EP3890830A2 - Procédés et compositions pour la prévention et/ou le traitement de l'ischémie et de la lésion d'ischémie-reperfusion - Google Patents

Procédés et compositions pour la prévention et/ou le traitement de l'ischémie et de la lésion d'ischémie-reperfusion

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Publication number
EP3890830A2
EP3890830A2 EP19816630.8A EP19816630A EP3890830A2 EP 3890830 A2 EP3890830 A2 EP 3890830A2 EP 19816630 A EP19816630 A EP 19816630A EP 3890830 A2 EP3890830 A2 EP 3890830A2
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EP
European Patent Office
Prior art keywords
apo
ischemia
glyc
glycosylated
reperfusion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19816630.8A
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German (de)
English (en)
Inventor
Lina Badimon Maestro
Judit CUBEDO RÀFOLS
Gemma VILAHUR GARCÍA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glycardial Diagnostics S L
Glycardial Diagnostics SL
Original Assignee
Glycardial Diagnostics S L
Glycardial Diagnostics SL
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Publication date
Application filed by Glycardial Diagnostics S L, Glycardial Diagnostics SL filed Critical Glycardial Diagnostics S L
Publication of EP3890830A2 publication Critical patent/EP3890830A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates, in general, to methods and compositions for the prevention and/or treatment of ischemia injury and of ischemia/reperfusion injury.
  • Mitochondria are the main source of ATP through oxidative phosphorylation via the electron transport chain. Because of the energy requirements of the heart the role of mitochondria is crucial, in fact they represent nearly one-third of its total mass. The correct maintenance of mitochondrial homeostasis is essential for cell survival because mitochondria are potent sources of free radicals and proapoptotic factors, but they can also reduce the detrimental effects of an excessive oxidative stress.
  • Myocardial ischemia affects the electron transport chain leading to an increase in cardiomyocyte death during reperfusion. Experimental approaches have demonstrated that chemical blockade of electron transport during ischemia inhibits the opening of the mitochondrial permeability transition pore (MPTP) decreasing cardiomyocyte injury during reperfusion.
  • MPTP mitochondrial permeability transition pore
  • IPost-Co Ischemic post-conditioning
  • RISK Reperfusion Injury Salvage Kinases
  • SAFE pro- survival Survivor Activating Factor Enhancement
  • the invention relates to glycosylated Apolipoprotein J (ApoJ- Glyc) for use in the prevention of ischemia injury.
  • the invention relates to glycosylated Apolipoprotein J (ApoJ- Glyc) or non-glycosylated Apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemia injury.
  • the invention relates to glycosylated Apolipoprotein J (ApoJ- Glyc) or non-glycosylated Apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemia-reperfusion injury, wherein the ApoJ-Glyc or the non-Glyc ApoJ is administered after the onset of the ischemia and before the reperfusion.
  • ApoJ- Glyc glycosylated Apolipoprotein J
  • non-Glyc ApoJ non-glycosylated Apolipoprotein J
  • FIG. 1 Scheme of the experimental design used in the pilot study, in the ischemia model and in the ischemia reperfusion model.
  • the schemes show a timeline of the different interventions in the animal model of myocardial infarction (MI).
  • MI myocardial infarction
  • AMI acute myocardial ischemia, i.p.: intraperitoneal.
  • Figure 3. Apo J total levels in serum (Commercial ELISA Kit). Apo J i.p. administration 5 minutes prior to ischemia induction is able to significantly increase Apo J circulating levels in the mouse model of MI.
  • Figure 6. Scheme of the experimental design used in the rat model of ischemia/ reperfusion. The scheme shows a timeline of the different interventions in the rat model of myocardial infarction (MI) 10 minutes after ischemia induction (45 minutes of ischemia followed by 24h of reperfusion). AMI: acute myocardial ischemia, i.v.: intravenous.
  • MI myocardial infarction
  • Figure 7 Effect of Apo J-Glyc and Non-Glyc Apo J on the infarct size in the rat model of acute MI by left anterior descending artery ligation.
  • Figure 8 Effect of Apo J-Glyc and Non-Glyc Apo J on functional parameters in the rat model of acute MI by left anterior descending artery ligation. Significant improvement of the left ventricular end systolic pressure (LVESP) after Apo J-Glyc and
  • Figure 9 Effect of Apo J-Glyc and Non-Glyc Apo J on functional parameters in the rat model of acute MI by left anterior descending artery ligation.
  • FIG. 10 Effect of Apo J-Glyc and Non-Glyc Apo J on necrosis biomarker in the rat model of acute MI by left anterior descending artery ligation.
  • FIG. 11 Scheme of the experimental design used in the rat model of ischemia reperfusion.
  • the scheme shows a timeline of the different interventions 10 minutes before ischemia induction in the rat model of myocardial infarction (MI; 45 minutes of ischemia followed by 24h of reperfusion).
  • Figure 12 Effect of Apo J-Glyc and Non-Glyc Apo J on the infarct size in the rat model of acute MI by left anterior descending artery ligation.
  • Figure 13 Effect of Apo J-Glyc and Non-Glyc Apo J on functional parameters in the rat model of acute MI by left anterior descending artery ligation.
  • LVESP left ventricular end systolic pressure
  • Figure 15 Effect of Apo J-Glyc and Non-Glyc Apo J on necrosis biomarker in the rat model of acute MI by left anterior descending artery ligation.
  • glycosylated Apo lipoprotein J is able to prevent ischemia injury in mice when administered previous to the onset of ischemia (fig. 2), that both glycosylated Apo J and non-glycosylated Apo J are able to reduce the infarcted area when administered after ischemia injury in mice (fig. 4) and that both glycosylated Apo J and non-glycosylated Apo J are able to reduce the infarcted area when administered just after ischemia injury and before reperfusion in mice (fig. 5).
  • the invention relates to glycosylated Apolipoprotein J (ApoJ- Glyc) for use in the prevention of ischemia injury.
  • “Apolipoprotein J” or“ApoJ”, as used herein, refers to a polypeptide also known as“clusterin’’,“testosterone-Repressed Prostate Message”,“complement- Associated Protein SP-40,40”, “complement cytolysis inhibitor”, complement Lysis Inhibitor”,“sulfated glycoprotein”,“Ku70-Binding Protein”,“NA1/NA2”,“TRPM-2”, “KUB1”,”CLI”.
