

[0025] As used herein, the term "therapeutically effective amount" means the amount of a pharmaceutical composition of the present invention administered to a patient sufficient to constitute treatment as defined above.

[0026] method: Accordingly, a first object of the present invention relates to a method for treating vascular permeability in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of i) an AREG polypeptide or ii) a polynucleotide encoding an AREG polypeptide.

[0027] The methods of the present invention are particularly suitable for reducing and / or preventing vascular permeability in patients in need thereof. More particularly, the methods of the present invention are suitable for reducing and / or preventing excessive vascular permeability and hyperpermeability. Even more particularly, the methods of the present invention are suitable for reducing and / or preventing capillary hyperpermeability.

[0028] In some embodiments, the patient suffers from a disease or condition associated with vascular permeability. Diseases and conditions associated with vascular permeability that are relevant to embodiments of the present invention include, but are not limited to, edema, cardiovascular disease, myocardial infarction, peripheral vascular disease, ischemia, stroke, cancer, atherosclerosis, psoriasis, diabetes, autoimmune diseases such as rheumatoid arthritis, thrombocytopenia, altitude sickness, barotrauma, iatrogenic disorders, bacterial infections, viral infections, and ophthalmic conditions associated with vascular leakage, such as non-proliferative and proliferative retinopathies (including diabetic retinopathy), macular edema (including diabetic macular edema), glaucoma, and macular degeneration (including age-related macular degeneration). The edema can be generalized or focal or organ-specific. The edema can be, for example, cardiac edema, pulmonary edema, renal edema, macular edema, cerebral edema, malnutrition edema, or lymphedema. The edema can result from surgical procedures, particularly major surgical procedures such as cardiac surgery, organ transplant surgery, knee and hip replacement surgery, dental surgery or limb amputation surgery (eg, associated with diabetic complications).

[0029] In some embodiments, the patient is suffering from vascular leak syndrome.

[0030] In some embodiments, the patient is suffering from systemic capillary leak syndrome.

[0031] In some embodiments, the vascular permeability is secondary to sepsis.

[0032] As used herein, the term "sepsis" has its general meaning in the art and is a syndrome of physiological, pathological, and biochemical abnormalities induced by infection (Singer, Mervyn, et al. "The third international consensus definitions for sepsis and septic shock (Sepsis-3)." Jama 315.8 (2016): 801-810).

[0033] In some embodiments, the patient is suffering from SIRS. As used herein, the term "SIRS" has its common meaning in the art and refers to systemic inflammatory response syndrome.

[0034] In some embodiments, the septic patient suffers from acute respiratory distress syndrome. As used herein, the term "acute respiratory distress syndrome" (abbreviated as ARDS) refers to a serious and life-threatening medical condition characterized by the presence of risk factors (e.g., pneumonia, pancreatitis, etc.), bilateral pulmonary infiltrates, and oxygen impairment that is not fully explained by heart failure. More specifically, the term ARDS used herein refers to the acute respiratory distress syndrome defined in the Berlin definition in 2011 (ARDS Definition Task Force et al. 2012 JAMA 307(23): 2526-2533).

[0035] In some embodiments, the patient is suffering from shock. As used herein, the term "shock," unless otherwise specified, is used to describe circulatory shock, cardiogenic shock, ischemic shock, hypervolemic shock, hemorrhagic shock, septic shock, or other types of shock associated with a reduction in blood volume in an organ or tissue or an insufficient blood supply to an organ or tissue (e.g., post-resuscitation syndrome). Shock that can be treated in accordance with the present invention can occur in many situations. For example, events that create a risk of shock can occur in the setting of civilian and military trauma, such as hemorrhage, which creates a risk of hemorrhagic shock. As another example, events such as planned surgery can create a risk of shock. Examples of such surgeries include heart valve replacement surgery, coronary artery bypass graft surgery, stent placement surgery, orthopedic surgery, organ repair surgery, organ transplant surgery, device implantation surgery, etc.

