JP2022510403A - Methods and compositions for the prevention and / or treatment of ischemic injury and ischemic reperfusion injury - Google Patents

Abstract

The present invention relates to the use of ApoJ-Glyc in the prevention of ischemic injury and the use of both ApoJ-Glyc and non-Glyc ApoJ in the treatment of ischemic injury and ischemia-reperfusion injury.

Description

The invention generally relates to methods and compositions for the prevention and / or treatment of ischemic injury and ischemic reperfusion injury.

During ischemia, reduced oxygen utilization causes significant changes in the heart's energy metabolism. Ischemia causes mitochondrial damage, increased reactive oxygen species (ROS) production, and oxidative DNA damage. Rapid reperfusion is the best way to rescue ischemic heart, but this process activates a detrimental signaling cascade that can cause cardiomyocyte damage and ultimately increase infarct size. Is associated with cell damage caused by. Imbalances in oxygen supply and consumption during ischemia and reperfusion coordinately alter gene and protein expression, as well as the activity of various proteins, leading to changes in cardiomyocyte structure and function.

Mitochondria are the major source of ATP by oxidative phosphorylation via the electron transport chain. The role of mitochondria is very important because the heart requires energy, and in fact mitochondria make up almost one-third of the total mass of the heart. Proper maintenance of mitochondrial homeostasis is responsible for cell survival, as mitochondria are not only a powerful source of free radicals and pro-apoptotic factors, but can also mitigate the harmful effects of excessive oxidative stress. It is essential. Myocardial ischemia affects the electron transport chain and causes increased myocardial cell death during reperfusion. It has been experimentally shown that chemically blocking electron transfer during ischemia inhibits the opening of mitochondrial permeability transition pores (MPTPs) and reduces cardiomyocyte damage during reperfusion. Ischemia post-conditioning (IPost-Co) is the application of short-term myocardial ischemia / reperfusion several times during reperfusion after a long-term ischemic attack, activating the endogenous survival-promoting signaling cascade. It has been shown that it suppresses reperfusion injury and reduces infarct size. There are several studies supporting changes in specific protective pathways in IPost-Co, such as activation of reperfusion injury salvage kinase (RISK) or survivor activator enhancement (SAFE). Furthermore, it has been reported that downregulation of the aryl hydrocarbon receptor (AhR) signaling pathway is thought to contribute to the cardioprotective effect provided by IPost-Co. Clinical trials examining cardioprotection against ischemia / direct reperfusion injury (IdR) have yielded controversial results (Cung TT, N. Engl. J. Med. 2015; 373: 1021-31). ), The need for further research is emphasized in order to clarify the mechanism that has not yet been elucidated.

Effective treatments to reduce or prevent ischemic reperfusion injury have not been established. Despite the growing understanding of the pathophysiology of this process and the encouragement of preclinical trials of multiple drugs, most clinical trials to prevent reperfusion injury have been disappointing. Therefore, given the status quo, there is still a need to develop strategies to prevent damage caused by ischemic reperfusion.

In a first aspect, the invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) for use in the prevention of ischemic injury.

In a second aspect, the invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemic injury.

In a third aspect, the invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemic reperfusion injury. Alternatively, non-Glyc ApoJ is characterized by being administered after the onset of ischemia and before reperfusion.

It is a schematic diagram of the experimental design used for the pilot study, the ischemia model, and the ischemia reperfusion model. These schematics show the various interventions in the animal model of myocardial infarction (MI) in chronological order. AMI: Acute myocardial ischemia, i. p. : In the abdominal cavity. It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the infarct size in the mouse model of acute MI by ligation of the left anterior descending artery. Administered 5 minutes before MI (vehicle n = 3, ApoJ-Glyc n = 8, non-Glyc ApoJ n = 8). It is a figure which shows the total ApoJ level (commercially available ELISA kit) in serum. In a mouse model of MI, intraperitoneal administration of ApoJ 5 minutes prior to ischemia induction can significantly increase ApoJ circulation levels. It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ administration on the infarct size in the mouse model of myocardial ischemia for 45 minutes by ligation of the left anterior descending artery. Administered 10 minutes after MI induction (vehicle, n = 12; ApoJ-Glyc, n = 12; non-Glyc ApoJ, n = 11). Results are expressed as mean ± SEM. It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ administration on the infarct size in the ischemia-reperfusion mouse model. Administered 10 minutes after MI induction (vehicle, n = 12; ApoJ-Glyc, n = 12; non-Glyc ApoJ, n = 12). Animals were subjected to ischemia for 45 minutes and reperfusion for 2 hours. Results are expressed as mean ± SEM. It is a schematic diagram of the experimental design used for the rat model of ischemia-reperfusion. This schematic shows the various interventions in the rat model of myocardial infarction (MI) 10 minutes after ischemia induction in chronological order (24 hours reperfusion after 45 minutes of ischemia). AMI: Acute myocardial ischemia, i. v. : Intravenous. It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the infarct size in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, 10 minutes after induction of ischemia, ApoJ-Glyc and non-Glyc ApoJ were administered i. v. Upon administration, the infarct size (ratio of infarct size (IS) to risk region (AAR)) was significantly reduced (45-minute ischemia / 24-hour reperfusion; vehicle n = 13, ApoJ-Glyc n = 11, non-Glyc. ApoJ n = 11). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the functional parameter in the rat model of acute MI by ligation of the left anterior descending artery. Intravenous administration of ApoJ-Glyc and non-Glyc ApoJ 10 minutes after induction of ischemia in a rat model of MI significantly improved left ventricular end-systolic pressure (LVESP) (45-minute ischemia, 24-hour reperfusion). Vehicle n = 13, ApoJ-Glyc n = 11, non-Glyc ApoJ n = 11). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the functional parameter in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, 10 minutes after induction of ischemia, ApoJ-Glyc and non-Glyc ApoJ were administered i. v. Administration significantly improved left ventricular relaxation (45-minute ischemia / 24-hour reperfusion; vehicle n = 13, ApoJ-Glyc n = 11, non-Glyc ApoJ n = 11). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the necrotic biomarker in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, plasma levels of myocardial troponin I were significantly reduced in rats treated with ApoJ-Glyc 10 minutes after ischemia induction (45-minute ischemia / 24-hour reperfusion; vehicle n = 13, ApoJ). -Glyc n = 11, non-Glyc ApoJ n = 11). This effect was not observed when non-Glyc ApoJ was administered. It is a schematic diagram of the experimental design used for the rat model of ischemia-reperfusion. This schematic shows the various interventions 10 minutes prior to ischemia induction in a rat model of myocardial infarction (MI; 45 minutes ischemia followed by 24 hours reperfusion) in chronological order. It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the infarct size in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, ApoJ-Glyc and non-Glyc ApoJ were i.y. 10 minutes prior to 45-minute ischemia and 24-hour reperfusion. v. Upon administration, the infarct size (ratio of infarct size (IS) to risk region (AAR)) was significantly reduced (vehicle n = 13, ApoJ-Glyc n = 13, non-Glyc ApoJ n = 13). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the functional parameter in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, ApoJ-Glyc and non-Glyc ApoJ were i.y. 10 minutes prior to 45-minute ischemia and 24-hour reperfusion. v. Administration significantly improved left ventricular end-systolic pressure (LVESP) (vehicle n = 13, ApoJ-Glyc n = 13, non-Glyc ApoJ n = 13). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the functional parameter in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, ApoJ-Glyc and non-Glyc ApoJ were i.y. 10 minutes prior to 45-minute ischemia and 24-hour reperfusion. v. Administration significantly improved left ventricular relaxation (vehicle n = 13, ApoJ-Glyc n = 13, non-Glyc ApoJ n = 13). It is a figure which shows the effect of ApoJ-Glyc and non-Glyc ApoJ on the necrotic biomarker in the rat model of acute MI by ligation of the left anterior descending artery. In a rat model of MI, significant changes in plasma levels of myocardial troponin I were observed in rats treated with ApoJ-Glyc and non-Glyc ApoJ 10 minutes prior to 45-minute ischemia and 24-hour reperfusion. No (vehicle n = 13, ApoJ-Glyc n = 13, non-Glyc ApoJ n = 13).