  • Human Apo J is the polypeptide provided under accession number PI 0909 in the UniProtKB/Swiss-Prot database (Entry version 212 of 12 September 2018).
  • glycosylated generally refers to any protein having covalently attached oligosaccharide chains.
  • glycosylated ApoJ or "Apo J containing GlcNAc residues", as used herein, refers to any Apo J molecule containing at least one repeat of N- acetylglucosamine (GlcNAc) in at least one glycan chain, although typically, the Apo J will contain at least one N-acetylglucosamine in each glycan chain.
  • GlcNAc N- acetylglucosamine
  • the glycosylated ApoJ contains a N-glycan at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the ApoJ preproprotein sequence as defined in the NCBI database entry with accession number NP 001822.3 (release of Sep 23, 2018).
  • the glycosylated ApoJ contains N-glycans at every N-glycosylation site within ApoJ, i.e.
  • the glycosylated ApoJ contains a N- glycan in least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the ApoJ preproprotein sequence as defined in the
  • NCBI database entry with accession number NP_001822.3 release of Sep 23, 2018.
  • Amo J containing GlcNAc residues include Apo J molecules containing at least one GlcNAc residue in high-mannose N-glycans, complex-type N-glycans or hybrid oligosaccharides N-glycans. Depending on the type of N-glycans, the GlcNAc may be found directly attached to the polypeptide chain or in a distal position in the N-glycan.
  • GlcNAc or“N-acetyl glucosamine” refers to a derivative of glucose resulting from the amidation of glucosamine by acetic acid and having the general structure:
  • the Apo J-containing GlcNAc residues contains two residues of GlcNAc and is referred herein as (GlcNAc)2.
  • Apo J molecules containing (GlcNAc)2 residues includes molecules wherein the (GlcNAc)2 is found in high-mannose N-glycans, in complex-type N-glycans, in hybrid oligosaccharides N-glycans or in O-glycans.
  • the (GlcNAc)2 may be found directly attached to the polypeptide chain or in a distal position in the N-glycan.
  • the “Apo J-containing GlcNAc residues” is substantially free of other types of N-linked or O-linked carbohydrates.
  • the“Apo J containing GlcNAc residues” does not contain N-linked or O- linked a-mannose residues.
  • the“Apo J-containing GlcNAc residues” does not contain N-linked or O-linked a-glucose residues.
  • the“Apo J-containing GlcNAc residues” does not contain N-linked or O- linked a-mannose residues or N-linked or O-linked a-glucose residues.
  • ApoJ-Glyc is performed by a single type of glycosylated Apo J molecule, glycosylated at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the ApoJ preproprotein sequence as defined in the NCBI database entry with accession number NP 001822.3 (release of Sep 23, 2018).
  • ApoJ- Glyc is provided as a combination of glycosylated Apo J molecules wherein each molecule may be glycosylated at different sites among the Asn N-glycosylation sites at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the ApoJ preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • ApoJ-Glyc is a single type of glycosylated ApoJ, glycosylated at every N-glycosylation site within ApoJ, i.e. at each Asn at positions 86, 103, 145, 291, 317, 354 and 374 with respect to the ApoJ preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • ApoJ-Glyc contains GlcNAc residues. In another preferred embodiment, ApoJ-Glyc contains GlcNAc residues and sialic acid residues.
  • Apo J-containing GlcNAc and sialic acid residues refers to any Apo J molecule containing at least one repeat of N-acetylglucosamine in its glycan chain and at least one repeat of sialic acid residue.
  • sialic acid refers to the monosaccharide known as N- acetylneuraminic acid (Neu5Ac) and having the general structure
  • the Apo J contains two residues of GlcNAc and one sialic acid residue (hereinafter referred to (GlcNAc) 2 -Neu5Ac).
  • GlcNAc sialic acid residue
  • the GlcNAc and sialic acid residues are connected by virtue of one or more monosaccharides.
  • the“Apo J-containing GlcNAc residues and sialic acid residues” is substantially free of other types of N-linked or O-linked carbohydrates.
  • the“Apo J-containing GlcNAc and sialic acid residues” does not contain N-linked or O-linked a-mannose residues.
  • the“Apo J- containing GlcNAc and sialic acid residues” does not contain N-linked or O-linked a- glucose residues.
  • the“Apo J-containing GlcNAc residues and sialic acid residues” does not contain N-linked or O-linked a-mannose residues or N- linked or O-linked a- glucose residues.
  • ApoJ-Glyc for use according to the invention is purified from human plasma.
  • ApoJ-Glyc Purification of ApoJ-Glyc from human plasma can be carried out using conventional methods known in the art such as those described by de Silva et al (J.Biol.Chem., 1990, 265: 24: 14292-14297), Kelso et al. (Biochemistry, 1994, 33: 832- 839) or Calero et al, (Biochem. J., 1999, 344: 375-383).
  • ApoJ-Glyc can be purified from human plasma by immunoaffinity chromatography using an anti- ApoJ specific antibody.
  • ApoJ-Glyc can be further purified by HPLC, preferably reverse phase HPLC.
  • prevention relates to the capacity to prevent, minimize or hinder the onset or development of a disease or condition before its onset.
  • Ischemia as used herein, relates to a restriction in blood supply to tissues or organs causing a shortage of oxygen and glucose needed for cellular metabolism. Ischemia may be transient or permanent.
  • ischemia injury relates to the damage due to a shortage of oxygen and glucose needed for cellular metabolism.
  • ischemia injury is due to a condition selected from the group consisting of infarction, atherosclerosis, thrombosis, thromboembolism, lipid- embolism, bleeding, stent, surgery, angioplasty, end of bypass during surgery, organ transplantation, total ischemia, and combinations thereof.
  • infarct or“infarction” relates to a localized area of ischemic necrosis produced by anoxia following occlusion of the arterial supply or venous drainage of a tissue or organ. More particularly, a myocardial infarction (MI), commonly known as heart attack, is related to an event by which blood stops flowing properly to part of the heart, and the heart muscle is injured due to not receiving enough oxygen.
  • MI myocardial infarction
  • an infarct is the result of blockage of one of the coronary arteries due to an unstable buildup of white blood cells, cholesterol and fat.