[0036] In particular, the method of the present invention is particularly suitable for improving the chances of return of spontaneous circulation (ROSC) after cardiac arrest.

[0037] In some embodiments, the methods of the present invention are particularly suited to treating vascular permeability induced by cardiac arrest.

[0038] In some embodiments, the methods of the present invention are particularly suited for reducing and / or preventing vascular permeability during the treatment of ischemic conditions.

[0039] In some embodiments, the methods of the present invention are particularly suited to reducing and / or preventing vascular permeability that can occur following acute myocardial infarction.

[0040] In some embodiments, the methods of the present invention are performed sequentially or simultaneously with standard methods for treating ischemic conditions. Typically, standard methods include reperfusion of the ischemic organ (e.g., the heart) via angioplasty (e.g., coronary, renal, or carotid angioplasty), thrombolysis, or coronary artery surgery. The term "percutaneous coronary intervention" refers to coronary angioplasty, a therapeutic method for treating stenosed (narrowed) coronary arteries in the heart, as seen in coronary heart disease. The term "thrombolysis" refers to the administration of a thrombolytic agent. Currently available thrombolytic agents include reteplase (r-PA or Retavase), alteplase (t-PA or Activase), urokinase (Abbokinase), pro-kinase, anisoylated purified streptokinase activator complex (APSAC), and streptokinase.

[0041] In some aspects, the present invention relates to a method for treating an ischemic condition in a patient in need thereof, comprising the steps of: i) restoring blood supply in the ischemic tissue; and ii) reducing and / or preventing vascular permeability by administering to the patient a therapeutically effective amount of an AREG polypeptide or a polynucleotide encoding an AREG polypeptide, wherein steps i) and ii) are performed sequentially or simultaneously.

[0042] AREG polypeptide: In some embodiments, the AREG polypeptide of the present invention comprises an amino acid sequence having at least 80% identity to the amino acid sequence ranging from amino acid residue 142 to amino acid residue 182 (the "EGF domain") of SEQ ID NO:1.

[0043] In some embodiments, the AREG polypeptide of the invention comprises an amino acid sequence ranging from amino acid residue 142 to amino acid residue 182 of SEQ ID NO:1 (the "EGF domain") and may differ from said amino acid sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 substitutions, deletions, and / or insertions.

[0044] According to the present invention, the AREG polypeptide of the present invention is a soluble polypeptide. As used herein, the term "soluble polypeptide" refers to a polypeptide that is not membrane-bound.

[0045] In some embodiments, the AREG polypeptide of the present invention is an 11.3 kDa glycoprotein consisting of 98 amino acid residues. In some embodiments, the AREG polypeptide of the present invention comprises or consists of an amino acid sequence having at least 80% identity with the amino acid residue ranging from amino acid residue 101 (S) to amino acid residue 198 (K) of SEQ ID NO:1.

[0046] In some embodiments, the AREG polypeptide of the present invention is fully or partially glycosylated. As used herein, the term "glycosylated" with respect to a polypeptide means that sugar chains are present at one or more sites of a protein molecule. In particular, a glycosylated protein typically refers to a protein that is modified by N-glycan or O-glycan addition. The term "fully glycosylated" indicates that all predetermined sites (i.e., amino acid residues) of a polypeptide are glycosylated. The term "partially glycosylated" indicates that one or more sites are glycosylated, but not all are glycosylated.

[0047] Commercial sources of AREG polypeptides are well known and typically include those available from R&D Systems (catalog number: 262-AR-100) or Preprotech (catalog number: 100-55B).