We can prevent ischemic injury when glycosylated apolipoprotein J is administered in mice prior to the onset of ischemia (FIG. 2), and both glycosylated ApoJ and non-glycosylated ApoJ in mice. The infarct area can be reduced when administered after ischemic injury (Fig. 4), and infarct when both glycosylated ApoJ and non-glycosylated ApoJ are administered immediately after ischemic injury and prior to reperfusion in mice. It was admitted that the area could be reduced (Fig. 5).

Prevention of ischemic injury In the first aspect, the present invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) for use in the prevention of ischemic injury.

The terms " apolipoprotein J" or "ApoJ ", as used herein, "crusterin", "testosterone-Repressed Prostate Message", "complement-related protein SP-40," 40 ”,“ Complement cell lysis inhibitor ”,“ Complement lysis inhibitor ”,“ Sulfated glycoprotein ”,“ Ku70 binding protein ”,“ NA1 / NA2 ”,“ TRPM-2 ”,“ KUB1 ”,“ A polypeptide also known as "CLI". Human ApoJ is a polypeptide provided in the UniProtKB / Swiss-Prot database with accession number P10909 (entry version 212, September 12, 2018).

The term "glycosylation" generally refers to any protein that has a covalently linked oligosaccharide chain.

The term "glycosylated ApoJ" or "ApoJ containing a GlcNAc residue", as used herein, comprises a repetition of at least one N-acetylglucosamine (GlcNAc) in at least one glycan chain. Any ApoJ molecule that contains at least one N-acetylglucosamine in each glycan chain. In one embodiment, glycosylated ApoJ is at position 86, 103, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). N-glycans are contained in a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another embodiment, glycosylated ApoJ is registered at all N-glycosylation sites within ApoJ, i.e., registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the defined ApoJ preproprotein sequence, each Asn at positions 86, 103, 145, 291 and 317, 354 and 374 contains an N-glycan. In another embodiment, glycosylated ApoJ is at positions 86 and 103 with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). At least one, at least two, at least three, at least four, at least five, at least six, selected from the group consisting of Asn residues 145, 291 317, 354 or 374. Alternatively, at least 7 Asn residues contain N-glycans.

Examples of the "ApoJ containing a GlcNAc residue" include ApoJ molecules containing at least one GlcNAc residue in a high mannose N-glycan, a complex N-glycan, or a hybrid oligosaccharide N-glycan. Depending on the type of N-glycan, GlcNAc may be directly attached to the polypeptide chain or may be present at the end of the N-glycan.

The term "GlcNAc" or "N-acetylglucosamine" refers to a derivative of glucose obtained by amidating glucosamine with acetic acid and having the following general structure:

In one embodiment, the ApoJ-containing GlcNAc residue contains two residues of GlcNAc and is referred to herein as (GlcNAc) ₂ . As ApoJ molecules containing (GlcNAc) ₂ residues, molecules in which (GlcNAc) ₂ is present in high mannose N-glycans, complex N-glycans, hybrid oligosaccharides N-glycans, or O-glycans are included. Can be mentioned. Depending on the type of N-glycan, (GlcNAc) ₂ may be directly attached to the polypeptide chain or may be present at the end of the N-glycan.

In a preferred embodiment, the "ApoJ-containing GlcNAc residue" is substantially free of other types of N-linked or O-linked carbohydrates. In one embodiment, "ApoJ containing a GlcNAc residue" does not contain an N-linked or O-linked α-mannose residue. In another embodiment, the "ApoJ-containing GlcNAc residue" does not contain an N-linked or O-linked α-glucose residue. In yet another embodiment, the "ApoJ-containing GlcNAc residue" comprises an N-linked or O-linked α-mannose residue or an N-linked or O-linked α-glucose residue. do not do.

In a preferred embodiment, ApoJ-Glyc is the 86th, 103rd, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). It is performed by a single glycosylated ApoJ molecule glycosylated with a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another preferred embodiment, ApoJ-Glyc is provided as a combination of glycosylated ApoJ molecules, each molecule registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the ApoJ preproprotein sequence defined as, it may be glycosylated at a different site of the N-glycosylation site of Asn at positions 86, 103, 145, 291 and 317, 354 or 374. .. In another preferred embodiment, ApoJ-Glyc is registered at all N-glycosylation sites within ApoJ, i.e., registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the defined ApoJ preproprotein sequence, it is a single type of glycosylated ApoJ glycosylated at each Asn at positions 86, 103, 145, 291 and 317, 354 and 374.

In a preferred embodiment, ApoJ-Glyc contains a GlcNAc residue. In another preferred embodiment, ApoJ-Glyc contains GlcNAc residues and sialic acid residues.

The term "ApoJ-containing GlcNAc residue and sialic acid residue", as used herein, refers to the repetition of at least one N-acetylglucosamine in the glycan chain of the ApoJ molecule and at least one sialic acid residue. Refers to any ApoJ molecule containing the repetition of.

The term "sialic acid" as used herein refers to a monosaccharide known as N-acetylneuraminic acid (Neu5Ac), which has the following general structure:

In one embodiment, ApoJ contains two GlcNAc residues and one sialic acid residue (hereinafter referred to as (GlcNAc) ₂ -Neu5Ac). In another embodiment, the GlcNAc residue and the sialic acid residue are linked by one or more monosaccharides.

In a preferred embodiment, "ApoJ-containing GlcNAc residues and sialic acid residues" are substantially free of other types of N-linked or O-linked carbohydrates. In one embodiment, "ApoJ-containing GlcNAc residues and sialic acid residues" do not contain N-linked or O-linked α-mannose residues. In another embodiment, "ApoJ-containing GlcNAc residues and sialic acid residues" do not contain N-linked or O-linked α-glucose residues. In yet another embodiment, the "ApoJ-containing GlcNAc residue and sialic acid residue" is an N-linked or O-linked α-mannose residue, or an N-linked or O-linked α-. Contains no glucose residues.

In a preferred embodiment, ApoJ-Glyc for use according to the invention is purified from human plasma.

Purification of ApoJ-Glyc from human plasma is performed 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. ( It can be performed using conventional methods known in the art, such as those described by Biochem. J., 1999, 344: 375-383). In one embodiment, ApoJ-Glyc can be purified from human plasma by immunoaffinity chromatography with anti-ApoJ-specific antibodies. In a more preferred embodiment, ApoJ-Glyc can be further purified by HPLC, preferably reverse phase HPLC.

As used herein, the term " prevention " refers to the ability to prevent, minimize, or prevent the onset or morbidity of a disease or condition before it develops.

As used herein, " ischemia " refers to the lack of oxygen and glucose required for cell metabolism due to restricted blood supply to tissues or organs. Ischemia may be transient or permanent.

As used herein, the term " ischemic injury " refers to injury due to a deficiency of oxygen and glucose required for cell metabolism.

In a preferred embodiment, the ischemic injury is infarction, atherosclerosis, thrombosis, thromboembolism, lipid embolism, bleeding, stent, surgery, angioplasty, termination of intraoperative bypass, organ transplantation, complete ischemia. , And a condition selected from the group consisting of combinations thereof.

" Infarction " or "infarction" refers to the area of local ischemic necrosis caused by anoxic conditions due to obstruction of arterial supply or venous drainage of tissue or organ. More specifically, myocardial infarction (MI), commonly known as a heart attack, relates to an event in which blood does not flow normally to a part of the heart and the heart muscle is damaged due to insufficient oxygen supply. .. Infarction usually results from the obstruction of one of the coronary arteries due to the unstable accumulation of white blood cells, cholesterol and fat. Important risk factors are history of cardiovascular disease, old age, smoking, high blood LDL cholesterol and triglyceride levels, low HDL cholesterol levels, diabetes, hypertension, lack of exercise, obesity, chronic kidney disease, excessive alcohol intake. , And the use of cocaine and amphetamine. Methods for determining whether a subject has an infarct are known in the art for tracking electrical signals of the heart by electrocardiogram (ECG) and for myocardial damage such as creatine kinase (CK-MB) and troponin. Blood sample tests for related substances include, but are not limited to. The ECG test is used to distinguish between the two types of myocardial infarction based on the shape of the trace. The ST segment above the baseline of the trace is called ST-segment elevation MI (STEMI) and usually requires more aggressive treatment. Methods for quantifying infarct size are known to those of skill in the art, measuring serum marker levels such as creatine kinase (CK) -MB levels in serum samples (Grande P et al., 1982 Circulation 65: 756-764), triphenyl. Tissue staining with tetrazolium chloride (Fishbein MC et al., 1981 Am Heart J 101 (5): 593-600), technetium (Tc) -99m technetium-99m technetium single photon emission computed tomography (SPECT) myocardial perfusion imaging, and magnetic resonance. Can be mentioned.