  • Important risk factors are previous cardiovascular disease, old age, tobacco smoking, high blood levels of LDL cholesterol and triglycerides, low levels of HDL cholesterol, diabetes, high blood pressure, lack of physical activity, obesity, chronic kidney disease, excessive alcohol consumption, and the use of cocaine and amphetamines.
  • Methods to determine whether a subject has suffered from infarction are known in the art and include, without limitation, tracing electrical signals in the heart by an electrocardiogram (ECG), and testing a blood sample for substances associated with heart muscle damage, including creatine kinase (CK-MB) and troponin.
  • ECG electrocardiogram
  • ECG testing is used to differentiate between two types of myocardial infarctions based on the shape of the tracing.
  • An ST section of the tracing higher than the baseline is called an ST elevation MI (STEMI) which usually requires more aggressive treatment.
  • ST elevation MI ST elevation MI
  • Methods to determine infarct size are known by the skilled person and include measurement of serum markers including creatine kinase (CK)-MB levels in a serum sample (Grande P et al. 1982 Circulation 65: 756-764), tissue staining with triphenyl tetrazolium chloride (Fishbein MC et al. 1981 Am Heart J 101(5): 593-600), technetium (Tc)-99m sestamibi single photon emission computed tomography (SPECT) myocardial perfusion imaging, and magnetic resonance.
  • CK creatine kinase
  • SPECT technetium
  • “Atherosclerosis” relates to any hardening of arteries secondary to atheroma or accumulation in the artery walls that is made up of inflammatory cells (mostly macrophage cells) and cell debris, that contain lipids.
  • the artery wall thickens as a result of the accumulation of calcium and fatty materials such as cholesterol and triglyceride. The elasticity of the artery walls is reduced, impairing blood flow.
  • Thrombosis relates to the formation of a blood clot or thrombus inside a blood vessel, obstructing the flow of blood through the circulatory system.
  • Thromboembolism relates to the formation in a blood vessel of a clot
  • the clot may plug a vessel in the lungs (pulmonary embolism), brain (stroke), gastrointestinal tract, kidneys, or leg.
  • Lipid-embolism or“fat embolism” refers to the often asymptomatic presence of fat globules in the lung parenchyma and peripheral circulation after long bone or other major trauma.
  • Blooding relates to the process of losing blood or having blood flow, especially surgically.
  • internal bleeding occurs when there is damage to an artery or vein allowing blood to escape the circulatory system and collect inside the body.
  • the internal bleeding may occur within tissues, organs, or in cavities of the body.
  • “Stent” relates to a dispositive such as a tube inserted into a natural passage/conduit in the body to prevent or counteract a disease-induced localized flow constriction.
  • “surgery” or“surgical treatment” is meant any therapeutic procedure that involves methodical action of the hand or of the hand with an instrument, on the body of a human or other mammal, to produce a curative or remedial.
  • Angioplasty relates to a technique of mechanically widening narrowed or obstructed arteries, the latter typically being a result of atherosclerosis.
  • An empty and collapsed balloon on a guide wire, known as a balloon catheter is passed into the narrowed locations and then inflated to a fixed size using water pressures some 75 to 500 times normal blood pressure (6 to 20 atmospheres).
  • the balloon forces expansion of the inner white blood cell/clot plaque deposits and the surrounding muscular wall, opening up the blood vessel for improved flow, and the balloon is then deflated and withdrawn.
  • a stent may or may not be inserted at the time of ballooning to ensure the vessel remains open.
  • “Bypass surgery” relates to a class of surgeries involving rerouting a tubular body part and includes cardiopulmonary bypass, partial ileal bypass surgery, ileojunal bypass, gastric bypass and vascular bypass such as coronary artery bypass surgery.
  • Cardiopulmonary bypass (CBP) temporarily takes over the function of the heart and lungs during surgery, maintaining the circulation of blood and the oxygen content of the body.
  • Partial ileal bypass surgery is a surgical procedure which involves shortening the ileum to shorten the total small intestinal length.
  • the ileojejunal bypass is a surgery designed as a remedy for morbid obesity.
  • a vascular bypass is a surgical procedure performed for inadequate or loss of blood flow to a region of the body.
  • coronary artery bypass surgery also known as coronary artery bypass graft (CABG) surgery, is a surgical procedure performed to relieve angina and reduce the risk of death from coronary artery disease.
  • CABG coronary artery bypass graft
  • Transplantation is meant a surgical procedure by which a cell, tissue or organ is transferred from a donor subject to a recipient subject or from one part of the body to another in the same subject.
  • The“donor subject” is the subject who gives blood, cells, tissues, or an organ for another subject by blood transfusion or an organ transplant.
  • the donor subject is a human or another mammal.
  • The“recipient subject” is the subject who receives blood, cells, tissues, or an organ from another subject by blood transfusion or an organ transplant.
  • the recipient subject is a human or another mammal.
  • Transplanted tissues comprise, but are not limited to, bone tissue, tendons, corneal tissue, heart valves, veins and bone marrow.
  • Transplanted organs comprise, but are not limited to, heart, lung, liver, kidney, pancreas and intestine.
  • the particular surgical procedure of transplantation wherein the donor subject and the recipient subject are genetically non-identical members of the same species is known as allotransplantation.
  • allotransplant also known as allograft, allogeneic transplant or homograft
  • allotransplantable refers to organs or tissues that are relatively often or routinely transplanted. Examples of allotransplantable organs include heart, lung, liver, pancreas, kidney and intestine.
  • xenotransplantation The particular surgical procedure of transplantation wherein the donor subject and the recipient subject are members of different species is known as xenotransplantation.
  • xenotransplant also known as xenograft, xenogeneic transplant or heterograft
  • xenotransplant is related to the transplantation of cells, tissues or organs sourced from a donor to a recipient, wherein donor and recipient are members of different species.
  • Total ischemia relates to ischemia in which arterial and/or venous blood supply are occluded.
  • the ischemia injury to be prevented according to the invention may occur in any organ or a tissue from a subject.
  • Organs include, without limitation, brain, heart, kidneys, liver, large intestine, lungs, pancreas, small intestine, stomach, muscles, bladder, spleen, ovaries and testes.
  • the organ is selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof.
  • the organ is heart.
  • Tissues include, without limitation, nerve tissue, muscle tissue, skin tissue and bone tissue.