[0048] In some embodiments, the AREG polypeptides of the present invention may be modified to improve their therapeutic effects. Such modifications of therapeutic compounds can be used to reduce toxicity, increase circulation time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by combining them with various drug carrier vehicles that modify biodistribution. A strategy for improving drug viability is the use of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change permeability through physiological barriers, and modify clearance rates from the body. Water-soluble polymers containing drug moieties as terminal groups, part of the backbone, or pendant groups on the polymer chain have been synthesized to achieve either targeted or sustained-release effects. Polyethylene glycol (PEG) is widely used as a drug carrier due to its high biocompatibility and ease of modification. Conjugation to various drugs, proteins, and liposomes has been shown to improve retention time and reduce toxicity. PEG can be attached to active agents via the hydroxyl groups at the ends of the chains and through other chemical methods; however, PEG itself is limited to a maximum of two active agents per molecule. In another approach, copolymers of PEG with amino acids have been explored as novel biomaterials that retain the biocompatible properties of PEG but have the added advantage of multiple attachment points per molecule (providing higher drug loading) and can be synthetically designed to suit a variety of applications.

[0049] In some embodiments, the AREG polypeptide of the present invention is fused to the Fc domain of an immunoglobulin. Suitable immunoglobulins include IgG, IgM, IgA, IgD, and IgE. IgG and IgA are preferred, and IgG is most preferred, for example, IgG1. The Fc domain may be a complete Fc domain or a function-conservative variant thereof. The AREG polypeptide of the present invention can be linked to the Fc domain via a linker. The linker may consist of approximately 1 to 100, preferably 1 to 10, amino acid residues.

[0050] According to the present invention, the AREG polypeptide of the present invention can be produced by conventional automated peptide synthesis methods or recombinant expression. General principles for designing and producing proteins are well known to those skilled in the art. The AREG polypeptide of the present invention can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols described in Stewart and Young; Tam et al., 1983; Merrifield, 1986, and Barany and Merrifield, Gross and Meienhofer, 1979. The AREG polypeptide of the present invention can also be synthesized by solid-phase techniques using an exemplary peptide synthesizer such as the Model 433A from Applied Biosystems Inc. The purity of any given protein produced by automated peptide synthesis or recombinant methods can be determined using reverse-phase HPLC analysis. The chemical certainty of each peptide can be established by any method well known to those skilled in the art. As an alternative to automated peptide synthesis, recombinant DNA technology can be used, in which a nucleotide sequence encoding a selected protein is inserted into an expression vector and transformed or transfected into a suitable host cell, which is cultured under conditions suitable for expression, as described below. Recombinant methods are particularly preferred for producing longer polypeptides. A variety of expression vector / host systems are available to contain and express the peptide or protein coding sequence.These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with viral expression vectors (e.g., baculovirus; see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of a variety of techniques for optimizing mammalian expression of proteins; see, e.g., Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful for recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those of skill in the art and are briefly described below. Mammalian host systems for recombinant protein expression are also well known to those of skill in the art. Host cell lines can be selected for their particular ability to process expressed proteins or produce specific post-translational modifications useful for providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves a "prepro" form of a protein, may also be important for correct insertion, folding, and / or function. Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific intracellular and characteristic mechanisms for such post-translational activities, which can be selected to ensure the correct modification and processing of the introduced foreign protein.In the recombinant production of the AREG polypeptide of the present invention, it may be necessary to use a vector containing a polynucleotide molecule for encoding the AREG polypeptide of the present invention. Methods for preparing such vectors and producing host cells transformed with such vectors are well known to those skilled in the art. The polynucleotide molecules used in such efforts are generally ligated to vectors containing selectable markers and origins of replication for propagation in a host. These elements of an expression construct are well known to those skilled in the art. Generally, an expression vector contains DNA encoding a given protein operably linked to appropriate transcriptional or translational control sequences, such as those derived from mammalian, microbial, viral, or insect genes. Examples of control sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences for controlling transcription and translation.

[0051] Polynucleotides: In some embodiments, a polynucleotide of the present invention is messenger RNA (mRNA).

[0052] In some embodiments, the polynucleotide is inserted into a vector, such as a viral vector.