" Atherosclerosis " refers to any secondary arteriosclerosis caused by atherosclerosis or accumulations in the arterial wall consisting of inflammatory cells (mainly macrophage cells) and cell debris containing lipids. .. The accumulation of calcium and fats such as cholesterol and triglycerides results in thickening of the arterial wall. The elasticity of the arterial wall is reduced and blood flow is obstructed.

" Thrombosis " refers to the formation of blood clots or thrombi in blood vessels that impede blood flow through the circulatory system.

"Thrombosis" refers to the formation of a blood clot ( thrombus ) in a blood vessel, which peels off and is carried into the bloodstream, blocking another blood vessel. Blood clots can block blood vessels in the lungs (pulmonary embolism), brain (stroke), gastrointestinal tract, kidneys, or legs.

"Fat embolism " or " fat embolism " refers to the often asymptomatic presence of fat globules in the lung parenchyma and peripheral circulation after severe trauma such as long canal bone.

" Bleeding " refers to the process of blood loss or blood flow, especially surgically. In particular, internal bleeding occurs when an artery or vein is damaged and blood leaks from the circulatory system and collects in the body. Internal bleeding may occur in tissues, organs, or body cavities.

A " stent " is one relating to a disease, such as a tube inserted into a natural tube / conduit in the body, to prevent or prevent disease-induced local channel narrowing.

" Surgery " or " surgical treatment " means any therapeutic procedure involving the methodical action of the hand or instrument for the purpose of healing or treatment of the human or other mammalian body. ..

" Angioplasty " relates to a technique for mechanically dilating a stenotic or occluded artery, which is typically the result of atherosclerosis. An empty crushed balloon attached to a guide wire, known as a balloon catheter, is inserted into the stenosis site and expanded to a certain size at a water pressure (6 to 20 atmospheres) about 75 to 500 times the normal blood pressure. The balloon forces the leukocyte / blood clot plaque deposits in the artery and the surrounding muscle wall to dilate, open the blood vessels to improve blood flow, and then contract and withdraw the balloon. Stents may or may not be inserted during ballooning to ensure that the vessels remain open.

" Bypass surgery " refers to a type of surgery that bypasses a portion of a tubular body, including vascular bypass grafting such as cardiopulmonary bypass, partial ileal bypass grafting, ileojunal bypass grafting, gastric bypass grafting, and coronary artery bypass grafting. Can be mentioned. Cardiopulmonary bypass (CBP) temporarily substitutes for the function of the heart and lungs during surgery to maintain blood circulation and oxygen content in the body. Partial ileal bypass surgery is a surgical procedure that shortens the ileum and shortens the overall length of the small intestine. Ileal jejunal bypass surgery is a surgery designed as a treatment for pathological obesity. Vascular bypass is a surgical procedure performed when blood flow to a part of the body is inadequate or lost. In particular, coronary artery bypass grafting, also known as coronary artery bypass grafting (CABG), is a surgical procedure performed to reduce angina and reduce the risk of death from coronary artery disease.

" Transplantation " means a surgical procedure that transfers cells, tissues or organs from a donor subject to a recipient subject, or from one part of the body of the same subject to another. A "donor subject" is a subject that provides blood, cells, tissues, or organs to another subject by blood transfusion or organ transplantation. The donor target is a human or other mammal. A "recipient subject" is a subject who receives blood, cells, tissues, or organs from another subject by blood transfusion or organ transplantation. The recipient target is a human or other mammal. Transplanted tissue includes, but is not limited to, bone tissue, tendons, corneal tissue, heart valves, veins and bone marrow. Transplanted organs include, but are not limited to, the heart, lungs, liver, kidneys, pancreas and intestines. A particular surgical procedure for transplantation in which the donor and recipient subjects are genetically non-identical members of the same species is known as allogeneic transplantation. Thus, the term allogeneic transplantation (also known as allograft, allogeneic transplantation, or homograft) relates to the transplantation of cells, tissues, or organs provided by non-genetically identical members of the same species as the recipient. The term "allograftable" refers to an organ or tissue that is transplanted relatively frequently or on a daily basis. Examples of allograftable organs include the heart, lungs, liver, pancreas, kidneys and intestines. A particular surgical procedure for transplantation in which the donor and recipient subjects are members of different species is known as xenograft. Therefore, the term xenograft (also known as xenograft, xenograft, or xenograft) is a cell, tissue, or organ donated by a donor to a recipient when the donor and recipient are members of different species. It is about the transplantation of.

" Complete ischemia " refers to ischemia when the blood supply in the arteries and / or veins is obstructed.

The ischemic injury prevented by the present invention can occur in any organ or tissue of interest. Organs include, but are not limited to, the brain, heart, kidneys, liver, large intestine, lungs, pancreas, small intestine, stomach, muscles, bladder, spleen, ovaries and testis. In a preferred embodiment, the organ is selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof. In a more preferred embodiment, the organ is the heart. Tissues include, but are not limited to, nerve tissue, muscle tissue, skin tissue and bone tissue.

In a preferred embodiment, the ischemic injury is an organ dysfunction (in an ischemic organ or any other organ), infarction, inflammation (in a damaged organ or tissue), oxidative damage, mitochondrial membrane potential damage, apoptosis, re-injury. It is selected from the group comprising perfusion-related arrhythmias, cardiac stunning, cardiac lipotoxicity, ischemic scar formation, and combinations thereof.

" Organ dysfunction " as used herein refers to a condition in which a particular organ does not perform its expected function. Organ dysfunction progresses to organ failure if normal homeostasis cannot be maintained without external clinical intervention. Methods of determining organ dysfunction are known to those of skill in the art and include monitoring and sequential organ dysfunction assessment (SOFA) scores, multiple organ dysfunction (MOD) scores and logistic organ dysfunction (LOD). ) Scores, etc., but are not limited to these.

" Infarction " is as already defined.

" Inflammation " or " inflammatory response " refers to a series of changes that occur in inflamed tissue. Inflammation, in particular, relates to a biological response to harmful stimuli such as pathogens, damaged cells or irritants. Methods for determining inflammation are known in the art and include measurement of erythrocyte sedimentation rate (ESR) (higher ESR indicates inflammation) and measurement of C-reactive protein (CRP) (higher CRP level indicates inflammation). ), And white blood cell count (which increases with inflammation), but is not limited to these.

" Oxidative damage " refers to damage to biomolecules that can be caused by the direct attack of active species during oxygen recovery. Oxidative damage can include lipid peroxidation, oxidative DNA damage and protein oxidative damage. Methods for measuring lipid peroxidation include MDA (malondialdehyde) -TBA (thiobarbituric acid) measurement by HPLC and isoprostane (polyunsaturated fatty acid peroxidation specific end product) by mass analysis. ), But is not limited to these. Methods for measuring DNA oxidative damage include, but are not limited to, measurement of 8-hydroxy-2'-deoxyguanosine (8OHdG). Methods for measuring oxidative damage to proteins include quinurenin (derived from tryptophan), dityrosine (which is considered metabolically stable and can be detected in urine), valine and leucine hydroxides, and L-dihydroxyphenylalanine (. L-DOPA), quantification of oxidation products of individual amino acids such as orthotyrosine, 2-oxohistidine, glutamate semialdehyde and adipate semialdehyde, as well as carbonyl assays (including measurement of the carbonyl group of proteins). , Not limited to these.