  • the ischemia injury is selected from the group comprising organ dysfunction (in the ischemic organ or in any other organ), infarct, inflammation (in the damaged organ or tissue), oxidative damage, mitochondrial membrane potential damage, apoptosis, reperfusion-related arrhythmia, cardiac stunning, cardiac lipotoxicity, ischemia-derived scar formation, and combinations thereof.
  • Organ dysfunction as used herein relates to a condition wherein a particular organ does not perform its expected function. An organ dysfunction develops into organ failure if the normal homeostasis cannot be maintained without external clinical intervention. Methods to determine organ dysfunction are known by the skilled person comprising, without limitation, monitorization and scores including sequential organ failure assessment (SOFA) score, multiple organ dysfunction (MOD) score and logistic organ dysfunction (LOD) score.
  • SOFA sequential organ failure assessment
  • MOD multiple organ dysfunction
  • LOD logistic organ dysfunction
  • inflammation relates to a set of changes occurring in a tissue that undergoes inflammation.
  • inflammation relates to the biological response to harmful stimuli, including pathogens, damaged cells or irritants.
  • Methods to determine inflammation include, without limitation, measure of erythrocyte sedimentation rate (ESR), wherein a higher ESR is indicative of inflammation, measure of C-reactive protein (CRP), wherein a higher level of CRP is indicative of inflammation, and leukocyte count (increased in inflammation).
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • Oxidative damage relates to the biomolecular damage that can be caused by direct attack of reactive species during oxygen restoration. Oxidative damage may involve lipid peroxidation, oxidative DNA damage and oxidative damage to proteins. Methods to determine lipid peroxidation include, without limitation, MDA (malondialdehyde)-TBA (thiobarbituric acid) determination by HPLC, and quantification of isoprostanes (which are specific end products of the peroxidation of polyunsaturated fatty acids) by mass spectrometry. Methods to determine DNA oxidative damage include, without limitation, measure of 8-hydroxy-2'-dcoxyguanosinc (80HdG).
  • Methods to determine oxidative damage to proteins include, without limitation, quantification of individual amino acid oxidation products including kynurenines (from tryptophan), bityrosine (which appears to be metabolically stable and can be detected in urine), valine and leucine hydroxides, L-dihydroxyphenylalanine (L-DOPA), ortho-tyrosine, 2-oxo- histidine, glutamate semialdehyde and adipic semialdehyde, as well as the carbonyl assay (involving measurement of protein carbonyl groups).
  • kynurenines from tryptophan
  • bityrosine which appears to be metabolically stable and can be detected in urine
  • valine and leucine hydroxides L-dihydroxyphenylalanine (L-DOPA)
  • ortho-tyrosine 2-oxo- histidine
  • glutamate semialdehyde and adipic semialdehyde as well as the carbonyl assay (involving measurement of protein carbonyl
  • The“mitochondrial membrane potential damage” relates to alterations in
  • mitochondrial membrane potential damage in the form of proton gradient across the mitochondrial inner membrane.
  • Methods for evaluation of mitochondrial membrane potential damage include the use of fluorescent probes for monitoring membrane potential including the JC1 dye (Cell Technology) and the measure of overall fluorescence at excitation and emission wavelengths allowing the quantification of green (485 nm and 535 nm) and red fluorescence (550 nm and 600 nm). Prolonged ischemia of any tissue or organ is known to induce mitochondrial membrane potential damage.
  • Apoptosis is related to a regulated network of biochemical events which lead to a selective form of cell suicide, and is characterized by readily observable morphological and biochemical phenomena, such as the fragmentation of the deoxyribonucleic acid (DNA), condensation of the chromatin, which may or may not be associated with endonuclease activity, chromosome migration, margination in cell nuclei, the formation of apoptotic bodies, mitochondrial swelling, widening of the mitochondrial cristae, opening of the mitochondrial permeability transition pores and/or dissipation of the mitochondrial proton gradient.
  • DNA deoxyribonucleic acid
  • condensation of the chromatin which may or may not be associated with endonuclease activity
  • chromosome migration chromosome migration
  • margination in cell nuclei the formation of apoptotic bodies
  • mitochondrial swelling widening of the mitochondrial cristae
  • Methods to determine cell apoptosis include, without limitation, assays that measure DNA fragmentation (including staining of chromosomal DNA after cell permeabilization), assays that measure the activation of caspases such as caspase 3 (including protease activity assays), assays that measure caspase cleavage products (including detection of PARP and cytokeratin 18 degradation), assays that examine chromatin chromatography (including chromosomal DNA staining), assays that measure DNA strand breaks (nicks) and DNA fragmentation (staggered DNA ends) (including active labeling of cell nick translation or ISNT and active labeling of cells by end labeling or TUNEL), assays that detect phosphatidylserine on the surface of apoptotic cells (including detection of translocated membrane component), assays that measure plasma membrane damage/leakage (including trypan blue exclusion assay and propidium iodide exclusion assay).
  • DNA fragmentation including staining of
  • Exemplary assays include analysis of scatter ' s parameters of apoptotic cells by flow cytometry, analysis of DNA content by flow cytometry (including DNA staining in a fluorochrome solution such as propidium iodide), fluorochrome labelling of DNA strand breaks by terminal deoxynucleotidyl transferase or TdT-assay, analysis of annexin-V binding by flow cytometry and TUNEL assay.
  • a fluorochrome solution such as propidium iodide
  • the ischemia injury may also involve a reperfusion-related arrhythmia.
  • “Arrhytmia” also known as cardiac dysrhythmia or irregular heartbeat, relates to a group of conditions in which the electrical activity of the heart is irregular, faster or slower, than normal.
  • the heartbeat may be too fast (tachycardia, over 100 beats per minute) or too slow (bradycardia, less than 60 beats per minute), and may be regular or irregular.
  • arrhytmia may cause cardiac arrest.
  • Arrhythmias can occur in the upper chambers of the heart (atria), or in the lower chambers of the heart (ventricles). Determination of arrhythmia is performed by the skilled in the art by means of electrocardiography (ECG).
  • ECG electrocardiography
  • “Cardiac stunning” involves different dysfunctional levels occurring after an episode of acute ischemia, despite blood flow is near normal or normal. After brief episodes of ischemia and reperfusion, prolonged mechanical dysfunction persists, although no histological signs of irreversible injury to cardiomyocytes exist, this phenomenon is called myocardial stunning. stunning involves different facets: in addition to post-ischemic ventricular dysfunction myocardial (myocardial stunning), there is evidence of vascular/microvascular/endothelial injury (vascular stunning), post-ischemic metabolic dysfunction (metabolic stunning), long-lasting impairment of neurotransmission (neural/neuronal stunning), and electrophysio logical alterations (electrical stunning).