[0053] As used herein, the term "vector" refers to a vehicle capable of introducing a polynucleotide into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. As used herein, the term "viral vector" encompasses vector DNA and the viral particles produced therefrom. Viral vectors can be replication-competent or can be genetically disabled to be replication-deficient or replication-impaired. As used herein, the term "replication-competent" encompasses replication-selective and conditionally replication-competent viral vectors engineered to replicate better or selectively in specific host cells (e.g., tumor cells). As used herein, the term "non-viral vector" particularly refers to vectors of plasmid origin, and optionally, such vectors are combined with one or more substances that improve the transfection efficiency and / or stability of the vector and / or the protection of the vector.

[0054] In some embodiments, the viral vector is an AAV vector. As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and variants thereof. The AAV vector can be deleted of all or part of one or more AAV wild-type genes, preferably the rep and / or cap genes, but retain functional adjacent ITR sequences.

[0055] In some embodiments, the viral vector is a retroviral vector. As used herein, the term "retroviral vector" refers to a vector containing structural and functional genetic elements derived primarily from a retrovirus. In some embodiments, the retroviral vector of the present invention is derived from a retrovirus selected from the group consisting of an alpharetrovirus (e.g., avian leukosis virus), a betaretrovirus (e.g., mouse mammary tumor virus), a gammaretrovirus (e.g., murine leukemia virus), a deltaretrovirus (e.g., bovine leukemia virus), an epsilonretrovirus (e.g., walleye dermal sarcoma virus), a lentivirus (e.g., HIV-1, HIV-2), and a spumavirus (e.g., human spumavirus).

[0056] In some embodiments, retroviral vectors of the invention are viral particles of replication-defective retroviruses capable of transferring exogenous import RNA of genes in place of retroviral mRNA.

[0057] In some embodiments, the retroviral vector of the present invention is a lentiviral vector.

[0058] As used herein, the term "lentiviral vector" refers to a vector containing structural and functional genetic elements primarily derived from a lentivirus. In some embodiments, the lentiviral vector of the present invention is selected from the group consisting of HIV-1, HIV-2, SIV, FIV, EIAV, BIV, VISNA, and CAEV vectors. In some embodiments, the lentiviral vector is an HIV-1 vector.

[0059] The structure and composition of the vector genome used to prepare the retroviral vector of the present invention are in accordance with those described in the art. In particular, minimal retroviral gene delivery vectors can be prepared from vector genomes that contain only the sequences of the non-coding regions of the retroviral genome necessary to provide recognition signals for DNA or RNA synthesis and processing, apart from the recombinant nucleic acid molecules of the present invention. In some embodiments, the retroviral vector genome contains all the elements necessary for nuclear import and accurate expression of the polynucleotide of interest (i.e., the transgene). Examples of elements that can be inserted into the retroviral genome of the retroviral vector of the present invention include at least one (preferably two) long terminal repeats (LTRs), such as LTR5' and LTR3', a psi sequence involved in encapsidation of the retroviral genome, and optionally at least one DNA flap containing the cPPT and CTS domains. In some embodiments of the present invention, the LTR, preferably LTR3', is deleted in favor of a U3 promoter and enhancer and replaced with a minimal promoter that allows transcription during vector production, while an internal promoter that allows transgene expression is added. In particular, the vector is a self-inactivating (SIN) vector containing a non-functional or modified 3' long terminal repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome during integration, resulting in inactivation of the promoter activity of both LTRs. Therefore, the vector genome can be a replacement vector in which all viral coding sequences between the two long terminal repeats (LTRs) are replaced by the recombinant nucleic acid molecule of the present invention.

[0060] In some embodiments, the retroviral vector genome lacks functional gag, pol, and / or env retroviral genes. "Functional" refers to genes that are correctly transcribed and / or correctly expressed. Thus, the retroviral vector genome of the invention in this embodiment contains at least one gag, pol, and env gene that is not transcribed or is incompletely transcribed; the term "incompletely transcribed" refers to an alteration in the transcript gag, gag-pro, or gag-pro-pol, one or more of which are not transcribed. In some embodiments, the retroviral genome lacks gag, pol, and / or env retroviral genes.