" Mitochondrial membrane potential (Δψm) damage " relates to changes in membrane potential in the form of a proton gradient across the inner mitochondrial membrane. Methods for assessing mitochondrial membrane potential damage are known to those of skill in the art and the use of fluorescent probes to monitor membrane potential such as JC1 dye (Cell Technology) and green fluorescence (485 nm and 535 nm) and red fluorescence (550 nm). And 600 nm) quantification includes measurement of total fluorescence at excitation and emission wavelengths. Prolonged ischemia of any tissue or organ is known to induce mitochondrial membrane potential damage.

" Apoptosis " relates to a controlled network of biochemical events that cause selective cell suicide, such as fragmentation of deoxyribonucleic acid (DNA), condensation of chromatin (endonuclease activity). (Maybe unrelated), chromosomal migration, marginal orientation of cell nuclei, formation of apoptotic bodies, mitochondrial swelling, dilation of mitochondrial cryste, opening of mitochondrial permeable transition holes, and / or disappearance of mitochondrial proton gradient. It is characterized by easily observable morphological and biochemical phenomena. Methods for determining cell apoptosis are known to those skilled in the art, such as an assay for measuring DNA fragmentation (such as staining of chromosomal DNA after cell permeation treatment) and an assay for measuring activation of caspase such as caspase 3. Assays for measuring caspase cleavage products (such as detection of PARP and cytokeratin 18 degradation), assays using chromatin chromatography (such as chromosomal DNA staining), DNA strand breaks (nicks) and DNA fragmentation (twisted type) Assays for measuring (the DNA ends of cells) (such as cell activation labeling with nick translation or ISNT, and terminal labeling or cell activation labeling with TUNEL), assays for detecting phosphatidylserine on the surface of apoptotic cells (detection of transposition membrane components, etc.) ), Assays for measuring cell membrane damage / leakage, such as, but not limited to, tripanblue exclusion assay and propidium iodide exclusion assay. Exemplary assays include analysis of the scattering parameters of apoptotic cells by flow cytometry, analysis of DNA content by flow cytometry (such as DNA staining in a fluorescent dye solution such as propidium iodide), terminal deoxynucleotidyl transferase. Fluorescent dye labeling or TdT assay for DNA strand breaks by flow cytometry, analysis of annexin V binding by flow cytometry, and TUNEL assay.

Ischemia injury may also mean reperfusion-related arrhythmias. " Arrhythmia ", also known as cardiac rhythm disorders or irregular heartbeats, relates to a group of states in which the electrical activity of the heart is more irregular, faster, or slower than normal. Arrhythmic heartbeats can be too fast (tachycardia, over 100 beats per minute), too slow (bradycardia, less than 60 beats per minute), regular or irregular. There is also. In some situations, arrhythmias can cause cardiac arrest. Arrhythmias can occur in the upper chamber (atria) of the heart or in the lower chamber (ventricular) of the heart. The determination of arrhythmia is performed by a person skilled in the art using an electrocardiogram (ECG).

"Cardiac stunning " means various levels of dysfunction that occur after an acute ischemic attack, despite near-normal or normal blood flow. After a brief ischemic attack and reperfusion, mechanical dysfunction may continue for a long time, even though the cardiomyocytes have no histological signs of irreversible damage, a phenomenon called myocardial stunning. .. Stunning includes post-ischemic ventricular dysfunction myocardium (myocardial stunning), blood vessel / microvessel / endothelial injury (vascular stunning), post-ischemic metabolic dysfunction (metabolic stunning), and long-term There are various aspects, including signs of impaired neurotransmission (nerve / nerve cell stunning) and electrophysiological changes (electrical stunning).

" Cardiac lipotoxicity " includes changes in fatty acid metabolism, intramyocardial lipid overload, and contractile dysfunction. Although it is unclear how lipids cause cardiac dysfunction, the accumulation of intramyocardial triglycerides is associated with altered gene expression. Specifically, the expression of the peroxisome proliferator-activated receptor α (PPARα) regulatory gene is increased. PPARα is a nuclear receptor that, when activated by long-chain fatty acids, induces the expression of proteins that promote fatty acid uptake and oxidation. Cardiac-specific overexpression of PPARα results in cardiac dysfunction in mice exposed to high concentrations of circulating fatty acids. Pharmacological activation of PPARα in pressure-loaded rat heart causes systolic dysfunction. In diabetic and obese patients, the expression of the inflammatory cytokine tumor necrosis factor α (TNF-α) is increased in lipid overloaded tissues, showing a positive correlation with insulin resistance. TNF-α can directly cause systolic dysfunction of the heart and has been implicated in the pathological remodeling of heart failure. Accumulation of excess lipids in cardiomyocytes can lead to the production of toxic lipid intermediates, which can lead to cell death.

" Scar " refers to any scar left on the tissue after healing the wound or injury. In particular, the term relates to traces left on ischemic tissue. In the context of the present invention, scar formation is due to ischemia.

Ischemia injury is, for example, one or more surgical procedures or other procedures that restore blood flow to a tissue or organ that is a natural phenomenon (eg, recovery of blood flow after myocardial infarction), trauma, or reduced blood supply. It can be caused by many causes, including therapeutic intervention in the bloodstream. Examples of such surgical procedures include coronary artery bypass grafting, graft surgery, coronary angioplasty, organ transplantation surgery, and the like (for example, cardiopulmonary bypass surgery).

Prevention of ischemic injury can be achieved by administering ApoJ-Glyc before the onset of ischemia and by administering ApoJ-Glyc after the onset of ischemia but before the onset of ischemic injury. Will be understood. In a preferred embodiment, ApoJ is administered prior to the initiation of ischemia induction. In a more preferred embodiment, ApoJ is at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours before the onset of ischemia. It is administered 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, or earlier.

In one embodiment, the damage caused by ischemic injury results from cerebral ischemia. In another embodiment, damage to cerebral tissue is caused by ischemic stroke. The term "ischemic stroke" refers to the sudden loss of brain function caused by the obstruction of blood vessels to the brain (causing a lack of oxygen to the brain), muscle loss, loss of sensation or consciousness, It is characterized by symptoms that vary depending on the degree and severity of brain damage, such as dizziness and obscure speech, and is also called a stroke or cerebrovascular accident. Also, the term "cerebral ischemia" (or "stroke") refers to a lack of blood supply to the brain, often resulting in a lack of oxygen to the brain.

In one embodiment, the damage caused by ischemia is due to cardiac ischemia. In an even more preferred embodiment, cardiac ischemia results from myocardial ischemia.

The term "myocardial ischemia" refers to circulatory disorders caused by coronary atherosclerosis and / or lack of oxygen supply to the myocardium. For example, acute myocardial infarction is an irreversible ischemic invasion of myocardial tissue. This invasion causes obstructive (eg, thrombotic or embolic) events in the coronary circulation, creating an environment in which myocardial metabolic demand exceeds the oxygen supply to myocardial tissue.

In certain embodiments, the injury is caused by microvascular angina. As used herein, the term "microvascular angina" refers to a condition that results from inadequate blood flow through the small blood vessels of the heart.

In a preferred embodiment, cardiac ischemia results from an acute myocardial infarction.

As used herein, " myocardial infarction " (MI) or "acute myocardial infarction" is ischemic necrosis of part of the myocardium due to obstruction of one or several coronary arteries or branches thereof. Myocardial infarction is characterized by the loss of functional cardiomyocytes, which irreversibly damages myocardial tissue. As coronary artery disease progresses, the myocardium becomes infarcted. As a specific example, infarction occurs when an atherosclerotic plaque present in a coronary artery ulcerates or ruptures, causing an acute occlusion of the blood vessel.

In certain embodiments, ischemic injury results from coronary artery occlusion (myocardial infarction), as well as revascularization and blood reperfusion.