  • Cardiac lipotoxicity involves a constellation of altered fatty acid metabolism, intramyo cardial lipid overload and contractile dysfunction. Although it is unclear how lipids induce cardiac dysfunction, accumulation of intramyocardial triglyceride is associated with altered gene expression. Specifically, there is increased expression of the peroxisome proliferator-activated receptor a (PPARa)-regulated genes. PPARa is a nuclear receptor that, when activated by long chain fatty acids, induces the expression of proteins that increase the uptake and oxidation of fatty acids. Cardiac-specific overexpression of PPARa induces cardiac dysfunction in mice exposed to high circulating fatty acid levels.
  • PPARa peroxisome proliferator-activated receptor a
  • TNF-a inflammatory cytokine tumor necrosis factor a
  • “Scar” relates to any mark left on a tissue following the healing of a wound or damage. Particularly, this term related to the marks left in ischemic tissue. In the context of the invention, scar formation is derived from ischemia.
  • An ischemia injury can be caused by a lot of causes, for example, by a natural event (e.g., restoration of blood flow following a myocardial infarction), a trauma, or by one or more surgical procedures or other therapeutic interventions that restore blood flow to a tissue or organ that has been subjected to a diminished supply of blood.
  • a natural event e.g., restoration of blood flow following a myocardial infarction
  • a trauma e.g., restoration of blood flow following a myocardial infarction
  • surgical procedures include, for example, coronary artery bypass, graft surgery, coronary angioplasty, organ transplant surgery and the like (e.g., cardiopulmonary bypass surgery).
  • the prevention of ischemia injury can be achieved by administering Apo J-Glyc prior to the onset of ischemia as well as by administering Apo J-Glyc after the onset of ischemia but before the damages caused by ischemia appear.
  • Apo J is administered previous to the onset of ischemia induction.
  • Apo J is administered at least 10 min,, 20 min,, 30 min,, 40 min., 50 min., lh, 2 h., 3 h., 4 h., 5 h., 6 h., 7 h., 8 h., 9 h. 10 h. 1 day, 2 days, 3 days, 4 days, 5 days or more before the onset of ischemia.
  • the damage caused by ischemia injury is due to cerebral ischemia.
  • the damage to the cerebral tissue is caused by ischemic stroke.
  • ischemic stroke refers to a sudden loss of brain function caused by a blockage or a blood vessel to the brain (resulting in the lack of oxygen to the brain), characterized by loss of muscular control, diminution or loss of sensation or consciousness, dizziness, slurred speech, or other symptoms that vary with the extent and the severity of the damage to the brain, also called cerebral accident, or cerebrovascular accident.
  • Cerebral ischemia (or “stroke”) also refers to a deficiency in blood supply to the brain, often resulting in a lack of oxygen to the brain.
  • the damage caused by ischemia is due to cardiac ischemia.
  • the cardiac ischemia is due to myocardial ischemia.
  • myocardial ischemia refers to circulatory disturbances caused by coronary atherosclerosis and/or inadequate oxygen supply to the myocardium.
  • an acute myocardial infarction represents an irreversible ischemic insult to myocardial tissue. This insult results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation and produces an environment in which the myocardial metabolic demands exceed the supply of oxygen to the myocardial tissue.
  • the damage is caused by a microvascular angina.
  • microvascular angina refers to a condition resulting from inadequate blood flow through the small cardiac blood vessels.
  • the cardiac ischemia is due to acute myocardial infarction.
  • Myocardial infarction (MI) or“acute myocardial infarction” as used herein is an ischemic necrosis of part of the myocardium due to the obstruction of one or several coronary arteries or their branches. Myocardial infarction is characterized by the loss of functional cardiomyocytes, the myocardial tissue being irreversibly damaged. The myocardium, or heart muscle, suffers an infarction when advanced coronary disease exists. In a particular case this occurs when an atheromatous plaque located inside a coronary artery ulcerates or ruptures, causing an acute obstruction of that vessel.
  • the ischemia injury is due to coronary obstruction (myocardial infarction) and further revascularization and blood re-flow.
  • Apo J-Glyc for use in the prevention of ischemia injury is administered prior to ischemia.
  • the time of administration before the onset of ischemia is nor particularly limiting.
  • the Apo J-Glyc is administered at least 1 second, at least 5, at least 10, at least 15, at least 30 or at least 45 seconds prior to ischemia, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes prior to ischemia, or even at least 1 hour, 2 hours or 3 hours prior to ischemia or even before.
  • Apo J-Glyc for use in the prevention of ischemia injury is administered at a therapeutically effective amount.
  • terapéuticaally effective amount in relation to the use of Apo J-Glyc in the prevention of ischemia injury, relates to the sufficient amount of Apo J-Glyc to achieve an appreciable prevention, cure, delay, reduction of severity or amelioration of one or more symptoms derived from a disease, and will generally be determined by, among other causes, the characteristics of the agent itself and the therapeutic effect to be achieved. It will also depend on the subject to be treated, the severity of the disease suffered by said subject, the chosen dosage form, etc. For this reason, the doses mentioned in this invention must be considered only as guides for the person skilled in the art, who must adjust the doses depending on the aforementioned variables.
  • the effective amount produces the amelioration of one or more symptoms of the disease that is being treated.
  • the dosage needed to provide an effective treatment which can be adjusted by one expert in the art, will vary depending on age, health, fitness, sex, diet, weight, degree of alteration of the receptor, frequency of treatment, nature and condition of the injury, nature and extent of impairment or illness, medical condition of the subject, route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profile of the particular compound used, if using a system drug delivery, and if the compound is administered as part of a combination of drugs.
  • the amount of the compound for use according to the invention that is therapeutically effective in the prevention of ischemia injury in a subject can be determined by conventional clinical techniques (see, for example, The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, NJ, 1995, and Drug Facts and Comparisons, Inc., St. Louis, MO, 1993).
  • Apo J-Glyc for use in the prevention of ischemia injury is administered at a dose range of between 0.1 mg/kg and 1 mg/kg.