[0061] In some embodiments, the retroviral vector genome also lacks the coding sequences for the Vif-, Vpr-, Vpu-, and Nef-accessory genes (in the case of HIV-1 retroviral vectors), or complete or functional genes thereof.

[0062] Typically, the retroviral vectors of the present invention are non-replicating. That is, the vector and retroviral vector genome are incapable of forming new particles that bud from infected host cells. This can be achieved by the absence of gag, pol, or env genes in the retroviral genome, as noted in the paragraph above; it can also be achieved by deleting other viral coding sequences and / or cis-acting gene elements necessary for particle formation.

[0063] Thus, the present invention encompasses the use of virus-like particles. As used herein, the term "virus-like particle" or "VLP" refers to a structure similar to a virus particle but lacking the viral genome, which is incapable of replication and lacks pathogenicity. The particle typically contains at least one structural protein derived from a virus. Preferably, only one structural protein is present. Most preferably, other nonstructural components of the virus are absent. Thus, virus-like particles can spontaneously self-assemble in vitro under appropriate conditions by viral structural proteins, while eliminating genetic material and potential replication hazards. Furthermore, virus-like particles, with diameters of approximately 20-150 nm, possess the properties of nanometer-sized materials, such as a large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), a non-irregular spatial structure, and good biocompatibility. Thus, assembled virus-like particles have great potential as delivery systems for specifically transporting diverse cargoes. In some embodiments, one or more zinc finger motifs of the Gag protein are replaced with one or more RNA-binding domains. In some embodiments, the RNA-binding domain is a coat protein of MS2 bacteriophage, PP7 phage, or Q3 phage, prophage HK022 Nun protein, U1A protein, or hPum protein. More preferably, the RNA-binding domain is a coat protein of MS2 bacteriophage or PP7 phage. Even more preferably, the RNA-binding domain is a coat protein of MS2 bacteriophage. These embodiments are particularly suitable for packaging mRNA encoding an apelin polypeptide into a VLP. Accordingly, in some embodiments, the mRNA encoding an apelin polypeptide encapsulated in the viral particles of the present invention comprises at least one encapsidation sequence. "Encapsidation sequence" refers to an RNA motif (sequence and three-dimensional structure) that is specifically recognized by the RNA-binding domain described above. Preferably, the encapsidation sequence is a stem-loop motif.More preferably, the encapsidation sequence of the retroviral particle is the stem-loop motif of MS2 bacteriophage or PP7 phage RNA. The stem-loop motif, and more particularly the stem-loop motif of MS2 bacteriophage RNA or PP7 phage RNA, may be used alone or repeated several times, preferably 2 to 25 times, more preferably 2 to 18 times, for example 6 to 18 times. In some embodiments, the invention encompasses the use of LentiFlash® technology, which is based on non-integrating lentiviral particles constructed using a bacteriophage coat protein and its cognate 19-nt stem loop to replace the native lentiviral Psi packaging sequence to achieve active mRNA packaging into lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouille P, Sensebe L, Pages JC. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol Ther Methods Clin Dev. 2015 Oct 21;2:15039. doi: 10.1038 / mtm.2015.39. PMID: 26528487; PMCID: PMC4613645).

[0064] The retroviral vectors of the present invention can be produced by any method known in the art, including transient transfection, in stable cell lines, and / or with helper viruses.

[0065] formulation: In some embodiments, the polypeptides or polynucleotides of the present invention can be conjugated to at least one other molecule. Typically, the molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs. In some embodiments, the polypeptides or polynucleotides of the present invention are formulated using one or more lipid-based structures, including, but not limited to, liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). Liposomes are artificially prepared vesicles that can be composed primarily of a lipid bilayer and can be used as delivery vehicles for administering pharmaceutical formulations. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be 50-500 nm in diameter. Liposome design can include, but is not limited to, opsonins or ligands to improve liposome attachment to unhealthy tissue or to activate events such as endocytosis. Liposomes can also contain low or high pH to improve delivery of pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus, and devices described in U.S. Patent Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372.In some embodiments, the liposomes are formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (described in US20100324120), and are capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA). The polypeptides of the polynucleotides of the present invention can be encapsulated by liposomes and / or contained in an aqueous core that can then be encapsulated by liposomes (see International Publication Nos. WO2012031046, WO2012031043, WO2012030901, and WO2012006378, and U.S. Patent Publication Nos. US20130189351, US20130195969, and US20130202684).In some embodiments, the polynucleotides of the invention are formulated in stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; and U.S. Patent Publication No. US20130122104).