In a preferred embodiment, ApoJ-Glyc for use in the prevention of ischemic injury is administered prior to ischemia. The administration time before the onset of ischemia is not particularly limited. In a preferred embodiment, ApoJ-Glyc is at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 30 seconds, or at least 45 seconds before ischemia, preferably ischemia. At least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours, 3 hours, or even before ischemia. Is administered to.

In a preferred embodiment, ApoJ-Glyc for use in the prevention of ischemic injury is administered in a therapeutically effective amount.

The term " therapeutically effective amount ", when used herein in connection with the use of ApoJ-Glyc in the prevention of ischemic injury, clearly prevents, cures, or delays one or more symptoms derived from a disease. It relates to an amount of ApoJ-Glyc sufficient to achieve a reduction or improvement in severity, which is generally determined in particular by the characteristics of the drug itself and the therapeutic effect to be achieved. It also depends on the subject being treated, the severity of the disease the subject is suffering from, the dosage form selected and the like. Therefore, the doses described in the present invention should be considered only as a guide for those skilled in the art, and those skilled in the art must adjust the dose according to the uncertainties mentioned above. In one embodiment, the effective amount ameliorate one or more symptoms of the disease to be treated.

The determination of the optimal range for a therapeutically effective amount of the above compounds for use according to the present invention, even if individual needs differ, is based on the routine experience of one of ordinary skill in the art. In general, the dosage required to provide effective treatment can be adjusted by those skilled in the art, including age, health, adaptability, gender, diet, weight, degree of receptor change, frequency of treatment, injury. Pharmacological considerations such as nature and condition, nature and extent of disorder or disease, subject's medical condition, route of administration, activity, efficacy, pharmacokinetic and toxicological profile of the particular compound used, drug delivery It depends on whether the system is used and whether the above compounds are administered as part of a drug combination. The amount of compound for use according to the invention that is therapeutically effective in the prevention of ischemic injury in a subject can be determined by conventional clinical techniques (eg, The Physician's Desk Reference, Medical Electricals Company, Inc., Inc. See Oradell, NJ, 1995, and Drug Factors and Comparisons, Inc., St. Louis, MO, 1993).

In a preferred embodiment, ApoJ-Glyc for use in the prevention of ischemic injury is administered in a dose range between 0.1 mg / kg and 1 mg / kg. In a more preferred embodiment, ApoJ-Glyc is 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 Is administered in the dose range of. In an even more preferred embodiment, ApoJ-Glyc is administered at a dose of 0.5 mg / kg.

Non-limiting routes of administration of ApoJ-Glyc include, in particular, non-invasive pharmacological routes of administration, such as oral, gastrointestinal, nasal, or sublingual routes, and invasive routes of administration, such as parenteral routes. Can be mentioned. In certain embodiments, ApoJ-Glyc is administered in therapeutically effective amounts by parenteral routes (eg, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intrathecal, etc.). "Administration by parenteral route" is understood as a route of administration consisting of administering the compound of interest by injection and therefore requires the use of a syringe and a needle. Intramuscular (injecting the compound into the muscle tissue), intravenously (injecting the compound into the vein), subcutaneously (injecting under the skin), and intradermally (injecting the layer of skin), depending on the tissue the needle reaches. There are various types of parenteral punctures, such as injections in between). The intrathecal route is used to administer a drug that does not sufficiently penetrate the blood-brain barrier to the central nervous system, thereby administering the drug to the space around the spinal cord (intrathecal space). In a preferred embodiment, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intrathecal administration. In a preferred embodiment, the route of administration is intravenous.

In a preferred embodiment, ApoJ-Glyc is administered as a single dose. As used herein, a "single dose" is ApoJ-, a physically separated unit administered in a single dose / single dose / single route / single contact. Glyc dose. The single dose may be administered once or several times daily. In certain embodiments, the single dose is administered once daily. In another embodiment, the single dose is administered twice daily. For example, a single dose may be divided into two doses and each half of the divided dose may be administered to the subject at different times of the day.

In a preferred embodiment, ApoJ-Glyc is at least 1 second before ischemia, typically at least 15 seconds, 30 seconds or 45 seconds before ischemia, preferably at least 1 minute before ischemia. It is administered 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours, 3 hours, or even before ischemia.

Treatment of Ischemia Injuries In a second aspect, the invention relates to glycosylated apolipoprotein J (ApoJ-Glyc) or non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemic injury.

The term " glycosylated ApoJ " is defined with respect to the first aspect of the invention and is used herein as well.

The term " non-glycosylated ApoJ " as used herein refers to an N-glycosylation site (with respect to an ApoJ preproprotein sequence defined as being registered in the NCBI database at accession number NP_001822.3). , 86th, 103rd, 145th, 291st, 317th, 354th and 374th amino acids) are not glycosylated ApoJ.

The term " therapeutic " as used herein refers to both therapeutic and prophylactic measures, the purpose of which is to prevent or prevent unwanted physiological changes or disorders such as ischemic reperfusion injury. It is to slow down (reduce). Beneficial or desired clinical outcomes, whether detectable or undetectable, are relief of symptoms, reduction of the extent of the disease, stabilization of the condition (ie, not exacerbated), delay in disease progression. Alternatively, it may include, but is not limited to, slowing, improving or alleviating the condition, and remission (whether partial or complete remission). "Treatment" may also mean prolongation of survival compared to the expected survival without treatment. Those in need of treatment include not only those who already have the symptoms or disability, but also those who are susceptible to the symptoms or disability, or who should prevent the symptoms or disability.

In a preferred embodiment, ApoJ-Glyc is a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue. In another preferred embodiment, ApoJ-Glyc is a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue and a sialic acid residue.

In a preferred embodiment, ApoJ-Glyc is the 86th, 103rd, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). N-glycans are contained in a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another preferred embodiment, glycosylated ApoJ is registered at all N-glycosylation sites within ApoJ, ie, in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the ApoJ preproprotein sequence defined as, each Asn at positions 86, 103, 145, 291 and 317, 354 and 374 contains an N-glycan. In another embodiment, glycosylated ApoJ is at positions 86 and 103 with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). At least one, at least two, at least three, at least four, at least five, at least six or at least selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. At least 7 Asn residues contain N-glycans.

In a preferred embodiment, ApoJ-Glyc is the 86th, 103rd, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). It is a single type glycosylated ApoJ molecule glycosylated with a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another preferred embodiment, ApoJ-Glyc is a combination of glycosylated ApoJ molecules, each molecule as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the defined ApoJ preproprotein sequence, it may be glycosylated at different sites selected from the group consisting of Asn residues at positions 86, 103, 145, 291 and 317, 354 or 374. .. In another preferred embodiment, ApoJ-Glyc is registered at all N-glycosylation sites within ApoJ, i.e., registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the defined ApoJ preproprotein sequence, it is a single type of ApoJ-Glyc glycosylated at each Asn at positions 86, 103, 145, 291 and 317, 354 and 374.

In a preferred embodiment, ApoJ-Glyc for use according to this aspect of the invention is purified from human plasma as described above. In another preferred embodiment, ApoJ-non-Glyc for use according to the invention is recombinant ApoJ produced recombinantly in an organism lacking the mechanism required for N-type glycosylation. In a preferred embodiment, ApoJ-non-Glyc is a well-known procedure in the art as described in Dubbs and Wilson (Plos One, 9 (1): e86989. Doi: 10.1371 / journal. Pone. 0086689). , Or E. as described in Rosano and Ceccarelli (Frontiers in Microbiology, 2014, doi: 10.3389 / fmicb. 2014.00172). Using any other method suitable for expression of the recombinant protein in coli , E. It is of recombinant origin obtained by expression with colli. In one embodiment, ApoJ-non-Glyc is expressed under conditions where this molecule is expressed in inclusion bodies. It is expressed in coli, its inclusion bodies are purified, and the protein is solubilized in the presence of a chaotropic agent such as urea and / or a disulfide group-containing reagent such as DTT.

In a preferred embodiment, the ischemic injury is infarction, atherosclerosis, thrombosis, thromboembolism, lipid embolism, bleeding, stent, surgery, angioplasty, termination of intraoperative bypass, organ transplantation, complete ischemia. , And a condition selected from the group consisting of combinations thereof.