  • Apo J-Glyc is administered at a dose range of between 0.1-0.2 mg/kg, 0.2- 0.3 mg/kg, 0.3-0.4 mg/kg, 0.4-0.5 mg/kg, 0.5-0.6 mg/kg, 0.6-0.7 mg/kg, 0.7-0.8 mg/kg, 0.8-0.9 mg/kg, 0.9-1 mg/kg.
  • Apo J-Glyc is administered at a dose of 0.5 mg/kg.
  • the administration routes for Apo J-Glyc include, among others, non-invasive pharmacological administration routes, such as the oral, gastroenteric, nasal, or sublingual route, and invasive administration routes, such as the parenteral route.
  • the Apo J-Glyc is administered in a therapeutically effective amount by means of a parenteral route (e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc.).
  • a parenteral route e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc.
  • intramuscular the compound is injected into the muscle tissue
  • intravenous the compound is injected into the vein
  • subcutaneous injected under the skin
  • intradermal injected between the layers of skin
  • the intrathecal route is used for administering into the central nervous system drugs which do not penetrate the blood-brain barrier well, such that the drug is administered into the space surrounding the spinal cord (intrathecal space).
  • the administration is an intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intrathecal administration.
  • the administration route is intravenous.
  • the Apo J-Glyc is administered as a single dose.
  • a "single dose" refers to a physically discrete unit a dose of Apo J-Glyc which administered in one dose/at one time/single route/single point of contact.
  • the single dose may be administered one or more times per day. In a particular embodiment, the single dose is administered once per day. In another embodiment, the single dose is administered twice per day. For example, a single dose may be split into two doses and each half of the split dose is administered to a subject at a different time during the day.
  • the Apo J-Glyc is administered at least 1 second prior to ischemia, typically at least 15, 30 or 45 seconds prior to ischemia, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes prior to ischemia, or even at least 1 hour, 2 hours or 3 hours prior to ischemia or even earlier.
  • Treatment of ischemia injury typically at least 15, 30 or 45 seconds prior to ischemia, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes prior to ischemia, or even at least 1 hour, 2 hours or 3 hours prior to ischemia or even earlier.
  • the invention relates to glycosylated Apolipoprotein J (Apo J- Glyc) or non-glycosylated Apolipoprotein J (Non-Glyc Apo J) for use in the treatment of ischemia injury.
  • glycosylated Apo J has been defined in respect of the first aspect of the invention and is equally used herein.
  • non-glycosylated Apo J refers to Apo J in which none of the N-glycosylation sites (amino acids at positions 86, 103, 145, 291, 317, 354 and 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822) are glycosylated.
  • treatment refers to both therapeutic measures and prophylactic or preventive measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as ischemia/reperfusion injury.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the Apo J-Glyc is glycosylated Apo J containing N- acetylglucosamine (GlcNAc) residues. In another preferred embodiment, the Apo J-Glyc is glycosylated Apo J containing N-acetylglucosamine (GlcNAc) residues and sialic acid residues.
  • Apo J-Glyc contains an N-glycan at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • the glycosylated Apo J contains N-glycans at every N- glycosylation site within Apo J, i.e.
  • the glycosylated Apo J contains a N-glycan in least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a single type of glycosylated Apo J molecule, glycosylated at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP 001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a combination of glycosylated Apo J molecules wherein each molecule may be glycosylated at different sites selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a single type of Apo J-Glyc, glycosylated at every N-glycosylation site within Apo J, i.e. at each Asn at positions 86, 103, 145, 291, 317, 354 and 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • ApoJ-Glyc for use according to the present aspect of the invention is purified from human plasma as described above.
  • ApoJ-non Glyc for use according to the present invention is a recombinant ApoJ which has been produced recombinantly in an organism that lacks the machinery required for N-type glycosylation.
  • ApoJ-non-Glyc is of recombinant origin obtained by expression in E.coli using procedures well known in the art such as those described in Dabbs and Wilson (Plos One, 9(1): e86989.
  • ApoJ-non- Glyc is expressed in E.coli under conditions resulting in the expression of the molecule in inclusion bodies, the inclusion bodies purified and the protein solubilized in the presence of chaotropic agents such as urea and/or disulphide group containing reagents such as DTT.
  • ischemia injury is due to a condition selected from the group consisting of infarction, atherosclerosis, thrombosis, thromboembolism, lipid- embolism, bleeding, stent, surgery, angioplasty, end of bypass during surgery, organ transplantation, total ischemia, and combinations thereof
  • ischemia injury is produced in an organ or a tissue selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof
  • the ischemia injury is selected from the group comprising organ dysfunction (in the ischemic organ or in any other organ), infarct, inflammation (in the damaged organ or tissue), oxidative damage, mitochondrial membrane potential damage, apoptosis, reperfusion-related arrhythmia, cardiac stunning, cardiac lipotoxicity, ischemia-derived scar formation, and combinations thereof
  • the damage caused by ischemia injury is due to cerebral ischemia.
  • the damage caused by ischemia is due to cardiac ischemia.
  • the cardiac ischemia is due to myocardial ischemia or acute myocardial ischemia.
  • Apo J-Glyc for use in the treatment of ischemia injury is administered during ischemia.
  • ApoJ-Glyc for use according to the invention is administered after the onset ischemia.
  • the Apo J-Glyc for use according to the invention is administered at a period of time which can vary broadly.
  • the Apo J-Glyc is administered at least 1 second, at least 5, at least 10, at least 15, at least 30 or at least 45 seconds after the onset of ischemia, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes after the onset ischemia, or even at least 1 hour, 2 hours or 3 hours after the onset of ischemia or even after.
  • Apo J-Glyc for use in the treatment of ischemia injury is administered at a therapeutically effective amount.
  • Apo J-Glyc and/or non-Glyc-Apo J for use in the treatment of ischemia injury is administered during ischemia.
  • Apo J-Glyc and/or non-Glyc-Apo J for use according to the invention is administered after the onset of ischemia.
  • the Apo J-Glyc and/or non-Glyc-Apo J for use according to the invention is administered at a period of time which can vary broadly. In a preferred embodiment, it is administered at least 1, at least 5, at least 10, at least 15, at least 30 or at least 45 seconds after ischemia. In another preferred embodiment at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes after ischemia, or even at least 1 hour, 2 hours or 3 hours after ischemia or even later.
  • Apo J-Glyc for use according to the invention is purified from human plasma.