[0066] Typically, the active ingredients (i.e., polypeptides or polynucleotides) of the present invention are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices such as biodegradable polymers, to form pharmaceutical compositions. The terms "pharmaceutical" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.

[0067] The present invention is further illustrated by the following figures and examples, which, however, should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawings]

[0068] [Figure 1] Relationship between plasma AREG concentration on ICU arrival and fluid balance over 72 hours in an independent cohort of 77 patients with cardiogenic and post-cardiac arrest. *** p<0.001. [Figure 2A] Total weight / dry weight ratios of various organs 1 hour after ROSC in a mouse model of cardiac arrest. [Figure 2B] Serum amphiregulin concentration 1 hour after ROSC. *p<0.05. [Figure 3] Survival rates of areg-KO mice, wild-type mice, and wild-type mice injected with recombinant mouse AREG at the time of resuscitation (n>10 per group). [Figure 4] Time course of left ventricular ejection fraction (LVEF) after cardiac arrest in wild-type mice injected with recombinant mouse AREG during resuscitation and their littermate controls. T0=ROSC. [Figure 5] Intra-organ fluorescence ratios of fluorescently labeled dextran (155 kDa) and cadaverine in organs from wild-type mice injected with recombinant mouse AREG during resuscitation and their littermate controls. Organs were harvested after 4 minutes of low flow (during resuscitation) and 4 minutes after the intravenously injected dye had circulated. Ratios for the heart, lung, right kidney, liver, and brain were normalized to the mean control value for each organ and pooled as a total score ratio. n = 4 per group. *p < 0.05 between groups. FI, fluorescence intensity.

[0069] Working Example: In a prospective translational study, we performed RNA sequencing on CD14+ circulating monocytes from 11 patients (with similar confounding factors) with very severe cardiogenic shock (mean age 55 years, IQR 33-66). All patients were on veno-arterial extracorporeal membrane oxygenation (ECMO) and had a SAPS II score of 84 (53 / 107), pH 7.0 (6.9-7.2), and lactateemia of 11 (9-13) mmol / L at the time of ECMO implantation. RNA sequencing was compared between patients with severe capillary leak (arbitrarily defined as a fluid balance >75 ml / kg in the first 72 hours; n = 7) and those without (n = 4). We identified 860 differentially expressed genes, 38 of which were retained after false discovery rate correction. Of particular interest was amphiregulin (Areg), which was 56-fold more highly expressed in monocytes from patients with massive vascular leak. Circulating AREG levels at ICU admission were confirmed to correlate with fluid balance levels in an independent cohort of 77 patients with circulatory failure (cardiogenic and postresuscitation syndrome) (FIG. 1).

[0070] We developed a mouse model of resuscitated cardiac arrest (CA). Briefly, CA was induced by injecting KCl into the jugular vein. Mice were intubated, and after a period of no blood flow, cardiac arrest was restored by resuscitation and epinephrine infusion. We observed significant organ edema and a significant increase in circulating AREG expression (Figure 2). In a porcine model of post-CA dysfunction involving extracorporeal resuscitation, which required a dramatic increase in body fluids to maintain blood pressure (a situation comparable to that demonstrated by RNA sequencing in humans), we also report massively induced expression of circulating AREG.