In another preferred embodiment, ischemic injury occurs in an organ or tissue selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof.

In another preferred embodiment, the ischemic injury is an organ dysfunction (in an ischemic organ or any other organ), infarction, inflammation (in a damaged organ or tissue), oxidative damage, mitochondrial membrane potential damage, apoptosis. , Reperfusion-related arrhythmias, cardiac stunning, cardiac lipotoxicity, ischemic scar formation, and combinations thereof.

In a preferred embodiment, the damage caused by ischemic injury results from cerebral ischemia.

In another preferred embodiment, the damage caused by ischemia is due to cardiac ischemia. In an even more preferred embodiment, cardiac ischemia results from myocardial ischemia or acute myocardial ischemia.

In a preferred embodiment, ApoJ-Glyc for use in the treatment of ischemic injury is administered during ischemia. In another embodiment, ApoJ-Glyc for use according to the invention is administered after the onset of ischemia.

More preferably, ApoJ-Glyc for use according to the invention is administered during a widely variable period. In a preferred embodiment, ApoJ-Glyc is at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 30 seconds or at least 45 seconds after the onset of ischemia, preferably from the onset of ischemia. Administered at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours, 3 hours, or even after the onset of ischemia. Ischemia.

In a preferred embodiment, ApoJ-Glyc for use in the treatment of ischemic injury is administered in a therapeutically effective amount.

In one embodiment, ApoJ-Glyc and / or non-Glyc-ApoJ for use in the treatment of ischemic injury is administered during ischemia. In a preferred embodiment, ApoJ-Glyc and / or non-Glyc-ApoJ for use according to the invention are administered after the onset of ischemia.

More preferably, ApoJ-Glyc and / or non-Glyc-ApoJ for use according to the invention are administered during a widely variable period. In a preferred embodiment, it is administered at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 30 seconds, 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, or even at least 1 hour, 2 hours or after ischemia. It is administered after 3 hours or even later.

In a preferred embodiment, ApoJ-Glyc for use according to the invention is purified from human plasma. In another preferred embodiment, the non-Glyc-ApoJ for use according to the invention is a human recombinant. In another preferred embodiment, ApoJ-Glyc for use according to the invention is purified from human plasma and non-Glyc-ApoJ is a human recombinant.

In a preferred embodiment, ApoJ-Glyc and / or non-Glyc-ApoJ for use in the treatment of ischemic injury is administered in a dose range between 0.1 mg / kg and 2 mg / kg. In a more preferred embodiment, ApoJ-Glyc is 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 Doses between ~ 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 Administered in a range. In a further preferred embodiment, ApoJ-Glyc is 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 an even more preferred embodiment, ApoJ-Glyc is administered at a dose of 0.5 mg / kg.

In a preferred embodiment, the route of administration is intravenous.

In a preferred embodiment, ApoJ-Glyc or non-Glyc-ApoJ is administered as a single dose. As used herein, a "single dose" is ApoJ-, a physically separated unit administered in a single dose / single dose / single route / single contact. Glyc or non-Glyc-ApoJ dose. The single dose may be administered once or several times daily. In certain embodiments, the single dose is administered once daily. In another embodiment, the single dose is administered twice daily. For example, a single dose may be divided into two doses and each half of the divided dose may be administered to the subject at different times of the day.

In a preferred embodiment, ApoJ-Glyc is at least 1 second after ischemia, typically at least 15 seconds, 30 seconds or 45 seconds after ischemia, preferably at least 1 minute and 5 minutes after ischemia. After, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours, 3 hours, or even after ischemia.

All the terms and embodiments mentioned above with respect to the prevention of ischemic injury are similarly applicable to this aspect of the invention.

Treatment of ischemia-reperfusion injury We also in a mouse ischemia-reperfusion model early after ischemia with glycosylated apolipoprotein J (ApoJ-Glyc) or non-glycosylated apolipoprotein J (non-Glyc-ApoJ). ) Was administered, and it was found that the infarct size was significantly reduced as compared with the placebo group. Accordingly, in another aspect, the invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc-ApoJ) for use in the treatment of ischemic reperfusion injury. Glyc or non-Glyc-ApoJ is administered after the onset of ischemia and before reperfusion.

Administration of glycosylated apolipoprotein J (ApoJ-Glyc) or non-glycosylated apolipoprotein J (non-Glyc-ApoJ) according to this embodiment by mitigating the damage caused by ischemia is the damage caused by subsequent reperfusion. It will be understood that it is even more advantageous in that it also leads to mitigation of. This prevents damage associated with reperfusion. Therefore, in another embodiment, the invention relates to a glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc-ApoJ) for use in the prevention of ischemic reperfusion injury. -Glyc or non-Glyc-ApoJ is administered after the onset of ischemia and before reperfusion.

The term " reperfusion " as used herein relates to the restoration of blood flow to ischemic tissue. Although reperfusion of blood into ischemic tissue is undoubtedly beneficial, it is known that reperfusion itself can paradoxically cause a cascade of adverse reactions that damage the tissue.

As used herein, the term "ischemic reperfusion injury" is also known as "ischemic reperfusion injury" and is an organ caused by the restoration of blood supply to an organ or tissue after an ischemic period. Or it is about tissue damage. The lack of oxygen and nutrients from the blood during the ischemic period leads to a condition in which restoration of circulation results in inflammation and oxidative damage due to the induction of oxidative stress rather than restoration of normal function. Oxidative stress associated with reperfusion may damage the affected tissue or organ. Ischemia reperfusion injury is characterized by oxygen depletion during ischemic events and subsequent reoxygenation during reperfusion and associated production of reactive oxygen species.

The injury caused by ischemic reperfusion is the result of the interaction between the substances accumulated during ischemia and the substances delivered during reperfusion. The cornerstone of these events is oxidative stress, which is defined as the imbalance between oxygen radicals and the endogenous exclusion system. The result is cytotoxicity and cell death, which is initially localized but eventually systemic if the inflammatory response is not suppressed.

In one embodiment, glycosylated apolipoprotein J (ApoJ-Glyc) and / or non-glycosylated apolipoprotein J (non-Glyc-ApoJ) are administered after the onset of ischemia and before reperfusion.

In a preferred embodiment, ApoJ-Glyc is a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue. In another preferred embodiment, ApoJ-Glyc is a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue and a sialic acid residue.

In a preferred embodiment, ApoJ-Glyc is the 86th, 103rd, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). N-glycans are contained in a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another preferred embodiment, glycosylated ApoJ is registered at all N-glycosylation sites within ApoJ, ie, in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the ApoJ preproprotein sequence defined as, each Asn at positions 86, 103, 145, 291 and 317, 354 and 374 contains an N-glycan. In another embodiment, glycosylated ApoJ is at positions 86 and 103 with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). At least one, at least two, at least three, at least four, at least five, at least six, selected from the group consisting of Asn residues 145, 291 317, 354 or 374. Alternatively, at least 7 Asn residues contain N-glycans.

In a preferred embodiment, ApoJ-Glyc is the 86th, 103rd, with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). It is a single type glycosylated ApoJ molecule glycosylated with a single Asn residue selected from the group consisting of Asn residues at positions 145, 291 and 317, 354 or 374. In another preferred embodiment, ApoJ-Glyc is a combination of glycosylated ApoJ molecules, each molecule as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). With respect to the defined ApoJ preproprotein sequence, it is glycosylated at any possible site selected from the group consisting of Asn residues at positions 86, 103, 145, 291 and 317, 354 or 374. May be. In another preferred embodiment, ApoJ-Glyc is registered at all N-glycosylation sites within ApoJ, i.e., registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). ApoJ is a single type of glycosylated ApoJ glycosylated at the 86th, 103rd, 145th, 291st, 317th, 354th and 374th Asns with respect to the ApoJ preproprotein sequence defined as.

Non-glycosylated ApoJ is 86th, 103rd, 145th, 291st with respect to the ApoJ preproprotein sequence defined as being registered in the NCBI database with accession number NP_001822.3 (published September 23, 2018). N-glycans are not contained in any of the Asn residues at positions 317, 354 and 374.