  • non-Glyc-Apo J for use according to the invention is human recombinant.
  • Apo J-Glyc for use according to the invention is purified from human plasma and non-Glyc- Apo J is human recombinant.
  • Apo J-Glyc and/or non-Glyc-Apo J for use in the treatment of ischemia injury is administered at a dose range of between 0.1 mg/kg and 2 mg/kg.
  • Apo J-Glyc is administered at a dose range of between 0.1 -0.2 mg/kg, 0.2-0.3 mg/kg, 0.3-0.4 mg/kg, 0.4-0.5 mg/kg, 0.5-0.6 mg/kg, 0.6- 0.7 mg/kg, 0.7-0.8 mg/kg, 0.8-0.9 mg/kg, 0.9-1 mg/kg, 1-1.1 mg/kg, 1.1-1.2 mg/kg, 1,2- 1.3 mg/kg, 1.3- 1.4 mg/kg, 1.4- 1.5 mg/kg.
  • Apo J- Glyc is administered at a dose range of between 0.1-1.4 mg/kg, 0.2-1.3 mg/kg, 0.3-1.2 mg/kg, 0.4-1.1 mg/kg, 0.5-1 mg/kg, 0.6-0.9 mg/kg, 0.7-0.8 mg/kg. In a still more preferred embodiment, ApoJ-Glyc is administered at a dose of 0.5 mg/kg.
  • the administration route is intravenous.
  • the Apo J-Glyc or non-Glyc-Apo J is administered as a single dose.
  • a single dose refers to a physically discrete unit a dose of Apo J-Glyc or non-Glyc-Apo J which administered in one dose/at one time/single route/single point of contact.
  • the single dose may be administered one or more times per day. In a particular embodiment, the single dose is administered once per day. In another embodiment, the single dose is administered twice per day. For example, a single dose may be split into two doses and each half of the split dose is administered to a subject at a different time during the day.
  • the Apo J-Glyc is administered at least 1 second after ischemia, typically at least 15, 30 or 45 seconds after ischemia, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes after ischemia, or even at least 1 hour, 2 hours or 3 hours after ischemia or even later.
  • the invention relates to glycosylated Apo lipoprotein J (Apo J-Glyc) or non-glycosylated Apo lipoprotein J (non-Glyc-Apo J) for use in the treatment of ischemia-reperfusion injury, wherein the Apo J-Glyc or the non-Glyc-Apo J is administered after the onset of the ischemia and before the reperfusion.
  • the administration of glycosylated Apolipoprotein J (Apo J-Glyc) or of non-glycosylated Apo lipoprotein J (non-Glyc-Apo J) is additionally advantageous in that it also results in a reduction in the damage caused by the subsequent reperfusion. Thereby preventing the damage associated with the reperfusion.
  • the invention relates to glycosylated Apolipoprotein J (Apo J-Glyc) or non-glycosylated Apolipoprotein J (non-Glyc-Apo J) for use in the prevention of ischemia-reperfusion injury, wherein the Apo J-Glyc or the non-Glyc-Apo J is administered after the onset of the ischemia and before the reperfusion.
  • reperfusion relates to the restoration of blood flow to the ischemic tissue.
  • reperfusion itself can elicit a cascade of adverse reactions that paradoxically injure tissue.
  • ischemia/reperfusion injury also known as “ischemia/reperfusion damage” relates to organ or tissue damage caused when blood supply returns to the organ or tissue after a period of ischemia.
  • the absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.
  • Oxidative stress associated with reperfusion may cause damage to the affected tissues or organs.
  • Ischemia/reperfusion injury is characterized biochemically by a depletion of oxygen during an ischemic event followed by reoxygenation and the concomitant generation of reactive oxygen species during reperfusion.
  • the injury that occurs with ischemia/reperfusion is the result of the interaction between the substances that accumulate during ischemia and those that are delivered on reperfusion.
  • the cornerstone of these events is oxidative stress, defined as the imbalance between oxygen radicals and the endogenous scavenging system.
  • the result is cell injury and death, which is initially localized, but eventually becomes systemic if the inflammatory reaction is unchecked.
  • glycosylated Apolipoprotein J (Apo J-Glyc) and/or non- glycosylated Apolipoprotein J (non-Glyc-Apo J) is administered after the onset of ischemia and before reperfusion.
  • the Apo J-Glyc is glycosylated Apo J containing N- acetylglucosamine (GlcNAc) residues. In another preferred embodiment, the Apo J-Glyc is glycosylated Apo J containing N-acetylglucosamine (GlcNAc) residues and sialic acid residues.
  • Apo J-Glyc contains a N-glycan at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • the glycosylated Apo J contains N-glycans at every N- glycosylation site within Apo J, i.e.
  • the glycosylated Apo J contains a N-glycan in least 1 , at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the ApoJ preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a single type of glycosylated Apo J molecule, glycosylated at a single Asn residue selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP 001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a combination of glycosylated Apo J molecules wherein each molecule may be glycosylated at any possible site selected from the group consisting of the Asn residues at positions 86, 103, 145, 291, 317, 354 or 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc is a single type of glycosylated Apo J, glycosylated at every N-glycosylation site within Apo J, i.e. at each Asn at positions 86, 103, 145, 291, 317, 354 and 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • the non-glycosylated Apo J does not contain N-glycans at any of the Asn residues at positions 86, 103, 145, 291, 317, 354 and 374 with respect to the Apo J preproprotein sequence as defined in the NCBI database entry with accession number NP_001822.3 (release of Sep 23, 2018).
  • Apo J-Glyc for use according to the present aspect of the invention is purified from human plasma.
  • another preferred non-glycosylated Apo J for use according to the present aspect of the invention is human recombinant.
  • ischemia/reperfusion injury is due to a condition selected from the group consisting of infarction, atherosclerosis, thrombosis, thromboembolism, lipid-embolism, bleeding, stent, surgery, angioplasty, end of bypass during surgery, organ transplantation, total ischemia, and combinations thereof.
  • the ischemia/reperfusion injury is produced in an organ or a tissue selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof.
  • the ischemia/reperfusion injury is selected from the group comprising organ dysfunction, infarct, inflammation, oxidative damage, mitochondrial membrane potential damage, apoptosis, reperfusion-related arrhythmia, cardiac stunning, cardiac lipotoxicity, ischemia-derived scar formation, and combinations thereof
  • the damage caused by ischemia/reperfusion injury is due to cerebral ischemia.