[0071] Most importantly, we demonstrated the important role of this protein in Areg- / - mice, as i) fewer KO mice achieved return of spontaneous circulation (ROSC) compared with wild-type mice (10% vs. 80%, n > 10 per group, p < 0.05), and ii) intravenous infusion of 10 μg of recombinant AREG during resuscitation showed beneficial effects, such as a higher ROSC rate and a trend toward significantly better survival rates compared with wild-type mice (Figure 3). Echocardiographic studies demonstrated better myocardial function after cardiac arrest in mice infused with 10 μg of recombinant AREG during resuscitation (Figure 4). Extravasation of fluorescently labeled dextran (155 kDa) and cadaverine in organs of wild-type mice and their littermate controls infused with recombinant mouse AREG during resuscitation confirmed reduced vascular leakage of large molecules in recombinant AREG-infused mice (Figure 5). These results demonstrate that AREG administration in patients suffering from vascular permeability is beneficial and that the pharmacokinetic profile of the protein appears very promising (i.e., the half-life of the protein after intraperitoneal injection of 10 μg reaches 110 minutes).

[0072] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure.

Claims

1. A method for treating vascular hyperpermeability in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) an AREG polypeptide or ii) a polynucleotide encoding an AREG polypeptide.

2. 2. The method of claim 1, wherein the patient is suffering from a disease or condition associated with increased vascular permeability selected from the group consisting of focal edema, cardiovascular disease, myocardial infarction, peripheral vascular disease, ischemia, stroke, cancer, atherosclerosis, psoriasis, diabetes, autoimmune diseases such as rheumatoid arthritis, thrombocytopenia, altitude sickness, barotrauma, iatrogenic injury, bacterial infection, viral infection, and ocular conditions associated with vascular leakage such as non-proliferative and proliferative retinopathies (including diabetic retinopathy), macular edema (including diabetic macular edema), glaucoma, or macular degeneration (including age-related macular degeneration).

3. 10. The method of claim 1, wherein the patient is suffering from vascular leak syndrome.

4. 10. The method of claim 1, wherein the patient is suffering from systemic capillary leak syndrome.

5. The method of claim 1 , wherein the vascular hyperpermeability is secondary to sepsis.

6. The method of claim 1, wherein the patient is suffering from SIRS.

7. 10. The method of claim 1, wherein the patient is suffering from shock.

8. 9. The method of claim 8, wherein the shock is selected from the group consisting of circulatory shock, cardiogenic shock, ischemic shock, hypervolemic shock, hemorrhagic shock, and septic shock.

9. 10. The method of claim 1 for improving the chances of return of spontaneous circulation after cardiac arrest (ROSC).

10. 10. The method of claim 1 for treating cardiac arrest-induced vascular hyperpermeability.

11. 10. The method of claim 1 for preserving the integrity of the vascular endothelial cell barrier during the treatment of an ischemic condition.

12. A method for treating an ischemic condition in a patient in need thereof, comprising the steps of: i) restoring blood supply in the ischemic tissue; and ii) preserving the integrity of the vascular endothelial cell barrier in the ischemic tissue by administering to the patient a therapeutically effective amount of an AREG polypeptide or a polynucleotide encoding an AREG polypeptide, wherein steps i) and ii) are performed sequentially or simultaneously.

13. 13. The method of any one of claims 1 to 12, wherein the AREG polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 142 to the amino acid residue at position 182 of SEQ ID NO:

1.

14. 15. The method of claim 14, wherein the AREG polypeptide comprises an amino acid sequence ranging from amino acid residue 142 to amino acid residue 182 of SEQ ID NO: 1, and may differ from said amino acid sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, deletions and / or insertions.

15. The method of claim 14, wherein the AREG polypeptide comprises or consists of an amino acid sequence having at least 80% identity with the amino acid residue ranging from amino acid residue 101 (S) to amino acid residue 198 (K) of SEQ ID NO:

1.

16. 15. The method of claim 14, wherein the AREG polypeptide is fully or partially glycosylated.

17. The method of any one of claims 1 to 14, wherein the polynucleotide is messenger RNA (mRNA).

Images