In a preferred embodiment, ApoJ-Glyc for use according to this aspect of the invention is purified from human plasma. In another preferred embodiment, the non-glycosylated ApoJ for use according to this aspect of the invention is a human recombinant.

In a preferred embodiment, the ischemic reperfusion injury is infarction, atherosclerosis, thrombosis, thromboembolism, lipid embolism, bleeding, stent, surgery, angioplasty, termination of intraoperative bypass, organ transplantation, complete. It is due to ischemia and a condition selected from the group consisting of combinations thereof.

In another preferred embodiment, ischemia-reperfusion injury occurs in an organ or tissue selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof.

In another preferred embodiment, ischemia-reperfusion injury is organ dysfunction, infarction, inflammation, oxidative damage, mitochondrial membrane potential damage, apoptosis, reperfusion-related arrhythmia, cardiac stunning, cardiac lipotoxicity. , Scar formation due to ischemia, and a combination thereof.

In one embodiment, the damage caused by ischemia-reperfusion injury is due to cerebral ischemia.

In one embodiment, the damage caused by ischemic reperfusion is due to cardiac ischemia. In an even more preferred embodiment, cardiac ischemia results from myocardial ischemia.

In a preferred embodiment, cardiac ischemia results from acute myocardial ischemia.

In a preferred embodiment, ApoJ-Glyc or non-Glyc-ApoJ for use in the prevention of ischemia-reperfusion injury is administered after the onset of ischemia and before reperfusion.

The timing of administration of ApoJ-Glyc or non-Glyc-ApoJ for use in the prevention of ischemic reperfusion injury is not particularly limited as long as it is administered before the start of reperfusion. In a preferred embodiment, ApoJ-Glyc or non-Glyc-ApoJ is administered at least 1 second, at least 5 seconds, at least 10 seconds, at least 15 seconds, at least 30 seconds, or at least 45 seconds after ischemia. To. In another preferred embodiment, at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours or after ischemia. It is administered after 3 hours or even later.

In a preferred embodiment, glycosylated apolipoprotein J (ApoJ-Glyc) and / or non-glycosylated apolipoprotein J (non-Glyc-ApoJ) are administered in therapeutically effective amounts.

In a preferred embodiment, ApoJ-Glyc for use in the prevention of ischemic injury is administered in a dose range between 0.1 mg / kg and 1 mg / kg. In a more preferred embodiment, ApoJ-Glyc is 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 Is administered in the dose range of. In an even more preferred embodiment, ApoJ-Glyc is administered at a dose of 0.5 mg / kg.

Suitable routes of administration are those defined above with respect to the first and second aspects of the invention.

In a preferred embodiment, the route of administration is intravenous.

In a preferred embodiment, ApoJ-Glyc for use according to the invention is purified from human plasma. In another preferred embodiment, the non-Glyc-ApoJ for use according to the invention is a human recombinant. In another preferred embodiment, ApoJ-Glyc for use according to the invention is purified from human plasma and non-Glyc-ApoJ is a human recombinant.

In a preferred embodiment, ApoJ-Glyc or non-Glyc-ApoJ is administered as a single dose. As used herein, a "single dose" is ApoJ-Glyc, a physically separated unit administered in a single dose / single dose / single route / single contact. Or the dose of non-Glyc-ApoJ. The single dose may be administered once or several times daily. In certain embodiments, the single dose is administered once daily. In another embodiment, the single dose is administered twice daily. For example, a single dose may be divided into two doses and each half of the divided dose may be administered to the subject at different times of the day.

In a preferred embodiment, ApoJ-Glyc or non-Glyc-ApoJ is at least 1 second prior to reperfusion, typically at least 15 seconds, 30 seconds or 45 seconds prior to reperfusion, preferably reperfusion. At least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or even at least 1 hour, 2 hours, 3 hours, or even before reperfusion. Be administered.

All the terms and embodiments mentioned above with respect to the prevention of ischemic injury are similarly applicable to this aspect of the invention.

The present invention will be described in detail below with reference to the following examples, but these are merely exemplary and do not limit the scope of the invention.

Materials and Methods Rodents Welfare Mouse Models: 11-week-old C3H male mice were obtained from Janvier Labs (France). Animals were acclimatized for one week and bred on free feeding. Approval of the Institutional Review Board for Animal Care was obtained in accordance with appropriate rules and procedures for animal rearing (Generalitat de Catalunya DOGC2073, 1995).

Rat model: Mature Sprague Dayley rats were used to receive protein (approximately 250 mg by weight) intravenously. Animals were acclimatized for 5 days according to standard procedures and fed freely. After obtaining the approval of the Animal Care and Use Ethics Committee, appropriate rules and procedures regarding animal breeding were followed.

Rodent anesthetized animals were deeply anesthetized with ketamine-xylazine (100 mg / kg and 10 mg / kg; IP) and buprenorphine (0.075 mg / kg) administered as an analgesic. The animals were then intubated and connected to a mouse ventilator using a 20 G cannula (Inspira asv, Harvard Apparatus). In addition, electrocardiograms (ECG, rodent surgical monitors, Indus Instruments) of each animal were obtained on a temperature controlled operating table after chest thoracotomy.

Myocardial ischemia surgery by ligation of the left anterior descending coronary artery Left thoracotomy was performed between the 4th intercostal space, and the ribs were separated with a retractor for microscopic surgery. The pericardium was opened, and the left anterior descending artery (LAD) was confirmed and ligated with 7-0 silk suture.

Ischemia mouse model mice were occluded for 45 minutes. To avoid dehydration, the chest was temporarily closed with animal skin and a small clamp. After 45 minutes of ischemia, blood was extracted using EDTA as an anticoagulant. Finally, the animals were sacrificed, the heart was removed and immersed in PBS for a few minutes, and the liver was frozen at −80 ° C.

Mouse model of ischemia-reperfusion In the ischemia -reperfusion model, LAD was restored 45 minutes after ischemia to restore blood flow. After 2 hours, LAD was religated and blood was extracted using EDTA as an anticoagulant. The risk area (AAR) was then defined by intravenous administration of 1% Evans Blue in 0.9% NaCl (Sigma-Aldrich, USA). As mentioned above, the animals were sacrificed, the heart was removed and immersed in PBS for a few minutes and the liver was frozen at −80 ° C.

Rat model of ischemia-reperfusion In the ischemia -reperfusion model, LAD was restored 45 minutes after ischemia to restore blood flow. After 24 hours, the LAD was religated and blood was extracted using EDTA as an anticoagulant. The risk area (AAR) was then defined by intravenous administration of 1% Evans Blue in 0.9% NaCl (Sigma-Aldrich, USA). As mentioned above, the animals were sacrificed, the heart was removed and immersed in PBS for a few minutes and the liver was frozen at −80 ° C.

In a pilot study of product-administered mice, ApoJ-Glyc or non-Glyc ApoJ (3 mg / kg, ip) was administered 5 minutes prior to MI induction. In the preclinical mouse study, 6 mg / kg of each treatment was applied to both the ischemia model and the ischemia reperfusion model 10 minutes after MI induction. p. I injected it. In the rat study, 0.75 mg / kg was administered intravenously (iv). The placebo group received the same dose of vehicle (PBS) (ip in mice, iv in rats).

Measurement of infarct area In the pilot mouse study, morphological evaluation of infarct size was performed by immunohistochemical analysis. After the procedure, the mouse heart was immersed in a fixed solution (4% paraformaldehyde) and embedded in an OCT compound to prepare cross-sections from the apex to the base (10 μm thick sections at 200 μm intervals). Sections were stained with hematoxylin and eosin, and morphometric analysis of infarct size was performed by a blinded, skilled operator using image analysis software (ImageJ, NIH). Infarct size was calculated as the sum of myocardial infarct areas between sections and expressed as a percentage of the total LV wall surface. Three measurements were made per section.