  • the damage caused by ischemia/reperfusion is due to cardiac ischemia.
  • the cardiac ischemia is due to myocardial ischemia.
  • the cardiac ischemia is due to acute myocardial infarction.
  • the Apo J-Glyc or the non-Glyc-Apo J for use in the prevention of ischemia/reperfusion injury is administered after the onset ischemia and before the reperfusion.
  • Apo J-Glyc or of non-Glyc-Apo J for use in the prevention of ischemia/reperfusion injury is not particularly limited provided that it is administered prior to the onset of reperfusion.
  • Apo J-Glyc or of non-Glyc-Apo J is administered at least 1, at least 5, at least 10, at least 15, at least 30 or at least 45 seconds after ischemia.
  • glycosylated Apolipoprotein J (Apo J-Glyc) and/or non-glycosylated Apolipoprotein J (non-Glyc-Apo J) are administered at a therapeutically effective amount.
  • Apo J-Glyc for use in the prevention of ischemia injury is administered at a dose range of between 0.1 mg/kg and 1 mg/kg.
  • Apo J-Glyc is administered at a dose range of between 0.1-0.2 mg/kg, 0.2- 0.3 mg/kg, 0.3-0.4 mg/kg, 0.4-0.5 mg/kg, 0.5-0.6 mg/kg, 0.6-0.7 mg/kg, 0.7-0.8 mg/kg, 0.8-0.9 mg/kg, 0.9-1 mg/kg.
  • Apo J-Glyc is administered at a dose of 0.5 mg/kg.
  • Suitable administration routes are those which have been defined above in respect to the first and second aspects of the invention.
  • the administration route is intravenous.
  • Apo J-Glyc for use according to the invention is purified from human plasma.
  • non-Glyc-Apo J for use according to the invention is human recombinant.
  • Apo J-Glyc for use according to the invention is purified from human plasma and non-Glyc- Apo J is human recombinant.
  • the Apo J-Glyc or non-Glyc-Apo J is administered as a single dose.
  • a single dose refers to a physically discrete unit a dose of Apo J-Glyc or non-Glyc-Apo J which administered in one dose/at one time/single route/single point of contact.
  • the single dose may be administered one or more times per day. In a particular embodiment, the single dose is administered once per day. In another embodiment, the single dose is administered twice per day. For example, a single dose may be split into two doses and each half of the split dose is administered to a subject at a different time during the day.
  • the Apo J-Glyc or non-Glyc-Apo J is administered at least 1 second prior to reperfusion, typically at least 15, 30 or 45 seconds prior to reperfusion, preferably at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes prior to reperfusion, or even at least 1 hour, 2 hours or 3 hours prior to reperfusion or even earlier.
  • Mouse model Eleven weeks old C3H male mice were obtained from Janvier Labs (France). Animals were acclimated for a week and were fed in ad libitum diet. Appropriate rules and procedures for animal care were followed (Generalitat de Catalunya DOGC 2073, 1995) and approved by the ethical committee for animal testing.
  • Rat model Adult Sprague Dawley rats were used for intravenous administration of the protein (250 mg weight approximately). Animals were acclimated for 5 days according the standard procedures and were fed in ad libitum diet. Appropriate rules and procedures for animal care were followed after ethical committee for animal testing approving.
  • Left thoracotomy was performed on the fourth intercostal space, ribs were separated with a retractor for microsurgery. Pericardium was opened, left anterior descending artery (LAD) was identified and ligated with 7-0 silk suture.
  • LAD left anterior descending artery
  • mice were submitted to 45 minutes of occlusion.
  • thorax was closed provisionally with the skin of the animal and a small clamp hollowing 45 minutes of ischemia, blood extraction was performed using EDTA as anticoagulant.
  • animals were sacrificed, heart was extracted and immersed in PBS for a few minutes and liver was frozen at -80°C.
  • infarct size was calculated by the sum of myocardial infarct areas between sections and expressed as a percentage of total LV wall surface. Three measurements per section were determined.
  • Example 2 Treatment with Apo J-Glyc and Non-Glyc Apo J after ischemia induction reduces ischemic heart injury in an ischemia mouse model: Potential therapeutic role against ischemic damage.
  • ischemia model (fig. 1 , middle panel), Apo J-Glyc or Non-Glyc Apo J were administered 10 minutes after induction of ischemia. Following 45 minutes of ischemia, blood extraction was performed, animals were sacrificed, heart was extracted and the was frozen at -80°C. Treatment with either Apo J-Glyc or Non-Glyc Apo J (6 mg/kg, i.p.) significantly reduced the infarct size when administered at an early stage after ischemia (15% and 12% respectively vs. Placebo group) in the murine model of acute myocardial infarction by left anterior descending artery ligation (table 1 and figure 4).
  • Example 3 Treatment with Apo J-Glyc and Non-Glyc Apo J after ischemia induction reduces ischemic heart injury in an ischemia-reperfusion mouse model: Potential therapeutic role against ischemia-reperfusion damage.
  • LAD was reversed and blood flow was restored with 2h of reperfusion.
  • AAR Area-at-risk
  • IS infarcted area
  • ratio IS/AAR in an ischemia/reperfusion mouse model after i.p. administration of 6 mg/kg of Apo J-Glyc / Non-Glyc ApoJ 10 minutes after ischemia onset.
  • Example 4 Treatment with Apo J-Glyc and Non-Glyc Apo J after ischemia induction reduces ischemic heart injury in an ischemia-reperfusion rat model: Potential therapeutic role against ischemia-reperfusion damage.
  • Example 5 Treatment with Apo J-Glyc and Non-Glyc Apo J prior to myocardial infarction reduces myocardial injury in an ischemia-reperfusion rat model: Potential preventive role against ischemia-reperfusion damage.

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Abstract

L'invention concerne l'utilisation d'Apo J-Glyc dans la prévention d'une lésion ischémique, et à la fois de l'Apo J-Glyc et de l'Apo J non Glyc dans le traitement d'une lésion ischémique et dans le traitement d'une lésion d'ischémie-reperfusion.
EP19816630.8A 2018-12-05 2019-12-04 Procédés et compositions pour la prévention et/ou le traitement de l'ischémie et de la lésion d'ischémie-reperfusion Withdrawn EP3890830A2 (fr)

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