In preclinical mouse and rat studies, after the procedure, the heart was frozen at −80 ° C. for 5 minutes and a 1 mm thick section was obtained using a blade. Samples were incubated with 1% TTC (tetrazolium chloride, Sigma-Aldrich, USA) in PBS at 37 ° C. for 15 minutes. After staining, both sides of the sample were imaged and quantification of the infarcted area was performed by a blinded skilled operator using ImageJ (NIH, Bethesda, Maryland, USA). Results were expressed as a percentage of infarcted area to total left ventricular area.

Determination of Risk Area (AAR) In preclinical mouse and rat studies, both sides of the sample were imaged after staining to quantify the infarct area (IS) by a blinded expert operator ImageJ (NIH, Bethesda). , Maryland, USA). Results are expressed as a percentage of infarcted area to total left ventricular area. Results were expressed as percentage of risk area (AAR), percentage of infarcted area (IS), and infarcted area / risk area (IS / AAR) for the left ventricle.

The normality of the statistical analysis data was tested by the Shapiro-Wilk test. After that, ANOVA post-hook analysis was also performed (ANOVA; StatView, SAS Institute).

Example 1: In an ischemic mouse model, treatment with ApoJ-Glyc prior to myocardial infarction reduces myocardial injury: possible prophylactic role against ischemic injury.
In the pilot study, ApoJ-Glyc or non-Glyc ApoJ (3 mg / kg, ip) was administered 5 minutes prior to MI induction (Fig. 1, top). In a mouse model of ischemia, administration of ApoJ-Glyc reduced infarct size (percentage of left ventricle assessed by immunohistochemical analysis) by an average of 17% compared to the placebo group, resulting in ischemic damage to the heart. I was able to prevent it. This effect was not observed when non-Glyc ApoJ was administered (Fig. 2).

Furthermore, when the total ApoJ level in serum was analyzed using a commercially available ELISA kit, the ApoJ circulation level was significantly increased (FIG. 3).

Example 2: In an ischemic mouse model, treatment with ApoJ-Glyc and non-Glyc ApoJ after ischemia induction reduces ischemic heart injury: possible therapeutic role for ischemic injury.
In the ischemia model (Fig. 1, middle), ApoJ-Glyc or non-Glyc ApoJ was administered 10 minutes after the induction of ischemia. After 45 minutes of ischemia, blood was extracted, the animals were sacrificed, the heart was removed and frozen at -80 ° C.

In a mouse model of acute myocardial infarction due to left anterior descending artery ligation, administration of either ApoJ-Glyc or non-Glyc ApoJ (6 mg / kg, ip) early after ischemia significantly reduced the infarct size (6 mg / kg, ip). 15% and 12%, respectively, relative to the placebo group) (Table 1 and Figure 4).

Example 3: In a mouse model of ischemia-reperfusion, treatment with ApoJ-Glyc and non-Glyc ApoJ after ischemia induction reduces ischemic heart injury: possible therapeutic role for ischemia-reperfusion injury ..
In the ischemia-reperfusion model (Fig. 1, lower row), LAD was restored 45 minutes after ischemia, and blood flow was restored by reperfusion for 2 hours. In this mouse model of 45-minute ischemia and 2-hour reperfusion by ligation of the left anterior descending artery, administration of either ApoJ-Glyc or non-Glyc ApJ early after ischemia (10 minutes for induction of ischemia) was compared with the placebo group. The significant reduction in infarct size resulted in effective treatment of ischemic heart injury and prevention of further injury caused by reperfusion.

Example 4: In a rat model of ischemia-reperfusion, treatment with ApoJ-Glyc and non-Glyc ApoJ after ischemia induction reduces ischemic heart injury: possible therapeutic role for ischemia-reperfusion injury ..
In the ischemia-reperfusion model (FIG. 6), LAD was restored 45 minutes after ischemia, and blood flow was restored by reperfusion for 24 hours. Administration of either ApoJ-Glyc or non-Glyc ApoJ after 45 minutes of ischemia and before 24 hours of reperfusion significantly reduced ischemic damage to the heart. This effect is due to a reduction in infarct size (ratio of infarct size (IS) to risk area (AAR), FIG. 7), as well as an improvement in left ventricular end-systolic pressure (LVESS, FIG. 8) and an improvement in left ventricular relaxation (FIG. 9). ) Proven. Plasma myocardial troponin I levels were significantly reduced in rats treated with ApoJ-Glyc, but not in rats treated with non-GlycApoJ (FIG. 10).

Example 5: In a rat model of ischemia-reperfusion, treatment with ApoJ-Glyc and non-Glyc ApoJ prior to myocardial infarction reduces myocardial injury: possible prophylactic role for ischemia-reperfusion injury.
In the ischemia-reperfusion model (FIG. 11), LAD was restored 45 minutes after ischemia, and blood flow was restored by reperfusion for 24 hours. Administration of either ApoJ-Glyc or non-Glyc ApoJ prior to 45-minute ischemia and 24-hour reperfusion significantly prevented ischemic damage to the heart. This effect is associated with reduced infarct size (ratio of infarct size (IS) to risk area (AAR), FIG. 12), as well as improved left ventricular end-systolic pressure (LVESS, FIG. 13) and improved left ventricular relaxation (FIG. 14). ) Proven. Administration of either ApoJ-Glyc or non-Glyc ApoJ did not significantly change plasma myocardial troponin I levels (FIG. 15).

Claims (12)

Glycosylated apolipoprotein J (ApoJ-Glyc) for use in the prevention of ischemic injury. Glycosylated apolipoprotein J (ApoJ-Glyc) or non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemic injuries. A glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use in the treatment of ischemia-reperfusion injury, which is administered after the onset of ischemia and before reperfusion. ApoJ-Glyc or non-Glyc ApoJ. Claimed that the glycosylated ApoJ is a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue or a glycosylated ApoJ containing an N-acetylglucosamine (GlcNAc) residue and a sialic acid residue. Glycosylated ApoJ for use according to item 1, 2 or 3. The glycosylated ApoJ for use according to any one of claims 1 to 4, wherein the glycosylated ApoJ is obtained by purification from human plasma. The ischemic injury or ischemia-reperfusion injury is infarction, atherosclerosis, thrombosis, thromboembolism, lipid embolism, bleeding, stent, surgery, angioplasty, termination of intraoperative bypass, organ transplantation, complete The glycosylated apolipoprotein J (ApoJ-Glyc) for use according to any one of claims 1 to 4, characterized in that it is due to ischemia and a condition selected from the group consisting of combinations thereof. ) Or non-glycosylated apolipoprotein J (non-Glyc ApoJ). The ischemic injury or ischemia-reperfusion injury is characterized in that it occurs in an organ or tissue selected from the group consisting of heart, liver, kidney, brain, intestine, pancreas, lung, skeletal muscle and combinations thereof. A glycosylated apolipoprotein J (ApoJ-Glyc) or a non-glycosylated apolipoprotein J (non-Glyc ApoJ) for use according to any one of claims 1 to 6. The ischemic injury or ischemia-reperfusion injury is organ dysfunction, infarction, inflammation, oxidative injury, mitochondrial membrane potential injury, apoptosis, reperfusion-related arrhythmia, cardiac stunning, cardiac lipotoxicity, imagination. The glycosylated apolipoprotein J (ApoJ-Glyc) for use according to any one of claims 1 to 7, characterized in that it is selected from the group comprising scar formation by blood, and combinations thereof. Non-glycosylated apolipoprotein J (non-Glyc ApoJ). Glycosylated ApoJ or non-glycosylated for use according to any one of claims 1-8, wherein the ischemic injury or ischemia-reperfusion injury is due to cerebral ischemia or cardiac ischemia. ApoJ. The glycosylated ApoJ or non-glycosylated ApoJ for use according to claim 9, wherein the cardiac ischemia is caused by an acute myocardial infarction (AMI). The glycosylated ApoJ or non-glycosylated ApoJ for use according to any one of claims 1 to 10, wherein the glycosylated ApoJ or non-glycosylated ApoJ is administered intravenously. The glycosylated ApoJ or non-glycosylated ApoJ for use according to any one of claims 1 to 11, wherein the glycosylated ApoJ or non-glycosylated ApoJ is administered as a single dose.

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